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Rebecca S Siegel, Daniel P DicksteinPediatric Mood, Imaging, and NeuroDevelopment Program, EP Bradley Hospital, East Providence, RI, USAAbstract: Anxiety disorders are the most prevalent mental health concern facing adolescents today, yet they are largely undertreated. This is especially concerning given that there are fairly good data to support an evidence-based approach to the diagnosis and treatment of anxiety, and also that untreated, these problems can continue into adulthood, growing in severity. Thus, knowing how to recognize and respond to anxiety in adolescents is of the utmost importance in primary care settings. To that end, this article provides an up-to-date review of the diagnosis and treatment of anxiety disorders geared towards professionals in primary care settings. Topics covered include subtypes, clinical presentation, the etiology and biology, effective screening instruments, evidence-based treatments (both medication and therapy), and the long-term prognosis for adolescents with anxiety. Importantly, we focus on the most common types of anxiety disorders, often known as phobias, which include generalized anxiety disorder, social anxiety/social phobia, separation anxiety disorder, panic disorder, and specific phobias. In summary, anxiety is a common psychiatric problem for adolescents, but armed with the right tools, primary care providers can make a major impact.Keywords: anxiety disorders, adolescents, presentation, etiology, assessment, treatment, primary care

Background : Patients with cholangiocarcinoma often have indwelling biliary stents or catheters which are prone to obstructions and/or infections; studies show that 20-40% present with fever and/or jaundice requiring urgent treatment in the outpatient setting for which there are no uniform guidelines. The goal was to develop an expert panel consensus on this topic using the modified RAND/UCLA Delphi process to rate treatment appropriateness. Methods : Thirteen expert physicians from relevant specialties, geography, and practice settings were recruited for the panel. Patient scenarios were developed and panelists rated the therapies before and after a face-to-face discussion. The appropriateness of various therapies was rated on a scale from 1-9 and classified as appropriate, inappropriate, or uncertain. Scenarios with greater than 2 (>2) ratings of 1-3 (inappropriate) and greater than 2 (>2) ratings of 7-9 (appropriate) were considered to have disagreement and were not assigned an appropriateness rating. Results : Panelists were from all US regions and the UK (8%) and had practiced for a mean 16.5 years (4-33 years). Panelists rated 480 scenarios before the meeting and re-rated 288 of the clinical scenarios after the meeting. The panelists agreed that ongoing treatment with chemotherapy did not influence decision-making and, therefore, 192 scenarios were excluded from the final list. Disagreement decreased from 37.5% before to 10.4% after the meeting. Consensus on stent/tube manipulation and inpatient antibiotic therapy was obtained and summarized in patients as "appropriate" or "maybe appropriate" based on a patient's bilirubin level at presentation. Conclusions : The Delphi process produced consensus guidelines to fill an unmet need in the urgent management of ascending cholangitis in patients with cholangiocarcinoma.

Background: Lymphovascular invasion and perineural invasion are histopathological features associated with higher-risk colon cancer., Objective: The purpose of this study was to quantify the impact of lymphovascular and perineural invasion on overall survival after diagnosis and to determine the protective effect of adjuvant chemotherapy for early adenocarcinoma with high-risk factors., Design: This was a retrospective database review of the 2010-2014 National Cancer Database for colon cancer., Settings: Individuals diagnosed with invasive adenocarcinoma of the colon (histology code 8140) with primary surgical resection with >12 nodes harvested and no positive nodes on pathological examination were included., Patients: A total of 32,493 patients underwent surgical resection for stage II adenocarcinoma of the colon., Interventions: The study involved multivariate Cox regression analysis of the impact of lymphovascular and perineural invasion and adjuvant chemotherapy on overall survival after a diagnosis of stage II adenocarcinoma of the colon., Main Outcome Measures: Survival after a diagnosis of stage II adenocarcinoma of the colon was measured., Results: Five-year survival after diagnosis and surgical resection without adjuvant chemotherapy was lower for patients with lymphovascular (60.0%), perineural (56.9%), and lymphovascular and perineural invasion (55.8%) compared with double-negative disease (66.1%). Log-rank testing confirmed that adjuvant chemotherapy improved 5-year survival after diagnosis for lymphovascular (85.5%), perineural (83.6%), and lymphovascular and perineural invasion (74.3%). After controlling for differences in cohorts, Cox regression analysis showed an increased HR for mortality of 14.0% for lymphovascular (HR = 1.141 (95% CI, 1.060-1.228)), 32.1% for perineural (HR = 1.321 (95% CI, 1.176-1.483)), and 41.0% for lymphovascular and perineural invasion (HR = 1.409 (95% CI, 1.231-1.612)) compared with having neither. Chemotherapy showed a 43% reduction in hazard for mortality (HR = 0.570 (95% CI, 0.513-0.633))., Limitations: The study was limited by its retrospective review and observational bias., Conclusions: Lymphovascular and perineural invasion have a detrimental effect on survival after diagnosis of stage II adenocarcinoma of the colon. Chemotherapy may be protective specifically when lymphovascular and perineural invasion are present. See Video Abstract at http://links.lww.com/DCR/A786.

Background: With advances in myelodysplastic syndromes (MDS), patient cohorts from different time periods might be different., Objectives: To compare presentation and outcomes between two cohorts., Methods: Data were collected from George Washington University Medical Center, Washington, DC, USA 1986-1987 (DC), and Tel Aviv Medical Center, Israel 1999-2009 (TA)., Results: The study comprised 227 patients (139 TA, 88 DC). TA patients were older (75.4 ± 9.8 vs. 63.8 ± 14.3 years, P < 0.001) and had more cardiovascular diseases (56.8% vs. 14.8%, P < 0.001), fewer cytopenias (1.67 ± 0.82 vs. 2.0 ± 0.93, P = 0.003), and lower mean corpuscular volume (94.3 ± 9.9 fl vs. 100.5 ± 15.3 fl, P < 0.001). Hemoglobin, leukocyte, neutrophil, and platelet counts were similar. More TA patients had dysplasias. Bone marrow cellularity and cytogenetics were similar, but more TA patients had blasts < 5% (73.4% vs. 50.6%, P = 0.003). More TA patients had early French-American-British (FAB) disease (66.9% vs. 40.9%, P < 0.001) and lower risk disease per International Prognostic Scoring System (81% vs. 50%, P < 0.001). The 5 year survival (5YS) of TA patients was not significantly greater (62% vs. 55%). 5YS by FAB was also slightly greater for TA patients (77% vs. 65% for early FAB; 43% vs. 37% for advanced FAB, P > 0.05)., Conclusions: Although patients diagnosed with MDS at a later period were older and had more cardiovascular co-morbidities, they had fewer cytopenias, tended to have earlier disease, and had minimally greater, but not significant, 5YS.

Background: The purpose of this study was to present our evaluation of the outcome of oropharyngeal cancer managed with neoadjuvant chemotherapy and transoral surgery (TOS) with neck dissection as definitive treatment., Methods: This is a case series of 17 patients with advanced oropharyngeal cancer who were treated with neoadjuvant chemotherapy followed by TOS. The treatment details and oncologic outcome are reported. The volumetric response of the tumor to neoadjuvant chemotherapy is evaluated and validated by histopathology., Results: Seventeen patients with TNM stages III and IV oropharyngeal cancer constitute this series for survival analysis. On a median and mean follow-up of 31 and 40 months, respectively, 16 of the 17 patients were alive without recurrence. Disease-specific survival (DSS) and overall survival (OS) at 3 years were 94.1%., Conclusion: Adjuvant chemotherapy followed by TOS and neck dissection is a feasible and efficacious novel therapeutic approach for definitive management of moderately advanced oropharyngeal cancer, reserving radiotherapy (RT) for salvage or adverse features. © 2016 Wiley Periodicals, Inc. Head Neck 38: 1837-1846, 2016., (© 2016 Wiley Periodicals, Inc.)

Increased expression of progesterone receptor (PR) has been reported in gastric cancer (GC). We have previously identified a functional T889C point mutation in DNA polymerase beta (POLB), a DNA repair gene in GC. To provide a detailed analysis of molecular changes associated with the mutation, human cDNA microarrays focusing on 18 signal transduction pathways were used to analyze differential gene expression profiles between GC tissues with T889C mutant in POLB gene and those with wild type. Among the differentially expressed genes, notably, PR was one of the significantly up-regulated genes in T889C mutant POLB tissues, which were subsequently confirmed in POLB gene transfected AGS cell line. Interestingly, patients with T889C mutation and PR positivity were associated with higher incidence of intraperitoneal metastasis (IM). In vitro studies indicate that PR expression was upregulated in AGS cell line when transfected with T889C mutant expression vector. Cotransfection of T889C mutant allele and PR gene induced cell migration in the cell line. These data demonstrated that T889C mutation-associated PR overexpression results in increased IM. Therefore, T889C mutation-associated PR overexpression may serve as a biomarker for an adverse prognosis for human GC.

MicroRNA (miRNA) dysfunction is associated with a variety of human diseases, including cancer. Our previous study showed that miR-671-5p was deregulated throughout breast cancer progression. Here, we report for the first time that miR-671-5p is a tumor-suppressor miRNA in breast tumorigenesis. We found that expression of miR-671-5p was decreased significantly in invasive ductal carcinoma (IDC) compared to normal in microdissected formalin-fixed, paraffin-embedded (FFPE) tissues. Forkhead Box M1 (FOXM1), an oncogenic transcription factor, was predicted as one of the direct targets of miR-671-5p, which was subsequently confirmed by luciferase assays. Forced expression of miR-671-5p in breast cancer cell lines downregulated FOXM1 expression, and attenuated the proliferation and invasion in breast cancer cell lines. Notably, overexpression of miR-671-5p resulted in a shift from epithelial-to-mesenchymal transition (EMT) to mesenchymal-to-epithelial transition (MET) phenotypes in MDA-MB-231 breast cancer cells and induced S-phase arrest. Moreover, miR-671-5p sensitized breast cancer cells to cisplatin, 5-fluorouracil (5-FU) and epirubicin exposure. Host cell reactivation (HCR) assays showed that miR-671-5p reduces DNA repair capability in post-drug exposed breast cancer cells. cDNA microarray data revealed that differentially expressed genes when miR-671-5p was transfected are associated with cell proliferation, invasion, cell cycle, and EMT. These data indicate that miR-671-5p functions as a tumor suppressor miRNA in breast cancer by directly targeting FOXM1. Hence, miR-671-5p may serve as a novel therapeutic target for breast cancer management.

Objective: Circulating tumor cells (CTCs) have known prognostic implications in metastatic castration-resistant prostate cancer, but little is known regarding its utility in biochemical recurrence (BR) of prostate cancer. The primary objectives were to determine whether CTCs are measurable in patients with BR and whether it can reliably predict prostate-specific antigen (PSA) increase and PSA doubling times (PSADTs)., Methods: BR was identified in patients after prostatectomy or radiation or both, with a PSA increase of ≥ 0.2 for prior prostatectomy or > 2 mg/dL increase for post-nadir in prior radiotherapy. CTCs were enumerated at baseline at the time of study entry using the CellSearch (Janssen Diagnostics, Raritan, NJ) test., Results: The median age for all 36 patients accrued was 69.5 years (range, 51-91) with a median PSA of 1.65 ng/mL (range, 0.2-65.8). Gleason scores ranged from 5 to 9 (median, 7). The majority had prostatectomy (n = 25), external beam radiotherapy (n = 9), CyberKnife (Accuray, Sunnyvale, CA) (n = 1), and combined radiohormonal therapy (n = 1). PSADT ranged from 0.35 to 55 months, with a median of 7.43 months. The incidence of positive CTCs was 8.3% (3 patients), of whom 2 had biopsy-proven bony lesions on presenting with equivocal scans and PSADTs of 2.27 and 3.08 months, respectively. The third CTC-positive patient had a PSADT of 4.99 months., Conclusions: Obtaining CTCs in unselected patients presenting with BR has a relatively low yield. However, obtaining a positive CTC raises the suspicion of the presence of metastatic disease and may have utility for longitudinal follow-ups of patients with BR., (Copyright © 2015 Elsevier Inc. All rights reserved.)

This study examined the longitudinal association between mood episode severity and relationships in youth with bipolar (BP) disorder. Participants were 413 Course and Outcome of Bipolar Youth study youth, aged 12.6 ± 3.3 years. Monthly ratings of relationships (parents, siblings, and friends) and mood episode severity were assessed by the Adolescent Longitudinal Interval Follow-up Evaluation Psychosocial Functioning Schedule and Psychiatric Rating Scales, on average, every 8.2 months over 5.1 years. Correlations examined whether participants with increased episode severity also reported poorer relationships and whether fluctuations in episode severity predicted fluctuations in relationships, and vice versa. Results indicated that participants with greater mood episode severity also had worse relationships. Longitudinally, participants had largely stable relationships. To the extent that there were associations, changes in parental relationships may precede changes in episode severity, although the magnitude of this finding was small. Findings have implications for relationship interventions in BP youth.

Study Objective: Pregnancy during adolescence is a risk factor for adverse medical and psychosocial outcomes, including psychiatric illness. Psychiatric illness is linked with obstetric complications along with impaired maternal functioning in the postpartum period. This article provides a comprehensive review of the research examining the intersection of psychopathology and adolescent pregnancy and the postpartum period., Design: A literature search was conducted using PubMed (Medline), PsycINFO, and CINAHL for articles published between 1990 and 2013 that examined depression, anxiety, bipolar disorder, and psychosis during pregnancy and the postpartum period in adolescents age 21 years or younger. Articles were selected that covered the following topics: Prevalence or incidence, comorbidity, psychosocial correlates, birth outcomes, parenting, child outcomes, and psychosocial treatment. Forty articles were found and reviewed., Results: There is a substantial research base examining self-reported depressive symptoms in adolescents during pregnancy and the postpartum period. Existing research suggests that pregnant and parenting adolescents are at greater risk for experiencing depressive symptoms than pregnant and postpartum adult women. Depression in the perinatal period is also a risk factor for substance and alcohol abuse and a harsher parenting style in adolescents. Areas for future research in this population include investigating the prevalence, psychosocial correlates, and outcomes of clinically diagnosed Major Depressive Disorder, developing and empirically validating psychotherapeutic treatments, and focusing upon other psychiatric diagnoses such as bipolar disorder, anxiety, and psychosis., (Copyright © 2014 North American Society for Pediatric and Adolescent Gynecology. Published by Elsevier Inc. All rights reserved.)

Bendamustine, active in multiple myeloma (MM), is a bifunctional mechlorethamine derivative with alkylating properties. Bortezomib, approved to treat MM, is effective in combination with alkylators. The tolerability and efficacy of bendamustine plus bortezomib in relapsed/refractory MM was assessed in an open-label, dose-escalating, phase I/II study. Patients aged ≥18 years received intravenous bendamustine 50, 70, or 90 mg/m(2) (days 1 and 4) plus bortezomib 1·0 mg/m(2) (days 1, 4, 8, and 11) for up to eight 28-day cycles. No dose-limiting toxicity was observed after cycle 1; bendamustine 90 mg/m(2) plus bortezomib 1·0 mg/m(2) was designated the maximum tolerated dose (MTD). The most common grade 3/4 adverse events were leucopenia (58%), neutropenia (50%), lymphopenia (45%), and thrombocytopenia (30%). Primary efficacy measure was overall response rate (ORR), which was the combined complete response (CR), very good partial response (VGPR), partial response (PR), and minimal response (MR). ORR was 48% (one CR, two VGPR, nine PR, and seven MR) for all 40 enrolled patients, 52% (16/31) at the MTD (90 mg/m(2) ), and 42% and 46% for prior use of bortezomib (n = 31) or alkylators (n = 28) respectively. Bendamustine plus bortezomib was well tolerated with promising efficacy in this heavily pretreated population., (© 2012 Blackwell Publishing Ltd.)

Background: Stomatal guard cells are the regulators of gas exchange between plants and the atmosphere. Ca(2+)-dependent and Ca(2+)-independent mechanisms function in these responses. Key stomatal regulation mechanisms, including plasma membrane and vacuolar ion channels have been identified and are regulated by the free cytosolic Ca(2+) concentration ([Ca(2+)](cyt))., Scope: Here we show that CO(2)-induced stomatal closing is strongly impaired under conditions that prevent intracellular Ca(2+) elevations. Moreover, Ca(2+) oscillation-induced stomatal closing is partially impaired in knock-out mutations in several guard cell-expressed Ca(2+)-dependent protein kinases (CDPKs) here, including the cpk4cpk11 double and cpk10 mutants; however, abscisic acid-regulated stomatal movements remain relatively intact in the cpk4cpk11 and cpk10 mutants. We further discuss diverse studies of Ca(2+) signalling in guard cells, discuss apparent peculiarities, and pose novel open questions. The recently proposed Ca(2+) sensitivity priming model could account for many of the findings in the field. Recent research shows that the stomatal closing stimuli abscisic acid and CO(2) enhance the sensitivity of stomatal closing mechanisms to intracellular Ca(2+), which has been termed 'calcium sensitivity priming'. The genome of the reference plant Arabidopsis thaliana encodes for over 250 Ca(2+)-sensing proteins, giving rise to the question, how can specificity in Ca(2+) responses be achieved? Calcium sensitivity priming could provide a key mechanism contributing to specificity in eukaryotic Ca(2+) signal transduction, a topic of central interest in cell signalling research. In this article we further propose an individual stomatal tracking method for improved analyses of stimulus-regulated stomatal movements in Arabidopsis guard cells that reduces noise and increases fidelity in stimulus-regulated stomatal aperture responses ( Box 1). This method is recommended for stomatal response research, in parallel to previously adopted blind analyses, due to the relatively small and diverse sizes of stomatal apertures in the reference plant Arabidopsis thaliana.

Introduction: Several lines of evidence point to an important role for BP1, an isoform of DLX4 homeobox gene, in breast carcinogenesis and progression. BRCA1 is a well-known player in the etiology of breast cancer. While familial breast cancer is often marked by BRCA1 mutation and subsequent loss of heterozygosity, sporadic breast cancers exhibit reduced expression of wild type BRCA1, and loss of BRCA1 expression may result in tumor development and progression., Methods: The Cister algorithm and Genomatix program were used to identify potential BP1 binding sites in BRCA1 gene. Real-time PCR, Western blot and immunohistochemistry analysis were performed to verify the expression of BRCA1 and BP1 in cell lines and breast cancer tissues. Double-stranded siRNA transfection was carried out for silencing BP1 expression. ChIP and EMSA were used to confirm that BP1 specifically binds to BRCA1., Results: A putative BP1 binding site was identified in the first intron of BRCA1, which was confirmed by chromatin immunoprecipiation and electrophoresis mobility shift assay. BP1 and BRCA1 expression were inversely correlated in breast cancer cell lines and tissues, suggesting that BP1 may suppress BRCA1 transcription through consensus sequence binding., Conclusions: BP1 homeoprotein represses BRCA1 expression through direct binding to its first intron, which is consistent with a previous study which identified a novel transcriptional repressor element located more than 500 base pairs into the first intron of BRCA1, suggesting that the first intron plays an important role in the negative regulation of BRCA1. Although further functional studies are necessary to confirm its repressor activity towards BRCA1, the elucidation of the role of BP1 in breast tumorigenesis holds great promise in establishing BP1 as a novel target for drug therapy.

Tumor invasion and metastasis remain a major cause of mortality in breast cancer patients. It was reported that BP1, a homeobox isoform of DLX4, is overexpressed in 80% of breast cancer patients and in 100% of estrogen receptor negative (ER-) tumors. The prevalence of BP1 positive cells and the intensity of BP1 immunoreactivity increased with the extent of ductal proliferation and tumorigenesis. These findings imply that BP1 may play an important role in ER- breast cancer. We sought to determine the effects and mechanisms of BP1 on cell proliferation and metastasis using ER- Hs578T cells as a model. Cells were transfected with either pcDNA3.2 plasmid containing BP1 gene, or pcDNA3.2 vector, then selected and cloned. Overexpression of BP1 increased cell proliferation rate by 2-5 fold (p<0.005), and enhanced the in vitro invasive activity by 25-65 fold (p<0.001). Microarray experiments were performed to identify differentially expressed genes when BP1 is overexpressed. The gene expression profile of the transfected cell lines were compared, resulting in 71 differentially expressed genes with a fold-change of >=2.0. Of those genes, 49 were up-regulated and 22 were down-regulated. Significant pathways were identified involving cell proliferation and metastasis. These data demonstrated that overexpression of BP1 significantly enhanced cell proliferation and metastatic potential in ER- Hs578T cells. Further analysis with more ER- cell lines and patient samples is warranted to establish BP1 as a therapeutic target for ER- breast cancer.

This study used a 2-month prospective research design to examine the bi-directional interplay between peer victimization and social anxiety among adolescents. Participants included 228 adolescents (58% female) in grades 10-12. Three types of peer victimization were examined: overt (physical aggression or verbal threats), relational (malicious manipulation of a relationship, such as by friendship withdrawal), and reputational (damaging another's peer relationships, such as through rumor spreading). Adolescents' self-reported feelings of social anxiety and peer victimization experiences were assessed at two time points, in November and January of the same school year. Peer victimization was strongly related to adolescents' social anxiety, and relational victimization explained additional unique variance. Moreover, peer victimization was both a predictor and consequence of social anxiety over time, with the most robust results found for relational victimization. Limited support was obtained for gender as a moderating variable. Findings highlight the deleterious effects of peer victimization, especially relational victimization, and suggest avenues for future research and clinical intervention for adolescents experiencing such victimization.

Stomatal closure in response to abscisic acid depends on mechanisms that are mediated by intracellular [Ca2+] ([Ca2+]i), and also on mechanisms that are independent of [Ca2+]i in guard cells. In this study, we addressed three important questions with respect to these two predicted pathways in Arabidopsis thaliana. (i) How large is the relative abscisic acid (ABA)-induced stomatal closure response in the [Ca2+]i-elevation-independent pathway? (ii) How do ABA-insensitive mutants affect the [Ca2+]i-elevation-independent pathway? (iii) Does ABA enhance (prime) the Ca2+ sensitivity of anion and inward-rectifying K+ channel regulation? We monitored stomatal responses to ABA while experimentally inhibiting [Ca2+]i elevations and clamping [Ca2+]i to resting levels. The absence of [Ca2+]i elevations was confirmed by ratiometric [Ca2+]i imaging experiments. ABA-induced stomatal closure in the absence of [Ca2+]i elevations above the physiological resting [Ca2+]i showed only approximately 30% of the normal stomatal closure response, and was greatly slowed compared to the response in the presence of [Ca2+]i elevations. The ABA-insensitive mutants ost1-2, abi2-1 and gca2 showed partial stomatal closure responses that correlate with [Ca2+]i-dependent ABA signaling. Interestingly, patch-clamp experiments showed that exposure of guard cells to ABA greatly enhances the ability of cytosolic Ca2+ to activate S-type anion channels and down-regulate inward-rectifying K+ channels, providing strong evidence for a Ca2+ sensitivity priming hypothesis. The present study demonstrates and quantifies an attenuated and slowed ABA response when [Ca2+]i elevations are directly inhibited in guard cells. A minimal model is discussed, in which ABA enhances (primes) the [Ca2+]i sensitivity of stomatal closure mechanisms.

Background: A common limitation in guard cell signaling research is that it is difficult to obtain consistent high expression of transgenes of interest in Arabidopsis guard cells using known guard cell promoters or the constitutive 35S cauliflower mosaic virus promoter. An additional drawback of the 35S promoter is that ectopically expressing a gene throughout the organism could cause pleiotropic effects. To improve available methods for targeted gene expression in guard cells, we isolated strong guard cell promoter candidates based on new guard cell-specific microarray analyses of 23,000 genes that are made available together with this report., Results: A promoter, pGC1(At1g22690), drove strong and relatively specific reporter gene expression in guard cells including GUS (beta-glucuronidase) and yellow cameleon YC3.60 (GFP-based calcium FRET reporter). Reporter gene expression was weaker in immature guard cells. The expression of YC3.60 was sufficiently strong to image intracellular Ca2+ dynamics in guard cells of intact plants and resolved spontaneous calcium transients in guard cells. The GC1 promoter also mediated strong reporter expression in clustered stomata in the stomatal development mutant too-many-mouths (tmm). Furthermore, the same promoter::reporter constructs also drove guard cell specific reporter expression in tobacco, illustrating the potential of this promoter as a method for high level expression in guard cells. A serial deletion of the promoter defined a guard cell expression promoter region. In addition, anti-sense repression using pGC1 was powerful for reducing specific GFP gene expression in guard cells while expression in leaf epidermal cells was not repressed, demonstrating strong cell-type preferential gene repression., Conclusion: The pGC1 promoter described here drives strong reporter expression in guard cells of Arabidopsis and tobacco plants. It provides a potent research tool for targeted guard cell expression or gene silencing. It is also applicable to reduce specific gene expression in guard cells, providing a method for circumvention of limitations arising from genetic redundancy and lethality. These advances could be very useful for manipulating signaling pathways in guard cells and modifying plant performance under stress conditions. In addition, new guard cell and mesophyll cell-specific 23,000 gene microarray data are made publicly available here.

Stomatal pores in the epidermis of plants enable gas exchange between plants and the atmosphere, a process vital to plant life. Pairs of specialized guard cells surround and control stomatal apertures. Stomatal closing is induced by abscisic acid (ABA) and elevated CO(2) concentrations. Recent advances have been made in understanding ABA signaling and in characterizing CO(2) transduction mechanisms and CO(2) signaling mutants. In addition, models of Ca(2+)-dependent and Ca(2+)-independent signaling in guard cells have been developed and a new hypothesis has been formed in which physiological stimuli are proposed to prime Ca(2+) sensors, thus enabling specificity in Ca(2+)-dependent signal transduction.

Objective: The effect of support, from dependable non-household relationships, on breast cancer progression was studied using a prospective, longitudinal design., Methods: Dependable social support was assessed in women with invasive breast carcinoma Stages II and III within 18 months after diagnosis. Disease outcome was monitored for 8 to 9 years. Cox regression analyses, including the Nottingham Prognostic Index (NPI) score of disease severity, tested the association between the number of dependable support persons and time to death., Results: Ninety participants listed between 1 and 16 dependable nonhousehold support persons (mean = 6, S.D. = 4). There were 21 recurrences and 16 deaths from breast cancer at the end of the study. The number of dependable supports predicted decreased mortality [RR = 0.41 (0.21-0.80), P = .01] after controlling for NPI. NPI predicted increased mortality [RR = 1.6 (1.0, 2.4), P = .05]., Conclusions: For patients with Stages II and III breast cancer, the number of dependable, nonhousehold relationships predicts decreased mortality, after accounting for disease severity.

Background: To examine the risk for psychopathology in offspring at risk for bipolar disorder and the course of psychiatric disorders in these youth., Methods: Using structured diagnostic interviews (Structured Clinical Interview for DSM-IV [SCID] and Kiddie Schedule for Affective Disorders and Schizophrenia [K-SADS]), psychiatric diagnoses of 117 nonreferred offspring of parents with diagnosed bipolar disorder were compared with those of 171 age- and gender-matched offspring of parents without bipolar disorder or major depression., Results: Compared with offspring of parents without mood disorders, high-risk youth had elevated rates of major depression and bipolar disorder, anxiety, and disruptive behavior disorders. High-risk offspring also had significantly more impaired Global Assessment of Functioning (GAF) scores, higher rates of psychiatric treatment, and higher rates of placement in special education classes. Disruptive behavior disorders, separation anxiety disorder, generalized anxiety disorder (GAD), social phobia, and depression tended to have their onset in early or middle childhood, whereas bipolar disorder, obsessive-compulsive disorder (OCD), panic disorder, and substance use disorder had onset most frequently in adolescence., Conclusions: These findings support the hypothesis that offspring of parents with bipolar disorder are at significantly increased risk for developing a wide range of severe psychiatric disorders and accompanying dysfunction. Early disruptive behavior and anxiety disorders, as well as early-onset depression, may be useful markers of risk for subsequent bipolar disorder in high-risk samples.

Human T-cell lymphotropic virus-I (HTLV-I)-related adult T-cell leukemia/lymphoma (ATL) is a model disease for proof of viral oncogenesis. HTLV-I infection is endemic in southern Japan and the Caribbean basin, and occurs sporadically in Africa, Central and South America, the Middle East, and the southeastern United States. ATL occurs in only 2% to 4% of HTLV-I-infected people [1-3]. When it does occur, it is usually aggressive and difficult to treat; most people survive for less than 1 year [1-3]. Combination chemotherapy with cytotoxic agents has yielded complete response rates of 20% to 45%, but responses usually last only a few months [3]. Recently, novel treatments, such as monoclonal antibodies directed at the interleukin-2 receptor and the combination of interferon alfa and zidovudine, have been shown to be active in the treatment of patients with ATL. A small percentage of patients achieve long-lasting remissions [2,3].

Purpose: Primary cutaneous T-cell lymphomas (CTCLs) encompass a wide variety of lymphomas that are characterized by the localization of the malignant lymphocytes to the skin at presentation. Advances in molecular biologic techniques, including immunophenotyping and gene rearrangement studies to determine clonality, have led to more frequent diagnosis of CTCL as well as more consistent subclassification of these entities. However, there continues to be confusion in the classification, prognosis, and management of patients with CTCL. The purpose of this review is to present a summary of the diagnosis, prognosis, and treatment of CTCL, with specific emphasis on mycosis fungoides (MF) and Sézary syndrome (SS). We also present a detailed discussion of the entities that make up the differential diagnosis of CTCL., Design: We reviewed the medical literature on CTCL and other diseases that make up the differential diagnosis of CTCL., Results and Conclusion: MF and SS are the most common forms of CTCL. The etiology of this disease is still unknown. Patients may go for months to years with skin abnormalities before being diagnosed. MF/SS is an indolent disease and patients with T1 disease have a normal life expectancy. Patients who undergo transformation to large-cell lymphoma (8% to 23% of patients) have a poor prognosis, with mean survival ranging from 2 to 19 months. Treatment for MF/SS continues to be palliative. There are many new therapies that are currently being investigated in clinical trials, and the DAB(389)IL-2 fusion protein was recently approved for the treatment of refractory MF/SS.

Purpose: Primary cutaneous B-cell lymphoma (PCBCL) has only recently been recognized as a distinct clinical entity. With the advent of improved immunophenotyping and immunogenotyping, increasing numbers of PCBCL cases are being diagnosed. However, there is much confusion regarding the classification, treatment, and prognosis of these patients. The purpose of this article is to review and analyze the available data to provide the clinician with a concise summary of the diagnosis, prognosis, and treatment of PCBCL., Design: We conducted a thorough review of the medical literature on PCBCL, with a focus on classification, prognosis, and treatment trials., Results and Conclusion: PCBCL is defined as a B-cell lymphoma originating in the skin. There is no evidence of extracutaneous disease at presentation and for 6 months after diagnosis, as assessed by adequate staging procedures. Currently, the European Organization for Research and Treatment of Cancer classification is the most concise disease classification scheme, dividing the subtypes of PCBCL by clinical behavior and histopathologic findings. Based on this classification, the most common subtype of PCBCL is follicular center cell lymphoma. PCBCL is generally an indolent form of lymphoma with a good prognosis. Although local cutaneous recurrences are observed in 25% to 68% of patients, dissemination to internal organs is rare. Five-year survival rates typically range from 89% to 96%. A specific subtype, large B-cell lymphoma of the leg, is noted to have a poorer prognosis, with a 5-year survival rate of 58%. Overly aggressive treatment of PCBCL has not been shown to improve survival or prevent relapse. The treatment of choice usually varies depending on the type of PCBCL, the body surface area, and the location of the involvement, as well as the age and general health condition of the patient. The majority of studies indicate that PCBCL is highly responsive to radiation therapy. Polychemotherapy should be reserved for involvement of noncontiguous anatomic sites or those with extracutaneous spread.

Effective treatment for cutaneous T-cell lymphomas (CTCL) requires an accurate and specific diagnosis based on the clinical presentation combined with evaluation of the histopathology, immunophenotyping, and gene rearrangement studies. Careful clinical and pathologic evaluation in centers familiar with the diverse forms of CTCL is most valuable for determining treatment options. The goals of treatment in mycosis fungoides (MF), which afflicts more than 50% of patients with CTCL, are the relief of symptoms and improvement in cosmetics. Despite some uncontrolled clinical trial results that have been reported to suggest "cures" in this disease, the general perception remains that this disease is not curable with standard therapies available today. Treatment is divided into topical (skin-directed) and systemic therapy. The most active systemic agent for the treatment of MF remains interferon-alpha, although many new modalities have recently been approved for the treatment of CTCL.

Neuronal nicotinic acetylcholine receptors (nAChRs) are a class of ion channels with significant potential as molecular targets for the design of drugs to treat a variety of CNS disorders. The discovery that neuronal nAChRs are further subdivided into multiple subtypes suggests that drugs which act selectively at specific nAChR subtypes might effectively treat Parkinson's disease (PD), Alzheimer's disease (AD), schizophrenia, ADHD, depression, anxiety or pain without the accompanying adverse side effects associated with non-selective agents such as nicotine (1) and epibatidine. Altinicline (SIB-1508Y) is a novel, small molecule designed to selectively activate neuronal nAChRs and is undergoing clinical evaluation for the treatment of PD. It was selected from a series of compounds primarily on the basis of results from functional assays, including (a) measurement of Ca2+ flux in stable cell lines expressing specific recombinant human neuronal nAChR subtypes; (b) determination of in vitro and in vivo neurotransmitter release; (c) in vivo models of PD. Biological data on both altinicline and the series of compounds from which it was selected are reported.

This study was conducted to assess the efficacy and toxicity of intravenous (i.v.) ceftazidime and ciprofloxacin in neutropenic febrile patients undergoing high dose myeloablative therapy and hematopoietic stem cell transplantation (HSCT). All patients undergoing HSCT for leukaemia, lymphoma, multiple myeloma and solid tumours received open-label ceftazidime 2 g i.v. every 8 h and ciprofloxacin 400 mg i.v. every 12 h if they developed fever while they were neutropenic. Success with or without modification of this regimen was defined as survival through the neutropenic period; failure was defined as death secondary to infection. Of 106 patients treated with this regimen, the success rate was 99%. Sixty-one of the patients (57.5%) defervesced within 48-72 h and remained afebrile without regimen modification. In 41.5% of the cases (44/106), the regimen was modified because of persistent fever. One patient died secondary to sepsis. The combination of ceftazidime and ciprofloxacin as initial empiric antibacterial therapy in febrile neutropenic patients undergoing myeloablative therapy and HSCT is highly effective and is associated with minimal toxicity.

It has been observed that some patients in complete remission (CR) after 2-chlorodeoxyadenosine (2-CdA) for hairy cell leukemia (HCL) have hypocellular bone marrow biopsies despite normal peripheral blood cell counts. This discrepancy between bone marrow cellularity and peripheral blood cell counts suggests the possibility of abnormal sites of hematopoiesis. To determine sites of hematopoiesis, 11 radionuclide scans using technetium-99m (99mTc) sulfur colloid were performed in eight patients. Although no single, pattern was observed on the 99mTc sulfur colloid scans, two of the eight patients, both with virtually aplastic marrows, had multiple areas of increased uptake in the distal appendicular skeleton, suggesting abnormal sites of hematopoiesis. The same two patients had magnetic resonance imaging (MRI), which confirmed the abnormal sites of hematopoiesis.

Purpose: Chronic thrombocytopenia is uncommon in children and frequently thought to be secondary to chronic idiopathic thrombocytopenic purpura (ITP), which is considered an immune disorder. However, not all children with chronic ITP respond to immunosuppressive therapy. Platelet survival and megakaryocyte growth were studied to determine if there is a failure of platelet production in children with chronic thrombocytopenia who carry a presumptive diagnosis of chronic ITP., Patients and Methods: In vitro megakaryocyte growth using a plasma clot system and in vivo survival of 111In-labeled autologous platelets were studied in seven patients (aged 2 days to 17 years at diagnosis; aged 2 to 28 years at time of megakaryocyte study) with chronic isolated thrombocytopenia (range 1,000 to 130,000/microl)., Results: All seven patients exhibited elevated platelet-associated immunoglobulin G early in the course of their disease and showed normal marrow morphology with normal numbers of morphologically typical megakaryocytes on initial marrow biopsy. Occasional dysplastic-appearing megakaryocytes were noted in three of the seven patients at diagnosis and all patients were noted to have dysplastic megakaryocytes, reduced megakaryocytes, or both during follow-up (range 5 to 16 years). Either morphologic or karyotypic abnormalities indicative of myelodysplasia subsequently developed in three patients. No patient exhibited any significant megakaryocyte colony growth under basal conditions. In one patient, megakaryocytic colonies significantly increased when grown in the presence of serum from aplastic patients and growth factors (granulocyte-macrophage colony-simulating factor, interleukin-3, and interleukin-6). Erythroid colony growth was markedly deficient in four of five patients studied and myeloid colonies were normal in two of three patients studied. Five of the seven patients underwent platelet survival studies. Platelet survival was < 6 days in the 4 patients with platelet counts < 100,000/microl (range 2 to 60,000/microl; survival range 92 to 137 hours) and was normal in the patient whose platelet count was > 100,000/microl (platelets 132,000/microl; survival 253 hours). All five patients had either overtly low or inappropriately low platelet turnovers (range 100 to 1423 platelets/microl per hour; normal range 1200 to 1600 platelets/microl per hour). The patient with the lowest platelet count and platelet turnover had previously undergone a splenectomy without benefit., Conclusions: Megakaryocyte dysfunction resulting in subnormal production of platelets may play a significant role in the thrombocytopenia noted in some patients who have an isolated thrombocytopenia and a clinical picture that suggests ITP. Determination of platelet turnover may help to identify these patients. These data suggest the presence of a stem cell defect which may progress to myelodysplasia or overt marrow failure in these patients.

The authors compared concentrations of soluble beta-amyloid protein precursor (s beta PP) in cerebrospinal fluid (CSF) in 45 patients diagnosed with probable Alzheimer disease (AD) and 26 normal older control volunteers. Soluble beta-amyloid protein precursor concentrations were measured in 125 CSF samples using an enzyme-linked immunosorbent assay. All subjects had Mini-Mental State Examination (MMSE) and Clinical Dementia Rating Scale (CDRS) scores and assessment of disease duration. The s beta PP concentrations in CSF in the probable AD group (mean +/- SD = 493 +/- 268 micrograms/L) were decreased significantly compared with the age-matched control group (mean = 831 +/- 302 micrograms/L; p < 0.0001). In the probable AD group, MMSE scores correlated positively with s beta PP concentrations (correlation coefficient r = 0.53, p < 0.0001), and CDRS ratings and disease duration correlated inversely with s beta PP concentrations (r = -0.59, p < 0.0001 and r = -0.479, p = 0.0006, respectively). Although the decrease in CSF s beta PP from levels found in healthy elderly controls was significant in AD subjects, there was substantial overlap. In AD, CSF s beta PP was most reduced in patients in later stages of the disease. The s beta PP concentrations reflect disease severity, but utility in differential diagnosis has not been determined.

Flow cytometric analysis was performed on 50 infiltrating labular carcinomas (ILCs) of the breast from 50 patients with clinical follow-up (average duration, 4.1 yr) who had been treated between 1976 and 1991. The patients were classified as alive with no evidence of disease, alive with disease, or dead of disease. Ploidy and S-phase fraction (SPF) were compared with clinical outcome, histologic pattern (classical versus variant patterns), nuclear grade 1 or 2, axillary lymph node status, tumor size, percentage of signet ring cells, and estrogen receptor status. There was no association between aneuploid or diploid ILC and disease recurrence (of those patients classified as alive with no evidence of disease plus dead of disease, 4 (40%) of 10 were aneuploid and 15 (38%) of 40 were diploid), survival (of those classified as alive with no evidence of disease plus alive with disease, 9 (90%) of 10 were aneuploid and 36 (90%) of 40 were diploid), or any of the other factors evaluated. However, ILCs with a high SPF were more likely to recur than those with a low SPF (a high SPF was found in 9 [56%] of 16; a low SPF was found in 9 [30%] of 30), but this relationship was not statistically significant (P = 0.08). When only diploid ILCs were considered, there was a statistically significant association between high SPF and recurrence (a high SPF was found in 9 [64%] of 14, a low SPF was found in 6 [24%] of 25, P = 0.033). After the stratification of the diploid ILCs by the stage of disease, this relationship persisted only in Stage 1 (recurrence: a high SPF was found in 3 [75%] of 4; a low SPF was found in 1 [9%] of 11, P = 0.033). There was no association between SPF and any of the other factors evaluated. Our study indicates that ILC is usually diploid and that SPF may be a prognostic indicator that is limited to Stage 1 disease.

To establish prognostically useful pathologic features for infiltrating lobular carcinoma, histologic pattern, nuclear Grade 1 or 2, lymphatic invasion, the presence and extent of lobular carcinoma in situ, estrogen and progesterone receptor status, axillary lymph node status, tumor size, and pathologic stage were assessed as prognostic variables in 92 cases of infiltrating lobular carcinoma. Clinical follow-up was obtained (mean duration, 5.2 yr), and patients were classified as alive with no evidence of disease, alive with disease, or dead of disease. Recurrence (alive with disease and dead of disease) was associated with axillary lymph node metastases (P = 0.04), tumors measuring > 1.0 cm (P = 0.008), and pathologic Stage III/IV disease (P = 0.033). Survival (no evidence of disease and alive with disease) was associated with Stage I/II disease (P = 0.003). Statistically insignificant associations with disease recurrence or survival follow: infiltrative pattern (classical, alveolar, solid, mixed), nuclear grade, lymphatic vessel invasion, presence of lobular carcinoma in situ, extent of lobular carcinoma in situ (< 25% or > or = 25%), and hormone receptor status. Many of the prognostic features used in ductal carcinoma do not appear to be applicable to infiltrating lobular carcinoma. However, tumor size, axillary node status, and pathologic stage are prognostically useful in infiltrating lobular carcinoma.

The Alzheimer's disease related protein, amyloid beta-protein precursor (A beta PP), contains a domain homologous to Kunitz-type serine protease inhibitors (KPI). The recombinant KPI domain of A beta PP is a potent inhibitor of coagulation factors XIa and IXa and functions as an anticoagulant in vitro. Here we report the expression, purification, and characterization of a reactive center lysine mutant of the KPI domain of A beta PP (KPI-Lys17). An expression plasmid for the KPI-Lys17 domain of A beta PP encoded amino acids 285-345 of the A beta PP cDNA containing a lysine substitution at arginine 17 in the KPI domain. The secreted 61-amino acid product was purified to homogeneity and functionally characterized. The protease inhibitory properties of the KPI-Lys17 domain were compared to those of the native KPI domain of A beta PP. Both KPI domains equally inhibited trypsin, chymotrypsin, and coagulation factors IXa and Xa. However, the KPI-Lys17 domain was an approximately 25-fold less effective inhibitor of coagulation factor XIa resulting in markedly less prolongation of the activated partial thromboplastin time compared to the native KPI domain of A beta PP. On the other hand, the KPI-Lys17 domain was an approximately 10- and 5-fold better inhibitor of plasmin in a chromogenic substrate assay and in a fibrinolytic assay, respectively, than the native KPI domain of A beta PP. Together, these studies suggest that the KPI-Lys17 domain has enhanced anti-fibrinolytic and diminished factor XIa inhibitory properties compared to the native KPI domain of A beta PP.

To determine the prognostic significance of signet ring cells in infiltrating lobular carcinomas, the percentage of signet ring cells in 99 infiltrating lobular carcinomas was correlated with the patients' clinical outcomes (mean follow-up interval of 4.8 years). When the carcinomas were divided into those with 0%, 1-9%, and 10% or more signet ring cells, 57% (26/46) of patients with 10% or more signet ring cells had experienced recurrences or metastases compared with 40% (2/5) and 31% (15/48) with 0% and 1-9%, respectively. A similar analysis performed with breakpoints at 20% or 30% failed to yield any statistically significant associations. When patients were stratified by pathologic stage, patients with stage I disease and 10% or more signet ring cells were more likely to have recurrences or metastases than those patients with stage I tumors and fewer than 10% signet ring cells. There was no relationship between signet ring cells and disease progression in stages II, III, and IV. These results indicate that the presence of 10% or more signet ring cells represents a poor individual prognostic factor in stage I infiltrating lobular carcinomas.

In this report we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the Kunitz-type proteinase inhibitor (KPI) domain of the amyloid beta-protein precursor-like protein-2 (APLP-2). The expression plasmid for the KPI domain of APLP-2 encoded amino acids 305-364 of the APLP-2 cDNA (Slunt et al. (1994) J. Biol. Chem. 269, 2637-2644). The secreted 60 amino-acid product was purified to homogeneity and biochemically characterized. Amino-acid sequencing of the expressed KPI domain of APLP-2 verified its integrity. The proteinase inhibitory properties of the KPI domain of APLP-2 were compared to those of the KPI domain of proteinase nexin-2/amyloid beta-protein precursor (PN-2/A beta PP). Both KPI domains potently inhibited trypsin and, to a lesser extent, chymotrypsin, plasmin, and coagulation factors XIa and IXa. However, the KPI domain of APLP-2 was a approximately 20-fold less effective inhibitor of coagulation factor XIa compared to the KPI domain of PN-2/A beta PP. Similarly, the KPI domain of APLP-2 was a less effective anticoagulant in coagulation based assays than the KPI domain of PN-2/A beta PP. These studies indicate that the KPI domains of PN-2/A beta PP and APLP-2 form a family of proteinase inhibitors although the former is a better inhibitor of factor XIa and a more potent anticoagulant than the latter.

There is evidence for increased factor VII turnover and the associated increased thrombin generation and fibrinolytic activities in sickle cell disease (SCD) that may affect in vivo platelet and endothelial cell reactivity. We studied the release of specific eicosanoids that are indicative of in vivo platelet activation and endothelial cell injury. The circulating and urinary levels of 2,3-dinor thromboxane B2(2,3-dinor-TxB2),TxB2,PGI2 [as 6-keto-PGF1 alpha], and PGE2 were measured in 15HbSS patients, eight HbAA non-haemolytic anaemic individuals and 12 healthy HbAA controls using specific RIAs. The mean urinary 2,3-dinor-TxB2 in the HbSS patients was significantly higher than in both the healthy HbAA and the anaemic controls. 6-keto-PGF1 alpha was undetected in the urines of the healthy HbAA controls, but was measured insignificant amounts in the HbSS and the HbAA anaemic patients. The urinary concentrations of PGE2 and TxB2 in HbSS patients' samples were also significantly higher than those of both control groups (P < 0.05). PGE2 and TxB2 levels were below the detection limit in the plasmas of the HbAA subjects, but were measurable in the HbSS and HbAA anaemic plasmas. The plasma level of 6-keto-PGF1 alpha in the HbSS patients was also significantly higher than in the control groups. The data indicates a persistent inflammatory process in the HbSS patients, and is consistent with the hypothesis that there is platelet and endothelial cell activation in SCD.

Although the mechanisms involved in the persistent clinical complications of sickle cell disease have not yet been fully delineated, previous studies suggest that sickle cell (HbSS) patients have a disposition to generate more thrombin and plasma in vivo than normal subjects. The reasons for the impaired regulation of haemostasis in HbSS patients is poorly understood. We report studies evaluating the extent to which in vivo coagulation and fibrinolysis are altered in HbSS patients in steady state. The concentrations of total factor VII (F(VII)t), factor VII zymogen (F(VII)z), thrombin-antithrombin III (TAT), fibrinopeptide A(FPA), and fibrin D-dimer in plasmas of 50 normal controls (HbAA) and 45 HbSS steady state patients, were measured using sensitive and specific enzyme-linked immunoassays. The average plasma concentration of F(VII)t, in sickle cell plasma was significantly lower than that of the control subjects (0.70 +/- 0.19 U/ml versus 1.16 +/- 0.41 U/ml), whereas F(VII)z in the patients and controls were 0.47 +/- 0.15 U/ml and 1.15 +/- 0.33 U/ml respectively, P < 0.001. Both measures of factor VII suggest a higher factor VII turnover in sickle cell disease. The mean concentration of TAT in the plasma of HbSS patients were significantly higher than those of HbAA controls (371 +/- 44 pM versus 42 +/- 2 pM) (P < 0.001), a difference that is strongly indicative of higher rates of in vivo thrombin generation by HbSS patients. Plasmas of HbSS patients had significantly higher concentrations of FPA compared to those of the control subjects (12.85 +/- 1.96 ng/ml versus 4.22 +/- 0.37 ng/ml) (P < 0.001). The D-dimer levels were also higher in the HbSS than control plasmas (1029.6 +/- 58.6 ng/ml versus 224.3 +/- 27.6 g/ml) (P < 0.001), with the patients' values being indicative of enhanced fibrinolysis. These results strongly suggest accelerated in vivo coagulation and fibrinolysis in HbSS patients even during steady state. They are consistent with the hypothesis that haemostasis is less tightly regulated in the HbSS patients than in HbAA controls. The altered regulation of haemostasis may contribute to the initiation of vaso-occlusive processes associated with sickle cell painful episodes.

The protease inhibitor, protease nexin-2 (PN-2), is the secreted isoform of the Alzheimer's amyloid beta-protein precursor (A beta PP) that contains the Kunitz-type protease inhibitor (KPI) domain. Here we describe the use of the methylotrophic industrial yeast Pichia pastoris as a host system for the large scale production of the KPI domain of PN-2/A beta PP. In addition to the 57 amino acid KPI domain, the expression product contained an additional four amino acid residues at its amino terminus that correspond to amino acids 285-288 of A beta PP (Ponte et al. 1988 Nature 311:525-527). This expression system generated yields of greater than 1.0 gram of KPI domain per liter of fermentation media. The secreted 61 amino acid product was purified to homogeneity and biochemically characterized. Amino acid analysis and sequencing of the entire expressed KPI domain verified its integrity. Similar to native PN-2/A beta PP, the purified KPI domain potently inhibited trypsin, chymotrypsin, and coagulation factor XIa. Although heparin augments the inhibition of factor XIa by native PN-2/A beta PP it had no effect on the inhibition of factor XIa by expressed KPI domain suggesting that heparin binds to regions on native PN-2/A beta PP outside of the protease inhibitory domain. This KPI domain expression product should be useful in studying the physiologic and pathophysiologic functions of PN-2/A beta PP.

A synthetic gene encoding aprotinin (bovine pancreatic trypsin inhibitor) was fused to the Saccharomyces cerevisiae prepro alpha mating factor leader sequence at the dibasic amino acid processing site. Pichia pastoris strains were developed to express one or multiple copies of a methanol-inducible expression cassette containing the gene fusion. P. pastoris containing a single copy of the vector secreted approximately 150 mg/l of immunoreactive protein. A construct bearing five copies of the expression cassette secreted 930 mg/l of aprotinin. The purified aprotinin molecule was equipotent with the native molecule in a trypsin inhibition assay. Protein sequence analysis showed that the alpha factor-aprotinin fusion was not processed at the basic amino acid residues Lys-Arg. Instead, recombinant aprotinin had additional N-terminal amino acids derived from prepro alpha factor. The N-terminal extension was variably 11 or 4 amino acids. Inclusion of the spacer DNA sequence encoding Glu and Ala between aprotinin and the Lys-Arg processing site led to the secretion of a biologically active aprotinin containing only a Glu-Ala N-terminal extension.

Operation of a continuous microbial fermentor with cell recycle can significantly reduce degradation-associated loss of a secreted protein product. Under continuous fermentation conditions, proteolysis of a recombinant growth hormone releasing factor (GRF) analog secreted by S. cerevisiae was first order with respect to GRF concentration. The maximal GRF concentration was increased from 5 mg/l to 30 mg/l by the use of a cell recycle reactor, and volumetric productivity was increased more than 10-fold to an average of 10 mg/l-1/h-1. A mathematical model shows that increased productivity in the cell recycle reactor results from a reduced degradation rate and a shorter residence time of the product in the fermentor.

Five hundred indium-111 labeled platelet imaging studies (387 donor and 113 autologous) were performed postoperatively in 473 patients who had undergone total hip replacement, total knee replacement, or internal fixation of a hip fracture to detect occult deep venous thrombosis. All patients had been anticoagulated prophylactically with aspirin, warfarin sodium (Coumadin), or dextran. Thirty-four possible cases of proximal deep venous thrombosis were identified in 28 asymptomatic patients. To verify the scan results, 31 venograms were performed in 25 patients (three refused). In 21 of 31 cases, totally occlusive thrombi were detected; in 5 cases, partially occlusive thrombi were detected; in 5 cases, no thrombus was seen. No patient who had a negative scan nor any patient who had a verified positive scan (and received appropriate heparin therapy) subsequently developed symptoms or signs of pulmonary embolism. One hundred forty-one indium study patients also underwent Doppler ultrasonography/impedance plethysmography (Doppler/IPG) as a comparative non-invasive technique. In 137 cases, the results of the indium study and Doppler/IPG studies were congruent. The indium study had no false negative results that were detected by Doppler/IPG. No patient had any clinically evident toxicity. These results suggest that indium-111 labeled platelet scanning is a safe, noninvasive means for identifying DVT in high risk patients.

The blood cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) have abnormal interactions with complement. The activity of the alternative pathway C3 convertase on the platelets of 9 out of 19 patients with PNH was elevated. 10 patients had C3 convertase activity within the normal range even though 80-95% of their platelets lacked the complement regulatory protein decay accelerating factor (DAF) that is absent from the affected blood cells in PNH. PNH and normal platelets released factor H when C3 was bound to their surfaces. This may account for the apparent regulation of C3 convertase activity on platelets that lack DAF. The abnormal uptake of the membrane attack complex of complement by PNH III erythrocytes was not seen in PNH platelets. 111Indium-labeled platelet survival times were normal in five of eight patients, which suggests that the lack of the membrane attack complex defect results in normal platelet survival in PNH.

The hydrogen-oxidizing bacterium, Alcaligenes eutrophus (ATCC 17707), was grown in chemostat culture with gas-phase (hydrogen, oxygen, and carbon dioxide) and liquid-phase (mineral nutrients) feedstreams; data were used to generate an analytical form for the specific growth rate equation. Model parameters obtained include Monod rate parameters for dissolved hydrogen and oxygen gases, yield coefficients, and specific maintenance rates under conditions of hydrogen or oxygen limitations. These values are similar to some obtained previously by Ohi et al. for another hydrogen bacterium. The observed increase in specific maintenance rates under hydrogen-versus-oxygen-limited culture may be associated with hydrogenase deactivation by oxygen.