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The γ-secretase protein complex executes the intramembrane proteolysis of amyloid precursor protein (APP), which releases Alzheimer disease β-amyloid peptide. In addition to APP, γ-secretase also cleaves several other type I membrane protein substrates including Notch1 and N-cadherin. γ-Secretase is made of four integral transmembrane protein subunits: presenilin (PS), nicastrin, APH1, and PEN2. Multiple lines of evidence indicate that a heteromer of PS-derived N- and C-terminal fragments functions as the catalytic subunit of γ-secretase. Only limited information is available on the domains within each subunit involved in the recognition and recruitment of diverse substrates and the transfer of substrates to the catalytic site. Here, we performed mutagenesis of two domains of PS1, namely the first luminal loop domain (LL1) and the second transmembrane domain (TM2), and analyzed PS1 endoproteolysis as well as the catalytic activities of PS1 toward APP, Notch, and N-cadherin. Our results show that distinct residues within LL1 and TM2 domains as well as the length of the LL1 domain are critical for PS1 endoproteolysis, but not for PS1 complex formation with nicastrin, APH1, and PEN2. Furthermore, our experimental PS1 mutants formed γ-secretase complexes with distinct catalytic properties toward the three substrates examined in this study; however, the mutations did not affect PS1 interaction with the substrates. We conclude that the N-terminal LL1 and TM2 domains are critical for PS1 endoproteolysis and the coordination between the putative substrate-docking site and the catalytic core of the γ-secretase.

Diabetes is characterized by several poorly understood phenomena including dysfunctional wound healing and impaired vasculogenesis. p53, a master cell cycle regulator, is upregulated in diabetic wounds and has recently been shown to play a regulatory roles in vasculogenic pathways. We have previously described a novel method to topically silence target genes in a wound bed with small interfering (si)RNA. We hypothesized that silencing p53 results in improved diabetic wound healing and augmentation of vasculogenic mediators. Paired 4-mm stented wounds were created on diabetic db/db mice. Topically applied p53 siRNA, evenly distributed in an agarose matrix, was applied to wounds at postwound day 1 and 7 (matrix alone and nonsense siRNA served as controls). Animals were sacrificed at postwound days 10 and 24. Wound time to closure was photometrically assessed, and wounds were harvested for histology, immunohistochemistry, and immunofluorescence. Vasculogenic cytokine expression was evaluated via Western blot, reverse transcription-polymerase chain reaction, and enzyme-linked immunosorbent assay. The ANOVA/t-test was used to determine significance (p≤ 0.05). Local p53 silencing resulted in faster wound healing with wound closure at 18±1.3 d in the treated group vs. 28±1.0 d in controls. The treated group demonstrated improved wound architecture at each time point while demonstrating near-complete local p53 knockdown. Moreover, treated wounds showed a 1.92-fold increase in CD31 endothelial cell staining over controls. Western blot analysis confirmed near-complete p53 knockdown in treated wounds. At day 10, VEGF secretion (enzyme-linked immunosorbent assay) was significantly increased in treated wounds (109.3±13.9 pg/mL) vs. controls (33.0±3.8 pg/mL) while reverse transcription-polymerase chain reaction demonstrated a 1.86-fold increase in SDF-1 expression in treated wounds vs. controls. This profile was reversed after the treated wounds healed and before closure of controls (day 24). Augmented vasculogenic cytokine profile and endothelial cell markers are associated with improved diabetic wound healing in topical gene therapy with p53 siRNA., (© 2010 by the Wound Healing Society.)

Objective: Radiotherapy, an essential modality in cancer treatment, frequently induces fibrotic processes in the skin, including accumulation of extracellular matrix. Transforming growth factor-β is essential in regulating extracellular matrix gene expression and is dependent on Smad3, an intracellular mediator/transcription factor. Our study characterized the genetic expression involved in extracellular matrix accumulation during radiation-induced fibrosis. We performed Smad3 gene silencing in an attempt to abrogate the effects of radiation., Study Design: Laboratory research., Setting: University laboratory., Subjects and Methods: C57 murine dermal fibroblasts were irradiated with 20 Gy RNA isolated (0, 6, 12, 24, 48, 72 hours postirradiation) and mRNA analyzed (reverse transcriptase polymerase chain reaction) for known regulators (Smad3, interleukin-13 [IL-13]), tumor necrosis factor-α [TNF-α]) and mediators of fibrosis (collagen 1A1 [Col1A1]), TGF-β, matrix metalloprotease-1 and -2 (MMP-1, MMP-2), and tissue inhibitor of metalloprotease-1 (TIMP-1). Smad3 gene expression was silenced using siRNA in an effort to restore an unirradiated gene profile., Results: Following irradiation, there was a steady increase in mRNA expression of Smad3, IL-13, TGF-β, Col1A1, MMP-2, TIMP-1, with peak at 12 to 24 hours and subsequent decline by 72 hours. TNF-α expression remained elevated throughout. MMP-1 showed minimal expression initially, which decreased to negligible by 72 hours. Inhibition of Smad3 significantly decreased expression of Col1A1, TGF-β, MMP-2, and TIMP-1. IL-13 and TNF-α expression was not affected by Smad3 silencing., Conclusion: We have characterized the early-phase mRNA expression profiles of the major mediators of radiation-induced fibrosis. Smad3 siRNA effectively abrogated the elevation of Col1A1, TGF-β, TIMP-1, and MMP-2. IL-13 and TNF-α were unaffected by Smad3 silencing and appear to be minor regulators in fibrosis. These findings suggest a therapeutic rationale for Smad3 silencing in vivo., (Copyright © 2010 American Academy of Otolaryngology–Head and Neck Surgery Foundation. Published by Mosby, Inc. All rights reserved.)

Objective: To attempt to mitigate the effects of irradiation on murine skin after high-dose radiation using a novel transcutaneous topical delivery system to locally inhibit gene expression with small interfering RNA (siRNA) against Smad3., Design: Laboratory investigation., Setting: University laboratory., Subjects: Twenty-five wild-type C57 mice., Intervention: In an isolated skin irradiation model, the dorsal skin of C57 wild-type mice was irradiated (45 Gy). Just before irradiation, Smad3 and nonsense siRNA were applied to 2 separate dorsal skin areas and then reapplied weekly. Skin was harvested after 1 and 4 weeks. Smad3 expression were assessed by immunohistochemistry, and collagen deposition and architecture was examined using picrosirius red collagen staining., Main Outcome Measures: Epidermal thickness was measured semiquantitatively at 4 weeks. Radiation-induced fibrosis was measured quantitatively via tensiometry. The Young modulus, a measure of cutaneous rigidity inversely related to elasticity, was determined, with normal irradiated skin serving as a control specimen., Results: Murine skin treated with topical Smad3 siRNA demonstrated effective Smad3 inhibition at 1 week and persistent suppression at 4 weeks. Collagen deposition and epidermal thickness were significantly decreased in skin treated with Smad3 siRNA compared with control irradiated skin. Tensiometry demonstrated decreased tension in Smad3 siRNA-treated skin, with a Young modulus of 9.29 MPa (nonirradiated normal skin, 7.78 MPa) compared with nonsense (control) siRNA-treated skin (14.68 MPa)., Conclusions: Smad3 expression can be effectively silenced in vivo using a novel topical delivery system. Moreover, cutaneous Smad3 inhibition mitigates radiation-induced changes in tissue elasticity, restoring a near-normal phenotype.

Background: Primary alveolar cleft repair has a 41 to 73 percent success rate. Patients with persistent alveolar defects require secondary bone grafting. The authors investigated scaffold-based therapies designed to augment the success of alveolar repair., Methods: Critical-size, 7 x 4 x 3-mm alveolar defects were created surgically in 60 Sprague-Dawley rats. Four scaffold treatment arms were tested: absorbable collagen sponge, absorbable collagen sponge plus recombinant human bone morphogenetic protein-2 (rhBMP-2), hydroxyapatite-tricalcium phosphate, hydroxyapatite-tricalcium phosphate plus rhBMP-2, and no scaffold. New bone formation was assessed radiomorphometrically and histomorphometrically at 4, 8, and 12 weeks., Results: Radiomorphometrically, untreated animals formed 43 +/- 6 percent, 53 +/- 8 percent, and 48 +/- 3 percent new bone at 4, 8, and 12 weeks, respectively. Animals treated with absorbable collagen sponge formed 50 +/- 6 percent, 79 +/- 9 percent, and 69 +/- 7 percent new bone, respectively. Absorbable collagen sponge plus rhBMP-2-treated animals formed 49 +/- 2 percent, 71 +/- 6 percent, and 66 +/- 7 percent new bone, respectively. Hydroxyapatite-tricalcium phosphate treatment stimulated 69 +/- 12 percent, 86 +/- 3 percent (p < 0.05), and 87 +/- 14 percent new bone, respectively. Histomorphometry demonstrated an increase in bone formation in animals treated with hydroxyapatite-tricalcium phosphate plus rhBMP-2 (p < 0.05; 4 weeks) compared with empty scaffold., Conclusions: Radiomorphometrically, absorbable collagen sponge and hydroxyapatite-tricalcium phosphate scaffolds induced more bone formation than untreated controls. The rhBMP-2 added a small but significant histomorphometric osteogenic advantage to the hydroxyapatite-tricalcium phosphate scaffold.

The aim was to assess the influence of treatment, tumor stages and sites on the severity of dysphagia following treatment. Sequential modified barium swallow (MBS) examinations were performed in patients who complained of chronic dysphagia following treatment of their head and neck cancer. Patients were selected if they were cancer free at their last MBS and had 2 or more MBS studies. Dysphagia severity was graded on a scale of 1 to 7. Dysphagia grade was compared between the first and last MBS to assess its evolution. Between 1996 and 2005, 63 patients with chronic dysphagia underwent MBS to assess dysphagia severity for nutritional support. Twenty-one patients (33%) had improvement of their dysphagia. Two of these patients (3%) achieved normalization of the swallowing. Twenty-five patients (40%) had no change of the dysphagia severity. Dysphagia grade increased in 17 patients (27%). Analysis of patient characteristics did not show any significant difference between these three groups of patients. MBS is a useful tool to monitor dysphagia severity and to identify aspiration risk. Stages of disease and treatment modality do not seem to impact on the course of dysphagia.

Our aim was to identify risk factors for aspiration following concurrent chemoradiation for oropharyngeal cancer. 46 patients with locally advanced oropharyngeal carcinoma underwent concurrent chemoradiation at our institution. All patients underwent modified barium swallow to assess dysphagia severity and to determine the need for continued tube feedings after treatment. Dysphagia severity was graded as 1-7. There were 5 Grade 2, 11 Grade 3, 5 Grade 4, 5 Grade 5, 10 Grade 6 and 10 Grade 7 scores. 25 patients (54%) developed aspiration (5 trace, 20 severe). The aspiration rate for T1-T2 and T3-T4 tumours was 31% and 67%, respectively (p = 0.03). There was no statistical difference in the aspiration rate between the base of the tongue and tonsillar carcinoma (p = 0.23). Despite anatomical organ preservation, most patients with locally advanced oropharyngeal carcinoma had moderate to severe dysphagia after chemoradiation. Patients with large tumours had a significant risk of developing aspiration following treatment.

Background: The study of human autologous fat grafting has been primarily anecdotal. In this study, the authors aim to develop a murine model that recapitulates human fat grafting to study the fate of injected fat and the cell populations contained within., Methods: The authors' method of fat harvesting and refinement has been described previously. The authors injected nude and tie2/lacZ mice with 2 ml of human lipoaspirate placed on the dorsal surface in a multipass, fan-like pattern. Fatty tissue was injected in small volumes of approximately 1/30 ml per withdrawal. The dorsal skin and associated fat was excised at various time points. Sections were stained with hematoxylin and eosin and cytochrome c oxidase IV. Transgenic tie2/lacZ samples were stained with X-galactosidase. At the 8-week time point, volumetric analysis was performed., Results: Volumetric analysis at the 8-week time point showed 82 percent persistence of the original volume. Gross analysis showed it to be healthy, nonfibrotic, and vascularized. Hematoxylin and eosin analysis showed minimal inflammatory or capsular reaction, with viable adipocytes. Fat grafted areas were vascularized with multiple blood vessels. Cytochrome c oxidase IV human-specific stain and beta-galactosidase expression revealed these vessels to be of human origin., Conclusions: The authors have developed a murine model with which to study the fate of injected lipoaspirate. There is a high level of persistence of the grafted human fat, with minimal inflammatory reaction. The fat is viable and vascularized, demonstrating human-derived vessels in a mouse model. This model provides a platform for studying the populations of progenitor cells known to reside in lipoaspirate.

We tested the hypothesis that the stable isotope [(13)C]pantoprazole is O-demethylated by cytochrome P450 CYP2C19 and that the (13)CO(2) produced and exhaled in breath as a result can serve as a safe, rapid, and noninvasive phenotyping marker of CYP2C19 activity in vivo. Healthy volunteers who had been genotyped for the CYP2C19(*)2, CYP2C19(*)3, and CYP2C19(*)17 alleles were administered a single oral dose of [(13)C]pantoprazole sodium-sesquihydrate (100 mg) with 2.1 g of sodium bicarbonate. Exhaled (13)CO(2) and (12)CO(2) were measured by IR spectroscopy before (baseline) and 2.5 to 120 min after dosing. Ratios of (13)CO(2)/(12)CO(2) after [(13)C]pantoprazole relative to (13)CO(2)/(12)CO(2) at baseline were expressed as change over baseline (DOB). Maximal DOB, DOB(15) to DOB(120), and area under the DOB versus time curve (AUC(0-120) and AUC(0-infinity)) were significantly different among three genotype groups (CYP2C19(*)1/(*)1, n = 10; CYP2C19(*)1/(*)2 or CYP2C19(*)1/(*)3, n = 10; and CYP2C19(*)2/(*)2, n = 5) with predicted extensive metabolizers (EMs), intermediate metabolizers (IMs), and poor metabolizers (PMs) of CYP2C19, respectively (Kruskal-Wallis test, p < 0.01); linear regression analysis indicated a gene-dose effect relationship (r(2) ranged between 0.236 and 0.522; all p < 0.05). These breath test indices were significantly lower in PMs than IMs (p < 0.05) or EMs (p < 0.01) of CYP2C19. [(13)C]Pantoprazole plasma exposure showed significant inverse correlation with breath test indices in the respective subjects (Pearson r = -0.74; p = 0.038). These feasibility data suggest that the [(13)C]pantoprazole breath test is a reliable, rapid, and noninvasive probe of CYP2C19 and seems to be a useful tool to optimize drug therapy metabolized by CYP2C19.

Background: Despite technical advancement, treatment of congenital alveolar clefts has remained controversial. Currently, primary alveolar cleft repair (i.e., gingivoperiosteoplasty) has a 41 to 73 percent success rate. However, the remaining patients have persistent alveolar bone defects requiring secondary grafting procedures. Morbidity of secondary procedures includes pain, graft resorption, extrusion or infection, and graft or tooth loss. The authors present a novel rat alveolar defect model designed to facilitate investigation of therapeutics aimed at improving bone formation following primary alveolar cleft repair in humans., Methods: Sixteen 8-week-old Sprague-Dawley rats underwent creation of a 7 x 4 x 3-mm complete alveolar defect from the maxillary incisors to the zygomatic arch. Four animals were humanely killed at each of the following time points: 0, 4, 8, and 12 weeks. Morphometric analysis of the alveolar defect was determined by means of micro-computed tomography and histology., Results: Micro-computed tomography demonstrated that new bone filled 43 +/- 5.6 percent of the alveolar defect at 4 weeks, 53 +/- 8.3 percent at 8 weeks, and 48 +/- 3.5 percent at 12 weeks. Histologically, at 4 weeks, proliferating fibroblasts and polymorphonuclear cells were scattered throughout the disorganized collagen in the intercalary gap. By 8 weeks, nascent woven bone spicules extended from the edges of the defect. At 12 weeks, the woven spicules had remodeled, with scant additional bone deposition., Conclusion: This model creates a critical-size alveolar defect that is similar in size and location to human alveolar defects and is suitable for studying proposed therapeutics.

Alzheimer disease beta-amyloid (Abeta) peptides are generated via sequential proteolysis of amyloid precursor protein (APP) by BACE1 and gamma-secretase. A subset of BACE1 localizes to cholesterol-rich membrane microdomains, termed lipid rafts. BACE1 processing in raft microdomains of cultured cells and neurons was characterized in previous studies by disrupting the integrity of lipid rafts by cholesterol depletion. These studies found either inhibition or elevation of Abeta production depending on the extent of cholesterol depletion, generating controversy. The intricate interplay between cholesterol levels, APP trafficking, and BACE1 processing is not clearly understood because cholesterol depletion has pleiotropic effects on Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this study, we used an alternate strategy to explore the function of BACE1 in membrane microdomains without altering the cellular cholesterol level. We demonstrate that BACE1 undergoes S-palmitoylation at four Cys residues at the junction of transmembrane and cytosolic domains, and Ala substitution at these four residues is sufficient to displace BACE1 from lipid rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null fibroblasts and neuroblastoma cells revealed that S-palmitoylation neither contributes to protein stability nor subcellular localization of BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1 did not have discernible influence on BACE1 processing of APP or secretion of Abeta. These results indicate that post-translational S-palmitoylation of BACE1 is not required for APP processing, and that BACE1 can efficiently cleave APP in both raft and non-raft microdomains.

Voriconazole is an effective antifungal drug, but adverse drug-drug interactions associated with its use are of major clinical concern. To identify the mechanisms of these interactions, we tested the inhibitory potency of voriconazole with eight human cytochrome P450 (CYP) enzymes. Isoform-specific probes were incubated with human liver microsomes (HLMs) (or expressed CYPs) and cofactors in the absence and the presence of voriconazole. Preincubation experiments were performed to test mechanism-based inactivation. In pilot experiments, voriconazole showed inhibition of CYP2B6, CYP2C9, CYP2C19, and CYP3A (half-maximal [50%] inhibitory concentrations, <6 microM); its effect on CYP1A2, CYP2A6, CYP2C8, and CYP2D6 was marginal (<25% inhibition at 100 microM voriconazole). Further detailed experiments with HLMs showed that voriconazole is a potent competitive inhibitor of CYP2B6 (K(i) < 0.5), CYP2C9 (K(i) = 2.79 microM), and CYP2C19 (K(i) = 5.1 microM). The inhibition of CYP3A by voriconazole was explained by noncompetitive (K(i) = 2.97 microM) and competitive (K(i) = 0.66 microM) modes of inhibition. Prediction of the in vivo interaction of voriconazole from these in vitro data suggests that voriconazole would substantially increase the exposure of drugs metabolized by CYP2B6, CYP2C9, CYP2C19, and CYP3A. Clinicians should be aware of these interactions and monitor patients for adverse effects or failure of therapy.

The anaerobic degradation of tetradecylamine and other long-chain alkylamines by a newly isolated denitrifying bacterium was studied. Strain ZN6 was isolated from a mixture of soil and active sludge and was identified as representing Pseudomonas stutzeri, based on partial 16S rRNA gene sequence analysis. Strain ZN6 was a mesophilic, motile, Gram-negative rod-shaped bacterium and was able to grow on a variety of compounds including even-numbered primary fatty amines with alkyl chains ranging from C(4) to C(18) coupled to nitrate reduction. Alkylamines were used as sole carbon, energy and nitrogen source and were completely mineralized. Nitrate was dissimilated by ZN6 to nitrite. When strain ZN6 was grown under nitrate limitation, nitrite was slowly dissimilated further. When cocultivated with the complete denitrifier Castellaniella defragens ZN3, anaerobic degradation under denitrifying of alkylamines by strain ZN6 was slightly faster. Strain ZN3 is a complete denitrifier, unable to convert tetradecylamine, and was copurified from the same enrichment culture as strain ZN6. The proposed pathway for the degradation of alkylamines in strain ZN6 starts with C-N cleavages to alkanals and further oxidation to the corresponding fatty acids.

Cardiac involvement in lung cancer is found in up to 25% of autopsy cases. However, despite the considerable mortality and morbidity associated with cardiac metastasis, antemortem diagnosis is unusual. A rare case of lung cancer presenting as a left atrial mass is reported.

Objective: We assessed the rate of aspiration following radiation for non-nasopharyngeal head and neck cancer., Design: Retrospective study., Setting: Veterans Administration Hospital., Patients: Thirty-three patients who underwent radiation for head and neck cancer. Modified barium swallow was performed prior to and following treatment to assess the persistence of dysphagia and aspiration risk. All patients were cancer free at the time of the swallowing study. Dysphagia severity was graded from 1 to 7., Results: Preradiation baseline dysphagia was observed as follows: 10 grade 1, 14 grade 2, 9 grade 3, and 1 grade 4. Following radiation, at a median follow-up of 3 months, nine patients had grade 1, eight patients had grade 2, six patients had grade 3, two patients had grade 4, three patients had grade 5, two patients had grade 6, and three patients had grade 7. Overall, 24% (8 of 33) of the patients developed aspiration (grades 5-7). Fifteen percent (5 of 33) of the patients had severe aspiration (grades 6-7) requiring tube feedings. All patients who developed severe aspiration continued to require tube feedings more than 1 year following treatment completion., Conclusion: Aspiration is a significant source of morbidity following radiation for non-nasopharyngeal head and neck cancer. Aspiration may develop for all tumour stages or sites. Diagnostic studies such as modified barium swallow should be included in future prospective head and neck cancer studies to assess the prevalence of aspiration because of its often silent nature.

The vacuolar-type H+ -ATPase (V-ATPase) is a multimeric enzyme with diverse functions in plants such as nutrient transport, flowering, stress tolerance, guard cell movement and development. A partial sequence of V-ATPase proteolipid was identified among the expressed sequence tags (ESTs) generated from Acanthus ebracteatus, and selected for full-length sequencing. The 876-nucleotide cDNA consists of an open reading frame of 165 amino acids. The deduced amino acid sequence displays high similarity (81%) with its homologs from Arabidopsis thaliana, Avecinnia marina and Gossypium hirsutum with the four transmembrane domains characteristics of the 16 kDa proteolipid subunit c of V-ATPase well conserved in this protein. Southern analysis revealed the existence of several members of proteolipid subunit c of V-ATPase in A. ebracteatus. The mRNA of this gene was detected in leaf, floral, stem and root tissues, however, the expression level was lower in stem and root tissues.

The effectiveness of the cough reflex in patients who aspirated following radiation for head and neck cancer was evaluated in 89 patients (49 chemoradiation, 33 postoperative radiation, and 7 radiation alone). All patients had modified barium swallow because of dysphagia. The cough reflex was graded as present and effective, ineffective, intermittently effective, or absent. All patients were cancer-free at the time of the swallowing study. The cough reflex was present and effective in 46 patients (52%), ineffective in 17 patients (19%), and absent in 26 patients (29%) on initial investigation. Among the 43 patients who had ineffective or absent cough reflex, their treatment was chemoradiation (26), postoperative radiation (13), and radiation alone (4). In 30 patients who had sequential modified barium swallow, the cough reflex was constantly effective, ineffective, or intermittently effective in 12 (40%), 13 (43%), and 5 (17%) patients, respectively. The cough reflex was frequently ineffective or absent in patients who aspirated following radiation for head and neck cancer. Cough may also be intermittently ineffective to protect the airways following radiation.

Aim: To assess the risk of developing aspiration during chemoradiation for head and neck cancer., Patients and Methods: A retrospective review of 114 patients who underwent concurrent chemoradiation for locally advanced head and neck cancer was undertaken. Patients were determined as having aspiration if they had pneumonia on chest-X-ray (CXR) and/or had documented aspiration on the modified barium swallow (MBS) during their treatment., Results: Fifteen patients (13%) developed aspiration during chemoradiation. Twelve patients (10%) had aspiration demonstrated on CXR alone (9 patients) or combined with MBS (3 patients). Three patients (3%) had aspiration on MBS alone. Three of the six patients with aspiration observed on MBS had normal swallowing on their pretreatment MBS. All 15 patients had severe mucositis and neutropenia at the time of the aspiration. Despite broad-spectrum antibiotics and supportive care, six patients (5%) died., Conclusion: Aspiration may develop during chemoradiation for head and neck cancer because of radiation-induced altered swallow.

This study examines the efficacy of swallowing therapy in cancer-free patients who developed aspiration following treatment for locally advanced head and neck cancer. The records of 41 patients who underwent swallowing therapy for aspiration were reviewed. All patients were cancer free at a median follow-up of 25 months (6-150 months). Their treatment were respectively chemoradiation (24), and postoperative radiation (17). All patients had two or more modified barium swallow (MBS). Dysphagia severity was graded from 1 to 7. Dysphagia grade was compared before and following swallowing therapy. Before swallowing therapy, there were 16 grade 5 (trace aspiration), and 25 grade 6-7 (severe aspiration). In the chemoradiation group, there were nine grade 5, five grade 6, and 10 grade 7. Corresponding numbers for the postoperative group were: seven grade 5, seven grade 6, and three grade 7. Following swallowing therapy, there were six grade 3, seven grade 4, 10 grade 5, six grade 6, and 12 grade 7. In the chemoradiation group, there were four grade 3, three grade 4, four grade 5, five grade 6, and eight grade 7. In the postoperative group, there were two grade 3, four grade 4, six grade 5, one grade 6, and four grade 7. Overall, 13 patients (32%) had improvement of their dysphagia severity. Seven of them were in the chemoradiation group (29%), and six (35%) were in the postoperative group. Among 25 patients who presented with grade 6-7 aspiration, only nine (36%) improved to grade 5 or less. Four of them (27%) were in the chemoradiation group, and five (29%) were in the postoperative group. Swallowing therapy is effective to improve dysphagia severity and reduce the need for tube feedings. However, a significant number of patients still suffered from chronic severe aspiration. New strategies must be devised to improve their outcome.

Background: Alzheimer's disease (AD) is characterized by cerebral deposition of beta-amyloid (Abeta) peptides. Abeta is released from ectodomain cleaved amyloid precursor protein (APP) via intramembranous proteolysis by gamma-secretase, a complex consisting of presenilin and a few other proteins. p23/TMP21, a member of the p24 family type I transmembrane proteins, was recently identified as a presenilin complex component capable of modulating gamma-secretase cleavage. The p24 family proteins form oligomeric complexes and regulate vesicular trafficking in the early secretory pathway, but their role in APP trafficking has not been investigated., Results: Here, we report that siRNA-mediated depletion of p23 in N2a neuroblastoma and HeLa cells produces concomitant knockdown of additional p24 family proteins and increases secretion of sAPP. Furthermore, intact cell and cell-free Abeta production increases following p23 knockdown, similar to data reported earlier using HEK293 cells. However, we find that p23 is not present in mature gamma-secretase complexes isolated using an active-site gamma-secretase inhibitor. Depletion of p23 and expression of a familial AD-linked PS1 mutant have additive effects on Abeta42 production. Knockdown of p23 expression confers biosynthetic stability to nascent APP, allowing its efficient maturation and surface accumulation. Moreover, immunoisolation analyses show decrease in co-residence of APP and the APP adaptor Mint3. Thus, multiple lines of evidence indicate that p23 function influences APP trafficking and sAPP release independent of its reported role in gamma-secretase modulation., Conclusion: These data assign significance to p24 family proteins in regulating APP trafficking in the continuum of bidirectional transport between the ER and Golgi, and ascribe new relevance to the regulation of early trafficking in AD pathogenesis.

The aim of this study was to evaluate the aspiration rate following nonsurgical therapy, i.e. chemoradiation or radiation alone for laryngeal cancer. Modified barium swallow was performed in 43 patients who complained of dysphagia following chemoradiation (n = 22) or radiation alone (n = 21) for laryngeal cancer. Patients were selected if they were cancer free at the time of the swallowing study. Dysphagia severity was graded on a scale of 1-7. Patients were grouped according to the dysphagia severity: no aspiration (grade 1-4), and severe (grade 5-7). Mean and median dysphagia grades were 4.4/5 and 3.5/3 for chemoradiation and radiation, respectively. Aspiration occurred in 12 patients (54%) of the chemoradiation group and 7 (33%) of the radiation alone group (p = 0.13). There was a higher proportion of patients with large tumor (T3-T4) in the chemoradiation group (64%) compared to the radiation group (5%) (p = 0.0001). Aspiration is a significant source of morbidity in patients treated for laryngeal cancer with chemoradiation or radiation alone. Aspiration occurred in both groups. Although the observed difference in aspiration rates did not achieve statistical significance, the higher aspiration rate in the chemoradiation group may be due to a higher proportion of large tumors, to the additional toxic effect of chemotherapy, or to the small number of patients in both groups. Diagnostic studies such as modified barium swallow should be part of future laryngeal cancer prospective studies to assess the prevalence of aspiration as it may be silent.

Background and Purpose: We would like to assess the prevalence of aspiration before and following chemoradiation for head and neck cancer., Patients and Methods: We reviewed retrospectively the Modified Barium Swallow (MBS) in 63 patients who underwent concurrent chemotherapy and radiation for head and neck cancer. MBS was performed prior to treatment to determine the need for immediate gastrostomy tube placement. MBS was repeated following treatment to assess the safety of oral feeding prior to removal of tube feeding. All patients were cancer free at the time of the swallowing study. No patient had surgery. Dysphagia severity was graded on a scale of 1-7. Tube feedings were continued if patients were diagnosed to have severe aspiration (grade 6-7) or continued weight loss. Patients with abnormal swallow (grade 3-7) received swallowing therapy following MBS., Results: Before treatment, there were 18 grade 1, 18 grade 2, 9 grade 3, 8 grade 4, 3 grade 5, 3 grade 6, and 4 grade 7. Following chemoradiation, at a median follow-up of 2 months (1-10 months), one patient had grade 1, eight patients had grade 2, nine patients had grade 3, eight patients had grade 4, 13 patients had grade 5, seven patients had grade 6, and 11 patients had grade 7. Six patients died from aspiration pneumonia (one before, three during, and two post-treatment), and did not have the second MBS. Overall, 37/63 (59%) patients developed aspiration, six of them (9%) fatal. If we excluded the 10 patients who had severe aspiration at diagnosis and the six patients who died from pneumonia, the prevalence of severe aspiration was 33% (21/63)., Conclusions: Aspiration remained a significant morbidity following chemoradiation for head and neck cancer. Its prevalence is underreported in the literature because of its often silent nature. Diagnostic studies such as MBS should be part of future head and neck cancer prospective studies to assess the prevalence of aspiration, and for rehabilitation.

Objective: The purpose of the study is to evaluate dysphagia severity following chemoradiation and postoperative radiation for head and neck cancer, and particularly the aspiration risk because of its potential life-threatening consequence., Materials and Methods: We reviewed retrospectively the modified barium swallow (MBS) results in 110 patients who complained of dysphagia following chemoradiation (57) and postoperative radiation (53) of their head and neck cancer. Patients were selected if they were cancer free at the time of the swallowing study. Dysphagia severity was graded on a scale of 1-7. Patients were grouped according to the dysphagia severity: mild (grades 2-3), moderate (grades 4-5), and severe (grades 6-7)., Results: Mean and median dysphagia grades were 4.84/5 and 4.12/4 for chemoradiation and postoperative radiation respectively. The mean difference between the two groups is statistically significant (p=0.02). Mild dysphagia occurred in 13 patients (22%) of the chemoradiation group and 17 (32%) of the postoperative group. Corresponding number for the moderate group was 25 (43%) and 25 (48%), respectively. Severe dysphagia was significant in the chemoradiation group (34%) compared to the postoperative group (19%). However, the difference was not statistically significant (p=0.29). There was a higher proportion of patients with large tumor (T3-T4) in the chemoradiation group who developed severe dysphagia., Conclusion: Dysphagia remained a significant morbidity of chemoradiation and postoperative radiation for head and neck cancer. Dysphagia may be more severe in the chemoradiation group because of the higher proportion of patients with large tumor, the high radiation dose, and a high number of oropharyngeal tumors. Aspiration occurred in both groups. Diagnostic studies such as MBS should be part of future head and neck cancer prospective studies to assess the prevalence of aspiration, as it may be silent.

Unlabelled: The purpose of this investigation was to evaluate chronic dysphagia (lasting 3 or more months) following treatment for head and neck cancer. Since dysphagia is a common sequela post therapy in cancer survivors, it may be helpful for the clinician to be aware of the persistence of dysphagia as well as its usual severity. Modified Barium Swallow (MBS) examinations were performed in cancer-free patients who complained of dysphagia following treatment for head and neck cancer. The severity of the dysphagia was graded on a scale of 1 to 7. Each patient had sequential MBS and underwent swallowing therapy in between. The severity of dysphagia was compared between the first and last MBS study to determine whether the swallowing function had returned to normal., Results: Between 1996 and 2004, 12 patients with dysphagia underwent repeated MBS following treatment. Swallowing function did not return to normal in all patients. At a median time of 29 months following treatment (range 8 to 94 months), the severity of dysphagia decreased in 8 patients (67%), remained unchanged in 3 patients (25%) and worsened in 1 patient (8%). Chronic dysphagia following treatment is unlikely to resolve with time despite rehabilitation therapy. Excessive scarring following treatment may be responsible for the persistence and severity of dysphagia. Physicians should be aware of the long-term effects of dysphagia on patient nutrition and psychological well-being.

E-cadherin is a cell surface adhesion molecule that is expressed in both epithelial and endothelial tissues. In this study, an improved method for the simple production of the human E-cadherin-derived first repeat E-CAD1 was developed by exporting it into the periplasmic space of Escherichia coli. Localization of the recombinant protein into the periplasm allowed the isolation of E-CAD1 without cell lysis. The N-terminus of E-CAD1 is fused to a streptavidin-derived peptide to allow single-step purification using a Streptag affinity column. Optimal expression in LB medium produced 3.2 mg/L while expression in minimal medium containing 15NH(4)Cl as the sole source of nitrogen produced 4.2 mg/L purified (15)N-labeled E-CAD1. Heteronuclear NMR spectroscopy confirmed that the purified E-CAD1 produced in this manner was correctly folded. The expression and purification protocol for unlabeled and isotopically labeled E-CAD1 permits rapid preparative production of this protein for mechanistic and structural studies.

Waglerin-1 (Wtx-1) is a 22-amino acid peptide that competitively antagonizes muscle nicotinic acetylcholine receptors (nAChRs). Previous work demonstrated that Wtx-1 binds to mouse nAChRs with higher affinity than receptors from rats or humans, and distinguished residues in alpha and epsilon subunits that govern the species selectivity. These studies also showed that Wtx-1 binds selectively to the alpha-epsilon binding site with significantly higher affinity than to the alpha-delta binding site. Here we identify residues at equivalent positions in the epsilon, gamma, and delta subunits that govern Wtx-1 selectivity for one of the two binding sites on the nAChR pentamer. Using a series of chimeric and point mutant subunits, we show that residues Gly-57, Asp-59, Tyr-111, Tyr-115, and Asp-173 of the epsilon subunit account predominantly for the 3700-fold higher affinity of the alpha-epsilon site relative to that of the alpha-gamma site. Similarly, we find that residues Lys-34, Gly-57, Asp-59, and Asp-173 account predominantly for the high affinity of the alpha-epsilon site relative to that of the alpha-delta site. Analysis of combinations of point mutations reveals that Asp-173 in the epsilon subunit is required together with the remaining determinants in the epsilon subunit to achieve Wtx-1 selectivity. In particular, Lys-34 interacts with Asp-173 to confer high affinity, resulting in a DeltaDeltaG(INT) of -2.3 kcal/mol in the epsilon subunit and a DeltaDeltaG(INT) of -1.3 kcal/mol in the delta subunit. Asp-173 is part of a nonhomologous insertion not found in the acetylcholine binding protein structure. The key role of this insertion in Wtx-1 selectivity indicates that it is proximal to the ligand binding site. We use the binding and interaction energies for Wtx-1 to generate structural models of the alpha-epsilon, alpha-gamma, and alpha-delta binding sites containing the nonhomologous insertion.

Cadherin is a cell adhesion molecule crucial for epithelial and endothelial cell monolayer integrity. The previously solved x-ray crystallographic structure of the E-CAD12 cis-dimer displayed an unpaired Cys(9), which protruded away from the Cys(9) on the other protomer. To investigate the possible biological function of Cys(9) within the first repeat (the E-cadherin-derived N-terminal repeat), E-CAD1 was overexpressed and secreted into the periplasmic space of Escherichia coli cells. Recombinant E-CAD1 produced a mixed monomer and dimer in an equilibrium fashion. The dimer was linked by a disulfide through Cys(9) pairing. Analysis by high pressure liquid chromatography and electron microscopy suggested the existence of oligomeric complexes. Mutation at Trp(2) appears to indicate that these oligomeric complexes trans-dimerize. Interestingly, mutation of Cys(9) affected not only the cis-dimerization, but also the trans-oligomerization of E-CAD1. Accordingly, it is plausible that, under oxidative stress, the homophilic interactions of E-cadherin through E-CAD1 may be promoted and stabilized by this disulfide bond.

One hundred and one pummelo plants representing eight different cultivars of pummelo were fingerprinted and evaluated for intra- and inter-varietal variability using five enzyme systems. Starch gel electrophoresis was used to analyse extracts from fresh young leaves collected from trees located in the provinces of Can Tho and Vinh Long. Isozymic analysis revealed that all cultivars are isozymically heterogeneous, and composed of two or more clones. A total of 26 clones were discriminated from eight cultivars. Unique combinations of isozymic variants of the five enzymes assayed identified 18 (69%) of the 26 clones. The remaining eight clones could be separated into four additional groups. Our results have proven the potential use of isozymic variability for cultivar identification and assessment of the uniformity or purity of a variety.

Rationale: There are increasing reports of sex differences in the etiology of drug abuse in humans. A nonhuman primate model is useful for examining sex as a variable in drug abuse., Objectives: To determine whether there are sex differences in the acquisition of oral phencyclidine (PCP) self-administration and to compare the effect of altered feeding conditions on drug self-administration in male and female monkeys., Methods: Acquisition of orally delivered PCP was studied using 7 female and 11 male adult rhesus monkeys. Initially, the monkeys were not food restricted, and they were given access to water under concurrent fixed-ratio (FR) 1 schedules during daily 3-h sessions. Each lip-contact response on a drinking spout resulted in a 0.3 ml liquid delivery. After baseline levels of water intake were obtained for 5 days, water was replaced with PCP (0.125 mg/ml) at both drinking spouts. Body weights were then reduced to 85% of free-feeding weights, and the monkeys were fed 30 min before the session began. The FR value was increased from 1 to 2, 4, and 8, at both drinking spouts. As a final step in the procedure, water and PCP were concurrently available at the two spouts under FR 8 schedules. Acquisition of PCP-reinforced behavior was considered to have occurred if PCP intake was consistently greater than water intake., Results: Lip-contact responses and liquid deliveries were not significantly different between the females and males throughout the acquisition period, but there was a significant increase in responding and decrease in liquid intake as FR increased, and a significant increase in PCP consumption due to food restriction that did not differ in males and females. On a milligram per kilogram basis, female monkeys consumed nearly twice as much PCP as the males; however, this effect was not significant. The females showed significantly higher PCP than water intake while the males consumed approximately equal amounts of PCP and water. Of the seven females, 100% met the acquisition criterion of significantly greater PCP than water intake, while only 36.4% of the males met the criterion., Conclusion: These results concur with previous rat studies and indicate that female monkeys are more likely than males to acquire drug-reinforced behavior.

The BIOMIC is a computerized system used to calculate the minimal inhibitory concentration (MIC) of an antimicrobial agent from a zone of inhibition generated by a disk diffusion test. This system was developed using bacterial pathogens of human origin. This study investigated the use of the BIOMIC system for determining MICs for bacterial pathogens from animals. The MICs generated by the BIOMIC system were compared with the MICs generated using a broth microdilution testing method. A total of 663 drug-organism combinations was tested. These combinations included 3 species of gram-positive bacteria, 5 species of gram-negative bacteria, and the antimicrobial agents ampicillin, gentamicin, cephalothin, ciprofloxacin, enrofloxacin, trimethoprim/sulfamethoxazole, tetracycline, and erythromycin. Overall, the MICs generated by the BIOMIC system correlated with the broth microdilution MICs for 72% of the total drug-organism combinations tested. The Pseudomonas aeruginosa strains tested showed the highest agreement between the 2 systems, with 100% for all antibacterial agents tested, whereas Pasteurella haemolytica, Pasteurella multocida, and enterococci showed the least agreement (76%, 57%, and 47%, respectively). Among these organisms, trimethoprim-sulfa showed the least agreement (31%) and ciprofloxacin showed the greatest (91%). These results indicate that the BIOMIC system could be a useful tool in veterinary medicine for producing quantitative antimicrobial susceptibility results. However, it is currently unreliable for some drug-bacteria combinations. This discrepancy possibly could be corrected by modification of the software using data points generated by a large-scale study.

Pyrimethamine and cycloguanil resistance of Plasmodium falciparum has been linked to mutations in the dihydrofolate reductase (dhfr) portion of the dhfr-ts gene. In this paper, the DNA sequence of the dhfr-ts gene of 50 isolates from Vietnam and 2 clones (T9/94 and T9/96) isolated from a malaria patient from Thailand have been analyzed. A comparison between these isolates and clones showed differential mutation patterns. Forty-eight isolates were found to consist of mutations associated with Pyr. A novel leucine mutation at position 140 was found in the isolate VP8 and in clone T9/94. The isolate VP8 and the clone T9/94 were found to also have the characteristic changes at positions 16 (Val) and 108 (Thr) that have been found in cycloguanil-resistant isolates. The isolate VP35 was shown to be resistant to both antifolates, while the clone T9/96 was found to be sensitive to both antifolates and to have a sequence identical to that of wild-type dhfr-ts. The two clones from a single patient showed the coexistence of resistant and sensitive clones in the absence of treatment by antifolates. Since cycloguanil resistance seems to be rare in Vietnam, cycloguanil alone or in combination with other antimalarial agents might be an alternative for treatment and prophylaxis, even in areas with high resistance to pyrimethamine.

Objectives: To determine whether changes in cardiac output, regional blood flow, and intracranial pressure during permissive hypercapnia are blood pH-dependent and can be attenuated by correction of intravascular acidemia., Design: Prospective, controlled study., Setting: Research laboratory., Subjects: Female Marino ewes., Interventions: Animals were instrumented with a pulmonary artery catheter, femoral arterial and venous catheters, a catheter in the third cerebral ventricle, and ultrasonic flow probes on the left carotid, superior mesenteric, and left renal arteries 1 wk before experimentation. At initiation of the protocol, ewes underwent endotracheal intubation and mechanical ventilation under general anesthesia. Minute ventilation was reduced to induce hypercapnia with a target PaCO2 of 80 torr (10.7 kPa). In the pH-uncorrected group (n = 6), arterial blood pH was allowed to decreased without treatment. In the pH-corrected group (n = 5), 14.4 mEq/kg of sodium bicarbonate was given intravenously as a bolus to correct arterial blood pH toward a target arterial pH of 7.40 (dose calculated by the Henderson-Hasselbalch equation)., Measurements and Main Results: Arterial blood pH, PCO2, cardiac output, intracranial pressure, and carotid, superior mesenteric, and renal artery blood flow rates were measured at normocapnic baseline and at every hour during hypercapnia for 6 hrs. In the pH-uncorrected group, arterial blood pH decreased from 7.41 +/- 0.03 at normocapnia to 7.14 +/- 0.01 (p < .01 vs. normocapnia) as blood PCO2 increased to 81.2 +/- 1.8 torr (10.8 +/- 0.2 kPa). In the pH-corrected group, arterial blood pH was 7.42 +/- 0.02 at normocapnia and was maintained at 7.37 +/- 0.01 while PaCO2 was increased to 80.3 +/- 0.9 torr (10.7 +/- 0.1 kPa). Significant increases in cardiac output occurred with the initiation of hypercapnia for both groups (pH-uncorrected group: 4.3 +/- 0.6 L/min at normocapnia vs. 6.8 +/- 1.0 L/min at 1 hr [p < .05]; pH-corrected group: 4.1 +/- 0.4 at normocapnia vs. 5.7 +/- 0.4 L/min at 1 hr [p < .05]). However, this increase was sustained only in the uncorrected group. Changes in carotid and mesenteric artery blood flow rates, as a percent of baseline values, showed sustained significant increases in the pH-uncorrected groups (p < .05) and only transient (carotid at 1 hr) or no (superior mesenteric) significant change in the pH-corrected groups. Conversely, significant increases in renal artery blood flow were seen only in the pH-uncorrected group during the last 2 hrs of the experiment (p < .05). Organ blood flow, as a percent of cardiac output, did not change significantly in either group. Intracranial pressure increased significantly in the pH-uncorrected group (9.0 +/- 1.5 mm Hg at normocapnia vs. 26.8 +/- 5.1 at 1 hr, p < .05), and remained increased, while showing no significant change in the pH-corrected group (8.5 +/- 1.6 mm Hg at normocapnia to 7.7 +/- 4.2 at 1 hr)., Conclusions: Acute hypercapnia, induced within 1 hr, is associated with significant increases in cardiac output, organ blood flow, and intracranial pressure. These changes can be significantly attenuated by correction of blood pH with the administration of sodium bicarbonate, without adverse effects on hemodynamics.

The intravascular oxygenator and carbon dioxide removal device (IVOX; CardioPulmonics, Salt Lake City, UT) has been shown to perform 30% of the gas exchange in animals and patients with acute respiratory failure. Among the factors that limit gas exchange is the mass transfer resistance in the blood phase. To determine if a reduction in mass transfer resistance by mixing venous blood can enhance the O2 transfer and CO2 removal by IVOX, a right atrium-pulmonary artery venovenous bypass circuit was used in sheep to model the adult vena cava. A size 9 IVOX (894 fibers with 0.41 m2 surface area, n = 5) was incorporated in the bypass circuit and the blood flow controlled by a roller pump ranging from 1 to 4 l/min. An intra-aortic balloon was placed near the shaft of the IVOX and pulsated at the rate adjusted to best improve CO2 removal (100-120 bpm). O2 transfer and CO2 removal were measured with balloon pulsation on and off at different flow rates. Results showed that blood mixing by pulsation of the balloon caused a 25-49% increase in O2 transfer by IVOX, and this increase remained relatively constant throughout the full flow range. CO2 removal was also increased by up to 35%, but at flows between 3.5 and 4 l/min, the effect of mixing was diminished. It is concluded that reduction in the mass transfer resistance by blood mixing improves gas exchange. Because O2 is more diffusion limited, it is more dependent upon mixing of blood for gas exchange than CO2. More design improvements to incorporate active mixing may further enhance the gas exchange performance of IVOX.

In this study, 638 patients with either Plasmodium falciparum or P. vivax malaria were treated with artemisinin (qinghaosu) that was isolated and formulated into tablets and capsules in Vietnam. In all cases, artemisinin treatment resulted in a rapid clearance of parasitemia and fever. Recrudescence rates were highest in those groups receiving treatment for five or less days (50%), but were between 10% and 23% for those groups receiving the drug for 5-10 days. A low recrudescent rate (9.5%) was also found when patients were treated with a combination of artemisinin for three days and tetracycline for five days. Thus, artemisinin represents a useful and economically feasible component of the malaria control program in Vietnam.

To assess the feasibility and effectiveness of combined therapy on locally advanced cervical cancer, we entered 38 patients into a study. The patients were treated with mitomycin-C (10 mg/m2) on Days 1 and 30 and 5-FU (1000 mg/m2) on Days 1 to 4 and Days 30 to 33. In 5 weeks 4500-5000 cGy was given concurrently, followed by radioactive implants. Twenty-six patients had an early-stage disease (IB-IIB) and twelve had a late-stage disease (IIIB-IVA). Eighty-seven percent (33/38) of the patients had a tumor measuring 5 cm or more. The other 5 patients with a tumor size under 5 cm had biopsy-proven positive pelvic nodes; 2 of these 5 patients had a pretherapy hysterectomy. Tumor response, complete (CR) vs partial (PR), was assessed in 36 patients 3 months after completion of therapy. A CR was noted in 80% (29/36) of the patients. The PR status conferred a detrimental effect on the pelvic disease control (PDC), disease-free survival (DFS), and survival (S) while late stage correlated with the development of distant metastases (DM) and a poor DFS. PDC was obtained in 93% (27/29) of the patients who had a CR, as compared to only 43% (3/7) of those with a PR (P = 0.0228). The DFS and S rates were 59 and 77% for patients with a CR and 21 and 19% for those with a PR; respective P values were 0.0340 and 0.0002. Eleven percent (3/26) of the patients with an early stage developed DM, as compared to 50% (6/12) of those with late stage, (P = 0.0016). The DFS rates were 80 and 37% for patients with an early and late stage, respectively (P = 0.0141). Four patients developed transient neutropenia and one had transient thrombocytopenia. The second dose of mitomycin-C was omitted in 4 patients due to persistent neutropenia in 3 and to transfusion-related hepatitis in 1. Two percent (5/21) of the patients who had a staging laparotomy developed wound dehiscence. Three patients developed non-cancer-related small bowel obstruction requiring surgery. We concluded that this combined regimen was well tolerated. Although it was effective in controlling the cancer in the pelvis, this regimen failed to control DM in late-stage patients.

The outcome of women with adenosquamous carcinoma of the cervix has been a controversial issue. To assess the prognostic significance of this cell type, the medical records of 29 patients with stage IB primary adenosquamous carcinoma of the cervix were reviewed. Twenty-six had cervical tumors measuring 3 cm or less. With a median follow-up of 60 months, the overall disease-free 5-year survival was 85%. Pelvic lymph node metastases were found in three of 19 patients (16%) with stage IB, and periaortic lymph nodes were negative in eight. A log-likelihood chi 2 test comparing lymphatic spread, recurrence rate, and 5-year disease-free survival in patients with stage IB disease and tumors less than 3 cm versus a similar group of 58 patients with pure squamous cell carcinoma of equivalent stage and comparable size treated during the same time disclosed no significant differences in any of the indicators analyzed. We conclude that stage IB adenosquamous carcinoma of the cervix does not differ in its metastatic potential or outcome from the more common pure squamous cell carcinoma.

The 3M Company recently produced a special version of the type 6702 seed for use in animal studies of ocular melanoma. The seed consists of a single I-125 impregnated ion exchange resin sphere encapsulated in a 3.0-mm-long titanium cylinder, as opposed to the normal 4.5-mm cylinder containing from three to five resin spheres. Monte Carlo simulations and measurements show that the dose distribution around the 3-mm capsule differs in places by up to 10% from that of the 4.5-mm seed. In addition, the two seed types differ by 12% in the ratio of dose in tissue along the transverse seed axis to apparent activity. These differences must be taken into account when using these seeds.

The aim of thrombolytic therapy for acute myocardial infarction with plasminogen activators such as streptokinase is to lyse the coronary thrombus and reestablish blood flow as quickly as possible so that heart tissue loss is minimized and mortality rates are improved. Streptokinase has been encapsulated in large unilamellar phospholipid vesicles and tested in an animal model of acute myocardial infarction. The time required to restore vessel patency has been reduced more than 50% when compared with findings for free streptokinase. The total dosage of streptokinase required was lower, and smaller remnant thrombi were observed with the encapsulated agent. Results from this initial unoptimized study may have significant implications for further reduction in mortality from heart attacks by therapy with plasminogen activators.

A modification of the Smoluchowski collision theory for platelet aggregation is proposed with additional kinetic terms to accommodate observed disaggregation behavior. This model, consisting of a set of coupled, nonlinear, first-order differential equations, approximates size distributions with time for normal human platelets in plasma after the addition of a stimulus. Parameters controlling the kinetics of the formation and breakup of aggregates are numerically investigated. The aggregation coefficient, predominant during the aggregation phase, is strongly dependent on both time and aggregating agent doses. For the disaggregation phase, the disaggregation rate constants are a function of aggregate size, with a time-dependent disaggregation coefficient. Numerical results generated by the model are compared with experimental volume-size distribution curves from the literature.

Citrated platelet-rich plasma was used to occlude 3-microns and 10-microns poresize Nuclepore membranes after recalcification as a thrombus model. Morphologic studies, using both light microscopy and scanning electron microscopy, indicated that over 90 percent of the number of pores available for filtration in hydrophilic and hydrophobic membranes were occluded either partially or completely. Results of transient and steady state pressure drop measurements supported the morphologic studies. It was found that the percentage of oxygen transported across the occluded membranes was greater for filtration of red blood cell suspensions diluted with a perfluorochemical emulsion than that of those diluted with Ringers. The findings in this study suggested that perfluorochemical emulsions might transport oxygen across a thrombus to maintain tissue viability during acute ischemic events.

The Syed-Neblett perineal template has been in use at our institution since 1984. We have occasionally encountered problems in inserting the needles through the peripheral rows of the template and have seen significant convergence of the needles cephalad to the template. To solve these problems we have modified the Syed-Neblett template. In the new design, the entrance holes are closer together than in the original template, but they are angled so as to achieve a more compact design while maintaining a dose distribution close to that of the Syed-Neblett design. We have seen significantly fewer clinical problems with this design. In addition, a smaller number of needles achieve the same dose distribution; this simplifies their visualization on radiographs.

The clot-lysing ability of streptokinase (SK) was examined using membrane-bound thrombi. Encapsulation of SK in large unilamellar phospholipid vesicles (liposomes) resulted in entrapping approximately 30% of its original activity. Measurements of streptokinase activity for liposomal-encapsulated streptokinase (LESK) indicated little loss of activity or leakage in Tris-buffered saline over a 24-hr period at temperatures of 4 and 23 degrees C. However, incubation of free SK and LESK in platelet-poor plasma (PPP) at 37 degrees C resulted in a decrease of SK activity. The retention of SK activity in LESK was considerably higher than that of unentrapped SK. Clot-dissolving time (CDT) was measured by monitoring the pressure drop during slow filtration in plasma through membrane-bound thrombi. The results indicated that both LESK and free SK were able to activate the fibrinolytic system. Without prior incubation in PPP at 37 degrees C, the CDT of a SK and PPP mixture (SK/PPP) was 10.7 +/- 1.9 min (n = 12), while that of a LESK and PPP mixture (LESK/PPP) was 12.4 +/- 1.7 min (n = 12). The CDT-detected clot-lysing abilities of both SK and LESK were diminished by incubation in PPP, but to different extents. After 15- and 30-min incubations, the CDT of SK/PPP increased significantly to 15.5 +/- 1.5 and 24.1 +/- 2.4 min (n = 5, P less than 0.05), respectively. In contrast, the CDT of LESK/PPP increased to 13.3 +/- 0.8 min (n = 5) after 15 min of incubation and to 16.0 +/- 1.1 min (n = 5, P less than 0.05) after a 30-min incubation. These results suggest that entrapment of SK in liposomes preserves the thrombolytic potential of the plasminogen activator by limiting its exposure to the components of the plasma.