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Introduction: The efficacy and economy of an alternative sparing method for posterior lumbar interbody fusion (PLIF) using a single cage fixed with pedicle screws placed on a single side (SS group, n=22) was compared to that of a standard bilateral protocol using two cages and pedicle screws placed bilaterally (BL group, n=15)., Methods: All PLIFs were non-compensation cases done at a single level by a single surgeon and were similar in most background characteristics. Significant differences were not found between the two groups in fusion rates, complications or in 2-year prospectively collected outcomes including percent improvement in back and leg pain (visual analog scales) and the Oswestry disability index., Results: Perioperative results significantly favored the SS group: BL patients lost 81% more blood, used 74% more time for surgery, stayed in hospital 1.7 days longer, and the hospital-related cost per procedure was twice as high. Currently, the SS procedure typically averages less than 1 h and blood loss less than 50 mL. In summary, the BL and SS groups had similar outcomes while the SS procedure provided substantially superior efficiency and economy., Conclusion: In conclusion, the results of this retrospective comparative level III study warrant further studies on the SS protocol which may lead to the adoption of this minimally invasive protocol in the standard practice of PLIF in selected cases., (Copyright Georg Thieme Verlag KG Stuttgart. New York.)

Background: Uncontrolled proliferation and increased motility are hallmarks of neoplastic cells, therefore markers of proliferation and motility may be valuable in assessing tumor progression and prognosis. MCM2 is a member of the minichromosome maintenance (MCM) protein family. It plays critical roles in the initiation of DNA replication and in replication fork movement, and is intimately related to cell proliferation. Ki-67 is a proliferation antigen that is expressed during all but G0 phases of the cell cycle. Gelsolin is an actin-binding protein that regulates the integrity of the actin cytoskeletal structure and facilitates cell motility. In this study, we assessed the prognostic significance of MCM2 and Ki-67, two markers of proliferation, and gelsolin, a marker of motility, in non-small cell lung cancer (NSCLC)., Methods: 128 patients with pathologically confirmed, resectable NSCLC (stage I-IIIA) were included. Immunohistochemistry was utilized to measure the expressions of these markers in formalin-fixed, paraffin-embedded tumor tissues. Staining and scoring of MCM2, Ki-67 and gelsolin was independently performed. Analyses were performed to evaluate the prognostic significance of single expression of each marker, as well as the prognostic significance of composite expressions of MCM2 and gelsolin. Cox regression and Kaplan-Meier survival analysis were used for statistical analysis., Results: Of the three markers, higher levels of gelsolin were significantly associated with an increased risk of death (adjusted RR = 1.89, 95% CI = 1.17-3.05, p = 0.01), and higher levels of MCM2 were associated with a non-significant increased risk of death (adjusted RR = 1.36, 95% CI = 0.84-2.20, p = 0.22). Combined, adjusted analyses revealed a significantly poor prognostic effect for higher expression of MCM2 and gelsolin compared to low expression of both biomarkers (RR = 2.32, 95% CI = 1.21-4.45, p = 0.01). Ki-67 did not display apparent prognostic effect in this study sample., Conclusion: The results suggest that higher tumor proliferation and motility may be important in the prognosis of NSCLC, and composite application of biomarkers might be of greater value than single marker application in assessing tumor prognosis.

Background: Gelsolin is an actin-binding protein that mediates cellular motility and maintains the integrity of cytoskeletal structure. Diminished expression of gelsolin has been observed in human cancer cell lines and tumors. Studies of the prognostic effect of gelsolin expression (GE) in non-small cell lung cancer (NSCLC) are rare and results are inconsistent to date. The present study used immunohistochemistry to evaluate the prognostic effect of gelsolin expression in 155 patients with resectable NSCLC., Methods: Detection of gelsolin in tumor cells was performed by immunohistochemistry, and two approaches to classification were used to describe expression: expression level (negative, reduced or high) and expression uniformity (uniform or variable). Expression level was determined by a weighted index of intensity of staining (i.e., overall tendency) in the specimen. Expression uniformity was based on the presence or absence of variability in immunostaining within the tumor section. Chi-square test, student t-test, Cox proportional hazards regression and Kaplan-Meier survival analysis were used in data analyses., Results: After controlling for covariates, high level gelsolin expression was significantly associated with poor survival compared with negative gelsolin expression in NSCLC, and this adverse prognostic effect was specific to patients with stage II tumors and for patients with squamous cell carcinomas. Similarly, variable gelsolin expression was significantly associated with poor survival compared with uniform gelsolin expression and this adverse prognostic effect was also specific to patients with stage II tumors and for patients with squamous cell carcinomas., Conclusion: High level gelsolin expression and variable gelsolin expression are adverse prognostic factors for NSCLC in this study, which might manifest the high motility and heterogeneity of tumor cells, two distinguishing characteristics for tumors with potentially enhanced invasive and dissemination capabilities.

Background Context: The use of metal cage prosthetic devices in anterior cervical discectomy with fusion (ACDF) has increased rapidly. One of these devices is the titanium Rabea cage (Signus, Alzenau, Germany) [correction]. There are no peer-reviewed objective reports on the use of these cages in cervical discectomy., Purpose: The authors present preliminary outcomes data on the Rabea cage. This study is intended to provide adjunct data for surgeons who are using or are considering the use of these devices., Study Design/setting: Patients in a private practice were studied prospectively as part of a long-term assessment of outcomes using several surgical procedures. Data were collated and analyzed by an independent researcher., Patient Sample: Rabea cages were used in consecutive candidates (n=37) for ACDF. The results using Rabea cages were compared with two prospectively studied control groups, one historical (n=66) and one concurrent (n=28), both groups using ACDF with bone allograft and no instrumentation., Outcome Measures: Success at 6 months after surgery was determined using six major patient-reported outcome measures, including visual analog scales (VAS) for arm and neck pain, the Oswestry pain and disability scale, four-part (excellent-through-poor) scales for measurement of return to activities of daily living or to work and satisfaction with the results of surgery. Perioperative complications, number of vertebral levels fused, and worker's compensation and smoking status were also compared among the study groups. Fusion and subsidence were evaluated for the Rabea cage group., Methods: Criteria for inclusion consisted of consecutive patients who presented with unremitting radicular arm pain, with or without neck pain, and/or a neurological deficit that correlated with appropriate level and side neural compression on magnetic resonance imaging or computed tomography. Questionnaire follow-up was at 6 months after surgery with 100% compliance., Results: A total of 50 cages were implanted in the 37 patients. At 6-month follow-up, 78% of patients reported successful arm pain relief (VAS scores below 5). Patient satisfaction was successful in 78% of the cases. Other success rates included neck pain relief in 73% and Oswestry pain and disability scale in 70%. There was a median improvement in the Oswestry scores of 28 points (61% change). Worker's compensation patients fared dramatically worse than did the noncompensation patients in all outcome measurements. Combining all three study groups resulted in significantly worse outcomes for multilevel than for single-level procedures and for smokers compared with nonsmokers, but low case numbers precluded conclusive analysis for the Rabea group alone. In the Rabea group there were two complications, neither cage related, whereas none were reported for the ACDF controls. Rabea group fusion rates were 84% at 3 months and 95% at 6 months, but the clinical relevance of this radiological evidence when metal prostheses are used is questionable. The outcomes results were clinically and statistically indistinguishable from those of our control groups and were similar to published studies using other titanium cages., Conclusions: This is the first prospective, independently conducted report on Rabea cages. Results of the short-term (6 months) follow-up did not differ from outcomes results in our patients who have undergone ACDF with bone allograft. Although this is a preliminary assessment, the Rabea cage may represent an alternative to bone dowels and hip graft. As is the case for other allografts, artificial or bone, the main advantage is elimination of donor site complications, and the disadvantages include difficulty in radiographic assessment of fusion and potential for cage subsidence.

Melatonin is a pineal hormone that strongly inhibits the growth of breast cancer cells in vitro and in vivo. We report thefirst use of immunohistochemical analysis to determine the distribution of the high-affinity melatonin receptor subtype, MTI, in human breast tissue, the hypothalamic suprachiasmatic nucleus, and skin. The MT1 antibody, which is specific for the cytoplasmic portion of the receptor, produced cytoplasmic staining in normal-appearing breast epithelial cells and ductal carcinoma cells; stromal cells, myoepithelial cells, and adipocytes were nonreactive. The majority of nonneoplastic samples (13/19 [68%]) were negative to weakly positive, while moderate to strong reactivity was seen in most cancer samples (49/65 [75%]). Thus, although MT1 receptors were detectable in normal and malignant breast epithelium, high receptor levels occurred more frequently in tumor cells (P < .001), and tumors with moderate or strong reactivity were more likely to be high nuclear grade (P < .045). These findings may have implications for the use of melatonin in breast cancer therapy.

Loss of gelsolin, a tumor suppressor, is one of the most frequently occurring molecular defects in breast cancers of diverse etiologies and across at least three animal species: human, mouse, and rat. Our previous analysis of breast cancer cells demonstrated that the deficiency is not due to mutation of the gelsolin gene, but instead to epigenetic factors, including decreased transcription of the gene. The study described herein provides the first functional characterization of the human gelsolin promoter and reveals a mechanistic basis for the reduced gelsolin transcription. In reporter gene assays, the gelsolin promoter was less active in low-gelsolin-expressing breast cancer cells. A cis-element mediating this reduced promoter activity was defined as a 27-bp sequence located approximately 135 bp upstream of the transcription start site. Gel shift and supershift assays and Southwestern blotting analysis indicated that activating transcription factor-1 (ATF-1) and a protein of approximately 100 kDa may have cancer cell-specific DNA-binding activity to the 27-bp gelsolin cis-element. Although the ATF-1 protein was highly expressed in both benign and tumorigenic breast cells, its DNA-binding activity was selectively abundant in the cancer cells and correlated inversely with the gelsolin mRNA level. Thus, our results suggest a role for ATF-1 in gelsolin promoter silencing in contrast to its transactivating effect on various other promoters., ((c) 2002 Elsevier Science (USA).)

Object: The authors assessed the efficacy and outcomes of lumbar microdiscectomy performed on an outpatient basis by administering six questionnaires before and at five time points after surgery. The results were compared with those reported in literature in which the success rates vary between 70% and 80% and in excess of 90%. The authors use the methodology and data derived from their study to evaluate critically the relevance of these two categories of success rates., Methods: This is a prospective study of 212 consecutive, eligible patients who underwent outpatient microscopic discectomy for the treatment of lumbar disc herniation: no previous lumbar lesion had been treated. Data were collected from questionnaires given to the patients before and at five time points after surgery, including at a variable final follow-up examination (mean 2 years postoperatively). Data were collated and analyzed independently by individuals other than the operating surgeons. In both bi- and multivariate analyses, only two preoperative parameters were prognostically significant. The first factor was Workers' Compensation status, which had a negative effect on outcome. The second factor was patient age, which also had a negative effect and was linear with increasing age between 25 years and 56 years--that is, the ages most commonly encountered in cases of herniated disc. Successful outcome rates were as follows: leg pain relief according to a visual analog scale (VAS), 80%; back pain relief (VAS), 77%; Oswestry Low Back Disability Index, 78%; satisfaction with the results of surgery, 76%; return to normal daily activities, 65%; and return to work, 61%., Conclusions: The findings of this study support the evidence that lumbar microdiscectomy performed on an outpatient basis is a very safe and effective means of treating sciatic pain due to disc herniation. The authors believe that their outcome success rates of 75 to 80% are more realistic than those of 90% or more found in some reports.

Mipp is a kelch-related, placental-specific gene that is ectopically expressed in many BALB/c mouse mammary carcinomas of various etiologies. The Kelch family encompasses proteins that are emerging as key links between microfilaments and a variety of cellular structures and functions. Mouse mammary tumors express two mipp transcripts (2.2 and 5.6 kb). We cloned the 2.2 kb mipp mRNA and analysed the product of its 1.7 kb ORF. The 584 residue MIPP protein has an N-terminal BTB domain and six C-terminal tandem Kelch repeats. Despite expression of two mipp RNAs, only a single MIPP protein is expressed in mammary tumors. MIPP protein binds to microfilaments in vitro and co-immunoprecipitates with actin. MIPP co-localized with concanavalin A at the endoplasmic reticulum, suggesting that MIPP might mediate interactions between microtubules and actin filaments. Because MIPP expression is widespread in mouse mammary tumors, it might contribute to tumorigenesis. Although MIPP had little effect on the growth rate of human breast cell lines following transfection, it greatly reduced the formation of duct-like structures on reconstituted basement membrane. Our results suggest that MIPP could contribute to malignant progression in the mouse mammary epithelial cells by perverting their response to cues from the extracellular matrix.

The production of heritable changes in gene expression is the driving force in the development and progression of breast cancer. Such changes can result from mutations or from epigenetic events such as hypermethylation of DNA and hypoacetylation of histones. Histone acetylation and DNA methylation are major determinants of chromatin structure, and chromatin structure is a primary regulator of gene transcription. Cancer cells frequently contain both mutated genes and genes with altered expression due to one or more epigenetic mechanisms. This review describes the epigenetic changes that disrupt normal chromatin architecture and modify the expression of key genes in breast cancer cells. The structural integrity of the latter genes is usually intact, but their expression has been substantially altered due to methylation in their promoter region or deacetylation of histones that interact with their promoter region or both mechanisms. Genes affected by epigenetic changes in breast cancers include HoxA5, p21WAF, gelsolin, BRCA1, BRCA2, E-cadherin, steroid hormone receptors, and retinoic acid receptor II. Because these epigenetic modifications are usually reversible by treatment with certain drugs, they represent vulnerabilities in the cancer cell that can be exploited as novel targets for new prevention and therapeutic strategies.

The actin cytoskeleton underlies several normal cellular functions and is deranged during carcinogenesis. Gelsolin, a multifunctional actin-binding protein, is downregulated in several types of tumors and its abnormal expression is one of the most common defects noted in invasive breast carcinoma (ICA). This study utilizes immunohistochemistry to examine the expression of gelsolin in 95 ICA, 59 ductal carcinoma in situ (DCIS) and 36 benign lesions, including 17 atypical ductal hyperplasia (ADH). Cytoplasmic staining was scored as positive, reduced or negative. Gelsolin expression was then correlated with patient's age, tumor size, histologic grade and lymph node status. All unremarkable breast biopsies, 88% of ADH, 44% of DCIS and 28% of ICA were positive for gelsolin. This represents a significant difference among the groups (p = < 0.0001) and the trend towards reduced gelsolin with the progression to ICA is significantly linear (p = < 0.0001). For invasive carcinoma, patients older than 44 years were significantly more likely to have decreased expression of gelsolin than patients 44 years old and younger (p = 0.007). Bivariate analysis showed no correlation of gelsolin expression with lymph node status (p = 0.62), tumor size (p = 0.10), histologic grade (p = 0.42), estrogen receptor status (p = 1.0) or other clinicopathologic parameters. In clinical follow-up, there were 18 breast tumor related deaths within a median follow-up time of 4.2 years. Survival analysis indicated that the level of gelsolin expression may be associated with survival (p = 0.06). In summary, the frequency of gelsolin deficiency increases significantly with progression from ADH to DCIS to ICA. Additionally, gelsolin expression may be an independent marker of prognosis.

Gelsolin is an actin-binding/severing protein expressed in intracellular and secreted forms. It is a major regulator of the form and function of the actin cytoskeleton in most all cells. Here we demonstrate that female mice with a targeted deletion of the gelsolin gene (Gsn-/-) have defects in mammary gland morphogenesis. Two distinct defects were identified in the gelsolin-null mammary gland. First, the mammary anlage from Gsn-/- mice failed to elongate at the onset of puberty and remained rudimentary until approximately 9 weeks of age, early block (Gsn-/-(EB)). Second, after the mammary epithelium had filled the mammary fat pad, a complete lack of terminal branching, or late block, was observed (Gsn-/-(LB)). The Gsn-/-(EB) was seen in 70% of Gsn-/- mice and appeared to be dependent on a modifier gene(s) in addition to the loss of gelsolin. Gsn-/-(LB) was observed in all Gsn-/- mice. Terminal end buds (TEBs) were not evident in the mammary anlage from Gsn-/-(EB) mice until approximately 9 weeks of age. Cellular proliferation in the terminal ductal regions of Gsn-/-(EB) females was detected by bromodeoxyuridine incorporation, but was less than that found in the TEBs of age-matched controls. In mice deficient for gelsolin, mammary gland architecture was unaltered at the histological level. Lobuloalveolar development was delayed in response to pregnancy in mammary glands of Gsn-/- mice but was otherwise normal. Lactation and involution in the gelsolin-null animals were similar to those of wild-type mice. Transplantation of epithelium devoid of gelsolin into a wild-type (GsnWT) mammary fat pad resulted in proper arborization of the ductal tree. Transplantation of GsnWT epithelium into the Gsn-/- fat pad recapitulated the lack of terminal branching seen in Gsn-/- females. These results indicate that gelsolin is required in the mammary stroma for proper ductal morphogenesis. Our results provide the first evidence of an actin regulatory protein affecting mammary ductal growth through stromal-epithelial communication., (Copyright 2000 Academic Press.)

Decreased gelsolin and increased cyclin D1 are among the most common defects found in human and rodent breast cancers. Our purpose was to determine the frequency of concurrence of these 2 alterations in this malignancy. Our results demonstrate that gelsolin protein and mRNA were significantly reduced in 80-100% of rodent mammary carcinomas that developed spontaneously, following oncogene introduction, or after treatment with viral, chemical or hormonal agents. The reduction in gelsolin most likely occurs during the transition from preneoplasia to carcinoma because hyperplasias had normal levels of gelsolin whereas microtumors had reduced expression. Southern analysis revealed no major mutations in the gelsolin gene of tumors with low expression. Cyclin D1 mRNA was increased in 50-100% of these rodent mammary tumors, although the cyclin D1 gene was not amplified. By nuclear runon assay, downregulation of gelsolin in both human and mouse mammary cancer cells involved diminished transcription and, conversely, human breast cancer cells expressing high levels of cyclin D1 had increased initiation of cyclin D1 transcription compared with cyclin D1 low expressors. Thus, alteration in the rate of transcription appears to be an important factor underlying the dysfunction of these genes. According to our data, concurrent deregulation of gelsolin and cyclin D1 is highly prevalent among breast cancers of humans and rodents, with both defects present in 89% of the neoplasms analyzed in this study. In fact, most tumors in every rodent model of mammary tumorigenesis examined had the 2 alterations.

Gelsolin is a multifunctional, actin-binding protein that is greatly decreased in many transformed cell lines and tumor tissues, including breast cancers. Downregulation of gelsolin RNA occurs in most breast cancers of rats, mice, and humans, but gross mutations of the gelsolin gene have not been found. Here we demonstrate by PCR and RT-PCR analysis that there are no point mutations in putative regulatory regions or the entire coding region of the cytoplasmic isoform of the gelsolin gene in human breast cancer cells (BCC). To determine if epigenetic modification is involved in downregulating gelsolin expression in MDA-MB-231 (MDA231), MCF7, and T47D BCC, we have used Southern blot analysis, 5-azacytidine (5aza) treatment, and trichostatin A (TSA) treatment. Southern blot analysis performed on genomic DNA demonstrated altered CpG methylation within intron 1 in DNA from all BCC compared to normal, mortal human mammary epithelial cells (HMEC). Treatment of the BCC with 5aza converted the DNA restriction pattern to that seen in untreated HMEC genomic DNA and caused modest increases in gelsolin RNA and protein. Incubation with TSA, an inhibitor of histone deacetylase, induced a dramatic upregulation of gelsolin RNA and protein levels which preceded apoptotic death of all BCC within 48-60 h. Our data support a role for epigenetic changes in chromatin structure leading to downregulation of gelsolin expression in human breast cancer. To our knowledge, this is the first example of a tumor suppressor gene downregulated in human breast cancer by changes in histone acetylation., (Copyright 1999 Academic Press.)

Expression of gelsolin, an actin filament regulatory protein, in human breast ductal carcinoma in situ (DCIS) was analyzed by immunohistochemistry using a monoclonal antibody. Formalin-fixed paraffin-embedded tissues from 59 pure DCIS specimens and 33 DCIS specimens with associated invasive components were evaluated for gelsolin reactivity and compared to eight normal breast cases and 76 invasive breast cancers. The proportion of cases exhibiting negative/low expression of gelsolin in the epithelium was as follows -- normal, 0%; pure DCIS, 56%; DCIS associated with invasion, 58% in the DCIS component and 66% in the invasive component; invasive carcinoma, 70%. These data demonstrate that down-regulation of gelsolin expression in breast epithelium frequently parallels progression to malignancy. Testing gelsolin expression (normal vs. negative/low levels) in the DCIS lesions for associations with patient age or any of the following histopathologic parameters revealed no significant (95% probability level) correlations -- tumor size; pathologic (Van Nuys system) grade; nuclear grade; necrosis; presence of histologic calcifications; presence or type of adjacent benign lesions; architectural histologic pattern; and mammographic extent. Gelsolin loss was more commonly associated with mammographic soft tissue lesions as compared to calcified lesions (P = 0.009). A positive trend of borderline significance (P = 0.06) found in the DCIS with invasion group was a correlation between down-regulated gelsolin expression in the DCIS component and size (< versus > or = 15 mm) of the invasive tumor. In conclusion, reduced gelsolin protein is detectable in at least half of breast lesions which have progressed to DCIS. The trend between increasing gelsolin loss and malignant progression from normal epithelium to DCIS to invasive breast cancer (P < 0.0001) suggests additional investigation is needed to determine the potential of altered gelsolin expression as a marker for prognosis and for therapeutic interventions in breast cancer.

This study retrospectively analysed 60 patients who had undergone microsurgical lumbar discectomy at an age of > or = 60 years. The results were compared with those obtained in 44 discectomy patients who were operated on during the same study period, but not selected for age. Sixty-five operations were performed on the elderly group and 49 on the age comparison group. Patients were scored for pain relief in a short-term follow-up (2 months) using office visit records. Long-term follow-ups [mean 6.5 years (elderly) vs 8.8 (comparison) years], obtained by a mailed questionnaire, quantified leg and back pain and scored success in return to normal activities (RTA) and satisfaction with the results of surgery. In the short-term, overall pain relief was highly successful and not significantly different in both group [94% (elderly), 98% (comparison)]. Long-term follow-up yielded the following successful outcomes (elderly, comparison groups): leg pain relief (91%, 86%), back pain relief (76%, 76%), RTA (68%, 87%), and satisfaction (81%, 91%). As with other pre- and postoperative parameters, these differences were not statistically significant. As the proportion of older individuals continues to rise in developed countries, physicians are increasingly faced with geriatric patients whose symptoms are caused by herniated lumbar discs. The present study indicates that microsurgical discectomy for relief of this condition can be performed safely and effectively on these older patients.

Down-regulation of gelsolin, an actin-binding protein, is frequently found in several types of transformed cells and tumors. The present study demonstrates that gelsolin protein and RNA were absent or markedly reduced in human breast cancer cell lines relative to "normal" mortal human mammary epithelial cells and benign, immortalized cell lines. Moreover, actin filaments were usually attenuated coincident with the reduction in gelsolin. Gelsolin was also missing or greatly decreased in 70% of 30 human sporadic, invasive breast carcinomas examined by immunocytochemistry and in 100% of virally induced mouse and chemically induced rat mammary carcinomas evaluated by Northern analysis. Southern analysis revealed no major mutations in the gelsolin gene of human breast cancer cells. Our results show that partial or total loss of gelsolin expression is common to the majority of breast cancers of diverse etiologies in three animal species and point to gelsolin as a candidate suppressor of breast cancer.

The objective of this pilot study was to evaluate the safety and efficacy of cervical discectomy with fusion performed on an outpatient basis. The experimental group (50 consecutive patients) was studied prospectively and the outcomes were compared with 53 consecutive, retrospectively analyzed, admitted controls who underwent the same procedure. Outcomes for both groups were assessed by patient-response questionnaires and clinical examination. At follow-up times of 1.3 (outpatient) and 1.6 (inpatient) years, outcomes (outpatient/inpatient) expressed as percent successful were as follows: Relief of arm pain (80/70%); relief of neck pain (78/68%); relief of arm muscle weakness and atrophy (94/96%); return to normal activities (64/70%); return to work (65/68%); and satisfaction with the results of surgery (86/83%). No statistically significant differences between outpatients and inpatients were found for any of the outcome parameters studied. There was no mortality and the operative complication rate was 2% for each study group. The results indicate that conversion of cervical discectomy with fusion from an admitted to an ambulatory practice did not compromise the safety or efficacy of the surgical procedure. Potential economic savings to overall health costs of the United States that might result from such conversion could exceed $100 million annually.

Endogenous murine leukemia virus-related elements (MLVEs) are often overexpressed in primary mammary carcinomas of BALB/c mice. We therefore searched for mutations associated with MLVEs and found amplified sequences of the ecotropic MLVE in hormonally and chemically induced mammary neoplasms. Restriction fragment length polymorphism (RFLP) analysis revealed DNA rearrangements consistent with 1-10 or more new copies of the ecotropic MLVE in the genome of these tumors. This is the first evidence of mutations involving an endogenous retrovirus other than mouse mammary tumor virus in mouse mammary carcinomas.

Human LINE-1 (L1Hs) retrotransposons can act as insertional mutagens and are expressed in a variety of tumors, including breast cancer. The purpose of the present study was to examine expression of the p40 protein encoded by the first open reading frame of a L1Hs element in normal human breast tissue of patients without malignant breast disease and in nontumor breast tissue adjacent to cancer and to compare it to expression in breast carcinomas. An antiserum specific for the L1Hs p40 protein was used to analyze its expression in 5 reduction mammoplasties, 16 primary breast cancers, 1 lymph node metastasis and 13 non-malignant breast tissues adjacent to matched primaries by western blotting and/or immunocytochemistry. The immunoreactive band observed on westerns consistently had a M(r) of approximately 46 kDa. Westerns detected some p40 protein expression in all malignant and nonmalignant tissues examined, although 4 of 5 reduction mammoplasties had very low or trace levels as compared with tumors. Nonmalignant breast tissues adjacent to cancers showed significant western band reactivity, and all 15 tumors were positive. Immunocytochemistry revealed staining specificity of the antibody for epithelial cells. Of 12 invasive carcinomas examined, 100% were positive for the p40 protein, whereas one reduction with benign proliferative disease was very weakly reactive, two histologically normal reductions were negative, and 4 of 6 nonmalignant tissues adjacent to cancers were negative. Our data indicated that expression of the L1Hs p40 protein was often elevated in tumor cells of human breast cancers compared to epithelium of normal mammary gland.

Study Design: This study retrospectively analyzed 15 patients in the rarely seen young (under 21 years) age group who had undergone discectomy without fusion for prolapsed (herniated) lumbar disc., Objectives: The results were analyzed for degree of success in several outcome parameters to relate the efficacy of this patient group/procedure pair to that of other studies., Summary of Background Data: Sixteen operations were performed on this patient group, including six by a conventional procedure and ten by a microsurgical technique. Although most previous studies tend to support the use of discectomy, some physicians have reportedly been reluctant to implement these procedures in young patients., Methods: Patients were followed in a short-term (median 3.3 months) assessment using records of post-operative office visits. Long-term (median, 10.5 years) follow-up was done by a mailed, self-report questionnaire that quantified pain in leg and back and scored for degree of success in ability to return to normal activities and satisfaction with the results of surgery., Results: The short-term results were excellent for all but one patient. Long-term follow-up yielded the following successful outcomes: relief of back pain, 77%; and relief of leg pain, return to normal activities, and satisfaction with surgery, each 85%., Conclusion: Despite some tendency to delay discectomy in children and young adults, physicians are urged to be aware of this rare condition and the excellent long-term outcomes and limited complications resulting from timely implementation of discectomy after a failed course of conservative therapy. Moreover, fusion should be avoided except in cases of vertebral instability.

Eighty-two patients who underwent reoperation using a lumbar microdiskectomy approach for recurrent back and leg pain were evaluated retrospectively. Patients were entered consecutively except for exclusion of those who had a spinal fusion in addition to the diskectomy or those where a diskectomy was performed as an adjunct to decompression for spinal stenosis. As a percentage of all diskectomies performed by us over the 13 years of the study, the overall rate of reoperation (including all patients who underwent more than one diskectomy procedure regardless of vertebral level and side) was 7.4%, with those having a reoperation on the same level and either the same or contralateral side as the initial procedure representing 4.5%. Long-term outcomes obtained from 68 questionnaire responses (83% compliance) included 56% who had successful leg pain relief, 54% who had successful back pain relief, 44% who successfully returned to work, 51% who successfully returned to normal activity, and 73% who were satisfied with the results of surgery. Poor outcomes correlated most significantly with reoperation on the same vertebral level, same side, and short (< or = 1 year) time interval between consecutive diskectomies. A major conclusion was that workers' compensation patients presenting within 1 year with recurrent complaints after diskectomy and whose radiologic findings indicate a same-level, same-side recurrence represent extremely poor outcome risks for repeat diskectomy.

Amplified expression of the endogenous retrotransposons, intracisternal A particles (IAPs) and murine leukemia virus-related elements (MLVEs), along with decreased expression of VL30 elements frequently occurs during mouse mammary tumorigenesis. We have now analyzed the expression of these retroelements during the normal developmental and differentiation cycle of the mammary gland as found in virgin, pregnant, lactating, and postlactation adult female BALB/c mice. Retrotransposon expression was either unchanged or decreased during the progressive stages of the cycle compared to virgin tissue. Likewise, growth of mammary epithelial cells in primary culture had little or no effect on expression of IAPs, MLVEs and VL30 sequences. Thus, the dramatic changes involving these retrotransposons in many mouse mammary tumors appear unrelated to any normal state.

Of the several families of endogenous retrovirus-like elements present in the mouse genome, only mouse mammary tumor virus has been analyzed for its role in mammary carcinogenesis. Very little is known about the expression and activities of other retro-elements in normal and malignant mammary epithelium. We have begun investigating the possible involvement of the 3 retrotransposons, intracisternal A particles (IAPs), murine-leukemia-virus-related (MuLVr) elements, and VL30 sequences, in neoplastic progression of the mammary gland in BALB/c mice. The purpose of the present study was to determine which of these elements was active in primary mammary carcinomas induced by chemical, hormonal and viral agents. Each of these cancers had aberrant expression of at least one of the latter retrovirus-like components. IAP and/or MuLVr sequences were over-expressed 3 to 100-fold in most of the tumors as compared with normal mammary tissue, whereas VL30 expression was markedly decreased by 5- to 35-fold in almost all of the neoplasms. Our results thus demonstrate that substantial changes in the expression of one or more of these 3 families of endogenous retrotransposons are triggered during mouse mammary tumorigenesis, regardless of etiology. Direct involvement of IAPs and MuLVr elements in neoplastic progression by transposition and insertional mutagenesis in the genome of several hematopoietic cell types has already been demonstrated. Their elevated expression in many mammary carcinomas suggests that these retrotransposons may also be potential participants in some pathways of mouse mammary carcinogenesis.

A total of 258 consecutive decompressive lumbar laminectomies performed on 244 individuals presenting with spinal stenosis were analyzed retrospectively. Spinal fusion was avoided in all but two patients. Outcome in terms of pain relief and return to normal activity was evaluated in two stages, one derived from patient charts and having a relatively short-term follow-up time (mean 8.4 months) and a second derived from patient responses to a questionnaire (which also scored for satisfaction with the results of surgery), which had a longer follow-up time (mean 4.7 years). More than 20 clinical and operative parameters were analyzed. Overall, a high degree of success (93% pain relief, 95% return to normal activity) was achieved in the short term, which was supported by the longer-term follow-up data (64% pain relief, 56% activity return, 75% satisfaction). The following factors were not significantly correlated with outcome: patient age; sex; worker's compensation or no-fault insurance status; employed versus not employed; a history of back surgery prior to the laminectomy studied; existence of degenerative spondylolisthesis or scoliosis; complete versus incomplete myelographic block; or the level of the lumbar spine undergoing surgery. The major conclusions arising from these data are: 1) for all age groups through at least the eighth decade of life, decompressive lumbar laminectomy is a relatively safe operation having a high medium-to-long-term success rate; 2) lumbar instability following laminectomy is rare, even in individuals presenting prior to surgery with degenerative instability conditions; and 3) lumbar fusion in addition to the decompressive laminectomy procedure is rarely required for degenerative spinal stenosis.

Keratin polypeptides obtained from mouse mammary epithelial cells (MMEC) were found to be modified by covalent attachment of lipids. MMEC in primary culture were incubated in 3H-palmitate and treated with 1.5M KCl/1% Triton X-100 to obtain a cytoskeletal (CS) fraction containing primarily keratin and actin filaments. After exhaustive extraction to remove labeled lipids, the CS proteins were separated by gel electrophoresis, and the labeled 46 kD (K18) and 55 kD (K8) keratin polypeptides were excised, subjected to acid hydrolysis and the chloroform-soluble products were resolved on thin layer chromatography. For both keratins, covalently bound lipid included major peaks which co-chromatographed with fatty acid standards. Also, unlabeled lipid resolving with fatty acid standards was found covalently bound to both keratins. The results are discussed in terms of keratin-lipid-membrane interactions.

Cytoskeletal proteins obtained from mouse mammary epithelial cells (MMEC) were found to be modified by covalent attachment of lipids. Primary cultures of MMEC were incubated in the presence of 3H-palmitate for 4 h. A cytoskeletal (CS) fraction was prepared by treatment of the cells with 1.5M KCl and 1% Triton X-100. The residual material, consisting primarily of keratin and actin filaments was exhaustively (10-20 rounds, including sonications) extracted with chloroform/methanol to remove non-covalently bound labeled lipids. The CS protein was then acid-hydrolyzed and the chloroform-soluble products subjected to thin layer chromatography (TLC). Two-thirds of the covalently bound radiolabel appeared as a very hydrophobic peak on a TLC system optimized for separation of neutral lipids. Ten percent separated into 4-5 peaks on a polar lipid TLC system. A small amount of label was traced to fatty acid-like components. Autoradiography of two-dimensional gels indicated that all the CS proteins resolvable by Coomassie blue staining were also radiolabeled. The results are discussed in terms of CS-lipid-membrane interactions.

Lipids noncovalently associated with cytoskeletal (CS) proteins of mouse mammary epithelial cells (MMEC) grown in primary culture were analyzed. A CS fraction, prepared by subjecting MMEC to 1.5 M KCl and 1% Triton X-100 in phosphate buffered saline (pH 7.4), was extracted 4-6 times with chloroform/methanol. Thin-layer chromatography (TLC) indicated that in comparison to whole cell lipid extracts, CS lipids consisted mostly of neutral lipids, especially triacylglycerols and, possibly cholesteryl esters. TLC analysis of chloroform/methanol CS extracts prepared from MMEC that had been incubated 4 h in [3H]palmitate revealed similar results, with the majority of label appearing in triacylglycerols and other neutral lipids. By autoradiography of sodium dodecyl sulfate polyacrylamide gels, all of the major CS proteins appeared labelled. The major regions of autoradiographic density of the gel were excised, the protein solubilized, and the lipids extracted and subjected to TLC. Most of the radiolabel appeared at the origin and ion front and resolved as neutral lipids. In contrast, keratins of 54-55 kDa and 46 kDa appeared to be associated noncovalently with a higher ratio of polar lipids (possibly phospholipids) to nonpolar (neutral lipids). Very little radioactivity, mostly neutral lipid, was associated with actin. A previously unidentified CS component of 30 kDa had primarily noncovalently bound neutral lipid. The results are discussed in terms of the apparent interactions of keratin filaments with the plasma membrane, nuclear envelope and cytoplasmic organelles.

In order to develop new probes for investigating cellular alterations accompanying neoplastic progression of mouse mammary epithelium, rat monoclonal antibodies were prepared against cytoskeletal extracts of cultured normal and malignant mouse mammary epithelial cells. One of these antibodies, VG10, reacted in immunofluorescent staining with the majority of mouse mammary epithelial cells from hyperplastic alveolar nodule outgrowth lines of hormonal, chemical, and viral etiologies and the primary adenocarcinomas arising spontaneously from the nodules. Moreover, most mammary carcinomas developing in BALB/cfC3H, BALB/cV, and 7,12-dimethylbenzanthracene-treated BALB/c mice also had cells recognized by VG10. In contrast, normal mouse mammary epithelial cells from virgin and pregnant mice were unreactive. Immunoelectron microscopy identified the reactive component in the abnormal cells as intracisternal A-particles (IAPs), endogenous retrovirions with the ability to transpose in the mouse genome. Western blotting experiments established that the VG10 antibody is specific for the Mr 73,000 gag protein of IAPs. The results indicate that expression of IAPs is a common occurrence in the evolution of malignancy in mammary epithelium of BALB/c mice and may be induced during the transformation of normal cells to the preneoplastic stage. Expression of IAPs is therefore a candidate marker for a step in neoplastic progression in this system and has the potential for contributing to the carcinogenic process.

The effects of gentamicin on three proteolytic enzymes were studied. Gentamicin was tested at concentrations of 0.5-500 microgram/ml. Trypsin was tested at 0.5 microgram/ml using p-tosyl-L-arginine methyl ester and at 0.1 and 0.5 microgram/ml using azocoll as the substrate. Chymotrypsin was tested at 0.1 and 0.5 microgram/ml with azocoll. A soluble [14C]collagen assay was used to measure activity of collagenase derived from Clostridium histolyticum. The profiles of proteolytic activity vs. gentamicin concentration were similar for all three enzymes. At lower concentrations of gentamicin (less than 70 microgram/ml), there were two peaks of enhanced protease activity generally followed by inhibition. These unusual multiphasic effects of gentamicin on three different proteases are not presently understood, but they imply a previously unreported mode of action for this antibiotic.

The keratins and other cytoskeletal proteins expressed by normal, preneoplastic, and malignant mammary tissues in BALB/c mice and by cells in primary cultures established from these tissues were analyzed and compared. The preneoplastic lesions were hyperplastic alveolar nodules (HAN) derived originally from mice treated by hormonal stimulation (D2), exposed to a chemical carcinogen (C4), or spontaneously expressing mouse mammary tumor virus (CV2) and maintained by serial transplantation. All tumors were mammary adenocarcinomas which developed as primary neoplasms from the HAN outgrowth lines. Cytoskeletal extracts were prepared from the tissues and cultured cells and subjected to two-dimensional polyacrylamide gel electrophoresis. Comparison of the major polypeptides in the normal and abnormal tissue extracts revealed considerable similarities in the cytoskeletal profiles. Three basic and seven acidic polypeptides ranging in molecular weight from 40,000 to 90,000 were regularly identified. However, notable differences were also found. A Mr 55,000 keratin (IEF 55) was prominent in one HAN, the D2, and all tumor tissues but not in normal gland. Likewise, a Mr 46,000 polypeptide (IEF 46), which has been tentatively identified previously as a keratin, was absent in normal epithelium but present in all abnormal tissues except the C4 and CV2 HAN. A Mr 58,000 polypeptide (NEPHGE 58) was not detected in normal gland or the C4 lesions but was found in all other abnormal tissues. The overall pattern of polypeptides in cytoskeletal extracts from normal and abnormal mammary cells in primary culture resembled that of the corresponding tissue but also had important differences. In all cell cultures, IEF 46 and IEF 55 were major species, while the larger and more basic components were markedly reduced. A Mr 56,000 polypeptide (NEPHGE 56) was detected only in C4 HAN and C4 and CV2 tumor cells. Trace or small, variable amounts of a Mr 57,000 basic keratin (NEPHGE 57) were present in normal and D2 tissues and cultured cells. NEPHGE 57 was dramatically increased in C4 and CV2 tissues and cultured cells and may be related to expression of squamous metaplasia and keratinization which are characteristic of these lesions. Although production of IEF 46 and IEF 55 may be associated with neoplastic progression of mammary epithelium, particularly in vivo, the association is not exclusive since normal cells express these polypeptides when grown in primary culture. In addition, correlations between altered keratin expression and the mode of induction of the mammary lesions were not obvious.

The sequence of heat shock-induced perturbations in protein synthesis and cytoskeletal organization was investigated in primary cultures of mouse mammary epithelial cells (MMEC). Exposure of the cells to 45 degrees C for 15 min caused a marked inhibition of protein synthesis through 2 h after heart. Resumption of protein synthesis began by 4 h, was complete by 8 h, and was accompanied by induction of four major heat shock proteins (HSPs) of 68, 70, 89, and 110 kD. Fluorescent cytochemistry studies indicated that heat shock elicited a reversible change in the organization of keratin filaments (KFs) and actin filaments but had a negligible effect on microtubules. Changes in the organization of KFs progressed gradually with maximal retraction and collapse into the perinuclear zone occurring at 1-2 h after heat followed by restoration to the fully extended state at 8 h. In contrast, actin filaments disappeared immediately after heat treatment and then rapidly returned within 30-60 min to their original appearance. The translocation of many organelles first into and then away from the juxtanuclear area along with the disruption and reformation of polyribosomes were concurrent with the sequential changes in distribution of KFs. The recovery of the arrangement of KFs coincided with but was independent of the resumption of protein synthesis and induction of HSPs. Thermotolerance could be induced in protein synthesis and KFs, but not in actin filaments, by a conditioning heat treatment. Neither protein synthesis nor induction of HSPs was necessary for the acquisition of thermotolerance in the KFs. The results are compatible with the possibility that protein synthesis may depend on the integrity of the KF network in MMEC. Heat shock thus can efficiently disarrange the KF system in a large population of epithelial cells, thereby facilitating studies on the functions of this cytoskeletal component.

Two monoclonal antikeratin antibodies, AE1 and AE3, were used in indirect immunocytochemistry to examine keratin expression in normal, benign proliferative, and malignant human breast epithelium. Both antibodies reacted strongly with most luminal cells in ducts and acini of normal gland. While AE1 did not stain myoepithelium, AE3 recognized myoepithelial cells of ducts but not acini, implying a cytoskeletal difference between the myoepithelium of these two components. Moreover, the antibodies reacted differently with the myoepithelium of intracanalicular as compared with pericanalicular types of fibroadenomas. Tumour cells of infiltrating ductal carcinomas with a prominent intraductal component stained more homogeneously with AE1 and AE3 than those without intraductal growth. The results provide evidence for two phenotypes of myoepithelial cells and for the presence of cryptic keratin epitopes in human breast epithelial cells. The finding that neither AE1 nor AE3 is a universal detector of these cells has important clinical and experimental implications.

Three monoclonal anti-keratin antibodies, AE1, AE3, and AE4, were used to compare the expression of keratins in normal, preneoplastic, and malignant mouse mammary epithelial cells growing in primary culture. In indirect immunofluorescence, AE1 did not stain normal cells but did stain a minority of preneoplastic and carcinoma cells. AE3 reacted with a subpopulation of epithelial cells in both the normal and abnormal cultures, except for certain cultures from one type of tumor wherein all of the epithelial cells were reactive. AE4 decorated an elaborate keratin filament network in all cultured mammary epithelial cells, regardless of neoplastic state. In double-label immunofluorescence, a guinea pig anti-keratin antiserum, which reacts preferentially with myoepithelial cells, exhibited coincident staining with AE1 in the tumor cultures and AE3 in the normal and most tumor cultures, indicating that the cells recognized by the antibodies in these populations were myoepithelial. Immunoblot experiments with cytoskeletal polypeptides extracted from the normal and tumor cells demonstrated that the set of keratins recognized by each monoclonal antibody was essentially the same in all of the cells except for a Mr 40,000 component that was present in normal cells but either absent or diminished in the cancer cells. Thus, while normal cells had Mr 40,000 and 50,000 keratins recognized by AE1, the epitope detected by this antibody was apparently concealed or "masked" in situ. AE3 reacted in immunoblots with a major keratin group (Mr 54,000-55,000) and a minor keratin (Mr 57,000), while AE4 reacted only with the Mr 54,000-55,000 keratin species. Because immunofluorescence with AE4 showed that the Mr 54,000-55,000 keratin group was present in all mammary epithelial cells, the AE3-reactive epitope must be masked in the majority of normal and tumor cells. The data therefore showed that epitopes on three major keratins, the Mr 40,000, 50,000, and 54,000-55,000 group, were "masked" in normal cells, whereas in tumor cells "masking" involved primarily the Mr 54,000-55,000 keratin. Attempts to "unmask" the epitopes recognized by AE1 in normal cells or to increase the number of cells reactive with AE3 in the normal and tumor cultures failed. Thus, certain cultured preneoplastic and neoplastic mammary cells with a myoepithelial phenotype have an altered organization of keratins that is manifested by a keratin antigenic determinant which is visible by immunocytochemistry in the abnormal cells but not in normal mouse mammary cells. This is the first demonstration that the immunoreactivity of keratins can be modified during neoplastic progression of epithelial cells.

Previous electrophoretic analysis has indicated that keratins 13 and 16 (K13 and K16) are not present in normal human breast epithelium, but K16 is expressed in some ductal carcinomas (Moll et al., Cell, 31: 11-24, 1982). K16 may thus represent a marker for identifying a subset of ductal carcinomas and for distinguishing such tumor cells from normal cells. To explore this possibility further, we have used the monoclonal antibody Ks8.12, reportedly specific for K13 and K16 (Huszar et al., Differentiation, 31: 141-153, 1986), to examine keratin expression in human breast tissues. In immunocytochemistry the antibody reacted with epithelial cells of all normal samples, hyperplasias, and fibroadenomas. The staining was moderate to strong and always heterogeneous, involving most but not all luminal cells of ducts and acini. Myoepithelial cells were never stained. Of twenty-one ductal carcinomas examined, 90% gave a very weak or no reaction. The remaining 10% of cancers exhibited moderate to strong staining in about 50% of tumor cells. Cytoskeletal polypeptides extracted from the tissues and separated by polyacrylamide gel electrophoresis were analyzed by Western blotting to identify the polypeptides recognized by Ks8.12. In samples from normal tissue and a fibroadenoma, the antibody recognized a major Mr 48,000 component, a size appropriate for K16. In extracts from a hyperplasia and two of four carcinomas, the antibody detected a Mr 54,000 polypeptide, as well as a Mr 48,000 band, properties consistent with K13 and K16, respectively. Our results provide the first evidence indicating that K16 is present in normal as well as abnormal human breast epithelium. In addition, the data suggest that K13 may be expressed in some benign lesions and ductal carcinomas of the breast. Accordingly, Ks8.12 may prove to be useful for subclassifying ductal carcinomas and for discriminating between normal and certain benign and malignant disorders of human mammary epithelium.

The antischistosomal activity of oral doses of amoscanate (4-isothiocyanato-4'-nitrodiphenylamine) was determined in infected Cebus apella (capuchin monkeys) and Macaca mulatta (rhesus monkeys). In C. apella infected with Schistosoma japonicum or S. mansoni an initial dose of 10 mg/kg body weight did not alter fecal egg counts, but a subsequent dose of 25 mg/kg markedly reduced both egg counts and worm burdens; in animals infected with S. haematobium, a single dose of 25 mg/kg of amoscanate was similarly effective. Comparable schistosomicidal effects were also produced in S. japonicum- and S. mansoni-infected M. mulatta by single oral doses of 20 and 35 mg/kg, respectively. In both C. apella and M. mulatta the coadministration of single oral doses of 50 or 75 mg/kg of erythromycin attenuated the appearance of mutagenic metabolites of amoscanate in the urine but did not interfere with the antischistosomal action of amoscanate. In non-infected monkeys single oral doses of 75 mg/kg of amoscanate with or without erythromycin (50 mg/kg in C. apella or 75 mg/kg in M. mulatta) did not cause any major organ toxicity as revealed by gross and histopathologic examination, hematology, blood chemistry, electrocardiograms and urinalysis. The data indicate that in C. apella and M. mulatta, amoscanate is a relatively non-toxic antischistosomal agent effective orally against a broad spectrum of schistosome species.

The effects of ZnCl2 on the viability and morphology of cercariae and schistosomules of Schistosoma mansoni were examined. The longevity of cercariae was inversely proportional to the zinc concentration from 0.05 to 5 micronM. Zinc caused body-tail separation and crenation of bodies but not of tails of cercariae, as well as alteration of the staining of cercariae in the indirect immunofluorescence test. Zinc was also effective in reducing penetration of cercariae into mice in a brief (10 min) exposure period. Although the effects on schistosomule morphology were not as conspicuous as with cercariae, 5mM ZnCl2 also affected this stage of the parasite.

1. Normal human sera and plasma were fractionated in order to identify inhibitors of the "penetration" proteases of Schistosoma mansoni cercariae. 2. The main inhibitor, accounting for 90% of the total activity of serum, appears to be alpha 1-antitrypsin (alpha 1-AT) as identified by separation on DEAE-cellulose and Sephadex, by immunoelectrophoresis and by anticercarial protease activity of purified alpha 1-AT preparations. 3. The inhibition profiles of purified preparations of the 6 known serum antiproteases suggest that the parasite protease is similar to vertebrate chymotrypsin. 4. On a molar basis, the order of inhibitory activity against the cercarial protease is: alpha 1-AT = alpha 2-macroglobulin; C'-1-inactivator; alpha 1-antichymotrypsin. No inhibition was obtained with inter-alpha-inhibitor or antithrombin III.

Mammary epithelium is composed of ductal, alveolar, and myoepithelial cells, and undergoes dramatic responses in growth, differentiation, and function to hormonal stimuli during the four stages of the mammary developmental cycle represented in virgin, pregnant, lactating, and involuting animals. To determine if progression of the epithelium through the cycle is accompanied by changes in cytoskeletal composition, particularly the keratins, the polypeptides in cytoskeletal extracts from BALB/c mouse mammary tissues were analyzed by one- and two-dimensional gel electrophoresis combined with immunoblots using polyclonal and monoclonal antikeratin antibodies. The major polypeptides in cytoskeletal fractions enriched in intermediate filaments included seven acidic and three basic components ranging in molecular weight from 40,000 to 90,000. Two major polypeptides of Mr 50,000 and 40,000, along with two minor components of Mr 57,000 and 55,000 were identified as keratins. The polypeptide profiles of mammary glands from virgin, pregnant, lactating, and involuting mice were very similar, indicating a remarkable stability of cytoskeletal composition during hormonal shifts and periods of minimal or maximal cell growth and differentiated function. The data also suggest that ductal and alveolar cells express the same set of cytoskeletal polypeptides, including keratins. Mammary cells grown in primary culture exhibited a loss or reduction in most of the basic polypeptides, a large increase in an acidic Mr 55,000 keratin, and the appearance of a prominent acidic polypeptide of Mr 46,000. The latter results demonstrate that keratin expression in mouse mammary epithelial cells is subject to regulation by certain environmental factors.

Extracts of the eggs of the human blood fluke, Schistosoma mansoni, exhibit proteolytic activity which requires the presence of added thiol reagents or cyanide. The pH optimum for hydrolysis of Azocoll and cartilage proteoglycan is 4.8--5.2 and the molecular weight of the major component is 25--26,000. The effects of inhibitors suggest this activity belongs to the acidic thiol proteinase class, with a similarity to Cathepsin B. These proteinases may be involved in nutrition of the egg or sporocyst, in penetration of eggs or miracidia through host tissues, or in the immunopathology of schistosomiasis.