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Axonal navigation during development requires that cues present in the extracellular environment be capable of modifying the structure of the cone in a dynamic way. Protein kinase C (PKC) has long been suspected to be one of the multiple molecular relays present in the terminal structure of the developing axon and involved in the transduction of extracellular signals. The latter proposal is, however, based on the use of drugs or of protocols leading to pleiotropic and often nonspecific effects. In the present study, we have taken advantage of the discovery of a peptide capable of translocating across biological membranes and to accumulate in the cytoplasm and nucleus of cells in culture, to internalize a highly specific peptidic inhibitor of PKC. We demonstrate that linking the two peptides (vector and PKC inhibitor) allows the internalization of the latter in live cells, specifically inhibits PKC and provokes a rapid modification of growth cone morphology. This set of data thus establishes that a peptidic inhibitor of PKC activity, once internalized, provokes a change in growth cone morphology, reminiscent of the collapse phenotype. In addition, the present study describes a new efficient and harmless way to introduce pharmacologically active substances in neural cells in culture.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanding polyglutamine repeat in the IT15 or huntingtin gene. Although this gene is widely expressed and is required for normal development, the pathology of HD is restricted to the brain, for reasons that remain poorly understood. The huntingtin gene product is expressed at similar levels in patients and controls, and the genetics of the disorder suggest that the expansion of the polyglutamine repeat induces a toxic gain of function, perhaps through interactions with other cellular proteins. Here we report the identification of a protein (huntingtin-associated protein (HAP)-1) that binds to huntingtin. This binding is enhanced by an expanded polyglutamine repeat, the length of which is also known to correlate with the age of disease onset. The HAP-1 protein is enriched in the brain, suggesting a possible basis for the selective brain pathology of HD.

A polyglutamine expansion (encoded by a CAG repeat) in specific proteins causes neurodegeneration in Huntington's disease (HD) and four other disorders, by an unknown mechanism thought to involve gain of function or toxicity of the mutated protein. The pathological threshold is 37-40 glutamines in three of these diseases, whereas the corresponding normal proteins contain polymorphic repeats of up to about 35 glutamines. The age of onset of clinical manifestations is inversely correlated to the length of the polyglutamine expansion. Here we report the characterization of a monoclonal antibody that selectively recognizes polyglutamine expansion in the proteins implicated in HD and in spinocerebellar ataxia (SCA) 1 and 3. The intensity of signal depends on the length of the polyglutamine expansion, and the antibody also detects specific pathological proteins expected to contain such expansion, in SCA2 and in autosomal dominant cerebellar ataxia with retinal degeneration, whose genes have not yet been identified.

Recent studies have confirmed that the 90 kDa heat-shock protein (hsp90) interacts both in vitro and in vivo with steroid receptors, encouraging further detailed physicochemical and functional analysis of its chaperone role. Thus, to explore the relationship between hsp90 and receptors, the baculovirus system was used to overexpress the chick hsp90 alpha (chsp90) along with the chick oestradiol receptor (cER) or the human mineralocorticosteroid receptor (hMR). These receptors were able to form 9 S complexes with chsp90, demonstrating the association of the co-expressed recombinant proteins. Three mutants of chsp90 (delta A, delta B and delta Z) have been created by deletion of the A (residues 221-290) and B (530-581) regions, rich in charged amino acids, and the Z (392-419) region, a putative leucine zipper. After co-expression, anti-receptor antibodies immunoprecipitated the cER or hMR complexed with the wild-type chsp90, the delta B or the delta Z mutant, but not with the delta A chsp90, indicating that deletion of the A region of chsp90 leads to a lack of interaction with these receptors. The hormone binding capacity of the cER was unaffected after its co-expression with each of the three mutants. In contrast, the hMR co-expressed with the delta B mutant failed to bind aldosterone, a finding confirmed in vivo by the absence of hormone-induced hMR nuclear translocation. Thus the B region is required for high-affinity ligand binding by the hMR. Our results suggest that the A region (but not the B or Z regions) is involved in binding of chsp90 to the cER and hMR, while the B region is essential for hormone binding by the hMR, consistent with a chaperone function for hsp90.

To test the hypothesis that selective pressure has led to the retention of cellulose-binding domains (CBDs) by hemicellulase enzymes from aerobic bacteria, four new xylanase (xyn) genes from two cellulolytic soil bacteria, Pseudomonas fluorescens subsp. cellulosa and Cellvibrio mixtus, have been isolated and sequenced. Pseudomonas genes xynE and xynF encoded modular xylanases (XYLE and XYLF) with predicted M(r) values of 68,600 and 65000 respectively. XYLE contained a glycosyl hydrolase family 11 catalytic domain at its N-terminus, followed by three other domains; the second of these exhibited sequence identity with NodB from rhizobia. The C-terminal domain (40 residues) exhibited significant sequence identity with a non-catalytic domain of previously unknown function, conserved in all the cellulases and one of the hemicellulases previously characterized from the pseudomonad, and was shown to function as a CBD when fused to the reporter protein glutathione-S-transferase. XYLF contained a C-terminal glycosyl hydrolase family 10 catalytic domain and a novel CBD at its N-terminus. C. mixtus genes xynA and xynB exhibited substantial sequence identity with xynE and xynF respectively, and encoded modular xylanases with the same molecular architecture and, by inference, the same functional properties. In the absence of extensive cross-hybridization between other multiple cel (cellulase) and xyn genes from P. fluorescens subsp. cellulosa and genomic DNA from C. mixtus, similarity between the two pairs of xylanases may indicate a recent transfer of genes between the two bacteria.

A trinucleotide repeat (CAG) expansion in the huntingtin gene causes Huntington's disease (HD). In brain tissue from HD heterozygotes with adult onset and more clinically severe juvenile onset, where the largest expansions occur, a mutant protein of equivalent intensity to wild-type huntingtin was detected in cortical synaptosomes, indicating that a mutant species is synthesized and transported with the normal protein to nerve endings. The increased size of mutant huntingtin relative to the wild type was highly correlated with CAG repeat expansion, thereby linking an altered electrophoretic mobility of the mutant protein to its abnormal function. Mutant huntingtin appeared in gray and white matter with no difference in expression in affected regions. The mutant protein was broader than the wild type and in 6 of 11 juvenile cases resolved as a complex of bands, consistent with evidence at the DNA level for somatic mosaicism. Thus, HD pathogenesis results from a gain of function by an aberrant protein that is widely expressed in brain and is harmful only to some neurons.

Detection of loss of heterozygosity (LOH) and DNA flow cytometry (FCM) were used to trace the origin of bilateral ovarian cancer from 16 patients. From each tumour the DNA index (DI) and LOH patterns for chromosomes 1, 3, 6, 11, 17, 18, 22 and X were determined with 36 microsatellite markers. Formalin-fixed, paraffin-embedded as well as frozen specimens were used. Flow cytometric cell sorting was used to enrich tumour cells for polymerase chain reaction (PCR)-driven LOH analysis. Analysis of the LOH data showed that in 12 of the 16 cases concordance was observed for all informative markers, namely retention of heterozygosity (ROH) or loss of identical alleles in both tumour samples. In four cases discordant LOH patterns were observed. In two cases the discordant LOH was found for one of the chromosomes tested while other LOH patterns clearly indicated a unifocal origin. This suggests limited clonal divergence. In the other two cases all LOH patterns were discordant, most likely indicating an independent origin. The number of chromosomes showing LOH ranged from 0 to 6. Comparison of DNA FCM and the LOH data showed that the latter technique has a higher sensitivity for the detection of a unifocal origin. In 14/16 cases evidence was found for a unifocal origin, while in two cases clonal divergence was found at LOH level and in two other cases clonal divergence at DNA ploidy level. In 12 cases the complete observed allelotype had developed before the formation of metastases, including the two cases showing a large DNA ploidy difference.

This study was undertaken to evaluate the relationship between serum tumor necrosis factor (TNF) and coagulopathy in patients with prostate cancer. TNF levels in 104 sera obtained from 101 prostate cancer patients were determined using an enzyme immunoassay. Serum levels of fibrin/fibrinogen degradation product E fragment (FDP) and plasma levels of fibrin degradation product D-dimer in patients with elevated serum TNF levels were 1221.95 +/- 375.94 ng/ml and 27.34 +/- 9.81 micrograms/ml, which were significantly higher than those (FDP, 94.35 +/- 13.17 ng/ml; D-dimer, 1.03 +/- 0.20 micrograms/ml) in patients with undetectable serum TNF levels (P < 0.01). In addition, patients with elevated serum TNF levels showed significant increases in plasma levels of thrombin-antithrombin-III complex and plasmin-alpha 2-antiplasmin inhibitor complex and a significantly higher incidence of positive plasma soluble fibrin monomer complex than did those with undetectable serum TNF levels. The percentage of prothrombin time was significantly decreased in the group with elevated serum levels of TNF. Serum levels of TNF were significantly elevated in patients with serum FDP levels of > or = 200 ng/ml than in those with serum FDP levels of < 200 ng/ml (3.91 +/- 0.45 versus 2.17 +/- 0.08 units/ml) and in patients with plasma D-dimer levels of > or = 2 micrograms/ml than in those with plasma D-dimer levels of < 2 micrograms/ml (3.82 +/- 0.48 versus 2.10 +/- 0.06 units/ml). These results suggest that TNF may be one of the pathogenetic factors that could explain the occurrence of coagulopathy in patients with prostate cancer.

A human papillomavirus (HPV) type 16 containing oral squamous cell carcinoma cell line 93VU147T at early passage was demonstrated to match its primary tumor with regard to HPV status and loss of heterozygosity at loci potentially involved in HPV-mediated carcinogenesis. DNA in situ hybridization of the cell line and the primary tumor revealed the presence of HPV 16 DNA clonally associated with the neoplastic cells. One- and two-dimensional Southern blot hybridization suggested HPV 16 to be integrated in the host genome at over hundred copies/cell. An identical restriction enzyme profile was observed for the tumor and the cell line. Viral DNA integration was confirmed by fluorescence in situ hybridization on metaphase spreads of the cell line, which revealed six stained loci comprising one at 15q14-15 and five at cytogenetically unidentifiable chromosomes. In addition, the tumor and the cell line displayed mRNA expression of the E6/E7 region encoding the viral oncoproteins, as determined by reverse transcription-PCR. Northern blot analysis of the cell line revealed three major and three minor transcripts harboring E6/E7 sequences. Both the primary tumor and cell line showed loss of heterozygosity at the 11q22 (D11S35) and 18q21 (DCC) loci. These data support a role for HPV 16 in the development of a subset of oral cancers, presumably in concert with loss of function of tumor suppressor genes at 11q and 18q.

Two key elements that are thought to be required for replication initiation in eubacteria are the DnaA protein, a trans-acting factor, and the replication origin, a cis-acting element. As a first step in studying the replication initiation process in mycobacteria, we have isolated a 4-kb chromosomal DNA fragment from Mycobacterium smegmatis that contains the dnaA gene. Nucleotide sequence analysis of this region revealed homologies with the rpmH gene, which codes for the ribosomal protein L34, the dnaA gene, which codes for the replication initiator protein DnaA, and the 5' end of the dnaN gene, which codes for the beta subunit of DNA polymerase III. Further, we provide evidence that when cloned into pUC18, a plasmid that is nonreplicative in M. smegmatis, the DNA fragment containing the dnaA gene and its flanking regions rendered the former capable of autonomous replication in M. smegmatis. We suggest that the M. smegmatis chromosomal origin of replication is located within the 4-kb DNA fragment.

PDGF-B released from colon tumor cells regulated tumor growth in athymic mice in a paracrine manner by inducing blood vessel formation. A positive correlation was found between expression of PDGF B-chain in cells grown in vitro and the number of factor VIII-positive blood vessels in tumors induced by three classes of colon carcinoma cell lines. Elevated expression of PDGF-B was also correlated with tumor size. Each cell line had the same mutations in the colon cancer genes APC, DCC, and p53 and had wild type c-K-ras genes (Huang et al. [1994] Oncogene, 9:3701-3706.) eliminating the possibility that any differences in tumor blood vessel formation were due to mutations and/or deletions in these genes. Colon carcinoma cells released biologically active PDGF capable of stimulating the growth of NIH3T3 cells, which was inhibited by neutralizing antisera to PDGF-AB chains. An inverse correlation was found between induction of factor VIII-positive blood vessels and expression of vascular endothelial growth factor (VEGF), while no correlation was seen with expression of either TGF alpha or k-FGF. Basic fibroblast growth factor (FGF) expression was not detected in these tumor cells. TGF beta 1 was capable of inducing PDGF-B expression in the undifferentiated U9 colon carcinoma cell line, but this sensitivity was not seen in differentiated cells. In contrast, TGF beta 1 inhibited VEGF expression in both undifferentiated cells and differentiated colon cancer cells. Thus, TGF beta 1 has two roles in the growth of undifferentiated U9 colon carcinoma cells in vivo: direct stimulation of cell proliferation as we have showed in earlier studies, and an increase in angiogenesis by inducing PDGF-B.

The APC gene is mutated in familial adenomatous polyposis (FAP) as well as in sporadic colorectal tumours. The product of the APC gene is a 300 kDa cytoplasmic protein associated with the adherence junction protein catenin. Here we show that overexpression of APC blocks serum-induced cell cycle progression from G0/G1 to the S phase. Mutant APCs identified in FAP and/or colorectal tumours were less inhibitory and partially obstructed the activity of the normal APC. The cell-cycle blocking activity of APC was alleviated by the overexpression of cyclin E/CDK2 or cyclin D1/CDK4. Consistent with this result, kinase activity of CDK2 was significantly down-regulated in cells overexpressing APC although its synthesis remained unchanged, while CDK4 activity was barely affected. These results suggest that APC may play a role in the regulation of the cell cycle by negatively modulating the activity of cyclin-CDK complexes.

Operation of esophageal cancer accompanies a big surgical stress and postoperative pulmonary complications such as respiratory failure are observed at high frequencies. On the other hand, when a big stress is added to a body, a state of hypercoagulation in which blood coagulation mechanism is abnormally enhanced appears and this state is closely related to organ failures but has many unknown points. So, we examined 39 patients given excision of esophageal cancer with respect to occurrence of postoperative respiratory failures, participation of coagulopathy in aggravation and their degrees before and after the operation to find out the relationship between postoperative respiratory failure and the state of hypercoagulation. We gave a diagnosis of respiratory failure to the patients whose respiratory index exceeded 1.5 on the day after operation but did not show atelectasis or hydrothorax. As a result, respiratory failures were observed in 7 out of 39 cases (17.9%). When a risk score (RS) of postoperative respiratory failure was determined using multivalent analysis (quantification type II) with preoperative factors such as age and function of heart, lung, liver, and kidney as well as preoperative blood coagulation factors, RS = 2.87 (antithrombin-III (AT-III) less than 75%) +1.89 (age over 70 years) +1.78 (respiratory index over 0.15) +1.44 (serum albumin less than 3.0 mg/dl) +1.28 (cardiac index less than 3.0 l/min/m2) was obtained and a drop in preoperative AT-III was considered a risk for occurrence of post operative respiratory failure. In contrast, referring changes in postoperative value of blood coagulation factors, a distinct rise in fibrinogen (FBG) appeared in early stage after operation in the respiratory failure group compared to the control group (p < 0.01). Moreover, recovery in AT-III of blood coagulation factors and in plasminogen and antiplasmin of fibrinolytic factors was delayed (p < 0.05) indicating promotion of postoperative hypercoagulation state and delay in recovery of coagulation-fibrinolysis factors. However, participation of platelet factors was absent.

Six novel transition state analogs (TSAs) of cocaine (10-14 and 17) and one non-cocaine, p-aminophenylphosphonyl ester of cyclohexanol (19), were synthesized and characterized by 1H- and 13C-NMR and FAB-MS. (1R)-ecgonine methyl ester or cyclohexanol were subjected to phenylphosphonylation in the presence of dicyclohexyl carbodiimde (DCC) and 4-N,N-dimethyl aminopyridine (4-DMAP). TSA-IV (10), however, was synthesized from norcocaine which was protected with dibromoethane to yield 4 before acid hydrolysis, esterification and phenylphosphonylation were carried out. TSA-III (11) TSA-I (12) and (19), using various length spacer arms, were coupled with the immunogenic protein, diphtheria toxoid (DT). The TSAs coupled with DT were used to immunize mice and after appropriate boosts their sera were tested for the presence and titer of anti-TSA polyclonal antibodies using ELISA. Preliminary results show that the mice immunized with these TSAs produced high titers of polyclonal catalytic antibodies, except for (19), with the ability to hydrolyze the substrate 125I-4'-iodococaine in an in vitro assay, even in the presence of noncatalytic anti-TSA antibodies.

Rapid, on-site human immunodeficiency virus (HIV) testing has the potential to improve the delivery of prevention services in publicly funded counseling and testing sites. The Single Use Diagnostic System (SUDS) HIV-1 is the only rapid enzyme immunoassay (EIA) approved for diagnostic use in the United States. To evaluate the feasibility of using SUDS in public clinics and to validate the test's performance in a public health laboratory, we conducted blinded SUDS testing on plasma sent for HIV testing. From 19 March through 30 June 1993, 1,923 consecutive samples from a sexually transmitted diseases clinic and an HIV counseling and testing clinic were tested on site with SUDS. Tests done in the first two weeks with a malfunctioning centrifuge n = 402) and those done when there were excessively high temperatures in the laboratory (n = 53) were analyzed separately. Of 1,466 tests, 39 were positive by both SUDS and EIA (with Western blot [immunoblot] confirmation) and 7 were SUDS positive and EIA negative. Western blotting was used as the "gold standard" to adjudicate these discrepancies. There were no SUDS-negative and EIA-positive tests. Compared with that of EIA (with Western blot confirmation), the sensitivity of SUDS was 100% (95% confidence interval, 88.8 to 100%) and the specificity was 99.5% (95% confidence interval, 98.9 to 99.8%). The positive predictive value of SUDS was 88% in the STD clinic and 81% in the HIV counseling and testing clinic. There was a 7.7-fold increase in false positives, from 0.48 to 3.7%, when there was inadequate centrifugation and when the temperature exceeded the manufacturer's recommendations. Rapid, on-site HIV testing by the SUDS assay is feasible and practical in public health settings. The test can be performed accurately, at reasonable cost, and within the time frame of a typical clinic visit. Caution should be used, however, as two conditions adversely affected the accuracy of this test: inadequate specimen preparation and elevated temperature.

The interaction between antithrombin III (ATIII) and albumin-heparin conjugates covalently coupled onto carboxylated polystyrene beads either in buffer containing albumin or in plasma was studied using 14C-labeled ATIII. Binding isotherms of ATIII were modeled using a summation of two Langmuir equations. These equations describe the binding of ATIII to two different sets of binding sites, one with a high, the other with a low affinity of ATIII to these sites are 9 x 10(6) L/mol and 0.3 x 10(6) L/mol, respectively. The binding of ATIII to surface binding sites with a high affinity for ATIII was correlated with the presence of specific ATIII binding sites in the immobilized heparin. Binding of ATIII from albumin solutions to binding sites with a low affinity for ATIII was dominated by nonspecific binding of ATIII to the immobilized heparin. A third small fraction of the surface bound. ATIII is probably adsorbed to sites on the surface not covered with heparin. In the case of the binding of ATIII to the heparinized surface from plasma solutions, a fraction of initially adsorbed ATIII was desorbed by other plasma proteins. This desorption in combination with direct competition between ATIII and other plasma proteins resulted in lower ATIII surface concentrations using plasma as compared to the ATIII surface concentrations obtained using albumin solutions. The binding of ATIII to nonspecific binding sites was almost completely inhibited in the presence of plasma proteins. The amount of ATIII bound to immobilized heparin via specific ATIII binding sites was 30% lower in plasma solutions as compared to the specific binding of ATIII using albumin solutions. It is concluded that the accessibility of immobilized heparin for ATIII in plasma decreases by binding of heparin-binding proteins onto the immobilized heparin and/or adsorption of other plasma proteins on the heparinized surface.

From a histologic and endoscopic standpoint, colon and rectal cancer (CRC) begins as a small neoplastic polyp which progressively enlarges and transforms through a dysplasia stage into invasive cancer. Recently, molecular abnormalities underlying the adenomacarcinoma progression have been defined. The adenomatous polyposis coli (APC) gene and mismatch repair genes are found to be dysfunctional early in the neoplastic process; either as inherited or somatic mutations. Subsequently, polyps progress to cancer along one of two paths depending on which gene is abnormal. When the APC gene is the initial mutation tumor development follows the "loss of heterozygocity" (LOH) pathway. If mismatch repair genes are altered, the "replication error" (RER) pathway is followed. Somatic mutations of the K-ras oncogene and the MCC, DCC, and p53 tumor suppressor genes accumulate in the LOH pathway and mark the progression through polyp stages. Microsatellite instability is a characteristic of the RER pathway but the precise genes involved in this pathway currently are not known. Defining these pathways has led to a new classification scheme for CRC with resultant changes in our clinical approach to screening, surveillance, and treatment.

<h4>Background</h4>Reports about the effects of progestins on cell proliferation are contradictory. We investigated the effect of progesterone, medroxyprogesterone acetate, megestrol acetate, ORG 2058 and the antiprogestin RU 486 on two hormone-dependent cell lines, T47D and MCF-7 (characterized by a different content of PgR). The aim of the study was to understand the eventual ability of progestins to interfere with cell proliferation stimulated by estradiol and various growth factors (TGF-a, IGF-I, IGF-II).<h4>Material and methods</h4>MCF-7 and T47D cells were maintained in DMEM/F12 medium supplemented with 2% FCS while experiments were carried out in the same culture medium using DCC-stripped FCS.<h4>Results</h4>In the absence of estradiol, all tested progestins generally tended to stimulate cell growth in the T47D cell line, but in the MCF-7 cell line only the highest concentrations (10(-6) M and 10(-7) M) were found to be stimulatory. In contrast, in the presence of 10(-8) M estradiol, progestins tended to inhibit cell growth stimulation in MCF-7 and T47D cell lines. The antiprogestin RU 486 exerted a stimulatory effect similar to that promoted by estradiol itself in MCF-7 cells. Instead, in T47D cells, RU 486 did not modify cell growth in the absence of estradiol, but tended to counteract the estradiol-promoted cell proliferation. In MCF-7 cells, medroxyprogesterone acetate and megestrol acetate were also able to effectively counteract the cell growth induced by TGF-alpha. However, none of these progestins was able to abolish cell proliferation promoted by IGF-I or IGF-II.<h4>Conclusion</h4>We therefore concluded that failure to respond to progestin treatment may be due to the very heterogeneous nature of human breast tumors and to the inability of these molecules to interfere with the IGF-R pathway.

<h4>Unlabelled</h4>Functional methods for ATIII determination are essential for the diagnosis of deficiencies of this important thrombin inhibitor.<h4>Purpose</h4>The aim of this work was standardize the method for ATIII level determination in plasma, in microplates.<h4>Method</h4>ATIII levels were measured, using the chromogenic substrate Tos-Gly-Pro-Arg-NAN, which is specific for thrombin, and which has been sinthesized at the Biophysical Department of the Escola Paulista de Medicina of the Federal University of São Paulo, Brazil.<h4>Results</h4>Among 21 patients with deep venous thrombosis (DVT), 20 had ATIII levels above 70% (113 +/- 22%). A 22 year-old female patient, who had recurrent DVT and a familiar DVT antecedent, had a congenital ATIII deficiency (56%). ATIII levels were normal in 6 patients with von Willebrand disease (109 +/- 28%), as expected. In 20 patients with severe hepatic failure, it has been found reduced ATIII levels (42 +/- 19%), since this inhibitor is produced by the liver. In 3 patients with sepsis and DIC, ATIII levels have also been reduced (45 +/- 5%) owing to consumption during blood coagulation activation. There was a significant correlation between ATIII levels and the prothrombin time, as well as the factor V levels, and both are good parameters to assess hepatic function and to monitor DIC. There was a significant correlation between ATIII levels measured using the chromogenic substrate Tos-Gly-Pro-Arg-NAN and those measured using S-2238, produced by Kabi Laboratories.<h4>Conclusions</h4>This method for ATIII determination in plasma is easy to perform and it can detect ATIII deficiency in patients with hepatic failure, disseminated intravascular coagulation and thrombophilia.