Fingerprinting the circulating repertoire of antibodies from cancer patients

Recognition of molecular diversity in disease is required for the development of targeted therapies. We have developed a screening method based on phage display to select peptides recognized by the repertoire of circulating tumor-associated antibodies. Here we isolated peptides recognized by antibodies purified from the serum of prostate cancer patients. We identified a consensus motif, NX(S/T)DK(S/T), that bound selectively to circulating antibodies from cancer patients over control antibodies from blood donors. We validated this motif by showing that positive serum reactivity to the peptide was specifically linked to disease progression and to shorter survival in a large patient population. Moreover, we identified the corresponding protein eliciting the immune response. Finally, we showed a strong and specific positive correlation between serum reactivity to the tumor antigen, development of metastatic androgen-independent disease, and shorter overall survival. Exploiting the differential humoral response to cancer through such an approach may identify molecular markers and targets for diagnostic and therapeutic intervention.     

Localization of carboxypeptidase A-like enzyme in rat kidney

In this study we demonstrate that carboxypeptidase A (CPA)-like enzyme is expressed in rat kidney. The major metabolites of angiotensin (Ang) I by the rat renal mesangial cell extract at 37 degrees C, pH 7.4, were Ang 1-9 and Ang II. Quinaprilat did not influence the formation of Ang 1-9, but it inhibited formation of Ang II. The formation of Ang 1-9 was inhibited by potato carboxypeptidase inhibitor, 1,10-phenanthroline or EDTA. Lowering the pH from 7.4 to 4.0 also inhibited the formation of this nonapeptide. These findings suggest that a metallocarboxypeptidase is responsible for Ang 1-9 production. Using monoclonal antibodies to CPA, Western blot showed the presence of CPA-like enzyme in the extracts prepared from the mesangial cells or kidney cortex of the rat. Immunohistochemistry showed that CPA-like enzyme is localized in the mesangial glomerular cells and adventitia of kidney blood vessels, whereas it was absent in the renal tubules. Our data suggest that a CPA-like enzyme could be added to a repertoire of enzymes present in the rat mesangial cells and adventitia of renal blood vessels.     

Roles of hnRNP A1, SR proteins,and p68 helicase in c-H-ras alternative splicing regulation

Human ras genes play central roles in coupling extracellular signals with complex intracellular networks controlling proliferation, differentiation, and apoptosis, among others processes. c-H-ras pre-mRNA can be alternatively processed into two mRNAs due to the inclusion or exclusion of the alternative exon IDX; this renders two proteins, p21H-Ras and p19H-RasIDX, which differ only at the carboxy terminus. Here, we have characterized some of the cis-acting sequences and trans-acting factors regulating IDX splicing. A downstream intronic silencer sequence (rasISS1), acting in concert with IDX, negatively regulates upstream intron splicing. This effect is mediated, at least in part, by the binding of hnRNP A1. Depletion and add-back experiments in nuclear extracts have confirmed hnRNP A1's inhibitory role in IDX splicing. Moreover, the addition of two SR proteins, SC35 and SRp40, can counteract this inhibition by strongly promoting the splicing of the upstream intron both in vivo and in vitro. Further, the RNA-dependent helicase p68 is also associated with both IDX and rasISS1 RNA, and suppression of p68 expression in HeLa cells by RNAi experiments results in a marked increase of IDX inclusion in the endogenous mRNA, suggesting a role for this protein in alternative splicing regulation.     

The eukaryotic nucleotide excision repair pathway

Nucleotide excision repair (NER) is the most versatile mechanism of DNA repair, recognizing and dealing with a variety of helix-distorting lesions, such as the UV-induced photoproducts cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4 PPs). In this review, we describe the main protein players and the different sequential steps of the eukaryotic NER mechanism in human cells, from lesion recognition to damage removal and DNA synthesis. Studies on the dynamics of protein access to the damaged site, and the kinetics of lesion removal contribute to the knowledge of how the cells respond to genetic insult. DNA lesions as well as NER factors themselves are also implicated in changes in cell metabolism, influencing cell cycle progression or arrest, apoptosis and genetic instability. These changes are related to increased mutagenesis and carcinogenesis. Finally, the recent collection of genomic data allows one to recognize the high conservation and the evolution of eukaryotic NER. The distribution of NER orthologues in different organisms, from archaea to the metazoa, displays challenging observations. Some of NER proteins are widespread in nature, probably representing ancient DNA repair proteins, which are candidates to participate in a primitive NER mechanism.     

Platelet-Activating Factor (PAF) Modulates Peritoneal Mouse Macrophage Infection by Leishmania amazonensis

The effects of platelet-activating factor (PAF) on the infection of peritoneal mouse macrophages by Leishmania amazonensis were investigated. Prior to the infection, the parasites and/or the macrophages were treated with PAF and/or one of the following modulators: WEB 2086 (PAF antagonist), and the modulators of protein kinase C, phorbol-12-myristate-13-acetate (PMA), and sphingosine. The infection was inhibited when the macrophages or both the parasites and the macrophages were treated with PAF, but stimulated by PAF-treated parasites. WEB 2086 abrogated PAF effects in both systems. The infection was stimulated when the macrophages were treated with sphingosine plus PAF, but inhibited when the macrophages were treated with sphingosine and the parasites with sphingosine plus PAF. The infection was inhibited by sphingosine-treated parasites, either in the presence or in the absence of PAF. Leishmania amazonensis–macrophage infection was inhibited by PMA in all systems tested. Received: 27 September 2000 / Accepted: 15 December 2000     

PCR identification of Salmonella cells in food and stool samples after immunomagnetic separation

The purpose of the present study was to investigate the application of various sample preparation methods (cell washing before lysis, purification of DNA using phenol extraction method, immunomagnetic separation-IMS) for the final PCR identification of Salmonellacells. The presence of PCR inhibitors in processed food products (milk powder and dried eggs) can be the cause of false-negative results in PCR without IMS of target cells. It was also demonstrated that IMS-PCR was successfully used for identification and quick confirmation of untypical Salmonella strains isolated from human stool samples and rabbit meat. However, IMS cannot eliminate intracellular PCR inhibitors present in immunoseparated Salmonella cells. These inhibitors must be taken into consideration in evaluation of PCR procedure.     

Glutathione S transferase M1 andT1 genotypes and susceptibility to smoking related larynx cancer

Susceptibility to smoking related larynx cancer has been suggested to be associated with genetically determined differences in the ability to detoxify carcinogens present in tobacco smoke. The genetic polymorphisms of glutathione S-transferases, involved in the metabolic inactivation of, for example, tobacco derived carcinogens, have been recognized as potential risk modifiers in various environmentally induced malignancies, including larynx cancer. We employed PCR-based methods to determine the distribution of the GSTM 1 and G STT1 null genotypes in 171 larynx cancer patients and 180 controls to examine further their potential role in individual susceptibility to this neoplasm. The GSTM 1 null genotype was found in 49 1 % of the cases and 57 7 % of the controls and the GSTT1 null genotype in 17 5 % of the cases and 21 7 % of the controls, respectively. Larynx cancer risk associated with the lack of GST M 1 (OR = 0 7; 95 % CI: 0 5-1 1) or GSTT1 (OR = 0 8; 95 % CI: 0 5-1 3) was not significantly affected by age, smoking status, or cancer progression. Although this study thus suggests no role for the G STM 1 and GSTT1 gene polymorphisms in individual susceptibility to smoking-related larynx cancer, due to its relatively small sample size more data are required before any definite conclusions can be drawn.     

Effect of nitric oxide synthase inhibition on hypercapnia-induced hypothermia and hyperventilation

Hypercapnia elicits hypothermia in a numberof vertebrates, but the mechanisms involved are not well understood. Inthe present study, we assessed the participation of the nitric oxide(NO) pathway in hypercapnia-induced hypothermia and hyperventilation bymeans of NO synthase inhibition by usingN-nitro-L-arginine(L-NNA). Measurements ofventilation, body temperature, and oxygen consumption were performed inawake unrestrained rats before and afterL-NNA injection(intraperitoneally) and L-NNA injection followed by hypercapnia (5%CO₂). Control animals received saline injections. L-NNA alteredthe breathing pattern during the control situation but not duringhypercapnia. A significant (P < 0.05) drop in body temperature was measured after bothL-NNA (40 mg/kg) and 5%inspired CO₂, with a drop inoxygen consumption in the first situation but not in the second.Hypercapnia had no effect onL-NNA-induced hypothermia. Theventilatory response to hypercapnia was not changed byL-NNA, even thoughL-NNA caused a drop in bodytemperature. The present data indicate that the two responses elicitedby hypercapnia, i.e., hyperventilation and hypothermia, do not share NOas a common mediator. However, theL-arginine-NO pathwayparticipates, although in an unrelated way, in respiratory function andthermoregulation.

     

Sam68 sequestration and partial loss of function are associated with splicing alterations in FXTAS patients

Fragile X‐associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder caused by expansion of 55–200 CGG repeats in the 5′‐UTR of the FMR1 gene. FXTAS is characterized by action tremor, gait ataxia and impaired executive cognitive functioning. It has been proposed that FXTAS is caused by titration of RNA‐binding proteins by the expanded CGG repeats. Sam68 is an RNA‐binding protein involved in alternative splicing regulation and its ablation in mouse leads to motor coordination defects. Here, we report that mRNAs containing expanded CGG repeats form large and dynamic intranuclear RNA aggregates that recruit several RNA‐binding proteins sequentially, first Sam68, then hnRNP‐G and MBNL1. Importantly, Sam68 is sequestered by expanded CGG repeats and thereby loses its splicing‐regulatory function. Consequently, Sam68‐responsive splicing is altered in FXTAS patients. Finally, we found that regulation of Sam68 tyrosine phosphorylation modulates its localization within CGG aggregates and that tautomycin prevents both Sam68 and CGG RNA aggregate formation. Overall, these data support an RNA gain‐of‐function mechanism for FXTAS neuropathology, and suggest possible target routes for treatment options.     

Detection of resistance genes and susceptibility patterns in Bacteroides and Parabacteroides strains

Susceptibility to five antimicrobials was determined for Bacteroides spp. (n = 52) and Parabacteroides distasonis (n = 8). All isolates were susceptible to metronidazole. The resistance rates to ampicillin, cefoxitin, tetracycline and clindamycin were 98%, 9.6%, 65.3% and 19.2% of the Bacteroides strains, respectively. The genes cepA, cfiA, cfxA, tetQ, ermF and nim were found in 69.2%, 17.3% 9.6%, 50%, 7.7% and 3.8% for these strains respectively. All P. distasonis strains were resistant to ampicilin. Cefoxitin, tetracycline and clindamycin resistance rates were 75%, 87.5% and 50%, respectively. The ermF and nim genes were absent and 37.5%, 12.5%, 12.5% and 87.5% of this strains possessed cepA, cfiA, cfxA and tetQ genes, respectively. Ten cfiA gene positive strains of Bacteroides and Parabacteroides were submitted to E-test with imipenem and amoxicillin–clavulanate. The resistance rate to imipenem was 4.1% and 8.3% to amoxicillin–clavulanate. This feature is for the first time described in Brazil.