Identification of genes expressed in the rat prostate that are modulated differently by castration and Finasteride treatment

In mammals, testosterone and 5alpha-dihydrotestosterone (DHT) are the principal male hormones (androgens). Testosterone is the most abundant circulating androgen, and is converted in specific tissues to DHT by the 5alpha-reductase enzymes. Although each of these androgens binds to the same receptor protein (androgen receptor, AR), each exerts biologically distinct effects. Theories to explain the specific effects of testosterone and DHT have centered on kinetic differences of binding of androgens to the receptor or differences in the metabolic fates of the two hormones. In the current experiments, differential display PCR (ddPCR) was used to identify genes regulated differently by testosterone and DHT. Adult male rats were treated as follows: castrated, treated with Finasteride (an inhibitor of 5alpha-reductase) or left intact for ten days. RNA was prepared from the dissected prostates of these animals and used for ddPCR. Genes exhibiting four distinct patterns of regulation were observed among the mRNAs. Class 1 genes showed equivalent expression in intact and Finasteride-treated animals, but were absent in castrated animals (mRNAs D1, D2, D6, D10). Class 2 genes showed higher expression in intact animals, intermediate levels following Finasteride treatment, but were absent in castrated animals (mRNA D8). Two classes of gene were particularly intriguing: class 3 showed gene expression only in the intact animal (mRNA D7, D9) and class 4 showed increased gene expression following Finasteride treatment (mRNA D3). While the patterns observed for some of these genes (e.g. D8) suggest that the different biological effects of testosterone and DHT may be due to the lower affinity of the AR for testosterone and limiting tissue concentrations of androgen, our results also suggest that some genes expressed in the rat prostate may be regulated in fundamentally different ways in response to testosterone and DHT.     

Bronchopulmonary dysplasia. Impact of surfactant replacement therapy

Many functional dyspepsia treatment trials have until recently suffered from important weaknesses in study design. A major problem has been the low number of studies that have used validated outcome measures. Fortunately, progress has been made in this area. The evidence for the efficacy of antacids, H2-receptor antagonists, omeprazole, domperidone, cisapride and anti-Helicobacter therapy is reviewed. Although several of these have shown benefit, it is unclear whether this may be a result of the inclusion of patients with unrecognized gastro-oesophageal reflux disease. The data on anti-Helicobacter therapy are conflicting.     

Intergroup biases and eyewitness testimony

The study examined how the in-group/out-group status of a perpetrator of a distinctly violent crime might influence an eyewitness's evaluation of his behavior and a witness's performance in an identification task. Immigrant and Swedish students saw a film showing a simulated robbery, with an immigrant or a Swede as the perpetrator. Results showed that both groups evaluated an ethnically dissimilar perpetrator as more culpable than an ethnically similar perpetrator. In a line-up task, both immigrant and Swedish participants mistakenly identified an innocent immigrant more often than an innocent Swede. Participants' biased evaluations of the perpetrator are discussed in terms of cognitive and motivational mechanisms. Expectations regarding the typical ethnicity of a perpetrator of this type of crime are suggested to account for the findings of the line-up task.     

Small wire external fixation of high energy tibial plateau fractures

Open plate osteosynthesis for high energy tibial plateau fractures with dissociation between the metaphysis and diaphysis has been plagued with frequent soft tissue complications. The Harbor-University of California at Los Angeles Medical Center's experience with small wire external fixation supplemented by limited internal fixation is examined. This alternative method of adequate stable fixation offers the advantage of minimal soft tissue compromise. Twenty-four patients with Schatzker Type VI tibial fractures were treated with small wire external fixation. Supplementary limited internal fixation was used with percutaneous screws in 10 patients and with open reduction in one patient. Sixteen patients had isolated fractures, and eight others suffered multiple injuries. Minimum followup was 12 months. All fractures healed. Complications included one septic knee, two infections at screw sites, and one 10 degrees knee flexion contracture. One knee had Grade 3 radiographic arthrosis, five had Grade 2, 10 had Grade 1, and eight showed no arthrosis. The outcomes (Knee Society clinical rating system) of this study compare favorably with outcomes described in reports published previously for this type of fracture, despite inclusion of eight multiply injured patients. This technique preserves the goals of early range of motion and stable fixation for these devastating injuries, while decreasing the observed major wound complications and nonunion rates. However, longer followup may reveal higher arthrosis rates, specifically in those fractures that were not anatomically reduced.     

An automated fluorescent PCR method for detection of shiga toxin-producing Escherichia coli in foods

An automated fluorescence-based PCR system (a model AG-9600 AmpliSensor analyzer) was investigated to determine whether it could detect Shiga toxin-producing Escherichia coli (STEC). The AmpliSensor PCR assay involves amplification-mediated disruption of a fluorogenic DNA signal duplex (AmpliSensor) that is homologous to conserved target sequences in a 323-bp amplified fragment of Shiga toxin genes stx1, stx2, and stxe. Using the Amplisensor assay, we detected 113 strains of STEC belonging to 50 different serotypes, while 18 strains of non-Shiga-toxin-producing E. coli and 68 strains of other bacteria were not detected. The detection limits of the assay were less than 1 to 5 CFU per PCR mixture when pure cultures of five reference strains were used and 3 CFU per 25 g of food when spiked ground beef samples that were preenriched overnight were used. The performance of the assay was also evaluated by using 53 naturally contaminated meat samples and 48 raw milk samples. Thirty-two STEC-positive samples that were confirmed to be positive by the culture assay were found to be positive when the AmpliSensor assay was used. Nine samples that were found to be positive when the PCR assay was used were culture negative. The system described here is an automated PCR-based system that can be used for detection of all serotypes of STEC in food or clinical samples.     

Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration

Human procathepsin L has been expressed in E. coli in the form of inclusion bodies. The recombinant protein was isolated, refolded and processed at pH 5.5 by the addition of dextran sulfate which increased the overall yield of cathepsin L almost 10-fold. After the auto-activation of the 38 kDa procathepsin L at least three processing sites were determined by N-terminal amino acid sequencing. After replacing the Ala205 residue by glutamic acid, cathepsin B-like specificity was introduced into cathepsin L. This mutation resulted in a 15-fold increased activity toward the substrate Z-Arg-Arg-AMC and in a 29-fold decreased activity toward Z-Phe-Arg-AMC. Residue 205 is thereby confirmed experimentally to be critical for the specificity of cathepsins B and L.     

Summary of the II International Symposium on Cytomegalovirus

Human cytomegalovirus (HCMV) is a highly species-specific DNA virus belonging to the Betaherpesvirinae subfamily of the herpesviridae family. Like other herpesviruses, primary infection with HCMV is followed by persistence of the virus in a latent form. The sites of latency are still largely undefined, but they probably include bone marrow progenitor cells and peripheral blood monocytes. From these sites, the virus can reactivate, resulting in renewed shedding of the virus, or, in immunocompromized persons, development of disease. Humans are the only reservoir of HCMV and transmission occurs by person-to-person contact. Infection with HCMV is common. In most developed countries, HCMV seroprevalence steadily increases after infancy and 10-20% of children are infected before puberty. In adults, the prevalence of antibodies ranges from 40 to 100%. Although HCMV has a world-wide distribution, infection with HCMV is more common in the developing countries and in areas of low socioeconomic conditions, which is predominantly related to the closeness of contacts within these populations. Except for a mononucleosis-like illness in some persons, infection with HCMV rarely causes disease in immunocompetent individuals. However, HCMV can cause severe morbidity and mortality in congenitally infected newborns and immunocompromized patients, most notably transplant-recipients and HIV-infected persons. This article provides a review of the information presented at the Second International Symposium on Cytomegalovirus organized and convened by The Macrae Group (New York City, NY) in Acapulco, Mexico on 24-28 April 1998. During this symposium, the state-of-the-art knowledge on diagnosis, treatment and prophylaxis of HCMV infections were discussed, and, based on this information, attempts to highlight the future directions in basic and clinical research areas that need to be stimulated to facilitate advancement in prevention and treatment of CMV disease.     

Kinetics of receptor-mediated radiotoxicity of 16alpha-[125I]-iodostradiol-3,17beta

AIM: The radiocytotoxic effects in estrogen receptor (ER) containing MCF-7 cells of a mamma carcinoma were investigated following incubation with [125I]E ranging from 1 h to 24 h. METHODS: The receptor status of the cells was confirmed by immunohistochemical staining. The accumulation of [125I]E in MCF-7 cells was tested in the presence and absence of radioinert E and [127I]E and in ER-negative cells in comparison to ER-positive cells. The subcellular distribution was investigated in 0.25 M Saccharose by ultra centrifugation. The radiocytotoxicity was assessed in ER-positive and negative cells by a standard colony forming assay after incubating with [125I]E (1.85 kBq/ml-55.5 kBq/ml) for 1, 2, 4, 8, 12, and 24 h. RESULTS: A significant cytotoxicity was observed only when ER-rich MCF-7-cells were incubated with [125I]E alone. The maximal cytotoxic effect was a reduction of survival fraction to 20-25%. This was achieved at radioactivity concentrations > 37 kBq/ml. Maximal effect was seen after 8 h incubation, extension of incubation time did not further increase toxicity. CONCLUSION: The results suggest that the radioactivity was bound to ER. Through their nuclear localization radioestrogens tagged with radionuclides emitting very low energy electrons (Auger electrons) bear potential for therapy by ER-mediated deposition of lethal doses of ionizing radiation to single cells without affecting neighbouring cells. But, instead of 125I the shorter-living 123I shall be used for labelling because the deciding radiation effectes occur within the first 8 h.     

The Golgi and endoplasmic reticulum remain independent during mitosis in HeLa cells

Partitioning of the mammalian Golgi apparatus during cell division involves disassembly at M-phase. Despite the importance of the disassembly/reassembly pathway in Golgi biogenesis, it remains unclear whether mitotic Golgi breakdown in vivo proceeds by direct vesiculation or involves fusion with the endoplasmic reticulum (ER). To test whether mitotic Golgi is fused with the ER, we compared the distribution of ER and Golgi proteins in interphase and mitotic HeLa cells by immunofluorescence microscopy, velocity gradient fractionation, and density gradient fractionation. While mitotic ER appeared to be a fine reticulum excluded from the region containing the spindle-pole body, mitotic Golgi appeared to be dispersed small vesicles that penetrated the area containing spindle microtubules. After cell disruption, M-phase Golgi was recovered in two size classes. The major breakdown product, accounting for at least 75% of the Golgi, was a population of 60-nm vesicles that were completely separated from the ER using velocity gradient separation. The minor breakdown product was a larger, more heterogenously sized, membrane population. Double-label fluorescence analysis of these membranes indicated that this portion of mitotic Golgi also lacked detectable ER marker proteins. Therefore we conclude that the ER and Golgi remain distinct at M-phase in HeLa cells. To test whether the 60-nm vesicles might form from the ER at M-phase as the result of a two-step vesiculation pathway involving ER-Golgi fusion followed by Golgi vesicle budding, mitotic cells were generated with fused ER and Golgi by brefeldin A treatment. Upon brefeldin A removal, Golgi vesicles did not emerge from the ER. In contrast, the Golgi readily reformed from similarly treated interphase cells. We conclude that Golgi-derived vesicles remain distinct from the ER in mitotic HeLa cells, and that mitotic cells lack the capacity of interphase cells for Golgi reemergence from the ER. These experiments suggest that mitotic Golgi breakdown proceeds by direct vesiculation independent of the ER.     

Increased density of microtubule associated protein 2-immunoreactive neurons in the prefrontal white matter of schizophrenic subjects

Recent studies have suggested that schizophrenia may be related to prenatal disturbances in the cortical subplate, a transient but essential structure in the formation of cerebral cortical circuitry. Although most subplate neurons die during later development, some remain as the interstitial neurons of the adult white matter. In this study we used a monoclonal antibody against the cytoskeletal protein, microtubule associated protein-2 (MAP2), to quantify the density and distribution of labeled neurons in postmortem brain specimens containing the prefrontal white matter from five schizophrenic cases and matched controls. In both schizophrenics and matched controls, the density of white matter neurons decreased with increasing white matter depth. However, the mean density of MAP2-immunoreactive neurons was greater in the superficial white matter of the schizophrenic subjects compared to the matched controls. In contrast, no difference in the density of labeled neurons was seen in the deeper white matter. These findings are consistent with an abnormality in the development of the cortical subplate in at least some cases of schizophrenia.