Experimental analysis of the annotation of promoters in the public database

The ability to identify and examine promoter elements is important to researchers who wish to understand how gene expression is regulated in normal and pathological states. Unfortunately, the number of human promoters that have been directly experimentally defined is small. In order to determine if promoter sequences can be identified by simply aligning mRNA and genomic sequences, we have used a reporter gene assay to assess the promoter activity of the immediate 5' region flanking 38 mRNAs mapping to chromosome 21. For comparison, we have measured the activities of 19 sequences not thought to be promoters and 39 sequences taken from the Eukaryotic Promoter Database. Our results suggest that alignment of reference mRNAs to genomic sequence allows promoters to be identified for at least 75% of genes. These data provide the first empirical evidence that the current state of annotation of the genome is sufficient to allow molecular geneticists to correctly identify promoter sequences for most genes for which reference mRNA and genomic sequences are available.     

Regulation of hepatocyte activator inhibitor-1 expression by androgen and oncogenic transformation in the prostate

Hepatocyte activator inhibitor-1 (HAI-1) is a transmembrane serine protease inhibitor that regulates the conversion of latent to active hepatocyte growth factor (HGF). Studies supporting a role for the HGF pathway in prostate carcinogenesis prompted an analysis of HAI-1 expression in the prostate. Here we analyze the regulation of HAI-1 expression by androgen, oncogenic transformation, and cancer progression. Immunohistochemical analysis revealed that HAI-1 expression was restricted to prostate epithelium, where staining occurred primarily in basal and atrophic luminal epithelial cells. Compared to normal glands, HAI-1 expression was significantly increased in localized prostate cancer and was present in most prostate cancer metastases. HAI-1 protein expression levels were sensitive to androgen in normal epithelium but not in cancer. Although androgen did not increase HAI-1 protein expression levels in LNCaP cells, it decreased HAI-1 surface expression, consistent with previous data from our group (Martin DB, Gifford DR, Wright ME, Keller A, Yi E, Goodlett DR, Aebersold R, Nelson PS: Quantitative proteomic analysis of proteins released by neoplastic prostate epithelium. Cancer Res 2004, 64:347-355). HAI-1 overexpression in cancer was predictive of prostate-specific antigen recurrence (relative risk, 1.24). These results suggest that HAI-1 regulates the HGF Met axis on prostate epithelial cells and influences HGF mediated tumor invasion and metastasis.     

High levels of estradiol impair spatial performance in the Morris water maze and increase 'depressive-like' behaviors in the female meadow vole

The present study investigated sex differences and the effect of a high level of estradiol in the female meadow vole on performance in the forced swim test (FST) and the Morris water maze in meadow voles. Female meadow voles were ovariectomized (OVX) and administered either vehicle (sesame oil) or estradiol for 2 days prior to performing the FST. Four days following the FST, all animals were run in the Morris water maze. Results indicated that estradiol-injected female meadow voles showed more 'depressive-like' behaviors in the FST (greater time spent immobile and less time spent swimming) than vehicle-treated female or male meadow voles. In addition, estradiol-treated females had impaired performance (greater latencies and distance swam to reach the hidden platform) than both vehicle-treated female and male meadow voles, consistent with previous data. Despite the fact that estradiol administration increased 'depressive-like' behaviors in the FST and impaired performance in the Morris water maze, there was no correlation between the two behaviors indicating that 'depressive-like' behaviors did not account for the differences seen in spatial performance in the Morris water maze. To our knowledge, this is the first demonstration in rodents indicating that estradiol-mediated changes in behavior in the FST is not indicative of subsequent performance in the Morris water maze.     

Entamoeba invadens contains the components of a classical adrenergic signaling system

Epinephrine (Epi) was previously found to bypass the need for galactose ligands during early steps in the initiation of Entamoeba encystment. Epinephrine is presumed to act on amoebae through a classical adrenergic signaling pathway that results in the increased production of cyclic adenosine monophosphate (cAMP). The object of this study was to verify the existence of an adrenergic like pathway and its response to Epi in both whole Entamoeba trophozoites and purified plasma membrane preparations. Whole trophozoite and purified membrane preparations from Entamoeba invadens responded to the presence of Epi by increasing the production of cAMP. The modulators of heterotrimeric G protein signaling, forskolin (FK), pertussis toxin (PTX) and cholera toxin (CTX), also increased cAMP levels in whole cells and membrane fragments. All of these increases in cAMP were inhibited by specific inhibitors of adenylyl cyclase (AC). Treatment of membrane fragments with epinephrine caused an increased binding of non-hydrolysable GTP analogs. Entamoeba trophozoites therefore appear to contain G-protein-regulated adenylyl cyclase that functions downstream of an adrenergic ligand receptor.     

Epitope mapping of a protective monoclonal antibody against Pneumocystis carinii with shared reactivity to Streptococcus pneumoniae surface antigen PspA

Pneumocystis carinii is an opportunistic fungal pathogen that causes pneumonia in the immunocompromised host. A protective monoclonal antibody (MAb) termed 4F11 generated against mouse-derived P. carinii was shown by indirect immunofluorescence assay (IFA) to bind surface antigens of P. carinii derived from multiple host species, including humans. We have identified multiple epitopes recognized by MAb 4F11 in two recombinant mouse P. carinii antigens. The epitopes mapped have similar proline content and positive charge distribution. The consensus 8-mer epitope recognized by MAb 4F11 is K/RPA/RPK/QPA/TP. Immune sera raised against intact mouse P. carinii recognized native antigens affinity purified with MAb 4F11 and a recombinant antigen reactive with MAb 4F11. Database searches for short, nearly exact matches to the mapped MAb 4F11 epitopes identified a bacterial surface antigen, Streptococcus pneumoniae PspA, with a similar proline-rich region. In an IFA, MAb 4F11 detected antigens on the S. pneumoniae surface, and Western blotting identified a protein in S. pneumoniae lysates consistent with the M(r) of PspA. A fragment of the S. pneumoniae PspA gene was cloned and sequenced, and the deduced amino acid sequence contained a region with strong similarity to the MAb 4F11 epitopes identified in P. carinii. The PspA recombinant polypeptide was recognized by MAb 4F11 in a Western blot. The ability of MAb 4F11 to recognize similar proline-rich epitopes may explain its ability to recognize P. carinii derived from multiple hosts and will permit testing of the epitopes recognized by this antibody in immunization against P. carinii.     

Construction and characterization of a live, attenuated aroA deletion mutant of Pseudomonas aeruginosa as a candidate intranasal vaccine

Antibodies to the lipopolysaccharide O antigen of Pseudomonas aeruginosa mediate high-level immunity, but protective epitopes have proven to be poorly immunogenic, while nonprotective or minimally protective O-antigen epitopes often elicit the best immune responses. With the goal of developing a broadly protective P. aeruginosa vaccine, we used a gene replacement system based on the Flp recombinase to construct an unmarked aroA deletion mutant of the P. aeruginosa serogroup O2/O5 strain PAO1. The resultant aroA deletion mutant of PAO1 is designated PAO1 Delta aroA. The aroA deletion was confirmed by both PCR and failure of the mutant to grow on minimal media lacking aromatic amino acids. When evaluated for safety and immunogenicity in mice, PAO1 Delta aroA could be applied either intranasally or intraperitoneally at doses up to 5 x 10(9) CFU per mouse without adverse effects. No dissemination of PAO1 Delta aroA to blood, liver, or spleen was detected after intranasal application, and histological evidence of pneumonia was minimal. Intranasal immunization of mice and rabbits elicited high titers of immunoglobulin G to whole bacterial cells and to heat-stable bacterial antigens of all seven prototypic P. aeruginosa serogroup O2/O5 strains. The mouse antisera mediated potent phagocytic killing of most of the prototypic serogroup O2/O5 strains, while the rabbit antisera mediated phagocytic killing of several serogroup-heterologous strains in addition to killing all O2/O5 strains. This live, attenuated P. aeruginosa strain PAO1 Delta aroA appears to be safe for potential use as an intranasal vaccine and elicits high titers of opsonic antibodies against multiple strains of the P. aeruginosa O2/O5 serogroup.     

Vector competence of Peruvian mosquitoes (Diptera: Culicidae) for epizootic and enzootic strains of Venezuelan equine encephalomyelitis virus

Mosquitoes collected in the Amazon Basin, near Iquitos, Peru, were evaluated for their susceptibility to epizootic (IAB and IC) and enzootic (ID and IE) strains of Venezuelan equine encephalomyelitis (VEE) virus. After feeding on hamsters with a viremia of approximately 10(8) plaque-forming units of virus per milliliter, Culex (Melanoconion) gnomatus Sallum, Huchings, & Ferreira, Culex (Melanoconion) vomerifer Komp, and Aedes fulvus (Wiedemann) were highly susceptible to infection with all four subtypes of VEE virus (infection rates > or = 87%). Likewise, Psorophora albigenu (Peryassu) and a combination of Mansonia indubitans Dyar & Shannon and Mansonia titillans (Walker) were moderately susceptible to all four strains of VEE virus (infection rates > or = 50%). Although Psorophora cingulata (Fabricius) and Coquillettidia venezuelensis (Theobald) were susceptible to infection with each of the VEE strains, these two species were not efficient transmitters of any of the VEE strains, even after intrathoracic inoculation, indicating the presence of a salivary gland barrier in these species. In contrast to the other species tested, both Culex (Melanoconion) pedroi Sirivanakarn & Belkin and Culex (Culex) coronator Dyar & Knab were nearly refractory to each of the strains of VEE virus tested. Although many of the mosquito species found in this region were competent laboratory vectors of VEE virus, additional studies on biting behavior, mosquito population densities, and vertebrate reservoir hosts of VEE virus are needed to incriminate the principal vector species.     

Pathways to ischemic neuronal cell death: are sex differences relevant?

We have known for some time that the epidemiology of human stroke is sexually dimorphic until late in life, well beyond the years of reproductive senescence and menopause. Now, a new concept is emerging: the mechanisms and outcome of cerebral ischemic injury are influenced strongly by biological sex as well as the availability of sex steroids to the brain. The principal mammalian estrogen (17 β estradiol or E2) is neuroprotective in many types of brain injury and has been the major focus of investigation over the past several decades. However, it is becoming increasingly clear that although hormones are a major contributor to sex-specific outcomes, they do not fully account for sex-specific responses to cerebral ischemia. The purpose of this review is to highlight recent studies in cell culture and animal models that suggest that genetic sex determines experimental stroke outcome and that divergent cell death pathways are activated after an ischemic insult. These sex differences need to be identified if we are to develop efficacious neuroprotective agents for use in stroke patients.     

Hepatische Extraktion und Clearance von Renin bei Lebercirrhosen

Zusammenfassung Bei 14 kompensierten und 8 dekompensierten Lebercirrhosen wurden Vergleichsbestimmungen der Renin-Aktivität (RA) im arteriellen und Lebervenenblut (Methode Kaneko et al.) durchgeführt. Die errechnete hepatische Renin-Extraktion (HE %) ist im Gegensatz zu Befunden anderer bei Cirrhosekranken nicht aufgehoben. Die Einzelwerte sind weit gestreut, die HE beträgt bei kompensierten Cirrhosen durchschnittlich 17,8±17,3%, bei dekompensierten Cirrhosen 30,5±5,8%. Ein signifikanter Unterschied der HE zu einer Vergleichsgruppe Hochdruckkranker (Mittelwert 28,5±20,4%,n=20) besteht nicht (p>0,05 bzw.>0,4).Aus den bei der Mehrzahl der Cirrhosekranken durchgeführten Bestimmungen leberhämodynamischer Größen geht hervor, daß die Renin-Extraktion der Tendenz nach mit dem Lebervenenverschlußdruck ansteigt (r=–0,393,p>0,05). Zum hepatischen Plasmafluß, bestimmt mit der Indocyaningrün-Methode, ergibt sich eine umgekehrte, statistisch allerdings nicht gesicherte Beziehung (r=–0,427,p>0,05). Es wird geschlossen, daß die hepatische Extraktion von Renin bei Lebercirrhosen vermutlich durch die intrahepatische Passagezeit beeinflußt wird.Unter Berücksichtigung der für die Vergleichsgruppe errechneten hepatischen Renin-Clearance (HRC) von 292±178 ml/min ist die HRC bei kompensierten Cirrhosen auf 68%, bei dekompensierten Cirrhosen auf 42,5% herabgesetzt. Im Rahmen der komplexen Regulationsstörung des Renin-Angiotensin-Systems bei fortgeschrittenen Lebercirrhosen dürften danach nicht allein Änderungen der renalen Sekretion, sondern außerdem eine Einschränkung der hepatischen Renin-Clearance für die Aufrechterhaltung einer erhöhten Plasma-Enzym-Aktivität von Bedeutung sein.     

A kinetic analysis of the in vitro sensitization of murine peritoneal mast cells with monoclonal IgE anti-DNP antibody.

Incubation of murine peritoneal cells with monoclonal IgE anti-DNP antibody in vitro led to sensitization of mast cells, measured as release of 5-HT upon challenge with DNP-HSA antigen. Sensitization was maximal at 0.3-3.0 micrograms/ml of IgE anti-DNP and declined above and below this concentration range. In kinetic studies, the time-course of sensitization was clearly divisible into an early slow phase of approximately 4 hr, followed by a more rapid linear phase from 4 to 48 hr. The early slow phase was more pronounced at lower concentrations of IgE anti-DNP (within the range 0.05-5.0 micrograms/ml). The degree of sensitization obtained after incubation of peritoneal cells with IgE anti-DNP for fixed periods (2, 4 and 8 hr) was markedly increased when the cells were washed and recultured in IgE-free medium, thus demonstrating that sensitization proceeds subsequent to an early stage of binding of IgE to receptors. Sensitization with IgE anti-DNP was blocked by addition of excess rat myeloma IgE, but only to a marked extent (greater than 50%) when the blocking immunoglobulin was added during the first 2 hr, thus providing further evidence that the major part of binding of the IgE antibody took place during this early stage, that is, prior to the phase of greatest sensitization. These findings indicate a period of delay between binding of IgE to receptors and functional sensitization, measured as mediator release in response to antigen.