Molecular dissection of 2B4 signaling: implications for signal transduction by SLAM-related receptors

2B4 is a SLAM-related receptor expressed on natural killer (NK) cells and cytotoxic T cells. It can regulate killing and gamma interferon secretion by NK cells, as well as T-cell-mediated cytotoxicity. There are conflicting data regarding the mechanism of action of 2B4. In these studies, we attempted to understand better the nature and basis of 2B4 signaling. Our studies showed that engagement of 2B4 on NK cells triggered a tyrosine phosphorylation signal implicating 2B4, Vav-1, and, to a lesser extent, SHIP-1 and c-Cbl. Structure-function analyses demonstrated that this response was defined by a series of tyrosine-based motifs in the cytoplasmic region of 2B4 and was not influenced by the extracellular or transmembrane segment of 2B4. In addition, the 2B4-induced signal was absolutely dependent on coexpression of SAP, a Src homology 2 (SH2) domain-containing adaptor associating with SLAM-related receptors and mutated in X-linked lymphoproliferative disease. It was also observed that 2B4 was detectably associated with the Src-related protein tyrosine kinase FynT in an immortalized NK cell line. Mutation of arginine 78 of SAP, a residue critical for binding of SAP to FynT, eliminated 2B4-mediated protein tyrosine phosphorylation, implying that SAP promotes 2B4 signaling most probably by recruiting FynT. Finally, despite the similarities in the signaling modalities of 2B4 and its relative SLAM, the natures of the tyrosine phosphorylation signals induced by these two receptors were found to be different. These differences were not caused by variations in the extent of binding to SAP but rather were dictated by the tyrosine-based sequences in the cytoplasmic domain of the receptors. Taken together, these data lead to a better understanding of 2B4 signaling. Furthermore, they provide firm evidence that the signals transduced by the various SLAM-related receptors are unique and that the specificity of these signals is defined by the distinctive arrays of intracytoplasmic tyrosines in the receptors.     

Efficacy of two rabeprazole/gatifloxacin-based triple therapies for Helicobacter pylori infection

OBJECTIVES: To evaluate the efficacy of two novel treatment regimens consisting of gatifloxacin (400 mg daily), amoxicillin (1 g twice daily), and rabeprazole 20 mg once (RAG20) or twice daily (RAG40) given for 7 days in the eradication of Helicobacter pylori. METHODS: Eligible patients undergoing endoscopy and having a positive rapid urease assay for H. pylori were enrolled in this open-label trial. Gastric biopsies from a random cohort of patients were cultured for H. pylori and in vitro susceptibility to gatifloxacin and amoxicillin was performed using the E-test. Compliance and side-effects were evaluated by phone calls. (14)C-urea breath tests were performed a minimum of 4 weeks after therapy and 3 weeks after any acid suppressive therapy. RESULTS: A total of 104 patients, 52 in each group (40 females and 64 males; mean age 45.7 years) were enrolled sequentially. Eradication occurred in 43 out of 52 patients in RAG20 group (both per-protocol and intention-to-treat analysis: 83%; 95% CI: 72-93%) and in 48 of 52 patients in the RAG40 group (both per-protocol and intention-to-treat analysis: 92%; 95% CI: 85-99%). Seven patients in the RAG40 group who had previously failed one or more treatment regimens for H. pylori were cured. No significant adverse effects were reported. All 50 recovered H. pylori strains were susceptible to amoxicillin and gatifloxacin in vitro. CONCLUSIONS: A 7-day regimen of gatifloxacin-rabeprazole-amoxicillin is effective eradication therapy for H. pylori. The use of rabeprazole twice daily results in superior eradication rates including cases of failed primary therapy. This new regimen is simple, well-tolerated, and may lead to higher compliance and lower costs.     

Fast determination of propranolol in urine and pharmaceutical preparations by stopped-flow and micellar-stabilized room-temperature phosphorescence: validation of the method

The stopped-flow mixing technique has been used to study the kinetic determination of propranolol by means of micellar-stabilized room-temperature phosphorescence. This mixing system diminishes the time required for the deoxygenation of micellar medium by sodium sulfite, allowing a kinetic curve that levels off within only 7s to be obtained. The phosphorescence enhancers thallium (I) nitrate, sodium dodecyl sulfate, and sodium sulfite were optimized to obtain maximum sensitivity and selectivity. A pH value of 6.54 was selected as adequate for phosphorescence development. The kinetic curves of propranolol phosphorescence were scanned at lambda(ex)=290 nm and lambda(em)=524 nm. The calibration graphs were linear for the concentration range from 25 to 400 ng mL(-1). The phosphorescence lifetime of propranolol is approximately 1210 micros. The detection limit calculated as proposed clayton was 13.53 ng mL(-1) and by applying the error propagation theory, the detection limit was 8.37 ng mL(-1). The repeatability was studied using 10 solutions of 200 ng mL(-1) of propranolol; if error propagation theory is assumed, the relative error is 1.94%. The standard deviation for a replicate sample was 4.0 ng mL(-1). This method was successfully applied to the determination of propranolol in commercial formulations and in urine. Suitable recovery values were obtained.     

Acetoclastic and hydrogenotrophic methane production and methanogenic populations in an acidic West-Siberian peat bog

Sites in the West Siberian peat bog 'Bakchar' were acidic (pH 4.2-4.8), low in nutrients, and emitted CH4 at rates of 0.2-1.5 mmol m(-2) h(-1). The vertical profile of delta13CH4 and delta13CO2 dissolved in the porewater indicated increasing isotope fractionation and thus increasing contribution of H2/CO2-dependent methanogenesis with depth. The anaerobic microbial community at 30-50 cm below the water table produced CH4 with optimum activity at 20-25 degrees C and pH 5.0-5.5 respectively. Inhibition of methanogenesis with 2-bromo-ethane sulphonate showed that acetate, phenyl acetate, phenyl propionate and caproate were important intermediates in the degradation pathway of organic matter to CH4. Further degradation of these intermediates indicated that 62-72% of the CH4 was ultimately derived from acetate, the remainder from H2/CO2. Turnover times of [2-14C]acetate were on the order of 2 days (15, 25 degrees C) and accounted for 60-65% of total CH4 production. Conversion of 14CO2 to 14CH4 accounted for 35-43% of total CH4 production. These results showed that acetoclastic and hydrogenotrophic methanogenesis operated closely at a ratio of approximately 2 : 1 irrespective of the incubation temperature (4, 15 and 25 degrees C). The composition of the archaeal community was determined in the peat samples by terminal restriction fragment length polymorphism (T-RFLP) analysis and sequencing of amplified SSU rRNA gene fragments, and showed that members of Methanomicrobiaceae, Methanosarcinaceae and Rice cluster II (RC-II) were present. Other, presumably non-methanogenic archaeal clusters (group III, RC-IV, RC-V, RC-VI) were also detected. Fluorescent in situ hybridization (FISH) showed that the number of Bacteria decreased (from 24 x 10(7) to 4 x 10(7) cells per gram peat) with depth (from 5 to 55 cm below the water table), whereas the numbers of Archaea slightly increased (from 1 x 10(7) to 2 x 10(7) cells per gram peat). Methanosarcina spp. accounted for about half of the archaeal cells. Our results show that both hydrogenotrophic and acetoclastic methanogenesis are an integral part of the CH4-producing pathway in acidic peat and were represented by appropriate methanogenic populations.     

A model of meningococcal bacteremia after respiratory superinfection in influenza A virus-infected mice

We developed a model of sequential influenza A virus (IAV)-Neisseria meningitidis serogroup C (Nm) infection in BALB/c mice. Mice infected intranasally with a sublethal IAV dose (260 pfu) were superinfected intranasally with Nm. Fatal meningococcal pneumonia and bacteremia were observed in IAV-infected mice superinfected with Nm on day 7, but not in those superinfected on day 10. The susceptibility of mice to Nm superinfection was correlated with the peak interferon-gamma production in the lungs and decrease in IAV load. After Nm challenge, both IAV-infected and uninfected control mice produced the inflammatory cytokines interleukin (IL)-1 and IL-6. However, IL-10 was detected in susceptible mice superinfected on day 7 after IAV infection, but not in resistant mice. This model of dual IAV-Nm infection was also used to evaluate the role of bacterial virulence factors in the synthesis of the capsule. A capsule-defective mutant was cleared from the lungs, whereas a mutant inactivated for the crgA gene, negatively regulating expression of the pili and capsule, upon contact with host cells, retained invasiveness. Therefore, this model of meningococcal disease in adult mice reproduces the pathogenesis of human meningococcemia with fatal sepsis, and is useful for analyzing known or new genes identified in genomic studies.     

Signaling lymphocytic activation molecule-associated protein controls NKT cell functions

X-linked lymphoproliferative disease (XLP) is a fatal immunological disorder that typically manifests following EBV infection. XLP patients exhibit a number of immune defects including abnormal T, B, and NK lymphocyte function. These defects have been attributed to mutations of Src homology 2 domain-containing gene 1A (SH2D1A), the gene encoding signaling lymphocytic activation molecule-associated protein (SAP), an intracellular adaptor molecule expressed in lymphocytes. We have observed that SAP knockout (SAPKO) mice and humans with XLP have a complete lack of CD1d-restricted NKT cells. As expected, SAPKO mice injected with the NKT cell agonist, alpha-galactosylceramide failed to generate NKT cell IFN-gamma or IL-4. Furthermore, in contrast to wild-type littermates, SAPKO mice coinjected with OVA and alpha-galactosylceramide failed to mount OVA-specific CTL responses. These data suggest that an absence of NKT cells may underlie part of the immune dysregulation seen in SAPKO mice and in XLP patients.     

Response of wheat cultivars to the presence of sodium salts at germination and in early development

The effect of various sodium salts at the early developmental stages of wheat cultivars was studied. Germination was not affected by the presence of salts but cv. H-68 was more tolerant than Mexipak during later growth. The roots were more sensitive than the shoots. The salts under study could be divided into two groups. Bicarbonate, carbonate, acetate and citrate were more toxic for growth than sulphate, nitrate, phosphate and chloride of sodium. The individual members of each group did not vary much from one another.     

Two new haemoglobins: haemoglobin Perspolis (alpha 64 (E13) Asp leads to Tyr) and haemoglobin J-Kurosh (alpha 19 (AB) Ala leads to Asp).

Two new haemoglobins are described which were found during a regular survey on voluntary blood donors in Iran. They are haemoglobin Perspolis [alpha 64 (E13) Asp leads to Tyr] and haemoglobin J-Kurosh [alpha 19 (AB) Ala leads to Asp]. The amino acid substitution in these two variants was determined by fingerprinting and amino acid analysis of the tryptic peptides and thermolytic peptides derived from abnormal tryptic peptides. Neither haemoglobin was associated with clinical symptoms.     

Prostate‐specific membrane antigen‐targeted photoacoustic imaging of prostate cancer in vivo

A sensitive, noninvasive method to detect localized prostate cancer, particularly for early detection and repetitive study in patients undergoing active surveillance, remains an unmet need. Here, we propose a molecular photoacoustic (PA) imaging approach by targeting the prostate‐specific membrane antigen (PSMA), which is over‐expressed in the vast majority of prostate cancers. We performed spectroscopic PA imaging in an experimental model of prostate cancer, namely, in immunocompromised mice bearing PSMA+ (PC3 PIP) and PSMA? (PC3 flu) tumors through administration of the known PSMA‐targeted fluorescence agent, YC‐27. Differences in contrast between PSMA+ and isogenic control tumors were observed upon PA imaging, with PSMA+ tumors showing higher contrast in average of 66.07‐fold with 5 mice at the 24‐hour postinjection time points. These results were corroborated using standard near‐infrared fluorescence imaging with YC‐27, and the squared correlation between PA and fluorescence intensities was 0.89. Spectroscopic PA imaging is a new molecular imaging modality with sufficient sensitivity for targeting PSMA in vivo, demonstrating the potential applications for other saturable targets relevant to cancer and other disorders.