Glutamate Metabolizing Enzymes in Prefrontal Cortex of Alzheimer’s Disease Patients

Amounts of glutamate metabolizing enzymes such as glutamate dehydrogenase (GDH), glutamine synthetase (GS), GS-like protein (GSLP), and phosphate-activated glutaminase (PAG) were compared in prefrontal cortex of control subjects and patients with Alzheimer disease (AD). The target proteins were quantified by ECL-Western immunoblotting in extracts from brain tissue prepared by two different techniques separating enzymes preferentially associated with cytoplasm (GDH I and II isoenzymes, GS, and partially GSLP) and membrane (GDH III, PAG, and partially GSLP) fractions. Amounts of all listed enzymes were found significantly increased in the patient group compared with controls. Some links between the measured values were observed in the control, but not in the AD patient group. The results may suggest for the pathological interruption of regulatory relations between distinct enzymes of glutamate metabolism in brain of AD patients.     

Distribution of spoligotyping defined genotypic lineages among drug-resistant Mycobacterium tuberculosis complex clinical isolates in Ankara, Turkey

Background

Investigation of genetic heterogeneity and spoligotype-defined lineages of drug-resistant Mycobacterium tuberculosis clinical isolates collected during a three-year period in two university hospitals and National Tuberculosis Reference and Research Laboratory in Ankara, Turkey.

Methods and Findings

A total of 95 drug-resistant M. tuberculosis isolates collected from three different centers were included in this study. Susceptibility testing of the isolates to four major antituberculous drugs was performed using proportion method on Löwenstein–Jensen medium and BACTEC 460-TB system. All clinical isolates were typed by using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) methods. Seventy-three of the 95 (76.8%) drug resistant M. tuberculosis isolates were isoniazid-resistant, 45 (47.4%) were rifampicin-resistant, 32 (33.7%) were streptomycin-resistant and 31 (32.6%) were ethambutol-resistant. The proportion of multidrug-resistant isolates (MDR) was 42.1%. By using spoligotyping, 35 distinct patterns were observed; 75 clinical isolates were grouped in 15 clusters (clustering rate of 79%) and 20 isolates displayed unique patterns. Five of these 20 unique patterns corresponded to orphan patterns in the SITVIT2 database, while 4 shared types containing 8 isolates were newly created. The most prevalent M. tuberculosis lineages were: Haarlem (23/95, 24.2%), ill-defined T superfamily (22/95, 23.2%), the Turkey family (19/95, 20%; previously designated as LAM7-TUR), Beijing (6/95, 6.3%), and Latin-America & Mediterranean (LAM, 5/95 or 5.3%), followed by Manu (3/95, 3.2%) and S (1/95, 1%) lineages. Four of the six Beijing family isolates (66.7%) were MDR. A combination of IS6110-RFLP and spoligotyping reduced the clustering rate from 79% to 11.5% among the drug resistant isolates.

Conclusions

The results obtained showed that ill-defined T, Haarlem, the Turkey family (previously designated as LAM7-TUR family with high phylogeographical specifity for Turkey), Beijing and LAM were predominant lineages observed in almost 80% of the drug-Resistant M. tuberculosis complex clinical isolates in Ankara, Turkey.     

Calreticulin Mutations in Myeloproliferative Neoplasms: Comparison of Three Diagnostic Methods

Calreticulin (CALR) mutations have recently been reported in 70–84% of JAK2V617F-negative myeloproliferative neoplasms (MPN), and this detection has become necessary to improve the diagnosis of MPN. In a large single-centre cohort of 298 patients suffering from Essential Thrombocythemia (ET), the JAK2V617F, CALR and MPL mutations were noted in 179 (60%), 56 (18.5%) and 13 (4.5%) respectively. For the detection of the CALR mutations, three methods were compared in parallel: high-resolution melting-curve analysis (HRM), product-sizing analysis and Sanger sequencing. The sensitivity for the HRM, product-sizing analysis and Sanger sequencing was 96.4%, 98.2% and 89.3% respectively, whereas the specificity was 96.3%, 100% and 100%. In our cohort, the product-sizing analysis was the most sensitive method and was the easiest to interpret, while the HRM was sometimes difficult to interpret. In contrast, when large series of samples were tested, HRM provided results more quickly than did the other methods, which required more time. Finally, the sequencing method, which is the reference method, had the lowest sensitivity but can be used to describe the type of mutation precisely. Altogether, our results suggest that in routine laboratory practice, product-sizing analysis is globally similar to HRM for the detection of CALR mutations, and that both may be used as first-line screening tests. If the results are positive, Sanger sequencing can be used to confirm the mutation and to determine its type. Product-sizing analysis provides sensitive and specific results, moreover, with the quantitative measurement of CALR, which might be useful to monitor specific treatments.     

Does Pattern Scan Laser (PASCAL) photocoagulation really induce less VEGF expression in murine retina than conventional laser treatment?

To investigate the differences in the mRNA and protein expression levels of vascular endothelial growth factor (VEGF) in murine retina between mice subjected to conventional laser (AG) and those subjected to Pattern Scan Laser (PASCAL) system. Male C57BL/6 mice were randomly assigned to one of three groups: Group 1 (G1) receiving retinal scatter laser photocoagulation using with AG photocoagulator (n = 16), Group 2 (G2) receiving retinal scatter laser photocoagulation using with PASCAL (n = 16) and Group 3 (G3) served as an untreated control group (n = 6). Molecular and morphological analyses of VEGF were performed on days 1, 2 and 5 by ELISA, real-time PCR and immuno-histochemical analysis. In samples which underwent AG (G1), when compared with the control group (G3), VEGF mRNA level increased 2.4 folds on day 2, whereas it decreased on day 5 (p □ 0.001). In samples which underwent PASCAL (G2), on the other hand, VEGF mRNA level increased 1.8 folds on day 1 and 2.2 folds on day 5 when compared with the control group (G3). In samples which underwent AG (G1), when compared with the control group (G3), VEGF protein level increased significantly on day 2, whereas it decreased on day 5 (p □ 0.001). In group G2, the VEGF levels in the sensory retina significantly increased as compared to control groups at both 2 and 5 days after laser photocoagulation using PASCAL laser (p = 0.012, both time points). The peak expressions of VEGF protein in samples which underwent PASCAL and conventional laser were found on day 5 and day 2 respectively. In retinas of PASCAL-treated mice, VEGF immunoreactivity gradually increased during the 5-day follow-up. However, in argon laser group, the strongest VEGF immunoreactivity was detected on day 2, then started to decrease on day 5. In summary, the expression of VEGF protein and mRNA gradually increase during a 5-day follow-up period in PASCAL-treated mouse eyes, whereas in AG group they reach their peak levels on the second day of follow-up and started decreasing after then. These results may also explain why the PASCAL system is less effective in regressing neovascularization in the clinic.     

alpha-bromoacetophenone derivatives as neutral protein tyrosine phosphatase inhibitors: structure-Activity relationship

A series of alpha-haloacetophenone derivatives was tested for inhibition of protein tyrosine phosphatases SHP-1 and PTP1B. The results show that the bromides are much more potent than the corresponding chlorides, whereas the phenyl ring is remarkably tolerant to modifications. Derivatization of the phenyl ring with a tripeptide Gly-Glu-Glu resulted in a potent, selective inhibitor against PTP1B.     

<Emphasis Type="Italic">FHIT</Emphasis> Gene Sequence Variants and Reduced Fhit Protein Expression in Glioblastoma Multiforme

Molecular studies have an important role in the elucidation of the mechanisms involved in Glioblastoma multiforme (GBM) development. The occurrence of FHIT gene alterations, which has an important role in different cancers, has not yet been studied well in GBM. We aimed to investigate the occurrence of alterations of FHIT gene sequence and protein expression in the GBMs. Sequence alterations in exons 5–9 of the FHIT gene were screened in 63 GBMs using the single-strand conformational polymorphism method, followed by DNA sequencing. Additionally, the level of Fhit protein expression in tissues of 48 tumors was assessed by immunohistochemistry (IHC). In our investigation, FHIT gene alterations in the coding region were detected in 11 of the 63 GBM cases (17.5%). Two different sequence variants were determined: one novel missense variant (G→C transition at codon 49) and one previously described silent alteration (C→T transition at codon 88). Using web-based programs, such as SIFT and ESEfinder, it was determined that both alterations might have caused significant modification on protein function. In addition, we identified a previously reported an intronic polymorphism (T→A transition at IVS8-17) in 47.5% of cases as a similar rate (45%) in the control group. Moreover, it was observed that Fhit protein expression was reduced in 87.5% of tumors. In conclusion, the reduction or loss of Fhit protein expression by genetic alterations or epigenetic mechanisms in GBM might be associated with brain tumorigenesis.     

Evidence of fast serotonin transmission in frog slowly adapting type 1 responses

The Merkel cell-neurite (MCN) complex generates slowly adapting type 1 (SA1) response when mechanically stimulated. Both serotonin (5-HT) and glutamate have been implicated in the generation of normal SA1 responses, but previous studies have been inconclusive as to what their roles are or how synaptic transmission occurs. In this study, excised dorsal skin patches from common water frogs (Rana ridibunda) were stimulated by von Frey hairs during perfusion in a tissue bath, and single-unit spike activity was recorded from SA1 fibres. Serotonin had no significant effect on the SA1 response at low (10?μM) concentration, significantly increased activity in a force-independent manner at 100?μM, but decreased activity with reduced responsiveness to force at 1?mM. Glutamate showed no effect on the responsiveness to force at 100?μM. MDL 72222 (100?μM), an ionotropic 5-HT3 receptor antagonist, completely abolished the responsiveness to force, suggesting that serotonin is released from Merkel cells as a result of mechanical stimulation, and activated 5-HT3 receptors on the neurite. The metabotropic 5-HT2 receptor antagonist, ketanserin, greatly reduced the SA1 fibre's responsiveness to force, as did the non-specific glutamate receptor antagonist, kynurenic acid. This supports a role for serotonin and glutamate as neuromodulators in the MCN complex, possibly by activation and/or inhibition of signalling cascades in the Merkel cell associated with vesicle release. Additionally, it was observed that SA1 responses contained a force-independent component, similar to a dynamic response observed during mechanical vibrations.