Isolation and Characterization of a Rat Brain Triakontatetraneuropeptide, a Posttranslational Product of Diazepam Binding Inhibitor: Specific Action at the Ro 5-4864 Recognition Site

This report describes the purification and characterization from rat brain of triakontatetraneuropeptide (TTN, DBI 17-50), a major biologically active processing product of diazepam binding inhibitor (DBI). Brain TTN was purified by immunoaffinity chromatography with polyclonal octadecaneuropeptide, DBI 33-50) antibodies coupled to CNBr-Sepharose 4B followed by two reverse-phase HPLC steps. The amino acid sequence of the purified peptide is: Thr-Gln-Pro-Thr-Asp-Glu-Glu-Met-Leu-Phe-Ile-Tyr-Ser-His-Phe-Lys-Gln-Ala-Thr-Val - Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys. Synthetic TTN injected intracerebroventricularly into rats induces a proconflict activity (IC50 0.8 nmol/rat) that is prevented by the specific "peripheral" benzodiazepine (BZ) receptor antagonist isoquinoline carboxamide, PK 11195, but not by the "central" BZ receptor antagonist imidazobenzodiazepine, flumazenil. TTN displaces [3H]Ro 5-4864 from synaptic membranes of olfactory bulb with a Ki of approximately 5 microM. TTN also enhances picrotoxinin inhibition of gamma-aminobutyric acid (GABA)-stimulated [3H]flunitrazepam binding. These data suggest that TTN, a natural DBI processing product acting at "Ro 5-4864 preferring" BZ binding site subtypes, might function as a putative neuromodulator of specific GABAA receptor-mediated effects.     

Photochemical and photophosphorylation activities of chloroplasts and leaf mesostructure in Chinese cabbage plants grown under illumination with light-emitting diodes

Leaf mesostructure, photochemical activity, and chloroplast photophosphorylation (PP) in the fourth true leaf of 28-day-old Chinese cabbage (Brassica chinensis L.) plants were investigated. Plants were grown under a light source based on red (650 nm) and blue (470 nm) light-emitting diodes (LED) with red/blue photon flux ratio of 7: 1 and under illumination with high-pressure sodium lamp (HPSL) at photon flux densities of 391 ± 24 μmol/(m² s) (“normal irradiance”) and 107 ± 9 μmol/(m² s) (“low irradiance”) in photosynthetically active range. At normal irradiance, the leaf area in plants grown under HPSL was twofold higher than in LED-illuminated plants; other parameters of leaf mesostructure were little affected by spectral quality of incident light. The lowering of growth irradiance reduced the majority of leaf mesostructure parameters in plants grown under illumination with HPSL, whereas in LED-illuminated plants the lowered irradiance reduced only specific leaf weight but increased the leaf thickness and dimensions of mesophyll cells and chloroplasts. The photochemical activity of isolated chloroplasts was almost independent of growth irradiance and light spectral quality. Light quality and intensity used for plant growing had a considerable impact on PP in chloroplasts. At normal light intensity, the highest activity of noncyclic PP in chloroplasts was observed for plants grown under HPSL; at low light intensity the highest rates of PP were noted for plants grown under LED. The P/2e⁻ ratio, which characterizes the degree of PP coupling to electron transport in the chloroplast electron transport chain, showed a similar pattern. Thus, the narrow-band spectrum of the light source had little influence on leaf mesostructure and electron transport rates. However, this spectrum significantly affected the chloroplast PP activity. The PP patterns at low and normal light intensities were opposite for plants grown under LED and HPSL light sources. We suppose that growing plants under LED array at normal light intensity disturbed the chloroplast coupling system, thus preventing the effective use of light energy for ATP synthesis. At low light intensity, chloroplast PP activity was significantly higher under LED illumination, but plant growth was suppressed because of impaired adaptation to low light intensity.     

Roles of ATM and NBS1 in chromatin structure modulation and DNA double-strand break repair

We developed a novel system to create DNA double-strand breaks (DSBs) at defined endogenous sites in the human genome, and used this system to detect protein recruitment and loss at and around these breaks by chromatin immunoprecipitation (ChIP). The detection of human ATM protein at site-specific DSBs required functional NBS1 protein, ATM kinase activity and ATM autophosphorylation on Ser 1981. DSB formation led to the localized disruption of nucleosomes, a process that depended on both functional NBS1 and ATM. These two proteins were also required for efficient recruitment of the repair cofactor XRCC4 to DSBs, and for efficient DSB repair. These results demonstrate the functional importance of ATM kinase activity and phosphorylation in the response to DSBs, and support a model in which ordered chromatin structure changes that occur after DNA breakage depend on functional NBS1 and ATM, and facilitate DNA DSB repair.     

Effect of the spaceflight radiation factor on the growth of Brassica pekinensis and micromycetes Fusarium oxysporum

The effects of chronic exposure in the combination of low-dose gamma- and neutron radiation on growth of Brassica pekinensis (lour) Rupr, var. Khibinskaya, and micromycetes of Fusarium oxysporum, a representative of soil microflora were stadied. Seeds, water and vegetating plants raised from previously irradiated seeds were subjected to radiation exposure. The gamma-neutron dose rate was -0.013 sGy/d and the neutron flux was -20 n/cm2 d. Growth of Brassica pekinensis and of Fusarium oxysporum on the background of weak chronic ionizing irradiation by radioactive nuclides was noticeably changed when compared with the growth in the normal radiation environment. The radiosensitivity of Fusarium oxysporum isolated from the artificial soil following 60-d exposure was far higher than the radioactivity of the culture isolated from non-irradiated soil. Fusarium oxysporum isolated from the irradiated soil failed to form spores normally even when cultivated in a non-irradiated nutrient substrate without further exposure. These peculiarities persisted at least for four months. 15-mo gamma- and neutron exposure of Brassica pekinensis seeds held in storage for 5 years retained their original germinating capacity, whereas the non-irradiated seeds suffered deprivation of this quality. It was noticed that the Brassica pekinensis reaction on chronic exposure of low doses was markedly dependent on the growth conditions non-related directly with the radiation background.     

I/D polymorphism in the angiotensin-converting enzyme gene in men with myocardial infarction at young age]

The rate of D allele did not differ between patients with ischemic heart disease (IHD) who had myocardial infarction before the age 45, and healthy males. The DD genotype of the ACE gene was much more frequently encountered in the patients than in healthy males. The findings suggest that the DD genotype is an independent risk factor of the IHD and myocardial infarction in young patients.     

Genetic variants of platelet ADP receptor P2Y12 associated with changed platelet functional activity and development of cardiovascular diseases

The key role in platelet aggregation is played by the platelet ADP receptor P2Y12, which is the target for antiaggregant drugs, clopidogrel and ticlopidine. At present, only sporadic data on genetic variants of platelet ADP receptor P2Y12 are available from literature, and their association with thromboembolic and cardiovascular diseases still remains obscure. Analysis of the group of subjects with high platelet reactivity resulted in identification of two nucleotide substitutions, C18T and G36T, in the coding region of the P2Y12 gene. The frequency of the P2Y12 T18 allele was higher in control group than in the group of patients survived from myocardial infarction at the age under 45 years (39% versus 28%, respectively, P = 0.04). Moreover, in the T18 carriers, platelet aggregation activity was lower than in the carriers of the wild-type genotype (0.84 ± 0.05%/s versus 1.01 ± 0.08%/s, respectively, P = 0.03). In the group of patients with early myocardial infarctions, a tendency towards the increased frequency of T36 allele in comparison with control group (20 and 12%, respectively, P = 0.07) was observed. The rate of ADP-induced platelet aggregation in the carriers of T36 allele from the control group was somewhat higher than in the subjects with the GG36 genotype (1.31 ± 0.16%/s versus 1.12 ± 0.06%/s, respectively, P = 0.07). The nucleotide substitutions identified were in lincage disequilibrium, i.e., allele T18 conformed to allele G36. On the contrary, allele C18 conformed to allele T36. Haplotype T18G36 was found to be responsible for the decreased risk of myocardial infarction and decreased platelet reactivity. It is suggested that polymorphisms of the P2Y12 gene identified can be used for determination of the risk group for myocardial infarction in the young males.     

Comparable Long-Term Efficacy of Lopinavir/Ritonavir and Similar Drug-Resistance Profiles in Different HIV-1 Subtypes

Background

Analysis of potentially different impact of Lopinavir/Ritonavir (LPV/r) on non-B subtypes is confounded by dissimilarities in the conditions existing in different countries. We retrospectively compared its impact on populations infected with subtypes B and C in Israel, where patients infected with different subtypes receive the same treatment.

Methods

Clinical and demographic data were reported by physicians. Resistance was tested after treatment failure. Statistical analyses were conducted using SPSS.

Results

607 LPV/r treated patients (365 male) were included. 139 had HIV subtype B, 391 C, and 77 other subtypes. At study end 429 (71%) were receiving LPV/r. No significant differences in PI treatment history and in median viral-load (VL) at treatment initiation and termination existed between subtypes. MSM discontinued LPV/r more often than others even when the virologic outcome was good (p = 0.001). VL was below detection level in 81% of patients for whom LPV/r was first PI and in 67% when it was second (P = 0.001). Median VL decrease from baseline was 1.9±0.1 logs and was not significantly associated with subtype. Median CD4 increase was: 162 and 92cells/µl, respectively, for patients receiving LPV/r as first and second PI (P = 0.001), and 175 and 98, respectively, for subtypes B and C (P<0.001). Only 52 (22%) of 237 patients genotyped while under LPV/r were fully resistant to the drug; 12(5%) were partially resistant. In48%, population sequencing did not reveal resistance to any drug notwithstanding the virologic failure. No difference was found in the rates of resistance development between B and C (p = 0.16).

Conclusions

Treatment with LPV/r appeared efficient and tolerable in both subtypes, B and C, but CD4 recovery was significantly better in virologically suppressed subtype-B patients. In both subtypes, LPV/r was more beneficial when given as first PI. Mostly, reasons other than resistance development caused discontinuation of treatment.