Vitamin A deficiency results in meiotic failure and accumulation of undifferentiated spermatogonia in prepubertal mouse testis

Vitamin A (retinol) is required for maintenance of adult mammalian spermatogenesis. In adult rodents, vitamin A withdrawal is followed by a loss of differentiated germ cells within the seminiferous epithelium and disrupted spermatogenesis that can be restored by vitamin A replacement. However, whether vitamin A plays a role in the differentiation and meiotic initiation of germ cells during the first round of mouse spermatogenesis is unknown. In the present study, we found that vitamin A depletion markedly decreased testicular expression of the all-trans retinoic acid-responsive gene, Stra8, and caused meiotic failure in prepubertal male mice lacking lecithin:retinol acyltransferase (Lrat), encoding for the major enzyme in liver responsible for the formation of retinyl esters. Rather than undergoing normal differentiation, germ cells accumulated in the testes of Lrat(-/-) mice maintained on a vitamin A-deficient diet. These results, together with our previous observations that germ cells fail to enter meiosis and remain undifferentiated in embryonic vitamin A-deficient ovaries, support the hypothesis that vitamin A regulates the initiation of meiosis I of both oogenesis and spermatogenesis in mammals.     

Arsenic content in the femur head of the residents of southern and central Poland

Arsenic content was assayed in the samples of the femur head of the people living in southern and central Poland (Kraków, n=13; Silesian region, n=13; Łódź, n=12). The average age being 68.7±8.7 yr. Arsenic content in the femur head was determined applying the hydride generation atomic absorption spectrometry (HG-AAS) method after microwave mineralization. The average arsenic contents in the femur head of the residents of the Łódź, Kraków, and Silesian regions were 0.41 μg/g, 0.37 μg/g, and 0.18 μg/g, respectively. No correlation has been found between arsenic content in the femur head and the content of other metals. Neither the age nor sex of the people tested affected the arsenic content in the femur head.     

Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters

BACKGROUND: The goal of this study was to design improved regulatable lentivirus vector systems. The aim was to design tetracycline (tet)-regulatable lentivirus vectors based on the Tet-on system displaying low background expression in the absence of the doxycycline (DOX) inducer and high transgene expression levels in the presence of DOX. METHODS: We constructed a binary lentivirus vector system that is composed of a self-inactivating (SIN) lentivirus vector bearing inducible first- or second-generation tet-responsive promoter elements (TREs) driving expression of a transgene and a second lentivirus vector encoding a reverse tetracycline-controlled transactivator (rtTA) that activates transgene expression from the TRE in the presence of DOX. RESULTS: We evaluated a number of different rtTAs and found rtTA2S-M2 to induce the highest levels of transgene expression. Regulated transgene expression was stable in human breast carcinoma cells implanted into nude mice for up to 11 weeks. In an attempt to minimize background expression levels, the chicken beta-globin cHS4 insulator element was cloned into the 3' long terminal repeat (LTR) of the transgene transfer vector. The cHS4 insulator element reduced background expression but expression levels following DOX addition were lower than those observed with vectors lacking an insulator sequence. In a second strategy, vectors bearing second-generation TREs harboring repositioned tetracycline operator elements were used. Such vectors displayed greatly reduced leakiness in the absence of DOX and induced transgene expression levels were up to 522-fold above those seen in the absence of DOX. CONCLUSIONS: Inducible lentivirus vectors bearing insulators or second-generation TREs will likely prove useful for applications demanding the lowest levels of background expression.     

Molecular discrimination of type-I over type-II methionyl aminopeptidases

Two residues that are conserved in type-I methionyl aminopeptidases (MetAPs) but are absent in all type-II MetAPs are the cysteine residues (Escherichia coli MetAP-I: C59 and C70) that reside at the back of the substrate recognition pocket. These Cys residues are 4.4 A apart and do not form a disulfide bond. Since bacteria and fungi contain only type-I MetAPs while all human cells contain both type-I and type-II MetAPs, type-I MetAPs represent a novel antibiotic/antifungal target if type-I MetAPs can be specifically targeted over type-II. Based on reaction of the thiol-specific binding reagent 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) with the type-I MetAP from E. coli and the type-II MetAP from Pyrococcus furiosus, the type-I MetAP can be selectively inhibited. Verification that DTNB covalently binds to C59 in EcMetAP-I was obtained by mass spectrometry (MS) from reaction of DTNB with the C59A and C70A mutant EcMetAP-I enzymes. In addition, two inhibitors of EcMetAP-I, 5-iodopentaphosphonic acid (1) and 6-phosphonohexanoic acid (2), were designed and synthesized. The first was designed as a selective-C59 binding reagent while the second was designed as a simple competitive inhibitor of EcMetAP. Indeed, inhibitor 1 forms a covalent interaction with C59 based on activity assays and MS measurements, while 2 does not. These data indicate that type-I MetAPs can be selectively targeted over type-II MetAPs, suggesting that type-I MetAPs represent a new enzymatic target for antibacterial or antifungal agents.     

A sensitive and specific HPLC method for the determination of total pentosidine concentration in plasma

Pentosidine is an advanced glycation end-product (AGE) appearing when arginine and lysine residues in proteins are cross-linked with carbonyl derivatives. This paper presents an improved method for the synthesis of pentosidine and reversed-phase chromatography of this substance with fluorometric detection that enables sensitive (0.01 pmol/mg protein) and specific determination of pentosidine in plasma. Separation is done twice on the same C(18) Vydac 218TP54 column, first with trifluoroacetic acid and next with heptafluorobutyric acid as ion pair. The inter-day coefficient of variation is 6.4% at pentosidine concentration in plasma of 25 pmol/mg protein and 8% at 1.7 pmol/mg protein. Spectral properties of pentosidine exploited during identification of the substance with UV absorption and fluorescence detectors are described. Maximum of absorbance was observed at 325 nm, maximum fluorescence at lambda(ex)/lambda(em)=330/373 nm. The method may prove useful for the study of processes associated with generation and accumulation of pentosidine in the body as a marker of AGE production in healthy subjects and patients with chronic renal failure.     

The non-synonymous SNP, R1150W, in SCN9A is not associated with chronic widespread pain susceptibility

Background

Chronic pain conditions are characterized by significant individual variability complicating the identification of pathophysiological markers. Leukocyte telomere length (TL), a measure of cellular aging, is associated with age-related disease onset, psychosocial stress, and health-related functional decline. Psychosocial stress has been associated with the onset of chronic pain and chronic pain is experienced as a physical and psychosocial stressor. However, the utility of TL as a biological marker reflecting the burden of chronic pain and psychosocial stress has not yet been explored.

Findings

The relationship between chronic pain, stress, and TL was analyzed in 36 ethnically diverse, older adults, half of whom reported no chronic pain and the other half had chronic knee osteoarthritis (OA) pain. Subjects completed a physical exam, radiographs, health history, and psychosocial questionnaires. Blood samples were collected and TL was measured by quantitative polymerase chain reaction (qPCR). Four groups were identified characterized by pain status and the Perceived Stress Scale scores: 1) no pain/low stress, 2) no pain/high stress, chronic pain/low stress, and 4) chronic pain/high stress. TL differed between the pain/stress groups (p = 0.01), controlling for relevant covariates. Specifically, the chronic pain/high stress group had significantly shorter TL compared to the no pain/low stress group. Age was negatively correlated with TL, particularly in the chronic pain/high stress group (p = 0.03).

Conclusions

Although preliminary in nature and based on a modest sample size, these findings indicate that cellular aging may be more pronounced in older adults experiencing high levels of perceived stress and chronic pain.     

Effects of ligand binding and oxidation on hinge-bending motions in S-adenosyl-L-homocysteine hydrolase

Domain motions of S-adenosyl-l-homocysteine (AdoHcy) hydrolase have been detected by time-resolved fluorescence anisotropy measurements. Time constants for reorientational motions in the native enzyme were compared with those for enzymes where key residues were altered by site-directed mutation. Mutations M351P, H353A, and P354A were selected in a hinge region for motion between the open and closed forms of the enzyme, as identified in a previous normal-mode study [Wang et al. (2005) Domain motions and the open-to-closed conformational transition of an enzyme: A normal-mode analysis of S-adenosyl-l-homocysteine hydrolase, Biochemistry 44, 7228-7239]. In wild-type, substrate-free AdoHcy hydrolase (NAD(+) cofactor in each subunit), reorientational motions were detected on time scales of 10-20 and 80-90 ns. The faster motion is attributed to the domain motion, and the slower motion is attributed to the tumbling of the enzyme. The domain motion was also detected for the enzyme complexes E(NADH/3'-keto-adenosine) and E(NAD(+)/3'-deoxyadenosine) but was absent for the complex E(NADH/3'-keto-neplanocin A). The results indicate that AdoHcy hydrolase exists in equilibrium of open and closed structures, with the equilibrium shifted toward the more mobile open form for the substrate-free enzyme, E(NAD(+)), and for intermediates formed early in the catalytic cycle after substrate binding or formed late prior to product release, E(NAD(+)/ligand). However, the strong inhibitor neplanocin A upon binding undergoes oxidation, forming the complex E(NADH/3'-keto-neplanocin). For this complex, which is analogous to the enzyme complex with the central catalytic intermediate, the equilibrium was shifted toward the more rigid closed form. A similar pattern was observed for M351P and P354A mutants. In contrast, the domain motion could not be detected, either in the absence or presence of ligands or with the cofactor in either the oxidized or reduced state, for the H353A protein, suggesting that this mutation changes the hinge-bending dynamics of the enzyme.     

The antiviral drug ribavirin is a selective inhibitor of S-adenosyl-L-homocysteine hydrolase from Trypanosoma cruzi

Ribavirin (1,2,4-triazole-3-carboxamide riboside) is a well-known antiviral drug. Ribavirin has also been reported to inhibit human S-adenosyl-L-homocysteine hydrolase (Hs-SAHH), which catalyzes the conversion of S-adenosyl-L-homocysteine to adenosine and homocysteine. We now report that ribavirin, which is structurally similar to adenosine, produces time-dependent inactivation of Hs-SAHH and Trypanosoma cruzi SAHH (Tc-SAHH). Ribavirin binds to the adenosine-binding site of the two SAHHs and reduces the NAD(+) cofactor to NADH. The reversible binding step of ribavirin to Hs-SAHH and Tc-SAHH has similar K(I) values (266 and 194 microM), but the slow inactivation step is 5-fold faster with Tc-SAHH. Ribavirin may provide a structural lead for design of more selective inhibitors of Tc-SAHH as potential anti-parasitic drugs.     

Characterization of chicken cystatin binding to rat renal brush-border membranes

Chicken cystatin, a homologue of human cystatin C, like other low-molecular-weight proteins is metabolized by renal proximal tubule cells. However, the precise mechanism(s) of this process has not been elucidated yet. To characterize chicken cystatin binding to renal brush-border membranes, the incubation of fluorescein labelled protein with rat cortical homogenate was performed. Saturation-dependent and reversible binding with low affinity (K_d = 3.67–4.07 μM) and high capacity (B_max = 2.32–2.79 nmol/mg) was observed. Bovine albumin was the most potent competitor (K_i = 0.7 μM) among other megalin/cubilin ligands tested. The presence of Ca^+ 2 ions was necessary to effective cystatin binding by brush-border membranes. Obtained data strongly support the hypothesis that chicken cystatin is a novel ligand for megalin/cubilin receptors tandem on proximal tubular cells.