Alpha‐Lipoic Acid Supplementation Reduces mTORC1 Signaling in Skeletal Muscle from High Fat Fed,Obese Zucker Rats

The mammalian target of rapamycin (mTOR) signaling pathway is hyperactive in liver, adipose and skeletal muscle tissues of obese rodents. Alpha‐lipoic acid (αLA) has been well accepted as a weight‐loss treatment, though there are limited studies on its effect on mTOR signaling in high‐fat fed, obese rodents. Therefore, the goal of this study was to determine mTOR signaling and oxidative protein alterations in skeletal muscle of high‐fat fed, obese rats after αLA supplementation. Phosphorylation of the mTOR substrate, eukaryotic initiation factor (eIF) 4E‐binding protein 1 (4E‐BP1) and eIF4B were significantly reduced (p < 0.05) in muscle from αLA supplemented rats. Activation of AMP‐activated protein kinase (AMPK), an mTOR inhibitory kinase, was higher (p < 0.05) in the αLA group. Protein expression of markers of oxidative metabolism, acetyl CoA carboxylase (ACC), cytochrome c oxidase IV (COX IV), peroxisome proliferator‐activated receptor (PPAR), and PPAR gamma coactivator 1‐alpha (PGC‐1α) were significantly higher (p < 0.05) after αLA supplementation compared to non‐supplemented group. Our findings show that αLA supplementation limits the negative ramifications of consuming a high fat diet on skeletal muscle markers of oxidative metabolism and mTORC1 signaling.     

Substrate Flexibility of the Flavin-Dependent Dihydropyrrole Oxidases PigB and HapB Involved in Antibiotic Prodigiosin Biosynthesis

In the biosynthesis of the tripyrrolic pigment prodigiosin, PigB is a predicted flavin-dependent oxidase responsible for the formation of 2-methyl-3-amylpyrrole (MAP) from a dihydropyrrole. To prove which dihydropyrrole is the true intermediate, both possibilities, 5-methyl-4-pentyl-3,4-dihydro-2H-pyrrole ( 5 a , resulting from transamination of the aldehyde of 3-acetyloctanal) and 2-methyl-3-pentyl-3,4-dihydro-2H-pyrrole ( 6 , resulting from transamination of the ketone), were synthesised. Only 5 a restored pigment production in a strain of Serratia sp. ATCC 39006 blocked earlier in MAP biosynthesis. PigB is membrane-associated and inactive when its transmembrane domain was deleted, but HapB, its homologue in Hahella chejuensis, lacks the transmembrane domain and is active in solution. Two colourimetric assays for PigB and HapB were developed, and the HapB-catalysed reaction was kinetically characterised. Ten analogues of 5 a were synthesised, varying in the C2 and C3 side chains, and tested as substrates of HapB in vitro and for restoration of pigment production in Serratia ΔpigD in vivo. All lengths of side chain tested at C3 were accepted, but only short side chains at C2 were accepted. The knowledge that 5 a is an intermediate in prodigiosin biosynthesis and the ease of synthesis of analogues of 5 a makes a range of prodigiosin analogues readily available by mutasynthesis.     

Sample suitability for the detection of minor white cell populations (microchimerism) by polymerase chain reaction

BACKGROUND: There is increasing use of highly sensitive testing with polymerase chain reaction (PCR) to study white cell microchimerism after transfusion and transplantation. This study investigated possible artifactual sources of allogeneic sample contamination before PCR testing. STUDY DESIGN AND METHODS: Quantitative Y-chromosome PCR was used to study microchimerism among transfused patients with sickle cell disease (SCD) and thalassemia by using residual specimens from the clinical laboratory. High levels of circulating male white cells among transfused patients with SCD but not thalassemia led to concern over the artifactual origin of male cells. To investigate, paired specimens were collected from 26 female SCD patients: one specimen underwent processing only for PCR, while the other underwent testing in the clinical laboratory before PCR as a process control. All laboratory instruments were also assessed for their ability to impart male allogeneic cells to aliquots of female blood. RESULTS: Thirty-three (31%) of 107 SCD samples, but 0 of 20 thalassemia samples, gave a high-level PCR signal. One of 26 paired samples that was not exposed to clinical laboratory equipment had low-level PCR positivity while 10 of the 26 became strongly positive after testing on a blood cell analyzer and a reticulocyte analyzer. Sixteen of 32 female samples became positive after reticulocyte analysis, while none became positive after blood cell analysis. Samples from thalassemia patients tested PCR-negative because reticulocyte counts had not been performed. CONCLUSION: Allogeneic cell contamination is common with clinical laboratory equipment. These samples may not be suitable for microchimerism studies. In addition to method controls, process controls should be employed where appropriate.     

The 70 kDa subunit of replication protein A is required for the G1/S and intra-S DNA damage checkpoints in budding yeast

The rfa1-M2 and rfa1-M4 Saccharomyces cerevisiae mutants, which are altered in the 70 kDa subunit of replication protein A (RPA) and sensitive to UV and methyl methane sulfonate (MMS), have been analyzed for possible checkpoint defects. The G1/S and intra-S DNA damage checkpoints are defective in the rfa1-M2 mutant, since rfa1-M2 cells fail to properly delay cell cycle progression in response to UV irradiation in G1 and MMS treatment during S phase. Conversely, the G2/M DNA damage checkpoint and the S/M checkpoint are proficient in rfa1-M2 cells and all the checkpoints tested are functional in the rfa1-M4 mutant. Preventing S phase entry by alpha-factor treatment after UV irradiation in G1 does not change rfa1-M4 cell lethality, while it allows partial recovery of rfa1-M2 cell viability. Therefore, the hypersensitivity to UV and MMS treatments observed in the rfa1-M4 mutant might only be due to impairment of RPA function in DNA repair, while the rfa1-M2 mutation seems to affect both the DNA repair and checkpoint functions of Rpa70.     

Growth inhibition of human colon carcinoma cells by combinations of anti-epidermal growth factor-related growth factor antisense oligonucleotides

To gain insight into the factors controlling the maintenance or loss of T cell self tolerance we produced beef insulin (BI)-transgenic BALB/c mice. Transgenic mice express BI under control of the human insulin promoter and secrete physiological amounts of beef insulin. Although these mice are tolerant to BI, as evidenced by the lack of insulin-specific IgG antibody production following intraperitoneal immunization, tolerance is not complete. Footpad immunization results in a weak antigen-specific T cell proliferative response, indicating the presence of self-reactive BI-specific T cell in the periphery. These T cells are functional in vivo, providing support for IgG1, IgG2a, and IgG2b BI-specific antibody production, but require higher higher concentrations of antigen than nontransgenic T cells (both in vivo and following recall responses in vitro) to become activated. In vitro, BI-specific T cell proliferation in BI-transgenic mice can be largely restored by addition of interleukin-2, indicating that a significant component of T cell tolerance is mediated by anergy. To characterize the autoreactive T cells that become activated when tolerance is broken, BI-specific T cell hybridomas were generated from transgenic mice and compared to a panel of hybridomas previously derived from nontransgenic BALB/c mice. The majority of BI-transgenic hybridomas recognized the immunodominant A1-14 beef insulin peptide but with lower avidity than BALB/c hybridomas. Consistent with this, none of the dominant T cell receptor rearrangements found in the BALB/c BI-specific T cell receptor repertoire were found in the transgenic hybridomas. These results indicate that, despite evidence for clonal inactivation of many BI-specific T cells in BI-transgenic mice, loss of tolerance results from activation of low-affinity antigen-specific T cells that appear to have escaped this process.     

L-733,060, a novel tachykinin NK1 receptor antagonist; effects in [Ca2+]i mobilisation, cardiovascular and dural extravasation assays

Short RNA species that encompass the psi domain of the retroviral genome spontaneously form dimers in vitro, and the retroviral nucleocapsid protein activates this dimerization in vitro. Addition of gag RNA sequences downstream of the 3' end of the psi domain decreases the level of spontaneous dimerization. Here, we report the effects of RNA length on dimerization in vitro, studied with RNA fragments from Moloney murine leukaemia virus that contain the psi domain and all or part of the gag sequence. Extension of the RNA leads to progressive inhibition of the in vitro dimerization process. Sequences located downstream of the 3' end of the psi domain seem to stabilize the monomeric structures. This stabilization participates in dimerization of the RNA sequences involved in the recognition of two RNA molecules. We studied the ability of nucleocapsid protein 10 to promote dimerization of such long RNA fragments, and found that the protein greatly enhances their dimerization in vitro. We propose that nucleocapsid protein 10 stimulates the overall dimerization process by reduction of the energy barrier that must be overcome to allow dimer formation. Our results show that dimerization of RNA form Moloney murine leukaemia virus in vitro is enhanced by nucleocapsid protein 10. This finding is in agreement with the involvement of the nucleocapsid protein in RNA dimerization in vivo.     

Partial characterization of an immortalized human trophoblast cell-line, TCL-1, which possesses a CSF-1 autocrine loop

High doses of morphine produce a state of behavioural inactivity and muscular rigidity. This type of 'catalepsy' is clearly different from the state which is produced by the administration of neuroleptics, e.g. haloperidol. While haloperidol-induced catalepsy can easily be antagonised by NMDA receptor antagonists, there has been a report that the non-competitive NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine (MK-801) potentiates morphine-induced catalepsy. The aim of this study was to further examine the role of glutamate receptors in the mediation of morphine-induced catalepsy. To this end we coadministered morphine (20, 40, 60 mg/kg i.p.) with MK-801 (0.1 and 0.3 mg/kg i.p.), the competitive NMDA receptor antagonist DL-(E)-2-amino-4-methyl-5-phosphono-3-pentoic acid (CGP 37849) (2 and 6 mg/kg i.p.), or 1-(4-aminophenyl)-4-methyl-7,8-methylen-dioxy-5H-2,3- benzodiazepine (GYKI 52466) (2 and 4 mg/kg), an antagonist of the AMPA type of glutamate receptors, respectively. The degree of catalepsy was assessed using two different methods, the 'bar/podium/grid' test which is commonly used to measure neuroleptic-induced catalepsy, and a test for the presence or absence of righting reflexes after turning the animals into a supine position. It was found that in the 'bar/podium/grid' test coadministration of both NMDA receptor antagonists significantly and dose-dependently augmented morphine-induced catalepsy. The results using the AMPA receptor antagonist were less clear since the lower dose of GYKI 52466 tended to attenuate the morphine effect whereas the higher dose augmented morphine-induced catalepsy in some cases. While placing the animals on the bar and on the podium produced essentially the same results, the grid was found to be inapplicable for the measurement of morphine-induced catalepsy since the animals did not cling to the grid and fell off almost immediately after being released from the experimenter's hand. With respect to the righting reflexes it was found that the number of animals not showing these responses increased when MK-801 or CGP 37849 was coadministered with morphine. In contrast, most of the animals treated with GYKI 52466 and morphine displayed intact righting reflexes. It is concluded that glutamatergic transmission plays an important role in the mediation of morphine-induced catalepsy, though different to that of haloperidol-induced catalepsy, and that NMDA and AMPA receptors are differentially involved in different aspects of the associated behavioural state.     

Effect of vitamin A supplementation on diarrhoea and acute lower-respiratory-tract infections in young children in Brazil

A beneficial effect of periodic vitamin A supplementation on childhood mortality has been demonstrated, but the effect on morbidity is less clear. We investigated the effect of vitamin A supplementation on diarrhoea and acute lower-respiratory-tract infections (ALRI) in children from northeastern Brazil in a randomised, double-blind, placebo-controlled community trial. 1240 children aged 6-48 months were assigned vitamin A or placebo every 4 months for 1 year. They were followed up at home three times a week, and data about the occurrence and severity of diarrhoea and ALRI were collected. Any child with cough and respiratory rate above 40 breaths per min was visited by a paediatrician. The overall incidence of diarrhoea episodes was significantly lower in the vitamin-A-supplemented group than in the placebo group (18.42 vs 19.58 x 10(-3) child-days; rate ratio 0.94 [95% Cl 0.90-0.98]). The benefit of supplementation was greater as regards severe episodes of diarrhoea; the incidence was 20% lower in the vitamin A group than in the placebo group (rate ratio 0.80 [0.65-0.98]). With the standard definition of diarrhoea (> or = 3 liquid or semi-liquid stools in 24 h) the effect of vitamin A on mean daily prevalence did not reach significance, but as the definition of diarrhoea was made more stringent (increasing number of stools per day), a significant benefit became apparent, reaching for diarrhoea with 6 or more liquid or semi-liquid stools in 24 h a 23% lower prevalence. We found no effect of vitamin A supplementation on the incidence of ALRI. The reduction in severity of diarrhoea may be the most important factor in the lowering of mortality by vitamin A supplementation.