Restriction of dietary energy and protein induces molecular changes in young porcine skeletal muscles

Little is known about the molecular changes in response to dietary restriction (energy and/or protein) in young growing skeletal muscles. To profile such changes and to gain insights into the signaling molecules that could mediate the diet effects, a dedicated porcine skeletal muscle cDNA-microarray approach was used to characterize differential muscle gene expression between conventionally fed and diet-restricted (20% less protein and 7% less energy) growing pigs, reared from 9 to 21 wk of age. In both red and white muscles, diet restriction resulted in the accumulation of significantly more intramuscular fat, and in the increased expression of genes involved in substrate (protein, glycogen, and lipid) turnover, in translation and mitochondrial function, and in raising glycolytic and oxidative phosphorylation potentials. The unexpected increase in intramuscular lipids in diet-restricted growing pigs could have important health implications for restricted diets in childhood. Despite reduced circulating insulin, more genes, including several novel growth modulatory genes, had higher expression levels, indicating that the cellular response to dietary restriction is an active process. One such responsive gene, P311, was most highly expressed in striated muscles and had a differentiation-dependent increase of expression in murine C2C12 cells, suggesting a role in differentiation/postdifferentiation phenotype determination.     

Comparison of the time to onset of action on myocardial ischaemia following intravenous administration of isosorbide dinitrate and 5-isosorbide mononitrate in Chinese patients

OBJECTIVE: The onset of action of intravenous isosorbide dinitrate (CAS 87-33-2, ISDN) and intravenous 5-isosorbide mononitrate (CAS 16051-77-7, 5-ISMN) were compared by measurement of the indicators of perfusion to ischaemic myocardium. METHODS: Twenty-five patients with coronary heart disease were randomly allocated to receive intravenous ISDN or 5-ISMN. The extent of myocardial ischaemia before infusion and at 3, 15 and 45 min after commencement of infusion was evaluated using 99mTc-MIBI myocardium tomography imaging and electrocardiograms. RESULTS: The perfusion defects were significantly reduced or resolved in 11 patients (84.1%) receiving ISDN and 2 patients (15.38%) receiving 5-ISMN at 3 min. At 15 min the improvement was significantly greater in the ISDN group than in the 5-ISMN group. The improvements of 99mTc-MIBI myocardial uptake ratio and electrocardiograms were statistically significant in the ISDN group at 3 min and 15 min compared to pre-infusion. Although a significant improvement appeared at 15 min in the 5-ISMN group, it was significantly less than that observed in the ISDN group (p < 0.05). After 45 min, there were improvements in ischaemia in both groups with the difference compared to pre-infusion being significant, and there was no statistically significant difference between the ISDN and 5-ISMN group. CONCLUSION: In patients with coronary heart disease with ischaemic episodes the onset of therapeutic activity was more rapid with intravenous ISDN compared to 5-ISMN. ISDN should be the preferred intravenous nitrate for acute ischaemic episodes where a rapid onset of therapeutic action is desired.     

Molecular cloning and pharmacological characterization of the guinea pig 5-HT1E receptor

The human 5-HT(1E) receptor gene was cloned more than a decade ago. Little is known about its function, and there have been no reports of its existence in the genome of small laboratory animals. In this study, attempts to clone the 5-HT(1E) gene from the rat and mouse were unsuccessful. In fact, a search of the mouse genome database revealed that the 5-HT(1E) receptor gene is missing from the mouse genome. However, the 5-HT(1E) gene was cloned from guinea pig genomic DNA and was characterized. The guinea pig 5-HT(1E) receptor gene encodes a protein of 365 amino acids. It shares 88% (nucleic acid) and 95% (amino acid) homology with the human receptor. The guinea pig 5-HT(1E) receptor showed similar pharmacology to the human 5-HT(1E) receptor in radioligand binding assays. Serotonin (5-hydroxytryptamine, 5-HT) dose-dependently stimulated [35S]GTPgammaS binding to the guinea pig 5-HT(1E) receptor with an EC(50) of 13.6+/-1.92 nM, similar to that of the human 5-HT(1E) receptor (13.7+/-1.78 nM). Activation of the guinea pig 5-HT(1E) receptor was also achieved by ergonovine, alpha-methyl-5-HT, 1-naphthylpiperazine, methysergide, tryptamine, and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Methiothepin exhibited antagonist activity. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that 5-HT(1E) mRNA was present in the guinea pig brain with the greatest abundance in the hippocampus, followed by the olfactory bulb. Lower levels were detected in the cortex, thalamus, pons, hypothalamus, midbrain, striatum, and cerebellum. Our current study marks the first identification of the 5-HT(1E) receptor gene in a commonly used laboratory animal species. This finding should allow the elucidation of the receptor's role(s) in the complex coordination of central serotonergic effects.     

Laulimalide and paclitaxel: a comparison of their effects on tubulin assembly and their synergistic action when present simultaneously

Previous work has shown that laulimalide, a sponge-derived natural product, resembles paclitaxel in enhancing tubulin assembly and in its effects on cellular microtubules. The two compounds, however, seem to have distinct binding sites on tubulin polymer. Nearly equimolar amounts of tubulin, laulimalide, and paclitaxel are recovered from microtubules formed with both drugs. In the present study, we searched for differences between laulimalide and paclitaxel in their interactions with tubulin polymer. Laulimalide was compared with paclitaxel and epothilone A, a natural product that competes with paclitaxel in binding to microtubules, for assembly properties at different temperatures and for effects of GTP and microtubule-associated proteins on assembly. Although minor differences were observed among the three drugs, their overall effects were highly similar, except that aberrant assembly products were observed more frequently with paclitaxel and that the polymers formed with laulimalide and epothilone A were more stable at 0 degrees C. The most dramatic difference observed between laulimalide and epothilone A was that only laulimalide was able to enhance assembly synergistically with paclitaxel, as would be predicted if the two drugs bound at different sites in polymer. Because stoichiometric amounts of laulimalide and paclitaxel can cause extensive tubulin assembly, maximum synergy was observed at lower temperatures under reaction conditions in which each drug alone is relatively inactive. Laulimalide-induced assembly, like paclitaxel-induced assembly, was inhibited by drugs that inhibit tubulin assembly by binding at either the colchicine- or vinblastine-binding site. When radiolabeled GTP is present in a reaction mixture with either laulimalide or paclitaxel, nucleotide hydrolysis occurs with incorporation of radiolabeled GDP into polymer.     

Pharmacology of the mouse-isolated cerebral artery

The routine availability of murine models of various cerebral circulatory disorders requires characterization of the regulation of cerebral artery tone in the mouse. Using vasoconstrictors and vasodilators with known efficacy in the cranial circulation of other species, we determined the pharmacological properties of the isolated pressurized mouse middle cerebral artery (MCA). The maximal pressure-induced myogenic constriction in isolated mouse MCA was 20.6+/-2.4%. Inhibition of nitric oxide (NO) and endothelin-1 (ET-1) altered the extent of pressure-induced myogenic tone. Isolated mouse MCA failed to either constrict or relax to 5-hydroxytryptamine (5-HT) and histamine; other vasoconstrictors demonstrated the following rank order of efficacy: ET-1>phenylephrine>U-46619. The rank order of endothelium-dependent vasodilator efficacy was bradykinin (BK)>acetylcholine (ACh)>substance P. The constriction produced by phenylephrine (PE) required a smaller increase in intracellular Ca(2+) elevation compared to constriction of a similar magnitude produced by membrane depolarization with potassium chloride (KCl). Pressure-induced myogenic tone (20-80 mm Hg) in mouse MCA was associated with smooth muscle cell membrane depolarization (-52.6+/-0.9 to -37.3+/-1.75 mV). Pressure-induced myogenic tone occurred with a smaller change in membrane potential compared to tone of a similar magnitude produced with KCl (-43.37+/-2.66 vs. -29.47+/-1.05 mV). The mouse MCA has a pharmacological profile that is distinct from other species including humans; however, similar to findings in other cerebral arteries, the mouse MCA shows intracellular sensitization to Ca(2+) following receptor activation.     

Constitutional activation of IL-6-mediated JAK/STAT pathway through hypermethylation of SOCS-1 in human gastric cancer cell line

The interleukin-mediated Janus kinase (JAK)/STAT pathway plays a crucial role in carcinogenesis. Recently, increased STAT3 activity was found in hepatocellular carcinoma and multiple myeloma in which there was silencing of SOCS-1 (suppressor of cytokine signalling-1) by gene promoter hypermethylation. We investigated the expression level of interleukin-6 (IL-6) and SOCS-1 in gastric cancer cell lines. Expression of SOCS-1 correlated with IL-6 level in most of the cell lines, except for AGS cells in which SOCS-1 was absent despite a high level of IL-6 production. Methylation analysis by methylation-specific polymerase chain reaction and bisulphite sequencing revealed that CpG island of SOCS-1 was densely methylated in AGS cells. Demethylation treatment by 5'aza-deoxycytidine restored SOCS-1 expression and also suppressed constitutive STAT3 phosphorylation in AGS cells. Moreover, methylation of SOCS-1 was detected in 27.5% (11 of 40) of primary gastric tumours samples, 10% (one of 10) of adjacent noncancer tissues but not in any (zero of nine) normal gastric mucosa. Methylation of SOCS-1 also correlated with the loss of mRNA expression in some primary gastric cancers. In conclusion, this is the first report to demonstrate that hypermethylation of SOCS-1 led to gene silencing in gastric cancer cell line and primary tumour samples. Downregulation of SOCS-1 cooperates with IL-6 in the activation of JAK/STAT pathway in gastric cancer.     

Cytology of human ovarian surface epithelial brushings

BACKGROUND: The human ovarian surface epithelium (HOSE) is the putative source of ovarian epithelial cancer, the most lethal gynecologic malignancy that affects women in the United States. The current study was designed to provide a database of normal HOSE cell features for diagnostic and research applications. METHODS: HOSE was harvested from 42 women undergoing laparoscopy or laparotomy for benign gynecologic disorders, infertility problems, or pregnancy. Of the 42 women, 12 were postovulatory and 20 were receiving hormonal regimens. Cells were harvested with a sterile brush inserted through a laparoscopic port or with a sterile cell scraper at laparotomy. RESULTS: Two HOSE populations were identified, ranging in size from 8 to 10 microm and from 15 to 20 microm, respectively. The cells measuring 15-20 microm exhibited slight anisonucleosis, more prominent nucleoli, fine cytoplasmic metachromasia, and an overall reparative or squamoid morphology. Cells were single or arranged in small clusters, sheets, or papillae. They coexpressed cytokeratin and vimentin but did not overexpress p53. Cellularity and proliferation (up to 3.2% +/- 0.8) were higher and papillae more frequent in postovulatory and cyst-bearing ovaries, including polycystic ovaries, suggesting underlying ovarian or hormonal influences. Representative HOSE brushings yielded a mean of 23,133 cells per patient (range, 4250-64,500 cells), equivalent to an estimated 0.58, 0.46, and 0.14 microg of nuclear protein, cell RNA, and nuclear DNA, respectively. Within 7-10 days of explantation, HOSE cells formed confluent monolayers with immunohistochemical and ultrastructural epithelial features. CONCLUSIONS: The current study defined baseline features of HOSE cells important to pathologists and clinicians evaluating women at risk for ovarian epithelial cancer and to researchers investigating the pathobiology of this aggressive gynecologic malignancy.     

Association analysis of a neural nitric oxide synthase gene polymorphism and antipsychotics-induced tardive dyskinesia in Chinese schizophrenic patients

Summary. Recent findings from rodent studies with chronic administration of antipsychotic drugs have indicated the role of neural nitric oxide synthase (NOS1) on the susceptibility of tardive dyskinesia (TD). In the present study, the association between a 3-untranslated region C267T (3-UTR C267T) polymorphism of the NOS1 gene and TD as well as TD severity was investigated in 251 Chinese schizophrenic patients with long-term antipsychotic treatment (TD: 128, non-TD: 123). After adjusting the effects of confounding factors, there was no significant association between NOS1 3-UTR C276T genotypes and TD occurrence (p=0.758). With in the TD group, we could not discover a significant correlation between NOS1 3-UTR C276T genotypes and the scores of abnormal involuntary movement scale (AIMS) (p=0.219 and 0.774). We concluded that the NOS1 3-UTR C276T polymorphism might not play a major role in the susceptibility of TD development, or on the severity of TD.     

Genotype determines phenotype in experimental Lyme borreliosis

Borrelia burgdorferi sensu lato, the causative organism of Lyme borreliosis, is a heterogeneous group of spirochetes, consisting of at least three pathogenic species. To test the hypothesis that the genetic heterogeneity is the reason for the clinical differences, we investigated whether the experimental disease induced by European isolates is different from that induced by American isolates. Two American isolates of species B. burgdorferi sensu stricto were compared with three European isolates, two of species B. garinii, and one of species B. afzelii. The patterns of infection, immunity, and inflammation induced by the different species was distinctive. Inflammatory cells and levels of antibody in B. garinii- and B. afzelii-infected animals were lower than in B. burgdorferi s.s.-infected animals, whereas levels of spirochetal infection in the skin and nervous system were higher in the former group of animals. These data demonstrate that B. burgdorferi s.s. strains are more infective and inflammatory, whereas B. garinii and B. afzelii strains can survive the adaptive immune response to a greater degree and persist at greater numbers in the skin and nervous system. The results explain to a large extent the disparities between LNB in humans in the United States and Europe.     

Labeling and dynamic imaging of synaptic vesicle-like microvesicles in PC12 cells using TIRFM

Total internal reflection fluorescence microscopy (TIRFM) was employed to study the trafficking and exocytosis of synaptic vesicle-like microvesicles (SLMVs) in PC12 cells. SLMVs were labeled with vesicular acetylcholine transporter (VAChT) tagged with enhanced green fluorescent protein (EGFP), which displayed punctuate distribution under TIRFM and confocal microscopy. Immunofluorescence analysis confirmed the colocalization of EGFP and VAChT. No significant difference was observed in the distribution or sorting of VAChT when fused either at the N- or the C-terminus. Thus, tagging with GFP does not appear to impair or change the traffic of the VAChT in PC12 cells. Under TIRFM, EGFP-labeled spots moved in a restrained fashion, which resembled that of secretory granules and underwent exocytosis upon stimulation. Together, these data indicate that EGFP-tagged VAChT can be used to explore SLMVs trafficking using TIRFM.