Determination of the angle between the acceptor and anticodon stems of a truncated mitochondrial tRNA

Significant departures from the canonical (cloverleaf) secondary structure of transfer (t)RNAs can be found among the mitochondrial (m)tRNAs of higher metazoans; these mtRNAs thus pose a challenge to the concept of an invariant, L-shaped tertiary conformation for all tRNAs. For bovine mtRNASer(AGY), which lacks the entire "dihydrouridine" (dhU) arm, two distinct tertiary models have been proposed: the first model preserves the L-shaped conformation at the expense of overall size; the second model preserves the absolute distance between the 3' terminus and the anticodon loop, while allowing the acceptor-anticodon interstem angle to vary. We have tested the central predictions of these two models by performing a series of transient electric birefringence measurements on bovine mtRNASer(AGY) constructs in which the aminoacyl-acceptor and anticodon stems were each extended by approximately 70 bp. This mtRNA species is particularly amenable to analysis, since the native bovine (heart) mtRNA is completely unmodified outside of the anticodon loop. For magnesium ion concentrations above 1 mM, the interstem angle for the extended mtRNA, 120(+/-5) degrees, is approximately 50% larger than the corresponding angle for yeast tRNAPhe (70-80 degrees) under the same ionic conditions. Furthermore, the interstem angles of the two tRNAs exhibit strikingly different responses to the addition of magnesium ions: the interstem angle for yeast tRNAPhe is reduced by nearly 50 % upon addition of 2 mM magnesium ions, whereas the angle for mtRNASer(AGY) increases by about 10%. Our data thus support a central prediction of the second model; namely, that truncated mtRNAs will possess more open interstem angles. In addition, we demonstrate that birefringence amplitude data can be used to provide model-independent estimates for the interstem angles.     

Seroprevalence survey of Egyptian tourism workers for hepatitis B virus, hepatitis C virus, human immunodeficiency virus, and Treponema pallidum infections: association of hepatitis C virus infections with specific regions of Egypt

Blood samples from 740 Egyptian Nationals working in the tourism industry at two sites in the South Sinai governorate were screened for markers of infection with hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Treponema pallidum. Study subjects included 467 individuals from a rural seashore tourist village and 273 persons at two hotels in a well-established resort town. Subjects' ages ranged from 15 to 70 years; 99.3% were male. The prevalence of serologic markers for currently asymptomatic or past HBV infection alone was 20.7% (n = 153), of markers for past or chronic HCV infection alone was 7.4% (n = 55), and of markers for both HBV and HCV was 6.9% (n = 51). Of the 204 individuals positive for anti-HBV core antibody, 12 (5.9%) were also positive for hepatitis B surface antigen. Two individuals (0.3%) had a serologic market suggestive of an active syphilitic infection. No subject was found to be HIV-seropositive. History of prior injections and number of injections were associated with infection with HCV. Primary residence in the Nile delta and valley areas where schistosomiasis is highly endemic, was also a statistically significant risk factor for HCV, but not HBV infection.     

Coexpression of leptin receptor and preproneuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus

Leptin, the protein product of the adipose tissue-specific ob (obese) gene (1), reduces the body weight, adiposity and food intake of obese ob/ob mice on peripheral or central injection (2, 3, 4). [125I]leptin binding has been detected in mouse choroid plexus (5), from which a leptin receptor gene was expression cloned (5). The gene has at least 6 splice variants (6, 7). Leptin receptor mRNA was localized in the hypothalamus by in situ hybridization being particularly abundantly expressed in the arcuate nucleus (8). There is evidence linking the physiological effects of injected leptin with hypothalamic neuropeptide Y (9, 10) (NPY), which has potent central effects on food intake and energy balance (11), and is also expressed in the arcuate nucleus. Here we report dual in situ hybridization studies for leptin receptor and NPY gene expression in the mouse arcuate nucleus, where the majority of cells examined expressed both genes. This provides the first direct evidence that leptin acts on cells that express NPY mRNA.     

The Pain Coping Questionnaire: preliminary validation

Breda virus (BRV), a member of the genus torovirus, is an established etiological agent of diarrhea of cattle, which is found as two separate serotypes, BRV-1 and BRV-2. In this study, a 7.5 kb fragment of the BRV-1 genome that bracketed the genes for the structural proteins of BRV was amplified by long RT-PCR and the amplicon purified and sequenced directly. Sequence analysis revealed the presence of four open reading frames (ORF) corresponding to the peplomer (S), envelope (M), and nucleocapsid (N) genes, and an ORF for a novel 1.2 kb gene located between the M and N genes. This new gene was identical in nucleotide sequence to the hemagglutinin-esterase (HE) gene of BRV-2. With the exception of this new ORF, BRV-1 manifests 80% nucleotide sequence identity with the torovirus prototype, Berne virus (BEV) in the 7.5 kb region from the 3' end of the genome that contains the genes for the structural proteins. A 504 base segment containing the ORF for the BRV-1 N gene was amplified by RT-PCR, and cloned into an Escherichia coli expression system. The resulting protein was purified by SDS-PAGE and used to immunize guinea pigs. Hyperimmune serum was reactive with bovine torovirus (BTV) and human torovirus (HTV) antigens. By immunoelectron microscopy, it was shown to aggregate broken but not intact torovirus particles from BTV-positive fecal specimens. By immunoblot, the hyperimmune serum reacted specifically with the 20 kD N proteins of both BTV and HTV, as well as with the expressed N protein. BRV-1 and BRV-2 immune sera from gnotobiotic calves, but not human convalescent sera from HTV-infected patients, reacted with the expressed N protein by immunoblot. These findings were applied to the design of a dot blot assay that could specifically detect BTV and HTV from fecal specimens.     

Role of multimerized porcine thyroglobulin in iodine storage

In rodents calorie restriction (CR) reduces cancer incidence, improves health by delaying age-related declines in physiologic measures, and extends both median and maximal life span. The mechanisms underlying the various beneficial effects of CR remain undefined. In this study, heterozygous p53-deficient (p53(+/-)) mice (in which the inactivation of one allele of the p53 tumor suppressor gene increases susceptibility to spontaneous and carcinogen-induced tumor development) and wild-type (WT) litter mates were subjected to a two-stage skin carcinogenesis protocol with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate. Instead of skin carcinomas, however, the chemical treatment protocol caused ulcerous skin lesions, and 89% of mice fed ad libitum died from infection/septicemia. When WT mice were restricted to 60% of the average calorie intake of the respective ad libitum group, however, only 33% developed such lesions, and the CR mice survived twice as long on average as the ad libitum mice. CR also extended life span in p53(+/-) mice, but 50% of p53(+/-) mice subjected to CR still developed skin ulcers and mean life span was shorter than that seen in WT mice. Differences in response to CR between WT and p53(+/-) mice may be due to the reduction in p53 gene dosage, dissimilarity in the application of the CR treatment, or both. These results suggest that some of the beneficial effects of CR may need full expression of p53 for complete realization.     

Bridging the GAP in inositol 1,3,4,5-tetrakisphosphate signalling

Recent advances in analyzing wnt signaling have provided evidence that frizzled proteins can function as wnt receptors. We have identified Xfz3, a Xenopus frizzled family member. The amino acid sequence is 89% identical to the product of the murine gene Mfz3, and is predicted to be a serpentine receptor with seven transmembrane domains. Xfz3 is a maternal mRNA with low levels of expression until the end of gastrulation. The expression level increases significantly from neurulation onward. Whole-mount in situ hybridization analysis shows that expression of Xfz3 is highly restricted to the central nervous system. High levels of expression are detected in the anterior neural folds. Low levels of expression are also detected in the optic and otic vesicles, as well as in the pronephros anlage. In addition, Xfz3 mRNA is concentrated in a large band in the midbrain. Overexpression of Xfz3 blocks neural tube closure, resulting in embryos with either bent and strongly reduced anteroposterior axis in a dose-dependent manner. However, it does not affect gastrulation, the expression and localization of organizer-specific genes such as goosecoid, chordin and noggin. Therefore, Xfz3 is not involved in early mesodermal patterning. Injection of RNA encoding GFP-tagged Xfz3 shows that overexpressed proteins can be detected on the cell surface until at least late neurula stage, suggesting that they can exert an effect after gastrulation. Our expression data and functional analyses suggest that the Xfz3 gene product has an antagonizing activity in the morphogenesis during Xenopus development.     

The effect of intravenous and oral fluconazole on the pharmacokinetics and pharmacodynamics of intravenous alfentanil

The growth-promoting activity of PDGF-BB was studied on the adult osteoclasts in the present study. The PDGF receptor beta was detected on the osteoclast membrane through immunohistochemistry (LSAB method) and immunomicroscopy. The PDGF-BB was exerted on the osteoclasts that adhered to the bone slice at concentrations of 0, 10, 20, 30, and 40 ng/ml. The volume of Howship's lacuna augmented significantly and the number of resorption pits also increased with its dose (p < 0.01). The activity of both total acid phosphatase (ACP) and tartrate-resistant acid phosphatase (TRAP) increased significantly. These results suggest that PDGF-BB promotes adult osteoclastic bone resorption directly through PDGF receptor beta and is believed to play important roles in the bone healing process and reconstruction.     

Pathogenic mechanisms in the rheumatoid nodule: comparison of proinflammatory cytokine production and cell adhesion molecule expression in rheumatoid nodules and synovial membranes from the same patient

OBJECTIVE: To investigate the production of proinflammatory cytokines and expression of cell adhesion molecules in the rheumatoid nodule. METHODS: Cytokine content (tumor necrosis factor alpha [TNFalpha], interleukin-1beta [IL-1beta], and IL-1 receptor antagonist [IL-1Ra]), at the messenger RNA (mRNA) and protein levels, and cell adhesion molecule expression were studied in 16 rheumatoid nodules and 6 synovial membranes. RESULTS: Macrophages in the rheumatoid nodules contained TNFalpha, IL-1beta, and IL-1Ra mRNA and protein, particularly in perivascular cells of the stroma and in the palisading layer. All cell adhesion molecules studied were expressed in both the rheumatoid nodules and synovial membranes, with increased expression of E-selectin in the rheumatoid nodule compared with the synovial membrane, and with the absence of vascular cell adhesion molecule 1 expression on cells of the palisading layer in the rheumatoid nodule. CONCLUSION: The presence of similar proinflammatory cytokines and cell adhesion molecules in the rheumatoid nodule and synovial membrane suggests that similar pathogenic processes result in the chronic inflammation and tissue destruction in these lesions.