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.     

Phosphatidylinositol 3-kinase is necessary and sufficient for insulin-stimulated stress fiber breakdown

Rat-1 fibroblasts overexpressing the human insulin receptor undergo rapid actin rearrangement in response to insulin. Breakdown of stress fibers present in quiescent cells is followed by transient membrane ruffling and a return of stress fibers. We investigated the signaling pathways that mediate this insulin-stimulated reorganization of the actin cytoskeleton, which was visualized with rhodamine-phalloidin. Treatment of cells with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin prevented insulin action at the preliminary step of stress fiber breakdown. Cellular microinjection of a polyclonal antibody directed against the p85 subunit of PI3-kinase as well as a purified recombinant p85-SH2 domain protein also inhibited actin reorganization. Transient expression of a constitutively active form of PI3-kinase (p110*) was sufficient to cause both stress fiber breakdown and membrane ruffling in the absence of insulin. Microinjection of a polyclonal anti-Shc antibody or dominant negative N17-Ras protein did not affect actin dynamics, and although constitutively active V12-Ras caused modest cytoskeletal reorganization, this effect was blocked by pretreatment with wortmannin. In summary, activation of PI3-kinase is necessary and sufficient to stimulate actin rearrangement, indicating that PI3-kinase may initiate the only signaling cascade required for insulin to induce cytoskeletal restructuring.     

More effective suppression of hemostatic system activation in patients undergoing cardiac surgery by heparin dosing based on heparin blood concentrations rather than ACT

This study was designed to determine whether the maintenance of higher than usual patient-specific heparin concentrations during cardiopulmonary bypass (CPB) was associated with more effective suppression of hemostasis system activation. Thirty-one patients scheduled for repeat cardiac surgery or combined procedures (i.e., coronary revascularization + valve repair/replacement) were consented and enrolled in this study. All patients received porcine heparin and protamine and were randomly assigned to monitoring of anticoagulation by either celite ACT alone (Control, n = 16) or by kaolin ACT combined with on-site measurements of whole blood heparin concentration (Intervention, n = 15). Blood specimens collected before administration of heparin, before weaning from CPB and after administration of protamine were analyzed with a battery of coagulation assays. Patients in the intervention cohort received appreciably greater heparin doses than control patients, resulting in higher anti-Xa heparin levels at the end of CPB. Fibrinopeptide A and D-dimer levels were higher in the control group before discontinuation of CPB. Percent decrease during CPB were greater in the control group for factors V and VIII, fibrinogen and antithrombin III. Percent decrease in complement 3 was greater in the control group after protamine and bleeding times measured in the Intensive Care Unit were significantly more prolonged in this group. Maintenance of higher patient-specific heparin concentrations during CPB more effectively suppresses excessive hemostatic system activation than do standard heparin doses chosen based on measurement of ACT. These findings may explain, at least in part, the significant reduction in perioperative blood loss and blood product use when higher heparin concentrations are maintained.     

The immunogenicity of three Haemophilus influenzae type B conjugate vaccines after a primary vaccination series in Philippine infants

Serum antibody responses to three Haemophilus influenzae type b (Hib) capsular polysaccharide-protein conjugate vaccine (PRP-OMP, PRP-T, and HbOC) were evaluated in 174 Philippine infants after a primary vaccination series. Children were randomized to receive one of the Hib vaccines (Hib groups) or into a control group. Vaccination was carried out at six, 10 and 14 weeks of age based on the local Expanded Program of Immunization schedule. Sera were collected at six weeks of age for the Hib groups and one month after the third dose for all subjects. Anti-Hib concentrations were determined by the Farr-type radioimmunoassay. There were no significant differences (P = 0.3626) in the prevaccination anti-Hib geometric mean concentration (GMC) among the three Hib groups. Differences in the GMC after the primary series of three doses were significant (P < 0.0001); GMC was highest for PRP-T (6.62 micrograms/ml), followed by HbOC (1.9 micrograms/ml), then PRP-OMP (1.06 micrograms/ml), and lowest for the control group (0.11 microgram/ml). We conclude that all three Hib conjugate vaccines (PRP-T, HbOC, and PRP-OMP) were immunogenic after three primary doses among Philippine infants.     

Mutation of serine-46 to aspartate in the histidine-containing protein of Escherichia coli mimics the inactivation by phosphorylation of serine-46 in HPrs from gram-positive bacteria

Histidine-containing protein (HPr) is a phosphocarrier protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system. HPr is phosphorylated at the active site residue, His15, by phosphoenolpyruvate-dependent enzyme I in the first enzyme reaction in the process of phosphoryl transfer to sugar. In many Gram-positive bacterial species HPr may also be phosphorylated at Ser46 by an ATP-dependent protein kinase but not in the Gram-negative Escherichia coli and Salmonella typhimurium. One effect of the phosphorylation at Ser46 is to make HPr a poor acceptor for phosphorylation at His15. In Bacillus subtilis HPr, the mutation Ser46Asp mimics the effects of phosphorylation. A series of mutations were made at Ser46 in E. coli HPr: Ala, Arg, Asn, Asp, Glu, and Gly. The two acidic replacements mimic the effects of phosphorylation of Ser46 in HPrs from Gram-positive bacteria. In particular, when mutated to Asp46, the His 15 phosphoacceptor activity (enzyme I Km/Kcat) decreases by about 2000-fold (enzyme I Km, 4 mM HPr; Kcat, approximately 30%). The alanine and glycine mutations had near-wild-type properties, and the asparagine and arginine mutations yielded small changes to the Km values. The crystallographic tertiary structure of Ser46Asp HPr has been determined at 1.5 A resolution, and several changes have been observed which appear to be the effect of the mutation. There is a tightening of helix B, which is demonstrated by a consistent shortening of hydrogen bond lengths throughout the helix as compared to the wild-type structure. There is a repositioning of the Gly54 residue to adopt a 3(10) helical pattern which is not present in the wild-type HPr. In addition, the higher resolution of the mutant structure allows for a more definitive placement of the carbonyl of Pro11. The consequence of this change is that there is no torsion angle strain at residue 16. This result suggests that there is no active site torsion angle strain in wild-type E. coli HPr. The lack of substantial change at the active center of E. coli HPr Ser46Asp HPr suggests that the effect of the Ser46 phosphorylation in HPrs from Gram-positive bacteria is due to an electrostatic interference with enzyme I binding.     

Cell surface chondroitin sulfate proteoglycans in tumor cell adhesion, motility and invasion

Genes that encode enzymes that convert inactive "prodrugs" into anticancer metabolites may be therapeutically useful against brain tumors. Unlike other genes tested to date in brain tumor models, the Escherichia coli gpt gene is unique in that it not only sensitizes cells to the prodrug 6-thioxanthine (6TX) but also encodes resistance to a different regimen (mycophenolic acid, xanthine, and hypoxanthine), thus providing a means to select for gpt-positive cells. In the present study, rat C6 glioma cells were infected with a retrovirus vector that transduces this gene. A clonal line (C6GPT-7) was derived that exhibited significant 6TX susceptibility in vitro with an ID50 of 2.5 mumol/L, whereas 50% growth inhibition of parental C6 cells was not achieved at concentrations tested (up to 50 mumol/L). This line also exhibited significant sensitivity to 6-thioguanine (6TG), with an ID50 of 0.05 mumol/L, whereas 50% growth inhibition of parental C6 cells was achieved at 0.5 mumol/L. In a "bystander" assay, C6GPT-7 tumor cells efficiently transferred 6TX sensitivity to C6 cells at ratios as low as 1:9 (C6GPT-7:C6). This in vitro bystander effect was abrogated when C6GPT-7 and C6 cells were separated by a microporous membrane, suggesting that it was not mediated by highly diffusible metabolites. In vivo both 6TX and 6TG significantly inhibited the growth of subcutaneously transplanted C6GPT-7 cells but not that of C6 cells in athymic mice. In an intracerebral model, both 6TX and 6TG exhibited significant antiproliferative effects against tumors formed by C6GPT-7 cells. These findings provide a basis for exploring further gene therapy strategies based on in vivo transfer of the E coli gpt gene to provide chemosensitivity against 6TX and 6TG.     

Image compression in digital mammography: effects on computerized detection of subtle microcalcifications

Our previous receiver operating characteristic (ROC) study indicated that the detection accuracy of microcalcifications by radiologists is significantly reduced if mammograms are digitized at 0.1 mm x 0.1 mm. Our recent study also showed that detection accuracy by computer decreases as the pixel size increases from 0.035 mm x 0.035 mm. It is evident that very large matrix sizes have to be used for digitizing mammograms in order to preserve the information in the image. Efficient compression techniques will be needed to facilitate communication and archiving of digital mammograms. In this study, we evaluated two compression techniques: full frame discrete cosine transform (DCT) with entropy coding and Laplacian pyramid hierarchical coding (LPHC). The dependence of their efficiency on the compression parameters was investigated. The techniques were compared in terms of the trade-off between the bit rate and the detection accuracy of subtle microcalcifications by an automated detection algorithm. The mean-square errors in the reconstructed images were determined and the visual quality of the error images was examined. It was found that with the LPHC method, the highest compression ratio achieved without a significant degradation in the detectability was 3.6:1. The full frame DCT method with entropy coding provided a higher compression efficiency of 9.6:1 at comparable detection accuracy. The mean-square errors did not correlate with the detection accuracy of the microcalcifications. This study demonstrated the importance of determining the quality of the decompressed images by the specific requirements of the task for which the decompressed images are to be used. Further investigation is needed for selection of optimal compression technique for digital mammograms.