Cytokine induction of heat shock protein in human granulosa-luteal cells

The infiltration of leukocytes is a characteristic feature ofluteolysis in humans. Leukocytes are known to generate physiologicalinducers of cell stress such as cytokines which have been implicatedas mediators of functional luteal regression. In cells exposedto stress, a response characterized by an increase in heat shockprotein (HSP) synthesis occurs. Recently, the induction of HSP-70in rat luteal cells has been shown to inhibit luteinizing hormone(LH) and cAMP-sensitive progesterone production, possibly byinterfering with the translocation of cholesterol to the mitochondrialcytochrome P450_SCC. We therefore investigated whether HSP-70is induced in human granulosa-luteal cells and its relationshipto steroidogenesis. [³⁵S]Methionine labelling showed an increasein a 70 kDa protein after heat treatment which was demonstratedto be HSP-70 by Western analysis using monoclonal antibodiesagainst the constitutive and inducible forms of HSP-70. Inductionof HSP-70 in human granulosa-luteal cells was also seen withinterferon (IFN) (10 ng/ml), tumour necrosis factor (TNF)-     

Colorimetric microwell plate hybridization assay for detection of amplified Mycobacterium tuberculosis DNA from sputum samples.

We developed a colorimetric microwell plate hybridization assay (CoMPHA) for the specific detection of 5'-biotinylated amplified Mycobacterium tuberculosis DNA. The optical densities of the CoMPHA corresponded to the initial amounts of purified template DNA. Here, we show that the CoMPHA is useful in distinguishing the PCR-positive and PCR-negative samples.     

Shwartzman reaction in streptozotocin-induced diabetic rats

The possibility that experimental diabetes could prepare for the generalized Shwartzman reaction was investigated in female Sprague-Dawley streptozotocin-induced diabetic rats. After 48 hours, 1 week, and 9 weeks of diabetes, the rats were injected with 2 mg/kg of endotoxin, and the animals were sacrificed 2, 4, 8, and 24 hours after endotoxin. Ninety percent of the diabetic animals given endotoxin developed massive glomerular capillary fibrin deposition accompanied by marked decrease in platelet count. The age- and sex-matched nondiabetic control rats had no such changes. This marked susceptibility to endotoxin, previously only reported in pregnant rats, was present as early as 1 week of diabetes. The degree of glycemic control greatly influenced the susceptibility of diabetic rats to the generalized Shwartzman reaction. Only 28% of the diabetic animals given insulin once daily (4.6 +/- 0.3 units, mean +/- SEM) and maintaining a blood glucose level of 269 +/- 19 mg/dl developed glomerular thrombi. In contrast, the diabetic animals that did not receive insulin and had a blood glucose level of 617 +/- 21 mg/dl all developed fibrin thrombi. We conclude that the diabetic state in rats induces a unique susceptibility to the generalized Shwartzman reaction following a single injection of endotoxin, which varies with the severity of the diabetic state. Although the pathogenesis is unclear, this phenomenon may reflect abnormalities in the glomerular capillary wall and/or the coagulation system that may be important in the development of microvascular complications. Furthermore, this phenomenon may, in the animal model, mirror the increased risk of the diabetic patient to intravascular coagulation with bacterial sepsis.     

Activation of complement by cell surface components of Staphylococcus aureus.

The abilities of intact Staphylococcus aureus H, crude cell walls (CCW), purified cell walls (PCW, peptidoglycan [PG] and covalently linked teichoic acid), peptidoglycan, and cell membranes (CM) to activate the complement system in normal human serum, C2-deficient serum, and immunoglobulin-deficient serum were compared. On a weight basis, PCW was the most active fraction; intact organisms and CCW were about equally effective; and PG was least active in causing complement consumption in normal serum. CM also activated complement but did not give a clear dose-response relationship in the concentrations used. Kinetic studies revealed that C3-C9 consumption occurred at a significantly slower rate in C2-deficient serum, indicating that intact organisms, PCW, and PG may activate the complement system via the classical and alternative pathways in normal serum. C3-C9 consumption was also slower in immunoglobulin-deficient serum than in normal serum, implying that immunoglobulins play a role in attaining maximum rates of complement activation. In all sera studied, PG was less active in complement activation than PCW. These results indicate that a number of cell surface components of S. aureus can play a role in complement activation by this organism and that the presence of teichoic acid has a significant enhancing effect in this regard.     

Sustained release of ascorbate-2-phosphate and dexamethasone from porous PLGA scaffolds for bone tissue engineering using mesenchymal stem cells

The purpose of this research was to develop porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds from which ascorbate-2-phosphate (AsAP) and dexamethasone (Dex) are continuously released for a month for osteogenesis of mesenchymal stem cells for bone tissue engineering. Porous PLGA matrices containing AsAP and Dex were prepared by solvent casting/particulate leaching method. In vitro release and water uptake studies were performed in Dulbecco's phosphate buffered saline at 37 degrees C and 15 rpm. Drug loading and release rates were determined by high performance liquid chromatography. Release studies of Dex and AsAP showed that, after an initial burst release lasting 4 and 9 days, respectively, release rates followed zero order kinetics with high correlation coefficients at least until 35 days. Incorporation of AsAP into the scaffolds increased the release rates of Dex and AsAP, and the scaffold water uptake. When mesenchymal stem cells (MSCs) were cultured in the AsAP and Dex containing scaffolds in vitro, the amount of mineralization was significantly higher than in control scaffolds. In conclusion, AsAP and Dex were incorporated into porous PLGA scaffolds and continuously released over a month and osteogenesis of MSCs was increased by culture in these scaffolds.     

Association of aortic atherosclerosis with cerebral beta-amyloidosis and learning deficits in a mouse model of Alzheimer's disease

High fat/high cholesterol diets exacerbate beta-amyloidosis in mouse models of Alzheimer's disease (AD). It has been impossible, however, to study the relationship between atherosclerosis and beta-amyloidosis in those models because such mice were on atherosclerosis-resistant genetic backgrounds. Here we report the establishment of AD model mice, B6Tg2576, that are prone to atherosclerosis. B6Tg2576 mice were produced by back-crossing Tg2576 mice, an AD mouse model overexpressing human amyloid beta-protein precursor with the Swedish double mutation, to C57BL/6 mice, a strain susceptible to diet-induced atherosclerosis. An atherogenic diet induced aortic atherosclerosis and exacerbated cerebral beta-amyloidosis in B6Tg2576 mice. Compared with age-matched non-transgenic littermates, B6Tg2576 mice developed significantly more diet-induced aortic atherosclerosis. Unexpectedly, normal diet-fed B6Tg2576 mice also developed fatty streak lesions (early atherosclerosis) in the aorta. The aortic atherosclerotic lesion area positively correlated with cerebral beta-amyloid deposits in B6Tg2576 mice on both atherogenic and normal diets. Furthermore, behavioral assessments demonstrated that B6Tg2576 mice fed an atherogenic diet had more spatial learning impairment than those fed a normal diet. Our results suggest that synergistic mechanisms may be involved in the pathogenesis of atherosclerosis and AD. These findings may have important implications in the prevention and treatment of cardiovascular diseases as well as AD.     

Evidence for linkage of nonsyndromic cleft lip with or without cleft palate to a region on chromosome 2

Results from a genome-wide screen of 10 multiplex families ascertained through probands with nonsyndromic cleft lip with or without cleft palate (CL/P) in Mexico, Argentina, and the United States yielded suggestive evidence of linkage to chromosomes 2, 6, 17 and 18. Fine mapping excluded all regions except chromosome 2. Subsequent analysis was performed on the original 10 families plus an additional 16 families using 31 markers on chromosome 2. This analysis showed intriguing evidence of linkage to 2q (Zlr=2.26, empirical P-value=0.028 in a chromosome-wide analysis). Transmission disequilibrium tests also revealed evidence of linkage and disequilibrium for two markers in this region (D2S168 and D2S1400 with P-values=0.022 and 0.006, respectively). A subset of these 26 families provided additional evidence for a susceptibility gene for CL/P on 2q, suggesting that further studies of genes in this region are warranted.     

Use of egg yolk-derived immunoglobulin as an alternative to antibiotic treatment for control of Helicobacter pylori infection

The present study evaluated the potential use of immunoglobulin prepared from the egg yolk of hens immunized with Helicobacter pylori (immunoglobulin Y [IgY]-Hp) in the treatment of H. pylori infections. The purity of our purified IgY-Hp was 91.3%, with a yield of 9.4 mg of IgY per ml of egg yolk. The titer for IgY-Hp was 16 times higher than that for IgY in egg yolk from nonimmunized hens, and IgY-Hp significantly inhibited the growth and urease activity of H. pylori in vitro. Bacterial adhesion on AGS cells was definitely reduced by preincubation of both H. pylori (10(8) CFU/ml) and 10 mg of IgY-Hp/ml. In Mongolian gerbil models, IgY-Hp decreased H. pylori-induced gastric mucosal injury as determined by the degree of lymphocyte and neutrophil infiltration. Therefore, in this experimental model, H. pylori-associated gastritis could be successfully treated by orally administered IgY-Hp. The immunological activity of IgY-Hp stayed active at 60 degrees C for 10 min, suggesting that pasteurization can be applied to sterilize the product. Fortification of food products with this immunoglobulin would significantly decrease the H. pylori infection. In conclusion, the IgY-Hp obtained from hens immunized by H. pylori could provide a novel alternative approach to treatment of H. pylori infection.     

Effects of neostigmine on the pharmacokinetics of intravenous parathion in rats

It was reported that the area under the plasma concentration-time curve from time zero to time infinity (AUC) of parathion was significantly smaller and the time-averaged total body clearance (CL) of parathion was significantly faster after intravenous administration of parathion to rats pretreated with dexamethasone than those in control rats. This was supported by significantly faster intrinsic clearance of parathion to form paraoxon in hepatic microsomal fraction of rats pretreated with dexamethasone. The above data suggested that parathion was metabolized to paraoxon by dexamethasone-inducible hepatic cytochrome P450 (CYP) 3A in rats. The purpose of this study is to explain the protective effects of neostigmine against paraoxon toxicity by suppressing CYP3A and hence decreasing formation of toxic metabolite, paraoxon by neostigmine. The pharmacokinetic changes of parathion and its active metabolite, paraoxon, were investigated after intravenous administration of parathion, 3 mg/kg, to control Sprague-Dawley rats and the rats pretreated with neostigmine (200 microg/kg, intraperitoneal injection 30 min before parathion administration). After 1-min intravenous infusion of parathion to rats pretreated with neostigmine, the AUC of parathion (65.1 versus 74.3 microg min/ml) was significantly greater and the CL of parathion (45.1 versus 40.4 ml/min/kg) was significantly slower than those in control rats. Based on in vitro hepatic microsomal studies, neostigmine inhibited significantly the erythromycin N-demethylase activity (1.03 versus 0.871 nmol/mg protein/min), mainly mediated by hepatic cytochrome P450 3A in rats. The above data suggested that the formation of paraoxon was inhibited in rats pretreated with neostigmine by inhibiting CYP3A.     

A new resin matrix for dental composite having low volumetric shrinkage

The applications of dental restorative composite resins containing 2,2 bis [4-(2-hydroxy-3-methacryloyloxy propoxy) phenyl] propane (Bis-GMA), as a base resin, and triethylene glycol dimethacrylate (TEGDMA), as a diluent, are often limited in dentistry due to the relatively large amount of volumetric shrinkage that occurs during the curing reaction. In this study, various new resin matrices were examined for use as dental composites in order to reduce the amount of volumetric shrinkage that occurs in dental composites as a result of curing. Bis-GMA derivatives were synthesized by substituting methyl groups for hydrogen on the phenyl ring. The derivatives of TEGDMA with different chain lengths or reactive groups were also examined. The molecular structural changes in the TEGDMA derivatives were not effective in reducing the level of volumetric shrinkage. The resin matrix containing a Bis-GMA derivative and TEGDMA showed a reduced amount of volumetric shrinkage in proportion to the number of methyl groups on the phenyl rings. Polymerization with a mixture of Bis-GMA, its derivatives and a diluent is a promising strategy for obtaining a polymer with a low amount of volumetric shrinkage. A comparison of the volumetric shrinkage of dental composites containing Bis-GMA, TMBis-GMA (2,2-bis[3,5-dimethyl, 4-(2-hydroxy-3-methacryloyloxy propoxy) phenyl] propane)), and TEGDMA with that prepared from a Bis-GMA and TEGDMA mixture showed that the volumetric shrinkage reduction in the new resin was approximately 50%. Furthermore, the mechanical strength of the former was higher than that of the latter.