Detection of spores of Bacillus anthracis from environment using polymerase chain reaction

A sensitive PCR based detection of Bacillus anthracis spores from environnment was standardized. Specific 1247bp amplicon could be detected with template concentration as low as 13 pg. Sensitivity was enhanced to 10 fold by nesting with second set of primers, forming 208bp amplicon. Extraction of DNA from spores purified from soil samples by aqueous polymer two-phase system followed by partial germination and freeze-thaw treatment yielded best results. Soil sample spiked with spores (8x10(2)/g of sample) could be detected with this method.     

Parathyroid hormone regulation of type II sodium-phosphate cotransporters is dependent on an A kinase anchoring protein

Parathyroid hormone inhibits sodium-phosphate cotransport in proximal renal tubule cells through activation of several kinases. We tested the hypothesis that the activity of these kinases was coordinated by an A kinase anchoring protein (AKAP) by demonstrating that the type II sodium-phosphate cotransporter (NaPi-4) physically associated with an AKAP and that this association was necessary for regulation of phosphate transport by parathyroid hormone. Immunoprecipitation with anti-NaPi-4 antiserum and glutathione S-transferase pull-down with GST-NaPi-4 showed that NaPi-4 associated with AKAP79, protein kinase A catalytic and regulatory subunits, and the parathyroid hormone receptor in opossum kidney cells. When the regulatory subunit of protein kinase A was uncoupled from the AKAP by a competing peptide, parathyroid hormone lost the ability to inhibit phosphate transport. This result was confirmed by co-transfecting HEK293 cells with the sodium-phosphate cotransporter and wild type AKAP, a mutant AKAP79, or the empty vector. 8-Bromo-cAMP was able to inhibit phosphate transport in cells expressing the wild type AKAP79 but not empty vector or mutant AKAP79. We conclude that parathyroid hormone inhibits proximal renal tubule sodium-phosphate cotransport through a signaling complex dependent upon an AKAP.     

Chitosan cross-linking with a water-soluble,blocked diisocyanate. 1. Solid state

The present investigation focuses on the synthesis and application of a cross-linking agent that is compatible with the solubility characteristics of chitosan. A water-soluble, blocked-diisocyanate was prepared as a bisulfite adduct to 1,6-hexamethylene diisocyanate, which proved to be stable for several weeks in aqueous, acidic chitosan solutions at room temperature. Thermal cross-linking of chitosan as cast, dried films was investigated by varying the NCO/NH(2) ratio from 0.0 to 1.2. Spectroscopic (IR), thermal (TGA), swelling, and structural (WAXD) studies indicated that chitosan was cross-linked in a concentration-dependent manner under mild thermal conditions: 60 degrees C for 24 h. Cross-linking inefficiency was concluded to be due to lack of mobility of the reacting species in the solid state. In a preliminary study, the enzymatic degradation with Chitinase (E. C. 3.2.1.14) from Streptomyces griseus was found to be the greatest for non-crosslinked chitosan, followed by chitin, and then by cross-linked samples.     

Immunohistochemical expression of pi class glutathione S-transferase and alpha-fetoprotein in hepatocellular carcinoma and chronic liver disease

OBJECTIVE: To determine whether tumor marker pi glutathione transferase (GST-pi) is expressed in hepatocellular carcinoma (HCC) and other chronic liver diseases and to compare its expression with that of alpha-fetoprotein (AFP). STUDY DESIGN: Samples used were formalin-fixed, paraffin-embedded liver tissues: normal (n = 3), chronic hepatitis B (n = 15), cirrhosis (n = 15) and HCC (n = 30). The expression of AFP and GST-pi was detected by using immunohistochemistry with the peroxidase-antiperoxidase method. AFP immunoreactivity was based on the cytoplasm of the hepatocytes, while GST-pi immunoreactivity was based on the nuclei of hepatocytes. RESULTS: In normal liver tissues, AFP was not expressed. However, there was strong staining of GST-pi in bile duct epithelium cells and weak staining in hepatocytes. Our results showed higher AFP immunoreactivity in cases of HCC (36.7%) as compared to cirrhosis (6.7%) and hepatitis B (0%), whereas GST-pi immunoreactivity was lower in cases of HCC (53.3%) as compared to cases of cirrhosis (100.0%) and hepatitis B (93.3%). Percent sensitivity of AFP determination for HCC was 36.7% as compared to 53.3% for GST-pi, thus making GST-pi a more sensitive marker for detection of HCC. This study showed a significant relationship between the intensity and percentage of cells stained in hepatitis B, cirrhosis and HCC for GST-pi immunoreactivity (P < .001, .001 and .05, respectively) but not for AFP (P > .05). Statistical analysis showed that there was no significant relationship between expression of AFP and GST-pi in cirrhosis and HCC cases. Hepatitis B virus infection in HCC cases showed a positive rate of 46.7%, with AFP staining positively in 42.9% of tissues and GST-pi staining positively in 57.1% of tissues. CONCLUSION: AFP is a diagnostic but rather insensitive tissue marker for HCC. However, the absence of AFP in benign chronic liver disease makes this marker useful in differentiating between HCC and other chronic liver diseases, whereas GST-pi can be used as a diagnostic marker for HCC as well as in detecting other chronic liver diseases.     

The optimal sequence of microvascular repair during prolonged clamping in free flap transfer

During free flap transfer, the surgeon may decide to begin with repair of the artery or the vein(s) and to unclamp the first vessel as soon as repair is completed or maintain the clamping of both vessels until completion of all repairs. Complications can lead to prolonged clamping times, potentially increasing the risk of tissue ischemia, vascular damage, and thrombosis. The goals of the present study were to determine whether the sequence of vessel repair and the duration of clamping affect the success of free flap transfer in cases requiring prolonged clamping. Sixty abdominal fasciocutaneous free flaps based on the superficial inferior epigastric vessels were created in Sprague-Dawley rats. To model clinical situations in which prolonged clamping is necessary, the study used a 1-hour delay before the repair of the second vessel. Flaps were randomized into four groups. In group I (n = 15), the artery was repaired first, and the arterial clamp was removed immediately to allow arterial inflow. In group II (n = 15), the arterial repair was first, and the arterial clamp was maintained until completion of venous repair. In group III (n = 15), venous repair was first, with venous clamping maintained until completion of the arterial repair. In group IV (n = 15), initial venous repair was followed by immediate unclamping, before arterial repair. On release of all clamps, the patency of arteries and veins was confirmed immediately and after 1 hour using a "milking" test. On the fifth postoperative day, each flap was assessed for necrosis and for patency of the anastomoses. Of 15 flaps in each group, five (33 percent) failed in group I, four (27 percent) failed in groups II and III, and six (40 percent) failed in group IV. Differences between groups were not statistically significant (p = 0.8). These results demonstrate that in cases requiring prolonged occlusive clamping (2 to 3 hours), factors such as venous congestion, possible clamp injury, and presence of static blood in contact with the new anastomosis have relatively equivalent contributions to the risk of failure. Accordingly, no advantage seems to be gained by beginning with the artery or the vein or by using early or delayed unclamping of the first vessel repaired.     

Human galanin expresses amphipathic properties that modulate its vasoreactivity in vivo

The purpose of this study was to determine whether human galanin, a pleiotropic 30-amino acid neuropeptide, expresses amphipathic properties in vitro and, if so, whether these properties modulate its vasoactive effects in the intact peripheral microcirculation. We found that human galanin aggregates in an aqueous solution and forms micelles with a critical micellar concentration (CMC) of 0.4 microM. In addition, the peptide interacted with model membrane as indicated by long and significant increase of the surface pressure of the biomimetic monolayer membrane in vitro. Interactions of human galanin with sterically stabilized phospholipid micelles (SMM) were not associated with a significant change in peptide conformation. Using intravital microscopy, we found that suffusion of human galanin alone elicited significant concentration-dependent vasoconstriction in the intact hamster cheek pouch. This response was amplified when human galanin in SSM was suffused onto the cheek pouch. The effects of human galanin alone and in SSM were mediated by galanin receptors because galantide, a galanin receptor antagonist, abrogated galanin-induced vasoconstriction. Collectively, these data show that human galanin expresses amphipathic properties in the presence of phospholipids which in turn amplifies its vasoactive effects in the intact peripheral microcirculation.     

Targeted expression of placental lactogen in the beta cells of transgenic mice results in beta cell proliferation, islet mass augmentation, and hypoglycemia

The factors that regulate pancreatic beta cell proliferation are not well defined. In order to explore the role of murine placental lactogen (PL)-I (mPL-I) in islet mass regulation in vivo, we developed transgenic mice in which mPL-I is targeted to the beta cell using the rat insulin II promoter. Rat insulin II-mPL-I mice displayed both fasting and postprandial hypoglycemia (71 and 105 mg/dl, respectively) as compared with normal mice (92 and 129 mg/dl; p < 0.00005 for both). Plasma insulin concentrations were inappropriately elevated, and insulin content in the pancreas was increased 2-fold. Glucose-stimulated insulin secretion by perifused islets was indistinguishable from controls at 7.5, 15, and 20 mm glucose. Beta cell proliferation rates were twice normal (p = 0. 0005). This hyperplasia, together with a 20% increase in beta cell size, resulted in a 2-fold increase in islet mass (p = 0.0005) and a 1.45-fold increase in islet number (p = 0.0012). In mice, murine PL-I is a potent islet mitogen, is capable of increasing islet mass, and is associated with hypoglycemia over the long term. It can be targeted to the beta cell using standard gene targeting techniques. Potential exists for beta cell engineering using this strategy.     

Identification of an N-Terminal Region of Sigma 54 Required for Enhancer Responsiveness

Sigma 54 associates with bacterial core RNA polymerase and converts it into an enhancer-responsive enzyme. Deletion of the N-terminal 40 amino acids is known to result in loss of the ability to respond to enhancer binding proteins. In this work PCR mutagenesis and genetic screens were used to identify a small patch, from amino acids 33 to 37, that is required for proper response to activator in vivo. Site-directed single point mutants within this segment were constructed and studied. Two of these were defective in responding to the enhancer binding protein in vitro. The mutants could still direct the polymerase to bind to DNA and initiate transient melting. However, they failed in directing activator-dependent formation of a heparin-stable open complex. Thus, amino acid region 33 to 37 includes critical activation response determinants. This region overlaps the larger leucine patch negative-control region, suggesting that anti-inhibition and positive activation are closely coupled events.     

Ovalbumin increases macromolecular efflux from the in situ nasal mucosa of allergic hamsters

Gao, Xiao-Pei, Syed R. Akhter, and Israel Rubinstein.Ovalbumin increases macromolecular efflux from the in situ nasal mucosa of allergic hamsters. J. Appl.Physiol. 84(1): 169-176, 1998.The purpose ofthis study was to determine whether bradykinin mediatesovalbumin-induced increase in macromolecular efflux from the nasalmucosa of ovalbumin-sensitized hamsters in vivo and, if so, whether theL-arginine/nitric oxidebiosynthetic pathway transduces, in part, this response. We found thatsuffusion of ovalbumin onto the in situ nasal mucosa ofovalbumin-sensitized hamsters, but not of controls, elicited asignificant time- and concentration-dependent increase in clearance offluorescein isothiocyanate-labeled dextran (mol mass, 70 kDa;P < 0.05). HOE-140, but notdes-Arg⁹,[Leu⁸]-bradykinin,andN^G-L-argininemethyl ester (L-NAME), but notN^G-D-argininemethyl ester, significantly attenuated ovalbumin-induced responses.L-Arginine, but notD-arginine, abolished the effects ofL-NAME.L-NAME also significantlyattenuated bradykinin-, but not adenosine- induced increase inmacromolecular efflux from the in situ nasal mucosa. Overall, thesedata suggest that ovalbumin increases macromolecular efflux from the insitu nasal mucosa of ovalbumin-sensitized hamsters, in part, byproducing bradykinin with subsequent activation of theL-arginine/nitric oxidebiosynthetic pathway.

     

A Ca2+‐binding protein with numerous roles and uses: parvalbumin in molecular biology and physiology

Parvalbumins (PVs) are acidic, intracellular Ca²⁺‐binding proteins of low molecular weight. They are associated with several Ca²⁺‐mediated cellular activities and physiological processes. It has been suggested that PV might function as a “Ca²⁺ shuttle” transporting Ca²⁺ from troponin‐C (TnC) to the sarcoplasmic reticulum (SR) Ca²⁺ pump during muscle relaxation. Thus, PV may contribute to the performance of rapid, phasic movements by accelerating the contraction–relaxation cycle of fast‐twitch muscle fibers. Interestingly, PVs promote the generation of power stroke in fish by speeding up the rate of relaxation and thus provide impetus to attain maximal sustainable speeds. However, immunological monitoring of diverse tissues demonstrated that PVs are also present in non‐muscle cells. The axoplasmic transport and various intracellular secretory mechanisms including the endocrine secretions seem to be controlled by the Ca²⁺ regulation machinery. Any defect in the Ca²⁺ handling apparatus may cause several clinical problems; for instance, PV deficiency alters the neuronal activity, a key mechanism leading to epileptic seizures. Moreover, atypical relaxation of the heart results in diastolic dysfunction, which is a major cause of heart failure predominantly among the aged people. PV may offer a unique potential to correct defective relaxation in energetically compromised failing hearts through PV gene transfer. Consequently, PV gene transfer may present a new therapeutic approach to correct cellular disturbances in Ca²⁺ signaling pathways of diseased organs. Hence, PVs appear to be amazingly useful candidate proteins regulating a variety of cellular functions through action on Ca²⁺ flux management.