Partial purification of a competence factor from Rhizobium japonicum

A competence factor (CF) from Rhizobium japonicum was partially purified to 43 fold on Sephadex G-100. This CF preparation was sensitive to heat, trypsin and pronase, was resistant to DNase 1, RNase A and lysozyme. It had an approximate mol. wt. of 82,000. Osmotic shock treatment of competent cells revealed that the CF is located in the periplasmic region of the cell.Abbreviations CF competence factor - BSA bovine serum albumin - YM yeast mannitol medium     

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.     

Analysis of 31P NMR spectra of enzyme-bound reactants and products of adenylate kinase using density matrix theory of chemical exchange

31P NMR spectra of equilibrium mixtures of enzyme-bound reactants and products of the adenylate kinase reaction (formula; see text) were analyzed by using computer simulations based on density matrix theory of chemical exchange. Since adenylate kinase has the unique feature that the reactants in the reverse direction are both ADP molecules, which are indistinguishable off the enzyme, the density matrix equations are formulated for the ABC + D in equilibrium A'B' + A"B" exchange appropriate for the reaction, in which the interchange of A'B' and A"B" is explicitly introduced. It is shown that the consideration of this interchange is essential to explain the experimentally observed line shapes. By comparison of the computer-simulated spectra with various values for the rates of the exchange with the experimental spectra for porcine adenylate kinase at pH 7.0 and T = 4 degrees C, the following characteristic rates were determined: interconversion rates, 375 +/- 30 s-1 (ATP formation) and 600 +/- 50 s-1 (ADP formation); interchange rates of donor and acceptor ADP's, 100 +/- 30 s-1 (in the presence of optimal Mg2+ concentration), 1500 +/- 100 s-1 (in the absence of Mg2+). It is shown that under the conditions of the experiments the interchange rate is the lower limit of the dissociation rate of ADP (or MgADP from the acceptor site if Mg2+ was present) from the enzyme complexes. The significance of these interchange rates and their values relative to the interconversion rates is discussed with special reference to the role of the Mg2+ ion in the differentiation of the two nucleotide binding sites on adenylate kinase.     

Developmental expression of Pop1/Bves.

Initial studies have suggested that Pop1/Bves protein is exclusively expressed in the smooth muscle walls of the coronary vessels, implying its possible importance in coronary diseases. However, the mRNA and activity of this gene are detected in both skeletal and cardiac muscles, not coronary smooth muscle, and Pop1/Bves knockout mice have defects in skeletal muscle regeneration. Here we used specific monoclonal antibodies (MAbs) raised against chicken Pop1/Bves and demonstrated the presence of this protein in cardiomyocytes through development and its apparent absence in coronary vessels. Immunostaining of cardiomyocytes cultured in vitro confirmed the membrane localization of this protein in cells that participate in cell adhesion, with significant intracellular staining seen in isolated cells. In skeletal muscle, Pop1 protein becomes detectable at embryonic day (E) 7, coincident with the differentiation of morphologically distinct muscle masses from the limb muscle blastema, but the protein is not found at high levels in the cell membrane of myotubes until E11, coincident with the formation of secondary myotubes from satellite cells. These data support the hypothesis that Pop1/Bves is a cell adhesion molecule present in skeletal and cardiac muscle.     

Unusual Pauses In Sinus rhythm And Intermittent 2 to 1 AV Block In A Patient With Concealed His Extra Systoles - A Rare Cause Of Bradycardia

A 56 year old male with a past medical history of hypertension and dyslipidemia presented with recurrent dizziness. Routine EKG was performed, which suggested frequent junctional extra systoles with compensatory pauses. During telemetry periods of 2:1 block with effective ventricular rate of 34 bpm was observed. His bundle study suggested frequent His extra systoles causing functional AV block. Treatment with anti-arrhythmic medication, paradoxically improved AV block and symptoms in our patient.     

Neck muscle paths and moment arms are significantly affected by wrapping surface parameters

In this paper, we studied the effects of wrapping surfaces on muscle paths and moment arms of the neck muscle, semispinalis capitis. Sensitivities to wrapping surface size and the kinematic linkage to vertebral segments were evaluated. Kinematic linkage, but not radius, significantly affected the accuracy of model muscle paths compared to centroid paths from images. Both radius and linkage affected the moment arm significantly. Wrapping surfaces that provided the best match to centroid paths over a range of postures had consistent moment arms. For some wrapping surfaces with poor matches to the centroid path, a kinematic method (tendon excursion) predicted flexion moment arms in certain postures, whereas geometric method (distance to instant centre) predicted extension. This occurred because the muscle lengthened as it wrapped around the surface. This study highlights the sensitivity of moment arms to wrapping surface parameters and the importance of including multiple postures when evaluating muscle paths and moment arm.     

Determination of dideoxyosone precursors of AGEs in human lens proteins

Dideoxyosones (DDOs) are intermediates in the synthesis of advanced glycation endproducts (AGEs), such as pentosidine and glucosepane. Although the formation of pentosidine and glucosepane in the human lens has been firmly established, the formation of DDOs has not been demonstrated. The aim of this study was to develop a reliable method to detect DDOs in lens proteins. A specific DDO trapping agent, biotinyl-diaminobenzene (3,4-diamino-N-(3-[5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl]aminopropyl)benzamide) (BDAB) was added during in vitro protein glycation or during protein extraction from human lenses. In vitro glycated human lens protein showed strong reaction in monomeric and polymeric crosslinked proteins by Western blot and ELISA. Glycation of BSA in the presence of BDAB resulted in covalent binding of BDAB to the protein and inhibited pentosidine formation. Mass spectrometric analysis of lysozyme glycated in the presence of BDAB showed the presence of quinoxalines at lysine residues at positions K1, K33, K96, and K116. The ELISA results indicated that cataractous lens proteins contain significantly higher levels of DDO than non-cataractous lenses (101.9±67.8 vs. 31.7±19.5AU/mg protein, p<0.0001). This study provides first direct evidence of DDO presence in human tissue proteins and establishes that AGE crosslink synthesis in the human lens occurs via DDO intermediates.