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.     

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.     

Head and neck anthropometry, vertebral geometry and neck strength in height-matched men and women

Women have an increased incidence of whiplash injury and neck pain compared to men. Physical and numerical models represent one avenue to explore and potentially explain these gender differences, but a valid model of the female neck does not yet exist. A fundamental question in the development of a female neck model is whether female necks are simply scaled versions of male necks, or whether there are significant inter-gender geometrical differences. The goal of this study was to quantify differences in head and neck geometry and neck strength in pairs of male and female subjects matched for standing height and neck length. Based on 14 matched pairs of men and women, we found that most head and neck anthropometric parameters were significantly smaller in females compared to males. Moreover, gender differences in a number of neck anthropometry parameters (an average of 9-16% smaller in females) were larger than differences in head anthropometry parameters (an average of 3-6% smaller in females). Female vertebrae between C3 and C7 were significantly smaller than male vertebrae in the anterior-posterior dimension (p < 0.012) but not in the medial-lateral dimension (p > 0.07). Female necks were also significantly weaker than male necks (32% weaker in flexion and 20% weaker in extension; p < 0.001), and these strength differences corresponded well to those predicted solely from the observed geometric differences. These results demonstrate that male and female necks are not geometrically similar and indicate that a female-specific model will be necessary to study gender differences in neck-related disorders.     

Identification of cis sequences controlling efficient position-independent tissue-specific expression of human major histocompatibility complex class I genes in transgenic mice.

We previously reported that genomic major histocompatibility complex class I human leukocyte antigen (HLA)-B7 gene constructs with as little as 0.66 kb of 5'- and 2.0 kb of 3'-flanking DNA were expressed efficiently and appropriately in transgenic mice. To identify and characterize the relevant cis-acting regulatory elements in more detail, we have generated and analyzed a series of transgenic mice carrying native HLA-B7 genes with further 5' truncations or intronic deletions and hybrid constructs linking the 5'-flanking region of B7 to a reporter gene. We were unable to detect a specific requirement for sequence information within introns 2 to 7 for either appropriate constitutive or inducible class I expression in adult animals. The results revealed the presence of cis-acting regulatory sequences between -0.075 kb and -0.66 kb involved in driving efficient copy number-dependent constitutive and gamma interferon-enhanced tissue-specific expression. The region from -0.11 to -0.66 kb is also sufficient to prevent integration site-specific "position effects," because in its absence HLA-B7 expression is frequently detected at significant levels at inappropriate sites. Conserved sequence elements homologous to the H-2 class I regulatory element, or enhancer A, and the interferon response sequence are located between about -151 and -228 bp of the B7 gene. Our results also indicate the existence of sequences downstream of -0.11 kb which can influence the pattern of tissue-specific expression of the HLA-B7 gene and the ability of this gene to respond to gamma interferon.     

Production of 3-acetyldeoxynivalenol by Fusarium graminearum R 2118 in submerged cultures

Summary Production of of 3-acetyldeoxynivalenol (3-ADN) by Fusarium graminearum R 2118 in submerged cultures was characterized for five different media. Toxin production was examined as a function of mycelial growth, sugar utilization, pH and phosphate concentration. In submerged cultures, 3-ADN appeared after 2 days of incubation at 25°C when mycelial growth had slowed down and the pH of the media had dropped to 4.5 or lower. A two stage process was developed for high and rapid production of 3-ADN, in which the biosynthetically active mycelium was grown in a yeast extract-peptone-sucrose medium and the toxin was produced by the mycelium in a sucrose containing minimal medium. Yields of 90–110 mg/l were obtained within 5 days in the production medium. Acidity (low pH) and low phosphate concentration in the minimal medium were both required for 3-ADN production, representing two independent regulating factors for the 3-ADN biosynthesis.     

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.     

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.     

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.