Immunohistochemical localization of porcine diazepam-binding inhibitor (DBI) to rat endocrine pancreas

Summary The occurrence of diazepam-binding inhibitor (DBI), isolated and characterized from porcine upper intestine, was examined in the pancreas of Sprague-Dawley albino rats using indirect immunofluorescence. The polypeptide was found in the endocrine Langerhans islets and, utilizing double-labelling controls, it was shown to be present within the peripherally located glucagon-containing cells. Regulation of islet hormone production may therefore be under DBI control.     

Dibutyryl cyclic AMP decreases the rate of lipoprotein lipase synthesis in cultured adipocytes

The regulation of avian lipoprotein lipase by dibutyryl cyclic AMP in cultured adipocytes was studied with quantitative and specific methods for the measurements of enzyme catalytic activity, enzyme protein mass, and immunoadsorption of labeled enzyme. Incubation of adipocytes in 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline results in a time-dependent decrease in cell lipoprotein lipase catalytic activity. The activity is decreased by 70% in 4 h and over 90% by 12 h. The decrease in cellular catalytic activity is due to a decrease in both enzyme content and enzyme catalytic efficiency. 4 h after exposure of adipocytes to cAMP, enzyme protein was decreased from 3.58 +/- 0.5 to 1.92 +/- 0.1 ng/dish and specific activity from 15.1 +/- 2.1 to 8.4 +/- 1.1 nmol/ng. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in lipoprotein lipase activity was half-maximal at less than 25 microM dibutyryl cyclic AMP. The rate of lipoprotein lipase synthesis was estimated by measuring the incorporation of L-[35S]methionine into enzyme protein during 30 min. A method for the quantitative immunoadsorption of lipoprotein lipase from cell lysates was developed. Utilizing this immunoadsorption technique, the rate of incorporation of L-[35S]methionine into lipoprotein lipase was 0.0026 +/- 0.002%, when expressed as a percentage of that incorporated into total trichloroacetic acid-precipitable counts. By 2 h after exposure of adipocytes to 0.5 mM dibutyryl cAMP, the relative synthesis rate had already decreased to 64 +/- 4% of the control rate. After 16 h the synthesis rate was 43.2 +/- 13.8% of the control rate. The observed decreased synthesis rate could account for most of the decreased cellular enzyme content and diminished enzyme secretion rate.     

Regulation of human leukocyte microsomal hydroxymethylglutaryl-CoA reductase activity by a phosphorylation and dephosphorylation mechanism

The activity of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), obtained from cultured human IM-9 lymphoid cells or freshly isolated human peripheral blood leukocytes, is modulated by a phosphorylation/dephosphorylation mechanism. Addition of MgATP + ADP to IM-9 cell microsomal reductase leads to a time-dependent loss of enzyme activity. Inactivated reductase is reactivated by rat liver reductase phosphatase. Kinase-dependent IM-9 cell microsomal reductase, prepared by heating IM-9 microsomes for 15 min at 50°C, is inactivated in the presence of MgATP and ADP only after addition of cytosolic reductase kinase from either IM-9 cells, freshly isolated leukocytes or rat liver. Inactivation is time-dependent and dependent on the cytosolic protein concentration. Inactivated reductase is reactivated by rat liver reductase phosphatase. For cultured IM-9 cells and freshly isolated leukocytes incubated with culture medium for 2 h, the ratios of active (unphosphorylated) to total (phosphorylated + unphosphorylated) reductase activity are 0.22 and 0.43, respectively. Thus, in addition to its regulation by changes in the amount of total enzyme protein, human leukocyte reductase activity is also modulated by a phosphorylation/dephosphorylation mechanism.     

Detection of Anorectal and Cervicovaginal Chlamydia Trachomatis Infections following Azithromycin Treatment: Prospective Cohort Study with Multiple Time-Sequential Measures of rRNA,DNA, Quantitative Load and Symptoms

Background

Determination of Chlamydia trachomatis (Ct) treatment success is hampered by current assessment methods, which involve a single post-treatment measurement only. Therefore, we evaluated Ct detection by applying multiple laboratory measures on time-sequential post-treatment samples.

Methods

A prospective cohort study was established with azithromycin-treated (1000 mg) Ct patients (44 cervicovaginal and 15 anorectal cases). Each patient provided 18 self-taken samples pre-treatment and for 8 weeks post-treatment (response: 96%; 1,016 samples). Samples were tested for 16S rRNA (TMA), bacterial load (quantitative PCR; Chlamydia plasmid DNA) and type (serovar and multilocus sequence typing). Covariates (including behavior, pre-treatment load, anatomic site, symptoms, age, and menstruation) were tested for their potential association with positivity and load at 3–8 weeks using regression analyses controlling for repeated measures.

Findings

By day 9, Ct positivity decreased to 20% and the median load to 0.3 inclusion-forming units (IFU) per ml (pre-treatment: 170 IFU/ml). Of the 35 cases who reported no sex, sex with a treated partner or safe sex with a new partner, 40% had detection, i.e. one or more positive samples from 3–8 weeks (same Ct type over time), indicating possible antimicrobial treatment failure. Cases showed intermittent positive detection and the number of positive samples was higher in anorectal cases than in cervicovaginal cases. The highest observed bacterial load between 3–8 weeks post-treatment was 313 IFU/ml, yet the majority (65%) of positive samples showed a load of ≤2 IFU/ml. Pre-treatment load was found to be associated with later load in anorectal cases.

Conclusions

A single test at 3–8 weeks post-treatment frequently misses Ct. Detection reveals intermittent low loads, with an unknown risk of later complications or transmission. These findings warrant critical re-evaluation of the clinical management of single dose azithromycin-treated Ct patients and fuel the debate on defining treatment failure. Clinicaltrials.gov Identifier: NCT01448876.     

Functional Characterization of Key Enzymes involved in l-Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

Bacillus methanolicus wild-type strain MGA3 secretes 59 g/liter⁻¹ of l-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in l-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low K_m value for ammonium (10 mM) and a high K_m value for l-glutamate (250 mM), and the V_max value is 7-fold higher for l-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar K_m values (1 to 1.4 mM) and V_max values (4 U/mg) for both l-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating l-glutamate production in this organism.     

Spatial and temporal coordination of traction forces in one-dimensional cell migration

ABSTRACT

Migration of a fibroblast along a collagen fiber can be regarded as cell locomotion in one-dimension (1D). In this process, a cell protrudes forward, forms a new adhesion, produces traction forces, and releases its rear adhesion in order to advance itself along a path. However, how a cell coordinates its adhesion formation, traction forces, and rear release in 1D migration is unclear. Here, we studied fibroblasts migrating along a line of microposts. We found that when the front of a cell protruded onto a new micropost, the traction force produced at its front increased steadily, but did so without a temporal correlation in the force at its rear. Instead, the force at the front coordinated with a decrease in force at the micropost behind the front. A similar correlation in traction forces also occurred at the rear of a cell, where a decrease in force due to adhesion detachment corresponded to an increase in force at the micropost ahead of the rear. Analysis with a bio-chemo-mechanical model for traction forces and adhesion dynamics indicated that the observed relationship between traction forces at the front and back of a cell is possible only when cellular elasticity is lower than the elasticity of the cellular environment.     

Quantitative detection of siRNA and single-stranded oligonucleotides: relationship between uptake and biological activity of siRNA

The quantitative detection of oligomeric nucleic acids including short double-stranded RNA in cells and tissues becomes increasingly important. Here, we describe a method for the detection of siRNA in extracts prepared from mammalian cells, which is based on liquid hybridization with a ³²P-labelled probe followed by a nuclease protection step. The limit of detection of absolute amounts of siRNA is in the order of 10–100 amol. This methodology is suited to quantitatively follow the spontaneous uptake of siRNA by mammalian cells, i.e. without the use of carrier substances. This protocol may also be used to detect extremely low amounts of other kinds of short nucleic acids, including antisense oligonucleotides.