Chemical Synthesis and Cloning of a Gene Coding for Bacillus Subjilis Hbsu Protein

Abstract

A gene coding for Bacillus subtilis non-specifically DNA-binding HBsu protein has been chemically synthesized and cloned. The gene was designed to permit expression of the encoded protein in several ways.     

Dietary Regimens Modify Early Onset of Obesity in Mice Haploinsufficient for Rai1

Smith-Magenis syndrome is a complex genomic disorder in which a majority of individuals are obese by adolescence. While an interstitial deletion of chromosome 17p11.2 is the leading cause, mutation or deletion of the RAI1 gene alone results in most features of the disorder. Previous studies have shown that heterozygous knockout of Rai1 results in an obese phenotype in mice and that Smith-Magenis syndrome mouse models have a significantly reduced fecundity and an altered transmission pattern of the mutant Rai1 allele, complicating large, extended studies in these models. In this study, we show that breeding C57Bl/6J Rai1^+/− mice with FVB/NJ to create F1 Rai1^+/− offspring in a mixed genetic background ameliorates both fecundity and Rai1 allele transmission phenotypes. These findings suggest that the mixed background provides a more robust platform for breeding and larger phenotypic studies. We also characterized the effect of dietary intake on Rai1^+/− mouse growth during adolescent and early adulthood developmental stages. Animals fed a high carbohydrate or a high fat diet gained weight at a significantly faster rate than their wild type littermates. Both high fat and high carbohydrate fed Rai1^+/− mice also had an increase in body fat and altered fat distribution patterns. Interestingly, Rai1^+/− mice fed different diets did not display altered fasting blood glucose levels. These results suggest that dietary regimens are extremely important for individuals with Smith- Magenis syndrome and that food high in fat and carbohydrates may exacerbate obesity outcomes.     

Evidence of residual force enhancement for multi-joint leg extension

Force enhancement is a well accepted property of skeletal muscle and has been observed at all structural levels ranging from single myofibrils to voluntarily activated m. quadriceps femoris in vivo. However, force enhancement has not been studied for multi-joint movements like human leg extension; therefore knowledge about its relevance in daily living remains limited. The purpose of this study was to determine whether there is force enhancement during maximal voluntary multi-joint leg extension. Human leg extension was studied (n=22) on a motor driven leg press dynamometer where external reaction forces under the feet as well as activity of 8 lower extremity muscles were measured. In addition, torque in the ankle and knee joints was calculated using inverse dynamics. The steady-state isometric force, joint torques, and muscle activation after active stretch (20° stretch amplitude at 60°/s) were compared with the corresponding values obtained during isometric reference contractions. There was consistent force enhancement during and following stretch for both forces and joint torques. Potentiation during stretch reached values between 26% and 30%, while a significant force enhancement of 10.5–12.3% and 4.3–7.4% remained 0.5–1 and 2.5–3 s after stretch, respectively. During stretch, EMG signals of m. gastrocnemius medialis and lateralis were significantly increased, while following stretch all analyzed muscles showed the same activity as during the reference contractions. We conclude from these results that force enhancement exists in everyday movements and should be accounted for when analyzing or modelling human movement.     

Suppression of Electron Transfer to Dioxygen by Charge Transfer and Electron Transfer Complexes in the FAD-dependent Reductase Component of Toluene Dioxygenase

The three-component toluene dioxygenase system consists of an FAD-containing reductase, a Rieske-type [2Fe-2S] ferredoxin, and a Rieske-type dioxygenase. The task of the FAD-containing reductase is to shuttle electrons from NADH to the ferredoxin, a reaction the enzyme has to catalyze in the presence of dioxygen. We investigated the kinetics of the reductase in the reductive and oxidative half-reaction and detected a stable charge transfer complex between the reduced reductase and NAD⁺ at the end of the reductive half-reaction, which is substantially less reactive toward dioxygen than the reduced reductase in the absence of NAD⁺. A plausible reason for the low reactivity toward dioxygen is revealed by the crystal structure of the complex between NAD⁺ and reduced reductase, which shows that the nicotinamide ring and the protein matrix shield the reactive C4a position of the isoalloxazine ring and force the tricycle into an atypical planar conformation, both factors disfavoring the reaction of the reduced flavin with dioxygen. A rapid electron transfer from the charge transfer complex to electron acceptors further reduces the risk of unwanted side reactions, and the crystal structure of a complex between the reductase and its cognate ferredoxin shows a short distance between the electron-donating and -accepting cofactors. Attraction between the two proteins is likely mediated by opposite charges at one large patch of the complex interface. The stability, specificity, and reactivity of the observed charge transfer and electron transfer complexes are thought to prevent the reaction of reductase_TOL with dioxygen and thus present a solution toward conflicting requirements.     

Breakdown of exogenous insulin by langerhans islets of the pancreas in vitro

Pancreatic islets were isolated from Wistar rats, albino mice, spiny mice and sand rats (Psammomys obesus). Evidence is presented that pancreatic islets contain an enzyme system degrading insulin in the presence of glutathione or other sulfhydryl-containing compounds. Apparent K_m values for insulin and glutathione (in the presence of EDTA) are 14.0 μM (mol. wt 5700) and 1.28 mM, respectively. Maximum breakdown of ¹²⁵I-labeled insulin was found at about pH 7.2. After ultracentrifugation of islet homogenates the microsomal fraction contained the greatest relative specific insulin-degrading activity. The specific insulin-degrading actvitity was found to be higher in Wistar rats and albino mice than in spiny mice and sand rats. Starvation of Wistar rats for 72 h caused a decrease inthe enzymatic activity.     

Investigations on isolated islets of Langerhans in vitro. Influence of temperature changes during preparation on some parameters of insulin metabolism.

Pancreatic islets of Wistar rats were prepared by digestion with collagenase and then washed and isolated at three different temperatures (4, 22 and 37 degrees C). The efficiency of washing with regard to proteolytic and collagenolytic activities in the wash buffer was not affected by the temperatures used. The islet thiol:protein-disulphide oxidoreductase activity (EC 1.8.4.2) was apparently unchanged, whereas washing temperatures lower than 37 degrees C resulted in a diminished insulin content. The insulin secretion of islets, isolated at 4 degrees C, is reduced in response to glucose without changing the sigmoidal shape of dose-response curve.     

Sindbis virus ts103 has a mutation in glycoprotein E2 that leads to defective assembly of virions.

Sindbis virus mutant ts103 is aberrant in the assembly of virus particles. During virus budding, proper nucleocapsid-glycoprotein interactions fail to occur such that particles containing many nucleocapsids are formed, and the final yield of virus is low. We have determined that a mutation in the external domain of glycoprotein E2, Ala-344----Val, is the change that leads to this phenotype. Mapping was done by making recombinant viruses between ts103 and a parental strain of the virus, using a full-length cDNA clone of Sindbis virus from which infectious RNA can be transcribed, together with sequence analysis of the region of the genome shown in this way to contain the ts103 lesion. A partial revertant of ts103, called ts103R, was also mapped and sequenced and found to be a second-site revertant in which a change in glycoprotein E1 from lysine to methionine at position 227 partially suppresses the phenotypic effects of the change at E2 position 344. An analysis of revertants from ts103 mutants in which the Ala----Val change had been transferred into a defined background showed that pseudorevertants were more likely to arise than were true revertants and that the ts103 change itself reverted very infrequently. The assembly defect in ts103 appeared to result from weakened interactions between the virus membrane glycoproteins or between these glycoproteins and the nucleocapsid during budding. Both the E2 mutation leading to the defect in virus assembly and the suppressor mutation in glycoprotein E1 are in the domains external to the lipid bilayer and thus in domains that cannot interact directly with the nucleocapsid. This suggests that in ts103, either the E1-E2 heterodimers or the trimeric spikes (consisting of three E1-E2 heterodimers) are unstable or have an aberrant configuration, and thus do not interact properly with the nucleocapsid, or cannot assembly correctly to form the proper icosahedral array on the surface of the virus.