Rapid chromosome territory relocation by nuclear motor activity in response to serum removal in primary human fibroblasts

Background  

Radial chromosome positioning in interphase nuclei is nonrandom and can alter according to developmental, differentiation, proliferation, or disease status. However, it is not yet clear when and how chromosome repositioning is elicited.     

Studies of the process of renaturation and assembly of Escherichia coli succinyl-CoA synthetase from its alpha and beta subunits

Succinyl-CoA synthetase catalyzes the substrate-level phosphorylation step of the tricarboxylic acid cycle. The enzyme, as isolated from Escherichia coli, has an alpha 2 beta 2 subunit structure. It is known that substrate-binding sites are distributed between both subunit types and that the active enzyme is the nondissociating tetramer. This paper describes a study of the process of assembly of the enzyme from its denatured constituent subunits. Starting with equimolar mixtures of the subunits that are prepared in denaturing conditions (6 M urea, 5% acetic acid), rapid renaturation to produce virtually a fully active enzyme occurs after neutralization and dilution under suitable conditions. This process occurs most efficiently in the presence of either ATP or Pi, indicating that occupation of the phosphoryl-binding site on the refolding alpha subunit facilitates productive intrasubunit interactions. We have determined conditions of protein concentration, pH, temperature, final urea concentration, and buffer compositions that optimize both the rate and extent of production of active enzyme. The final refolded product is indistinguishable from the native species with respect to its specific catalytic activity, size, and other physical properties. To probe further the mechanism and route of renaturation, we have shown that the rate of appearance of activity has first-order dependence on each of the two subunits. The step that determines the rate of assembly is thus bimolecular, such as the association of structural monomers to form a dimeric transient species. The highly specific mutual interactions between the refolding transient species of subunits must be essential for the correct assembly of this enzyme from the two gene products in vivo.     

Place conditioning with morphine and phencyclidine: dose dependent effects

In a place conditioning paradigm, rats were exposed to one of two distinctive environments following injection of drug or vehicle. Preference was measured under drug free conditions by allowing subjects free access to both settings and measuring where they spent more time. Comparisons were made between morphine and saline; PCP and saline; and one of several doses of morphine and a standard dose. Morphine was preferred over saline and, when compared to the reference dose, lower doses of morphine were less preferred and higher doses more preferred. PCP was never preferred over saline and under some conditions produced a conditioned place aversion. The ability to generate dose dependent effects with morphine should allow more sophisticated studies in which shifts in dose response curves are required.     

Classical conditioning, decay and extinction of cocaine-induced hyperactivity and stereotypy

Following 10 daily pairings of multiple conditioned stimuli with injection of cocaine (15 mg/kg), the presentation of the stimuli alone elicited behaviors in rats similar to those induced by cocaine. The behaviors included increased duration or frequency of rearing, sniffing, head bobbing, and horizontal locomotor activity (crossing). The level of the conditioned response for several of these behaviors approximated that induced by the drug itself. The conditioned drug effect showed decay over 15 days but little extinction during 4 daily trials. Brain concentrations of the dopamine metabolites, homovanillic acid and dihydroxyphenylacetic acid, were similar in the conditioned and pseudoconditioned control groups in both the caudate and mesolimbic areas. The behavioral results demonstrate that, in a classical conditioning paradigm, previously neutral stimuli can elicit behaviors similar to those induced by cocaine and that certain conditioned responses show time related decline. This agrees with the reported conditioning of amphetamine's behavioral effects but differs in terms of the action on brain dopamine turnover.     

Preadipocyte Recruitment in Stromal Vascular Cultures After Depletion of Committed Preadipocytes by Immunocytotoxicity

Glucocorticoids or the glucocorticoid analog dexamethasone (DEX) enhances the differentiation of preadipocytes in the presence of insulin and influences preadipocyte proliferation. The purpose of the present study was to determine if DEX can induce the recruitment of preadipocytes. Using monoclonal antibodies for complement-mediated cytotoxicity, preadipocytes were removed from porcine stromal vascular (S-V) cell cultures. Our experiments demonstrated for the first time that after removal of preadipocytes by cytotoxicity, preadipocytes or fat cells could be induced by DEX or DEX plus insulin but not by insulin alone. However, many more fat cells were induced (258 ± 15/unit area) when DEX was added with fetal bovine serum (FBS) followed with insulin treatment, compared to DEX with insulin (21.3 ± 5.1/ unit area) after removal of preadipocytes. Immunocyto-chemistry with AD-3, a preadipocyte marker, showed that DEX with FBS for 3 days after seeding (i.e., the proliferation phase) produced many more preadipocytes (AD-3 positive, 223 ± 45/unit area) than FBS alone (10.5 ± 1.4/unit area). Bromodeoxyuridine (BrdU) incorporation assays demonstrated that the efficiency of DEX with FBS (i.e., during proliferation) was mitosis dependent. Accordingly, we conclude that: porcine S-V cultures contain preadipocytes at different stages of differentiation and that DEX induced early preadipocyte differentiation depends on mitosis.     

On the monomeric structure and proposed regulatory properties of phosphoenolpyruvate carboxykinase of Escherichia coli.

Phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating)) (EC 4.1.1.49) has been purified to homogeneity from Escherichia coli. The enzyme shows the same molecular weight (ca. 65000) either by sedimentation equilibrium under nondenaturing conditions or by polyacrylamide gel electrophoresis in the presence of detergent, indicating that the enzyme has a monomeric structure. We have confirmed the previous observation that NADH is an inhibitor of this enzyme, but we have failed to detect the previously reported appearance of homotropic cooperativity with respect to substrate binding the presence of this inhibitor. Lack of such homotropic interactions is in harmony with our conclusion that the enzymes is a monomer. Replacement of Mg2+ by Mn2+ in the assay medium lowers the Km for phosphoenolpyruvate by an order of magnitude, but does not affect the characteristics of inhibition by NADH.     

Concordance of BAI and BMI with DXA in the Newfoundland Population

Background:

Body adiposity index (BAI), indirect method proposed to predict adiposity, was developed using Mexican Americans and very little data are available regarding its validation in Caucasian populations to date.

Objective:

The study objectives were to validate the BAI with dual‐energy X‐ray absorptiometry (DXA) body fat percentage (%BF), taking into consideration the gender and adiposity status.

Design and Methods:

A total of 2,601 subjects (Male 662, Female 1939) from our Complex Diseases in the Newfoundland population: Environment and Genetics (CODING) study participated in this investigation. Pearson correlations, with the entire cohort along with men and women separately, were used to compare the correlation of both BAI and BMI with %BF. Additionally, the concordance between BAI and BMI with %BF were also performed among normal‐weight (NW), overweight (OW), and obese (OB) groups. Adiposity status was determined by the Bray Criteria according to DXA %BF.

Results:

BAI performs better than BMI in our Caucasian population by: (1) reflecting the gender difference in total %BF between women and men, (2) correlating better with DXA %BF than BMI when women and men are combined, and (3) performing better in NW and OW subjects for both the sexes. However, BAI performs less effectively than BMI in OB men and women.

Conclusion:

In summary, the BAI method is a better estimate of adiposity than BMI in non‐OB subjects in our Caucasian population. A measurement sensitive to the changes in adiposity for both men and women is suggested to be incorporated into the present BAI equation to increase accuracy.