Flow cytometric measurement of telomere length

The regulation of telomere length may be involved in the cellular physiology of senescence, reproduction, cancer, immune response to infection, and possibly immune deficiency. The measurement of telomere length, critical to research in this area, has traditionally been performed by Southern blot analysis, which is cumbersome and time consuming. Several alternative methods have been described in recent years. Some, such as pulsed-field electrophoresis, slot blots, and centromere-to-telomere ratio measurements are essentially improvements to the Southern blot technique. However, other methods such as fluorescent in situ hybridization on metaphase chromosome spreads and flow cytometry-based fluorescent in situ hybridization represent a completely new technical approach to the problem. In this review, we compare methods, with particular emphasis placed on flow cytometric techniques for measuring telomere length in situ and identifying potential areas where improvements may still be made.     

Transformation of Internal Extracellular Bacteria Isolated from Rhagoletis completa Cresson Gut with Enhanced Green Fluorescent Protein

We discovered Zeocin™ is an effective antibiotic against Enterobacter agglomerans and Klebsiella pneumoniae strains isolated from the walnut husk fly (Rhagoletis completa Cresson: Family Tephritidae) and that bleomycin resistance can be used as a selective marker in transforming plasmids. We transformed Ent. agglomerans and K. pneumoniae strains originally isolated from their close association with R. completa gut to produce enhanced green fluorescent protein, a variant of green fluorescent protein in the first demonstration of genetic transformation of internal extracellular bacteria isolated from a tephritid pest. We report methods for plasmid-mediated transformation of these bacteria, the expression of fluorescent marker protein from the transforming plasmids, and the stability of the transforming plasmid in the bacteria. We also discuss applications of this technology in the study of pest biology and control implementation. Received: 5 November 1999 / Accepted: 15 December 1999     

Synthesis and antimicrobial activity of 2-fluorophenyl-4,6-disubstituted [1,3,5]triazines

A series of 2-fluorophenyl-4,6-disubstituted [1,3,5]triazines (1) and (2) were synthesized and evaluated for their antimicrobial activity against three representative gram-positive bacteria and two fungi. The structure–activity relationship (SAR) demonstrates that the 3- or 4-fluorophenyl component attached directly to the triazine ring was essential for activity. Of these compounds, 14, 15, and 25 demonstrated significant activity against all selected organisms compared to control. These compounds were generally nontoxic and may prove useful as antimicrobial agents.     

Similar hypotensive responses to resistance exercise with and without blood flow restriction

Low intensity resistance exercise (RE) with blood flow restriction (BFR) has gained attention in the literature due to the beneficial effects on functional and morphological variables, similar to those observed during traditional RE without BFR, while the effects of BFR on post-exercise hypotension remain unclear. The aim of the present study was to compare the blood pressure (BP) response of trained normotensive individuals to RE with and without BFR. In this cross-over randomized trial, eight male subjects (23.8 ± 4 years, 74 ± 3 kg, 174 ± 4 cm) completed two exercise protocols: traditional RE (3 x 10 repetitions at 70% one-repetition maximum [1-RM]) and low intensity RE (3 x 15 repetitions at 20% 1-RM) with BFR. Blood pressure measurements were performed after 15 min of seated rest (0), immediately after and 10 min, 20 min, 30 min, 40 min, 50 min and 60 min after the experimental sessions. Similar hypotensive effects for systolic BP (SBP) were observed for both protocols (P < 0.05) after exercise, with no differences between groups (P > 0.05) and no statistically significant difference for diastolic BP (P > 0.05). These results suggest that in normotensive trained individuals, both traditional RE and RE with BFR induce hypotension for SBP, which is important to prevent cardiovascular disturbances.     

Phosphate and ADP Differently Inhibit Coordinated Smooth Muscle Myosin Groups

Actin filaments propelled in vitro by groups of skeletal muscle myosin motors exhibit distinct phases of active sliding or arrest, whose occurrence depends on actin length (L) within a range of up to 1.0 μm. Smooth muscle myosin filaments are exponentially distributed with ≈150 nm average length in vivo—suggesting relevance of the L-dependence of myosin group kinetics. Here, we found L-dependent actin arrest and sliding in in vitro motility assays of smooth muscle myosin. We perturbed individual myosin kinetics with varying, physiological concentrations of phosphate (P_i, release associated with main power stroke) and adenosine diphosphate (ADP, release associated with minor mechanical step). Adenosine triphosphate was kept constant at physiological concentration. Increasing [P_i] lowered the fraction of time for which actin was actively sliding, reflected in reduced average sliding velocity (ν) and motile fraction (f_mot, fraction of time that filaments are moving); increasing [ADP] increased the fraction of time actively sliding and reduced the velocity while sliding, reflected in reduced ν and increased f_mot. We introduced specific P_i and ADP effects on individual myosin kinetics into our recently developed mathematical model of actin propulsion by myosin groups. Simulations matched our experimental observations and described the inhibition of myosin group kinetics. At low [P_i] and [ADP], actin arrest and sliding were reflected by two distinct chemical states of the myosin group. Upon [P_i] increase, the probability of the active state decreased; upon [ADP] increase, the probability of the active state increased, but the active state became increasingly similar to the arrested state.     

Renin inhibitors for the treatment of hypertension: design and optimization of a novel series of tertiary alcohol-bearing piperidines

The design and optimization of a novel series of renin inhibitor is described herein. Strategically, by committing the necessary resources to the development of synthetic sequences and scaffolds that were most amenable for late stage structural diversification, even as the focus of the SAR campaign moved from one end of the molecule to another, highly potent renin inhibitors could be rapidly identified and profiled.     

Melanocortin-4 receptors expressed by cholinergic neurons regulate energy balance and glucose homeostasis

Melanocortin-4 receptor (MC4R) mutations cause dysregulation of energy balance and hyperinsulinemia. We have used mouse models to study the physiological roles of extrahypothalamic MC4Rs. Re-expression of MC4Rs in cholinergic neurons (ChAT-Cre, loxTB MC4R mice) modestly reduced body weight gain without altering food intake and was sufficient to normalize energy expenditure and attenuate hyperglycemia and hyperinsulinemia. In contrast, restoration of MC4R expression in brainstem neurons including those in the dorsal motor nucleus of the vagus (Phox2b-Cre, loxTB MC4R mice) was sufficient to attenuate hyperinsulinemia, while the hyperglycemia and energy balance were not normalized. Additionally, hepatic insulin action and insulin-mediated suppression of hepatic glucose production were improved in ChAT-Cre, loxTB MC4R mice. These findings suggest that MC4Rs expressed by cholinergic neurons regulate energy expenditure and hepatic glucose production. Our results also provide further evidence of the dissociation in pathways mediating the effects of melanocortins on energy balance and glucose homeostasis.     

Modeling the oscillation dynamics of activated airway smooth muscle strips

When strips of activated airway smooth muscle are stretched cyclically, they exhibit force-length loops that vary substantially in both position and shape with the amplitude and frequency of the stretch. This behavior has recently been ascribed to a dynamic interaction between the imposed stretch and the number of actin-myosin interactions in the muscle. However, it is well known that the passive rheological properties of smooth muscle have a major influence on its mechanical properties. We therefore hypothesized that these rheological properties play a significant role in the force-length dynamics of activated smooth muscle. To test the plausibility of this hypothesis, we developed a model of the smooth muscle strip consisting of a force generator in series with an elastic component. Realistic steady-state force-length loops are predicted by the model when the force generator obeys a hyperbolic force-velocity relationship, the series elastic component is highly nonlinear, and both elastic stiffness and force generation are adjusted so that peak loop force equals isometric force. We conclude that the dynamic behavior of airway smooth muscle can be ascribed in large part to an interaction between connective tissue rheology and the force-velocity behavior of contractile proteins.