The development of new triazole based inhibitors of tumor necrosis factor-alpha (TNF-alpha) production

4-Aryl-5-pyridyl and 4-aryl-5-pyrimidyl based inhibitors of TNF-alpha production, which contain a novel triazole 5-member heterocyclic core, are described. Many pyridyl triazoles containing either an alkyl ether or a substituted aryl side chain on the triazole core showed sub-micromolar activity against LPS-induced TNF-alpha, while pyrimidyl triazoles containing an ethoxymethyl side chain exhibited even better inhibitory activity. Secondary screening data are presented for the pyrimidyl triazoles. Triazole 14e combined excellent potency with good oral bioavailability in the rat.     

The effect of density-dependent treatment and behavior change on the dynamics of HIV transmission

In this work, we propose a model for heterosexual transmission of HIV/AIDS in a population of varying size with an intervention program in which treatment and/or behavior change of the infecteds occur as an increasing function of the density of the infected class in the population. This assumption has socio-economic implications which is important for public health considerations since density-dependent treatment/behavior change may be more cost-saving than a program where treatment/behavior change occurs linearly with respect to the number of infecteds. We will make use of the conservation law of total sexual contacts which enables us to reduce the two-sex model to a simpler one-sex formulation. Analytical results will be given. Unlike a similar model with linear treatment/behavior change in Hsieh (1996) where conditions were obtained for the eradication of disease, we will show that density-dependent treatment/behavior change cannot eradicate the disease if the disease is able to persist without any treatment/behavior change. This work demonstrates the need to further understand how treatment/behavior change occurs in a society with varying population.     

Bladder injection of "naked" hSlo/pcDNA3 ameliorates detrusor hyperactivity in obstructed rats in vivo

The goal of these studies was to examine the potential utility of bladder instilled K+ channel gene therapy with hSlo cDNA (i.e., the maxi-K channel) to ameliorate bladder overactivity in a rat model of partial urinary outlet obstruction. Twenty-two female Sprague-Dawley rats were subjected to partial urethral (i.e., outlet) obstruction, with 17 sham-operated control rats run in parallel. After 6 wk of obstruction, suprapubic catheters were surgically placed in the dome of the bladder in all rats. Twelve obstructed rats received bladder instillation of 100 microg of hSlo/pcDNA in 1 ml PBS during catheterization, and another 10 obstructed rats received 1 ml PBS (7 rats) or 1 ml PBS containing pcDNA only (3 rats). Two days after surgery cystometry was performed on all animals to examine the characteristics of the micturition reflex in conscious and unrestrained rats. Obstruction was associated with a three- to fourfold increase in bladder weight and alterations in virtually every micturition parameter estimate. PBS-injected obstructed rats routinely displayed spontaneous bladder contractions between micturitions. In contrast, hSlo injection eliminated the obstruction-associated bladder hyperactivity, without detectably affecting any other cystometric parameter. Presumably, expression of hSlo in rat bladder functionally antagonizes the increased contractility normally observed in obstructed animals and thereby ameliorates bladder overactivity. These initial observations indicate a potential utility of gene therapy for urinary incontinence.     

Intercellular communication in cultured human vascular smooth muscle cells

Intercellular communication through gap junction channelsplays a fundamental role in regulating vascular myocyte tone. We investigated gap junction channel expression and activity in myocytes from the physiologically distinct vasculature of the human internal mammary artery (IMA, conduit vessel) and saphenous vein (SV,capacitance vessel). Northern and Western blots documented the presenceof connexin43 (Cx43) in frozen tissues and cultured cells from both vessels. Northern blots also confirmed the presence of Cx40 mRNA incultured IMA and SV myocytes. Dual whole cell patch-clamp experiments revealed that macroscopic junctional conductance was voltage dependent and characteristic of that observed for Cx43. In the majority ofrecords, in both vessels, single-channel activity was dominated by amain-state conductance of 120 pS, with subconducting events comprisingless than 10% of the amplitude histograms. However, some recordsshowed "atypical" unitary events that had a conductance similar toCx40 (~140-160 pS), but gating behavior like that of Cx43. Assuch, it is conceivable that the presence and coexpression of Cx40 andCx43 in IMA and SV myocytes may result in heteromeric channelformation. Nonetheless, in terms of gating, Cx43-like behavior clearly dominates.

     

Nonhepatic response to portal glucose delivery in conscious dogs

The glycemic and hormonal responses and net hepatic and nonhepatic glucose uptakes were quantified in conscious 42-h-fasted dogs during a 180-min infusion of glucose at 10 mg. kg(-1). min(-1) via a peripheral (Pe10, n = 5) or the portal (Po10, n = 6) vein. Arterial plasma insulin concentrations were not different during the glucose infusion in Pe10 and Po10 (37 +/- 6 and 43 +/- 12 microU/ml, respectively), and glucagon concentrations declined similarly throughout the two studies. Arterial blood glucose concentrations during glucose infusion were not different between groups (125 +/- 13 and 120 +/- 6 mg/dl in Pe10 and Po10, respectively). Portal glucose delivery made the hepatic glucose load significantly greater (36 +/- 3 vs. 46 +/- 5 mg. kg(-1). min(-1) in Pe10 vs. Po10, respectively, P < 0.05). Net hepatic glucose uptake (NHGU; 1.1 +/- 0. 4 vs. 3.1 +/- 0.4 mg. kg(-1). min(-1)) and fractional extraction (0. 03 +/- 0.01 vs. 0.07 +/- 0.01) were smaller (P < 0.05) in Pe10 than in Po10. Nonhepatic (primarily muscle) glucose uptake was correspondingly increased in Pe10 compared with Po10 (8.9 +/- 0.4 vs. 6.9 +/- 0.4 mg. kg(-1). min(-1), P < 0.05). Approximately one-half of the difference in NHGU between groups could be accounted for by the difference in hepatic glucose load, with the remainder attributable to the effect of the portal signal itself. Even in the absence of somatostatin and fixed hormone concentrations, the portal signal acts to alter partitioning of a glucose load among the tissues, stimulating NHGU and reducing peripheral glucose uptake.     

Distribution of CAG repeat size in the dentatorubral and pallidoluysian atrophy (DRPLA) gene in a normal population in Taiwan

Dentatorubral and pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder with expansion of trinucleotide CAG repeats in the coding region of the gene. Expansion of the repeat tract beyond the normal range produces gene products with extended polyglutamine tracts. In this study, we analyzed the distribution of the CAG repeats in the DRPLA alleles in a normal Taiwanese population. We observed 15 different alleles and found that the range of the CAG repeat number was from 7-21. The most frequent allele contained 15 CAG repeats that represented 20% of the total analyzed alleles, followed by the 17 repeats (15.8%). The heterozygosity rate of this locus was 88%. Twelve parents-to-children transmissions of the DRPLA alleles in a Machado-Joseph disease family appeared to be normal without any alteration of the CAG repeat numbers. Phenotypes of DRPLA overlapped those of autosomal dominant cerebellar ataxia (ADCA). In order to identify DRPLA patients in Taiwan, we screened six autosomal dominant cerebellar ataxia patients without expansion in known spinocerebellar ataxia genes. All six patients had the repeat numbers within the normal range; thus, the possibility of DRPLA could be excluded.     

Rational design of landmark probes for quantitative DNA fiber mapping (QDFM)

Rapid construction of high-resolution physical maps requires accurate information about overlap between DNA clones and the size of gaps between clones or clone contigs. We recently developed a procedure termed ‘quantitative DNA fiber mapping’ (QDFM) to help construct physical maps by measuring the overlap between clones or the physical distance between non-overlapping contigs. QDFM is based on hybridization of non-isotopically labeled probes onto DNA molecules that were bound to a solid support and stretched homogeneously to ~2.3 kb/µm. In this paper, we describe the design of probes that bind specifically to the cloning vector of DNA recombinants to facilitate physical mapping. Probes described here delineate the most frequently used cloning vectors such as BACs, P1s, PACs and YACs. As demonstrated in representative hybridizations, vector-specific probes provide valuable information about molecule integrity, insert size and orientation as well as localization of hybridization domains relative to specifically-marked vector sequences.     

Requirement for Phe36 for DNA binding and mismatch repair by Escherichia coli MutS protein

The MutS family of DNA repair proteins recognizes base pair mismatches and insertion/deletion mismatches and targets them for repair in a strand-specific manner. Photocrosslinking and mutational studies previously identified a highly conserved Phe residue at the N-terminus of Thermus aquaticus MutS protein that is critical for mismatch recognition in vitro. Here, a mutant Escherichia coli MutS protein harboring a substitution of Ala for the corresponding Phe36 residue is assessed for proficiency in mismatch repair in vivo and DNA binding and ATP hydrolysis in vitro. The F36A protein is unable to restore mismatch repair proficiency to a mutS strain as judged by mutation to rifampicin or reversion of a specific point mutation in lacZ. The F36A protein is also severely deficient for binding to heteroduplexes containing an unpaired thymidine or a G:T mismatch although its intrinsic ATPase activity and subunit oligomerization are very similar to that of the wild-type MutS protein. Thus, the F36A mutation appears to confer a defect specific for recognition of insertion/deletion and base pair mismatches.