Characterization of acyclovir susceptibility and genetic stability of varicella-zoster viruses isolated during acyclovir therapy.

We have characterized the susceptibility and genetic stability of varicella-zoster viruses (VZV) isolated from skin lesions of three patients with herpes zoster and six patients with varicella treated with conventional short-term acyclovir (ACV) administration. The susceptibilities to ACV of the serial isolates from the patients were examined, and there was no significant difference in the susceptibility to ACV among the isolates before and during the ACV treatment, indicating that conventional short-term ACV treatment did not generate ACV-resistant VZV infections. Polymerase chain reaction (PCR) analyses of these as well as seven thymidine kinase-deficient VZV strains derived from in vitro ACV treatment were carried out to examine their genomic stability. Five regions containing tandem direct reiterations (R1-R5) were amplified by PCR and compared, and the region containing the Pst I-site was also examined. PCR analyses demonstrated that the R1, R5 and the Pst I-sites were stable and useful in epidemiological studies even after ACV treatment. The R2, R3 and R4 sites were far less stable in these experimental conditions. In this paper we discuss the results of the PCR analyses with regard to the dynamics of VZV population in patients with VZV infection treated with conventional short-term ACV administration.

Generation and transit pathway of H+ is critical for inhibition of palmar sweating by iontophoresis in water.

Passing galvanic current across the skin (known as "tap water iontophoresis" or TWI) inhibits sweating; however, its mechanism of action is unclear. Using improved methods, we confirmed that anodal current has more of an inhibitory effect than cathodal current, water is superior to saline, and the inhibitory effect is a function of the amperage used. To address the importance of current flowing through the pores, a layer of silicone grease was placed on the skin to reduce the shunt pathway across the epidermis. With silicone, total skin conductance decreased 60% without the sweat pores being occluded, swelling of the stratum corneum and collapse of the poral lumen was prevented, and current-induced inhibition of sweating was enhanced, most likely because of an increase in current density in the pores. The pH of anodal water, but not of saline, dropped to 3, whereas that of cathodal water increased to 10 during passage of current through the skin. Acidified anodal water was superior to alkaline water. Sweat glands isolated from TWI-induced anhidrotic palmar skin responded to methacholine in vitro, but the sweat rate and pharmacological sensitivity were slightly lowered. Thus the strong acidity generated by hydrolysis of water in the anodal bath and the further accumulation of H+ in the sweat duct by anodal current may be responsible for TWI-induced inhibition of sweating due to an unknown lesion(s) in the duct or sweat pore. The secretory coil function may also be altered because of exposure to intense acidity during TWI. The importance of H+ movement into the sweat pore for inhibition of sweating could be further exploited to develop new strategies for the control of sweating.

Seijo-bofu-to, Jumi-haidoku-to and Toki-shakuyaku-san suppress rashes and incidental symptoms in acne patients.

Seijyo-bofu-to, Jumi-haidoku-to and Toki-shakuyaku-san effectively suppressed acne rashes as well as incidental symptoms. The synergistic activities of the ingredients in the Kampo formulations might produce these effects. In contrast, distinct suppression of incidental symptoms was not found with antimicrobials. The cause of adverse effects in antimicrobials has not yet been clarified and different degrees of suppression of incidental symptoms among the Kampo formulations exist.

Effects of topically applied spironolactone on androgen stimulated sebaceous glands in the hamster pinna.

The effects of spironolactone (5% SYC-201G, a preparation developed for clinical use in acne vulgaris by Searle Yakuhin K.K.), which is known to have antiandrogenic effects by competitively inhibiting dihydrotestosterone at androgen receptor sites, was topically applied to the androgen stimulated sebaceous glands of adult female golden hamsters. Androgen stimulation, induced by intramuscular injection of testosterone propionate (TP) every other day over a two week period, resulted in a 2.5 to 2.7 time increase in the size of the sebaceous glands of the hamster pinna. Once-daily treatment with 5% SYC-201G or matching placebo was applied to androgen-stimulated hamsters on one pinna only during the same period as TP injection. Comparison between the treated and untreated sides revealed a significant suppression in the sebaceous gland size (p < 0.05) by 5% SYC-201G; no such effect was observed with placebo. The difference in the suppression rate of the sebaceous gland size between 5% SYC-201G (23%) and matching placebo (-4.7%) was significant (p < 0.01).

Stimulation of Cl conductance by minoxidil sulfate and K conductance by minoxidil in eccrine clear cells.

Minoxidil sulfate (MXS), an antihypertensive agent and hair growth promoter, has been reported to stimulate K channels in vascular smooth muscle cells. We now report that MXS stimulates whole cell Cl currents, whereas minoxidil (MX) stimulates K currents in dissociated eccrine clear cells. Using whole cell clamp techniques we observed that: 1) 1 mM MXS stimulated sweat secretion in vitro; 2) MXS depolarized the membrane potential by as much as 40 mV; 3) MXS stimulated membrane conductance, increased inward current and shifted the reversal potential to the right when physiological electrolyte solutions were used; 4) in symmetrical Cl (Cl/Cl) solutions without permeable cations, MXS induced outwardly rectifying current-voltage (I-V) relationships; 5) in the Cl/Cl solutions, the MXS-induced current responses to imposed voltage pulses showed time-dependent activation, especially at the depolarizing potentials; 6) the reversal potential of the MXS-stimulated I-V curves in the Cl/Cl solutions shifted to the right by 55 mV when [Cl] in the bath was decreased from 157 to 7 mM; 7) MXS did not elevate cytosolic Ca or cAMP, although prolonged exposure to a Ca-free solution abolished the effect of MXS and 8) MXS-stimulated conductance was partially inhibited by diphenylamine-2-carboxylic acid, a blocker of Cl channels. The data suggest that MXS stimulates Cl channels, most likely depolarization-activated, outwardly rectifying channels. In contrast, the parent compound MX hyperpolarized the membrane potential and stimulated outward current without elevating cytosolic Ca and was independent of extracellular Ca, suggesting that MX stimulates Ca-insensitive K currents.

Na-K-2Cl cotransporters are present and regulated in simian eccrine clear cells.

In freshly dissociated rhesus palm eccrine clear cells, regulatory volume increase (RVI) was studied using image analysis as a measure of Na-K-2Cl cotransport activity. Pseudo-RVIs, as well as RVI during methacholine (MCh)-induced cell shrinkage, were observed in clear cells and were inhibited by 100 microM bumetanide or in Na-free medium, but were not inhibited by amiloride or ouabain. RVI in hypertonic medium and RVI after MCh-induced cell shrinkage were accelerated by adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents (forskolin+isoproterenol) and inhibited by phorbol ester. RVI in hypertonic medium was enhanced by a phosphatase inhibitor, okadaic acid. mRNA for Na-K-2Cl cotransporter (NaKCC) was demonstrated in freshly isolated rhesus sweat secretory coils by polymerase chain reaction (PCR) after reverse transcription using a set of primers derived from the published human NaKCC (hNaKCC) 1 sequence, i.e., nucleotides 2,043-2,810. The deduced amino acid sequence of the PCR-amplified 767-base pair segment was identical to that of hNaKCC 1 from a human colon cell line (T84). The data are interpreted to indicate that NaKCC, showing strong homology to secretory type hNaKCC 1, is present in rhesus eccrine secretory coils and may participate in the cotransport component of eccrine sweat secretion and cell volume regulation, especially during cholinergic stimulation. The data also raise the possibility that sweat gland NaKCC may be upregulated by cAMP-mediated protein phosphorylation and downregulated by protein kinase C.

Terminal deoxynucleotidyltransferase directly interacts with a novel nuclear protein that is homologous to p65.

BACKGROUND: Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances Ig and TcR gene diversity in the N region in B- and T-cells. TdT is found as a member of a large protein complex in the lysate of the thymocytes. To elucidate the molecular mechanism of the synthesis of the N region, we first attempted to isolate the genes with products that are interacting directly with TdT. RESULTS: Using a yeast two-hybrid system, we isolated a cDNA clone encoding a novel nuclear protein that interacts with TdT. This protein was designated as TdT interacting factor 1 (TdIF1). TdIF1 has a high degree of homology to the transcription factor p65, which belongs to the nuclear receptor superfamily. TdIF1 contains HMG-I and HMG-Y DNA binding domains (AT-hooks) and can bind to single- and double-stranded DNA. TdT and TdIF1 were co-eluted at position 232 kDa by gel filtration of MOLT4 lysate. TdIF1 can enhance TdT activity fourfold in vitro assay system using oligo(dT)16 as primers. CONCLUSIONS: TdIF1 binds directly to TdT, both in vitro and in vivo. TdIF1 and TdT exist as the members of a 232 kDa protein complex. TdIF1 can enhance TdT activity maximum fourfold in vitro assay system, suggesting that it positively regulates the synthesis of the N region during V(D)J recombination in the Ig and TcR genes.

Terminal deoxynucleotidyltransferase is negatively regulated by direct interaction with proliferating cell nuclear antigen.

BACKGROUND: The repertoires of Ig and TcR are generated by a combinatorial rearrangement of variable (V), diversity (D), and joining (J) segments (V(D)J recombination) in B- and T-cells. Terminal deoxynucleotidyltransferase (TdT) adds extra nucleotides (N nucleotides) at the junctions of the gene segments to enhance the Ig and TcR genes diversity. Using an anti-TdT antibody column, TdT has been purified as a member of a megadalton protein complex from rat thymus. The N region would be synthesized with the large protein complex. RESULTS: The cDNAs for proliferating cell nuclear antigen (PCNA) were isolated by yeast two-hybrid screening as the gene products which directly interacted with TdT. The interaction between PCNA and TdT was confirmed by co-immunoprecipitation, both in vitro and in vivo. TdT binds directly to a PCNA trimer, as shown by gel filtration. TdT interacts with PCNA in its DNA polymerization domain (DPD), but not in its BRCA-1 C-terminal (BRCT) domain. TdT activity was reduced to 17% of the maximum value by TdT/PCNA complex formation. CONCLUSION: TdT interacts directly with PCNA through its DPD. A functional consequence of this interaction is the negative regulation of TdT activity. These findings suggest that TdT catalyses the addition of N nucleotides under the negative control of PCNA during V(D)J recombination.