Rapid, simple and sensitive high-performance liquid chromatographic method for detection and determination of acyclovir in human plasma and its use in bioavailability studies

A rapid, simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the measurement of acyclovir concentrations in human plasma and its use in bioavailability studies is evaluated. Unchanged acyclovir has been quantified without the introduction of an internal standard using the present method. Human plasma proteins were selectively precipitated by the addition of 7% perchloric acid to spiked plasma samples or to the plasma samples obtained after acyclovir administration to human volunteers and the mixture was spun at 1000 g for 10 min. The supernatant was directly injected into a Novaflex C₁₈ column and detected at 254 nm. The mobile phase consisted of octane sulfonic acid buffer (pH 2.5) and methanol (92:08). The limit of quantitation for acyclovir in plasma was 20 ng/ml, which enabled the determination of the area under the curve (AUC) more precisely, that is, it is much closer to its extrapolated value. The present method has been successfully applied to samples from bioavailability studies.     

Mlc1p Is a Light Chain for the Unconventional Myosin Myo2p in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the unconventional myosin Myo2p is of fundamental importance in polarized growth. We explore the role of the neck region and its associated light chains in regulating Myo2p function. Surprisingly, we find that precise deletion of the six IQ sites in the neck region results in a myosin, Myo2-Δ6IQp, that can support the growth of a yeast strain at 90% the rate of a wild-type isogenic strain. We exploit this mutant in a characterization of the light chains of Myo2p. First, we demonstrate that the localization of calmodulin to sites of polarized growth largely depends on the IQ sites in the neck of Myo2p. Second, we demonstrate that a previously uncharacterized protein, Mlc1p, is a myosin light chain of Myo2p. MLC1 (YGL106w) is an essential gene that exhibits haploinsufficiency. Reduced levels of MYO2 overcome the haploinsufficiency of MLC1. The mutant MYO2-Δ6IQ is able to suppress haploinsufficiency but not deletion of MLC1. We used a modified gel overlay assay to demonstrate a direct interaction between Mlc1p and the neck of Myo2p. Overexpression of MYO2 is toxic, causing a severe decrease in growth rate. When MYO2 is overexpressed, Myo2p is fourfold less stable than in a wild-type strain. High copies of MLC1 completely overcome the growth defects and increase the stability of Myo2p. Our results suggest that Mlc1p is responsible for stabilizing this myosin by binding to the neck region.     

Computer analysis of regulatory signals in complete bacterial genomes. Translation initiation of ribosomal protein operons]

Signals of translation initiation of operons of Haemophilus influenzae ribosomal proteins were predicted. This process is regulated by the formation of secondary RNA structures to which one of the proteins encoded in a particular operon binds. In some cases, these structures imitate the region of protein binding to rRNA. Predictions are made by comparing with homologous operons of Escherichia coli and analogous regions of rRNA and by estimating the energy of secondary structure formation. It is shown that this regulatory mechanism occurs: in operons L11, S10, S15, spc, and alpha of H.influenzae and, probably, in operon S15 of Helicobacter pylori, Bacillus subtilis, and Mycoplasma genitalium.     

The Ca2+ channel β2 subunit is selectively targeted to the axon terminals of supraoptic neurons

The assembly of high voltage-activated Ca²⁺ channels with different β subunits influences channel properties and possibly subcellular targeting. We studied β subunit expression in the somata and axon terminals of the magnocellular neurosecretory cells, which are located in the supraoptic nucleus (SON) and neurohypophysis, respectively. Antibodies directed against the 4 Ca_Vβ subunits (Ca_Vβ₁-Ca_Vβ₄) were used for immunoblots and for immunostaining of slices of these two tissues. We found that all 4 β subunits are expressed in both locations, but that Ca_Vβ₂ had the highest relative expression in the neurohypophysis. These data suggest that the Ca_Vβ₂ subunit is selectively targeted to axon terminals and may play a role in targeting and/or regulating the properties of Ca²⁺ channels.     

Clutch size relative to tree cavity size in Northern Flickers

We analysed clutch size versus nest size in 153 broods of the Northern Flicker Colaptes auratus , a woodpecker using natural cavities in British Columbia, Canada. Larger volume cavities were less susceptible to predation and cavity size was positively associated with the age and body size of males and with the body condition of female parents. Although clutches varied between 4 and 11 eggs, and the floor area of cavities varied about 5-fold, we found no relationship between clutch size and floor area or cavity volume. To see if there were fitness consequences to clutch size relative to nest size, we examined hatching success and nestling mortality in flicker broods. Hatching success was not related to cavity size, but crowding slightly reduced nestling survival even when clutch size was controlled statistically. However, there was no effect of cavity size on the total number of nestlings fledged. Newly excavated flicker cavities were smaller than reused cavities suggesting a cost to excavation. This cost, coupled with the minimal fitness consequences of overcrowding, may explain why flickers do not adjust clutch size to cavity size.     

The metabolism of inositol 1,3,4-trisphosphate to inositol 1,3-bisphosphate

We previously demonstrated a pathway for the metabolism of inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) to inositol 3,4-bisphosphate (Ins(3,4)P2) in calf brain extracts. Inositol polyphosphate 1-phosphatase, a Mg2+-dependent, lithium ion-inhibited enzyme, specifically hydrolyzes Ins(1,3,4)P3 to Ins(3,4)P2 and Ins(1,4)P2 to Ins 4-P (Inhorn, R. C., Bansal, V. S., and Majerus, P. W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2170-2174). Now we have found an alternative pathway for the metabolism of Ins(1,3,4)P3 in crude calf brain extracts. Along this pathway, Ins(1,3,4)P3 is first converted to Ins(1,3)P2 which is further hydrolyzed to Ins 1-P. This pathway involves a 4-phosphatase and a 3-phosphatase which do not require Mg2+ and are not inhibited by lithium ions. A similar 4-phosphatase also degrades Ins(3,4)P2 to Ins 3-P. Three different inositol bisphosphates formed from calf brain supernatant are each further metabolized by a separate enzyme. The three inositol monophosphates, i.e. Ins 1-P, Ins 3-P, and Ins 4-P, are converted to inositol by inositol monophosphate phosphatase (Ackermann, K. E., Gish, B. G., Honchar, M. P., and Sherman, W. R. (1987) Biochem. J. 242, 517-524).     

Molecular mechanics studies on poly(purine).poly(pyrimidine) sequences in DNA: polymorphism and local variability

Energy minimization has been carried out on three poly(purine).poly(pyrimidine) sequences--d(G)10.d(C)10, d(A)10.d(T)10, and d(AG)5.d(CT)5--using the molecular mechanics program AMBER (Assisted Model Building and Energy Refinement). In order to extensively scan the conformational space available, five different helical models were studied, three of them being right-handed helices while the other two were left helical. For all three sequences the right-handed A- and B-type helices are energetically slightly preferred over the left helices, but the energy difference between the various right-handed helices is only marginal. A detailed analysis has been carried out to characterize the local structural variability in the refined structures, both in terms of torsion angles as well as other parameters such as base-pair tilt, wedge roll, and wedge tilt, etc. All three sequences exhibit similar structural features for a particular form, but both the forms A and B show significant deviations from fiber models. In particular, the A-form structures have higher unit rise (2.7 A), and lower unit twist (31 degrees) and base-pair tilt (12 degrees), compared to the fiber model, which has corresponding values of 2.56 A, 32.7 degrees, and 20 degrees, respectively. All these changes indicate that the refined models are closer to the A-form structure observed in crystals of oligonucleotides. In the refined B-for models, the helical parameters are close to the fiber B-form, although the torsion angles show considerable variations. None of the three sequences examined, including the d(A)n.d(T)n sequence, show any pronounced curvature for the B-form structure.     

Levels and 75Se-labeling of specific proteins as a consequence of dietary selenium concentration in mice and rats

Selenium-labeled proteins (SLP) distinct from glutathione peroxidase (GSH-PX) recently have been purified and partially characterized. Antisera to two SLP, a 56-kDa and a 14-kDa protein, were generated in rabbits and used to examine expression of these proteins as a consequence of dietary selenium concentration (0.02, 0.2, 2.0 ppm) in mice and rats. Additionally, the kinetics of 75Se labeling in plasma, liver, kidney, and mammary gland were examined over a 40-hr time period as a function of dietary selenium concentration. A plasma 57-kDa protein was labeled by 30 min after 75Se injection and reached maximum labeling by 4 hr. The cellular 56-kDa and 14-kDa proteins, as well as GSH-Px, labeled progressively over 40 hr starting between 1 and 4 hr after injection. In general, the 56-kDa and GSH-Px followed similar labeling patterns, whereas the 14-kDa protein was labeled less and was not labeled in discernible quantities until 40 hr. The extent of labeling of all proteins was inversely proportional to the dietary selenium concentration and was probably a reflection of different endogenous selenium body pools. The most important observation was generated by the immunoblot data. The amount of 56-kDa and 14-kDa proteins as detected and measured on immunoblots was not a function of dietary selenium concentration. This result suggests that the synthesis and maintenance of the 56-kDa and 14-kDa proteins are not selenium dependent, a characteristic which distinguishes the two proteins from GSH-Px. The single exception to the above results was the 40% decrease of liver 14-kDa protein concentration in carcinogen-treated rats fed 2.0 ppm of selenium. An organic selenium compound, selenobetaine, did not lead to a decrease under similar conditions. In 15 rat mammary tumors induced by 7,12-dimethylbenzanthracene and analyzed on immunoblots, the SLP-56 was undetected in 5 cases and appeared as two bands (56,000 Da, 50,000 Da) in 10 cases. This latter result raises the possibility that the expression of SLP-56 may be altered in mammary tumors as compared with normal mammary gland.     

Population genetics of glyoxalase I (GLO) polymorphism in India

Phenotype and gene frequency data are presented on the glyoxalase I (GLO) polymorphism in seven endogamous caste groups: Jat Sikh, Ramdasia Sikh, Ramgarhia Sikh, Khatri, Brahmin and Bania of Patiala district, and Jat Sikh of Faridkot district of Punjab, North-West India. Apparently, there is considerable heterogeneity in the frequency distribution of the GLO1 gene that varies from 0.168 in Bania to 0.287 in Brahmin. However, these differences are not statistically significant, and the overall GLO1 frequency in Punjab is well within the North Indian range.