The disposition of venlafaxine enantiomers in dogs, rats, and humans receiving venlafaxine.

A stereospecific high-performance liquid chromatographic (HPLC) method was developed for the quantitation of the enantiomers of venlafaxine, an antidepressant, in dog, rat, and human plasma. The procedure involves derivatization of venlafaxine with the chiral reagent, (+)-S-naproxen chloride, and a postderivatization procedure. The method was linear in the range of 50 to 5,000 ng of each enantiomer per ml of plasma. No interference by endogenous substances or known metabolites of venlafaxine occurred. Studies to characterize the disposition of the enantiomers of venlafaxine were conducted in dog, rat, and human, following oral administration of venlafaxine. The Cmax, area under the curve (AUC) and (S)/(R) concentration ratios of the (R)- and (S)-enantiomers were compared. In rats, the mean plasma ratio of (S)-venlafaxine to that of (R)-venlafaxine over 0.5 to 6.0 h varied from 2.97 to 8.50 with a mean value of 5.51 +/- 2.45. The Cmax, AUC0-infinity, and t 1/2 values of the (R)- and (S)-enantiomers in dogs were not significantly different from one another (P greater than 0.1). The mean ratios [(S)/(R)] of enantiomers of venlafaxine in human over a 2 to 6 h interval ranged from 1.33 to 1.35 with an overall ratio of 1.34 +/- 0.26 (n = 12). These ratios of the enantiomers [(S)/(R)] were not statistically different from unity (P greater than 0.1) indicating that the disposition of venlafaxine enantiomers in humans is not stereoselective and is more similar to that in dogs than that in rats.     

Heterogeneity of soil and plant N and C associated with individual plants and openings in North American shortgrass steppe

Small-scale spatial heterogeneity of soil organic matter (SOM) associated with patterns of plant cover can strongly influence population and ecosystem dynamics in dry regions but is not well characterized for semiarid grasslands. We evaluated differences in plant and soil N and C between soil from under individual grass plants and from small openings in shortgrass steppe. In samples from 0 to 5 cm depth, root biomass, root N, total and mineralizable soil N, total and respirable organic C, C:N ratio, fraction of organic C respired, and ratio of respiration to N mineralization were significantly greater for soil under plants than soil from openings. These differences, which were consistent for two sites with contrasting soil textures, indicate strong differentiation of surface soil at the scale of individual plants, with relative enrichment of soil under plants in total and active SOM. Between-microsite differences were substantial relative to previously reported differences associated with landscape position and grazing intensity in shortgrass steppe. We conclude that microscale heterogeneity in shortgrass steppe deserves attention in investigation of controls on ecosystem and population processes and when sampling to estimate properties at plot or site scales.     

On-line thermal monitoring of aspartic acid separation in a high voidage ion-exchange column at high flow rate

A simple thermal monitoring technique has been successfully applied to an adsorption system using a novel ion exchanger with a large internal void volume (voidage) which can be operated at high superficial velocity (SV). Temperature changes resulting from heats of adsorption could be followed effectively using semi-conductor thermistor devices inserted into the resin through the column wall. Results show that, despite the high feed rates adopted, the thermal signals generated can be consistently related to the position of the breakthrough front within the bed.     

Targeting and fusion in vesicular transport

Vesicular transport of proteins and lipids between distinct subcellular compartments is directly responsible for generating and maintaining the structure of the organelles of the secretory and endocytic pathways in eukaryotic cells. Rapid advances in a variety of experimental systems have resulted in the identification of molecules involved in late steps of the transport process. This article presents a general paradigm for vesicular fusion and reviews the available experimental evidence.     

Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement.

The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectable marker. Verification of the delta fol::kan strain has been accomplished using genetic and biochemical criteria, including Southern analysis of the chromosomal DNA. The delta fol::kan mutation is stable in E. coli K549 [thyA polA12 (Ts)] and can be successfully transduced to other E. coli strains providing they have mutations in their thymidylate synthetase (thyA) genes. A preliminary investigation of the relationship between fol and thyA gene expression suggests that a Fol- cell (i.e., a dihydrofolate reductase deficiency phenotype) is not viable unless thymidylate synthetase activity is concurrently eliminated. This observation indicates that either the nonproductive accumulation of dihydrofolate or the depletion of tetrahydrofolate cofactor pools is lethal in a Fol- ThyA+ strain. Strains containing the thyA delta fol::kan lesions require the presence of Fol end products for growth, and these lesions typically increase the doubling time of the strain by a factor of 2.5 in rich medium.     

Organization and expression of a second chromosome follicle cell gene cluster in Drosophila

Four genes expressed during the period of vitelline membrane formation are clustered within 8 kb of DNA in region 26A of the second chromosome. Temporal and quantitative difference in the profiles of accumulated RNA suggest that the genes are independently regulated although they are selectively expressed during the stages of vitelline membrane biosynthesis. In situ hybridization and S1 analyses of RNAs from fractionated eggchambers established that these genes are active only in the follicle cells. S1 mapping with in vitro synthesized RNA probes shows that three of the genes are tandemly oriented. All four appear to be intronless. In vitro translation products from hybrid-selected RNAs indicate that two of these genes code for major vitelline membrane proteins. Sequence analysis of these two genes support this conclusion. The cell- and stage-specific expression of the other two genes, encoding less abundant RNAs, suggests that they also play a role in early eggshell production.     

Superficial and deeper layers of dog normal articular cartilage. Role of hyaluronate and link protein in determining the sedimentation coefficients distribution of the nondissociatively extracted proteoglycans

Proteoglycans were extracted under nondissociative conditions from superficial and deeper layers of dog normal articular cartilage. The purified a-A1 preparations were characterized by velocity gradient centrifugation. Superficial specimens exhibited an abundant population of slow sedimenting aggregates whereas the aggregates of deeper preparations sedimented as two well-defined families of molecules. These dissimilarities in the size distribution of the aggregates observed between superficial and deeper a-A1 preparations derived most of all from differences in their content of hyaluronate and link proteins: (a) superficial preparations contained twice as much hyaluronate as deeper specimens; (b) superficial aggregates were link-free and unstable at pH 5.0 whereas deeper preparations contained link-proteins and their faster sedimenting aggregates were stabilized against dissociation at pH 5.0. In these proteoglycan preparations from different cartilage layers, the monomers exhibited an identical capacity for aggregation and the hyaluronate molecules displayed quite similar molecular weight (Mr = 5 x 10(5] and aggregating capacity. These observations as well as aggregating studies conducted with highly purified link protein and purified hyaluronate specimens of different molecular weights support the following conclusions: (a) link protein not only stabilizes proteoglycan aggregates but also enhances the aggregating capacity of hyaluronate; (b) for all practical purposes, the slow sedimenting aggregates represent a secondary complex of hyaluronate and proteoglycan monomers whereas the fast sedimenting aggregates may be considered as a ternary complex wherein link protein stabilizes the hyaluronate-proteoglycans interaction; (c) the distinctive heterogeneity of articular cartilage can be related to structurally different proteoglycan aggregates. The structural dissimilarities observed between superficial and deeper aggregates could reflect the different macromolecular organization of the proteoglycan molecules in the territorial and interterritorial matrices, respectively.     

Stability and substructure of cardiac myosin subfragment 1 and isolation and properties of its heavy-chain subunit

The substructure and the thermal stability of the subunit interactions of bovine cardiac myosin subfragment 1 (SF1) have been examined. The results are in agreement with previous reports that the cardiac protein is cleaved in a very similar manner [Flink, I. L., & Morkin, E. (1982) Biophys. J. 37, 34; Korner, M., Thiem, N. V., Cardinaud, R., & Lacombe, G. (1983) Biochemistry 22, 5843-5847] but at a much faster rate [Applegate, D., Azarcon, A., & Reisler, E. (1984) Biochemistry 23, 6626-6630] than the skeletal protein. Additionally, it is found that the long-lived, steady-state intermediates formed by these proteins with MgATP at high ionic strength differ in their susceptibilities to tryptic attack especially at the 27K/50K junction of the associated heavy chains, suggesting a different conformation for these intermediates of the cardiac and skeletal SF1's. The thermal stability of the subunit interactions under conditions approaching the physiological state was examined by thermal ion-exchange chromatography of cardiac SF1 at 39.5 degrees C in the presence of MgATP. This results in the separation of part of the protein as the isolated heavy chain which is found to exhibit high levels of ATPase activity in the absence and presence of actin. Tryptic digestion of cardiac SF1 prior to thermal ion-exchange chromatography produces greater dissociation, with the heavy chain in this case being isolated as a complex of 27K, 50K, and 18-20K fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. IV. Recognition and activation by cloned glycoproteins gB and gD

Results of studies in mice and clinical observations in man indicate that T cell-mediated immunity is important in resistance to herpes simplex virus (HSV) infections. This study was undertaken to elucidate the viral antigen specificity of human HSV-immune T cells. Purified HSV-1 glycoproteins gB-1 and gD-1, cloned and expressed in mammalian cells, were found to stimulate proliferation of, and interleukin 2 (IL 2) production by, peripheral blood lymphocytes (PBL) of HSV seropositive individuals, indicating the presence of memory T cells to gB-1 and gD-1 in individuals with serologic evidence of immunity to HSV. Second, T cell clones, generated by stimulation of PBL with HSV-1, were found to recognize gB-1 or gD-1, as evidenced by the ability of the clones to proliferate in response to stimulation with gB-1 or gD-1 in the absence of exogenous IL 2. Third, HSV-specific T cell clones, lytic for HSV-1 or both HSV-1- and HSV-2-infected autologous target cells, were generated after stimulation of PBL with purified cloned gB-1 or gD-1. Our findings, that human HSV-specific T cells can recognize and be activated by HSV subunit antigens gB-1 or gD-1, imply that these glycoproteins play a role in human T cell-mediated immunity to HSV and support the contention that a gB-1 or gD-1 subunit vaccine may be protective in man.     

The functional domains of coagulation factor VIII:C

A lack of factor VIII:C, manifested as a bleeding disorder due to the absence of clot formation, is known as hemophilia A, an X chromosome-linked inherited disease afflicting 1-2 males/10,000. To determine the minimum functional domain(s) essential for factor VIII:C activity, we have expressed the amino-terminal (92-kDa) and carboxyl-terminal (80-kDa) proteolytic cleavage products as individual, secreted polypeptides in monkey cells without the 909-residue central region. We have found that neither terminal domain alone is able to promote coagulation in factor VIII:C-deficient plasma. However, when the 92- and 80-kDa peptides are co-expressed, clotting activity is readily detected. Thus, these two chains alone constitute an active or activatable complex. The central domain is required neither for activity nor for the assembly of an active complex from two chains expressed in trans. These results suggest that a truncated derivative of factor VIII:C may be useful in coagulation therapy.