Regulation of N-acetylglucosaminyltransferase V activity. Kinetic comparisons of parental, Rous sarcoma virus-transformed BHK, and L-phytohemagglutinin-resistant BHK cells using synthetic substrates and an inhibitory substrate analog

Baby hamster kidney (BHK) cells transformed with Rous sarcoma virus, RS-BHK cells, demonstrate a 2.5-fold increase in the activity of N-acetylglucosaminyltransferase V (GlcNAc-T V, EC 2.4.1.155), and this increase in activity appears to be specific for this enzyme. By contrast, a lectin-resistant BHK cell line selected for its ability to grow in high levels of L-phytohemagglutinin, LP3.3, is characterized by a specific decrease in its GlcNAc-T V activity. To test if these alterations in the apparent Vmax of GlcNAc-T V are due to changes in the efficiency of populations of enzymes in RS-BHK and LP3.3 cells compared to the parental BHK cells, we have compared the kinetic properties of the enzymes from these three sources. The Km constants observed for both the sugar nucleotide donor (UDP-GlcNAc) and two synthetic trisaccharide acceptors were indistinguishable. The Vmax values toward three synthetic acceptors were also determined first for the BHK GlcNAc-T V, and they varied by over 5-fold. When these values were measured for the variant and transformed cell enzymes, however, similar 5-fold differences were still observed, although the absolute values for these acceptors were all higher or lower for the RS-BHK and LP3.3 enzymes, respectively. In addition, we have synthesized a deoxygenated analog of the specific GlcNAc-T V acceptor, beta GlcNAc(1,2) alpha Man(1,6) beta ManOR, where the reactive 6'-OH group has been removed, and the resulting trisaccharide was found to be a competitive inhibitor of the enzyme. The Ki for this inhibitor was near 70 microM for the GlcNAc-T V from all three sources. These kinetic comparisons demonstrate that the enzymes from the three cell types have kinetically indistinguishable active sites. These results suggest that the differences in the apparent Vmax values among the cell types are most likely due to alterations in the number of active molecules rather than in the modulation of either their catalytic activities or specificities.     

Mechanism of action of macrophage-derived suppressor factor produced by soluble immune response suppressor-treated macrophages

After a 2-hr incubation with soluble immune response suppressor (SIRS), a product of concanavalin A-activated murine T cells, macrophages release a factor, M phi-derived suppressor factor (M phi-SF), which nonspecifically suppresses immune responses in vitro. The mechanism(s) of action of M phi-SF and range of cell types affected by M phi-SF have been investigated. M phi-SF suppressed antibody responses to background levels if added at culture initiation and by 80 to 90% if added as late as 2 hr before assay. Primary and secondary IgM and IgG antibody responses, proliferative responses to T cell and B cell mitogens, antibody and protein secretion, and the division of several tumor cell lines in culture were inhibited by M phi-SF. Division of synchronized tumor cells was inhibited when M phi-SF was added at any point prior to and during mitosis; this inhibition could be reversed with 2-mercaptoethanol. In the presence of M phi-SF, asynchronous tumor cells accumulated in the cell cycle just prior to cell division and could be released into mitosis by 2-mercaptoethanol. These data indicate that M phi-SF inhibits cell division by causing a block at or in mitosis and suggest that M phi-SF may be a general inhibitor of cellular proliferation and possibly of protein secretion.     

The distribution of individual heterozygosity in natural populations

Estimation of the distribution of the level of individual heterozygosity within natural populations is explored with both Monte-Carlo simulation studies and data from natural populations. Simulations indicate that heterozygosities estimated from as few as a dozen randomly chosen loci may, to some degree, reflect (r = 0.35) heterozygosity determined by 100 independent loci. The shape of the expected distribution of heterozygosity is heavily dependent upon levels of heterozygosity at the loci. Complete genetic data for 12 loci from 997 Fundulus heteroclitus are used to describe the distributions of heterozygosity for different localities, for age classes and for sexes. The distributions deviate from normality. Distributions from different localities are not different, but the distributions are heterogeneous among age classes at one of two localities and are heterogeneous between the sexes.     

Distribution-free regression analysis of grouped survival data

Methods based on regression models for logarithmic hazard functions, Cox models, are given for analysis of grouped and censored survival data. By making an approximation it is possible to obtain explicitly a maximum likelihood function involving only the regression parameters. This likelihood function is a convenient analog to Cox's partial likelihood for ungrouped data. The method is applied to data from a toxicological experiment.     

Cytosolic location of an endo-N-acetyl-beta-D-glucosaminidase activity in rat liver and kidney.

Endo-N-acetyl-beta-D-glucosaminidase activity towards an oligomannosidic type glycoamino acid substrate was found in the soluble fraction of rat liver and kidney. No evidence for a lysosomal form of the activity was found.     

MA 160 and EB 33 cell lines: HeLa cell contaminants,hybrids or prostatic epithelial cells?

Summary Studies of acid phosphates produced by cell lines MA 160 and EB 33 demonstrated immunochemically their prostatic origin. MA 160 and EB 33, rather than being HeLa contaminants, may be hybrids of prostatic epithelial and HeLa cells or true prostatic cell lines with chromosomal changes common to all long-term cultivated cell lines. This research was supported by NIH (Cancer) Research Grants Nos. 18748 and 16426; and Detroit General Hospital Research Corporation.     

Measurement of adriamycin (doxorubicin) and its metabolites in human plasma using reversed-phase high-performance liquid chromatography and fluorescence detection

We describe a method for measuring adriamycin and its major metabolite, adriamycinol, in plasma, using reversed-phase high-performance liquid chromatography and fluorescence detection. The lower limit of detection is approximately 1 ng/ml for both compounds; within-day coefficients of variation are 3.6% and 4.4% for adriamycin and adriamycinol, respectively. A slight modification of this procedure also allows measurement of aglycone metabolites.     

Persistence and Biodegradation of Spilled Residual Fuel on an Estuarine Beach

The enrichment of hydrocarbon-degrading bacteria and the persistence of petroleum hydrocarbons on an estuarine beach after a spill of residual fuel oil on 11 April 1973 in Upper Narragansett Bay, R.I. was investigated. A rapid enrichment occurred during days 4 to 16 after the oil spill and a significant population of hydrocarbon-degrading bacteria was maintained in the beach sand for at least a year. The concentration of petroleum hydrocarbons in the mid-tide area declined rapidly during the bacterial enrichment period, remained fairly constant throughout the summer, and then declined to a low concentration after 1 year. An increased concentration of branched and cyclic aliphatic hydrocarbons in the low-tide sediment 128 days after the spill suggested a migration of hydrocarbons during the summer. Hydrocarbon biodegradation was apparent during the winter months at a rate of less than 1 mug of hydrocarbon per g of dry sediment per day.     

Stomatal closure in response to xanthoxin and abscisic acid

Summary The stomata of detached leaves of Commelina communis L., Hordeum vulgare L., Zea mays L., Vicia faba L., Phaseolus vulgaris L. and Xanthium strumarium L. closed when xanthoxin (XAN) was added to the transpiration stream. XAN was approximately half as active as (+)-abscisic acid (ABA) at an equivalent concentration. XAN, like ABA, sensitized stomata of Xanthium strumarium to CO₂. In contrast to ABA, XAN was ineffective in closing stomata of isolated epidermal strips of C. communis or V. faba. This may be because XAN added to the transpiration stream is converted to ABA during passage from the xylem to the epidermis.Abbreviations ABA Abscisic acid - XAN xanthoxin