Polymer fractionation in aqueous two-phase polymer systems.

We consider the effects of the addition of poly(ethylene glycol) (PEG) of different molecular weights to aqueous two-phase system of PEG 8000 and dextran 500. The first purpose of this study was to determine the molecular weight partitioning of the polymers themselves so that, for example, aqueous two-phase separations using affinity ligands can be improved. The second purpose was to examine whether this molecular weight partitioning could be predicted by using solution thermodynamic models so that it would be possible to optimize affinity partitioning without extensive laboratory work. Experimentally, we find that, by increasing the PEG concentration of any molecular weight in the feed, the high molecular weight PEG concentration in the dextran-rich phase is reduced. This observation can be used to reduce the loss of expensive ligated PEG used in affinity partitioning. Further, there is generally good agreement between our experimental data and the predictions of a solution thermodynamic model.     

On the Genetic Control of Genes Located in the Sex-Chromosome Heterochromatin of DROSOPHILA MELANOGASTER

The genetic properties of a recessive autosomal point mutant, "daughterless" (symbol: da), are described. They are: (1) da maps in the euchromatin of 2L at about position 39 on the genetic map and between 27D and 31E on the salivary map; (2) when homozygous in females, da causes the production of unisexual male progenies owing to sex differential zygote mortality in the egg stage; (3) da has no effect on the progeny of mutant males; (4) female zygotes die, while at least some male zygotes survive, irrespective of the number of Y chromosomes or the amount of X-chromosome heterochromatin carried by either the mutant female or her progeny; (5) homozygous da progeny of heterozygous parents also show sex differential survival favoring males indicating a da effect in development as well as in oogenesis, but with the developmental effect much less pronounced; (6) extra heterochromatin from either the X or Y chromosome in either the mother or her progeny can reduce the mortality caused by da in development (and, therefore, conceivably can also reduce the abnormality in oogenesis, although this is obscured by the severity of the maternal effect.)—From these results, it is suggested that (1) da regulates either the activity of structural genes in the sex chromosome heterochromatin or the activity or stability of their products, and (2) it is a defective product of sex chromosome heterochromatic genes that causes the abnormalities resulting in the observed mortality of heterozygous progeny of homozygous mutant mothers and of homozygous mutant progeny of heterozygous mothers.—The striking parallels with the properties of the gene, "abnormal oocyte" are noted as is the close linkage between the two loci. The possibility of a special sex-chromosome-heterochromatin-regulator region on chromosome 2 is considered.     

Histamine modulation of eosinophil migration.

Preincubation of eosinophils with 10(-5) M or higher concentrations of histamine inhibited the eosinophil chemotactic response to endotoxin-activated serum whether by using the nucleopore filter assay and counting the cells migrating through the filter, or by using the Zigmond-Hirsch assay and counting the cells at each 10-mum interval. When the H2-receptor sites on the eosinophils were blocked by metiamide, the inhibitory capacity of histamine was prevented. Preincubation of eosinophils with 10(-6) M histamine increased the number of responding eosinophils to endotoxin-activated serum and this enhancement was blocked by an H1-receptor antagonist. Isoproteronol and aminophylline inhibited eosinophil movement and increasing concentrations of dibutryl cyclic AMP inhibited eosinophil migration. Concentrations of histamine that consistently resulted in inhibition of eosinophil movement stimulated an increase in cyclic AMP that was prevented by blocking the H2-receptor but not the H1-receptor. Thus, histamine-dependent inhibition of the eosinophil chemotactic response to other agents is mediated through the H2-receptor and is associated with an increase in the intracellular level of cyclic AMP whereas histamine dependent enhancement of eosinophil migration to other agents appears to be mediated through the H1-receptor. Eosinophils behave as a heterogeneous population as assessed by the ability of histamine to augment or inhibit cell migration. This may reflect differences in H1 to H2 receptor density or cell responsiveness to receptor stimulation. The chemoattractant activity of histamine itself is not influenced by H1 or H2 receptor antagonists, thus it is possible that an eosinophil has a third type of histamine receptor.     

A dnaT mutant with phenotypes similar to those of a priA2::kan mutant in Escherichia coli K-12

The ability to repair damaged replication forks and restart them is important for cell survival. DnaT is essential for replication restart in vitro and yet no definite genetic analysis has been done in Escherichia coli K-12. To begin, dnaT822, an in-frame six-codon (87-92) deletion was constructed. DnaT822 mutants show colony size, cell morphology, inability to properly partition nucleoids, UV sensitivity, and basal SOS expression similar to priA2::kan mutants. DnaT822 priA2::kan double mutants had phenotypes similar to those of the single mutants. DnaT822 and dnaT822 priA2::kan mutant phenotypes were fully suppressed by dnaC809. Previously, a dominant temperature-sensitive lethal mutation, dnaT1, had been isolated in E. coli 15T(-). DnaT1 was found to have a base-pair change relative to the E. coli 15T(-) and E. coli K-12 dnaT genes that led to a single amino acid change: R152C. A plasmid-encoded E. coli K-12 mutant dnaT gene with the R152C amino acid substitution did not display a dominant temperature-sensitive lethal phenotype in a dnaT(+) strain of E. coli K-12. Instead, this mutant dnaT gene was found to complement the E. coli K-12 dnaT822 mutant phenotypes. The significance of these results is discussed in terms of models for replication restart.     

Genetic diversity of dodder (<Emphasis Type="Italic">Cuscuta</Emphasis> spp.) collected from commercial cranberry production as revealed in the<Emphasis Type="Italic">trn</Emphasis>L (UAA) intron

Swamp dodder (Cuscuta gronovii) is a parasitic plant detrimental to cranberries. Observation of emergence of dodder seeds collected from a cultivated cranberry bog in Massachusetts revealed 2 or more peak emergence times during 4 consecutive growing seasons. Molecular methods were used to investigate genetic variation among the emerging dodder seedlings. On emergence, dodder seedlings were collected and analyzed for DNA sequence diversity in thetrnL (UAA) intron, a noncoding region of chloroplast DNA. DNA sequence analysis of 87 seedlings collected during the 1999 and 2000 growing seasons revealed the presence of 2 dodder ecotypes, designated A and B. Comparative DNA sequence analysis indicated that in thetrnL (UAA) intron, the sequence of ecotype A is identical to that ofCuscuta gronovii, whereas the sequence of ecotype B is closest to that ofCuscuta attenuata (99.3% sequence identity; 293 bases considered). ABg/II restriction enzyme cut site was identified that distinguished between thetrnL (UAA) introns of ecotypes A and B. Restriction fragment length polymorphism (RFLP) was used to analyze the sequences of 100 seedlings collected during the growing seasons of 2001 and 2002. Only 10 of the 187 samples were ecotype A, all of which emerged on or before May 7 in the growing seasons. Therefore, the predominant dodder haplotype found in this study may be a close relative ofC. attenuata and notC. gronovii, the common species found in cranberry bogs.     

Melatonin,immune function and aging

Aging is associated with a decline in immune function (immunosenescence), a situation known to correlate with increased incidence of cancer, infectious and degenerative diseases. Innate, cellular and humoral immunity all exhibit increased deterioration with age. A decrease in functional competence of individual natural killer (NK) cells is found with advancing age. Macrophages and granulocytes show functional decline in aging as evidenced by their diminished phagocytic activity and impairment of superoxide generation. There is also marked shift in cytokine profile as age advances, e.g., CD3+ and CD4+ cells decline in number whereas CD8+ cells increase in elderly individuals. A decline in organ specific antibodies occurs causing reduced humoral responsiveness. Circulating melatonin decreases with age and in recent years much interest has been focused on its immunomodulatory effect. Melatonin stimulates the production of progenitor cells for granulocytes-macrophages. It also stimulates the production of NK cells and CD4+ cells and inhibits CD8+ cells. The production and release of various cytokines from NK cells and T-helper lymphocytes also are enhanced by melatonin. Melatonin presumably regulates immune function by acting on the immune-opioid network, by affecting G protein-cAMP signal pathway and by regulating intracellular glutathione levels. Melatonin has the potential therapeutic value to enhance immune function in aged individuals and in patients in an immunocompromised state.