Determination of surface immunoglobulin density on frozen-thawed mononuclear cells analyzed by the fluorescence-activated cell sorter

Recent data have demonstrated that differences in sIg density on B lymphocytes distinguish functionally distinct subpopulations of these cells. Other reports suggest that cyropreservation may change the frequency of sIg-bearing lymphocytes. To determine if cryopreservation alters either the frequency of sIg cells or the distribution of sIg density, PBM from normals and patients with CLL and LCL were analyzed using the FACS. Aliquots of Ficoll-Hypaque-separated PBM were controlled-rate frozen (1 °C/min) in 7.5% Me₂SO in RPMI 1640 and thawed in a 37 °C water bath on the same day. Fresh and frozen-thawed PBM aliquots were labeled with fluorescein conjugates of F(ab′) fragments of affinity chromatography-purified anti-Fab or class-specific anti-μ, anti-δ, anti-γ, or anti-α. Histograms of relative cell fluorescence, reflecting sIg density, were prepared for each aliquot with the FACS. The frequency of sIg-bearing PBM labeled with each reagent was not significantly altered by freezing. Likewise, FACS profiles demonstrated that the distribution of sIg density on normal and CLL PBM was unchanged after freezing. However, the fluorescence peak produced by frozen-thawed unlabeled cells was occasionally slightly broader than that of fresh cells, suggesting increased autofluorescence induced by freezing. These data indicate that frozen cell preparations may be utilized for the study of B-lymphocyte subsets as determined by sIg density.     

Phenotypic identification of memory cytolytic T lymphocytes in a subset of Lyt-2+ cells

Murine peripheral Lyt-2+ T cells could be subdivided according to surface expression of the Pgp-1 glycoprotein into major (71%) Pgp-1- and minor (29%) Pgp-1+ subsets. A striking correlation was observed between Pgp-1 expression and enrichment for antigen-specific memory cytolytic T lymphocyte precursors (CTLp). After immunization with the male minor transplantation antigen H-Y, virtually all the H-Y-specific CTLp were found in the minor Pgp-1+ subset of Lyt-2+ cells. In addition, after alloimmunization the frequency of allospecific CTLp resistant to inhibition by anti-Lyt-2 antibody was markedly enriched within the Pgp-1+ cells, suggesting an enrichment for CTLp bearing high avidity antigen receptors. Taken together, these data suggest that surface Pgp-1 expression is stably acquired at the time of primary antigenic stimulation by virgin T cells. As such, Pgp-1 represents an important marker for identifying a subset of Lyt-2+ T cells with the quantitative and qualitative properties of memory CTLp.     

Allometric shape change and heterochrony in the freeliving coral Trachyphyllia bilobata (Duncan)

Regression analysis has been used to study the relationship between age, size, shape, and surface area in two ancestral-descendant populations of the Neogene Caribbean coral Trachyphyllia bilobata. Analyses of the relationship between size and age show that the relationship is isometric and that little difference occurs between populations in mean corallite length or height and in their rates of growth. Onset of columella growth is significantly earlier, however, in the descendant population. Studies of the relationship between size and shape show that growth is allometric, with shape change occurring in both corallum elongation and pinching of the corallite wall during ontogeny. In the descendant population, pinching and elongation initiate earlier in the ontogeny of the coral. These results suggest that the evolutionary development of the meandroid form in freeliving corals has been accomplished by heterochrony, involving a complex set of disassociated peramorphic changes in ontogeny accompanied by paedomorphic changes in astogeny. Further analyses show that the observed heterochronic changes serve to decrease corallum surface area which may in turn enhance sediment removal and nutrition in unstable habitats.     

Purification of DNA polymerase II, a distinct DNA polymerase, from Saccharomyces cerevisiae

Yeast DNA polymerases I and III have been well characterized physically, biochemically, genetically and immunologically. DNA polymerase II is present in very small amounts, and only partially purified preparations have been available for characterization, making comparison with DNA polymerases I and III difficult. Recently, we have shown that DNA polymerases II and III are genetically distinct (Sitney et al., 1989). In this work, we show that polymerase II is also genetically distinct from polymerase I, since polymerase II can be purified in equal amounts from wild-type and mutant strains completely lacking DNA polymerase I activity. Thus, yeast contains three major nuclear DNA polymerases. The core catalytic subunit of DNA polymerase II was purified to near homogeneity using a reconstitution assay. Two factors that stimulate the core polymerase were identified and used to monitor activity during purification and analysis. The predominant species of the most highly purified preparation of polymerase II is 132,000 Da. However, polymerase activity gels suggest that the 132,000-Da form of DNA polymerase II is probably an active proteolytic fragment derived from a 170,000-Da protein. The highly purified polymerase fractions contain a 3'----5'-exonuclease activity that purifies at a constant ratio with polymerase during the final two purification steps. However, DNA polymerase II does not copurify with a DNA primase activity.     

Cardiovascular and metabolic responses to noradrenaline in men acclimatized to cold baths

The purpose of this study was to see whether artificial acclimatization to cold would reduce the pressor response to noradrenaline (NA) as natural acclimatization has been shown to do, and whether it would induce nonshivering thermogenesis. Three white men were infused with NA at four dosage levels between 0.038 and 0.300 g·kg^–1·min^–1 (2–23 g·min^–1), before and after artificial acclimatization to cold and again 4 months later when acclimatization had decayed. Acclimatization was induced by ten daily cold (15°Q baths of 30–60 min followed by rapid rewarming in hot (38–42°C) water, and was confirmed by tests of the subjects responses to whole-body cooling in air. Three control subjects also underwent the first and third tests. Acclimatization substantially reduced the pressor response to NA at 0.150 and 0.300 g·kg^–1·min^–1, confirming earlier findings by the same technique in naturally acclimatized men, and its decay increased this response to beyond its initial levels (P<0.05 for both changes). Acclimatization did not change the response to NA of heart rate, subjective impressions, skin temperature of finger and toe, pulmonary ventilation, or plasma free fatty acids and ketone bodies. At no time did NA increase oxygen consumption, or increase skin temperature or heat flow over reported sites of brown fat. These findings would seem to show that acclimatization to cold reduces sensitivity to the pressor effect of NA but does not induce nonshivering thermogenesis, and that the reduced sensitivity is replaced by a hypersensitivity to NA when acclimatization decays.     

Mortality of young-of-the-year rainbow smelt (Osmerus mordax) in Lake Erie associated with the occurrence of Glugea hertwigi

An extensive mortality of young-of-the-year rainbow smelt (Osmerus mordax) occurred in Lake Erie during the fall of 1969. Dead and dying smelt were observed along the north shore from west of Long Point in the central basin to Port Maitland in the eastern basin. No other fish species was involved. Glugea hertwigi, a microsporidan parasite, was observed in 90% of the distressed smelt examined, and is believed to have been a major contributing factor to the mortality.     

Effects of diet,habitat, and phylogeny on the fecal microbiome of wild African savanna (Loxodonta africana) and forest elephants (L. cyclotis)

The gut microbiome, or the community of microorganisms inhabiting the digestive tract, is often unique to its symbiont and, in many animal taxa, is highly influenced by host phylogeny and diet. In this study, we characterized the gut microbiome of the African savanna elephant (Loxodonta africana) and the African forest elephant (Loxodonta cyclotis), sister taxa separated by 2.6–5.6 million years of independent evolution. We examined the effect of host phylogeny on microbiome composition. Additionally, we examined the influence of habitat types (forest versus savanna) and diet types (crop‐raiding versus noncrop‐raiding) on the microbiome within L. africana. We found 58 bacterial orders, representing 16 phyla, across all African elephant samples. The most common phyla were Firmicutes, Proteobacteria, and Bacteroidetes. The microbiome of L. africana was dominated by Firmicutes, similar to other hindgut fermenters, while the microbiome of L. cyclotis was dominated by Proteobacteria, similar to more frugivorous species. Alpha diversity did not differ across species, habitat type, or diet, but beta diversity indicated that microbial communities differed significantly among species, diet types, and habitat types. Based on predicted KEGG metabolic pathways, we also found significant differences between species, but not habitat or diet, in amino acid metabolism, energy metabolism, and metabolism of terpenoids and polyketides. Understanding the digestive capabilities of these elephant species could aid in their captive management and ultimately their conservation.     

Dna2 Is Involved in CA Strand Resection and Nascent Lagging Strand Completion at Native Yeast Telomeres

Post-replicational telomere end processing involves both extension by telomerase and resection to produce 3′-GT-overhangs that extend beyond the complementary 5′-CA-rich strand. Resection must be carefully controlled to maintain telomere length. At short de novo telomeres generated artificially by HO endonuclease in the G₂ phase, we show that dna2-defective strains are impaired in both telomere elongation and sequential 5′-CA resection. At native telomeres in dna2 mutants, GT-overhangs do clearly elongate during late S phase but are shorter than in wild type, suggesting a role for Dna2 in 5′-CA resection but also indicating significant redundancy with other nucleases. Surprisingly, elimination of Mre11 nuclease or Exo1, which are complementary to Dna2 in resection of internal double strand breaks, does not lead to further shortening of GT-overhangs in dna2 mutants. A second step in end processing involves filling in of the CA-strand to maintain appropriate telomere length. We show that Dna2 is required for normal telomeric CA-strand fill-in. Yeast dna2 mutants, like mutants in DNA ligase 1 (cdc9), accumulate low molecular weight, nascent lagging strand DNA replication intermediates at telomeres. Based on this and other results, we propose that FEN1 is not sufficient and that either Dna2 or Exo1 is required to supplement FEN1 in maturing lagging strands at telomeres. Telomeres may be among the subset of genomic locations where Dna2 helicase/nuclease is essential for the two-nuclease pathway of primer processing on lagging strands.