Effects of vitamin D3 and IFN-gamma on the synthesis of the second complement component, C2, by a human myeloid leukemia (HL-60) cell line

HL-60 cells, a human promyelocytic cell line, can be induced to differentiate along either monocytic or granulocytic pathways. The production of the second complement component, C2, is a marker of monocytic differentiation and can be up-regulated by cytokine stimulation. We studied the effects of IFN-gamma and vitamin D3, two factors previously shown to induce monocytic differentiation of HL-60 cells, on C2 production and C2 mRNA content. We found that HL-60 cells produce little if any C2 but can be induced to synthesize C2 by IFN-gamma. Vitamin D3 pretreatment followed by IFN-gamma stimulation resulted in earlier and greater production of C2. HL-60 cells did not contain detectable amounts of C2 mRNA unless they were stimulated with IFN-gamma. Pretreatment with vitamin D3 followed by IFN-gamma stimulation resulted in a 147% increase in C2 mRNA content compared with IFN-gamma stimulation alone. These results indicate that the up-regulation of C2 production by IFN-gamma and vitamin D3 is pretranslational although additional posttranslational effects were not excluded. C2 production by these cells is a useful marker of monocytic differentiation.     

Molecular evolution modeled as a fractal Poisson process in agreement with mammalian sequence comparisons

The fractal doubly stochastic Poisson process (FDSPP) model of molecular evolution, like other doubly stochastic Poisson models, agrees with the high estimates for the index of dispersion found from sequence comparisons. Unlike certain previous models, the FDSPP also predicts a positive geometric correlation between the index of dispersion and the mean number of substitutions. Such a relationship is statistically proven herein using comparisons between 49 mammalian genes. There is no characteristic rate associated with molecular evolution according to this model, but there is a scaling relationship in rates according to a fractal dimension of evolution. The FDSPP is a suitable replacement for the homogeneous Poisson process in tests of the lineage dependence of rates and in estimating confidence intervals for divergence times. As opposed to other fractal models, this model can be interpreted in terms of Darwinian selection and drift.      

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.     

The modulation of choline phosphoglyceride metabolism in human colon cancer

Colorectal cancer has a high incidence of morbidity and mortality in the North American population. Elevated levels of plasmalogens have been reported in some neoplastic tissues including colon tumors, but the mechanism for this increase has not been defined. Since changes in plasmalogen level are usually associated with changes in the other phospholipid subclasses, a general increase in all phospholipid subclasses may also be found in colonic neoplasms. In this study, the levels of the major phospholipids, including their plasmalogen and diacylphospholipid subclasses, were found to be elevated in human malignant colonic tissues. Since phosphatidylcholine is the most prominent type of phospholipid found in both malignant and control tissues, the mechanism for its accumulation during malignancy was investigated. Decreases in phospholipase C and D activities were observed in tumor samples, but an enhancement of the CTP: phosphocholine cytidylyltransferase activity was also detected. Immunoblotting analysis revealed that the elevated cytidylyltransferase activity was caused by a three-fold increase in the level of enzyme protein during tumor development. Based on these enzyme studies, we conclude that the high level of phosphatidylcholine in colon tumors resulted from a decrease in its turnover and an increase in its expression.     

Packaging system for rapid production of murine leukemia virus vectors with variable tropism.

A method for rapidly producing helper-free murine leukemia virus (MLV) without using packaging cell lines is described. Viruses bearing ecotropic or amphotropic MLV or Rous sarcoma virus envelope glycoprotein and containing various retroviral vector genomes have been prepared with titers 30 to 40-fold higher than those produced by transient transfection of standard packaging cells. This system can be used to alter the cellular tropism of MLV by incorporating other envelope glycoproteins and to prepare retroviral vector stocks without establishing stable producer cell lines. This method will be particularly useful for preparing viruses that encode toxic proteins and for the rapid analysis of panels of mutant envelope glycoproteins.     

On the distribution of Na+ pump sites in the frog skin

Exposure of the outside of the isolated frog skin to a Ringer's solution, made hypertonic by the addition of mannitol, causes a rapid and sustained increase in transepithelial permeability through a structural distortion-a focal blistering-of the "tight" junctions of the outermost living cell layer. [(3)H]ouabain, used as an autoradiographic marker for the Na+-pump (Na+-K+-adenosine triphosphatase), is usually unable to penetrate the frog skin from the outside solution, but when added to a hypertonic mannitol- Ringer's solution in the outside bath it readily penetrates the epithelium, presumably through the opened shunt pathway. Radioautographic analysis of [(3)H]ouabain binding sites revealed that most of ouabain enters from the outside solution binds to the sites on the cell membranes of the stratum spinosum, as was the case when it was applied from the inside bath in an earlier study. The outer living cell layer, the first to be exposed to ouabain, does not appear to be the major site for the Na+-pump, and therefore, is not likely to be responsible for most of the active pumping of Na+. This result demonstrates that previous failure to show a high density of Na+-pump sites on the cells of the outermost layer, when [(3)H]ouabain was applied from the inside solution, was not due to the inability of the marker to reach these cells at a sufficient concentration to reveal all pump sites. These results provide further support for a model of Na+-transport across the frog skin which distributes the active pump step on the inward facing membranes of all living cells.     

Acquired cellular immunity: extracellular killing of Listeria monocytogenes by a product of immunologically activated macrophages

A listericidal factor was released by monolayers of peritoneal cells from BCG-immune guinea pigs following incubation with PPD in cell culture. Significantly less listericidal factor was released by monolayers of BCG-immune cells incubated without PPD, or by monolayers of nonimmune cells incubated in the presence or absence of antigen. Culture supernatants containing listericidal factor were active at a dilution of 1:10, but supernatants diluted 1:100 were not listericidal. Dialysis of supernatants against fresh tissue culture medium did not affect their ability to kill Listeria, although heating at 60 °C for 30 min destroyed all activity. Supernatants from cultures of guinea pig fibroblasts or ascites-form hepatoma were not listericidal.     

Envelope glycoproteins from human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus can use human CCR5 as a coreceptor for viral entry and make direct CD4-dependent interactions with this chemokine receptor.

Several members of the chemokine receptor family have recently been identified as coreceptors, with CD4, for entry of human immunodeficiency virus type 1 (HIV-1) into target cells. In this report, we show that the envelope glycoproteins of several strains of HIV-2 and simian immunodeficiency virus (SIV) employ the same chemokine receptors for infection. Envelope glycoproteins from HIV-2 use CCR5 or CXCR4, while those from several strains of SIV use CCR5. Our data indicate also that some viral envelopes can use more than one coreceptor for entry and suggest that some of these coreceptors remain to be identified. To further understand how different envelope molecules use CCR5 as an entry cofactor, we show that soluble purified envelope glycoproteins (SU component) from CCR5-tropic HIV-1, HIV-2, and SIV can compete for binding of iodinated chemokine to CCR5. The competition is dependent on binding of the SU glycoprotein to cell surface CD4 and implies a direct interaction between envelope glycoproteins and CCR5. This interaction is specific since it is not observed with SU glycoprotein from a CXCR4-tropic virus or with a chemokine receptor that is not competent for viral entry (CCR1). For HIV-1, the interaction can be inhibited by antibodies specific for the V3 loop of SU. Soluble CD4 was found to potentiate binding of the HIV-2 ST and SIVmac239 envelope glycoproteins to CCR5, suggesting that a CD4-induced conformational change in SU is required for subsequent binding to CCR5. These data suggest a common fundamental mechanism by which structurally diverse HIV-1, HIV-2, and SIV envelope glycoproteins interact with CD4 and CCR5 to mediate viral entry.     

Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype.

The biological phenotype of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to the severity of the HIV infection. Here we show that the two previously described groups of rapid/high, syncytium-inducing (SI) and slow/low, non-syncytium-inducing (NSI) isolates are distinguished by their ability to utilize different chemokine receptors for entry into target cells. Recent studies have identified the C-X-C chemokine receptor CXCR4 (also named fusin or Lestr) and the C-C chemokine receptor CCR5 as the principal entry cofactors for T-cell-line-tropic and non-T-cell-line-tropic HIV-1, respectively. Using U87.CD4 glioma cell lines, stably expressing the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, we have tested chemokine receptor specificity for a panel of genetically diverse envelope glycoprotein genes cloned from primary HIV-1 isolates and have found that receptor usage was closely associated with the biological phenotype of the virus isolate but not the genetic subtype. We have also analyzed a panel of 36 well-characterized primary HIV-1 isolates for syncytium induction and replication in the same series of cell lines. Infection by slow/low viruses was restricted to cells expressing CCR5, whereas rapid/high viruses could use a variety of chemokine receptors. In addition to the regular use of CXCR4, many rapid/high viruses used CCR5 and some also used CCR3 and CCR2b. Progressive HIV-1 infection is characterized by the emergence of viruses resistant to inhibition by beta-chemokines, which corresponded to changes in coreceptor usage. The broadening of the host range may even enable the use of uncharacterized coreceptors, in that two isolates from immunodeficient patients infected the parental U87.CD4 cell line lacking any engineered coreceptor. Two primary isolates with multiple coreceptor usage were shown to consist of mixed populations, one with a narrow host range using CCR5 only and the other with a broad host range using CCR3, CCR5, or CXCR4, similar to the original population. The results show that all 36 primary HIV-1 isolates induce syncytia, provided that target cells carry the particular coreceptor required by the virus.