Therapeutic Restoration of Endothelial Glycocalyx in Sepsis.

Endothelial glycocalyx (EG) is disintegrated during sepsis. We have previously shown that this occurs very early in the course of sepsis and its prevention improves the survival of mice with sepsis. Here, we sought to investigate the possibility of pharmacologically accelerating the restoration of disintegrated EG in sepsis. We used a soilage injection model to induce polymicrobial sepsis in C57/BL6 mice and measured total body EG. En face aortic preparations were used for staining of markers of EG and atomic force microscopy was used to measure EG in vitro. In vitro studies were conducted in cultured endothelial cells either exposed to a lipopolysaccharide or enzymatically denuded of EG. Sulodexide (SDX), a heparin sulfate-like compound resistant to degradation by heparanase, accelerated EG regeneration in vitro and in vivo. The total volume of EG was drastically reduced in septic mice. Administration of SDX produced a dramatic acceleration of EG restoration. This effect, unrelated to any SDX-induced differences in microbial burden, was associated with better control of vascular permeability. Notably, SDX demonstrated not only a remarkable capacity for EG regeneration in vitro and in vivo but was also associated with improved animal survival, even when instituted 2 hours after induction of severe sepsis. In conclusion, 1) EG is disintegrated in sepsis, the event which contributes to high animal mortality; 2) pharmacologic acceleration of EG restoration can be achieved using SDX; and 3) SDX reduces vascular permeability, which is elevated in septic mice, and improves animal survival.

Transcriptional repression mediated by repositioning of genes to the nuclear lamina.

Nuclear compartmentalization seems to have an important role in regulating metazoan genes. Although studies on immunoglobulin and other loci have shown a correlation between positioning at the nuclear lamina and gene repression, the functional consequences of this compartmentalization remain untested. We devised an approach for inducible tethering of genes to the inner nuclear membrane (INM), and tested the consequences of such repositioning on gene activity in mouse fibroblasts. Here, using three-dimensional DNA-immunoFISH, we demonstrate repositioning of chromosomal regions to the nuclear lamina that is dependent on breakdown and reformation of the nuclear envelope during mitosis. Moreover, tethering leads to the accumulation of lamin and INM proteins, but not to association with pericentromeric heterochromatin or nuclear pore complexes. Recruitment of genes to the INM can result in their transcriptional repression. Finally, we use targeted adenine methylation (DamID) to show that, as is the case for our model system, inactive immunoglobulin loci at the nuclear periphery are contacted by INM and lamina proteins. We propose that these molecular interactions may be used to compartmentalize and to limit the accessibility of immunoglobulin loci to transcription and recombination factors.

The effects of message framing and ethnic targeting on mammography use among low-income women.

The authors examined the effects that differently framed and targeted health messages have on persuading low-income women to obtain screening mammograms. The authors recruited 752 women over 40 years of age from community health clinics and public housing developments and assigned the women randomly to view videos that were either gain or loss framed and either targeted specifically to their ethnic groups or multicultural. Loss-framed, multicultural messages were most persuasive. The advantage of loss-framed, multicultural messages was especially apparent for Anglo women and Latinas but not for African American women. These effects were stronger after 6 months than after 12 months.

Resistance of primary isolates of human immunodeficiency virus type 1 to neutralization by soluble CD4 is not due to lower affinity with the viral envelope glycoprotein gp120.

Recombinant soluble CD4 (rsCD4) has potent antiviral activity against cell line-adapted isolates of the human immunodeficiency virus type 1 (HIV-1) but low activity toward HIV-1 primary isolates from patients. A simple hypothesis proposed to explain this discrepancy, which questions the therapeutic utility of soluble CD4-based approaches, is that the major envelope glycoprotein, gp120, of patient virus has lower affinity for CD4 than does gp120 from laboratory viruses. To test this hypothesis, we have produced pairs of low- and high-passage HIV-1 isolates which, depending on culture passage history, display dramatically different sensitivities to neutralization by rsCD4. Here, we present evidence that the HIV-1 major envelope glycoprotein cDNAs cloned from one such isolate pair show only minor differences in their deduced gp120 primary structures, and these occur outside regions previously shown to be involved in CD4 interactions. In addition, recombinant gp120 from a low-passage rsCD4-resistant patient virus binds rsCD4 with high affinity, equal to that previously measured for recombinant gp120 from high-passage cell line-adapted virus isolates. These data indicate that differences in CD4-gp120 affinity do not account for rsCD4 resistance in HIV-1 recently isolated from patients.

P21 v-ras inhibits induction of c-myc and c-fos expression by platelet-derived growth factor.

The viral oncogene v-ras inhibited the platelet-derived growth factor (PDGF)-induced upregulation of c-myc and c-fos proto-oncogene expression in fibroblast monolayers. These v-ras-containing cells proliferated in the absence of c-myc induction and no longer required PDGF to support growth. Fibroblasts expressing v-ras continued to express the same number of functional PDGF receptors on their surface as uninfected cells, yet the usual induction of transcription of the genes c-myc, c-fos, and JE in response to PDGF stimulation did not occur in the presence of newly introduced v-ras or chronic v-ras gene expression, and synthesis of c-myc protein did not occur. This inhibitory effect on growth factor-mediated induction of cellular proto-oncogenes was specific for PDGF in that induction of the c-myc and c-fos genes by certain other factors was not impaired.

Regulation of c-myc and c-fos mRNA levels by polyomavirus: distinct roles for the capsid protein VP1 and the viral early proteins.

The levels of c-myc, c-fos, and JE mRNAs accumulate in a biphasic pattern following infection of quiescent BALB/c 3T3 mouse cells with polyomavirus. Maximal levels of c-myc and c-fos mRNAs were seen within 1 hr and were nearly undetectable at 6 hr after infection. At 12 hr after infection mRNA levels were again maximal and remained elevated thereafter. Empty virions (capsids) and recombinant VP1 protein, purified from Escherichia coli, induced the early but not the late phase of mRNA accumulation. Virions, capsids, and recombinant VP1 protein stimulated [3H]thymidine nuclear labeling and c-myc mRNA accumulation in a dose-responsive manner paralleling their affinity for the cell receptor for polyoma. The second phase of mRNA accumulation is regulated by the viral early gene products, as shown by polyomavirus early gene mutants and by a transfected cell line (336a) expressing middle tumor antigen upon glucocorticoid addition. These results suggest that polyomavirus interacts with the cell membrane at the onset of infection to increase the levels of mRNA for cellular genes associated with cell competence for DNA replication, and subsequently these levels are maintained by the action of the early viral proteins.

Platelet-derived growth factor and double-stranded ribonucleic acids stimulate expression of the same genes in 3T3 cells.

Platelet-derived growth factor (PDGF) stimulates expression of a "competence" gene family in Balb/c-3T3 cells. The competence family contains the c-myc and c-fos genes together with several functionally uncharacterized genes (JE, KC, and r-fos) that have been isolated as cDNA clones. We show that double-stranded ribonucleic acid is a potent inducer of the competence gene family. Infection with vesicular stomatitis virus also induces expression of this gene family. Conversely, PDGF stimulates expression of genes hitherto characterized as responsive to double-stranded ribonucleic acids, including the beta-fibroblast interferon and (2'-5')-oligoadenylate synthetase genes. These PDGF-inducible genes could conceivably function in a feedback loop to control 3T3 cell growth. Some of the genes, such as c-fos and c-myc, are induced quickly by PDGF and may initiate a round of cell division. Others, such as beta-fibroblast interferon and (2'-5')-oligoadenylate synthetase, are induced more slowly and may function as feedback inhibitors of the growth response to PDGF.

Expression of the c-fos gene and of an fos-related gene is stimulated by platelet-derived growth factor.

Complementary DNA clones of genes induced by platelet-derived growth factor (PDGF) in BALB/c-3T3 cells were isolated; one such clone contains a domain having nucleotide sequence homology with the third exon of c-fos. This nucleotide sequence homology is reflected in the predicted amino acid sequences of the gene products. Under low stringency conditions, the mouse v-fos gene cross-hybridizes with the PDGF-inducible complementary DNA clone. However, the messenger RNA transcripts of mouse c-fos and the new fos-related gene can be distinguished by gel electrophoresis and by S1 nuclease analysis. Expression of the authentic c-fos gene is induced by PDGF and superinduced by the combination of PDGF and cycloheximide.

Mechanism of reduced water intake in rats at high altitude.

Water intake was reduced during the 1st day of hypobaric hypoxia (inspired O2 pressure of 75 Torr) to 35-40% of the normoxic level in both normal rats (N) and rats with diabetes insipidus (DI). Analysis of water intake under graded saline loads at several inspired O2 levels (inspired O2 fractional concentrations of 0.105, 0.120, and 0.2095) indicated that hypoxia increased the threshold for osmotic stimulation of drinking without changing the sensitivity of the response in both N and DI rats. Nephrectomized N rats reduced water intake during hypoxia to 33% of the nephrectomized normoxic level of intake, and nephrectomized DI rats reduced intake to 47% of the nephrectomized normoxic intake. From these results it is concluded that reduced angiotensin II formation was not the factor responsible for reduced water intake during hypoxia. Polyethylene glycol-induced hypovolemia resulted in increased water intake during normoxia, but during hypoxia it was reduced to 29% of the normoxic rate. Reduced body temperature and hyperventilation were not the source of hypoxic attenuation of thirst. The mechanism may reside beyond the central integration of osmotic and nonosmotic information, or at the osmotic sensing mechanism itself.