Immunization with Th-CTL fusion peptide and cytosine-phosphate-guanine DNA in transgenic HLA-A2 mice induces recognition of HIV-infected T cells and clears vaccinia virus challenge

We evaluated immunogenicity of a novel Th-CTL fusion peptide composed of the pan DR Th epitope and a CTL epitope derived from HIV-pol in two transgenic HLA-A*0201/K(b) mouse models. The immunogenicity of peptides of this structure is highly dependent on coadministered cytosine-phosphate-guanine DNA. Initial evaluations of peptide-specific immunity are based on results of chromium release assay, intracellular cytokine, and tetramer staining. Significant cytotoxic T cell responses are found upon a single immunization with as low as 0.1 nmol both peptide and cytosine-phosphate-guanine DNA. Splenocytes from immunized mice recognize naturally processed HIV-pol expressed from vaccinia virus (pol-VV). Translation of immunologic criteria into more relevant assays was pursued using systemic challenge of immunized mice with pol-VV. Only mice receiving both peptide and DNA together successfully cleared upward of 6 logs of virus from ovaries, compared with controls. Challenge with pol-VV by intranasal route of intranasal immunized mice showed a significant reduction in the levels of VV in lung compared with naive mice. A convincing demonstration of the relevance of these vaccines is the robust lysis of HIV-infected Jurkat T cells (JA2/R7/Hyg) by immune splenocytes from peptide- and DNA-immunized mice. This surprisingly effective immunization merits consideration for clinical evaluation, because it succeeded in causing immune recognition and lysis of cells infected with its target virus and reduction in titer of highly pathogenic VV.     

Augmentation of the push-pull effect by terminal aortic occlusion during head-down tilt.

Tolerance to positive vertical acceleration (Gz) gravitational stress is reduced when positive Gz stress is preceded by exposure to hypogravity, which is called the "push-pull effect." The purpose of this study was to test the hypothesis that baroreceptor reflexes contribute to the push-pull effect by augmenting the magnitude of simulated hypogravity and thereby augmenting the stimulus to the baroreceptors. We used eye-level blood pressure as a measure of the effectiveness of the blood pressure regulatory systems. The approach was to augment the magnitude of the carotid hypertension (and the hindbody hypotension) when hypogravity was simulated by head-down tilt by mechanically occluding the terminal aorta and the inferior vena cava. Sixteen anesthetized Sprague-Dawley rats were instrumented with a carotid artery catheter and a pneumatic vascular occluder cuff surrounding the terminal aorta and inferior vena cava. Animals were restrained and subjected to a control gravitational (G) profile that consisted of rotation from 0 Gz to 90 degrees head-up tilt (+1 Gz) for 10 s and a push-pull G profile consisting of rotation from 0 Gz to 90 degrees head-down tilt (-1 Gz) for 2 s immediately preceding 10 s of +1 Gz stress. An augmented push-pull G profile consisted of terminal aortic vascular occlusion during 2 s of head-down tilt followed by 10 s of +1 Gz stress. After the onset of head-up tilt, the magnitude of the fall in eye-level blood pressure from baseline was -20 +/- 1.3, -23 +/- 0.7, and -28 +/- 1.6 mmHg for the control, push-pull, and augmented push-pull conditions, respectively, with all three pairwise comparisons achieving statistically significant differences (P < 0.01). Thus augmentation of negative Gz stress with vascular occlusion increased the magnitude of the push-pull effect in anesthetized rats subjected to tilting.     

Gestation, thermoregulation, and metabolism in a viviparous snake, Vipera aspis: evidence for fecundity-independent costs

Oxygen consumption of gestating Aspic vipers, Vipera aspis (L.), was strongly dependent on body temperature and mass. Temperature-controlled, mass-independent oxygen consumption did not differ between pregnant and nonpregnant females. Maternal metabolism was not influenced during early gestation by the number of embryos carried but was weakly influenced during late gestation. These results differ from previous investigations that show an increase in mass-independent oxygen consumption in reproductive females relative to nonreproductive females and a positive relationship between metabolism and litter size. These data also conflict with published field data on V. aspis that show a strong metabolic cost associated with reproduction. We propose that, under controlled conditions (i.e., females exposed to precise ambient temperatures), following the mobilisation of resources to create follicles (i.e., vitellogenesis), early gestation per se may not be an energetically expensive period in reproduction. However, under natural conditions, the metabolic rate of reproductive females is strongly increased by a shift in thermal ecology (higher body temperature and longer basking periods), enabling pregnant females to accelerate the process of gestation. Combining both laboratory and field investigation in a viviparous snake, we suggest that reproduction entails discrete changes in the thermal ecology of females to provide optimal temperatures to the embryos, whatever their number. This results in the counterintuitive notion that metabolism may well be largely independent of fecundity during gestation, at least in an ectothermic reptile.     

Insights into cyclin groove recognition: complex crystal structures and inhibitor design through ligand exchange

Inhibition of CDK2/CA (cyclin-dependent kinase 2/cyclin A complex) activity through blocking of the substrate recognition site in the cyclin A subunit has been demonstrated to be an effective method for inducing apoptosis in tumor cells. We have used the cyclin binding motif (CBM) present in the tumor suppressor proteins p21(WAF1) and p27(KIP1) as a template to optimize the minimal sequence necessary for CDK2/CA inhibition. A series of peptides were prepared, containing nonnatural amino acids, which possess nano- to micromolar CDK2-inhibitory activity. Here we present X-ray structures of the protein complex CDK2/CA, together with the cyclin groove-bound peptides H-Ala-Ala-Abu-Arg-Ser-Leu-Ile-(p-F-Phe)-NH(2) (peptide 1), H-Arg-Arg-Leu-Ile-Phe-NH(2) (peptide 2), Ac-Arg-Arg-Leu-Asn-(m-Cl-Phe)-NH(2) (peptide 3), H-Arg-Arg-Leu-Asn-(p-F-Phe)-NH(2) (peptide 4), and H-Cit-Cit-Leu-Ile-(p-F-Phe)-NH(2) (peptide 5). Some of the peptide complexes presented here were obtained through the novel technique of ligand exchange within protein crystals. This method may find general application for obtaining complex structures of proteins with surface-bound ligands.     

Tyrosine 537 within the Na+,K+-ATPase alpha-subunit is essential for AP-2 binding and clathrin-dependent endocytosis

In renal epithelial cells endocytosis of Na(+),K(+)-ATPase molecules is initiated by phosphorylation of its alpha(1)-subunit, leading to activation of phosphoinositide 3-kinase and adaptor protein-2 (AP-2)/clathrin recruitment. The present study was performed to establish the identity of the AP-2 recognition domain(s) within the Na(+),K(+)-ATPase alpha(1)-subunit. We identified a conserved sequence (Y(537)LEL) within the alpha(1)-subunit that represents an AP-2 binding site. Binding of AP-2 to the Na(+),K(+)-ATPase alpha(1)-subunit in response to dopamine (DA) was increased in OK cells stably expressing the wild type rodent alpha-subunit (OK-WT), but not in cells expressing the Y537A mutant (OK-Y537A). DA treatment was associated with increased alpha(1)-subunit abundance in clathrin vesicles from OK-WT but not from OK-Y537A cells. In addition, this mutation also impaired the ability of DA to inhibit Na(+),K(+)-ATPase activity. Because phorbol esters increase Na(+),K(+)-ATPase activity in OK cells, and this effect was not affected by the Y537A mutation, the present results suggest that the identified motif is specifically required for DA-induced AP-2 binding and Na(+),K(+)-ATPase endocytosis.     

Modification of fatty acid composition in tomato (Lycopersicon esculentum) by expression of a borage delta6-desaturase

The improvement of nutritional quality is one potential application for the genetic modification of plants. One possible target for such manipulation is the modification of fatty acid metabolism. In this work, expression of a borage Δ⁶-desaturase cDNA in tomato (Lycopersicon esculentum L.) has been shown to produce γ-linolenic acid (GLA; 18:3 Δ^6,9,12) and octadecatetraenoic acid (OTA; 18:4 Δ^6,9,12,15) in transgenic leaf and fruit tissue. This genetic modification has also, unexpectedly, resulted in a reduction in the percentage of linoleic acid (LA 18:2 Δ^9,12) and a concomitant increase in the percentage of α-linolenic acid (ALA; 18:3 Δ^9,12,15) in fruit tissue. These changes in fatty acid composition are thought to be beneficial for human health.     

SVR angiosarcomas can be rejected by CD4 costimulation dependent and CD8 costimulation independent pathways

PURPOSE: We wished to determine whether virally- induced endothelial tumors are rejected by CD4 and CD8 lymphocytes, and whether there are differences in requirements for costimulation in the rejection of these tumors by lymphocyte subsets. EXPERIMENTAL DESIGN: We have developed a model of endothelial tumorigenesis through the sequential introduction of SV40 large T antigen and oncogenic H-ras into endothelial cells. These cells (SVR cells) form highly aggressive angiosarcomas in immunocompromised mice, but do not grow in syngeneic C57BL/6 mice. Using both acute blockade with systemic administration of antibodies and mice genetically deficient in the costimulatory molecules CD28, CD40, and CD40L, we have delineated the requirements of costimulation required to reject this virally-induced endothelial tumor. RESULTS: Control of SVR angiosarcoma is mediated through T lymphocytes, and both CD4 and CD8 lymphocytes are capable of controlling SVR angiosarcoma growth in vivo. Mice genetically deficient in CD28, CD40, and CD40L were able to reject SVR tumors, but depletion of these mice of CD8, but not CD4 cells led to rapid tumor growth. This data suggests that CD4 mediated rejection has a greater dependence of costimulation than CD8 mediated rejection. Surprisingly, acute depletion of costimulatory molecules in immunocompetent C57BL/6 mice led to rapid tumor growth. CONCLUSIONS: Significant differences exist in the immune status of mice acutely depleted of costimulatory molecules versus genetically deficient mice. Our results suggest that acute depletion is more immunosuppressive than genetic depletion. Humans who undergo costimulatory blockade may require periodic surveillance for virally-induced tumors.     

Comparison of PGE2, prostacyclin and leptin release by human adipocytes versus explants of adipose tissue in primary culture

The present studies were designed to investigate the sites of PGE(2), prostacyclin and leptin formation in human adipose tissue. Most of the PGE(2) and prostacyclin formation by adipose tissue explants from obese humans after 48 h in primary culture was due to blood vessels and other tissues not digested by collagenase. However, there was appreciable PGE(2) formation by adipocytes over a 48 h incubation and leptin formation was only seen in adipocytes. An increase in COX-2 immunoreactive protein was also seen after incubation of isolated human adipocytes for 48 h. The release of PGE(2) by adipocytes incubated for 48 h was about 4% that by intact adipose tissue explants while the release of prostacyclin was about 1.5% that by tissue. However, in a different experimental design where PGE(2) formation was measured over 2 h in the presence of 20 microM arachidonic acid the formation of PGE(2) by adipocytes after 48 h prior incubation in primary culture was 38% of that by tissue explants. Dexamethasone enhanced leptin release by adipocytes while inhibiting PGE(2) release and COX-2 up-regulation. The mechanisms involved in up-regulation of COX-2 activity during primary culture of adipocytes and the inhibition of this by dexamethasone do not appear to involve p38 MAPK or p42-44 MAPK. Interleukin I(beta) further enhanced PGE(2) formation by adipocytes but did not affect leptin formation. In conclusion, these data indicate that leptin release is exclusively a function of adipocytes while prostanoids are made by both adipocytes and the other cells present in human adipose tissue