Activation of brain renin-angiotensin-aldosterone system by central sodium in Wistar rats

Functional studies indicate that the sympathoexcitatory and pressor responses to an increase in cerebrospinal fluid (CSF) [Na+] by central infusion of Na+-rich artificial cerebrospinal fluid (aCSF) in Wistar rats are mediated in the brain by mineralocorticoid receptor (MR) activation, ouabain-like compounds (OLC), and AT1-receptor stimulation. In the present study, we examined whether increasing CSF [Na+] by intracerebroventricular infusion of Na+-rich aCSF activates MR and thereby increases OLC and components of the renin-angiotensin system in the brain. Male Wistar rats received via osmotic minipump an intracerebroventricular infusion of aCSF or Na+-rich aCSF, in some groups combined with intracerebroventricular infusion of spironolactone (100 ng/h), antibody Fab fragments (to bind OLC), or as control gamma-globulins. After 2 wk of infusion, resting blood pressure and heart rate were recorded, OLC and aldosterone content in the hypothalamus were assessed by a specific ELISA or radioimmunoassay, and angiotensin-converting enzyme (ACE) and AT1-receptor binding densities in various brain nuclei were measured by autoradiography using 125I-labeled 351 A and 125I-labeled ANG II. When compared with intracerebroventricular aCSF, intracerebroventricular Na+-rich aCSF increased CSF [Na+] by approximately 5 mmol/l, mean arterial pressure by approximately 20 mmHg, heart rate by approximately 65 beats/min, and hypothalamic content of OLC by 50% and of aldosterone by 33%. Intracerebroventricular spironolactone did not affect CSF [Na+] but blocked the Na+-rich aCSF-induced increases in blood pressure and heart rate and OLC content. Intracerebroventricular Na+-rich aCSF increased ACE and AT1-receptor-binding densities in several brain nuclei, and Fab fragments blocked these increases. These data indicate that in Wistar rats, a chronic increase in CSF [Na+] may increase hypothalamic aldosterone and activate CNS pathways involving MR, and OLC, leading to increases in AT1-receptor and ACE densities in brain areas involved in cardiovascular regulation and hypertension.     

Stimulation of brain Na+ channels by FMRFamide in Dahl SS and SR rats

Stimulation of brain Na+ channels by Phe-Met-Arg-Phe-NH2 (FMRFamide) increases sympathetic nerve activity and blood pressure (BP) in Wistar rats. Blockade of brain ouabain-like compounds (OLC) by specific antibody Fab fragments prevents these responses to intracerebroventricular FMRFamide. In the present study, we evaluated the effects of high-salt intake on brain FMRFamide levels and the responses of BP and brain OLC to intracerebroventricular infusion of FMRFamide in Dahl salt-sensitive (SS) and salt-resistant (SR) rats. FMRFamide and OLC content was measured with the use of RIA and ELISA, respectively. A high-salt diet (1,370 micromol Na+/g) for 2 wk significantly increased BP in Dahl SS but not in SR rats. On a regular salt diet, Dahl SS and SR rats showed similar FMRFamide levels in the whole hypothalamus, pons and medulla, and spinal cord. A high-salt diet for 2 wk did not affect FMRFamide levels in these tissues in both Dahl SS and SR rats. In Dahl SS but not in SR rats, chronic intracerebroventricular infusion of FMRFamide (200 nmol. kg(-1).day(-1)) for 2 wk significantly increased BP (mean arterial pressure: 116 +/- 5 vs. 100 +/- 2 mmHg; P < 0.01). Chronic intracerebroventricular infusion of FMRFamide significantly increased hypothalamic and pituitary OLC in Dahl SS but not SR rats. These results indicate that Dahl SS rats exhibit enhanced central responses to FMRFamide. In Dahl SS but not in SR rats on a high-salt diet, enhanced Na+ entry through FMRFamide-activated brain Na+ channels may increase brain OLC release, thereby leading to hypertension.     

Cardiac adaptation to endurance exercise in rats

Endurance exercise is widely assumed to improve cardiac function in humans. This project has determined cardiac function following endurance exercise for 6 (n = 30) or 12 (n = 25) weeks in male Wistar rats (8 weeks old). The exercise protocol was 30 min/day at 0.8 km/h for 5 days/week with an endurance test on the 6th day by running at 1.2 km/h until exhaustion. Exercise endurance increased by 318% after 6 weeks and 609% after 12 weeks. Heart weight/kg body weight increased by 10.2% after 6 weeks and 24.1% after 12 weeks. Echocardiography after 12 weeks showed increases in left ventricular internal diameter in diastole (6.39 ± 0.32 to 7.90 ± 0.17 mm), systolic volume (49 ± 7 to 83 ± 11 μl) and cardiac output (75 ± 3 to 107 ± 8 ml/min) but not left wall thickness in diastole (1.74 ± 0.07 to 1.80 ± 0.06 mm). Isolated Langendorff hearts from trained rats displayed decreased left ventricular myocardial stiffness (22 ± 1.1 to 19.1 ± 0.3) and reduced purine efflux during pacing-induced workload increases. 31P-NMR spectroscopy in isolated hearts from trained rats showed decreased PCr and PCr/ATP ratios with increased creatine, AMP and ADP concentrations. Thus, this endurance exercise protocol resulted in physiological hypertrophy while maintaining or improving cardiac function. (Mol Cell Biochem 251: 51–59, 2003)     

Herpes simplex virus type 1 evades the effects of antibody and complement in vivo

Herpes simplex virus type 1 (HSV-1) encodes a complement-interacting glycoprotein, gC, and an immunoglobulin G (IgG) Fc binding glycoprotein, gE, that mediate immune evasion by affecting multiple aspects of innate and acquired immunity, including interfering with complement components C1q, C3, C5, and properdin and blocking antibody-dependent cellular cytotoxicity. Previous studies evaluated the individual contributions of gC and gE to immune evasion. Experiments in a murine model that examines the combined effects of gC and gE immune evasion on pathogenesis are now reported. Virulence of wild-type HSV-1 is compared with mutant viruses defective in gC-mediated C3 binding, gE-mediated IgG Fc binding, or both immune evasion activities. Eliminating both activities greatly increased susceptibility of HSV-1 to antibody and complement neutralization in vitro and markedly reduced virulence in vivo as measured by disease scores, virus titers, and mortality. Studies with C3 knockout mice indicated that other activities attributed to these glycoproteins, such as gC-mediated virus attachment to heparan sulfate or gE-mediated cell-to-cell spread, do not account for the reduced virulence of mutant viruses. The results support the importance of gC and gE immune evasion in vivo and suggest potential new targets for prevention and treatment of HSV disease.     

HveA (Herpesvirus Entry Mediator A), a Coreceptor for Herpes Simplex Virus Entry, also Participates in Virus-Induced Cell Fusion

The purpose of this study was to determine whether a cell surface protein that can serve as coreceptor for herpes simplex virus type 1 (HSV-1) entry, herpesvirus entry mediator (previously designated HVEM but renamed HveA), also mediates HSV-1-induced cell-cell fusion. We found that transfection of DNA from KOS-804, a previously described HSV-1 syncytial (Syn) strain whose Syn mutation was mapped to an amino acid substitution in gK, induced numerous large syncytia on HveA-expressing Chinese hamster ovary cells (CHO-HVEM12) but not on control cells (CHO-C8). Antibodies specific for gD as well as for HveA were effective inhibitors of KOS-804-induced fusion, consistent with previously described direct interactions between gD and HveA. Since mutations in gD determine the ability of HSV-1 to utilize HveA for entry, we examined whether the form of virally expressed gD also influenced the ability of HveA to mediate fusion. We produced a recombinant virus carrying the KOS-804 Syn mutation and the KOS-Rid1 gD mutation, which significantly reduces viral entry via HveA, and designated it KOS-SR1. KOS-SR1 DNA had a markedly reduced ability to induce syncytia on CHO-HVEM12 cells and a somewhat enhanced ability to induce syncytia on CHO-C8 cells. These results support previous findings concerning the relative abilities of KOS and KOS-Rid1 to infect CHO-HVEM12 and CHO-C8 cells. Thus, HveA mediates cell-cell fusion as well as viral entry and both activities of HveA are contingent upon the form of gD expressed by the virus.     

Mutational evidence of internal fusion loops in herpes simplex virus glycoprotein B

Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is one of four glycoproteins necessary and sufficient for HSV cellular entry. Recently, the crystal structures of HSV-1 gB and vesicular stomatitis virus glycoprotein G were determined. Surprisingly, the two proteins share remarkable structural homology. Both proteins are homotrimeric and center about a long alpha-helix, features reminiscent of class I fusion proteins, such as influenza virus hemagglutinin or paramyxovirus F. However, these structures revealed that G has internal fusion loops, similar to the fusion loops of the class II fusion proteins, and that these loops are structurally conserved in gB. To examine whether these putative fusion loops are important for gB function, we mutated potential membrane-interacting (hydrophobic) residues to charged amino acids. Of most interest were mutant gB proteins that were expressed on the cell surface and were recognized by monoclonal antibodies against conformational epitopes but lacked the ability to function in cell-cell fusion assays. We find that three of the five hydrophobic amino acids targeted in these loops, tryptophan 174, tyrosine 179, and alanine 261, are integral in the function of gB. Our data suggest that they are part of an important functional domain. We hypothesize that two loops in domain 1 of HSV gB function as fusion loops. Our data are further evidence that gB is a viral fusogen and suggest clues as to how gB may function.     

N-terminal mutants of herpes simplex virus type 2 gH are transported without gL but require gL for function

Glycoprotein H (gH) is conserved among all herpesviruses and is essential for virus entry and cell fusion along with gL, gB, and, in most alphaherpesviruses, gD. Within the gH/gL heterodimer, it is thought that gH accounts for the fusion function and gL acts as a chaperone for the folding and transport of gH. Here, we found that the N terminus of gH2 contains important elements involved in both its folding and its transport. Our conclusions are based on the phenotypes of a series of gH deletion mutants in which the signal sequence (residues 1 to 18) was retained and N-terminal residues were removed up to the number indicated. The first mutant, gH2Delta29 (deletion of residues 19 to 28), like wild-type (WT) gH, required gL for both transport and function. To our surprise, two other mutants (gH2Delta64 and gH2Delta72) were transported to the cell surface independent of gL but were nonfunctional, even when complexed with gL. Importantly, a fourth mutant (gH2Delta48) was transported independent of gL but was functional only when complexed with gL. Using a panel of monoclonal antibodies against gH2, we found that when gH2Delta48 was expressed alone, its antigenic structure differed from that of gH2Delta48/gL or gH2-WT/gL. Mutation of gH2 residue R39, Y41, W42, or D44 allowed gL-independent transport of gH. Our results also show that gL is not merely required for gH transport but is also necessary for the folding and function of the complex. Since gH2Delta64/gL and gH2Delta72/gL were nonfunctional, we hypothesized that residues critical for gH/gL function lie within this deleted region. Additional mutagenesis identified L66 and L72 as important for function. Together, our results highlight several key gH residues: R39, Y41, W42, and D44 for gH transport and L66 and L72 for gH/gL structure and function.     

Antigenic and mutational analyses of herpes simplex virus glycoprotein B reveal four functional regions

Glycoprotein B (gB), along with gD, gH, and gL, is essential for herpes simplex virus (HSV) entry. The crystal structure of the gB ectodomain revealed it to be an elongated multidomain trimer. We generated and characterized a panel of 67 monoclonal antibodies (MAbs). Eleven of the MAbs had virus-neutralizing activity. To organize gB into functional regions within these domains, we localized the epitopes recognized by the entire panel of MAbs and mapped them onto the crystal structure of gB. Most of the MAbs were directed to continuous or discontinuous epitopes, but several recognized discontinuous epitopes that showed some resistance to denaturation, and we refer to them as pseudo-continuous. Each category contained some MAbs with neutralizing activity. To map continuous epitopes, we used overlapping peptides that spanned the gB ectodomain and measured binding by enzyme-linked immunosorbent assay. To identify discontinuous and pseudocontinuous epitopes, a purified form of the ectodomain of gB, gB(730t), was cleaved by alpha-chymotrypsin into two major fragments comprising amino acids 98 to 472 (domains I and II) and amino acids 473 to 730 (major parts of domains III, IV, and V). We also constructed a series of gB truncations to augment the other mapping strategies. Finally, we used biosensor analysis to assign the MAbs to competition groups. Together, our results identified four functional regions: (i) one formed by residues within domain I and amino acids 697 to 725 of domain V; (ii) a second formed by residues 391 to 410, residues 454 to 475, and a less-defined region within domain II; (iii) a region containing residues of domain IV that lie close to domain III; and (iv) the first 12 residues of the N terminus that were not resolved in the crystal structure. Our data suggest that multiple domains are critical for gB function.     

Using archaeogenomic and computational approaches to unravel the history of local adaptation in crops

Our understanding of the evolution of domestication has changed radically in the past 10 years, from a relatively simplistic rapid origin scenario to a protracted complex process in which plants adapted to the human environment. The adaptation of plants continued as the human environment changed with the expansion of agriculture from its centres of origin. Using archaeogenomics and computational models, we can observe genome evolution directly and understand how plants adapted to the human environment and the regional conditions to which agriculture expanded. We have applied various archaeogenomics approaches as exemplars to study local adaptation of barley to drought resistance at Qasr Ibrim, Egypt. We show the utility of DNA capture, ancient RNA, methylation patterns and DNA from charred remains of archaeobotanical samples from low latitudes where preservation conditions restrict ancient DNA research to within a Holocene timescale. The genomic level of analyses that is now possible, and the complexity of the evolutionary process of local adaptation means that plant studies are set to move to the genome level, and account for the interaction of genes under selection in systems-level approaches. This way we can understand how plants adapted during the expansion of agriculture across many latitudes with rapidity.