Reducing arginase activity via dietary manganese deficiency enhances endothelium-dependent vasorelaxation of rat aorta

L-Arginine is a common substrate for the enzymes arginase and nitric oxide synthase (NOS). Acute inhibition of arginase enzyme activity improves endothelium-dependent vasorelaxation, presumably by increasing availability of substrate for NOS. Arginase is activated by manganese (Mn), and the consumption of a Mn-deficient (Mn-) diet can result in low arginase activity. We hypothesize that endothelium-dependent vasorelaxation is greater in rats fed Mn- versus Mn sufficient (Mn+) diets. Newly weaned rats fed Mn+ diets (0.5 microg Mn/g; n = 12) versus Mn+ diets (45 microg Mn/g; n = 12) for 44 +/- 3 days had (i) lower liver and kidney Mn and arginase activity (P < or = 0.05), (ii) higher plasma L-arginine (P < or = 0.05), (iii) similar plasma and urine nitrate + nitrite, and (iv) similar staining for endothelial nitric oxide synthase in thoracic aorta. Vascular reactivity of thoracic aorta (approximately 720 microm i.d.) and small coronary arteries (approximately 110 microm i.d.) was evaluated using wire myographs. Acetylcholine (ACh; 10(-8)-10(-4) M) produced greater (P < or = 0.05) vasorelaxation in thoracic aorta from Mn- rats (e.g., maximal percent relaxation, 79 +/- 7%) versus Mn + rats (e.g., maximal percent relaxation, 54 +/- 9%) at 5 of 7 evaluated doses. Tension produced by NOS inhibition using N(G) monomethyl-L-arginine (L-NMMA; 10(-3) M) and vasorelaxation evoked by (i) arginase inhibition using difluoromethylornithine (DFMO; 10(-7) M), (ii) ACh (10(-8)-10(-4) M) in the presence of DFMO, and (iii) sodium nitroprusside (10(-9)-10(-4) M) were unaffected by diet. No differences existed between groups concerning these responses in small coronary arteries. These findings support our hypothesis that endothelium-dependent vasorelaxation is greater in aortic segments from rats that consume Mn- versus Mn+ diets; however, responses from small coronary arteries were unaffected.     

Zinc-deficient rats have more limited bone recovery during repletion than diet-restricted rats

The objective of this study was to investigate the effects of dietary zinc deficiency and diet restriction on bone development in growing rats, and to determine whether any adverse effects could be reversed by dietary repletion. Weanling rats were fed either a zinc-deficient diet ad libitum (ZD; <1 mg zinc/kg) or nutritionally complete diet (30 mg zinc/kg) either ad libitum (CTL) or pair-fed to the intake of the ZD group (DR; diet-restricted) for 3 weeks (deficiency phase) and then all groups were fed the zinc-adequate diet ad libitum for 3, 7, or 23 days (repletion phase). Excised femurs were analyzed for bone mineral density (BMD) using dual-energy x-ray absorptiometry, and plasma was analyzed for markers of bone formation (osteocalcin) and resorption (Ratlaps). After the deficiency phase, ZD had lower body weight and reduced femur BMD, zinc, and phosphorus concentrations compared with DR; and these parameters were lower in DR compared with CTL. Femur calcium concentrations were unchanged among the groups. Reduced plasma osteocalcin in ZD and elevated plasma Ratlaps in DR suggested that zinc deficiency limits bone formation while diet restriction accelerates bone resorption activity. After 23 days of repletion, femur size, BMD, and zinc concentrations remained lower in ZD compared with DR and CTL. Body weight and femur phosphorus concentrations remained lower in both ZD and DR compared with CTL after repletion. There were no differences in plasma osteocalcin concentrations after the repletion phase, but the plasma Ratlaps concentrations remained elevated in DR compared with CTL. In summary, both ZD and DR lead to osteopenia during rapid growth, but the mechanisms appear to be due to reduced modeling in ZD and higher turnover in DR. Zinc deficiency was associated with a greater impairment in bone development than diet restriction, and both deficiencies limited bone recovery during repletion in growing rats.     

Characterization of the 70S Ribosome from Rhodopseudomonas palustris using an integrated "top-down" and "bottom-up" mass spectrometric approach

We present a comprehensive mass spectrometric approach that integrates intact protein molecular mass measurement ("top-down") and proteolytic fragment identification ("bottom-up") to characterize the 70S ribosome from Rhodopseudomonas palustris. Forty-two intact protein identifications were obtained by the top-down approach and 53 out of the 54 orthologs to Escherichia coli ribosomal proteins were identified from bottom-up analysis. This integrated approach simplified the assignment of post-translational modifications by increasing the confidence of identifications, distinguishing between isoforms, and identifying the amino acid positions at which particular post-translational modifications occurred. Our combined mass spectrometry data also allowed us to check and validate the gene annotations for three ribosomal proteins predicted to possess extended C-termini. In particular, we identified a highly repetitive C-terminal "alanine tail" on L25. This type of low complexity sequence, common to eukaryotic proteins, has previously not been reported in prokaryotic proteins. To our knowledge, this is the most comprehensive protein complex analysis to date that integrates two MS techniques.     

Solution structure of the human Grb14-SH2 domain and comparison with the structures of the human Grb7-SH2/erbB2 peptide complex and human Grb10-SH2 domain

Grb14 is an adapter protein that is known to be overexpressed in estrogen receptor positive breast cancers, and in a number of prostate cancer cell lines. Grb14 has been demonstrated to bind to a number of activated receptor tyrosine kinases (RTKs) and to modulate signals transduced through these receptors. The RTKs to which Grb14 binds include the insulin receptor (IR), the fibroblast growth factor receptor (FGFR), the platelet-derived growth factor receptor (PDGFR), and the tunica endothelial kinase (Tek/Tie2) receptor. Grb14 has been shown to bind to these activated RTKs through its Src homology 2 (SH2) domain, with the exception of the insulin receptor, where the primary binding interaction is via a small domain adjacent to the SH2 domain (the BPS or PIR domain). Grb14 is a member of the Grb7 family of proteins, which also includes Grb7 and Grb10. We have solved the solution structure of the human Grb14-SH2 domain and compared it with the recently determined Grb7-SH2 and Grb10-SH2 domain structures.     

The quiet revolution

Sixty years have passed since Avery, MacLeod and McCarty published their landmark paper revealing DNA as the genetic material, writes Heather Dawes.     

Homoepiboxidines: further potent agonists for nicotinic receptors

Homoepiboxidine (3) and the corresponding N-methyl (4) and N-benzyl (5) derivatives were prepared from a 6beta-carbomethoxynortropane (8). Affinities and functional activities at neuromuscular, central neuronal and ganglionic-type nicotinic receptors were compared to those of epibatidine 1, and epiboxidine 2. Homoepiboxidine had equivalent affinity/activity to epiboxidine at neuromuscular, neuronal alpha4beta2, and most alpha3-containing ganglionic-type nicotinic receptors. The N-substituted derivatives showed reduced affinity/activity at most receptor subtypes. Replacement of the methylisoxazole moiety of 3 and 4 with a methyloxadiazole moiety provided analogues 6 and 7, which had greatly reduced affinity/activity in virtually all assays at nicotinic receptors. Marked analgetic activity in mice occurred at the following ip doses: epibatidine 10 microg/kg; epiboxidine 25 microg/kg; homoepiboxidine 100 microg/kg; N-methylhomoepiboxidine 100 microg/kg; the methyloxadiazole (6) 100 microg/kg. The time course at such ip doses was significantly longer for homoepiboxidine 3 with marked analgesia still manifest at 30 min post-injection. Epiboxidine and the homoepiboxidines were less toxic than epibatidine.     

Representation of spectrotemporal sound information in the ascending auditory pathway

The representation of sound information in the central nervous system relies on the analysis of time-varying features in communication and other environmental sounds. How are auditory physiologists and theoreticians to choose an appropriate method for characterizing spectral and temporal acoustic feature representations in single neurons and neural populations? A brief survey of currently available scientific methods and their potential usefulness is given, with a focus on the strengths and weaknesses of using noise analysis techniques for approximating spectrotemporal response fields (STRFs). Noise analysis has been used to foster several conceptual advances in describing neural acoustic feature representation in a variety of species and auditory nuclei. STRFs have been used to quantitatively assess spectral and temporal transformations across mutually connected auditory nuclei, to identify neuronal interactions between spectral and temporal sound dimensions, and to compare linear vs. nonlinear response properties through state-dependent comparisons. We propose that noise analysis techniques used in combination with novel stimulus paradigms and parametric experiment designs will provide powerful means of exploring acoustic feature representations in the central nervous system.     

NO mediates effects of estrogen on central regulation of blood pressure in restrained,ovariectomized rats

We tested the hypotheses that estrogen replacement in ovariectomized (OVX) rats attenuates cardiovascular responses to psychological stress and that nitric oxide (NO) in the brain mediates these effects. Female rats were OVX; one group received 17beta-estradiol (OVX-E) for 11-12 days and the other received vehicle (OVX-V). Seven days after OVX, OVX-E and OVX-V rats were chronically instrumented for arterial pressure measurements and intracerebroventricular injections. Later (4-5 days), OVX-E and OVX-V rats received intracerebroventricular injections of NG-nitro-l-arginine (88 microg/kg), an inhibitor of constitutive NO production, or vehicle. Mean arterial pressure (MAP) and heart rate responses were then measured in conscious rats exposed to two cycles of 1-h restraint/1-h rest. We show that MAP responses in restrained OVX-E rats were attenuated both during restraint and during rest. Although inhibition of NO production in the brain had no effect on MAP responses to restraint in OVX-V rats, it augmented responses in restrained OVX-E rats, especially during periods of rest, so that MAPs in restrained OVX-E and OVX-V rats were indistinguishable. Finally, NO levels in hypothalami and brain stems were elevated in restrained OVX-E, but not OVX-V, rats compared with their respective unrestrained controls. These results show that estrogen replacement in OVX rats reduces arterial pressure responses to psychological stress and that these effects are mediated, at least in part, by NO.