Congenic strains reveal the effect of the renin gene on skeletal muscle angiogenesis induced by electrical stimulation.

Previous studies have indicated the importance of angiotensin II (ANG II) in skeletal muscle angiogenesis. The present study explored the effect of regulation of the renin gene on angiogenesis induced by electrical stimulation with the use of physiological, pharmacological, and genetic manipulations of the renin-angiotensin system (RAS). Transfer of the entire chromosome 13, containing the physiologically regulated renin gene, from the normotensive inbred Brown Norway (BN) rat into the background of an inbred substrain of the Dahl salt-sensitive (SS/Mcwi) rat restored renin levels and the angiogenic response after electrical stimulation. This restored response was significantly attenuated when SS-13(BN)/Mcwi consomic rats were treated with lisinopril or high-salt diet. The role of ANG II on this effect was confirmed by the complete restoration of skeletal muscle angiogenesis in SS/Mcwi rats infused with subpressor doses of ANG II. Congenic strains derived from the SS-13(BN)/Mcwi consomic were used to further verify the role of the renin gene in this response. Microvessel density was markedly increased after stimulation in congenic strains that contained the renin gene from the BN rat (congenic lines A and D). This angiogenic response was suppressed in control strains that carried regions of the BN genome just above (congenic line C) or just below (congenic line B) the renin gene. The present study emphasizes the importance of maintaining normal renin regulation as well as ANG II levels during the angiogenesis process with a combination of physiological, genetic, and pharmacological manipulation of the RAS.     

Genetic determinants on rat chromosome 6 modulate variation in the hypercapnic ventilatory response using consomic strains.

To understand the genetic basis of pathways involved in the control of breathing, a large scale, high-throughput study using chromosomal substitution strains of rats is underway. Eight new consomic rat stains (SS-2(BN), SS-4(BN), SS-6(BN), SS-7(BN), SS-8(BN), SS-11(BN), SS-12(BN), SS-14(BN), SS-Y(BN)), containing one homozygous BN/NHsdMcwi (BN) chromosome on a background of SS/JrHsdMcwi (SS), were created by PhysGen (http://pga.mcw.edu) Program for Genomic Applications. Male and female rats were studied using standard plethysmography under control conditions and during acute hypoxia (inspired oxygen fraction = 0.12) and hypercapnia (inspired CO(2) fraction = 0.07). The rats were also studied during treadmill exercise. Both male and female BN rats had a significantly lower ventilatory response during 7% CO(2) compared with SS rats of the same gender. SS-6(BN) female rats had a significantly reduced ventilatory response, similar to BN rats due primarily to a reduced tidal volume. Male SS-6(BN) rats had a significantly reduced tidal volume response to hypercapnia but a slightly increased frequency response during hypercapnia. Gene(s) on the Y chromosome may play a role in this increased frequency response in the male rats because the SS-Y(BN) hypercapnic ventilatory response involves a significantly increased frequency response. Several chromosomal substitutions slightly altered the ventilatory responses to hypoxia and exercise. However, genes on chromosomes 6 and Y of those studied are of primary importance in aspects of ventilatory control currently studied.     

Introgression of chromosome 13 in Dahl salt-sensitive genetic background restores cerebral vascular relaxation

To evaluate the potential role of impaired renin-angiotensin system (RAS) function in contributing to reduced vascular relaxation in Dahl salt-sensitive (S) rats, responses to ACh (10(-6) mol/l) and hypoxia (Po(2) reduction to 40-45 mmHg) were determined in isolated middle cerebral arteries of Dahl S rats, Brown Norway (BN) rats, and consomic rats having chromosome 13 (containing the renin gene) or chromosome 16 of the BN rat substituted into the Dahl S genetic background (SS-13(BN) and SS-16(BN), respectively). Arteries of BN rats on a low-salt (LS) diet (0.4% NaCl) dilated in response to ACh and hypoxia, whereas dilation in response to these stimuli was absent in Dahl S rats on LS diet. Vasodilation to ACh and hypoxia was restored in SS-13(BN) rats on an LS diet but not in SS-16(BN) rats. High-salt diet (4% NaCl), to suppress ANG II, eliminated vasodilation to hypoxia and ACh in BN and in SS-13(BN) rats. Treatment of SS-13(BN) rats with the AT(1) receptor antagonist losartan also eliminated the restored vasodilation in response to ACh and hypoxia. These studies suggest that restoration of normal RAS regulation in SS-13(BN) consomic rats restores vascular relaxation mechanisms that are impaired in Dahl S rats.     

Sex-specific differences in chromosome-dependent regulation of vascular reactivity in female consomic rat strains from a SSxBN cross

High-throughput studies in the Medical College of Wisconsin Program for Genomic Applications (Physgen) were designed to link chromosomes with physiological function in consomic strains derived from a cross between Dahl salt-sensitive SS/JrHsdMcwi (SS) and Brown Norway normotensive BN/NHsdMcwi (BN) rats. The specific goal of the vascular protocol was to characterize the responses of aortic rings from these strains to vasoconstrictor and vasodilator stimuli (phenylephrine, acetylcholine, sodium nitroprusside, and bath hypoxia) to identify chromosomes that either increase or decrease vascular reactivity to these vasoactive stimuli. Because previous studies demonstrated sex-specific quantitative trait loci (QTLs) related to regulation of cardiovascular phenotypes in an F2 cross between the parental strains, males and females of each consomic strain were included in all experiments. As there were significant sex-specific differences in aortic sensitivity to vasoconstrictor and vasodilator stimuli compared with the parental SS strain, we report the results of the females separately from the males. There were also sex-specific differences in aortic ring sensitivity to these vasoactive stimuli in consomic strains that were fed a high-salt diet (4% NaCl) for 3 wk to evaluate salt-induced changes in vascular reactivity. Differences in genetic architecture could contribute to sex-specific differences in the development and expression of cardiovascular diseases via differential regulation and expression of genes. Our findings are the first to link physiological traits with specific chromosomes in female SS rats and support the idea that sex is an important environmental variable that plays a role in the expression and regulation of genes.     

Differential expression of cardiac mitochondrial proteins

Mitochondria were isolated from whole hearts of Dahl salt sensitive (SS) and chromosome 13 consomic control (SS.13BN/Mcwi) rats using a mechanical homogenization process followed by density centrifugation. The proteins present in the two mitochondria preparations were quantified; equal amounts of protein from each sample were taken and trypsinized in the presence of either 16O or 18O before pooling. Incorporation of one or two 18O atoms at the C-terminus of the peptide cleaved by trypsin allows the distinction between the two samples. The proteins were identified by automated MS/MS sequencing and relative amounts of each protein assessed by comparison of the intensities of the constituent peptides. Relative quantification was performed using the ZoomQuant (v1.24) software. Nine proteins were found to be differentially expressed. Electron transfer flavoprotein alpha (P13803, ETFA) protein expression was two-fold lower in the SS compared to the SS.13BN. This was confirmed by Western blot and 2-DE gel quantification. Potential functional implications of this differential expression include an impaired capacity of the heart to oxidize fatty acids in the SS strain compared to the control. Mathematical modeling of mitochondrial electron transport predicted that the observed change in ETFA expression may result in decreased activity of the electron transport chain.     

Role of 20-HETE in the antihypertensive effect of transfer of chromosome 5 from Brown Norway to Dahl salt-sensitive rats

This study examined whether substitution of chromosome 5 containing the CYP4A genes from Brown Norway rat onto the Dahl S salt-sensitive (SS) genetic background upregulates the renal production of 20-HETE and attenuates the development of hypertension. The expression of CYP4A protein and the production of 20-HETE were significantly higher in the renal cortex and outer medulla of SS.5(BN) (chromosome 5-substituted Brown Norway rat) consomic rats fed either a low-salt (LS) or high-salt (HS) diet than that seen in SS rats. The increase in the renal production of 20-HETE in SS.5(BN) rats was associated with elevated expression of CYP4A2 mRNA. MAP measured by telemetry rose from 117 ± 1 to 183 ± 5 mmHg in SS rats fed a HS diet for 21 days, but only increased to 151 ± 5 mmHg in SS.5(BN) rats. The pressure-natriuretic and diuretic responses were twofold higher in SS.5(BN) rats compared with SS rats. Protein excretion rose to 354 ± 17 mg/day in SS rats fed a HS diet for 21 days compared with 205 ± 13 mg/day in the SS.5(BN) rats, and the degree of glomerular injury was reduced. Baseline glomerular capillary pressure (Pgc) was similar in SS.5(BN) rats (43 ± 1 mmHg) and Dahl S (44 ± 2 mmHg) rats. However, Pgc increased to 59 ± 3 mmHg in SS rats fed a HS diet for 7 days, while it remained unaltered in SS.5(BN) rats (43 ± 2 mmHg). Chronic administration of an inhibitor of the synthesis of 20-HETE (HET0016, 10 mg·kg(-1)·day(-1) iv) reversed the antihypertensive phenotype seen in the SS.5(BN) rats. These findings indicate that the transfer of chromosome 5 from the BN rat onto the SS genetic background increases the renal expression of CYP4A protein and the production of 20-HETE and that 20-HETE contributes to the antihypertensive and renoprotective effects seen in the SS.5(BN) consomic strain.     

Effect of renal medullary H2O2 on salt-induced hypertension and renal injury

Dahl salt-sensitive (SS) and consomic, salt-resistant SS-13(BN) rats possess substantial differences in blood pressure salt-sensitivity even with highly similar genetic backgrounds. The present study examined whether increased oxidative stress, particularly H2O2, in the renal medulla of SS rats contributes to these differences. Blood pressure was measured using femoral arterial catheters in three groups of rats: 1) 12-wk-old SS and consomic SS-13(BN) rats fed a 0.4% NaCl diet, 2) SS rats fed a 4% NaCl diet and chronically infused with saline or catalase (6.9 microg x kg(-1) x min(-1)) directly into the renal medulla, and 3) SS-13(BN) fed high salt (4%) and infused with saline or H2O2 (347 nmol x kg(-1) x min(-1)) into the renal medullary interstitium. After chronic blood pressure measurements, renal medullary interstitial H2O2 concentration ([H2O2]) was collected by microdialysis and analyzed with Amplex red. Blood pressure and [H2O2] were both significantly higher in SS (126 +/- 3 mmHg and 145 +/- 17 nM, respectively) vs. SS-13(BN) rats (116 +/- 2 mmHg and 56 +/- 14 nM) fed a 0.4% diet. Renal interstitial catalase infusion significantly decreased [H2O2] (96 +/- 41 vs. 297 +/- 52 nM) and attenuated the hypertension (146 +/- 2 mmHg catalase vs. 163 +/- 4 mmHg saline) in SS rats after 5 days of high salt (4%). H2O2 infused into the renal medulla of consomic SS-13(BN) fed high salt (4%) for 7 days accentuated the salt sensitivity (145 +/- 2 mmHg H2O2 vs. 134 +/- 1 mmHg saline). [H2O2] was also increased in the treated group (83 +/- 1 nM H2O2 vs. 44 +/- 9 nM saline). These data show that medullary production of H2O2 may contribute to salt-induced hypertension in SS rats and that chromosome 13 of the Brown Norway contains gene(s) that protect against renal medullary oxidant stress.     

Narrowing a region on rat chromosome 13 that protects against hypertension in Dahl SS-13BN congenic strains

Transfer of chromosome 13 from the Brown Norway (BN) rat onto the Dahl salt-sensitive (SS) genetic background attenuates the development of hypertension, but the genes involved remain to be identified. The purpose of the present study was to confirm by telemetry that a congenic strain [SS.BN-(D13Hmgc37-D13Got22)/Mcwi, line 5], carrying a 13.4-Mb segment of BN chromosome 13 from position 32.4 to 45.8 Mb, is protected from the development of hypertension and then to narrow the region of interest by creating and phenotyping 11 additional subcongenic strains. Mean arterial pressure (MAP) rose from 118 ± 1 to 186 ± 5 mmHg in SS rats fed a high-salt diet (8.0% NaCl) for 3 wk. Protein excretion increased from 56 ± 11 to 365 ± 37 mg/day. In contrast, MAP only increased to 152 ± 9 mmHg in the line 5 congenic strain. Six subcongenic strains carrying segments of BN chromosome 13 from 32.4 and 38.2 Mb and from 39.9 to 45.8 Mb were not protected from the development of hypertension. In contrast, MAP was reduced by ～30 mmHg in five strains, carrying a 1.9-Mb common segment of BN chromosome 13 from 38.5 to 40.4 Mb. Proteinuria was reduced by ～50% in these strains. Sequencing studies did not identify any nonsynonymous single nucleotide polymorphisms in the coding region of the genes in this region. RT-PCR studies indicated that 4 of the 13 genes in this region were differentially expressed in the kidney of two subcongenic strains that were partially protected from hypertension vs. those that were not. These results narrow the region of interest on chromosome 13 from 13.4 Mb (159 genes) to a 1.9-Mb segment containing only 13 genes, of which 4 are differentially expressed in strains partially protected from the development of hypertension.     

Restoration of normal vascular relaxation mechanisms in cerebral arteries by chromosomal substitution in consomic SS.13BN rats

This study sought to identify the mechanisms of vascular relaxation that are rescued in middle cerebral arteries (MCA) of SS.13BN consomic rats by substituting chromosome 13 containing the renin gene from Brown Norway (BN) rats into the Dahl salt-sensitive (SS) genetic background. Isolated MCA from SS rats exhibited an indomethacin-sensitive constriction in response to acetylcholine (ACh) and hypoxia. ACh-induced dilation was NO dependent and hypoxic dilations were cyclooxygenase (COX) dependent in BN and SS.13BN rats. In SS rats, hypoxic dilation was restored by indomethacin and abolished by inhibiting cytochrome P-450 epoxygenases, suggesting a role for epoxyeicosatrienoic acids. MCA from SS and SS.13BN rats constricted and MCA from BN rats dilated in response to the stable prostacyclin analog iloprost. MCA from SS.13BN and BN rats (but not SS rats) dilated in response to the prostaglandin E2 receptor agonist butaprost. Hypoxia increased prostacyclin release in cerebral arteries from all the strains, whereas thromboxane A2 production was reduced in BN rat vessels only. These data suggest that SS rats may be less sensitive to vasodilator prostaglandins and that normalization of renin-angiotensin system regulation causes a switch from production of COX-derived vasoconstrictor metabolites (in SS rats) toward NO-dependent relaxation in response to ACh- and prostaglandin-dependent dilation in response to hypoxia in SS.13(BN) rats.     

Resistance to myocardial ischemia in five rat strains: is there a genetic component of cardioprotection?

There is a need to develop new and more consistent animal models of cardioprotection. Traditionally, outbred dogs, rabbits, and rats have been studied. We determined resistance to ischemia in isolated hearts from inbred strains of rats. Hearts from inbred rats: SS/Mcw (Dahl S, Dahl salt-sensitive), DA/Hsd (Dark Agouti), LEW/Hsd (Lewis), and BN/SsN/Mcw (Brown Norway); and from an outbred rat: Hsd:WIST (Wistar) were subjected to 27 min of global, no-flow ischemia, followed by 3 h of reperfusion. Infarct size in the Brown Norway rat was 2.5 times less than that observed in the Dahl S rat, with the Dark Agouti, Lewis, and Wistar rats intermediate in response. Hearts from Brown Norway rats were also most resistant to ischemia in terms of postischemic enzyme leakage and contractile and vascular function compared with other strains. The average polymorphism rate between strains revealed that such strains were genetically diverse. This study demonstrates strain differences in resistance to myocardial ischemia, suggesting these rats could be used to study a genetic and/or environmental basis for these differences and to provide new animal models for the physiological study of cardioprotection.     

Genetic isolation of quantitative trait loci for blood pressure development and renal mass on chromosome 5 in the spontaneously hypertensive rat

Total genome scans of genetically segregating populations derived from spontaneously hypertensive rats (SHR) and other rat models of essential hypertension suggested a presence of quantitative trait loci (QTL) regulating blood pressure on multiple chromosomes, including chromosome 5. The objective of the current study was to test directly a hypothesis that chromosome 5 of the SHR carries a blood pressure regulatory QTL. A new congenic strain was derived by replacing a segment of chromosome 5 in the SHR/Ola between the D5Wox20 and D5Rat63 markers with the corresponding chromosome segment from the normotensive Brown Norway (BN/Crl) rat. Arterial pressures were directly monitored in conscious, unrestrained rats by radiotelemetry. The transfer of a segment of chromosome 5 from the BN strain onto the SHR genetic background was associated with a significant decrease of systolic blood pressure, that was accompanied by amelioration of renal hypertrophy. The heart rates were not significantly different in the SHR compared to SHR chromosome 5 congenic strain. The findings of the current study demonstrate that gene(s) with major effects on blood pressure and renal mass exist in the differential segment of chromosome 5 trapped within the new SHR.BN congenic strain.     

Evidence that Somatostatin Enhances Endogenous Acetylcholine Release in the Rat Hippocampus

The present experiments show that somatostatin (SS)-like immunoreactive material is present in the hippocampus and that its release can be increased by K+ stimulation of rat hippocampal slices, suggesting that SS-like peptides may be of significance to neurotransmission in the hippocampus. Exogenous SS-28 and SS-14 enhanced the K(+)-evoked release of endogenous acetylcholine (ACh) from rat hippocampal slices, whereas amino-terminal fragments of SS-28 did not. The increased ACh release in the presence of either peptide appeared to be mediated by an interaction with SS receptors because cyclo-SS, a putative SS antagonist, abolished the effects of both SS-28 and SS-14. In addition, the increase in ACh release induced by SS-14 or SS-28 was antagonized by the calcium channel antagonists omega-conotoxin GVIA, nifedipine, and cinnarizine, implicating voltage-sensitive calcium channels in this effect. Moreover, the effect was sensitive to tetrodotoxin, suggesting an indirect action of the peptides at a site distal to cholinergic nerve terminals. Cysteamine, which has been reported to deplete SS content and to increase SS release in brain, augmented the basal and evoked release of ACh from hippocampal slices, without affecting SS-like content and release. Finally, neuropeptide Y, which is colocalized with SS in many neurons of the hippocampal formation, did not alter ACh release, nor did it facilitate the SS-induced increase. The results suggest that in the rat hippocampus, both SS-28 and SS-14 interact with SS receptors to regulate ACh release indirectly by a mechanism that involves alterations of calcium influx during depolarization.     

Somatostatin inhibits hepatic growth hormone receptor and insulin-like growth factor I mRNA expression by activating the ERK and PI3K signaling pathways

Previously, we reported that somatostatins (SS) inhibit organismal growth by reducing hepatic growth hormone (GH) sensitivity and by inhibiting insulin-like growth factor I (IGF-I) production. In this study, we used hepatocytes isolated from rainbow trout to elucidate the mechanism(s) associated with the extrapituitary growth-inhibiting actions of SS. SS-14, a predominant SS isoform, stimulated tyrosine phosphorylation of several endogenous proteins, including extracellular signal-regulated kinase (ERK), a member the mitogen-activated protein kinase (MAPK) family, and protein kinase B (Akt), a downstream target of phosphatidylinositol 3-kinase (PI3K). SS-14 specifically stimulated the phosphorylation of both ERK 1/2 and Akt in a concentration-dependent fashion. This activation occurred within 5-15 min, then subsided after 1 h. The ERK inhibitor U0126 retarded SS-14-stimulated phosphorylation of ERK 1/2, whereas the PI3K inhibitor LY294002 blocked SS-14-stimulated phosphorylation of Akt. SS-14-inhibited expression of GH receptor (GHR) mRNA was blocked by U0126 but not by LY294002. By contrast, U1026 had no effect on SS-14 inhibition of GH-stimulated IGF-I mRNA expression, whereas LY294002 partially blocked the inhibition of GH-stimulated IGF-I mRNA expression by SS-14. These results indicate that SS-14-inhibited GHR expression is mediated by the ERK signaling pathway and that the PI3K/Akt pathway mediates, at least in part, SS-14 inhibition of GH-stimulated IGF-I expression.     

Establishment of consomic strains derived from A/J and SM/J mice for genetic analysis of complex traits

Consomic strains, in which one chromosome is derived from a donor strain and the other chromosomes are derived from the recipient strain, provide a powerful tool for the dissection of complex genetic traits. In this study we established ten consomic strains (A-2^SM, A-6^SM, A-11^SM, A-12^SM, A-13^SM, A-15^SM, A-17^SM, A-18^SM, A-19^SM, A-Y^SM) using the SM/J strain as the donor and the A/J strain as the recipient; these are the parental strains of a set of SMXA recombinant inbred (RI) strains that we had developed previously. We analyzed body weights and blood lipid levels in the consomic and parental strains. The mean values for each trait showed a continuous range of variation in the consomic strains suggesting that they are controlled by multiple genes. We previously identified suggestive QTLs for body weight on chromosome 6 in SMXA RI strains and (SM/J?×?A/J)F₂ mice. The observation that the A-6^SM consomic strain had a significantly lower mean body weight than the A/J strain supports the presence of this QTL on chromosome 6. Similarly, the higher blood triglyceride level in the A-11^SM strain shows the existence of a previously mapped QTL on chromosome 11, and the A-12^SM strain provides evidence of a QTL for blood total cholesterol level on chromosome 12. These consomic strains, along with the previously developed set of SMXA RI strains from A/J and SM/J mice, offer an invaluable and powerful resource for the analysis of complex genetic traits in mice.     

Establishment of consomic strains derived from A/J and SM/J mice for genetic analysis of complex traits

Consomic strains, in which one chromosome is derived from a donor strain and the other chromosomes are derived from the recipient strain, provide a powerful tool for the dissection of complex genetic traits. In this study we established ten consomic strains (A-2^SM, A-6^SM, A-11^SM, A-12^SM, A-13^SM, A-15^SM, A-17^SM, A-18^SM, A-19^SM, A-Y^SM) using the SM/J strain as the donor and the A/J strain as the recipient; these are the parental strains of a set of SMXA recombinant inbred (RI) strains that we had developed previously. We analyzed body weights and blood lipid levels in the consomic and parental strains. The mean values for each trait showed a continuous range of variation in the consomic strains suggesting that they are controlled by multiple genes. We previously identified suggestive QTLs for body weight on chromosome 6 in SMXA RI strains and (SM/J × A/J)F₂ mice. The observation that the A-6^SM consomic strain had a significantly lower mean body weight than the A/J strain supports the presence of this QTL on chromosome 6. Similarly, the higher blood triglyceride level in the A-11^SM strain shows the existence of a previously mapped QTL on chromosome 11, and the A-12^SM strain provides evidence of a QTL for blood total cholesterol level on chromosome 12. These consomic strains, along with the previously developed set of SMXA RI strains from A/J and SM/J mice, offer an invaluable and powerful resource for the analysis of complex genetic traits in mice.

     

Receptor binding of somatostatin-14 and somatostatin-28 in rat brain: differential modulation by nucleotides and ions

The tissue-selective binding of the two principal bioactive forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28), their ability to modulate cAMP-dependent and -independent regulation of post-receptor events to different degrees and the documentation of specific labelling of SS receptor subtypes with SS-28 but not SS-14 in discrete regions of rat brain suggest the existence of distinct SS-14 and SS-28 binding sites. Receptor binding of SS-14 ligands has been shown to be modulated by nucleotides and ions, but the effect of these agents on SS-28 binding has not been studied. In the present study we investigated the effects of adenine and guanine nucleotides as well as monovalent and divalent cations on rat brain SS receptors quantitated with radioiodinated analogs of SS-14 ([125I-Tyr11]SS14, referred to in this paper as SS-14) and SS-28 ([Leu8, D-Trp22, 125I-Tyr25] SS-28, referred to as LTT* SS-28) in order to determine if distinct receptor sites for SS-14 and SS-28 could be distinguished on the basis of their modulation by nucleotides and ions. GTP as well as ATP exerted a dose-dependent inhibition (over a concentration range of 10(-7)-10(-3) M) of the binding of the two radioligands. The nucleotide inhibition of binding resulted in a decrease the Bmax of the SS receptors, the binding affinity remaining unaltered. GTP (10(-4) M) decreased the Bmax of LTT* SS-28 binding sites to a greater extent than ATP (145 +/- 10 and 228 +/- 16 respectively, compared to control value of 320 +/- 20 pmol mg-1). Under identical conditions GTP was less effective than ATP in reducing the number of T* SS-14 binding sites (Bmax = 227 +/- 8 and 182 +/- 15, respectively, compared to 340 +/- 15 pmol mg-1 in the absence of nucleotides). Monovalent cations inhibited the binding of both radioligands, Li+ and Na+ inhibited the binding of T* SS-14 to a greater extent than K+. The effect of divalent cations on the other hand was varied. At low concentration (2 mM) Mg2+, Ba2+, Mn2+, Ca2+ and Co2+ augmented the binding of both T* SS-14 and LTT* SS-28, while higher than 4 mM Co2+ inhibited binding of both ligands. LTT* SS-28 binding was reduced in the presence of high concentrations of Ba2+ and Mn2+ also. Interestingly Ca2+ at higher than 10 mM preferentially inhibited LTT* SS-28 binding and increased the affinity of SS-14 but not SS-28 for LTT* SS-28 binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

High affinity binding sites for somatostatin to rat pituitary

Binding sites for somatostatin (SS) are described in rat pituitary membranes using either [¹²⁵I-Tyr¹¹]-SS-14 or [Leu⁸, D-Trp²², ¹²⁵I-Tyr²⁵]-SS-28 as radioligands; in each case saturable and high affinity binding sites with K_D's for SS of 1.09 and 0.95 nM respectively have been characterized. The binding capacity is 100 f mols/mg protein. The potencies of various SS analogs measured in the radioreceptor assay are in agreement with the potencies in a bioassay measuring inhibition of growth hormone release; in particular, SS-28 is slightly less potent than SS-14. A comparison of these data with those describing SS binding in brain and pancreas suggests that some pharmacological differences may exist between pituitary, brain and pancreas binding sites for SS.     

Phylogenetic analysis of a gene cluster encoding an additional, rhizobial-like type III secretion system that is narrowly distributed among Pseudomonas syringae strains

ABSTRACT: BACKGROUND: The central role of Type III secretion systems (T3SS) in bacteria-plant interactions is well established, yet unexpected findings are being uncovered through bacterial genome sequencing. Some Pseudomonas syringae strains possess an uncharacterized cluster of genes encoding putative components of a second T3SS (T3SS-2) in addition to the well characterized Hrc1 T3SS which is associated with disease lesions in host plants and with the triggering of hypersensitive response in non-host plants. The aim of this study is to perform an in silico analysis of T3SS-2, and to compare it with other known T3SSs. RESULTS: Based on phylogenetic analysis and gene organization comparisons, the T3SS-2 cluster of the P. syringae pv. phaseolicola strain is grouped with a second T3SS found in the pNGR234b plasmid of Rhizobium sp. These additional T3SS gene clusters define a subgroup within the Rhizobium T3SS family. Although, T3SS-2 is not distributed as widely as the Hrc1 T3SS in P. syringae strains, it was found to be constitutively expressed in P. syringae pv phaseolicola through RT-PCR experiments. CONCLUSIONS: The relatedness of the P. syringae T3SS-2 to a second T3SS from the pNGR234b plasmid of Rhizobium sp., member of subgroup II of the rhizobial T3SS family, indicates common ancestry and/or possible horizontal transfer events between these species. Functional analysis and genome sequencing of more rhizobia and P. syringae pathovars may shed light into why these bacteria maintain a second T3SS gene cluster in their genome.     

Isolation of a Genomic Region Affecting Most Components of Metabolic Syndrome in a Chromosome-16 Congenic Rat Model

Metabolic syndrome is a highly prevalent human disease with substantial genomic and environmental components. Previous studies indicate the presence of significant genetic determinants of several features of metabolic syndrome on rat chromosome 16 (RNO16) and the syntenic regions of human genome. We derived the SHR.BN16 congenic strain by introgression of a limited RNO16 region from the Brown Norway congenic strain (BN-Lx) into the genomic background of the spontaneously hypertensive rat (SHR) strain. We compared the morphometric, metabolic, and hemodynamic profiles of adult male SHR and SHR.BN16 rats. We also compared in silico the DNA sequences for the differential segment in the BN-Lx and SHR parental strains. SHR.BN16 congenic rats had significantly lower weight, decreased concentrations of total triglycerides and cholesterol, and improved glucose tolerance compared with SHR rats. The concentrations of insulin, free fatty acids, and adiponectin were comparable between the two strains. SHR.BN16 rats had significantly lower systolic (18–28 mmHg difference) and diastolic (10–15 mmHg difference) blood pressure throughout the experiment (repeated-measures ANOVA, P < 0.001). The differential segment spans approximately 22 Mb of the telomeric part of the short arm of RNO16. The in silico analyses revealed over 1200 DNA variants between the BN-Lx and SHR genomes in the SHR.BN16 differential segment, 44 of which lead to missense mutations, and only eight of which (in Asb14, Il17rd, Itih1, Syt15, Ercc6, RGD1564958, Tmem161a, and Gatad2a genes) are predicted to be damaging to the protein product. Furthermore, a number of genes within the RNO16 differential segment associated with metabolic syndrome components in human studies showed polymorphisms between SHR and BN-Lx (including Lpl, Nrg3, Pbx4, Cilp2, and Stab1). Our novel congenic rat model demonstrates that a limited genomic region on RNO16 in the SHR significantly affects many of the features of metabolic syndrome.