The angiotensin IV analog Nle-Tyr-Leu-psi-(CH2-NH2)3-4-His-Pro-Phe (norleual) can act as a hepatocyte growth factor/c-Met inhibitor.

The angiotensin (Ang) IV analog norleual [Nle-Tyr-Leu-psi-(CH2-NH2)(3-4)-His-Pro-Phe] exhibits structural homology with the hinge (linker) region of hepatocyte growth factor (HGF) and is hypothesized to act as a hinge region mimic. Norleual competitively inhibited the binding of HGF to its receptor c-Met in mouse liver membranes, with an IC(50) value of 3 pM. Predictably, norleual was able to inhibit HGF-dependent signaling, proliferation, migration, and invasion in multiple cell types at concentrations in the picomolar range. Ex vivo studies demonstrated that norleual exhibited potent antiangiogenic activity, an attribute that would be predicted for a HGF/c-Met antagonist. Furthermore, norleual suppressed pulmonary colonization by B16-F10 murine melanoma cells, which are characterized by an overactive HGF/c-Met system. Together, these data suggest that AngIV analogs exert at least some of their biological activity through interference with the HGF/c-Met system and may have utility as therapeutic agents in disorders that are dependent on an intact HGF/c-Met system. Finally, the ability of norleual to induce marked biological responses in human embryonic kidney cells, which do not express insulin-responsive aminopeptidase (IRAP), coupled with the observed effects of norleual on the HGF/c-Met system, casts doubt on the physiological significance of AngIV-dependent inhibition of IRAP. [Corrected]

Mitochondrial DNA analyses and the origin and relative age of parthenogenetic lizards (genus Cnemidophorus).

Morphological, karyological, and allozyme analyses indicate that the parthenogenetic lizards Cnemidophorus neomexicanus and diploid C. tesselatus are hybrids formed, respectively, by crosses involving the bisexual species C. tigris and C. inornatus, and C. tigris and C. septemvittatus. Mitochondrial DNA, which is inherited maternally, was obtained from each of these species. Analyses of the mitochondrial DNA's and their restriction endonuclease digestion products by electron microscopy and agarose gel electrophoresis support the hybridization hypothesis by indicating that C. tigris (specifically the subspecies marmoratus) was the maternal parent species for both C. neomexicanus and C. tesselatus. Furthermore, these data imply that these two parthenogenetic species are younger than some races of C. tigris.

Recovery of olfactory function after bilateral bulbectomy.

Mice were trained to discriminate between scented and unscented air. After olfactory bulbs were removed, discrimination was lost, but returned with the formation of synaptic connections between regenerated primary olfactory neurons and the cortex of the forebrain. The acquisition of a second olfactory-mediated task by long-term bulbectomized mice and controls was indistinguishable. The results emphasize the plasticity of the nervous system, correlate the presence of neural connections between olfactory mucosa and forebrain with the recovery of olfactory function, suggest that olfactory-mediated memory resides at least in part outside the olfactory bulbs, and demonstrate that the bulbs are not required for the acquisition of olfactory tasks.

Sex chromosomes in lizards.

Karyotypes of many species of the genus Sceloporus support the generalization that there are no morphologically recognizable sex chromosomes in lizards; however, there is a marked sexual dimorphism in the karyotypes of Sceloporus jarrovi and Sceloporus poinsetti. During meiosis in males, whose diploid number of chromosomes is 31, preferential segregation of chromosomes from a trivalent results in heterogamety.

AT4 receptor activation increases intracellular calcium influx and induces a non-N-methyl-D-aspartate dependent form of long-term potentiation.

The angiotensin 4 receptor (AT4) subtype is heavily distributed in the dentate gyrus and CA1-CA3 subfields of the hippocampus. Neuronal pathways connecting these subfields are believed to be activated during learning and memory processing. ur laboratory previously demonstrated that application of the AT4 agonist, Norleucine1-angiotensin IV, enhanced baseline synaptic transmission and long-term potentiation, whereas perfusion with the AT4 antagonist, Norleucine1-Leu3-psi(CH2-NH2)3-4-angiotensin IV disrupted long-term potentiation stabilization in area CA1. The objective of the present study was to identify the mechanism(s) responsible for Norleucine1-angiotensin IV-induced increase in hippocampal long-term potentiation. Hippocampal slices perfused with Norleucine1-angiotensin IV for 20 min revealed a notable increase in baseline responses in a non-reversible manner and were blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt. Infusions of Norleucine1-angiotensin IV prior to, but not after theta burst stimulation, significantly enhanced long-term potentiation compared with control slices. Further, N-methyl-D-aspartate receptor-independent long-term potentiation could be induced by tetanization during the perfusion of Norleucine1-angiotensin IV in the presence of the N-methyl-D-aspartate antagonist, D,L-2-amino-5-phosphonovaleric acid. Blockade of select voltage dependent calcium channels significantly reduced Norleucine1-angiotensin IV-induced increase in baseline responses and subsequent long-term potentiation suggesting that AT4 receptor activation increases intracellular calcium levels via altering voltage dependent calcium channels and triggers an N-methyl-D-aspartate-independent form of long-term potentiation. In support of this notion the application of Nle1-angiotensin IV to cultured rat hippocampal neurons resulted in increased intracellular calcium derived exclusively from extracellular sources. Consistent with these observations Nle1-angiotensin IV was capable of augmenting the uptake of 45Ca2+ into rat hippocampal slices. Taken together, these data indicate that increased calcium influx through postsynaptic calcium channels contribute to Norleucine1-angiotensin IV-induced enhancement of long-term potentiation.

Contributions of the brain angiotensin IV-AT4 receptor subtype system to spatial learning.

The development of navigational strategies to solve spatial problems appears to be dependent on an intact hippocampal formation. The circular water maze task requires the animal to use extramaze spatial cues to locate a pedestal positioned just below the surface of the water. Presently, we investigated the role of a recently discovered brain angiotensin receptor subtype (AT4) in the acquisition of this spatial learning task. The AT4 receptor subtype is activated by angiotensin IV (AngIV) rather than angiotensins II or III, as documented for the AT1 and AT2 receptor subtypes, and is heavily distributed in the CA1-CA3 fields of the hippocampus. Chronic intracerebroventricular infusion of a newly synthesized AT4 agonist (Norleucine1-AngIV) via osmotic pump facilitated the rate of acquisition to solve this task, whereas treatment with an AT4 receptor antagonist (Divalinal) significantly interfered with the acquisition of successful search strategies. Animals prepared with bilateral knife cuts of the perforant path, a major afferent hippocampal fiber bundle originating in the entorhinal cortex, displayed deficits in solving this task. This performance deficit could be reversed with acute intracerebroventricular infusion of a second AT4 receptor agonist (Norleucinal). These results suggest that the brain AngIV-AT4 system plays a role in the formation of spatial search strategies and memories. Further, application of an AT4 receptor agonist compensated for spatial memory deficits in performance accompanying perforant path knife cuts. Possible mechanisms underlying this compensatory effect are discussed.

Calpain II induced insolubilization of lens beta-crystallin polypeptides may induce cataract.

Addition of calpain II (EC 3.4.22.17) to soluble proteins from 10-day-old rat lens caused an increase in turbidity and production of water-insoluble protein. The insolubilization increased with higher concentrations of both lens protein and calpain II, it could be prevented by the cysteine protease inhibitor E-64; it required at least 0.5 mM Ca2+, it was limited to 6% of the soluble protein present and resulted from precipitation of proteolyzed beta-crystallin polypeptides. When compared by two-dimensional electrophoresis, the insoluble beta-crystallin polypeptides produced by calpain II were similar to insoluble beta-crystallin polypeptides found in cataractous lenses. Trypsin also caused insolubilization of beta-crystallin polypeptides, but these polypeptides were unlike polypeptides produced during cataract formation. These data suggested that the loss of solubility was due to a specific removal of N/or C-terminal extensions from beta-crystallin polypeptides by calpain II, and that a similar process may occur in vivo during cataract formation. It is hypothesized that the insoluble protein produced by calpain II causes cataract by increasing light scatter in the lens.

Length variation and heteroplasmy are frequent in mitochondrial DNA from parthenogenetic and bisexual lizards (genus Cnemidophorus).

Samples of mtDNA isolated from each of 92 lizards representing all color pattern classes of Cnemidophorus tesselatus and two populations of C. tigris marmoratus were digested with the restriction endonucleases MboI, TaqI, RsaI and MspI. The mtDNA fragment sizes were compared after radioactive labeling and gel electrophoresis. Three features were notable in the comparisons: there was little variation due to gain or loss of cleavage sites, two fragments varied noticeably in length among the samples, one by a variable amount up to a maximum difference of approximately 370 base pairs (bp) and the other by a discrete amount of 35 bp, these two fragments occasionally varied within, as well as between, samples. Two regions that corresponded in size to these variants were identified by restriction endonuclease cleavage mapping. One of these is adjacent to the D-loop. Heteroplasmy, heretofore rarely observed, occurred frequently in these same two regions. Variability in the copy number of a tandemly repeated 64-bp sequence appears to be one component of the variation, but others (e.g., base substitutions or small additions/deletions) must also be involved. The frequent occurrence of these length variations suggests either that they can be generated rapidly or that they were inherited from a highly polymorphic ancestor. The former interpretation is favored.

Brain angiotensin receptor subtypes in the control of physiological and behavioral responses.

This review summarizes emerging evidence that supports the notion of a separate brain renin-angiotensin system (RAS) complete with the necessary precursors and enzymes for the formation and degradation of biologically active forms of angiotensins, and several binding subtypes that may mediate their diverse functions. Of these subtypes the most is known about the AT1 site which preferentially binds angiotensin II (AII) and angiotensin III (AIII). The AT1 site appears to mediate the classic angiotensin responses concerned with body water balance and the maintenance of blood pressure. Less is known about the AT2 site which also binds AII and AIII and may play a role in vascular growth. Recently, an AT3 site was discovered in cultured neoblastoma cells, and an AT4 site which preferentially binds AII(3-8), a fragment of AII now referred to as angiotensin IV (AIV). The AT4 site has been implicated in memory acquisition and retrieval, and the regulation of blood flow. In addition to the more well-studied functions of the brain RAS, we review additional less well investigated responses including regulation of cellular function, the modulation of sensory and motor systems, long term potentiation, and stress related mechanisms. Although the receptor subtypes responsible for mediating these physiologies and behaviors have not been definitively identified research efforts are ongoing. We also suggest potential contributions by the RAS to clinically relevant syndromes such as dysfunctions in the regulation of blood flow and ischemia, changes in cognitive affect and memory in clinical depressed and Alzheimer's patients, and angiotensin's contribution to alcohol consumption.

Variations in angiotensin-II release from the rat brain during the estrous cycle.

To investigate the hypothesis that the release of angiotensin-II (AII) in the rat brain increases on the day of proestrus, samples of cerebrospinal fluid (CSF) and interstitial fluid from the general region of the bed nucleus of the stria terminalis pars ventralis in the preoptic-anterior hypothalamic area were monitored for AII-immunoreactive material (AII-ir) using push-pull cannulas. Samples of CSF were obtained on the day of proestrus and diestrus day 1 at 30-min intervals from 1200-1600 h. Samples of interstitial fluid were obtained at 25-min intervals from 0930-1600 h. The rate of release of AII-ir into CSF was significantly greater on proestrus compared to diestrus day 1, and in the early afternoon of proestrus compared to the late afternoon. In five of seven rats and in the overall comparison of AII-ir release from the preoptic-anterior hypothalamic area, significantly more AII-ir was released on the day of proestrus vs. diestrus day 1. These observations are consistent with previous studies suggesting that brain AII may play a role in the regulation of LH release on the day of proestrus.

Leucine aminopeptidase M-induced reductions in blood pressure in spontaneously hypertensive rats.

Leucine aminopeptidase M significantly reduced blood pressure for up to 40 minutes when infused intracerebroventricularly into anesthetized spontaneously hypertensive rats (SHR) from a mean +/- SEM of 190 +/- 4 to 94 +/- 7 mm Hg and also in normotensive Wistar-Kyoto (WKY) rats from 138 +/- 5 to 68 +/- 8 mm Hg. Cerebrospinal fluid levels of angiotensin II (Ang II) and III were measured by radioimmunoassay and indicated drops with leucine aminopeptidase M infusion in SHR (from 36 +/- 6 to 11 +/- 1 pg/100 microliters) and in WKY rats (from 33 +/- 9 to 13 +/- 1 pg/100 microliters). Pretreatment with the specific angiotensin receptor antagonist [Sar1, Thr8]Ang II (sarthran) significantly diminished the subsequent leucine aminopeptidase M-induced decreases in blood pressure in SHR and facilitated recovery to base level blood pressure and heart rate in blood strains. Thus, exogenous application of leucine aminopeptidase M into the brain lateral ventricles of SHR is temporarily effective at reducing blood pressure, and this effect appears dependent on the brain angiotensinergic system. This treatment also reduced blood pressure in WKY rats; however, pretreatment with sarthran was reasonably ineffective at preventing subsequent leucine aminopeptidase M-induced decreases in blood pressure.

Differences in glucose-dependent insulinotrophic polypeptide hormone and hepatic lipase in subjects of southern and northern Europe: implications for postprandial lipemia.

BACKGROUND: This study was an extension of a previous study that showed different lipemic responses to standard test meals in subjects from southern and northern Europe. OBJECTIVE: The aim was to determine in 32 healthy young men from northern and southern Europe whether differences in the secretion of insulin and glucose-dependent insulinotrophic polypeptide (GIP) might explain these findings through the actions of these hormones on lipoprotein lipase. DESIGN: We investigated in a randomized, single-blind, crossover study the effects of 2 test meals of identical macronutrient composition but different saturated fatty acid (SFA) and monounsaturated fatty acid (MUFA) contents on postprandial GIP, insulin, the ratio of incremental triacylglycerol to apolipoprotein B-48 (a marker of chylomicron size), and the activity of postheparin lipases. RESULTS: Fasting and postprandial GIP concentrations and postheparin hepatic lipase activities were significantly higher in the southern Europeans (P < 0.001 and P < 0.02, respectively). Lipoprotein lipase activity after the SFA-rich meal was significantly higher in the northern Europeans (P < 0.01). HL activity 9 h after the SFA-rich meal and the area under the curve (AUC) for the postprandial insulin response correlated with the AUC for the postprandial GIP response [r = 0.44 (P < 0.04) and r = 0.46 (P < 0.05), respectively]. There were no significant differences in chylomicron size between the 2 groups for either meal, but when the groups were combined there was a significant difference in chylomicron size between the SFA- and MUFA-rich meals (P < 0.05), which could be due to the formation of larger chylomicrons after the MUFA-rich meal. CONCLUSION: The significantly higher GIP and insulin responses and HL activities in southern Europeans may provide an explanation for our previous report of attenuated postprandial triacylglycerol and apolipoprotein B-48 responses in them.

Effect of long-term olive oil dietary intervention on postprandial triacylglycerol and factor VII metabolism.

Although the beneficial effects of Mediterranean-type diets, which are rich in olive oil, a good source of monounsaturated fatty acids (MUFAs), are generally accepted, little is known about the effects of long-term dietary MUFA intake on postprandial lipoprotein metabolism and hemostasis. This study used a single-blind, randomized, crossover design to investigate the relative effects of a long-term dietary olive oil intervention and a control [saturated fatty acid (SFA)-enriched] diet on postprandial triacylglycerol metabolism and factor VII activity. The postprandial response to a standard test meal was investigated in 23 healthy men who adhered to both diets for 8 wk. cis-MUFAs were successfully substituted for SFAs in the MUFA diet without affecting total dietary fat or energy intakes. The long-term dietary MUFA intervention significantly reduced plasma and LDL-cholesterol concentrations (P = 0.01). Postprandial triacylglycerol concentrations were significantly greater in the early postprandial period after the MUFA diet (P = 0.003). Postprandial factor VII activation and the concentration of the factor VII antigen were significantly lower after the MUFA diet (P = 0.04 and P = 0.006, respectively). This study showed that isoenergetic substitution of MUFAs for SFAs reduces plasma cholesterol and reduces the degree of postprandial factor VII activation. The alterations in the postprandial triacylglycerol response suggest a greater rate of dietary fat absorption and postprandial triacylglycerol metabolism after a diet rich in MUFAs. This study presents new insights into the biochemical basis of the beneficial effects associated with long-term dietary MUFA consumption, which may explain the lower rates of coronary mortality in Mediterranean regions.

Aminopeptidase-induced elevations and reductions in blood pressure in the spontaneously hypertensive rat.

In vitro results indicated that human placenta-derived aminopeptidase A (APA) was very effective at hydrolyzing aspartate from the angiotensin molecule, thus converting angiotensin II to angiotensin III, but was not active against angiotensin III. In vivo experiments revealed significant elevations in blood pressure when APA was intracerebroventricularly infused into anesthetized spontaneously hypertensive rats (SHR) and Wistar-Kyoto normotensive control rats (WKY), with maximum mean (+/- s.e.m.) increases of 30.0 +/- 2.5 and 32.5 +/- 3.7 mmHg, respectively. By contrast, in vitro incubation results utilizing leucine aminopeptidase M (LAP-M) indicated very active degradation of angiotensin III, with less rapid degradation of angiotensin II. The intracerebroventricular infusion of LAP-M significantly reduced blood pressure, particularly in the SHR, but also in WKY, -65.8 +/- 5.1 and -42.5 +/- 6.1 mmHg, respectively. Pretreatment with the specific angiotensin receptor antagonist, Sar1, Thr8 angiotensin II (sarthran) significantly diminished the subsequent APA-induced increase in blood pressure in members of both strains. Pretreatment with sarthran has previously been shown to significantly diminish LAP-M-induced decreases in blood pressure in SHR. Thus, the effects of these aminopeptidases appear to be primarily dependent upon the brain angiotensinergic system, and are consistent with the hypothesis that angiotensin III is the primary active form of central angiotensin.

The action of porcine glucagon on the motility of the canine duodenum and jejunum.

1 Intravenous bolus doses of porcine glucagon of 0.001-0.05 mg kg-1 caused intense stimulation of the duodenum and jejunum of the dog. 2 Intravenous infusion of porcine glucagon at 0.025-0.05 mg kg-1 h-1 caused similar stimulation. In both cases the stimulation was phasic in nature. 3 Stimulation of the duodenum and jejunum following glucagon was accompanied by a decrease in frequency of the intestinal basic electrical rhythm (BER). No change was seen in the intervals between successive periods of phase III motor activity.

Effects of hormone replacement therapy on platelet membrane fatty acid composition.

Oestrogen replacement therapy has been shown to protect postmenopausal women from ischaemic heart disease, strokes and hypertension. The mechanism of protection conferred by oestrogen, although partly attributable to changes in serum lipoproteins, is not fully understood. The present study was undertaken to assess the effect of hormone replacement therapy on the composition of platelet membrane fatty acids in postmenopausal women. These were analysed by gas-liquid chromatography before and six weeks after continuous conjugated equine oestrogen therapy (0.625 mg daily) combined with cyclical therapy with 75 micrograms L-norgestrel from day 17 to 28 of a 28-day cycle. Each subject acted as her own control. The principal findings of the study were that, following treatment, there was a 16.2% reduction in platelet membrane polyunsaturated fatty acids (P < 0.001), an increase of 9.1 and 7.1% in saturated fatty acids and monounsaturated fatty acids respectively (P < 0.001) and a 17.8% reduction in arachidonic acid (P < 0.003). There was no correlation between changes in membrane fatty acids and serum lipoproteins. This suggests that the changes in membrane composition noted in this study may be a primary effect of hormone replacement therapy, especially oestrogen.

Gas flow through a bronchopleural fistula. Measuring the effects of high-frequency jet ventilation and chest-tube suction.

High-frequency jet ventilation (HFJV) is FDA-approved for ventilating patients with bronchopleural fistulae (BPF), yet little is known about its effect on the fistula airleak. We quantitated a patient's BPF airleak during both conventional volume-cycled ventilation and HFJV. The effect of chest-tube suction (CTS) on BPF flow was also studied. Despite a significant reduction in peak airway pressure, the HFJV resulted in a 50-70 percent increase in BPF flow. CTS also significantly increased the airleak. HFJV may not always be the preferential method for ventilating patients with BPF and we recommend measuring the fistula airleak when attempting to optimize a patient's ventilatory parameters.

Angiotensin III-induced dipsogenic and pressor responses in rodents.

Subcutaneous injections of [des-Asp1]-angiotensin I [( des-Asp1]-AI), angiotensin II (AII), and angiotensin III (AIII) induced drinking in the laboratory rat and the South American rodent Octodon degus, but not in the gerbil. In a second experiment, pretreatment with captopril, an angiotensin converting enzyme inhibitor, prevented the endogenous conversion of subcutaneously injected [des-Asp1]-AI to AIII and prevented drinking in rats and degus. The pharmacological artifact of hypovolemia caused by angiotensin-induced increases in vascular permeability was not observed in members of these species. In a final experiment blood pressure changes resulting from subcutaneous injections of AII and AIII in rats and gerbils were measured. Significant pressor elevations were seen following the administration of both analogues, although AII was more potent. These results demonstrate that AIII is dipsogenic in rats and degus and serves as a pressor agent in rats and gerbils. No ready explanation is available for the gerbil's relative lack of dipsogenicity to the presently tested angiotensins.

Angiotensin's contribution to dipsogenic additivity in several rodent species.

Significant positive correlations were obtained between plasma angiotensin levels and drinking following the administration of polyethylene glycol (PEG) in rats, gerbils, and hamsters and following isoproterenol (ISOP) in rats and gerbils, but not in a South American rodent, Octodon degus (degus). Hamsters revealed elevations in plasma angiotensin following ISOP but no drinking, whereas degus failed to show changes in plasma angiotensin to either treatment, although drinking occurred following PEG. All species drank to 2.5 M NaCl injections with no measurable changes in plasma angiotensin concentrations. A second experiment addressed our previous inability to measure [125I] angiotensin II (AII) specific binding in the brains of gerbils and degus and indicated that these animals possess circumventricular organ (CVO) angiotensin III (AIII) receptors; thus, circulating AIII may be the ligand in these species. A final experiment examined members of these species for dipsogenic additivity following the pairing of extracellular and intracellular thirst challenges. Rats and gerbils revealed additivity when challenged with either PEG or ISOP paired with 2.5 M NaCl. Degus and hamsters indicated additivity only with PEG and 2.5 M NaCl combined. Despite the presence of CVO angiotensin receptors in degus and hamsters, it is concluded that the important component of the hypovolemic dipsogenic stimulus in members of these species may be activation of volume receptors rather than brain angiotensin receptors.

Surgical glove perforation in obstetrics.

Perforation of surgical gloves places the obstetrician at risk for blood-borne infectious diseases. Seven hundred fifty-four surgical gloves used in vaginal and cesarean deliveries and postpartum tubal ligations were examined for evidence of perforation by the air inflation-water submersion technique. The overall glove perforation rate was 13.3%, with 62% of the perforations remaining unrecognized during the surgical procedure. The majority of perforations occurred on the fingers of the nondominant hand. Multivariate analysis with logistic regression indicated that cesarean delivery (odds ratio 3.52), any vaginal laceration or episiotomy (odds ratio 4.95), and chief resident status (odds ratio 3.00) were the major risk factors for surgical glove perforation. Surgical technique by assistants, especially in complex cases, is as important as that of the primary surgeon in regard to glove perforations.