Change in the functional properties of actin by its glycation in vitro

The influence of glycation (non-enzymatic glycosylation) on structural and functional properties of actin of rabbit skeletal muscle and the effects of the natural anti-glycating dipeptide carnosine were studied. Glucose (0.5 M), fructose (0.5 M), and glyceraldehyde (0.05 M) were used as glycating agents. Marked changes in the structural and functional properties were observed in the presence of glyceraldehyde when high-molecular-weight components appear. This was followed by a decrease in the ability of actin to activate myosin ATPase, to polymerize, and to inhibit DNase I. In the presence of 0.05 M carnosine, the quantity of high-molecular-weight products decreased and myosin ATPase activation was retained. Since muscle tissue contains millimolar quantities of carnosine, glycation of actin associated with changes in its properties is evidently more likely to occur in non-muscle cells.     

Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery after experimental subarachnoid hemorrhage

BACKGROUND AND PURPOSE: Gene transfer with recombinant viral vectors encoding vasodilator proteins may be useful in therapy of cerebral vasospasm after subarachnoid hemorrhage (SAH). Relaxations mediated by nitric oxide are impaired in cerebral arteries affected by SAH. The present study was designed to determine the effect of SAH on the efficiency of ex vivo adenovirus-mediated gene transfer to canine basilar arteries and to examine whether expression of recombinant endothelial nitric oxide synthase (eNOS) gene may have functional effects on vasomotor reactivity of spastic arteries affected by SAH. METHODS: Replication-deficient recombinant adenovirus vectors encoding bovine eNOS (AdCMVeNOS) and Escherichia coli beta-galactosidase (AdCMVbeta-Gal) genes were used for ex vivo gene transfer. Rings of basilar arteries obtained from control dogs and dogs exposed to SAH were incubated with the vectors in minimum essential medium. Twenty-four hours after gene transfer, expression and function of the recombinant genes were evaluated by (1) histochemical or immunohistochemical staining, (2) beta-galactosidase protein measurement, and (3) isometric tension recording. RESULTS: Transduction with AdCMVbeta-Gal and AdCMVeNOS resulted in the expression of recombinant beta-galactosidase and eNOS proteins mostly in the vascular adventitia. The expression of beta-galactosidase protein was approximately 2-fold higher in SAH arteries than in normal arteries. Endothelium-dependent relaxations caused by bradykinin and substance P were suppressed in SAH arteries. The relaxations to bradykinin were significantly augmented in both normal and SAH arteries after AdCMVeNOS transduction but not after AdCMVbeta-Gal transduction. The relaxations to substance P were augmented by AdCMVeNOS transduction only in normal arteries. Bradykinin and substance P caused relaxations even in endothelium-denuded arteries, when the vessels were transduced with AdCMVeNOS. These endothelium-independent (adventitia-dependent) relaxations to bradykinin observed after AdCMVeNOS transduction were similar between normal and SAH arteries, whereas those to substance P were significantly reduced in SAH arteries compared with normal arteries. CONCLUSIONS: These results suggest that expression of recombinant proteins after adenovirus-mediated gene transfer may be enhanced in cerebral arteries affected by SAH and that successful eNOS gene transfer to spastic arteries can at least partly restore the impaired nitric oxide-mediated relaxations through local (adventitial) production of nitric oxide.     

A genetic defect resulting in mild low-renin hypertension

Severe low-renin hypertension has few known causes. Apparent mineralocorticoid excess (AME) is a genetic disorder that results in severe juvenile low-renin hypertension, hyporeninemia, hypoaldosteronemia, hypokalemic alkalosis, low birth weight, failure to thrive, poor growth, and in many cases nephrocalcinosis. In 1995, it was shown that mutations in the gene (HSD11B2) encoding the 11beta-hydroxysteroid dehydrogenase type 2 enzyme (11beta-HSD2) cause AME. Typical patients with AME have defective 11beta-HSD2 activity, as evidenced by an abnormal ratio of cortisol to cortisone metabolites and by an exceedingly diminished ability to convert [11-3H]cortisol to cortisone. Recently, we have studied an unusual patient with mild low-renin hypertension and a homozygous mutation in the HSD11B2 gene. The patient came from an inbred Mennonite family, and though the mutation identified her as a patient with AME, she did not demonstrate the typical features of AME. Biochemical analysis in this patient revealed a moderately elevated cortisol to cortisone metabolite ratio. The conversion of cortisol to cortisone was 58% compared with 0-6% in typical patients with AME whereas the normal conversion is 90-95%. Molecular analysis of the HSD11B2 gene of this patient showed a homozygous C-->T transition in the second nucleotide of codon 227, resulting in a substitution of proline with leucine (P227L). The parents and sibs were heterozygous for this mutation. In vitro expression studies showed an increase in the Km (300 nM) over normal (54 nM). Because approximately 40% of patients with essential hypertension demonstrate low renin, we suggest that such patients should undergo genetic analysis of the HSD11B2 gene.     

The effect of hormone replacement therapy alone and in combination with simvastatin on plasma lipids of hypercholesterolemic postmenopausal women with coronary artery disease

Follicular growth, lifespan of the corpus luteum, and antioxidant status of lactating Holsteins that experienced heat stress were monitored. Eleven multiparous cows, 60 to 110 d in milk, were maintained from 0800 to 1800 h daily in environmental chambers from d 11 to 21 of the estrous cycle. Cows were randomly assigned to a heat stress (mean dry bulb temperature peaked at 38.3 degrees C) or control treatment (mean dry bulb temperatures varied from 20.8 to 25.6 degrees C). Rectal temperature and respiration rates of heat-stressed cows were higher at 1600 h than were those of control cows. The length of the estrous cycle and the interval from estrus until luteolysis were not different between treatments. Two of 6 control cows and 1 of 5 heat-stressed cows had extended cycles (> 24 d). Heat-stressed cows had more class 1 (2 to 5 mm) follicles from d 11 to 15 of the estrous cycle. Numbers of class 2 (6 to 9 mm) and class 3 (> or = 9 mm) follicles were similar between treatments. Plasma progesterone concentrations were higher for heat-stressed cows until d 19 of the estrous cycle. Treatment did not affect concentrations of alpha-tocopherol, beta-carotene, retinol, retinyl palmitate, or total protein in plasma or concentrations of malondialdehyde in muscle. In conclusion, heat stress did not extend luteal function or the length of the estrous cycle of lactating Holstein cows but did affect follicular growth and progesterone concentrations in plasma. Heat stress did not appear to increase lipid peroxidation or decrease lipid-soluble antioxidant concentrations in blood.     

The connection between absence-like seizures and hypothermia induced by penicillin: possible implication on other animal models of petit mal epilepsy

In this study we investigated the relationship between penicillin-induced hypothermia and petit mal epilepsy induced by this proconvulsant antibiotic. In order to find a possible dose-dependent relationship, we used two doses: 1500.000 and 1000.000 U/kg b.wt., both known as being sufficient to induce absence-like attacks with subsequent spike and wave discharges (SWD) in electrocorticogram (ECoG). Because of experimental data suggesting penicillin binding to benzodiazepine receptor recognition site, we also studied penicillin-induced changes in body temperature after diazepam pretreatment. Results of this study clearly show that penicillin in doses known to induce petit mal-like epilepsy concomitantly induces statistically significant dose-dependent decrease in body temperature. Pretreatment with diazepam completely prevents both penicillin-induced hypothermia and SWDs. On the other hand, both the diazepam and mixed diazepam + penicillin treatments did not significantly alter body temperature. These results suggest, however, that at least some of the penicillin effects described could be assigned to its binding to the benzodiazepine receptor recognition site at GABA(A) ionophore. This may have an important clinical implication because the inhibitory action of penicillin at the benzodiazepine receptor recognition site could account for the mechanism of penicillin-induced unspecific encephalopathies in humans. The relationship between petit mal epilepsy and hypothermia sheds new light on the action mechanisms of penicillin-induced absence seizures.     

Increased aggression in rats after withdrawal of long term used oxazepam

The withdrawal of oxazepam (5 mg/kg i.p.) applied for 1 year in rats, increased shock-induced aggression of animals. This phenomenon is interpreted as a sign of abstinence and suggests that long-term treatment causes dependence to oxazepam in rats.     

Mast cell-dependent neutrophil and mononuclear cell recruitment in immunoglobulin E-induced gastric reactions in mice

BACKGROUND & AIMS: Immunoglobulin (Ig) E-dependent reactions elicit an immediate response and can also result in a late-phase reaction that is characterized by the infiltration of leukocytes. This study assessed whether IgE-dependent late-phase responses can be elicited in the stomach wall of mice and examined the role of mast cells in this reaction. METHODS: IgE-dependent gastric inflammation was elicited in genetically mast cell-deficient KitW/KitW-v mice, the congenic normal (+/+) mice, and mast cell-deficient KitW/KitW-v mice that had undergone local and selective reconstitution of gastric mast cell populations. RESULTS: IgE-dependent gastric reactions were associated with mast cell degranulation and the infiltration of both neutrophils and mononuclear cells in normal mice, but no significant leukocyte infiltration was observed in mast cell-deficient KitW/KitW-v mice. By contrast, in mast cell-reconstituted KitW/KitW-v mice, IgE-dependent reactions were associated with the infiltration of neutrophils and mononuclear cells. CONCLUSIONS: These results show that late-phase reactions can occur during IgE-dependent gastric inflammation in the mouse and that the infiltration of both neutrophils and mononuclear cells that are observed during this reaction are mast cell dependent.     

Cloning, sequencing, and expression in escherichia coli of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes

OxlT is the oxalate/formate exchange protein that represents the vectorial component of a proton-motive metabolic cycle in Oxalobacter formigenes. Here we report the cloning and sequencing of OxlT and describe its expression in Escherichia coli. The OxlT amino acid sequence specifies a polytopic hydrophobic protein of 418 residues with a mass of 44,128 daltons. Analysis of hydropathy and consideration of the distribution of charged residues suggests an OxlT secondary structure having 12 transmembrane segments, oriented so that the N and C termini face the cytoplasm. Expression of OxlT in E. coli coincides with appearance of a capacity to carry out the self-exchange of oxalate and the heterologous, electrogenic exchange of oxalate with formate. The unusually high velocity of OxlT-mediated transport is also preserved in E. coli. We conclude that the essential features of OxlT are retained on its expression in E. coli.     

Elevated tissue inhibitor of metalloproteinase 1 RNA in colorectal cancer stroma correlates with lymph node and distant metastases

Tissue inhibitor of metalloproteinase (TIMP) inhibits the proteolytic activity of several matrix metalloproteinases centrally involved in tumor invasion and metastases. The purpose of this study was to determine the origin of TIMP-1 mRNA production in both human colorectal cancer (CRC) and metastatic liver lesions as well as define the relationships between TIMP-1 RNA expression and standard clinicopathological variables of CRC. Total cellular RNA, extracted from 56 CRC and 10 liver metastases, were examined by Northern blot hybridization. The mean/normal mucosa fold increase of TIMP-1 RNA was significantly elevated in both CRC (12.1 +/- 1.7) and liver metastases (10.0 +/- 3.6). No relationship was noted between TIMP-1 expression and tumor size, location nor differentiation. Based on lymph node metastases status, significantly higher TIMP-1 RNA levels were found in CRC with metastases than in those without metastases (15.6 +/- 3.3 versus 7.9 +/- 1.3) (P = 0.04). Similarly, TIMP-1 RNA levels were higher in primary CRC with distant metastases than those without distant metastases (17.6 +/- 4.1 versus 9.3 +/- 1.9) (P = 0. 04). In situ hybridization localized TIMP-1 mRNA predominantly in tumor stroma within spindle fibroblast-like cells rather than in cancer cells themselves. The correlation between the increased TIMP-1 mRNA level and advanced CRC stage noted in this study reflects a possible growth-promoting function for TIMP-1 in human CRC.