Upregulation of adrenomedullin and its receptor components during cardiomyocyte hypertrophy induced by chronic inhibition of nitric oxide synthesis in rats

Adrenomedullin may provide a compensatory mechanism to attenuate left ventricular hypertrophy (LVH). Nitric oxide synthase inhibition, induced by chronic administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) to rats, induces cardiac hypertrophy in some, but not all cases; there are few reports of direct assessment of cardiomyocyte parameters. The objective was to characterize hypertrophic parameters in left (LV) and right ventricular (RV) cardiomyocytes after administration of L-NAME to rats for 8 wk and to determine whether adrenomedullin and its receptor components were upregulated. After treatment with L-NAME (20 and 50 mg x kg(-1) x day(-1)), compared with nontreated animals, 1) systolic blood pressure increased (by 34.2 and 104.9 mmHg), 2) heart weight-to-body wt ratio increased 24.1% at the higher dose (P

Neuropeptide Y Y(1) receptor regulates protein turnover and constitutive gene expression in hypertrophying cardiomyocytes.

Increased levels of neuropeptide Y correlate with severity of left ventricular hypertrophy in vivo. At cardiomyocyte level, hypertrophy is characterised by increased mass and altered phenotype. The aims were to determine the contributions of increased synthesis and reduced degradation of protein to neuropeptide Y-mediated increase in mass, assess effects on gene expression, and characterise neuropeptide Y Y receptor subtype involvement. Neuropeptide Y (10 nM) increased protein mass of adult rat ventricular cardiomyocytes maintained in culture (24 h) (16%>basal) and de novo protein synthesis (incorporation of [14C]phenylalanine) (18%>basal). Neuropeptide Y (100 nM) prevented degradation of existing protein at 8 h. Actinomycin D (5 µM) attenuated increases in protein mass to neuropeptide Y (=1 nM) but not to neuropeptide Y (10 nM). [Leu31, Pro34]neuropeptide Y (10 nM), an agonist at neuropeptide Y Y1 receptors, increased protein mass (25%>basal) but did not stimulate protein synthesis. Neuropeptide Y-(3–36) (10 nM), an agonist at neuropeptide Y Y2 receptors, increased protein mass (29%>basal) and increased protein synthesis (13%>basal), respectively. Actinomycin D (5 µM) abolished the increase in protein mass elicited by neuropeptide Y-(3–36) but not that by [Leu31, Pro34]neuropeptide Y. BIBP3226 [(R)-N2-(diphenylacetyl)-N-(4-hydroxyphenylmethyl)-d-arginine amide] (1 µM), a neuropeptide Y Y1 receptor subtype-selective antagonist, and T4 [neuropeptide Y-(33–36)]4, a neuropeptide Y Y2 receptor subtype-selective antagonist, attenuated the increase in protein mass to 100 nM neuropeptide Y by 68% and 59%, respectively. Neuropeptide Y increased expression of the constitutive gene, myosin light chain-2 (MLC-2), maximally at 12 h (4.7-fold>basal) but did not induce (t=36 h) expression of foetal genes (atrial natriuretic peptide (ANP), skeletal-a-actin and myosin heavy chain-ß). This increase was attenuated by 86% and 51%, respectively, by BIBP3226 (1 µM) and T4 [neuropeptide Y-(33–36)]4 (100 nM). [Leu31, Pro34]neuropeptide Y (100 nM) (2.4-fold>basal) and peptide YY-(3–36) (100 nM) (2.3 fold>basal) increased expression of MLC-2 mRNA at 12 h. In conclusion, initiation of cardiomyocyte hypertrophy by neuropeptide Y requires activation of both neuropeptide Y Y1 and neuropeptide Y Y2 receptors and is associated with enhanced synthesis and attenuated degradation of protein together with increased expression of constitutive genes but not reinduction of foetal genes.

Differential expression of components of the cardiomyocyte adrenomedullin/intermedin receptor system following blood pressure reduction in nitric oxide-deficient hypertension.

Adrenomedullin (AM) and intermedin (IMD; adrenomedulln-2) are vasodilator peptides related to calcitonin gene-related peptide (CGRP). The actions of these peptides are mediated by the calcitonin receptor-like receptor (CLR) in association with one of three receptor activity-modifying proteins. CGRP is selective for CLR/receptor activity modifying protein (RAMP)1, AM for CLR/RAMP2 and -3, and IMD acts at both CGRP and AM receptors. In a model of pressure overload induced by inhibition of nitric-oxide synthase, up-regulation of AM was observed previously in cardiomyocytes demonstrating a hypertrophic phenotype. The current objective was to examine the effects of blood pressure reduction on cardiomyocyte expression of AM and IMD and their receptor components. Nomega-nitro-L-arginine methyl ester (L-NAME) (35 mg/kg/day) was administered to rats for 8 weeks, with or without concurrent administration of hydralazine (50 mg/kg/day) and hydrochlorothiazide (7.5 mg/kg/day). In left ventricular cardiomyocytes from L-NAME-treated rats, increases (-fold) in mRNA expression were 1.6 (preproAM), 8.4 (preproIMD), 3.4 (CLR), 4.1 (RAMP1), 2.8 (RAMP2), and 4.4 (RAMP3). Hydralazine/hydrochlorothiazide normalized systolic blood pressure (BP) and abolished mRNA up-regulation of hypertrophic markers sk-alpha-actin and BNP and of preproAM, CLR, RAMP2, and RAMP3 but did not normalize cardiomyocyte width nor preproIMD or RAMP1 mRNA expression. The robust increase in IMD expression indicates an important role for this peptide in the cardiac pathology of this model but, unlike AM, IMD is not associated with pressure overload upon the myocardium. The concordance of IMD and RAMP1 up-regulation indicates a CGRP-type receptor action; considering also a lack of response to BP reduction, IMD may, like CGRP, have an anti-ischemic function.

Myocardial expression of the endothelin system in endotoxin-treated rats.

Although circulating plasma levels of endothelin (ET)-1 are elevated in endotoxemia, little is known about the myocardial expression of the ET system in endotoxic shock. We assessed the temporal mRNA expression pattern of key components of the ET system (pre-pro ET (ppET) -1, -2, ET-converting enzyme-1, ETA and ETB receptors) by reverse transcription polymerase chain reaction in a rat model of early endotoxic shock. Lipopolysaccharide (5 mg/kg, i.p.) caused a transient increase (p 12-fold increase; p

Kinestatin: a novel bradykinin B2 receptor antagonist peptide from the skin secretion of the Chinese toad, Bombina maxima

We have isolated a novel bradykinin B2-receptor antagonist peptide, kinestatin, from toad (Bombina maxima) defensive skin secretion. Mass spectroscopy established a molecular mass of 931.56 Da and a provisional structure: pGlu-Leu/Ile-Pro-Gly-Leu/Ile-Gly-Pro-Leu/Ile-Arg.amide. The unmodified sequence, -QIPGLGPLRG-, was located at the C-terminus of a 116-amino-acid residue open-reading frame following interrogation of a sequenced B. maxima skin cDNA library database. This confirmed the presence of appropriate primary structural attributes for the observed post-translational modifications present on the mature peptide and established residue 2 as Ile and residues 5/8 as Leu. Kinestatin represents a prototype novel peptide from amphibian skin.

Distribution of the Receptor for Advanced Glycation End Products (RAGE) in the Human Male Reproductive Tract. Prevalence in Men with Diabetes Mellitus.

BACKGROUND: Diabetics have a significantly higher percentage of sperm with nuclear DNA (nDNA) fragmentation and increased levels of advanced glycation end products (AGEs), in their testis, epididymis and sperm. As the receptor for AGEs (RAGE) is important to oxidative stress and cell dysfunction, we hypothesise, that it may be involved in sperm nDNA damage. METHODS: Immunohistochemistry was performed to determine the presence of RAGE in the human testis and epididymis. A comparison of the receptor's incidence and localisation on sperm from 10 diabetic and 11 non-diabetic men was conducted by blind semi-quantitative assessment of the immunostaining. ELISA analysis ascertained RAGE levels in seminal plasma and sperm from 21 diabetic and 31 non-diabetic subjects. Dual labelling immunolocalisation was employed to evaluate RAGE's precise location on the sperm head. RESULTS: RAGE was found throughout the testis, caput epididymis, particularly the principle cells apical region, and on sperm acrosomes. The number of sperm displaying RAGE and the overall protein amount found in sperm and seminal plasma were significantly higher in samples from diabetic men (p

Chemical ablation of gastric inhibitory polypeptide receptor action by daily (Pro3) GIP administration improves glucose tolerance and ameliorates insulin resistance and abnormalities of islet structure in obesity-diabetes.

Glucose-dependent insulinotropic polypeptide (gastric inhibitory polypeptide [GIP]) is an important incretin hormone secreted by endocrine K-cells in response to nutrient ingestion. In this study, we investigated the effects of chemical ablation of GIP receptor (GIP-R) action on aspects of obesity-related diabetes using a stable and specific GIP-R antagonist, (Pro3)GIP. Young adult ob/ob mice received once-daily intraperitoneal injections of saline vehicle or (Pro3)GIP over an 11-day period. Nonfasting plasma glucose levels and the overall glycemic excursion (area under the curve) to a glucose load were significantly reduced (1.6-fold; P <0.05) in (Pro3)GIP-treated mice compared with controls. GIP-R ablation also significantly lowered overall plasma glucose (1.4-fold; P <0.05) and insulin (1.5-fold; P <0.05) responses to feeding. These changes were associated with significantly enhanced (1.6-fold; P <0.05) insulin sensitivity in the (Pro3)GIP-treated group. Daily injection of (Pro3)GIP reduced pancreatic insulin content (1.3-fold; P <0.05) and partially corrected the obesity-related islet hypertrophy and ß-cell hyperplasia of ob/ob mice. These comprehensive beneficial effects of (Pro3)GIP were reversed 9 days after cessation of treatment and were independent of food intake and body weight, which were unchanged. These studies highlight a role for GIP in obesity-related glucose intolerance and emphasize the potential of specific GIP-R antagonists as a new class of drugs for the alleviation of insulin resistance and treatment of type 2 diabetes.

Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets: Report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n¼56), mantle cell lymphoma (n¼54), marginal zone lymphoma (n¼41) and follicular lymphoma (n¼109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.

Molecular basis for estrogen receptor alpha deficiency in BRCA1-linked breast cancer

Background BRCA1-mutant breast tumors are typically estrogen receptor alpha (ER alpha) negative, whereas most sporadic tumors express wild-type BRCA1 and are ER alpha positive. We examined a possible mechanism for the observed ER alpha-negative phenotype of BRCA1-mutant tumors.

Methods We used a breast cancer disease-specific microarray to identify transcripts that were differentially expressed between paraffin-embedded samples of 17 BRCA1-mutant and 14 sporadic breast tumors. We measured the mRNA levels of estrogen receptor 1 (ESR1) ( the gene encoding ER alpha), which was differentially expressed in the tumor samples, by quantitative polymerase chain reaction. Regulation of ESR1 mRNA and ER alpha protein expression was assessed in human breast cancer HCC1937 cells that were stably reconstituted with wild-type BRCA1 expression construct and in human breast cancer T47D and MCF-7 cells transiently transfected with BRCA1-specific short-interfering RNA ( siRNA). Chromatin immunoprecipitation assays were performed to determine if BRCA1 binds the ESR1 promoter and to identify other interacting proteins. Sensitivity to the antiestrogen drug fulvestrant was examined in T47D and MCF-7 cells transfected with BRCA1-specific siRNA. All statistical tests were two-sided.

Results Mean ESR1 gene expression was 5.4-fold lower in BRCA1-mutant tumors than in sporadic tumors ( 95% confidence interval [CI]=2.6-fold to 40.1-fold, P =.0019). The transcription factor Oct-1 recruited BRCA1 to the ESR1 promoter, and both BRCA1 and Oct-1 were required for ER alpha expression. BRCA1-depleted breast cancer cells expressing exogenous ER alpha were more sensitive to fulvestrant than BRCA1-depleted cells transfected with empty vector ( T47D cells, the mean concentration of fulvestrant that inhibited the growth of 40% of the cells [IC40] for empty vector versus ER alpha: > 10(-5) versus 8.0 x 10(-9) M [ 95% CI=3.1x10(-10) to 3.2 x 10(-6) M]; MCF-7 cells, mean IC40 for empty vector versus ER alpha : > 10(-5) versus 4.9 x 10(-8) M [ 95% CI=2.0 x 10(-9) to 3.9 x 10(-6) M]).

Conclusions BRCA1 alters the response of breast cancer cells to antiestrogen therapy by directly modulating ER alpha expression.

Cyclin G associated kinase interacts with interleukin 12 receptor beta2 and suppresses interleukin 12 induced IFN-gamma production.

BACKGROUND: Although severe encephalopathy has been proposed as a possible contraindication to the use of noninvasive positive-pressure ventilation (NPPV), increasing clinical reports showed it was effective in patients with impaired consciousness and even coma secondary to acute respiratory failure, especially hypercapnic acute respiratory failure (HARF). To further evaluate the effectiveness and safety of NPPV for severe hypercapnic encephalopathy, a prospective case-control study was conducted at a university respiratory intensive care unit (RICU) in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) during the past 3 years. METHODS: Forty-three of 68 consecutive AECOPD patients requiring ventilatory support for HARF were divided into 2 groups, which were carefully matched for age, sex, COPD course, tobacco use and previous hospitalization history, according to the severity of encephalopathy, 22 patients with Glasgow coma scale (GCS) <10 served as group A and 21 with GCS = 10 as group B. RESULTS: Compared with group B, group A had a higher level of baseline arterial partial CO2 pressure ((102 +/- 27) mmHg vs (74 +/- 17) mmHg, P <0.01), lower levels of GCS (7.5 +/- 1.9 vs 12.2 +/- 1.8, P <0.01), arterial pH value (7.18 +/- 0.06 vs 7.28 +/- 0.07, P <0.01) and partial O(2) pressure/fraction of inspired O(2) ratio (168 +/- 39 vs 189 +/- 33, P <0.05). The NPPV success rate and hospital mortality were 73% (16/22) and 14% (3/22) respectively in group A, which were comparable to those in group B (68% (15/21) and 14% (3/21) respectively, all P > 0.05), but group A needed an average of 7 cm H2O higher of maximal pressure support during NPPV, and 4, 4 and 7 days longer of NPPV time, RICU stay and hospital stay respectively than group B (P <0.05 or P <0.01). NPPV therapy failed in 12 patients (6 in each group) because of excessive airway secretions (7 patients), hemodynamic instability (2), worsening of dyspnea and deterioration of gas exchange (2), and gastric content aspiration (1). CONCLUSIONS: Selected patients with severe hypercapnic encephalopathy secondary to HARF can be treated as effectively and safely with NPPV as awake patients with HARF due to AECOPD; a trial of NPPV should be instituted to reduce the need of endotracheal intubation in patients with severe hypercapnic encephalopathy who are otherwise good candidates for NPPV due to AECOPD.