Spatiotemporal expression patterns of natriuretic peptides in rat testis and epididymis during postnatal development.

Natriuretic peptide (NP) family is composed of atrial, brain and C-type NP (NPPA, NPPB and NPPC). Here, we aimed to investigate NP expression in testis and epididymis during postnatal development. NPPA expression was observed in gonocytes at prepubertal period but in only spermatocytes in pachytene and leptotene/zygotene stage at pubertal period. In prepubertal and pubertal periods, we detected NPPB expression in only Leydig cells. However, NPPC expression was detected in all of the gonocytes and Sertoli cells, spermatocytes and some interstitial cells in prepubertal and pubertal periods. In postpubertal and mature periods, NPPA and NPPB staining were detected in Leydig cells, elongated and round spermatids but not in spermatogonia and spermatocytes. However, we observed NPPC expression in all cells of the seminiferous tubules and Leydig cells in the postpubertal and mature periods. Epididymal epithelium showed intense NPPC expression during postnatal period but weak NPPA and NPPB expression in prepubertal and pubertal periods. The expression of three NPs in the testis significantly increased after puberty. In conclusion, puberty had a significant effect on NP expression in testis. Unlike NPPA and NPPB, expression of NPPC in all cells of the seminiferous tubule suggests that NPPC is effective in each step of spermatogenesis.

[Natriuretic peptides as markers of development and prognosis of pulmonary hypertension severity in patients with chronic obstructive disease.]

Studied the diagnostic and prognostic significance of N-terminal precursor of natriuretic peptide С-type (NT-proCNP) and brain natriuretic peptide (NT-proBNP) in patients with COPD with pulmonary hypertension (PH). The study included 47 patients with COPD (II - IV degrees of severity, 2016 GOLD, men - 44, women -3, mean age 59,3±9.12 years, disease duration of 13.7±5.93 years, the index of Smoking at 23.1±10,93 pack-years, BMI of 27.2±12,06 m/kg2.). Criteria of pulmonary hypertension on the basis of the doppler-echocardiography was an increase of pulmonary artery systolic pressure (PASP) > 40 mmHg alone. Depending on the presence and degree of enhancement PASP patients were divided into three groups: 1 - without pulmonary hypertension (PASP < 40 mmHg, n=168), 2 - moderate pulmonary hypertension (PASP 40 - 55 mmHg, n=101), 3-group with severe pulmonary hypertension (PASP > 55 mmHg, n=19). There was a statistically significant intergroup differences (p1-2 0,001, p 2-3 0,001, p1-3 < 0,001) values of NT-proCNP and NT-proBNP. There was a significant correlation relationship SDLA with the concentration of NT-proCNP (r=0,53, p<0,05) and NT-proBNP (r=0,67; p=0,05). A high diagnostic value of determination of NT-proCNP and NT-proBNP to predict the development and severity of PH in patients with COPD. Cox regression analysis showed that elevated levels of NT-proCNP and NT-proBNP in COPD patients of PH c are the predictors of hospital mortality.

Asociación de valores elevados de péptido natriurético auricular y copeptina con riesgo de mortalidad / High levels of atrial natriuretic peptide and copeptin and mortality risk

OBJETIVO: Determinar si los niveles plasmáticos de región media del péptido natriurético proauricular (RM-proPNA), copeptina y procalcitonina (PCT) se asocian con aumento del riesgo de mortalidad. MÉTODOS: Estudio prospectivo observacional que incluyó a 254 niños críticamente enfermos. Se compararon los niveles de RM-proPNA, copeptina y PCT entre niños con alto (grupo A; n=33) y bajo (grupo B; n=221) riesgo de mortalidad y entre pacientes con un número de órganos en fallo mayor de 1 (grupo 1; n=71) y menor de 2 (grupo 2; n=183). RESULTADOS: Las medianas (rangos) de RM-proPNA, copeptina y PCT en grupo A vs. grupo B fueron, respectivamente: 209,4 (30,5-1.415,8) vs. 75,0 (14,6-867,2) pmol/l (p < 0,001); 104,4 (7,4-460,9) vs. 26,6 (0,00-613,1) pmol/l (p < 0,001) y 7,8 (0,3-552,0) vs. 0,3 (0,02-107,0) ng/ml (p < 0,001). El área bajo la curva (AUC) para diferenciar grupo A y B fue (intervalo de confianza del 95%): 0,764 (0,674-0,854) para RM-proPNA; 0,735 (0,642-0,827) para copeptina y 0,842 (0,744-0,941) para PCT, sin diferencias significativas. Las AUC para diferenciar los grupos 1 y 2 fueron: 0,837 (0,784-0,891) para RM-proPNA, 0,735 (0,666-0,804) para copeptina y 0,804 (0,715-0,892) para PCT, con diferencias significativas entre RM-proPNA y copeptina, p = 0,01. CONCLUSIONES: Los niveles elevados de RM-proPNA, copeptina y PCT se asocian con aumento de las puntuaciones de riesgo de mortalidad. RM-proPNA mostró mayor asociación que la copeptina con el número de órganos en fallo

OBJECTIVE: To determine whether high levels of mid-regional pro-atrial natriuretic peptide (MR-proANP), copeptin, and procalcitonin (PCT) plasma concentrations are associated with increased mortality risk. METHODS: Prospective observational study including 254 critically ill children. MR-proANP, copeptin and PCT were compared between children with high (Group A; n=33) and low (Group B; n=221) mortality risk, and between patients with failure of more than 1 organ (Group 1; n=71) and less than 2 (Group 2; n=183). RESULTS: Median (range) of MR-proANP, copeptin, and PCT levels in group A vs B were, respectively: 209.4 (30.5-1415.8) vs. 75.0 (14.6-867.2) pmol/L (P<.001); 104.4 (7.4-460.9) vs. 26.6 (0.00-613.1) pmol/L (P<.001), and 7.8 (0.3-552.0) vs. 0.3 (0.02-107.0) ng/mL (P<.001). The area under the curve (AUC) for the differentiation of group A and B was 0.764 (95% CI: 0.674-0.854) for MR-proANP; 0.735 (0.642-0.827) for copeptin, and 0.842 (0.744-0.941) for PCT, with no statistical differences. The AUCs for the differentiation of group 1 and 2 were: 0.837 (0.784-0.891) for MR-proANP, 0.735 (0.666-0.804) for copeptin, and 0.804 (0.715-0.892) for PCT, with statistical differences between MR-proANP and copeptin, P=.01. CONCLUSIONS: High levels of MR-proANP, copeptin and PCT were associated with increased mortality risk scores. MR-proANP showed a higher association than copeptin with number of organs in failure

B-type natriuretic peptide and acute heart failure: Fluid homeostasis, biomarker and therapeutics / Péptido natriurético de tipo B e insuficiencia cardiaca aguda: homeostasis de fluidos, biomarcadores y terapéutica

Natriuretic peptides are a family of peptides with similar structures, but are genetically distinct with diverse actions in cardiovascular, renal and fluid homeostasis. The family consists of an atrial natriuretic peptide (ANP) and a brain natriuretic peptide (BNP) of myocardial cell origin, a C-type natriuretic peptide (CNP) of endothelial origin, and a urodilatin (Uro) which is processed from a prohormone ANP in the kidney. Nesiritide, a human recombinant BNP, was approved by the Federal Drug Administration (FDA) for the management of acute heart failure (AHF) in 2001. Human recombinant ANP (Carperitide) was approved for the same clinical indication in Japan in 1995, and human recombinant Urodilatin (Ularitide) is currently undergoing phase III clinical trial (TRUE AHF). This review will provide an update on important issues regarding the role of BNP in fluid hemostasis as a biomarker and therapeutics in AHF (AU)

Los péptidos natriuréticos son una familia de péptidos con estructura semejante, pero distintos genéticamente, con múltiples acciones en la homeostasis cardiovascular, renal y de fluidos. Esta familia está formada por un péptido natriurético auricular y un péptido natriurético cerebral que se originan en las células miocárdicas, un péptido natriurético de tipo C de origen endotelial y una urodilatina que se procesa de una prohormona del péptido natriurético auricular en el riñón. La nesitirida, un péptido natriurético cerebral recombinante humano, fue aprobada por la Administración de Medicamentos y Alimentos (FDA) en el año 2001 para el tratamiento de la insuficiencia cardiaca aguda. El péptido natriurético auricular recombinante humano (carperitida) fue aprobado con las mismas indicaciones clínicas en Japón en el año 1995, y la urodilatina recombinante humana (ularitida) forma parte ahora mismo de un ensayo de fase iii (TRUE-AHF). Esta revisión permitirá actualizar aspectos importantes relativos al papel que desempeña el péptido natriurético cerebral en la homeostasis de fluidos como biomarcador y fármaco para las insuficiencias cardiacas agudas (AU)

Serial Evaluation of Abdominal Fluid and Serum Amino-terminal pro-C-type Natriuretic Peptide in Dogs with Septic Peritonitis.

BACKGROUND: Serum N-terminal pro-C-natriuretic peptide (NT-proCNP) has shown promise as a diagnostic biomarker for sepsis. Its sensitivity to detect dogs with septic peritonitis (SP) is reportedly low, perhaps attributable to the compartmentalization of NT-proCNP in the abdominal cavity. OBJECTIVES: To evaluate the use of an ELISA for the measurement of NT-proCNP in canine abdominal fluid and to describe the peri-operative pattern of abdominal fluid and serum NT-proCNP concentrations in dogs with SP. ANIMALS: Five client-owned dogs with nonseptic abdominal effusion of varying etiologies and 12 client-owned dogs with SP undergoing abdominal surgery and placement of a closed-suction abdominal drain (CSAD). Six dogs were included upon hospital admission; 6 were included the day after surgery. METHODS: Prospective pilot study. A commercially available ELISA kit was analytically validated for use on canine abdominal fluid. The NT-proCNP concentrations were measured in the abdominal fluid of control dogs, and in serum and abdominal fluid of dogs with SP from admission for CSAD removal. RESULTS: In dogs with SP, admission abdominal fluid NT-proCNP concentrations were lower than the concurrent serum concentrations (P = 0.031), and lower than control canine abdominal fluid concentrations (P = 0.015). Postoperatively, abdominal fluid NT-proCNP concentrations remained lower than serum concentrations (P < 0.050), except on day 4. CONCLUSIONS AND CLINICAL IMPORTANCE: The ELISA kit was able to measure NT-proCNP in canine abdominal fluid. In dogs with SP, low serum NT-proCNP concentrations cannot be explained by abdominal compartmentalization.

C-type natriuretic peptide and its receptors in atherosclerotic plaques of the carotid artery of clinically asymptomatic patients.

OBJECTIVES: C-type natriuretic peptide (CNP) has anti-inflammatory, anti-proliferative and anti-migratory properties. No data exist on the presence of CNP in human atherosclerotic plaques of the carotid artery. Therefore, this study aimed to analyse qualitatively the distribution pattern and characteristics of CNP and its receptors in both, early and advanced human carotid plaques, as well as in stable and unstable lesions. In addition, the aim of this study was to evaluate CNP and its receptors as possible biomarkers to predict plaque stability in advanced lesions. METHODS: Advanced carotid artery plaques of 40 asymptomatic patients (20 histologically stable and 20 histologically unstable) and early arteriosclerotic lesions of three patients were analysed. RESULTS: Serum level of CNP was similar in patients with stable and unstable plaques (196 ± 19 pg ml(-1) vs. 198 ± 25 pg ml(-1), p = 0.948). Expression level of natriuretic peptide receptor 3 (NPR3) was significantly higher in unstable plaques compared to stable plaques (5.6 ± 1.8% vs. 1.7 ± 0.5%, p = 0.045). Expression levels of CNP and NPR2 were higher in unstable plaques but the differences were not statistically significant. The distribution pattern of CNP, NPR2 and NPR3 varied qualitatively between early and advanced carotid plaques. No relevant histological differences were observed with respect to plaque stability. CONCLUSIONS: This study shows the presence of CNP and its receptors in atherosclerotic plaques of human carotid artery, with increased expression of NPR3 in histologically unstable plaques. In this study, serum CNP was not associated with histological plaque stability. In future, larger studies are required to further evaluate whether proteins of the CNP axis would be useful as biomarkers.

Presence of dendroaspis natriuretic peptide and its binding to NPR-A receptor in rabbit kidney.

Natriuretic peptides help to maintain sodium and fluid volume homeostasis in a healthy cardio-renal environment. Since the identification of Dendroaspis natriuretic peptide (DNP) as a new member of the natriuretic peptide family, DNP has been considered as an important regulator of natriuresis and dieresis. The present study was undertaken to investigate the presence of immunoreactive Dendroaspis natriuretic peptide (DNP) and its specific receptor in rabbit. DNP was detected in heart, kidney, liver, brain, and plasma by radioimmunoassay (RIA). DNP contents of cardiac atrium and ventricle, renal cortex and medulla, liver, and brain were 1.42 ± 0.15, 1.0 6 ± 0.08, 2.55 ± 0.21, 1.81 ± 0.16, 1.36 ± 0.22, and 0.69 ± 0.15 pg/mg of wet weight, respectively. The concentration of DNP in plasma was 235.44 ± 15.44 pg/ml. By quantitative in vitro receptor autoradiography, specific ¹²5I-DNP binding sites were revealed in glomeruli, interlobular artery, acuate artery, vasa recta bundle, and inner medulla of the kidney with an apparent dissociation constant (K(d)) of 0.29 ± 0.05, 0.36 ± 0.03, 0.84 ± 0.19, 1.18 ± 0.23, and 10.91 ± 1.59 nM, respectively. Basal rate of 3', 5'-cyclic guanosine monophosphate (cGMP) production by particulate guanylyl cyclase (GC) activation of glomerular membranes was basally 13.40 ± 1.70 pmol/mg protein/min. DNP caused an increment of cGMP production in similar magnitude to that caused by ANP, BNP, and urodilatin, while the production of cGMP by CNP was significantly lower than that by DNP. Our results show that plasma levels of DNP were higher when compared to other tissues. DNP produces cGMP via the NPR-A receptor subtype in the kidney, similarly to ANP and BNP, suggesting that plasma DNP could have similar functions as ANP and BNP.

Valor pronóstico postoperatorio del extremo aminoterminal del propéptido natriurético tipo B en el trasplante cardíaco / Prognostics value of NT-proBNP after heart transplantation

Introducción. El trasplante cardíaco es una opción terapéutica disponible para algunos pacientes con insuficiencia cardíaca avanzada. Es importante establecer el pronóstico que presentan estos pacientes para poder diferenciar aquéllos con peor expectativa de vida y adoptar medidas adicionales en estos casos. Los péptidos natriuréticos cardíacos han demostrado su utilidad diagnóstica y pronóstica en diferentes enfermedades. El objetivo de este estudio es valorar la utilidad del extremo aminoterminal del propéptido natriurético tipo B (NT-proBNP) postoperatorio en el pronóstico de los pacientes trasplantados cardíacos a corto plazo. Materiales y métodos. determinó la concentración de NT-proBNP a los 15 días postoperatorios en 50 pacientes que recibieron trasplante cardíaco para valorar la utilidad pronóstica de mortalidad a 6 meses de seguimiento. Resultados. Los pacientes que fallecieron mostraron concentraciones de NT-proBNP significativamente superiores que los que sobrevivieron, con una mediana de la concentración de NT-proBNP a los 15 días postrasplante de 20.632ng/l (intervalo interculartílico: 6.183 a 37.925ng/l) frente a 3.923ng/l (intervalo interculartílico: 1.752 a 6.890ng/l) respectivamente. Se observó que el hazard-ratio de mortalidad se multiplica por 8,5 veces (IC del 95%: 1,7–44,2) en el grupo de pacientes con concentraciones de NT-proBNP, cuantificadas a los 15 días postrasplante, superiores al valor discriminatorio de 7.500ng/l (AU)

Introduction. Cardiac transplantation is a widely accepted option for the treatment of end-stage congestive heart failure. In order to identify those patients at risk of short life expectancy, it could be worthwhile to establish an individual prognosis factor. The prognostic and diagnostic values of natriuretic peptides have been studied in different areas of clinical practice. The objective of this study was to evaluate the short-term prognostic ability of NT-proBNP concentration in heart transplantation patients. Materials and methods. The group studied consisted of 50 adult heart transplant patients. NT-proBNP concentration was measured in each patient 15 days after surgery to evaluate the prognostic value at 6 months follow up. Results. The non-survivor patients showed higher NT-proBNP concentrations than survivors, with a median of NT-proBNP concentration on the 15th day post-transplantation of 20632ng/L (IIC: 6183 to 37925ng/L) and 3923ng/L (IIC: 1752 to 6890ng/L) in the non-survivors and the survivors groups, respectively. The hazard ratio of mortality was 8.5 times higher (95% CI: 1.7 to 44.2) in those patients with NT-proBNP concentrations over 7500ng/L on the 15th day post-transplantation (AU)

Molecular characterisation and functional interrogation of a local natriuretic peptide system in rodent pituitaries, alphaT3-1 and LbetaT2 gonadotroph cells.

In the pituitary, C-type natriuretic peptide (CNP) has been implicated as a gonadotroph-specific factor, yet expression of the CNP gene (Nppc) and CNP activity in gonadotrophs is poorly defined. Here, we examine the molecular expression and putative function of a local gonadotroph natriuretic peptide system. Nppc, along with all three natriuretic peptide receptors (Npr1, Npr2 and Npr3), was expressed in both alphaT3-1 and LbetaT2 cells and primary mouse pituitary tissue, yet the genes for atrial-(ANP) and B-type natriuretic peptides (Nppa and Nppb) were much less abundant. Putative processing enzymes of CNP were also expressed in alphaT3-1 cells and primary mouse pituitaries. Transcriptional analyses revealed that the proximal 50 bp of the murine Nppc promoter were sufficient for GNRH responsiveness, in an apparent protein kinase C and calcium-dependent manner. Electrophoretic mobility shift assays showed Sp1/Sp3 proteins form major complexes within this region of the Nppc promoter. CNP protein was detectable in rat anterior pituitaries, and electron microscopy detected CNP immunoreactivity in secretory granules of gonadotroph cells. Pharmacological analyses of natriuretic peptide receptor activity clearly showed ANP and CNP are potent activators of cGMP production. However, functional studies failed to reveal a role for CNP in regulating cell proliferation or LH secretion. Surprisingly, CNP potently stimulated the human glycoprotein hormone alpha-subunit promoter in LbetaT2 cells but not in alphaT3-1 cells. Collectively, these findings support a role for CNP as the major natriuretic peptide of the anterior pituitary, and for gonadotroph cells as the major source of CNP expression and site of action.

Atrial natriuretic peptide and related peptides.

In recent years, biomarkers have been recognized as important tools for diagnosis, risk stratification, and therapeutic decision-making in cardiovascular diseases. Currently, the clinical potential of several natriuretic peptides is under scientific investigation. The well-known counter-regulatory hormones are atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), dendroaspis natriuretic peptide (DNP) and urodilatin, which play an important role in the homeostasis of body fluid volume. ANP and BNP have already been demonstrated to have diagnostic usefulness in a great number of studies, which have progressed from bench to bedside. This article summarizes existing data on ANP and related peptides in cardiovascular and other disorders, and outlines the potential clinical usefulness of these markers.

Lack of circadian change of concentration of C-type natriuretic peptide in rabbit aqueous humor.

PURPOSE: To determine whether there is a circadian change of the concentration of C-type natriuretic peptide (CNP) in rabbit aqueous humor. METHODS: Forty-one male white New Zealand rabbits were submitted to a 12 h light and 12 dark lighting schedule; lights on was at 0 h, lights off at 12 h. C-type natriuretic peptide was assayed at 3 light (2 h, 6 h and 10 h) and 3 dark (14,18 and 22 h) times. All groups consisted of four animals but two had more animals to increase the power of tests (6 h, n = 12; 22 h, n = 13). Dependence between the two eyes was tested by Pearson's correlation. The mean of two eyes was considered for analysis. Differences in concentration in pg/200 ml were evaluated by one-way ANOVA, t test and COSINOR analysis. RESULTS: There were no significant differences between the 6 analyzed groups (one-way ANOVA p = 0.157). Pooled into two groups (light and dark), the C-type natriuretic peptide concentration of the light group was lower, 2.626 +/- 0.92 pg/200 ml compared with the dark group 3.02 +/- 1.16 pg/200 ml but did not reach a significant difference (t test; p = 0.23). COSINOR analysis was not statistically significant (R% = 7.72 p = 0.209). CONCLUSION: These data demonstrate that the concentration of CNP in aqueous humor did not show a statistically significant circadian change in rabbits entrained to a 12 h light: 12 h dark lighting schedule.

Expression and localization of C-type natriuretic peptide in human vascular smooth muscle cells.

OBJECTIVES: C-type natriuretic peptide (CNP) released by vascular endothelium relaxes smooth muscle and is important in the maintenance of vascular tone. Since it is not known whether other human vascular cell types produce CNP, we investigated its expression in human vascular smooth muscle. METHODS: CNP expression was examined by RT-PCR in vascular smooth muscle cells (SMC) cultured from human saphenous vein (SV), internal mammary artery (IMA) and radial artery (RA), and CNP protein was probed using immunostaining, in tissue sections and in SMCs cultured from these vessels, respectively. RESULTS: PCR for CNP produced a 334 bp product in all SMC cultures, as expressed in endothelial cells, although the band intensity was markedly less in SMCs. Myocardium from CNP-knockout mouse did not express CNP, while there was expression in wild-type mouse. CNP protein was detected by immunostaining in 100% of SMC cultures. By immunostaining of tissue sections, CNP was detected throughout the medial layer, but not adventitia, of all vessel types. CONCLUSIONS: Expression of CNP at gene and protein level by human vascular SMCs suggests that CNP may have the capacity to regulate vascular tone independently of the endothelium.

Los péptidos natricuréticos en insuficiencia cardíaca (Revisión) / The natriuretics peptides in cardiac insuficiente (Review)

El enfoque clínico y fisiopatológico de la insuficiencia cardíaca va variando conforme se conocen nuevos detalles. A inicios del siglo XX su enfoque partía de un exceso de líquidos dentro del organismo que obligaba a los médicos en la eliminación de líquidos y cambios en su distribución a través de torniquetes o sangrías. Ya ha mediados del siglo pasado este sufre un cambio de perspectiva al verse al ICC como un fenómeno de falla de bomba; pero el mayor cambio es el que vivimos actualmente en el cual es vista la ICC como un fenómeno neurohormonal, con participación de hormonas y sus receptores en la génesis fisiopatología de la IC. Es menester de este artículo presentar a la familia de los péptidos natriuréticos atriales y su participación en los eventos fisiopatológicos de la ICC y su estado actual como marcador de severidad de la misma.

Identification of novel bradykinin-potentiating peptides and C-type natriuretic peptide from Lachesis muta venom

The generation of expressed sequence tags (ESTs) from the pit-viper snake Lachesis muta venom glands allowed us to identify two cDNA isoforms which encode the precursors for bradykinin-potentiating peptides (BPPs) and a C-type natriuretic peptide (CNP). The sequence data derived from these cDNAs combined with the venom peptides identification using MALDI-TOF mass spectrometry analysis predicted that these molecules are the precursor protein isoforms that are further processed to produce five novel BPPs and a CNP. They were identified directly in crude venom using MALDI-TOF. The BPPs sequences were further confirmed by MALDI-TOF/TOF de novo sequencing, and an unusual BPP with a residue of tryptophan at the N-terminus (usually it is pyroglutamate) was identified. The putative processing steps required to form the mature BPPs and CNP seem to be similar to those proposed for the ones found in the venom of Bothrops jararaca and Glodyus blomhoffi.

Preparation of recombinant thioredoxin fused N-terminal proCNP: Analysis of enterokinase cleavage products reveals new enterokinase cleavage sites.

C-type natriuretic peptide (CNP) acts as a paracrine hormone to dilate blood vessels and is also required for the growth of long bones. In vivo, CNP is produced by cleavage from the C-terminal end of a larger proCNP peptide. The remaining N-terminal proCNP fragment (NT-proCNP) escapes into the circulation where its concentration is much higher than that of CNP due presumably to a lower clearance rate. Our strategy to obtain large quantities of pure NT-proCNP for further physiological investigations was to express it as a fusion protein with His(6)-tagged thioredoxin followed by cleavage using enterokinase to yield NT-proCNP alone. We have successfully designed and artificially synthesized the coding sequence specifying both mouse and human NT-proCNP with built-in codon bias towards Escherichia coli codon preference. An enterokinase recognition sequence was incorporated immediately upstream of the NT-proCNP coding sequence to allow the fusion protein to be cleaved without leaving any extra residues on the NT-proCNP peptide. High levels of fusion proteins were obtained, constituting 50-58% of total bacterial proteins. Greater than 90% of recombinant thioredoxin/NT-proCNP was expressed in the soluble form and purified to near homogeneity in a single chromatographic step using nickel as the metal ion in IMAC. A time course analysis of the products released from enterokinase cleavage of the recombinant proteins by ESI-MS revealed three sensitive secondary cleavage sites: two were located on vector-associated sequences linking the thioredoxin moiety and NT-proCNP, and one at the C-terminal end of NT-proCNP. Clearly, substrate specificity of both the native and recombinant forms of enterokinase for the recognition sequence DDDDK was by no means exclusive. Hydrolysis at the unexpected LKGDR site located towards the carboxyl end on NT-proCNP was significantly more efficient than at the internally sited DDDDK target sequence. However, when this same sequence was sited internally replacing the DDDDK in another construct of thioredoxin/mouse NT-proCNP, it was found to be poorly processed by enterokinase. Our results showed that non-target sequences can be preferentially recognized over the canonical DDDDK sequence when located accessibly at the ends of proteins.

Identification of novel bradykinin-potentiating peptides and C-type natriuretic peptide from Lachesis muta venom.

The generation of expressed sequence tags (ESTs) from the pit-viper snake Lachesis muta venom glands allowed us to identify two cDNA isoforms which encode the precursors for bradykinin-potentiating peptides (BPPs) and a C-type natriuretic peptide (CNP). The sequence data derived from these cDNAs combined with the venom peptides identification using MALDI-TOF mass spectrometry analysis predicted that these molecules are the precursor protein isoforms that are further processed to produce five novel BPPs and a CNP. They were identified directly in crude venom using MALDI-TOF. The BPPs sequences were further confirmed by MALDI-TOF/TOF de novo sequencing, and an unusual BPP with a residue of tryptophan at the N-terminus (usually it is pyroglutamate) was identified. The putative processing steps required to form the mature BPPs and CNP seem to be similar to those proposed for the ones found in the venom of Bothrops jararaca and Glodyus blomhoffi.

Reconstruction of the patterns of gene expression in the developing mouse heart reveals an architectural arrangement that facilitates the understanding of atrial malformations and arrhythmias.

Firm knowledge about the formation of the atrial components and of the variations seen in congenital cardiac malformations and abnormal atrial rhythms is fundamental to our understanding of the normal structure of the definitive atrial chambers. The atrial region is relatively inaccessible and has continued to be the source of disagreement. Seeking to resolve these controversies, we made three-dimensional reconstructions of the myocardial components of the developing atrium, identifying domains on the basis of differential expression of myocardial markers, connexin40, and natriuretic precursor peptide A. These reconstructions, made from serial sections of mouse embryos, show that from the outset of atrial development, the systemic and pulmonary veins are directly connected to the atrium. Relative to the systemic junctions, however, the pulmonary venous junction appears later. Our experience shows that three-dimensional reconstructions have three advantages. First, they provide clear access to the combined morphological and molecular data, allowing clarification and verification of morphogenetic concepts for nonmorphological experts and setting the scene for further discussion. Second, they demonstrate that, from the outset, the myocardium surrounding the pulmonary veins is distinct from that clothing the systemic venoatrial junctions. Third, they reveal an anatomical and molecular continuity between the entrance of the systemic venous tributaries, the internodal atrial myocardium, and the atrioventricular region. All these regions are derived from primary myocardium, providing a molecular basis for the observed nonrandom distribution of focal right atrial tachycardias.

Effect of exercise on natriuretic peptides in plasma and urine in chronic heart failure.

BACKGROUND: Plasma atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are elevated in chronic heart failure (CHF). ANP is known to be increased during exercise in healthy subjects and CHF, while the response in BNP during exercise is less clear and does not exist in C-type natriuretic peptide (CNP) and aquaporin-2 (AQP2) in either healthy subjects or CHF. METHODS: Eleven patients with CHF and eleven healthy subjects performed a maximal aerobic exercise test. ANP and BNP in plasma were determined every 3 min and at maximum exercise by radioimmunoassay (RIA) and CNP and AQP2 in urine were determined before and after the exercise test by RIA. RESULTS: The absolute increase in BNP during exercise was higher in patients with CHF (CHF: 4.1 pmol/l; healthy subjects: 1.3 pmol/l, P<0.05) and was positively correlated to BNP at rest (P<0.05), while the absolute increase in ANP during exercise was the same in the two groups (CHF: 4.2 pmol/l; healthy subjects: 6.8 pmol/l, not significant, NS). In CHF, exercise did not change either u-CNP excretion (rest: 9.8 ng/mmol creatinine; after exercise: 8.8 ng/mmol, NS) or u-AQP2 (rest: 466 ng/mmol creatinine; after exercise: 517 ng/mmol creatinine, NS) as well as in healthy subjects where u-CNP (rest: 9.7 ng/mmol creatinine; after exercise: 9.2 ng/mmol creatinine) and u-AQP2 (rest: 283 ng/mmol creatinine; after exercise: 307 ng/mmol creatinine) were the same at rest and after exercise. CONCLUSION: The absolute increase in BNP during exercise is higher in patients with CHF compared to healthy subjects. It is suggested that this is a compensatory phenomenon to improve the exercise capacity in CHF, and that BNP is a more important factor in cardiovascular homeostasis during exercise in CHF than ANP.