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A Negro

Publications and source records attributed to A Negro.

At least 19 recordsLinked to original sources
PubMed2001

Evidence for FSH-dependent upregulation of SPATA2 (spermatogenesis-associated protein 2).

Here we report the cloning and characterization of a novel cDNA named spata 2. SPATA2 is the ortholog of PD1, a human testicular protein which has been suggested to play a role in spermatogenesis. The spata 2 sequence reveals an open reading frame encoding a protein of 511 amino acids. Northern blot analysis with rat mRNA demonstrated two distinct transcripts of 2.2 and 4.0 kb. Tagging recombinant SPATA2 with the green fluorescent protein (GFP) and expressing the chimeric polypeptide in HLtat transfected cells indicated that SPATA2 is located in the nucleus. RT-PCR analysis revealed that spata 2 mRNA is expressed in the testis and to a lesser extent in the brain while skeletal muscle and kidney showed a barely visible signal. The same analysis demonstrated that isolated Sertoli cells express spata 2 mRNA. Treating Sertoli cells with FSH in vitro induced remarkable changes in the steady-state level of spata 2 mRNA in a time-dependent manner. In developing testis spata 2 transcripts were first detected 10 days post partum and expression levels increased steadily with age. The ability of FSH to stimulate spata 2 mRNA expression as well as its developmental expression suggests that this protein might play a role in regulating spermatogenesis and thus, according to the Gene Nomenclature Committee, we propose the name SPATA2 (Spermatogenesis associated protein 2) for this protein (or gene).

Amino Acid Sequence↗
PubMed2001

The metabolism and imaging in live cells of the bovine prion protein in its native form or carrying single amino acid substitutions.

Prion diseases are probably caused by an abnormal form of a cellular glycoprotein, the prion protein. Recent evidence suggests that the prion strain causing BSE has been transmitted to humans, thereby provoking a variant form of Creutzfeldt-Jacob disease. In this work, we analyzed the behavior of normal and malformed isoforms of the bovine PrP in transfected mammalian cell lines. Biochemical and immunocytochemical assays were complimented with imaging of live cells expressing fusion constructs between PrP and GFP. Bovine homologues of human E200K and D178N (129M) mutations were used as models of pathogenic isoforms. We show that the GFP does not impair the metabolism of native and mutant bPrPs and is thus a valid marker of PrP cellular distribution. We also show that each amino acid replacement provokes alterations in the cell sorting and processing of bPrP. These are different from those ascribed to both murine mutant homologues. However, human and bovine PrPs carrying the D178N genotype had similar cellular behavior.

Amino Acid Substitution↗
PubMed2001

-344C/T polymorphism of CYP11B2 gene in Italian patients with idiopathic low renin hypertension.

Most patients with low renin essential hypertension are not qualitatively different from patients with idiopathic hyperaldosteronism, as in both conditions aldosterone secretion is not appropriately reduced. The aim of the study was to investigate allele and genotype frequencies of the -344C/T polymorphism, located in the promoter region of the aldosterone synthase gene, in 83 patients with idiopathic low renin hypertension characterized by an increased aldosterone to renin ratio, including both patients with low renin essential hypertension (n=53) and subjects with idiopathic hyperaldosteronism (n=30), compared with 78 patients with normal to high renin essential hypertension and 126 normotensive control subjects. The relationship of -344C/T genotypes to basal and postcaptopril plasma aldosterone/plasma renin activity ratio was also examined in the entire hypertensive population. An increased frequency of the T allele and a relative excess of TT homozygosity over CC homozygosity were found in patients with idiopathic low renin hypertension in comparison with both normal to high renin hypertensives and normotensive controls. A higher post-captopril aldosterone to renin ratio was found in the hypertensives with TT genotype than in those with CC genotype, and TT+TC genotypes were associated with a smaller decrease in the aldosterone-to-renin ratio elicited by captopril administration. The present study suggests that the -344C/T polymorphism, or a functional variant in linkage disequilibrium with it, may play a role in the abnormal regulation of aldosterone secretion in idiopathic low renin hypertension.

Aldosterone↗
PubMed2000

KDC1, a novel carrot root hair K+ channel. Cloning, characterization, and expression in mammalian cells.

Potassium is an essential nutrient which plays an important role in many aspects of plant growth and development. Plants have developed a number of highly specific mechanisms to take up potassium from the soil; these include the expression of K(+) transporters and potassium channels in root cells. Despite the fact that root epidermal and hair cells are in direct contact with the soil, the role of these tissues in K(+) uptake is not well understood. Here we report the molecular cloning and functional characterization of a novel potassium channel KDC1 which forms part of a new subfamily of plant K(in) channels. Kdc1 was isolated from carrot root RNA and in situ hybridization experiments show Kdc1 to be highly expressed in root hair cells. Expressing the KDC1 protein in Chinese hamster ovary cells identified it as a voltage and pH-dependent inwardly rectifying potassium channel. An electrophysiological analysis of carrot root hair protoplasts confirmed the biophysical properties of the Kdc1 gene product (KDC1) in the heterologous expression system. KDC1 thus represents a major K(+) uptake channel in carrot root hair cells.

Amino Acid Sequence↗
PubMed2000

Crystal structure and refolding properties of the mutant F99S/M153T/V163A of the green fluorescent protein.

The mutant F99S/M153T/V163A of the Green Fluorescent Protein (c3-GFP) has spectral characteristics similar to the wild-type GFP, but it is 42-fold more fluorescent in vivo. Here, we report the crystal structure and the refolding properties of c3-GFP and compare them with those of the less fluorescent wt-GFP and S65T mutant. The topology and the overall structure of c3-GFP is similar to the wild-type GFP. The three mutated residues, Ser99, Thr153, and Ala163, lie on the surface of the protein in three different beta-strands. The side chains of Ser99 and Thr153 are exposed to the solvent, whereas that of Ala163 points toward the interior of the protein. No significant deviation from the structure of the wild-type molecule is found around these positions, and there is not clear evidence of any distortion in the position of the chromophore or of the surrounding residues induced by the mutated amino acids. In vitro refolding experiments on urea-denatured c3-GFP reveal a renaturation behavior similar to that of the S65T molecule, with kinetic constants of the same order of magnitude. We conclude that the higher fluorescence activity of c3-GFP can be attributed neither to particular structural features nor to a faster folding process, as previously proposed.

Crystallography↗
PubMed2000

Bovine prion protein as a modulator of protein kinase CK2.

On the basis of far-Western blot and plasmon resonance (BIAcore) experiments, we show here that recombinant bovine prion protein (bPrP) (25-242) strongly interacts with the catalytic alpha/alpha' subunits of protein kinase CK2 (also termed 'casein kinase 2'). This association leads to increased phosphotransferase activity of CK2alpha, tested on calmodulin or specific peptides as substrate. We also show that bPrP counteracts the inhibition of calmodulin phosphorylation promoted by the regulatory beta subunits of CK2. A truncated form of bPrP encompassing the C-terminal domain (residues 105-242) interacts with CK2 but does not affect its catalytic activity. The opposite is found with the N-terminal fragment of bPrP (residues 25-116), although the stimulation of catalysis is less efficient than with full-size bPrP. These results disclose the potential of the PrP to modulate the activity of CK2, a pleiotropic protein kinase that is particularly abundant in the brain.

Animals↗
PubMed2000

Temperature-dependent functional expression of a plant K(+) channel in mammalian cells.

The Arabidopsis thaliana potassium channel KAT1 was expressed and characterized in Chinese hamster ovary cells. KAT1-GFP fusion protein was successfully targeted to the plasma membrane and electrophysiological analysis revealed functional expression of KAT1 only in cells cultured at 30 degrees C. The main biophysical characteristics of KAT1 are similar to those described for the channel expressed in other systems. CHO cells represent an advantageous expression system and may be the system of choice to study the expression, assembly, function, and regulation of plant potassium channels in general.

Animals↗
PubMed2000

Full length alpha-synuclein is present in cerebrospinal fluid from Parkinson's disease and normal subjects.

Several clues suggest that alpha-synuclein, a presynaptic protein, plays a central role in the pathogenesis of idiopathic Parkinson's disease (PD). To search a peripheral marker of PD, we analyzed presence and amount of alpha-synuclein in CSF from 12 PD patients and 10 neurologically normal subjects. The protein was extracted from CSF samples through immunoprecipitation and immunoblotting with different specific anti-alpha-synuclein antibodies. We identified a 19 kDa band that corresponds to monomeric alpha-synuclein, given its comigration with homologue human recombinant peptide as well as with the protein extracted from cerebral cortex of normal subjects. The amount of CSF 19 kDa alpha-synuclein did not significantly vary in PD and normal cases. These findings have two implications: (a) full length alpha-synuclein is released by neurons in the extracellular space; (b) alpha-synuclein does not appear a peripheral marker of PD pathology.

Biomarkers↗
PubMed2000

Susceptibility of the prion protein to enzymic phosphorylation.

Ten protein kinases have been assayed for their ability to phosphorylate in vitro the recombinant bovine PrP (25-242) (rbPrP). Substantial phosphorylation was observed with PKC, CK2, and two tyrosine kinases, Lyn and c-Fgr. With regard to CK2, phosphorylation occurs at Ser 154 with a stoichiometry of about 0.1 mol phosphate/mol rbPrP, which is doubled by mild heat treatment of rbPrP. Heat also reduces the overall protein ellipticity, suggesting that reversibly unfolded conformers are more susceptible to phosphorylation. Our data disclose the possibility that phosphorylation might modulate PrP biological activity.

Animals↗
PubMed2000

Determination by capillary zone electrophoresis of berenil, phenamidine, diampron and dibromopropamidine in serum and urine.

A quick, simple and reliable analysis method has been developed in order to determine berenil, phenamidine, diampron and dibromopropamidine by capillary zone electrophoresis in samples of serum and urine. In order to define the operation parameters in CZE, we have carried out a study on how the apparent electrophoretic mobility (mu(app)) varies when pH, buffer concentration, voltage and temperature are modified. Ohm's law plot has been studied, too. With the data obtained from this study we have determined the optimum work conditions, which are: citrate buffer 25 mM, pH=3.70, 14 kV, 30 degrees C, wavelength of the UV detector: 200 nm, capillary tube: 570 mm x 75 microm. Under these conditions, all the products appear in times between: 7.6 min phenamidine and 8.8 min dibromopropamidine, limits of detection being: berenil: 0.50, phenamidine: 0.25, diampron: 0.40 and dibromopropamidine: 0.80 microg ml(-1). We have carried out a recovery study with three kinds of extraction cartridges: Sep-pak C-18 plus, Sep-pak C-8 plus and Oasis HBL for each one of the products in blood and urine.

Animals↗
PubMed2000

The immunosuppressant steroid cholesterylphosphoserine inhibits tumour necrosis factor-alpha secretion in vitro and in vivo.

The synthetic steroid cholesterylphosphoserine (CPHS) inhibited the secretion of TNF-alpha in lipopolysaccharide-challenged human monocytes. CPHS (5-20 microM) was effective when added together with the endotoxin, or after an interval (1-2 h) sufficient to have allowed for initiation of TNF-alpha synthesis. Consistently, CPHS did not alter TNF-alpha gene transcription. In contrast to its action on TNF-alpha, CPHS showed only marginal effects on interleukin 1beta secretion. Given intraperitoneally to mice 2 h before lipopolysaccharide CPHS prevented the rise in plasma TNF-alpha (IC(50): 5 mg/kg). The inhibition of TNF-alpha secretion by CPHS may contribute to the immunosuppressive activity of this steroid.

Animals↗
PubMed2000

Heart rate variability in patients with Sjögren's syndrome.

Heart rate variability (HRV) gives information about sympathetic parasympathetic autonomic balance. Our purpose was to determine whether HRV is abnormal in patients with Sjögren's syndrome. In 16 patients with Sjögren's syndrome and 30 matched controls, a short time analysis of HRV was performed for both the frequency and the time domain. In the time domain, patients tended to display a slower heart rate, greater R-R variability and higher standard deviation of the mean (SDNN) than did healthy subjects, but the differences were not statistically significant. In the frequency domain the spectral measures of HRV showed a slight reduction of LF and an increase of HF; as a result, the ratio between high and low frequencies, representative of sympathovagal modulation, was significantly reduced. Our data suggest an increase in the parasympathetic control of heart rate in patients with Sjögren's syndrome. This predominance in vagal tone could exert a protective and antiarrhythmic role in patients with primary Sjögren's syndrome, and may be relevant with reference to the lower incidence of sudden death in this disorder compared to other major autoimmune diseases.

Arrhythmias, Cardiac↗
PubMed1999

cDNA cloning and characterization of PD1: a novel human testicular protein with different expressions in various testiculopathies.

A novel human cDNA sequence has been isolated from human testis cDNA library. This sequence, named PD1, reveals an open reading frame encoding a protein of 520 amino acids. A partial sequence similarity has been found with the RBM gene involved in the regulation of human spermatogenesis. Northern blot analysis for PD1 mRNA from several human tissues demonstrated two distinct transcripts of 2.7 (more abundant) and 4.0 kb and revealed that PD1 is expressed in testis and to a lesser extent also in spleen, thymus, and prostate. Immunohistochemical analysis of human testis showed that this protein is detected in the cytoplasm of Sertoli cells. Antibodies against a rhPD1 fragment were used for Western blot analysis, which confirmed the presence of a 60-kDa molecule in crude extract of human testicular cells obtained from fine-needle aspiration and showed different patterns in various testiculopathies, suggesting a role for such gene in human spermatogenesis.

Amino Acid Sequence↗
PubMed1998

Expression study on D123 gene product: evidence for high positivity in testis.

A novel 44-kDa gene product (D123) has been proposed as necessary for S-phase entry of the cell cycle: a point mutation resulted in a temperature-sensitive arrest in G1-phase. From human fibrosarcoma cDNA library, we have isolated an identical gene and studied its sequence and mRNA and protein expression. Compared with D123, three nucleotide differences within the human coding sequence, plus others, result in a change of two amino acids. A partial sequence similarity has been found with a yeast gene of unknown function. The protein has several potential phosphorylation sites, is highly hydrophilic, and may be highly structured in alpha-helix. The mRNA is abundantly expressed by a variety of normal and transformed cells and by all tissues examined, being most highly expressed in testis. Specific antibodies, raised against a rhD123 polypeptide, recognize a major 42- to 44-kDa molecule in crude extract of various human cell lines. Immunohistochemistry reveals that D123 protein is not homogeneously expressed: it is detected, often in granular vescicles, in the cytoplasm of some epithelial, stromal, and sperm cells and in varicosities lining nervous fibers, while it appears to be absent in nuclei, endothelial, and smooth muscle cells. The precise link between cytoplasmic occurrence of D123 and cell cycle progression still remains to be clarified.

Amino Acid Sequence↗
PubMed1998

Acute effects of intravenous sodium chloride load on calcium metabolism and on parathyroid function in patients with primary aldosteronism compared with subjects with essential hypertension.

To elucidate the mechanisms involved in increased parathyroid function in primary aldosteronism (PA), we evaluated the effects of an intravenous NaCl load on Ca metabolism and plasma level of intact parathyroid hormone (PTH) in patients with PA compared with that in patients with essential hypertension (EH). Sixteen PA patients and 16 EH patients who were well matched for age, gender, body mass index, renal function, and systolic (SBP) and diastolic blood pressure (DBP) were examined. In each subject, after 6 days of a controlled intake of Na, K, and Ca, isotonic saline was infused at a rate of 500 mL/h for 4 h. At baseline, in spite of similar BP values and urinary Na excretion (U[Na]V), urinary excretion of Ca (U[Ca]V) and PTH were higher in the PA group than in the EH group. In both groups, the NaCl load caused a decrease of serum ionized Ca (Ca2+) and an increase in PTH, U(Na)V, and U(Ca)V. However, these changes were significantly greater in the PA group. The increased baseline U(Ca)V in PA could be due to reduced reabsorption of sodium in aldosterone insensitive tubular sites, as a result of the "escape phenomenon." The increased U(Ca)V may explain the higher basal PTH in PA patients, which is needed for maintaining a normal Ca2+. The greater changes in the Ca2+/PTH profile elicited by the saline load in PA patients are apparently due to a higher calciuretic response following a more exaggerated natriuresis in PA.

Adult↗
PubMed1998

Increased plasma levels of platelet-derived growth factor (PDGF-BB + PDGF-AB) in patients with never-treated mild essential hypertension.

Platelet-derived growth factor (PDGF) could play a role in both vascular hypertrophy and atherosclerotic disease associated with hypertension. To assess whether plasma PDGF level is increased in mild essential hypertension, we measured plasma PDGF concentration in 25 never-treated patients with uncomplicated mild essential hypertension and in 22 normotensive healthy subjects. To evaluate the contribution of platelets to plasma PDGF in the two groups, we also measured plasma beta-thromboglobulin (BTG). Measurement of PDGF was carried out through an enzyme-linked immunoadsorbent assay, which detects two PDGF dimers, namely PDGF-BB and PDGF-AB. Both plasma PDGF and BTG were higher in the hypertensive than in the normotensive subjects. The ratio of PDGF to BTG was similar in the two groups. Plasma PDGF was weakly correlated with plasma BTG in the normotensive subjects, whereas this relationship was lost in the hypertensive patients. Our results suggest that the increase in plasma PDGF (PDGF-AB + PDGF-BB) in never-treated essential hypertension is mainly due to platelet activation. The increased circulating level of PDGF could play a role in the vascular structural changes associated with hypertension.

Adult↗
PubMed1998

Neurotrophins rescue cerebellar granule neurons from oxidative stress-mediated apoptotic death: selective involvement of phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathway.

Cerebellar granule neurons maintained in medium containing serum and 25 mM K+ reliably undergo an apoptotic death when switched to serum-free medium with 5 mM K+. New mRNA and protein synthesis and formation of reactive oxygen intermediates are required steps in K+ deprivation-induced apoptosis of these neurons. Here we show that neurotrophins, members of the nerve growth factor gene family, protect from K+/serum deprivation-induced apoptotic death of cerebellar granule neurons in a temporally distinct manner. Switching granule neurons, on day in vitro (DIV) 4, 10, 20, 30, or 40, from high-K+ to low-K+/serum-free medium decreased viability by >50% when measured after 30 h. Treatment of low-K+ granule neurons at DIV 4 with nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3, or neurotrophin-4/5 (NT-4/5) demonstrated concentration-dependent (1-100 ng/ml) protective effects only for BDNF and NT-4/5. Between DIV 10 and 20, K+-deprived granule neurons showed decreasing sensitivity to BDNF and no response to NT-4/5. Cerebellar granule neuron death induced by K+ withdrawal at DIV 30 and 40 was blocked only by neurotrophin-3. BDNF and NT-4/5 also circumvented glutamate-induced oxidative death in DIV 1-2 granule neurons. Granule neuron death caused by K+ withdrawal or glutamate-triggered oxidative stress was, moreover, limited by free radical scavengers like melatonin. Neurotrophin-protective effects, but not those of antioxidants, were blocked by selective inhibitors of phosphatidylinositol 3-kinase or the mitogen-activated protein kinase pathway, depending on the nature of the oxidant stress. These observations indicate that the survival-promoting effects of neurotrophins for central neurons, whose cellular antioxidant defenses are challenged, require activation of distinct signal transduction pathways.

Animals↗