[Characterization of mycobacterial antigens of sea lions].
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Animals↗
Biomedical subjects
F Bigi
Publications and source records attributed to F Bigi.
Molecular epidemiology of Mycobacterium bovis isolates from South America.
One hundred seventy-eight isolates of Mycobacterium bovis were subjected to DNA restriction fragment length polymorphism (RFLP) analysis, using the direct repeat element (DR) and the polymorphic GC-rich repeat sequence (PGRS) as probes. By combining the patterns generated by the two repeat DNA elements, 93 different patterns were observed. One hundred-one isolates were grouped in clusters, which include 25 different clusters. One pattern was the most frequently observed, clustering 18.5% of isolates. It was only found in the Center and northeast regions of Argentina and in one isolate from Paraguay. The isolates from Brazil analyzed here presented exclusive patterns (only found in a particular region). The number of exclusive patterns was high in all argentine regions: northeast 78%, center 81%, and Buenos Aires 81%.
Animals↗
An mpb-64 flanking sequence specific for Mycobacterium bovis.
A clone carrying a plasmid with the mpb-64 gene and 3' flanking sequences (plasmid pMBA122) was detected during the screening of a Mycobacterium bovis genomic library with sera from infected cattle. When the pMBA122 insert was used as a probe in Southern blots against PvuII-digested mycobacterial DNA, it distinguished the different M. tuberculosis complex species. This probe hybridized with a 7-kb band in M. tuberculosis, a 5-kb band in M. bovis and a 3-kb band in M. tuberculosis complex strains from wild seals. Smal genomic digestions enabled us to locate this polymorphic region downstream of the mpb-64 gene. In order to clone this particular region, we designed a pair of PCR primers. Unexpectedly, these primers amplified only M. bovis DNA; no amplification was seen in M. tuberculosis DNA. When the annealing temperature was lowered from 70 to 55 degrees C, an amplification product of the same size was obtained with M. tuberculosis. This product was cloned and sequenced, and showed partial homology to the M. bovis amplified fragment. Therefore, this region comprises M. bovis sequences with a lower homology with M. tuberculosis than other compared sequences. This suggests that a more precise differentiation method at the species level for the M. tuberculosis complex could be achieved using PCR directed to this region.
Animals↗
A novel 27 kDa lipoprotein antigen from Mycobacterium bovis.
A novel Mycobacterium bovis antigen was identified from an expression library using sera from naturally infected cattle. The Escherichia coli recombinant clone expressed a 27 kDa protein, named P27. A rabbit serum against the recombinant antigen recognized a protein of 27 kDa in cellular extracts from M. bovis and M. tuberculosis. No protein was recognized in the culture supernatant. Sequence analysis indicated that P27 has a molecular mass of 24 kDa, showing a characteristic signal sequence for lipoprotein modification (a signal peptidase type II site). The gene is identical to a gene identified in the M. tuberculosis genome sequencing project. Cellular fractionation experiments suggested that P27 is an integral membrane protein. The antigen was recognized by individual sera and peripheral blood mononuclear cells (PBMC) from diseased cattle. PCR experiments with specific primers directed to the P27 structural gene indicated that it is only present in the M. tuberculosis species complex. In conclusion, a novel immunogenic lipoprotein in M. bovis/M. tuberculosis has been identified. The results presented here and elsewhere suggest that mycobacterial lipoproteins should be considered in the design of new recombinant vaccines and diagnostic methods.
Amino Acid Sequence↗
Comparison of different genetic markers for molecular epidemiology of bovine tuberculosis.
In the present study three different genetic markers were used in an RFLP study to differentiate Mycobacterium bovis (M. bovis) isolated in Argentina. The markers were: the insertion sequence IS6110, the direct repeat (DR) sequence flanking IS6110, and a polymorphic GC-rich repetitive sequence (PGRS), called pMBA2. Two restriction enzymes were used, PvuII for IS6110 and DR (DR/PvuII) and AluI for DR (DR/AluI) and pMBA2. DNA from 85 of M. bovis isolates was digested with PvuII and hybridized with IS6110 and Dr. IS6110 was not useful to differentiate M. bovis because most of the isolates contain a single monomorphic copy. The use of DR allowed a limited degree of differentiation. DNA from 44 of these isolates was also digested with AluI and hybridized with DR and pMBA2. In this condition these probes differentiated the isolates in many different RFLP types. By combining the patterns generated with DR/AluI and pMBA2 it was possible to increase the differentiation up to obtain 30 different RFLP types and 54% of the isolates were differentiated because they showed a unique pattern. Six isolates of M. bovis involved in two different outbreaks of bovine tuberculosis were correctly identified. Thus, DR and pMBA2 could be, at the moment, the probes of choice for comparisons of M. bovis isolates in different regions and for epidemiological surveillance of bovine tuberculosis.
Animals↗
Genetic characterization of mycobacteria from South American wild seals.
Tuberculosis has been recently diagnosed in four wild seals found stranded in the Atlantic coast of Argentina. By bacteriological studies and IS6110 hybridization, these isolates were characterized as belonging to the Mycobacterium tuberculosis complex. A genetic characterization using RFLP (Restriction fragment length polymorphism) and a species-specific probe of M. tuberculosis, called mtp40, showed hybridization with this probe on a single band. A similar band was also found in M. tuberculosis H37Rv. This showed a relationship between M. tuberculosis and the wild seal isolates. However these would also seem to belong to a different genetic group in the M. tuberculosis complex, since they do not grow on glycerol-egg containing medium (Lowenstein-Jensen) as typical M. tuberculosis strains usually do. Repeated sequences pMBA2, pTNB12, DR and IS6110 were used as probes to evaluate the epidemiological relationships between the 4 cases of tuberculosis. A low degree of polymorphism was observed, that suggested that these isolates were epidemiologically related.
Animals↗
Cloning of a novel polymorphic GC-rich repetitive DNA from Mycobacterium bovis.
A repetitive DNA from a wild-type Mycobacterium bovis isolate was cloned and characterized. The repeated segment was present in M. bovis and M. tuberculosis but was absent from the six other mycobacteria tested. Sequence analysis demonstrated that this repetitive element belonged to the polymorphic GC-rich repeat sequence type, a family of interspersed repeated DNA. This fragment, when used as a probe in restriction fragment length polymorphism analyses, was able to detect polymorphism in M. bovis genotypes that went undetected when the established IS6110 was used as a probe. This repetitive element should be useful in epidemiological studies of bovine tuberculosis.
Base Sequence↗
Characterization of a novel Mycobacterium bovis secreted antigen containing PGLTS repeats.
Serum from naturally infected cattle was used to identify a novel Mycobacterium bovis antigen from an expression library. The first recombinant product identified was a fusion protein with lacZ (55 kDa). A clone containing the whole gene was also obtained. This clone expressed a 38-kDa protein. A rabbit serum against the recombinant antigen reacts in M. bovis supernatants with two proteins of 36 and 34 kDa. The new protein was called P36/P34. The gene cloned has a deduced amino acid sequence with a predicted molecular mass of 28 kDa, showing a characteristic signal sequence for exportation. The protein bears partial homology to a 28-kDa protein from M. leprae. An interesting feature of the P36/P34 sequence is that it contains several PGLTS repeats, which are not present in the M. leprae protein. Antigenic determinants seem also to be conserved between the two proteins because sera from leprosy patients recognized the recombinant M. bovis protein. The discrepancy among the molecular mass deduced from the sequence (28 kDa), that of the recombinant protein in Escherichia coli (38 kDa), and that of the native protein in M. bovis (36 and 34 kDa) could be attributed to posttranslational modifications or to the high proline content that may alter the migration properties of the protein. This antigen seems to be immunodominant during bovine tuberculosis, because 8 of 9 serum specimens from diseased cattle are reactive. The homology among the M. leprae 28-kDa protein, the protein described in this article, and a recently described M. tuberculosis protein suggests the existence of a new protein family in mycobacteria.
Amino Acid Sequence↗
Two-site assay of intact parathyroid hormone in primary hyperparathyroidism: studies in basal conditions, following adenoma removal and during calcium and EDTA infusion.
This study has been carried out in order to investigate parathyroid hormone secretion in patients with primary hyperparathyroidism in basal conditions, during stimulation and suppression tests and following successful surgery. Parathyroid gland secretory activity has been evaluated by a highly sensitive immunoradiometric assay (IRMA) which detects only the biologically intact active hormone and with a well established midmolecule (MM) PTH RIA. There was a good correlation between the two assays in basal state (r = 0.779); however the correlation found between serum PTH levels and total calcium values was better for the intact hormone (P less than 0.001) than for the radioimmunoassay (P less than 0.05). Twenty-four hours following surgery, serum intact PTH levels were in all patients less than 10 pg/ml while midmolecule PTH was still detectable, thereafter remaining at a higher level during the next six days. Serum IRMA PTH levels fell rapidly in response to the increase in serum calcium, then there was a trend to reach a plateau; serum midregion PTH levels fell, although slower than those of intact hormone. The percent increase obtained for serum intact hormone levels was higher than that observed for MM RIA, following EDTA stimulation. The results obtained indicate that the assays of intact and midmolecule parathyroid hormone clearly reflect different aspects of hormone metabolism 'in vivo' and may prove therefore to be useful for its investigation in various calcium disorders.
Adenoma↗
Studies on the clinical significance of serum alpha 2 HS-glycoprotein measurement in metabolic bone diseases.
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Adult↗
Effects of salmon calcitonin on the bone loss induced by ovariectomy.
We present the results of a 12-month clinical study assessing the effects of synthetic salmon calcitonin (sCT) on a group of fertile white women who had undergone ovariectomy for uterine fibromatosis. The study was performed to verify whether CT can prevent the loss of bone mass and the changes in calcium-phosphorus metabolism associated with acute estrogen deficiency. The study consisted of an initial double-blind phase of 6 months, followed by a 6-month open period. Treated patients were given 100 MRC U of synthetic salmon CT injected i.m. in the morning, every other day, starting on the 7th day after the operation and continued for 12 months. Control patients received a placebo injection for the first 6 months; subsequently, they too were treated with sCT i.m. every other day for 6 months at the same dose as the 12 month-treated group. All patients received 500 mg of elementary calcium p.o., b.i.d. Bone mineral content (BMC) was measured at the extreme distal radius of the nondominant arm by a dual photon bone densitometer which utilizes two radio nuclides, 241 Am and 125 I, with energies of about 60 and 30 KeV. Biochemical parameters of the calcium-phosphorus metabolism were also measured. After 12 months of study, no significant changes of BMC were observed in the 12 months sCT treated group, while control patients, treated 6 months after the ovariectomy, showed a significant decrease in BMC values.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Acute hypocalcemia enhances prolactin levels: a study before and after oophorectomy.
The effect of acute hypocalcemia on serum prolactin values was investigated in 13 female subjects before and after oophorectomy. An infusion of disodium-EDTA (50 mg/kg b.w., for 120 min) was administered to each patient. Ionized calcium, immunoreactive parathyroid hormone and prolactin serum levels were determined 15 min prior to the infusion, at the beginning of each infusion and thereafter at every 30 min interval. The decrease in serum ionized calcium induced by Na2-EDTA infusion was not influenced by surgery. Serum prolactin and parathyroid hormone values before and after oophorectomy showed a significant (p less than 0.01) increase at 90 and 120 min. The maximum serum prolactin increase was achieved at 120 min and was not influenced by oophorectomy. The results demonstrate that acute hypocalcemia increases serum prolactin levels in female subjects and suggest a possible role for parathyroid hormone and dopaminergic system, even if their interaction remains speculative.
Adult↗
Pathogenetic aspects of involutional osteoporosis.
The pathogenetic factors of postmenopausal and senile osteoporosis are reviewed. Both postmenopausal and senile osteoporosis occur as a result of a defective regulation of bone remodelling which leads to a negative uncoupling between bone resorption and bone formation. Systemic and local factors contribute to the development of the phenomenon. While studies of the systemic factors involved in age-related bone loss are well advanced, although still incomplete, the study of the local factors responsible at a tissue level for the negative skeletal balance has barely begun.
Adult↗
Clinical value of the measurement of bone remodelling markers in primary hyperparathyroidism.
This study was performed in order to evaluate the clinical usefulness of the measurement of total serum alkaline phosphatase activity (AP), serum osteocalcin (BGP) and urinary hydroxyproline (OHPr) in assessing bone remodelling in primary hyperparathyroidism. Thirty-two patients with primary hyperparathyroidism were included in the study. No statistically significant differences were observed between the mean values of Z-scores obtained for each marker. Furthermore, an inverse correlation was found between percentage of bone mineral content at the distal radius and both BGP (r = -0.57; p less than 0.05) and AP (r = -0.49; p less than 0.05). The results obtained demonstrate that, contrary to other metabolic bone diseases (e.g. Paget's disease of bone), all three markers examined may be used in clinical practice to evaluate the entity of skeletal turnover in primary hyperparathyroidism.
Adult↗
Studies on citrate metabolism in normal subjects and kidney stone patients.
The study was carried out in order to investigate citrate metabolism (i.e. serum levels, filtered load, tubular reabsorption, total and fasting urinary excretion) in a large group of normal subjects (n = 39) and kidney stone patients (n = 45). We found that mean serum citrate levels and filtered loads were similar in the two groups studied. On the other hand, both the 24-hour urinary citrate and the fasting citrate/creatinine ratio were significantly reduced, whereas the mean tubular reabsorption of citrate appeared to be significantly increased in kidney stone patients in respect to control subjects. A good correlation was found between the fasting and the 24-hour citrate/creatinine ratio in both normal subjects (r = 0.84, p less than 0.001) and kidney stone patients (r = 0.89, p less than 0.001). Our data indicate that the kidney appears to be involved in the pathogenesis of hypocitraturia in kidney stone patients and that a substantial proportion of these patients show a reduced urinary excretion of citrate. Finally, the evaluation of the fasting citrate/creatinine ratio may replace the measurement of the substance on the basis of a 24-hour urine collection.
Adolescent↗
Treatment for mild hyperparathyroidism.
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Humans↗
Serum osteocalcin in primary hyperparathyroidism: short-term effect of surgery.
This study has been carried out in order to evaluate both serum osteocalcin levels in primary hyperparathyroidism and their changes following surgery. Twenty-one consecutive patients were studied (12 females and 9 males, aged 46 +/- 17 years). Preoperatively, a better correlation was found between serum osteocalcin and serum alkaline phosphatase activity (r = 0.79, p less than 0.001) than between serum osteocalcin and the 24-hour urinary hydroxyproline/creatine ratio (r = 0.55, p less than 0.05). Following the surgical removal of hyperfunctioning parathyroid tissue, a modest but significant decrease was observed in the serum levels of osteocalcin; this reached a nadir during the 1st or 2nd day after the removal of the adenoma. The mean levels then tended to rise, so that the values measured on the 7th day after parathyroidectomy (12.4 +/- 2.5 ng/ml) were not significantly different in respect to basal values (13.6 +/- 2.7 ng/ml). A parallel pattern was also noted as concerns the serum alkaline phosphatase activity. On the contrary, mean values of serum immunoreactive parathyroid hormone (243 +/- 78 vs. 58 +/- 11 pmol/l; p less than 0.02) and serum calcium (12.4 +/- 0.5 vs. 9.2 +/- 0.3 mg/dl; p less than 0.01) were significantly reduced and mean values of serum phosphorus (2.4 +/- 0.2 vs. 3.1 +/- 0.2 mg/dl; p less than 0.001) significantly higher in comparison to basal values.(ABSTRACT TRUNCATED AT 250 WORDS)
1-Carboxyglutamic Acid↗
Citric acid and calcium nephrolithiasis.
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Absorption↗