Biomedical subjects
Publications and source records attributed to V Singh.
A case of hypothyroidism presenting with cutaneous vasculitis is described. Apart from typical findings of hypothyroidism, the patient had hypertension (HT). With thyroxine replacement therapy patient showed an improvement in hypothyroidism, HT and cutaneous vasculitis.
Molecular epidemiology of rotavirus infections was studied in children with acute gastroenteritis attending the Nehru Hospital, Chandigarh. A total of 87 stool samples were studied for RNA migration pattern by polyacrylamide gel electropherosis. Only 33 strains could be electropherotyped and two different RNA types 'Short' and 'Long' were observed. Fifteen had a Short RNA pattern and 18 had a Long RNA pattern. There were no intergenomic variations between the Short or Long RNA types. On analysis of subgroup specificity by ELISA using monoclonal antibodies, all Long strains belonged to subgroup 2 and all Short strains to subgroup 1. Our results show that only two electropherotypes were prevalent in the area studied and during a particular peak of infection, only one electropherotype was predominant.
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The cervico-vaginal secretions from 51 women with various grades of dysplastic lesions of uterine cervix were assessed for mutagenic potential by Ames test using histidine deficient mutant strain of Salmonella typhimurium TA-98: with S-9 mix. Twenty three per cent of samples from women with cervical dysplasia were found significantly positive (P less than 0.001) for mutagenic activity compared to 3% positive from control. The frequency of mutagenic secretions detected were almost uniform, irrespective of the severity of cervical lesions. None of cervico-vaginal secretions, positive for mutagenicity could revert the tester strain when tested in absence of S-9 mix (liver microsomal enzymes). This indicates that mutagens in cervico-vaginal secretions are effective only when activated enzymatically.
Coleonol, a diterpine prevented biochemical changes induced by coronary artery ligation in rabbits at a dose of 10 mg/kg, iv. It increased the heart mitochondrial oxygen uptake and O ratio, which may be responsible for the stabilization of heart membrane. The decrease in serum creatine phosphokinase, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase phospholipase and lipid peroxide and increase in cytochrome P450, glycogen and superoxide dismutase activity by coleonol treatment could have contributed to restore myocardial integrity and cardiac function disturbed by coronary artery ligation. The cardioprotective activity of coleonol was found to be comparable to propranolol.
Serum and 24 hours' urinary copper levels were studied in 71 patients with acute viral hepatitis including 35 with encephalopathy. Thirty age and sex matched healthy controls were also studied. Copper estimation was done by atomic absorption spectrophotometry. Serum and 24 hours' urinary copper levels (164.85 +/- 29.31 micrograms/dl and 123.54 +/- 7.87 micrograms/24 h respectively) were significantly (P less than 0.001) increased in acute viral hepatitis patients. There was no significant difference in levels between patients with and without encephalopathy.
On the basis of the observation that in the hormonotoxin oLH-gelonin conjugation of the toxin occurs via the alpha-subunit of lutropin, an attempt was made to develop a general method for generation of similar hybrid proteins involving other glycoprotein hormones. In this approach sites suitable for conjugation would be first introduced into the alpha-subunit, hybridized with any native hormone specific beta-subunit of choice (LH-beta, FSH-beta, TSH-beta) following which the toxic component gelonin would be added on in the form of gelonin-S-S-alpha---beta complex. Thus, thiolated lutropin alpha-subunit recombined well with free lutropin beta-subunit, yielding a hybrid which was active in terms of receptor binding, immunoreactivity, and steroidogenic properties. However, subsequent conjugation with thiolated gelonin, a ribosome inactivating protein, resulted in dissociation of beta-subunit from its non-covalent union with the thiolated alpha-subunit. It is concluded that the addition of positively charged gelonin at particular sites on the alpha-subunit led to the destabilization of the lutropin quaternary structure.
The product of protein/creatinine ratio (in a random urine specimen) and estimated daily urinary creatinine excretion were evaluated as a function of 24 h urinary protein excretion in 40 patients with pathology proteinuria having varying grades of renal function. An excellent correlation was found between the results obtained by this method and those from the standard 24 h urine collection method in healthy controls (r = 0.99) and in patients with normal (r = 0.88) or mild to moderately impaired renal function (r = 0.99). However, this method did not give a good measure of quantitative proteinuria in patients with advanced renal failure (r = 0.56), possibly due to decreased urinary excretion of creatinine in patients with advanced renal failure as compared to the estimated value. The product of protein/creatinine ratio and estimated daily urinary creatinine excretion thus gave a quick and alternate reliable method of estimating 24 h proteinuria in patients having normal or mild to moderately impaired renal function. However, this method is not suitable for patients with advanced renal failure.
The chromosomal complements of five species of mammals, Mus musculus (Linn.), Rattus rattus rattus (Linn.), Bandicota bengalensis (Gray and Hardw.), Funambulus pennanti (Wr.) and Lepus ruficaudatus (Geoff.) were elaborated. The karyotypes of all the species were compared. The diploid number (2n) in Mus is 40, Rattus 42, Bandicota 42, Funambulus 54 and Lepus 48. An extra large X-chromosome was observed in one population of male Rattus rattus rattus (Linn.), which might have been formed as a result of duplication of the terminal segments of both the arms of the chromosomes.
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Gelonin purified from the seeds of Gelonium multiflorum using cation-exchange and gel-filtration chromatography was characterized for its purity, homogeneity and Mr by reverse-phase h.p.l.c. and SDS/polyacrylamide-gel electrophoresis analysis and judged to be 98% pure. As the cross-linking agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) has been used for linking gelonin via its epsilon-NH2 group to its carrier antibodies or hormones for immunotoxin or hormonotoxin respectively, an attempt was made to study the effect of this modification of gelonin on its immunoreactivity. A radioimmunoassay was developed for this purpose. By sequential modification, four categories of amino group modifications on immunoreactivity were observed. Even one or two modifications, representing one-twentieth to one-tenth of available epsilon-NH2 groups in the protein caused about 75% loss in immunoreactivity, with additional reactions contributing to further deteriorations. By using a gelonin radioimmunoassay, the immunoreactivity of gelonin in three hormonotoxins was determined with gelonin and modified gelonin as standards. The gelonin equivalent in our hormonotoxins was in agreement with the values determined by spectrophotometric and gel-electrophoresis methods. As the immunoreactivity of gelonin-SPDP was not further altered after conjugation to its carrier protein ovine lutropin, a specific radioimmunoassay of gelonin could be used to evaluate the molar ratio of the conjugates prepared by using SPDP as cross-linker and gelonin-SPDP as a standard.
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With the aim of targeting toxins to selected cells in the gonad, we have prepared conjugates of ovine luteinizing hormone (oLH) with a single chain ribosome-inactivating protein called gelonin. The two proteins were thiolated by using N-succinimidyl-3-(2-pyridyldithio)propionate and subsequently reacted under appropriate conditions to form oLH-S-S-gelonin complex. A complete biochemical analysis of thiolated oLH and oLH-gelonin conjugates has been performed. The linkage of the hormone to the toxin probably occurred through a single amino group in the alpha-subunit, with the beta-subunit remaining free. Modification of a single amino group on the alpha-subunit reduced receptor binding and immunological reactivity of the thiolated oLH, but subsequent complexing with the toxin-gelonin did not seriously compromise these activities. oLH and gelonin were calculated to be present in a 1:1 ratio in the hormonotoxin preparation. The conjugate retained significant steroidogenic activity in rat granulosa cells. Upon reaction with mouse tumor Leydig cells (MA-10 cells), the toxin component of the complex became internalized to a sufficient degree to effectively inhibit protein synthesis. The studies provide a rational basis for the design and study of large hormonotoxins.
We have studied the reaction of epsilon-amino groups of ovine lutropin with N-succinimidyl-3-(2-pyridyl-dithio) propionate (SPDP) and observed that modification of up to 1/3 of all available residues primarily occurs in the alpha-subunit. The 12 lysines available could be classified into three categories, as highly, moderately and less accessible. The three lysine residues of the hormone that appeared to resist modification may be in the intersubunit contact region. Sequential modifications of lysine residue in the alpha-subunit led to progressive reduction in receptor binding and immunological activities, but not in full steroidogenic potential. The discrepancy between reduced receptor binding and full steroidogenic activity may be related to the introduction of addition hydrophobicity to the ovine lutropin molecule. Total inactivation resulted only when the majority (9/12) of available amino groups were altered by this reaction. Thus, our study shows that by a limited reaction it is possible to create conjugation site(s) in ovine lutropin with significant retention of hormonal activity.
The properties of two hormonotoxins, human choriogonadotropin and ovine pituitary lutropin, each conjugated to the ribosome inactivating protein gelonin, have been compared. In both cases the linkage is effected through a disulfide bond provided by the cross-linker N-succinimidyl-3-(2-pyridyldithio)propionate. In each conjugate the hormone and toxin were calculated to be present in a ratio of 1:1. Immunological reactivity, receptor binding activity and steroidogenic activity of the hormonotoxins in radioimmunoassay or in membranes or cells containing LH receptors were reduced but not abolished in comparison with the thiolated hormones. Purified hormonotoxins were characterized by immunoassays specific for both interaction components, viz. the hormone (LH/hCG) and gelonin. Binding of the toxic component (gelonin) to the mouse Leydig tumour cells (MA-10 cells) was shown to occur via the hormone part of the hormonotoxins. This was effectively competed by the native hormone but not free gelonin. Both hormonotoxins were cytotoxic to MA-10 cells as protein synthesis was inhibited in their presence. This was associated with destruction of tumor cells in culture.
Nomadic tribes and pastoral communities dwelling in the North-West and Trans-Himalaya are reputed to have mastered their traditional practices and knowledge about plants used to combat different diseases of their livestock. Ethnobotanical explorations carried out during the past 3 years have brought to light 18 plant species which have been used to alleviate the common sufferings of livestock in this region. The botanical identity of these plants is given along with their local names, methods of preparation and administration.
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The amino groups in the beta-subunit of ovine luteinizing hormone (oLH) were modified by thiolation using N-succinimidyl-3-(2-pyridyldithio) propionate so that it may be coupled in a disulfide linkage to similarly modified ribosome inactivating protein, gelonin. The modified beta-subunit was able to hybridize with free LH alpha-subunit and the complex retained full biological activity. However, when gelonin was coupled to the beta-subunit, the resulting conformational changes masked or eliminated the sites necessary for intersubunit recognition of the free alpha-subunit. This has important implications for the design in the synthesis of gonadotropin-toxin/drug conjugates.