Determination of urinary urate with alkylamine glass-bound uricase.
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Amines↗
Biomedical subjects
A K Bhargava
Publications and source records attributed to A K Bhargava.
Discrete analysis of serum uric acid with immobilized uricase and peroxidase.
Commercially available uricase and peroxidase have been immobilized onto alkylamine glass and arylamine glass beads respectively. A discrete method has been developed to determine uric acid in serum using immobilized uricase and peroxidase. The method is based on generation of H2O2 from serum uric acid by immobilized uricase and its measurement by a colour reaction catalyzed by immobilized peroxidase. The minimum detection limit of the method was 8 microg/0.1 ml sample. The mean analytical recovery of added uric acid in serum was 87.5%. The within and between assay coefficient of variation (C.V.) were <6.58% and <10.77% respectively. The serum uric acid in apparently healthy adults and persons suffering from different disease was found to be 25-55 microg/ml, 32+/-2.25 (range, mean+/-S.D.) and 55-200 microg/ml; 52+/-6.4 (range, mean+/-S.D.) respectively by our method. A good correlation (r = 0.8170) was obtained between the serum urate values by this method and with those obtained by commercial Enzo-kit method.
Colorimetry↗
Parotid tuberculosis simulating malignancy.
An interesting case of parotid tumour simulating malignancy is reported. The rarity of this lesion and the associated clinical and diagnostic problems are emphasized together with the relevant literature.
Adult↗
Immobilization of sorghum leaf oxalate oxidase onto alkylamine and arylamine glass.
An oxalate oxidase (EC 1.2.3.4) purified from grain sorghum leaves was immobilized onto alkylamine and arylamine glass beads through glutaraldehyde coupling and diazotization with a conjugation yield of 10.8 mg/g support and 9.2 mg/g support, respectively. The enzyme retained 67.5% and 34.1% of its initial specific activity after immobilization onto alkylamine and arylamine glass, respectively. The enzyme exhibited an increase in optimum pH, temperature for maximum activity, energy of activation (Ea) and time for linearity but decrease in thermal stability at 60 degrees C after immobilization on both types of beads. The K(m) value for oxalate was increased by 9- to 10-fold but Vmax remained unaltered after immobilization. Both alkyl and arylamine glass bound enzyme was unaffected by physiological concentrations of Cl- and NO3-. The analytic importance of this work is demonstrated.
Adult↗
Immobilization of Amaranthus leaf oxalate oxidase on alkylamine glass.
A membrane bound oxalate oxidase from leaves of Amaranthus spionsus has been partially purified and immobilized on alkylamine glass with a yield of 9.2 mg protein/g support. The enzyme retained 99.4% of initial activity of free enzyme after immobilization. There was no change in the optimum pH (3.5) and Vmax but the temperature for maximum activity was slightly decreased (35 degrees C) and energy of activation (Ea) and Km for oxalate were increased after immobilization. The immobilized enzyme preparation was stable for 6 months, when stored in distilled water at 4 degrees C. Presence of Cl- did not affect the activity of immobilized enzyme.
Adult↗
Determination of urinary oxalate with oxalate oxidase and peroxidase immobilized on to glass beads.
We have reported the immobilization of barley oxalate oxidase on to alkylamine glass beads through glutaraldehyde coupling (Pundir, C. S., Satyapal and Kuchhal, N. K. (1993) Clinical Chemistry 39, 1750-1751). The present report describes the immobilization of commercially available horseradish peroxidase on to zirconia-coated arylamine glass beads through diazotization and a new method for the discrete assay of urinary oxalate using both immobilized oxalate oxidase and peroxidase. In the method, urinary oxalate is precipitated with CaCl2, redissolved in HCl and then assayed using immobilized enzymes. The oxalate in 24 h urine samples from apparently healthy male adults was measured by this method and found to be in the range of 12.2-28.0 mg with a mean of 19.8 mg. The percentage recovery of added oxalate (17.5 mg/l) was 96.7 +/- 3.4 (mean +/- SD). The mean value of urinary oxalate by our method is comparable with those obtained by the Sigma kit method. The cost of oxalate determination in 100 urine samples by the present method has been compared with that of the Sigma kit method.
Biochemistry↗
Chemical analysis of urinary calculi in Haryana.
Quantitative chemical analysis of 225 urinary calculi (128 Renal, 57 bladder and 40 ureter) collected from hospitals of different districts of Haryana was carried out. CaOxM was found to be present as a major component in all the stones analysed, while MAP, HA, CA and UA were found in 83.7%, 83.6%, 13% and 78.9% cases, respectively. The content of CaOxM was found to be higher in renal stones as compared to ureter and bladder stones, while the concentration of MAP was higher in bladder stones as compared to kidney and ureter stones. HA content was higher in ureter and UA was higher in bladder stones as compared to other urinary stones.
Adolescent↗
Isolation of human ear specific cDNAs and construction of cDNA libraries from surgically removed small amounts of inner ear tissues.
We have used representational difference analysis (RDA) for subtractive hybridization of oligo dT primed directionally cloned cDNA libraries from human inner ear tissue and a B-lymphoblast cell line. Two rounds of subtraction-amplification, followed by differential hybridization of selected clones led to the isolation of genes which were specific to the ear. Sequence analysis of randomly chosen clones revealed the presence of a histidine rich Ca2+ binding protein, human dynamin, collagen type 1A1, collagen type 2A1, SPARC, human growth hormone, and several specific genes which had no sequence homology in the data base. Furthermore, to apply these techniques for isolating genes specific to distinct inner ear structures and/or cell types of inner ear for which the starting tissue material is limiting, we have used a modified PCR based protocol to construct representative cDNA libraries. We have characterized a cDNA library constructed from small amounts of inner ear tissues recovered by ablative surgical procedure involving labyrinthectomy. The potential application of these protocols for isolating genes involved in hearing and deafness is discussed.
Adult↗
Expression of protein kinase regulator genes in human ear and cloning of a gamma subtype of the 14-3-3 family of proteins.
We have used oligonucleotides corresponding to conserved regions of protein kinase regulators of 14-3-3 gene family as primers to amplify these genes from cDNAs constructed from the human fetal inner ear. Sequence characterization of clones revealed that the 14-3-3 cDNA library from the fetal inner ear has high abundance of clones encoding a protein kinase regulator (theta subtype), a member of 14-3-3 gene family, and relatively lower abundance of clones for two other members of the gene family. One of these genes is identical to the eta subtype of human 14-3-3 genes; there is no cloned gene for the other subtype in the human 14-3-3 gene family in the nucleic acid data bases. A sequence homology search revealed that the latter shared significant homology with the gamma subtype of the rat 14-3-3 family. On the basis of the sequence data, it appears that this clone represents a human homolog of the rat gamma subtype. The results demonstrate the expression of 14-3-3 genes in the inner ear, characterize a human homolog of the rat gamma subtype of 14-3-3, implicate these proteins in ear development, and indicate the utility of gene family polymerase chain reaction (PCR) for investigating gene expression in specific tissues.
14-3-3 Proteins↗
Localized neurofibromatosis associated with chronic post-mastectomy lymphoedema--a case report.
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Axilla↗
Uniform amplification of a mixture of deoxyribonucleic acids with varying GC content.
A PCR method for uniform amplification of a mixture of DNA templates differing in GC content is described using the two enzyme approach (Klentaq1 and Pfu DNA polymerase) and a combination of DMSO and betaine. This method was applied to amplify the CGG repeat region from the fragile X region.
Base Composition↗
Differential expression of four members of the POU family of proteins in activated and phorbol 12-myristate 13-acetate-treated Jurkat T cells.
The POU family of proteins binds specifically to octamer DNA motifs present in the promoters of several genes and regulates their expression. We identified the presence of four members of the POU family of proteins, Oct-1, Oct-2, Oct-T1, and Oct-T2, in the human T-cell line Jurkat. To obtain insight into the physiological role played by these proteins in T cells, we studied the time course of expression of these genes in resting, activated, and phorbol 12-myristate 13-acetate (PMA)-treated cells. The expression of the gene encoding Oct-1 (now assigned the name OTF1 for octamer-binding transcription factor 1) remained unchanged and the levels of Oct-T2 mRNA decreased with increasing time of incubation to undetectable amounts in all three states of T-cell growth. The levels of Oct-2 mRNA and protein were increased in activated cells, were increased to a lesser extent in the PMA-treated cells, and were undetectable in resting cells. The levels of the Oct-T1 transcripts increased dramatically in PMA-treated cells but not in resting or activated cells. Sequence analysis of the Oct-T1 cDNA showed an open reading frame coding for a POU domain-containing protein of 42.7 kDa. Transient transfection of the gene encoding Oct-T1 decreased the activity of the interleukin 2 gene promoter in activated Jurkat cells. Further, there is evidence for an additional octamer-binding protein, Oct-T3, in Jurkat T cells.
Amino Acid Sequence↗
Salivary duct carcinoma.
Salivary duct carcinoma is an uncommon malignant tumor that occurs mainly in the parotid gland of elderly men. The 11 cases of salivary duct carcinoma which are included in this study occurred in older men (mean age 56 years) and were located in the parotid (7), submandibular salivary gland (2), and the minor salivary glands in the maxilla (2). The maximum tumor dimension ranged from 3 to 9 cm. Microscopically, all had infiltrating margins, with circumscribed groups of epithelial cells arranged in various patterns; the invasive component was embedded in a desmoplastic stroma. Perineural invasion and lymph node metastasis were noted in seven and three cases, respectively, at the time of initial surgery. Radical surgery was offered to ten patients and postoperative radiotherapy to nine patients. Salivary duct carcinoma appears to be an aggressive tumor with distinctive histological features, which has not been described in the minor salivary glands of the maxilla to date. The clinicopathologic features of these tumors are presented, with a review of the literature.
Adenocarcinoma↗
Scalp flap--a utility and reconstructive option for head and neck surgeons.
Regional scalp flaps are invaluable in resurfacing defects resulting from resections in the area of the upper face, orbit and scalp itself. The tissue lies adjacent to the defect, is easy to harvest, and can be termed as a 'durable one-stage' procedure. Scalp defects posterior to the vertex lend themselves to resurfacing by a posterior flap based on the occipital arteriovenous system. Anterior scalp defects including upper face and orbit can be resurfaced by an anterior scalp flap based on the superficial temporal arteriovenous system. While large areas can be resurfaced and the donor site effectively camouflaged, this flap finds less of an acceptance amongst patients with defects in the upper facial region when compared to the scalp defects. We recount our experience with these flaps and describe the high points of the reconstructive procedure and the results obtained in ten consecutive cases carried out over a three year period at the Department of Head and Neck Surgery, Kidwai Memorial Institute of Oncology, Bangalore, India. The short healing time and minimal morbidity make this reconstructive option an attractive one for the practising Head and Neck Surgeon.
Carcinoma, Squamous Cell↗
Clinical spectrum of myasthenia gravis and problems of management.
Seventeen cases of myasthenia gravis, aged 20-79 years, including one case of ocular myasthenia gravis, seen over a 3 year period, were analysed. Dysphagia was the presenting symptom in two cases. One patient had ectopic thymus gland in relation to the cardiac border, which posed difficulty in diagnosis. A majority of the patients were on medical treatment, including neostigmine, prednisolone and azathioprim. Plasmapheresis was done in 4 cases during crisis, with significant benefit. One patient was put on intermittent ventilatory support for 14-16 hours a day for over 2 years; she died during a power failure. Seven patients were subjected to thymectomy.
Adult↗
Cloning and cDNA sequence of a bovine submaxillary gland mucin-like protein containing two distinct domains.
A lambda gt11 cDNA library prepared from bovine submaxillary gland mRNA was screened with polyclonal anti-apo-bovine submaxillary mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core protein. One of the positive clones had a 1.8 kilobase (kb) cDNA insert and coded for an incomplete protein. A 2.0-kb cDNA clone was isolated by rescreening the library with the 1.8-kb cDNA. Nucleotide sequencing of the full-length 2.0-kb cDNA revealed an open reading frame that coded for a 563-amino acid protein. A striking feature of the cloned protein is the skewed distribution of the amino acids, most notably that of the hydroxy amino acids and cysteine. The amino-terminal domain of 339 residues is very rich in threonine, serine, and glycine and poor in cysteine, aspartic acid, tyrosine, phenylalanine, and tryptophan. In contrast, the carboxyl-terminal domain of 224 residues is rich in cysteine, aspartic acid, tyrosine, lysine, and asparagine and relatively poor in threonine, serine, and glycine. A search of the protein data bank for homologies to the deduced amino acid sequence revealed statistically significant matches to several proteins, including the porcine submaxillary apomucin fragment. The cysteine-rich domain by itself was not statistically homologous with any of the registered polypeptide sequences. RNA blot analysis using DNA probes corresponding to the mucin-like and cysteine-rich regions detected a nearly identical pattern of transcripts, demonstrating that the characterized clones are not artifacts of cDNA library construction. The blots also showed the presence of polydisperse transcripts in bovine submaxillary gland but no detectable hybridization signals in liver or brain RNA.
Amino Acid Sequence↗
Expression of human globin genes in transgenic mice carrying the beta-globin gene cluster with a mutation causing G gamma beta + hereditary persistence of fetal hemoglobin.
We have introduced into the mouse germ line the 40-kilobase (kb) Kpn I fragment containing the beta-globin gene cluster from an individual with a non-deletion form of hereditary persistence of fetal hemoglobin (HPFH) believed to be due to a point mutation at position -202 of the G gamma-globin gene. The G gamma-globin gene, as well as the beta-globin gene, was expressed in adult erythroid tissues of the resulting transgenic mice. The level of expression of the G gamma-globin gene was about 20% of that of the beta-globin gene. Others have previously shown that cloned individual normal human beta- and gamma-globin genes containing a limited amount of 5'- and 3'-flanking DNA are expressed in a manner similar to that of their corresponding murine homologs during development in transgenic mice. In contrast, we have observed that the pattern of expression of the normal (non-mutated) A gamma- and beta-globin genes in the 40-kb insert was different from that of their corresponding murine homologs. The beta-globin gene remained inactive at the fetal stage, whereas the normal A gamma-globin gene was expressed beyond the embryonic (yolk sac) stage into the fetal stage of development and then became inactive in adult erythroid cells. The pattern of expression of the human globin transgenes during mouse development resembles that observed during human development. These results suggest that the gross organization of the human beta-like globin gene cluster is important for stage-specific expression of each human globin gene during development.
Animals↗
Serum levels of cancer-associated antigen CA-195 in gastrointestinal cancers and its comparison with CA19-9.
The antibody against CA-195 binds to an epitope that consists of both Lewis A and sialylated Lewis A blood group antigen, whereas CA19-9 has shown specificity for sialylated Lewis A blood group antigen. CA19-9 and CA-195 levels were measured in the sera of 52 normal subjects; 65 benign disease patients; and 74 non-gastrointestinal, 149 colorectal, and 119 upper gastrointestinal cancer patients to correlate their levels with disease status of the patients. Low incidence and levels were found among normal subjects for both markers; however, in the benign disease group a slightly higher incidence of elevation was seen for CA19-9. Among colorectal cancer patients CA-195 appeared to show higher sensitivity for primary as well as advanced disease. Levels of both markers showed similar incidences of elevation among upper gastrointestinal cancer patients. Based on these results the contribution of Lea specificity of CA-195 cannot be ruled out, and it may be used alone or in combination with other markers for monitoring of patients with colorectal, pancreatic, gastric, gall bladder, bile duct, and liver cancers.
Antigens, Tumor-Associated, Carbohydrate↗