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Biomedical subjects

A K Mitra

Publications and source records attributed to A K Mitra.

At least 19 recordsLinked to original sources
PubMed1992

Pulmonary biotransformation of insulin in rat and rabbit.

In vitro biodegradation of insulin in rabbit and rat lung homogenates was investigated. Insulin can be sequentially metabolized into two primary fragments in rabbit lung homogenate by an aminopeptidase. The amino acid sequences of the fragments were found to be the des-Phe-InsulinB1 (Metabolite I) and des-Phe-Val-InsulinB1-2 (Metabolite II). However, only the former metabolite (Metabolite I) was identified in the rat lung homogenate. The km and Vm values associated with rabbit lung homogenate were 0.29 +/- 0.14 mM and 16.4 +/- 6.9 microM/hr/mg protein, respectively, whereas those for a rabbit lung preparation containing both microsomes and cytosol were 0.22 +/- 0.07 mM and 17.9 +/- 5.4 microM/hr/mg protein, respectively. The km and Vm associated with the cytosolic fraction of rabbit lung were 0.32 +/- 0.16 and 20.6 +/- 6.1 microM/hr/mg protein, respectively. The results indicate that the lung aminopeptidase may be a cytosolic enzyme. The degradation of dimeric insulin in the lung homogenate was faster than that of hexameric insulin due to the difference in collision frequency between the enzyme and insulin aggregates. The major metabolites in the lungs reportedly retain almost the same bioactivity of insulin, suggesting that the pulmonary route of insulin delivery will not adversely affect its hypoglycemic activity.

Animals↗
PubMed1992

Enhanced delivery of 5-iodo-2'-deoxyuridine to the brain parenchyma.

5'-Ester derivatives of 5-iodo-2'-deoxyuridine (IDU) with varying degrees of lipophilicity were examined to evaluate the effectiveness of lipophilic ester prodrugs for enhanced and sustained delivery of IDU to the brain parenchyma. Approximately 1.0% (1.0 +/- 0.19; n = 4) of the total radioactivity was found in the brain at 30 min following intravenous administration of the lipophilic benzoyl-5'-ester of 125I-labeled IDU, whereas IDU per se yielded only 0.01% (0.01 +/- 0.06; n = 4). Since the IDU 5'-esters generated significantly higher levels of IDU in the brain, an HPLC analysis of IDU in the presence of 5'-esters and the metabolite 5-iodouracil was developed to characterize IDU uptake in the brain. The drug was detected at levels of 6.6 and 9.5 micrograms/g of brain tissue at 3 hr following intravenous administration of valeryl and benzoyl IDU, respectively, at a dose level of 40 mg/kg IDU equivalent each. IDU, on the other hand, when injected at a similar dose level, produced concentration levels below 0.01 micrograms/g of brain tissue, which was too low to be detected accurately by the HPLC assay. These results suggest that the 5'-ester derivatives cross the blood-brain barrier effectively and generate significantly higher brain levels of the parent drug in the brain parenchyma. The regenerated hydrophilic drug because of its polarity is "locked in" the brain and is subsequently metabolized by pyrimidine phosphorylase to 5-iodouracil.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Nasal membrane and intracellular protein and enzyme release by bile salts and bile salt-fatty acid mixed micelles: correlation with facilitated drug transport.

The effects of four bile salts, one fusidate derivative, and one mixed micellar formulation of bile salt-fatty acid combination on the nasal mucosal protein and enzyme release have been investigated in rats using an in situ nasal perfusion technique. Deoxycholate (NaDC) was found to possess the maximum protein solubilizing activity, followed by taurodihydrofusidate (STDHF), cholate, glycocholate (NaGC), and taurocholate (NaTC) in a descending order. The difference in protein solubilization of NaDC and NaGC was further characterized by the release of 5'-nucleotidase (5'-ND), a membrane-bound enzyme, and lactate dehydrogenase (LDH), an intracellular enzyme, in the perfusate. While both NaDC and NaGC caused comparable 5'-ND release from nasal membrane, intracellular LDH release was significantly higher with NaDC. The greater protein and LDH solubilizing effects of NaDC corresponded well with its faster rate of disappearance from the nasal perfusate. Therefore, the dihydroxy bile salt NaDC tends to cause intracellular damage and cell lysis, whereas the trihydroxy bile salt NaGC appears to produce primarily mucosal membrane perturbations. Linoleic acid in the form of soluble mixed micelles with glycocholate caused a further increase in nasal protein release. However, the rate and extent of nasal membrane protein release by the mixed micelles composed of 15 mM glycocholate and 5 mM linoleic acid were significantly lower than those caused by either deoxyholate or STDHF at the same concentrations. Nasal absorption of acyclovir, a non-absorbable hydrophilic model antiviral agent, was found to be enhanced in the presence of conjugated trihydroxy bile salts and bile salt-fatty acid mixed micelles.(ABSTRACT TRUNCATED AT 250 WORDS)

5'-Nucleotidase↗
PubMed1992

Kinetics and mechanism of degradation of a cyclic hexapeptide (somatostatin analogue) in aqueous solution.

A highly active cyclic hexapeptide analogue of somatostatin, Cyclo(N-Me-L-Ala-L-Tyr-D-Trp-L-Lys-L-Val-L-Phe), L-363,586, was found to improve the control of postprandial hyperglycemia in diabetic animals when given in combination with insulin. The compound is reported to be relatively stable in blood, nasal cavity, and intestinal lumen but undergoes rapid degradation in aqueous solution. The objective of this study was to elucidate the degradation mechanisms based on the kinetic data and the structure of the degradation products. Both pH and temperature had a profound influence on the instability of the peptide in aqueous solution. The data indicated that the peptide was most stable at a pH of about 4.7. The pH-rate profile exhibited specific acid catalysis at a pH less than 3.0 and base catalysis above pH 10.5. The kinetic pKa was determined to be 9.7. This pKa could be attributed to the tyrosine residue. The mechanisms of degradation under acidic and alkaline conditions appear to be different. Identification of the fragments obtained using mass spectrometry and amino acid sequencing suggest that the cyclic compound was cleaved to yield a linear fragment, which underwent further cleavage at both peptide linkages alpha to the tryptophanyl residue. The indole group of that residue is probably the potential nucleophile attacking the adjacent carbonyls. A rate equation for the degradation of the hexapeptide has been proposed.

Amino Acid Sequence↗
PubMed1992

Acyclovir permeation enhancement across intestinal and nasal mucosae by bile salt-acylcarnitine mixed micelles.

The purpose of this study was to investigate the absorption enhancement of acyclovir, an antiviral agent, by means of bile salt-acylcarnitine mixed micelles. The specificity, site dependence, palmitoyl-DL-carnitine chloride (PCC) concentration dependence, and effects of absorption promoters on acyclovir absorption via the nasal cavity (N) and four different intestinal segments of the rat, i.e., duodenum (D), upper jejunum (UJ), combined lower jejunum and ileum (LJ), and colon (C) were evaluated. The present study employed the rat in situ nasal and intestinal perfusion techniques and utilized sodium glycocholate (NaGC), three acylcarnitines, and their mixed micelles as potential nasal and intestinal absorption promoters. Acylcarnitines used were DL-octanoylcarnitine chloride (OCC), palmitoyl-DL-carnitine chloride (PCC), and DL-stearoylcarnitine chloride (SCC). All acylcarnitines and NaGC by themselves produced negligible enhancement of acyclovir absorption in the rat intestine, while OCC and SCC were totally ineffective in the nasal cavity. However, the mixed micellar solutions of NaGC with PCC or SCC could significantly increase the mucosal membrane permeability of acyclovir in the colon and nasal cavity. On the other hand, NaGC-OCC mixed micelles slightly increased the absorption of acyclovir by both routes. When a mixed micellar solution of NaGC with PCC was used, the rank order of apparent acyclovir permeability (Papp; cm/sec), corrected for surface area of absorption, was N (10.54 +/- 0.62 x 10(-5)) > D (6.82 +/- 0.30 x 10(-5)) > LJ (2.90 +/- 0.08 x 10(-5)) > C (2.54 +/- 0.14 x 10(-5)) > UJ (2.30 +/- 0.22 x 10(-5)).(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1992

Cyclodextrins as nasal absorption promoters of insulin: mechanistic evaluations.

The safety and effectiveness of cyclodextrins (CD) as nasal absorption promoters of peptide-like macromolecules have been investigated. The relative effectiveness of the cyclodextrins in enhancing insulin nasal absorption was found to be in the descending order of dimethyl-beta-cyclodextrin (DM beta CD) greater than alpha-cyclodextrin (alpha-CD) greater than beta-cyclodextrin (beta-CD), hydroxypropyl-beta-cyclodextrin (HP beta CD) greater than gamma-cyclodextrin (gamma-CD). A direct relationship linking absorption promotion to nasal membrane protein release is evident, which in turn correlates well with nasal membrane phospholipid release. The magnitude of the membrane damaging effects determined by the membrane protein or phospholipid release may provide an accurate, simple, and useful marker for predicting safety of the absorption enhancers. In order to estimate further the magnitude of damage and specificity of cyclodextrin derivatives in solubilizing nasal membrane components, the enzymatic activities of membrane-bound 5'-nucleotidase (5'-ND) and intracellular lactate dehydrogenase (LDH) in the perfusates were also measured. HP beta CD at a 5% concentration was found to result in only minimal removal of epithelial membrane proteins as evidenced by a slight increase in 5'-ND and total absence of LDH activity. On the other hand, 5% DM beta CD caused extensive removal of the membrane-bound 5'-ND. Moreover, intracellular LDH activity in the perfusate increased almost linearly with time. The cyclodextrins are also capable of dissociating insulin hexamers into smaller aggregates, and this dissociation depends on cyclodextrin structure and concentration. Enhancement of insulin diffusivity across nasal membrane through dissociation may provide an additional mechanism for cyclodextrin promotion of nasal insulin absorption.

5'-Nucleotidase↗
PubMed1992

Brain parenchymal metabolism of 5-iodo-2'-deoxyuridine and 5'-ester prodrugs.

In an attempt to generate derivatives of 5-iodo-2'-deoxyuridine (IDU) with enhanced blood-brain barrier (BBB) permeability, a series of 5' ester prodrugs of IDU was synthesized and their metabolism studied in rat brain homogenate and its different subcellular fractions. The rate of hydrolysis was dependent on the steric and polar nature of the ester substituent. Ester hydrolyzing activities were associated primarily with the cytosolic fraction and were due mainly to the presence of cholinesterases as confirmed by inhibition experiments performed with different esterase inhibitors. The metabolism of IDU to 5-iodouracil (5-IU) by the cytosolic fraction, in the presence and absence of specific pyrimidine nucleoside phosphorylase inhibitors, also suggests that there are two specific enzyme systems catalyzing two different metabolic processes. IDU 5'-esters competitively inhibit the metabolism of IDU and the inhibitory effect depends on the affinity of a particular ester toward the enzyme and also on the rate by which the ester itself undergoes hydrolysis. In the absence of any 5'-ester, 95% IDU was metabolized within 6 hr. However, in the presence of an eightfold molar excess of butyryl-IDU, the hydrolysis of IDU was completely inhibited over a 6-hr time period.

Animals↗
PubMed1992

Mechanism and site dependency of intestinal mucosal transport and metabolism of thymidine analogues.

This study has been undertaken to investigate the mechanisms of intestinal mucosal transport and metabolism of thymidine analogues and to identify any optimal site(s) of the rat intestine particularly involved in the absorption of thymidine analogues. The intestinal absorption of 3'-azido-3'-deoxythymidine (AZT) was studied at three initial concentrations in four segments of the rat intestine using an in situ recirculating perfusion technique. Disappearance of AZT followed first-order kinetics throughout the gastrointestinal (GI) tract at all tested concentrations. The apparent first-order rate constants were found to be relatively invariant over a broad range of concentrations from 0.01 to 1.0 mM. Corrected for the length of each segment, the apparent permeability (Papp) of AZT was 3.01 +/- 0.32 x 10(-5) cm/sec (mean +/- SE) in the duodenum, 2.06 +/- 0.24 x 10(-5) cm/sec in the upper jejunum, 0.76 +/- 0.13 x 10(-5) cm/sec in the combined lower jejunum and ileum, and 0.32 +/- 0.10 x 10(-5) cm/sec in the colon, which indicated that intrinsic absorptivity was greater in the upper GI tract than in the lower portions possibly due to the differences in surface area for absorption. No AZT metabolite appeared in any part of the GI tract. On the other hand, thymidine and other analogues, i.e., 5-iodo-2'-deoxyuridine and 2'-deoxyuridine, were rapidly metabolized into nucleobase and sugar in the upper GI tract, whereas in the colon no metabolite appeared. A free 3'-OH group appears to be necessary for the metabolism (catabolism) of thymidine analogues in the rat intestine mainly by pyrimidine nucleoside phosphorylase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Dissociation of insulin oligomers by bile salt micelles and its effect on alpha-chymotrypsin-mediated proteolytic degradation.

Bile salts have been found to be effective absorption promoters of insulin across mucosal barriers, i.e., nasal and gastrointestinal. One of the mechanisms proposed for absorption enhancement is the dissociation of insulin oligomers to monomers, rendering a higher insulin diffusivity. alpha-Chymotryptic degradation and circular dichroism studies were used to characterize such a transition. When zinc insulin (hexamers) and sodium insulin (dimers) were subjected to alpha-chymotryptic degradation, a 3.2-fold difference in the apparent first-order rate constants was observed (zinc insulin being slower than sodium insulin), representing the intrinsic difference in the concentration of total associated species in solution (three times). In the presence of a bile salt, sodium glycocholate (NaGC), the rate of degradation of both zinc and sodium insulin increased in an asymptotic manner. A maximum increase of 5.4-fold was observed for zinc insulin at a 30 mM NaGC concentration and a 2.1-fold increase was noted for sodium insulin at 10 mM NaGC, both values being close to the theoretical numbers of 6- and 2-fold as predicted by the complete dissociation of hexamers and dimers to monomers. The result indicates dissociation of insulin oligomers to monomers by bile salt micelles, probably by hydrophobic micellar incorporation of monomeric units. Circular dichroism studies also revealed progressive attenuation of molecular ellipticities at negative maxima of 276, 222, and 212 nm for zinc insulin solution in the presence of NaGC. Therefore, both alpha-chymotryptic degradation and circular dichroism studies have consistently demonstrated that the bile salts may be capable of dissociating insulin oligomers to monomers, a fact which may play an important role in enhancing insulin bioavailability.

Bile Acids and Salts↗
PubMed1992

Pharmacodynamics of insulin following intravenous and enteral administrations of porcine-zinc insulin to rats.

Previous work from this laboratory showed site-dependent variations in the apparent permeability of insulin as measured using the everted rat gut sac technique, with the greatest permeability in the distal jejunum and the lowest in the duodenum (5). To quantify better the rate and extent of insulin absorption from the small intestine, closed-loop in situ experiments were performed in nondiabetic rats. Results correlated with the everted gut sac technique in that the absolute bioavailability determined in situ was higher for insulin solution administered to the more distal region of the intestine (0.133%) than that absorbed from an earlier portion of the intestine (0.059%). While the difference in regional bioavailabilities was not significant (P = 0.08), the blood glucose response showed highly significant differences (P = 0.0015), with severe and prolonged hypoglycemia resulting from insulin delivered to the distal jejunum/proximal ileum. Insulin administered iv followed a two-compartment pharmacokinetic model. Whole-body elimination rate constants were similar for both iv and enteral insulin. Although therapeutic quantities of insulin were absorbed from the distal small intestine, absorption enhancers would be necessary to decrease the dose of insulin required.

Animals↗
PubMed1992

Metal uptake by mycelia during submerged growth and by sporocarps of an edible fungus Volvariella volvacea.

Uptake of a few metals by V. volvacea was determined during submerged growth of the organism in sublethal concentration of each metal salt. The uptake of Pb2+ and Hg2+ was 5 and 5.23 micrograms g-1 respectively while that of Cu2+ was 500 micrograms g-1 under experimental conditions. Treatment of spawned substrate separately with different metal salts showed maximum and minimum uptake of Pb2+ (100 micrograms g-1) and Cd2+ (2.93 micrograms g-1) respectively by sporocarps. All metal salts at test concentrations reduced biological efficiency of sporocarp production but markedly by Co2+. Cd2+ and Co2+ were highly toxic to mycelia and sporocarps respectively. The uptake of Cu2+ by mycelia and Pb2+ by sporocarps were highest among the five metals tested. Metal toxicity, tolerance and uptake capacity of V. volvacea differ considerably with concentration of metal ions.

Agaricales↗
PubMed1992

Nosocomial rotavirus diarrhea in two medical wards of a pediatric hospital in Calcutta.

One hundred eighty nine children suffering from different medical problems were admitted in two wards of a pediatric hospital in Calcutta during the period between November 18, 1985 and February 10, 1986. Amongst them, 36 children developed nosocomial diarrhea and rotavirus was detected from 80.5% of the cases. The nosocomial rotavirus diarrhea cases had lesser frequency of stools and only mild dehydration but the course of illness was longer in comparison to that of the hospitalized rotavirus diarrhea cases. There is a possibility of spread of infection via fomites, environmental surfaces and most likely mothers.

Child, Preschool↗
PubMed1991

Epidermal growth factor (EGF) induces oligomerization of soluble, extracellular, ligand-binding domain of EGF receptor. A low resolution projection structure of the ligand-binding domain.

Ligand-induced oligomerization is a universal phenomenon among growth factor receptors. Although the mechanism involved is yet to be defined, much evidence indicates that receptor oligomerization plays a crucial role in receptor activation and signal transduction. Here we show that epidermal growth factor (EGF) is able to stimulate the oligomerization of a recombinant, soluble, extracellular ligand-binding domain of EGF receptor. Covalent cross-linking experiments, analysis by sodium dodecyl sulfate-gel electrophoresis, size exclusion chromatography, and electron microscopy demonstrate that receptor dimers, trimers and larger multimers are formed in response to EGF. This establishes that receptor oligomerization is an intrinsic property of the extracellular ligand-binding domain of EGF receptor. Ligand-induced conformational change in the extracellular domain will stimulate receptor-receptor interactions. This may bring about the allosteric change involved in signal transduction from the extracellular domain across the plasma membrane, resulting in the activation of the cytoplasmic kinase domain. Electron microscopic images of individual extracellular ligand-binding domains appear as clusters of four similarly-sized stain-excluding areas arranged around a central, relatively less stain-excluded area. This suggests that the extracellular ligand-binding domain is structurally composed of four separate domains.

Base Sequence↗
PubMed1991

Wild-type and mutant bacterioopsins D85N, D96N, and R82Q: high-level expression in Escherichia coli.

The integral membrane protein bacterioopsin, found in the extremely halophilic archaebacterium Halobacterium halobium, was expressed in Escherichia coli as a fusion protein containing 13 heterologous amino acids at the amino terminus. The expressed protein was localized primarily to the E. coli cytoplasmic membrane (greater than 80%) and had an in vivo half-life of 26 min. The amount of bacterioopsin in E. coli crude lysates was quantitated immunologically from Western blots and was expressed at 10-20-fold higher levels than seen previously (i.e., 17 mg/L; 5.6% of the total protein). Three distinct forms of the protein were detected immunologically: two of the forms were generated by the removal of either one or four amino acid residues at the amino terminus; the third form remained unaltered.

Amino Acid Sequence↗
PubMed1991

Wild-type and mutant bacteriorhodopsins D85N, D96N, and R82Q: purification to homogeneity, pH dependence of pumping, and electron diffraction.

Bacterioopsin, expressed in Escherichia coli as a fusion protein with 13 heterologous residues at the amino terminus, has been purified in the presence of detergents and retinylated to give bacteriorhodopsin. Further purification yielded pure bacteriorhodopsin, which had an absorbance ratio (A280/A lambda max) of 1.5 in the dark-adapted state in a single-detergent environment. This protein has a folding rate, absorbance spectrum, and light-induced proton pumping activity identical with those of bacteriorhodopsin purified from Halobacterium halobium. Protein expressed from the mutants D85N, D96N, and R82Q and purified similarly yielded pure protein with absorbance ratios of 1.5. Proton pumping rates of bacteriorhodopsins with the wild-type sequence and variants D85N, D96N, and R82Q were determined in phospholipid vesicles as a function of pH. D85N was inactive at all pH values, whereas D96N was inactive from pH 7.0 to pH 8.0, where wild type is most active, but had some activity at low pH. R82Q showed diminished proton pumping with the same pH dependence as for wild type. Bacteriorhodopsin purified from E. coli crystallized in two types of two-dimensional crystal lattices suitable for low-dose electron diffraction, which permit detailed analysis of structural differences in site-directed variants. One lattice was trigonal, as in purple membrane, and showed a high-resolution electron diffraction pattern from glucose-sustained patches. The other lattice was previously uncharacterized with unit cell dimensions a = 127 A, b = 67 A, and symmetry of the orthorhombic plane group pgg.

Bacteriorhodopsins↗
PubMed1991

Systemic absorption of L- and D-phenylalanine across the rat nasal mucosa.

Nasal absorption of L-phenylalanine (L-Phe) and D-phenylalanine (D-Phe) have been investigated in rats using an in vivo absorption technique. L-Phe was effectively absorbed into systemic circulation with peak plasma level at 45 min after nasal administration. The absolute nasal bioavailability was calculated to be 96.3% when 5 mg/kg dose was given to fasted rats. The absolute bioavailability declined to 66.8% when 12.5 mg/kg of L-Phe was given intranasally while the time to reach peak concentration remained at 45 min. The results substantiated previous in situ data that, despite its high polarity, L-Phe was transported across the rat nasal mucosa into blood stream by the large neutral amino acid (LNAA) carrier. The carrier showed partial saturation at higher doses. On the other hand, when 5 mg/kg of D-Phe was given intranasally, slow and incomplete absorption was observed resulting in a peak time of 60 min and an absolute bioavailability of only 35.2%, suggesting specificity of the carrier for natural L-amino acids.

Administration, Intranasal↗