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Biomedical subjects

H M Patel

Publications and source records attributed to H M Patel.

At least 19 recordsLinked to original sources
PubMed2002

Modulation of murine liver macrophage clearance of liposomes by diethylstilbestrol. The effect of vesicle surface charge and a role for the complement receptor Mac-1 (CD11b/CD18) of newly recruited macrophages in liposome recognition.

We have studied the blood clearance and reticuloendothelial organ distribution of intravenously injected neutral (egg phosphatidylcholine, egg PC/cholesterol, mol ratio 7:2), anionic (egg PC/cholesterol/dicetylphosphate, mol ratio 7:2:1), and cationic (egg PC/cholesterol/stearylamine, mol ratio 7:2:1) liposomes of approximately the same size distribution in mice 3 days after treatment with the synthetic oestrogen diethylstilbestrol (DES). Male mice administered DES intraperitoneally at a dose of 1 mg per mouse (body weight 22-25 g) manifested an increase in the vascular clearance rate of liposomes irrespective of the initial vesicle surface charge. The enhancement in the vascular clearance of liposomes in DES-treated animals was associated with a concomitant increase in liver weight as well as hepatic phagocytosis. However, DES treatment significantly enhanced the hepatic sequestration (on the basis of % of injected dose of liposomes per g of liver tissue) of positively charged liposomes when compared to both neutral and negatively charged vesicles of similar size distribution. This observation was also confirmed by the 'hepatic-blockade' experiments where blockade was induced by prior intravenous injection of liposomes of the same size distribution and charge to that of test vesicles. The in vitro cell suspension studies suggested that the enhanced liposome uptake (irrespective of the initial vesicle surface charge) by Kupffer cells of DES-treated mice was independent of changes in the blood opsonization processes. Furthermore, in vitro studies also showed the operation of multiple mechanisms and involvement of different populations of liver macrophages (resident and recruited cells) in liposome recognition following DES treatment. For example, in DES-treated animals, the newly recruited liver macrophages were found to play a major role in the clearance of stearylamine incorporated liposomes via complement receptors (Mac-1). The resident Kupffer cells seem to recognize cationic vesicles via other receptors as the expression of Mac-1 is virtually absent in these cells. On the other hand, complement receptors seem to play a minor role in the uptake of anionic DCP vesicles by hepatic macrophages of DES-treated mice. DES appears to offer a new approach in dissecting the mechanisms of liposome-macrophage interaction.

Animals↗
PubMed2001

In vitro reconstitution of the Pseudomonas aeruginosa nonribosomal peptide synthesis of pyochelin: characterization of backbone tailoring thiazoline reductase and N-methyltransferase activities.

During iron starvation the Gram-negative pathogenic bacterium Pseudomonas aeruginosa makes the nonribosomal peptide siderophore pyochelin by a four protein, 11 domain assembly line, involving a cascade of acyl-S-enzyme intermediates on the PchE and PchF subunits that are elongated, heterocyclized, reduced, and N-methylated before release. Purified PchG is shown to be an NADPH-dependent reductase for the hydroxyphenylbisthiazoline-S-PchF acyl enzyme, regiospecifically converting one of the dihydroheterocyclic thiazoline rings to a thiazolidine. The K(m) for the PchG protein is 1 microM, and the k(cat) for throughput to pyochelin is 2 min(-1). The nitrogen of the newly generated thiazolidine ring can be N-methylated upon addition of SAM, to yield the mature pyochelin chain still tethered as a pyochelinyl-S-PchF at the PCP domain. A presumed methyltransferase (MT) domain embedded in the PchF subunit catalyzes this N-methylation. Mutation of a conserved G to R in the MT core motif abolishes MT activity and subsequent chain release from PchF. The thioesterase (TE) domain of PchF catalyzes hydrolytic release of the fully mature pyochelinyl chain to produce the pyochelin siderophore at a rate of 2 min(-1), at least 30-40-fold faster than in the absence of hydroxyphenylbisthiazolinyl-COOH (HPTT-COOH) chain reduction and N-methylation. A mutation in the PchF TE domain does not catalyze autodeacylation and release of the pyochelinyl-S-enzyme. Thus, full reconstitution of the nonribosomal peptide synthetase assembly line by purified protein components has been obtained for production of this tandem bisheterocyclic siderophore.

Amino Acid Sequence↗
PubMed2001

Toward efficient analysis of >70 kDa proteins with 100% sequence coverage.

For complete characterization of larger proteins, primary structural analysis by mass spectrometry must be made more efficient. A straightforward approach is illustrated here using two proteins of 159 and 199 kDa with five and nine Lys residues, respectively. These proteins were degraded by Lys-C to mixtures of peptides ranging in size from 5 to 48 kDa, whose multiply charged ions (from electrospray ionization) are far more amenable than the intact proteins to direct interrogation in a Fourier-transform mass spectrometer. For the 199 kDa PchF of approximately 60% purity, an unfractionated Lys-C digest gave 106 isotopic distributions from 71 components (most of which were below 6 kDa); 15% sequence coverage was obtained. For the > 90% pure PchE (159 kDa), complete sequence coverage was obtained from six Lys-C peptides of 5, 8, 26, 32, 40 and 48 kDa, with all but the largest of these measured at isotopic resolution on a 4.7 Tesla instrument. Practical strategies for implementing this characterization strategy on a proteomic scale are considered.

Amino Acid Sequence↗
PubMed2001

Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines.

Nonribosomal peptide synthetases are large enzyme complexes that synthesize a variety of peptide natural products through a thiotemplated mechanism. Assembly of the peptides proceeds through amino acid loading, amide-bond formation and chain translocation, and finally thioester lysis to release the product. The final products are often heavily modified, however, through methylation, epimerization, hydroxylation, heterocyclization, oxidative cross-linking and attachment of sugars. These activities are the province of specialized enzymes (either embedded in the multidomain nonribosomal peptide synthetase structure or standalone).

Bacteria↗
PubMed2001

Essential PchG-dependent reduction in pyochelin biosynthesis of Pseudomonas aeruginosa.

The biosynthetic genes pchDCBA and pchEF, which are known to be required for the formation of the siderophore pyochelin and its precursors salicylate and dihydroaeruginoate (Dha), are clustered with the pchR regulatory gene on the chromosome of Pseudomonas aeruginosa. The 4.6-kb region located downstream of the pchEF genes was found to contain three additional, contiguous genes, pchG, pchH, and pchI, probably forming a pchEFGHI operon. The deduced amino acid sequences of PchH and PchI are similar to those of ATP binding cassette transport proteins with an export function. PchG is a homolog of the Yersinia pestis and Y. enterocolitica proteins YbtU and Irp3, which are involved in the biosynthesis of yersiniabactin. A null mutation in pchG abolished pyochelin formation, whereas mutations in pchH and pchI did not affect the amounts of salicylate, Dha, and pyochelin produced. The pyochelin biosynthetic genes were expressed from a vector promoter, uncoupling them from Fur-mediated repression by iron and PchR-dependent induction by pyochelin. In a P. aeruginosa mutant lacking the entire pyochelin biosynthetic gene cluster, the expressed pchDCBA and pchEFG genes were sufficient for salicylate, Dha, and pyochelin production. Pyochelin formation was also obtained in the heterologous host Escherichia coli expressing pchDCBA and pchEFG together with the E. coli entD gene, which provides a phosphopantetheinyl transferase necessary for PchE and PchF activation. The PchG protein was purified and used in combination with PchD and phosphopantetheinylated PchE and PchF in vitro to produce pyochelin from salicylate, L-cysteine, ATP, NADPH, and S-adenosylmethionine. Based on this assay, a reductase function was attributed to PchG. In summary, this study completes the identification of the biosynthetic genes required for pyochelin formation from chorismate in P. aeruginosa.

ATP-Binding Cassette Transporters↗
PubMed2001

Evaluation of the effects of nicorandil on experimentally induced gastric ulcers.

The present study was designed to evaluate the effects of a potassium channel opener, nicorandil, and to elucidate its possible mechanism of action in aspirin plus pylorus ligation induced and ethanol-induced gastric ulcers in rats. In an attempt to ascertain the involvement of K(ATP) channels in the modulation of gastric ulcers, the effects of nicorandil alone as well as in the presence of the K(ATP) channel blocker glibenclamide were studied. Nicorandil and glibenclamide were administered orally at a dose of 2 mg/kg throughout the study. Nicorandil showed significant protection in all the selected models that was evident from a significant reduction in the ulcer index. The results of nicorandil treatment were comparable with those of cimetidine treatment in both models. Glibenclamide was found to inhibit this effect of nicorandil. Further, glibenclamide showed proulcerogenic potential in ethanol and aspirin plus pylorus ligation models. In the aspirin plus pylorus ligation model, nicorandil showed a significant reduction in total acidity, pepsin activity, and protein content and a significant rise in mucin activity. The effect of nicorandil was also studied on gastric mucosal blood flow (GMBF). The GMBF was found to be more increased in the test group than in the control group, indicating enhancement of GMBF by nicorandil. Glibenclamide reversed this effect of nicorandil as well. It is concluded from our study that nicorandil possesses antiulcer activity in the models employed in the present study. This may be attributed to the opening of K(ATP) channels, inhibition of acid secretion, enhancement of mucin activity, and improvement in GMBF.

Animals↗
PubMed2000

Cost-effective management of acute atrial fibrillation: role of rate control, spontaneous conversion, medical and direct current cardioversion, transesophageal echocardiography, and antiembolic therapy.

Management strategies for the acute treatment of atrial fibrillation (AF) include: (1) the use of intravenous drugs for rate control, (2) drug termination, or (3) direct current (DC) cardioversion. Delays in cardioversion can promote atrial remodeling and add thromboembolic risk. Rate control awaiting spontaneous or pharmacologic conversion may be a cost-effective strategy in patients presenting with recent onset of symptoms. Early DC cardioversion can be cost-effective and minimize antiembolic therapy issues in the acute setting. In patients presenting with AF of unknown or >48 hours' duration, rate control and therapeutic warfarin for 3-4 weeks followed by medical or DC cardioversion is standard practice. However, delays in conversion promote atrial remodeling that makes restoration of sinus rhythm more difficult and increases the likelihood of postcardioversion AF recurrence. Transesophageal echocardiography can identify patients at low risk for a cardioversion-related embolic event and allows cardioversion to be performed earlier, thereby minimizing atrial remodeling.

Algorithms↗
PubMed2000

Amiodarone: what have we learned from clinical trials?

Amiodarone is an antiarrhythmic agent commonly used in the treatment of supraventricular and ventricular tachyarrhythmias. This paper reviews clinical trials in which amiodarone was used in one of the treatment arms. Key post-myocardial infarction trials include EMIAT and CAMIAT, both of which demonstrated that amiodarone reduced arrhythmic but not overall mortality. In patients with congestive heart failure (CHF), amiodarone was associated with a neutral survival in CHF/STAT and improvement in survival in GESICA. In patients with nonsustained ventricular tachycardia, the MADIT trial demonstrated that therapy with an implantable cardioverter-defibrillator (ICD) improved survival compared with the antiarrhythmic drug arm in such patients, most of whom were taking amiodarone. In sustained VT/VF patients, the CASCADE trial demonstrated that empiric amiodarone lowered arrhythmic recurrence rates compared with other drugs guided by serial Holter or electrophysiologic studies. Several trials including AVID, CIDS, and CASH have demonstrated the superiority of ICD therapy compared with empiric amiodarone in improving overall survival. Clinical implications of these trials are discussed.

Amiodarone↗
PubMed2000

Amiodarone: clinical trials.

Amiodarone is an antiarrhythmic agent commonly used in the treatment of supraventricular and ventricular tachyarrhythmias. This article reviews the results and clinical implications of primary and secondary prevention trials in which amiodarone was used in one of the treatment arms. Key post-myocardial infarction primary prevention trials include the European Myocardial Infarct Amiodarone Trial (EMIAT) and the Canadian Amiodarone Myocardial Infarction Trial (CAMIAT), both of which demonstrated that amiodarone reduced arrhythmic but not overall mortality. In congestive heart failure patients, amiodarone was studied as a primary prevention strategy in two pivotal trials: Grupo de Estudio de la Sobrevida en la Insuficiencia Cardiac en Argentina (GESICA) and Amiodarone in Patients With Congestive Heart Failure and Asymptomatic Ventricular Arrhythmia (CHF-STAT). Amiodarone was associated with a neutral overall survival and a trend toward improved survival in nonischemic cardiomyopathy patients in CHF/STAT and improved survival in GESICA. In post-myocardial infarction patients with nonsustained ventricular tachycardia and a depressed ejection fraction, the Multicenter Automatic Defibrillator Implantation Trial (MADIT) demonstrated that implantable cardioverter-defibrillators (ICD) statistically improved survival compared to the antiarrhythmic drug arm, most of whose patients were taking amiodarone. In patients with histories of sustained ventricular tachycardia or ventricular fibrillation, the Cardiac Arrest Study in Seattle: Conventional Versus Amiodarone Drug Evaluation (CASCADE) trial demonstrated that empiric amiodarone lowered arrhythmic recurrence rates compared to other drugs guided by serial Holter or electrophysiologic studies. However, arrhythmic death rates were high in both treatment arms of the study. Several secondary prevention trials, including the Antiarrhythmics Versus Implantable Defibrillators Study (AVID), the Canadian Implantable Defibrillator Study (CIDS), and the Cardiac Arrest Study Hamburg (CASH), have demonstrated the superiority of ICD therapy compared to empiric amiodarone in improving overall survival. Based on the above findings, amiodarone is safe to use in post-myocardial infarction and congestive heart failure patients that need antiarrhythmic therapy. Although amiodarone is effective in treating malignant arrhythmias, high-risk patients should be considered for an ICD as frontline therapy.

Amiodarone↗
PubMed1999

Assembly of the Pseudomonas aeruginosa nonribosomal peptide siderophore pyochelin: In vitro reconstitution of aryl-4, 2-bisthiazoline synthetase activity from PchD, PchE, and PchF.

Three Pseudomonas aeruginosa proteins involved in biogenesis of the nonribosomal peptide siderophore pyochelin, PchD, PchE, and PchF, have been expressed in and purified from Escherichia coli and are found to produce the tricyclic acid hydroxyphenyl-thiazolyl-thiazolinyl-carboxylic acid (HPTT-COOH), an advanced intermediate containing the aryl-4,2-bis-heterocyclic skeleton of the bithiazoline class of siderophores. The three proteins contain three adenylation domains, one specific for salicylate activation and two specific for cysteine activation, and three carrier protein domains (two in PchE and one in PchF) that undergo posttranslational priming with phosphopantetheine to enable covalent tethering of salicyl and cysteinyl moieties as acyl-S-enzyme intermediates. Two cyclization domains (Cy1 in PchE and Cy2 in PchF) create the two amide linkages in the elongating chains and the cyclodehydrations of acylcysteine moieties into thiazolinyl rings. The ninth domain, the most downstream domain in PchF, is the chain-terminating, acyl-S-enzyme thioester hydrolase that releases the HPTT-S-enzyme intermediate to the observed tandem bis-heterocyclic acid product. A PchF-thioesterase domain active site double mutant fails to turn over, but a monocyclic hydroxyphenyl-thiazolinyl-cysteine (HPT-Cys) product continues to be released from PchE, allowing assignment of the cascade of acyl-S-enzyme intermediates involved in initiation, elongation, and termination steps.

Adenosine Triphosphate↗
PubMed1998

A critical period for reduced brain vulnerability to developmental injury. II. Volumetric study of the neocortex and thalamus in cats.

Groups of young adult cats with a left hemineodecortication at postnatal (P) ages (in days) 5-15 (P10), 30 (P30) 60 (P60), 90 (P90), 120 (P120) and in adulthood, were used to measure the volume of the thalamus, bilaterally, and of the remaining neocortex (right hemisphere). The same subjects were employed for the behavioral studies reported in the preceding paper. There was a bilateral, age-dependent, thalamic volume decrease. Ipsilateral to the resection, the thalamic shrinkage was the largest for the adult-lesioned cats (by 56.7%) and it was the smallest for the P30 group (43.4%), with a tendency towards a greater atrophy as the age at lesion increased. A similar pattern of atrophy was seen for the contralateral thalamus but the volume reduction was much less pronounced such that it was significant only for the four older age-at-lesion groups (ranging from 18.2% to 11.2% for the P120 and P90 groups respectively). Once again, the shrinkage was the smallest for the P30 group (5.3%). The remaining neocortex also shrunk in these animals, but the volume decrease was significant only for the adult-lesioned (17.8%) and the P120 group (15.4%), while the P30 group had practically no shrinkage (2.4%). The frontal cortex had no atrophy or it was minimal but the shrinkage gradually increased caudally such that all lesioned groups had some size reduction of the occipital cortex. The present results, together with the main conclusion of the preceding paper, indicate that there is a critical maturation period (CMP) of reduced forebrain vulnerability to neocortical injury which, in cats, tends to end between 30 to 60 days postnatally. The implications for developmental brain damage in other higher mammal species as well as the possible morphological ontogenetical underpinnings of this period are discussed.

Aging↗
PubMed1998

A decade of clinical trial developments in postmyocardial infarction, congestive heart failure, and sustained ventricular tachyarrhythmia patients: from CAST to AVID and beyond. Cardiac Arrhythmic Suppression Trial. Antiarrhythmic Versus Implantable Defibrillators.

Multiple trials using antiarrhythmic drugs, pharmacologic therapy, and implantable cardioverter defibrillators have been performed in an attempt to improve survival in patients: (1) postmyocardial infarction; (2) with congestive heart failure, with and without nonsustained ventricular tachycardia; and (3) with sustained ventricular tachycardia and those who have survived an out-of-hospital cardiac arrest. This article reviews some of the key findings and limitations of completed and ongoing trials. We also make recommendations for the current treatment of such patients based on the results of these trials.

Adrenergic beta-Antagonists↗
PubMed1997

Abbreviated junctional sequences impoverish antibody diversity in urodele amphibians.

Of the six complementarity-determining regions (CDR) forming the structure of the Ab combining site, CDR3 of heavy chain is the most variable in length and sequence. Diversity of this loop is determined by the number of gene segments involved, extent of addition to or deletion from the joining genes, and imprecision of the site of recombination. In neonatal mice and Xenopus tadpoles, the last two factors occur less frequently than in adults, which in tadpoles result in low affinity Ab responses that do not mature. In contrast, adult urodele amphibians make larval-like responses and are notorious for lifelong poor immunocompetence. The mechanism for this is not known, and in this study we cloned germline VH genes from the axolotl and obtained rearrangements to these VH gene segments by reverse-transcriptase PCR. These sequences were analyzed for heavy chain junctional diversity and found to be even less variable than that in newborn mouse or Xenopus tadpoles, although for different reasons. Only 29% of the CDR3 loop in the axolotl consisted of somatically generated sequences, compared with 44% in tadpole, 39% in newborn mice, and 57% in both adult mice and Xenopus. This distinguishing feature of axolotl CDR3 results not only from shorter junctional sequences, but also unusually extensive integration of germline JH sequence. As the CDR3 loop is the most important portion of the Ig sequence for determining Ab combining site diversity, our data provide the molecular basis for a contributing factor in the deficient urodele amphibian Ab responses.

Aging↗
PubMed1996

Altered tissue-specific opsonic activities and opsono-recognition of liposomes in tumour-bearing rats.

Reticuloendothelial phagocytic and serum opsonic activity was evaluated at terminal stages of tumour growth in rats transplanted subcutaneously with chondrosarcoma in an attempt to evaluate the role of opsonic protein(s) in governing liposome recognition and clearance by the macrophage system. The liver of the tumour-bearing animals manifested a decline in the uptake of multilamellar vesicles composed of egg phosphatidylcholine: cholesterol: dicetyl phosphate (mole ratio 7:2:1) from the blood when compared to healthy animals. In contrast, an increase in splenic clearance of liposomes was encountered in tumour-bearing rats. Studies with isolated liver non-parenchymal cells suggested that liposome recognition in both health and at terminal stages of cancer growth is influenced by a serum opsonin, which can be precipitated by 35-50% ammonium sulphate, as well as the concentration of calcium levels in serum. Serum of healthy animals equally enhanced liposome recognition by the hepatic macrophages of both normal and tumour-bearing rats. In contrast, both cell populations manifested poor liposome recognition in the presence of serum pooled from tumour-bearing animals and the results were comparable to the corresponding liposome-cell interaction in the absence of serum. The opsonic activity of serum derived from tumour-bearing rats could be demonstrated either by prior dialysis of serum against de-ionized water or by addition of EGTA. Liver phagocytes of healthy animals recognized more liposome in the presence of dialysed or EGTA-chelated tumour-serum than that of liver cells derived from tumour transplanted rats. A significant increase in serum calcium concentration was found in all tumour-bearing rats. When the concentration of calcium in the serum of normal animals was increased to the level that is encountered in tumour-bearing rats, a sharp drop in liposome recognition by liver phagocytes was observed. This drop in opsonic activity was not related to changes in the ionic strength of serum. The ammonium sulphate precipitated opsonin was also calcium-sensitive and its opsonic activity was abolished in the presence of calcium. Studies with isolated splenic phagocytes suggested that an increase in the opsonic activity of serum, but not the elevated calcium level, was responsible for hyperphagocytosis of liposomes by the splenic phagocytes of tumour-transplanted animals. The opsonic molecule which enhanced liposome recognition by liver non-parenchymal cells failed to enhance liposome clearance by the splenic phagocytes. These findings suggest that the alteration in macrophage clearance of liposomes during the terminal growth of cancer may be mediated in part by changes in the opsonic capacity of serum.

Animals↗
PubMed1995

Uptake of drug-carrier liposomes by placenta: transplacental delivery of drugs and nutrients.

Liposomes with different charge, size and lipid composition were injected i.v. into pregnant rats and rabbits and their uptake and transport across the placenta were studied. Results show that the placenta is very efficient in removing liposomes from the maternal circulation and that it takes up more liposomes per gram of tissue than the liver. Liposomes are degraded intracellularly in the placenta and the entrapped material is then transported across to the fetus as free molecules. Under the experimental conditions described in this article, no intact liposomes were found to be transported across the placenta. In comparison to the i.v. injection, infusion of liposomes into pregnant rabbits had greater effect on the localization of liposomal drug in the placenta and fetal blood. In summary, these results show that small unilamellar vesicles enhance delivery of drugs and nutrients to the placenta. They also suggest the possible danger of toxicity to the fetus if the expectant female is undergoing liposomal drug treatment.

Animals↗