Diversity of killer cell immunoglobulin-like receptor genes in the Bengali population of northern West Bengal, India.

The Indian Subcontinent exhibits extensive diversity in its culture, religion, ethnicity and linguistic heritage, which symbolizes extensive genetic variations within the populations. The highly polymorphic Killer cell Immunoglobulin-like Receptor (KIR) family plays an important role in tracing genetic differentiation in human population. In this study, we aimed to analyse the KIR gene polymorphism in the Bengali population of northern West Bengal, India. To our knowledge, this is the first report on the KIR gene polymorphism in the Bengalis of West Bengal, India. Herein, we have studied the distribution of 14 KIR genes (KIR3DL1-3DL3, KIR2DL1-2DL5, KIR2DS1-2DS5 AND KIR3DS1) and two pseudogenes (KIR3DP1 and 2DP1) in the Bengalis. Apart from the framework genes (KIR2DL4, 3DL2, 3DL3 and 3DP1), which are present in all the individuals, the gene frequencies of other KIR genes varied between 0.34 and 0.88. Moreover, upon comparing the KIR polymorphism of the Bengalis with the available published data of other world populations, it has been found that the Indo-European-speaking Bengalis from the region share both Dravidian and Indo-Aryan gene pool with considerable influences of mongoloid and European descents. Furthermore, evidences from previously published data on human leucocyte antigen and Y-chromosome haplogroup diversity support the view. Our results will help to understand the genetic background of the Bengali population, in illustrating the population migration events in the eastern and north-eastern part of India, in explaining the extensive genetic admixture amongst the different linguistic groups of the region and also in KIR-related disease researches.

A study of the association of childhood asthma with HLA alleles in the population of Siliguri, West Bengal, India.

Asthma is a heterogeneous disease for which a strong genetic basis is firmly established. It is a complex disorder influenced by gene-environment interaction. Human leukocyte antigen (HLA) genes have been shown to be consistently associated with asthma and its related phenotypes in various populations. The aim of this study was to determine the frequency of the selected HLA classes I and II allelic groups in asthmatic and control groups. HLA typing was performed using polymerase chain reaction-sequence-specific typing (PCR-SSP) method. The allele frequency was estimated by direct counting. Frequency of each HLA allelic group was compared between asthmatic group and control group using χ(2) test. P-value was corrected by multiplying with the number of the allelic groups studied. Odds ratio (OR) and its corresponding 95% confidence interval (CI) for each allelic group were calculated using graphpad instat 3.10. The results of this study showed a significantly higher frequency of HLA-DRB1*03 in asthmatics than in controls (11.43% vs 3.64%, OR = 3.78, 95% CI = 1.61-8.85, P = 0.0025, Pcorr < 0.05). Analysis of HLA alleles in low and high total serum immunoglobulin E (IgE) level in asthmatics revealed no significant association. HLA-DRB1*03 may be implicated in the susceptibility to asthma in the pediatric population.

Chicken domestication: an updated perspective based on mitochondrial genomes.

Domestic chickens (Gallus gallus domesticus) fulfill various roles ranging from food and entertainment to religion and ornamentation. To survey its genetic diversity and trace the history of domestication, we investigated a total of 4938 mitochondrial DNA (mtDNA) fragments including 2843 previously published and 2095 de novo units from 2044 domestic chickens and 51 red junglefowl (Gallus gallus). To obtain the highest possible level of molecular resolution, 50 representative samples were further selected for total mtDNA genome sequencing. A fine-gained mtDNA phylogeny was investigated by defining haplogroups A-I and W-Z. Common haplogroups A-G were shared by domestic chickens and red junglefowl. Rare haplogroups H-I and W-Z were specific to domestic chickens and red junglefowl, respectively. We re-evaluated the global mtDNA profiles of chickens. The geographic distribution for each of major haplogroups was examined. Our results revealed new complexities of history in chicken domestication because in the phylogeny lineages from the red junglefowl were mingled with those of the domestic chickens. Several local domestication events in South Asia, Southwest China and Southeast Asia were identified. The assessment of chicken mtDNA data also facilitated our understanding about the Austronesian settlement in the Pacific.

Total serum immunoglobulin e in children with asthma.

Immunoglobulin (Ig) E has been shown to be a major contributing factor for the development of bronchial hyperresponsiveness in asthma. An elevation in serum IgE levels contributes to asthma and is considered a potent predictor of the development of asthma. The objectives of the present study were to estimate the levels of total serum IgE in asthmatic and healthy control subjects and to investigate the relationship of various demographic and clinical characteristics with the total serum IgE level in asthmatics. We measured the levels of total serum IgE using the ELISA kits (AccuBind, Monobind Inc., USA). The relevant demographic and clinical data were obtained using the questionnaire. The results showed that asthmatic children had significantly elevated level of total serum IgE compared to that of the healthy controls. The levels of total IgE and IL-4 in sera of 44 asthmatic children showed a significant positive correlation. Total serum IgE >150 IU/mL was found to be significantly associated with the age, exposure to cigarette smoke, and raised eosinophil count in asthmatic children. In conclusion, the elevated level of total serum IgE may demonstrate the allergic etiology of asthma in the subjects studied.

Monitoring protein folding and unfolding pathways through surface hydrophobicity changes using fluorescence and circular dichroism spectroscopy.

In the present study we have investigated the characteristics of folding and unfolding pathways of two model proteins, ovalbumin and alpha-lactalbumin, monitored through the changes in surface hydrophobicity using fluorescence and circular dichroism spectroscopy. In the unfolding process, it was observed that ovalbumin and alpha-lactalbumin followed a three state transition pathway involving an intermediate state having high surface hydrophobicity. The intermediate state has also been characterized by circular dichroism spectroscopy, and it was found that the intermediate retained almost the same secondary structure as the native proteins, and therefore it can be referred to as molten globule state. The refolding process was monitored using fluorescence and circular dichroism spectroscopy, and it was observed that the refolding of alpha-lactalbumin was reversible and proceeded through the accumulation of similar type of intermediates as observed during its unfolding pathway. However, on refolding from the guanidine hydrochloride-denatured state, ovalbumin reached a different folded state.

Synthesis and evaluation of dimethyl tin 4-cyclohexyl thiosemicarbazone as a novel antitumor agent.

AIM: To develop a rationally designed new organotin compound namely dimethyl tin 4-cyclohexyl thiosemicarbazone (D4-t) and evaluate its putative antitumor activity. METHODS: Starting from 4-cyclohexyl thiosemicarbazone, a three step synthetic procedure was followed to obtain the title compound. In vivo lymphocyte activation property of the compound at three different doses was assayed by measuring the blastogenesis. Concanavalin A (ConA) was used as standard mitogen for murine T cells stimulation in vivo . Also, the synthesis of DNA by the activated lymphocytes was measured after injecting the D4-t. The lymphocyte activation property and antitumor efficacy of D4-t were assessed in Sarcoma-180 (S-180) bearing mice. The organization of lymphoid cells was studied in the histological preparations of spleen and mesenteric lymph node. Tumor neutralization assay (Winn assay) was conducted to examine whether immune responses were associated with the manifestation of antitumor efficacies of this compound in S-180 in vivo . The DNA synthesis inhibitory effect of the compound in S-180 cells was studied in vitro, and was found significant (P < 0.001). RESULTS: Different doses of the new compound caused differential response of blastogenesis and DNA synthesis. In comparison to ConA, the title compound showed a good number of blast cells at its optimum dose of 5 mg/kg. It caused maximum synthesis of DNA by the lymphoid cells. In histological preparations, the gradual transformation of lymphocytes into blasts was observed without any visible toxicity. Winn assay revealed that 5 mg/kg of D4-t was able to reduce tumor mass without severe toxicity. This organotin compound also inhibits the synthesis of DNA in S-180 tumor cells in comparison to Platin10 and ConA. CONCLUSION: The title compound has the lymphocyte activation property and stimulates immune response of the lymphoid cells, which in turn express the antitumor activity without any significant toxicity. Results indicate promising therapeutic potential of D4-t.

Genetic affinities of north and northeastern populations of India: inference from HLA-based study.

India is like a microcosm of the world in terms of its diversity; religion, climate and ethnicity which leads to genetic variations in the populations. As a highly polymorphic marker, the human leukocyte antigen (HLA) system plays an important role in the genetic differentiation studies. To assess the genetic diversity of HLA class II loci, we studied a total of 1336 individuals from north India using DNA-based techniques. The study included four endogamous castes (Kayastha, Mathurs, Rastogies and Vaishyas), two inbreeding Muslim populations (Shias and Sunnis) from north India and three northeast Indian populations (Lachung, Mech and Rajbanshi). A total of 36 alleles were observed at DRB1 locus in both Hindu castes and Muslims from north, while 21 alleles were seen in northeast Indians. At the DQA1 locus, the number of alleles ranged from 11 to 17 in the studied populations. The total number of alleles at DQB1 was 19, 12 and 20 in the studied castes, Muslims and northeastern populations, respectively. The most frequent haplotypes observed in all the studied populations were DRB1*0701-DQA1*0201-DQB1*0201 and DRB1*1501-DQA1*0103-DQB1*0601. Upon comparing our results with other world populations, we observed the presence of Caucasoid element in north Indian population. However, differential admixturing among Sunnis and Shias with the other north Indians was evident. Northeastern populations showed genetic affinity with Mongoloids from southeast Asia. When genetic distances were calculated, we found the north Indians and northeastern populations to be markedly unrelated.

Chaperone mediated solubilization of 69-kDa recombinant maltodextrin glucosidase in Escherichia coli.

AIMS: To investigate the factors affecting expression and solubilization of Escherichia coli maltodextrin glucosidase in E. coli. METHODS AND RESULTS: Expression level and solubilization of the recombinant E. coli maltodextrin glucosidase was studied in E. coli at different temperatures, in presence of overexpressed GroEL, GroES and externally supplemented glycerol. Aggregation of maltodextrin glucosidase in the cytoplasm was partially prevented by the co-expression of GroEL and GroES, and using externally supplemented glycerol or lowering the culture temperature. Co-expression of GroEL and GroES or simultaneous presence of overexpressed GroEL, GroES and externally supplemented glycerol together resulted significant increase of the activity of maltodextrin glucosidase. The growth rate of E. coli was inhibited by the formation of inclusion bodies whereas the presence of overexpressed GroEL, GroES alone or together with glycerol enhanced the growth rate of E. coli substantially. CONCLUSIONS: The results indicated that lowering the temperature, use of GroEL, GroES and glycerol could be few controlling factors for the solubilization of recombinant aggregation-prone maltodextrin glucosidase in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study could help in developing the strategy for enhancing the production of soluble industrial enzymes and finding the therapeutic agents against protein misfolding diseases.

Laccase of Cyathus bulleri: structural, catalytic characterization and expression in Escherichia coli.

Cyathus bulleri, a ligninolytic fungus, produces a single laccase the internal peptides (3) of which bear similarity to laccases of several white rot fungi. Comparison of the total amino acid composition of this laccase with several fungal laccases indicated dissimilarity in the proportion of some basic and hydrophobic amino acids. Analysis of the circular dichroism spectrum of the protein indicated 37% alpha-helical, 26% beta-sheet and 38% random coil content which differed significantly from that in the solved structures of other laccases, which contain higher beta-sheet structures. The critical role of the carboxylic group containing amino acids was demonstrated by determining the kinetic parameters at different pH and this was confirmed by the observation that a critical Asp is strongly conserved in both Ascomycete and Basidiomycete laccases. The enzyme was denatured in the presence of a number of denaturing agents and refolded back to functional state with copper. In the folding experiments under alkaline conditions, zinc could replace copper in restoring 100% of laccase activity indicating the non-essential role of copper in this laccase. The laccase was expressed in Escherichia coli by a modification of the ligation-anchored PCR approach making it the first fungal laccase to be expressed in a bacterial host. The laccase sequence was confirmed by way of analysis of a 435 bp sequence of the insert.

HLA-A and HLA-B distribution in Toto - a vanishing sub-Himalayan tribe of India.

The frequency of HLA-A and HLA-B locus alleles was studied by using polymerase chain reaction-based sequence-specific primer method in a very primitive and vanishing sub-Himalayan Indian Tribe, the Toto population of North Bengal. The Toto, a Mongoloid tribe with a population size of 1172 reside only in the Totopara of Jalpaiguri district of North Bengal. We studied 40 individuals and observed some high frequency alleles when compared to other Indian tribal, non-tribal, and major world populations. Particularly, the frequency of HLA-B14 was 32.5% in the Toto population, the highest known frequency reported in any population in the world. This indigenous tribal population may harbour novel HLA alleles and unique haplotypes which extensive HLA genotyping will help to reveal, and thus further our understanding of their genetic admixture and migration patterns.

The role of HLA-G in cytokine homeostasis during early pregnancy complicated with maternal infections: a novel etiopathological approach to the neurodevelopmental understanding of schizophrenia.

Schizophrenia is perhaps the most enigmatic and tragic psychotic disorder with remarkable mortality and morbidity. Schizophrenia is complex and clinically a heterogeneous disorder. The etiological basis of schizophrenia ranges from autoimmune to neurodevelopmental hypothesis in one hand and involvement of different major gene segment with susceptibility loci on the other. Recently, neurodevelopmental hypothesis gained much impetus over the other domain. To support the neurodevelopmental basis, a number of investigations have shown that maternal infections during pregnancy increases the risk of the offspring developing schizophrenia and other neurodevelopmental disorders. The pathological mechanisms underlying this phenomenon is largely unknown. Many have suggested the involvement of different immune markers and shown that cytokines generated in response to maternal infection alter early brain development through their inflammatory activity. However, these findings have escaped discussion on various important issues related to cytokine homeostasis which depends on a large number of immune parameters including non-classical HLA-G molecules. Infections during early stages of pregnancy may alter cytokine regulation by disturbing the whole uterine immune milieu. To elucidate this issue, authors have tried to correlate the possible relationships between maternal infections and aberration of immune networking at the feto-maternal interface and their subsequent influence on the structural and functional abnormalities of the developing brain. The authors hypothesize that there exists a counter regulatory interaction among proinflammatory cytokines like TNF-alpha, HLA-G molecules and different immune cells like NK cells. We emphasize that HLA-G molecules are the novel immune players which maintain the immune homeostasis during early pregnancy in a manner that it can protect developing fetus from maternal immune attack. However, maternal infections may lead to the disturbance of HLA-G expression which in turn may fail to maintain its otherwise inhibitory potential to down regulate the detrimental inflammatory cytokines. Investigation on such interaction may unravel novel molecular mechanisms of neurodevelopmental basis of schizophrenia. Testing of our proposed hypothesis on animal models and on in vitro derived extravillous trophoblast cell lines holds promise of great insights to usher a new dimension of schizophrenia research and for developing new therapeutic strategies for better treatment and to adopt genetic prediction in schizophrenia management paradigm.

GroEL/GroES-mediated folding of a protein too large to be encapsulated.

The chaperonin GroEL binds nonnative proteins too large to fit inside the productive GroEL-GroES cis cavity, but whether and how it assists their folding has remained unanswered. We have examined yeast mitochondrial aconitase, an 82 kDa monomeric Fe(4)S(4) cluster-containing enzyme, observed to aggregate in chaperonin-deficient mitochondria. We observed that aconitase folding both in vivo and in vitro requires both GroEL and GroES, and proceeds via multiple rounds of binding and release. Unlike the folding of smaller substrates, however, this mechanism does not involve cis encapsulation but, rather, requires GroES binding to the trans ring to release nonnative substrate, which likely folds in solution. Following the phase of ATP/GroES-dependent refolding, GroEL stably bound apoaconitase, releasing active holoenzyme upon Fe(4)S(4) cofactor formation, independent of ATP and GroES.

Equilibrium and kinetic studies on folding of the authentic and recombinant forms of human alpha-lactalbumin by circular dichroism spectroscopy.

The equilibrium and kinetics of the unfolding and refolding of authentic and recombinant human alpha-lactalbumin, the latter of which had an extra methionine residue at the N-terminus, were studied by circular dichroism spectroscopy, and the results were compared with the results for bovine and goat alpha-lactalbumins obtained in our previous studies. As observed in the bovine and goat proteins, the presence of the extra methionine residue in the recombinant protein remarkably destabilized the native state, and the destabilization was entirely ascribed to an increase in the rate of unfolding. The thermodynamic stability of the native state against the unfolded state was lower, and the thermodynamic stability of the molten globule state against the unfolded state was higher for the human protein than for the other alpha-lactalbumins previously studied. Thus, the population of the molten globule intermediate was higher during the equilibrium unfolding of human alpha-lactalbumin by guanidine hydrochloride. Unlike the molten globule states of the bovine and goat proteins, the human alpha-lactalbumin molten globule showed remarkably more intense circular dichroism ellipticity than the native state in the far-ultraviolet region below 225 nm. During refolding from the unfolded state, human alpha-lactalbumin thus exhibited overshoot kinetics, in which the alpha-helical peptide ellipticity exceeded the native value when the molten globule folding intermediate was formed in the burst phase. The subsequent folding involved reorganization of nonnative secondary structures. It should be noted that the rate constant of the major refolding phase was approximately the same among the three types of alpha-lactalbumin and that the rate constant of unfolding was accelerated 18-600 times in the human protein, and these results interpreted the lower thermodynamic stability of this protein.

Acute mesenteric ischemia and malpractice claims.

BACKGROUND: Acute mesenteric ischemia can be a difficult diagnosis to make, but delay contributes directly to infarction, and this may provide a setting for malpractice claims. METHODS: We reviewed 180 consecutive malpractice claims submitted by attorneys for medical expert (ME) review during the 12 years ending in late 1998. Seven cases involved acute mesenteric ischemia. RESULTS: Alleged failure to make a timely diagnosis was the basis for 5 of these claims, failure to provide anticoagulant protection for 1, and failure to prevent nonocclusive ischemic infarction for 1. Six claims were closed after ME review and 1 claim involving late diagnosis was settled before trial. CONCLUSIONS: The risk of a malpractice claim is reduced by consideration of computed tomography (CT), angiography, and surgical consultation as soon as a patient is seen whose differential diagnosis includes acute mesenteric ischemia.

Role of hla class-I antigens in delusional disorder.

The investigation was conducted to find out whether there is any association between delusional disorder and HLA antigens. The sample comprised 50 patients with delusional disorder and 282 control samples collected from normal controls. Statistical analysis revealed that the frequency of A3 antigen of the locus A are significantly higher. In case of HLA - B locus significantly higher frequency of B5 and B21 antigens have also been observed. The present study shows that there may be some association of HLA class-1 antigens with delusional disorder.

Necrotizing fasciitis and malpractice claims.

BACKGROUND: Necrotizing fasciitis (NF) is an aggressive bacterial infection of the superficial fascia and subcutaneous tissues that is increasing in incidence. The high toll exacted by this illness provides a setting for malpractice claims. METHOD: We reviewed 180 consecutive malpractice claims submitted by attorneys for medical expert review between 1987 and late 1997. Four cases involved NF. RESULTS: Alleged failure to obtain timely surgical consultation was the basis for three claims, and alleged failure to prevent NF by proper nursing care was the basis for the fourth. Three cases were closed and one was settled. CONCLUSIONS: The cornerstone of risk management for a clinical presentation compatible with NF is immediate surgical consultation, with other diagnostic steps a secondary consideration.

Effect of the extra n-terminal methionine residue on the stability and folding of recombinant alpha-lactalbumin expressed in Escherichia coli.

The structure, stability, and unfolding-refolding kinetics of Escherichia coli-expressed recombinant goat alpha-lactalbumin were studied by circular dichroism spectroscopy, X-ray crystallography, and stopped-flow measurements, and the results were compared with those of the authentic protein prepared from goat milk. The electric properties of the two proteins were also studied by gel electrophoresis and ion-exchange chromatography. Although the overall structures of the authentic and recombinant proteins are the same, the extra methionine residue at the N terminus of the recombinant protein remarkably affects the native-state stability and the electric properties. The native state of the recombinant protein was 3.5 kcal/mol less stable than the authentic protein, and the recombinant protein was more negatively charged than the authentic one. The recombinant protein unfolded 5.7 times faster than the authentic one, although there were no significant differences in the refolding rates of the two proteins. The destabilization of the recombinant protein can be fully interpreted in terms of the increased unfolding rate of the protein, indicating that the N-terminal region remains unorganized in the transition state of refolding, and hence is not involved in the folding initiation site of the protein. A comparison of the X-ray structures of recombinant alpha-lactalbumin determined here with that of the authentic protein shows that the structural differences between the proteins are confined to the N-terminal region. Theoretical considerations for the differences in the conformational and solvation free energies between the proteins show that the destabilization of the recombinant protein is primarily due to excess conformational entropy of the N-terminal methionine residue in the unfolded state, and also due to less exposure of hydrophobic surface on unfolding. The results suggest that when the N-terminal region of a protein has a rigid structure, expression of the protein by E. coli, which adds the extra methionine residue, destabilizes the native state through a conformational entropy effect. It also shows that differences in the electrostatic interactions of the N-terminal amino group with the side-chain atoms of Thr38, Asp37, and Asp83 bring about a difference in the pKa value of the N-terminal amino group between the proteins, resulting in a greater negative net charge of the recombinant protein at neutral pH.

Pulmonary embolism and malpractice claims.

BACKGROUND: Deep venous thrombosis (DVT) with pulmonary embolization (PE) often occurs as an unexpected event with fatal consequences. This provides a setting for malpractice claims. METHODS: We reviewed 160 consecutive malpractice claims submitted by attorneys for medical expert review during the 11-year period ending in 1997. Seven cases involved DVT with PE. RESULTS: Alleged failure to anticipate and reduce the chance of PE was the basis for six of the claims. All six patients were at risk for lower extremity DVT, and one had a history of DVT 6 months earlier. The PE was manifested by sudden death in three cases. The seventh case represented a complication of heparin therapy for PE. CONCLUSIONS: We conclude that risk management for PE should focus primarily on DVT. Physicians should perform and document an examination for DVT whenever there is a history of lower extremity stasis or it is likely to occur. They should also consider documenting a concurring second opinion when making anticoagulant-related decisions.