Degradation of hexachlorocyclohexane isomers by two strains of alcaligenes faecalis isolated from a contaminated site.
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Alcaligenes↗
Biomedical subjects
A Kaushik
Publications and source records attributed to A Kaushik.
Cloning and characterization of the human GFRA2 locus and investigation of the gene in Hirschsprung disease.
The glial-cell-line-derived neurotrophic factor (GDNF) family receptors alpha (GFRalpha) are cell surface bound glycoproteins that mediate interactions of the GDNF ligand family with the RET receptor. These interactions are crucial to the development of the kidney and some peripheral nerve lineages. In humans, mutations of RET or RET ligands are associated with the congenital abnormality Hirschsprung disease (HSCR) in which nerves and ganglia of the hind gut are absent. As the GFRalpha family are required for normal activation of the RET receptor, they are also candidates for a role in HSCR. The GFRA2 gene, which is required for the development of the myenteric nerve plexus, is an excellent candidate gene for HSCR. In this study, we cloned the human GFRA2 locus, characterized the gene structure, and compared it with other GFRA family members. We further investigated the GFRA2 gene for mutations in a panel of HSCR patients. GFRA2 has nine coding exons that are similar in size and organization to those of other GFRA family genes. We identified six sequence variants of GFRA2, four of which did not affect the amino acid sequence of the GFRalpha-2 protein. Two further changes that resulted in amino acid substitutions were found in exon 9 and were predicted to lie in the amino acid sequence encoding the glycosylphosphatidylinositol-linkage signal of GFRalpha-2. There was no difference in frequency of any of the sequence variants between control and HSCR populations. Our data indicate that members of the GFRA gene family are closely related in intron/exon structure and in sequence. We have not detected any correlation between sequence variants of GFRA2 and the HSCR phenotype.
Base Sequence↗
Origin of bovine IgM structural variants.
The structure of IgM determined from two cDNAs isolated from a Holstein (BLV7G1) and an Angus x Hereford cross-bred (B5D8) cow reveals high sequence similarity both at nucleotide (98.7%) and amino acid (97.9%) level and is closest to sheep (89.4%). Three bovine IgM allotypes, designated as IgMa, IgMb and IgMc, are classified based on nucleotide substitutions in all the Cmu exons resulting in amino acid replacements. Further, insertion of three in-frame codons at Cmu1 and Cmu2 junction of B5D8 IgM from the intervening intron, via cleavage of pre-mRNA at an alternate cryptic 5' splice donor site, leads to generation of additional bovine IgM variants. The C1q-binding site, involved in classical complement pathway, is identified in bovine IgM where ten amino acids are conserved across species. Interestingly, bovine IgM has the lowest number of proline residues (5) in the Cmu2 domain in comparison to other species (7-9) and this is likely to impose structural constraints on mobility of Fab arms of the bovine IgM during antigen recognition. The rigidity in the bovine IgM Cmu2 domain may, however, facilitate exposure of C1q-binding site subsequent to antigen binding and enhance its complement fixing ability. The restricted mobility of bovine IgM Fab arms may possibly favor generation of an antigen-combining site requiring an unusually long third complementarity determining region of the heavy chain (CDR3H), apart from antigen selection of variable domains. This is consistent with the fact that an exceptionally long CDR3H has not been observed in bovine IgG which bears a long and more flexible hinge region. Additional hydrophilic threonine and serine residues in the Cmu2 domain of bovine IgM, as compared to other species, however, enhance its ability to extend into the solvent. Finally, restricted fragment length polymorphism analysis of genomic DNA from four cattle breeds reveals the presence of; at least, four allelic variants of bovine Cmu gene.
Amino Acid Sequence↗
[Reconstruction of the chain of the auditory ossicles after radical operation on the middle ear].
As restoration of hearing in patients after radical surgery on the middle ear for otitis media purulenta chronica remains topical, we tried to increase the efficacy of ossiculoplasty in such patients by staged reconstruction of the middle ear. A total of 102 patients were examined and operated on. A modified classification of osseous chain defects was used to evaluate the degree of damage to the osseous system. The following policy was employed for reconstruction of the middle ear: in patients with large trepanation cavities (more than 5 ml) mastoidoplasty was carried out as the first stage using a musculo-periosteal flap with an anterior or inferior pedicle; at the second stage, myringoplasty was performed using a two-layer graft followed by ossiculoplasty with autotransplants (fingernail plate, auricular or tragal cartilage, cortical layer bone). Long-term follow-up of the patients showed that good and satisfactory hearing levels were obtained in 79.4% of the patients. The air-bone gap was closed by 10 dB or more in the speech frequency range in 80.4% of the patients. Staged reconstruction of the middle ear using autotransplant materials significantly decreased the number of postoperative complications (perforation of the plastic flap, cicatrical displacement of the prosthesis and graft rejection).
Adolescent↗
Leuvectin Vical Inc.
Vical's Leuvectin, a plasmid DNA expression vector encoding interleukin-2 (IL-2) complexed with a lipid delivery vehicle (DMRIE/DOPE)for intratumoral injection, is under development as a potential gene therapy for cancer. In 1997, it was in phase II trials for kidney and prostate cancer [249445], [270243]. In April 2001, the phase II trial in kidney cancer was discontinued, with a new phase II trial planned for this indication [406405]. In May 2001, prostate cancer trials were ongoing [409071]. By March 1999, testing for other indications had been suspended, with the company's priority being to work toward the most promising indications [316709]. In November 2000, it was announced that Vical has been issued US-06147055 for the use of gene-based, non-viral delivery of IL-2 for the treatment of cancer. The company was also issued US-05641665 covering the Leuvectin construct in 1997 [389677].
Animals↗
Exceptionally long CDR3H region with multiple cysteine residues in functional bovine IgM antibodies.
We analyzed VDJ and VJ rearrangements in IgM-secreting B lymphocytes from a cow infected with bovine leukemia virus (BLV). BLV causes expansion of CD5(+) and IgM(+) B lymphocytes regardless of antigen specificity. The data showed that single point mutations contribute to the diversification of IgM antibodies. The most striking observation, however, is that approximately 9% of theVDJ rearrangement in IgM-secreting B cells encode an exceptionally long third complementarity-determining region of the heavy chain (CDR3H; 56 to 61 amino acids) with multiple cysteine residues. Such an exceptionally long CDR3H is the first ever to be documented for an antibody in a species. These VDJ rearrangements encode functional IgM antibodies as some of these show polyspecific reactivity. The presence of even-numbered cysteine residues in the CDR3H may provide hitherto unknown configurational ability to the antigen combining site via intra-CDR3H disulfide bridging. In addition, the VDJ rearrangements encoding exceptionally long CDR3H paired with either novel V(lambda)1 or V(x)1x genes, earlier noted not to be expressed. Overall, these experiments provide evidence that somatic hypermutations and generation of an exceptionally long CDR3H contribute to the diversification of IgM antibodies in cattle.
Amino Acid Sequence↗
Need for research in companion animal vaccinology.
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Animal Welfare↗
The effects of prednisone and azathioprine on circulating immunoglobulin levels and lymphocyte subpopulations in normal dogs.
This study investigates serum immunoglobulin (SIg) levels and lymphocyte subpopulations in normal dogs in response to putative immunosuppressive doses of prednisone and/or azathioprine. The objectives were to quantify SIg levels and lymphocyte subpopulations, including Thy-1+, CD4+, CD8+ and B cells, in normal dogs both before and after the administration of prednisone and/or azathioprine at 2 mg/kg, PO, each. Eighteen beagles were divided into 3 groups of 6 dogs each. Blood samples for radial immunodiffusion assay of IgG, IgM and IgA, complete blood count (CBC)and flow cytometry were collected prior to the administration of any drugs and again after 14 d of azathioprine, prednisone or azathioprine and prednisone. Peripheral blood mononuclear cells were isolated using density centrifugation and were incubated with monoclonal antibodies reacting with CD4+, CD8+, Thy-1+ and membrane immunoglobulin. Lymphocyte subsets were quantified using flow cytometry. Azathioprine-treated dogs had no significant changes in SIg levels or lymphocyte subpopulations. Prednisone-treated dogs had significant (P < 0.05) decreases in all SIg levels, all lymphocyte subpopulations and erythrocyte numbers, and had an increase in neutrophil counts. Prednisone and azathioprine-treated dogs had significant (P < 0.05) decreases in serum IgG levels and Thy-1+ and CD8+ lymphocyte subpopulations, with an increase in the CD4:CD8. These dogs also had a significant decrease in erythrocyte number and a significant increase in the monocyte count. These findings suggest that azathioprine and prednisone in combination or prednisone alone may be useful for the treatment of T cell-mediated diseases since decreased circulating T cell levels were demonstrated following treatment. The combination of drugs or azathioprine alone may not be appropriate for treatment of acute or autoantibody-mediated immune disease, because SIg levels were minimally affected by treatment.
Animals↗
The primary antibody repertoire of kappa-deficient mice is characterized by non-stochastic Vlamda1 + V(H) gene family pairings and a higher degree of self-reactivity.
We have investigated the primary antibody repertoire of genetically manipulated 129/Sv kappa-deficient (JCkappaD) mice, in order to understand the contributions of the lambda-light chain, in the absence of an otherwise predominant kappa-light chain, to the development of humoral immunity. The expression of Vlambda1 gene (lambda1 and lambda3 subtypes) and the Vlambda1 + V(H) (J558, 36-60, V(H)11 and S107) gene family associations were studied in 7.43 x 10(3) mitogen-activated splenic B-lymphocyte clones of JCkappaD origin. Furthermore, the functional significance of the exclusive expression of the lambda-light chain, in the peripheral B-cell repertoire of JCkappaD mice, was analysed by determining natural autoantibody specificities in the circulating serum immunoglobulin and the frequency of autoreactive B-lymphocyte clones in the peripheral B-lymphocyte repertoire. These experiments revealed that: first, of the three available Vlambda genes at the lambda locus, the Vlambda1 gene is the one that is expressed most frequently (59.9%); second, non-random Vlambda1 + V(H) (J558, 36-60) gene family pairings occur in kappa-deficient mice; and third, a higher degree of self-reactivity is generated as a result of exclusive use of the lambda-light chain, as evidenced by higher levels of serum natural autoantibodies as well as a high frequency of autoreactive B-lymphocyte clones in kappa-deficient (129/Sv JCkappaD) mice. These observations suggest that the high murine kappa/lambda ratio in mice may, apart from high sequence diversity at the kappa-locus, be a result of endogenous selection against the lambda-light chain to restrict self-reactivity within the homeostatic threshold.
Animals↗
CDRH3 length is the target of selection of disease-associated IgM autoantibodies.
The experiments outlined here provide evidence for positive selection of B-1 lymphocytes secreting IgM autoantibodies in the autoimmune mev mice as a result of the reduced threshold of responsiveness to autoantigens because of PTP1C deficiency, which is targeted at the CDR3 length of the variable region of the heavy chain. In addition, characteristic differences in the size, hydrophobicity pattern, and relative local charge of the CDR3 of the heavy chain of disease-associated IgM autoantibodies from moth-eaten mice help distinguish them from physiological natural autoantibodies present in normal mice.
Animals↗
A single predominantly expressed polymorphic immunoglobulin VH gene family, related to mammalian group, I, clan, II, is identified in cattle.
In order to understand the generation of antibody diversity in cattle, seven cDNAs, from heterohybridomas secreting bovine IgM and IgG1 antibodies, were cloned and structurally analyzed for rearranged bovine VDJ genes. All of the seven bovine VH genes, together with four available bovine VH gene sequences, shared a high nucleotide sequence homology (84.2-93.5%). Based upon the criteria of nucleic acid homology > or =80%, all of the bovine VH gene sequences isolated from the expressed antibody repertoire constitute a single VH gene family, which we have designated as bovine VH1 (Bov VH1). An analysis of 44 bovine IgM-secreting mouse x cattle heterohybridomas, originating from polyclonally-activated PBLs from bovine leukemia virus-infected cattle, revealed that all of these expressed Bov VH1 (100%) based upon DNA sequencing and Northern dot blot. The bovine VH genes showed highest DNA sequence similarity, ranging between 81.5 and 87.6%, with a single sheep VH gene family (related to human VH4) and are, thus, closest to the VH genes from another ruminant species. The Bov VH1 gene family is most homologous to the murine VH Q-52 (71.8-78%) and human VH4 (67.4-69.8%) gene families, which belong to mammalian group, I, clan, II, VH genes. The CDR3 length of rearranged bovine VDJ genes is characteristically long (15-23 amino acids). The bovine JH gene segments were most homologous to human JH4 (82.1-87.2%) and JH5 (84.6-89.7%) genes, suggesting the existence of at least two JH gene segments. An analysis of CDRs provides evidence that somatic hypermutations contribute significantly to the generation of antibody diversity in cattle. Southern blot analysis of BamH I, EcoR I and Hind III digested genomic DNA from four cattle breeds (Holstein, Jersey, Hereford and Charolais) revealed three RFLP patterns; the genomic complexity of Bov VH1 ranged between 13 and 15 genes. These observations provide evidence for polymorphism at the bovine Ig-VH locus, similar to that seen in mice and humans.
Amino Acid Sequence↗
Restricted CDR3 length of the heavy chain is characteristic of six randomly isolated disease-associated VH J558+ IgM autoantibodies in lupus prone motheaten mice.
To investigate the origin of disease-associated IgM autoantibodies (AAb), we compared the genetic and structural characteristics of IgM AAb from autoimmune prone motheaten (mev) mice with natural autoantibodies (NAAb) from normal background C57/BL6 strain. Six hybridoma-derived IgM molecules each were obtained both from mev mice, at the terminal stage of systemic autoimmune disease, and from mitogen-stimulated C57/BL6 mice. These were randomly selected for VH J558 gene expression (aberrantly expressed in mev mice). The variable regions of the IgM molecules, both from autoimmune and normal mice, were encoded by unmutated germline VH genes. Disease-associated AAb from mev mice were predominantly encoded by the J558 subfamily 186.2, whereas five J558 subfamilies were utilized in NAAb originating from normal mice. Junctional diversity as a result of N or P nucleotide insertions and D-D fusions was noted among IgMs originating from both mev (mostly B-1 lymphocytes) and C57BL/6 (mostly B-2 lymphocytes) mice. Interestingly, all six J558+ IgMs from mev mice showed a restricted CDR3 length of 10 amino acids, with similar hydrophobicity indices. Four unique V-D-J rearrangements were observed among these IgMs. None of the IgMs were polyreactive and three of the six were subsequently observed to express monospecific autoreactivity with synthetic peptides (residues 81-92 and 37-53) representing segments of the T cell CD4-accessory molecule. Three IgM antibodies had hydrophilic arginine residues in their CDR3 heavy chain region. By contrast, all six J558+ IgMs from C57/BL6 mice had variable CDR3 length, distinct VDJ rearrangements and a local negative charge in the CDR3 region. Four of these IgMs demonstrated polyreactivity with multiple conserved autoantigens and, hence, were classified as NAAb. These findings provide evidence for either positive or impaired negative selection of B-1 lymphocytes secreting disease-associated IgM AAb in mev mice. This likely results from a reduced threshold of responsiveness to autoantigens due to PTP1C deficiency, which is targeted at the CDR3 length of the variable region of the heavy chain. In addition, characteristic differences in the size and hydrophobicity pattern of the CDR3 of the heavy chain allow structural distinction between monospecific disease-associated IgM AAb and the polyreactive IgM NAAb.
Amino Acid Sequence↗
Association of cerebral palsy with epilepsy.
Fifty patients of various types of cerebral palsy were studied to find out an association between cerebral palsy, EEG abnormalities and development quotient. Seventy-six per cent patients had spastic cerebral palsy. Hypotonic cerebral palsy was the next common type (14%). Athetosis and ataxic forms were found to be rare (2% each). Epilepsy was associated with 56% patients. Clinical types of seizures observed were: Generalised tonic-clonic (43%), myoclonic (17.9%), generalised tonic (10.7%), partial simple (10.7%) and partial complex (17.9%). The incidence of seizures was highest in hypotonic type in which 85.7% had epilepsy. Mean developmental quotient of cerebral palsy patients was 34.9% with maximum retardation in hypotonic cerebral palsy (25.14%). Sixty per cent of patients had abnormal EEG, out of these hypotonic patients had maximum (70%) chances of EEG abnormality followed by spastic patients (55%). Developmental retardation was more severe statistically in the patients with abnormal EEG than normal EEG.
Cerebral Palsy↗
The primary antibody repertoire of normal, immunodeficient and autoimmune mice is characterized by differences in V gene expression.
During the last decade, the structure and organization of the immunoglobulin heavy and light chain locis have been defined in mice and humans. Studies on VH gene expression at different stages of development, in different organs and disease states have provided useful insight into the construction of a primary antibody repertoire in mice. Clearly, 3'VH genes 7183, Q52 and Vh11, which are conserved during evolution, are preferentially expressed during early development of the B-lymphocyte repertoire. A preferential use for the V kappa 4 gene family is evident during early B-cell development. The initial development of the primary antibody repertoire is therefore influenced by a restricted set of VH and V kappa gene elements. The restricted B-cell repertoire is subsequently normalized in the periphery, as revealed by stochastic VH gene expression, as a result of exposure to environmental antigens. Obviously, the peripheral B-cell pool characterized by stochastic VH gene expression is selectively replenished by newly generated B cells in bone marrow that preferentially expresses 3'VH genes. The V kappa genes are, however, expressed in a non-random manner in the neonatal and adult B-lymphocyte repertoire that is probably related to VH and V kappa association dynamics and/or positive or negative selection. Interestingly, these characteristics of neonatal and adult primary repertoire are noted in both B1 and B2 lymphocytes. No remarkable age-related differences are evident for VH and V kappa gene expression. In healthy mice, both the mitogen responsive (available) and unstimulated (expressed) B-cell repertoire show similar VH gene expression. Interestingly, VH gene expression varies in different organs which may reflect, or occur as a result of, the specialized function of each organ. For example, J558 gene expression is higher in the peripheral LN where B cells continuously encounter exogenous antigens. The skewed VH and V kappa gene expression noted in immunodeficient and autoimmune lupus-prone mice reflects the impairment of the primary antibody repertoire associated with immunodeficiency and autoimmune disorders.
Animals↗
V lambda-light chain genes reconstitute immune responses to defined carbohydrate antigens or haptens by utilizing different VH genes.
The contribution of the lambda-light chain to the development of peripheral B cell repertoire and generation of specific antibodies to haptens and polysaccharide antigens was studied in genetically manipulated kappa-deficient and lambda 2-transgenic mice. The results clearly demonstrate a non-stoichiometric VH gene family expression in the absence of k-light chain and suggest a non-stochastic pairing between VH and V lambda genes, expressed in the peripheral B cell repertoire. A shift in VH gene utilization in the case of VI lambda + antibodies was evident in response to beta 2-6 fructosan and TNP hapten. These observations demonstrate the availability of compensatory mechanisms in the absence of VK genes and are consistent with the hypothesis that VH gene family expression is controlled by genetic factors from inside the VH locus. Furthermore, genetic factors from outside the VH locus, namely restricted available light chain diversity, may lead to a shift in VH gene utilization in the peripheral B cell repertoire.
Animals↗
Homologues of murine Vh11 gene are conserved during evolution.
Since the murine Vh11 gene family shows 75% homology with group III VH gene families and is preferentially expressed in B-1 lymphocytes, we have analyzed if corresponding Vh11 genes existed across phylogenetically distant mammalian lineages. In a Southern blot, homologues of the murine Vh11 gene were detected in the genomic DNA from rats, pigs, sheep, cattle, horses, dogs and man. These observations suggest conservation of corresponding sequences of the Vh11 gene family during evolution, similar to group III VH genes, either because of strong selection pressures essential for species fitness and survival or molecular drive leading to these consequences.
Animals↗
Diverse VH and V kappa genes encode antibodies to Pseudomonas aeruginosa LPS.
The molecular nature of the murine Ab response to Pseudomonas aeruginosa LPS was examined using a panel of 10 well-defined anti-LPS mAbs. Abs to P. aeruginosa LPS are encoded by diverse V-genes, with at least five VH and four V kappa gene families represented in these Abs. The Abs that bind to hydrophilic O-polysaccharide side chains of B-band LPS and A-band LPS are encoded by VH J558, SM7, and J606 gene families, while Abs to hydrophobic core and lipid A regions are encoded by X24, SM7, and Q52 gene families. All active JH and only two J kappa (J kappa 2 and J kappa 5) germ-line genes are utilized in the anti-LPS Abs examined. Four of six anti-P. aeruginosa mAbs used diversity genes of the DSP2 gene family. Interestingly, JH1 and JH2 use was observed in three mAbs that reacted with hydrophilic LPS epitopes (O-polysaccharide, A-band LPS), whereas JH3 and JH4 use was observed in three mAbs that bound to the more hydrophobic regions of LPS (core, lipid A). Point mutations were observed in framework and complementarity-determining regions (CDRs) of VH and VL genes, suggesting an Ag-driven maturation process in response to P. aeruginosa LPS. Mutations occurred in all heavy chain CDRs, as well as in CDR1 and CDR3 of the light chain, indicating an important role of these regions in binding to LPS. These data suggest that diverse VH and V kappa genes encode Abs to LPS from P. aeruginosa.
Amino Acid Sequence↗
Contribution of the V lambda light chain to the development of the primary antibody repertoire.
The experiments outlined here provide evidence that most VH gene families are expressed in association with either of three V lambda genes available in kappa-deficient mice, though their expression is nonrandom. An exclusive use of V lambda L-chain genes, where predominantly expressed V lambda 1 and V lambda 2 differ by only seven amino acid substitutions in the germline, results in a higher degree of self-reactive antibodies to conserved natural antigens like albumin, thyroglobulin, and stress proteins. The higher levels of self-reactive antibodies, characteristic of preimmune B-cell repertoire, are likely to be a product of nonrandom VH gene family expression and their restricted pairing with three V lambda L-chain genes. These data suggest that a low lambda L-chain expression in normal mice is due, in part, to endogenous selection against lambda L chain in order to restrict self reactivity within the homeostatic threshold.
Animals↗