Screening of C-kit gene Mutation in Acute Myeloid Leukaemia in Northern India.

BACKGROUND: Acute Myeloid Leukaemia (AML) is a cancer of blood-forming cells in bone marrow. C-kit gene is a Receptor Tyrosine Kinase class III (RTK) that is expressed by early hematopoietic progenitor cells and plays an important role in hematopoietic stem cell proliferation, differentiation and survival. It is known that c-kit is a proto-oncogene and the activating c-kit mutations are likely to contribute in the development of leukaemia in humans. Exon 11 of c-Kit gene is the frequent site for mutations in different kinds of tumours. METHODS: In order to determine the frequency and prevalence of exon 11 mutations in 51 AML cases, we have done polymerase chain reaction-single-strand conformational polymorphism followed by direct DNA sequencing. RESULTS: The c-kit mutations in exon 11 were detected in 15.68% (8/51) in AML cases. We have detected totally ten missense mutations in eight AML cases those include Lys550Asn, Tyr568Ser, Ile571Leu, Tyr578Pro, Trp582Ser and Arg588Met and novel missense mutations at codons Ile563Lys and Val569Leu. Mutations at codons Ile571Leu and Trp582Ser was found in two independent cases. CONCLUSION: The presence of c-kit mutations in our study adds to investigative spectrum of AML cases. Since the c-kit mutations are seen in other malignancies, mutations in exon 11 of the c-kit gene might be involve in pathogenesis and represent useful predictive genetic marker in AML. Further studies in larger group of cases possibly will be required to determine the prognostic implications and to investigate how these mutations are co-related to the progression and pathogenesis of AML.

Diabetic neuroarthropathy.

OBJECTIVE: This study was undertaken to assess the incidence of diabetic neuroarthropathy and its related morbidity. METHODS: The medical records of 296 diabetic patients were analyzed retrospectively between June of 1998 and July of 1999. The patients with long standing, poorly controlled diabetes mellitus and associated peripheral neuropathy were evaluated clinically and radiographically for the presence of arthropathic changes in the feet. Clinically, neuropathy was considered if there was absence of ankle jerk or glove and stocking sensory loss, or both. Radiographically, the presence of stress fractures, dislocation/subluxation, lytic or arthritic lesions of the bone and joints were taken as indicative of the disease. They were treated conservatively by total contact casting or surgically in the form of ray excision, amputation and skin grafting. They were followed up for an average period of 13 months. Results were evaluated clinically and radiographically. RESULTS: The maximum incidence of diabetes mellitus was in the age group of 41-80 years. Diabetic neuropathy was present in 37 patients (12.5%). Male to female ratio was 23:14 with an average age of 70.42 years. The mean duration of diabetes mellitus was 14.2 years. Seventeen feet in 11 patients (4%) were found to have diabetic neuroarthropathy. The joints involved were tarsometatarsal (76%), metatarsophalangeal (59%), subtalar (47%) and interphalangeal joints (41%). Two patients underwent foot amputations. Patients treated with total contact casting resulted in satisfactory progress. CONCLUSION: Diabetic neuroarthropathy, a less recognized complication of diabetes mellitus needs greater attention in Saudi Arabia. High-risk feet should be subjected to routine radiographs or preferably a computerized tomography examination. The timely detection of this problem can save many patients from disastrous complications.

Diabetic neuroarthropathy.

OBJECTIVE: This study was undertaken to assess the incidence of diabetic neuroarthropathy and its related morbidity. METHODS: The medical records of 296 diabetic patients were analyzed retrospectively between June of 1998 and July of 1999. The patients with long standing, poorly controlled diabetes mellitus and associated peripheral neuropathy were evaluated clinically and radiographically for the presence of arthropathic changes in the feet. Clinically, neuropathy was considered if there was absence of ankle jerk or glove and stocking sensory loss, or both. Radiographically, the presence of stress fractures, dislocation/subluxation, lytic or arthritic lesions of the bone and joints were taken as indicative of the disease. They were treated conservatively by total contact casting or surgically in the form of ray excision, amputation and skin grafting. They were followed up for an average period of 13 months. Results were evaluated clinically and radiographically. RESULTS: The maximum incidence of diabetes mellitus was in the age group of 41-80 years. Diabetic neuropathy was present in 37 patients (12.5%). Male to female ratio was 23:14 with an average age of 70.42 years. The mean duration of diabetes mellitus was 14.2 years. Seventeen feet in 11 patients (4%) were found to have diabetic neuroarthropathy. The joints involved were tarsometatarsal (76%), metatarsophalangeal (59%), subtalar (47%) and interphalangeal joints (41%). Two patients underwent foot amputations. Patients treated with total contact casting resulted in satisfactory progress. CONCLUSION: Diabetic neuroarthropathy, a less recognized complication of diabetes mellitus needs greater attention in Saudi Arabia. High-risk feet should be subjected to routine radiographs or preferably a computerized tomography examination. The timely detection of this problem can save many patients from disastrous complications.

Cloning and expression of beta-glucosidase genes in Escherichia coli and Saccharomyces cerevisiae using shuttle vector pYES 2.0.

Genes for beta-glucosidase (Bgl) isolated from a genomic library of the cellulolytic bacterium, Cellulomonas biazotea, were cloned in pUC18 in its SacI cloning site and transformed to E. coli. Ten putative recombinants showed blackening zones on esculin plates, yellow zones on pNPG plates, in liquid culture and on native polyacrylamide gel electrophoresis activity gels. They fell into three distinct groups. Three representative E. coli clones carried recombinant plasmids designated pRM54, pRM1 and pRM17. The genes were located on 5.6-, 3.7- and 1.84-kb fragments, respectively. Their location was obtained by deletion analysis which revealed that 5.5, 3.2, and 1.8 kb fragments were essential to code for BglA, BglB, and BglC, respectively, and conferred intracellular production of beta-glucosidase on E. coli. Expression of the bgl genes resulted in overproduction of beta-glucosidase in the three clones. Secretion occurred into the periplasmic fractions. Three inserts carrying bgl genes from the representative recombinant E. coli were isolated with SacI, ligated in the shuttle vector pYES 2.0 in its SacI site and transformed to E. coli and S. cerevisiae. The recombinant plasmids were redesignated pRPG1, pRPG2 and pRPG3 coding for BglA1, BglB1 and BglC1. The cloned genes conferred extracellular production of beta-glucosidase on S. cerevisiae and enabled it to grow on cellobiose and salicin. The gall promoter of shuttle vector pYES 2.0 enabled the organisms to produce twice more beta-glucosidase than that supported by the lacZ-promoter of pUC18 plasmid in E. coli. The cloned gene can be used as a selection marker for introducing recombinant plasmids in wild strains of S. cerevisiae. The enzyme produced by bgl+ yeast and E. coli recombinants resembles that of the donor with respect to temperature and pH requirement for maximum activity. Other enzyme properties of the beta-glucosidases from S. cerevisiae were substantially the same as those from C. biazotea.

Probing the mechanism of action of Bacillus thuringiensis insecticidal proteins by site-directed mutagenesis--a minireview.

The current model of the mechanism of action of several Bacillus thuringiensis insecticidal crystal proteins (Cry) is reviewed and tested by site-directed mutagenesis experiments. Amino acid (aa) residues were substituted in each of the three domains of Cry toxins and the effects on toxin stability, binding to receptors, irreversible insertion into the membrane, and ion channel activity were examined. Mutant proteins with aa altered on the putative membrane-proximal surface of domain I are affected in insertion into the membrane and toxicity, but not in binding to the receptor. Alterations in the putative receptor-binding loops of domain II show an effect on the initial (reversible) binding to the receptor when certain aa are altered, while affecting irreversible binding when other aa are altered. Mutant proteins with aa altered in a conserved track of aa of domain III have altered ion channel properties, as measured by the voltage clamping of insect midguts and the K+ permeability of brush border membrane vesicles. In summary, domain I is involved in insertion into the membrane and affects ion channel function, domain II is involved in receptor binding and insertion into the membrane, and domain III is involved ion channel function, receptor binding, and insertion into the membrane.

Mutations at domain II, loop 3, of Bacillus thuringiensis CryIAa and CryIAb delta-endotoxins suggest loop 3 is involved in initial binding to lepidopteran midguts.

Alanine substitutions of loop 3 residues, 438SGFSNS443, of CryIAb toxin were constructed to study the functional role of these residues in receptor binding and toxicity to Manduca sexta and Heliothis virescens. Experiments with trypsin and insect gut juice enzyme digestions of mutant toxins showed that these mutations did not produce any gross structural changes to the toxin molecule. Bioassay data showed that mutant G439A (alanine substitution of residue Gly439) and F440A significantly reduced toxicity toward M. sexta and H. virescens. In contrast, mutants S438A, S441A, N442A, and S443A were similar or only marginally less toxic (2-3 times) to the insects compared to the wild-type toxin. Binding studies with brush border membrane vesicles prepared from M. sexta and H. virescens midgut membranes revealed that the loss of toxicity of mutants G439A and F440A was attributable to substantially reduced initial binding. Consistent with the initial binding, mutants G349A and F440A showed 3.5 times less binding to M. sexta and H. virescens brush border membrane vesicles, although the off-rate of bound toxins was not affected. The role of hydrophobic residue, Phe440, is distinctly different from our previous observation that alanine substitution of Phe371 at loop 2 of CryIAb did not affect initial binding but reduced irreversible association of the toxin to the receptor or membrane toward M. sexta (Rajamohan, F., Alcantara, E., Lee, M. K., Chen, X. J., and Dean, D. H. (1995) J. Bacteriol. 177, 2276-2282). Likewise, deletion of relatively hydrophobic CryIAa loop 3 residues, 440AAGA443 (D3a), resulted in reduced toxicity to Bombyx mori (>62 times less) and M. sexta (28 times less). The loss of toxicity was correlated with reduced initial binding to midgut vesicles prepared from these insects. However, alanine substitution of residues 437LSQ439 (A3a), contiguous to loop 3, altered neither toxicity nor receptor binding toward B. mori or M. sexta. These results suggest that the loop 3 residues of CryIAb and CryIAa toxins establish hydrophobic interactions with the receptor molecule, and mutations at these hydrophobic residues affect initial binding.

Substitution of residues on the proximal side of Cry1A Bacillus thuringiensis delta-endotoxins affects irreversible binding to Manduca sexta midgut membrane.

Substitution of a positively charged residue (R93F) or addition of a negatively charged residue (A92D) at the N-terminal of alpha 3 helix of domain I of the Cry1Ac delta-endotoxin resulted in a substantial reduction in toxicity against Manduca sexta. The N-terminal residues of helix 3 are considered to be on the same (proximal) surface of the toxin as the loops in domain II which are involved in the binding of the toxin to the receptor. The loss of toxicity was not caused by a decrease in the initial binding but rather by reduced irreversible binding. Only 65 and 75% of the A92D and R93F mutant toxin, respectively, bound to midgut vesicles irreversibly, compared to 94% of the wild type toxin. On the other hand, replacing A119 in a loop on the distal side of the helices with negatively charged residues (A119D or A119E) did not affect toxicity or irreversible binding.