A case of acute myeloid leukemia-M2 with trisomy 4 in addition to t(8;21).

t(8;21)(q22;q22) is the most frequently observed karyotypic abnormality associated with acute myeloid leukemia (AML), specifically in FAB-M2. Short-term unstimulated bone marrow (BM) and peripheral blood lymphocyte culture showed 47,XX, +4,t(8;21) in all metaphase plates; and interphase and metaphase results of AML-ETO fusion was positive and trisomy of 4 was confirmed with WCP probes. Trisomy 4 in AML with t(8;21) is a rare numerical abnormality. Here we present such case of patient which may constitute a distinctive subtype.

Diabetes mellitus in Dombivli--an urban population study.

OBJECTIVE: The present study was undertaken with the following aims and objectives. a) To determine the prevalence of diabetes. b) To compare the prevalence of newly diagnosed diabetic subjects, diagnosed by the ADA and WHO criteria. c) To determine to what extent prevalence rates are affected by gender, age and body mass index. METHODS: The study population consisted of 520 subjects aged 20 years and above selected by random sampling. Blood glucose both fasting and post glucose (75 gms) two hours sample were analyzed. Baseline clinical data like height, weight and blood pressure were recorded. Known cases of diabetes were excluded from the study. RESULTS: The prevalence of diabetes (fasting blood glucose) as per WHO criteria was 4.61% while it was 7.5% as per ADA criteria. The prevalence according to the two hour glucose reading was 6.15%. The mean glucose level increase with age. With increasing BMI the percentage of diabetic subjects also increases. The prevalence of impaired glucose tolerance (IGT) was 8.6% in subjects below 50 years and 13.4% in subjects older than 50 years. IGT values also increase with increasing age and BMI. 1.3% of subjects had normal fasting glucose level (< 126 mgm%) but high post glucose levels (> 200 mgm%). CONCLUSIONS: The prevalence of diabetes and IGT is higher as compared to earlier studies. The mean blood glucose and IGT increase with increasing age and BMI. Several subjects had normal fasting blood glucose but increased two hour blood glucose level indicating that fasting glucose alone should not be used to diagnose DM. Urbanization and life style of residents of Dombivli contribute to the high prevalence.

GGA*TCC-interrupted triplets in long GAA*TTC repeats inhibit the formation of triplex and sticky DNA structures, alleviate transcription inhibition, and reduce genetic instabilities.

Large expansions of GAA.TTC repeats in the first intron of the frataxin (X25) gene are the principal mutation responsible for Friedreich's ataxia (FRDA). Sticky DNA, based on R.R.Y triplexes, was found at the expanded GAA.TTC repeats from FRDA patients. The (GAAGGA.TCCTTC)(65) repeat occurs in the same frataxin locus but is nonpathogenic and does not form sticky DNA. To elucidate the behavior of sticky DNA, we introduced various extents of GGA.TCC interruptions into the long GAA.TTC repeat. More than 20% of GGA.TCC interruptions abolished the formation of sticky DNA. However, the GAA.TTC repeats with less than 11% of GGA.TCC interruptions formed triplexes and/or sticky DNA similar to the uninterrupted repeat sequence. These triplexes showed different P1 nuclease sensitivities, and the GGA.TCC interruptions were slightly more sensitive than the surrounding GAA.TTC repeats. Furthermore, genetic instability investigations in Escherichia coli revealed that a small number (4%) of interruptions substantially stabilized the long GAA.TTC tracts. Furthermore, the greater the extent of interruptions of the GAA.TTC repeats, the less inhibition of in vitro transcription was observed, as expected, based on the capacity of interruptions to inhibit the formation of sticky DNA. We propose that the interruptions introduce base mismatches into the R.R.Y triplex, which explains the observed chemical and biological properties.

Tandem duplication. A novel type of triplet repeat instability.

Triplet repeat sequence (TRS) inserts containing (CTG.CAG)(n) (17-175 units in length) were tandemly duplicated when propagated in plasmids in Escherichia coli. The products of this novel type of TRS genetic instability are tracts of as many as 34 multiple units, which contain the entire TRS as well as 129 base pairs of nonrepetitive flanking sequence. The duplication process required the presence of two or more TRS-containing units. Close proximity (170 base pairs) of the TRS to the R6K gamma origin of replication of the pUTminiTn5Cm-derived constructs stimulated the tandem duplication process. These events are proposed to occur due to secondary structure formation, stalling of DNA synthesis, and slippage-mediated misalignment of the complementary strands relative to each other during DNA replication. This mechanism may account for the TRS-associated duplications in protein kinase and metalloprotease genes in neuroblastomas and melanomas, as well as the massive repeat expansions in type II triplet repeat neurological diseases.

DNA polymerase III proofreading mutants enhance the expansion and deletion of triplet repeat sequences in Escherichia coli.

The influence of mutations in the 3' to 5' exonucleolytic proofreading epsilon-subunit of Escherichia coli DNA polymerase III on the genetic instabilities of the CGG.CCG and the CTG.CAG repeats that cause human hereditary neurological diseases was investigated. The dnaQ49(ts) and the mutD5 mutations destabilize the CGG.CCG repeats. The distributions of the deletion products indicate that slipped structures containing a small number of repeats in the loop mediate the deletion process. The CTG.CAG repeats were destabilized by the dnaQ49(ts) mutation by a process mediated by long hairpin loop structures (>/=5 repeats). The mutD5 mutator strain stabilized the (CTG.CAG)(175) tract, which contained two interruptions. Since the mutD5 mutator strain has a saturated mismatch repair system, the stabilization is probably an indirect effect of the nonfunctional mismatch repair system in these strains. Shorter uninterrupted tracts expand readily in the mutD5 strain, presumably due to the greater stability of long CTG.CAG tracts (>100 repeats) in this strain. When parallel studies were conducted in minimal medium, where the mutD5 strain is defective in exonucleolytic proofreading but has a functional MMR system, both CTG.CAG and CGG.CCG repeats were destabilized, showing that the proofreading activity is essential for maintaining the integrity of TRS tracts. Thus, we conclude that the expansion and deletion of triplet repeats are enhanced by mutations that reduce the fidelity of replication.

Expansion and deletion of triplet repeat sequences in Escherichia coli occur on the leading strand of DNA replication.

Expansions and deletions of triplet repeat sequences that cause human hereditary neurological diseases were previously suggested to be mediated by the formation of DNA hairpins on the lagging strand during replication. The replication properties of CTG.CAG, CGG.CCG, and TTC.GAA repeats were studied in Escherichia coli using an in vivo phagemid system as a model for continuous leading strand synthesis. The repeats were substantially deleted when the CTG, CGG, and GAA repeats were the templates for rolling circle replication from the f1 phage origin. The deletions may be mediated by hairpins formed by these repeat tracts. The distributions of the deletion products of the CTG.CAG and CGG.CCG tracts indicated that hairpins of discrete sizes mediate deletions during complementary strand synthesis. Deletions during rolling circle synthesis are caused by larger hairpins of specific sizes. Thus, most deletion products were of defined lengths, suggesting a preference for specific hairpin intermediates. Small expansions of the CTG.CAG and CGG.CCG repeats were also observed, presumably due to the formation of CTG and CGG hairpins on the nascent complementary strand. Since rolling circle replication has been established in vitro as a model for leading strand synthesis, we conclude that triplet repeat instability can also occur on the leading strand of DNA replication.

Sickle cell syndromes in and around Bardoli.

The study comprised of 2 groups. In group I sickling test was done in students studying in a school which mainly caters to the educational needs of the backward community. Out of 130 students examined 24 were found to be sicklers. The distribution of this cases among various castes/tribes were as follows--Choudharys (Cd)-13, Gamits (Gt)-4, Dhodhia Patels (DP)-4, Koknis (K)-2 and Koli Patel (KP)-1. In group II, patients admitted in the hospital between Jan '81 to June '82 were studied. The prevalence of sickle cell syndrome was 1.74%. The most common mode of presentation were limb pains and weakness. Hemoglobin values ranged from 3.0 gram% to 12 gms%. 35 cases of HbSS, 149 cases of HbAS and 1 case of Sickle Beta thalassemia were seen. The distribution of the cases amongst the various tribes and castes were as follows-Cd-93, Gt-56, DP-23, KP-7, K-4 and Rathods (R)-2. No cases were found in Anavil Brahmins or Patidar Patels. Clinical and pathological observations included palpable splenomegaly in 54 cases, splenic abscess in 1 case, isothenuria in large number of patients, microscopic hematuria in 6 cases and frank hematuria in 1 case. Osteomyelitis and cholecystitis were seen in one case each.

Definition of interferon gamma-response elements in a novel human Fc gamma receptor gene (Fc gamma RIb) and characterization of the gene structure.

The human Fc gamma RI (CD64) is a high affinity receptor for the Fc portion of immunoglobulin (Ig), and its constitutively low expression on the cell surface of monocyte/macrophage and neutrophils is selectively upregulated by interferon gamma (IFN-gamma) treatment (Perussia, B., E. T. Dayton, R. Lazarus, V. Fanning, and G. Trinchieri. 1983. J. Exp. Med. 158:1092). Three distinct cDNAs have been cloned and code for proteins that predict three extracellular Ig-like domains (Allen, J.M., and B. Seed. 1989. Science [Wash. DC]. 243:378). Several differences in the coding region of these cDNAs suggest that in addition to polymorphic differences a second Fc gamma RI gene could possibly exist. This alternative Fc gamma RI gene (Fc gamma RIb) was defined by the lack of a genomic HindIII restriction site (van der Winkel, J. G. J., L. U. Ernst, C. L. Anderson, and I. M. Chiu. 1991. J. Biol. Chem. 266:13449). We describe the characterization a second gene (Fc gamma RIb) that has a termination codon in the third extracellular domain and therefore predicts a soluble form of a termination codon in the third extracellular domain and therefore predicts a soluble form of the receptor. We also define two distinct IFN-gamma-responsive regions in the 5' flanking sequence of Fc gamma RIb that resemble motifs that have been defined in the class II major histocompatibility complex promoter. The Fc gamma RIb promoter does not possess canonical TATA or CCAAT boxes, but does possess a palindromic motif that closely resembles the initiator sequence identified in the terminal deoxynucleotidyl transferase/human leukocyte IFN/adeno-associated virus type II P5 gene promoters (Smale, S. T., and D. Baltimore. 1989. Cell. 57:103; Seto, E., Y. Shi, and T. Shenk. 1991. Nature [Lond.]. 354:241; Roy, A. L., M. Meisterernst, P. Pognonec, and R. C. Roeder. 1991. Nature [Lond.]. 354:245) virus type II P5 gene promoters raising interesting questions as to its role in the basal and myeloid-specific transcription of this gene.

A matched set of cat vectors for rapid mutational analysis of eukaryotic promoters and enhancers.

The eukaryotic cat expression vectors, pBRAMScat1 and pBRAMScat2, were constructed to simplify the analysis of genomic fragments containing putative transcriptional regulatory elements. These vectors contain the f1 filamentous phage origin of replication for single-stranded DNA rescue, and permit site-directed mutagenesis, and dideoxy sequencing of nested deletion mutants using commercial T3, T7 and M13 universal forward/reverse primers. The above features eliminate the need to shuttle back and forth between a conventional cloning vector and the cat expression vector during the analysis of putative eukaryotic gene regulatory elements. Plasmid pBRAMScat1 contains the bacterial chloramphenicol acetyltransferase-encoding gene (cat) and no eukaryotic promoter and was designed for the analysis of eukaryotic promoters. Plasmid pBRAMScat2 contains the cat gene under the control of the Herpes simplex virus thymidine kinase promoter and was designed for the analysis of eukaryotic enhancers.

Modulation of human lepromatous monocyte-macrophage functions in vitro by tuftsin.

Human peripheral blood monocytes/macrophages derived from normal donors, patients of tuberculoid leprosy (BT/TT) and lepromatous leprosy (BL/LL) were assayed for stimulated phagocytic responses to the potent macrophage stimulator "Tuftsin" (NH2-Thr-Lys-Pro-Arg-OH) after varying periods (6 h to 14 days) of culture in vitro. The assays consisted of visual scoring of ingested Mycobacterium leprae and radiometric measurement of ingested 14C-acetate labelled Staphylococcus aureus and Mycobacterium tuberculosis (H37Ra). While normal and BT/TT macrophages showed a progressively increasing ability for tuftsin-stimulated phagocytosis with increasing age of culture in vitro, BL/LL macrophages showed the opposite response so that 14-day cultures were refractory to a stimulatory dose of up to 7.0 microM (10 to 20 times the optimal dose for normal and BT/TT macrophages). The 14-day BL/LL macrophage cultures were, however, responsive to 35 microM tuftsin (100 times the optimal dose for normal macrophages). Analysis of the dose-response curves also indicates that BT/TT cultures despite exhibiting an apparent similarity to normal macrophages demonstrate a rightward shift for a maximal stimulated phagocytosis. Finally SEM photo-micrographs of 14-day macrophage cultures of the three groups revealed that while normal and BT/TT cultures demonstrated an increase in membrane ruffling and filopodia on stimulation with 0.8 microM tuftsin, BL/LL cultures exhibited none of the features associated with stimulation. From the above findings, we conclude that lepromatous macrophages may display an aberrant differentiation profile leading to a terminal state of unresponsiveness and that the defect may possibly lie at the level of tuftsin receptor expression or transmembrane signal transduction.

Effect of tuftsin stimulation on the microbicidal activity exerted by blood monocyte-macrophages of leprosy patients.

The ability of blood monocyte/macrophages from normal donors, tuberculoid leprosy (BT/TT) and lepromatous leprosy (BL/LL) patients to exert enhanced microbicidal activity was assayed after stimulating with 0.8 microM tuftsin, as a function of the duration of cultures in vitro. Normal and BT/TT macrophage cultures showed a statistically significant increase in microbicidal activity against Staphylococcus aureus at all ages of culture (6 h to 14 days), though the overall magnitude of the enhancement shows a decrease with increasing culture age in the same populations. However, 14-day old BL/LL macrophage cultures were unable to undergo tuftsin-mediated stimulation of microbicidal activity against S. aureus and even, fresh 6 h-old cultures exhibited a tuftsin-stimulated response profile similar to 14-day old normal and BT/TT cultures. Also, 7 and 14-day cultures of normal, BT/TT and BL/LL macrophages were unable to inhibit/kill intracellular Mycobacterium leprae after a single stimulation with 0.8 microM tuftsin. However, serial, daily stimulation with 0.8 microM tuftsin resulted in 77-140% inhibition of 3H-thymidine uptake by the 12th day of cultures in vitro in the three groups. These results suggest that BL/LL macrophages exhibit a premature inability to undergo tuftsin stimulated microbicidal activity, which may possibly be reversed by serial dosage of tuftsin.