Cholera in India: an analysis of reports, 1997-2006.

OBJECTIVE: To more accurately define the annual incidence of cholera in India, believed to be higher than reported to the World Health Organization (WHO). METHODS: We searched the biomedical literature to extract data on the cases of cholera reported in India from 1997 to 2006 and compared the numbers found to those reported annually to WHO over the same period. The latter were obtained from WHO's annual summaries of reported cholera cases and National health profile 2006, published by India's Central Bureau of Health Intelligence. FINDINGS: Of India's 35 states or union territories, 21 reported cholera cases during at least one year between 1997 and 2006. The state of West Bengal reported cases during all 10 years, while the state of Maharashtra and the union territory of Delhi reported cases during nine, and Orissa during seven. There were 68 outbreaks in 18 states, and 222 038 cases were detected overall. This figure is about six times higher than the number reported to WHO (37 783) over the same period. The states of Orissa, West Bengal, Andaman and Nicobar Islands, Assam and Chhattisgarh accounted for 91% of all outbreak-related cases. CONCLUSION: The reporting of cholera cases in India is incomplete and the methods used to keep statistics on cholera incidence are inadequate. Although the data are sparse and heterogeneous, cholera notification in India is highly deficient.

Uracil-initiated base excision DNA repair synthesis fidelity in human colon adenocarcinoma LoVo and Escherichia coli cell extracts.

The error frequency of uracil-initiated base excision repair (BER) DNA synthesis in human and Escherichia coli cell-free extracts was determined by an M13mp2 lacZ alpha DNA-based reversion assay. Heteroduplex M13mp2 DNA was constructed that contained a site-specific uracil target located opposite the first nucleotide position of opal codon 14 in the lacZ alpha gene. Human glioblastoma U251 and colon adenocarcinoma LoVo whole-cell extracts repaired the uracil residue to produce form I DNA that was resistant to subsequent in vitro cleavage by E. coli uracil-DNA glycosylase (Ung) and endonuclease IV, indicating that complete uracil-initiated BER repair had occurred. Characterization of the BER reactions revealed that (1) the majority of uracil-DNA repair was initiated by a uracil-DNA glycosylase-sensitive to Ugi (uracil-DNA glycosylase inhibitor protein), (2) the addition of aphidicolin did not significantly inhibit BER DNA synthesis, and (3) the BER patch size ranged from 1 to 8 nucleotides. The misincorporation frequency of BER DNA synthesis at the target site was 5.2 x 10(-4) in U251 extracts and 5.4 x 10(-4) in LoVo extracts. The most frequent base substitution errors in the U251 and LoVo mutational spectrum were T to G > T to A >> T to C. Uracil-initiated BER DNA synthesis in extracts of E. coli BH156 (ung) BH157 (dug), and BH158 (ung, dug) was also examined. Efficient BER occurred in extracts of the BH157 strain with a misincorporation frequency of 5.6 x 10(-4). A reduced, but detectable level of BER was observed in extracts of E. coli BH156 cells; however, the mutation frequency of BER DNA synthesis was elevated 6.4-fold.

K(+) and Mg(2+) ions promote the self-splicing of the td intron RNA inhibited by spectinomycin.

The effects of Mg(2+) and K(+) ions on the self-splicing inhibition of the td (thymidylate synthase gene) intron RNA by spectinomycin were investigated. The maximum splicing activity occurred at 20 mM KCl. The K(m) and V(max) values for GTP in the presence of 5 mM Mg(2+) are 2.25 microM and 0.55 min(-1), whereas those for GTP both in the presence of 5 mM Mg(2+) and 5 mM K(+) are 1.23 microM and 0. 46 min(-1), respectively. Spectinomycin at 10 mM concentration inhibited the splicing by about 10%, but at 20 mM concentration, the splicing rate was inhibited by about 63%. The splicing inhibition by the low concentration of spectinomycin was overcome markedly as the concentration of Mg(2+) ion was raised. At 30 mM spectinomycin, however, the splicing inhibition was not significantly affected by increasing the concentration of Mg(2+). A similar activation of the splicing rate was observed as the concentration of K(+) ion was increased. The concentration of K(+) ion required for the normal recovery of the splicing was much higher than that of Mg(2+) ion. Unlike Mg(2+) ion, 30 mM K(+) ion effectively alleviated the splicing inhibition by spectinomycin at its high concentration. The results indicate that K(+) and Mg(2+) ions may show mechanistically different interactions with spectinomycin in the self-splicing reaction of the td intron RNA.

Serum immunoglobulin levels in vasectomized men: preliminary report.

Seventy-five men undergoing vasectomy were tested by radial immunodiffusion assay prevasectomy and at 6 weeks and 3, 6, and 12 months postvasectomy for possible changes in serum immunoglobulin (Ig) levels. No significant changes occurred when the results were analyzed on a group basis per time period. However, when each individual's prevasectomy Ig level was considered as his norm, significant changes appeared to occur in serum IgG levels at 6 weeks, 6 months, and 12 months postvasectomy; in serum IgM levels at 6 weeks; but not, to date, in serum IgA levels. Seasonal, environmental, and infectious factors do not seem to be associated with the changes found. No significant seminal Ig changes have been demonstrated. Consideration of these findings and implications for possible immunopathology are briefly discussed.

Escherichia coli double-strand uracil-DNA glycosylase: involvement in uracil-mediated DNA base excision repair and stimulation of activity by endonuclease IV.

Escherichia coli double-strand uracil-DNA glycosylase (Dug) was purified to apparent homogeneity as both a native and recombinant protein. The molecular weight of recombinant Dug was 18 670, as determined by matrix-assisted laser desorption-ionization mass spectrometry. Dug was active on duplex oligonucleotides (34-mers) that contained site-specific U.G, U.A, ethenoC.G, and ethenoC.A targets; however, activity was not detected on DNA containing a T.G mispair or single-stranded DNA containing either a site-specific uracil or ethenoC residue. One of the distinctive characteristics of Dug was that the purified enzyme excised a near stoichiometric amount of uracil from U.G-containing oligonucleotide substrate. Electrophoretic mobility shift assays revealed that the lack of turnover was the result of strong binding by Dug to the reaction product apyrimidinic-site (AP) DNA. Addition of E. coli endonuclease IV stimulated Dug activity by enhancing the rate and extent of uracil excision by promoting dissociation of Dug from the AP. G-containing 34-mer. Catalytically active endonuclease IV was apparently required to mediate Dug turnover, since the addition of 5 mM EDTA mitigated the effect. Further support for this interpretation came from the observations that Dug preferentially bound 34-mer containing an AP.G target, while binding was not observed on a substrate incised 5' to the AP-site. We also investigated whether Dug could initiate a uracil-mediated base excision repair pathway in E. coli NR8052 cell extracts using M13mp2op14 DNA (form I) containing a site-specific U.G mispair. Analysis of reaction products revealed a time dependent appearance of repaired form I DNA; addition of purified Dug to the cell extract stimulated the rate of repair.

Fidelity of uracil-initiated base excision DNA repair in Escherichia coli cell extracts.

The error frequency and mutational specificity associated with Escherichia coli uracil-initiated base excision repair were measured using an M13mp2 lacZalpha DNA-based reversion assay. Repair was detected in cell-free extracts utilizing a form I DNA substrate containing a site-specific uracil residue. The rate and extent of complete uracil-DNA repair were measured using uracil-DNA glycosylase (Ung)- or double-strand uracil-DNA glycosylase (Dug)-proficient and -deficient isogenic E. coli cells. In reactions utilizing E. coli NR8051 (ung(+) dug(+)), approximately 80% of the uracil-DNA was repaired, whereas about 20% repair was observed using NR8052 (ung(-) dug(+)) cells. The Ung-deficient reaction was insensitive to inhibition by the PBS2 uracil-DNA glycosylase inhibitor protein, implying the involvement of Dug activity. Under both conditions, repaired form I DNA accumulated in conjunction with limited DNA synthesis associated with a repair patch size of 1-20 nucleotides. Reactions conducted with E. coli BH156 (ung(-) dug(+)), BH157 (ung(+) dug(-)), and BH158 (ung(-) dug(-)) cells provided direct evidence for the involvement of Dug in uracil-DNA repair. The rate of repair was 5-fold greater in the Ung-proficient than in the Ung-deficient reactions, while repair was not detected in reactions deficient in both Ung and Dug. The base substitution reversion frequency associated with uracil-DNA repair was determined to be approximately 5.5 x 10(-)(4) with transversion mutations dominating the mutational spectrum. In the presence of Dug, inactivation of Ung resulted in up to a 7.3-fold increase in mutation frequency without a dramatic change in mutational specificity.

Fidelity of uracil-initiated base excision DNA repair in DNA polymerase beta-proficient and -deficient mouse embryonic fibroblast cell extracts.

Uracil-initiated base excision DNA repair was conducted using homozygous mouse embryonic fibroblast DNA polymerase beta (+/+) and (-/-) cells to determine the error frequency and mutational specificity associated with the completed repair process. Form I DNA substrates were constructed with site-specific uracil residues at U.A, U.G, and U.T targets contained within the lacZalpha gene of M13mp2 DNA. Efficient repair was observed in both DNA polymerase beta (+/+) and (-/-) cell-free extracts. Repair was largely dependent on uracil-DNA glycosylase activity because addition of the PBS-2 uracil-DNA glycosylase inhibitor (Ugi) protein reduced ( approximately 88%) the initial rate of repair in both types of cell-free extracts. In each case, the DNA repair patch size was primarily distributed between 1 and 8 nucleotides in length with 1 nucleotide repair patch constituting approximately 20% of the repair events. Addition of p21 peptide or protein to DNA polymerase beta (+/+) cell-free extracts increased the frequency of short-patch (1 nucleotide) repair by approximately 2-fold. The base substitution reversion frequency associated with uracil-DNA repair of M13mp2op14 (U.T) DNA was determined to be 5.7-7.2 x 10(-4) when using DNA polymerase beta (+/+) and (-/-) cell-free extracts. In these two cases, the error frequency was very similar, but the mutational spectrum was noticeably different. The presence or absence of Ugi did not dramatically influence either the error rate or mutational specificity. In contrast, the combination of Ugi and p21 protein promoted an increase in the mutation frequency associated with repair of M13mp2 (U.G) DNA. Examination of the mutational spectra generated by a forward mutation assay revealed that errors in DNA repair synthesis occurred predominantly at the position of the U.G target and frequently involved a 1-base deletion or incorporation of dTMP.

The lymphocyte transformation response of fetal hemolymphatic tissue to mitogens and antigens.

Lymphocyte transformation responses to three different mitogens (phytohemagglutinin (PHA), pokeweed mitogen (PWM), and concanavalin A (Con A)) as well as four antigens (streptolysin O (SLO), keyhole limpet hemocyanin (KLH), streptokinase-streptodornase (SKSD) and tuberculin purified protein derivative (PPD)) were studied ontogenetically in 30 human fetuses ranging in gestational age from 6-19 weeks. In most organs lymphocyte responsiveness to Con A in human fetuses seemingly develops in concert with responsiveness to PHA and PWM. One 19 week fetus had an apparent antigen specific response to SLO with stimulation of cord blood and bone marrow lymphocytes. The same fetus also had mitogen responsiveness in bone marrow.

A radiomicroassay for cytotoxic antibody to human spermatozoa. Quantification by tritiated actinomycin d.

A radiomicroassay for titration of spermocytotoxic antibody is described. The assay used [3H]AACTINOMYCIN D ([3H]Act D) to label damaged spermatozoa in a fashion analogous to penetration by vital dye. Optimal conditions for and some kinetics of the assay are presented. The assay is sensitive, reliable, simple to perform and uses only small amounts of serum and spermatozoa. Applied to sperm antibody positive human postvasectomy sera, the assay compared favourably in sensitivity eith vital dye microscopic observations and with parallel titration by the Isojima's immobilization tests.

Dengue carrier culture and antigen production in human lymphoblastoid lines.

Replication of dengue type 2 (D2) viruses was studied in four lymphoblastoid cell lines; Raji, HR1, EB3 and RPMI 6410. The HR1 cell line failed to support D2 growth while the other cell lines showed varying susceptibility. Both D2 strain 16681 and strain New Guinea C (NGC) passaged in LLC-MK2 cells replicated readily in Raji cells, while a high mouse-brain-passaged NGC strain did not. A soluble complement-fixing (SCF) antigen from D2-infected Raji cells showed lines of identity with a D2SCF antigen prepared from infected suckling mouse brains. Electron microscopic studies of a D2 Raji carrier culture showed that virions were located in a membrane-vesicle complex in the cytoplasm of the cells. Precursors of viral particles were more frequently observed in the parts of the vesicle which had rough endoplasmic reticular membranes. Mature virus particles were observed often at the boundary of the other part of the vesicle, which consisted of smooth endoplasmic reticulum. Occasionally, a large crystalloid structure consisting of incomplete viral particles was seen in degenerative cells. Dengue carrier cultures in human lymphoblastoid lines may provide a convenient in vitro system for study of aspects of dengue virus-leukocyte interactions.

Intravenous 5-fluorouracil versus oral doxifluridine as preoperative concurrent chemoradiation for locally advanced rectal cancer: prospective randomized trials.

BACKGROUND: Preoperative radiation treatment with concomitant intravenous infusion of 5-fluorouracil (5-FU) is known to be effective in shrinking and downstaging of tumors. However, chemotherapy has often been limited by its toxicity and poor patient compliance. Oral 5-FU is known to have several advantages over conventional intravenous 5-FU infusion such as lower toxicity and higher quality of life without compromising the efficacy of the treatment. The aim of this study was to compare intravenous 5-FU with oral doxifluridine with respect to tumor response, toxicity and quality of life. METHODS: Twenty-eight patients with rectal cancer, staged as over T3N1 or T4 by transrectal ultrasonography between July 1997 and December 1998, were included in this study. Intravenous 5-FU (450 mg/m2) and leucovorin (20 mg/m2) were given for five consecutive days during the first and fifth weeks of radiation therapy (50.4 Gy) (n = 14). Oral doxifluridine (700 mg/m2/day) and leucovorin (20 mg/m2) were given daily during radiation treatment (n = 14). Quality of life was scored according to 22 activity items (good, >77; fair, >58; poor, <57). Surgical resection was performed 4 weeks after completion of concurrent chemoradiation treatment. Tumor response was classified into CR (complete remission), PR (partial response; 50% diminution of tumor volume or downstaging ) and NR (no response). RESULTS: Tumor response was CR 3/14 (21.4%), PR 7/14 (50%) and NR 4/14 (28.6%) in the IV arm versus CR 2/14 (14.2%), PR 6/14 (42.9%) and NR 6/14 (42.9%) in the Oral arm (p = 0.16, 0.23, 0.24), respectively. The quality of life was poor (36.4% versus 33.3%), fair and good (63.6% versus 66.7%) between the IV arm and Oral arm, respectively. Gastrointestinal toxicity was 2/14 (14.3%) in the IV arm versus 5/14 (35.7%) in the Oral arm, respectively. Stomatitis was only observed in the IV arm (1/14, 7.1%). Hematological toxicity was 3/14 (21.4%) in the IV arm versus 4/14 (28.5%) in the Oral arm, respectively. Systemic recurrence during the follow-up periods were 1/14 (7.1%) in the IV arm and 2/14 (14.3%) in the Oral arm, respectively (p = 0.307). One local recurrence was observed in the Oral arm. CONCLUSION: Even though the results were not entirely reliable owing to the small number of patients enrolled, oral doxifluridine-based chemotherapy as preoperative chemoradiation for advanced rectal cancer did not show any significant advantages over intravenous infusion.