SYNTHESIS AND BIOLOGICAL EVALUATION OF ANTITUMOR ALDOPHOSPHAMIDE ACETALS

Although the major metabolic pathways of cyclophosphamide are well established, the mechanism of antitumor drug selectivity is highly controversial. However, it is widely accepted that aldophosphamide, one of the primary metabolites, plays a crucial role in drug selectivity. In an attempt to gain a better understanding of the mechanism of selectivity of cyclophosphamide, a series of aldophosphamide analogs have been synthesized.^ The new analogs, unlike aldophosphamide, are relatively stable in neutral solution; however, they are converted rapidly to aldehydo intermediates in the presence of carboxylate esterase. Due to structural differences, these analogs may be classified into three different groups, arbitrarily designated as A, B, C, depending upon the facility with which the intermediate aldehydes form 4-hydroxy cyclic tautomers. The half-life of the aldehydo/4-hydroxy cyclic tautomeric mixture is longer for bis(acetoxy)aldophosphamide acetal I (a representative of group A), shorter for the n-ethyl analog III (B), and shortest for the N,N-dimethyl analog IV (C). The ratio of aldophosphamide: 4-hydroxycyclophosphamide at pseudoequilibrium is 1: 4 for compound I, 1: 2 for compound III and 0: 1 for compound IV. The therapeutic efficacy of these compounds are group A $>$ group B $>$ group C. It is apparent that the equilibrium position between the aldehydo and 4-hydroxy cyclic tautomers, which determines their stability, is a crucial determinant of both the cytotoxicity and antitumor selectivity. These findings, taken in conjunction with the aldehyde dehydrogenase selectivity hypothesis, may provide an explanation for the unique antitumor activity of cyclophosphamide. ^

Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II.

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.

A case-control study of hepatitis C virus and its interaction with hepatitis B virus on the development of hepatocellular carcinoma in the USA

Objective. The aim of this study was to assess the independent risk of hepatitis C virus (HCV) infection in the development of hepatocellular carcinoma (HCC). The independent risk of hepatitis B virus (HBV), its interaction with hepatitis C virus and the association with other risk factors were examined.^ Methods. A hospital-based case-control study was conducted between January 1994 and December 1995. We enrolled 115 pathologically confirmed HCC patients and 230 nonliver cancer controls, who were matched by age ($\pm$5 years), gender, and year of diagnosis. Both cases and controls were recruited from The University of Texas M. D. Anderson Cancer Center at Houston. The risk factors were collected through personal interviews and blood samples were tested for HCV and HBV markers. Univariate and multivariate analyses were performed through conditional logistic regression.^ The prevalence of anti-HCV positive is 25.2% in HCC cases compared to 3.0% in controls. The univariate analysis showed that anti-HCV, HBsAg, alcohol drinking and cigarette smoking were significantly associated with HCC, however, family history of cancer, occupational chemical exposure, and use of oral contraceptive were not. Multivariate analysis revealed a matched odds ratio (OR) of 10.1 (95% CI 3.7-27.4) for anti-HCV, and an OR of 11.9 (95% CI 2.5-57.5) for HBsAg. However, dual infection of HCV and HBV had only a thirteen times increase in the risk of HCC, OR = 13.9 (95% CI 1.3-150.6). The estimated population attributable risk percent was 23.4% for HCV, 12.6% for HBV, and 5.3% for both viruses. Ever alcohol drinkers was positively associated with HCC, especially among daily drinkers, matched OR was 5.7 (95% CI 2.1-15.6). However, there was no significant increase in the risk of HCC among smokers as compared to nonsmokers. The mean age of HCC patients was significantly younger among the HBV(+) group and among the HCV(+)/HBV(+) group, when compared to the group of HCC patients with no viral markers. The association between past histories of blood transfusion, acupuncture, tattoo and IVDU was highly significant among the HCV(+) group and the HBV(+)/HCV(+) group, as compared to HCC patients with no viral markers. Forty percent of the HCC patients were pathologically or clinically diagnosed with liver cirrhosis. Anti-HCV(+) (OR = 3.6 95% CI 1.5-8.9) and alcohol drinking (OR = 2.7 95% CI 1.1-6.7), but not HBsAg, are the major risk factors for liver cirrhosis in HCC patients.^ Conclusion. Both hepatitis B virus and hepatitis C virus were independent risk factors for HCC. There was not enough evidence to determine the interaction between both viruses. Only daily alcoholic drinkers showed increasing risk for HCC development, as compared to nondrinkers. ^

Alpha C protein of group B Streptococcus as a potential vaccine candidate

Group B Streptococcus (GBS) is a leading cause of life-threatening infection in neonates and young infants, pregnant women, and non-pregnant adults with underlying medical conditions. Immunization has theoretical potential to prevent significant morbidity and mortality from GBS disease. Alpha C protein (α C), found in 70% of non-type III capsule polysaccharide group B Streptococcus, elicits antibodies protective against α C-expressing strains in experimental animals and is an appealing carrier for a GBS conjugate vaccine. We determined whether natural exposure to α C elicits antibodies in women and if high maternal α C-specific serum antibody at delivery is associated with protection against neonatal disease. An ELISA was designed to measure α C-specific IgM and IgG in human sera. A case-control design (1:3 ratio) was used to match α C-expressing GBS colonized and non-colonized women by age and compare quantified serum α C-specific IgM and IgG. Sera also were analyzed from bacteremic neonates and their mothers and from women with invasive GBS disease. Antibody concentrations were compared using t-tests on log-transformed data. Geometric mean concentrations of α C-specific IgM and IgG were similar in sera from 58 α C strain colonized and 174 age-matched non-colonized women (IgG 245 and 313 ng/ml; IgM 257 and 229 ng/ml, respectively). Delivery sera from mothers of 42 neonates with GBS α C sepsis had similar concentrations of α C-specific IgM (245 ng/ml) and IgG (371 ng/ml), but acute sera from 13 women with invasive α C-expressing GBS infection had significantly higher concentrations (IgM 383 and IgG 476 ng/ml [p=0.036 and 0.038, respectively]). Convalescent sera from 5 of these women 16-49 days later had high α C-specific IgM and IgG concentrations (1355 and 4173 ng/ml, respectively). In vitro killing of α C-expressing GBS correlated with total α C-specific antibody concentration. Invasive disease but not colonization elicits α C-specific IgM and IgG in adults. Whether α C-specific IgG induced by vaccine would protect against disease in neonates merits further investigation. ^

The association between diabetes and hepatitis C: A systematic review of epidemiological evidence

Background. Several studies have proposed a link between type 2 Diabetes mellitus (DM2) and Hepatitis C infection (HCV) with conflicting results. Since DM2 and HCV have high prevalence, establishing a link between the two may guide further studies aimed at DM2 prevention. A systematic review was conducted to estimate the magnitude and direction of association between DM2 and HCV. Temporality was assessed from cohort studies and case-control studies where such information was available. ^ Methods. MEDLINE searches were conducted for studies that provided risk estimates and fulfill criteria regarding the definition of exposure (HCV) and outcomes (DM2). HCV was defined in terms of method of diagnosis, laboratory technique and method of data collection; DM2 was defined in terms of the classification [World Health Organization (WHO) and American Diabetes Association (ADA)] 1-3 used for diagnosis, laboratory technique and method of data collection. Standardized searches and data abstraction for construction of tables was performed. Unadjusted or adjusted measures of association for individual studies were obtained or calculated from the full text of the studies. Template designed by Dr. David Ramsey. ^ Results. Forty-six studies out of one hundred and nine potentially eligible articles finally met the inclusion and exclusion criteria and were classified separately based on the study design as cross-sectional (twenty four), case-control (fifteen) or cohort studies (seven). The cohort studies showed a three-fold high (confidence interval 1.66–6.29) occurrence of DM2 in individuals with HCV compared to those who were unexposed to HCV and cross sectional studies had a summary odds ratio of 2.53 (1.96, 3.25). In case control studies, the summary odds ratio for studies done in subjects with DM2 was 3.61 (1.93, 6.74); in HCV, it was 2.30 (1.56, 3.38); and all fifteen studies, together, yielded an odds ratio of 2.60 (1.82, 3.73). ^ Conclusion. The above results support the hypothesis that there is an association between DM and HCV. The temporal relationship evident from cohort studies and proposed pathogenic mechanisms also suggest that HCV predisposes patients to development of DM2. Further cohort or prospective studies are needed, however, to determine whether treatment of HCV infections prevents development of DM2.^