Diagnostic criteria for diabetes revisited: making use of combined criteria

Background  

Existing cut-offs for fasting plasma glucose (FPG) and post-load glucose (2hPG) criteria are not equivalent in the diagnosis of diabetes and glucose intolerance. Adjusting cut-offs of single measurements have not helped so we undertook this project to see if they could be complementary.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Characterization of interactions of 4-nitrophenylpropyl-N-alkylamine with ς receptors

Sigma receptors are small membrane proteins implicated in a number of pathophysiological conditions, including drug addiction, psychosis, and cancer; thus, small molecule inhibitors of sigma receptors have been proposed as potential pharmacotherapeutics for these diseases. We previously discovered that endogenous monochain N-alkyl sphingolipids, including d-erythro-sphingosine, sphinganine, and N,N-dimethylsphingosine, bind to the sigma-1 receptor at physiologically relevant concentrations [Ramachandran, S., et al. (2009) Eur. J. Pharmacol. 609, 19-26]. Here, we investigated several N-alkylamines of varying chain lengths as sigma receptor ligands. Although the K(I) values for N-alkylamines were found to be in the micromolar range, when N-3-phenylpropyl and N-3-(4-nitrophenyl)propyl derivatives of butylamine (1a and 1b, respectively), heptylamine (2a and 2b, respectively), dodecylamine (3a and 3b, respectively), and octadecylamine (4a and 4b, respectively) were evaluated as sigma receptor ligands, we found that these compounds exhibited nanomolar affinities with both sigma-1 and sigma-2 receptors. A screen of high-affinity ligands 2a, 2b, 3a, and 3b against a variety of other receptors and/or transporters confirmed these four compounds to be highly selective mixed sigma-1 and sigma-2 ligands. Additionally, in HEK-293 cells reconstituted with K(v)1.4 potassium channel and the sigma-1 receptor, these derivatives were able to inhibit the outward current from the channel, consistent with sigma receptor modulation. Finally, cytotoxicity assays showed that 2a, 2b, 3a, and 3b were highly potent against a number of cancer cell lines, demonstrating their potential utility as mixed sigma-1 and sigma-2 receptor anticancer agents.     

Electron-donating para-methoxy converts a benzamide-isoquinoline derivative into a highly Sigma-2 receptor selective ligand

The sigma-2 (σ2) receptor has been suggested to be a promising target for pharmacological interventions to curb tumor progression. Development of σ2-specific ligands, however, has been hindered by lack of understanding of molecular determinants that underlie selective ligand-σ2 interactions. Here we have explored effects of electron donating and withdrawing groups on ligand selectivity for the σ2 versus σ1 receptor using new benzamide-isoquinoline derivatives. The electron-donating methoxy group increased but the electron-withdrawing nitro group decreased σ2 affinity. In particular, an extra methoxy added to the para-position (5e) of the benzamide phenyl ring of 5f dramatically improved (631 fold) the σ2 selectivity relative to the σ1 receptor. This para-position provided a sensitive site for effective manipulation of the sigma receptor subtype selectivity using either the methoxy or nitro substituent. Our study provides a useful guide for further improving the σ2-over-σ1 selectivity of new ligands.     

Characterization of the cocaine binding site on the sigma-1 receptor

The cocaine photoaffinity label 3-iodo-4-azidococaine ([125I]IACoc) binds to the sigma-1 receptor with an affinity that is 2-3 orders of magnitude higher than the parent compound cocaine [Kahoun, J. R., and Ruoho, A. E. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1393-1397]. In the present study, the binding properties of several cocaine derivatives to the guinea pig liver sigma-1 receptor were determined. The results from assessing the affinity of various derivatives of cocaine which were substituted on the phenyl ring indicated that an important determinant of binding to the guinea pig sigma-1 receptor binding site may be the development of a dipole in the ring in which the pi electron density of the phenyl ring is reduced. This implies that an electron-rich source is present in the sigma-1 receptor binding site, such as the pi system of an aromatic ring or other electron-rich side chains, which interact with the phenyl ring of cocaine. The precise [125I]IACoc derivatization site in the guinea pig sigma-1 receptor was identified using chemical cleavage and purification of the resulting labeled peptides. Cyanogen bromide cleavage of the [125I]IACoc photolabeled sigma-1 receptor followed by radiosequencing identified Asp188, which is located in the putative steroid binding domain-like II (SBDL II) near the carboxyl terminus, as the site of [125I]IACoc insertion. Systematic truncation of the C-terminus indicated the requirement for the last 15 amino acid residues of the receptor for [125I]IACoc photolabeling.     

Out of Africa and back again: nested cladistic analysis of human Y chromosome variation

We surveyed nine diallelic polymorphic sites on the Y chromosomes of 1,544 individuals from Africa, Asia, Europe, Oceania, and the New World. Phylogenetic analyses of these nine sites resulted in a tree for 10 distinct Y haplotypes with a coalescence time of approximately 150,000 years. The 10 haplotypes were unevenly distributed among human populations: 5 were restricted to a particular continent, 2 were shared between Africa and Europe, 1 was present only in the Old World, and 2 were found in all geographic regions surveyed. The ancestral haplotype was limited to African populations. Random permutation procedures revealed statistically significant patterns of geographical structuring of this paternal genetic variation. The results of a nested cladistic analysis indicated that these geographical associations arose through a combination of processes, including restricted, recurrent gene flow (isolation by distance) and range expansions. We inferred that one of the oldest events in the nested cladistic analysis was a range expansion out of Africa which resulted in the complete replacement of Y chromosomes throughout the Old World, a finding consistent with many versions of the Out of Africa Replacement Model. A second and more recent range expansion brought Asian Y chromosomes back to Africa without replacing the indigenous African male gene pool. Thus, the previously observed high levels of Y chromosomal genetic diversity in Africa may be due in part to bidirectional population movements. Finally, a comparison of our results with those from nested cladistic analyses of human mtDNA and beta-globin data revealed different patterns of inferences for males and females concerning the relative roles of population history (range expansions) and population structure (recurrent gene flow), thereby adding a new sex-specific component to models of human evolution.      

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

The phylogeny of the hominoid primates: a statistical analysis of the DNA-DNA hybridization data

Sibley and Ahlquist compared the single-copy nuclear DNA sequences of the hominoid primates using DNA-DNA hybridization. From this data set they estimated a phylogeny that clusters man and chimpanzees using a distance Wagner procedure. However, no assessment of statistical confidence in this estimated phylogeny was made, despite the fact that their data set contains internal inconsistencies concerning the correct branching order. This paper presents a modification of Pielou's Q- statistic that allows one to make nonparametric tests of phylogenetic relationship from distance data. The results of this analysis indicate that the estimated phylogeny of Sibley and Ahlquist is without statistical significance owing to the internal inconsistencies of the data set. A survey and additional analyses of other types of molecular data indicate that the phylogeny that clusters chimpanzees and gorillas and has the human lineage splitting off earlier is statistically consistent with all the molecular data (including the DNA-DNA hybridization data), whereas the phylogeny estimated by Sibley and Ahlquist can be rejected at the 5% level using the data on restriction- endonuclease sites in the mitochondrial genome.