Uncialamycin as a novel payload for antibody drug conjugate (ADC) based targeted cancer therapy

Uncialamycin analogs were evaluated as potential cytotoxic agents in an antibody-drug conjugate (ADC) approach to treating human cancer. These analogs were synthesized using Hauser annulations of substituted phthalides as a key step. A highly potent uncialamycin analog 3c with a valine-citrulline dipeptide linker was conjugated to an anti-mesothelin monoclonal antibody (mAb) through lysines to generate a meso-13 conjugate. This conjugate demonstrated subnanomolar potency (IC50?=?0.88?nM, H226 cell line) in in vitro cytotoxicity experiments with good immunological specificity to mesothelin-positive lung cancer cell lines. The potency and mechanism of action of this uncialamycin class of enediyne antitumor antibiotics make them attractive payloads in ADC-based cancer therapy.     

Kinetic FP-TDI assay for SNP allele frequency determination

Strategies for identifying genetic risk factors in complex diseases by association studies require the comparison of allele frequencies of numerous SNPs between affected and control populations. Theoretically, hundreds of thousands of SNP markers across the genome will have to be genotyped in these studies. Genotyping SNPs one sample at a time is extremely costly and time consuming. To streamline whole genome association studies, some have proposed to screen SNPs by pooling the DNA samples initially for allele frequency determination and perform individual genotyping only when there is a significant discrepancy in allele frequencies between the affected and control populations. Here we describe a new method for determining the allele frequency of SNPs in pooled DNA samples using a two-color primer extension assay with real-time monitoring of fluorescence polarization (named kinetic FP-TDI assay). By comparing the ratio of the rate of incorporation of the two allele-specific dye-terminators, one can calculate the relative amounts of each allele in the pooled sample. The accuracy of allele frequency determination with pooled samples is within 3.3 +/- 0.8% of that determined by genotyping individual samples that make up the pool.     

Phospholipid transfer protein (PLTP) mRNA expression is stimulated by developing embryos in the oviduct

In mammal, fertilization and early preimplantation embryo development occurs in the oviduct. Evidence is accumulating that the oviductal epithelia secrete various biomolecules to the lumen during the secretory phase of the estrus cycle to enhance embryo development. This secretory activity of the oviduct is under the regulation of steroid hormones. Observations also suggested that the gametes and embryos modulate the physiology and gene-expressing pattern of the oviduct. However, the underlying molecular changes remain elusive. We hypothesize that the developing embryos interact with the surrounding environment and affect the gene expression patterns of the oviduct, thereby modulating the oviductal secretory activity conducive to the preimplantation embryo development. To test this hypothesis, suppression subtractive hybridization (SSH) was used to compare the gene expressions in mouse oviduct containing transferred in vitro cultured preimplantation embryos with that of oviduct containing oocytes during the preimplantation period. We reported here the identification and characterization of phospholipids transfer protein (PLTP), which is highly expressed in the embryo-containing oviduct and localized at the oviductal epithelium by in situ hybridization. PLTP contains signal peptide putative for secretory function. More importantly, PLTP mRNA increases in the oviductal epithelia of pregnant, but not pseudo-pregnant mice when assayed by real-time PCR. Taken together, our data suggested that PLTP may play important role(s) during in vivo preimplantation embryo development. This molecule would be a target to delineate the mechanisms and the roles of oviductal secretory proteins on early embryonic development.     

Associations of Birth Order with Early Adolescent Growth,Pubertal Onset,Blood Pressure and Size: Evidence from Hong Kong’s “Children of 1997” Birth Cohort

BackgroundBirth order has been proposed as a cardiovascular risk factor, because the lower birth weight and greater infant weight gain typical of firstborns could programme metabolism detrimentally.MethodsWe examined the associations of birth order (firstborn or laterborn) with birth weight-for-gestational age, length/height and body mass index (BMI) z-scores during infancy, childhood, and puberty using generalized estimating equations, with age at pubertal onset using interval-censored regression and with age-, sex- and height-standardized blood pressure, height and BMI z-scores at 13 years using linear regression in a population-representative Chinese birth cohort: “Children of 1997” (n = 8,327).ResultsCompared with laterborns, firstborns had lower birth weight-for-gestational age (mean difference = -0.18 z-score, 95% confidence interval (CI) -0.23, -0.14), lower infant BMI (-0.09 z-score, 95% CI -0.14, -0.04), greater childhood height (0.10 z-score, 95% CI 0.05, 0.14) and BMI (0.08 z-score, 95% CI 0.03, 0.14), but not greater pubertal BMI (0.05 z-score, 95% CI -0.02, 0.11), adjusted for sex, parental age, birthplace, education and income. Firstborns had earlier onset of pubic hair (time ratio = 0.988, 95% CI 0.980, 0.996), but not breast or genitalia, development. Firstborns had greater BMI (0.07 z-score, 95% CI 0.002, 0.15), but not height (0.05 z-score, 95% CI -0.01, 0.11), at 13 years, but similar blood pressure.ConclusionsDifferences by birth order continue into early adolescence with firstborns being heavier with earlier pubic hair development, which could indicate long-term cardiovascular risk.     

Glucose-6-Phosphate Dehydrogenase Deficiency and Physical and Mental Health until Adolescence

BackgroundTo examine the association of glucose-6-phosphate dehydrogenase (G6PD) deficiency with adolescent physical and mental health, as effects of G6PD deficiency on health are rarely reported.MethodsIn a population-representative Chinese birth cohort: “Children of 1997” (n = 8,327), we estimated the adjusted associations of G6PD deficiency with growth using generalized estimating equations, with pubertal onset using interval censored regression, with hospitalization using Cox proportional hazards regression and with size, blood pressure, pubertal maturation and mental health using linear regression with multiple imputation and inverse probability weighting.ResultsAmong 5,520 screened adolescents (66% follow-up), 4.8% boys and 0.5% girls had G6PD deficiency. G6PD-deficiency was not associated with birth weight-for-gestational age or length/height gain into adolescence, but was associated with lower childhood body mass index (BMI) gain (-0.38 z-score, 95% confidence interval (CI) -0.57, -0.20), adjusted for sex and parental education, and later onset of pubic hair development (time ratio = 1.029, 95% CI 1.007, 1.050). G6PD deficiency was not associated with blood pressure, height, BMI or mental health in adolescence, nor with serious infectious morbidity until adolescence.ConclusionsG6PD deficient adolescents had broadly similar physical and mental health indicators, but transiently lower BMI gain and later pubic hair development, whose long-term implications warrant investigation.     

Extensive restriction site polymorphism at the human phenylalanine hydroxylase locus and application in prenatal diagnosis of phenylketonuria.

A total of 10 restriction site polymorphisms have been identified at the human phenylalanine hydroxylase locus using a full-length human phenylalanine hydroxylase cDNA clone as a hybridization probe to analyze human genomic DNA. These polymorphic patterns segregate in a Mendelian fashion and concordantly with the disease state in various PKU kindreds. The frequencies of the restriction site polymorphisms at the human phenylalanine hydroxylase locus among Caucasians are such that the observed heterozygosity in the population is 87.5%. Thus, most families with a history of classical phenylketonuria can take advantage of the genetic analysis for prenatal diagnosis and carrier detection of the hereditary disorder.     

Homology between phenylalanine and tyrosine hydroxylases reveals common structural and functional domains

Phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TYH) are mixed-function oxidases that share many characteristic biochemical and immunological properties. The recent cloning and sequencing of full-length cDNAs for both human PAH and rat TYH allow detailed comparison of their primary structures. There is a high degree of homology between PAH and TYH on nucleic acid and amino acid levels. The pattern of homology suggests that these molecules are comprised of a homologous core containing the determinants for enzymatic activity and a nonhomologous region that contributes to substrate specificity and regulation. The degree of homology also suggests that these two proteins evolved from a common ancestor.     

Purification and chemical modification of a phosphatidylinositol kinase from sheep brain.

A membrane-bound phosphatidylinositol (PtdIns) kinase has been purified approximately 9500-fold to apparent homogeneity from sheep brains. The purification procedure involves: solubilisation of the membrane fraction with Triton X-100, ammonium sulphate fractionation and a number of ion-exchange and gel-filtration chromatography steps. The purified enzyme exhibited a final specific activity of 1149 nmol.min-1.mg-1. The molecular mass of the enzyme was estimated to be 55 kDa by SDS/PAGE and 150 +/- 10 kDa by HPLC gel filtration in the presence of Triton X-100. Kinetic measurements have shown that the apparent Km value of PtdIns kinase for the utilisation of PtdIns is 22 microM and for ATP 67 microM. Mg2+ was the most effective divalent cation activator of PtdIns kinase, with maximal enzymatic activity reached at a concentration of 10 mM Mg2+. In addition to adenosine and ADP, the 2'(3')-O-(2,4,6-trinitrophenyl) derivative of ATP was found to be a strong competitive inhibitor of the enzyme, with a Ki of 32 microM. Enzymatic activity was found to be stimulated by Triton X-100 but inhibited by deoxycholate.     

The concentration of dopamine, 5-hydroxytryptamine,and some of their acid metabolites in the brain of genetically diabetic rats

This is a report of the concentrations of DA, DOPAC, HVA, 5-HT, and 5-HIAA in the brain ofspontaneously diabetic male Wistar rats. These rats showed a marked increase in blood and urine glucose, polydipsia, polyuria, and weight loss that had an onset 11–23 days earlier. Controls were litter mates with no hyperglycemia, glucosuria or weight reduction. Thespontaneously diabetic rats showed a significant reduction of DOPAC in the striatum, and DOPAC, HVA, and 5HIAA in the olfactory tubercles (to 69, 61, 62, and 65% of their respective controls). No changes were found in the concentrations of DA or 5-HT. Thus thespontaneously diabetic rats showed a marked reduction in striatal and mesolimbic DA and mesolimbic 5-HT metabolism. This reduction in metabolism could be the consequence of a reduction in the formation of DA and 5-HT.