Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.      

Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.      

The interaction of 1-alkyl-4, 4-diphenylpiperidines with the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine receptor binding site

Summary 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) is a selective neurotoxin which produces degeneration of the nigrostriatal bundles in the central nervous system of man and animals. In these areas of the brain are concentrated the receptor binding sites for [³H]MPTP. 1-Alkyl-4, 4-diphenylpiperidines displace [³H]MPTP from these binding sites with K₁ values in the micromolar range. The t-butyl analogue in this class of substances, budipine, is a novel therapeutic agent for Parkinsonism whose mechanism has not yet been fully clarified. The affinity of budipine for the MPTP receptor binding site was determined as a K₁ value of 2.2M. Other 4, 4-diphenylpiperidine derivatives such as 1-methyl-4, 4-diphenylpiperidine and 1-i-propyl-4, 4-diphenylpiperidine have substantially lower affinities. Monoamine oxidase inhibitors such as deprenyl, pargyline and harmaline have affinities to the MPTP receptors which parallel their affinity for the B type of monoamine oxidase (MAO B). This supports the theory that the MPTP receptor binding sites is identical with membrane bound MAO B.     

Inhaled environmental combustion particles cause myocardial injury in the Wistar Kyoto rat.

Epidemiologists have associated particulate matter (PM) air pollution with cardiovascular morbidity and premature mortality worldwide. However, experimental evidence demonstrating causality and pathogenesis of particulate matter (PM)-induced cardiovascular damage has been insufficient. We hypothesized that protracted, repeated inhalation by rats of oil combustion-derived, fugitive emission PM (EPM), similar in metal composition to selected sources of urban air PM, causes exposure duration- and dose-dependent myocardial injury in susceptible rat strains. Zinc was the only primary water-leachable/bioavailable element of this EPM. Male Sprague-Dawley (SD), Wistar Kyoto (WKY), and spontaneously hypertensive (SH) rats were exposed nose-only to EPM (2, 5, or 10 mg/m(3), 6 h/day for 4 consecutive days or 10 mg/m(3), 6 h/day, 1 day/week for 4 or 16 consecutive weeks). Two days following the last EPM exposure, cardiac and pulmonary tissues were examined histologically. The results showed that particle-laden alveolar macrophages were the only pulmonary lesions observed in all three rat strains. However, WKY rats exposed to EPM (10 mg/m(3) 6 h/day, 1 day/week for 16 weeks) demonstrated cardiac lesions with inflammation and degeneration. To further characterize the nature of EPM-associated lesions, more rigorous histopathological and histochemical techniques were employed for WKY and SD rats. We examined the hearts for myocardial degeneration, inflammation, fibrosis, calcium deposits, apoptosis, and the presence of mast cells. Decreased numbers of granulated mast cells, and multifocal myocardial degeneration, chronic-active inflammation, and fibrosis were present in 5 of 6 WKY rats exposed to EPM for 16 weeks. None of these lesions were present in WKY exposed to clean air. EPM-related cardiac lesions were indistinguishable from air-exposed controls in SD and SH rats. This study demonstrates that long-term inhalation exposures to environmentally relevant PM containing bioavailable zinc can cause myocardial injury in sensitive rats. These findings provide supportive evidence for the epidemiological associations of cardiovascular morbidity and ambient PM.     

A prospective study of methylenetetrahydrofolate reductase and methionine synthase gene polymorphisms, and risk of colorectal adenoma

We examined the relationship between a functional polymorphism (667C-- >T, ala-->val) of the methylenetetrahydrofolate reductase gene (MTHFR) and the risk of colorectal adenomas in the prospective Nurses' Health Study. Among 257 incident polyp cases and 713 controls, the MTHFR val/val polymorphism [relative risk (RR) = 1.35, 95% confidence interval (CI) 0.84-2.17] was not significantly associated with risk of adenomas. This lack of association was observed for both small (RR = 1.36, 95% CI 0.76-2.45) and large (RR = 1.32, 95% CI 0.66-2.66) adenomas. Furthermore, there was no significant interaction between this polymorphism and consumption of either folate, methionine or alcohol. We also examined the relationship of a newly identified polymorphism (asp919gly) of the methionine synthase gene (MS) with the risk of colorectal adenomas in the same population. The MS gly/gly polymorphism was also not significantly associated with risk of colorectal adenomas (RR = 0.66, 95% CI 0.26-1.70). These results, which need to be confirmed in other studies, suggest that the MTHFR val/val polymorphism, which has been previously inversely associated with risk of colorectal cancer, plays a role only in a late stage (adenoma-- >carcinoma) of colorectal tumorigenesis, and/or may protect against malignant transformation in the subset of benign adenomas, which may progress to malignancy.      

Molecular weights of poly(butyl cyanoacrylate) nanoparticles determined by mass spectrometry and size exclusion chromatography

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and size exclusion chromatography (SEC) were employed to elucidate the chemical composition, mean number average molecular weight (M_n), mean weight average molecular weight (M_w), and polydispersity (PD) of poly(butyl cyanoacrylate) (PBCA) manufactured by emulsion polymerisation. Both methods gave similar results for M_n, but substantial differences were observed for M_w and PD, with MALDI producing consistently lower values which could not be improved by off-line coupling of SEC and MALDI. MALDI gave a more detailed view on the chemical composition of the cyanoacrylate and revealed the presence of two additional polymer series with different end groups besides the expected PBCA series, which showed different retention in SEC. Their formation is explained by the secession/addition of formaldehyde from/to the regular polymer via (reverse) Knoevenagel reaction. In additional experiments, the influence of different pH on PBCA-NP during polymerisation was examined by comparison of polymerisation yield and particle diameter to their chemical composition as revealed by the MALDI spectra. The most uniform nanoparticles, with the highest polymerisation yield, narrowest particle size, and mass distribution were produced at pH 1.