A new biscoumarin glucoside ester from Ruta chalepensis cell cultures

The investigation of a plant cell culture of RUTA CHALEPENSIS L. yielded three compounds: isorutarin, rutacridone, and rutarensin, a so far unknown ester of daphnoretin glucoside ("daphnorin"), as well as 3-hydroxy-3-methylglutaric acid.     

The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity

The integrity of sperm DNA is crucial for the maintenance of genetic health. A major source of damage is reactive oxygen species (ROS) generation; therefore, antioxidants may afford protection to sperm DNA. The objectives of the study were, first, to measure the effects of antioxidant supplementation in vitro on endogenous DNA damage in spermatozoa using the single cell gel electrophoresis (comet) assay and, second, to assess the effect of antioxidant supplementation given prior to X-ray irradiation on induced DNA damage. Spermatozoa from 150 patients were prepared by Percoll centrifugation in the presence of ascorbic acid (300, 600 microM), alpha tocopherol (30, 60 microM), urate (200, 400 microM), or acetyl cysteine (5, 10 microM). DNA damage was induced by 30 Gy X-irradiation. DNA strand breakage was measured using the comet assay. Sperm DNA was protected from DNA damage by ascorbic acid (600 microM), alpha tocopherol (30 and 60 microM) and urate (400 microM). These antioxidants provided protection from subsequent DNA damage by X-ray irradiation. In contrast, acetyl cysteine or ascorbate and alpha tocopherol together induced further DNA damage. Supplementation in vitro with the antioxidants ascorbate, urate and alpha tocopherol separately has beneficial effects for sperm DNA integrity.      

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.      

Polymers containing enzymatically degradable bonds. VI.Hydrophilic gels cleavable by chymotrypsin

New types of hydrophilic gels based on N-(2-hydroxypropyl)methacrylamide which contain oligopeptide sequences in the crosslinks were prepared. These gels are enzymatically degradable by chymotrypsin. The rate of their degradation may be varied within a broad range by changes in the length and detailestructure of the oligopeptide sequence in the crosslinks and by changing their network density.     

Poly(ethylene glycol)s containing enzymatically degradable bonds

The possibility to use poly(ethylene glycol) (PEG) as a synthetic, water-soluble polymeric carrier of biologically active compounds was investigated. The relationship between structure of oligopeptide (amino acid)-PEG conjugates and their hydrolysis catalyzed by chymotrypsin was studied. Two types of derivatives were synthesized: Derivatives of PEG 2000, containing an oligopeptide sequence terminating in p-nitroaniline (drug model) and higher molecular weight derivatives of PEG 2000 containing in the main chain enzymatically and hydrolytically degradable bonds. The rates of chymotrypsin catalyzed release of p-nitroaniline at pH 8,0 and 25°C were determined over a range of substrate concentrations to derive values for k_cat and K_M. The influence of the length and detailed structure of the oligopeptide sequence on the rate of hydrolysis was demonstrated. Comparison with copolymers of N-(2-hydroxypropyl)methacrylamide and poly(maleic anhydride-co-vinylpyrrolidone) showed that the sterical hindrance of the formation of the enzyme-substrate complex is much less pronounced in the PEG-oligopeptide conjugates. The latter conclusion is also valid in the case of cleavage of PEG derivatives which contain enzymatically degradable bonds in the main chain. The incorporation of a single amino acid residue (phenylalanine) into the main PEG chain is sufficient to make the polymer susceptible to chymotrypsin catalyzed hydrolysis.     

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.      

Reduced cardiotoxicity of doxorubicin given in the form ofN-(2-hydroxypropyl) methacrylamide conjugates: an experimental study in the rat

Summary A rat model was used to evaluate the general acute toxicity and the late cardiotoxicity of 4 mg/kg doxorubicin (DOX) given either as free drug or in the form of threeN-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates. In these HPMA copolymers, DOX was covalently bound via peptide linkages that were either non-biodegradable (Gly-Gly) or degradable by lysosomal proteinases (Gly-Phe-Leu-Gly). In addition, one biodegradable conjugate containing galactosamine was used; this residue was targeted to the liver. Over the first 3 weeks after the i.v. administration of free and polymer-bound DOX, all animals showed a transient reduction in body weight. However, the maximal reduction in body weight seen in animals that received polymer-bound DOX (4 mg/kg) was significantly lower than that observed in those that received free DOX (4 mg/kg) or a mixture of the unmodified parent HPMA copolymer and free DOX (4 mg/kg;P<0.01). Throughout the study (20 weeks), deaths related to cardiotoxicity were observed only in animals that received either free DOX or the mixture of HPMA copolymer and free DOX; in these cases, histological investigations revealed marked changes in the heart that were consistent with DOX-induced cardiotoxicity. Sequential measurements of cardiac output in surviving animals that received either free DOX or the mixture of HPMA copolymer and free DOX showed a reduction of 30% in function beginning at the 4th week after drug administration. The heart rate in these animals was 12% lower than that measured in age-matched control rats (P<0.05). Animals that were given the HPMA copolymer conjugates containing DOX exhibited no significant change in cardiac output throughout the study (P<0.05). In addition, no significant histological change was observed in the hearts of animals that received DOX in the form of HPMA copolymer conjugates and were killed at the end of the study. However, these animals had shown a significant increase in heart rate beginning at 8 weeks after drug administration (P<0.01). This study demonstrates that covalent binding of DOX to HPMA copolymer conjugates via both stable and biodegradable peptidyl linkages considerably reduces both the general acute toxicity and the late cardiotoxicity of DOX in the rat and could offer the potential for improving the therapeutic index in the clinical application of DOX.