Molecular structure and properties of protonated and methylated derivatives of cytosine

Ab initio techniques were used to determine the effects of protonation and methylation on cytosine's molecular geometry, molecular affinities, relative stability, and structural rigidity. The geometries of local minima were fully optimized by the gradient procedure at DFT and MP2 levels of theory with the medium size 6-31G(d,p) basis set. The results of energetic analysis indicate that N(3)-methyl-cytosine and C(5)-methyl-cytosine are the most stable derivatives for monocationic and neutral species, respectively. The structural rigidity of each species was assessed by an analysis of normal out-of-plane frequencies, the amplitudes, and by the contribution of internal coordinates to the potential energy distributions. The obtained evidence suggests that methylation increases the overall structural flexibility of cytosine and that all molecules in this study populate a non-planar conformation 50% of the time.     

Circulating proangiogenic cytokines and angiogenesis inhibitor endostatin in untreated patients with chronic lymphocytic leukemia

The serum concentration of two pro-angiogenic cytokines: basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1), and anti-angiogenic factor endostatin in the serum of 80 never treated B-cell chronic lymphocytic leukemia (CLL) patients and 27 healthy volunteers was measured using an enzyme linked immunosorbent assay. The serum levels of both bFGF and TGF-beta1 were found to be significantly higher in the CLL group (median 40.5 pg/ml and 38.6 ng/ml respectively) when compared to the control group (median 9.4 pg/ml and 18.9 ng/ml, respectively) (p<0.001). The levels of endostatin were not significantly different in CLL and control groups (median 12.3 ng/ml and 8.4 ng/ml, respectively) (p=0.09). In the group of CLL patients the level of bFGF was significantly higher in patients with progressive disease as compared with patients with stable disease (median 90.5 pg/ml and 40.5 pg/ml respectively) (p<0.001). Patients in Rai stage III and IV also had significantly higher levels of bFGF than patients in Rai stage 0-II (median 100.1 pg/ml and 29.3 pg/ml respectively) (p<0.001). The levels of both TGF-beta1 and endostatin were lower in patients in Rai stage III and IV (median 28.9 ng/ml and 9.1 ng/ml respectively) than in patients in Rai stage 0-II (42.8 ng/ml and 13.1 ng/ml respectively) (p<0.001 and p=0.002 respectively). The level of endostatin was also lower in the group of CLL patients with progressive disease (median 10.0 ng/ml) as compared to patients with stable disease (median 20.5 ng/ml) (p=0.008). In conclusion, the disturbance in the balance between pro- and anti-angiogenic factors may have an important influence on the course of CLL.     

Synthesis of (3,4) beta-methylenecepham and (3,4) beta-methylene-carbacepham via intramolecular carbene addition to double bond

A series of new (3,4) beta-methylenecepham and carbacepham analogues were synthesised as potential antibacterial agents. The key step of the synthesis included presumed generation of the carbene species from the oxalimide substrate effected by triethylphosphite and its intramolecular addition to the double bond. The stereochemistry of the tricyclic system has been elucidated by NMR and X-ray crystallography. In preliminary screening, two of the synthesised compounds exhibited modest antibacterial activity at 1.5-2.0 mg/mL against a number of bacterial strains.     

Comprehensive analysis of two Alu Yd subfamilies

Alu elements have inserted in the human genome throughout primate evolution. A small number of Alu insertions have occurred after the divergence of humans from nonhuman primates and therefore should not be present in nonhuman primate genomes. Most of these recently integrated Alu elements are contained with a series of discrete Alu subfamilies that are related to each other based upon diagnostic nucleotide substitutions. We have extracted members of the Alu Yd subfamily that are derivatives of the Alu Y subfamily that share a common 12-bp deletion that defines the Yd lineage from the draft sequence of the human genome. Analysis of the Yd Alu elements resulted in the recovery of two new Alu subfamilies, Yd3 and Yd6, which contain a total of 295 members (198 Yd3 and 97 Yd6). DNA sequence analysis of each of the Alu Yd subfamilies yielded age estimates of 8.02 and 1.20 million years old for the Alu Yd3 and Yd6 subfamilies, respectively. Two hundred Alu Yd3 and Yd6 loci were screened using polymerase chain reaction (PCR) assays to determine their phylogenetic origin and associated levels of human genomic diversity. The Alu Yd3 subfamily appears to have started amplifying relatively early in primate evolution and continued propagating albeit at a low level as many of its members are found in a variety of hominoid (humans, greater and lesser ape) genomes. Only two of the elements are polymorphic in the human genome and absent from the genomes of nonhuman primates. By contrast all of the members of the Alu Yd6 subfamily are restricted to the human genome, with 12% of the elements representing insertion polymorphisms in human populations. A single Alu Yd6 locus contained an independent parallel forward insertion of a paralogous Alu Sq sequence in the owl monkey. These Alu subfamilies are a source of genomic fossil relics for the study of primate phylogenetics and human population genetics.     

Tissue distribution of pneumadin immunoreactivity in the rat

Pneumadin (PNM) is a decapeptide, originally isolated from mammalian lungs, which exerts a potent stimulating effect on arginine-vasopressin (AVP) release, thereby evoking an antidiuretic effect. We have established a specific radioimmunoassay (RIA) method for rat PNM determination, the sensitivity of which is sufficient for measuring tissue content of the peptide. Moreover, raised antibodies have been used for the immunocytochemical detection of PNM in several rat organs. As expected, high concentrations of PNM were detected by RIA in newborn and adult rat lungs and immunocytochemistry (ICC) localized PNM immunoreactivity (IR) in the bronchial and bronchiolar epithelium. Very high concentrations of PNM were measured by RIA in the prostate, and ICC showed that PNM-IR is contained in the epithelial cells. RIA and ICC demonstrated the presence of low amounts of PNM in the thymus. The highest content of radioimmunoassayable PNM was found in the kidneys and intestinal tract, but dilution test suggested the presence of some interfering substances in these tissues. Accordingly, ICC-detectable PNM-IR was absent in the kidneys and present only in the duodenal criptae and Brunner's glands of the intestinal tract. RIA did not measure sizeable PNM concentrations in the thyroid gland, but ICC showed PNM-IR in C-cells. RIA and ICC did not detected PNM in testes, seminal vesicles, ovaries, uterus, pancreas, liver, spleen, adrenal glands, and heart. Taken together, our findings suggest that PNM, in addition to its role as hypothalamo-pituitary AVP secretagogue, may be involved in the autocrine-paracrine functional regulation of other peripheral organs, like lungs and prostate and perhaps duodenum, thymus and thyroid gland.     

Pneumadin in the ventral prostate and seminal vesicles of estradiol-treated rats: RIA and immunocytochemical studies

Pneumadin (PNM) is a decapeptide (the rat peptide: Tyr-Gly-Glu-Pro-Lys-Leu-Asp-Ala-Gly-Val-NH2) isolated from mammalian lungs. Human and rat PNM differ only by substitution of one amino acid--Tyr/Ala. PNM evokes an antidiuretic effect via a potent stimulation of arginine-vasopressin (AVP) release. By means of recently established, highly specific RIA method, high concentration of PNM had been found in the rat ventral prostate. Castration resulted in a profound drop in PNM concentration, an effect prevented by testosterone replacement. The present studies were aimed at investigating the effect of prolonged estradiol administration on PNM concentration, content and localization in the prostate and seminal vesicles of the rat. Depo estradiol (estradiolum valerianicum) administration to adult male rats resulted in a notable atrophy of ventral prostate and seminal vesicles. During the entire experiment (till day 30 after administration), PNM concentration in ventral prostate was similar to that seen in intact animals, while peptide content per gland was markedly lowered. PNM immunostaining was observed in prostate epithelium of estradiol-treated rats and its localization resembled that observed in intact animals. Nearly 40 times lower PNM concentration than in ventral prostate was found in seminal vesicles. In contrast to prostate, on days 20 and 30 of estradiol treatment PNM concentration in seminal vesicles was higher than in intact rats. However, due to profound seminal vesicle atrophy, PNM content per entire gland was notably lowered in estradiol-injected rats. By immunocytochemistry, PNM-immunoreactive substances were not found in seminal vesicles of either intact or estradiol-administered rats. High PNM concentration in the rat prostate suggests its important role in the function of the gland.     

NPY: its occurrence and relevance in the female reproductive system

Neuropeptide Y (NPY), an amidated peptide composed of 36 amino acid residues, is the most widely distributed neuropeptide that performs a broad spectrum of physiological functions in both the central and peripheral nervous systems. Among numerous other actions, this peptide is involved, at the periphery, in the neural regulation of blood pressure and blood flow through the organs, and also, acting via Y2 and/or Y5 receptors, in the regulation of angiogenesis. NPY influences blood vessels via its own Y receptors, predominantly of the Y1 subtype. As a sympathetic co-transmitter NPY causes vasoconstriction, stimulates vascular growth and potentiates the contractile activity of noradrenaline (NA), and as a parasympathetic neurotransmitter it is involved in the regulation of vasodilatation within e.g. the uterine artery. In the female reproductive system, NPY not only regulates the blood flow, but also the contractile activity of non-vascular smooth muscle cells of the uterus and oviduct, as well as the secretory function of the ovary. Both the concentration of NPY and its influence on the blood flow through the female reproductive organs are finely tuned by fluctuations in the concentration of ovarian steroid hormones. Thus, the present review was aimed at summarizing the current knowledge dealing with the physiological relevance of NPY in the regulation of female gonad and genital tract function, with a special regard to the pig as a model animal.     

Application of STS markers for leaf rust resistance genes in near-isogenic lines of spring wheat cv. Thatcher

Sequence tagged site (STS) markers for eight resistance genes against Puccinia recondita f. sp. tritici were used to screen a set of near-isogenic lines of wheat cv. Thatcher containing in total 40 different Lr genes and their alleles. Polymerase chain reaction (PCR) analysis was carried out by using STS, SCAR and CAPS primers specific for the leaf rust resistance genes Lr1, Lr9, Lr10, Lr19, Lr24, Lr28, Lr37 and Lr47. The STS, CAPS and SCAR markers linked to resistance genes Lr9, Lr10, Lr19, Lr24, Lr37 and Lr47 were found to be reliable in diverse genetic backgrounds. The amplification product of the Lr1 gene marker was detected in the susceptible cv. Thatcher and in all of the near-isogenic lines examined except Lr2a, Lr2b, Lr2c and Lr19. The sequence analysis of PCR products amplified in lines Lr1, Lr10, Lr28 and in cv. Thatcher indicated that the near-isogenic lines and cv. Thatcher contained in the targeted chromosome region an allele that differed from the original alleles corresponding to Lr1/6*Thatcher (TLR621) and susceptible Thatcher (TH621). The amplification product specific to the STS marker of the Lr1 gene was amplified in almost all Thatcher near-isogenic lines and in cv. Thatcher because their alleles possessed primer sequences identical to the original allele TLR621. The marker for the Lr28 resistance gene was identified in line Lr28, carrying gene Lr28, and in 21 other near-isogenic lines. The sequencing of PCR products specific to Lr28 and generated in lines Lr1, Lr10 and Lr28 indicated that the lines Lr1, Lr10 and Lr28 are heterozygous in this region.     

Resistance genes in barley (Hordeum vulgare L.) and their identification with molecular markers

Current information on barley resistance genes available from scientific papers and on-line databases is summarised. The recent literature contains information on 107 major resistance genes (R genes) against fungal pathogens (excluding powdery mildew), pathogenic viruses and aphids identified in Hordeum vulgare accessions. The highest number of resistance genes was identified against Puccinia hordei, Rhynchosporium secalis, and the viruses BaYMV and BaMMV, with 17, 14 and 13 genes respectively. There is still a lot of confusion regarding symbols for R genes against powdery mildew. Among the 23 loci described to date, two regions Mla and Mlo comprise approximately 31 and 25 alleles. Over 50 R genes have already been localised and over 30 mapped on 7 barley chromosomes. Four barley R genes have been cloned recently: Mlo, Rpg1, Mla1 and Mla6, and their structures (sequences) are available. The paper presents a catalogue of barley resistance gene symbols, their chromosomalocation and the list of available DNA markers useful in characterising cultivars and breeding accessions.