Bronchodilatory properties of 2-decarboxy-2-hydroxymethyl prostaglandin E1

We evaluated in a double-blind study the bronchodilatory properties of 2-decarboxy-2-hydroxymethyl prostaglandin E₁ (PGE₁-carbinol), described recently as a nonirritant bronchodilator in animals. Fifteen asthmatic patients received by inhalation single doses of 1, 10, and 30 μg PGE₁-carbinol, 55 μg PGE₂, and placebo (10% ethanol in normal saline, which was also used as diluent for the PGs). Such pulmonary function tests as forced expiratory volume in 1 second, forced vital capacity, and maximal expiratory flow were monitored during 2 hours following inhalation of each compound. 10 and 30 μg PGE₁-carbinol produced significant but short-acting bronchodilation, similar to that caused by 55 μg PGE₂. One-third of the patients reported mild cough and throat irritation during and shortly after inhalation of 30 μg PGE₁-carbinol or 55 μg PGE₂. Placebo and 1 μg PGE₁-carbinol produced minimal side effects, but neither agent caused bronchodilation. In an adjunctive, unblinded trial, the same patients received 400 μg fenoterol. Fenoterol caused greater bronchodilation 15 and 30 minutes after inhalation than did the PGs in the double-blind study.     

Maturation of somatic embryos of Pinus strobus is promoted by a high concentration of gellan gum

Application of somatic embryogenesis to Pinus strobus clonal propagation and genetic improvement was hampered by the difficulty in achieving synchronous maturation of a large number of somatic embryos that would germinate and produce plants. Media containing abscisic acid (80 μ M ) and osmotic agents such as sucrose, polyethylene glycol and/or dextran did not sustain development of mature somatic embryos from plated embryonal masses. This indicated that factors other than osmotic agents might be involved in sustaining development of Pinus strobus somatic embryos to maturity. It was subsequently found that media lacking osmotica but containing a high concentration of gellan gum (1%) induced significant improvement in the development of mature somatic embryos in the presence of 80 or 120 μ M abscisic acid. This positive effect was independent of the genotype and all four tested lines displayed similar responses. Media containing gellan gum at concentrations from 0.4 to 1.2% formed gels that varied in their strength. Gel strength was proportional to the concentration of gellan gum in the specific medium but varied depending on the medium formulation. Gel strength increased with the duration of storage of the culture medium by 46% (SD 14) after 14 days of storage. Preliminary results showed that embryos matured on high gellan gum media displayed improved germination frequencies. These results indicate that in Pinus strobus the water status and possibly other medium characteristics that are influenced by increased concentration of gelling agent have stimulatory effects on maturation of somatic embryos.     

Biosynthesis,glycosylation, and enzymatic processing in vivo of human tripeptidyl-peptidase I

Human tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal serine protease that removes tripeptides from the free N termini of small polypeptides and also shows a minor endoprotease activity. Due to various naturally occurring mutations, an inherited deficiency of TPP I activity causes a fatal lysosomal storage disorder, classic late infantile neuronal ceroid lipofuscinosis (CLN2). In the present study, we analyzed biosynthesis, glycosylation, transport, and proteolytic processing of this enzyme in stably transfected Chinese hamster ovary cells as well as maturation of the endocytosed proenzyme in CLN2 lymphoblasts, fibroblasts, and N2a cells. Human TPP I was initially identified as a single precursor polypeptide of approximately 68 kDa, which, within a few hours, was converted to the mature enzyme of approximately 48 kDa. Compounds affecting the pH of intracellular acidic compartments, those interfering with the intracellular vesicular transport as well as inhibition of the fusion between late endosomes and lysosomes by temperature block or 3-methyladenine, hampered the conversion of TPP I proenzyme into the mature form, suggesting that this process takes place in lysosomal compartments. Digestion of immunoprecipitated TPP I proenzyme with both N-glycosidase F and endoglycosidase H as well as treatment of the cells with tunicamycin reduced the molecular mass of TPP I proenzyme by approximately 10 kDa, which indicates that all five potential N-glycosylation sites in TPP I are utilized. Mature TPP I was found to be partially resistant to endo H treatment; thus, some of its N-linked oligosaccharides are of the complex/hybrid type. Analysis of the effect of various classes of protease inhibitors and mutation of the active site Ser(475) on human TPP I maturation in cultured cells demonstrated that although TPP I zymogen is capable of autoactivation in vitro, a serine protease that is sensitive to AEBSF participates in processing of the proenzyme to the mature, active form in vivo.     

Modulation of antibiotic resistance in bacterial pathogens by oleanolic acid and ursolic acid

Antibiotic resistance among bacterial pathogens is a serious problem for human and veterinary medicine, which necessitates the development of novel therapeutics and antimicrobial strategies. Some plant-derived compounds, e.g. pentacyclic triterpenoids such as oleanolic acid (OA) and ursolic acid (UA), have potential as a new class of antibacterial agents as they are active against many bacterial species, both Gram-positive and Gram-negative, and specifically target the cell envelope. The aim of the present study was to investigate the influence of OA and UA on the susceptibility of four bacterial pathogens (Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus and Staphylococcus epidermidis) to the β-lactam antibiotics ampicillin (Ap) and oxacillin (Ox). Antimicrobial assays were conducted with bacteria growing in liquid suspension cultures (planktonic cells) or as biofilms. Using FICI value estimation and the time-kill method it was demonstrated that in some combinations, the tested compounds acted in synergy to lower the susceptibility of S. aureus, S. epidermidis and L. monocytogenes to ampicillin and oxacillin, but no synergy was observed for P. aeruginosa. These results indicate that OA and UA may be useful when administered in combination with β-lactam antibiotics to combat bacterial infections caused by some Gram-positive pathogens.     

PSMC analysis of effective population sizes in molecular ecology and its application to black‐and‐white Ficedula flycatchers

Climatic fluctuations during the Quaternary period governed the demography of species and contributed to population differentiation and ultimately speciation. Studies of these past processes have previously been hindered by a lack of means and genetic data to model changes in effective population size (N_e) through time. However, based on diploid genome sequences of high quality, the recently developed pairwise sequentially Markovian coalescent (PSMC) can estimate trajectories of changes in N_e over considerable time periods. We applied this approach to resequencing data from nearly 200 genomes of four species and several populations of the Ficedula species complex of black‐and‐white flycatchers. N_e curves of Atlas, collared, pied and semicollared flycatcher converged 1–2 million years ago (Ma) at an N_e of ≈ 200 000, likely reflecting the time when all four species last shared a common ancestor. Subsequent separate N_e trajectories are consistent with lineage splitting and speciation. All species showed evidence of population growth up until 100–200 thousand years ago (kya), followed by decline and then start of a new phase of population expansion. However, timing and amplitude of changes in N_e differed among species, and for pied flycatcher, the temporal dynamics of N_e differed between Spanish birds and central/northern European populations. This cautions against extrapolation of demographic inference between lineages and calls for adequate sampling to provide representative pictures of the coalescence process in different species or populations. We also empirically evaluate criteria for proper inference of demographic histories using PSMC and arrive at recommendations of using sequencing data with a mean genome coverage of ≥18X, a per‐site filter of ≥10 reads and no more than 25% of missing data.     

The capacity of UL49.5 proteins to inhibit TAP is widely distributed among members of the genus Varicellovirus

The lifelong infection by varicelloviruses is characterized by a fine balance between the host immune response and immune evasion strategies used by these viruses. Virus-derived peptides are presented to cytotoxic T lymphocytes by major histocompatibility complex (MHC) class I molecules. The transporter associated with antigen processing (TAP) transports the peptides from the cytosol into the endoplasmic reticulum, where the loading of MHC-I molecules occurs. The varicelloviruses bovine herpesvirus 1 (BoHV-1), pseudorabies virus, and equid herpesviruses 1 and 4 have been found to encode a UL49.5 protein that inhibits TAP-mediated peptide transport. To investigate to what extent UL49.5-mediated TAP inhibition is conserved within the family of Alphaherpesvirinae, the homologs of another five varicelloviruses, one mardivirus, and one iltovirus were studied. The UL49.5 proteins of BoHV-5, bubaline herpesvirus 1, cervid herpesvirus 1, and felid herpesvirus 1 were identified as potent TAP inhibitors. The varicella-zoster virus and simian varicellovirus UL49.5 proteins fail to block TAP; this is not due to the absence of viral cofactors that might assist in this process, since cells infected with these viruses did not show reduced TAP function either. The UL49.5 homologs of the mardivirus Marek's disease virus 1 and the iltovirus infectious laryngotracheitis virus did not block TAP, suggesting that the capacity to inhibit TAP via UL49.5 has been acquired by varicelloviruses only. A phylogenetic analysis of viruses that inhibit TAP through their UL49.5 proteins reveals an interesting hereditary pattern, pointing toward the presence of this capacity in defined clades within the genus Varicellovirus.     

Frequency of lactase persistence genotype in a healthy Polish population

The majority of mammals are unable to digest lactose due to post-weaning deactivation of the LCT gene, which is responsible for encoding the enzyme lactase (i.e., adult-type hypolactasia). A substitution of C with T at position −13910 bp upstream of the LCT gene has been linked to the lactase persistence phenotype in European populations. We investigated the frequency of LCT-13910C>T polymorphism in 223 blood donors from central Poland. All samples were genotyped by polymerase chain reaction and direct sequencing. The LCT-13910 T allele (lactase persistence) was present in 51% of individuals sampled from the Polish population. We did not find any non-European variants associated with lactase persistence (LCT-13907C>G, LCT-13913T>C, LCT-13915T>G), or any new polymorphisms within the sequenced region. Allele frequencies obtained are in agreement with results from other countries and confirm the unique pattern of distribution of the LCT-13910C>T genotype in Europe.     

A regulatory role for NBS1 in strand-specific mutagenesis during somatic hypermutation

Activation-induced cytidine deaminase (AID) is believed to initiate somatic hypermutation (SHM) by deamination of deoxycytidines to deoxyuridines within the immunoglobulin variable regions genes. The deaminated bases can subsequently be replicated over, processed by base excision repair or mismatch repair, leading to introduction of different types of point mutations (G/C transitions, G/C transversions and A/T mutations). It is evident that the base excision repair pathway is largely dependent on uracil-DNA glycosylase (UNG) through its uracil excision activity. It is not known, however, which endonuclease acts in the step immediately downstream of UNG, i.e. that cleaves at the abasic sites generated by the latter. Two candidates have been proposed, an apurinic/apyrimidinic endonuclease (APE) and the Mre11-Rad50-NBS1 complex. The latter is intriguing as this might explain how the mutagenic pathway is primed during SHM. We have investigated the latter possibility by studying the in vivo SHM pattern in B cells from ataxia-telangiectasia-like disorder (Mre11 deficient) and Nijmegen breakage syndrome (NBS1 deficient) patients. Our results show that, although the pattern of mutations in the variable heavy chain (V(H)) genes was altered in NBS1 deficient patients, with a significantly increased number of G (but not C) transversions occurring in the SHM and/or AID targeting hotspots, the general pattern of mutations in the V(H) genes in Mre11 deficient patients was only slightly altered, with an increased frequency of A to C transversions. The Mre11-Rad50-NBS1 complex is thus unlikely to be the major nuclease involved in cleavage of the abasic sites during SHM, whereas NBS1 might have a specific role in regulating the strand-biased repair during phase Ib mutagenesis.     

Somatic embryogenesis and plantlet development in Pinus sylvestris and Pinus pinaster on medium with and without growth regulators

To initiate somatic embryogenesis in Pinus sylvestris and Pinus pinaster, immature seeds were collected from June to August and the developmental stage of the zygotic embryos was determined. Four developmental stages were distinguished and the response of the zygotic embryos at each of the four developmental stages was compared intra- and inter-species. For this study, modified Litvay's medium (LM), with or without growth regulators, was chosen. Somatic embryogenesis was initiated and maintained on both media but the two species displayed different propensities. In P. sylvestris, the highest initiation frequency was obtained with intact megagametophytes containing embryos at the four-cell stage to the stage of cleavage polyembryony (up to 22 and 9%, respectively). The culture medium had no significant effect on the initiation and proliferation of embryogenic cultures. In P. pinaster, however, the best response occurred from excised zygotic embryos at the stage prior to elongation of cotyledon primordia (up to 40% explants responded), on medium with growth regulators. Another characteristic distinguishing the two species in culture was that in some embryogenic cell lines of P. sylvestris, somatic embryos matured spontaneously when initiated and maintained on medium without growth regulators. Some of these embryos developed into plantlets on the same medium at the frequency of 40%. Therefore, in P. sylvestris all the stages of somatic embryogenesis were achieved on the medium without growth regulators. However, in both species, maturation of a large number of somatic embryos was greatly improved on medium containing high concentration of gellan gum (Gelrite 10 g l^?1) and abscisic acid (60 μM). Cotyledonary somatic embryos subsequently germinated (72 and 80% for P. sylvestris and P. pinaster, respectively) and developed into plantlets (48 and 29%, for P. sylvestris and P. pinaster, respectively). This represents a significant improvement in plantlet recovery from somatic embryos of both species.     

Hemoglobin adducts and urinary mercapturic acids in rats as biological indicators of butadiene exposure

Binding of 1,2-epoxy-3-butene, the primary metabolite of butadiene, to hemoglobin (Hb) and excretion of its mercapturic acid in urine were studies as potential indicators of butadiene exposure. Four groups of Wistar rats were exposed to butadiene at 0, 250, 500 and 1000 ppm 6 h/day, 5 days/week, during 2 weeks. Blood was collected at the end of exposure and 17 days later for analysis of hemoglobin adducts and adduct stability. Urine was collected each day during exposure (afternoon samples) and in between exposures (morning samples). Adducts of 1,2-epoxy-3-butene to N-terminal valine in Hb were measured using the N-alkyl Edman procedure and GC/MS of the thiohydantoin derivatives. The corresponding mercapturic acid was analysed, after deacetylation, through derivatization with phthaldialdehyde and HPLC with fluorescence detection. The Hb adducts proved to be stable and are therefore useful for dosimetry of long-term exposure to butadiene. The adduct levels increased linearly with exposure dose up to 1000 ppm (3 nmol/g Hb at 1000 ppm). The increase with exposure dose of the mercapturic acid concentration in urine was also compatible with a linear does response up to 1000 ppm. The sensitivity of both analytical methods needs to be improved for their application to human samples.