Mapping nonrecombining regions in barley using multicolor FISH

Fluorescence in situ hybridization (FISH) is a widely used method to localize DNA sequences on chromosomes. Out of the many uses, FISH facilitates construction of physical maps by ordering contigs of large-insert DNA clones, typically bacterial artificial chromosome (BAC) and establishing their orientation. This is important in genomic regions with low recombination frequency where genetic maps suffer from poor resolution. While BAC clones can be mapped directly by FISH in plants with small genomes, excess of repetitive DNA hampers this application in species with large genomes. Mapping single-copy sequences such as complementary DNA (cDNA) is an attractive alternative. Unfortunately, localization of single-copy sequences shorter than 10 kb remains a challenging task in plants. Here, we present a highly efficient FISH technique that enables unambiguous localization of single copy genes. We demonstrated its utility by mapping 13 out of 15 full-length cDNAs of variable length (2,127–3,400 bp), which were genetically defined to centromeric and pericentromeric regions of barley chromosome 7H. We showed that a region of 1.2 cM (0.7 %) on genetic map represented more than 40 % of the physical length of the chromosome. Surprisingly, all cDNA probes occasionally revealed hybridization signals on other chromosomes, indicating the presence of partially homologous sequences. We confirmed the order of 10 cDNA clones and suggested a different position for three cDNAs as compared to published genetic order. These results underline the need for alternative approaches such as FISH, which can resolve the order of markers in genomic regions where genetic mapping fails.     

Lipophosphonoxins: new modular molecular structures with significant antibacterial properties

Novel compounds termed lipophosphonoxins were prepared using a simple and efficient synthetic approach. The general structure of lipophosphonoxins consists of four modules: (i) a nucleoside module, (ii) an iminosugar module, (iii) a hydrophobic module (lipophilic alkyl chain), and (iv) a phosphonate linker module that holds together modules i-iii. Lipophosphonoxins displayed significant antibacterial properties against a panel of Gram-positive species, including multiresistant strains. The minimum inhibitory concentration (MIC) values of the best inhibitors were in the 1-12 μg/mL range, while their cytotoxic concentrations against human cell lines were significantly above this range. The modular nature of this artificial scaffold offers a large number of possibilities for further modifications/exploitation of these compounds.     

High-frequency jet ventilation improves gas exchange in extremely immature infants with evolving chronic lung disease

Extremely preterm infants often develop chronic lung disease (CLD) characterized by heterogeneous aeration; poorly supported, floppy airways; and air trapping. High-frequency jet ventilation (HFJV) with high end-expiratory pressure (optimal lung volume strategy [OLVS]) may improve airway patency, lead to better gas distribution, improve gas exchange, and facilitate extubation. In a pilot trial, this study sought to explore the effect of HFJV on oxygenation, ventilation, and ease of extubation in preterm infants with evolving CLD and refractory respiratory failure (RRF). From September 2002 to October 2004, 12 episodes of RRF developed in 10 ventilated extremely immature infants with evolving CLD (10 on conventional and two on high-frequency oscillation). Chorioamnionitis was confirmed in all infants, patent ductus arteriosus was ligated in five patients, and UREAPLASMA UREALYTICUM was cultured from trachea in four patients. HFJV with OLVS was initiated when oxygenation index (OI) > 10 or exhaled tidal volume (V TE) >or= 7 mL/kg were required to maintain partial pressure of carbon dioxide, arterial (Pa CO2) < 60 mm Hg. Inspiratory time (0.02/s) and frequency (310 to 420/min) were set initially with adjustment of pressure amplitude to keep Pa CO2 between 45 and 55 mm Hg. Ventilatory stabilization and weaning from mechanical ventilation with extubation to nasal continuous positive airway pressure (CPAP) were the goals of this approach. Gas exchange data were analyzed by Analysis of variance for repeated measures. Ten patients on 11 occasions of RRF were extubated to nasal CPAP successfully in a median of 15.5 days. Nine of 10 patients survived (one died of pentalogy of Cantrell), all required supplemental O2 at 36 weeks. Pa CO2 decreased within 1 hour after the initiation of HFJV, and OI decreased by 24 hours. Both remained significantly lower until successful extubation ( P < 0.02). Compared with conventional ventilation or high-frequency oscillatory ventilation, HFJV used with OLVS appears to improve gas exchange and may facilitate weaning from mechanical ventilation (MV) in extremely immature infants with evolving CLD. These encouraging pilot data need to be confirmed in a larger clinical trial.     

Anti-tumour necrosis factor-alpha activity in Ixodes ricinus saliva

Tumour necrosis factor-alpha (TNF-alpha) is one of the most prominent inflammatory mediators playing a central role in starting off the inflammatory reactions of the innate immune system. We identified a TNF-alpha-inhibitory activity in the saliva and salivary gland extract (SGE) from partially fed Ixodes ricinus ticks. Using mouse and human TNF-alpha specific ELISA, we showed that tick saliva or SGE markedly reduced the level of detectable cytokine. Both saliva and SGE inhibited the cytotoxic effect of TNF-alpha in a bioassay. Elimination of the TNF-alpha-inhibitory activity in SGE by trypsin digestion demonstrated that the anti-TNF-alpha factor is a protein. Fast protein liquid chromatography fractionation of SGE showed one peak of TNF-alpha-inhibitory activity corresponding to a protein with estimated molecular mass 23 kDa. The likely mechanism of the inhibitory effect is a direct binding of the cytokine. The TNF-alpha-inhibitory molecule seems to play an important role in the anti-inflammatory effect of tick saliva at the tick feeding site, providing a gateway to the host for tick-borne pathogens.     

Cultivation of HIV-1 virus on mononuclear peripheral blood cells of HIV-1 seropositive individuals

Using the method of co-cultivation with phytohaemagglutinin-stimulated lymphocytes from healthy donors, the author isolated the HIV-1 virus from peripheral mononuclear blood cells of three patients with the AIDS symptomatology and one patient with the ARC symptomatology. The presence of the virus in infected cells was proved by detection of the viral antigen p 24 in enzymatic immunoassays and in the immunofluorescence test. Three of the isolated strains were adapted to sensitive continual tissue cultures, where the isolates caused chronic infection of the cells associated with the development of a cytopathic effect. In the investigated patients no relationship was proved between viraemia and antigenaemia. The author discusses the importance of virus cultivation for laboratory diagnosis, epidemiology and research of HIV infection and AIDS.     

Efficiency of embryotoxicity testing procedures: I. A compromise approach

As the alternative to current embryotoxicity screening techniques, an attempt was made to utilize the principles of a procedure used in basic teratological research, for simplification, speed and economy. The effects of substances, administered to rats on days 10 and 11 of pregnancy, on the activity of the caudal morphogenetic system were examined by trunk measurements of embryos harvested on day 13. The caudal morphogenetic system of rat embryos reflected some of the changes induced in the maternal-embryonic complex.     

Effect of adrenergic blocking agents on the acute tolbutamide response in dogs

Summary Four normal adult dogs were given a single i.v. dose of tolbutamide (sodium salt, 50 mg/kg), and the plasma levels of insulin and glucose were followed for 2 hrs. The drug produced a 2 to nearly 6 fold increase in the concentration of plasma insulin and a 40 % reduction in plasma glucose. A single injection of mg 5 phentolamine, an -adrenergic blocking agent, given 30 min prior to the administration of tolbutamide, enhanced the insulin release by a factor of 2. Pretreatment with the -adrenergic blocker Kö 592 (0.1 mg/kg and 1 mg/kg) produced the opposite effect; it inhibited the insulinogenic response to tolbutamide. Identical results were reported by us with glybenclamide (HB 419) when tested under similar experimental conditions. The results of the present experiments with tolbutamide indicate that the two oral hypoglycemic agents differ in potency, but not in the mechanism of action, as judged by identical changes in response to adrenergic blocking agents.Supported by a grant-in-aid from Hoechst Pharmaceuticals of Canada.