Determination of methotrexate in human urine at nanomolar levels by high-performance liquid chromatography with column switching

An high-performance liquid chromatographic method with column switching for the detection of less than 4 ng of methotrexate in the urine of oncologic nurses is described. Urine samples were purified by solid-phase extraction on silica-bonded phenyl columns, eluting impurities with ethyl acetate. After elution from the column, the analyte was concentrated ten-fold, evaporating the solvent. On a strong anion-exchange column (Nucleosil 100 SB), methotrexate was separated from the remaining interfering substances, was then switched to a reversed-phase column (LiChrospher 100 RP-18e), and finally eluted by a linear gradient in a solvent system consisting of ammonium formate buffer (pH 2.7) and acetonitrile. Absorbance was monitored at 310 nm. This method has proved to be suitable for detecting traces of methotrexate in urine in order to individualize risks and to reduce further the occupational safety hazard for hospital personnel.     

Pectinolytic enzymes of anaerobic fungi

Pectinolytic enzymes of four rumen fungi have been described. Three fungal species were monocentric Neocallimastix spp. H15, JL3 and OC2, and one isolate was a polycentric strain of Orpinomyces joyonii , A4. They differed in degree of pectin degradation and utilization. Only the strain Neocallimastix sp. H15 and partially Orpinomyces joyonii A4 were able to utilize pectin to a higher extent. The most important pectinolytic activity in all these isolates represented pectin lyase (EC 4.2.2.10) and polygalacturonase (EC 3.2.1.15). Their specific activities were in the range of 100–900 and 10–450 μg galacturonic acid h^-1 mg protein^-1 for pectin lyase and polygalacturonase, respectively. Polygalacturonase, located mainly in the endocellular fraction, was inhibited by calcium ions and had the main pH optimum at pH 6.0. All strains produced pectate lyase (EC 4.2.2.2). None of the strains tested produced pectinesterase (EC 3.1.1.11).     

Growth characteristics of atypical mycobacteria in water and their comparative resistance to disinfectants.

With the increasing significance of group IV atypical mycobacteria as etiological agents in a variety of infections, studies were conducted to determine their growth capabilities in water and their comparative resistance to disinfectants used to decontaminate hospital equipment. Isolates of Mycobaterium chelonei (TM strains) from peritoneal fluids of patients and peritoneal dialysis machines were able to multiply in commercial distilled water, with generation times at 25 degrees C ranging from 8 to 15 h. Levels of 10(5) to 10(6) cells per ml were attained, and these stationary-phase populations declined only slightly over a 1-year period. Results of studies to determine resistance to disinfectants showed the following. (i) TM strains of M. chelonei cultured in commercial distilled water showed survivors in 2% aqueous formaldehyde (HCHO) solutions up to 24 h; in 8% HCHO, only a 2-log reduction in viable counts was observed over a 2-h sampling period. Reference ATCC strains of M. chelonei and M. fortuitum were rapidly inactivated, with no survivors after 2 h of exposure to 2% HCHO or 15 min of exposure to 8% HCHO. (ii) In 2% alkaline glutaraldehyde, TM strains survived 60 min. whereas ATCC strains showed no survivors after 2 min of contact time. (iii) All M. chelonei and M. fortuitum strains survived 60 min of exposure to concentrations of 0.3 and 0.7 microgram of free chlorine per ml at pH 7.     

Preparation and preliminary biological screening of cholic acid-juvenoid conjugates

Steroidal compounds have been utilized as carriers and for modification of physico-chemical properties of model biologically active secondary alcohols - juvenoids. Juvenoids are juvenile hormone analogues - environmentally safe insecticides, possessing significant biological activity towards different arthropods groups in focus on insect pest species. Structure modification of juvenoids plays important role to control the rate of liberation and decomposition of juvenoid in digestive system and can also play important role in the mode of action towards selected insect. This study presents an approach to the synthesis of steroidal monomers and dimers carrying one and two molecules of a juvenoid in their structures. The prepared compounds were tested for their inhibition activity on reproduction of the blowfly Neobellieria (Sarcophaga) bullata. These steroid-juvenoid conjugates showed promising possibilities in synthesis of new unique biochemical insecticides. Preliminary biological test results of prepared compounds are presented.     

Natural variation in colony inbreeding does not influence susceptibility to a fungal pathogen in a termite

Reduced genetic diversity through inbreeding can negatively affect pathogen resistance. This relationship becomes more complicated in social species, such as social insects, since the chance of disease transmission increases with the frequency of interactions among individuals. However, social insects may benefit from social immunity, whereby individual physiological defenses may be bolstered by collective‐level immune responses, such as grooming or sharing of antimicrobial substance through trophallaxis. We set out to determine whether differences in genetic diversity between colonies of the subterranean termite, Reticulitermes flavipes, accounts for colony survival against pathogens. We sampled colonies throughout the United States (Texas, North Carolina, Maryland, and Massachusetts) and determined the level of inbreeding of each colony. To assess whether genetically diverse colonies were better able to survive exposure to diverse pathogens, we challenged groups of termite workers with two strains of a pathogenic fungus, one local strain present in the soil surrounding sampled colonies and another naïve strain, collected outside the range of this species. We found natural variation in the level of inbreeding between colonies, but this variation did not explain differences in susceptibility to either pathogen. Although the naïve strain was found to be more hazardous than the local strain, colony resistance was correlated between two strains, meaning that colonies had either relatively high or low susceptibility to both strains regardless of their inbreeding coefficient. Overall, our findings may reflect differential virulence between the strains, immune priming of the colonies via prior exposure to the local strain, or a coevolved resistance toward this strain. They also suggest that colony survival may rely more upon additional factors, such as different behavioral response thresholds or the influence of a specific genetic background, rather than the overall genetic diversity of the colony.