Application of stains in clinical microbiology

Stains have been used for diagnosing infectious diseases since the late 1800s. The Gram stain remains the most commonly used stain because it detects and differentiates a wide range of pathogens. The next most commonly used diagnostic technique is acid-fast staining that is used primarily to detect Mycobacterium tuberculosis and other severe infections. Many infectious agents grow slowly on culture media or may not grow at all; stains may be the only method to detect these organisms in clinical specimens. In the hands of experienced clinical microscopists, stains provide rapid and cost-effective information for preliminary diagnosis of infectious diseases. A review of the most common staining methods used in the clinical microbiology laboratory is presented here.     

Combinatorial synthetic design.

The range and diversity of solid-phase synthetic methodology continues to develop very rapidly, allowing the generation of previously inaccessible compound libraries. The design of such libraries requires consideration of linkage strategies, newly developed solid-phase reactions and emerging solution-phase processes.     

cDNA cloning and sequencing of rat monoamine oxidase A: comparison with the human and bovine enzymes.

1. The nucleotide and deduced amino acid sequences of rat liver MAO A were determined, and sequence identities among MAO A and B from rat, human and bovine were compared. 2. MAO A from rat exhibited greater than 85% sequence identity with bovine and human MAO A, and 70% identity with rat MAO B. 3. Rat adrenal cDNAs were restriction mapped, partially sequenced and found to be identical to rat liver MAO A, suggesting that these two tissues express the same polypeptide.     

Pentalenene biosynthesis and the enzymatic cyclization of farnesyl pyrophosphate: Proof that the cyclization is catalyzed by a single enzyme

A cell-free preparation from Streptomyces UC5319 has been developed which catalyzes the conversion of trans, trans-farnesyl pyrophosphate (1) to pentalenene (2), the parent hydrocarbon of the pentalenolactone family of sesquiterpene antibiotics. Incubation of [9-³H₂, 12,13-¹⁴C]farnesyl pyrophosphate with the pentalenene synthetase gave [1,8-³H₂, 14,15-¹⁴C]pentalenene, as established by a combination of chemical and microbial degradation methods. The retention of both equivalents of tritium in the enzymatically derived pentalenene establishes that the cyclization of trans, trans-farnesyl pyrophosphate to 2 is catalyzed by a single enzyme.     

An improved method for monitoring parasitism and establishment of Oobius agrili (Hymenoptera: Encyrtidae), an egg parasitoid introduced for biological control of the emerald ash borer (Coleoptera: Buprestidae) in North America

Oobius agrili Zhang and Huang (Hymenoptera: Encyrtidae) is a solitary egg parasitoid that has been released in the United States since 2007 for biocontrol of the invasive emerald ash borer (EAB), Agrilus planipennis Fairmaire (Coleoptera: Buprestidae). Field and laboratory trials with ash logs infested with EAB eggs were conducted in Michigan between 2009 and 2010 to improve methods for monitoring the establishment of O. agrili. Naturally occurring EAB eggs were collected in both parasitoid-release and control (non-parasitoid-release) plots to compare with the EAB egg-sentinel log (ESL) technique. In three parasitoid-release plots, >50% of ESLs had O. agrili-parasitized eggs ranging from 3.9% to 48.2% egg parasitism after one week of field exposure. No EAB eggs were attacked by O. agrili on the ESLs deployed in control plots. In the laboratory, 100% of ESLs exposed to O. agrili inside rearing jars for one week had parasitized-eggs (68.5% egg parasitism). Deployment of ESLs detected low levels of parasitism by O. agrili in all three ash stands where O. agrili was released in previous years. In contrast, collection of naturally occurring EAB eggs detected the parasitism in only one of these three parasitoid-release ash stands. No parasitism was detected in control ash stands with either method. These findings indicate that populations of O. agrili released in previous years had successfully overwintered and established in the released ash stands by 2010, but had not yet dispersed to the control stands.     

The requirement for cell surface galactosyl residues during oxidative activation of normal and chronic lymphocytic leukemia lymphocytes.

Galactose oxidase stimulated normal and leukemic lymphocytes to undergo DNA synthesis and cell division. Although the response of normal lymphocytes to galactose oxidase was enhanced with neuraminidase pretreatment, substantial activation of leukemic lymphocytes required pretreatment with neuraminidase. Leukemic lymphocytes exhibited maximal response to neuraminidase-galactose oxidase later than that observed in normal lymphocytes. Treatment of lymphocytes with trypsin diminished their response to galactose oxidase. When lymphocytes were pretreated with β-galactosidase to specifically remove cell surface galactosyl residues, the response to galactose oxidase was prevented. The response of normal and leukemic lymphocytes to sodium periodate was also reduced after treatment with galactose oxidase. These data support the concept that oxidation of cell surface galactosyl residues is critical during lymphocyte activation.     

Study of Receptor-Chaperone Interactions Using the Optical Technique of Spectroscopic Ellipsometry

This work describes a detailed quantitative interaction study between the novel plastidial chaperone receptor OEP61 and isoforms of the chaperone types Hsp70 and Hsp90 using the optical method of total internal reflection ellipsometry (TIRE). The receptor OEP61 was electrostatically immobilized on a gold surface via an intermediate layer of polycations. The TIRE measurements allowed the evaluation of thickness changes in the adsorbed molecular layers as a result of chaperone binding to receptor proteins. Hsp70 chaperone isoforms but not Hsp90 were shown to be capable of binding OEP61. Dynamic TIRE measurements were carried out to evaluate the affinity constants of the above reactions and resulted in clear discrimination between specific and nonspecific binding of chaperones as well as differences in binding properties between the highly similar Hsp70 isoforms.