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Dear Editor, The importance of the microbiology laboratory in hospital-based infection control programmes is well established. The role of the laboratory in infection surveillance, in the management of antimicrobial resistance [...]

Study design:This is a Delphi study.Objectives:Defining variables that potentially influence the outcomes of an animal study regarding pathophysiology of traumatic spinal cord injury (TSCI).Setting:This study was conducted in Iran.Methods:A modified two-round Delphi study was conducted. As the first round, an initial questionnaire was developed on the basis of literature and a series of focus group discussions. In the second round, the participants were asked to score the items through a 10-point scale. Consensus was achieved through the following criteria: (1) the median of scores has to be at 7.5 or higher, and (2) at least 70% of participants need to rate 7 or higher. Also, the inter-rater reliability analysis was performed to determine consistency among raters using the Kappa coefficient and Cronbach's alpha.Results:Twenty-one experts participated in our study. From the first round of the study, a 47-item checklist was developed. By considering the aforementioned criteria for consensus building on extremely important factors, we reached a 15-item checklist including species, strain, method and level of injury, control group, genetic background, severity of injury, attrition, use of appropriate test, blindness, method of allocation to treatments, regulation and ethics, age/weight, bladder expression, number of animals/group and statistics. The inter-rater reliability for the raters was found to be Kappa=0.82 (P<0.001). A Cronbach's alpha of 0.9 for all the questions indicated high internal consistency.Conclusion:This study introduces a checklist of variables that potentially influence the outcomes of animal studies regarding TSCI pathophysiology and describe its validity and reliability.

A co-operative project between the Association of Learned and Professional Society Publishers and Queen's University, Belfast, supported by the Electronic Libraries Programme, was set up to create a website and host the publications of five learned societies on an experimental basis. In the light of the experience gained, societies who have not taken the first steps in providing electronic services for their members and subscribers are offered advice on the structure, setup, and service requirements for their first website.

The dipyruvylated glycolipid from Mycobacterium smegmatis (Saadat, S., and Ballou, C.E. (1983) J. Biol. Chem. 258, 1813-1818) has been shown to have the following structure in which FA1 is tetra- or hexadecanoic acid and FA2 is 2,4-dimethyl-2-eicosenoic acid. (formula; see text) The fast atom bombardment mass spectrum showed two major ions [M - H]- at m/z 1511 and 1539 (Mr 1512 and 1540) in a ratio of 1.4:1, suggesting that the glycolipid was a mixture of homologs that differed in fatty acid composition by 2 methylene groups. Analysis revealed C14, C16, and C22 fatty acids in ratios of 0.6:0.4:1.0, indicating that 60% of the molecules contained a C14 and C22 fatty acid whereas 40% contained a C16 and C22 fatty acid. The fragmentation pattern showed that a single glucose unit along with the smaller fatty acid could be lost to yield a tetrasaccharide with attached C22 fatty acid, and a second fragmentation yielded a trisaccharide containing 2 pyruvic acids but without attached fatty acid. The C14 and C16 fatty acids were identified as myristic and palmitic acid, whereas the C22 fatty acid was 2,4-dimethyl-2-eicosenoic acid. Precise localization of the fatty acids came from periodate oxidation and methylation analysis.

We have studied the regulation of tyrosine hydroxylase by cell-cell contact in primary cultures of bovine adrenal chromaffin cells. Preparation of dissociated chromaffin cells from bovine adrenal medullae or the harvesting of cultured cells resulted in a rapid decrease of the specific mRNA(TH) (defined as the amount of mRNA(TH) (where TH represents tyrosine hydroxylase) per microgram of total RNA). The decrease in mRNA(TH) levels appears to be stimulated by the loss of cell contact, as it occurs much more rapidly than would be expected from the turnover rate of mRNA(TH) in cultured cells. Similarly an enhanced rate of tyrosine hydroxylase enzyme degradation was observed in chromaffin cells when brought into low contact cultures. In contrast to the decrease in mRNA(TH) levels, however, the decrease in tyrosine hydroxylase enzyme levels was not so rapid and could be prevented in high contact cultures. The mRNA(TH) increased 4-fold in high contact cultures relative to dissociated cells within 1 day after plating. Similarly the rate of synthesis of tyrosine hydroxylase enzyme molecules was maximal after 1 day, although the increase in the absolute amount of tyrosine hydroxylase occurred only slowly. The increase in specific mRNA(TH) by cell contact was inhibited by alpha-amanitin, indicating that cell contact evokes an increase in tyrosine hydroxylase levels by increasing the transcription of mRNA(TH), in addition to inhibiting the degradation of tyrosine hydroxylase molecules.

As a first step towards the identification and purification of the molecule(s) that are involved in cell contact-mediated tyrosine hydroxylase (TH) induction in cultures of bovine adrenal chromaffin cells, we have prepared plasma membranes (PM) from bovine adrenal medulla and tested their ability to mimick cell contact-mediated TH induction in low density chromaffin cultures. PM indeed induced TH in a manner similar to that observed in high density cultures. The maximal TH induction reached by PM corresponded to 69% of that of high density cultures, and half-maximal TH induction was obtained with 12 micrograms of PM per ml of medium. The induction of TH by PM was blocked by alpha-amanitin as observed in high density cultures. Since acetylcholinesterase was neither induced in high density nor in PM-treated low density cultures, an induction of TH as a result of a general increase in protein synthesis was excluded. The cell contact molecule(s) appear to be intrinsic membrane proteins. They were not removed by high or low salt extraction, but solubilized by 50 mM octylglucoside. They were resistant to 0.1% trypsin and heat denaturation but inactivated by 0.01% chymotrypsin. PM isolated from the adrenal cortex, kidney, and liver also induced TH in low density chromaffin cell cultures, although to a smaller extent than PM of the adrenal medulla. In contrast, muscle and erythrocyte PM were inactive. This shows that the cell contact molecule(s) are not restricted to the adrenal medulla, but are also present in some other but not all tissues.

A crude glycolipid fraction was isolated from Mycobacterium smegmatis ATCC 356 by ethanolic extraction and silica gel chromatography. After deacylation of the glycolipid fraction, a dipyruvylated pentasaccharide (acidic oligosaccharide A) and a monopyruvylated tetrasaccharide (acidic oligosaccharide B1) were purified by ion exchange and gel filtration chromatography. Methylation analysis, proton nuclear magnetic resonance, mass spectrometry, and periodate oxidation suggested that oligosaccharide A was 4,6-(1-carboxyethylidene)-3-O-methyl-beta-D Glcp-(1-3)-4,6-(1-carboxyethylidene)-beta-D-Glcp-(1-4)-beta-D-Glcp-(1-6)-alpha- D Glcp-(1-1)-alpha-D-Glcp and that oligosaccharide B1 was 4,6-(1-carboxyethylidene)-beta-D-Glcp-(1-4)-beta-D-Glcp-(1-6)-alpha-D-Glcp-(1-1) -alpha-D-Glcp. Both compounds contain a trehalose unit as a part of the oligosaccharide structure, and B1 appears to be a biosynthetic precursor of A because the two differ only in a pyruvylated 3-O-methylglucose unit. A third component, acidic oligosaccharide B2, differs from oligosaccharide A only in lacking 1 of the 2 pyruvate residues.

Ciliary neurotrophic factor (CNTF) influences the levels of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) in cultures of dissociated sympathetic neurons from newborn rats. In the presence of CNTF both the total and specific activity of ChAT was increased 7 d after culture by 15- and 18-fold, respectively, as compared to cultures kept in the absence of CNTF. Between 3 and 21 d in culture in the presence of CNTF the total ChAT activity increased by a factor of greater than 100. Immunotitration demonstrated that the elevated ChAT levels were due to an increased number of enzyme molecules. In contrast to the increase in ChAT levels, the total and specific activity levels of TH were decreased by 42 and 36%, respectively, after 7 d in culture. Half-maximal effects for both ChAT increase and TH decrease were obtained at CNTF concentrations of approximately 0.6 ng and maximal levels were reached at 1 ng of CNTF per milliliter of medium. The effect of CNTF on TH and ChAT levels were seen in serum-containing medium as well as in serum-free medium. CNTF was shown to have only a small effect on the long-term survival of rat sympathetic neurons. We therefore concluded that the effects of CNTF on ChAT and TH are not due to selective survival of cells that acquire cholinergic traits in vitro, but are rather due to the induction of cholinergic differentiation of noradrenergic sympathetic neurons.