Phytanyl-pyrophosphate-linked substrate for a bacterial alpha-mannosyltransferase.

The biochemical characterization of bacterial glycosyltransferases involved in the assembly of cell-wall-associated polysaccharides is often hindered by the lack of the appropriate undecaprenyl-pyrophosphate-linked acceptor substrate. In order to find a suitable synthetic substrate for the alpha1,3-mannosyltransferase AceA from Acetobacter xylinum, phytanyl-pyrophosphate-linked cellobiose was prepared. In the presence of GDP-[14C]mannose and recombinant AceA, the phytanyl-pyrophosphate-linked cellobiose afforded a 14C-labeled trisaccharide that was sensitive to alpha-mannosidase degradation in a fashion analogous to the natural undecaprenyl-pyrophosphate-linked cellobiose substrate. These results suggest that phytanyl-pyrophosphate-linked oligosaccharides may be useful substrates for other important bacterial glycosyltransferases.

Development of recombinant, immobilised beta-1,4-mannosyltransferase for use as an efficient tool in the chemoenzymatic synthesis of N-linked oligosaccharides.

The preparation of the conserved core structure of asparagine-linked oligosaccharides found in eukaryotic glycoproteins is an important step towards the synthesis of homogeneous neoglycoproteins. So far, however, the convenient generation of the Manbeta4GlcNAcbeta4GlcNAc (Gn2M) core trisaccharide has proved to be a major obstacle because of the inherent difficulties associated with the synthesis of beta-mannosides. Here we report the overproduction in Escherichia coli of full-length and transmembrane-deleted yeast beta-1, 4-mannosyltransferases as novel N-terminal fusions bearing a decahistidinyl sequence and the minimal human Myc epitope. The recombinant enzymes were highly active and were amenable to immobilisation by nickel(II) chelation and to immunodetection with an anti-Myc monoclonal antibody. The immobilised, transmembrane-deleted enzyme exhibited an apparent Km of 14 microM for the synthetic acceptor substrate analogue, phytanyl-pyrophosphoryl-alpha-N,N'-diacetylchitobioside (PPGn2), under saturating donor conditions. This figure is comparable to those previously reported for native and recombinant yeast beta-1, 4-mannosyltransferases with, respectively, the natural dolichyl-linked acceptor and PPGn2. The validity of the reaction product was confirmed by chromatographic and spectroscopic analysis.

Enzyme-catalyzed formation of glycosidic linkages.

Significant progress has recently been achieved in the use of glycosidases and glycosyltransferases as synthetic tools. Glycosidases have been used to synthesize trisaccharides with a reasonable overall yield, as well as high-mannose neoglycoconjugates. Studies on glycosyltransferases have defined reaction mechanisms and demonstrated reasonable substrate tolerance of these enzymes. Effective methodology for the synthesis of defined glycoproteins has also been demonstrated.

The chemoenzymatic synthesis of the core trisaccharide of N-linked oligosaccharides using a recombinant beta-mannosyltransferase.

The chemical synthesis of the beta-mannosyl linkage of N-glycans has presented a great challenge to synthetic carbohydrate chemists. We have therefore investigated the application of beta-mannosyltransferases to the preparative synthesis N-linked core oligosaccharides. In this paper we report the chemoenzymatic synthesis of beta-D-mannopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranosyl- (1-->4)-2-acetamido-2-deoxy-alpha-D-glucopyranose on a preparative scale using a phytanyl-linked acceptor in the presence of a recombinant beta-(1-->4)-mannosyltransferase.

Preventing equestrian injuries. Locking the stable door.

The medical and sports literature databases were searched for equestrian sports-related injury published in English since 1980, together with conference abstracts and discussions with equestrian sporting bodies. This literature was critically reviewed, with emphasis on measures to prevent or control injury i.e. countermeasures. While there is considerable literature available on the epidemiology of injury incurred in most equestrian sports, there is little on the prevention of these injuries. Case-control or other studies evaluating the effectiveness of the countermeasures suggested by authors do not seem to exist. There is a good body of epidemiology that supports the proper use of approved helmets as a means of preventing injury in these sports. However, protective helmets do not always prevent injury as expected, and many riders do not choose to wear them because of perceived poor design. The search for the ideal equestrian helmet should continue. Ideally the effectiveness of helmets should be assessed scientifically. Among the other countermeasures discussed are the use of rules and regulations for conduct of events, knowledge of horse behaviour, well-conducted lessons, contraindicated medical conditions, public education, rider education, appropriate equipment and clothing, the riding environment, rider experience, safety stirrups, body protectors, falling techniques, and first aid measures. Even though the injury rate for equestrians is relatively low when compared with other sports, the injuries that are incurred are usually severe. prevention is often difficult because the behaviour of the horse is unpredictable. Countermeasures used for prevention should be evaluated for the effectiveness to reduce the frequency and severity of injuries to equestrians.

Trends in public hospital injury admission rates, Victoria, July 1987 to June 1993.

Admissions to Victorian public hospitals because of injury for the six years from July 1987 to June 1993 were analysed to ascertain the frequencies, rates and trends over time in rates. This information is important for epidemiological purposes, to determine priorities for injury prevention and, as baseline data, to monitor the success of interventions. The major causes of injury, and their rates, did not change markedly. Falls or transport-related injuries were still the major cause of hospitalisation. The mean annual all-age frequency over the six years was 67,902, an all-age, all-cause rate of 1620 per 100,000. The highest frequencies of injury occurred in the young age groups, 15-19, 20-24 and 25-29 years, but the highest rates occurred in the over-60 age groups. The all-age, all-cause injury rate increased significantly at 39 per 100,000 per year (2.4 per cent per year), notwithstanding the decreasing rate of transport-related injury of 17 per 100,000 per year (7.5 per cent). Significant increases were also observed for injuries from falls in all age groups and intentional self-inflicted injuries. For the 15-24 years age group, transport-related injury rates declined by 35 per 100,000 per annum but intentional injury rates were increasing. In the over-65 years age group, rates of injury from falls were increasing by 3.3 per cent per annum. The benefits of investment in prevention of transport-related injury are apparent. With high rates, frequencies and increases over time for many injury causes, similar resources for prevention of other causes of injury are justified and necessary.

Non-fatal injuries to young Victorians, 1986-1991.

OBJECTIVES: To describe the epidemiology of non-fatal injuries sustained by young Victorians requiring hospitalisation, and to provide baseline data to establish and evaluate injury prevention programs. DATA SOURCE: The Victorian Inpatient Minimum Database, as modified for use by Monash University Accident Research Centre. DESIGN: A five-year (July 1986 to June 1991) retrospective epidemiological study of 56,209 15-24-year-olds admitted to Victorian public hospitals because of injury. RESULTS: The annual Victorian hospital admission rate of 15-24-year-olds with injuries was 1609 per 100,000. The major causes of admission were, in order of frequency, transport injuries, falls (excluding falls in sport), sports injuries, assault, and self-inflicted injuries. The cost to the Victorian community in hospital expenditure is at least $31.5M per year. Of these injuries, 73.9% were unintentional; however, intentional self-inflicted and assault injury rates were higher for both adolescents (15-19 years) and young adults (20-24 years) compared with other age groups. CONCLUSION: High rates of injury for young people have been found, imposing a considerable burden on the community. As many injuries are preventable, the risk factors warrant further investigation. In particular, control measures could help reduce sports, transport-related and intentionally inflicted injuries.

Cell populations in the human early pregnancy decidua: natural killer activity and response to interleukin-2 of CD56-positive large granular lymphocytes.

Large granular lymphocytes (LGL) have been shown previously to be the most abundant cell type in the first trimester human decidua. Purified populations of decidual LGL were prepared by flow cytometry of cell dispersions labelled with NKH1 (CD56), an antibody specific for peripheral blood LGL, and the functional properties of CD56-positive cells, CD56-negative and unsorted decidual cells compared. Both CD56-positive cells and unsorted decidual cells have cytotoxic activity against the natural killer (NK) cell target K562 which was weak compared with that of peripheral blood mononuclear cells (PBMC). The CD56-negative cells had no cytotoxic activity against K562. All three decidual cell populations proliferated in response to recombinant human interleukin-2 (rIL-2), but none produced detectable levels of IL-2 in culture. When unsorted decidual cells were cultured for 7 days in rIL-2 the proportion of CD56-positive cells increased and NK activity against K562 was augmented. The NK activity of purified CD56-positive decidual cells was also augmented by culturing in rIL-2. The potential role of decidual LGL in regulating the development of the semi-allogeneic placenta is discussed.