[Causes, injuries, therapy and possibilities of prevention of equine-related accidents. Results of a 2-center-study]. / Ursachen, Verletzungen, Therapie und Präventionsmöglichkeiten von Unfällen im Reitsport. Ergebnisse einer 2-Center-Studie.

BACKGROUND: Horse riding is associated with a high rate of injuries. The possibilities of prevention are limited because of deficient knowledge about the causes and mechanisms of equine-related accidents. In the present study 198 equine-related accidents were analyzed and based on these data risk groups were identified and guidelines to improve safety in horse riding were formulated. MATERIAL AND METHODS: In this 2-center study the accidents of 169 riders over a period of 12 months were analyzed. Data on equine-related patterns of injury and the resulting treatment were collated from the clinical records. Additionally, a questionnaire was completed on the day of trauma by the patients, which covered age, gender, the cause and mechanism of injury and the use of safety equipment at the time of the accident. RESULTS: There were 169 (85.5%) female and 29 (14.5%) male riders with a median age of 27.2 years (range 5-74 years). Of the riders 55 (27.8%) were aged 18 years or younger, 134 (67.7%) riders were treated as outpatients while 64 (32.3%) required hospitalization, 51 (25.8%) required surgical treatment, 66 (33.3%) used a helmet and 14 (7.1%) a body protector at the time of accident. DISCUSSION: Equestrians wear helmets increasingly more when riding but the willingness to wear body protectors is disappointing. Protective headgear has been proven to reduce the risk of injuries but based on these data a positive effect of body protectors could not be shown. In this study safety vest users suffered from injuries of the upper body more often than those who did not wear a body protector. Children and adolescents often overestimate their skills; therefore, teaching and supervision of inexperienced young riders along with the use of protective equipment can prevent major injuries.

Effectiveness of safety vests in pediatric horseback riding.

BACKGROUND: Despite the benefit of safety vests to the reduction of torso injuries in children and adolescents is unclear, its' use is recommended. The aim of the present study is to determine the effectiveness of safety vests actually used in pediatric equestrian activities. PATIENTS AND METHOD: In this case-control-study, we analyzed the accidents of 92 riders aged 18 or younger who fell off a horse onto his/her torso during a period of 18 months. Data were gathered from the clinical records. Additionally, a questionnaire was administered on the day of trauma by the patients and/or their parents. RESULTS: The cases comprised 31 patients who sustained torso injuries. The controls were 61 riders with injuries of other body parts than to the torso. Safety vest use was not associated with a lower risk of torso injuries (OR=1.18, 95% CI (0.50, 2.81), p=0.707). Post hoc power analysis revealed that within such a setting an odds ratio of 0.266 could be found with a power of 80%. CONCLUSION: This study is not able to show an association between wearing a torso protector and protection from torso injuries, probably due to confounding. We did not detect a high effect of safety vest usage in our study population. Whether the development of a new generation of safety vests might be more effective remains unclear. An effective vest should be adapted to the requirements of children and adolescents and should protect the thorax and abdomen, but also the cervical and the lumbar spine.

Safety monitoring of a polyvalent immunoglobulin preparation: documentation of 15,548 administrations.

UNLABELLED: Between 1997 and 2002, a post-marketing surveillance study was conducted throughout Germany to evaluate Intraglobin F in a replacement therapy for primary and secondary immunodeficiency diseases. A total of 15,548 individual administrations in 1,705 patients were documented. METHODS: The study was conducted as a multicenter project involving 72 outpatient and inpatient treatment centers in Germany. The study variables were recorded during the routine treatment of patients with congenital or acquired immunodeficiencies. No additional variables outside the normal routine were recorded as is mandatory in post-marketing surveillance studies. RESULTS: The rate of adverse drug reactions (ADR) was 0.064% in 15,548 administrations or 0.59% with reference to 1,705 treated patients; eight non-serious adverse events (AE) were considered to have a "probable" and one further AE a "possible" causal association with the use of Intraglobin F. Only one AE assessed as "serious" was classified as "probably" treatment-related. The efficacy of Intraglobin F was rated by the treating physicians as "very good" or "good" in 91.8% of the evaluated patients. CONCLUSIONS: This post-marketing surveillance study has demonstrated the safety of Intraglobin F. The statistical results obtained with the data are supported by the overall assessment of the treating physicians who rated the tolerability of Intraglobin F as "very good" or "good" in 98.5% of the patients. No new or unexpected risks were observed.

The Schizosaccharomyces pombe GPI8 gene complements a Saccharomyces cerevisiae GPI8 anchoring mutant.

The final step in glycosylphosphatidylinositol (GPI) anchoring of cell surface proteins consists of a transamidation reaction, in which preassembled GPI donors are substituted for C-terminal signal sequences in nascent polypeptides. The Saccharomyces cerevisiae GPI8 gene (ScGPI8) encodes a protein which is involved in the GPI transamidation reaction. We have cloned and isolated the Schizosaccharomyces pombe GPI8 homologous gene (SpGPI8). The SpGPI8 gene encodes a protein of 411 amino acids with a calculated molecular weight of about 47 kDa. It shows 53.5% identity with the ScGPI8 and complements a S. cerevisiae GPI8 anchoring mutant.

Processing and localisation of a GPI-anchored Plasmodium falciparum surface protein expressed by the baculovirus system.

We describe the expression, in insect cells using the baculovirus system, of two protein fragments derived from the C-terminus of merozoite surface protein 1(MSP-1) of the human malaria parasite Plasmodium falciparum, and their glycosylation and intracellular location. The transport and intracellular localisation of the intact C-terminal MSP-1 fragment, modified by addition of a signal sequence for secretion, was compared with that of a similar control protein in which translation of the GPI-cleavage/attachment site was abolished by insertion of a stop codon into the DNA sequence. Both proteins could only be detected intracellularly, most likely in the endoplasmic reticulum. This lack of transport to the cell surface or beyond, was confirmed for both proteins by immunofluorescence with a specific antibody and characterisation of their N-glycans. The N-glycans had not been processed by enzymes localised in post-endoplasmic reticulum compartments. In contrast to MSP-1, the surface antigen SAG-1 of Toxoplasma gondii was efficiently transported out of the endoplasmic reticulum of insect cells and was located, at least in part, on the cell surface. No GPI-anchor could be detected for either of the MSP-1 constructs or SAG-1, showing that the difference in transport is a property of the individual proteins and cannot be attributed to the lack of a GPI-anchor. The different intracellular location and post-translational modification of recombinant proteins expressed in insect cells, as compared to the native proteins expressed in parasites, and the possible implications for vaccine development are discussed.

An early step of glycosylphosphatidyl-inositol anchor biosynthesis is abolished in lepidopteran insect cells following baculovirus infection.

The expression of recombinant proteins in their native state has become a prerequisite for a variety of functional and structural studies, as well as vaccine development. Many biochemical properties and functions of proteins are dependent on or reside in posttranslational modifications, such as glycosylation. The baculovirus system has increasingly become the system of choice due to it capabilities of performing posttranslational modifications and usually high yields of recombinant proteins. The Toxoplasma gondii surface antigen SAG1 was used as a model for a glycosylphosphatidyl-inositol (GPI)-anchored protein and expressed in insect cells using the baculovirus system. We show that the T. gondii SAG1 surface antigen expressed in this system was not modified by a GPI-anchor. In vitro and in vivo studies demonstrate that uninfected insect cells are able to produce GPI-precursors and to transfer a mature GPI-anchor to nascent proteins. These cells however are not capable to produce GPI-precursors following infection. We also show that the biosynthesis of the early GPI intermediate GlcNH(2)-PI is blocked in baculovirus-infected H5 cells, thus preventing the subsequent mannosylation steps for the synthesis of the conserved GPI-core-glycan. We therefore conclude that the baculovirus system is not appropriate for the expression of GPI-anchored proteins.

Chromosomal promoter replacement in Saccharomyces cerevisiae: construction of conditional lethal strains for the cloning of glycosyltransferases from various organisms.

Heterologous complementation in yeast has been a successful tool for cloning and characterisation of genes from various organisms. Therefore we constructed conditionally lethal Saccharomyces cerevisiae strains by replacing the endogenous promoter from the genes of interest (glycosyltransferases) by the stringently regulated GAL1-promoter, by a technique called chromosomal promoter replacement. Such yeast strains were constructed for the genes Alg 1, Alg7, Sec59, Wbp1 involved in N-Glycosylation, the genes Gpi2, Gpi3/Spt14, Gaal, Pis1, involved in GPI-anchor biosynthesis and Dpm involved in both pathways. All strains show the expected conditionally lethal phenotype on glucose-containing medium when expression of the respective gene is turned off.

Cloning and functional expression of the human GlcNAc-1-P transferase, the enzyme for the committed step of the dolichol cycle, by heterologous complementation in Saccharomyces cerevisiae.

The gene for the human dolichol cycle GlcNAc-1-P transferase (ALG7/GPT) was cloned by screening a human lung fibroblast cDNA library. The library was constructed in a Saccharomyces cerevisiae expression vector, and the positive clone was identified by complementation of the conditional lethal S.cerevisiae strain YPH-A7-GAL. This strain was constructed by replacing the endogenous promoter of the GPT-gene by the stringently regulated GAL1-promoter. This construct allows to specifically suppress the endogenous enzyme activity. The insert of the positive clone displayed an open reading frame of 1200 nucleotides, coding for a putative protein of 400 amino acids with a calculated molecular weight of 44.7 kDa. The deduced protein sequence shows a homology of over 90% when compared with other mammalian GPT sequences, thus resembling the close phylogenetic relationship between mammalian species. This homology however decreases to 40-50% when compared to more distantly related organisms such as S.cerevisiae , Schizosaccharomyces pombe , or Leishmania amazonensis . Biochemical characterization of the recombinant protein showed that it is functionally expressed in the S.cerevisiae strain YPH-A7-GAL. GlcNAc- and GlcNAc2-PP-Dolichol biosynthesis could be shown with isolated S.cerevisiae membranes from cells harboring the recombinant plasmid and grown on glucose thus suppressing transcription of the endogenous gene. Synthesis could be stimulated by dolicholphosphate and was inhibited by tunicamycin. These results show that we have cloned the human GlcNAc-1-P transferase by heterologous complementation in S. cerevisiae, a strategy that may be useful for the cloning and characterization of glycosyltransferases from a variety of organisms.

Glycosyl-phosphatidylinositols in protozoa structure, biosynthesis and intracellular localisation.

We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in the protozoa Toxoplasma gondii, Plasmodium falciparum, Plasmodium yoelii and Paramecium primaurelia. This comparison of structural and biosynthesis data should lead us to common and individual features of the GPI-biosynthesis and transport in different organisms.

Cloning and functional expression of glycosyltransferases from parasitic protozoans by heterologous complementation in yeast: the dolichol phosphate mannose synthase from Trypanosoma brucei brucei.

The gene for the enzyme dolichol phosphate mannose (Dol-P-Man) synthase from the parasitic protozoan Trypanosoma brucei brucei (T. brucei) was cloned by screening a T. brucei cDNA library and then sequenced. The library was constructed in a yeast expression vector and the positive clone was identified by complementation of a temperature-sensitive defect in the yeast strain DPM 1-6 [Orlean, Albright and Robbins (1988) J. Biol. Chem. 263, 17499-17507]. The insert of this clone displayed an open reading frame of 801 nucleotides coding for a putative protein of 267 amino acids. The deduced protein sequence showed an identity of 49% and a similarity of 69% with the published yeast sequence. Additional features of the T. brucei sequence are the presence of a putative signal sequence, a C-terminal transmembrane domain, a consensus sequence for phosphorylation by cAMP-dependent protein kinase and a stretch of five nucleotides immediately upstream from the putative initiation codon that could function as a prokaryotic ribosome binding site. A consensus sequence for dolichol binding (FI/VXF/YXXIPFXF/Y) found in the yeast protein could not be detected in the putative transmembrane domain of the T. brucei sequence. Biochemical characterization of the recombinant protein showed that it is functionally expressed in the yeast strain DPM 1-6 and Escherichia coli. In both constructs Dol-P-Man synthesis was shown in a cell-free system. Synthesis was stimulated by exogenous dolichol phosphate and inhibited by amphomycin. These results confirm that we have cloned the T. brucei Dol-P-Man synthase by heterologous complementation in yeast, an approach that might be applicable for other glycosyltransferases from various sources.

The phorbol 12-myristate 13-acetate (PMA)-induced oxidative burst in rat peritoneal neutrophils is increased by a 0.1 mT (60 Hz) magnetic field.

Magnetic fields (MF) may affect biological systems by increasing free radical concentrations. To test this, we have investigated whether low frequency (60 Hz) low intensity (0.1 mT) MF can modulate the phorbol 12-myristate 13- acetate (PMA) induced respiratory burst in primed rat peritoneal neutrophils, followed in real time using the dye 2',7'-dichlorofluorescin (DCFH), which reacts with free radical-derived oxidants such as H2O2 (which is formed from the dismutation of superoxide) to become 2',7'-dichlorofluorecein (DCF), a highly fluorescent compound. In the presence of the MF, a 12.4% increase in the fluorescence signal was observed in PMA-stimulated neutrophils (n = 5, P < 0.02, 18 pairs of measurements). We believe this represents the first experimental observation of MF influencing events involving free radical species generated during signal transduction in living cells.

Plasmodium cynomolgi: the hsp 70 gene.

The hsp 70 gene of Plasmodium cynomolgi was isolated and characterized. As expected the gene is highly similar to that of the hsp 70 gene of Plasmodium falciparum (98% at the protein level, 82% at the nucleotide level). Surprisingly, the hsp 70 gene appears to be present in a single copy in all the P. cynomolgi strains tested, a finding that has implications for the parasite's ability to undergo a heat shock response.

The effect of oligonucleotides complementary to the 3' end of the 16S rRNA on the formation of the bacterial 30S initiation complex.

Although much attention was focused on the role of the 16S RNA in mRNA selection by the 30S ribosomal subunit no true consensus site has emerged as yet. Oligonucleotides such as GAGG, UGAU and CCAA which are complementary to the 3' end of the 16S RNA stimulate the AUG-dependent binding of fMet-tRNA to 30S subunits. If those tetranucleotides are used in combination or if the octanucleotides GAGGUGAU and UGAUCCAA are applied, the degree of stimulation remains unchanged. Effects are strictly dependent on the presence of initiation factor 2 (IF-2) and cannot be produced by using A4 or U4. With sequences covalently linked to the AUG as in CCAAAUG and UGAUCCAAAUG, the efficiency of the initiation complex formation decreases significantly as compared to AUG with UGAUCCAAAUG being the least efficient mRNA analogue. The pentadecanucleotide GAGGUGAUCCAAAUG, however, shows the highest efficiency in directing the binding of the fMet-tRNA to 30S subunits and is clearly superior to AUG. Initiation factor 2 (IF-2), which stimulates tRNA binding significantly with AUG and CCAAAUG, both in terms of slope and plateau values of the binding curves, does not effect the initial rate of tRNA binding to GAGGUGAUCCAAAUG. In another set of experiments, where GAGG and AUG are separated by oligo(U) or oligo(A) sequences, the effect of chain length was investigated. mRNA analogues with a spacer of 6-9 nucleotides show the highest binding efficiencies, with a U spacer being superior to an A spacer, indicating that a more flexible spacer favours tRNA binding.