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Biomedical subjects

R Braun

Publications and source records attributed to R Braun.

At least 91 records · Page 5Linked to original sources
PubMed1995

Expression of GARP, a major surface glycoprotein of Trypanosoma congolense, on the surface of Trypanosoma brucei: characterization and use as a selectable marker.

Procyclic and epimastigote forms of Trypanosoma congolense express an immunodominant glutamic acid/alanine-rich protein (GARP) that covers the parasite surface. Although GARP shows no sequence similarity to procyclins from T. brucei, the general characteristics of the two sets of surface glycoproteins suggest that they have analogous functions, in much the same way that variant surface glycoproteins with unrelated primary sequences fulfil the same function in bloodstream form trypanosomes. Since T. brucei and T. congolense do not follow the same pathway through the tsetse fly, one possible function of procyclins might be to direct parasites to the correct compartments. As a first step towards testing this hypothesis, we have produced stably transformed procyclic forms of T. brucei in which the GARP coding region has been integrated into a procyclin expression site. GARP can be detected on the surface of these transgenic trypanosomes, uniformly distributed within the endogenous procyclin coat, but there are differences in post-translational modification when it is expressed in T. brucei rather than in T. congolense. The fact that GARP is readily accessible to antibodies which were raised against a bacterial fusion protein led us to examine its potential as a selectable surface marker for transfection. We have established a rapid and simple procedure for isolating stable transformants that provides an alternative to conventional methods of selection for antibiotic resistance.

Animals↗
PubMed1995

Eimeria maxima gametocyte antigens: potential use in a subunit maternal vaccine against coccidiosis in chickens.

Affinity-purified gametocyte antigens (APGA) from Eimeria maxima, emulsified in Freund's adjuvant, were injected intramuscularly into breeding hens on two or three occasions. As a result, progeny of the immunized hens were partially immune to infection with E. maxima, Eimeria tenella and Eimeria acervulina (with a reduction in total oocyst output of 45-63% as compared with progeny of untreated hens). Thus, APGA in Freund's adjuvant appears to have great potential as part of a maternally applied vaccine against coccidiosis. The ability of APGA to induce partial cross-species protection is most probably due to the existence of conserved epitopes in the different species as indicated by comparative Western blots of E. maxima and E. tenella. Surprisingly, Freund's adjuvant by itself also induced significant levels of maternal immunity to coccidiosis (with a 12-35% reduction in oocyst output in the progeny). In contrast to the purified antigens or Freund's alone, crude extracts from gametocytes as well as other developmental stages, induced little if any significant maternal immunity despite provoking the production of large amounts of parasite-specific IgG, including antibodies to APGA. This result indicates that a successful maternal vaccine against coccidiosis requires, in addition to good recognition of protective antigens, the exclusion of irrelevant antigens from the vaccine preparation.

Animals↗
PubMed1994

TUBIS, a fossilized retroposon in the tubulin gene cluster of Trypanosoma brucei.

The genome of Trypanosoma brucei contains many copies of TRS/ingi, a retroposon like element of about 5 kb length. One vestigial member of this family, TUBIS, is found in the tubulin gene cluster. The element has sequence homologies to reverse transcriptase. The similarity to TRS/ingi is lost after 4 kb, resulting in a 3' truncated element. The sequence following downstream shows similarities to 5' flanking regions of VSG genes.

Amino Acid Sequence↗
PubMed1994

Control of polyadenylation and alternative splicing of transcripts from adjacent genes in a procyclin expression site: a dual role for polypyrimidine tracts in trypanosomes?

The procyclin-associated genes (PAGs) of Trypanosoma brucei are located downstream of tandemly repeated procyclin genes and belong to the same alpha-amanitin-resistant polycistronic transcription units. In procyclic form trypanosomes the PAG 1 pre-mRNA is alternatively spliced to give rise to three transcripts of 2.7 kb, 1.8 kb and 1.3 kb. The two larger transcripts contain additional short open reading frames (ORFs) upstream of the major ORF. Trans-splicing to generate these transcripts occurs downstream of three different polypyrimidine tracts. A minor population of procyclin mRNAs is also generated by alternative splicing at a polypyrimidine tract that begins 524 bp upstream of the major splice acceptor site of the procyclin beta-gene. The same polypyrimidine tract is also required for accurate polyadenylation of mRNAs from the upstream procyclin alpha-gene (1). Alternatively polyadenylated forms of PAG 1 mRNAs can also be detected. All polyadenylation sites are found at a similar distance upstream of splice-acceptor sites, in each case with a polypyrimidine tract between them. Our results point to a dual role for polypyrimidine tracts in the maturation of trypanosome mRNAs.

Alternative Splicing↗
PubMed1994

A conserved stem-loop structure in the 3' untranslated region of procyclin mRNAs regulates expression in Trypanosoma brucei.

African trypanosomes that cycle between mammalian hosts and the tsetse fly vector must be poised to survive in different environments. The control of stage-specific gene expression is undoubtedly one of the keys to successful adaptation, but no regulatory elements have been defined to date. Procyclins (also known as procyclic acidic repetitive proteins) are specifically expressed on the surface of procyclic and epimastigote forms in the fly. Procyclin genes are already transcribed in bloodstream forms, but stable mRNA, and later the protein, are first detected when the parasites begin to differentiate into procyclic forms. We have now identified a region of 16 bases that forms part of a predicted stem-loop structure in the 3' untranslated regions of different procyclin mRNAs; both the sequence and the secondary structure of this 16-mer appear to be required for efficient translation of a reporter gene in procyclic forms. The level of steady-state mRNA, its polyadenylylation, and its distribution in the cell are all unaffected by the presence or absence of this element. Deletion of the 16-mer alone reduces expression more than removal or reversal of the entire 3' untranslated region and flanking region, suggesting that there are additional negative regulatory elements in the same 3' untranslated region.

Animals↗
PubMed1994

Virus-like particles in Eimeria nieschulzi are associated with multiple RNA segments.

RNA preparations from sporulated oocysts of Eimeria nieschulzi were found to contain 2 double-stranded RNA segments of 5.0 kb and 5.7 kb that were not present in other species of Eimeria. Treatment of crude lysates with RNase A revealed that in addition to these two segments, 3 other segments of 0.57 kb, 0.72 kb and 11.5 kb were protected from digestion, suggesting that they were enclosed within particles. Virus-like particles with a diameter of approximately 39 nm were purified by caesium chloride buoyant density centrifugation. Four of the five RNA segments copurified with these particles. In keeping with the nomenclature generally adopted for protozoan viruses, we have named this new isolate ENV 1. The largest RNA segment does not cosediment with ENV 1 particles and may be derived from another RNA-protein complex that is unstable under the conditions used. The particle size and genome structure of ENV 1 both differ from that of the Eimeria stiedae virus (ESV), which is the only other virus to have been isolated from Eimeria to date. Short cDNA clones derived from ENV 1 show significant homology to a region of the Leishmania virus (LRV 1) genome that encodes an RNA-dependent RNA polymerase. The polymerase sequences from ENV 1 and LRV 1 are more closely related to each other than to any other protein sequences in the GenEMBL Database. This raises intriguing questions about the origins of the two viruses, since Eimeria and Leishmania normally infect different hosts and also show different cell tropisms within these hosts.

Amino Acid Sequence↗
PubMed1994

Maternal transfer of antibodies induced by infection with Eimeria maxima partially protects chickens against challenge with Eimeria tenella.

Infection of breeding hens with Eimeria maxima induces production of Eimeria-specific IgG antibodies which are transferred to hatchlings via the egg yolk and confer a high degree of maternal immunity against homologous challenge and partial immunity to infection with another important species, Eimeria tenella. As an example, in an experiment using hatchlings from eggs collected between days 28 and 39 after infection of the hens with 20,000 sporulated E. maxima oocysts, control chicks (challenged with 100 sporulated oocysts) excreted 6.8 +/- 1.2 million (mean +/- S.E., n = 10) or 5.8 +/- 1.2 million (n = 8) oocysts of E. maxima or E. tenella, respectively, compared to 0.9 +/- 0.4 million (n = 5) E. maxima oocysts or 2.2 +/- 0.4 million (n = 9) E. tenella oocysts excreted by hatchlings of infected hens. This represents an 87% reduction in oocyst excretion with regard to E. maxima and a 62% reduction in oocyst excretion with regard to E. tenella in the progeny of the infected hens. In another experiment, eggs were collected from days 28 to 37 and again from days 114 to 123 after infection of the hens with E. maxima and hatchling oocyst excretion rates were 82% and 62%, respectively, reduced for E. maxima and 43% and 41%, respectively, reduced for E. tenella in the progeny of hens infected with E. maxima compared to the progeny of uninfected hens. ELISA and Western blot analyses of maternally-derived IgG revealed a high degree of cross-reactivity to antigens of E. maxima and E. tenella.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

[Pneumothorax in laparoscopic cholecystectomy (II)].

This case report deals with pneumothorax during elective laparoscopic cholecystectomy in a young woman with no history of severe pulmonary disease. After inflating the capnoperitoneum, pulse oximetry and capnography raised suspicion of pneumothorax whereas the physical examination showed no irregularities. Surgical drapes provided a lack of information from percussion and auscultation. Changing respiratory parameters including the use of pressure-controlled ventilation in absence of tension pneumothorax enabled sufficient ventilation until the insertion of a chest tube. There were no further postoperative complications. Pulse oximetry, capnography and relaxometry proved helpful in monitoring. There may be a benefit from the use of pressure-controlled-ventilation in certain situations, if all changes in volume-controlled-ventilation fail.

Carbon Dioxide↗
PubMed1994

Maternal transmission of immunity to Eimeria maxima: western blot analysis of protective antibodies induced by infection.

Infection of breeding hens with Eimeria maxima induces production of parasite-specific antibodies which are transferred, via the egg yolk, to hatchling chicks. These antibodies (immunoglobulin G) are highly protective, mediating up to a 97% reduction in oocyst excretion in challenged hatchlings. However, the degree of maternally derived immunity transferred by the hens to their offspring declines with increasing time after infection of the hens. This decline in immunity is directly related to declining immunoglobulin G titers. However, sera from highly protected hatchlings recognize only a very few E. maxima proteins on Western blots (immunoblots). In particular, a 230-kDa protein band is outstanding for its association with maternally derived immunity to E. maxima in hatchlings. This band was excised from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) preparative gel of crude merozoite protein extract. The SDS-PAGE cutout was emulsified in Freund's adjuvant and injected, intramuscularly, into six breeding hens on two occasions, 2 weeks apart. Eggs were collected from these hens 28 to 39 days after the second injection, and the hatchlings from these eggs were challenged with 150 sporulated oocysts of E. maxima. Subsequent oocyst excretion in these hatchlings was, on average, 54% lower than oocyst excretion by control chicks but only 37% lower (significant at P < 0.05) than that by chicks from hens sham immunized with Freund's adjuvant. The latter result is apparently due to the ability of the adjuvant to induce production of antibodies which recognize Eimeria spp. and thereby transfer some degree of protection to hatchlings. These experiments indicate that protective, maternally derived immunoglobulin G antibodies may be useful for the identification of putative anticoccidial vaccine candidates.

Animals↗
PubMed1994

Maternal transmission of immunity to Eimeria maxima: enzyme-linked immunosorbent assay analysis of protective antibodies induced by infection.

Vaccination of broiler chickens against Eimeria infection is problematic because of the need to ensure that birds are protected from the time of hatching. We have therefore investigated the feasibility of protecting hatchling broilers via maternal transfer of protective antibodies from hens to their offspring. Oral infection of broiler breeder hens with 20,000 sporulated Eimeria maxima oocysts caused production of antibodies which were passed into the egg yolk and subsequently to hatchlings. The level of specific antibodies in the yolks to unsporulated oocysts, sporulated oocysts, merozoites, and gametocytes was assessed by enzyme-linked immunosorbent assays. The levels in yolks of antibodies to all developmental stages peaked 3 to 4 weeks after infection of the hens. Groups of 10 hatchlings were challenged at 3 days of age by oral infection with 100 sporulated E. maxima oocysts. In the first experiment, the mean 4-day (days 6 to 9 post-infection) total number of oocysts excreted in the feces of chicks from eggs collected 3 weeks after infection of the hens was (0.6 +/- 0.4) x 10(6) (mean +/- standard error) compared with (9.9 +/- 1.4) x 10(6) for the progeny of uninfected hens, which represents a greater than 90% reduction. However, oocyst excretion by chicks from eggs collected 7 or 8 weeks after infection of the hens was only 47 or 68% lower than control values, reflecting declining levels of protective antibodies. In a second experiment, in which the hens were somewhat older and pretreated by intramuscular injection of saline in the emulsifying agent, Arlacel A, the period for which protective antibodies were transferred to hatchlings was prolonged. Thus, oocyst excretion by challenged hatchlings from eggs collected for an 8-week period after infection of the hens was more than 90% lower than oocyst excretion by control chicks, and even hatchlings of eggs collected 19 weeks after infection of the hens showed a 60% reduction in oocyst output. In both experiments, the levels of immunoglobulin G (IgG) antibodies to all developmental stages in yolks or hatchling sera were very strongly correlated with maternally derived immunity to E. maxima. In contrast, parasite-specific IgM or IgA was not detectable, either in egg yolk or egg white. These results demonstrate the ability of IgG antibodies to protect against E. maxima in poultry, thus raising the possibility of using protective maternally derived IgG antibodies to identify potentially protective parasite antigens and indicating the feasibility of using maternal immunization as a means for parasite control.

Animals↗
PubMed1994

Accurate polyadenylation of procyclin mRNAs in Trypanosoma brucei is determined by pyrimidine-rich elements in the intergenic regions.

Polycistronic precursor RNAs from trypanosomes are processed into monocistronic mRNAs by the excision of intergenic sequences and the addition of a 39-nucleotide spliced leader by trans splicing. These mRNAs are also polyadenylated, yet they do not contain the hexamer AAUAAA within their 3' untranslated regions (UTRs). To identify the signals required for the accurate polyadenylation of mRNAs, we tested the effects of deletions in either the procyclin 3' UTR or the downstream intergenic region on the polyadenylation of transcripts from a reporter gene. Deletion of the entire 3' UTR does not affect polyadenylation, but a crucial element is located in the intergenic region and includes a pyrimidine-rich sequence from positions 79 to 112 followed by an AG dinucleotide. Related motifs are also found a similar distance downstream of other genes in both the procyclin and the variant surface glycoprotein expression sites. These sequences bear a strong resemblance to splice acceptor sites, but they are generally several hundred base pairs upstream of the major splice acceptor site of the next gene in the transcription unit. There is evidence, however, that some of them can give rise to alternatively spliced transcripts with unusually long 5' UTRs.

Animals↗
PubMed1993

Eimeria tenella: characterization of a 5S ribosomal RNA repeat unit and its use as a species-specific probe.

The oocysts of Eimeria tenella, one of the most pathogenic of several species causing chicken coccidiosis, are difficult to distinguish microscopically from several other infective Eimeria species. One copy of a gene coding for 5S ribosomal RNA has been cloned from E. tenella and sequenced. A coding region of 120 nucleotides and an intergenic region of 608 nucleotides together make up a 5S rRNA repeat unit, of which there are many copies tandemly repeated in the genome. The intergenic region is species-specific and sequences derived from it can be used for species identification by polymerase chain reaction (PCR). The PCR procedure described here is particularly sensitive, with fewer than 10 oocysts sufficing to give a positive result. This method may also be useful for the identification by PCR of other cyst-forming parasites.

Animals↗
PubMed1993

Genotoxicity studies in semiconductor industry. 1. In vitro mutagenicity and genotoxicity studies of waste samples resulting from plasma etching.

Solid waste samples taken from the etching reactor, the turbo pump, and the waste air system of a plasma etching technology line in semiconductor production were studied as to their genotoxic properties in a bacterial repair test, in the Ames/Salmonella microsome assay, in the SOS chromotest, in primary mouse hepatocytes, and in Chinese hamster V79 cell cultures. All three waste samples were found to be active by inducing of unscheduled DNA-synthesis in mouse hepatocytes in vitro. In the bacterial rec-type repair test with Proteus mirabilis, waste samples taken from the turbo pump and the vacuum pipe system were not genotoxic. The waste sample taken from the chlorine-mediated plasma reactor was clearly positive in the bacterial repair assay and in the SOS chromotest wit Escherichia coli. Mutagenic activity was demonstrated for all samples in the presence and absence of S9 mix made from mouse liver homogenate. Again, highest mutagenic activity was recorded for the waste sample taken from the plasma reactor, while samples collected from the turbo pump and from the waste air system before dilution and liberation of the air were less mutagenic. For all samples chromosomal damage in V79 cells was not detected, indicating absence of clastogenic activity in vitro. Altogether, these results indicate generation of genotoxic and mutagenic products as a consequence of chlorine-mediated plasma etching in the microelectronics industry and the presence of genotoxins even in places distant from the plasma reactor. Occupational exposure can be expected both from the precipitated wastes and from chemicals reaching the environment with the air stream.

Animals↗
PubMed1992

Evolution of the retrotransposons TRS/ingi and of the tubulin genes in trypanosomes.

The African trypanosomes have genomes of high plasticity, as demonstrated for instance by their ability to shuffle their genes around, coding for variant-specific surface glycoproteins (VSGs). Another indication of their genome plasticity is the presence of multiple retro-elements. The retrotransposon-like element TRS/ingi is present in many copies in the genome of trypanosomes. One particular derivative of TRS/ingi, called TUBIS, had previously been found to interrupt a tubulin gene in a particular strain of T. brucei. Here both TRS/ingi and TUBIS were studied by hybridizing genomic DNA of various strains and species of trypanosomes with suitable probes in order to elucidate the evolution of this family of retro-elements. The TSR/ingi elements are highly repeated and have very long open reading frames, while TUBIS clearly is a truncated, inactivated form of this element, found in only one particular chromosomal location. Both elements were shown to be present in several strains and species of the subgenus Trypanozoon, in particular in T. brucei brucei, T. gambiense, T. rhodesiense, T. equiperdum and T. evansi. They could not be detected in species of other subgenera, in particular in T. congolense and T. cruzi. These findings suggest that the retrotransposon TRS/ingi was acquired by trypanosomes only after divergence of present day subgenera. The TUBIS element was found in exactly the same chromosomal location (at the 3' end of the tubulin gene cluster) in many different strains and species of the subgenus Trypanozoon. This shows that the element was transposed to this location before speciation of the subgenus. Although, TRS/ingi is unlikely to be involved directly in VSG switching, it may have contributed to the genome plasticity of trypanosomes.

Animals↗
PubMed1991

Detection of 6-thioguanine-resistant spleen lymphocytes in different mouse strains by autoradiography.

The variant frequencies for 6-thioguanine-resistant spleen cells in different mouse strains have been estimated by autoradiography for animals without chemical treatment and in cases of in vivo mutagen dosage with ethylnitrosourea and cyclophosphamide, respectively. In untreated mice, the following variant frequencies have been found: C57Bl/6J, 2.84 x 10(-5);NMRI, 3.04 x 10(-5);DBA/2J, 5.91 x 10(-5). The selective concentration of 6-thioguanine was 100 microM for strains NMRI and DBA, while in the case of C57Bl with this concentration, no variant cells could be counted and a selective concentration of 50 microM was chosen. Treatment with 70, 140, and 210 mg/kg ethylnitrosourea resulted in increased variant frequencies in cells isolated 8 or 15 days later. On the other hand, doses of 20, 60, and 120 mg/kg cyclophosphamide did not result in a clear dose-response relationship of variant frequency in cells isolated 1, 8, 15, 22, and 29 days after treatment. These data are discussed with respect to findings in human populations exposed occupationally to cyclophosphamide.

Animals↗