Tetanic fatigue and proximate post-tetanic recuperation in sartorius and flexor carpi radialis muscles of the male frog. Effects of iodoacetic acid (author's transl)]

The time-course of the isometric tension output, at 20 degrees C, during a long tetanus and after a short period of rest, was investigated in two isolated frog muscles : the sartorius and flexor carpi radialis muscles. To prevent aerobie and glycolytic recovery processes, some muscles were poisoned with 0,4 mM iodoacetic acid (IAA) and nitrogen, for 20 or 40 min. 1. For the unpoisoned sartorius muscle, tetanic tension declined quickly, but after a 0,8 sec period of rest, the muscle was able to develop high tension. Poisoning with IAA-N2 increased fatigue without suppressing the property of a proximate post-tetanic recuperation. 2. In the flexor carpi radialis muscle resistance to fatigue was very large before poisoning and diminished after poisoning. Proximate recuperation was very weak. 3. The results show that the recovery processes are not a primary factor of the development of the short-term fatigue ; they enhance the hypothesis that a failure of the electromechanical coupling can explain the rate of the tension fall in tetanized sartorius muscles.     

Detection of Mycoplasma hyopneumoniae by Air Sampling with a Nested PCR Assay

This article describes the first successful detection of airborne Mycoplasma hyopneumoniae under experimental and field conditions with a new nested PCR assay. Air was sampled with polyethersulfone membranes (pore size, 0.2 μm) mounted in filter holders. Filters were processed by dissolution and direct extraction of DNA for PCR analysis. For the PCR, two nested pairs of oligonucleotide primers were designed by using an M. hyopneumoniae-specific DNA sequence of a repeated gene segment. A nested PCR assay was developed and used to analyze samples collected in eight pig houses where respiratory problems had been common. Air was also sampled from a mycoplasma-free herd. The nested PCR was highly specific and 10⁴ times as sensitive as a one-step PCR. Under field conditions, the sampling system was able to detect airborne M. hyopneumoniae on 80% of farms where acute respiratory disease was present. No airborne M. hyopneumoniae was detected on infected farms without acute cases. The chance of successful detection was increased if air was sampled at several locations within a room and at a lower air humidity.     

Prion gene sequence variation within diverse groups of U.S. sheep,beef cattle,and deer

Prions are proteins that play a central role in transmissible spongiform encephalopathies in a variety of mammals. Among the most notable prion disorders in ungulates are scrapie in sheep, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer. Single nucleotide polymorphisms in the sheep prion gene (PRNP) have been correlated with susceptibility to natural scrapie in some populations. Similar correlations have not been reported in cattle or deer; however, characterization of PRNP nucleotide diversity in those species is incomplete. This report describes nucleotide sequence variation and frequency estimates for the PRNP locus within diverse groups of U.S. sheep, U.S. beef cattle, and free-ranging deer (Odocoileus virginianus and O. hemionus from Wyoming). DNA segments corresponding to the complete prion coding sequence and a 596-bp portion of the PRNP promoter region were amplified and sequenced from DNA panels with 90 sheep, 96 cattle, and 94 deer. Each panel was designed to contain the most diverse germplasm available from their respective populations to facilitate polymorphism detection. Sequence comparisons identified a total of 86 polymorphisms. Previously unreported polymorphisms were identified in sheep (9), cattle (13), and deer (32). The number of individuals sampled within each population was sufficient to detect more than 95% of all alleles present at a frequency greater than 0.02. The estimation of PRNP allele and genotype frequencies within these diverse groups of sheep, cattle, and deer provides a framework for designing accurate genotype assays for use in genetic epidemiology, allele management, and disease control.     

Immune responses to Mycoplasma conjunctivae in alpine ibex, alpine chamois, and domestic sheep in Switzerland

The humoral immune response of three alpine chamois (Rupicapra rupicapra rupicapra), two alpine ibex (Capra ibex ibex) and three domestic sheep naturally affected with infectious keratoconjunctivitis (IKC), and four ibex and two sheep experimentally infected with Mycoplasma conjunctivae was analysed. In addition, the local immune response to M. conjunctivae was analysed using conjunctival washes from chamois and sheep. Immunoblot analysis of sera using whole cell antigens of M. conjunctivae revealed the major immunogenic proteins which had molecular masses of 175, 83, 68, 60, 50, 42, 36, and 33 kDa. Major antigens were found at 83, 68, 60, and 42 kDa in both sera and conjunctival washes from naturally infected animals of all three Caprinae species. In experimentally infected animals, antibodies to the 68 and 60 kDa antigens were dominant. Naturally infected animals showed much stronger immune reactions than those experimentally infected, and specific antibodies appeared 2 to 4 wk after experimental infection. To evaluate possible cross-reactions, whole cell antigen of M. conjunctivae was analysed by immunoblot against hyperimmune sera of closely related Mycoplasma spp. Antibodies to the 175, 73, 68, 60, and 33 kDa antigens appeared to be specific to M. conjunctivae. Cross-reactions mainly with 83, 50, and 42 kDa antigens were detected, in particular with M. ovipneumoniae and M. bovoculi hyperimmune sera, but also with antisera against M. capricolum capricolum and M. putrefaciens.     

Towards the biocontrol of bindweeds with a mycoherbicide

Within the framework of the European COST Action 816, afive-year collaboration between scientists from five Europeancountries has made an important contribution to biologicalcontrol of field and hedge bindweeds (Convolvulus arvensis andCalystegia sepium, respectively). A fungus Stagonosporaconvolvuli strain LA39, able to infect both field and hedgebindweed, was found in the UK and its biocontrol efficacyimproved by optimising mass production, formulation and storagetechniques. This fungus controlled bindweeds in both a cemeteryand in maize crops. Its use fits best in an integrated pestmanagement system where a green cover controls most of the weedsexcept the bindweeds. DNA marker analyses indicate that thefungus reproduces sexually, which could be used to furtherimprove this mycoherbicide. In addition, the insect Melanagromyzaalbocilia, which itself exhibits biocontrol potential againstbindweeds, may be used in combination with LA39 to improve theability of the fungus to penetrate the stem of bindweeds.Overall, the results suggest that S. convolvuli LA39 haspromising potential as a bioherbicide for control of field andhedge bindweed.     

Single nucleotide polymorphism genotyping in polyploid wheat with the Illumina GoldenGate assay

Single nucleotide polymorphisms (SNPs) are indispensable in such applications as association mapping and construction of high-density genetic maps. These applications usually require genotyping of thousands of SNPs in a large number of individuals. Although a number of SNP genotyping assays are available, most of them are designed for SNP genotyping in diploid individuals. Here, we demonstrate that the Illumina GoldenGate assay could be used for SNP genotyping of homozygous tetraploid and hexaploid wheat lines. Genotyping reactions could be carried out directly on genomic DNA without the necessity of preliminary PCR amplification. A total of 53 tetraploid and 38 hexaploid homozygous wheat lines were genotyped at 96 SNP loci. The genotyping error rate estimated after removal of low-quality data was 0 and 1% for tetraploid and hexaploid wheat, respectively. Developed SNP genotyping assays were shown to be useful for genotyping wheat cultivars. This study demonstrated that the GoldenGate assay is a very efficient tool for high-throughput genotyping of polyploid wheat, opening new possibilities for the analysis of genetic variation in wheat and dissection of genetic basis of complex traits using association mapping approach. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Globular and asymmetric acetylcholinesterase in frog muscle basal lamina sheaths

After denervation in vivo, the frog cutaneus pectoris muscle can be led to degenerate by sectioning the muscle fibers on both sides of the region rich in motor endplate, leaving, 2 wk later, a muscle bridge containing the basal lamina (BL) sheaths of the muscle fibers (28). This preparation still contains various tissue remnants and some acetylcholine receptor-containing membranes. A further mild extraction by Triton X-100, a nonionic detergent, gives a pure BL sheath preparation, devoid of acetylcholine receptors. At the electron microscope level, this latter preparation is essentially composed of the muscle BL with no attached plasmic membrane and cellular component originating from Schwann cells or macrophages. Acetylcholinesterase is still present in high amounts in this BL sheath preparation. In both preparations, five major molecular forms (18, 14, 11, 6, and 3.5 S) can be identified that have either an asymmetric or a globular character. Their relative amount is found to be very similar in the BL and in the motor endplate-rich region of control muscle. Thus, observations show that all acetylcholinesterase forms can be accumulated in frog muscle BL.     

Carboxy-terminal processing of the large subunit of [Fe] hydrogenase from Desulfovibrio desulfuricans ATCC 7757

hydA and hydB, the genes encoding the large (46-kDa) and small (13. 5-kDa) subunits of the periplasmic [Fe] hydrogenase from Desulfovibrio desulfuricans ATCC 7757, have been cloned and sequenced. The deduced amino acid sequence of the genes product showed complete identity to the sequence of the well-characterized [Fe] hydrogenase from the closely related species Desulfovibrio vulgaris Hildenborough (G. Voordouw and S. Brenner, Eur. J. Biochem. 148:515-520, 1985). The data show that in addition to the well-known signal peptide preceding the NH2 terminus of the mature small subunit, the large subunit undergoes a carboxy-terminal processing involving the cleavage of a peptide of 24 residues, in agreement with the recently reported data on the three-dimensional structure of the enzyme (Y. Nicolet, C. Piras, P. Legrand, E. C. Hatchikian, and J. C. Fontecilla-Camps, Structure 7:13-23, 1999). We suggest that this C-terminal processing is involved in the export of the protein to the periplasm.