BKCa-Cav channel complexes mediate rapid and localized Ca2+-activated K+ signaling

Large-conductance calcium- and voltage-activated potassium channels (BKCa) are dually activated by membrane depolarization and elevation of cytosolic calcium ions (Ca2+). Under normal cellular conditions, BKCa channel activation requires Ca2+ concentrations that typically occur in close proximity to Ca2+ sources. We show that BKCa channels affinity-purified from rat brain are assembled into macromolecular complexes with the voltage-gated calcium channels Cav1.2 (L-type), Cav2.1 (P/Q-type), and Cav2.2 (N-type). Heterologously expressed BKCa-Cav complexes reconstitute a functional "Ca2+ nanodomain" where Ca2+ influx through the Cav channel activates BKCa in the physiological voltage range with submillisecond kinetics. Complex formation with distinct Cav channels enables BKCa-mediated membrane hyperpolarization that controls neuronal firing pattern and release of hormones and transmitters in the central nervous system.     

Effects of different media on proliferation and differentiation capacity of canine, equine and porcine adipose derived stem cells

Adult stem cells are of particular interest for therapeutic use in the field of regenerative medicine. Adipose-derived mesenchymal stem cells (ASCs) are an attractive stem cell source for all fields of regenerative medicine because adipose tissue - and therewith cells - can easily be harvested from each donor. However, common expansion using fetal bovine serum (FBS) can not be used for clinical applications as xenogenic proteins must be avoided. Adipose tissue from equine, canine and porcine donors was digested with collagenase to isolate ASCs. ASCs were either expanded in a cell culture medium supplemented with FBS or in a serum-free medium (UltraCulture; UC) supplemented with a serum substitute (UltroserG). From all three animal species, the adipogenic and osteogenic differentiation potential of ASCs cultured with different media was analyzed in vitro. Cell proliferation analysis showed a population doubling time of 48-68 h for canine cells, 54-65 h for porcine cells and 54-70 h for equine cells, expanded in different media. Except for porcine ASCs, cells cultured in media supplemented with FBS grew faster than cells expanded in UC medium with UltroserG. Yet, all cells maintained their potential to differentiate into adipocytes and osteoblasts. UltraCulture medium containing UltroserG can for all examined species be recommended if FBS needs to be avoided in the expansion of donor-derived (stem) cells.     

The association between teat end hyperkeratosis and teat canal microbial load in lactating dairy cattle

Most pathogens that cause bovine mastitis invade the udder lumen through the teat canal. Amino acids and intercellular lipids may support microbial colonisation of the teat canal epithelium by pathogenic microorganisms. The aim of this study was to investigate the association between teat end hyperkeratosis, which is induced by machine milking, and teat canal microbial load. Contralateral teats, which differed in teat end hyperkeratosis scores, were identified in a split-udder experiment. The teat canal's microbial load was evaluated using the wet and dry swab technique. Staphylococcus (S.) aureus, Streptococcus (Sc.) uberis, Escherichia (E.) coli and other coliforms were detected by agar plate cultures. The positive detection of E. coli and the log(10)-transformed E. coli load of a teat canal were significantly associated with the teat end hyperkeratosis score (P<0.05). There were significant differences with respect to positive findings for E. coli, as well as the microbial load of E. coli and Sc. uberis, between the less-calloused and the more-calloused teat of a pair. For S. aureus, no significant associations between hyperkeratosis score and teat canal microbial load were detected. In general, a teat with a highly calloused teat end had an increased teat canal microbial load compared with the contralateral teat, characterised by a lower callosity. The results of the present study indicate that the environmental pathogen load is associated with teat end hyperkeratosis. Further research is needed to identify factors that may affect teat canal microbial load in lactating dairy cattle.     

Obtaining raw material: plants as tool sources for nigerian chimpanzees

We investigated the acquisition of plant materials from which Nigerian chimpanzees manufacture wooden tools to harvest insects and honey from nests of army ants, honey bees and stingless bees. Slender trunks of juvenile trees and branches are most commonly used, and bendable vines rarely, probably reflecting the need to work with relatively sturdy tools to extract resources. While several tools are sometimes sourced from the same plant, there is also evidence for a depletion effect, as multiple tool sources at the same site are often spaced several metres apart. Identified tool sources belong to 27 species of at least 13 families. Honey-gathering implements are often chewed upon by chimpanzees. Interestingly, twigs of the most commonly used honey-gathering species possess antibacterial propensities and are favoured by Nigerians as chewing sticks. This suggests that extractive tools might possess associated medicinal or stimulatory properties. We do not know if chimpanzees actively select specific plant parts or species as we cannot compare observed with expected frequencies. Nevertheless, about three quarters of tools are picked from plants more than 6 m away from the extraction site, potentially indicating some degree of forward planning.     

Porcine intestinal epithelial cell lines as a new in vitro model for studying adherence and pathogenesis of enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) infections result in large economic losses in the swine industry worldwide. The organism causes diarrhea by adhering to and colonizing enterocytes in the small intestines. While much progress has been made in understanding the pathogenesis of ETEC, no homologous intestinal epithelial cultures suitable for studying porcine ETEC pathogenesis have been described prior to this report. In the current study, we investigated the adherence of various porcine ETEC strains to two porcine (IPEC-1 and IPEC-J2) and one human (INT-407) small intestinal epithelial cell lines. Each cell line was assessed for its ability to support the adherence of E. coli expressing fimbrial adhesins K88ab, K88ac, K88ad, K99, F41, 987P, and F18. Wild-type ETEC expressing K88ab, K88ac, and K88ad efficiently bound to both IPEC-1 and IPEC-J2 cells. An ETEC strain expressing both K99 and F41 bound heavily to both porcine cell lines but an E. coli strain expressing only K99 bound very poorly to these cells. E. coli expressing F18 adhesin strongly bound to IPEC-1 cells but did not adhere to IPEC-J2 cells. The E. coli strains G58-1 and 711 which express no fimbrial adhesins and those that express 987P fimbriae failed to bind to either porcine cell line. Only strains B41 and K12:K99 bound in abundance to INT-407 cells. The binding of porcine ETEC to IPEC-J2, IPEC-1 and INT-407 with varying affinities, together with lack of binding of 987P ETEC and non-fimbriated E. coli strains, suggests strain-specific E. coli binding to these cell lines. These findings suggest the potential usefulness of porcine intestinal cell lines for studying ETEC pathogenesis.     

Experimental infection of domestic pigeons with pigeon circovirus

Pigeon circovirus (PiCV) infection and young pigeon disease syndrome (YPDS), associated with high morbidity and mortality, have been recognized in young racing pigeons from large portions of Central Europe. There exist a number of data indicating that YPDS is a consequence of PiCV infection and subsequent immunosuppression. In order to prove PiCV to be one of the crucial factors of YPDS, an experimental infection with PiCV was performed under controlled conditions. Twenty-four domestic pigeons (Columba livia forma domestica) were divided into two groups with 12 pigeons each; an infection group and a control group. All birds were between their fourth to eighth week of life. Pigeons in the infection group were infected both intramuscularly and orally with PiCV purified from naturally infected birds, while pigeons in the control group received a placebo. To test a possible influence of the PiCV infection on the immune system, the animals in both groups were vaccinated simultaneously, on the same day, against PMV-1 (Lasovac plus, IDT, Dessau-Tornau, Germany). Weekly virologic testing showed a viraemic period, and excretion of the infection virus, in pigeons in the infection group. Replication of PiCV could be proved on the basis of histologic findings of multiglobular inclusion bodies, mainly observed in macrophages of the bursa of Fabricius. A PiCV, genetically distinct from the experimental virus, was detected in the control group by polymerase chain reaction (PCR) testing, but any histologic findings comparable to the infection group were absent. None of the pigeons revealed clinical signs of illness, or hints that immunosuppression had occurred, regardless of their group. The absence of stressful conditions, considered as a trigger for the development of YPDS, may be responsible for the failure of disease reproduction in our infection model.     

Rapid Gas Chromatographic Technique for Quantifying 2‐Acetyl‐1‐Pyrroline and Hexanal in Rice (Oryza sativa,L.)

The aroma of rice plays a role in its consumer acceptability. The popcorn‐like smell of aromatic rice stemming primarily from its 2‐ acetyl‐1‐pyrroline (2‐AP) content is considered desirable by many consumers. Conversely, hexanal has been correlated with off odors in rice that develop from lipid oxidation. A rapid method for 2‐AP and hexanal quantification suitable for use in breeding programs, large‐scale research efforts, and quality assurance programs is needed. While developing such a method, sample preparation (degree of milling, particle size), solvent extraction time and temperature, and gas chromatographic parameters were studied. Particle size had no influence on 2‐AP or hexanal recovered. One extraction solubilized ≈80% of the 2‐AP and 56% of the hexanal present in milled rice. The optimum extraction method was assessed to require 0.3 g of ground brown or milled rice in methylene chloride held at 85°C for 2.5 hr. The complete gas chromatographic run requires ≈25 min, and 50 samples can be analyzed per day. The optimized method's linear response (R² = 0.99) and reproducibility was demonstrated. The stability of 2‐AP and hexanal in frozen milled rice and in refrigerated methylene chloride extracts was excellent for at least six months. Milled and unmilled commercial and breeders' aromatic rice samples contained 10–1,104 ng/g of 2‐AP and 148–2,541 ng/g of hexanal. Genotype had the greatest effect on the 2‐AP and hexanal content of two lines grown over four years and in four states.     

Thiamine (vitamin B1) deficiency in germinating seeds of Phaseolus vulgaris L. exposed to soaking injury

Exogenous application of thiamine (vitamin B1) during imbibition improved germination and seedling development of bean (Phaseolus vulgaris L. cv. Lasso) seeds, which were exposed to soaking injury by submergence. Leaching of the vitamin into the incubation medium was not increased in submerged seeds. However, translocation of the thiamine reserves from the cotyledons to the seedling axis was reduced in response to soaking injury. After exogenous application of free thiamine to the seeds, thiaminedi-phosphate (TDP), which is the physiologically active coenzyme form of thiamine, accumulated in the seedling axis of submerged seeds, suggesting an increased demand for thiamine-dependent metabolic reactions in these tissues. Limited oxygen supply of the seeds during the soaking period induces a shift from respiration to fermentation of carbohydrates. Neither thiamine-dependent ethanolic fermentation, nor ATP production or adenylate energy charge (AEC) of the seedling axis were affected by exogenous thiamine application, suggesting that there is no limitation of thiamine-dependent reactions in the energy metabolism of the seedlings. Thus the physiological mechanisms improving germination and seedling development of submerged seeds in response to thiamine seed treatment are still not clear.