Importance of tetrahydrofolate and ATP in the anaerobic O-demethylation reaction for phenylmethylethers.

DL-Tetrahydrofolate (THF) and ATP were necessary for the anaerobic O-demethylation of phenylmethylethers in cell extracts of the type strain (ATCC 29683) of the homoacetogen Acetobacterium woodii. The reactants for this enzymatic activity have not been previously demonstrated in any system, nor has the mediating enzyme been studied. An assay using reaction mixtures containing 1 mM THF, 2 mM ATP, and 2 mM hydroferulate (i.e., 4-hydroxy,3-methoxyphenylpropionate) was developed and was performed under stringent anaerobic conditions. Pyridine nucleotides and several other possible cofactors were tested but had no effect on the activity. After centrifugation of disrupted cells at 27,000 x g, the activity was found primarily in the supernatant, which had a specific activity of 14.2 +/- 0.5 nmol/min/mg of protein. At saturating levels of each of the other two substrates, apparent Km values for the variable substrate were 0.65 mM hydroferulate, 0.27 mM ATP, and 0.17 mM THF. Activity was significantly decreased when extract was preincubated at 60 degrees C and was completely lost after preincubation in air for 30 min. Thus, the soluble anaerobic O-demethylating enzyme system of A. woodii is oxygen sensitive. The THF- and ATP-dependent activity measurable in the soluble fraction of cell extracts constituted about 34% of the activity seen with intact cells.     

SDS-PAGE characterization of the proteins in equine seminal plasma

The aims of this project were to document the protein profile of equine seminal plasma and determine the variability between stallions in the relative composition of proteins in the ejaculate. A single ejaculate was obtained from 14 stallions of varying breed and age. The gel fraction was removed by an in-line filter. The semen was centrifuged and the supernatant seminal plasma aspirated without disturbing the sperm pellet. The seminal plasma was recentrifuged and stored in cryovials at -70 degrees C. Samples were thawed, recentrifuged, assayed for protein concentration (BCA protein assay), divided into aliquots, then stored at -70 degrees C. A standard protein concentration of 50 microg was loaded in each 10 microl sample. SDS-PAGE was performed using 15% polyacrylamide and a mixture of molecular weight standards. The electrophoresed gel was stained for proteins with Coomassie blue, air-dried, then scanned by a megapixel camera interfaced to a computer. Image analysis software calculated integrated optical density (IOD) values for each lane, and bands within a lane. Each band IOD was expressed as a percentage of the total lane IOD, thus reflecting the relative concentration of each protein within the ejaculate. A total of 14 bands were identified, ranging from a large 120 kDa protein down to a small 14 kDa protein. No sample contained all 14 protein bands. Seven protein bands (101 kDa, 32 kDa, 26 kDa, 22 kDa, 18 kDa, 16 kDa, 14 kDa) were present in all samples, however the relative concentrations of protein within those bands varied between stallions. We demonstrated that although there is a characteristic equine seminal plasma protein profile on SDS-PAGE gels, there is between stallion variability in the relative amounts of each protein.     

Testicular prosthesis in a Quarterhorse stallion: a case report

A testicular prosthesis was surgically removed from the scrotum of a Quarterhorse stallion presented for evaluation of a large, firm, unilateral scrotal mass. The prosthesis was constructed from methyl methacrylate moulded around a roll of fiberglass casting tape. The prosthesis had been surgically implanted in the scrotum approximately 4 yr prior to presentation in order to give the appearance of 2 testicles in the scrotum for showing and breeding purposes. The horse had been used to successfully breed mares prior to presentation and produced 4.046 x 10(9) progressively motile, morphologically normal spermatozoa in an ejaculate collected 4 mo after surgery to remove the prosthesis. Ethical issues raised by this case are discussed.     

Two-dimensional polyacrylamide gel electrophoresis of bovine semen after cryopreservation in half-milliliter straws

One of the problems encountered with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the streaking of proteins so that individual spot identification is compromised. This study was conducted to determine whether a low loading dose (50 microg) of protein would permit resolution of more discrete protein spots using megapixel camera technology, and if so, to present a nomenclature for future comparisons of the identified proteins. If the major proteins could be identified in a 50-microg sample we aimed to determine whether they could be identified in the supernatant (seminal plasma plus extender) of cryopreserved semen. Two ejaculates were obtained from each of 6 bulls and bovine seminal plasma (BSP) protein concentration was standardized to 50 microg/10 microl. Isoelectric points (pI) and molecular weights (MWt) of BSP proteins were determined by measuring spot mobility on 2-D PAGE (15% polyacrylamide). Three distinct protein spot constellations (a,b,c) could be readily seen by the naked eye and a faintly stained constellation "d" was identified by the megapixel camera. The image analysis software located 6 protein spots in both constellation "a" (MWt 26 kDa; pI 4.2 to 4.8) and "b" ( MWt 27 kDa; pI 6.6 to 8.0). Constellation "c" contained 13 protein spots distributed in a right-angled triangle with its base towards the acidic end of the gel (MWt 14.7 to 18.8 kDa; pI 5.3 to 7.4). Only spots c(2), c(3), c(5), c(8), and c(13) were present in all 12 samples. Streaking can be eliminated by using 50 microg protein for 2-D PAGE, and the major protein spots are readily identified by megapixel camera technology. Protein spots c(3), c(5), c(13) and constellation "a" appear to correspond with Manjunath's proteins (BSP-A(1), -A(2); -A(3); -30 kDa). Killian's 2 low fertility proteins may lie in the "c" constellation, and 1 of the high fertility proteins may lie in the "b" constellation. The 3 major BSP proteins can be visualized in the supernatant of cryopreserved semen. We believe that the technique may prove useful for retrospective analysis of processed semen batches that achieve less than satisfactory results in the field.     

Seasonal movements of river dolphins (Inia geoffrensis) in a protected Amazonian floodplain

Deliberate killing for use as bait in a regional catfish (Calophysus macropterus) fishery is the primary threat affecting the survival of the Amazon river dolphin, or boto (Inia geoffrensis). Establishing and improving freshwater protected areas has been suggested as a possible course of action to protect the species. However, the ecology of the boto is poorly understood and more information is needed on the species’ habitat use and movement patterns to ensure that spatial protection initiatives meet conservation needs. In this study, mark‐recapture/resight data collected in and near the Mamirauá Sustainable Development Reserve, a várzea in the Brazilian Amazon, was used to examine transition probabilities of botos between various habitat types. Our findings suggest that it is imperative that spatial protection initiatives focused on the boto take into account the importance of várzea habitat and bay systems for individuals in early life stages. Moreover, because botos frequently occupy the main rivers, particularly during the low water period, protecting areas of the main river waters adjacent to várzeas could aid in the protection of hunted populations.     

Specific foetal growth rates of cetaceans

The numerous data in the International Whaling Statistics relating foetal length to date of catch have been studied. They can be used as alternatives to cube roots of foetal weights to give rates of foetal growth and for the computation of data of conception and foetal ages. There are major differences between the Mysticeti and Odontoceti. The main gestation period for the former is 10.8.±1-2 months and for the latter 14 ± 2 months. In both groups the greatest birth-weight is attained by an increased foetal growth rate, to such an extent in the rorquals that the gestation period in the largest is actually shorter than in the smaller species. There are dietetic and metabolic differences between the two sub-cohorts. The outstanding one is that pregnant mysticetes appear to stop feeding and lose body fat to the foetus during late pregnancy, at the time when the foetal increase in size is maximal. The causes of the differences in growth and metabolism of the two groups are unknown, apart from those imposed by differences in their ecology.     

Anaerobic c(1) metabolism of the o-methyl-C-labeled substituent of vanillate

The O-methyl substituents of aromatic compounds constitute a C(1) growth substrate for a number of taxonomically diverse anaerobic acetogens. In this study, strain TH-001, an O-demethylating obligate anaerobe, was chosen to represent this physiological group, and the carbon flow when cells were grown on O-methyl substituents as a C(1) substrate was determined by C radiotracer techniques. O-[methyl-C]vanillate (4-hydroxy-3-methoxy-benzoate) was used as the labeled C(1) substrate. The data showed that for every O-methyl carbon converted to [C]acetate, two were oxidized to CO(2). Quantitation of the carbon recovered in the two products, acetate and CO(2), indicated that acetate was formed in part by the fixation of unlabeled CO(2). The specific activity of C in acetate was 70% of that in the O-methyl substrate, suggesting that only one carbon of acetate was derived from the O-methyl group. Thus, it is postulated that the carboxyl carbon of the product acetate is derived from CO(2) and the methyl carbon is derived from the O-methyl substituent of vanillate. The metabolism of O-[methyl-C]vanillate by strain TH-001 can be described as follows: 3CH(3)OC(7)H(5)O(3) + CO(2) + 4H(2)O --> CH(3)COOH + 2CO(2) + 10H + 10e + 3HOC(7)H(5)O(3).     

A mathematical description of pressures in alveolar pores of Kohn

Small interalveolar holes within the lung are called pores of Kohn. Some researchers have correlated enlarged pore size with diseases, e.g. emphysema, that are characterized by tissue destruction. Mathematical models of the pressures generated in closed, fluid-filled and open, fluid-lined pores demonstrate that pressures capable of rupturing lung tissue can be developed in a pore due to the surface tension and shape of the air-liquid interface. Pore enlargement accompanied by tissue destruction is presented as a possible mechanism for the disease process observed during aging and the development of emphysema in the lung.     

Dispersed growth of Streptomyces in liquid culture

Summary The study of the physiology of the filamentous bacterium Streptomyces is inhibited by its formation of mycelial pellets in liquid cultures. It is demonstrated that dispersed growth may be achieved by the addition of polymers to the culture medium. Uncharged polymers, such as polyethylene glycol, are relatively ineffective but polyanions such as agar, Carbopol and Junlon produce dispersed cultures when included in a defined growth medium at low concentrations. Junlon-containing media enable optical density measurements to be used to follow batch growth of Streptomyces. Improvements in both biomass yield and product yield of the pigmented antibiotic actinorhodin were found to result from the incorporation of Junlon into minimal medium.     

Growth and reproduction ofPorphyra columbina Mont. (Bangiales,Rhodophyceae) from southern New Zealand

Changes in biomass and chemical composition, and the reproductive phenology ofPorphyra columbina Mont. were monitored at three sites in southern New Zealand over two growing seasons. Both temporal and spatial variations were found. Seasonal changes in biomass and chemical components were correlated with seawater nitrate concentrations and temperature. The summer decline in biomass was a result of the onset of unsuitable environmental conditions and the release of reproductive tissue. Under more suitable conditions, the decline in biomass was delayed. There was an inverse relationship between vegetative growth and reproduction. Reproductive plants first appeared in August at a time of increasing temperature, irradiance and daylength. Only larger plants which were mainly found in subsites low on the shore became reproductive. Plants sampled from high subsites had a shorter growth season, were generally smaller, had lower nitrogen and pigment content and were non-reproductive.Presented at the XIIIth International Seaweed Symposium, University of British Columbia, Vancouver, Canada, August 1989.