Cryopreservation and post-thawed fertility of ram semen frozen in different trehalose concentrations

We evaluated freeze-thawing tolerance of heterospermic ram spermatozoa (Pampinta breed) in a base diluent (Tris, citric acid, fructose, egg yolk, glycerol) with the addition of different trehalose concentrations (0-400 mOsm). We chose sperm motility, acrosome integrity and hypo-osmotic swelling test as parameters to evaluate cryopreservation capacity. We obtained the best results for 50 and 100 mOsm trehalose-supplemented extenders, with values (referred to fresh semen values) of 65% for motility, 75% for acrosome integrity and 50% for hypo-osmotic swelling test, while freeze-thawing tolerance diminished significantly for 200 and 400 mOsm of the disaccharide. Fertility values measured at lambing were 47.1 and 44.6% (2 consecutive years), using semen cryopreserved in 100 mOsm trehalose-containing diluent, which is 2.5 times greater than those obtained with the base diluent (18.5 and 14.5%). We conclude that the membrane-protecting disaccharide trehalose confers a greater cryoprotective capacity to the base extender, when added up to 100 mOsm. This action is reflected in the different sperm membranes, the motile activity and in vivo fertility.     

Function and cytochemical characteristics of postural limb muscles of the rhesus monkey: A telemetered EMG and immunofluorescence study

Using telemetered electromyography and immunocytochemical fibre typing (of both fresh frozen and preserved specimens), the present paper demonstrates clearly that at the elbow, knee, and ankle joints, the rhesus monkey (Macaca mulatta) is endowed with one extensor-muscle head specialized for posture. These postural heads are distinguished by (a) recruitment at low levels to maintain joint position against the effect of gravity, (b) recruitment near maximum levels during walking, and (c) high content and relatively large size of slow, fatigue-resistant (type I) muscle fibres. The nonpostural heads of the investigated muscles were recruited at levels correlated to the strenuousness of the effort and are notable by the small percentage and size of slow muscle fibres. The postmortem stability of the structural properties of myosin makes immunocytochemical fibre typing suitable for the study of preserved cadavers.     

In vitro study of proteolytic degradation of rat histidine decarboxylase.

Mammalian ornithine decarboxylase (ODC) is a very unstable protein which is degraded in an ATP-dependent manner by proteasome 26S, after making contact with the regulatory protein antizyme. PEST regions are sequences described as signals for protein degradation. The C-terminal PEST region of mammalian ODC is essential for its degradation by proteasome 26S. Mammalian histidine decarboxylase (HDC) is also a short-lived protein. The full primary sequence of mammalian HDC contains PEST-regions at both the N- and C-termini. Rat ODC and different truncated and full versions of rat HDC were expressed in vitro. In vitro degradation of rat ODC and rat 1-512 HDC were compared. Like ODC, rat 1-512 HDC is degraded mainly by an ATP-dependent mechanism. However, antizyme has no effect on the degradation of 1-512 HDC. The use of the inhibitors MG-132 and lactacystine significantly inhibited the degradation of 1-512 HDC, suggesting that a ubiquitin-dependent, proteasome 26S proteolytic pathway is involved. Results obtained with the different modifications of rat HDC containing all three PEST regions (full version, 1-656 HDC), only the N-terminal PEST region (1-512 HDC), or no PEST region (69-512 HDC), indicate that the N-terminal (1-69) fragment, but not the C-terminal fragment, determines that the HDC protein is a proteasome substrate in vitro.     

Stretch-activated ion channels modulate the resting membrane potential during early embryogenesis

By using the patch-clamp technique, stretch-activated ionic channels were found in the membrane of cleaving freshwater fish embryos at the early stages of embryogenesis (2-256 cells). The application of negative pressure to the pipette increased the frequency of activation and the duration of bursts. This type of channel has a preferential K+ selectivity. When bathed on both membrane surfaces with 140 mM KCl the channel conductance was 71 pS. The kinetic behaviour did not depend markedly on either membrane potential (in the range from -70 to +70 mV) or calcium concentration on the cytoplasmic side of the membrane. On continuous recording, the probability of the channel being open was found to change periodically over a 5- to 20-fold range for different cells. These variations correlated with changes in resting potential and membrane conductance during the cell cycle. These results suggest that the oscillation of resting potential within the cell cycle is associated with the operation of stretch-activated ion channels.     

Complexation of Lead by Bermuda Grass Root Exudates in Aqueous Media

Exudates produced from Bermuda grass roots were collected in deionized water from sterilized Bermuda grass sod at 3-day intervals over a period of 15 days. Exudates were analyzed for total organic carbon, and characterized via Fourier Transform Infrared Spectroscopy. Exudate samples were adjusted to pH values of 4.5, 6.5, and 7.5, amended with lead and quantified for soluble and complexed lead via Inductively Coupled Plasma—Optical Emission Spectrometry. Data obtained from total organic carbon measurements indicated compositional changes in Bermuda grass root exudates as organic carbon concentrations increased over time. Analysis of the infrared spectroscopy data indicated that carboxylic acids and amine functional groups were present in root exudates. Also, the ability of root-exuded compounds to solubilize lead in aqueous media was demonstrated as exudate samples dissolved an average of 60% more lead than deionized water. At pH values 4.5 and 7.5, lead complexation by Bermuda grass root exudates increased with decreasing molecular weight size fractions, while an opposite trend was observed at pH 6.5. Results from this study demonstrated the ability of Bermuda grass root exudates to complex lead in aqueous media.