Native Enzyme Mobility Shift Assay (NEMSA): a new method for monitoring carboxyl group modification of carboxymethylcellulase from Aspergillus niger

A simple, sensitive, accurate and more informative assay for determining the number of modified groups during the course of carboxyl group modification is described. Monomeric carboxymethylcellulase (CMCase) from Aspergillus niger was modified by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide. The different time-course aliquots were subjected to non-denaturing PAGE and the gel stained for CMCase activity. The number of carboxyl groups modified are directly read from the ladder of the enzyme bands developed at given time. This method showed that after 75 min of modification reaction there were five major species of modified CMCases in which 6 to 10 carboxyls were modified.     

Effect of perfusate pH on coronary flow and adenosine release in isolated rabbit heart

This study was undertaken in an attempt to further understand the relationship between adenosine and H+ ion. Using Langendorff hearts from male rabbits, the perfusion fluid pH was lowered from 7.4 to 7.1 and 6.8 with CO2. A 31 and 86% increase in coronary flow with a simultaneous increase in the release of adenosine by 61 and 128% was observed at pH 7.1 and 6.8, respectively. A direct relationship between adenosine release and coronary flow with a correlation coefficient of 0.99 was found at pH values of 7.4, 7.1, and 6.8. The degradation products of adenosine namely inosine and hypoxanthine were unchanged at 7.1 and 6.8 from 7.4. These data support a role for adenosine in the regulation of coronary flow and suggest a relationship between adenosine and H+ ion.     

Variation in the free amino acid content of skeletal muscle of Ophicephalus punctatus (Bloch)

The skeletal muscle of Ophicephalus punctatus contains nine essential free amino acids, arginine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, valine and lysine, and eight non-essential amino acids, alanine, aspartic acid, cystine, glutamic acid, glycine, tyrosine, proline and serine. Histidine and lysine dominated the free amino acids pool. Seasonal variation was detected in the levels of histidine, arginine, leucine, phenylalanine, glycine, cystine and serine with highest values occurring in April and again in November. Changes were also detected in the concentrations of certain amino acids as the fish grew in size. Levels of free amino acids did not significantly differ between sexes. Factors effecting variation are discussed.     

Reproductive biology, length-weight relationship and relative condition of Tilapia leucosticta (Trewavas) in Lake Naivasha, Kenya

In Lake Naivasha Tilapia leucosticta (Trewavas) spawns throughout the year. Males attain maturity at 180 mm and females at 160 mm total length. Males predominate over females in the catches (sex ratio 1-00 : 0-51). Fecundity ranged from 320 to 1328 eggs with an average of 751. The relationship between fecundity (F) and total length (L), body weight (W) and gonad weight (GW) is:
The length-weight regression coefficient is near to the cube. There is little seasonal fluctuation in relative condition of the fishes.     

Differential uptake of liposomes varying in size and lipid composition by parenchymal and kupffer cells of mouse liver

Using liposomes differing in size and lipid composition, we have studied the uptake characteristics of the liver parenchymal and Kupffer cells. Desferal labeled with iron-59 was chosen as a radiomarker for the liposomal content, because Desferal in its free form does not cross cellular membranes. At various time intervals after an intravenous injection of liposomes into mice, the liver was perfused with collagenase, and the cells were separated in a Percoll gradient. It was found that large multilamellar liposomes (diameter of about 0.5 μm) were mainly taken up by the Kupffer cells. For these large liposomes, the rate of uptake by Kupffer cells was rapid, with maximum uptake at around 2 hours after liposome injection. Unexpectedly, small unilamellar liposomes (diameter of about 0.08 μm) were less effectively taken up by Kupffer cells, and the rate of uptake was slow, with a maximum uptake at about 10 hours after liposome injection. In contrast, parenchymal cells were more effective in taking up small liposomes and the uptake of large liposomes was negligible. In addition, liposomes made with a galactolipid as part of the lipid constituents appeared to have higher affinity to parenchymal cells than liposomes made without the galactolipid. These findings should be of importance in designing suitable liposomes for drug targeting.     

Effects of decreased glutathione peroxidase activity on the pentose phosphate cycle in mouse lung

The effects of reducing glutathione peroxidase activity in the lung by changing dietary selenium intake has been investigated. In animals that were exposed to room air, selenium effects were confined to glutathione peroxidase activity, whereas under conditions of oxidant stress (ozone) the decrease in glutathione peroxidase activity prevented the stimulation of the pentose phosphate cycle (assayed by measuring glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities) which has been reported to increase in response to oxidant stress. The suppression of glutathione peroxidase activity was found to depend on dietary selenium concentration. The physiological significance of this observation may be related to the process of injury and repair in the lung.     

Evidence of synergy between Thy-1 and CD3/TCR complex in signal delivery to murine thymocytes for cell death.

The potential role of Thy-1 in CD3/TCR complex-mediated signal delivery to murine thymocytes was studied. Ag-mimicking cross-linked anti-CD3 mAb stimulated suspension of thymocytes from adult (6 to 8 wk old) mice for a brisk free cytoplasmic calcium ion ([Ca2+]i) rise, low level of inositol phosphate production, and marginal increase in tyrosine-specific phosphorylation of 110/120-kDa and 40-kDa cellular proteins. Weak but sustained [Ca2+]i rise, low inositol phosphate production, and weak protein tyrosine phosphorylation were also induced by the cross-linked anti-Thy-1 mAb that mimicked the putative natural ligand. The signal delivered via either of these two pathways was however insufficient for definitively promoting cell death and DNA fragmentation in the adult thymocytes. Here we demonstrated that anti-Thy-1 mAb synergized with anti-CD3 mAb for inducing a long-lasting prominent [Ca2+]i rise, definite inositol 1,4,5-triphosphate and inositol 1,3,4,5-tetrakiphosphate production, and extensive tyrosine-specific phosphorylation of 110/120-, 92-, 75-, and 40-kDa proteins, which resulted in marked promotion of cell death and DNA fragmentation in the adult thymocytes. This unique anti-Thy-1 antibody activity was confirmed to be directed to glycosylphosphatidylinositol-anchored Thy-1, and was distinguished from the known anti-L3T4 activity that augmented the CD3-mediated signal transduction in a different manner. The synergistic actions of anti-CD3 and anti-Thy-1 mAb obligatorily required the cross-linking of the two mAb together. The anti-CD3 and anti-Thy-1 mAb cross-linked together acted on immature thymocytes from newborn (less than 24 h after birth) mice for rather more extensive promotion of protein tyrosine phosphorylation and cell death. In addition, they affected peripheral T lymphocytes for accelerating protein tyrosine phosphorylation but not cell death. These results suggest a novel function of glycosylphosphatidylinositol-anchored Thy-1 as a possible unique intrathymic intensifier of the CD3/TCR complex-delivered signal for negative thymocyte selection.     

Detection of Vibrio cholerae O1 in the aquatic environment by fluorescent-monoclonal antibody and culture methods.

Vibrio cholerae O1 in plankton samples collected from ponds and rivers between February 1987 and January 1990 in Matlab, Bangladesh, was detected by the fluorescent-monoclonal antibody (FA) technique. Samples were collected at sites which were monitored fortnightly (fixed sites) as well as at sites that were part of a case-control study. FA results were compared with those obtained by conventional culture methods (CM). A total of 876 samples were collected; V. cholerae O1 was detected in 563 samples (64.27%) by the FA method and in 3 samples (0.34%) by CM. Of the fixed-site plankton samples, 439 (63.62%) were positive by FA and none were positive by CM. Of the 93 case sites sampled on the day after the occurrence of a case of cholera, 73 (78.49%) were positive for V. cholerae O1 by FA and 3 (3.2%) were positive by CM. In comparison, of the 93 first-day sample collections at control sites at the time a case of cholera occurred, only 51 (54.83%) were positive by FA and none were positive by CM. From the data, it is concluded that V. cholerae O1 is present throughout the year in the ponds and rivers of Bangladesh that were examined in this study and that V. cholerae can be detected by FA but not always by CM. The FA procedure was found to be very useful in detecting V. cholerae in plankton, with which it was associated and often occurred in large numbers in the nonculturable stage. Thus, studies investigating the significance of the role of environmental factors in the epidemiology of cholera can be performed effectively by using FA. Such studies are in progress.     

A Technique for Quick Fixation of Whole Drosophila and Other Small Insects for Scanning Electron Microscopy

A procedure for fixing small insects in natural postures for scanning electron microscopy is reported. Anesthetized insects are partially restrained using a depression slide and a coverslip while preliminary fixation is carried out and wings and legs are positioned with a fine brush. Following this, fixation is completed and the insect is prepared for scanning electron microscopy by essentially standard procedures, which may include critical point drying. Figures illustrate, however, that critical point drying is not necessary for more rigid parts of the exoskeleton. Use of this procedure assures naturally disposed parts even when only a single specimen is available.