The Feulgen Reaction after Glutaraldehyde Fixation

A technique is described for performing the Feulgen reaction for DNA on cells and tissues fixed in glutaraldehyde. Blockade free aldehydes by reducing them with fresh 0.5% NaBH₄ in 1% NaH₂PO₄ for 1 hr at room temperature, then rinse in water. Follow by a Feulgen reaction (hydrolysis at room temperature in 6 N HCI for 20 min, Schiff's reagent for 60 min.). Controls assure the completeness and irreversibility of the borohydride blockade. Cytophotometry shows that the DNA content per nucleus is unaffected by the blockade procedure.     

Possible common origin of glucosyltransferases in Oscillatoria princeps

Gel electrophoresis of the glucosyltransferases of the blue-green alga, Oscillatoria princeps, followed by immunodiffusion against anti-phosphorylase rabbit serum, showed cross-reactions of the two phosphorylase isozymes and the two synthetase isozymes of the alga. Weak cross-reactions were also obtained with the branching isozymes. Apparent extensive similarities in the structure of the phosphorylases and the synthetases were indicated. However, only partial structural similarities between the two groups of α-1,4-glucosidic bond formers and the branching isozymes exist as indicated by the weak immunological reactions obtained and the formation of “spurs” on the immunoprecipitin lines. If the synthesis of α-1,4-glucosidic linkages and the formation of α-1,6 cross linkages were at one time due to the bifunctional action of a single catalytic protein, then the separation of these two enzymatic activities took place prior to the derivation of the synthetases from the phosphorylases.     

Changes in the host natural killer cell population in mice during tumor development. 2. The mechanism of suppression of NK activity

Our earlier studies revealed that a rapid and progressive loss of splenic NK activity in mice during the development of a number of transplanted tumors as well as of spontaneous tumors was due to an inactivation of natural killer (NK) lineage cells rather than to their disappearance. The mechanism of this inactivation have now been explored in CBA/J mice receiving transplanted Ehrlich ascites tumors and in C3H/HeJ mice bearing spontaneous mammary tumors or receiving transplants of syngeneic mammary tumor lines of recent origin. A poor activation state or maturation arrest of NK lineage cells due to a low interferon level in vivo was ruled out, since the host NK activity could not be restored after administration of either an interferon inducer poly(I:C) or interferon-alpha, although such treatments enhanced the activity in tumor-free mice by four- to eightfold. Possible presence of host suppressor cells acting on the effector or preeffector stage of NK cells was explored by mixing spleen cells from tumor bearers with normal spleen cells either during the NK assay, or for a 20-hr period of in vitro short-term culture prior to the NK assay. Mixing during the NK assay led to a reduction of NK activity explicable by a simple dilution of active NK cell concentration rather a suppression of active NK cells. On the other hand, a 20-hr coculture of the mixed population at various ratios led to a complete abrogation of the NK activity, indicating that the suppressor cells acted on the preeffector stage of the NK Lineage. A further characterization of suppressor cells revealed that they were (1) contained in the adherent fraction of the spleen of tumor bearers, (2) of monocyte/macrophage morphology, (3) capable of phagocytosing latex particles, and (4) positive for surface Mac-1 antigen, as noted from a radioautographic binding of 125I-labeled monoclonal anti-Mac-1 antibody. The mechanism of the suppression was identified, at least in part, as being mediated by prostaglandin-like molecules, since the presence of indomethacin, a prostaglandin-inhibitor, during the 20-hr coculture period completely abrogated the suppression. Indomethacin exerted no direct effect on the recruitment or killer activity of NK lineage cells in vitro. NK cell suppression may be another normal immunoregulatory mechanism which alters the host-tumor balance in favor of the tumor rather than the host.     

In situ localization and characterization of bone marrow-derived cells in the decidua of normal murine pregnancy.

The study reported here was designed to examine the in situ distribution and characteristics of hemopoietically derived decidual cells during normal pregnancy in mice prenatally reconstituted with bone marrow cells carrying a transgenic marker. Bone marrow cells from a transgenic CD-1 strain (CD-1 beta; carrying 1000 copies of beta-globin genes in tandem) were injected into the yolk sac of Day 17 conventional CD-1 embryos. The pregnant females were allowed to deliver normally, and the female offspring raised to puberty were mated with CD-1 males and then killed on Day 12 of gestation. The extent of chimerism in sections of their spleens, uteri, and other organs was evaluated by in situ hybridization of the sections with a biotinylated cDNA probe specific for the beta-globin genes followed by avidin-biotin-peroxidase staining. Tissue controls were provided by CD-1 beta and CD-1 mice, respectively. Tissues were also processed without the application of the probe or with the application of biotinylated lambda DNA as specificity controls. Reconstituted mice exhibited variable degrees of hemopoietic chimerism as indicated by labeling of their splenic lymphocytes (18-54%; mean 42%) as well as hemopoietic cells in other organs. Variable cellular labeling was also noted in their decidua basalis and metrial glands. Labeled cells in these tissues were identified as typical decidual cells, macrophages, and granulated metrial gland (GMG) cells. Labeling of typical decidual cells varied extensively among implantation sites in the same chimera, the average labeling ranging from 17% to 33% (mean 24%) in various chimeras. Labeling was also noted in GMG cells, lymphocytes, and some decidual cells migrating out of metrial gland explants after 24-h culture. The non-pregnant uterus of a chimeric mouse revealed significant labeling of endometrial stromal cells indicative of their hemopoietic origin. These results revealed a hemopoietic origin of certain typical decidual cells and GMG cells identified in situ during normal murine pregnancy and a hemopoietic origin of certain endometrial stromal cells that may represent precursors of decidual cells. The precise timing of the predecidual stem cell migration from the bone marrow to the uterus remains to be defined.     

Evaluation of population status and foraging ecology of Sable antelope (Hippotragus niger roosevelti,Heller, 1910) in Shimba Hills National Reserve,Kenya

The Sable antelope (Hippotragus niger roosevelti, Heller, 1910) is nationally endemic to Shimba Hills National Reserve (SHNR) in Kenya. In the past few decades, its population has declined considerably. Despite the alarming decline and resultant localised distribution, a little information exists on the species population status and foraging ecology. Different ecological research techniques were used to collect relevant data and information on the species population structure and feeding habits. The results showed that Sable population comprised of sex and age structures that are skewed towards females and adult, respectively, whereas young and subadult populations were not significantly different. Seasonal change did not have significant influence on the diversity of food plants selected by Sable. Although crude protein and phosphorous levels in Sable faecal samples differed significantly between the seasons, they were within the recommended minimum maintenance requirements for wild herbivores. The study concludes that Sable has good survival rate and potential to breed but lacks stability in the population. Additionally, Sable forage quality and availability may not be limiting its population growth. There is need to establish management strategies for improving reproduction in Sable and understand the level of competition of the species with other mega herbivores in SHNR.     

Design and Analysis of a Mutant form of the Ice-Binding Protein from Choristoneura fumiferana

The Protein Journal - Ice-binding proteins are expressed in the cells of some cold adapted organisms, helping them to survive at extremely low temperatures. One of the problems in studying such...     

Depth-dependent photolabelling of membrane hydrophobic core with 9-diazofluorene-2-butyric acid

Hydrophobic photoactivable reagents, which readily partition into membranes, have proved very useful for studying membrane hydrophobic core. These reagents have been linked to fatty acids in order to obtain amphipathic photoactivable reagents which label membranes more effectively. By varying the length of these amphipathic reagents, an attempt to label membrane hydrophobic core at different depths can be made. We report here 9-diazofluorene-2-butyric acid as a new photoactivable reagent which labels the single bilayer vesicles prepared from egg phosphatidylcholine. The labelling site on the fatty acyl chains could be traced to be between the carbon atom 4 and 6. The new probe thus labels the membrane at a site which is proximal to what can be predicted from its length and transverse location in membranes.