A Simple, Inexpensive Metabolism Cage for Small Mammals

By utilizing ordinary laboratory equipment and a spherical feces-urine separator, a simple, inexpensive metabolism cage for small mammals can be constructed. A hardware cloth animal cage over a cylindrical battery jar containing a spherical feces-urine separator affords the following advantages not commonly found in commercial metabolism cages: 1) complete separation of feces and urine through minimal contact, 2) minimal evaporation of urine due to proximity of storage vessel and lack of exogenous air currents, and 3) extremely low cost of less than five dollars. The metabolism cage is designed to allow measurement of fluid intake, and to separate and collect feces and urine for numerous qualitative and quantitative determinations. In addition, the metabolism cage permits observation of the animal, feces, and urine at all times, is readily cleaned or sterilized, and is easily fashioned from common laboratory equipment.     

Effects of pH conditions on Ca2+ transport catalyzed by ionophores A23187, 4-BrA23187, and ionomycin suggest problems with common applications of these compounds in biological systems.

Phospholipid vesicles loaded with Quin-2 and 2'',7''-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) have been used to investigate the effects of pH conditions on Ca2+ transport catalyzed by ionophores A23187, 4-BrA23187, and ionomycin. At an external pH of 7.0, a delta pH (inside basic) of 0.4-0.6 U decreases the rate of Ca2+ transport into the vesicles by severalfold under some conditions. The apparent extent of transport is also decreased. In contrast, raising the pH by 0.4-0.6 U in the absence of a delta pH increases both of these parameters, although by smaller factors. The relatively large effects of a delta pH on the transport properties of Ca2+ ionophores seem to reflect a partial equilibration of the transmembrane ionophore distribution with the H+ concentration gradient across the vesicle membrane. This unequal distribution of ionophore can cause a very slow or incomplete ionophore-dependent equilibration of delta pCa with delta pH. A second factor of less certain origin retards full equilibration of delta pCa when delta pH = 0. These findings call into question several ionophore-based methods that are used to investigate the regulatory activities of Ca2+ and other divalent cations in biological systems. Notable among these are the null-point titration method for determining the concentration of free cations within cells and the use of ionophores plus external cation buffers to calibrate intracellular cation indicators. The present findings also indicate that the transport mode of Ca2+ ionophores is more strictly electroneutral than was thought, based upon previous studies.     

Structural basis for differential receptor binding of cholera and Escherichia coli heat-labile toxins: influence of heterologous amino acid substitutions in the cholera B-subunit

The closely related B-subunits of cholera toxin (CTB) and Escherichia coli heat-labile enterotoxin (LTB) both bind strongly to GM1 ganglioside receptors but LTB can also bind to additional glycolipids and glycoproteins. A number of mutant CT B-subunits were generated by substituting CTB amino acids with those at the corresponding positions in LTB. These were used to investigate the influence of specific residues on receptor-binding specificity. A mutated CTB protein containing the first 25 residues of LTB in combination with LTB residues at positions 94 and 95, bound to the same extent as native LTB to both delipidized rabbit intestinal cell membranes, complex glycosphingolipids (polyglycosylceramides) and neolactotetraosylceramide, but not to non-GM1 intestinal glycosphingolipids. In contrast, when LTB amino acid substitutions in the 1–25 region were combined with those in the 75–83 region, a binding as strong as that of LTB to intestinal glycosphingolipids was observed. In addition, a mutant LTB with a single Gly-33→Asp substitution that completely lacked affinity for both GM1 and non-GM1 glycosphingolipids could still bind to receptors in the intestinal cell membranes and to polyglycosylceramides. We conclude that the extra, non-GM1 receptors for LTB consist of both sialylated and non-sialylated glycoconjugates, and that the binding to either class of receptors is influenced by different amino acid residues within the protein.     

TPO1, a Member of a Novel Protein Family, Is Developmentally Regulated in Cultured Oligodendrocytes

Abstract: Although the myelin membrane contains only a small set of major proteins, more sensitive assays indicate the presence of a plethora of uncharacterized proteins. We have used an antibody perturbation approach to reversibly block the differentiation of prooligodendroblasts into myelinating cells, and, in combination with a differential screening procedure, identified novel mRNAs that are activated during this period. One cDNA, TPO1, recognizes a 5.5-kb mRNA that is strongly up-regulated in oligodendrocytes after release of the differentiation block and that is expressed at high levels in brain tissue during active myelination. This cDNA represents at least two mRNAs differing from each other in their 5'-termini. The TPO1 cDNA contains an open reading frame of 1,380 bp, encoding a protein of 51.8 kDa with a predicted pl of 9.1 that contains two regions homologous to nonclassic zinc finger motifs. Subcellular localization studies suggest the enriched presence of TPO1 in spherical structures along the major cytoplasmic processes of oligodendrocytes. TPO1, along with homologues expressed in testis, placenta, and PC12 cells, form a novel family of proteins with multiple hydrophobic domains possibly serving as membrane spanning regions. We postulate that in oligodendrocytes, TPO1 encodes a protein factor involved in myelin biogenesis.     

Comparison of the killer toxin of several yeasts and the purification of a toxin of type K2

A total of 13 killer toxin producing strains belonging to the genera Saccharomyces, Candida and Pichia were tested against each other and against a sensitive yeast strain. Based on the activity of the toxins 4 different toxins of Saccharomyces cerevisiae, 2 different toxins of Pichia and one toxin of Candida were recognized. The culture filtrate of Pichia and Candida showed a much smaller activity than the strains of Saccharomyces. Extracellular killer toxins of 3 types of Saccharomyces were concentrated and partially purified. The pH optimum and the isoelectric point were determined. The killer toxins of S. cerevisiae strain NCYC 738, strain 399 and strain 28 were glycoproteins and had a molecular weight of M_r=16,000. The amino acid composition of the toxin type K₂ of S. cerevisiae strain 399 was determined and compared with the composition of two other toxins.     

Effect of bombesin upon plasma somatostatin-like immunoreactivity, insulin and glucagon in normal and chemically sympathectomized dogs

The present study was designed to determine the effects of intravenously infused bombesin (10 ng/kg/min) upon basal and postprandial plasma somatostatin-like immunoreactivity (SLI), glucagon, insulin and triglyceride levels in normal (n = 12) and chemically sympathectomized (n = 11) dogs. Basal plasma SLI, glucagon and insulin levels rose significantly during the infusion of bombesin in the normal dogs, and this was not altered by chemical sympathectomy. Bombesin infusion enhanced the postprandial SLI response, while attenuating the postprandial glucagon response by 50% and the insulin response in the early postprandial phase of the meal. Sympathectomy did not significantly alter the basal levels of these polypeptides, but augmented the postprandial plasma SLI response during the first 90 min, and reduced the postprandial glucagon response during the infusion of bombesin. The postprandial insulin response was not affected by sympathectomy. In both normal and chemically sympathectomized dogs the rise in postprandial triglyceride levels was attenuated by bombesin infusion.     

Stem cells and immunological parameters in mice during the latency period and after the development of chemically induced leukemia

T-cell leukemias have been induced in adult BDF1 mice by 12 or 15 weeks of exposure to butylnitrosourea (BNU) in the drinking water. This led to a depression of CFU-S numbers and reduced T- and B-cell responses to mitogens. These parameters were then studied during the BNU-free preleukemic latency period in individual mice. At the same time, leukemic cells were traced in the thymus, the spleen, and the bone marrow by transplantation. In mice without leukemia and mice with leukemic cells in only one organ, there was a general tendency to normal CFU-S numbers and T- and B-cell responses with time after BNU, although control levels were reached in only a few of the mice. The reaction of mixed lymphocyte cultures (MLC) remained low during the latency period. In the thymus an imbalance of the Con A, PHA, and MLC responses was observed. Out of 25 mice with induced leukemia, 8 had leukemic cells in the thymus only and 2 in the marrow only. In mice with leukemic cells in all 3 hemopoietic organs and an enlargement of the spleen, a shift of CFU-S from the marrow to the spleen was observed.     

Combination of microdialysis and glucosensor permits continuous (on line) SC glucose monitoring in a patient operated device. II. Evaluation in animals.

The microdialysis technique was used for following the glucose content of the extracellular subcutaneous (SC) fluid under varying blood glucose levels in rats. The glucose content in the microdialysis perfusion fluid was continuously analyzed by means of the measuring flow chamber of an ex vivo glucose monitor. In six ChBB rats blood glucose levels were varied between 40 mg/dl and 575 mg/dl by intravenous (IV) infusion of glucose and by SC injections of insulin, respectively. After a running-in period of about half an hour, the glucose content in the perfusion fluid was closely related to the blood glucose concentration (r > 0.92) up to a time period of 6 hrs. The "relative recovery" rate of glucose by the microdialysis probe in the SC tissue varied within the 6 experimental sessions. The relative recovery rate could be shown to be not dependent on the absolute blood glucose levels in the individual rat within the glucose concentration range tested.     

Socially stimulated androgen surges in male hamsters: the roles of vaginal secretions, behavioral interactions, and housing conditions.

The sources of cues necessary for elicitation of androgen surges and sexual behavior in male golden hamsters (Mesocricetus auratus) were investigated. Circulating androgen levels were measured in males after interactions with other males or several types of estrous females: intact females, vaginectomized females, or vaginectomized females scented with vaginal secretions. All groups of males that interacted with estrous females demonstrated significant elevations in androgens whereas those that interacted with other males did not. Thus, the presence of vaginal secretions is not necessary for the elicitation of androgen surges in sexually experienced male hamsters. Individual differences in sexual performance were not correlated with the degree of change in androgen levels, suggesting that such hormonal responses are not graded but are all-or-none. Housing males in isolation from females did not alter either baseline androgen levels or the magnitude of androgen responses caused by interactions with females.