Discrete charge calculations of potentiometric titrations for globular proteins: sperm whale myoglobin, hemoglobin alpha chain, cytochrome c.

The modified Tanford-Kirkwood theory of Shire et al. for intramolecular electrostatic interactions has been applied to hydrogen ion equilibria of sperm whale ferrimyoglobin, human hemoglobin α-chain and horse cytochrome c. The model employs two sets of parameters derived from the crystalline protein structures, first, the atomic coordinates of charged amino acid residues and, second, static accessibility factors to reflect their solvent exposure. In addition, a consistent set of intrinsic pK values (pK_int) for the individual groups is employed. The theoretical pK values at half-titration for individual groups in each protein correspond to the available observed pK values, and the theoretical titration curves compare closely with experimental potentiometric curves.     

Effects of cytochalasin B on low-density lipoproteins metabolism by cultured human fibroblasts.

The role of cytoplasmic microfilaments in the metabolism of low-density lipoprotein by human fibroblasts was studied with the aid of cytochalasin B. At concentrations of 5--40 nmol/ml cytochalasin increased the surface binding but decreased the endocytosis of 125I-labelled low-density lipoprotein. Subsequent studies indicated that these changes reflected a reduction of the rate of internalisation of low-density lipoprotein receptors. Independent inhibitory effects were also observed on low-density lipoprotein degradation and on the cellular release of the trichloroacetic acid-soluble degradation products.     

Heterogeneity of human alpha-fetoprotein as revealed by isoelectric focusing in urea-containing gels.

The heterogeneity of human alpha-fetoprotein has been studied by analytical isoelectric focusing in polyacrylamide gel slabs in the presence of 8 M urea. Six major isoelectric variants could be identified over a pH range of 6.0--6.2. Verification of their identity was achieved by crossed immunoelectrophoresis into agarose gel containing monospecific antiserum to human alpha-fetoprotein. Complete desialylation of the protein did not abolish the heterogeneity; a complex pattern of major alpha-fetoprotein bands persisted over a more alkaline pH range. We have been able to correlate the pattern of alpha-fetoprotein heterogeneity seen following extended agarose gel electrophoresis with that obtained during isoelectric focusing in the presence of urea. The quantity of certain alpha-fetoprotein charge isomers in various alpha-fetoprotein isolates may be important in considering certain biological functions of this protein.     

Free proline content and sensitivity to desiccation and heat during yeast sporulation and spore germination.

Ascospores of a strain of Saccharomyces cerevisiae Hansen were less sensitive to desiccation and heat than vegetative cells. Desiccation resistance was acquired earlier during sporulation and lost later during spore germination than heat resistance. As spores matured, resistance to both stresses increased. With the exception of the first few hours in sporulation medium, when proline appeared to be utilized, the intracellular free proline content increased during sporulation and decreased during spore germination. Not all the proline lost could be detected in the germination medium, indicating that some was metabolically utilized by the germinating spores. Since exogenous proline supplied to vegetative or sporulating cells before desiccation increased their survival, it is suggested that the high level of free proline in mature spores may protect against desiccation stress.     

The protective capacities of fractionated immune thoracic duct lymphocytes against Nippostrongylus brasiliensis

Thoracic duct lymphocytes (TDL) obtained from rats either on the tenth day of a primary infection (Day 10 TDL) or 1 or 5 weeks after a tertiary infection (hyperimmune TDL) with Nippostrongylus brasiliensis were fractionated into cells lacking (sIg^?) or bearing (sIg⁺) surface immunoglobulin by a rosetting procedure. The abilities of unfractionated TDL, of the two subpopulations, and of the reconstituted cells to confer protection against the parasite were examined. The effector cells which cause worm expulsion were found only in (sIg^?) cells from Day 10 TDL and also predominantly in (sIg^?) cells from hyperimmune TDL. However, a small but significant degree of protection was conferred by (sIg⁺) cells from hyperimmune TDL. These results suggest that the mechanisms involved in worm expulsion are regulated by (sIg^?) cells but that (sIg⁺) cells from hyperimmune rats can also contribute to the mechanisms of worm expulsion.     

Protection against Nippostrongylus brasiliensis by adoptive immunization with immune thoracic duct lymphocytes

The protective capacities of different sources of immune lymphocytes against Nippostrongylus brasiliensis infection were examined. Thoracic duct lymphocytes (TDL) drained from donors on the tenth day of a primary infection (Day 10 TDL) conferred greater protection against adult worms established by larval infection than either mesenteric lymph node cells (MLNC) or TDL drained from hyperimmune donors. Day 10 TDL also conferred a high degree of protection against intraduodenally implanted “normal” and “damaged” worms. These results suggest that the different susceptibilities of “normal” and “damaged” worms to adoptive protection is a quantitative rather than a qualitative phenomenon. The results also emphasise that kinetic and dose-response experiments are important in evaluating the protective capacities of transferred cells.     

Thermoregulatory responses to temperature manipulation of the spinal cord in the marmot

Yellow-bellied marmots (M. flaviventris) acclimated to T_a = 20 °C were implanted with U-shaped polyethylene thermodes in the peridural space of the spinal cord. Decreasing the temperature of the cervical, thoracic, or lumbar areas of the cord increased heart rate, electromyographic activity, and oxygen consumption in the animals. These responses differed qualitatively from those elicited by heating the same cord areas, indicating specificity of the response to the temperature change.Increases in heat production were proportional to the amount of cooling of the cord. The thoracic area was found to be more thermosensitive than the lumbar area. No behavioral or physical thermoregulation was apparent when the spinal cord temperature was changed in these animals.In addition to the conclusion that regulation of spinal cord temperature may be important in the euthermic marmot, it was postulated that the temperature receptors located in the thoracic cord of the marmot may be important in maintaining shivering thermogenesis during arousal from hibernation.     

F9 embryonal carcinoma cells can differentiate into endoderm-like cells

The mouse teratocarcinoma cell line, F9, has been used in many laboratories as the epitome of the “nullipotent” embryonal carcinoma cell line. However, careful inspection of F9 cultures reveals the presence of small numbers of cells which possess several properties of endoderm, particularly parietal endoderm, and which can be shown to derive from the embryonal carcinoma component. Furthermore, tumors of F9 cells include isolated patches of endoderm-like cells surrounded by a thick secretion resembling Reichert's membrane. The proportion of endoderm-like cells in F9 cultures can be increased to varying degrees by causing the cells to form aggregates and/or maintaining them at high density for several days, although the endoderm-like cells produced in these ways contribute very little to the formation of subcutaneous tumors from the resultant mixed cultures. Differentiated cell types other than endoderm are rarely observed in F9 monolayer or aggregate cultures, even after several weeks. Cloning studies support the view that most, if not all, F9 cells can differentiate, albeit at very low incidence.