Identification of a distinct phase during melanogenesis that is sensitive to extracellular pH and ionic strength

The cell line B16/C3 will undergo melanogenesis at a specific time after plating. We have found that this time can be modulated by varying the pH of the culture medium. At high pH levels (8.2–8.6) the onset of melanogenesis occurs in 3 or 4 days, while at lower pH (6.7–7.2) it occurs in 7 or 8 days. Furthermore, the time of onset is also sensitive to the extracellular ionic strength. The addition of sodium lactate, sodium chloride, or any other salt tested delays or blocks completely the onset of melanogenesis. These effects are not simply consequence of growth inhibition, nor can they be correlated with patterns of lactate accumulation. These cells are sensitive to pH or ionic strength after entering the stationary phase just prior to the time of onset of melanogenesis. The existence of a specific pH-and ionic-strength-sensitive phase may provide an important clue to the events responsbile for differentiation in this system.     

Age as a factor in 6-hydroxydopamine-induced plasticity in the hypothalamus

Summary The question of age as a possible factor influencing the regenerative response of catecholaminergic varicosities in the hypothalamus was investigated in the supraoptic commissure and the paraventricular, periventricular, and dorsomedial hypothalamic nuclei of rats that had received intraventricular injections of the neurotoxin 6-hydroxydopamine when they were (1) neonates, (2) young adults, or (3) senescent adults. After postneurotoxin survival for 4, 21, 56, or 180 days, the animals were perfused, and the hypothalamic tissue sections were cut and processed using a glyoxylic acid method for localizing catecholamines. Four days following neurotoxin administration, counts of fluorescent varicosities showed a significant loss of catecholamine varicosities in each of the four areas. Subsequently, at least partial restoration of numbers of catecholamine varicosities occurred in all hypothalamic areas in all three age groups. It is concluded that, following selective lesions induced by the neurotoxin 6-OH-DA, catecholamine varicosities were restored both in immature and mature groups. According to the evidence obtained experimentally, the rate of restoration was greater in the neonate group, whereas the percentage restoration attained varies according to the hypothalamic area studied and the age of the animal.     

Selectivity in endocytosis of serum and cytosol proteins by macrophages in culture

It was found that cultured mouse macrophages preferentially endocytose and degrade short half-life serum and cytosol proteins. With increasing duration of endocytosis, the components of longer half-life are increasingly endocytosed and degraded. The relevance of these observations to the selectivity of protein turnover is discussed.     

Affinity chromatography on immobilised nucleotides. Some applications to the purification of thermophilic dehydrogenases and kinases.

The effect of pH and temperature on the capacity and binding of Bacillus stearothermophilus, alcohol dehydrogenase and phosphofructokinase to N6-(6-aminohexyl)-5'-AMP-Sepharose has been examined. Specific elution from the substituted AMP-Sepharose was examined using a variety of cofactors, fragments of cofactors and substrates. A purification scheme for each enzyme on the substituted AMP-Sepharose using nucleotides and gradients of pH and salt is presented. Interestingly, elevated temperature increased the affinity of both enzymes for N6-(6-aminohexyl)-5'-AMP-Sepharose, however, the Michaelis constant for nucleotide determined at various temperatures remained constant. The effect of pH and salt concentration on the binding of B. stearothermophilus glyceraldehyde-3-phosphate dehydrogenase to 6-aminohexanoyl-NAD+-Sepharose was also examined; raising the pH above 7.5 lowers the capacity of the matrix and the effect of a range of ammonium sulphate concentrations on the adsorption of the enzyme was examined. A specific purification of glyceraldehyde-3-phosphate dehydrogenase from partially purified extracts of this organism was achieved.     

Enhancement of Mycoplasma Virus Plaque Visibility by Tetrazolium

The plating of mycoplasma virus MVL-1 is greatly improved by the use of tetrazolium to enhance the visibility of the lawn.     

Amino acid sequence of neurotoxin I from Centruroides sculpturatus Ewing

The further characterization of toxin I from venom of the scorpion Centruroides sculpturatus Ewing (region, Southwestern United States) is reported. Toxin I is a single polypeptide chain of 64 amino acid residues crosslinked by four disulfide bridges. The complete amino acid sequence of toxin I was deduced from the sequence of its tryptic peptides and overlaps provided by its chymotryptic peptides. Toxin I has an amino terminal lysyl residue and a carboxyl terminal threonyl residue.The amino acid sequences of toxin I and neurotoxic variants 1, 2, and 3, likewise isolated from C. sculpturatus venom, differ at 26 positions.The sequences of toxin I from C. sculpturatus and toxins I and II from the North African scorpion, Androctonus australis Hector, are also compared.     

Separation of a proteoglycan fraction from Kurloff cells stimulating protein synthesis in macrophages.

Proteoglycan from Kurloff cells, when present in the medium in low concentrations, increased the incorporation of (3-H)leucine into trichloracetic acid-precipitable material by macrophages from peritoneal exudates, in addition to inhibiting their migration from capillary tubes, as observed previously. After treatment with 0.5 M-NaOH, followed by dialysis or ultrafiltration, material with the distinctive u.v. and i.r. spectra of the whole proteoglycan appeared in the diffusate, and biological activity was lost from the proteoglycan which remained in the dialysis residue. The diffusible material absorbed near 260 nm and had i.r. bands at 805 cm-minus-1 and 1260 cm-minus-1, but did not display the i.r. bands characteristic of chondrotin 4-sulphate. It contained little sulphate, no hexosamine and less than 1% of the uronic acid present in the whole proteoglycan, and there were only trace amounts of amino acids, xylose and galactose. However, significant amounts of ribose and organic phosphate were present, each representing about 1% of the whole proteoglycan. After proteolysis and chondroitanase digestion of the proteoglycan, a fraction with absorbance at 260 mn was eluted from Dowex 1 with water which stimulated the incorporation of (3H)leucine by macrophages and inhibited their migration from capillary tubes.