Histochemical Methods for Dissociated Muscle Fibers

Skeletal or cardiac muscle fibers can be separated by brief (3-5 second) dissociation of formalin-fixed pieces with a Willems Polytron (Brinkmann Instrument Co.). Such separated fibers are useful for demonstration of abnormal accumulations of lipids, carbohydrates, proteins and minerals in metabolic diseases. Staining techniques for demonstration of various stored materials include: 1) toluidine blue at pH 2.8 for acid mucopolysaccharide in skeletal muscle fibers in Pompe's glycogenesis 2,2) one-step trichrome stain for nemaline myopathy and for abnormal mitochondria in X-linked infantile cardiomyopathy, 3) periodic acid-methenamine silver stain for glycolipid-containing lysosomes in I-cell disease (mucolipidosis 2), 4) Sudan black B stain for lipid in skeletal muscle fibers in Reye's syndrome, infantile lactic acidosis, Leigh's infantile subacute necrotizing encephalopathy and Jansky-Bielschowsky late infantile ceroid lipofuscinosis, 5) iron stain for iron in cardiac and skeletal muscle fibers in thalassemia with advanced hemosiderosis, and 6) autofluorescence for “ceroid” in skeletal muscle fibers in Jansky-Bielschowsky disease.     

Reciprocal Allogrooming in Dam-reared and Hand-reared Impala Fawns

Among adult females and males of African antelope impala are unique in their performance of reciprocal allogrooming. The occurrence of this behaviour in neonatal impala fawns was explored in a free-ranging impala herd at the San Diego Wild Animal Park where 5 dam-reared fawns were observed from birth through 10 weeks of age. One-way maternal grooming and reciprocal allogrooming with the dam and non dam partners emerged as distinct behavioural systems. Maternal grooming, directed mostly to the anogenital area, was typical of that seen in other ungulates, and sharply declined over the first two weeks. Reciprocal allogrooming, characterized by alternate exchanges of grooming bouts with a partner in the same manner as in adults, was seen as early as 3–8 d after birth. All fawns were grooming with unrelated adult females by the end of the second week. By week 2 virtually every measure of reciprocal allogrooming by fawns (grooming delivered per hour, reciprocity, and percent of encounters initiated) was as high as for adults. The appearance of this reciprocal allogrooming pattern, especially at such an early age, appears to be unique among ungulates, and possibly mammals in general. Three hand-reared impala fawns, deprived of the opportunity to interact with older herdmates, but having access to impala fawns and heterospecific fawns, were observed from 1–3 mo of age. The hand-reared impala showed no alteration in the occurrence of reciprocal allogrooming behaviour compared with the dam-reared control fawns, indicating that allogrooming experience with older animals was not required for the appearance of reciprocal allogrooming at an early age. Interestingly, hand-reared fawns persisted in grooming heterospecific fawns despite the fact that heterospecifics rarely reciprocated grooming. We postulate that the strong predisposition for impala young to groom others may be related to the threat of tick infestation in the impala's ecotone habitat.     

DNase I hypersensitivity and expression of the Shrunken-1 gene of maize

The local chromatin structure of the Shrunken-1 (Sh) gene of maize was probed by analyzing DNase I hypersensitivity. Sh encodes the gene for sucrose synthetase, a major starch biosynthetic enzyme, which is maximally expressed in the endosperm during seed maturation. In addition to general DNase I sensitivity, specific DNase I hypersensitive sites were identified in endosperm chromatin that mapped near the 5 end of the Sh gene. The pattern of hypersensitive sites and their relative sensitivity were altered in other non-dormant tissues that produce little or no enzyme. However, some changes in chromatin structure appear to be independent of Sh gene expression and may reflect general alterations associated with plant development. The chromatin structure of several sh mutations, induced by Ds controlling element insertions, was also analyzed. Although the insertions perturbed expression of the gene, there were no notable effects on local chromatin structure.     

Carboxylmethylation of Calmodulin in Cultured Pituitary Cells

We have used fast protein liquid chromatography (FPLC) and reverse-phase HPLC to rapidly resolve carboxylmethylated proteins in cultured pituitary GH3 cells. This procedure preserves labile carboxylmethyl esters, which are lost under the usual procedures employed for protein fractionation. GH3 cells were incubated with [methyl-3H]-methionine in culture and incorporation of label into the soluble fraction, total cell protein, and protein carboxylmethyl esters was determined; protein carboxylmethyl ester formation was shown to be resistant to cycloheximide. Fractionation of protein carboxylmethyl esters from GH3 cells by gel permeation FPLC, anion-exchange FPLC, and reverse-phase HPLC in the presence of calcium and in the presence of EGTA identified two proteins that are major substrates for protein carboxylmethyltransferase and indicated that one of these proteins is calmodulin. Similar results were obtained when a cytosolic fraction from GH3 cells was incubated with S-adenosyl-L-[methyl-3H]methionine. These results indicate that rapid chromatography at low temperature and low pH is useful for the analysis of eucaryotic carboxylmethylated proteins and that contrary to reports obtained in other systems, calmodulin is carboxylmethylated in intact pituitary cells.