Microanalysis of lactose in tissue culture medium using an enzymatic-fluorometric method

A fluoroenzymatic microdetermination of lactose in tissue culture medium is described. The method is based on the conversion of lactose to galactonic acid by the enzymes galactosidase and galactose dehydrogenase. The resultant reduction of NAD to NADH is measured fluorometrically. Before assaying for lactose the high background fluorescence of tissue culture medium is reduced by deproteinization and adsorption of fluorescent substances with either Florisil or dextran-charcoal. As little as 0.05 nmol of lactose in medium with or without phenol red can be detected after this treatment, whereas for untreated medium the limit of sensitivity of the assay is 1.0 nmol. This method should prove useful for the detection of lactose produced by small numbers of mammary epithelial cells in culture.     

Quinacrine fluorescence and Giemsa banding in trisomy 22

Summary Using quinacrine fluorescence and Giemsa banding techniques we have identified an extra chromosome 22 in three non-mongoloid children with similar phenotypes and 47 chromosomes. In one of the children, the long arm of the extra 22 was shorter than usual. This 22q—chrcmcscme was observed in 4 normal family members with 46 chromosomes. In a fourth child, with similar physical findings, the extra G chromosome was shown to be neither a normal 21 nor 22. It must have arisen from a rearrangement in a parental gamete since it was not present in either parent's karyotype.No constellation of clinical findings, in association with an extra G chromosome, is sufficient evidence for the diagnosis of trisomy 22. The positive identification of the extra chromosome must be made using fluorescence and banding.This paper is dedicated to Professor Marcus M. Rhoades on his 70th birthday in grateful recognition of his friendship, help and advice.Supported in part by U.S. Public Health Service Grants HD1313, RR-75 and TI-HD-66 from the National Institutes of Health. A portion of this paper was presented at the annual meeting of the Society for Pediatric Research, Atlantic City, N.J. May 1, 1971.     

Neuron to glia signaling triggers myelin membrane exocytosis from endosomal storage sites

During vertebrate brain development, axons are enwrapped by myelin, an insulating membrane produced by oligodendrocytes. Neuron-derived signaling molecules are temporally and spatially required to coordinate oligodendrocyte differentiation. In this study, we show that neurons regulate myelin membrane trafficking in oligodendrocytes. In the absence of neurons, the major myelin membrane protein, the proteolipid protein (PLP), is internalized and stored in late endosomes/lysosomes (LEs/Ls) by a cholesterol-dependent and clathrin-independent endocytosis pathway that requires actin and the RhoA guanosine triphosphatase. Upon maturation, the rate of endocytosis is reduced, and a cAMP-dependent neuronal signal triggers the transport of PLP from LEs/Ls to the plasma membrane. These findings reveal a fundamental and novel role of LEs/Ls in oligodendrocytes: to store and release PLP in a regulated fashion. The release of myelin membrane from LEs/Ls by neuronal signals may represent a mechanism to control myelin membrane growth.     

Semiautomated enzymic micro methods for blood glucose and lactic acid on a single filtrate

Methods have been modified and adapted to permit enzymic automated micro analysis of glucose on 0.021 ml of blood, and of lactic acid on 0.0115 ml of blood. By means of a single 1:20 Somogyi filtrate with a final volume of 1–1.4 ml prepared from 0.1–0.15 ml blood, both determinations can be accomplished individually on the autoanalyzer.The range of concentrations of each method has been extended so that glucose may be determined between 5 and $\text{200 mg}\text{100 ml}$ (or higher depending on dilution) and lactic acid between 5 and $\text{125 mg}\text{100 ml}$.The methods are sensitive, reproducible, and stable. The recoveries and the comparison studies with manual methods are well within the limits of clinical investigation procdures.     

Structural and enzymatic properties of the AAA protein Drg1p from Saccharomyces cerevisiae. Decoupling of intracellular function from ATPase activity and hexamerization

The AAA protein Drg1 from yeast was affinity-purified, and its ATPase activity and hexamerization properties were analyzed. The same parameters were also determined for several mutant proteins and compared in light of the growth characteristics of the corresponding cells. The protein from a thermosensitive mutant exhibited reduced ATPase activity and hexamerization. These defects were not reversed by an intragenic suppressor mutation, although this allele supported growth at the nonpermissive temperature. A different set of mutants was generated by site-specific mutagenesis intended to adjust the Walker A box of the D2 domain of Drg1p to that of the D1 domain. A S562G exchange in D2 produced a nonfunctional protein that did not hexamerize but showed above-normal ATPase activity. The C561T mutant protein, on the other hand, was functional but hexamerized less readily and had reduced ATPase activity. In contrast, the C561T/S562G protein hexamerized less than wild type but had much higher ATPase activity. We distinguished strong and weak ATP-binding sites in the wild type protein but two weak sites in the C561T/S562G protein, indicating that the stronger site resides in D2. These observations are discussed in terms of the inter-relationship of ATPase activity per se, oligomeric status, and intracellular function for AAA proteins.     

Effect of administration of 5-hydroxytryptophan and an inhibitor ofl-aromatic amino acid decarboxylase on glucose metabolism in rat brain

The effect of 5-hydroxytryptophan (5-HTP)—the precursor of serotonin (5-hydroxytryptamine, 5-HT)—and of an inhibitor,N-(dl-seryl)-N-(2,3,4-trihydroxybenzyl)hydrazine (Ro4-4602), ofl-aromatic amino acid decarboxylase on the metabolism of glucose to amino acids in brain tissue was investigated. Labeled glucose (20 Ci, 0.24 mg in 0.2 ml 0.9% saline) was injected intravenously into fed rats pretreated with Ro4-4602 (50 mg/kg intraperitoneally) either alone or in combination with 5-HTP (30 mg/kg intravenously) or with the appropriate vehicle. After the injection of Ro4-4602 plus 5-HTP, the concentrations of 5-HT and 5-HTP in brain were increased, but the increase of 5-HTP that Ro4-4602 slightly inhibits the reaction of decarboxylation in the brain, although at the dose used the drug is usually considered to act only peripherally. After administration of Ro4-4602 alone or combined with 5-HTP, the concentration of glucose in plasma was not significantly increased. However, the concentration of glucose in brain was markedly increased with such treatments. The administration of Ro4-4602 alone or combined with 5-HTP reduced the flux of¹⁴C from labeled glucose to amino acids in brain. The concentrations of amino acids in brain were little changed by these treatments.     

Über Musterbildung in der Rhizodermis und Exodermis bei einigen Angiospermen und einer Polypodiacee

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