An examination and correction of plant tissue culture basal medium formulations

The inorganic formulations of fourteen common plant tissue culture basal media were examined from the primary literature. Inaccuracies and errors were found for molecular formulae, chemical hydrations, and molar equivalences for iron/EDTA complexation. A comparison with published basal medium formulations from six commercial suppliers uncovered additional inaccuracies, modifications, and errors, thereby emphasizing the need for investigators to examine and describe medium formulations precisely in future publications.     

The alpha subunit of type II Ca2+/calmodulin-dependent protein kinase is highly conserved in Drosophila

A monoclonal antibody against rat brain type II Ca2+/calmodulin-dependent protein kinase (CaM kinase) precipitates three proteins from Drosophila heads with apparent molecular weights similar to those of the subunits of the rat brain kinase. Fly heads also contain a CaM kinase activity that becomes partially independent of Ca2+ after autophosphorylation, as does the rat brain kinase. We have isolated a Drosophila cDNA encoding an amino acid sequence that is 77% identical to the sequence of the rat alpha subunit. All known autophosphorylation sites are conserved, including the site that controls Ca(2+)-independent activity. The gene encoding the cDNA is located between 102E and F on the fourth chromosome. The protein product of this gene is expressed at much higher levels in the fly head than in the body. Thus, both the amino acid sequence and the tissue specificity of the mammalian kinase are highly conserved in Drosophila.     

Histochemical studies of intestinal epithelial goblet cell glycoproteins during the development of the human foetus

Summary Histochemical studies performed on specimens of intestine from 12 to 37-week human foetuses showed that the epithelial glycoproteins of the goblet cells of the small intestine are non-sulphated sialoglycoproteins containing neutral sugar (hexose, 6-deoxy hexose or N-acetyl hexosamine residues with Periodic acid-Schiff (PAS) reactive vicinal diols), sialic acids without O-acyl substituents, smaller and variable quantities of sialic acids with O-acyl substituents at positions C8 or C9 (or with two or three side chain substituents) and O-acyl sugars (neutral sugars with an ester substituent blocking PAS reactivity). In the lower small intestine glycoproteins containing 8 (or 9)-O-acyl sialic acids are first observed in goblet cells at the tips of the villi. As the foetus matures their quantity increases and they are found in goblet cells located along the length of the villi. Smaller quantities of O-acyl sialic acids and traces of O-acyl sugars occur in the goblet cells of the upper small intestine. The colonic goblet cells contain sulphosialoglycoproteins of two types. The first type, found in the majority of specimens, contains O-sulphate ester, neutral sugar, O-acyl sugars and 8 (or 9)-O-acyl sialic acids. The second type contains O-sulphate ester, neutral sugars, and sialic acids which are either without side chain O-acyl substituents or are a mixture of such acids and 8 (or 9)-O-acyl sialic acids; O-acyl sugars are reduced or absent. The degree of sulphation of the foetal colonic goblet cell epithelial glycoproteins differs with the region of the colon, the level of the crypt and the gestational age of the foetus in a manner consistent with that described by Lev & Orlic (1974). The detection of O-acyl sugars in foetal intestinal glycoproteins adds to the known examples of such sugars and strengthens the suggestion that they are a normal constituent of colonic epithelial glycoproteins.Part of this work was presented at the 32nd meeting of the Canadian Federation of Biological Sciences, Calgary, Alberta, June 1989 (abstract # 336).     

The association of FAD with the cytochrome b−245 of human neutrophils

A plasma membrane fraction prepared from human neutrophils had a fluorescence resembling that of a fluorescent flavoprotein, with emission maximum near 520nm and excitation maxima near 380 and 460nm. The fluorescence emission and excitation properties of Triton N-101-solubilized membrane fraction resembled those of FAD. FAD was present in the membranes at a concentration of 417pmol/mg of protein and cytochrome b₋₂₄₅ at a concentration of 407pmol/mg of protein. In a 110-fold purified preparation of cytochrome b₋₂₄₅ the ratio of FAD:cytochrome b was 1:1. Analytical gradient centrifugation of neutrophil homogenates shows a coincidence of two cytochrome b peaks and two peaks of fluorescence, corresponding with plasma membrane and specific granule fractions; most of the FAD was non-fluorescent and located in fractions lighter than the plasma membrane. Plasma membrane fractions prepared from neutrophils of patients suffering from the X-linked form of chronic granulomatous disease lacked cytochrome b and contained 194pmol of FAD/mg of protein; plasma membrane fractions prepared from neutrophils of patients with the autosomal recessive form of chronic granulomatous disease contained both cytochrome b₋₂₄₅ and FAD in the normal range of concentrations in a ratio of 1:1. Phagocytic vesicles were prepared from normal neutrophils and found to contain FAD and cytochrome b in a ratio 2.22:1, suggesting that activation of neutrophils many involve the incorporation of an additional flavin into the membrane. Under anaerobic conditions in the presence of EDTA to act as an electron donor to a flavin, the cytochrome b₋₂₄₅ of neutrophil membranes was partly (12%) photoreducible, an effect increased to 100% by the addition of FMN. The extent of reduction of cytochrome b in an anaerobic neutrophil homogenate containing NADH increased from 30% to 70% on illumination. We suggest that these results indicate a close association between FAD and cytochrome b₋₂₄₅ and support a scheme for electron transport thus: [Formula: see text]     

Fermentative Conversion of Cellulose to Acetic Acid and Cellulolytic Enzyme Production by a Bacterial Mixed Culture Obtained from Sewage Sludge

A simple procedure that uses a cellulose-enriched culture started from sewage sludge was developed for producing cellulolytic enzymes and converting cellulose to acetic acid rather than CH₄ and CO₂. In this procedure, the culture which converts cellulose to CH₄ and CO₂ was mixed with a synthetic medium and cellulose and heated to 80°C for 15 min before incubation. The end products formed were acetic acid, propionic acid, CO₂, and traces of ethanol and H₂. Supernatants from 6- to 10-day-old cultures contained 16 to 36 mM acetic acid. Cellulolytic enzymes in the supernatant were stable at 2°C under aerobic conditions for up to 4 weeks and had the ability to hydrolyze carboxymethyl cellulose, a microcystalline cellulose, cellobiose, xylan, and filter paper to reducing sugars.     

Sciatin Is a Transferrin-Like Polypeptide

Abstract: Sciatin, an acidic glycoprotein from chicken sciatic nerve, has myotrophic effects on avian skeletal muscle cells in culture. As sciatin was found to have certain structural similarities to transferrin, we further investigated the physicochemical characteristics of sciatin in order to determine the relationship between these two proteins. Sciatin was found to be strikingly similar to ovotransferrin in amino acid composition. In addition, amino acid sequence analysis revealed that sciatin and ovotransferrin had identical amino-terminal sequences for at least the first 20 amino acid residues. Chicken ovotransferrin, but not human serum transferrin, cross-reacted with rabbit antisciatin antibodies upon rocket immunoelectrophoresis and double immunodiffusion in agar. In addition, in the presence of bicarbonate, sciatin bound approximately 2 mol ferrous iron/mol protein. Using the purification procedure developed for sciatin, we purified a protein from chicken serum that cross-reacted with antisciatin serum, migrated at a position identical to that of sciatin or ovotransferrin on two-dimensional gel electrophoresis, had an amino composition very similar to ovotransferrin and sciatin, and had myotrophic effects on cultured muscle cells. From these data, we conclude that sciatin is a growth-promoting polypeptide closely related in structure to transferrin.     

Isolation and preliminary characterization of cryptic satellite DNA in pea

Optimum conditions have been established for isolation of ‘cryptic’ satellite DNA from the genome of pea (Pisum sativum), using gradients of CS₂SO₄ containing silver ions. At an Ag⁺ :DNA-P ratio (R) of 0.1, and at alkaline pH, four fractions are obtained: mainband (buoyant density 1.437 g cm³; 67% of total DNA), satellite I (buoyant density 1.582 g/cm³; 7% of total DNA), satellite II (buoyant density 1.520 g/cm³, 11% of total) and satellite III (buoyant density variable between 1.45 and 1.51 g/cm³; 15% of total). The reiterated DNA content of these four fractions has been investigated by reassociation experiments conducted over a Cot range of 1 × 10^?5 to 2.0. All four fractions contain at least two kinetic components; each fraction, including the mainband, consists at least partly of highly reiterated DNA. Ribosomal RNA hybridizes only to the mainband.