High growth rate and regeneration capacity of hypocotyl protoplasts in some Brassicaceae

Protoplasts isolated from 4-day-old hypocotyls of various species of Brassica (Brassica napus, B. campestris and B. oleracea ) produced callus with high efficiency in media containing casein hydrolysate and high concentrations of the auxin 2,4-D (4.5 μM). Cell division began after 24 h and 60% of the cells had divided after 48 h. In contrast, protoplasts isolated from stem and mesophyll of plants grown in vitro or in the greenhouse began to divide after a delay of 3–5 days. In these cases 40–50% of the cells had divided after 5 days as compared to 70% for hypocotyl protoplasts. To obtain a high frequency of regeneration, rapidly growing calli were transferred to media having a high cytokinin:auxin ratio as early as possible, usually 3 weeks after protoplast isolation. The average regeneration frequency for calli obtained from mesophyll protoplasts was 50%, while as many as 70% of the calli derived from hypocotyl protoplasts of B. napus regenerated plantlets on a medium containing zeatin (9.1 μM) and IAA (0.6 μM). On the same medium regeneration of Brassica oleracea was obtained. A low percentage of calli (1%) from Brassica campestris formed shoots when cultured on a combination of zeatin (4.6 μM), BA (4.4 μM) and IAA (0.6 μM).     

Abnormal high density lipoproteins from patients with liver disease regulate cholesterol metabolism in cultured human skin fibroblasts

Apolipoprotein B (apoB) of plasma low density lipoproteins (LDL) binds to high affinity receptors on many cell types. A minor subclass of high density lipoproteins (HDL), termed HDL1, which contains apoE but lacks apoB, binds to the same receptor. Bound lipoproteins are engulfed, degraded, and regulate intracellular cholesterol metabolism and receptor activity. The HDL of many patients with liver disease is rich in apoE. We tested the hypothesis that such patient HDL would reduce LDL binding and would themselves regulate cellular cholesterol metabolism. Normal HDL had little effect on binding, uptake, and degradation of 125I-labeled LDL by cultured human skin fibroblasts. Patient HDL (d 1.063-1.21 g/ml) inhibited these processes, and in 15 of the 25 samples studied there was more than 50% inhibition at 125I-labeled LDL and HDL protein concentrations of 10 micrograms/ml and 25 micrograms/ml, respectively. There was a significant negative correlation between the percentage of 125I-labeled LDL bound and the apoE content of the competing HDL (r = -0.54, P less than 0.01). Patient 125I-labeled HDL was also taken up and degraded by the fibroblasts, apparently through the LDL-receptor pathway, stimulated cellular cholesterol esterification, increased cell cholesteryl ester content, and suppressed cholesterol synthesis and receptor activity. We conclude that LDL catabolism by the receptor-mediated pathway may be impaired in liver disease and that patient HDL may deliver cholesterol to cells.     

Stimulation of respiration and nitrogenase in bacteroids of Siratro (Macroptilium atropurpureum) by plant nodule cytosol

Nitrogenase activity (acetylene reduction) of isolated Siratro (Macroptilium atropurpureum) bacteroids was stimulated by addition of plant cytosol fractions which also preserved activity at high (up to 3%) O₂ tensions. These effects were not due to leghaemoglobin. Boiling removed some, but not all, of the protective capacity of the cytosol. Heat treated cytosol substantially stimulated the respiration of siratro bacteroids. Of a wide variety of compounds tested, only ascorbate could mimic the cytosol. Ascorbate was present in the cytosol fraction, in significant quantities. The effect of ascorbate was evident at low O₂ concentrations and in purified bacteroids, and was inhibited by cyanide. Siratro bacteroids appear to possess an oxidase which could serve a protective role in vivo.     

Childhood gastroenteritis: a population study.

A prospective study of gastroenteritis based on a population was carried out for 12 months on over 7000 children in general practice. The incidence of gastroenteritis was highest in the first year (127.7 children affected per 1000) and second year (90.8) of life, and gastroenteritis was rare after six years of age. Children from urban areas had gastroenteritis more commonly than children from semirural areas. A potential pathogen was isolated from half of the specimens: 78% were viruses, and rotavirus was identified most often.     

Purification and properties of the β-fructofuranosidase from Kluyveromyces fragilis

The β-fructofuranosidase from Kluyveromyces fragilis was purified to one band on electrophoresis by 3 different methods. Two of the preparations were found to be impure by isoelectric focusing. This demonstrates the need for more than one criteria of homogeneity when purifying this enzyme. The enzyme was found to be a glycoprotein, stable at 50°C, with a pH optimum of 4.5. The cations Hg²⁺, Ag⁺, Cu²⁺ and Cd²⁺ exhibited a marked inhibition of the enzyme. Competitive inhibition was observed with the fructose analog 2,5-anhydro-D-mannitol suggesting that the enzyme is inhibited by the furanose form of fructose.     

Bacillus cereus 569/H penicillinase serine-44 acylation by diazotized 6-aminopenicillanic acid

Penicillinase from Bacillus cereus 569/H was purified to homogeneity. Its active site was probed by use of an affinity label generated in situ by the diazotization of 6-aminopenicillanic acid, a catalytically poor substrate for this enzyme. The loss of activity arising during the inactivation is dependent upon pH and the penicillin:sodium nitrite ratio used. Optimal inactivation was obtained at pH 4.7 and reactivation could be prevented if subsequent purification and manipulations were performed at low pH. Inactivation by diazotized 6-aminopenicillanic acid was characterized further by tryptic and chymotryptic digestion of the inactivated enzyme and peptide mapping of the resulting digests. Amino acid analysis of the chymotryptic labeled peptide yielded a composition which corresponds to residues 41-46 (Ala-Phe-Ala-Ser-Thr-Tyr) in the published partial sequence of the enzyme (Thatcher, D. (1975) Biochem. J. 147, 313-326). Further digestion of this chymotryptic peptide with carboxypeptidase A reveals that serine-44 is modified in this affinity labeling procedure. Mass spectral analysis of the modified serine residue and alkali-released label, and comparison with spectra of model compounds indicates that the inactivation occurs with rearrangement of the beta-lactamthiazolidine structure to a dihydrothiazine.     

Cloning of Pf3, a filamentous bacteriophage of Pseudomonas aeruginosa, into the pBD214 vector of Bacillus subtilis.

The genome of Pf3, a filamentous single-stranded DNA bacteriophage of Pseudomonas aeruginosa (a gram-negative organism) was cloned into pBD214, a plasmid cloning vector of Bacillus subtilis (a gram-positive organism). Cloning in the gram-positive organism was done to avoid anticipated lethal effects. The entire Pf3 genome was inserted in each orientation at a unique Bc/I site within a thymidylate synthetase gene (from B. subtilis phage beta 22) on the plasmid. Additional clones were made by inserting EcoRI fragments of Pf3 DNA into a unique EcoRI site within this gene.     

Distinct distribution of immunoreactive dynorphin and leucine enkephalin in various populations of isolated adrenal cromaffin cells

The distribution of immunoreactive-dynorphin (ir-Dyn) in isolated subpopulations of bovine adrenal chromaffin cells was examined and compared with that of adrenaline (A), noradrenaline (NA) and ir-Leucine-Enkephalin (ir-Leu-Enk). Using a stepwise bovine serum albumin (BSA) gradient, various populations of catecholamine-storing cells were separated and designated as cell layers I, II and III. Cell layer I contained more NA than A; cell layer II contained slightly more A than NA whereas cell layer III was highly enriched in A. The original cell preparation contained 2.9 times more ir-Leu-Enk than ir-Dyn (4.7 and 1.6 pmoles per 10(6) cells, respectively). After separation of the cells on BSA gradient, ir-Dyn was mainly detected in cell layer I (4.0 pmoles/10(6) cells) whereas ir-Leu-Enk was concentrated in cell layer III (8.3 pmoles/10(6) cells). Both peptides were secreted in response to acetylcholine (5 x 10(-5) M), but the amount secreted was in accordance with the cell content in each peptide. After subcellular fractionation of the adrenal medulla, the neuropeptides were found in close association with catecholamines in the secretory granules. These results indicate that bovine adrenal chromaffin cells can be isolated according to their specific content in A, NA and opioid peptides and are consistent with the hypothesis of distinct biosynthetic pathways for Dyn and the Enk.     

Exogenous NAD Effects on Plant Mitochondria: A Reinvestigation of the Transhydrogenase Hypothesis

Addition of NAD⁺ to purified potato (Solanum tuberosum L.) mitochondria respiring α-ketoglutarate and malate in the presence of the electron transport inhibitor rotenone, stimulated O₂ uptake. This stimulation was prevented by incubating mitochondria with N-4-azido-2-nitrophenyl-aminobutyryl-NAD⁺ (NAP₄-NAD⁺), an inhibitor of NAD⁺ uptake, but not by 1 mm EGTA, an inhibitor of external NADH oxidation. NAD⁺-stimulated malate-cytochrome c reductase activity, and reduction of added NAD⁺ by intact mitochondria, could be duplicated by rupturing the mitochondria and adding a small quantity to the cuvette. The extent of external NAD⁺ reduction was correlated with the amount of extra mitochondrial malate dehydrogenase present. Malate oxidation by potato mitochondria depleted of endogenous NAD⁺ by storing on ice for 72 hours, was completely dependent on added NAD⁺, and the effect of NAD⁺ on these mitochondria was prevented by incubating them with NAP₄-NAD⁺. External NAD⁺ reduction by these mitochondria was not affected by NAP₄-NAD⁺. We conclude that all effects of exogenous NAD⁺ on plant mitochondrial respiration can be attributed to net uptake of the NAD⁺ into the matrix space.