Dissipation of the Proton Electrochemical Potential in Intact Chloroplasts (II. The pH Gradient Monitored by Cytochrome f Reduction Kinetics)

The potency of various uncouplers for collapsing the light-induced pH gradient across thylakoid membranes in intact chloroplasts was investigated by time-resolved optical spectroscopy. The thylakoid transmembrane pH gradient ([delta]pH) was monitored indirectly by measuring the rate of cytochrome (Cyt) f reduction following a light flash of sufficient duration to create a sizable [delta]pH. The results show that the rate of Cyt f reduction is controlled in part by the internal pH of the thylakoid inner aqueous space. At pH values from 6.5 to 8.0, the Cyt f reduction rate was maximal, whereas at lower pH values from 6.5 to 5.5 the reduction rate decreased to 25% of the maximal rate. The ability of three uncouplers, nigericin, carbonylcyanide m-chlorophenylhydrazone, and gramicidin, to accelerate the rate of Cyt f reduction was determined for intact chloroplasts isolated from spinach (Spinacia oleracea). The efficacy of the uncouplers for collapsing the [delta]pH was determined using the empirical relationship between the [delta]pH and the Cyt f reduction rate. For intact chloroplasts, nigericin was the most effective uncoupler, followed by carbonylcyanide m-chlorophenylhydrazone, which interacted strongly with bovine serum albumin. Gramicidin D, even at high gramicidin:chlorophyll ratios, did not completely collapse the pH gradient, probably because it partitions in the envelope membranes and does not enter the intact chloroplast.     

A rice (Oryza sativa L.) mutant having a low content of glutelin and a high content of prolamine

Among the mutant lines of rice that have been selected for morphological characters, one line, NM67, was found to have a low content of glutelin and a higher content of prolamine in its seed protein than other Japanese cultivars. This mutant is a semi-dwarf and partially sterile line, and its leaves turn yellow before heading. Genetic analysis after backcross to the original cultivar, Nihonmasari, revealed the following: (1) the character of low glutelin content was always accompanied by the character of high prolamine content; (2) the low glutelin (and high prolamine) character seemed to be manifested by a single dominant gene; and (3) semi-dwarfness, low fertility and early yellowing leaf of the mutant, which might also be pleiotropy, were controlled by a single recessive gene independent of the gene for protein content. The protein character of NM67 was genetically separated from semi-dwarfness and low fertility, and a new line having low glutelin content and high prolamine content with normal morphological characters comparable to those of the original cultivar was obtained from the progenies of the cross. The possible use of this line as a low protein rice cultivar is discussed.     

Inhibitory effect of oxygen accumulation on the growth of Spirulina platensis

Summary The growth of Spirulina platensis was studied in a light-limited culture under various dissolved oxygen (DO) concentrations. At high DO concentration, e.g. at 1.25 mM DO, the growth rate was decreased up to 36 % compared with that of 0.063 mM DO. The retarded growth rate at high DO concentrations seemed to be coupled with the degeneration of photosynthetic activity in terms of O₂ evolution. Under higher DO concentrations, superoxide dismutase and ascorbate peroxidase activities tended to increase, while the contents of photosynthetic pigment, such phycocyanin, carotenoid and chlorophyll-a decreased distinctly.     

Genetic Variation in VP7 Gene of Human Rotavirus Serotype 2 (G2 Type) Isolated in Japan,China, and Pakistan

Sequence analysis of the gene encoding the major neutralization glycoprotein (VP7) was performed on sixteen human isolates of serotype 2 of rotavirus in Japan, China, and Pakistan and their genetic variations were examined. Comparative studies of their nucleotide and deduced amino acid sequences between the sixteen isolates and the HU5 strain revealed an overall homology of more than 94%. A higher degree of homology in nucleotides was observed among the sixteen isolates than between HU5 and the isolates. A total of thirteen amino acid residues frequently converted to another amino acid. Out of the thirteen, five amino acid residues belonging to the major neutralizing epitope regions (C, E, and F in this communication) converted frequently. From the amino acid sequences three subtypes, subtype 1, subtype 2, and intermediate, were suggested to be classified as previously reported for serotype 1 (Xin et al, Virology, 1993, 197: 813-816).     

High-resolution solution structure of siamycin II: Novel amphipathic character of a 21-residue peptide that inhibits HIV fusion

Summary The 21-amino acid peptides siamycin II (BMY-29303) and siamycin I (BMY-29304), derived from Streptomyces strains AA3891 and AA6532, respectively, have been found to inhibit HIV-1 fusion and viral replication in cell culture. The primary sequence of siamycin II is CLGIGSCNDFAGCGYAIVCFW. Siamycin I differs by only one amino acid; it has a valine residue at position 4. In both peptides, disulfide bonds link Cys¹ with Cys¹³ and Cys⁷ with Cys¹⁹, and the side chain of Asp⁹ forms an amide bond with the N-terminus. Siamycin II, when dissolved in a 50:50 mixture of DMSO and H₂O, yields NOESY spectra with exceptional numbers of cross peaks for a peptide of this size. We have used 335 NOE distance constraints and 13 dihedral angle constraints to generate an ensemble of 30 siamycin II structures; these have average backbone atom and all heavy atom rmsd values to the mean coordinates of 0.24 and 0.52 Å, respectively. The peptide displays an unusual wedge-shaped structure, with one face being predominantly hydrophobic and the other being predominantly hydrophilic. Chemical shift and NOE data show that the siamycin I structure is essentially identical to siamycin II. These peptides may act by preventing oligomerization of the HIV transmembrane glycoprotein gp41, or by interfering with interactions between gp41 and the envelope glycoprotein gp120, the cell membrane or membrane-bound proteins [Frèchet, D. et al. (1994) Biochemistry, 33, 42–50]. The amphipathic nature of siamycin II and siamycin I suggests that a polar (or apolar) site on the target protein may be masked by the apolar (or polar) face of the peptide upon peptide/protein complexation.Abbreviations ABNR adopted basis Newton Raphson - AIDS acquired immunodeficiency syndrome - CW continuous wave - DMSO dimethylsulfoxide - DQF-COSY two-dimensional double-quantum-filtered correlation spectroscopy - HIV human immunodeficiency virus - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser enhancement spectroscopy - ppm parts per million - P.E.-COSY two-dimensional primitive exclusive correlation spectroscopy - REDAC redundant dihedral angle constraint - rf radio frequency - rmsd root-mean-square difference - SIV simian immunodeficiency virus - sw spectral width - _m mixing time - TOCSY two-dimensional total correlation spectroscopy - TSP trimethylsilyl-2,2,3,3-²H₄-propionate - 2D two-dimensional     

Single cell protein production from mandarin orange peel

Summary As the hydrolysis of mandarin orange peel with macerating enzyme (40°C, 24 h) produced 0.59 g g^–1 reducing sugar per dry peel compared to 0.36 by acid-hydrolysis (15 min at 120°C with 0.8 N H₂SO₄), the production of single cell protein (SCP) from orange peel was studied mostly using enzymatically hydrolyzed orange peel.When the enzymatically hydrolyzed peel media were used, the utilization efficiency of reducing sugars (%) and the growth yield from reducing sugars (g g^–1) were: 63 and 0.51 for Saccharomyces cerevisiae; 56 and 0.48 for Candida utilis; 74 and 0.69 for Debaryomyces hansenii and 64 and 0.70 for Rhodotorula glutinis. SCP production from orange peel by D. hansenii and R. glutinis were further studied. Batch cultures for 24 h at 30°C using 100 g dried orange peel produced 45 g of dried cultivated peel (protein content, 33%) with D. hansenii and 34 g (protein content, 50%) with R. glutinis, and 38 g (protein content, 44%) with a mixture of both yeasts.     

Inhibition of DNA synthesis in permeabilized L cells by novobiocin

Novobiocin was equipotent in inhibiting DNA and RNA synthesis in cultured mouse L cells. It also suppressed in vitro DNA and RNA synthesis in permeabilized L cells and nuclei; 50 percent inhibition of DNA and RNA synthesis was obtained by 1 mM and 20 mM novobiocin, respectively. ATP antagonized the effect of novobiocin. Nalidixic acid had a weak inhibitory effect on in vitro DNA synthesis; 10 mM nalidixic acid showed 60 percent inhibition. ATP did not antagonize nalidixic acid. The inhibitory effect of novobiocin exceeded that of aphidicolin. These findings suggest a participation of a gyrase- and/or type II topoisomerase-like enzyme in the DNA replication machinery in L cells.     

Macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates: definition of critical amino acids involved in cell tropism.

Previous experiments indicate that the V3 hypervariable region of the human immunodeficiency virus (HIV) envelope protein influences cell tropism of infection; however, so far no consistent V3 sequence can account for macrophage or T-cell tropism. In these experiments, we studied infectious recombinant HIV clones constructed by using V3 region sequences of HIV isolates from 16 patients to search for sequences associated with cell tropism. Remarkable homology was seen among V3 sequences from macrophage-tropic clones from different patients, and a consensus V3 region sequence for patient-derived macrophage-tropic viruses was identified. In contrast, V3 sequences of T-cell-tropic clones from different patients were highly heterogeneous, and the results suggested that sequence diversity leading to T-cell tropism might be generated independently in each patient. Site-specific mutations identified amino acids at several positions on each side of the GPGR motif at the tip of the V3 loop as important determinants of tropism for T cells and macrophages. However, a wide variety of mutant V3 sequences induced macrophage tropism, as detected in vitro. Therefore, the homogeneity of macrophage-tropic patient isolates appeared to be the result of selection based on a biological advantage in vivo.     

Accumulation,elimination, release and metabolism of pipecolic acid in the mouse brain following intraventricular injection

Following i.c.v. (intracerebral ventricular) injections ofd,l-[³H]pipecolic acid (PA), it is reabsorbed from the ventricles and redistributed to various brain regions. The highest accumulation is found in three brain regions ipsilateral to the injection site, hippocampus, neocortex, striatum, and in the diencephalon. Following preloading in vivo, the radioactivity is released from hippocampus slices in the perfusion medium after depolarization induced by high K⁺. During perfusion with a Ca⁺⁺ free medium containing EGTA, a significant reduction of release is observed.The radioactivity ofd,l-[³H]PA in the brain shows a more rapid phase of decrease from 0 to 2 hours and a slower phase from 2 to 5 hours. At 5 hours, only 28% radioactivity, represented mainly by PA, is left in the brain. Kidney secretion represents the major route of elimination of the injected PA. The presence of -aminoadipic acid both in brain and urine was observed. Probenecid (200 mg/kg) significantly increases the accumulation of i.c.v. injectedd,l-[³H]PA in brain and kidney. The presence of a regional accumulation of PA in certain brain regions, its metabolism in brain, its enhanced retention following probenecid administration and its Ca⁺⁺ dependent release following high K⁺ stimulation, all constitute indirect evidence for a neuronal localization of this brain endogenous iminoacid.