Conformational changes of α-lactalbumin induced by the stepwise reduction of its disulfide bridges: The effect of the disulfide bridges on the structural stability of the protein in sodium dodecyl sulfate solution

Four disulfide bridges of bovineα-lactalbumin (α-lact) were selectively reduced to obtain its derivatives with three, two, and zero disulfide bridges (designated as 3SS, 2SS, and OSSα-lact, respectively). The original helicity was almost maintained in 3SSα-lact missing only the Cys6-Cysl20 bridge. Upon the reduction of both Cys28-Cys111 and Cys6-Cys120 bridges, various changes occurred in the protein. In particular, the maximum fluorescence of 1-anilinonaphthalene-8-sulfonic acid was observed in this stage. Upon the reduction of all disulfide bridges, the hydrophobic box of the protein, formed by Trp60, Ile95, Tyr103, and Trp104, was disrupted and an internal helical structure was destroyed. The conformation of each derivative was examined mainly in a solution of sodium dodecyl sulfate. In the surfactant solution, the helicity increased from 33% to 37% in 3SSα-lact, from 26% to 31% in 2SSα-lact, and from 18% to 37% in OSSα-lact, as against from 34% to 44% in intactα-lact. On the other hand, the tryptophan fluorescence of each derivative was affected in very low surfactant concentrations, suggesting that the tertiary structure considerably changed prior to the secondary structural change in the surfactant solution.     

Growth pattern of isolated zoospores in Hydrodictyon reticulatum (Chlorococcales, Chlorophyceae)

Zoospores at various developmental stages in Hydrodictyon reticulatum were isolated from parent cells and cultured in Waris medium. Isolated zoospores grew to mature vegetative cells, and were able to reproduce zoo-spores that formed daughter hexagonal nets. Three types of shape appeared in cells 24 h after isolation: cylindrical, Y-shaped and 4-armed type. Protrusions of Y-shaped or 4-armed cells were formed at an angle of about 120° to the long axis of the cell. When cells were isolated at later stages, more cells became cylindrical in shape and fewer ceils became Y-shaped or 4-armed, Direction of cell growth also seemed to depend largely on the developmental stages of the zoospores. The later the isolated stages were, the more the cells elongated along the long axis of the zoospores.     

Organization of calsequestrin-positive sarcoplasmic reticulum in rat cardiomyocytes in culture

The sarcoplasmic reticulum (SR) regulates the levels of cytoplasmic free Ca²⁺ ions in muscle cells. Calsequestrin is a major Ca²⁺ -storing protein and is localized at special sites in the SR. To investigate the development of calsequestrin-positive SR and its interaction with the cytoskeleton, we examined the distribution of calsequestrin in cultured cardiomyocytes from newborn rats by immunofluorescence with anticalsequestrin and antitubulin antibodies and rhodamine-phalloidin. In frozen sections of neonatal rat heart, anticalsequestrin immunostaining was apparent as cross-striations at Z-lines. When newborn cardiomyocytes were isolated, calsequestrin-positive SR was disorganized and was apparent as small vesicles beneath the sarcolemma, whereas myofibrils accumulated in the center of the cells. As the cells spread in culture, calsequestrin-positive vesicles spread to the periphery of the cytoplasm, becoming associated with the developing myofibrils. In mature cells, calsequestrin was closely associated with myofibrils, showing cross-striations at the Z-lines. Double-labeling using anticalsequestrin and antitubulin antibodies demonstrated that the distribution of calsequestrin-positive structures was similar to that of the microtubular arrays. When the microtubules were depolymerized by nocodazole at an early stage, the extension of the SR to the cell periphery was inhibited. In mature cardiomyocytes, nocodazole appeared not to affect the distribution of the SR. These results indicate that the calsequestrin-positive SR in cardiomyocytes is organized at the proper sites of myofibrils during myofibrillogenesis and that the microtubules might serve as tracts for the transport of components of the SR. © 1994 Wiley-Liss, Inc.     

Autotrophic picoplankton in southern Lake Baikal: abundance, growth and grazing mortality during summer

Autotrophic picoplankton were highly abundant during the thermalstratification period in late July in the pelagic area (waterdepth 500–1300 m) of southern Lake Baikal; maximum numberswere 2 x 10⁶ cells ml^–1 in the euphotic zone ({small tilde}15m). Unicellular cyanobacteria generally dominated the picoplanktoncommunity, although unidentified picoplankton that fluorescedred under blue excitation were also abundant (maximum numbers4 x 10⁵ cells ml^–1) and contributed up to {small tilde}40%of the total autotrophic picoplankton on occasions. Carbon andnitrogen biomasses of autotrophic picoplankton estimated byconversion from biovolumes were 14–84 µg C l^–1and 3.6–21 µg N l^–1. These were comparableto or exceeded the biomass of heterotrophic bacteria. Autotropicpicoplankton and bacteria accounted for as much as 33% of paniculateorganic carbon and 81% of nitrogen in the euphotic zone. Measurementsof the photosynthetic uptake of [^l4C]bicarbonate and the growthof picoplankton in diluted or size-fractionated waters revealedthat 80% of total primary production was due to picoplankton,and that much of this production was consumed by grazers inthe <20 µ.m cell-size category. These results suggestthat picoplankton-protozoan trophic coupling is important inthe pelagic food web and biogeochemical cycling of Lake Baikalduring summer.     

Stereochemistry during aflatoxin biosynthesis: cyclase reaction in the conversion of versiconal to versicolorin B and racemization of versiconal hemiacetal acetate.

(1'R,2'S)-(-)-aflatoxins are produced from racemic versiconal hemiacetal acetate (VHA) through complicated pathways, including a metabolic grid involving VHA, versiconol acetate (VOAc), versiconol, and versiconal (VHOH), and a reaction sequence from VHOH to versicolorin A (VA) through (-)-versicolorin B (VB) [or (+/-)-versicolorin C] (K. Yabe, Y. Ando, and Y. Hamasaki, J. Gen. Microbiol. 137:2469-2475, 1991; K. Yabe, Y. Ando, and T. Hamasaki, Agric. Biol. Chem. 55:1907-1911, 1991). In this study, we examined stereochemical changes of substances formed during the conversion of VHA to VA by using chiral high-performance liquid chromatography. In cell-free experiments using the cytosol of Aspergillus parasiticus NIAH-26, both (2'S)- and (2'R)-VOAc enantiomers were formed at about a 1:2 ratio from racemic VHA in the presence of NADPH and dichlorvos (dimethyl 2,2-dichlorovinylphosphate). Also, the esterase activity catalyzing the conversion of VHA to VHOH or of VOAc to versiconol did not show the stereospecificity for the 2' carbon atom of VHA or VOAc. However, when racemic VHA or racemic VHOH was incubated with the cytosol, (1'R,2'S)-(-)-VB was formed exclusively. Furthermore, only (1'R,2'S)-(-)-VB, and not (1'S,2'R)-(+) antipode, served as a substrate for desaturase activity in the microsome fraction catalyzing the conversion of VB to VA. These results demonstrate that the stereoconfiguration of bis-furan moiety in aflatoxin molecules is determined by the cyclase enzyme catalyzing the reaction from VHOH to VB, and the (1'R,2'S)-(-) configuration was further confirmed by the subsequent desaturase reaction. Remarkably, we found nonenzymatic racemization in both the (2'R)- and (2'S)-VHA enantiomers, and it was dependent upon the temperature and alkaline conditions.     

Analyses of beta-1 syntrophin, syndecan 2 and gem GTPase as candidates for chicken muscular dystrophy.

Despite intensive studies of muscular dystrophy of chicken, the responsible gene has not yet been identified. Our recent studies mapped the genetic locus for abnormal muscle (AM) of chicken with muscular dystrophy to chromosome 2q using the Kobe University (KU) resource family, and revealed the chromosome region where the AM gene is located has conserved synteny to human chromosome 8q11-24.3, where the beta-1 syntrophin (SNTB1), syndecan 2 (SDC2) and Gem GTPase (GEM) genes are located. It is reasonable to assume those genes might be candidates for the AM gene. In this study, we cloned and sequenced the chicken SNTB1, SDC2 and GEM genes, and identified sequence polymorphisms between parents of the resource family. The polymorphisms were genotyped to place these genes on the chicken linkage map. The AM gene of chromosome 2q was mapped 130 cM from the distal end, and closely linked to calbindin 1 (CALB1). SNTB1 and SDC2 genes were mapped 88.5 cM distal and 27.6 cM distal from the AM gene, while the GEM gene was mapped 18.5 cM distal from the AM gene and 9.1 cM proximal from SDC2. Orthologues of SNTB1, SDC2 and GEM were syntenic to human chromosome 8q. SNTB1, SDC2 and GEM did not correspond to the AM gene locus, suggesting it is unlikely they are related to chicken muscular dystrophy. However, this result also suggests that the genes located in the proximal region of the CALB1 gene on human chromosome 8q are possible candidates for this disease.     

Immunological characterization of a 36 kDa Fe deficiency specific peptide in barley roots

In a previous paper we reported that an acidic 36 kDa peptide is the most strongly induced peptide among several peptides induced by Fe deficiency in barley roots. In this paper, polyclonal antibodies were raised against the 36 kDa peptide. This peptide appeared in the roots of all the graminaceous species tested (barley, rye, wheat, oat, maize, sorghum and rice) in response to Fe deficiency. More of the peptide was found in the roots of graminaceous species which secrete higher amounts of mugineic acids (MAs) under Fe deficient nutrition status. Induction of the 36 kDa peptide was first observed on the third day of Fe deficiency, rising to a maximum value on the seventh day. The trend has a positive correlation with secretion of MAs during Fe deficiency. Further, resupply of Fe resulted in a decrease in peptide production on the second day, reaching a control level on the seventh day. The rate of decrease in peptide production was observed to be slower than that of MA secretion. Other nutrient stresses such as B excess, B deficiency, Cu excess, Cu deficiency, Mn excess, Mn deficiency, Zn excess and Zn deficiency induced far less of the peptide. The specific expression of the 36 kDa peptide in roots of graminaceous species under Fe deficiency suggested the positive association of the peptide with a specific Fe deficiency tolerance mechanism in graminaceous plants.     

Accumulation of phosphoenolpyruvate in red cells incubated with the phosphate ester in an acidified isotonic sucrose medium.

Accumulation of exogenous phosphoenolpyruvate against the concentration gradient was observed when human red cells were incubated in an acidified isotonic sucrose medium. Fluoride increased the apparent accumulation by inhibition of the intracellular metabolic interconversion of the phosphate compound. The accumulation appeared to be specific for phosphoenolpyruvate and the accumulation rate for 3-phosphoglycerate, which has a molecular size and pKa similar to those of phosphoenolpyruvate, was less than one-tenth of the rate of phosphoenolpyruvate. Red cells incubated in the acidified sucrose medium tended to adhere to each other when examined with a scanning electron microscope.     

Clinical analysis on steroids. XII. Occurrence of D-homoannulation during the hot acid hydrolysis of 5β-pregnane-3α,20α-diol disulfate

Two D-homosteroids were isolated from the hydrolyzate of 5β-pregnane -3α,20α-diol disulfate (II) when it was refluxed in 3N hydrochloric acid. The structures of these steroids have been elucidated as 17α-methyl-D-homo-5β-androstane-3α, 17aβ-diol (VI) and 17α-methyl-17aγb-chloro-D-homo-5β-androstan-3α-ol (VIII) by instrumental analyses. The former was identical with a synthetic specimen derived from 5β-pregnane-3α,20β-diol di-sulfate (IV) by uranediol rearrangement. The main hydrolyzates obtained were 17α-ethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol (V) and 5β-pregnane-3α, 20α-diol (III).