Morphological change and enhanced pigment production of monascus when cocultured with saccharomyces cerevisiae or aspergillus oryzae

When a Monascus isolate, a producer of Monascus pigments, was cocultured with either Saccharomyces cerevisiae or Aspergillus oryzae in a solid sucrose medium, there were significant morphological changes in Monascus culture. Cocultures exhibited cell mass increases of 2 times and pigment yield increases of 30 to 40 times compared to monocultures of Monascus. However, enhanced cell growth, an increase in pigment production, and morphological change did not occur in coculture with Bacillus cereus. Saccharomyces cerevisiae was more effective at enhancing pigment production than Asp. oryzae. Enhanced cell growth and increased pigment production occurred only in conjunction with morphological changes. Culture filtrates of S. cerevisiae were also effective in inducing morphology change in Monascus, similar to culture broths of S. cerevisiae. The hydrolytic enzymes produced by S. cerevisiae, such as amylase, and chitinase, are thought to be the effectors. The commercial enzymes alpha-amylase and protease from Asp. oryzae both caused a morphological change in Monascus and were effective in enhancing pigment production. However, lysozyme, alpha-amylase and protease from Bacillus species, protease from Staphylococcus, and chitinase from Streptomyces were not effective. The hydrolytic enzymes which cause a morphological change of Monascus culture and enhancement of pigment production are thought to be capable of degrading Monascus cell walls. An approximate 10-fold increase in pigment production was observed in liquid cocultures with S. cerevisiae. Copyright 1998 John Wiley & Sons, Inc.     

Integration of the temperate phage phiU into the putative tRNA gene on the chromosome of its host Rhizobium leguminosarum biovar trifolii

The plasmid pCI6, carrying the attP site of the temperate phage phiU, integrates into the attB site on the chromosome of Rhizobium leguminosarum biovar trifolii strain 4S. The 4 kb EcoRI-HindIII region of pCI6 involved in site-specific integration was subcloned as the attP fragment of phage phiU and sequenced. The attL fragment, one of the new DNA junctions generated from the insertion of pCI6 into the chromosome of the host Rhizobium, was used as a hybridization probe for isolation of the attB fragment of strain 4S. The nucleotide sequence of the 2 kb PstI fragment of strain 4S, which hybridized with the attL fragment, was decided and compared with that of the attP fragment. A 53 bp common sequence was expected to be the core sequence of site-specific integration between phage phiU and strain 4S. One of the ORFs on the attP fragment, which was located adjacent to the core sequence, had structural homology to the integrase family. However, the attB fragment showed high homology with the tRNA genes of Agrobacterium tumefaciens and E. coli. A 47 bp sequence of the 53 bp core sequence overlapped with this tRNA-like sequence. This indicates that the target site of phage phiU integration is the putative tRNA gene on the chromosome of the Rhizobium host.     

Cloning and Bacterial Expression of Sesquiterpene Cyclase, a Key Branch Point Enzyme for the Synthesis of Sesquiterpenoid Phytoalexin Capsidiol in UV-Challenged Leaves of Capsicum annuum

Sesquiterpene cyclase, a branch point enzyme in the generalisoprenoid pathway for the synthesis of phytoalexin capsidiol,was induced in detached leaves of Capsicum annuum (pepper) byUV treatment. The inducibility of cyclase enzyme activitiesparalleled the absolute amount of cyclase protein(s) of pepperimmunodetected by monoclonal antibodies raised against tobaccosesquiterpene cyclase. A cDNA library was constructed with poly(A)⁺RNA isolated from 24 h UV-challenged leaves of pepper. A cDNAclone for sesquiterpene cyclase in pepper was isolated by usinga tobacco 5-epi aristolochene synthase gene as a hetero-logousprobe. The predicted protein encoded by this cDNA was comprisedof 559 amino acids and had a relative molecular mass of 65,095.The primary structural information from the cDNA clone revealedthat it shared 77%, 72% and 49% identity with 5-epi aristolochene,vetispiradiene, and cadinene synthase, respectively. The enzymaticproduct catalyzed by the cDNA clone in bacteria was identifiedas 5-epi aristolochene, as judged by argentation TLC. RNA blothybridization demonstrated the induction of an mRNA consistentwith the induction of cyclase enzyme activity in UV-treatedpepper. (Received March 2, 1998; Accepted June 15, 1998)     

Composite and clinal distribution of Glycine soja in Japan revealed by RFLP analysis of mitochondrial DNA

 Wild soybean (Glycine soja Sieb. et Zucc.), regarded as the progenitor of cultivated soybean [G. max (L.) Merr.], is widely distributed in East Asia. We have collected 1097 G. soja plants from all over Japan and analyzed restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA) in them. Based on the RFLPs detected by gel-blot analysis, using coxII and atp6 as probes, the collected plants were divided into 18 groups. Five mtDNA types accounted for 94% of the plants examined. The geographic distribution of mtDNA types revealed that, in many regions, wild soybeans grown in Japan consisted of a mixture of plants with different types of mtDNA, occasionally even within sites. Some of the mtDNA types showed marked geographic clines among the regions. Additionally, some wild soybeans possessed mtDNA types that were identical to those widely detected in cultivated soybeans. Our results suggest that the analysis of mtDNA could resolve the maternal lineage among plants of the genus Glycine subgenus Soja. Received: 16 June 1997/Accepted: 5 August 1997     

Free D- and L-amino acids in ventricular cerebrospinal fluid from Alzheimer and normal subjects

Summary Free D-Ser, D-Asp and total D-amino acids were significantly higher (p < 0.05) in Alzheimer (AD) ventricular CSF than in normal CSF. There was no significant difference in the total L-amino acids between AD and normal CSF, but L-Gln and L-His were significantly higher (p < 0.05) in ADCSF. The higher concentrations of these D- and L-amino acids in AD ventricular CSF could reflect the degenerative process that occurs in Alzheimer's brain since ventricular CSF is the repository of amino acids from the brain.     

An 8 kb Nucleotide Sequence at the 3' Flanking Region of the sspC Gene (184{degrees}) on the Bacillus subtilis 168 Chromosome Containing an Intein and an Intron

As part of the Bacillus subtilis genome sequencing project,we determined the complete nucleotide sequence of an 8000-bpfragment downstream of the sspC gene (184°) of the B. subtilis168 chromosome. The sequence analysis shows that the sspC geneis located inside of the SPß region, which differsfrom the current genetic map of B. subtilis 168. This regioncontains 12 putative ORFs (yojQ through yojZ and sspC). A homologysearch for the deduced products of the ORFs shows signi.cantsimilarities to enzymes involved in deoxyribonucleotide metabolism:ribonucleotide reductase (Nrd) E, NrdF, thioredoxinand dUTPase.Interestingly, this DNA fragment includes two split genes, yojPcontaining conserved motifs of an intein and yojQ and yojS withan 808-bp intervening sequence for a putative intron structure.In addition, the yojR gene includes a putative new DNA replicationterminator.     

Analysis of intracellular pH in the yeast Saccharomyces cerevisiae under elevated hydrostatic pressure: a study in baro- (piezo-) physiology

Hydrostatic pressure is a distinctive feature of deep-sea environments, and this thermodynamic parameter has potentially inhibitory effects on organisms adapted to living at atmospheric pressure. In the yeast Saccharomyces cerevisiae, hydrostatic pressure causes a delay in or cessation of growth. The vacuole is a large acidic organelle involved in degradation of cellular proteins or storage of ions and various metabolites. Vacuolar pH, as determined using the pH-sensitive fluorescent dye 6-carboxyfluorescein, was analyzed in a hydrostatic chamber with transparent windows under elevated hydrostatic pressure conditions. A pressure of 40–60 MPa transiently reduced the vacuolar pH by approximately 0.33. A vma3 mutant defective in vacuolar acidification showed no reduction of vacuolar pH after application of hydrostatic pressure, indicating that the transient acidification is mediated through the function of vacuolar H⁺-ATPase. The vacuolar acidification was observed only in the presence of fermentable sugars, and never observed in the presence of ethanol, glycerol, or 3-o-methyl-glucose as the carbon source. Analysis of a glycolysis-defective mutant suggested that glycolysis or CO₂ production is involved in the pressure-induced acidification. Hydration and ionization of CO₂ is facilitated by elevated hydrostatic pressure because a negative volume change (ΔV < 0) accompanies the chemical reaction. Moreover the glucose-induced cytoplasmic alkalization is inhibited by elevated hydrostatic pressure, probably because of inhibition of the plasma membrane H⁺-ATPase. Therefore, the cytoplasm tends to become acidic under elevated hydrostatic pressure conditions, and this could be crucial for cell survival. To maintain a favorable cytoplasmic pH, the yeast vacuoles may serve as proton sequestrants under hydrostatic pressure. We are investigating the physiological effects of hydrostatic pressure in the course of research in a new experimental field, baro- (piezo-) physiology. Received: January 22, 1998 / Accepted: February 16, 1998     

Kin-related social organization in a winter population of the voleClethrionomys rufocanus

Kinship amongClethrionomys rufocanus was investigated during the winter of 1992/93 in a 3-ha enclosure using both molecular and catch-mark-release techniques. Forty-six adult voles (22 males and 24 females) having high heterozygosities, which were collected from several natural populations, were released into the enclosure on 29 September 1992. Most fall-born individuals of both sexes stayed in their natal site during the non-breeding period (December–March), although reproductively active females dispersed during the fall breeding season (October–November). These philopatric individuals aggregated and formed an maternal family in the winter. Several females which failed to reproduce were solitary during this season. Some individuals which were derived from several families also aggregated into a mixed lineage group. Survival rate of fall-born voles from earlier litters was higher than that from later ones. Maternal families broke up soon after the onset of spring reproduction. Most females established a territory near the wintering site and made a kincluster, in which close relatives neighbored each other. Maternal families in winter bring about female kin-clusters in spring, which may influence reproductive output in the breeding season.     

Correlation analysis between fatty acid compositions of zooplankter individuals, fed on different phytoplankton species by means of pyrolysis–gas chromatography combined with on-line methylation

Pyrolysis–gas chromatography (Py–GC) combined with on-line methylation was applied to a correlation analysis between the distributions of fatty acid components in the lipids of zooplankter individuals and those of ingested algae using principal component analysis (PCA). Py–GC in the presence of organic alkali, tetramethylammonium hydroxide (TMAH), was used to estimate the apparent distributions of fatty acid components contained in a single individual zooplankter weighing several tens of micrograms and a small sample size of ingested algae samples in the order of 10 μg. The observed fatty acid compositions were used as a database for the PCA in order to discriminate the zooplankton and ingested algae samples. The result obtained indicated that the fatty acid compositions of zooplankton individuals used in this work were significantly reflected in those of their ingested food in spite of some contribution from isomerization and/or elongation of fatty acid components during digestion of the ingested algae phytoplankton in living zooplankters.     

Molecular Analyses of the Cu/Zn Superoxide Dismutase Gene in Patients with Familial Amyotrophic Lateral Sclerosis (ALS) in Japan

1. Amyotrophic lateral sclerosis (ALS) is a degenerative disorder characterized by selective damage to the neural system that mediates voluntary movement. Although the pathophysiologic process of ALS remains unknown, about 5 to 10% of cases are familial. According to genetic linkage studies, the familial ALS (FALS) gene has been mapped on chromosome 21 in some families and recent work identified some different missense mutations in the Cu/Zn superoxide dismutase gene in FALS families.2. We recently identified five mutations in six FALS families. The mutations identified in our FALS families are H46R, L84V, I104F, S134N, and V148I. The H46R mutation that locates in the active site of Cu/Zn SOD gene is associated with two Japanese families with very slow progression of ALS. On the other hand, the L84V mutation associated with a rapidly progressive loss of motor function with predominant lower motor neuron manifestations.3. In the family with the V148I, the phenotype of the patient varied very much among the affected members. One case had weakness of the lower extremities at first and died without bulbar paresis. The second case first noticed wasting of the upper limbs with bulbar symptoms, but the third had weakness of upper extremities without developing dysarthria nor dysphagia until death. These mutations account for 50% of all FALS families screened, although Cu/Zn SOD gene mutations are responsible for less than about 13–21% in the Western population.4. Our results indicate that the progression of disease with mutations of Cu/Zn SOD is well correlated with each mutation. The exact mechanism by which the abnormal Cu/Zn SOD molecules selectively affect the function of motor neurons is still unknown.