Configuration of actin microfilaments during sporangium development inAchlya bisexualis: comparison of two staining protocols

Summary The spatial organization of actin microfilaments during the asexual life cycle ofAchlya bisexualis has been examined by two methods. One is the standard procedure described by Heath [Eur J Cell Biol (1987) 44: 10–16], in which specimens are fixed with formaldehyde and then stained with rhodamine-phalloidin. In the other, introduced by Sonobe and Shibaoka [Protoplasma (1989) 148: 80–86], specimens are treated with the protein crosslinking agent MBS (m-maleimidobenzoyl-N-hydroxysuccinimide) before fixation and staining. Both methods display the actin-rich cleavage zones that outline the developing zoospores. However, in extending hyphae and young sporangia the images are significantly different. Specimens pretreated with MBS display more prominent axial microfilament cables than do standard specimens, while peripheral actin plaques are sparse or absent. The results suggest that actin microfilaments occur in several configurations, some of which may be obscured by the standard fixation procedure. Pretreatment with MBS, though probably subject to artefacts of its own, may help preserve some features that would otherwise be missed.Abbreviations Rh-Phal rhodamine phalloidin - MBS m-maleimidobenzoyl-N-hydroxysuccinimide - PIPES piperazine-N,N-bis [2-ethanesulfonic acid] - EGTA ethylene glycol-bis (-aminoethyl ether) N,N-tetraacetic acid - DMSO dimethyl sulfoxide     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

ATP synthesis associated with the conversion of hexachlorocyclohexane related compounds

Clostridium rectum strain S-17 converts -1,2,3,4,5,6-hexachlorocyclohexane (HCH) related compounds to chlorobenzenes. The metabolites from -1,2,3,4,5,6-hexachlorocyclohexene and -1,3,4,5,6-pentachlorocyclohexene are identified as 1,2,4-trichlorobenzene and 1,4-dichlorobenzene, respectively. ATP synthesis, converting these chlorinated compounds, is observed in the cell suspension of C. rectum as indicated by luciferase-luciferin reaction and phosphorylation of ³²P-labeled phosphate. These observation lead to the conclusion that HCH and related compounds serve as artificial electron acceptors of the Stickland reaction, and therefore, the reductive dechlorination is associated with ATP synthesis.Abbreviations HCH -1,2,3,4,5,6-hexachlorocyclohexane - HCCH -1,2,3,4,5,6-hexachlorocyclohexene - PCCH -1,3,4,5,6-pentachlorocyclohexene - TCCH -3,4,5,6-tetrachlorocyclohexene - 1,2,4-TCB 1,2,4-trichlorobenzene - 1,4-DCB 1,4-dichlorobenzene - MCB monochlorobenzene - DTT 1,4-dithiothreitol - IAA monoiodoacetic acid     

Comparative effects of sorbitol and sucrose as main carbon energy sources in micropropagation of apricot

In vitro proliferation and rooting capacity of San Castrese and Portici apricots (Prunus armeniaca L.) were tested on modified MS medium enriched with varying growth regulator concentrations and sucrose (58.4 mM) or sorbitol (116.8 mM) as main carbon energy sources. The interaction of proliferation and rooting media was also studied.Proliferation of both cultivars was proportional to benzyladenine (BA) concentration and enhanced with sorbitol media. However, 8.8 M BA was often associated with hyperhydricity, particularly when shoots were grown on sucrose media. Newly proliferated shoots elongated better on sorbitol media. The positive influence of sorbitol on proliferation and shoot growth was not due to osmotic effects. Moreover, sorbitol showed a positive carryover effect in hastening rooting of Portici. By contrast, when transferred to sorbitol rooting media, the shoots of both cultivars generally showed low rooting, with short, thick roots.Up to 70% of the plantlets that produced roots in sucrose media enriched with indolebutyric acid were successfully acclimatized when they were dipped in a benomyl (0.075% w/v) suspension before being transplanted with care being taken to prevent over-wetting of soil.Abbreviations BA 6-benzyladenine - IBA indolebutyric acid - GA₃ gibberellic acid - SEM standard error of mean     

The catalysis of nucleotide polymerization by compounds of divalent lead

Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A adenosine - U uridine - Im imidazole - MeIm 1-methyl-imidazole - EDTA ethylenediaminetetraacetic acid - pA adenosine 5-phosphate - pU uridine 5-phosphate - Ap adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - AppA P₁,P₂-diadenosine 5-diphosphate - pNp (N = A,U) nucleotide 2(3), 5-diphosphate - ImpA adenosine 5-phosphoreimidazolide - ImpU uridine 5-phosphorimidazolide - A ²pA adenylyl-[25]-adenosine - A ³pA adenylyl-[35]-adenosine - pA ²pA 5-phospho-adenylyl-[25]-adenosine - pA ³pA 5-phospho-adenylyl-[35]-adenosine - pUpU 5-phospho-uridylyl-uridine - pApU 5-phospho-adenylyl-uridine - pUpA 5-phospho-uridylyladenine - (pA)n (n, 2,3,4,) oligoadenylates with 5 terminal phosphate - ImpApA 5-phosphorimidazolide of adenylyl adenosine - (pA) ₅₊ pentamer and higher oligoadenylates with 5 terminal phosphate - (Ap)nA (n = 2,3,4) oligoadenylates without terminal phosphates In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage     

Sterols of male and female compound inflorescences of Zea mays L.

A comparison has been made between the sterols of male and female inflorescences and of pollen from Zea mays. The female inflorescence was shown to contain cholesterol, 24-methylcholesterol, 24-ethyl-5,22-cholestadien-3-ol, 24-ethylcholesterol and (28Z)-24-ethylidenecholesterol. Themale inflorescence contained the same five compounds together with 24-methylenecholesterol. Pollen contained 24-methylenecholesterol as its main sterol together with lesser amounts of cholesterol, 24-ethylcholesterol, (28Z)-24-ethylidenecholesterol, 24-methylene-5-cholest-7-en-3-ol and 4-methyl-24-methylene-5-cholest-7-en-3-ol.     

Molecular cloning and expression of multiple cellulase genes of Ruminococcus flavefaciens strain 186 in Escherichia coli

Summary Cellulase genes of the ruminant micro-organism Ruminococcus flavefaciens strain 186 have been cloned and expressed in Escherichia coli using the bacteriophage vector NM1149. Twenty-six clones showed expression of endo--1,4-d-glucanases and were divided into four groups according to their insert sizes of approximately 2, 3, 4 or 9 kilobases (kb). Two of the clones with 4 kb inserts also showed exo--1,4-d glucanase activity while two clones with 9 kb inserts showed -glucosidase activity. One of the clones with 9 kb inserts (M903) showed the activities of all three cellulase activities. In addition, two of the 4 kb-insert clones and one 9 kb-insert clone degraded Avicel (PH101).     

Cytoplasmic streaming in the cell model ofNitella

Summary The plasmalemma ofNitella internode was made freely permeable to solutes by treating the cell with detergent and EGTA under plasmolysis. After the treatment, the cytoplasmic streaming was stopped by bathing the cell in a medium lacking ATP. The streaming was reactivated by perfusing the exterior of the permeabilized cell with a medium containing both Mg²⁺ and ATP. The reactivated streaming could be reversibly stopped by depletion of ATP. However, depletion of Mg²⁺ irreversibly inhibited the streaming.Cytochalasin B at 5 g/ml irreversibly inhibited the reactivated streaming within a minute, showing that microfilaments are involved in the streaming.Abbreviations ATP adenosine-5-triphosphoric acid - CB cytochalasin B - CyDTA cyclohexanediamine-N,N-tetraacetic acid - DMSO dimethylsulfooxide - DTT dithiothreitol - EGTA ethyleneglycol-bis(-aminoethylether)-N,N tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - PMSF phenylmethyl-sulfonylfluoride     

Synthesis ofNeoglycoproteins containing the 3,6-di-O-methyl-β-d-glucopyranosyl epitope and their use in serodiagnosis of leprosy

A stratagem for the synthesis ofneoglycoproteins suitable for the selective serodiagnosis of leprosy is described in which synthetic 3,6-di-O-methyl--d-glucopyranose, the epitope of phenolic glycolipid I fromMycobacterium leprae, was used. Condensation of 8-methoxycarbonyloctanol with the acetobromo derivative of 3,6-di-O-methylglucose gave 8-methoxycarbonyloctyl 2,4-di-O-acetyl-3,6-di-O-methyl--d-glucopyranoside in 65% yield, and with absolute stereospecificity for the anomer. The deacylated product was converted to the crystalline hydrazide and coupled to bovine gamma globulin, bovine serum albumin and poly-d-lysinevia intermediate acyl azide formation to produce the 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranosyl polypeptides. Theneoglycoproteins were highly sensitive in ELISA and emulated the specificity of the native glycolipid in analysis of sera from patients throughout the spectrum of leprosy and from different geographical regions. The 8-carbonyloctyl 3,6-di-O-methyl--d-glucopyranoside-bovine serum albumin was also synthesized and shown to have about one-half the activity of the -linkedneoglycoprotein. A different synthetic approach produced the 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhamnopyranoside-bovine serum albumin which was also highly sensitive and specific for the serodiagnosis of leprosy. The presence of the second sugar unit, similar to that in the native glycolipid but for the absence ofO-methyl groups, seemed to provide a probe with greater felicity for the serological detection of tuberculoid leprosy.Thus, the results indicate that highly sensitive and specific antigen probes for the serodiagnosis of leprosy can be constructed based only on the terminal one or two sugars of phenolic glycolipid I, and the synthetic approach leads to the formation of haptens with absolute stereospecificity.Nomenclature BGG bovine gamma globulin - PGL-I phenolic glycolipid I - PDL poly-d-lysine - PBS phophate-buffered saline - 3,6-Me₂-Glc-Link-BSA 8-carbonyloctyl 3,6-di-O-methyl-glucopyranoside-bovine senalbumin - 3,6-Me₂-Glc-Rha-Link-BSA 8-carbonyloctyl 4-O-(3,6-di-O-methyl--d-glucopyranosyl)--l-rhan pyranoside-BSA     

DNA variations in regenerated plants of pea (Pisum sativum L.)

Summary The aim of this study was to determine whether DNA variations could be detected in regenerated pea plants. Two different genotypes were analyzed by cytogenetic and molecular techniques: the Dolce Provenza cultivar and the 5075 experimental line. Dolce Provenza regenerated plants showed a reduction in DNA content, particularly at the level of unique sequences and ribosomal genes. Moreover, regeneration was associated with an increase in DNA methylation of both internal and external cytosines of the CCG sequence. On the other hand, the DNA content of the 5075 line remained stable after regeneration. DNA reduction was found only in 5075 plants regenerated from callus cultures maintained for long incubation periods (about a year). The DNA variations observed are discussed both in relation to the genotype source and the role of tissue-culture stress.     

Occurrence of pppApp-synthesizing activity in actinomycetes and isolation of purine nucleotide pyrophosphotransferase

The occurrence of adenosine 5-triphosphate-3-diphosphate-synthesizing activity was detected in five strains of actinomycetes; Streptomyces morookaensis, Streptomyces aspergilloides, Streptomyces hachijoensis, Actinomyces violascens and Streptoverticillium septatum, out of 825 strains of actinomycetes, bacteria, fungi and imperfecti. Purine nucleotide pyrophosphotransferase were extracellularly excreted associating with the cell growth, and were purified partially or to apparent homogeniety from the culture filtrate. The enzymes are a monomeric protein with a molecular weight of 18000–26000 and synthesize adenosine, guanosine and inosine 5-phosphate (mono, di or tri)-3-diphosphate such as pApp, ppApp, pppApp, pGpp, ppGpp, pppGpp and pppIpp by transferring a pyrophosphoryl group from the 5-position of ATP, dATP and pppApp to the 3-position of purine nucleotides in the presence of a divalent cation and in alkaline state.Abbreviations pppApp adenosine 5-triphosphate 3-diphosphate - ppApp adenosine 5-diphosphate 3-diphosphate - pApp adenosine 5-monophosphate 3-diphosphate - pppGpp guanosine 5-triphosphate 3-diphosphate     

Molecular evolution of the nicotinic acetylcholine receptor: An example of multigene family in excitable cells

An extensive phylogenetic analysis of the nicotinic-acetylcholine-receptor subunit gene family has been performed by cladistic and phenetic methods. The conserved parts of amino acid sequences have been analyzed by CLUSTAL V and PHYLIP software. The structure of the genes was also taken in consideration. The results show that a first gene duplication may have occurred before the appearance of Bilateria. Three subfamilies then appeared: I-the neuronal -bungarotoxin binding-site subunits (7, 8); III-the neuronal nicotinic subunits (2–6, 2–4), which also contain the muscle acetylcholine-binding subunit (1); and IV—the muscle non- subunits (1, , ). The Insecta subunits (subfamily II) could be orthologous to family III and IV. Several tissular switches of expression from neuron to muscle and the converse can be inferred from the extant expression of subunits and the reconstructed trees. The diversification of the neuronal nicotinic subfamily begins in the stem lineage of chordates, the last duplications occurring shortly before the onset of the mammalian lineage. Such evolution parallels the increase in complexity of the cholinergic systems.Abbreviations -Bgt -bungarotoxin - ACh acetylcholine - MP maximum of parsimony - MYA million years ago - NJ neighbor-joining - nAChR nicotinic acetylcholine receptor Correspondence to: N. Le Novère     

Ellagic acid toxicity and interaction with benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol in human bronchial epithelial cells

Ellagic acid, a plant phenol present in various foods consumed by humans, has been reported to have both anti-mutagenic and anti-carcinogenic potential. To evaluate the potential anti-carcinogenic property of ellagic acid, we tested its effects on the toxicity of ben-zo[a]pyrene and benzo[a]pyrene, 7,8-dihydrodiol and binding of benzo[a]yrene to DNA in cultured human bronchial epithelial cells. The toxicity of ellagic acid itself for human bronchial epithelial cells was also determined. Using a colony-forming efficiency assay, it was found that a nontoxic concentration of ellagic acid (5 g/ml) enhanced the toxicity of benzo[a]pyrene.7,8-dihydrodiol in human bronchial epithelial cells. In contrast, ellagic acid at concentrations of l.5 and 3.0 g/ml inhibited binding of benzo[a]pyrenemetabolites to DNA in these cells. An explanation for the potentiating effect of ellagic acid on the toxicity of benzo[a]pyrene, 7,8-dihydrodiol will require further investigation into the possible mechanisms of interaction between these two compounds.Abbreviations B[a]P benzo[a]pyrene - B[a]P 7,8-DHD (±)trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene - B[a]PDE-1 (±)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-2 (±) 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-1:dG N²-]10{7,8,9-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - B[a]PDE-2:dG NZ-{10-[7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - CFE colony forming efficiency - EA ellagic acid - HBE human bronchial epithelial     

Structure of a new nonasaccharide isolated from human milk: VI2Fuc,V4Fuc,III3Fuc-p-lacto-N-hexaose

The structure of a new nonasaccharide isolated from human milk has been investigated. By using methylation analysis, FAB-MS and¹H-and¹³C-NMR spectroscopy as basic methods of structural investigation, this oligosaccharide was identified as VI²--Fuc,V⁴-Fuc,III³--Fuc-p-lacto-n-hexaose: Fuc1-2Gal1-3[Fuc1-4]GlcNAc1-3Gal1-4[Fuc1-3]GlcNAc1-3Gal1-4Glc.Abbreviations COSY correlation spectroscope - DP degree of polymerisation - FAB-MS fast atom bombardment-mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - GLC gas-liquid chromatography     

Immunofluorescence localization of the epidermolytic toxin target in mouse epidermal cells and tissue

Summary An epidermolytic toxin target was observed in keratohyalin granules of sectioned epidermis by a direct fluorescence procedure using FTC-toxin, but not by an indirect procedure using sequential reaction with toxin, anti-toxin and FTC-secondary antibody. The investigation of the two procedures was extended to keratinocytes. A dispase digestion procedure yielded three fractions which corresponded to basal, spinous and granular cells according to biochemical and morphological criteria. It was shown that the direct and indirect procedures both detected the toxin target in the keratohyalin granules of granular cells, but that the indirect procedure was very insensitive. In control experiments, the profilaggrin of keratohyalin granules was detected readily in cells by a direct procedure using FTC-antiprofilaggrin but only weakly by an indirect double antibody procedure. Insensitivity to indirect procedures thus appears to be a particular property of the keratohyalin granule site. It was shown that the toxin target was readily accessible in permeable (trypsin-isolated) granular cells but inaccessible in impermeable (dispase-isolated) cells.     

Isolation and characterization of a multienzyme complex containing DNA replicative enzymes from mitochondria ofS. cerevisiae

A 40 S multienzyme complex containing mtDNA polymerase was isolated from mitochondria ofS. cerevisiae by density gradient centrifugation and by gel filtration chromatography. Besides DNA polymerase, RNA polymerase, primase, 35 exonuclease and an ATPase activities were found to be associated with it. The presence of some of these enzymes were confirmed by Western blot. This high molecular weight multienzyme complex containing DNA has most of the attributes of a putative replisome.Abbreviations BCIP 5 bromo 4 chloro 3 indolyl phosphate - mtDNA mitochondrial DNA - NBT Nitroblue tetrazolium - PBS phosphate buffered saline     

The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update

Mitochondrial dysfunction has been shown to participate in the induction of apoptosis and has even been suggested to be central to the apoptotic pathway. Indeed, opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential (_m), release of apoptogenic factors and loss of oxidative phosphorylation. In some apoptotic systems, loss of _m may be an early event in the apoptotic process. However, there are emerging data suggesting that, depending on the model of apoptosis, the loss of _m may not be an early requirement for apoptosis, but on the contrary may be a consequence of the apoptotic-signaling pathway. Furthermore, to add to these conflicting data, loss of _m has been demonstrated to not be required for cytochrome c release, whereas release of apoptosis inducing factor AIF is dependent upon disruption of _m early in the apoptotic pathway. Together, the existing literature suggests that depending on the cell system under investigation and the apoptotic stimuli used, dissipation of _m may or may not be an early event in the apoptotic pathway. Discrepancies in this area of apoptosis research may be attributed to the fluorochromes used to detect _m. Differential degrees of sensitivity of these fluorochromes exist, and there are also important factors that contribute to their ability to accurately discriminate changes in _m.     

Viability and activity in readily culturable bacteria: a review and discussion of the practical issues

In microbiology the terms viability and culturability are often equated. However, in recent years the apparently self-contradictory expression viable-but-nonculturable (VBNC) has been applied to cells with various and often poorly defined physiological attributes but which, nonetheless, could not be cultured by methods normally appropriate to the organism concerned. These attributes include apparent cell integrity, the possession of some form of measurable cellular activity and the apparent capacity to regain culturability. We review the evidence relating to putative VBNC cells and stress our view that most of the reports claiming a return to culturability have failed to exclude the regrowth of a limited number of cells which had never lost culturability. We argue that failure to differentiate clearly between use of the terms viability and culturability in an operational versus a conceptual sense is fuelling the current debate, and conclude with a number of proposals that are designed to help clarify the major issues involved. In particular, we suggest an alternative operational terminology that replaces VBNC with expressions that are internally consistent.     

Natural fermentation of soybean (glycine max) with added salt for ‘soy-daddawa’ production

Microbial growth, pH and titratable acidity decreased with increasing NaCl concentration during the fermentation of soybeans. NaCl at 1% (w/w) in the fermenting mash improved the organoleptic quality of soy-daddawa.The author is with the Department of Microbiology, Obafemi Awolowo University, lle-lfe, Nigeria.