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.     

An alfalfa (Medicago sativa L.) cDNA encoding an acidic leghemoglobin (MsLb3)

We have found an alfalfa cDNA clone that encodes an acidic leghemoglobin. To date, 14 alfalfa leghemoglobin clones have been identified. Five different leghemoglobin components have been biochemically defined on the basis of their pI. A higher-resolution comparison, provided by sequence data analysis, identifies six leghemoglobin classes. All 14 leghemoglobins are assigned to the six classes, which can be distributed among the five leghemoglobin components. The newly identified leghemoglobin is the only member of a sixth class of leghemoglobins, and it also is the only member of one of the acidic leghemoglobin components IV or V.     

High crossability of wild barley (Hordeum spontaneum C. Koch) with bread wheat and the differential elimination of barley chromosomes in the hybrids

Four bread wheat (Triticum aestivum L.) cultivars, Aobakomugi, Chinese Spring, Norin 61 and Shinchunaga, were pollinated with five barley lines/cultivars consisting of three cultivated barley (Hordeum vulgare L.) lines, Betzes, Kinai 5 and OHL089, and two wild barley (Hordeum spontaneum C. Koch) lines, OUH602 and OUH324. Crossability, expressed as the percentage of embryo formation, varied from 0 to 55.4% among the cross combinations. The two wild barley lines generally had a higher crossability than the previously reported best pollinator, Betzes, and some Japanese wheat cultivars were better as the female parent than Chinese Spring. Ninety four hybrid plants were obtained from 250 embryos cultured, and their somatic chromosome numbers ranged from 21 to 36. Eighteen plants were mosaic in chromosome number. Twenty one-chromosome plants appeared most frequently (45.7%) followed by 28-chromosome plants (14.9%). C-banding analysis revealed that elimination of barley chromosomes was mainly responsible for the occurrence of aneuploid plants. In hypoploids derived from Betzes-crosses, chromosome 5 was preferentially eliminated as previously reported, while in hypoploids derived from OUH602-crosses, chromosome 4 was preferentially eliminated. The wild barley line OUH602 may be a useful parent for producing a new wheat-barley addition set because of its high crossability with wheat and a different pattern of chromosome elimination.     

On morphological variation in Keratella cochlearis populations from Holstein lakes (Northern Germany)

Keratella cochlearis occurs in many Holstein lakes (northern Germany) as three well defined and separated forms: cochlearis, hispida, and tecta, each showing very little variation between the lakes. The present data show that the tecta form did not originate from a Lauterborn cycle.     

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).     

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.     

Occurrence of reducing terminal N-acetylglucosamine 3-sulfate and fucosylated outer chains in acidic N-glycans of porcine zona pellucida glycoproteins

Structures of acidic N-glycans released from porcine zona pellucida glycoproteins by hydrazinolysis were studied. The results indicated that the acidic glycans are of mono- to tetraantennary complex-type with and without N-acetyllactosamine repeating units. Sulfated residues are not only located at the C-6 position of GlcNAc included in the N-acetyllactosamine repeating units, but also at the C-6 position of GlcNAc in the non-repeated antennae and at the C-3 position of reducing terminal GlcNAc residue. Analysis of the oligosaccharide fragments released by endo--galactosidase digestion and by hydrazine/nitrous acid treatment also revealed that various sulfated and non-sulfated forms of fucosylated structures such as Fuc12Gal14(±SO–36)GlcNAc (type 2H), Gal14(Fuc13)(±SO–36)GlcNAc(Lex) and Fuc13 or 4(±SO–36)GlcNAc, are expressed in the repeated outer chain moieties.     

Diallel analysis of submergence tolerance in rice,Oryza sativa L.

Summary The genetics of submergence tolerance in rice was studied in a 10 × 10 half-diallel cross set involving 10 lowland rice varieties, four of which were tolerant (FR13A, FR43B, Kurkaruppan, and Goda Heenati) and the remaining six were nontolerant (RD19, IR42, IR17494-32-1, IR19672-24-3, Jagannath, and CR1009). Estimates of genetic parameters following Hayman's method showed significant additive and nonadditive gene action and the latter appeared to be solely due to dominance. Narrow sense heritability (0.70) indicated that additive gene effects were more important in the inheritance of the trait. Tolerance was dominant over nontolerance and the average dominance was within the range of incomplete dominance. Dominant alleles were more concentrated in the three tolerant parents, FR13A, Kurkaruppan, and FR43B in that order. Wr/Vr graphic analysis suggested the involvement of both major and minor genes. Combining ability analysis by Griffing's method also indicated significance of both additive and nonadditive effects, and the former appeared to be more important than the latter. The hybrids involving FR13A with RD19, IR42, and IR17494-32-1, and those of Kurkaruppan with RD19 and CR1009 appeared to be promising for incorporating an adequate level of tolerance to submergence into lowland rice cultivars.     

Effect of giving insulin and adrenalin alternately on changes in the level of blood glucose in chickens of different breeds

Summary Differences in reaction to exogenous insulin and adrenalin were studied among chickens of Leghorn, White Rock and Rhode Island Red breeds, using level of blood glucose as indicator of the reaction to hormones. It was found that: 1.) The physiological quantity of glucose in blood in chickens of the three breeds ranged from 157 to 194 mg %. 2.) The reaction of chickens to insulin and adrenalin given alternately, measured by changes of blood glucose, differed according to the breed. The smallest reaction to hormones was in Leghorn, the biggest in White Rock. 3.) The chickens of Leghorn and Rhode Island Red breeds, as well as Leghorn and White Rock breeds, differed significantly in maximal glucose level after adrenalin, 4.) The correlation between the normal quantity of blood glucose and the body weight of birds appeared highly significant in cockerels of all three races combined.     

Modulation of cytokine responses of murine CD8+ αβ intestinal intraepithelial lymphocytes by IL-4 and IL-12

The immune responses of the intestine mucosa feature the noninflammatory type, such as IgA production and oral tolerance. Th2 type cytokines have been implicated in the induction of these noninflammatory responses. In the present study, cytokine responses of CD8+ and CD4+ TCR+ intestinal intraepithelial lymphocyte ( iIEL) subsets to TCR stimulation under the influence of IL-12, IL-4, or CD28 costimulation were examined. IL-12 enhanced production of IL-10 and IFN- by the CD8+ iIEL significantly but only marginally affected the CD8+ subset, whereas IL-4 induced IL-4, IL-5, and IL-10 production and augmented TGF- production by both subsets. CD28 costimulation induced production of Th2 cytokines by CD4+ iIEL in the absence of exogenous IL-4. Unlike lymph node CD4+ cells, the CD28 costimulation-induced Th2 differentiation of CD4+ iIEL was not inhibited by IFN-. These results demonstrate active cytokine production by CD4+, CD8+, as well as CD8+ iIEL. The Th2-skewed cytokine profile of CD8+ iIEL and the IFN--resistance of Th2 differentiation of the CD4+ iIEL suggest that both iIEL subsets contribute to the induction of noninflammatory mucosal immune responses.     

Wheat chromosome 2D carries genes controlling the activity of two DNA-degrading enzymes

DNA-degrading enzymes of 24.0 kDa and 27.0 kDa were observed to have different activities in two common wheat (Triticum aestivum L.) cultivars, Wichita and Cheyenne. A substrate-based SDS-PAGE assay revealed that these two enzymes were much more active in Wichita than in Cheyenne. Genes controlling the activities of these two enzymes were localized on chromosome 2D by testing DNA-degrading activities in reciprocal chromosome substitution lines between Wichita and Cheyenne. While the allele on Wichita chromosome 2D stimulated the activities of the 24.0- and 27.0-kDa enzymes in Cheyenne, the allele on Cheyenne chromosome 2D did not reduce the activities of the 24-kDa and 27-kDa enzymes in Wichita. Whether these genes code for the DNA-degrading enzymes themselves or for factors that regulate the enzyme activities remains unknown.This work was supported in part by USDA-Competitive Research Grants Office grant No. 90-37140-5426 to P.S.B. Contribution from Agricultural Research Division, University of Nebraska. Journal Series Number 10304     

Nucellar callus of ‘Femminello’ lemon,selected for tolerance toPhoma tracheiphila toxin,shows enhanced release of chitinase and glucanase into the culture medium

Phoma tracheiphila is the causative agent of the disease mal secco. Citrus cultivars differ substantially in respect to their sensitivity to the pathogenP. tracheiphila and its toxin. Some cultivars (e.g., Femminello lemon) are inherently sensitive while others (e.g., Tarocco orange) are tolerant. Cell lines derived from nucellar tissue of Femminello, Tarocco and a cell line selected for tolerance to the fungal toxin (Femminello-S) were used to study host-pathogen interaction. Our results showed that calli or conditioned media of Tarocco and Femminello-S inhibited the size of co-cultivated fungal colonies when compared to Femminello. In addition, conditioned medium of Tarocco as well as FemminelloS, but not Femminello, promoted bursting of hyphal tips. A ten-fold increase in chitinase and glucanase enzymatic activity, as evaluated by radiometric assay and laminarin hydrolysis respectively, was detected in Femminello-S extracellular extracts as compared to Femminello. An increase in chitinase was also shown by immunoblot analysis. Our findings suggest a positive correlation between the presence of chitinase and glucanase in the conditioned media of the cultured cells and the tolerance of those cells toP. tracheiphila toxin.     

Measurement of frequency-dependent sexual activity in Drosophila Melanogaster

In experiments on sexual competition in Drosophila melanogaster, the course of mating succes with time is represented by a sigmoid curve. By logarithmic transformation such curves are changed into straight lines that can be compared by covariance analysis. This method allows discrimination of the behaviour of the two types in competition, and allows us to follow it in the course of time. From a sexual competition experiment between the wild type Canton S and the mutant white-ebony we conclude that sexual activity of males and females of both types is generally frequency dependent, with evidence of rare-female advantage as well as rare-male advantage.     

Full 1H NMR assignment of a 24-nucleotide RNA hairpin: Application of the 1H 3D-NOE/2QC experiment

The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the ¹H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5/H5 for RNA plus H2/H2 for DNA), as well as for H5/H6, for H3/H4 in sugars with substantial populations of the N-pucker, for H1/H2 for S-puckered sugars, and usually for H2/H3. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectrum allowed assignment of all of the non-exchangeable protons, eliminating the need for stable-isotope labeling.     

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     

Isoform-Specific Membrane Translocation of Protein Kinase C After Ischemic Preconditioning

Mild cerebral anoxic/ischemic/stress insults promote tolerance and thereby protect the brain from subsequent lethal anoxic/ischemic insults. We examined whether specific activation of PKC , , , or isoforms is associated with ischemic preconditioning (IPC) in rat brain. IPC was produced by a 2-minute global cerebral ischemia. Membrane and cytosolic fractions of the hippocampi were immunoblotted using specific antibodies for PKC, , , and . PKC showed a significant translocation to the membrane fraction from 30 min to 4 h and PKC at 4 h following IPC. In contrast, the membrane/cytosol ratio of PKC showed a tendency to decrease at 30 min and 8 h, and the membrane/cytosol ratio of PKC was significantly decreased from 30 min to 24 h following IPC. These findings indicate PKC isoform-specific membrane translocations in the hippocampus after brief global brain ischemia and suggest that activation of PKC and PKC may be associated with IPC-induced tolerance in the rat hippocampus.     

The level of free amino acids in erythrocytes of different breeds of hen

Summary The content of free amino acids was determined in erythrocytes of adult Leghorn (Lg, White Rock (WR) and Cornish (Cr) hens, bred under identical conditions. The concentration of total amino acids was twice as high in the erythrocytes as in plasma, amounting to 396 m/100 ml, 424 m/100 ml and 475m/100 ml in White Rock, Cornish and Leghorn hens, respectively.Significant differences were found in the ratio of basic amino acids to acidic amino acids. These values were 0.76, 1.75 and 3.19 in White Rick, Leghorn and Cornish hens, respectively; in the plasma of all 3 breeds the ratio was 1. Statistically significant interbreed differences were expressed more distinctly in erythrocyte than in plasma amino acid concentrations. For absolute concentrations the differences were significant in the case of 9 amino acids.     

Endogeneous interferon α/β produced by murine Kupffer cells augments liver-associated natural killing activity

Summary Nonparenchymal liver cells from untreated C3HeB/FeJ mice, when incubated in medium containing-10% fetal bovine serum or portal serum, produced significant amounts of interferon alpha/beta (IFN/). In contrast, other cell populations (spleen, mononuclear blood cells and peritoneal cells) from C3HeB/FeJ mice or nonparenchymal liver cells from other strains of mice (C3H/HeJ, germ-free C3H/HeN and C57Bl/6J) produced little or no detectable IFN in fetal bovine serum under the same culture conditions. The cells in the nonparenchymal liver cell population responsible for IFN/ production were adherent, phagocytic, silica-sensitive, carbonyl-iron-sensitive, and Thy1.2^–, presumably Kupffer cells or resident liver macrophages. IFN/ production by cultured Kupffer cells was not observed if medium containing fetal bovine serum or portal serum was treated with polymyxin B or if Kupffer cells were cultured in serum-free medium. This suggested that small amounts of endotoxin in fetal bovine or portal serum stimulated Kupffer cells to produce IFN/. Possibly, Kupffer cells are in a different state of activation/maturation than peritoneal and splenic macrophages since the sensitivity of resident Kupffer cells from C3HeB/FeJ mice to the stimulatory effects of endotoxin. The endogenous production of IFN/ by Kupffer cells from C3HeB/FeJ mice can augment liver-associated natural killer (NK) activity against YAC-1 cells (4 h) and induce liver-associated cytotoxic activity, not restricted by the major histocompatibility complex, against NK resistant P815 mastocytoma cells (18 h).This work was supported by National Institutes of Health grant CA28835, VA Merit Grant and by the Margaret Duffy and Robert Cameron Troup Fund Abbreviations used: NPC, nonparenchymal liver cells; FBS, fetal bovine serum; IFN, interferon; -AsGm-1, anti-asialo-GM1; -Thy1.2, anti-Thy1.2; Hepes, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid; HBSS, Hanks' balanced salt solution; GBSS, Gey's balanced salt solution; SRBC, sheep red blood cells; Ab, mouse anti-SRBC; NK, natural killer; MHC, major histocompatibility complex; polyI·polyC, polyinosinec·polycytidylic acid     

Stablizing crude and ultrafiltrated α-amylase and α-glucosidase from Aspergillus oryzae by trehalose

Thermal resistance of freeze-dried -amylase and -glucosidase in trehalose matrices (1 to 20 % w/v) stored at 90 °C and relative humidities (RH) between 0 and 44 % was studied. At RH values up to 33 %, 10 % (w/v) trehalose was necessary to retain at least 50 % of -amylase activity. For -glucosidase, 10 % (w/v) trehalose was effective only at 0 % RH. Ultrafiltration of the crude enzymatic fermentation extracts enhanced enzyme stability per se. However, ultrafiltration in combination with 1 % (w/v) trehalose retained 74 % of -glucosidase and 95 % of -amylase activities. © Rapid Science Ltd. 1998     

A novel PH-CT-COSY methodology for measuring JPH coupling constants in unlabeled nucleic acids. Application to HIV-2 TAR RNA

A quantitative analysis of J_PH scalar couplings in nucleic acids is difficult due to small couplings to phosphorus, the extreme overlap of the sugar protons and the fast relaxation of the spins involved in the magnetization transfer. Here we present a new methodology that relies on heteronuclear Constant Time Correlation Spectroscopy (CT-COSY). The three vicinal ³J_PH3, ³J_PH5 and ³J_PH5 scalar couplings can be obtained by monitoring the intensity decay of the P_i-H3_{i – 1} peak as a function of the constant time T in a 2D correlation map. The advantage of the new method resides in the possibility of measuring the two ³J_PH5 and ³J_PH5 scalar couplings even in the presence of overlapped H5/H5 resonances, since the quantitative information is extracted from the intensity decay of the P-H3 peak. Moreover, the relaxation of the H3 proton is considerably slower than that of the H5/H5 geminal protons and the commonly populated conformations of the phosphate backbone are associated with large ³J_PH3 couplings and relatively small ³J_{PH5 / H5}. These two facts lead to optimal signal-to-noise ratio for the P-H3 correlation compared to the P-H5/H5 correlation.The heteronuclear CT-COSY experiment is suitable for oligonucleotides in the 10–15 kDa molecular mass range and has been applied to the 30mer HIV-2 TAR RNA. The methodology presented here can be used to measure P-H dipolar couplings (D_PH) as well. We will present qualitative results for the measurement of P-H_base and P-H2 dipolar couplings in the HIV-2 TAR RNA and will discuss the reasons that so far precluded the quantification of the D_PHs for the 30mer RNA.