Aroma-forming substance from a culture of the mold fungus Penicillium roqueforti during growth on milk curds

Production of aroma-forming substances by Penicillium roqueforti strains no. 31 and no. 541-A grown on curd was studied. The data showed that the strain no. 541-A is the most promising producer of cheese flavor. The flavor acquired a soily scent after a long-term (4-5 days) cultivation, which may hamper the use of these cultures (particularly, no. 31) in the food industry.     

Isolation of Two Streptothricin-like Antibiotics,Nos. 6241-A and B,as Inhibitors of de novo Starch Synthesis and Their Herbicidal Activity

Two streptothricin-like antibiotics, Nos. 6241-A and B, were obtained as inhibitors of de novo starch synthesis in excised leaf segments of barnyard millet (Panicum crus-galli). No. 6241-B was identified as SF-701, and No. 6241-A was a new antibiotic, in which N,N-dimethylglycine was substituted for the N-methylglycine of No. 6241-B (SF-701). Both antibiotics also inhibited plant growth. The inhibitory activity of No. 6241-B (SF-701) was approximately ten times that of No. 6241-A. By foliar treatment, No. 6241-B (SF-701) showed remarkable herbicidal activity against barnyard millet at a concentration of more than 500 ppm with little phytotoxicity for the rice plant (Oryza sativa L. cv. Nihonbare).     

Polymorphism of prion protein gene in Arctic fox (<Emphasis Type="Italic">Vulpes lagopus</Emphasis>)

Prion diseases are fatal neurodegenerative disorders of humans and certain other mammals. Prion protein gene (Prnp) is associated with susceptibility and species barrier to prion diseases. No natural and experimental prion diseases have been documented to date in Arctic fox. In the present study, coding region of Prnp from 135 Arctic foxes were cloned and screened for polymorphisms. Our results indicated that the Arctic fox Prnp open reading frame (ORF) contains 771 nucleotides encoding 257 amino acids. Four single nucleotide polymorphisms (SNPs) (G312C, A337G, C541T, and A723G) were identified. SNPs G312C and A723G produced silent mutations, but SNPs A337G and C541T resulted in a M–V change at codon 113 and R–C at codon 181, respectively. The Arctic fox Prnp amino acid sequence was similar to that of the dog (XM 542906). In short, this study provides preliminary information about genotypes of Prnp in Arctic fox.     

A new epitope peptide derived from hepatitis C virus 1b possessing the capacity to induce cytotoxic T-lymphocytes in HCV1b-infected patients with HLA-A11, -A31, and -A33

Background Hepatitis C virus (HCV) frequently causes chronic hepatitis, cirrhosis, and hepatocellular carcinoma after long-term persistent infection. Among various genotypes of HCV, HCV1b is resistant to standard interferon therapy, and thus the development of new treatment modality is needed. Results To provide a scientific basis for specific immunotherapy for HCV1b, we investigated HCV1b-derived epitope peptides recognized by human leukocyte antigen (HLA)-A11, -A31, or -A33-restricted cytotoxic T-lymphocytes (CTLs), and report here three novel vaccine candidate peptides selected by both antibody screening and CTL-inducing capacity from among 46 peptides of conserved regions of HCV1b sequences with binding motifs to HLA-A11, -A31, and -A33. Significant levels of IgG reactive to each of the three peptides were detected in the plasma of more than 50% of the HCV1b⁺ patients. One peptide at positions 30–39 of the core protein induced peptide-specific CTLs from peripheral blood mononuclear cells (PBMCs) of HLA-A11⁺, -A31⁺, and -A33⁺ patients. The other two peptides at positions 35–43 of the core protein and at positions 918–926 of the non-structural protein 2 also induced peptide-specific CTLs from the PBMCs of HLA-A11⁺ and -A33⁺ patients. Conclusion Therefore, the peptide at positions 30–39 of the core protein could be an appropriate target molecule of specific immunotherapy for all HLA-A11⁺, -A31⁺, and -A33⁺ patients with HCV1b-related diseases.     

IS<Emphasis Type="Italic">Rm31</Emphasis>, a new insertion sequence of the IS<Emphasis Type="Italic">66</Emphasis> family in<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti pSymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, ISRm31. ISRm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that ISRm31 is a member of the IS66 family. ISRm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying ISRm31 suggested that ISRm31 inserts randomly into S. meliloti genomes. Moreover, analysis of ISRm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, ISRm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.     

A novel reporter rat strain that expresses LacZ upon Cre‐mediated recombination

The recent widespread application of Cre/loxP technology has resulted in a new generation of conditional animal models that can better recapitulate many salient features of human disease. These models benefit from the ability to monitor the expression and functionality of Cre protein. We have generated a conditional (Cre/loxP dependent) LacZ reporter rat (termed the LacZ541 rat) to monitor Cre in transgenic rats. When LacZ541 rats were bred with another transgenic rat line expressing Cre recombinase under the control of the CAG promoter, LacZ/Cre double transgenic embryos displayed ubiquitous expression of LacZ, and when LacZ541 rats were bred with transgenic rats expressing Cre/loxP‐dependent oncogenic H‐ or K‐ras, LacZ was expressed in the lesions resulting from the activation of the oncogene. The LacZ541 rat enables evaluation of the performance of Cre‐expressing systems which are based upon transgenic rats or somatic gene transfer vectors and provides efficient and simple lineage marking. genesis 51:268–274. © 2013 Wiley Periodicals, Inc.     

Identification and Growth Characteristics of α-Galactosidase-producing Microorganisms

Two kinds of α-galactosidase-producing microorganisms, strain No. 31–2 and strain No. 7–5, have been isolated from soil and subjected to a determinative study. On the basis of the morphological and physiological characters, the strain No. 31–2 was identified to be belonged to genus Micrococcus and the strain No. 7–5 to genus Bacillus. The former strain, Micrococcus sp. No. 31–2, produced exclusively an intracellular α-galactosidase, and the latter one, Bacillus sp. No. 7–5, secreted the enzyme into culture medium. The cell growth and enzyme production of both strains were observed to reach the maximum under an alkaline culture condition. The intracellular α-galactosidase of Micrococcus sp. No. 31–2 was inducible by galactose, melibiose, and raffinose, while the α-galactosidase of Bacillus sp. No. 7–5 was produced constitutively.     

Antimicrobial Peptide JH-3 Effectively Kills Salmonella enterica Serovar Typhimurium Strain CVCC541 and Reduces Its Pathogenicity in Mice

Salmonella is an important zoonotic pathogen and is a major cause of gastrointestinal diseases worldwide. The current serious problem of antibiotic abuse has prompted the search for new substitutes for antibiotics. JH-3 is a small antimicrobial peptide with broad-spectrum bactericidal activity. In this study, we showed that JH-3 has good bactericidal activity towards the clinical isolate Salmonella enterica serovar Typhimurium strain CVCC541. The minimum inhibitory concentration (MIC) of JH-3 against this bacterium was determined to be 100 μg/mL, which could decrease the number of CVCC541 cells by 1000-fold in vitro within 5 h. The transmission electron microscopy (TEM) results showed that JH-3 can damage the cell wall and membrane of CVCC541, leading to the leakage of cell contents and subsequent cell death. To measure the bactericidal activity of CVCC541-infected mice were treated intraperitoneally 40 or 10 mg/kg JH-3 at 2 h or 3 days postinfection. Our results showed that treatment with 40 mg/kg JH-3 at 2 h postinfection had the best therapeutic effect and could significantly protect mice from a lethal dose of CVCC541. Furthermore, the clinical symptoms, bacterial burden in blood and organs, and intestinal pathological changes were all decreased and were close to normal. This study examined the therapeutic effect of the antimicrobial peptide JH-3 against S. enterica CVCC541 infection for the first time and determined the therapeutic effect of different JH-3 doses and treatment times, laying the foundation for studies of new antimicrobial agents.

     

Epitope Mapping of Antibodies Suggests the Novel Membrane Topology of B-Cell Receptor Associated Protein 31 on the Cell Surface of Embryonic Stem Cells: The Novel Membrane Topology of BAP31

When located in the endoplasmic reticulum (ER) membrane, B-cell receptor associated protein 31 (BAP31) is involved in the export of secreted proteins from the ER to the plasma membrane. In a previous study, we generated two monoclonal antibodies (mAbs), 297-D4 and 144-A8, that bound to surface molecules on human embryonic stem cells (hESCs), but not to surface molecules on mouse embryonic stem cells (mESCs). Subsequent studies revealed that the mAbs recognized BAP31 on the surface of hESCs. To investigate the membrane topology of BAP31 on the cell surface, we first examined the epitope specificity of 297-D4 and 144-A8, as well as a polyclonal anti-BAP31 antibody (α-BAP31). We generated a series of GST-fused BAP31 mutant proteins in which BAP31 was serially deleted at the C- terminus. GST-fused BAP31 mutant proteins were then screened to identify the epitopes targeted by the antibodies. Both 297-D4 and 144-A8 recognized C-terminal residues 208–217, while α-BAP31 recognized C-terminal residues 165–246, of BAP31 on hESCs, suggesting that the C-terminal domain of BAP31 is exposed on the cell surface. The polyclonal antibody α-BAP31 bound to mESCs, which confirmed that the C-terminal domain of BAP31 is also exposed on the surface of these cells. Our results show for the first time the novel membrane topology of cell surface-expressed BAP31 as the extracellular exposure of the BAP31 C-terminal domain was not predicted from previous studies.     

Cleavage of lambda repressor and synthesis of RecA protein induced by transferred UV-damaged F sex factor

Summary Transfer of a UV-damaged F sex factor to a recipient lysogen induces prophage development. Under these conditions RecA protein synthesis was induced and repressor cleaved, as observed upon direct induction, that is, when the recipient lysogen was directly exposed to UV-light. The efficiency of induction of RecA protein synthesis in recipient bacteria which had received an irradiated F-lac factor was about 80% of that measured upon direct induction. We observed the simultaneous disappearance of repressor and a slight production of cleavage fragments; quantitation by densitometric scanning of the autoradiogram after correction for the efficiency of transfer indicated that 55% of repressor was cleaved. Transfer of UV-damaged Hfr DNA failed to induce RecA protein synthesis. A phage vector carrying oriF, the cloned origin of F plasmid replication, after exposure to UV-light and infection of a recipient lysogen, induced RecA protein synthesis and a moderate but significant cleavage of repressor. Indirect induction by UV-damaged F sex factor or phage oriF resulted in biochemical cellular reactions similar to those observed upon direct induction. LexA repressor that negatively controls RecA protein synthesis appeared more susceptible to cleavage than did repressor.     

Large-scale comparative mapping of the MHC class I region of predominant haplotypes in Japanese

 In order to further our understanding of major histocompatibility complex (MHC) class I gene organization, we began a comparative analysis of the large scale organization of the class I region in diverse haplotypes. For these studies, the MHC in healthy Japanese donors who have the predominant MHC haplotypes and/or HLA-A or -B alleles was examined by pulsed field gel electrophoresis and Southern analysis using probes spanning the class I region. Hybridization with probes from the HLA-A to HLA-G region revealed that individuals expressing HLA-A30, -A31, or -A33 have an approximately 70 kilobase (kb) insertion near the HLA-A gene as compared with haplotypes containing the HLA-A11 or -A26 allele. Conversely, HLA-A24-containing haplotypes appear to have an approximately 50 kb deletion from the same region. Further, it appears that chromosomes carrying closely related alleles are similar to each other in this region, consistent with their presumed evolutionary relationship. While little is known about the gene content between the HLA-A and HLA-G region, it will be interesting to examine the prospect that functional genes do in fact reside within the inserted or deleted portions, thereby raising the possibility that distinct functional differences are conferred by different haplotypes. Overall, the results reported here should contribute to furthering our understanding of the association between diseases and HLA as well as provide new insights into the evolution of the MHC. Received: 11 December 1996     

Identification of the possible new odor-active compounds “12-methyltridecanal and its analogs” responsible for the characteristic aroma of ripe Gouda-type cheese

The aroma concentrates of the three maturation stages of Gouda-type cheeses were prepared by combining the solvent extraction and the solvent assisted flavor evaporation techniques. The aroma extract dilution analysis applied to the volatile fraction revealed 31 odorants that were identified or tentatively identified from the 38 odor-active peaks with FD factors between 4³ and 4⁸. By comparison with the FD factors in the three maturation stages of the cheeses, 16 odorants, including 12-methyltridecanal, which is a newly identified odorant from the cheese, increased with the increasing maturation stage of the cheese. In addition, many iso- and anteiso-methyl-branched long-chain aliphatic aldehydes could be identified as the analogs of 12-methyltridecanal, which have a unique odor note. It may be then expected that these aldehydes were able to influence the flavor of the highly ripened Gouda cheese, since these compounds also increased with the increasing maturation stage.     

Poly β-hydroxybutyrate depolymerase (PhaZ) in<Emphasis Type="Italic"> Azospirillum brasilense</Emphasis> and characterization of a<Emphasis Type="Italic"> phaZ</Emphasis> mutant

Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly--hydroxybutyrate (PHB) under sub-optimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments. PHB depolymerase (PhaZ) is an essential enzyme in PHB degradation. The phaZ gene was identified in Azospirillum brasilense, cloned, sequenced, and shown to be located on the chromosome. Insertion of a kanamycin-resistant cassette within phaZ of A. brasilense resulted in a phaZ mutant that was unable to degrade PHB; however, carbon source utilization was similar in both the wild-type and the mutant strain. The ability of the wild-type to endure starvation conditions, ultraviolet irradiation, heat, and osmotic shock, and to grow in the presence of hydrogen peroxide was higher than that of the mutant strain. By contrast, the ability of the phaZ mutant strain to endure desiccation was higher than that of the wild-type strain. No differences between the strains were seen in their ability to endure sonication, or to survive in carrier materials used for soil inoculants. In addition, motility was the same between the two strains, whereas cell aggregation and exopolysaccharide production were higher in the wild-type than in the phaZ mutant strain.     

Encoding genes for endosperm proteins in Hordeum chilense

Summary The endosperm proteins encoded by the genome Hch in Hordeum chilense, Tritordeum (amphiploid Hordeum chilense x Triticum turgidum), common wheat-H. chilense addition lines, and the segregating plants resulting from the cross Tritordeum x T. turgidum, were fractionated by three electrophoretical techniques: SDS-PAGE, A-PAGE, and bidimensional PAGE. Prolamin subunits with a high molecular weight (HMW) were well visualized by SDS-PAGE, the A-PAGE technique permitted good resolution for many hordeins and gliadins, and two-dimensional electrophoresis allowed new sets of bands coded by gene complexes from H. chilense chromosomes to be distinguished. The loci Hor-Hch1 (up to 11 subunits belonging to the -, — and -hordeins), Glu-Hch1 (one HMW prolamin subunit), Hor-Hch2 (one -hordein), and Hor-Hch3 (up to four -hordeins) were located on the H. chilense chromosomes 1Hch, 5Hch, and 7Hch.     

Studies on the histochemical „Leucine aminopeptidase“ reaction

Summary Since it was recently observed that purified cathepsin B will hydrolyse the chromogenic substrate LNA the obvious possibility of demonstrating the activity of this enzyme in tissue sections was explored. Chemical and histochemical experiments suggested that all aminopeptidase activity could be excluded by choosing a sufficiently acid pH for incubation, whereby the selective demonstration of cathepsin B activity was possible. This conclusion was further supported by different attempts to influence the histochemical reaction by preincubation with suitable activating and/or inhibiting reagents. This more specific procedure was used on sections from liver, kidney and granulomas rich in active macrophages. The suggested cathepsin B activity was demonstrated in autophagic vacuoles, and in other lysosome-like enlarged vacuoles or granules, appearing as a consequence of cellular injury as well as in actively phagocytosing macrophages.The following Abbreviations have been used AM Pase(s) aminopeptidase(s) in general - NA naphthylamides in general. — Substrates - LA Leucine amide - GPA Gly-Phe-amide - LNA L-leucyl--naphthylamide - Arg-NA L-arginyl--naphthylamide - BANA Benzoyl-dl-arginine--naphthylamide     

Is a gene for microcephaly located on chromosome 1?

Summary A 3-month-old boy with true microcephaly showed the same balanced reciprocal translocation 1q4p as his carrier mother. This reciprocal translocation had been transmitted for at least four generations. Different banding techniques allowed one to describe the rearrangement as: rcp t(1;4)(1pter 1q31::4p161 4pter; 4qter 4p153::1q321 1qter). On the other hand, the proband's father seemed to be a border-line mentally retarded and one of his relatives suffered from mental retardation of unknown origin. Taking into account all these results together with the current literature, it was concluded that the microcephaly appearing in our case could be due to the following two facts: (a) the father was an heterozygote for the gene for microcephaly, and (b) damage or a minute deletion on chromosome 1 between 1q31 and 1q321 bands could occur in the mother's family resulting in a mutation for microcephaly. If this was so, the gene for microcephaly should be located on chromosome 1 at the level of the 1q31–1q321 junction.     

Molecular mapping of a new public HLA class I epitope shared by all HLA-B and HLA-C antigens and defined by a monoclonal antibody

It has previously been shown that a mouse monoclonal antibody, designated 4E, reacts with an epitope common to all HLA-B and -C antigens and those of the HLA-Aw19 cross-reactive group, namely, HLA-A29,-A30, -A31, -A32, -Aw33, and -Aw74. In order to pinpoint the amino acid residues which comprise the public specificity recognized by 4E, an HLA-A29 cDNA clone was isolated and its predicted amino acid sequence compared with those of other clonedHLA class I genes. The isolated HLA-A29 cDNA corresponded to the rarer of the twoA29 variant alleles,A29.1. Two amino acid residues of HLA-A29.1, gln-144 and arg-151, were found in all 24HLA-B andHLA-C alleles examined but were present in only one of 15HLA-A alleles for which sequence data are available. Importantly, this exceptional allele wasHLA-A32, another member of the HLA-Aw19 cross-reactive group. Gln-144 and arg-151 should be capable of jointly contributing to the binding site for 4E, as they are situated in successive alpha-helical subregions and are predicted to be juxtaposed in the three-dimensional HLA molecule. Four other residues in the first or second external domains of HLA-A29.1 (thr-9, leu-62, gln-63, and his-102) were unique among theHLA-A alleles, but none of these was found in corresponding positions ofHLA-B or-C alleles and thus failed to correlate with presence or absence of the 4E determinant. These observations are consistent with the notion that gln-144 and arg-151 define a determinant common to HLA-B, HLA-C, and the HLA-Awl9 cross-reactive group and the binding site of the monoclonal antibody 4E.     

An HLA-A3-binding prostate acid phosphatase-derived peptide can induce CTLs restricted to HLA-A2 and -A24 alleles

We previously reported peptide vaccine candidates for HLA-A3 supertype (-A3, -A11, -A31, -A33)-positive cancer patients. In the present study, we examined whether those peptides can also induce cytotoxic T lymphocyte (CTL) activity restricted to HLA-A2, HLA-A24, and HLA-A26 alleles. Fourteen peptides were screened for their binding activity to HLA-A*0201, -A*0206, -A*0207, -A*2402, and -A*2601 molecules and then tested for their ability to induce CTL activity in peripheral blood mononuclear cells (PBMCs) from prostate cancer patients. Among these peptides, one from the prostate acid phosphatase protein exhibited binding activity to HLA-A*0201, -A*0206, and -A*2402 molecules. In addition, PBMCs stimulated with this peptide showed that HLA-A2 or HLA-A24 restricted CTL activity. Their cytotoxicity toward cancer cells was ascribed to peptide-specific and CD8⁺ T cells. These results suggest that this peptide could be widely applicable as a peptide vaccine for HLA-A3 supertype-, HLA-A2-, and -A24-positive cancer patients.     

Mapping and tagging of seed coat colour and the identification of microsatellite markers for marker-assisted manipulation of the trait in Brassica juncea

Microsatellite marker technology in combination with three doubled haploid mapping populations of Brassica juncea were used to map and tag two independent loci controlling seed coat colour in B. juncea. One of the populations, derived from a cross between a brown-seeded Indian cultivar, Varuna, and a Canadian yellow-seeded line, Heera, segregated for two genes coding for seed coat colour; the other two populations segregated for one gene each. Microsatellite markers were obtained from related Brassica species. Three microsatellite markers (Ra2-A11, Na10-A08 and Ni4-F11) showing strong association with seed coat colour were identified through bulk segregant analysis. Subsequent mapping placed Ra2-A11 and Na10-A08 on linkage group (LG) 1 at an interval of 0.6 cM from each other and marker Ni4-F11 on LG 2 of the linkage map of B. juncea published previously (Pradhan et al., Theor Appl Genet 106:607–614, 2003). The two seed coat colour genes were placed with markers Ra2-A11 and Na10-A08 on LG 1 and Ni4-F11 on LG 2 based on marker genotyping data derived from the two mapping populations segregating for one gene each. One of the genes (BjSC1) co-segregated with marker Na10-A08 in LG 1 and the other gene (BjSC2) with Ni4-F11 in LG 2, without any recombination in the respective mapping populations of 130 and 103 segregating plants. The identified microsatellite markers were studied for their length polymorphism in a number of yellow-seeded eastern European and brown-seeded Indian germplasm of B. juncea and were found to be useful for the diversification of yellow seed coat colour from a variety of sources into Indian germplasm.