A modular domain of NifU,a nitrogen fixation cluster protein,is highly conserved in evolution

HnifU, a gene exhibiting similarity tonifU genes of nitrogen fixation gene clusters, was identified in the course of expressed sequence tag (EST) generation from a human fetal heart cDNA library. Northern blot of human tissues and polymerase chain reaction (PCR) using human genomic DNA verified that the hnifU gene represented a human gene rather than a microbial contaminant of the cDNA library. Conceptual translation of the hnifU cDNA yielded a protein product bearing 77% and 70% amino acid identity to NifU-like hypothetical proteins fromHaemophilus influenzae andSaccharomyces cerevisiae, respectively, and 40–44% identity to the N-terminal regions of NifU proteins from several diazatrophs (i.e., nitrogen-fixing organisms). Pairwise determination of amino acid identities between the NifU-like proteins of nondiazatrophs showed that these NifU-like proteins exhibited higher sequence identity to each other (63–77%) than to the diazatrophic NifU proteins (40–48%). Further, the NifU-like proteins of non-nitrogenfixing organisms were similar only to the N-terminal region of diazatrophic NifU proteins and therefore identified a novel modular domain in these NifU proteins. These findings support the hypothesis that NifU is indeed a modular protein. The high degree of sequence similarity between NifU-like proteins from species as divergent as humans andH. influenzae suggests that these proteins perform some basic cellular function and may be among the most highly conserved proteins. Correspondence to: C.-C. Liew     

Production of a polysaccharide, methylan, in Methylobacterium organophilum by controlling ammonium ion

Summary Cell growth increased proportionally to the initial concentration of ammonium ion, however, methylan production was significantly inhibited at the high concentration of ammonium ion. The control of ammonium ion within the desired level(usually 0.45 g/l) was needed to reduce the inhibition. Methylan production was increased to 12.5 g/l by maintaining ammonium ion below 0.15 g/l.     

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.     

A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum.

Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes. Transport was restored by co-reconstitution of the ATP eluate. This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5). In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose. Purified BiP did not stimulate transport activity. Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates.     

New biologically active hybrid bacteriocins constructed by combining regions from various pediocin-like bacteriocins: the C-terminal region is important for determining specificity.

The pediocin-like bacteriocins, produced by lactic acid bacteria, are bactericidal polypeptides with very similar primary structures. Peptide synthesis followed by reverse-phase and ion-exchange chromatographies yielded biologically active pediocin-like bacteriocins in amounts and with a purity sufficient for characterizing their structure and mode of action. Despite similar primary structures, the pediocin-like bacteriocins, i.e., pediocin PA-1, sakacin P, curvacin A, and leucocin A, differed in their relative toxicities against various bacterial strains. On the basis of the primary structures, the polypeptides of these bacteriocins were divided into two modules: the relatively hydrophilic and well conserved N-terminal region, and the somewhat more diverse and hydrophobic C-terminal region. By peptide synthesis, four new biologically active hybrid bacteriocins were constructed by interchanging corresponding modules from various pediocin-like bacteriocins. All of the new hybrid bacteriocin constructs had bactericidal activity. The relative sensitivity of different bacterial strains to a hybrid bacteriocin was similar to that to the bacteriocin from which the C-terminal module was derived and quite different from that to the bacteriocin from which the N-terminal was derived. Thus, the C-terminal part of the pediocin-like bacteriocins is an important determinant of the target cell specificity. The synthetic bacteriocins were more stable than natural isolates, presumably as a result of the absence of contaminating proteases. However, some of the synthetic bacteriocins lost activity, but this was detectable only after months of storage. Mass spectrometry suggested that this instability was due to oxidation of methionine residues, resulting in a 10- to 100-fold reduction in activity.     

RELATIONSHIPS AMONG MORPHOLOGICAL STATUS, STEROID HORMONES, AND POST-THAWING VIABILITY OF FROZEN SPERMATOZOA OF MALE MINKE WHALES (BALAENOPTERA ACUTOROSTRATA)

Spermatozoa from 21 mature minke whales ( Balaenoptera acutorostrata ) taken in the Antarctic Ocean for Japanese research were recovered from vasa deferentia, diluted 1:9 in a Tris-based diluent, and frozen at - 80°C on board the vessel. After a period ranging from 45 to 125 d, the samples were transferred to liquid nitrogen and transported to the laboratory. After thawing at 37°C the motility (percentage of motile spermatozoa), vitality (proportion of live spermatozoa), and sperm concentration were determined for each sample. These values were tested for correlations with morphological measurements (body size, body weight, testis weight) and serum concentrations of progesterone (Pd), estradiol-17β (E2), and testosterone (T). Ten of 21 samples had motile spermatozoa (2%-40%). Although no motile spermatozoa were observed in 1.1 samples, all sperm samples were examined by eosinnigrosin staining and showed vitality levels of 3%44%. It was found that the motility (Y = 0.54) and vitality (r = 0.53) of the spermatozoa were significantly (P < 0.01) correlated with the E₂ levels (8.50 ± 1.80 pg/ml). Serum T levels (0.07 ± 0.02 ngml) were significantly correlated with the E₂ levels (r = 0.58, P < 0.01>, but sperm concentrations were not correlated with either Ea or T levels. The present study demonstrates that spermatozoa of minke whales can be successfully cryopreserved.     

The carboxylate type siderophore rhizoferrin and its analogs produced by directed fermentation

Summary Rhizoferrin is a novel carboxylate-type siderophore which has recently been isolated fromRhizopus microsporus and other fungi of the Mucorales (Zygomycetes). The present investigation shows that a variety of rhizoferrin analogs can be produced by directed fermentation. Thus both the diaminobutane backbone and the citric acid side chains of rhizoferrin have been substituted by diamine and citric acid analogs added to the culture medium. The new ligands as well as their iron complexes have been characterized by physicochemical methods. Conditions of precursor incorporation and implications for the biosynthesis of the new siderophores are discussed.     

Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by theE. coli lac repressor

Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.     

Kinetics of the Competitive Degradation of Deoxyribose and Other Molecules by Hydroxyl Radicals Produced by the Fenton Reaction in the Presence of Ascorbic Acid

The competition method in which the Fenton reaction is employed as an OH radical generator and deoxyribose as a detecting molecule, has been used to determine the rate constants for reactions of the OH radical with its scavengers. Nonlinear competition plots were obtained for those scavengers which reacted with the Fenton reagents (Fe²⁺ or H₂O₂). Ascorbic acid is believed to overcome this problem. We have investigated the kinetics of deoxyribose degradation by -OH radicals generated by the Fenton reaction in the presence of ascorbic acid, and observed that the inclusion of ascorbic acid in the Fenton system greatly increased the rate of OH radical generation. As a result, the interaction between some scavengers and the Fenton reagents became negligeable and linear competition plots of A7A vs scavenger concentrations were obtained. The effects of experimental conditions such as, the concentrations of ascorbic acid, deoxyribose, H₂O₂ and Fe²⁺-EDTA, the EDTA/Fe²⁺ ratio as well as the incubation time, on the deoxyribose degradation and the determination of the rate constant for mercaptoethanol chosen as a reference compound were studied. The small standard error, (6.76± 0.21) ±' 10⁹M^-1s^-1 observed for the rate constant values for mercaptoethanol determined under 13 different experimental conditions, indicates the latter did not influence the rate constant determination. This is in fact assured by introducing a term, k_x, into the kinetic equation. This term represents the rate of-OH reactions with other reagents such as ascorbic acid, Fe²⁺-EDTA, H₂O₂ etc. The agreement of the rate constants obtained in this work with that determined by pulse radiolysis techniques for cysteine, thiourea and many other scavengers, suggests that this simple competition method is applicable to a wide range of compounds, including those which react with the Fenton reagents and those whose solubility in water is low.