Effect of Processing on the Microflora of Norwegian Seaweed Meal, with Observations on Sporendonema minutum (Høye) Frank and Hess

Meal from the brown seaweed Ascophyllum nodosum (L.) Le Jol. is mainly used as an animal feed supplement. Since moist weed often develops a marked mold growth and since little was known about the microflora of seaweed meal, a cultural procedure was developed to enumerate the populations of bacteria, yeasts, and molds of seaweed meals manufactured by different drying processes. The microflora could be supported by a variety of media varying in levels of nutrition and in the source and concentration of salts. Fresh weed contained less than 10(3) bacteria and less than 10(2) yeasts and molds per g (dry weight). The type and extent of microbial populations in seaweed meal appeared to be dependent upon the method of seaweed drying. Rotary drum-drying at temperatures decreasing from 800 to 80 C maintained or reduced the microbial populations to 10(3) organisms per g (dry weight). Although meals with high nutritional quality can be obtained with warm air- or rock-dried weed, these conditions can also permit bacterial and mold development. Extended rock-drying in variable weather conditions and prolonged storage of moist weed, both of which decrease the nutritional quality, also lead to high bacterial numbers and to a marked development of the halophilic brown mold Sporendonema minutum which attained populations of 10(8) viable spores per g of dried weed. A poultry diet containing 5% badly molded weed had no apparent toxic or growth-depressing effect when fed to chicks.     

Comparative control of a branch-point enzyme in microorganisms

Thirty-two genera of microorganisms were identified with one of six distinctive control patterns for the enzyme 3-deoxy-d-arabino-heptulosonate-7-phosphate synthetase. These patterns included sequential feedback inhibition, isoenzyme feedback inhibition, cumulative feedback inhibition, and three (apparent) simple one-effector patterns. Documentation is provided of an overwhelming tendency for control patterns to be strongly conserved among the member species of the various genera that were examined.     

Apparatus for monitoring the oxygen uptake and carbon dioxide production of fermentations

The requirements of the continuous analysis of effluent gas streams from aerated flash and tank fermentors are described, as are instrumental devices for measuring the oxygen and carbon dioxide content of fermentor gases. The use of a specially designed sequential gas sample for monitoring four fermentations simultaneously and a system for precise control of low air flow and pressure is explained. Equations for calculating carbon dioxide production or oxygen consumption rates and respiratory quotients are given. A discussion of the operating characteristics of a device for automatic translation of aeration data between fermentors is presented.     

A model for the stepwise radiation inactivation of the alpha 2-dimer of Na,K-ATPase

This study is a direct continuation of Jensen, J., and N?rby, J. G., (1988) J. Biol. Chem. 263, 18063-18070. A new model in which we propose that the in situ organization of the Na,K-ATPase alpha-subunit is an alpha 2-dimer and which describes the stepwise degradation by radiation inactivation of this assembly is presented on the basis of the following findings. Radiation inactivation size for alpha-peptide integrity, normal nucleotide, vanadate and ouabain binding, and K-pNPPase activity is close to m(alpha) = 112 kDa; for Na-ATPase activity it is 135 kDa and for Na,K-ATPase activity it increases from 140 to about 195 kDa with increasing assay ATP concentration (equal to increasing average turnover). Normal Tl+ occlusion had the same radiation inactivation size as Vmax for Na,K-ATPase, i.e. about 195 kDa. The binding experiments disclosed radiation-produced molecules with active binding sites but with a lower than normal affinity. Radiation inactivation size for the total binding capacity of ADP and ouabain was therefore smaller than the size of an alpha-peptide, namely about 70 kDa, and for total Tl+ occlusion it was down to 40 kDa. We can explain all these observations by using a new approach to target size analysis and by assuming a dimeric organization of the alpha-subunit. Each alpha-peptide is degraded stepwise by first destruction of either a 42- or a 70-kDa domain, and the partly damaged peptide may retain biochemical activity. We conclude that there is no role for the beta-subunit in catalysis and that the alpha-peptide is organized as an alpha 2-dimer in the membrane with each alpha-subunit being able to perform complete catalytic cycles (and probably also active transport), provided that it is stabilized by an adjacent alpha-peptide or a sufficiently large fragment thereof.     

Ca2+/calmodulin-dependent protein kinase enriched in cerebellar granule cells. Identification of a novel neuronal calmodulin-dependent protein kinase

Ca2+-sensitive protein kinases are thought to play a pivotal role in Ca2+-mediated neuronal communication. We describe here the cloning, purification, and characterization of a major Ca2+/calmodulin-dependent, brain-specific protein kinase which is particularly enriched in cerebellar granule cells. The enzyme is comprised of Mr 65,000 and 67,000 polypeptides which copurify to homogeneity and phosphorylate synapsin I. The protein kinase is coded for by two poly(A+) RNAs of 2.0 and 3.5 kilobases which probably derive from a single gene. Two cDNA inserts, one of 198 base pairs and one of 1225 base pairs, contain a total of 677 base pairs of the protein coding sequence which includes sequences homologous to other calmodulin-dependent protein kinases including part of the calmodulin-binding domain. The surprising presence of extended sequences which are enriched in glutamate residues may influence the subcellular distribution of this kinase. Immunohistochemical localization with an affinity-purified antibody reveals that whereas the enzyme is expressed in several neuronal subpopulations, it is exceptionally enriched in the granule cells of the cerebellum. The relevance of the biochemical, molecular, and histologic properties of this enzyme is discussed in the context of neuronal Ca2+ signaling.     

The major 45-kDa protein of the yeast mitochondrial outer membrane is not essential for cell growth or mitochondrial function

As part of an analysis of the function and assembly of the mitochondrial outer membrane, we have cloned and characterized the yeast gene encoding a 45-kDa polypeptide (OM45) which is a major constituent of this membrane. The nuclear gene was isolated by immunological screening of plaques of recombinant phage lambda gt11 containing fragments of yeast genomic DNA using an antibody against OM45. Determination of the nucleotide sequence of the DNA fragment isolated by this approach revealed a single open reading frame of 1179 base pairs which encodes a protein having a predicted molecular mass of 44.6-kDa. Disruption of the OM45 gene in haploid yeast cells eliminated the expression of OM45. The mutant strain showed no apparent defect in cell viability, growth, mitochondrial function, or mitochondrial protein import.     

Purification and partial characterization of a major outer-membrane protein of Fusobacterium nucleatum

The major outer-membrane proteins of 40-41 kDa were identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in Fusobacterium nucleatum strains ATCC 10953, ATCC 25586, F3, F6 and Fev1. The proteins were purified by preparative gel electrophoresis. Their behaviour in gel filtration and gel electrophoresis, their sensitivity to proteolytic enzymes, and their amino acid composition were investigated. The purified proteins were partly sequenced from the N-terminal end. A 36.5 kDa portion was protected against extrinsic proteolytic (trypsin, chymotrypsin or pronase) digestion of whole cells. This polypeptide was isolated and partially sequenced from the N-terminal end. From these data and data from extrinsic iodination it was concluded that the N-terminal end of the protein is probably exposed on the surface of the cell. A database search revealed amino acid sequence similarity in an Ala-Pro-rich region of outer-membrane protein A (OmpA) in other Gram-negative bacteria.     

High-level expression of the Streptomyces clavuligerus isopenicillin N synthase gene in Escherichia coli.

The pcbC gene, which encodes isopenicillin N synthase (IPNS), was subcloned from Streptomyces clavuligerus into Escherichia coli by using the pT7 series of plasmid vectors. The polymerase chain reaction was used to introduce an NdeI site at the translation initiation codon of pcbC, allowing the gene to be inserted behind an E. coli type of ribosome binding site. This construction directed high-level expression of IPNS, but the IPNS was in an inactive form in inclusion bodies. Active IPNS was recovered by solubilizing and renaturing the protein.