Neurocalcin: a novel calcium-binding protein from bovine brain.

A novel calcium-binding protein (molecular weight 23,000-24,000, pI 5.3-5.5), which we term neurocalcin, was identified in bovine brain. Using calcium-dependent drug affinity chromatography ((S)-P-(2-aminoethyloxy)-N-[2-(4-benzyloxycarbonylpiperazinyl++ +)-1-(P- methoxybenzyl)ethyl]-N-methylbenzene-sulfonamide dihydrochloride, W-77, -coupled Sepharose 6B), we purified neurocalcin from bovine brain. The partial amino acid sequence of neurocalcin revealed it to be an as yet unidentified protein with three putative calcium binding sites (EF-hands). Further purification and sequence analysis demonstrated the presence of four isoprotein forms designated alpha, beta, gamma 1, and gamma 2. When the 165 sequenced residues of neurocalcin beta are compared with sequences of other proteins, neurocalcin beta has a 38.2% sequence homology with visinin and 45.5% with recoverin (Yamagata, K., Goto, K., Kuo, C.-H., Kondo, H., and Miki, N. (1990) Neuron 2, 469-476; Dizhoor, A. M., Ray, S., Kumar, S., Niemi, G., Spencer, M., Brolley, D., Walsh, K. A., Philipov, P. P., Hurley, J. B., and Stryer, L. (1991) Science 251, 915-918). Both visinin and recoverin are expressed specifically in retinal photoreceptors and are not found in brain. Unlike visinin and recoverin, neurocalcin is purified not only from retina but also from bovine brain. Our results suggest that neurocalcin is a recoverin-like protein expressed in bovine brain.     

Testosterone potentiation of the effectiveness of ACTH1-24 on the induction of the stretch-yawning syndrome (SYS) in male guinea pigs

Ventricular administration of ACTH^1–24 stimulated stretch-yawning in castrated male guinea pigs in a dose-related manner. Daily subcutaneous treatment with testosterone propionate (TP) facilitated the effects of ACTH^1–24 on this response. TP given without ACTH^1–24 stimulated yawning, but not stretching or combined stretch-yawning. Unlike most other species, guinea pigs did not display auto-grooming, scratching, or wet-dog shaking in response to intracranial ACTH^1–24. The results suggest that testosterone may alter the sensitivity of neural mechanisms which are responsive to ACTH^1–24.     

Oxygen evolution in dark-developed spruce chloroplasts

Chloroplasts isolated from dark-grown seedlings of Picea abiesshowed activities of DCIP and Fecy photoreductions widiout additionof an electron donor to PS-II. In addition to this, when light-inducedoxygen evolution was measured with an oxygen electrode, a significantamount of oxygen was found. These results indicate that thephotoreductions are coupled to the oxygen-evolving reaction.Furthermore, thylakoid membranes were functional in the protonuptake and the 515-nm absorbance change as parameters of theirphysicochemical functions. Electron microscopy showed that thylakoidswere well-developed with prolamellar bodies and partially stackedto from grana. We conclude that oxygen-evolving ability and the physicochemicalfunction of thylakoid membranes develop in chloroplasts of dark-grownspruce seedlings. (Received September 21, 1974; )     

Cooperative conformational change and aggregation of concanavalin A in alkaline solutions

Three properties, the binding activity to Sephadex G-75, conformation, and the extent of aggregation, of concanvalin A. (con A) in alkaline pH solutions were examined with special attention to the time course and their time-independent final values. Highly cooperative conformational changes among four subunits were suggested which were coupled either with protonation in the case of demetallized con A or with metal binding in the case of metal-liganded con A. Midpoints of the conversions of the metal-liganded con A were about pH 8.8, 9.1 and 9.1 with respect to the activity, the conformational change and the aggregation, respectively. These values were about 1 pH higher than the corresponding values of demetallized con A: 7.9, 8.05 and 8.2. Each conversion took place in narrow pH ranges. The pH range for the loss of activity was found to be significantly lower than those of the other two. The aggregation was suggested not to be coupled with the conformational change. Dissociation into subunits did not take place indicating strong interactions among four subunits in the tetramer.     

Altered reactivity of immunoglobulin produced by human-human hybridoma cells transfected by pSV2-neo gene

The HB4C5 and HF10B4 cell lines are human-human hybridomas producing human IgM monoclonal antibodies (MAbs) reactive to porcine carboxypeptidase A (CPase), but not to double stranded DNA (ds DNA). We obtained G418-resistant HB4C5 and HF10B4 cells by an introduction of pSV2-neo DNA. Almost all of the G418-resistant clones produced MAbs reactive to not only the CPase but the ds DNA. The results of the inhibition ELISA suggested that the cross-reactivity of the antibodies from G418-resistant clones to CPase and ds DNA was responsible for the alteration on their antigen specificity. HB4C5 and HF10B4 cells and their G418-resistant clones produced antibodies having glycosylated chain. The antibodies produced by tunicamycin-treated G418-resistant subclones of HB4C5 and HF10B4 lost the ability to bind to ds DNA, but retained the ability to bind to CPase. These results suggest that an introduction of pSV2-neo DNA into these hybridomas alters the specificities of their MAbs, and that the alteration to antigen binding specificities of their MAbs may be associated with glycosylation of the MAbs by these hybridomas.     

Aerobic denitrifying bacteria that produce low levels of nitrous oxide

Most denitrifiers produce nitrous oxide (N(2)O) instead of dinitrogen (N(2)) under aerobic conditions. We isolated and characterized novel aerobic denitrifiers that produce low levels of N(2)O under aerobic conditions. We monitored the denitrification activities of two of the isolates, strains TR2 and K50, in batch and continuous cultures. Both strains reduced nitrate (NO(3)(-)) to N(2) at rates of 0.9 and 0.03 micro mol min(-1) unit of optical density at 540 nm(-1) at dissolved oxygen (O(2)) (DO) concentrations of 39 and 38 micro mol liter(-1), respectively. At the same DO level, the typical denitrifier Pseudomonas stutzeri and the previously described aerobic denitrifier Paracoccus denitrificans did not produce N(2) but evolved more than 10-fold more N(2)O than strains TR2 and K50 evolved. The isolates denitrified NO(3)(-) with concomitant consumption of O(2). These results indicated that strains TR2 and K50 are aerobic denitrifiers. These two isolates were taxonomically placed in the beta subclass of the class Proteobacteria and were identified as P. stutzeri TR2 and Pseudomonas sp. strain K50. These strains should be useful for future investigations of the mechanisms of denitrifying bacteria that regulate N(2)O emission, the single-stage process for nitrogen removal, and microbial N(2)O emission into the ecosystem.     

DNA Inversion Regulates Outer Membrane Vesicle Production in Bacteroides fragilis

Phase changes in Bacteroides fragilis, a member of the human colonic microbiota, mediate variations in a vast array of cell surface molecules, such as capsular polysaccharides and outer membrane proteins through DNA inversion. The results of the present study show that outer membrane vesicle (OMV) formation in this anaerobe is also controlled by DNA inversions at two distantly localized promoters, IVp-I and IVp-II that are associated with extracellular polysaccharide biosynthesis and the expression of outer membrane proteins. These promoter inversions are mediated by a single tyrosine recombinase encoded by BF2766 (orthologous to tsr19 in strain NCTC9343) in B. fragilis YCH46, which is located near IVp-I. A series of BF2766 mutants were constructed in which the two promoters were locked in different configurations (IVp-I/IVp-II = ON/ON, OFF/OFF, ON/OFF or OFF/ON). ON/ON B. fragilis mutants exhibited hypervesiculating, whereas the other mutants formed only a trace amount of OMVs. The hypervesiculating ON/ON mutants showed higher resistance to treatment with bile, LL-37, and human β-defensin 2. Incubation of wild-type cells with 5% bile increased the population of cells with the ON/ON genotype. These results indicate that B. fragilis regulates the formation of OMVs through DNA inversions at two distantly related promoter regions in response to membrane stress, although the mechanism underlying the interplay between the two regions controlled by the invertible promoters remains unknown.     

Purification and characterization of organellar DNA polymerases in the red alga Cyanidioschyzon merolae

DNA polymerase gamma, a mitochondrial replication enzyme of yeasts and animals, is not present in photosynthetic eukaryotes. Recently, DNA polymerases with distant homology to bacterial DNA polymerase I were reported in rice, Arabidopsis, and tobacco, and they were localized to both plastids and mitochondria. We call them plant organellar DNA polymerases (POPs). However, POPs have never been purified in the native form from plant tissues. The unicellular thermotrophic red alga Cyanidioschyzon merolae contains two genes encoding proteins related to Escherichia coli DNA polymerase I (PolA and PolB). Phylogenetic analysis revealed that PolB is an ortholog of POPs. Nonphotosynthetic eukaryotes also have POPs, which suggested that POPs have an ancient origin before eukaryotic photosynthesis. PolA is a homolog of bacterial DNA polymerase I and is distinct from POPs. PolB was purified from the C. merolae cells by a series of column chromatography steps. Recombinant protein of PolA was also purified. Sensitivity to inhibitors of DNA synthesis was different in PolA, PolB, and E. coli DNA polymerase I. Immunoblot analysis and targeting studies with green fluorescent protein fusion proteins demonstrated that PolA was localized in the plastids, whereas PolB was present in both plastids and mitochondria. The expression of PolB was regulated by the cell cycle. The available results suggest that PolB is involved in the replication of plastids and mitochondria.