GROWTH RESPONSES OF SEVERAL DIATOM SPECIES ISOLATED FROM VARIOUS ENVIRONMENTS TO TEMPERATURE

Eight diatom species (Chaetoceros pseudocurvisetus Mang ., Stephanodiscus hantzschii Grun ., Skeletonema costatum ( Grev.) Cleve , Asterionella formosa Hass ., Thalassiosira nordenskioeldii Cleve , Detonula confervacea ( Cleve) Gran , Chaetoceros sp., and Nitzschia frigida Grun.) were isolated from various temperature environments ranging from temperate to the Arctic, and their growth responses to temperature were determined. Each species grew over a different temperature range. The lower and upper limits of each species varied from −1.8° to 20° C and from 2° to 30° C, respectively. The width of the growth range of each species. also varied from 3.8° to 25° C, and the growth of these species was observed, as a whole, between a wide temperature range from −1.8° to 30° C .
Within the growth temperature ranges, the growth rate of each species increased with temperature until reaching a maximum, which was followed by a steep decrease up to the upper limit of the growth range. As a result, each species showed a maximum rate at the temperature very near to the upper limit, which was generally higher than the isolation temperature. The specific growth rates were compared among the eight species. The interspecific maximum rate at each temperature exhibited an exponential increase with a Q₁₀ = 1.48. The relative growth rates of each species were calculated by normalizing the specific growth rates with the interspecific maximum rate at each respective temperature. The higher relative growth rates tended to occur at the isolation temperature of each species, suggesting that temperature is a significant control on species distributions in nature .     

Molecular cloning of two tandemly arranged peroxidase genes from Populus kitakamiensis and their differential regulation in the stem

A genomic library was prepared from Populus kitakamiensis and screened with the cDNA for an anionic peroxidase from P. kitakamiensis. One genomic clone was isolated that contained two tandemly oriented genes for anionic peroxidases, prxA3a and prxA4a. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, namely, GT and AG, at their 5 and 3 ends, respectively. The prxA3a and prxA4a genes encoded 347 and 343 amino acid residues, respectively, including putative signal sequences at the amino-termini. Putative promoters and polyadenylation signals were found in the flanking regions of both genes. The sequence of the coding region of prxA3a was completely identical to that of the cDNA clone pA3, whereas the sequence of the coding region of prxA4a was only 73% identical to that of the cDNA clone pA3. Northern blot analysis showed that the patterns of expression of the mRNAs that corresponded to prxA3a and prxA4a differed in stems of P. kitakamiensis.     

Purification and characterization of the Pseudomonas aeruginosa NfxB protein, the negative regulator of the nfxB gene.

The protein NfxB, involved in conferring resistance to quinolones in Pseudomonas aeruginosa, has a helix-turn-helix motif which is similar to that of other DNA-binding proteins. It appears to affect the membrane-associated energy-driven efflux of some antibiotics (H. Nikaido, Science 264:382-388, 1994). We constructed a plasmid that overproduced NfxB in Escherichia coli and purified the protein. Two species of NfxB (23 and 21 kDa), which are probably translated from different initiation codons, were isolated. Both proteins are also expressed in vivo in P. aeruginosa, with the 23-kDa NfxB being the major species. NfxB specifically binds upstream of the nfxB coding region as demonstrated by gel retardation and DNase I footprinting. Expression of the phi (nfxB'-lacZ+) (Hyb) gene was repressed in the presence of the nfxB gene product provided by a second compatible plasmid in E. coli. In the P. aeruginosa wild-type strain (PAO2142), NfxB was undetectable by immunoblotting; however, it was detected in the nfxB missense mutant (PK1013E). These results suggested that NfxB negatively autoregulates the expression of nfxB itself. Since the 54-kDa outer membrane protein (OprJ) (N. Masuda, E. Sakagawa, and S. Ohya, Antimicrob. Agents Chemother. 39:645-649, 1995) was overproduced in nfxB mutants, NfxB may also regulate the expression of membrane proteins that are involved in the drug efflux machinery of P. aeruginosa.     

Unique tissue distribution of a mouse macrophage C-type lectin

We examined mouse tissue for the expression of macrophage galactose/N-acetylgalactosamine-specificC-type lectin using a rat monoclonal antibody (mAb) specificfor this lectin (mAb LOM-14). The binding of mAb LOM-14 wasdetected in detergent extracts from tissue by means of immunoblottinganalysis. It was shown that this mAb did not cross-react withmouse hepatic lectins, a structural homologue. The macrophagelectin was widely distributed among various mouse tissues asjudged by the affinity isolation followed by the immunochemicaldetection. The exceptions were brain, liver, kidney, small intestine,and peripheral blood. Extracts from these organs exhibited,at best, very weak signals upon mAb LOM-14 binding, despitethe presence of cells expressing macrophage markers. The mostintense signal was observed in the extract from skin, suggestingthat cells expressing this lectin are abundant in skin. Thetissues shown to contain this lectin were further investigatedby immunohistochemical staining of the sections. Cells weredistributed in the connective tissue and in the interstice,particularly the dermis and subcutaneous layer of skin. Cellslocalized in the epithelium of skin (epidermis) or other epitheliathat we examined were not stained. Perivascular localizationof cells stained with mAb LOM-14 was also demonstrated in cardiacand skeletal muscle tissues. Immunoelectron microscopy revealedthe presence of this lectin along the rough endoplasmic reticulum.In conclusion, the distribution of C-type lectin specific forgalactose/N-acetylgalactosamine in mice was unique. The connectivetissue-specific distribution should provide important informationon the biological role of this lectin. lectin macrophage calcium-type lectin connective tissue     

Endotoxin Induces Severe Inflammatory Reactions with Necrosis at Sites Primed with Delayed-Type Hypersensitivity Reactions in Guinea Pigs

Guinea pigs immunized with Freund's complete adjuvant received challenge injection of the purified protein derivative of Mycobacterium tuberculosis in the flanks and the corneas to prepare delayed-type hypersensitivity (DTH) reactions. The animals were injected subcutaneously with lipopolysaccharide (LPS) or a synthetic lipid A (LA-15-PP). At the skin site primed with DTH reaction, increased swelling and hemorrhagic reaction followed by a definite necrotic reaction occurred. Severe corneal reactions were also observed in the animals. These findings indicate that bacterial endotoxin modulates DTH reactions and induces severe inflammatory reactions.     

Purification,primary structure,and evolution of cytochrome c-550 from the magnetic bacterium,Magnetospirillum magnetotacticum

Cytochrome c-550 was purified from Magnetospirillum magnetotacticum to an electrophoretically homogeneous state, and some of its properties were determined. The cytochrome showed absorption peaks at 528 and 409 nm in the oxidized form, and at 550, 521, and 414 nm in the reduced form. Its midpoint redox potential at pH 7.0 was determined to be +289 mV. The primary structure of cytochrome c-550 was determined. Cytochrome c is composed of 97 amino acid residues, and its molecular weight was calculated to be 10,873, including heme c. Its primary structure is very similar to those of Rhodospirillum fulvum and Rhodospirillum molischianum cytochromes c ₂, suggesting that M. magnetotacticum is phylogenetically related to photosynthetic bacteria.     

Mucopolysaccharidosis IVA: polymorphic haplotypes and informative RFLPs in the Japanese population

Seven different restriction fragment length polymorphisms (RFLPs) at the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) locus were analyzed using Southern blotting and polymerase chain reaction based techniques to search for the frequency of each RFLP produced by StyI, SphI, HaeIII, StuI, HapII, XhoI, and BamHI restriction endonucleases, respectively, in 36 mutant alleles, including two sibling cases and 100 normal alleles. Calculation of heterozygosity indexes showed that these RFLPs were polymorphic, ranging from 0.31 to 0.69 in mucopolysaccharidosis IVA (MPS IVA) patients compared with 0.21 to 0.65 in normal individuals. There was some significant difference in several RFLPs and in the combination with four kinds of RFLPs (SphI, StuI, HapII, XhoI polymorphisms). The normal alleles were composed of 13 different RFLPs haplotypes; the most common among the Japanese population carrying normal alleles was haplotype 8 (bDEF1) (31.3%), the others being dispersed. The same haplotype 8 was the most frequent in the mutant alleles (44.4%), with seven further haplotypes. These findings revealed the striking variety of polymorphic haplotypes in the MPS IVA gene. By using these five kinds of RFLPs, we examined the theoretical informativity of haplotype analysis in heterozygote detection in nine unrelated MPS IVA families and ten unrelated normal families. All the members of the MPS IVA families studied were diagnosed as a patient, carrier, or noncarrier. We propose that prenatal diagnosis or family analysis in cases in which mutations have not been characterized is now feasible.     

Electron microscopic study of endogenous peroxidase activity in human liver macrophages

We evaluated the conditions of fixation for ultrastructurally demonstrating the endogenous peroxidase (PO) activity of macrophages in biopsied human liver. The application of microwaving and immersion fixation with tannic acid and aldehydes allowed excellent visualization of PO activity in the nuclear envelope (NE), rough endoplasmic reticulum (rER), and cytoplasmic granules (CG), with good preservation of cellular ultrastructures. The macrophages with PO activity showed one of the following five patterns of PO localization: positive in both the NE and rER but negative in the CG (type 1); negative in both the NE and rER but positive in the CG (type 2); negative in the NE but positive in both the rER and CG (type 3); positive in all three (type 4); PO negative (type 5). The type 1 cells resembled typical Kupffer cells, type 2 cells monocytes, and type 3 and 4 cells the exudate-resident macrophages considered to be a transitional form between exudate and resident macrophages. Type 5 cells may also be a transitional form between the exudate and resident macrophage, or an end-stage macrophage derived from exudate macrophages which have lost their PO activity. Tannic-acid-aldehyde immersion fixation with microwaving may be a useful method in the study of the PO activities of macrophages in biopsied human liver specimens.     

Carbonic Anhydrase from the Blue-Green Alga (Cyanobacterium) Anabaena variabilis

Carbonic anhydrase (CA) activity was detected in homogenatesfrom Anabaena variabilis ATCC 29413, M-2 and M-3, but not inthe suspension of the intact cells. Activity was higher in cellsgrown in ordinary air (low-CO₂ cells) than in those grown inair enriched with 2–4% CO₂ (high-CO₂ cells). Fractionationby centrifugation indicated that the CA from A. variabilis ATCC29413 is soluble, whereas both soluble and insoluble forms existin A. variabilis M-2 and M-3. The addition of dithiothreitoland Mg^{2 $} greatly decreased the CA activity of A. variabilisATCC 29413. The specific activity of the CA from A. variabilis ATCC 29413was increased ca. 200 times by purification with ammonium sulfate,DEAE-Sephadex A-50 and Sephadex G-100. Major and minor CA peaksin Sephadex G-100 chromatography showed respective molecularweights of 48,000 and 25,000. The molecular weight of the CAdetermined by polyacrylamide disc gel electrophoresis was 42,000?5,000.The activity of CA was inhibited by ethoxyzolamide (I₅₀=2.8?10^-9M), acetazolamide (I₅₀=2.5?10^-7 M) and sulfanilamide (I₅₀=2.9?10^-6M). (Received January 5, 1984; Accepted April 26, 1984)     

Nitrobacter agilis Cytochrome c-550: Isolation, Physicochemical and Enzymatic Properties, and Primary Structure

Nitrobacter agilis cytochrome c-550 was purified to an electrophoreticallyhomogeneous state, and some of its properties were determined.The cytochrome showed an absorption peak at 410 nm in the oxidizedform, and peaks at 416, 521 and 550 nm in the reduced form.Its isoelectric point was 8.1 at 5?C. Analysis of the aminoacid composition showed that the cytochrome molecule was composedof 108 amino acid residues, 16 of which were lysine residues. The cytochrome reacted rapidly with N. agilis cytochrome c oxidaseand yeast cytochrome c peroxidase and more slowly with Pseudomonasaeruginosa nitrite reductase and bovine cytochrome c oxidase.The reactivities with these redox enzymes suggested that thecytochrome might be an evolutionary stage between bacterialand eukaryotic cytochromes c. The primary structure of the cytochrome from the N-terminusto the 85th residue was determined. The N-terminal sequencewas homologous to the corresponding portion of the primary structureof horse cytochrome c. ¹ Present adress: Department of Chemistry, Faculty of Science,Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo,152, Japan. (Received December 3, 1981; Accepted January 28, 1982)