Enclosure experiment on the control mechanism of planktonic bacterial standing stock

In order to understand the control mechanisms of a large, stable bacterial standing stock, enclosure experiments were conducted in a eutrophic lake, where both bacterial productivity and grazing pressure were very high. Total bacterial number in the different enclosures ranged from 1.2 to 2.7×10⁷ cells mL⁻¹ throughout the experiment. The average bacterial cell production rate estimated from a grazer eliminating experiment was 6.3×10⁵ cells mL⁻¹ h⁻¹. Difference in the bacterial cell production rate between shaded and unshaded enclosures was not apparent. Bacteria showed a reduction in standing stock of only about 25–30% even after the supply of light was cut to 1%. Bacteria in the shaded enclosures then recovered their production rate in the first 12 days of perturbation. Grazing pressure in the shaded enclosures was not less than that for the control. Thus, it was considered a control mechanism of bacterial stable standing stock that the bacteria shifted their organic substrate from extracellular dissolved organic carbon freshly released from phytoplankton to that already stocked in the water column, though it is not known whether the dominant bacteria were the same.     

Blocking of human immunodeficiency virus infection depends on cell density and viral stock age.

Quantitative infectivity assays were used to study how the blocking activity of soluble CD4 (sCD4) is affected by sCD4 concentration, target cell density, and viral stock age. During incubation with 20 nM sCD4, human immunodeficiency virus type 1 (HIV-1) stocks underwent irreversible inactivation. In contrast, inactivation with 2 nM sCD4 was almost entirely reversible. At lower sCD4 concentrations (less than or equal to 2 nM) and target cell densities of 6.25 x 10(4) ml-1, sCD4 blocking activity for HIV-1 gave a gp120-sCD4 association constant (Kassoc) of 1.7 x 10(9) M-1, which agrees with chemical measurements. At the higher density of 1.6 x 10(7) cells ml-1, however, the blocking activity was 20-fold less. During incubation of HIV-1 stock optimized for infectivity by rapid harvest, sCD4 blocking activity increased 20-fold during a 3-h window. These results show that competitive blocking activity depends strongly on target cell density and virion age. Thus, unappreciated variations in HIV stocks and assay conditions may hinder comparisons of blockers from laboratory to laboratory, and the age of HIV challenge stocks may influence studies of drug and vaccine efficacy. The results also suggest that blocking of viral particles in lymphoid compartments will require very high competitive blocker concentrations, which may explain the refractory outcomes from sCD4-based drug trials in humans.     

Morphological effects of cadmium on proximal tubular cells in rats

Twenty-four male rats of the Wistar strain divided into four groups were injected sc with a dose of 0.8, 1.5, and 3.0 mg Cd/kg body wt as CdCl₂ in saline, and saline alone to the control rats, three times a week for 3 wk. Cadmium levels of whole kidney homogenate, supernatant (cytosol), precipitate, and metallothionein (MT) fraction were measured. Histological changes of the renal proximal tubules were investigated by optical and electron microscopy. In the kidneys, Cd levels were increased with the increment of Cd dosage; 80–90% of Cd was contained in cytosol, and 55–75% was in MT fraction. Non-MT-Cd reached a maximum in the 1.5 mg Cd group, whereas that of the 3.0 mg Cd group showed some decline. With increasing Cd doses, the size of nuclei and nucleoli in the cells of proximal tubule showed significant enlargement and also an increase in the number of nucleoli on light microscopy. At higher doses, chromatin condensation of the tubular nuclei and vacuolar degeneration of the tubular cells were evident. On electron microscopy, perichromatin granules of the proximal tubular nuclei were increased in number, especially in the rats of Cd 0.8 mg and 1.5 mg/kg groups. As the Cd doses increased, ring-shaped nucleoli were increased in number and nucleolar segregation was observed more clearly. Moreover, in the 3.0 mg/kg Cd group, nuclear indentation and nucleoli containing compact dense granules were observed. In the cytoplasm, there was an increase of lysosomes, myelin bodies, ring-shaped mitochondria, and vesiculation; ultimate changes were degeneration and cell necrosis. The injured cells were heterogenously distributed in each nephron and this heterogeneity was attributed in the difference in Cd content and cell cycle in each cell of the nephron.     

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene inEscherichia coli

Summary The application of an inducible regulation system using the trytophanase operon promoter (TPase promoter; P_tna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of P_tna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, l-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-P_tnaTS was increased 500-fold compared to that of the cultured host strain. About 1 mol l-tryptophan/l reaction mixture was formed from indole and l-serine at 37° C for 3.5 h. Offprint requests to: H. Yukawa     

Complementation analysis of the wild-type and mutant ompR genes exhibiting different phenotypes of osmoregulation of the ompF and ompC genes of Escherichia coli

Expression of the ompF and ompC genes, which encode the major outer membrane proteins, OmpF and OmpC, respectively, is affected in a reciprocal manner by the osmolarity of the growth medium. This osmoregulation is mediated by the OmpR protein, a positive regulator of both genes, which is encoded by the ompR gene. Structural and functional properties of this regulatory protein were studied through complementation analysis of the wild-type and five mutant ompR genes that exhibited differences in osmoregulation of the expression of the OmpF and OmpC proteins. Complementation was carried out with combinations of a host strain and a plasmid, each of which carried either the wild-type or a mutant ompR gene. In some combinations, negative complementation was observed. For example, ompR1, a deletion mutation with an OmpF- OmpC- phenotype, was dominant to OmpF+ or OmpC+ phenotypes conferred by other ompR genes. Positive complementation of two mutant ompR genes was also observed in other combinations, when the two mutations were distantly located from each other on the OmpR protein. These results, together with other observations, support the view that the OmpR protein has a two-domain structure, each domain exhibiting a different role in the expression of the OmpF and OmpC proteins, and that this protein takes a multimeric structure as a functional unit.     

Changes in mRNA of Vigna mungo Cotyledons during Seed Germination

Quantitative and qualitative changes of mRNA in Vigna mungocotyledons during seed germination have been investigated. TotalRNA is higher in dry cotyledons and declines during germination.Poly(A)⁺ RNA also is present at a relatively high level in drycotyledons, increases slightly during the first day of germination,and then decreases. Polysomal RNA is very low in dry cotyledonsbut increases rapidly during the first day of germination, andthen declines. The translational activity of the mRNA in a wheatgerm cell-free system is low on day 0 but increases rapidlyon day 1 of germination. Two-dimensional gel electrophoresisof in vitro translation products reveals that many new peptidesare synthesized on day 1 of germination. Synthesis of most ofthese polypeptides continue throughout 5 days of germination. Change in the mRNA population during germination has been investigatedusing cDNA against poly(A)⁺ RNA from 3-day-old cotyledons. Withtotal RNA of day 3 and 5, the cDNA strongly hybridized withRNA similar in size to 25 S ribosomal RNA, but no specific bandsare detected with samples of day 0 or 1. With poly(A)⁺ RNA ofday 5 or 1, the cDNA tends to hybridize with RNAs of relativelysmall molecular size. Cordycepin and -amanitin prevent the increasein poly (A)⁺ RNA content and the appearance of new mRNAs duringthe first day of germination. ¹Present address: Division of Regulation of Macromolecular Function,Institute for Protein Research, Suita City, Osaka 565, Japan. (Received January 13, 1986; Accepted June 10, 1986)     

Purification and Properties of Sweet Potato NADPH-Cytochrome c (P-450) Reductase

NADPH-cytochrome c reductase, strictly NADPH-cytochrome P-450reductase, was purified by chromatography through DEAE-cellulose,2',5'-ADP-Sepharose, and Sephadex G-100 columns after solubilizationfrom microsomes from Ceratocystis fimbriata-infected sweet potatoroot tissue with Emulgen 913. The enzyme existed in three formsafter solubilization which migrated to positions correspondingto molecular weights of 81,000, 75,000 and 72,000 on an SDS-polyacrylamidegel. Trypsin treatment of the enzyme species with the largestpolypeptide yielded the species with the smallest one. Aftersucrose density gradient centrifugation of the pellet fractionobtained by centrifugation at 100,000?g of the crude extract,the enzyme species with the largest polypeptide was presentin the particulate fractions, whereas that with the smallestone was only found at the top of the gradient. We conclude thatthe enzyme species with the largest polypeptide is in an intact,amphipathic form, whereas that with the smallest one, and probablyalso the other species, is its hydrophilic domain produced byan endogenous protease(s). The K_m values of the enzyme in theintact form for NADPH and cytochrome c were 7.7 and 2.3 µM,respectively. ¹ Present address: Laboratory of Food Hygienics, Faculty ofAgriculture, Kagawa University, Miki-cho, Kida-gun, Kagawa 761-07,Japan. (Received September 6, 1984; Accepted December 27, 1984)     

Isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato

Optimum conditions for the isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato (Ipomoea batatas) were investigated. Growth phase of callus and the ratio of callus-enzyme solution affected the yield of protoplasts. Composition of the medium and protoplast density were examined for protoplast culture.Small colonies were regenerated from the protoplasts. Upon transfer to light a high amount of anthocyanin was accumulated in these colonies.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-(N-morpholino)-ethanesulfonic acid     

Occurrence of anthocyanoplasts in cell suspension cultures of sweet potato

Intensely pigmented and spherical vesicles (anthocyanoplasts) were found in anthocyanin-containing cells of sweet potato (Ipomoea batatas) suspension cultures. Anthocyanin synthesis began to first occur 24–48 h after exposure to light, and then numerous small red vesicles were detected under a microscope. The frequency of anthocyanoplast-containing cells rapidly increased to finally about 80% of the total cultured cells after 5 days of irradiation. Fully developed anthocyanoplasts reached 10–15 m in diameter. On the other hand, neither anthocyanin synthesis nor development of anthocyanoplasts was induced in the dark-cultured cells. 2,4-D also inhibited anthocyanin synthesis and development of these vesicles. The results suggest that anthocyanoplasts might be a site of anthocyanin synthesis and/or accumulation.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid