Secondary structural changes of large and small fragments of bovine serum albumin in thermal denaturation and in sodium dodecyl sulfate denaturation

The helicities in various fragments of bovine serum albumin (BSA) were examined in the thermal denaturation and in sodium docecyl sulfate (SDS) denaturation. The thermal denaturation was examined in a temperature range between 2 and 65°C. The helicity decreased with a rise of temperature and it recovered to some degree upon cooling temperature. A rather high reversibility was observed in the BSA fragments, which were located in the N-terminal of the parent protein and then contained the first large loop with no disulfide bridge. The high reversibility was available also for the helicity in the first large loop of the fragment, disulfide bridges of which were reduced. The fragments, which were smaller than one domain, became unstable in the SDS denaturation. The helicities of such fragments decreased in lower SDS concentrations compared with those of the intact BSA and the large fragments, which contained one or more domains. A resistance to the SDS denaturation appeared in the helices of every large loop even after the fragmentation. On the other hand, helicities of the fragments decreased to 20–25% upon the reduction of disulfide bridges. However, the helicities of these fragments increased to 35–40% in the SDS denaturation.     

Effect of retinyl acetate on the assembly of the fibronectin extracellular matrix and the processing of the fibronectin receptor β subunit of confluent C3H/10T1/2 mouse embryo fibroblasts

The mouse embryo fibroblast cell line, C3H/10T1/2, synthesized and deposited a large amount of fibronectin especially in the pericellular matrix. Confluent cultures of these cells cultured in the presence of 0.3 μg/ml of retinyl acetate released cell surface fibronectin and the extracellular matrix fibronectin fibrils were disorganized. The immunoblot analysis demonstrated that the number of the fibronectin receptor was decreased in the prolonged culturing of retinyl acetate-treated cells. Immunoprecipitation of ³⁵S-methionine pulse-chase labeled cell extracts by antifibronectin receptor antibody indicated that about one-half of the pre-β subunit was processed and converted to the mature form in control cells, and only about one-fourth of the pre-β subunit was processed in the retinyl acetate-treated confluent cells. 1-deoxymannojirimycin (MNJ), which is an inhibitor of oligosaccharide processing, induced disorganization of the extracellular matrix fibronectin assembly similar to that observed with retinyl acetate. The results of this study suggest that a mechanism of action of retinyl acetate is inhibition of the glycosylation during processing of the fibronectin receptor, a step necessary for fibronectin binding and for assembly of the extracellular matrix. © 1993 Wiley-Liss, Inc.     

Induction of berberine biosynthesis by cytokinins in Thalictrum minus cell suspension cultures

Production of berberine could be induced by adding 6-benzylaminopurine (BAP) to Thalictrum minus cells, cultured in suspension in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), early in the growth cycle. In the presence of BAP, the precursor, L-tyrosine, was rapidly converted into berberine which was then released into the medium, whereas substantial amounts of the intermediates, tyramine and dopamine, accumulated in non-berberine-producing cells grown in the same 2,4-D-containing medium without BAP. These results suggest that BAP activated enzymatic reactions subsequent to the formation of the amines in the biosynthesis of berberine.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - IAP 6-isopentenylaminopurine - LS medium Linsmaier-Skoog medium - Growth medium LS medium containing 10^-6 M 2,4-D     

Effects of low culture temperature on the induction of hsp70 mRNA and the accumulation of hsp70 and hsp105 in mouse FM3A cells.

We have shown that heat shock does not induce the synthesis of hsp70 in FM3A cells maintained at a low culture temperature of 33 degrees C although it does so in cells maintained at 37 degrees C [T. Hatayama et al. (1991) Biochem. Int. 24, 467-474]. In this paper, we show that FM3A cells maintained at 37 degrees C produced hsp70 mRNA during continuous heating at 42 degrees C or during postincubation at either 37 or 33 degrees C after being heated at 45 degrees C for 15 min, whereas cells maintained at 33 degrees C did not produce hsp70 mRNA during continuous heating at 37, 39, 42, or 45 degrees C, or during postincubation after being heated at any temperature. Thus the lack of hsp70 synthesis in cells maintained at 33 degrees C seemed to be due to the absence of hsp70 mRNA induction. Also, hsp70 was accumulated in cells maintained at 37 degrees C during continuous heating at 42 degrees C and during postincubation at 37 degrees C after heat shock at 45 degrees C, but not during postincubation at 33 degrees C. The cellular level of the constitutive hsp73 as well as the mRNA level were both similar in cells maintained at 33 and 37 degrees C. On the other hand, the cellular level of the constitutive hsp105 in cells maintained at 33 degrees C was only half of that in cells maintained at 37 degrees C. These hsp105 levels increased significantly in both types of cells after continuous heating at 39 degrees C. These findings indicate that the culture temperature affects not only the induction of hsp70 mRNA but also the accumulation of hsp70 and hsp105 in the cells.     

Upregulation of uncoupling proteins by oral administration of capsiate, a nonpungent capsaicin analog.

Capsiate is a nonpungent capsaicin analog, a recently identified principle of the nonpungent red pepper cultivar CH-19 Sweet. In the present study, we report that 2-wk treatment of capsiate increased metabolic rate and promoted fat oxidation at rest, suggesting that capsiate may prevent obesity. To explain these effects, at least in part, we examined uncoupling proteins (UCPs) and thyroid hormones. UCPs and thyroid hormones play important roles in energy expenditure, the maintenance of body weight, and thermoregulation. Two-week treatment of capsiate increased the levels of UCP1 protein and mRNA in brown adipose tissue and UCP2 mRNA in white adipose tissue. This dose of capsiate did not change serum triiodothyronine or thyroxine levels. A single dose of capsiate temporarily raised both UCP1 mRNA in brown adipose tissue and UCP3 mRNA in skeletal muscle. These results suggest that UCP1 and UCP2 may contribute to the promotion of energy metabolism by capsiate, but that thyroid hormones do not.     

Growth Characteristics and Intraspecies Host Specificity of a Large Virus Infecting the Dinoflagellate Heterocapsa circularisquama

The growth characteristics and intraspecies host specificity of Heterocapsa circularisquama virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate H. circularisquama, were examined. Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells. HcV was infective over a wide range of temperatures, 15 to 30°C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively. Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20°C. Compared to Heterosigma akashiwo virus, HcV was more widely infectious to H. circularisquama strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection. The present results are helpful in understanding the ecology of algal host-virus systems in nature.     

Changes in levels of mRNAs of transforming growth factor (TGF)-β1, -β2, -β3, TGF-β type II receptor and sulfated glycoprotein-2 during apoptosis of mouse uterine epithelium

To examine the roles played by transforming growth factors (TGF)-β1, -β2, -β3, and TGF-β type II receptors in the induction of apoptosis in the mouse uterine epithelium after estrogen deprivation, we investigated the expression of their mRNAs and the mRNA of sulfated glycoprotein-2 (SGP-2). Pellets containing 100 μg estradiol-17β (E₂) were implanted into ovariectomized mice and removed four days later. Apoptotic indices (percentage of apoptotic cells) of both luminal and glandular epithelia increased after E₂ pellets were removed, but administration of progesterone (P), 5-dihydrotestosterone (DHT), or continued implantation of E₂ pellets suppressed this increase. Levels of mRNAs of TGF-β1, -β2, and -β3, and SGP-2 did not increase after estrogen deprivation. However, estrogen deprivation caused a gradual increase in the level of TGF-β type II receptor mRNA, and its level increased about six-fold six days later. Moreover, E₂, P, and DHT markedly decreased the level of TGF-β type II receptor mRNA. In situ hybridization demonstrated that mRNAs of TGF-β1, -β2, -β3 and TGF-β type II receptor were localized to the epithelium. Exogenous administration of TGF-β1 into the uterine stroma induced apoptosis in the epithelium, a finding that suggests that signals produced by TGF-βs can induce apoptosis. Therefore, the present results suggest that increased sensitivity of uterine epithelial cells to TGF-βs, as demonstrated by an increase in TGF-β type II receptor mRNA, is involved in the induction of apoptosis after estrogen deprivation, although signals produced by TGF-βs do not appear sufficient to induce apoptosis.     

Compilation of All Genes Encoding Bacterial Two-component Signal Transducers in the Genome of the Cyanobacterium, Synechocystis sp. Strain PCC 6803

Bacteria have devised sophisticated signaling systems for elicitinga variety of adaptive responses to their environment, whichare generally referred to as the "two-component regulatory system."The widespread occurrence of the two-component systems in bothprokaryotes and eukaryotes implies that it is a powerful devicefor a wide variety of adaptive responses of cells to their environment.The two-component signal transducers contain one or more ofthree conserved and characteristic phosphotransfer signalingdomains, named the "transmitter, receiver, and alternative transmitter."The recently determined entire genomic sequence of Synechocystissp. strain PCC 6803 allowed us to compile systematically a completelist of genes encoding such two-component signal transductionproteins. The results of such an effort, made in this study,revealed that at least 80 ORFs were identified as members ofthe two-component signal transducers in this single speciesof cyanobacteria.