Expression of proteins containing disulfide bonds in an insect cell-free system and confirmation of their arrangements by MALDI-TOF MS

Escherichia coli alkaline phosphatase (AP) and human lysozyme (h-LYZ), which contain two and four disulfide bonds, respectively, were expressed in a cell-free protein synthesis system constructed from Spodoptera frugiperda 21 (Sf21) cells. AP was expressed in a soluble and active form using the insect cell-free system under non-reducing conditions, and h-LYZ was expressed in a soluble and active form under non-reducing conditions after addition of reduced glutathione (GSH), oxidized glutathione (GSSG), and protein disulfide isomerase (PDI). The in vitro synthesized proteins were purified by means of a Strep-tag attached to their C termini. Approximately 41 microg AP and 30 microg h-LYZ were obtained from 1 mL each of the reaction mixture. The efficiency of protein synthesis approached that measured under reducing conditions. Analysis of the disulfide bond arrangements by MALDI-TOF MS showed that disulfide linkages identical to those observed in the wild-type proteins were formed.     

Reductive genome evolution from the mother of Rickettsia

The Rickettsia genus is a group of obligate intracellular α-proteobacteria representing a paradigm of reductive evolution. Here, we investigate the evolutionary processes that shaped the genomes of the genus. The reconstruction of ancestral genomes indicates that their last common ancestor contained more genes, but already possessed most traits associated with cellular parasitism. The differences in gene repertoires across modern Rickettsia are mainly the result of differential gene losses from the ancestor. We demonstrate using computer simulation that the propensity of loss was variable across genes during this process. We also analyzed the ratio of nonsynonymous to synonymous changes (Ka/Ks) calculated as an average over large sets of genes to assay the strength of selection acting on the genomes of Rickettsia, Anaplasmataceae, and free-living γ-proteobacteria. As a general trend, Ka/Ks were found to decrease with increasing divergence between genomes. The high Ka/Ks for closely related genomes are probably due to a lag in the removal of slightly deleterious nonsynonymous mutations by natural selection. Interestingly, we also observed a decrease of the rate of gene loss with increasing divergence, suggesting a similar lag in the removal of slightly deleterious pseudogene alleles. For larger divergence (Ks > 0.2), Ka/Ks converge toward similar values indicating that the levels of selection are roughly equivalent between intracellular α-proteobacteria and their free-living relatives. This contrasts with the view that obligate endocellular microorganisms tend to evolve faster as a consequence of reduced effectiveness of selection, and suggests a major role of enhanced background mutation rates on the fast protein divergence in the obligate intracellular α-proteobacteria.     

Rapid SNP diagnostics using asymmetric isothermal amplification and a new mismatch-suppression technology

We developed a rapid single nucleotide polymorphism (SNP) detection system named smart amplification process version 2 (SMAP 2). Because DNA amplification only occurred with a perfect primer match, amplification alone was sufficient to identify the target allele. To achieve the requisite fidelity to support this claim, we used two new and complementary approaches to suppress exponential background DNA amplification that resulted from mispriming events. SMAP 2 is isothermal and achieved SNP detection from whole human blood in 30 min when performed with a new DNA polymerase that was cloned and isolated from Alicyclobacillus acidocaldarius (Aac pol). Furthermore, to assist the scientific community in configuring SMAP 2 assays, we developed software specific for SMAP 2 primer design. With these new tools, a high-precision and rapid DNA amplification technology becomes available to aid in pharmacogenomic research and molecular-diagnostics applications.     

Circumnutational movement in rice coleoptiles involves the gravitropic response: analysis of an agravitropic mutant and space‐grown seedlings

Plants exhibit helical growth movements known as circumnutation in growing organs. Some studies indicate that circumnutation involves the gravitropic response, but this notion is a matter of debate. Here, using the agravitropic rice mutant lazy1 and space‐grown rice seedlings, we found that circumnutation was reduced or lost during agravitropic growth in coleoptiles. Coleoptiles of wild‐type rice exhibited circumnutation in the dark, with vigorous oscillatory movements during their growth. The gravitropic responses in lazy1 coleoptiles differed depending on the growth stage, with gravitropic responses detected during early growth and agravitropism during later growth. The nutation‐like movements observed in lazy1 coleoptiles at the early stage of growth were no longer detected with the disappearance of the gravitropic response. To verify the relationship between circumnutation and gravitropic responses in rice coleoptiles, we conducted spaceflight experiments in plants under microgravity conditions on the International Space Station. Wild‐type rice seeds were germinated, and the resulting seedlings were grown under microgravity or a centrifuge‐generated 1 g environment in space. We began filming the seedlings 2 days after seed imbibition and obtained images of seedling growth every 15 min. The seed germination rate in space was 92–100% under both microgravity and 1 g conditions. LED‐synchronized flashlight photography induced an attenuation of coleoptile growth and circumnutational movement due to cumulative light exposure. Nevertheless, wild‐type rice coleoptiles still showed circumnutational oscillations under 1 g but not microgravity conditions. These results support the idea that the gravitropic response is involved in plant circumnutation.     

Development of the catecholaminergic innervation of the song system of the male zebra finch

Catecholamines (CA) have been proposed to have neuromodulatory actions, particularly on attention and learning, in a number of neural systems. Because several of the interconnected brain nuclei that mediate song learning and production in the adult male zebra finch (Taeniopygia guttata) contain these neurotransmitters, we investigated the appearance of the catecholaminergic innervation of the song nuclei of male zebra finches during posthatch development, specifically during the period in which song learning occurs. We studied the development of immunoreactivity for tyrosine hydroxylase (TH) in the song nuclei HVc, RA, NIf, LMAN, and Area X in young males aged 20, 35, and 60 days as well as in adults (>90 days). We also visualized catecholamines directly in Area X using CA histofluorescence. Both TH immunoreactivity and CA histofluorescence were initially low in Area X relative to their levels in the surrounding parolfactory lobe (LPO), and then increased during development to become more intense than in LPO by days 60–90. Similarly, TH immunoreactivity in HVc was initially low relative to that in the surrounding neostriatum, then increased during development to become more intense than that in the surround by day 60. TH immunostaining also increased markedly in NIf, RA, and LMAN over the same period. These results show that the levels of catecholamines and their major synthetic enzyme increase in song nuclei during development and thus raise the possibility that these transmitters contribute to the development of the song system or to song learning. © 1996 John Wiley & Sons, Inc.     

Liposome-encapsulated superoxide dismutase suppresses liposome-mediated augmentation of TNF-alpha production from peripheral blood leucocytes

Liposome-encapsulated hemoglobin (LEH), a candidate for a red cell substitute, has been reported to be cleared from circulation primarily by the phagocytic system and modulate the production of inflammatory cytokines, such as TNF-alpha and IL-6, both in vivo and in vitro. In this study, we investigated the effects of liposome vesicles on the LPS-induced TNF-alpha production using a whole blood culture system. We also studied the effects of superoxide dismutase (SOD) encapsulated in liposome on the cytokine production. The pre-treatment of whole blood with liposome vesicles potentiated the LPS-induced TNF-alpha production. The encapsulation of SOD in the liposome vesicles suppressed the liposome-mediated augmentation of TNF-alpha production in a dose-dependent manner. These results suggest that encapsulation of SOD in LEH decreases the production of inflammatory cytokines from the phagocytic system which may be caused or augmented by LEH infusion in vivo.     

Constructing a local district telepathology network in Japan. Diagnosis of intraoperative frozen sections via telepathology over an integrated service digital network and the National Television Standard Committee System

OBJECTIVE: To construct a local telepathology network between the Department of Pathology, Tohoku University Hospital, and Koritu Kesennuma Hospital, about 150 km away. STUDY DESIGN: Tohoku University Hospital is connected with Koritu Kesennuma Hospital by an integrated service digital network for telepathology using the National Television Standard Committee system. The cases submitted for telepathology were limited to those in which a rapid intraoperative diagnosis was made on frozen sections. RESULTS: At this writing, more than 200 cases were diagnosed during a period of 2.5 years. The cases submitted increased with time, amounting to 150 in 1996. In some cases the use of telepathology proved to be fairly advantageous. For example, in one case a radical operation was avoided because of a diagnosis on intraoperative frozen sections. DISCUSSION: There are problems to be solved before telepathology becomes available for practical use: (1) misdiagnosis due to poor quality of instruments, including the transmission cable and pictures; (2) cost-benefit ratio, (3) protection of patients' privacy, and (4) overwork for pathologists. The Japanese government will officially accept telepathology as a means of medical examination in the future. Despite some problems left, telepathology is a promising technology.