An empirical approach for structure-based prediction of carbohydrate-binding sites on proteins

A computer program system was developed to predict carbohydrate-bindingsites on three-dimensional (3D) protein structures. The programssearch for binding sites by referring to the empirical rulesderived from the known 3D structures of carbohydrate–proteincomplexes. A total of 80 non-redundant carbohydrate–proteincomplex structures were selected from the Protein Data Bankfor the empirical rule construction. The performance of theprediction system was tested on 50 known complex structuresto determine whether the system could detect the known bindingsites. The known monosaccharide-binding sites were detectedamong the best three predictions in 59% of the cases, whichcovered 69% of the polysaccharide-binding sites in the targetproteins, when the performance was evaluated by the overlapbetween residue patches of predicted and known binding sites. Received April 24, 2003; revised June 2, 2003; accepted June 10, 2003.     

Genetically engineered active Qbeta replicase in rabbit reticulocyte cell-free system: a fusion protein of EF-Tu and EF-Ts is functional as the subunit of Qbeta replicase

Qbeta replicase functioning in Escherichia coli is an RNA-dependent RNA polymerase composed of one phage-coded subunit and three host-coded proteins: ribosomal protein S1, and protein elongation factors EF-Tu and EF-Ts. Qbeta replicase lacking ribosomal protein S1 (alpha-less replicase) is capable of replicating some small RNAs. We attempted to create functional alpha-less replicase by co-expression of the mRNAs that code for the subunits of alpha-less replicase in a rabbit reticulocyte cell-free translation system. Replicase activity, however, could not be detected when both EF-Tu and EF-Ts were co-expressed with the phage-coded subunit. On the other hand, active alpha-less replicase was obtained when an EF-Ts-EF-Tu fusion protein was co-expressed with the phage-coded subunit. Consequently, we succeeded in generating genetically engineered active alpha-less Qbeta replicase which functions in a eukaryotic cell-free system.     

Immunohistochemical demonstration of 3,2''-dimethyl-4-aminobiphenyl DNA adduct formation in various organs of Syrian golden hamsters

We attempted to purify endogenous growth factors of chick embryonal retinal pigment epithelial cells from bovine neural retina. Ammonium sulfate precipitation, gel filtration, and anion exchange column chromatography were used. Though only partially purification, bovine retinal extract had two effects, growth and transformation, on chick embryonal pigment epithelial cells. We think that these purified factors may be novel from the estimation of their molecular weight by gel filtration.     

A light-transmitting two-dimensional photodetector array using a-Sipin photodiodes and poly-Si TFT's integrated on a transparent substrate

A light-transmitting two-dimensional photodetector array (32×32 cells) using amorphous silicon (a-Si) pin photodiodes and polysilicon (poly-Si) thin film transistors (TFT's) integrated on a transparent substrate has been developed for use in a free-space optical switching network. The fabrication and the characteristics of the photodetector array are discussed. With driving circuits and sensing amplifiers, this photodetector array shows a minimum detectable power of -25 dBm and an insertion loss of 0.4 dB for an incident optical beam with a diameter of 550 μm. By monitoring the positions and the states of input optical beams, this photodetector array can be used to control the optical paths in photonic switching systems, such as a 1024-input-port optical concentrator     

Abnormal metabolism of polyunsaturated fatty acids and phospholipids in diabetic glomeruli

Studies were done on changes in phospholipid content and fatty acid composition of phospholipids and on the role of the acylation pathway in synthesis of phospholipids in the development of abnormal fatty acid composition in the glomeruli of rats 2 and 10 mo after induction of diabetes with streptozotocin. The proportions of individual phospholipids in the glomeruli of rats were not changed 2 mo after induction of diabetes, but the proportion of phosphatidylethanolamine (PE) decreased and that of sphingomyelin increased 10 mo after induction of diabetes. In contrast, in liver the proportion of PE was increased and that of phosphatidylcholine was decreased. These results showed that changes of individual phospholipids in glomeruli were time-dependent and tissue-specific. Two mo after induction of diabetes, the main change in the phospholipid fatty acid composition of diabetic glomeruli was a decrease in arachidonic acid (AA); the main change in serum free fatty acids (FFA) was an increase in linoleic acid (LA) and a decrease in AA. Ten mo after induction of diabetes, the main changes in the phospholipid fatty acid composition of glomeruli were an increase in LA and a decrease in AA; the main change of the serum FFA composition was a decrease in AA. Thus, the fatty acid composition of glomerular phospholipids was not directly correlated to that of the serum in diabetic rats. Acyl-CoA synthetase and acyltransferase activities increased in diabetic glomeruli with either AA or LA as substrate, but activity toward LA increased more at 2 mo after induction of diabetes. Acyl-CoA synthetase activity increased in diabetic glomeruli with LA as substrate, but that did not change with AA as substrate at 10 mo after induction of diabetes. Furthermore, acyltransferase activity decreased in diabetic glomeruli with AA as substrate, although that did not change with LA as substrate at 10 mo after induction of diabetes.     

Multiple pathways are involved in the extracellular processing of MHC class I-restricted peptides

T cell stimulation by certain class I-restricted antigenic peptides, such as the HIV 1 gp160-derived peptide, P18, requires peptide processing by angiotensin-1 converting enzyme (ACE) in FCS. We observed that longer versions of P18 and the murine cytomegalovirus pp89-derived core peptide, pMCMV, which could stimulate T cell hybridomas in FCS, were not as sensitive to the ACE inhibitor captopril as P18. Using cell-free soluble murine class I MHC molecules and protease inhibitors, we found that there are pathways of differing efficiency that use enzymes other than ACE for the proteolytic processing of peptides in serum. The kinetics of the generation of T cell stimulatory activity among P18 variant peptides in serum differed with peptide length, and with the nature of amino and COOH-terminal extensions. Such processing occurs in human plasma as well as in FCS. The understanding of this processing, its kinetics, and its inhibitors can lead to better design of peptide-based therapies, including vaccines.     

Development of a strain for efficient degradation of polychlorinated biphenyls by patchwork assembly of degradation pathways

Rhodococcus jostii RHA1 accumulates chlorobenzoates (CBA) during the degradation of polychlorinated biphenyls (PCBs). CBA degradation is considered one of the rate-limiting steps in the complete degradation of PCBs. To reduce the accumulation of CBAs, the upper pathway enzyme genes for PCB degradation of RHA1 were introduced into a CBA-degrading bacterium, Burkholderia sp. NK8. The resulting recombinant strain exhibited no biphenyl 2,3-dioxygenase (BphA) activity encoded by bphAaAbAcAd genes, which encode the large and small subunits of the terminal oxygenase component and the ferredoxin and reductase subunits responsible for electron transfer from NADH to the large subunit. The remaining enzyme genes involved in the transformation of biphenyl to benzoate, bphB2C1D1, which encode dehydrogenase, ring-cleavage dioxygenase and hydrolase, conferred activities to NK8. To obtain the BphA activity of RHA1 in NK8, sets of BphA genes were constructed by combining the bphAaAbAcAd genes of RHA1 and bphA3A4 of Pseudomonas pseudoalcaligenes KF707, encoding the ferredoxin and reductase subunits. Hybrid derivatives of BphA containing the KF707 bphA3 conferred BphA activity to NK8, and a derivative containing the RHA1 bphAaAb and KF707 bphA3A4 genes exhibited the highest BphA activity. A plasmid containing the RHA1 bphAaAb and KF707 bphA3A4 genes plus the RHA1 bphB2C1D1 genes was constructed and introduced into NK8. The resulting recombinant strain efficiently degraded 2-, 3- and 4-chlorobiphenyls with an apparent reduction in CBA accumulation in comparison to the recombinant mutant strain, which had an insertion in the cbeA gene to inactivate CBA dioxygenase.     

A novel gene delivery system in plants with calcium alginate micro-beads

We have produced micrometer-sized calcium alginate beads referred to as "bio-beads" that encapsulate plasmid DNA molecules carrying a reporter gene. In order to evaluate the efficiency of the bio-beads in mediating genetic transfection, protoplasts isolated from cultured tobacco cells (BY-2) were transfected with bio-beads containing a plasmid that carries the modified green fluorescent protein gene CaMV35S-sGFP. With the bio-beads treatment, approximately ten-fold higher GFP expression was observed after 24 h incubation compared to that with the conventional method using a naked plasmid solution. Transfection was up to 0.22% efficient. These results indicate that bio-beads have a possibility for efficient transformation in plants.     

Functional metal-porphyrazine derivatives and their polymers, 10. Secondary fuel cells based on oxygen reduction on platinum electrode modified by polymers and metal-phthalocyanines

Secondary fuel cells based on oxygen reduction of platinum electrode modified by polymers and metal-phthalocyanine (Mt = Fe(III), Co(II), Ni(II), and Cu(II)) were studied. The discharge curves for the platinum electrode modified by poly(2-vinylpyridine) (or polystyrene) and Co-phthalocyanine in 30% KOH aqueous solution, for a 30 min charge at 500 μA, followed by a 100 μA discharge showed a stable plateau at about ?0.24 V SCE (Saturated Calomel Electrode). The open circuit voltage (vs. Zn) of the cell was 1.2 V, and the discharge capacity was of 46 A · h/kg. For this battery there was no significant decay in its characteristics after more than 30 charge-discharge cycles. In Mt-phthalocyanines, the values decreased in the order of Co(II) > Fe(III) > > Cu(II) > Ni(II). From a cyclic voltammogram for the electrode modified by the polymer and Co-Pc, the cathodic reactions were discussed.     

Quantitative risk assessment of noroviruses in drinking water based on qualitative data in Japan

Noroviruses are one of the major causes of viral gastroenteritis in Japan. A quantitative risk assessment was conducted to evaluate the health risk caused by this virus in drinking water. A Monte Carlo analysis was used to calculate both the probability of infection and the disease burden using disability-adjusted life years (DALYs). The concentration of noroviruses in tap water was estimated based on qualitative data and a most probable number (MPN) method with an assumed Poisson lognormal distribution. This numerical method was evaluated using two sets of available count data of Cryptosporidium: that collected from a river and that found in tap water in Japan. The dose-response relationships for noroviruses were estimated using assumed ID50 (10 or 100). The annual risk was higher than the US-EPA acceptable level (10(-4) [infection/ person-year]) but around the WHO level (10(-6) [DALYs/ person-year]). As suggested by others, since microbial concentrations are generally lognormally distributed, the arithmetic mean was directly related to the annual risk, suggesting that the arithmetic mean is more useful in representing the degree of microbial contamination than the geometric mean.