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The accumulation of intramyocellular lipid (IMCL) and extramyocellular lipid (EMCL) is associated with arterial stiffness in middle-aged and older adults. Habitual aerobic exercise induces the improvement of arterial stiffness with reduction in fat accumulation. However, the relationship between aerobic exercise-induced changes in muscular lipids and arterial stiffness remains unclear. The purpose of this study was to investigate whether habitual aerobic exercise-induced changes in IMCL and EMCL content would lead to an improvement of arterial stiffness. First, in a cross-sectional study, we investigated whether cardiorespiratory fitness level affects the association between IMCL or EMCL content and arterial stiffness in 60 middle-aged and older subjects (61.0±1.3 years). Second, in an intervention study, we examined whether aerobic exercise training-induced changes in IMCL and EMCL content are associated with a reduction in arterial stiffness in 18 middle-aged and older subjects (67.0±1.7 years). In the cross-sectional study, IMCL content was negatively correlated with brachial-ankle pulse wave velocity (baPWV) (r=−0.47, P<0.05), whereas EMCL content was positively correlated with baPWV (r=0.48, P<0.05) in the low-fitness group, but was not correlated in the high-fitness group. Furthermore, 8-week aerobic exercise training in older adults increased IMCL content and reduced EMCL content. The training-induced change in baPWV was negatively correlated with training-induced changes in IMCL but was positively correlated with training-induced changes in EMCL. These findings suggest that aerobic exercise training-induced changes in IMCL and EMCL content may be related to a reduction in arterial stiffness in middle-aged and older adults.

Our aim was to determine whether variant bile duct (BD) anatomy is associated with portal vein (PV) and/or hepatic artery (HA) anatomy. We examined the associations between BD anatomy and PV and/or HA anatomy in 407 living donor transplantation donors. We also examined whether the right posterior BD (RPBD) course was associated with the PV and/or HA anatomy. Variant PV, HA and BD anatomies were found in 11%, 25% and 25%, respectively, of 407 donors enrolled in this study. The presence of a variant BD was more frequently associated with a variant PV than with a normal PV (61% vs. 20%, p < 0.0001). By contrast, the presence of a variant HA was not associated with a variant BD. A supraportal RPBD was found in 357 donors (88%) and an infraportal RPBD was found in 50 donors (12%). An infraportal RPBD was significantly more common in donors with a variant PV than in donors with a normal PV (30% vs. 10%, p = 0.0004). Variant PV, but not variant HA, anatomies were frequently associated with variant BD anatomy. Additionally, an infraportal RPBD was more common in donors with a variant PV than in donors with a normal PV.

In Japan, drivers over the age of 65 years are classified as ‘older drivers’. On 1 October 2006, people over the age of 65 years comprised 20.8 per cent of the population, and 13.1 per cent of these are licensed drivers. Ageing causes a decrease in both physical and psychomotor abilities. Older drivers tend to cause traffic accidents owing to complex traffic conditions. The major goals of this study are to investigate the characteristics of older drivers' eye fixation movement and responses under heavy traffic volume at an intersection with a traffic signal. A personal-computer-based driving simulator was used to evaluate driver behaviours and to reproduce unexpected roadway hazards and realistic four-step traffic volume. An eye position recorder on a near-infrared system was used to collect eye movement data. The drivers' initial cognition ability was determined on the basis of reaction time tests under three different conditions. The older driver group in this study shows the following characteristics: first, as the task complexity increases, the reaction time increases (i.e. a slowed reaction); second, as the drivers become older, the time interval between recognition of a hazard and avoidance of the hazard increases; third, as the traffic volume increases, the driver has fewer eye fixations, shorter mean fixation time, and slower fixation speed compared with the younger drivers' group; fourth, traffic volume is one of the most important factors that complicates the driving task and causes hesitation in decision making. These results provide basic data to improve traffic services to make them suitable for older drivers in an ageing society.

AbstractIn 1994, the water quality standard of dialysate and substitution fluid for on-line HDF was established by the Kyushu Society for HDF. On the other hand, with the widespread use of high-flux membrane, reverse filtration and reverse diffusion became evident, and purification of the dialysate has become essential even for usual hemodialysis. By using ultrapure dialysate, new blood purification methods can be performed, such as internal filtration-enhanced hemodialysis, on-line HDF, and on-line HF. As a result, various clinical effects have been reported, such as improvement in anemia and in chronic inflammatory reactions. Suppression of complications involving long-term dialysis is expected, and even the prolongation of life expectancy. By using ultrapure dialysate as substitution fluid for saline solution, a fully automated dialysis machine has been developed. Furthermore, if fully automated consoles can be made smaller in size, they will contribute to the widespread use of home hemodialysis.Copyright © 2004 S. Karger AG, Basel

A series of 16 compounds related to chiral 4(5)-(5-aminomethyltetrahydrofuran-2-yl)imidazoles (1) have been designed, synthesized, and examined in vitro by radioligand displacement studies and functional assays for both the human H3- and H4-receptors expressed in SK-N-MC cells. Among them, the (2S,5S)-isomer 1d of amino compounds showed approximately 300-fold higher selectivity at the H3-receptor than the H4-receptor. On the other hand, (2R,5S)- and (2R,5R)-cyanoguanidines 3b and 3c, in which the amino group of the compounds 1b and 1c was substituted by the cyanoguanidino moiety, bound to the H4-receptor with a pEC50 value of 6.65 and 7.11, respectively, and had >40-fold selectivities over the H3-receptor. As such, 3b and 3c are the first selective H4 receptor agonists.

Abstract: A 42-year-old woman, who had received right breast-conserving therapy 5 years previously, was admitted with hemoptysis. Chest X-rays showed a tumor shadow in the left pulmonary hilus with partial atelectasis in the left lower lobe. Bronchofiberscopic examination showed a polypoid tumor arising from the endobronchial wall, with bleeding and a white surface coat. Although, microscopically, the tumor resembled the breast cancer resected previously, as we could not rule out primary lung carcinoma, and as the tumor seemed to be localized, thoracotomy was performed. During posterolateral thoracotomy, however, pleural metastatic nodules were found; therefore, only sampling of these nodules and the administration of 50 mg of cisplatin (CDDP) into the thoracic cavity were performed. From a comparison of the histopathological features of the original tumor and the intrathoracic tumor, and from the hormone receptor positivity of the pleural metastasis, we diagnosed the intrathoracic tumor as being highly suspicious of metastasis from breast cancer. The measurement of hormone receptor was informative for the diagnosis of and for selecting a therapeutic strategy for the metastatic breast cancer.

Selenocysteine lyase (SCL) (EC 4.4.1.16) is a pyridoxal 5'-phosphate-dependent enzyme that specifically catalyzes the decomposition of L-selenocysteine to L-alanine and elemental selenium. The enzyme was proposed to function as a selenium delivery protein to selenophosphate synthetase in selenoprotein biosynthesis (Lacourciere, G. M., and Stadtman, T. C. (1998) J. Biol. Chem. 273, 30921-30926). We purified SCL from pig liver and determined its partial amino acid sequences. Mouse cDNA clones encoding peptides resembling pig SCL were found in the expressed sequence tag data base, and their sequences were used as probes to isolate full-length mouse liver cDNA. The cDNA for mouse SCL (mSCL) was determined to be 2,172 base pairs in length, containing an open reading frame encoding a polypeptide chain of 432 amino acid residues (M(r) 47, 201). We also determined the sequence of the N-terminal region of putative human SCL. These enzymes were shown to be distantly related in primary structure to NifS, which catalyzes the desulfurization of L-cysteine to provide sulfur for iron-sulfur clusters. The recombinant mSCL overproduced in Escherichia coli was a homodimer with the subunit M(r) of 47,000. The enzyme was pyridoxal phosphate-dependent and highly specific to L-selenocysteine (the k(cat)/K(m) value for L-selenocysteine was about 4,200 times higher than that for L-cysteine). Reverse transcriptase-polymerase chain reaction and Western blot analyses revealed that mSCL is cytosolic and predominantly exists in the liver, kidney, and testis, where mouse selenophosphate synthetase is also abundant, supporting the view that mSCL functions in cooperation with selenophosphate synthetase in selenoprotein synthesis. This is the first report of the primary structure of mammalian SCL.

A novel perichromosomal protein, which we have named chmadrin, was identified from rat kangaroo PtK2 cells. The deduced amino acid sequence revealed structural homologies in several limited regions to the Ki-67 antigen (pKi-67). The subcellular localization of chmadrin was found to be similar to that of pKi-67 throughout the cell cycle, that is, predominantly nucleolar during interphase and perichromosomal in the mitotic phase. In addition, a certain population of the protein was found to be localized in heterochromatic foci in interphase nuclei. Transient expression analysis of the truncated proteins corresponding to the conserved regions clearly demonstrated the structural basis for the characteristic cellular localization. Residues 494-778, which show extensive similarity to the corresponding region of pKi-67, were efficiently targeted to nucleoli, whereas a repetitive structure found at the C-terminal portion, whose similarity to pKi-67 is weak, was localized precisely to mitotic chromosomes. The C-terminal portion was designated the 'LR domain' since several LR (leucine and arginine) pairs commonly appear in chmadrin and pKi-67. When overproduced in the interphase nuclei, the LR domain induced the formation of aberrant heterochromatin as a structural constituent. These are the first empirical data suggesting the involvement of perichromosomal proteins in the organization of chromatin structure.

Selenocysteine lyase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the exclusive decomposition of L-selenocysteine to L-alanine and elemental selenium. An open reading frame, named csdB, from Escherichia coli encodes a putative protein that is similar to selenocysteine lyase of pig liver and cysteine desulfurase (NifS) of Azotobacter vinelandii. In this study, the csdB gene was cloned and expressed in E. coli cells. The gene product was a homodimer with the subunit Mr of 44,439, contained 1 mol of PLP as a cofactor per mol of subunit, and catalyzed the release of Se, SO2, and S from L-selenocysteine, L-cysteine sulfinic acid, and L-cysteine, respectively, to yield L-alanine; the reactivity of the substrates decreased in this order. Although the enzyme was not specific for L-selenocysteine, the high specific activity for L-selenocysteine (5.5 units/mg compared with 0.019 units/mg for L-cysteine) supports the view that the enzyme can be regarded as an E. coli counterpart of mammalian selenocysteine lyase. We crystallized CsdB, the csdB gene product, by the hanging drop vapor diffusion method. The crystals were of suitable quality for x-ray crystallography and belonged to the tetragonal space group P43212 with unit cell dimensions of a = b = 128.1 A and c = 137.0 A. Consideration of the Matthews parameter Vm (3.19 A3/Da) accounts for the presence of a single dimer in the crystallographic asymmetric unit. A native diffraction dataset up to 2.8 A resolution was collected. This is the first crystallographic analysis of a protein of NifS/selenocysteine lyase family.

The lysine residue binding with the cofactor pyridoxal 5'-phosphate (PLP) plays an important role in catalysis, such as in the transaldimination and abstraction of alpha-hydrogen from a substrate amino acid in PLP-dependent enzymes. We studied the role of Lys39 of alanine racemase (EC 5.1.1.1) from Bacillus stearothermophilus, the PLP-binding residue of the enzyme, by replacing it site-specifically with alanine and characterizing the resultant K39A mutant enzyme. The mutant enzyme turned out to be inherently inactive, but gained an activity as high as about 0.1% of that of the wild-type enzyme upon addition of 0.2 M methylamine. The amine-assisted activity of the mutant enzyme depended on the pKa values and molecular volumes of the alkylamines used. A strong kinetic isotope effect was observed when alpha-deuterated D-alanine was used as a substrate in the methylamine-assisted reaction, but little effect was observed using its antipode. In marked contrast, only L-enantiomer of alanine showed a solvent isotope effect in deuterium oxide in the methylamine-assisted reaction. These results suggest that methylamine serves as a base not only to abstract the alpha-hydrogen from D-alanine but also to transfer a proton from water to the alpha-position of the deprotonated (achiral) intermediate to form D-alanine. Therefore, the exogenous amine can be regarded as a functional group fully representing Lys39 of the wild-type enzyme. Lys39 of the wild-type enzyme probably acts as the base catalyst specific to the D-enantiomer of alanine. Another residue specific to the L-enantiomer in the wild-type enzyme is kept intact in the K39A mutant.

DL-2-Haloacid dehalogenase from Pseudomonas sp. 113 (DL-DEX 113) catalyzes the hydrolytic dehalogenation of D- and L-2-haloalkanoic acids, producing the corresponding L- and D-2-hydroxyalkanoic acids, respectively. Every halidohydrolase studied so far (L-2-haloacid dehalogenase, haloalkane dehalogenase, and 4-chlorobenzoyl-CoA dehalogenase) has an active site carboxylate group that attacks the substrate carbon atom bound to the halogen atom, leading to the formation of an ester intermediate. This is subsequently hydrolyzed, resulting in the incorporation of an oxygen atom of the solvent water molecule into the carboxylate group of the enzyme. In the present study, we analyzed the reaction mechanism of DL-DEX 113. When a single turnover reaction of DL-DEX 113 was carried out with a large excess of the enzyme in H(2)(18)O with a 10 times smaller amount of the substrate, either D- or L-2-chloropropionate, the major product was found to be (18)O-labeled lactate by ionspray mass spectrometry. After a multiple turnover reaction in H(2)(18)O, the enzyme was digested with trypsin or lysyl endopeptidase, and the molecular masses of the peptide fragments were measured with an ionspray mass spectrometer. No peptide fragments contained (18)O. These results indicate that the H(2)(18)O of the solvent directly attacks the alpha-carbon of 2-haloalkanoic acid to displace the halogen atom. This is the first example of an enzymatic hydrolytic dehalogenation that proceeds without producing an ester intermediate.

Fluoroacetate dehalogenase (EC 3.8.1.3) catalyzes the dehalogenation of fluoroacetate and other haloacetates. The amino acid sequence of fluoroacetate dehalogenase from Moraxella sp. B is similar to that of haloalkane dehalogenase (EC 3.8.1.5) from Xanthobacter autotrophicus GJ10 in the regions around Asp-105 and His-272, which correspond to the active site nucleophile Asp-124 and the base catalyst His-289 of the haloalkane dehalogenase, respectively (Krooshof, G. H., Kwant, E. M., Damborský, J., Koca, J., and Janssen, D. B. (1997) Biochemistry 36, 9571-9580). After multiple turnovers of the fluoroacetate dehalogenase reaction in H218O, the enzyme was digested with trypsin, and the molecular masses of the peptide fragments formed were measured by ion-spray mass spectrometry. Two 18O atoms were shown to be incorporated into the octapeptide, Phe-99-Arg-106. Tandem mass spectrometric analysis of this peptide revealed that Asp-105 was labeled with two 18O atoms. These results indicate that Asp-105 acts as a nucleophile to attack the alpha-carbon of the substrate, leading to the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule on the carbonyl carbon atom. A His-272 --> Asn mutant (H272N) showed no activity with either fluoroacetate or chloroacetate. However, ion-spray mass spectrometry revealed that the H272N mutant enzyme was covalently alkylated with the substrate. The reaction of the H272N mutant enzyme with [14C]chloroacetate also showed the incorporation of radioactivity into the enzyme. These results suggest that His-272 probably acts as a base catalyst for the hydrolysis of the covalent ester intermediate.

Flow of Latex suspension with high concentration between plate glasses was observed through an optical microscope. Size of aggregates of Latex particles was estimated by measuring area of a region without particles behind an obstacle in the flow region (this technique had been developed by one of the authors). At the same time, viscosity and elasticity of the suspension were measured by a cone-plate viscometer, and relations between the aggregate size and these properties were obtained. A simple model based on the Stokes law of resistance and the Maxwell model for viscoelastic materials is proposed to explain these relations. The theoretical results agree with the present experiment.

1. In the isolated spinal cord of the neonatal rat, repetitive electrical stimulation of the upper cervical region elicited a prolonged depolarization of lumbar motoneurones (L3‐5) lasting 1‐2 min, which was recorded extracellularly from ventral roots, or intracellularly. 2. This depolarizing response was markedly depressed by the excitatory amino acid receptor antagonists D‐(‐)‐2‐amino‐5‐phosphonovaleric acid (D‐APV, 30 microM) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 10 microM). The remaining response was further depressed by a 5‐hydroxytryptamine (5‐HT) receptor antagonist, ketanserin (3 microM). 3. In the presence of these antagonists, a small part of the depolarizing response of slow time course remained, and this response was partially blocked by the tachykinin NK1 receptor antagonists GR71251 (0.3‐5 microM) and RP67580 (0.3‐1 microM). In contrast, RP68651 (0.3‐1 microM), the inactive enantiomer of RP67580, had no effect on the depolarizing response. 4. The slow depolarizing response in the presence of D‐APV, CNQX and ketanserin was markedly potentiated by a peptidase inhibitor, thiorphan (1 microM). 5. This descending fibre‐evoked slow depolarization became smaller after prolonged treatment (5‐7 h) with 5,7‐dihydroxytryptamine (10 microM), a neurotoxin for 5‐HT neurones. Under such conditions, the effects of thiorphan and GR71251 on the slow depolarization were virtually absent. 6. Under the action of D‐APV, CNQX and ketanserin, applications of tachykinins, substance P and neurokinin A produced depolarizing responses of lumbar motoneurones, and the responses were depressed by GR71251 and potentiated by thiorphan. 7. These results suggest that tachykinins contained in serotonergic fibres serve as neurotransmitters mediating the descending fibre‐evoked slow excitatory postsynaptic potentials in motoneurones.

The amino acid composition, isoelectric point, specificity of the antibody raised and various catalytic properties were determined for 2′:3′-cyclic nucleotide 3′-phosphodiesterase (EC 3.1.4.37) purified from bovine brain white matter by a procedure involving solubilization with elastase (EC 3.4.21.11).

Crystal structures of L-2-haloacid dehalogenase from Pseudomonas sp. YL complexed with monochloroacetate, L-2-chlorobutyrate, L-2-chloro-3-methylbutyrate, or L-2-chloro-4-methylvalerate were determined at 1.83-, 2.0-, 2.2-, and 2.2-A resolutions, respectively, using the complex crystals prepared with the S175A mutant, which are isomorphous with those of the wild-type enzyme. These structures exhibit unique structural features that correspond to those of the reaction intermediates. In each case, the nucleophile Asp-10 is esterified with the dechlorinated moiety of the substrate. The substrate moieties in all but the monochloroacetate intermediate have a D-configuration at the C2 atom. The overall polypeptide fold of each of the intermediates is similar to that of the wild-type enzyme. However, it is clear that the Asp-10-Ser-20 region moves to the active site in all of the intermediates, and the Tyr-91-Asp-102 and Leu-117-Arg-135 regions make conformational changes in all but the monochloroacetate intermediates. Ser-118 is located near the carboxyl group of the substrate moiety; this residue probably serves as a binding residue for the substrate carboxyl group. The hydrophobic pocket, which is primarily composed of the Tyr-12, Gln-42, Leu-45, Phe-60, Lys-151, Asn-177, and Trp-179 side chains, exists around the alkyl group of the substrate moiety. This pocket may play an important role in stabilizing the alkyl group of the substrate moiety through hydrophobic interactions, and may also play a role in determining the stereospecificity of the enzyme. Moreover, a water molecule, which is absent in the substrate-free enzyme, is present in the vicinities of the carboxyl carbon of Asp-10 and the side chains of Asp-180, Asn-177, and Ala-175 in each intermediate. This water molecule may hydrolyze the ester intermediate and its substrate. These findings crystallographically demonstrate that the enzyme reaction proceeds through the formation of an ester intermediate with the enzyme's nucleophile Asp-10.

Mutations in the SEC63 gene are associated with defects in protein translocation into the endoplasmic reticulum (ER) as well as in nuclear protein localization in Saccharomyces cerevisiae. To identify proteins that might interact and/or function with SEC63p, we cloned a high copy suppressor (HSS1) of the temperature-sensitive lethal phenotype of the sec63-101 mutant. HSS1 is an allele-specific sec63 suppressor that encodes an integral ER membrane glycoprotein of 206 amino acids with the N-terminus in the ER lumen and C-terminal region in the cytoplasm. Haploid strains disrupted for HSS1 are temperature-sensitive for growth and accumulate precursor forms of Kar2p and invertase. The HSS1 null allele is synthetically lethal in combination with mutations affecting ER translocation. We propose that HSS1p is important for ER translocation and interacts with previously identified components of the yeast translocation apparatus. HSS1 is identical to SEC66, which encodes a glycoprotein complexed with SEC62p and SEC63p.

Suspension cultures of Triptergium wildordii require oxygen at a rate of 3.2 to 0.8 uL of oxygen/mg dry wt of cells/hr. A recycling air lift fermenter was developed in which the major volume of air required to lift the cells is recycled air. Sufficient new air is fed to meet the oxygen requirements of the cells. Such a fermenter design supports the growth of a large production of cells suitable for batch or continuous feeding.

The big toe donor site of the wrap-around flap has been reconstructed immediately after elevation of the flap with a snuff @? flap from the dorsum of the recipient hand in 6 patients. Rapid wound healing and a satisfactory appearance were achieved. .

Selenocysteine lyase (EC 4.4.1.16) exclusively decomposes selenocysteine to alanine and elemental selenium, whereas cysteine desulfurase (NIFS protein) of Azotobacter vinelandii acts indiscriminately on both cysteine and selenocysteine to produce elemental sulfur and selenium respectively, and alanine. These proteins exhibit some sequence homology. The Escherichia coli genome contains three genes with sequence homology to nifS. We have cloned the gene mapped at 63.4 min in the chromosome and have expressed, purified to homogeneity, and characterized the gene product. The enzyme comprises two identical subunits with 401 amino acid residues (Mr 43,238) and contains pyridoxal 5'-phosphate as a coenzyme. The enzyme catalyzes the removal of elemental sulfur and selenium atoms from L-cysteine, L-cystine, L-selenocysteine, and L-selenocystine to produce L-alanine. Because L-cysteine sulfinic acid was desulfinated to form L-alanine as the preferred substrate, we have named this new enzyme cysteine sulfinate desulfinase. Mutant enzymes having alanine substituted for each of the four cysteinyl residues (Cys-100, Cys-176, Cys-323, and Cys-358) were all active. Cys-358 corresponds to Cys-325 of A. vinelandii NIFS, which is conserved among all NIFS-like proteins and catalytically essential (Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714-4720), is not required for cysteine sulfinate desulfinase. Thus, the enzyme is distinct from A. vinelandii NIFS in this respect.

A new member of the serine protease inhibitor (serpin) superfamily with megakaryocyte maturation activity was purified, and its cDNA was cloned and characterized. The predicted amino acid sequence consisting of 380 residues was unique and was 38% identical to the serpin plasminogen activator inhibitor type 2 (PAI-2). The recombinant factor expressed in Chinese hamster ovary cells showed species-specific activity on the induction of megakaryocyte maturation in vitro. When injected into mice, the factor indeed elicited an increase in the number of platelets in plasma. The sequence alignment indicated that the factor possessed a lysine residue at the P1 position, suggesting that it might function as an inhibitor of Lys-specific proteases. Although we could not show any inhibitory activities toward several known Lys-specific proteases, we detected the activity toward protease activity present in the culture supernatant of COLO 201 cells. These results suggested that the protein might influence the maturation of megakaryocytes via action as a serpin.

The C-C chemokines human monocyte chemoattractant protein-1 and -3 (MCP-1 and MCP-3) and mouse JE and FIC are potent activators of monocytes. Several receptors for MCP-1 and MCP-3 have been cloned from human monocytic cell lines, and one of these receptors, CCR2B, binds both MCP-l and MCP-3. Thus far, no murine receptors for JE or FIC have been reported. We have cloned a novel murine C-C chemokine receptor, designated mouse CCR2 (mCCR2), from the mouse monocyte cell line WEHI265.1. The predicted 373-amino acid sequence of mCCR2 shows highest identity (80%) with CCR2B. When stably expressed in human embryonic kidney 293 cells, mCCR2 specifically bound 125I-JE with high affinity. FIC was less potent than JE in competing 125I-JE binding to mCCR2-expressing cells, while three other mouse chemokines, MIP-1alpha, C10, and N51/KC, did not compete. mccr2 mRNA expression was detected in elicited peritoneal macrophages as well as in several mouse organs. The cloning of mCCR2 provides an important tool to investigate monocyte/macrophage responses to JE and FIC, to identify other targets for their action, and potentially to study models of CCR2 function in the mouse.

Abstract Some manufacturers have sited their factories in urban areas, others prefer to have them dispersed in rural areas. They should consider carefully how they should operate from their chosen locations. Factories of both types are discussed and suggestions are made for dealing with various matters, and for what corporations or factories should do in the 21st century. The NKK Corporation and Kirin Brewery Co. Ltd are described as examples of factories or plants located in urban areas and the Hoshizaki Electric Co. Ltd as a good example of a factory located in a rural area.

When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome c1, a mitochondrial inner membrane protein, the resulting hybrid proteins do not assemble into mitochondria when synthesized in the yeast Saccharomyces cerevisiae. Cells lacking mitochondrial cytochrome c1, but expressing the hybrid NLS-cytochrome c1 proteins, are unable to grow on glycerol since the hybrid proteins are associated primarily with the nucleus. A similar hybrid protein with a mutant NLS is transported to and assembled into the mitochondria. To identify proteins that might be involved in recognition of nuclear localization signals, we isolated conditional-lethal mutants (npl, for nuclear protein localization) that missorted NLS-cytochrome c1 to the mitochondria, allowing growth on glycerol. The gene corresponding to one complementation group (NPL1) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein. npl1-1 is allelic to sec63, a gene that affects transit of nascent secretory proteins across the endoplasmic reticulum. Rothblatt, J. A., R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman. 1989. J. Cell Biol. 109:2641-2652. The npl1 mutants reported here also weakly affect translocation of preprocarboxypeptidaseY across the ER membrane. A normally nuclear hybrid protein containing a NLS fused to invertase and a nucleolar protein are not localized to the nucleus in npl1/sec63 cells at the nonpermissive temperature. Thus, NPL1/SEC63 may act at a very early common step in localization of proteins to the nucleus and the ER. Alternatively, by affecting ER and nuclear envelope assembly, npl1 may indirectly alter assembly of proteins into the nucleus.

A new class of Escherichia coli mutants, referred to as grn, has been isolated by localized mutagenesis. These mutations affect the sigma subunit of DNA-dependent RNA polymerase (ribonucleoside 5'-triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) by abolishing the expression of the lambda N gene, and they are closely lniked to dnaG in the order dnaG-grn-uxaA. Detailed study of one such mutant, grn1, yielded the following results: (i) grn1 is a single mutation and the mutant cell shows cold-sensitivity in growth; (ii) the Grn phenotype of the mutant can easily be suppressed by secondary mutations in the beta subunit gene of RNA polymerase; (iii) purified holoenzyme of RNA polymerase isolated from the mutant showed an altered salt-dependency in vitro, and the mixed reconstitution of the mutant with the wild-type subunits showed that the sigma subunit of the grn1 mutant is altered; (iv) lambda phage mutants (lambda grg), which overcome the grn mutation, can be classified into two groups, the "nin-deletion" and the "N-mutant" groups (both of these are also able to grow on the previously described groN mutant of Georgopoulos and nusAB of Friedman); (iv) the mutant polymerase transcribed 12S as well as 7S RNA from lambda DNA in the presence of the rho factor in vitro. These results indicate that the grn mutation alters the sigma subunit of RNA polymerase and that the sigma subunit participates in activating the N-mediated antitermination mode of lambda phage transcription.

2',3'-Cyclic-nucleotide 3'-phosphodiesterase (EC 3.1.4.37) has been widely used as a marker for myelin-oligodendrocytes in the central nervous system. Evidence has been provided that the enzyme is identical with one of the Wolfgram proteins of central nervous system myelin. The amino acid sequence of bovine 2',3'-cyclic-nucleotide 3'-phosphodiesterase was determined by both protein and cDNA sequence analyses. Protein sequence analysis was done on bovine elastase 2',3'-cyclic-nucleotide 3'-phosphodiesterase, a low molecular weight enzyme obtained by solubilization with pancreatic elastase (EC 3.4.21.36) (Nishizawa, Y., Kurihara, T., and Takahashi, Y. (1980) Biochem. J. 191, 71-82; Kurihara, T., Nishizawa, Y., Takahashi, Y., and Odani, S. (1981) Biochem. J. 195, 153-157). Based on the carboxyl-terminal sequence of bovine elastase 2',3'-cyclic-nucleotide 3'-phosphodiesterase, synthetic oligodeoxyribonucleotides were prepared and used as probes for screening a cDNA library of bovine brain. A cDNA of 2305 base pairs was obtained and sequenced, and the complete amino acid sequence of bovine 2',3'-cyclic-nucleotide 3'-phosphodiesterase was deduced. Bovine 2',3'-cyclic-nucleotide 3'-phosphodiesterase deduced contains 400 amino acids including initiation methionine and has a molecular weight of 44,850. Bovine elastase 2',3'-cyclic-nucleotide 3'-phosphodiesterase corresponds to the 236 amino acids of bovine 2',3'-cyclic-nucleotide 3'-phosphodiesterase. RNA blot analysis revealed a single-species mRNA of about 2600 bases.

This paper describes the electromagnetic wave diffraction from a metallic Fourier grating based on the T-matrix analysis and the extended boundary condition. The grating consists of a basic sinusoidal wave and a second-harmonic wave. The analytical form of the expression of matrix elements is presented in the term of the Bessel functions. The error of power conservation versus the truncated number of the basic functions has been examined; thus a high accuracy result is obtained. Diffraction efficiencies versus incident angle, groove depth and wavelength have also been discussed for both perfect and lossy metallic (Au) gratings. Numerical results are in good agreement with those obtained from other methods and experimental values. Reasonable numerical results are presented for a groove depth per period of the grating less than 0.28.