Exaggerated response of arginine vasopressin-enhanced green fluorescent protein fusion gene to salt loading without disturbance of body fluid homeostasis in rats.

We examined the effects of chronic salt loading on the hypothalamic expressions of the enhanced green fluorescent protein (eGFP), arginine vasopressin (AVP) and oxytocin (OXT) genes in AVP-eGFP transgenic rats that expressed eGFP in the hypothalamic AVP-containing neurones. In these rats, salt loading for 5 days caused a marked increase of the eGFP fluorescence in the magnocellular divisions of the paraventricular nucleus (PVN), the supraoptic nucleus (SON) and the internal layer of the median eminence. Expression of the eGFP gene was increased seven- to eight-fold in the PVN and SON of salt-loaded rats in comparison with euhydrated rats. By contrast, none of these changes were observed in the suprachiasmatic nucleus. The expression of the AVP and OXT genes was increased 1.5- to two-fold in the PVN and SON of salt-loaded nontransgenic (control) and transgenic rats. There were no differences in the expression levels of the AVP and OXT genes in the PVN and SON between nontransgenic (control) and transgenic animals under normal conditions and after salt loading. In the posterior pituitary gland, the intensity of the eGFP fluorescence did not change after salt loading for 5 days, but increased after 10 days of salt loading. Upon salt loading, significant increases in the plasma AVP concentrations, plasma osmolality and plasma Na+ were observed. Furthermore, there were no significant differences in changes of water intake, food intake, urine volume, urine osmolality, urine Na+ concentrations, and the body weights in both models under normal or salt-loaded conditions. Our results show that the response of the AVP-eGFP fusion gene to chronic salt loading is exaggerated, and humoral responses such as AVP and OXT and the body fluid homeostasis are maintained in AVP-eGFP transgenic rats. The AVP-eGFP transgenic rat gives us a new opportunity to study the dynamics of the AVP system in vivo.

Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases.

We have purified GST-fused recombinant mouse Dnmt3a and three isoforms of mouse Dnmt3b to near homogeneity. Dnmt3b3, an isoform of Dnmt3b, did not have DNA methylation activity. Dnmt3a, Dnmt3b1 or Dnmt3b2 showed similar activity toward poly(dG-dC)-poly(dG-dC) for measuring de novo methylation activity, and toward poly(dI-dC)-poly(dI-dC) for measuring total activity. This indicates that the enzymes are de novo-type DNA methyltransferases. The enzyme activity was inhibited by NaCl or KCl at concentrations >100 mM. The kinetic parameter, K(m)(AdoMet), for Dnmt3a, Dnmt3b1 and Dnmt3b2 was 0.4, 1.2 and 0.9 microM when poly(dI-dC)-poly(dI-dC) was used, and 0.3, 1.2 and 0.8 microM when poly(dG-dC)-poly(dG-dC) was used, respectively. The K(m)(DNA) values for Dnmt3a, Dnmt3b1 and Dnmt3b2 were 2.7, 1.3 and 1.5 microM when poly(dI-dC)-poly(dI-dC) was used, and 3.5, 1.0 and 0.9 microM when poly(dG-dC)-poly(dG-dC) was used, respectively. For the methylation specificity, Dnmt3a significantly methylated CpG >> CpA. On the other hand, Dnmt3b1 methylated CpG > CpT >/= CpA. Immuno-purified Dnmt3a, Myc-tagged and overexpressed in HEK 293T cells, methylated CpG >> CpA > CpT. Neither Dnmt3a nor Dnmt3b1 methylated the first cytosine of CpC.

Revisiting the supratrigeminal nucleus in the rat.

The supratrigeminal nucleus (Vsup), originally proposed as a premotoneuron pool in the trigeminal reflex arc, is a key structure of jaw movement control. Surprisingly, however, the location of the rat Vsup has not precisely been defined. In light of our previous cat studies, we made two hypotheses regarding the rat Vsup: (1) the Vsup is cytoarchitectonically distinguishable from its surrounding structures; (2) the Vsup receives central axon terminals of the trigeminal mesencephalic nucleus (Vmes) neurons which are primary afferents innervating muscle spindles of jaw-closing muscles and periodontal ligaments around the teeth. To test the first hypothesis, we examined the cytoarchitecture of the rat Vsup. The Vsup was identified as an area medially adjacent to the dorsomedial part of trigeminal principal sensory nucleus (Vp), and extended from the level just rostral to the caudal two-thirds of the trigeminal motor nucleus (Vmo) to the level approximately 150 µm caudal to the Vmo. Our rat Vsup was much smaller and its location was considerably different in comparison to the Vsup reported previously. To evaluate the second hypothesis, we tested the distribution patterns of Vmes primary afferent terminals in the cytoarchitectonically identified Vsup. After transganglionic tracer applications to the masseter, deep temporal, and medial pterygoid nerves, a large number of axon terminals were observed in all parts of Vsup (especially in its medial part). After applications to the inferior alveolar, infraorbital, and lingual nerves, a small number of axon terminals were labeled in the caudolateral Vsup. The Vsup could also be identified electrophysiologically. After electrical stimulation of the masseter nerve, evoked potentials with slow negative component were isolated only in the Vsup. The present findings suggest that the rat Vsup can be cytoarchitectonically and electrophysiologically identified, receives somatotopic termination of the trigeminal primary afferents, and principally receives strong termination of the spindle Vmes primary afferents.

Malonate decarboxylase of Pseudomonas putida is composed of five subunits.

Two different forms of malonate decarboxylase were purified from Pseudomonas putida. The active form was composed of the five different subunits alpha (60 kDa), beta (33 kDa), gamma (28 kDa), delta (13 kDa), and epsilon (30 kDa) and the inactive form was composed of the four subunits lacking the epsilon subunit. The former catalyzed the decarboxylation of malonate to acetate, but the latter could not, although it retained both activities of acetyl-CoA:malonate CoA transferase and malonyl-CoA decarboxylase. The delta subunit of the active form was acylated by the incubation with [2-14C]malonyl-CoA, but the delta subunit of the inactive form was not labeled. From the above results and the N-terminal amino acid sequence analysis, it was concluded that the epsilon subunit was an essential subunit to function as malonyl-CoA:ACP transacylase, which was an indispensable component of the enzyme for the cyclic decarboxylation of malonate.

[The epidemiological study of hepatolithiasis in the Kamigoto district, Nagasaki where hepatolithiasis is high incidence].

Regional distribution, age, sex, type of disease and history of surgery of the biliary tract were examined in 134 patients with hepatolithiasis in the Kamigoto district. The ratio of patient to population was 1:126 in the region showing the highest incidence. When classified by age at establishment of the diagnosis, patient in their sixties comprised the largest portion in both sexes. The ratio of men to women was 1:1.2. When classified in accordance with the draft by Research Group for the Study of Hepatolithiasis, 103 cases (76.9%) were of the intrahepatic type, and 31 cases (23.1%) were of the intra-extrahepatic type. The stones were found in the right lobe in 58 patients (43.3%), the left lobe in 59 patients (44.0%) and the both lobes in 17 patients (12.7%). Forty-four patients (32.8%) had a history of surgery of the biliary tract. Ultrasonographic examination of the abdomen in junior high school students revealed hepatolithiasis in one. As a result, the intrahepatic type and the right lobe type are high frequency in this district, and epidemiological survey of the family history and the food habit in this district is necessary.

Projections from the dorsal peduncular cortex to the trigeminal subnucleus caudalis (medullary dorsal horn) and other lower brainstem areas in rats.

This study has revealed direct projections from the dorsal peduncular cortex (DP) in the medial prefrontal cortex (mPfC) to the trigeminal brainstem sensory nuclear complex and other lower brainstem areas in rats. We first examined the distribution of mPfC neurons projecting directly to the medullary dorsal horn (trigeminal subnucleus caudalis [Vc]) and trigeminal subnucleus oralis (Vo) which are known to receive direct projections from the lateral prefrontal cortex (insular cortex). After injections of the retrograde tracer Fluorogold (FG) into the rostro-dorsomedial part of laminae I/II of Vc (rdm-I/II-Vc), many neurons were labeled bilaterally (with an ipsilateral predominance) in the rostrocaudal middle level of DP (mid-DP) and not in other mPfC areas. After FG injections into the lateral and caudal parts of laminae I/II of Vc, or the Vo, no neurons were labeled in the mPfC. We then examined projections from the mid-DP by using the anterograde tracer biotinylated dextranamine (BDA). After BDA injections into the mid-DP, many axons and terminals were labeled bilaterally (with an ipsilateral predominance) in the rdm-I/II-Vc, periaqueductal gray and solitary tract nucleus, and ipsilaterally in the parabrachial nucleus and trigeminal mesencephalic nucleus. In addition, the connections of the mid-DP with the insular cortex were examined. Many BDA-labeled axons and terminals from the mid-DP were also found ipsilaterally in the caudalmost level of the granular and dysgranular insular cortex (GI/DI). After BDA injections into the caudalmost GI/DI, many axons and terminals were labeled ipsilaterally in the mid-DP. The projections from the mid-DP to the rdm-I/II-Vc and other brainstem nuclei suggest that mid-DP neurons may regulate intraoral and perioral sensory processing (including nociceptive processing) of rdm-I/II-Vc neurons directly or indirectly through the brainstem nuclei. The reciprocal connections between the mid-DP and caudalmost GI/DI suggest that this regulation may involve mid-DP interactions with the caudalmost GI/DI neurons.

Enzymatic production of pyrimidine nucleotides using Corynebacterium ammoniagenes cells and recombinant Escherichia coli cells: enzymatic production of CDP-choline from orotic acid and choline chloride (Part I).

Enzymatic production of cytidine diphosphate choline (CDP-choline) using orotic acid and choline chloride as substrates was investigated using a 200-ml beaker as a reaction vessel. When Cornybacterium ammoniagenes KY13505 cells were used as the enzyme source, UMP was accumulated up to 28.6 g/liter (77.6 mM) from orotic acid after 26 h of reaction. In this reaction, UDP and UTP were also accumulated, but CTP, a direct precursor of CDP-choline, was not accumulated sufficiently. Escherichia coli JF646/pMW6 cells, which overproduce CTP synthetase by selfcloning of the pyrG gene, were used together with cells of KY12505 for the enzymatic reaction using orotic acid as a substrate. CTP was produced at 8.95 g/liter (15.1 mM) after 23 h of this reaction. To produce CDP-choline, two additional enzyme activities were needed. E. coli MM294/pUCK3 and MM294/pCC41 cells, which express a choline kinase from Saccharomyces cerevisiae (CKIase; encoded by the CKI gene) and a cholinephosphate cytidylyltransferase from S. cerevisiae (CCTase; encoded by the CCT gene) respectively, were added to this CTP-producing reaction system. After 23 h of the reaction using orotic acid and choline chloride as substrates, 7.7 g/liter (15.1 mM) of CDP-choline was accumulated without addition of ATP or phosphoribosylpyrophosphate (PRPP). ATP and PRPP required in the CDP-choline forming reaction system are biosynthesized by those cells using glucose as a substrate.

ATP production from adenine by a self-coupling enzymatic process: high-level accumulation under ammonium-limited conditions.

To improve ATP production from adenine, we optimized cultivation and reaction conditions for the ATP producing strain, Corynebacterium ammoniagenes KY13510. In the conventional method, 28% NH4OH has been used both to adjust pH during cultivation and reaction, and to provide nitrogen for cell growth. In the ATP-producing reaction, high concentrations of inorganic phosphate and magnesium ion are needed, which form magnesium ammonium phosphate (MgNH4PO4) precipitate. To keep inorganic phosphate and magnesium ions soluble in the reaction mixture, it was indispensable to add phytic acid as a chelating agent of divalent metal ions. Under such conditions, 37 mg/ml (61.2 mM) ATP was accumulated in 13 h (Appl. Microbiol. Biotechnol. 21, 143 1985). If ammonium ion was depleted from the reaction mixture to avoid MgNH4 PO4 formation, we expected that there was no need to add phytic acid and ATP accumulation might be improved. Therefore, we obtained the cultured broth of C. ammoniagenes KY13510 strain with low ammonium ion content (less than 1 mg/ml as NH3) by the method that a part of alkali solution (28% NH4OH) for pH control was replaced with 10 N KOH. Using this culture broth, ATP producing reaction was done in 2-liter jar fermentor, controlling the pH of the reaction mixture with 10 N KOH. Under these conditions, the rate of ATP accumulation improved greatly, and 70.6 mg/ml (117 mM) ATP was accumulated in 28 h. The molar conversion ratio from adenine to ATP was about 82%. Phytic acid was slightly inhibitory to ATP formation under these ammonium-limited conditions.

Cloning of FAD synthetase gene from Corynebacterium ammoniagenes and its application to FAD and FMN production.

The cloning of a bifunctional FAD synthetase gene, which shows flavokinase and FMN adenylyltransferase activities, from Corynebacterium ammoniagenes was tried by hybridization with synthetic DNAs corresponding to the N-terminal amino acid sequence. The cloned PstI-digested 4.4 x 10(3)-base (4.4-kb) fragment could not express the FAD synthetase activity in E. coli, but could increase the two activities by the same factor of about 20 in C. ammoniagenes. The FAD-synthetase-gene-amplified C. ammoniagenes cells were applied to the production of FAD from FMN or riboflavin. The productivity of FAD from FMN was increased four to five times compared with the parent strain, and reached a 90% molar yield. The productivity of FAD from riboflavin was increased about eight times, with a 50% molar yield. The addition of Zn2+ to the reaction mixtures for the conversion from riboflavin to FAD brought about the specific inhibition of adenylyl-transferase activity and resulted in the accumulation of FMN.

Construction of a plasmid carrying both CTP synthetase and a fused gene formed from cholinephosphate cytidylyltransferase and choline kinase genes and its application to industrial CDP-choline production: enzymatic production of CDP-choline from orotic acid (Part II).

A new method for enzymatic production of cytidine diphosphate choline (CDP-choline) from orotic acid and choline chloride was developed. To establish an industrial manufacturing process, we constructed a plasmid, pCKG55, which simultaneously expressed in Escherichia coli the three following enzymes; CTP synthetase (encoded by the pyrG gene from E. coli), cholinephosphate cytidylyltransferase (encoded by the CCT gene from Saccharomyces cerevisiae), and choline kinase (encoded by the CKI gene from S. cerevisiae). CCT and CKI genes on pCKG55 were designed to be expressed as a single CCT/CKI fused protein. This CCT/CKI fused protein retained both activities and the thermal stability of its cholinephosphate cytidylyltransferase activity was nearly the same as the native CCT enzyme. Corynebacterium ammoniagenes KY13505 and E. coli MM294/pCKG55 were cultured in 5-liter jar fermentor independently. Equal volumes of each broth were mixed in a 2-liter jar fermentor, and then the enzymatic reaction was done using 47 mM orotic acid and 60 mM choline chloride as substrates. After 23 h of the reaction at 32 degrees C, 21.5 mM (11 g/liter) of CDP-choline was accumulated.

Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the purified gene product.

We attempted to clone an inosine kinase gene of Escherichia coli. A mutant strain which grows slowly with inosine as the sole purine source was used as a host for cloning. A cloned 2.8-kbp DNA fragment can accelerate the growth of the mutant with inosine. The fragment was sequenced, and one protein of 434 amino acids long was found. This protein was overexpressed. The overexpressed protein was purified and characterized. The enzyme had both inosine and guanosine kinase activity. The Vmaxs for guanosine and inosine were 2.9 and 4.9 mumol/min/mg of protein, respectively. The Kms for guanosine and inosine were 6.1 microM and 2.1 mM, respectively. This enzyme accepted ATP and dATP as a phosphate donor but not p-nitrophenyl phosphate. These results show clearly that this enzyme is not a phosphotransferase but a guanosine kinase having low (Vmax/Km) activity with inosine. The sequence of the gene we have cloned is almost identical to that of the gsk gene (K.W. Harlow, P. Nygaard, and B. Hove-Jensen, J. Bacteriol. 177:2236-2240, 1995).

High level expression of XMP aminase in Escherichia coli and its application for the industrial production of 5'-guanylic acid.

To improve the efficiency of the enzymatic conversion of 5'-xanthylic acid (XMP) to 5'-guanylic acid (GMP), we attempted to increase the activity of the conversion enzyme, XMP aminase (GMP synthetase) encoded by the guaA gene in Escherichia coli. By connecting the PL promoter of lambda phage, the SD sequence of trpL of E. coli, and ATG, at a suitable position upstream of the guaA gene, we obtained plasmid pPLA66. Sequencing of the nucleotides of the upstream region of the guaA gene on pPLA66 showed that the C-terminal region of the guaB gene, which encodes IMP dehydrogenase, was conserved and a short peptide consisted of 14 amino acids was coded. E. coli MP347/pPLA66 showed an increase in the activity of approximately 370 times when compared with that of the strain MM294, and the amount of the enzyme protein represented approx. 34% of the total cellular protein. Strain MP347/pPLA66 was cultivated in a 5-liter jar fermentor using a medium which contained mainly corn steep liquor. The culture broth had high XMP aminase activity. In the conversion reaction using mixed broths consisted of 600 ml of XMP-fermentation broth of Corynebacterium ummoniagenes KY13203 and 30 ml of cultured broth of E. coli MP347/pPLA66, a surfactant, Nymeen S-215 and xylene were added to the reaction mixture to make the cell membrane permeable to nucleotides. After 23 h of the reaction, 70 mg/ml (131 mM) of GMP.Na2.7H2O was accumulated from 83 mg/ml (155 mM) of XMP.Na3.7H2O, without addition of ATP. The molar conversion yield was approx. 85%. The facts that the cell membrane was treated to allow nucleotides to permeate and that the conversion reaction proceeded well enough in spite of a small amount of E. coli cells indicate ATP was regenerated from AMP by C. ammoniagenes cells and supplied to E. coli cells. Therefore, it was considered that the coupling reaction between these two kind of strains was established.

A novel process of inosine 5'-monophosphate production using overexpressed guanosine/inosine kinase.

A novel process for producing inosine 5'-monophosphate (5'-IMP) has been demonstrated. The process consists of two sequential bioreactions; the first is a fermentation of inosine by a mutant of Corynebacterium ammoniagenes, and the second is a unique phosphorylating reaction of inosine by guanosine/inosine kinase (GIKase). GIKase was produced by an Escherichia coli recombinant strain, MC1000(pIK75), which overexpressed the enzyme up to 50% of the total cellular protein. The overproducing plasmid, pIK75, which was randomly screened out from deletion plasmids with various lengths of intermediate sequence between the E. coli trpL Shine-Dalgarno sequence, derived from the vector plasmid, and the start codon of the GIKase structural gene. In pIK75, the start ATG was placed 16 bp downstream of the trpL Shine-Dalgarno sequence under the control of the E. coli trp promoter. Fermentation of inosine and its phosphorylation were sequentially performed in a 5-1 jar fermenter. At the end of inosine fermentation by C. ammoniagenes KY13761, culture broth of MC1000(pIK75) was mixed with that of KY13761 to start the phosphorylating reaction. Inosine in the reaction mixture was stoichiometrically phosphorylated, and 91 mM 5'-IMP accumulated in a 12-h reaction. This new biological process has advantages over traditional methods for producing 5'-IMP.

Nucleotide sequence of the FAD synthetase gene from Corynebacterium ammoniagenes and its expression in Escherichia coli.

The nucleotides of a bifunctional enzyme FAD synthetase gene, which showed both flavokinase and ATP:FMN adenylyltransferase activities, from Corynebacterium ammoniagenes were sequenced. The FAD synthetase gene product consisted of 338 amino acids and had a calculated molecular weight of 37,712. The deduced protein sequence of the FAD synthetase shared a homology with those of the protein X of Escherichia coli, which has been reported to have both flavokinase and ATP:FMN adenylyltransferase activities like the FAD synthetase of C. ammoniagenes, and the protein X of Pseudomonas fluorescens. From the analysis of the flanking sequences of the FAD synthetase gene, the gene organization and the operon structure around the FAD synthetase gene of C. ammoniagenes were thought to be different from those of Gram-negative bacteria. An over-expression system of the FAD synthetase of C. ammoniagenes was constructed in E. coli to study the structure and function of the protein. Under the tandem tryptophan promoter, the FAD synthetase activity increased 2231 times compared to that of non-transformed C. ammoniagenes.

Changes in the size and composition of intracellular pools of nonesterified coenzyme A and coenzyme A thioesters in aerobic and facultatively anaerobic bacteria.

Intracellular levels of three coenzyme A (CoA) molecular species, i.e., nonesterified CoA (CoASH), acetyl-CoA, and malonyl-CoA, in a variety of aerobic and facultatively anaerobic bacteria were analyzed by the acyl-CoA cycling method developed by us. It was demonstrated that there was an intrinsic difference between aerobes and facultative anaerobes in the changes in the size and composition of CoA pools. The CoA pools in the aerobic bacteria hardly changed and were significantly smaller than those of the facultatively anaerobic bacteria. On the other hand, in the facultatively anaerobic bacteria, the size and composition of the CoA pool drastically changed within minutes in response to the carbon and energy source provided. Acetyl-CoA was the major component of the CoA pool in the facultative anaerobes grown on sufficient glucose, although CoASH was dominant in the aerobes. Therefore, the acetyl-CoA/CoASH ratios in facultatively anaerobic bacteria were 10 times higher than those in aerobic bacteria. In Escherichia coli K-12 cells, the addition of reagents to inhibit the respiratory system led to a rapid decrease in the amount of acetyl-CoA with a concomitant increase in the amount of CoASH, whereas the addition of cerulenin, a specific inhibitor of fatty acid synthase, triggered the intracellular accumulation of malonyl-CoA. The acylation and deacylation of the three CoA molecular species coordinated with the energy-yielding systems and the restriction of the fatty acid-synthesizing system of cells. These data suggest that neither the accumulation of acetyl-CoA nor that of malonyl-CoA exerts negative feedback on pyruvate dehydrogenase and acetyl-CoA carboxylase, respectively.