Physiological role of gap-junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation.

Hemichannels in the overlapping regions of apposing cells plasma membranes join to form gap junctions and provide an intercellular communication pathway. Hemichannels are also present in the nonjunctional regions of individual cells and their activity is gated by several agents, including calcium. However, their physiological roles are unknown. Using techniques of atomic force microscopy (AFM), fluorescent dye uptake assay, and laser confocal immunofluorescence imaging, we have examined the extracellular calcium-dependent modulation of cell volume. In response to a change in the extracellular physiological calcium concentration (1.8 to

Nucleotide sequence of levansucrase gene (levU) of Zymomonas mobilis ZM1 (ATCC10988).

The extracellular levansucrase gene (levU) was cloned from the genomic DNA of Zymomonas mobilis ZM1 and the nucleotide sequence of the levU structural gene was determined. The 3.1 kb EcoRV-polylinker fusion DNA fragment containing the levU gene had an open reading frame of 1272 bps and the deduced amino acid sequence was 423 residues long with a molecular weight of 46,725. Although this protein exhibited little similarity with other known levansucrases, several well-conserved regions were observed (1-5 regions).

Nucleotide and derived amino acid sequences of an extracellular sucrase gene (invB) of Zymomonas mobilis ZM1 (ATCC10988).

DNA sequence analysis of a previously cloned 4.5 kb DNA fragment showed that the extracellular sucrase gene (invB) of Zymomonas mobilis was located in the 155 bp downstream of levansucrase gene (levU). The invB gene had an open reading frame of 1242 bp and the deduced amino acid sequence was 413 residues with a molecular weight of 46,107. The translated sequence of Z. mobilis invB was in good agreement with the 10 N-terminal amino acid residues determined by amino acid sequencing. The amino acid sequence of sucrase showed low similarity with that of other sucrases, but higher similarity with that of levansucrases.

Transcriptional analysis of levU operon encoding saccharolytic enzymes and two apparent genes involved in amino acid biosynthesis in Zymomonas mobilis.

Extracellular levansucrase (LevU) and sucrase (InvB) are two of the three saccharolytic enzymes involved in the sucrose metabolism of Zymomonas mobilis. The levU and invB genes were clustered with a 155bp interval on the chromosome. Both genes were transcribed constitutively at the basal level and the transcription of both genes was induced significantly when sucrose was added to the medium. These genes were transcribed as a bicistronic mRNA and the expression was modulated by a single promoter, which is located upstream of the levU gene. The transcriptional initiation site was mapped to -64bp from the translation start site of levU gene. These results indicated that two genes are most likely to constitute an operon. The glk operon, which encodes four glycolytic enzymes, was located close to the levU operon on the chromosome. Two apparent ORFs (ORF3 and 4) were found at the intervening sequence located between the glk and levU operons. These ORFs were transcribed divergently and showed high homology at the amino acid level with the bacterial global regulatory protein (Lrp) and aspartate racemase.

Desaturation and interconversion of dietary stearic and palmitic acids in human plasma and lipoproteins.

Dietary saturated fatty acids are implicated as a risk factor for atherosclerosis. The conversion of the major dietary saturated fatty acids stearic acid (18:0) and palmitic acid (16:0) to monounsaturated fatty acids in whole plasma and lipoprotein fractions is reported for seven healthy adult humans over 6 d using [U-13C]stearic acid (18:0*) and [U-13C]palmitic acid (16:0*) and high-precision mass spectrometry. A tracer dose (28-32 mg) of 18:0* or 16:0* was loaded into an emulsion and orally administered before breakfast. Serial blood samples were collected on day 1 and fasting blood was drawn daily until day 7. Overall conversion of 18:0 to 18:1 was approximately 14%, whereas that of 18:0 to 16:0 was approximately 2% in plasma up to 144 h. Conversion of 16:0 to 16:1 was < 2%, whereas conversion of 16:0 to 18:0 was approximately 6%. No other fatty acid metabolites were detected for 18:0* or 16:0*. The conversion products were observed mainly in chylomicrons and very-low-density lipoproteins, indicating that the intestine and liver have comparable roles in desaturating 18:0 and 16:0. Overall, these data indicate that dietary 18:0 desaturation is severalfold greater than 16:0 desaturation. The low level (14%) of 18:0 desaturation in omnivorous adults may have little influence on blood lipid profiles relevant to atherosclerosis risk.

Secretory production of Arthrobacter levan fructotransferase from recombinant Escherichia coli.

Levan fructotransferase (LFTase) from Arthrobacter ureafaciens K2032 was expressed with N-terminal fusion of a LacZ-derived secretion motif (TMITNSSSVP) using the lac promoter system in recombinant Escherichia coli JM109 [pUDF-A81]. In flask cultures, recombinant enzyme activity was detected in culture media, and sequence analysis of N-terminal residues showed that about 40% of the extracellular recombinant LFTase had an authentic N-terminus. In a fed-batch bioreactor containing recombinant E. coli at high cell concentrations (OD(600)>200), the extracellular LFTase accumulated to 46000 U ml(-1) (approximately 2.0 g l(-1)) which was almost 40% of total (intra- and extracellular) recombinant LFTase. The synthesized recombinant enzyme was secreted soon after gene expression was induced by IPTG. Prolonged high secretion caused cell lysis and growth inhibition during the production phase in fed-batch cultures. When lactose was added by continuous feed mode, the secretion of recombinant LFTase and hence the cell lysis were significantly delayed in spite of the increased synthesis level. Therefore the induced cell culture of recombinant E. coli could grow up to a much higher cell concentration with continuing recombinant enzyme synthesis. In the case of the controlled feed of lactose, the maximum activities (U ml(-1)) of total and extracellular LFTase were nearly 100% and 70% higher, respectively.

H+-pumping ATPase has little stimulatory effect on in vitro translocation of a model protein into Vibrio alginolyticus inside-out membrane vesicles.

We studied the effect of a H+ electrochemical potential generated by F1F0 ATPase on in vitro translocation of a model protein into Vibrio alginolyticus inside-out membrane vesicles. The F1F0 ATPase of V. alginolyticus catalyzed the pumping of H+ coupled to ATP hydrolysis as measured in fluorescence quenching experiments. Consequently, this enzyme leads to the generation of a H+ electrochemical potential. The H+ electrochemical potential generated by F1F0 ATPase was completely abolished by 30 microM N,N'-dicyclohexylcarbodiimide (DCCD) or 5 microM carbonylcyanide m-chlorophenylhydrazone (CCCP) at 30 degrees C. The treatment of membrane vesicles with 30 microM DCCD at 30 degrees C had little influence on the translocation activity of uncleavable OmpF-Lpp, a model secretory protein, as compared to the intact membrane vesicles. On the other hand, the NADH:quinone oxidoreductase of V. alginolyticus is known to be a Na+ pump that leads to generation of a Na+ electrochemical potential. This Na+ electrochemical potential stimulates protein translocation into inside-out membrane vesicles prepared from V. alginolyticus in the presence of Escherichia coli SecA [Tokuda, H., Kim, Y. J., and Mizushima, S. (1990) FEBS Lett. 264, 10-12] From these results, it is evident that the stimulatory effect of the Na+ electrochemical potential on protein translocation in V. alginolyticus is not affected by the H+ electrochemical potential influence of F1F0 ATPase.

Heteromeric gap junction channels in rat hepatocytes in which the expression of connexin26 is induced.

More than one isotype of the gap junction channel-forming protein subunit, known as connexin, are synthesized in most mammalian cells. Using a modified primary cell culture of rat hepatocytes, in which both connexin32 and connexin26 were expressed in a comparable degree, the molecular composition of connexin subtypes in a gap junction channel (i.e., homomeric or heteromeric) was studied. A fluorescent dye Lucifer Yellow-coupling among hepatocytes was blocked in the presence of 20 microM beta-glycyrrhetinic acid, and antagonist for the gap junction channel. Similarly, either one of the antibodies raised against connexin32 or connexin26 completely inhibited dye-coupling activity among hepatocytes. In addition, we studied the dye-coupling properties at different extracellular pHs in two primary rat hepatocytes: one in which connexin26 was induced, and the other which expresses connexin32 as a major gap junction channel protein. The dye-coupling among the connexin26-induced hepatocytes was more sensitive to lowering pHs than among the normal hepatocytes, in which less than 5% of gap junction protein is connexin26. Taken together, data obtained in our study strongly suggest that the connexins (32 & 26) are assembled to form heteromeric, rather than homomeric, gap junction channels in the connexin226-induced rat hepatocytes.

Enhanced production of anticoagulant hirudin in recombinant Saccharomyces cerevisiae by chromosomal delta-integration.

Recombinant Saccharomyces cerevisiae strains were developed to overproduce an anticoagulant hirudin. The delta-sequences of the yeast retrotransposon Ty1 and URA3 were used as target sites for a hirudin expression cassette. High copy-number transformants were successfully selected using a dominant selection antibiotic, G418. The copy numbers of the hirudin expression cassette integrated into delta-sequences of the yeast chromosome ranged from five to ten copies per cell. Production of hirudin in the delta-integrated recombinant S. cerevisiae system increased over two-fold compared with the YEp-based episomal hirudin expression system. A linear relationship between the copy number of the hirudin expression cassette and hirudin expression level was observed up to 10 copies. The hirudin expression cassettes integrated into the yeast chromosome were stably maintained in non-selective culture conditions.

Molecular cloning of a gene encoding the thermoactive levansucrase from Rrahnella aquatilis and its growth phase-dependent expression in Eescherichia coli.

A levansucrase gene (lsrA) from Rahnella aquatilis ATCC33071 was isolated from a genomic library and the nucleotide sequence of the lsrA structural gene was determined. lsrA is composed of 1248 bp and encodes 415 amino acid residues with a calculated molecular mass of 45.9 kDa. Although the amino acid sequence of lsrA gene showed good conservation with the sequences of reported levansucrases and of the conserved regions thought to be implicated in the enzyme activity, comparison of the deduced amino acid sequences certified the dissimilarity of the proteins from Gram-negative and Gram-positive bacteria. The lsrA gene was expressed from its own promoter in Escherichia coli in an active form. The lsrA expression in E. coli-pRL1CPR was affected by the growth phase of cells: it was repressed in the early phase of growth, but was significantly stimulated during the entrance of cells into the late phase of growth. The growth-phase-dependent fashion of lsrA expression was altered in a constitutive-like fashion by the deletion of an upstream region of lsrA (pNd137), suggesting that the growth-phase dependent expression of lsrA was mediated by the deleted upstream region.

In vitro inhibition of gap junctional intercellular communication by chemical carcinogens.

This study was conducted to assess the effects of chemical carcinogens on the gap junction-mediated intercellular communication in cultured mammalian cells. The method of scrape-loading dye transfer of lucifer yellow was adapted as a measure of gap junctional communication. Clone 9 cells derived from rat liver were treated with a model chemical carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the gap junctional communication was assessed by measuring the transfer of scrape-loaded lucifer yellow dye. When cells were treated with the carcinogen at 0.3 mg/ml, the fluorescent dye transfer was inhibited by 90% in 60 min. Other chemical agents, which include direct or indirect carcinogens and antitumor drugs, were also examined for their effects on the gap junctional communication. Direct carcinogens, such as MNNG, hydroxylamine and ethidium bromide, exhibited strong inhibition of intercellular communication, while indirect carcinogens, such as aflatoxin B1 and ethionine, exerted minor effects. Effects of test chemicals on the cell communication through gap junctions were readily quantitated by counting the number of cells stained with the fluorescent dye.

Desulfurization of diesel oils by a newly isolated dibenzothiophene-degrading Nocardia sp. strain CYKS2.

A dibenzothiophene (DBT)-degrading bacterial strain was isolated from dyeing industry wastewater and identified as Nocardia sp. CYKS2. The newly isolated bacterial strain Nocardia sp. CYKS2 was able to convert DBT to 2-hydroxybiphenyl (2-HBP) as the dead-end metabolite through a sulfur-specific pathway. Other organic sulfur compounds, such as thiophene derivatives, thiazole derivatives, sulfides, and disulfides were also desulfurized by Nocardia sp. CYKS2. In batch culture, 0.2 mM DBT was completely desulfurized in 60 h. After DBT was depleted, neither cell growth nor 2-HBP production was observed. When a model oil which DBT was dissolved in hexadecane was treated with growing cells, DBT was desulfurized from 10 mM to about 2 mM in 80 h. In this case, desulfurization rate was 0.279 mg-sulfur/(L-dispersion.h), which was about 2.5 times higher than that in the previous case of batch culture. When diesel oil was treated, the sulfur content decreased from 0.3 to 0.24 wt % in 48 h. A volumetric phase ratio of oil to water was 1/10 in this case. The sulfur decreased from 0.3 to 0.2 wt % in 48 h, when the volumetric phase ratio was 1/20. The desulfurization rates were 0.909 and 0.992 mg-sulfur/(L-dispersion.h), respectively.

Fatty acid carbon isotope ratios in humans on controlled diets.

Carbon stable isotope ratios for six serum fatty acids (FA) are reported for human subjects on controlled fat diets to determine the range of natural isotope abundance and to demonstrate the leveling effect of a well-controlled diet. Twenty-nine subjects were randomly assigned to one of three controlled diets containing high, medium, or low fat. Diets were consumed for 8 wk. Serum samples were collected at baseline (0), 5, 6, 7, and 8 wk. FA were extracted and methylated. Isotope ratios were analyzed by high-precision gas chromatography combustion-isotope ratio mass spectrometry. At baseline, mean delta 13C for 16:0b, 16:1a, 18:0a, 18:1c,18:2n-6d and 20:4n-6bc were -24.1, -21.7, -21.6, -25.6, -29.6, and -25.0/1000, respectively, with an average standard deviation of 1.9/1000. Most delta 13C decreased during the diet period and appeared to have stabilized by week 5 at -25.3, -21.9, -22.3, -26.5, -30.1, and -24.5/1000, respectively. Between-subjected variability decreased from 1.74 to 1.20/1000 on the controlled diets. Measurement variability was 0.53/1000. The within-subject variability during weeks 5-8 was 0.57/1000 (range of 0.32-0.84/1000), showing a minimum biological fluctuation on controlled diets. There was no diet group effect on delta 13C of serum FA. Except for 18:2, the delta 13C of experimental diets was lower than that of serum FA, consistent with observations in animals. These data show that carbon isotope ratios stabilize in response to controlled diets within 5 wk, reflecting the isotope ratio of their dietary source, and establish isotope ratio fluctuations for endogenous compounds for future studies.

Independent exponential feeding of glycerol and methanol for fed-batch culture of recombinant Hansenula polymorpha DL-1.

As a novel feeding strategy for optimizing human epidermal growth factor (hEGF) production with a recombinant Hansenula polymorpha DL-1 using the methanol oxidase (MOX) promoter in H. polymorpha DL-1, independent exponential feeding of two substrates was used. A simple kinetic model considering the cell growth on two substrates was established and used to calculate the respective feeding rates of glycerol and methanol. In the fedbatch culture with methanol-only feeding, the optimal set point of specific growth rate on methanol was found to be 0.10 h-1. When the fed-batch cultures were conducted by the independent feeding of glycerol and methanol, the actual specific growth rate on glycerol and methanol was slightly lower than the set point of specific growth rate. By the uncoupled feeding of glycerol and methanol the volumetric productivity of hEGF increased from 6.4 to 8.0 mg/(L.h), compared with methanol-only feeding.

Varus and internal rotational deformity of the ankle secondary to distal tibial physeal injury.

Nine children who sustained Lauge-Hansen's supination-inversion injury of distal tibial physeal injury with intact distal fibular physis, were followed until their maturity. The average varus deformity was 39 degrees (maximum, 80 degrees) with 23 degrees of internal rotational deformity. The longitudinal growth of the fibula was retarded compared with opposite normal leg. There was early closure of the medial distal tibial physis, gradual upward migration of medial malleolus, and eventually medial subluxation of the ankle; these resulted in gradual varus and internal rotational deformities of the injured ankle. It is thought that the resultant disabling deformity of ankle should be prevented by any means, though presently there are no available effective methods of treatment. It is suggested that the repeated corrective osteotomy should be carried out before epiphyseal deformity of the distal tibia and subluxation of the ankle joint develop.

Valgus ankle secondary to acquired fibular pseudoarthrosis in children. Long-term results of the Langenskiöld operation.

Nine cases of acquired absence of the fibular shaft were studied to determine the growth contribution of the distal fibula; in 6 cases the absence was caused by osteomyelitis and in 3 cases by trauma. The average valgus and external rotational deformities were 15.2 degrees and 10 degrees, respectively. In 3 of 7 cases surgically treated with Langenskiöld operation or supramalleolar corrective osteotomy, the valgus deformity recurred during the postoperative growth period. The speculated causes of gradual valgus deformity are the loss of physiologic thrust from the proximal to distal fibula, the tethering effect of contracted soft tissue on distal fibula and early physeal closure of the lateral part of the distal tibia due to continuous, uneven axial overloading. The Langenskiöld operation was found effective for the stability of ankle joint in the initial period, but could not prevent the postoperative revascularization of the ankle. However, it is strongly recommended that any types of prophylactic surgery should be carried out before the development of an epiphyseal deformity of distal tibia, and to prevent secondary osteoarthritis of the ankle joint.

Intratendinous ganglion cyst of the semimembranosus tendon.

Intratendinous ganglion cyst is a very rare lesion with an unknown aetiology that originates within the tendon. We encountered a case of 43-year-old woman who complained of a palpable, non-tender mass in the thigh with increasing swelling. An intratendinous ganglion cyst in the semimembranosus tendon of the lower extremity was diagnosed and located by ultrasound and MRI. Nine months after a surgical excision, there were recurrent ganglion cysts along the semimembranosus tendon. We describe this case with a review of the relevant literature.

Molecular cloning of levan fructotransferase gene from Arthrobacter ureafaciens K2032 and its expression in Escherichia coli for the production of difructose dianhydride IV.

AIMS: To clone and overexpress a novel levan fructotransferase gene lftA from Arthrobacter ureafaciens K2032. METHODS AND RESULTS: The lftA gene, encoding a levan fructotransferase (LFTase) of 521 amino acids (aa) residues, was cloned from the genomic DNA of A. ureafaciens K2032, and overexpressed in Escherichia coli. The recombinant LFTase overexpressed in E. coli was then used to produce a difructose dianhydride (DFA IV) from levan. DFA IV crystals with 97% purity could be obtained from the reaction mixture in 83.7% yield by using a natural crystallization method. CONCLUSIONS: The lftA gene cloned from A. ureafaciens K2032 encode a novel levan fructotransferase which produces difructose dianhydride (DFA IV) from levan. SIGNIFICANCE AND IMPACT OF THE STUDY: Levan fructotransferase is a useful enzyme with great promise in the production of DFA IV and various fructosides.