Serum VEGF increases in diabetic polyneuropathy, particularly in the neurologically active symptomatic stage.

AIM: To identify the relationship between vascular endothelial growth factor (VEGF) and diabetic polyneuropathy (DPN). METHODS: Two hundred and twenty diabetic patients participated, 113 with DPN and 107 without DPN. All patients were also classified according to the four stages of DPN (no neuropathy: stage 0; asymptomatic neuropathy: stage 1; symptomatic neuropathy: stage 2; disabling neuropathy: stage 3). Serum VEGF concentration was measured using an enzyme-linked immunosorbent assay (ELISA) and levels between the patients with and without DPN and also between the different stages of DPN, were compared. RESULTS: The mean serum VEGF level in all patients was 264.6 +/- 218.8 pg/ml. The mean serum VEGF level was higher in patients with DPN (310.1 +/- 224.3 pg/ml) than in the patients without DPN (216.5 +/- 204.0 pg/ml, P = 0.0014). Serum VEGF was higher in the 'symptomatic' stage (stage 2, 364.8 +/- 225.9 pg/ml) in comparison with the 'asymptomatic' (stage 1, 256.7 +/- 224.4 pg/ml, P = 0.015) and 'disabling' (stage 3, 180.3 +/- 109.4 pg/ml, P = 0.042) stages. The mean serum VEGF level in patients with diabetic retinopathy (261.1 +/- 210.6 pg/ml) and in patients with diabetic nephropathy (241.5 +/- 185.7 pg/ml) was not increased. CONCLUSIONS: The serum VEGF level is increased in patients with DPN, particularly in patients in the neurologically active 'symptomatic' stage.

Regulation of SV40 large T-antigen stability by reversible acetylation.

Reversible acetylation on protein lysine residues has been shown to regulate the function of both nuclear proteins such as histones and p53 and cytoplasmic proteins such as alpha-tubulin. To identify novel acetylated proteins, we purified several proteins by the affinity to an anti-acetylated-lysine antibody from cells treated with trichostatin A (TSA). Among the proteins identified, here we report acetylation of the SV40 large T antigen (T-Ag). The acetylation site was determined to be lysine-697, which is located adjacent to the C-terminal Cdc4 phospho-degron (CPD). Overexpression of the CBP acetyltransferase acetylated T-Ag, whereas HDAC1, HDAC3 and SIRT1 bound and deacetylated T-Ag. The acetylation and deacetylation occurred independently of p53, a binding partner of T-Ag, but the acetylation was enhanced in the presence of p53. T-Ag in the cells treated with TSA and NA or the acetylation mimic mutant (K697Q) became unstable in COS-7 cells, suggesting that acetylation regulates stability of T-Ag. Indeed, NIH3T3 cells stably expressing K697Q showed decreased anchorage-independent growth compared with those expressing wild type or the K697R mutant. These results demonstrate that acetylation destabilizes T-Ag and regulates the transforming activity of T-Ag in NIH3T3 cells.

Potent and specific inhibition of p60v-src protein kinase both in vivo and in vitro by radicicol.

A fungal metabolite, radicicol, with a macrocyclic ring induced the reversal of transformed phenotypes of v-src-transformed fibroblasts (Rous sarcoma virus-transformed 3Y1 rat fibroblast) at a quite low concentration of 0.1 microgram/ml. Actin stress fibers reappeared in the transformed cells after treatment with radicicol. Radicicol reduced the intracellular level of autophosphorylation of p60v-src as well as the level of other tyrosine-phosphorylated proteins in a dose-dependent manner. In vitro kinase assay revealed that radicicol effectively inhibited not only autophosphorylation but also transphosphorylation activities of purified p60v-src with a concentration producing 50% inhibition of 0.1 microgram/ml. However, radicicol showed no inhibitory effect on protein kinase C or protein kinase A. These results suggest that radicicol is a novel and specific protein-tyrosine kinase inhibitor and that the decreased level of tyrosine kinase activity of p60v-src causes reversion of transformed phenotypes of Rous sarcoma virus-transformed 3Y1 rat fibroblast. Furthermore, differentiation of Friend leukemia cells, which is one of the known characteristic phenomena associated with the inhibition of tyrosine kinase, was also induced in the concentration range of 0.05-0.5 microgram/ml, suggesting that the agent is useful for the analysis of differentiation as well as the kinase-mediated signal transduction.

Cyclic hydroxamic-acid-containing peptide 31, a potent synthetic histone deacetylase inhibitor with antitumor activity.

Cyclic hydroxamic-acid-containing peptide 1 (CHAP1), designed as a hybrid of trichostatin A and trapoxin, is a lead compound for the development of potent inhibitors of histone deacetylase (HDAC). In this study, we synthesized a series of CHAP derivatives and evaluated their biological activities by monitoring the potency of their inhibition of HDAC activity, their ability to augment the expression of MHC class-I molecules in B16/BL6 cells, and their effect on cell proliferation. A structure-activity relationship study using these three assay systems revealed several requirements of their structure for the strong inhibition of HDAC not only in the cell-free situation, but also in cells. When the structures of CHAP derivatives are represented as cyclo(-Asu(NHOH)-AA(2)-AA(3)-Pro or Pip-)(n), where Asu(NHOH) and Pip are zeta-hydroxamide-alpha-aminosuberic acid and pipecolic acid, respectively, (a) the tetrapeptide structure (n = 1) was better than the octapeptide one (n = 2); (b) AA(2) and AA(3) should be hydrophobic; and (c) the combination of amino acid chirality should be LDLD for the strongest inhibition of HDAC in cells (LDLD > LLLD, LDLL > LLDL). cyclo(-L-Asu(NHOH)-D-Tyr(Me)-L-Ile-D-Pro-) or CHAP31 was selected as one of the strongest CHAPs, and its biological activity was characterized further. CHAP31 was much more stable in the presence of cultured cells (t(1/2) > 3000 h) than trichostatin A (t(1/2) = 14.7 h) or trapoxin A (t(1/2) = 2.10 h). CHAP31 exhibited antitumor activity in C57BL x DBA/2 F(1) (BD2F(1)) mice bearing B16/BL6 tumor cells. Furthermore, CHAP31 inhibited the growth in four of five human tumor lines implanted into nude mice. These results suggest CHAP31 to be promising as a novel therapeutic agent for cancer treatment.

Oxamflatin is a novel antitumor compound that inhibits mammalian histone deacetylase.

Oxamflatin [(2E)-5-[3-[(phenylsufonyl) aminol phenyl]-pent-2-en-4-ynohydroxamic acid] induces transcriptional activation of junD and morphological reversion in various NIH3T3-derived transformed cell lines. We found that oxamflatin showed in vitro antiproliferative activity against various mouse and human tumor cell lines with drastic changes in the cell morphology and in vivo antitumor activity against B16 melanoma. Oxamflatin caused an elongated cell shape with filamentous protrusions as well as arrest of the cell cycle at the G1 phase in HeLa cells. These phenotypic changes of HeLa cells were apparently similar to those by trichostatin A (TSA), a specific inhibitor of histone deacetylase (HDAC). The effect of oxamflatin on the transcriptional activity of the cytomegalovirus (CMV) promoter was examined and compared with known HDAC inhibitors, TSA, sodium n-butyrate, and FR901228. Oxamflatin as well as all these inhibitors greatly enhanced the transcriptional activity of the CMV promoter in a dose-dependent manner. Oxamflatin, like TSA, inhibited intracellular HDAC activity, as a result of which marked amounts of acetylated histone species accumulated. Finally, effects on expression of several endogenous genes involved in cell morphology and cell cycle control in HeLa cells were analysed. Expression of gelsolin, cyclin E and Cdk inhibitors including p21WAF1/Cip1 was highly augmented, while that of cyclin A and cyclin D1 was decreased by oxamflatin. These results suggest that changes in the expression pattern of the genes regulating cell morphology and the cell cycle due to histone hyperacetylation are responsible for the antitumor activity, the morphological change and the cell cycle arrest induced by oxamflatin.

Suppression of morphological transformation by radicicol is accompanied by enhanced gelsolin expression.

Radicicol, an inhibitor of Src-family protein-tyrosine kinases, causes morphological reversion of v-src- and v-Ha-ras-transformed fibroblasts and arrest of the cell cycle at both the G1 and the G2 phases. Radicicol was found to inhibit the growth of several other oncogene-transformed cell lines and human carcinoma cell lines and to revert their cell morphology to be flat. In the radicicol-treated flat cells, actin stress fiber bundles were reorganized. Since this effect of radicicol on these cell lines was inhibited by cycloheximide, de novo protein synthesis is required for the morphological reversion. Screening of cellular proteins enhanced in response to radicicol by two-dimensional gel electrophoresis suggested that the amount of gelsolin, an actin regulatory protein, was distinctly increased upon radicicol treatment. Western blot and Northern blot analyses showed that radicicol enhanced transcription of the gelsolin gene in human carcinoma cell lines, as a result of which the amount of gelsolin was increased several folds. Injection with an anti-gelsolin antibody into cells and successive treatment with radicicol resulted in approximately 80% reduction of the number of flat cells with stress fibers in comparison with controls treated with an irrelevant antibody. These results show that elevated expression of gelsolin is associated, at least in part, with the suppression of transformation and the restoration of actin stress fibers in human carcinoma cells by radicicol.

Organization and nucleotide sequence of the secE-nusG region of Streptomyces griseus.

The nusG genes of Streptomyces griseus and Streptomyces coelicolor A3(2) were cloned by the DNA-probing method with synthetic oligonucleotides designed on the basis of the nucleotide sequence of the nusG gene of Streptomyces virginiae. The amino acid sequences of the NusG proteins deduced from the nucleotide sequences showed significant homologies to those from a variety of microorganisms. Nucleotide sequence analysis of the region upstream of the nusG gene of S. griseus revealed the presence of the secE gene, suggesting that secE and nusG are organized as an operon as is found in other microorganisms.

Cloning, nucleotide sequence and expression in Escherichia coli of two cobalt-containing nitrile hydratase genes from Rhodococcus rhodochrous J1.

Rhodococcus rhodochrous J1 produces two kinds of cobalt-containing nitrile hydratases (NHases); one is a high molecular mass-NHase (H-NHase) and the other is a low molecular mass-NHase (L-NHase). Both NHases are composed of two subunits of different sizes (alpha and beta subunits). The H- and L-NHase genes were cloned into Escherichia coli by a DNA-probing method using the NHase gene of Rhodococcus sp. N-774, a ferric ion-containing NHase producing strain, as the hybridization probe and their nucleotide sequences were determined. In each of the H- and L-NHase genes, the open reading frame for the beta subunit was located just upstream of that for the alpha subunit, which probably belongs to the same operon. The amino acid sequences of each subunit of the H- and L-NHases from R. rhodochrous J1 showed generally significant similarities to those from Rhodococcus sp. N-774, but the arrangement of the coding sequences for two subunits is reverse of the order found in the NHase gene of Rhodococcus sp. N-774. Each of the NHase genes was expressed in E. coli cells under the control of lac promoter, only when they were cultured in the medium supplemented with CoCl2.

Cloning and characterization of an amidase gene from Rhodococcus species N-774 and its expression in Escherichia coli.

For investigation of an unknown open reading frame which is present upstream of the nitrile hydratase (NHase) gene from Rhodococcus sp. N-774, a longer DNA fragment covering the entire gene was cloned in Escherichia coli. Nucleotide sequencing and detailed subcloning experiments predicted a single open reading frame consisting of 521 amino acid residues of Mr 54,671. The amino acid sequence, especially its NH2-terminal portion, showed significant homology with those of indoleacetamide hydrolases from Pseudomonas savastanoi and Agrobacterium tumefaciens, and acetamidase from Aspergillus nidulans. The 521-amino acid coding region was therefore expressed by use of the E. coli lac promoter in E. coli, and was found to direct a considerable amidase activity. This amidase hydrolyzed propionamide efficiently, and also hydrolyzed, at a lower efficiency, acetamide, acrylamide and indoleacetamide. These data clearly show that the unknown open reading frame present upstream of the NHase coding region encodes an amidase. Because the TAG translational stop codon of the amidase is located only 75 base pairs apart from the ATG start codon of the alpha-subunit of NHase, these genes are probably translated in a polycistronic manner.

Cloning and nucleotide sequence of a bacterial cytochrome P-450VD25 gene encoding vitamin D-3 25-hydroxylase.

The gene encoding an enzyme that catalyzes the hydroxylation at position 25 of vitamin D-3 was cloned from an actinomycete strain, Amycolata autotrophica, by use of a host-vector system of Streptomyces lividans. The amino acid sequence deduced from the nucleotide sequence revealed that this enzyme, tentatively named P-450VD25, contains several regions of strong similarity with amino acid sequences of cytochromes P-450 from a variety of organisms, primarily in the regions of an oxygen-binding site and a heme ligand pocket. Especially, P-450VD25 shows end-to-end similarity in amino acid sequence to P-450dNIR of Fusarium oxysporum and P-450SU2 of Streptomyces griseolus. The recombinant S. lividans strain containing the P-450VD25 gene on a multicopy plasmid converted vitamin D-3 in the medium into 25-hydroxyvitamin D-3 at a maximum yield of 10%.

Purification of cohesive-end-producing restriction endonuclease from Bacillus subtilis G.

A new restriction endonuclease was partially purified from Bacillus subtilis G (IAM1247). This restriction endonuclease (endonuclease RBsuG) seems to produce cohesive ends at its cleavage site.

Cloning and sequencing of the gene cluster encoding two subunits of membrane-bound alcohol dehydrogenase from Acetobacter polyoxogenes.

The membrane-bound alcohol dehydrogenase (ADH) from Acetobacter polyoxogenes NBI1028 is composed of a 72 kDa subunit and a 44 kDa cytochrome c subunit. The amino acid sequences of the two regions of the 72 kDa subunit were determined to prepare oligonucleotides for the purpose of amplification of a DNA fragment corresponding to the intermediate region by the polymerase chain reaction. A 0.5 kb DNA fragment thus amplified was used as the probe to clone a 7.0 kb PstI fragment coding for the whole 72 kDa subunit. Nucleotide sequencing and immunoblot analysis revealed that the cloned fragment contained the full structural genes for the 72 kDa and the 44 kDa subunits and they were clustered with the same transcription polarity. The predicted amino acid sequence of the gene for the 72 kDa subunit showed homology with that of the 72 kDa subunit from ADH of A. aceti and those of methanol dehydrogenase from methylotrophic bacteria. The 72 and 44 kDa subunits contained one and three typical haem binding sequences, respectively.

Regulation of secondary metabolism and cell differentiation in Streptomyces: A-factor as a microbial hormone and the AfsR protein as a component of a two-component regulatory system.

A-factor is a microbial hormone that functions as a key switch for secondary metabolite formation and morphogenesis in Streptomyces griseus. Genetic and biochemical studies on the A-factor-binding protein have implied that the binding protein present in the cytoplasm plays a role in repressing streptomycin (Sm) production and sporulation while the binding of A-factor to the binding protein releases this repression. The A-factor signal is transferred, probably via some additional regulatory proteins in the A-factor-regulatory cascade, to the strR gene, a regulator for Sm biosynthesis. A positive regulatory protein binds about 430-330 bp upstream from the transcription start point of the strR promoter and activates its transcription. The StrR product, in turn, activates the other Sm-biosynthesis genes. A global regulatory gene, afsR, of Streptomyces coelicolor A3(2) encodes a 993-amino acid protein that is phosphorylated by a specific phosphokinase, AfsK, encoded by the region just upstream from the afsR gene. Site-directed mutagenesis of afsR has revealed that phosphorylated AfsR globally stimulates transcription of antibiotic-production genes. It is most likely that AfsR and AfsK compose a two-component regulatory system. Although AfsR shows no significant homology with typical regulators of the two-component systems in other prokaryotes, such as OmpR and PhoB of Escherichia coli, it shows considerable homology with regulatory proteins in antibiotic biosynthetic gene clusters of Streptomyces spp., such as actII ORF4, dnrR1 ORF1 and redD ORF1.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and nucleotide sequence of a cellulase gene, casA, from an alkalophilic Streptomyces strain.

A gene encoding an endo-type semi-alkaline cellulase was cloned from an alkalophilic Streptomyces strain in Streptomyces lividans, and its nucleotide sequence was determined. Downstream from the transcriptional start point, which was determined by high-resolution S1 mapping, an open reading frame of 388 amino acids (aa) was present. The N-terminal amino acid sequence of the mature enzyme determined by an Edman degradation procedure suggested that the cellulase had an extraordinarily long leader sequence of about 70 aa. Comparison with the leader sequences of endoglucosidase H from Streptomyces plicatus and the cellulase from Cellulomonas fimi suggested that the semi-alkaline cellulase was processed in two steps during maturation.

Isolation and characterization of human pro-urokinase and its mutants accumulated within the yeast secretory pathway.

Human pro-urokinase (pro-UK) and two pro-UK deletion mutants, one lacking the epidermal growth factor(EGF)-like domain, and the other lacking both the EGF-like domain and the kringle domain, were produced in Saccharomyces cerevisiae. This was done using the yeast GAL7 promoter and the prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR). Although biologically active and heavily glycosylated pro-UKs were secreted into the culture medium, the amounts were extremely small. On the other hand, large amounts of pro-UKs of a single-chain form were accumulated inside the cells, exceeding 3-4% of total cellular proteins. The intracellular pro-UKs were N-glycosylated, probably with a single core carbohydrate unit, and amino acid sequencing of their N termini revealed that the secretion signal of MPR was correctly processed. Biologically active pro-UKs were recovered in high yields by means of solubilization with 4.5 M guanidine.HCl and subsequent dialysis for refolding. The refolded yeast pro-UK was indistinguishable from human kidney-derived pro-UK in terms of specific enzymatic activity and its secondary structure, as determined by circular dichroism spectroscopy.

Genetic and biochemical characterization of a protein phosphatase with dual substrate specificity in Streptomyces coelicolor A3(2).

A gene encoding a protein phosphatase (SppA) with a phosphoesterase motif, which was predicted by the genome project of the Gram-positive bacterium Streptomyces coelicolor A3(2), was cloned by PCR in pET32a(+) and expressed in Escherichia coli. SppA fused to thioredoxin (TRX-SppA) showed distinct heat-stable phosphatase activity toward p-nitrophenyl phosphate with optimal pH 8.0 and optimal temperature 55 degrees C. Mn2+ greatly enhanced enzyme activity, as is found with other protein Ser/Thr phosphatases. TRX-SppA was not inhibited by sodium orthovanadate or okadaic acid, both of which are known to be specific inhibitors of protein phosphatases. TRX-SppA showed phosphatase activity toward not only phosphoThr (pThr) and pTyr but also oligopeptides containing pSer, pThr, and pTyr, indicating that SppA is a protein phosphatase with dual substrate specificity. Disruption of the chromosomal sppA gene resulted in severe impairment of vegetative growth. All of these observations show that SppA, a protein phosphatase with dual specificity, plays an important, but not essential, role in vegetative growth of S. coelicolor A3(2). The presence of a single copy of sppA in all the 13 Streptomyces species examined, as determined by Southern hybridization, suggests a common role of SppA in general in Streptomyces species.

The aerial mycelium-defective phenotype of Streptomyces griseus resulting from A-factor deficiency is suppressed by a Ser/Thr kinase of S. coelicolor A3(2).

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is essential for aerial mycelium formation and streptomycin (Sm) production in Streptomyces griseus. A protein Ser/Thr kinase (AfsK), the product of the Streptomyces coelicolor A3(2) afsK gene, controlling secondary metabolism in this strain, reversed the aerial mycelium-negative phenotype of an A-factor-deficient mutant strain, S. griseus HH1, and induced sporulation without affecting A-factor productivity or Sm production. A mutant AfsK protein lacking kinase activity failed to induce aerial mycelium formation which indicates the importance of the kinase activity for suppression in S. griseus. These data suggest that a Ser/Thr kinase functionally similar to S. coelicolor A3(2) AfsK plays a regulatory role in aerial mycelium formation in S. griseus, either as a member in the A-factor regulatory network or independently of this network.

Cloning and sequences of two macrolide-resistance-encoding genes from mycinamicin-producing Micromonospora griseorubida.

Two macrolide-resistance determinants were cloned from a mycinamicin (Mm)-producing Micromonospora griseorubida strain in Streptomyces lividans and Streptomyces parvulus. One of the cloned genes, designated myrA, was cloned as a gene which conferred strong resistance to Mm and tylosin (Ty), but not to erythromycin (Er) or josamycin (Jm) on S. lividans. Another gene, named myrB, was cloned as an ErR-encoding gene which conferred MLS resistance (to macrolide, lincosamide and streptogramine B antibiotics) on S. parvulus. Both myrA and myrB were sequenced and the corresponding ORFs were determined. The deduced amino acid (aa) sequence of myrA showed no similarity to proteins in the available databases, suggesting that an unknown mechanism of macrolide resistance is exerted by the MyrA protein. The deduced aa sequence of myrB exhibited high similarity to 23S rRNA methyltransferases (MTases), such as ErmE and CarB, from a variety of microorganisms.

Cloning of a streptomycin-production gene directing synthesis of N-methyl-L-glucosamine.

A Streptomyces bikiniensis DNA fragment complementing a deficiency in streptomycin (Sm) production was cloned on the plasmid vector pIJ385. Host strain S. bikiniensis SD 1 used as the recipient in cloning displayed deficiency in biosynthesis of N-methyl-L-glucosamine, one of the moieties of Sm. The cloned fragment on the multicopy plasmid pIJ385 conferred sevenfold increase in Sm production in comparison with the wild-type parental strain. By subcloning, the region complementing the Sm deficiency of SD 1 was narrowed to a 3.0-kb fragment.

Site-directed mutagenesis of the A-factor receptor protein: Val-41 important for DNA-binding and Trp-119 important for ligand-binding.

The A-factor receptor protein (ArpA) plays a key role in the regulation of secondary metabolism and cellular differentiation in Streptomyces griseus. ArpA binds the target DNA site forming a 22 bp palindrome in the absence of A-factor, and exogenous addition of A-factor to the ArpA-DNA complex immediately releases ArpA from the DNA. An amino acid (aa) replacement at Val-41 to Ala in an alpha-helix-turn-alpha-helix (HTH) motif at the N-terminal portion of ArpA abolished DNA-binding activity but not A-factor-binding activity, suggesting the involvement of this HTH in DNA-binding. On the other hand, an aa replacement at Trp-119 to Ala generated a mutant ArpA that was unable to bind A-factor, thus resulting in an A-factor-insensitive mutant that bound normally to its target DNA in both the presence and absence of A-factor. These data suggest that ArpA consisting of two functional domains, one for HTH-type DNA-binding at the N-terminal portion and one for A-factor-binding at the C-terminal portion, is a member of the LacI family. Consistent with this, two ArpA homologues, CprA and CprB, from Streptomyces coelicolor A3(2), each of which contains a very similar aa sequence of the HTH to that of ArpA, also recognized and bound the same DNA target. However, neither CprA nor CprB recognized A-factor, probably due to much less similarity in the C-terminal domains.