Significant expression of vascular endothelial growth factor/vascular permeability factor in mouse ascites tumors.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is believed to be a potent mediator of peritoneal fluid accumulation and angiogenesis and of tumor growth in ascites tumor. Such roles, however, have not been generally established because of insufficient quantitative and systemic analyses. To address this, we examined the expression of VEGF in 13 mouse ascites tumors (5 sarcomas, 3 carcinomas, 2 lymphomas, 1 leukemia, 1 mastocytoma, and 1 plasmacytoma). Using a newly developed sensitive and specific radioreceptor binding assay and functional assays, we found that active VEGF was significantly accumulated (6-850 ng/ml) in the ascites fluids of all 13 tumors. VEGF concentrations are higher in the tumors of sarcoma and carcinoma origin (430.4 +/- 234.2 ng/ml) than in those of lymphoma and hematological tumor origin (19.2 +/- 10.45 ng/ml). VEGF that accumulated in the peritoneal fluids or expressed in the ascites tumor cells was easily visualized with immunoprecipitation Western blot analysis with a rough correlation to the expression levels of VEGF gene in these tumor cells, suggesting that the tumor cells, at least in part, contributed to the production of the VEGF that accumulated in the ascites fluid. Most ascites tumors expressed VEGF; the 164-amino acid isoform was predominant, the 120-amino acid isoform was less abundant, and the 188-amino acid isoform was least abundant. Several representative ascites tumors expressed similar, if not higher, levels of VEGF when they were cultured at normoxic states, suggesting that they expressed VEGF at high levels in a constitutive manner. The microvessel densities in the peritoneal walls of tumor-bearing mice, which are significantly higher than those in normal mice, basically correlated to but did not parallel the VEGF concentrations in their respective ascites fluids. Thus, a complicated relationship may exist between the VEGF production and angiogenesis associated with ascites tumor in vivo. Taken together, our observations suggest that VEGF plays a fundamental role in ascites tumor formation; however, its importance may vary according to tumor origin.

Prokaryotic DNA polymerase I: evolution, structure, and "base flipping" mechanism for nucleotide selection.

Accurate transmission of DNA material from one generation to the next is crucial for prolonged cell survival. Following the discovery of DNA polymerse I in Escherichia coli, the DNA polymerase I class of enzymes has served as the prototype for studies on structural and biochemical mechanisms of DNA replication. Recently, a series of genomic, mutagenesis and structural investigations have provided key insights into how Pol I class of enzymes function and evolve. X-ray crystal structures of at least three Pol I class of enzymes have been solved in the presence of DNA and dNTP, thus allowing a detailed description of a productive replication complex. Rapid-quench stop-flow studies have helped define individual steps during nucleotide incorporation and conformational changes that are rate limiting during catalysis. Studies in our laboratory have generated large libraries of active mutant enzymes (8000) containing a variety of substitutions within the active site, some of which exhibit altered biochemical properties. Extensive genomic information of Pol I has recently become available, as over 50 polA genes from different prokaryotic species have been sequenced. In light of these advancements, we review here the structure-function relationships of Pol I, and we highlight those interactions that are responsible for the high fidelity of DNA synthesis. We present a mechanism for "flipping" of the complementary template base to enhance interactions with the incoming nucleotide substrate during DNA synthesis.

Substitutions of Ser for Asn-163 and Pro for Leu-264 are important for stabilization of lipase from Pseudomonas aeruginosa.

The lipase gene from Pseudomonas aeruginosa was randomly mutated by error-prone PCR to obtain thermostable mutants, followed by screening for thermostable mutant lipases. Out of about 2,600 transformants, four thermostable clones were obtained. Their nucleotide sequences showed that they had two or three amino acid substitutions. Analysis of the thermal stabilization of these mutant lipases indicated that Asn-163 to Ser and Leu-264 to Pro mutations were essential for the increased stability of the lipase. We expressed a mutant lipase (StLipA-5) having only the Asn-163 to Ser mutation and another (StLipA-6) having only the Leu-264 to Pro mutation in P. aeruginosa PAO1161, purified them, and then confirmed that the temperature which causes a 50% decrease in the activity of the non-treated enzyme on treatment for 30 min was increased by 1.5 and 3 degrees C, respectively, compared to the wild-type enzyme. However, the thermal stability of the mutant lipase (StLipA-7) having both mutations was increased only by 2.5 degrees C. These mutant lipases were stabilized through a decrease in activation entropy. Kinetic studies showed that the Kcat/K(m) values of StLipA-5, StLipA-6, and StLipA-7 were decreased by 14.4, 52.9, and 26.0%, respectively. Interestingly, the pH-stabilities of StLipA-6 and StLipA-7 were also increased, especially at alkaline pH. Based on these results, the tertiary structure and mechanism of stabilization of the lipase were discussed.

Insertion of a signal peptide-derived hydrophobic segment into the mature domain of OmpC, an outer membrane protein, does not interfere with the export of the following polypeptide chain across the cytoplasmic membrane of E. coli.

The effects of a hydrophobic peptide segment inserted into the amino-terminal region of the mature domain of OmpC, an outer membrane protein, on its translocation across the cytoplasmic membrane was studied. Both the intact OmpC and central domain-deleted OmpC were examined. The hydrophobic segment was derived from the signal peptide of OmpF. Secretory translocation across the cytoplasmic membrane was examined by means of proteinase K treatment. Four monoclonal antibodies that recognize different regions of OmpC were used to characterize proteinase K-resistant fragments. Insertion of the hydrophobic segment did not appreciably prevent the translocation of these proteins across the cytoplasmic membrane, larger parts of them being found as mature forms, which were mostly localized outside the cytoplasmic membrane. Circumstantial evidence supports the view, on the other hand, that the inserted hydrophobic domain was retained in the cytoplasmic membrane. It is concluded, therefore, that the hydrophobic segment, although it is not exported across the cytoplasmic membrane, does not prevent the secretion of the following polypeptide chain. The secretion was dependent on the amino-terminal signal peptide. Insertion of positive charges immediately after the hydrophobic segment resulted in suppression of the translocation. Based on these results possible mechanisms by which the secretion of the polypeptide chain after the hydrophobic segment are discussed.

Changes in microsomal lysophospholipid acyltransferase activity are correlated with rat parotid gland enlargement induced by chronic administration of isoproterenol.

Chronic (5- and 10-day) administration of isoproterenol, an agent that induces the proliferation of salivary gland cells, produced increases in microsomal 1-acyl-sn-glycero-3-phosphate acyltransferase and 1-acyl-sn-glycero-3-phosphocholine acyltransferase activity in rat parotid glands in parallel with gland enlargement. This increased activity was reduced when the treatment was stopped, the reduction corresponding to the reduction in gland weight. There were significant correlations between lysophospholipid acyltransferase activity and gland weight, and between the activities of the two types of lysophospholipid acyltransferase. However, isoproterenol treatment did not affect any of the steps of the subsequent phospholipid N-methylation. These results suggest that the cell proliferation induced by chronic administration of isoproterenol in the parotid gland is accompanied by reversible and selective increases in microsomal lysophospholipid acyltransferases.

Osmoregulatory expression of porin genes in Escherichia coli: a comparative study on strains B and K-12.

Escherichia coli K-12 produces both the OmpF and OmpC porins, the relative amounts of which in the outer membrane are affected in a reciprocal manner by the osmolarity of the growth medium. In contrast, E. coli B produces only the OmpF porin, regardless of the medium osmolarity. In this study, it was revealed that there is an extensive deletion within the ompC locus of the E. coli B chromosome. Cloning and nucleotide sequencing of the regulatory gene, ompR, of E. coli B revealed that there are two amino acid alterations (Lys-6 to Asn and Ala-130 to Thr) in the amino acid sequence of the OmpR protein, as compared with that of E. coli K-12. It is suggested that these particular amino acid alterations are responsible for the constitutive expression of the ompF gene observed in E. coli B.

[Study on the effects of influenza virus vaccines--relation between the rate of two-fractional vaccination and the overall rate of absenteeism].

In 1987 and 1988, in 9 elementary schools, the percentage of children who received two sessions of vaccination and the overall rate of absenteeism resulting from influenza were determined for each class, and their relationship was investigated. The following results were obtained. 1) The mean vaccination rate was 58.6% among 157 classes in 1987, whereas it was 29.9% among 151 classes in 1988, the rate being significantly higher in 1987. 2) The mean overall rate of absenteeism was 1.524% in 1987, which was significantly lower than the corresponding rate, 2.802%, in 1988. 3) There was a significant negative correlation between the vaccination rate and the overall rate of absenteeism in 7 of the 9 schools in 1987; the overall rate of absenteeism became significantly low with an increase in the vaccination rate. 4) No such trend, however, was noted in any of the schools in 1988. 5) The difference between the results in 1987 and those in 1988 seems to be attributable to the facts that variability of the prevailing strains of influenza was low (V0, 82%) in 1987, in addition to the high vaccination rate in that year, and that influenza virus type B having a high variability (V3, or more, 78%) prevailed in 1988, when the vaccination rate was low.

In vivo mutagenesis by Escherichia coli DNA polymerase I. Ile(709) in motif A functions in base selection.

The fidelity of DNA replication by Escherichia coli DNA polymerase I (pol I) was assessed in vivo using a reporter plasmid bearing a ColE1-type origin and an ochre codon in the beta-lactamase gene. We screened 53 single mutants within the region Val(700)-Arg(712) in the polymerase active-site motif A. Only replacement of Ile(709) yielded mutator polymerases, with substitution of Met, Asn, Phe, or Ala increasing the beta-lactamase reversion frequency 5-23-fold. Steady-state kinetic analysis of the I709F polymerase revealed reductions in apparent K(m) values for both insertion of non-complementary nucleotides and extension of mispaired primer termini. Abolishment of the 3'-5' exonuclease activity of wild-type pol I increased mutation frequency 4-fold, whereas the combination of I709F and lack of the 3'-5' exonuclease yielded a 400-fold increase. We conclude that accurate discrimination of the incoming nucleotide at the polymerase domain is more critical than exonucleolytic proofreading for the fidelity of pol I in vivo. Surprisingly, the I709F polymerase enhanced mutagenesis in chromosomal DNA, although the increase was 10-fold less than in plasmid DNA. Our findings indicate the feasibility of obtaining desired mutations by replicating a target gene at a specific locus in a plasmid under continuous selection pressure.

Quantitative renaturation from a guanidine-denatured state of the SecA dimer, a 200 KDa protein involved in protein secretion in Escherichia coli.

SecA is an essential component of the protein secretory machinery of Escherichia coli. SecA denatured in 6 M guanidine hydrochloride was quantitatively renatured through dilution and dialysis. The renatured SecA was the same as native SecA as to the CD spectrum, fluorescence spectrum for tryptophan residues and dimeric structures. It was as functionally active as native SecA as to interactions with ATP and presecretory proteins, and in vitro translocation. SecA-N95, which lacks the carboxyl-terminal 70 amino acid residues including three of four cysteine residues and yet is as active as intact SecA as to in vitro translocation, was also renatured to an active form from the guanidine solution. Furthermore, the renaturation of SecA took place in the presence of 1 mM dithiothreitol. It is concluded that disulfide bridges, both intra- and intermolecular ones, do not play a role in the folding and functioning of the SecA molecule.