Impairment of dendritic cells (DC), the most effective activators of anti-cancer immune responses, is one mechanism for defective anti-tumor immunity, but the causes of DC impairment are incompletely understood. We evaluated the association of impaired DC differentiation with angiogenesis-associated molecules D-dimer, vascular endothelial growth factor (VEGF), urokinase plasminogen activator and plasminogen activator inhibitor (PAI-1) in peripheral blood from 41 patients with lung, breast, and colorectal carcinoma. Subsequently, we studied the effect of administration of the anti-VEGF antibody (bevacizumab) on DC maturation and function in vivo. Compared with healthy volunteers, cancer patients had a bias towards the immunoregulatory DC2, had deficits in DC maturation after overnight in vitro culture, and had a significant increase in immature myeloid cell progenitors of DC (0.50 +/− 0.31% vs. 0.32 +/− 0.16%, respectively, p=0.011). A positive correlation was found between the percentage of DC2 and PAI-1 (R=0.50) and between immature myeloid cells and VEGF (R=0.52). Bevacizumab administration to cancer patients was associated with a decrease in the accumulation of immature progenitor cells (0.39 +/− 0.30 % vs. 0.27 +/− 0.24 %, p=0.012) and induced a modest increase in the DC population in peripheral blood (0.47 +/− 0.23 % vs. 0.53 +/− 0.30 %). Moreover, anti-VEGF antibody treatment enhanced allo-stimulatory capacity of DC and T cell proliferation against recall antigens. These data suggest that DC differentiation is negatively associated with VEGF levels and may be one explanation for impaired anti-cancer immunity, especially in patients with advanced malignancies.

The Escherichia coli gene coding for dihydropteroate synthase (DHPS) has been cloned and sequenced. The protein has 282 amino acids and a compositional molecular mass of 30,314 daltons. Increased expression of the enzyme was realized by using a T7 expression system. The enzyme was purified and crystallized. A temperature-sensitive mutant was isolated and found to express a DHPS with a lower specific activity and lower affinities for para-aminobenzoic acid and sulfathiazole. The allele had a point mutation that changed a phenylalanine codon to a leucine codon, and the mutation was in a codon that is conserved among published DHPS sequences.Dihydropteroate synthase (DHPS) (EC 2.5.1.15) catalyzes the condensation of para-aminobenzoic acid (pAB) with 7,8-dihydro-6-hydroxymethylpterin-pyrophosphate, forming 7,8-dihydropteroate (39, 44). This intermediary metabolite is subsequently converted to tetrahydrofolic acid, essential for the syntheses of purines, thymidylate, glycine, methionine, pantothenic acid, and N-formylmethionyl-tRNA. Sulfonamides are pAB analogs that are recognized by DHPS as alternate substrates (4,7,45,57). In the presence of sulfonamides, DHPS forms a sulfa-pterin adduct that is metabolically inert and diffuses from the cell (40). Folate cofactor depletion results in growth inhibition and in the appropriate environment, cell death (55).DHPS activity was first identified in crude cell extracts of several organisms by Shiota and coworkers (44, 45) and was identified in Escherichia coli by Brown et al. (8). The kinetic characteristics of DHPS have been studied by using partially purified extracts. Recently, a purification procedure for DHPS was published which showed that this enzyme constituted less than 0.01% of all the proteins in a cell (54). DHPS was purified to homogeneity, the sequence of the first 28 amino acids was determined, and the protein was shown to be a homodimer of two 30-kDa subunits. However, the purification procedure yielded less than 2 mg of purified protein from 1 kg of starting material, emphasizing the need to clone and overexpress this important chemotherapeutic target.Recently, the chromosomal gene that codes for DHPS in Streptococcus pneumoniae was cloned, sequenced, and shown to code for a protein of 34 kDa (28). A similar sequence was also identified in a Bacillus subtilis folic acid biosynthetic operon (48). Two other genes (sulI and sulII) that code for plasmid-borne sulfonamide-resistant DHPSs have also been sequenced (38, 52). However, there is no information about the E. coli DHPS gene. In this communication, we report the cloning, sequencing, and enhanced expression of the E. coli DHPS gene, designated folP. We also report conditions that allow crystallization of this enzyme.

The degradation of the prolipoprotein signal peptide in vitro by membranes, cytoplasmic fraction, and two purified major signal peptide peptidases from Escherichia coli was followed by reverse-phase liquid chromatography (RPLC). The cytoplasmic fraction hydrolyzed the signal peptide completely into amino acids. In contrast, many peptide fragments accumulated as final products during the cleavage by a membrane fraction. Most of the peptides were similar to the peptides formed during the cleavage of the signal peptide by the purified membrane-bound signal peptide peptidase, protease IV. Peptide fragments generated during the cleavage of the signal peptide by protease IV and a cytoplasmic enzyme, oligopeptidase A, were identified from their amino acid compositions, their retention times during RPLC, and knowledge of the amino acid sequence of the signal peptide. Both enzymes were endopeptidases, as neither dipeptides nor free amino acids were formed during the cleavage reactions. Protease IV cleaved the signal peptide predominantly in the hydrophobic segment (residues 7 to 14). Protease IV required substrates with hydrophobic amino acids at the primary and the adjacent substrate-binding sites, with a minimum of three amino acids on either side of the scissile bond. Oligopeptidase A cleaved peptides (minimally five residues) that had either alanine or glycine at the P'1 (primary binding site) or at the P1 (preceding P'1) site of the substrate. These results support the hypothesis that protease IV is the major signal peptide peptidase in membranes that initiates the degradation of the signal peptide by making endoproteolytic cuts; oligopeptidase A and other cytoplasmic enzymes further degrade the partially degraded portions of the signal peptide that may be diffused or transported back into the cytoplasm from the membranes.

Analysis of mutant Escherichia coli thymidylate synthases (EC 2.1.1.45) with various amino acids substituted for cysteine at position 146 revealed the cysteine to be involved in the binding of 2'-deoxyuridylate as well as initiating the catalytic process. The substitution of a serine or alanine residue at position 146 did not appreciably alter the binding affinity for 2'-deoxyuridylate but the serine mutant enzyme was less active by a factor of 5000, whereas the alanine mutant enzyme was catalytically inactive. In contrast, the substitution of a glycine or threonine at position 146 created inactive enzymes with higher 2'-deoxyuridylate dissociation constants. The dissociation constant values for 2'-deoxyuridylate were used to estimate the overall contribution of the side chain of the amino acid at position 146 to substrate binding. The results suggested that the side chains of cysteine, alanine, and serine make nonspecific but effective van der Waals contacts with 2'-deoxyuridylate, thereby contributing about 0.82 kcal.mol-1 (1 cal = 4.184 J) to the apparent binding energy of the substrate.