Effects of phthalate esters on dendritic cell subsets and interleukin-4 production in fluorescein isothiocyanate-induced contact hypersensitivity

Phthalate esters with short alkyl chains, such as di-ethyl (DEP), di-n-propyl (DPP), and di-butyl phthalate (DBP), have adjuvant effects on an FITC-induced contact hypersensitivity mouse model. The adjuvant effects of DPP and DBP are associated with enhanced trafficking of FITC-presenting CD11b(+) dendritic cells (DC). DEP has relatively weak activity as to FITC-positive cell migration. Here we demonstrated that DBP and DPP also increased the number of FITC-positive CD8alpha(+) DC in draining lymph nodes. We also found enhanced production of interleukin-4 in draining lymph nodes after FITC sensitization with DEP, DPP, or DBP, suggesting an additional adjuvant mechanism of phthalate esters.     

Overproduction, purification, and ATPase activity of the Escherichia coli RuvB protein involved in DNA repair.

The ruvA and ruvB genes of Escherichia coli constitute an operon which belongs to the SOS regulon. Genetic evidence suggests that the products of the ruv operon are involved in DNA repair and recombination. To begin biochemical characterization of these proteins, we developed a plasmid system that overproduced RuvB protein to 20% of total cell protein. Starting from the overproducing system, we purified RuvB protein. The purified RuvB protein behaved like a monomer in gel filtration chromatography and had an apparent relative molecular mass of 38 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the value predicted from the DNA sequence. The amino acid sequence of the amino-terminal region of the purified protein was analyzed, and the sequence agreed with the one deduced from the DNA sequence. Since the deduced sequence of RuvB protein contained the consensus sequence for ATP-binding proteins, we examined the ATP-binding and ATPase activities of the purified RuvB protein. RuvB protein had a stronger affinity to ADP than to ATP and weak ATPase activity. The results suggest that the weak ATPase activity of RuvB protein is at least partly due to end product inhibition by ADP.     

Comparison of three microscopic techniques for diagnosis of Cyclospora cayetanensis

Cyclospora cayetanensis oocysts in the feces of humans from Kathmandu, Nepal were identified on the basis of their size and other morphological characteristics. We compared the detection of C. cayetanensis oocysts in the feces using three microscopic techniques such as formalin-ether sedimentation, sucrose centrifugal floatation, and direct smear. Standard procedures were used for the formalin-ether sedimentation and the sucrose centrifugal floatation techniques using 0.5 g of feces, however, the direct smear technique was performed using 10 microl of fecal suspension (0.005 g of feces) and observed under the fluorescent microscope. Of the 403 samples examined, 21 samples were positive for oocysts by all three techniques. Therefore, in these 21 samples, the number of oocysts recovered by the three techniques were compared. The highest number of oocyst was obtained by the sucrose centrifugal floatation technique. In contrast, the formalin-ether sedimentation technique was found to be the least reliable concentration technique for the detection of Cyclospora in human feces. Surprisingly, the direct smear technique was found to be an effective and rapid technique for diagnosis of C. cayetanensis making it a technique of choice for routine epidemiological investigation of the prevalence of this infection in human populations.     

Structural and mechanistic insights into homocysteine degradation by a mutant of methionine γ‐lyase based on substrate‐assisted catalysis

Methionine γ‐lyse (MGL) catalyzes the α, γ‐elimination of l ‐methionine and its derivatives as well as the α, β‐elimination of l ‐cysteine and its derivatives to produce α‐keto acids, volatile thiols, and ammonia. The reaction mechanism of MGL has been characterized by enzymological studies using several site‐directed mutants. The Pseudomonas putida MGL C116H mutant showed drastically reduced degradation activity toward methionine while retaining activity toward homocysteine. To understand the underlying mechanism and to discern the subtle differences between these substrates, we analyzed the crystal structures of the reaction intermediates. The complex formed between the C116H mutant and methionine demonstrated that a loop structure (Ala51–Asn64) in the adjacent subunit of the catalytic dimer cannot approach the cofactor pyridoxal 5′‐phosphate (PLP) because His116 disrupts the interaction of Asp241 with Lys240, and the liberated side chain of Lys240 causes steric hindrance with this loop. Conversely, in the complex formed between C116H mutant and homocysteine, the thiol moiety of the substrate conjugated with PLP offsets the imidazole ring of His116 via a water molecule, disrupting the interaction of His116 and Asp241 and restoring the interaction of Asp241 with Lys240. These structural data suggest that the Cys116 to His mutation renders the enzyme inactive toward the original substrate, but activity is restored when the substrate is homocysteine due to substrate‐assisted catalysis.     

On the role of periodism in the origin of proteins

Two different views have been proposed for origins of genes (or proteins). One is that primordial genes evolved from random sequences. This view underlies the concept of modern in vitro evolution experiments that functional molecules (even proteins) evolved from random sequence-libraries. On the contrary, the second view reminds that "random sequences" would be an unusual state in which to find RNA or DNA, because it is their inherent nature to yield periodic structures during the course of semi-conservative replication. In this second view, the periodicity of DNA (or RNA) is responsible for emergence of primordial genes. Although recent reports on the variety of periodicities present in proteins, genes and genomes are consistent with the second view, it has yet to be experimentally tested. We assessed the significance of periodicities of DNA in the origin of genes by constructing such periodic DNAs. The results showed that periodic DNA produced ordered proteins at very high rates, which is in contrast to the fact that proteins with random sequences lack secondary structures. We concluded that periodicity played a pivotal role in the origin of many genes. The observation should pave the way for new experimental evolution systems for proteins.     

Structural specificity of synthetic peptide adjuvant for induction of experimental allergic encephalomyelitis

The peptide N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), which has adjuvant activities, and 17 of its derivatives and analogs were synthesized and assayed to elucidate the structure necessary for adjuvant activity in induction of experimental allergic encephalomyelitis (EAE) in guinea pigs. The results revealed the importance of the d configuration and the α-carboxamide group of the isoglutaminyl residue of MDP for adjuvant activity. Replacement of the l-alanyl residue of MDP by d-alanine, but not by l-serine or glycine, resulted in a marked decrease in the activity. The β-methyl glycoside of MDP was found to be more active than the α-methyl derivative. 6-O-Stearoyl-N-acetylmuramyl-l-alanyl-d-isoglutamme showed activity.     

Pharmacophore Modeling for Anti-Chagas Drug Design Using the Fragment Molecular Orbital Method

BackgroundChagas disease, caused by the parasite Trypanosoma cruzi, is a neglected tropical disease that causes severe human health problems. To develop a new chemotherapeutic agent for the treatment of Chagas disease, we predicted a pharmacophore model for T. cruzi dihydroorotate dehydrogenase (TcDHODH) by fragment molecular orbital (FMO) calculation for orotate, oxonate, and 43 orotate derivatives.Conclusions/SignificanceFMO-based interaction energy analyses revealed a pharmacophore model for TcDHODH inhibitor. Hydrogen bond acceptor pharmacophores correspond to Lys43 and Lys214, hydrogen bond donor and acceptor pharmacophores correspond to Asn67 and Asn194, and the aromatic ring pharmacophore corresponds to FMN, which shows important characteristics of compounds that inhibit TcDHODH. In addition, the Lys214 residue is not conserved between TcDHODH and human DHODH. Our analysis suggests that these orotate derivatives should preferentially bind to TcDHODH, increasing their selectivity. Our results obtained by pharmacophore modeling provides insight into the structural requirements for the design of TcDHODH inhibitors and their development as new anti-Chagas drugs.     

Altered translation initiation factor 2 in the cold-sensitive ssyG mutant affects protein export in Escherichia coli.

The Escherichia coli gene secY (pr1A) codes for an integral membrane protein that plays an essential role in protein export. We previously isolated cold-sensitive mutations (ssy) as extragenic suppressors of temperature-sensitive secY24 mutation. Now we show that the ssyG class of mutations are within infB coding for the translation initiation factor IF2. The mutants produce altered forms of IF2 with a cold-sensitive in vitro activity to form a translation initiation complex. The mutation suppresses not only secY24 but also other secretion-defective mutations such as secA51 and rp10215. The beta-galactosidase enzyme activity of the MalE-LacZ 72-47 hybrid protein is strikingly reduced in the ssyG mutant at the permissive high temperature, while the hybrid protein itself is normally synthesized. This effect, which was observed only for the hybrid protein with a functional signal sequence, may result from some alteration in the cellular localization of the protein. These results suggest that IF2 or the translation initiation step can modulate protein export reactions. The isolation of cold-sensitive ssyG mutations in infB provides genetic evidence that IF2 is indeed essential for normal growth of E. coli cells.     

Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure.

Genetic evidence suggests that the Escherichia coli ruvC gene is involved in DNA repair and in the late step of RecE and RecF pathway recombination. To study the biochemical properties of RuvC protein, we overproduced and highly purified the protein. By employing model substrates, we examined the possibility that RuvC protein is an endonuclease that resolves the Holliday structure, an intermediate in genetic recombination in which two double-stranded DNA molecules are linked by single-stranded crossover. RuvC protein cleaves cruciform junctions, which are formed by the extrusion of inverted repeat sequences from a supercoiled plasmid and which are structurally analogous to Holliday junctions, by introducing nicks into strands with the same polarity. The nicked ends are ligated by E.coli or T4 DNA ligases. Analysis of the cleavage sites suggests that DNA topology rather than a particular sequence determines the cleavage site. RuvC protein also cleaves Holliday junctions which are formed between gapped circular and linear duplex DNA by the function of RecA protein. However, it does not cleave a synthetic four-way junction that does not possess homology between arms. The active form of RuvC protein, as studied by gel filtration, is a dimer. This is mechanistically suited for an endonuclease involved in swapping DNA strands at the crossover junctions. From these properties of RuvC protein and the phenotypes of the ruvC mutants, we infer that RuvC protein is an endonuclease that resolves Holliday structures in vivo.