Chicken mim-1 protein, P33, is a heterophil chemotactic factor present in Salmonella enteritidis immune lymphokine.

Lymphokine (ILK) secreted from concanavalin A-stimulated T cells from Salmonella Enteritidis-immune chickens is an undefined mixture of proteins that confers protection against Salmonella infectivity when administered to day-old chicks. It has previously been shown that polyclonal antibodies raised against human granulocyte colony-stimulating factor (GCSF) can neutralize the heterophil activation that is responsible for ILK's protective effect. Western blot analysis of ILK probed with anti-GCSF antibodies detects a prominent protein of mass 33 kDa. We have sequenced the first 20 amino acids of this protein and found it to be identical to residues 24 to 43 of P33, a 326-amino acid protein of unknown function encoded by the chicken mim-1 gene. The primary structure of P33 consists of two 140-residue imperfect repeats that are each homologous to a mammalian neutrophil chemotactic factor termed leukocyte cell-derived chemotaxin 2 (LECT2). We have expressed mim-1 in Escherichia coli and demonstrated in vitro that recombinant P33 is chemotactic for heterophils, the avian equivalent of mammalian neutrophils. We have also constructed a derivative of P33 that consists of residues 33 to 165 (P33[33-165]), the first repeat sequence of P33 that is homologous to LECT2. P33(33-165) is chemotactic for heterophils both in vitro and in vivo, inducing an influx of heterophils into the peritoneum in a response similar to that observed with ILK. These results suggest that P33 functions as a chemotactic factor in chickens and that it plays an active role in ILK-mediated protection against Salmonella infection.

Modulation of functional activities of chicken heterophils by recombinant chicken IFN-gamma.

The objective of the present studies was to examine the in vitro effects of recombinant chicken interferon-gamma (rChIFN-gamma) on shape change, phagocytosis, and the oxidative/nonoxidative killing activities of day-old chicken heterophils. Heterophils (4 x 10(6)/ml) were incubated with various concentrations of recombinant ChIFN-gamma from both Escherichia coli and transfected Cos cells for 2 h at 39 degrees C. The incubation of the neonatal heterophils with rChIFN-gamma resulted in significantly greater numbers of cells with membrane shape change when compared with the mock-treated heterophils. Both Cos cell-derived and E. coli-derived ChIFN-gamma significantly increased (p < 0.01) the phagocytosis of opsonized or nonopsonized Salmonella enteritidis by the neonatal heterophils in a concentration-dependent manner. Incubation with ChIFN-gamma induced no direct stimulation of the respiratory burst by the chicken heterophils but did prime the heterophils for a significantly strengthened respiratory burst to subsequent stimulation with opsonized zymosan (OZ). Lastly, incubation of the heterophils with ChIFN-gamma primed the cells for a significant increase in the release of beta-D-glucuronidase following stimulation with OZ. These results show that neonatal avian heterophils can respond to cytokine modulation with enhanced functional competence, suggesting that ChIFN-gamma can enhance the immune competence of the innate defenses of chickens during the first week of life.

Separation and quantification of two fluoroquinolones in serum by on-line high-performance immunoaffinity chromatography.

To demonstrate that two structurally similar chemicals can be extracted from a complex matrix and then separated from each other on the basis of their relative affinities for an antibody, an automated column-switching system was used, incorporating on-line, high-performance immunoaffinity chromatography (HPIAC). A high-affinity monoclonal antibody (Mab Sara-95) against the fluoroquinolone sarafloxacin was covalently cross-linked to a protein G column and used to capture fluoroquinolones in fortified serum samples. Interference from matrix components adhering nonspecifically to the column was minimized by the insertion of a protein G cleanup column between the injection port and the Mab Sara-95 derivatized HPIAC column. Upon injection, serum samples containing the fluoroquinolones passed through both columns. The cleanup column detained serum components, that otherwise would bind nonspecifically to the HPIAC column, but allowed the fluoroquinolones to pass through unhindered to the HPIAC column. The fluoroquinolones were then eluted from the HPIAC column according to their relative affinities for the antibody, and individual peaks were monitored using fluorescence detection. By using an on-line cleanup column in tandem with an HPIAC column, the fluoroquinolones could be separated from the serum matrix and then separated from each other on the basis of their affinity for Mab Sara-95 without the use of organic solvents or reversed-phase liquid chromatography (RPLC). This method demonstrates true immunoaffinity separation of structurally related compounds in a complex matrix.

The hsp60 gene of the human pathogenic fungus Coccidioides immitis encodes a T-cell reactive protein.

A heat shock protein-encoding gene (hsp60) from the human respiratory fungal pathogen, Coccidioides immitis (Ci), was cloned, sequenced, chromosome-mapped, expressed and immunolocalized in parasitic cells. Both the genomic and cDNA sequences are presented. The transcription start point and poly (A) addition site were confirmed. The hsp60 gene contains two introns and a 1782-bp ORF which translates a 594-amino acid (aa) protein of 62.4 kDa and pI of 5.6. The translated protein revealed two potential N-glycosylation sites. The deduced HSP60 showed 78-83% aa sequence similarity to reported fungal HSP60 proteins. The hsp60 gene was mapped to chromosome III of Ci and was shown to be a single copy gene by Southern and Northern hybridization. Expression of a 1737-bp cDNA fragment of the hsp60 gene in E. coli resulted in production of a recombinant protein. Amino acid sequence analysis of the recombinant protein confirmed that it was encoded by the Ci hsp60 gene. Antiserum raised in mice against the isolated recombinant protein immunolocalized HSP60 in the cytoplasm and wall of parasitic cells of Ci. The recombinant HSP60 was used to immunize BALB/c mice and was shown to induce proliferation of T cells isolated from lymph nodes of these animals. The hsp60 gene of Ci is the first reported heat-shock protein gene of this human pathogen.

Engineered complementation in Escherichia coli aspartate transcarbamoylase. Heterotropic regulation by quaternary structure stabilization.

Escherichia coli aspartate transcarbamoylase regulates pyrimidine biosynthesis by altering its activity homotropically in response to one of its substrates and heterotropically in response to nucleotide effectors. The mechanism of this regulation involves two structurally and functionally different forms of the enzyme, one with low activity and low affinity for substrates (T state) and the other with high activity and high affinity for substrates (R state). Heterotropic regulation may be due to the direct transmission of a regulatory "signal" between the regulatory site and the active site some 60 A away and/or may involve altering the relative stability of the two forms of the enzyme. By combining a T state-stabilized mutant version of the enzyme, previously thought to have a defect in a heterotropic transmission pathway, with a known R state-stabilized mutant enzyme, we were able to restore some properties of the wild-type enzyme. These data imply that the relative stabilization of the T and R states of the enzyme is in part responsible for the homotropic and heterotropic properties of aspartate transcarbamoylase and that direct pathways for transmission of the heterotropic signals are unlikely.

Isolation and characterization of two chitinase-encoding genes (cts1, cts2) from the fungus Coccidioides immitis.

Two chitinase (CTS)-encoding genes (cts) from Coccidioides immitis (Ci), a respiratory fungal pathogen of humans, were cloned and sequenced. Both the genomic and cDNA sequences are presented. The transcription start points and poly(A)-addition sites were confirmed. The cts1 gene contains five introns and a 1281-bp ORF which translates a 427-amino-acid (aa) protein of 47.4 kDa. The cts2 gene contains two introns and a 2580-bp ORF which translates a 860-aa protein of 91.4 kDa. The deduced CTS1 protein showed highest homology to the Aphanocladium album and Trichoderma harzianum CTS (74% and 76%, respectively), while CTS2 showed highest homology to the CTS of Saccharomyces cerevisiae (Sc) and Candida albicans (47% and 51%, respectively). The putative N-terminal sequence of the mature CTS1 protein also showed 89% homology to the reported N-terminal sequence of a 48-kDa complement fixation antigen (CF-Ag) of Ci which has demonstrated chitinase activity. The CF-Ag is a clinically important antigen used in serodiagnosis of this fungal disease. CTS2 showed several of the conserved features of the Sc CTS, including putative catalytic and Ser/Thr-rich domains, and a C-terminal Cys-rich region. We propose that CTS1 and CTS2 of Ci are members of two distinct classes of fungal chitinases, an observation not previously reported for a single fungus.

Cloning and expression of a gene encoding a T-cell reactive protein from Coccidioides immitis: homology to 4-hydroxyphenylpyruvate dioxygenase and the mammalian F antigen.

The gene which encodes a previously described T-cell reactive protein (TCRP) of the human fungal pathogen Coccidioides immitis (Ci) was cloned and sequenced. Both the genomic and cDNA sequences were determined. The transcription start point was confirmed. The tcrP gene has three introns and a 1197-bp ORF which translates to a 399-amino-acid (aa) protein (45.2 kDa). The predicted protein has approx. 50% aa sequence identity and 70% similarity to mammalian 4-hydroxyphenylpyruvate dioxygenase (HPPD) proteins and mammalian F-antigens. Expression of the Ci tcrP in Escherichia coli resulted in production of a deep brown pigment, consistent with E. coli expression of the bacterial HPPD homolog from Shewanella colwelliana. The TCRP is likely the Ci form of HPPD.

Site-directed mutagenesis of intersubunit boundary residues in histidine decarboxylase, a pH-dependent allosteric enzyme.

Histidine decarboxylase (HDC) from Lactobacillus 30a forms a trimer around a central cavity or well. Three active sites are formed around the well at the interface of each of two adjacent molecules. HDC exhibits cooperative kinetics at pH 7.6 and can be described in terms of a two-state, T and R, model. At pH 4.8, protons stabilize HDC in the R form. Asp 198 and Asp 53, from a neighboring molecule, are the core of the pH-sensitive mechanism controlling the shift in quaternary state. Eight site-directed mutations have been made to analyze the region. Several mutants, including the conversion of Asp 53 to Asn, cause HDC to exhibit sigmoidal kinetics even at pH 4.8. Others lock the enzyme into the T state. Kinetic analysis suggests that kcat values for T and R states are similar. The Km for the T state, near 8 mM, exceeds that for the R state by 40-fold and shows HDC is primarily regulated by altering its affinity for substrate.

Site-directed alteration of three active-site residues of a pyruvoyl-dependent histidine decarboxylase.

The active site of histidine decarboxylase (HDC) from Lactobacillus 30a contains a pyruvoyl cofactor sitting at the interface of two molecules in a trimer. Although exhibiting hyperbolic kinetics at pH 4.8, near its optimum, HDC is cooperative at pH 7.6, indicating that the units of the trimer communicate. A Hill plot analysis shows that HDC, at pH 7.6, can be described by a two-state model. The tense (T) state has an apparent Km for histidine of 50 mM, while the relaxed (R) state has a Km of 5 mM. To explore the catalytic mechanism, three of the cross-boundary active-site residues were altered by site-directed mutagenesis and their effects observed. Ile-59 is known to act as lid on the substrate binding pocket; it was converted to Ala (I59A) and to Val (I59V). The former was inactive, attesting to the importance of this residue in the mechanism. The I59V mutant showed a decrease in Km and in kcat at pHs 4.8 and 7.6. Ile-59 appears to help orient substrate properly for catalysis; decreasing its size expands the binding site. This may allow the substrate to bind more readily, but in a number of conformations which are not optimal for catalysis. Conversion of Tyr-62 to Phe (Y62F) had no effect on catalysis but raised the Km 7-fold at pH 4.8. Asp-63 appears to form an ion pair to the substrate imidazolium. Conversion to the neutral amide (D63N) had no effect on the kcat, but raised the Km 240-fold at pH 4.8. This is consistent with the notion that the ion pair is up to 3 kcal/mol stronger than a simple hydrogen bond with the substrate. The mutant had no detectable activity at pH 7.6.