Proteomic analysis of Rickettsia prowazekii.

Rickettsia prowazekii is an obligate intracellular gram-negative bacterium. Comparative proteomics study of a virulent strain (Breinl) versus an avirulent strain (Madrid E) was performed using an integrated liquid chromatography and mass spectrometer. About 30% of predicted proteins were detected and identified. Among the detected proteins, more than 30 proteins were of unknown function in both strains. Although several proteins were detected in only one strain, the overall distribution of detected proteins in different COGs (clusters of orthologs groups) was very similar between the two strains. Functional analysis of differentially expressed proteins, either qualitatively or quantitatively, may lead to the discovery of pathogenesis-related factors.

Expression, purification and characterization of the structure and disulfide linkages of insulin-like growth factor binding protein-4.

Insulin-like growth factor binding protein-4 (IGFBP-4), like the other five IGFBPs, is a critical regulator of the activity of insulin-like growth factor (IGF)-I and IGF-II. However IGFBP-4 seems to be the only IGFBP with no potential to enhance the mitogenic actions of the IGFs. IGFBP-1 to -3 and -5 each contain 18 conserved cysteine residues, IGFBP-6 lacks two of the twelve N-terminal cysteines, while IGFBP-4 has two additional cysteines in the central region. A plasmid was constructed to express rat IGFBP-4 as a thioredoxin fusion protein that included a hexahistidine sequence to permit affinity purification. The fusion protein was expressed in E.coli, purified using nickel-chelate affinity chromatography and cleaved by tobacco etch virus (TEV) protease to produce mature rat IGFBP-4 with an additional glycine residue at the N-terminus. Final purification was achieved by further nickel affinity chromatography and reverse phase HPLC. The isoelectric points of the recombinant IGFBP-4 were the same as those of the non-glycosylated isoforms of IGFBP-4 in rat serum. The binding affinities of the recombinant protein and IGFBP-4 secreted by rat cells to IGF-I were compared using a newly developed binding assay. No significant difference could be detected, consistent with proper folding of the recombinant protein. This indicates that glycosylation of IGFBP-4 does not affect its binding to IGF-I. Using mass spectrometry and tandem mass spectrometry no differences between authentic and recombinant IGFBP-4 could be detected. Eight of the ten disulfide linkages have been determined, including linkages of conserved cysteine residues not previously identified in other IGFBPs. Numbering the cysteine residues sequentially from the N-terminus only the disulfide connectivity of C1, C2, C5 and C6 could not be determined. However, C1 is not linked to C1 and C5 is not linked to C6. The established linkages were C3 to C8, C4 to C7, C9 to C 11, and C10 to C12. The two cysteines in the non-conserved mid-region unique to IGFBP-4 (C13 and C14) are linked together. Linkage of the C-terminal cysteine residues is identical to that of IGFBP-2, -5 and -6 (C15 to C16, C17 to C18 and C19 to C20). The central flexible core of IGFBP-4, containing two additional cysteines may contribute to its unique biological action.

A novel approach to reducing postoperative intraperitoneal adhesions through the inhibition of insulinlike growth factor I activity.

HYPOTHESIS: Interference with insulinlike growth factor I (IGF-I) activity, both systemically and intraperitoneally, reduces postoperative intraperitoneal adhesion severity. SETTING: Experimental animal model. DESIGN, INTERVENTIONS, AND MAIN OUTCOME MEASURES: Adult female rats were subjected to hypophysectomy, sham hypophysectomy (control), IGF binding protein 4 (IGFBP-4) treatment, or albumin treatment (control). All rats underwent laparotomy and uterine horn abrasion with adjacent parietal peritoneal trauma for the purpose of creating postoperative intraperitoneal adhesions. Glucocorticoids and thyroid hormone were replaced in the hypophysectomy group. On postoperative day 10, rats were weighed, subjected to phlebotomy, and killed. Postmortem laparotomies were performed and blinded observers scored uterine-peritoneal adhesions on a 0 to 3 scoring system. Plasma IGFBP-4 levels and organ weights were measured in the IGFBP-4 and albumin treatment groups. Blood samples in all rats were analyzed for IGF-I levels. RESULTS: Rats with low IGF-I levels (hypophysectomy) and inhibited IGF-I activity (IGFBP-4 treatment) formed significantly less severe adhesions than their control counterparts. As expected, rats in the hypophysectomy group displayed greater weight loss and lower plasma IGF-I levels than sham-treated rats. Rats treated with IGFBP-4 and those treated with albumin demonstrated no differences in body weight, organ weights, IGF-I levels, and IGFBP-4 levels. CONCLUSIONS: Both the reduction of systemic IGF-I levels via hypophysectomy and the inhibition of local intraperitoneal IGF-I activity via IGFBP-4 treatment resulted in diminished postoperative adhesion severity. Treatment with IGFBP-4 may play a role in postoperative adhesion prophylaxis in the future.

A new radioimmunoassay to measure rat insulin-like growth factor binding protein-4 (IGFBP-4) in serum, wound fluid and conditioned media.

Insulin-like growth factor binding protein-4 (IGFBP-4) is, like the other five IGFBPs, a critical regulator of the activity of insulin-like growth factor-I (IGF-I) and IGF-II. Because of the possible role of IGFBP-4 in multiple systems including reproduction, pregnancy, bone formation and wound healing, we developed a radioimmunoassay (RIA) to measure the concentration of IGFBP-4 in the serum, extracellular fluid and conditioned media of cells of rodents so that these animal models could be better exploited. Rat IGFBP-4 was expressed in Escherichia coli and purified to homogeneity. The recombinant material was used to raise a rabbit polyclonal antibody against rat IGFBP-4 and for iodination and standards. The RIA developed was sensitive to less than 1 ng/mL of rat IGFBP-4 and IGF-I did not interfere. There was no cross-reactivity with other rat IGFBPs on immuno-Western analysis of serum and wound fluid. IGFBP-4 from mouse serum did cross-react in our assay; however, serum from horse, pig or human showed no cross-reaction. Human IGFBP-1 and IGFBP-3 showed a very weak cross-reactivity. Serum IGFBP-4 levels showed no gender difference but did reveal a significant 66% increase in older rats. During the course of wound healing, which is IGF-I dependent, IGFBP-4 showed no changes. In conclusion an RIA for rat IGFBP-4 has been developed that specifically measures the concentration of IGFBP-4 in the sera, extracellular fluid and conditioned media of rodents. With this assay, the role of IGFBP-4 and IGFs in growth, development and the function of multiple systems can be further investigated using rodent models.

Characterization of the enzymatic specificity of the IGF-dependent insulin-like growth factor binding protein-4 (IGFBP-4) protease.

The insulin-like growth factor (IGF)-dependent IGF binding protein-4 protease secreted by cultured adult human fibroblasts was recently identified as pregnancy-associated plasma protein-A (PAPP-A). In this study we showed that in addition to human IGFBP-4 the IGF-dependent IGFBP-4 protease also digests recombinant rat IGFBP-4 into two fragments by specifically cleaving at the carboxyl-terminal side of methionine at position 131 for rat IGFBP-4. Thus the cleavage site is at the KMKV site, which is not represented in other IGFBPs. While kallikrein may cleave at this site, its action is not specific.

Phosphatidylcholine activation of human heart (R)-3-hydroxybutyrate dehydrogenase mutants lacking active center sulfhydryls: site-directed mutagenesis of a new recombinant fusion protein.

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme with a specific requirement of phosphatidylcholine (PC) for function. A plasmid has been constructed to express human heart (HH) BDH in Escherichia coli as a hexahistidine-tagged fusion protein (HH-Histag-BDH). A rapid two-step affinity purification yields active HH-Histag-BDH (and six mutants) with high specific activity ( approximately 130 micromol of NAD(+) reduced.min(-1).mg(-1)). HH-Histag-BDH has no activity in the absence of phospholipid and exhibits a specific requirement of PC for function. The HH-Histag-BDH-PC complex (and HH-BDH derived therefrom by enterokinase cleavage) has apparent Michaelis constants (K(m) values) for NAD(+), NADH, (R)-3-hydroxybutyrate (HOB), and acetoacetate (AcAc) similar to those for bovine heart or rat liver BDH. A computed structural model of HH-BDH predicts the two active center sulfhydryls to be C69 (near the adenosine moiety of NAD) and C242. With both sulfhydryls derivatized, BDH has minimal activity, but site-directed mutagenesis of C69 and/or C242 now shows that neither of these cysteines is required for PC activation or catalysis (the double mutant, C69A/C242A, is highly active with essentially normal kinetic parameters). Six cysteine mutants each have an increased K(m)(NADH) (2-6-fold) but an unchanged K(m)(NAD)+. The C242S and C69A/C242S enzymes (but not the analogous C242A mutants nor the C69A or C69S mutants) exhibit approximately 10-fold increases in K(m)(HOB) and K(m)(AcAc), reflecting an altered substrate binding site. Thus, although C242 (in the C-terminal lipid binding domain of BDH) is close to the active site, it appears to be in a hydrophobic environment and only indirectly defines the substrate binding site at the catalytic center of BDH.