Ligand binding by recombinant domains from insect ecdysone receptors.

The ligand binding domains (LBDs) from the EcR and USP proteins of four insect pests (Lucilia cuprina, Myzus persicae, Bemisia tabaci, Helicoverpa armigera) were purified as recombinant heterodimers. The K(d) values for [(3)H]-ponasterone A binding by LBD heterodimers that included the hinge regions (i.e., DE/F heterodimers) ranged 0.7-2.5 nM, with K(i) values for ecdysteroid and dibenzoylhydrazine ligands ranging from 0.1 nM to >448 microM. The K(d) and K(i) values for a recombinant H. armigera LBD heterodimer that lacked D-regions (i.e., an E/F heterodimer) were approximately 4 times higher than those for its DE/F counterpart. Rate constants were estimated for the L. cuprina LBD heterodimer. A fluorescein-inokosterone conjugate (K(i)~40 nM) was used to develop a novel binding assay based on fluorescence polarization. This assay, which ranked the affinity of competitor ecdysteroids in the same order as the [(3)H]-ponasterone A binding assay, is well suited to high-throughput screening. Ponasterone A had a higher affinity than muristerone A for the recombinant hemipteran LBD heterodimers, whereas the reverse was true for the recombinant dipteran one. The same preference was observed when these ligands were tested as inducers of ecdysone receptor-controlled gene expression in transfected mammalian cells. The binding data obtained in vitro using recombinant LBD heterodimers reflects the ability of agonists to induce transgene expression in recombinant mammalian cells, and can also reflect their efficacy as larvicides.

Linkers for improved cleavage of fusion proteins with an engineered alpha-lytic protease.

Addition of an N-terminal fusion partner can greatly aid the expression and purification of a recombinant protein in Escherichia coli. We investigated two genetically engineered proteases designed to remove the fusion partner after the protein of interest has been expressed. Recombinant human insulin-like growth factor-II (hIGF-II) has been produced from E. coli-derived fusion proteins using a novel enzymatic cleavage system that uses a mutant of alpha-lytic protease. Initially, two potential fusion protein linkers were designed, Pro-Ala-Pro-His (PAPH) and Pro-Ala-Pro-Met (PAPM), and were tested as substrates in the form of synthetic dodecapeptides. Using mass spectrometry and reverse-phase HPLC, the position of cleavage was confirmed and the kinetics of synthetic peptide cleavage were examined. Use of the linkers in hIGF-II fusion proteins produced in E. coli was then evaluated. The fusion proteins constructed consist of the first 11 amino acids of porcine growth hormone linked N-terminally to hIGF-II by six amino acids that include the dipeptide Val-Asn followed by a variable tetrapeptide protease cleavage motif. Mass spectrometry and N-terminal sequencing confirmed that proteolytic cleavage of the fusion proteins had occurred at the predicted sites. Using the fusion proteins as substrates, the cleavage of the rationally designed motifs by the alpha-lytic protease mutant was compared. The fusion protein containing the motif PAPM had a k(cat)/K(M) ratio indicating a 1.6-fold preference over the PAPH fusion protein for cleavage by this enzyme. Furthermore, when hIGF-II fusion proteins containing the designed cleavable linkers were processed with the engineered alpha-lytic protease, they gave greatly improved yields of native hIGF-II compared to an analogous fusion protein cleaved by H64A subtilisin. Comparison of the peptide and protein cleavage studies shows that the efficient proteolysis of the cleavage motifs is an inherent property of the designed sequences and is not determined by secondary or tertiary structure in the fusion proteins.

Combinatorial strategies for the discovery of novel protease specificities.

This article discusses proven and possible ways to generate novel cleavage specificities in serine proteases using combinatorial mutagenesis, compares the different ways of screening or selecting for desirable mutants, and examines the ways in which combinatorial substrate libraries can be used to gain a more comprehensive insight into protease cleavage preferences. The use of bacteriophage to display both combinatorial protease libraries and combinatorial substrate libraries will be discussed.

Human perlecan immunopurified from different endothelial cell sources has different adhesive properties for vascular cells.

Perlecan, a major heparan sulfate proteoglycan of vascularized tissues, was immunopurified from media conditioned by human endothelial cells of both arterial and venous origin. The heparan sulfate moiety of perlecan from cultured arterial cells differed in amount and/or composition from that produced by a transformed cell line of venous origin. Both forms of perlecan bound basic fibroblast growth factor with Kd approximately 70 nM. In ELISA experiments, perlecan and its protein core bound to various extracellular matrix components in a manner that was strongly influenced by the format of the assay. Human vascular smooth muscle cells and human endothelial cells adhered to perlecan-coated surfaces, and both cell types adhered better to the venous cell-derived than to the arterial cell-derived perlecan. Removal of the heparan sulfate chains abolished this difference and increased the ability of both types of perlecan to adhere vascular cells. Denaturation of perlecan and its protein core also rendered each of them more adhesive, indicating the presence of conformation-independent adhesion determinants in the polypeptide sequence. Their location was investigated using recombinant perlecan domains. Overall, our results represent the first demonstration of human perlecan acting as an adhesive molecule for human vascular cells and suggest that it may play a role in vascular wound healing.

Expression of human perlecan domain I as a recombinant heparan sulfate proteoglycan with 20-kDa glycosaminoglycan chains.

Recombinant forms of human perlecan domain I were secreted as proteoglycans by stably transfected human 293 cells. A recombinant domain I-only proteoglycan spanned the 95- to 265-kDa region in SDS-PAGE and appeared to be 160 kDa in denaturing gel filtration. Its glycosaminoglycan (GAG) content was approximately 67% heparan sulfate, and its average GAG chain size of 20 kDa suggested that the true molecular mass of the proteoglycan was 90 kDa. Domain I with enhanced green fluorescent protein fused to its C-terminus had an apparent molecular mass of 210-220 kDa and contained approximately 100% heparan sulfate. Its average GAG chain size (also 20 kDa) suggested a true molecular mass of 117 kDa for this proteoglycan. Its sulfate content of 53-77 mol SO2-4 per mole of protein indicated the presence of one sulfate group per 4-7 GAG sugar residues.

Cloning of a Lysobacter enzymogenes gene that encodes an arginyl endopeptidase (endoproteinase Arg-C).

Screening an expression library of Lysobacter enzymogenes DNA allowed us to clone a gene encoding a serine protease that cleaves synthetic substrates C-terminal to Arg and, to a lesser extent, Lys residues. The gene product, which shares sequence homology with the lysyl endopeptidases from L. enzymogenes and Achromobacter lyticus, consists of a signal sequence (24 residues), pro-region ( approximately 195 residues), and catalytic domain ( approximately 244 residues). Downstream of this gene is an open reading frame that lacks a promoter and appears to encode an inactive type I subtilase.

Inhibition of phenotype modulation, growth, and migration of vascular smooth muscle cells by a guanosine-rich 30-mer phosphorothioate oligodeoxynucleotide.

The testing of a 30-mer dG-rich phosphorothioate oligodeoxynucleotide (LG4PS) for effects on the behaviour of vascular smooth muscle cells (VSMC) in vitro and in vivo is described. LG4PS at 0.3 microM inhibited significantly the phenotype modulation of freshly isolated rabbit VSMC, and cell outgrowth from pig aortic explants was inhibited approximately 80% by 5 microM LG4PS. The growth of proliferating rabbit and pig VSMC was inhibited approximately 70% by 0.3 microM and 5 microM LG4PS, respectively. Though less marked, the antiproliferative effects of LG4PS on human VSMC were comparable to those obtained with heparin. The cytotoxic effects of LG4PS on VSMC in vitro were low. Despite these promising results, adventitial application of 2-200 nmol LG4PS in pluronic gel failed to reduce vascular hyperplasia in balloon-injured rabbit carotid arteries, and the highest dose caused extensive mortality.

Comparison of the heparanase enzymes from mouse melanoma cells, mouse macrophages, and human platelets.

The intracellular heparanases from mouse macrophage and melanoma cells are very similar in terms of their size (60-80 kDa), pI (5.3-4.1), pH optimum (< or = 5.5), and interactions with heparin. These proteins are therefore likely to be identical, suggesting that tumour and blood cells utilise the same heparanase enzyme. The human platelet enzyme is similar to the mouse enzymes in terms of pH optimum (< or = 5.5) and pI value (5.3-4.8), but appears to be smaller in size (40-60 kDa). It also seems to differ from the mouse enzymes in aspects of its surface charge, and in its interactions with heparin. There was no indication that proteolysis was of significance for the enzymes, nor that they contained any sialic acid residues.

Activation of platelet heparitinase by vascular cell lysates.

Tumour cells have been reported to contain an activator of platelet heparitinase which is absent from normal cells. Using an assay which measures the degradation of radiolabelled heparin, we observed that lysates of metastatic melanoma cells did activate platelet radiolabelled heparin, we observed that lysates of metastatic melanoma cells did activate platelet heparitinase but that lysates of arterial endothelial and smooth muscle cells did likewise, with the latter being particularly effective. The activator largely survived a 10 min preincubation of the cell lysates at 70 degrees C, but not at 100 degrees C. Experimental results indicated that the contents of 10(5) vascular smooth muscle cells could increase platelet heparitinase activity in vitro to 6 times its initial value. We suggest such activation may have physiological relevance and may even assist the development of certain cardiovascular diseases in man.

Inhibition of platelet heparitinase by phosphorothioate DNA oligonucleotides.

In view of the role that heparitinase and heparanase enzymes are thought to play in regulating the proliferation of smooth muscle cells, inhibitors of these enzymes may have therapeutic value in the treatment of vascular hyperplasia. Here we report that phosphorothioate oligodeoxynucleotides inhibit platelet heparitinase and related enzymes in vitro. The inhibition is greatly enhanced by the presence of a GGG motif in the oligonucleotide, and also increases with oligonucleotide for two phosphorothioate DNA 30-mers consisting solely of guanosine and thymidine nucleotides. Their inhibitory efficacy was greater when heparan sulphate was used as substrate.

Metastatic papillary thyroid carcinoma presenting as a primary renal neoplasm.

Clinically detectable thyroid cancer metastatic to the kidney is rare, with only six cases reported in the medical literature. Four of these have been follicular carcinoma, one papillary carcinoma, and one described as a thyroid adenoma. All of these had known thyroid neoplasms prior to development of their renal metastases. We report herein a seventh case of thyroid carcinoma metastatic to the kidney, unique in that the diagnosis of the kidney metastasis preceded the knowledge of the primary thyroid neoplasm. Furthermore, the follicular variant of papillary cancer, present in this case, has not been previously described in renal metastases from thyroid cancer. Treatment of the kidney metastases led to the subsequent discovery and treatment of the primary thyroid cancer. The patient underwent nephrectomy followed by total thyroidectomy, and is alive and disease-free 3 years postoperatively. Thyroid cancer metastatic to the kidney is rare clinically, but can be amenable to treatment with good long term results.

Purification and characterization of S1 mutants of alpha-lytic protease having altered catalytic properties.

A procedure is described for purifying alpha-lytic protease and its mutants from culture supernatants of recombinant Escherichia coli. The method affords substantial amounts (approx. 80 mg) of homogeneous enzyme. We compared the cleavage preferences of wild-type alpha-lytic protease and of mutants containing the substitutions Ala190 ("parent"), Ala190/Val192/His213/Met218 (mutant 1), Ala190/His213/Leu218 (mutant 9), and Ala190/Thr213/Leu218 (mutant 55), and for each enzyme we found broad agreement between the results obtained with synthetic ester and amide substrates. Kinetic constants were determined for the purified enzymes using selected tetrapeptide p-nitroanilide substrates. Mutant 55 had broad specificity and high activity. In terms of kcat/Km it cleaved at Met and Phe residues two to three times as effectively as the Ala190 enzyme and cleaved at Ala 7 times more effectively than the wild-type protease. The Ala190/His213 enzymes showed a preference for cleavage at His and Met residues. Not only were their kcat values for cleavage at His increased (in relation to the Ala190 enzyme) by an order of magnitude, but they also exhibited large decreases in kcat/Km for cleavage at other residues; for example, the value for cleavage at Phe was 400- to 600-fold lower. Mutant 9 cleaved a recombinant IGF-II fusion protein at a unique His residue and also at a nearby Asn residue.

Cost-effectiveness of preoperative localization studies in primary hyperparathyroid disease.

OBJECTIVE: To evaluate the effect of preoperative localization studies on the surgical management of patients with primary hyperparathyroid disease (PHPT). SUMMARY BACKGROUND DATA: Reported cure rates of initial surgical exploration for PHPT are close to 95%. Preoperative localization studies are frequently obtained to improve surgical success and decrease operative time. METHODS: Initial cervical exploration was performed in 113 patients with PHPT from 1981 to 1993. Twenty-four patients (21%) had surgery without preoperative localization studies. The remaining 89 patients (79%) had 132 noninvasive preoperative localization studies. Success of the localization studies in tumor localization, pathologic findings, postoperative serum calcium levels, and operative times were compared. Patient costs of the studies were calculated. RESULTS: Disease was identified during operation in 23 of 24 patients (96%) having cervical exploration without preoperative localization studies, and they had normal calcium levels after surgery. Eighty-seven of 89 patients (98%) having preoperative localization studies were surgically cured. The highest sensitivity rate (60%) and highest positive predictive value (79%) of the localization studies were found with thallium-technetium scintiscanning. Average cost of the localization studies was $901 per patient. Combination studies were obtained in 32 patients at an average cost of $1,314 per patient without improving sensitivity. Mean operating time did not differ for localized and nonlocalized patients. CONCLUSIONS: Preoperative localization studies did not improve parathyroid localization or cure rate and did not substantially shorten operating time in initial cervical exploration for PHPT. The economic burden of routine preoperative localization studies in these patients is not justified.

A new library of alpha-lytic protease S1 mutants generated by combinatorial random substitution.

Four positions in the S1 site of alpha-lytic protease (positions 190, 192, 213 and 218) were subjected to targeted random mutagenesis. In the resulting library we found active mutant proteases whose cleavage preferences could be grouped into three distinct families. Some potentially useful enzymes (such as one that prefers to cleave at Asn and Cys residues) were identified. In addition, we discuss instances where it was possible to relate changes in substrate specificity to alterations in the structure of the substrate binding site.

Calciphylaxis: early recognition and management.

Calciphylaxis, a syndrome of disseminated calcification found in chronic renal failure patients with secondary hyperparathyroidism, results in soft tissue calcification and vascular medial calcinosis leading to subsequent ischemic tissue necrosis. It is a rarely occurring condition in which patients present with painful, violaceous, mottled lesions of the extremities and/or trunk that progress to skin and subcutaneous tissue necrosis, non-healing ulcers, and gangrene. We reviewed the clinical course of seven patients (aged 24-69) with calciphylaxis treated at our institution over a 4-year period (October 1988-June 1992). All seven patients underwent parathyroidectomy, with a mean time of 8 weeks (range 3-20 weeks) between the onset of calciphylactic symptoms and parathyroidectomy. Four patients died, three secondary to wound-related sepsis. Of the three survivors, two healed soft tissue lesions primarily. The other required extremity amputation and wound excision before healing. Neither anatomical location of the soft tissue lesions nor post-parathyroidectomy serum calcium and phosphorus levels had any bearing on wound healing or mortality. Lesion severity at the time of parathyroidectomy appeared to best correlate with clinical course. Although treatment with phosphate-binding antacids, total or subtotal parathyroidectomy, and avoidance of challengers such as Vitamin D or local tissue trauma remain the mainstays of therapy, the uniform cure for calciphylaxis remains elusive. Prognosis for patients with calciphylaxis is dismal, even following late surgical intervention. Earlier recognition of the signs and symptoms of calciphylaxis should lead to timely parathyroidectomy in the hopes of ameliorating the symptoms and preventing or retarding its progressive sequelae.

Laparoscopic cholecystectomy in biliary pancreatitis.

Laparoscopic cholecystectomy has emerged as the treatment of choice for uncomplicated cholelithiasis. Despite early concerns, many surgeons have applied this new technique to more complicated biliary tract disease states, including biliary pancreatitis. To evaluate the safety of laparoscopic cholecystectomy in this setting, we retrospectively reviewed 29 patients with clinical and laboratory evidence of biliary pancreatitis who underwent this procedure between March 1990 and December 1992. The severity of pancreatitis was determined by Ranson's criteria. Two patients had a Ranson's score of 6, one of 5, one of 4, five scored 3, nine scored 2, nine also scored 1, and two patients scored 0. The mean serum amylase level on admission was 1,610 (range 148 to 7680). All patients underwent laparoscopic cholecystectomy during the same hospital admission for biliary pancreatitis, with the mean time of operation being 5.5 days from admission. Operative time averaged 123 minutes (range 60-220 minutes). Intraoperative cholangiography was obtained in 76 per cent of patients. Three patients had choledocholithiasis on intraoperative cholangiography and were treated with choledochoscopy, laparoscopic common bile duct exploration, and saline flushing of the duct. The mean length of hospital stay was 11 days (range 5-32 days). There were seven postoperative complications requiring prolonged hospitalization with all but one treated non-operatively. One patient with a preoperative Ranson score of 6 developed necrotizing pancreatitis and subsequently required operative pancreatic debridement and drainage. There were no deaths in this series and no postoperative wound infections. The average recovery period for return to work was 2 weeks. These statistics compare favorably with literature reports for open cholecystectomy in biliary pancreatitis.(ABSTRACT TRUNCATED AT 250 WORDS)

Over-expression of the yeast multifunctional arom protein.

The pentafunctional arom protein of Saccharomyces cerevisiae is encoded by the ARO1 gene. Substantial elevation of the levels of the arom protein (25-fold) was achieved in yeast using a vector that exploited the ubiquitin-fusion cleavage system of yeast. However, attempts to express the N-terminal 3-dehydroquinate synthase domain (E1) or the internal 3-dehydroquinase domain (E2) using the same system did not succeed. The yeast arom protein was successfully purified from the over-expressing transformant, and was found to possess all five enzymatic activities in a ratio similar to that observed in crude cell extracts. The purified material consisted mainly of a polypeptide that co-migrated in SDS-PAGE with intact arom proteins from other species.

Random mutagenesis of the substrate-binding site of a serine protease can generate enzymes with increased activities and altered primary specificities.

In the past, several point mutations have been introduced individually into the substrate-binding site of alpha-lytic protease (EC 3.4.21.12) and shown to affect its specificity in a predictable manner [Bone, R., Silen, J. L., & Agard, D. A. (1989) Nature 339, 191-195]. One of the resulting mutant enzymes (Met190Ala in the numbering system of Fujinaga et al.) [Fujinaga, M., Delbaere, L. T. J., Brayer, G. D., & James, M. N. G. (1985) J. Mol. Biol. 183, 479-502] cleaves at large hydrophobic residues. We chose this enzyme as the parent for a library of mutant proteases. The library was constructed by effecting combinatorial random substitution of up to four other residues (Gly191, Arg192, Met213, and Val218) thought likely to influence the primary specificity of the protease. Active enzymes in the library were screened with a range of synthetic substrates (encompassing 19 different amino acids in the P1 position) in order to evaluate their primary cleavage preferences. The amino acid sequences of active mutants revealed a strong preference for the replacement of Met213 with a His residue. This substitution also had the greatest observed effect on specificity, conferring a greatly increased and, in some cases, dominant ability to cleave at His residues in synthetic amide substrates. Mutant enzymes with greatly increased proteolytic activity were also found in the library.