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.

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.

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.

Purification of liver glutamate dehydrogenase by affinity precipitation and studies on its denaturation.

In the presence of glutaric acid, N2,N2'-adipodihydrazido-bis(N6-carbonylmethyl-NAD+)(bis-NAD+ ) forms cross-links between molecules of glutamate dehydrogenase, resulting in precipitation. The dependence of this process on bis-NAD+ and enzyme concentration has been investigated. This procedure has been shown to be effective in the purification of glutamate dehydrogenase from rat and ox liver, and a procedure is presented in which this affinity precipitation procedure is used instead of the affinity chromatography used in an earlier method (McCarthy, A.D., Walker, J.M. and Tipton, K.F. (1980) Biochem. J. 191, 605-611). The ox liver enzyme prepared in this way had not suffered the limited proteolysis that occurs during the preparation of the enzyme by other commonly used procedures. After the purified enzyme had been denatured by treatment with urea, guanidine hydrochloride, or low pH, no recovery of activity could be demonstrated following dilution or, in the last case, dialysis.

Genetic reconstruction and functional analysis of the repeating lipoyl domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli.

The dihydrolipoamide acetyltransferase component (E2p) of the pyruvate dehydrogenase complex of Escherichia coli contains three highly homologous sequences of about 100 residues that are tandemly repeated to form the N-terminal half of the polypeptide chain. All three sequences include a lysine residue that is a site for lipoylation and they appear to form independently folded functional domains. These lipoyl domains are in turn linked to a much larger (about 300 residues) subunit-binding domain of the E2p chain that aggregates to form the octahedral inner core of the complex and also contains the acetyltransferase active site. In order to investigate whether individual lipoyl domains play different parts in the enzymic mechanism, selective deletions were made in vitro in the dihydrolipoamide acetyltransferase gene (aceF) so as to excise one or two of the repeating sequences. This was facilitated by the high degree of homology in these sequences, which allowed the creation of hybrid lipoyl domains that closely resemble the originals. Pyruvate dehydrogenase complexes incorporating these genetically reconstructed E2p components were purified and their structures were confirmed. It was found that the overall catalytic activity, the system of active site coupling, and the ability to complement pyruvate dehydrogenase complex mutants, were not significantly affected by the loss of one or even two lipoyl domains per E2p chain. No special role can be attached thus far to individual lipoyl domains. On the other hand, certain genetic deletions affecting the acetyltransferase domain caused inactivation of the complex, highlighting particularly sensitive areas of that part of the E2p chain.

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.

Interactions of lipoyl domains with the E1p subunits of the pyruvate dehydrogenase multienzyme complex from Escherichia coli.

Equilibrium binding experiments were carried out with lipoyl domains and the pyruvate decarboxylase [pyruvate dehydrogenase (lipoamide), E1p, EC 1.2.4.1)] component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The dissociation constant (Ks) was estimated to be not less than 0.3 mM, exceeding the Km value (33 microM) for reductive acetylation of the domains by an order of magnitude. Thus, the lipoyl domain, which is required to promote reductive acetylation of the lipoyl group, does not appear to do this simply by enhancing the binding to E1p. The difference between Ks and Km suggests that the formation and release of reductively acetylated lipoyl domains from the enzyme may be a relatively rapid step in the mechanism.

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.

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.

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)

Safety, efficacy, cost, and morbidity of laparoscopic versus open cholecystectomy: a prospective analysis of 228 consecutive patients.

Laparoscopic cholecystectomy has become the procedure of choice in most hospitals for the resolution of surgically treatable gallbladder disease. Few reports address the results of laparoscopic cholecystectomy in comparison to open cholecystectomy during the same time interval within the same institution. One hundred ninety-six laparoscopic cholecystectomies were performed from April 1990 through February 1991. Initial patient selection was restricted to elective procedures for chronic cholecystitis with expanded indications as experience was gained. Of the 196 cases, 11 required conversion to open cholecystectomy, leaving 185 laparoscopic cholecystectomies for comparison. During the same period, 82 open cholecystectomies were performed. Thirty-nine of these were complicated cases and would not have been considered for laparoscopic cholecystectomy early in the study, leaving 43 routine open cholecystectomies for comparative purposes. In the laparoscopic group, 1.1 per cent of the patients had major operative complications as opposed to the open group, which had none. There were no common bile duct injuries in either group. To provide a true cost-benefit analysis, a group of patients was identified that would qualify for elective, same-day admission for either an open or laparoscopic procedure. Laparoscopic cholecystectomy (LC) was performed on 70 patients, and open cholecystectomy (OC) was performed on 26 patients. A comparison of data from these groups showed no significant difference in age or sex. Hospitalization costs averaged $5,390 for the LC group versus $5,392 for the OC group. Postoperative hospital stay averaged 1.3 days for the LC group versus 3.7 days for the OC group (P < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

Kinetics and specificity of reductive acylation of lipoyl domains from 2-oxo acid dehydrogenase multienzyme complexes.

Lipoamide and a peptide, Thr-Val-Glu-Gly-Asp-Lys-Ala-Ser-Met-Glu lipoylated on the N6-amino group of the lysine residue, were tested as substrates for reductive acetylation by the pyruvate decarboxylase (E1p) component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The peptide has the same amino acid sequence as that surrounding the three lipoyllysine residues in the lipoate acetyltransferase (E2p) component of the native enzyme complex. Lipoamide was shown to be a very poor substrate, with a Km much higher than 4 mM and a value of kcat/Km of 1.5 M-1.s-1. Under similar conditions, the three E2p lipoyl domains, excised from the pyruvate dehydrogenase complex by treatment with Staphylococcus aureus V8 proteinase, could be reductively acetylated by E1p much more readily, with a typical Km of approximately 26 microM and a typical kcat of approximately 0.8 s-1. The value of kcat/Km for the lipoyl domains, approximately 3.0 x 10(4) M-1.s-1, is about 20,000 times higher than that for lipoamide as a substrate. This indicates the great improvement in the effectiveness of lipoic acid as a substrate for E1p that accompanies the attachment of the lipoyl group to a protein domain. The free E2o lipoyl domain was similarly found to be capable of being reductively succinylated by the 2-oxoglutarate decarboxylase (E1o) component of the 2-oxoglutarate dehydrogenase complex of E. coli. The 2-oxo acid dehydrogenase complexes are specific for their particular 2-oxo acid substrates. The specificity of the E1 components was found to extend also to the lipoyl domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of the active-site lysine residues of two biosynthetic 3-dehydroquinases.

The lysine residues involved in Schiff-base formation at the active sites of both the 3-dehydroquinase component of the pentafunctional arom enzyme of Neurospora crassa and of the monofunctional 3-dehydroquinase of Escherichia coli were labelled by treatment with 3-dehydroquinate in the presence of NaB3H4. Radioactive peptides were isolated by h.p.l.c. following digestion with CNBr (and in one case after further digestion with trypsin). The sequence established for the N. crassa peptide was ALQHGDVVKLVVGAR, and that for the E. coli peptide was QSFDADIPKIA. An amended nucleotide sequence for the E. coli gene (aroD) that encode 3-dehydroquinase is also presented, along with a revised alignment of the deduced amino acid sequences for the biosynthetic enzymes.