A Smoothened receptor agonist is neuroprotective and promotes regeneration after ischemic brain injury.

Ischemic stroke occurs as a result of blood supply interruption to the brain causing tissue degeneration, patient disabilities or death. Currently, treatment of ischemic stroke is limited to thrombolytic therapy with a narrow time window of administration. The sonic hedgehog (Shh) signaling pathway has a fundamental role in the central nervous system development, but its impact on neural cell survival and tissue regeneration/repair after ischemic stroke has not been well investigated. Here we report the neuroprotective properties of a small-molecule agonist of the Shh co-receptor Smoothened, purmorphamine (PUR), in the middle cerebral artery occlusion model of ischemic stroke. We found that intravenous administration of PUR at 6 h after injury was neuroprotective and restored neurological deficit after stroke. PUR promoted a transient upregulation of tissue-type plasminogen activator in injured neurons, which was associated with a reduction of apoptotic cell death in the ischemic cortex. We also observed a decrease in blood-brain barrier permeability after PUR treatment. At 14 d postinjury, attenuation of inflammation and reactive astrogliosis was found in PUR-treated animals. PUR increased the number of newly generated neurons in the peri-infarct and infarct area and promoted neovascularization in the ischemic zone. Notably, PUR treatment did not significantly alter the ischemia-induced level of Gli1, a Shh target gene of tumorigenic potential. Thus our study reports a novel pharmacological approach for postischemic treatment using a small-molecule Shh agonist, providing new insights into hedgehog signaling-mediated mechanisms of neuroprotection and regeneration after stroke.

Comparison of fracture rate, deformation rate, and efficiency between rotary endodontic instruments driven at 150 rpm and 350 rpm.

Two groups of 30 mature molars each (S and F) were instrumented with Profile .04 taper Series 29 rotary instruments. Group S molars were instrumented at 150 rpm and group F at 350 rpm. The number of fractures, deformed files, and instrumentation time were recorded for each tooth. No instrument fractures occurred in either group. In group S the mean deformation rate and instrumentation time were 1.1 deformed files and 8.0 min per molar. In group F they were 0.57 deformed files and 4.6 min per molar. Both differences were significant (p < 0.05). The results indicate that Profile .04 taper Series 29 rotary instruments should be used at 350 rpm to nearly double efficiency and halve the deformation rate, compared with 150 rpm. Because no instrument fractures occurred while instrumenting 60 mature molars, both speeds should be considered safe.

Artificial chaperone-assisted refolding of citrate synthase.

The power of genetic engineering methods, along with increasing genomic information, makes heterologous expression of proteins an extremely important biochemical tool. Unfortunately, proteins obtained in this way often are not in their native form, and folding becomes a crucial step in protein production. We have recently developed a strategy that promotes the folding of chemically denatured proteins via the sequential addition of low molecular weight "artificial chaperones." Here we describe in detail the application of this method to porcine heart citrate synthase. Refolding yields of as high as 65% have been achieved. Mechanistic studies indicate that there are significant differences between artificial chaperone-assisted refolding of citrate synthase and artificial chaperone-assisted refolding of two other proteins that have been examined, carbonic anhydrase B (Rozema, D., and Gellman, S. H. (1996) J. Biol. Chem. 271, 3478-3487) and lysozyme (Rozema, D., and Gellman, S. H. (1996) Biochemistry 35, 15760-15771). The differences among these three test proteins reveal the range of procedural variation that must be considered in the application of the artificial chaperone method to new proteins.

Degree of integration can influence organizational economic decisions.

When healthcare providers join to form contracting organizations to negotiate managed care contracts, the resulting organizations can be characterized by their degree of integration: tightly integrated (ie, a single bottom line) or loosely integrated (ie, economic independence of each affiliated entity is preserved). Tightly integrated organizations generally are more attractive to payers and better able to optimize returns on their managed care contracts.

Managing for Biodiversity: Emerging Ideas for the Electric Utility Industry-Summary Statement

/ The conference entitled "Managing for Biodiversity: Emerging Ideas for the Electric Utility Industry" was held in Williamsburg, Virginia, USA, during 19-20 March 1996. This paper provides an overview of the key points, conclusions, and recommendations from both the presentations/papers and the discussions throughout the conference.KEY WORDS: Biodiversity; Partnerships; Utilities; Ecosystem management; Conservation; Electrification

Histopathologic effects of kinetic cavity preparation for the removal of enamel and dentin. An in vivo animal study.

Recent developments in technology, direct placement restorative materials, and cavity preparation design have renewed interest in kinetic cavity preparation, a term to describe the use of air-abrasion for removal of tooth structure. This study compared the pulpal response of 120 teeth in mixed-breed dogs treated with four kinetic cavity preparation combinations of pressure (80 psi and 160 psi) and aluminum oxide particle sizes (27 microns and 50 microns) to those treated with high-speed rotary burs. Class V buccal preparations were made and restored with an interim material. Teeth were collected 72 hours after surgery, decalcified, sectioned, stained with hematoxylin and eosin, and blindly evaluated by two examiners at the minimal dentin thickness. Samples were graded for extent of displacement, disruption, inflammation, and necrosis of pulpal structures. Differences between groups were analyzed with the use of Bonferroni-adjusted multiple Mann-Whitney-Wilcoxon tests with p < 0.05 being significant. Higher pressures and smaller particles yielded significantly fewer pulpal effects than the high-speed treated teeth whereas lower pressures and larger particles were not significantly different for most effects evaluated. No adverse soft tissue effects were noted when kinetic cavity preparation was directed at attached gingiva.

Degradation of 2,4-dichlorophenoxyacetic acid by Pseudomonas cepacia DBO1(pRO101) in a dual-substrate chemostat.

To determine the effect of a secondary carbon source on biodegradation of a chloroaromatic compound, Pseudomonas cepacia DBO1(pRO101) was grown in continuous cultures on basal salts media containing various mixtures of 2,4-dichlorophenoxyacetic acid (2,4-D) and succinate. Both succinate and 2,4-D were metabolized over the entire range of dilution rates and compositions analyzed (0.05 to 0.6 h-1). 2,4-Dichlorophenol (DCP), the only intermediate detected, accumulated to significant amounts (10 to 21 mg/liter) in the chemostat only when the dilution rate was 0.4 h-1 or greater. At these concentrations, DCP reduced the apparent growth rate of P. cepacia DBO1(pRO101) in batch cultures by 15 to 35% over the apparent growth rate on succinate alone. Succinate fed to the chemostat increased the cell density as well as the percentage of 2,4-D that was consumed at each dilution rate. When the amount of succinate in the feed exceeded the amount of 2,4-D, the specific rates of 2,4-D degradation in the chemostat or by washed cells were significantly lower than the specific rates for cells grown on 2,4-D alone, suggesting repression by succinate. However, when the amount of 2,4-D in the feed exceeded the amount of succinate, the specific rates of 2,4-D degradation remained at values equivalent to or higher than the specific rate for cells grown on 2,4-D alone. DCP accumulated significantly in the washed-cell assay, suggesting that the level of DCP hydroxylase is rate limiting.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate specificity of the gastrin-amidating enzyme.

As is the case with many other peptide hormones of the brain and gut, gastrin requires a carboxyl-terminal amide moiety for optimal biological activity. In the structure of progastrin, the carboxyl-terminal Phe of gastrin is followed by the sequence Gly93-Arg94-Arg95, which must be processed sequentially by an endoprotease, a carboxypeptidase, and an amidating enzyme to produce amidated bioactive gastrin. To examine the molecular determinants of peptide amidation in vivo, we mutated the wild-type Gly93 residue of progastrin to Ala93 and Ser93 and expressed the three progastrin DNAs in GH3 and MTC 6-23 endocrine cell lines. Although substantial quantities of amidated gastrin were seen in cells expressing wild-type progastrin, replacement of Gly93 with Ala93 completely abolished production of amidated gastrin when the cells were incubated in standard medium containing only L-alanine. In a similar fashion, cells expressing [Ser93]progastrin also demonstrated no production of amidated gastrin. When cells expressing [Ala93]- or [Ser93]progastrin were incubated in the presence of 1 mg/ml D-alanine or D-serine, respectively, a small but consistent amount of amidated gastrin production was detected (< 1% of wild type). These data lead us to conclude that the amidating enzyme has a rigid substrate specificity for a glycine-extended precursor. Furthermore, this in vivo substrate specificity confirms the importance of the pro-S-alpha-hydrogen of the carboxyl-terminal glycine for enzyme-substrate recognition.

Molecular analysis of dibasic endoproteolytic cleavage signals.

Biologically active peptide hormones are synthesized from larger precursor proteins by a variety of post-translational processing reactions. To characterize these processing reactions further we have expressed preprogastrin in two endocrine cell lines and examined the molecular determinants involved in endoproteolysis at dibasic cleavage sites. The Gly93-Arg94-Arg95 carboxyl-terminal processing site of progastrin must be processed sequentially by an endoprotease, a carboxypeptidase, and an amidating enzyme to produce bioactive gastrin. For these studies the dibasic Arg94-Arg95 residues that serve as signals for the initiation of this processing cascade were mutated to Lys94-Arg95, Arg94-Lys95, and Lys94-Lys95. In the GH3 cells the Lys94-Arg95 mutation slightly diminished synthesis of carboxyl-terminally amidated gastrin, whereas in the MTC 6-23 cells this mutation had no effect on amidated gastrin synthesis. In contrast, both Arg94-Lys95 and Lys94-Lys95 mutations resulted in significantly diminished production of amidated gastrin in both cell lines. A specific hierarchy of preferred cleavage signals at this progastrin processing site was demonstrated in both cell lines, indicating that cellular dibasic endoproteases have stringent substrate specificities. Progastrins with the Lys94-Arg95 mutation in GH3 cells also demonstrated diminished processing at the Lys74-Lys75 dibasic site, thus single amino acid changes at one processing site may alter cleavage at distant sites. These studies provide insight into the post-translational processing and biological activation of not only gastrin but other peptide hormones as well.

Common bile duct obstruction in cystic fibrosis: utility of hepatobiliary scintigraphy.

Abdominal pain occurs commonly in patients with cystic fibrosis, and is the result of a variety of causes, including hepatobiliary disorders. With the increasing duration of survival in these patients, diagnostic investigations for abdominal pain, including hepatobiliary scanning, may be utilized more frequently than in the past. Difficulties in the interpretation of scintigraphic studies may arise because of associated gallbladder anomalies that occur in more than 50% of patients with cystic fibrosis. Hypoplasia of the gallbladder (microgallbladder) occurs commonly. A case is presented in which Tc-99m disofenin hepatobiliary scanning proved diagnostic in a patient with cystic fibrosis and cholelithiasis leading to common bile duct obstruction. The hepatobiliary abnormalities and causes of common bile duct obstruction encountered in cystic fibrosis are reviewed.

Expression and processing of human preprogastrin in murine medullary thyroid carcinoma cells.

Gastrin, the primary hormonal mediator of postprandial gastric acid secretion, is produced from its precursor progastrin by a series of posttranslational processing reactions including dibasic residue cleavage and carboxyl-terminal alpha-amidation. Progastrin contains three dibasic cleavage signals, Arg57Arg58, Lys74Lys75, and Arg94Arg95, that appear to be cleaved differently in different tissues. Differential processing is a potential means by which the production of biologically active peptides may be regulated in a tissue-specific manner. To study these reactions further, we used the pZipNeo SV(X) retroviral vector to express human gastrin cDNA in a heterologous cell line (MTC 6-23) known to be capable of processing other peptide precursors. The psi 2 packaging cell line transfected with the gastrin cDNA-retroviral construct (pSVXgas) produced progastrin, but no substantial amounts of processed amidated gastrin were detected. amounts of processed amidated gastrin were detected. In contrast, MTC 6-23 cells infected with the viral stock obtained from the supernatant of pSVXgas-transfected psi 2 cells produced carboxyl-terminally amidated gastrin in all of its standard molecular forms, including sulfated and nonsulfated forms of tetratriacontagastrin (G-34), heptadecagastrin (G-17), and tetradecagastrin (G-14). These studies indicate that heterologous endocrine cell lines infected with a retroviral-peptide cDNA construct can serve as useful models for peptide hormone posttranslational processing.

Molecular pathogenesis of human immunodeficiency virus infection.

Molecular studies of the pathogenesis of human immunodeficiency virus (HIV) infections have proceeded rapidly following the molecular cloning and nucleotide sequence analysis of the HIV genome. Correlation of biochemical and functional studies of HIV-infected cells with the HIV nucleotide sequence has allowed the identification and preliminary functional characterization of many HIV proteins. These include structural proteins (gag), viral enzymes (pol), and viral regulatory proteins (tat, art). Cloned HIV DNA segments have been utilized as probes for in situ nucleic acid hybridization to study the distribution of HIV-infected cells in acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) patients. These studies have demonstrated the infection of macrophages as an important component of HIV-induced neurologic disease. Only very low numbers of HIV-infected lymphocytes can be identified in the peripheral blood of infected individuals. Thus, the mechanism of CD4 cell depletion in the pathogenesis of AIDS remain obscure.

The HIV 'A' (sor) gene product is essential for virus infectivity.

The genome of the human immunodeficiency virus (HIV) contains several open reading frames (ORFs) not present in other viruses. The 'A' gene, also known as Q2 P'3, ORF-1(4) or sor5, partially overlaps the pol gene; its protein product has a relative molecular mass of 23,000 (Mr 23K) and is present in productively infected cells. The function of this protein is unclear; mutant viruses deleted in 'A' replicate in and kill CD4+ lymphocyte lines, but the high degree of conservation of the deduced amino-acid sequence in nine different HIV isolates (80%) and the presence of analogous genes in HIV-2 and other lentiviruses suggest that the gene function is an important one. Here we describe a mutant virus deficient in the 'A' gene which produces virion particles normally; however, the particles are approximately 1,000 times less infective than wild type. Transcomplementation experiments partially restore infectivity. The mutant virus spreads efficiently when virus-producing cells are co-cultivated with CD4+ lymphocytes, however, indicating that HIV can spread from cell to cell in a mechanism that does not require the 'A' gene product and probably does not require the production of infective virus particles.

Productive, persistent infection of human colorectal cell lines with human immunodeficiency virus.

Thirteen adherent human non-lymphocyte cell lines were tested for their susceptibility to infection by human immunodeficiency virus. Productive infection could be demonstrated in three of five colorectal carcinoma cell lines examined; the other eight human non-lymphocyte cell lines were uninfectible. A susceptible colon carcinoma cell line (HT29), as well as normal colonic mucosa, was shown to contain a 3.0-kilobase species of poly(A)+ CD4 RNA, whereas uninfectible colon carcinoma and rhabdomyosarcoma cell lines synthesized no detectable T4 RNA. A persistently infected colon carcinoma cell line was established that continued to produce progeny human immunodeficiency virus for more than 10 weeks postinfection.

Central, stereoselective receptors mediate the acute effects of opiate antagonists on luteinizing hormone secretion.

Although a central site of acute opiate action in regulating luteinizing hormone (LH) secretion has been suggested by the ability of centrally implanted opiate antagonists to increase LH levels, opiate antagonists are lipophilic and could influence the pituitary in situ. Also, the physiological significance of opiate receptor blockade with antagonists rests on the assumed, but untested, stereoselectivity of these receptors. Therefore, a lipophobic quaternized derivative of naltrexone (MRZ 2663-Naltrexone methobromide) and dextro- (+) and levo- (-) stereoisomers of naloxone were used to study the site- and stereoselectivity of gonadotropin responses to opiate antagonists in vivo. Male rats were injected intracerebroventricularly (icv) or intravenously (iv) with the quaternary or tertiary congeners of naltrexone and subcutaneously (sc) with (-) or (+)-naloxone. Rats injected icv with 20 ug of quaternary naltrexone displayed significant increases in serum luteinizing hormone (LH). The onset of the response was rapid with serum LH levels being significantly elevated 15 minutes after the injection and returning to basal levels 30 minutes later. Rats injected iv with 10 mg/kg of quaternary naltrexone failed to show significant LH responses. Rats injected either centrally or periphally with equivalent doses of tertiary naltrexone showed LH responses that were similar to those found in animals injected icv with quaternary naltrexone. As little as 0.5 mg/kg of (-)-naloxone resulted in significant elevations in serum LH that were higher than those elicited by up to 10 mg/kg of (+)-naloxone, indicating that this effect of naloxone is stereoselective. These data support the argument that opioids can acutely modulate LH secretion through actions at stereoselective opioid receptors in the central nervous system.

Biological and biochemical characterization of a cloned Leu-3- cell surviving infection with the acquired immune deficiency syndrome retrovirus.

Leu-3- cells that survive infection with the acquired immune deficiency syndrome (AIDS) retrovirus can be induced with IUdR to express infectious virus. A cellular clone (8E5), isolated by limiting dilution of a mass culture of survivor cells, was found to contain a single, integrated provirus that was constitutively expressed. Although IUdR treatment of 8E5 cells failed to induce infectious virus, cocultivation with Leu-3+ cells generated the characteristic syncytia associated with acute AIDS retrovirus infection. The single integrated copy of proviral DNA directs the synthesis of all major viral structural proteins except p64, as monitored by immunoblotting. The relationship of the 8E5 clone to viral latency and persistence is discussed.