Mapping gene expression changes in the fetal rat testis following acute dibutyl phthalate exposure defines a complex temporal cascade of responding cell types

Phthalates are chemical plasticizers used in a variety of consumer products; in rodents, they alter testicular development, leading to decreased testosterone synthesis and maldevelopment of the reproductive tract. Here, our goals were to discover a set of biomarker genes that respond early after relatively low-dose-level dibutyl phthalate (DBP) exposure and map the responding testicular cell types. To identify testicular phthalate biomarker genes, 34 candidate genes were examined by quantitative PCR at 1, 2, 3, or 6 h after exposure of Gestational Day 19 rats to DBP dose levels ranging from 0.1 to 500 mg/kg body weight. Twelve genes (Ctgf, Cxcl10, Dusp6, Edn1, Egr1, Fos, Ier3, Junb, Nr4a1, Stc1, Thbs1, and Tnfrsf12a) were identified with increased expression by 1-3 h at 100 or 500 mg/kg DBP, and 7 of these 12 genes had increased expression by 6 h at 10 mg/kg DBP. Using in situ hybridization of fetal testis cryosections from DBP-exposed rats, the temporal cellular expression of 10 biomarker genes was determined. Genes with a robust response at 1 h (Dusp6, Egr1, Fos, and Thbs1) were induced in peritubular myoid cells. For Egr1 and Fos, the interstitial compartment also showed increased expression at 1 h. Cxcl10 and Nr4a1 were induced by 1-3 h in both sparsely located interstitial cells and peritubular myoid cells. By 3 h, Stc1 was induced in Leydig cells, and Edn1, Ier3, and Tnfrsf12a were increased in Sertoli cells. These data reveal a complex early cascade of phthalate-induced cellular responses in the fetal testis, and for the first time suggest that peritubular myoid cells are an important proximal phthalate target cell.     

Alpha-galactosidase production and use in a hollow-fiber reactor.

Soybean milk serves as a base for a variety of beverages designed for consumption in developing countries. Soybean flour contains raffinose and stachyose considered to be responsible for flatulence often associated with these products (J.J. Rackis, D.H. Honig, D.J. Sessa, and F.R. Steggerda, 1970). alpha-Galactosidase, produced on wheat bran, hydrolyzes the galactooligosaccharides of soybean milk.     

Overexpression inEscherichia coli,Folding, Purification, and Characterization of the First Three Short Consensus Repeat Modules of Human Complement Receptor Type 1

We have developed a simple expression, isolation, and folding protocol for an SCR oligomer comprising the first three SCRs of complement receptor Type 1 (C3b/C4b receptor, CD35). A T7 RNA polymerase expression system inEscherichia coliwas used to express the oligomer as inclusion bodies. The oligomer was recovered from solubilized inclusion bodies using batch adsorption on SP–Sepharose. The oligomer was folded by one-step dilution in 20 m ethanolamine/1 m EDTA supplemented with 1 m GSH/0.5 m GSSG. The folded material was processed to a concentrated (>20 mg/ml), usable product of greater than 98% purity using a combination of ultrafiltration, ammonium sulfate treatment, hydrophobic interaction, and size-exclusion chromatography. The yield of folded material varied between 6 and 15 mg/liter culture. The oxidation states of the 12 cysteine residues in SCR(1–3) were identified by HPLC of peptide fragments from a tryptic digest using dual UV/fluorescence detection, collection of selected peaks, and N-terminal sequencing. This methodology confirmed the expected location of disulfide bridges. Equilibrium and velocity sedimentation studies are interpreted in terms of a single sedimenting species with molecular weights of 21,629 and 21,063 by these respective techniques. These values compare to the predicted molecular weight, from amino acid composition, of 21,817. The hydrodynamic properties of the molecule indicate that it is asymmetric with an axial ratio of 1:5.2 or equivalent dimensions of 21 × 110 Å. SCR(1–3) has an unusual CD spectrum exhibiting a broad maximum at 220–230 nm and a minimum at 190 nm. There was little evidence of classical secondary structure. The product exhibited concentration-dependent inhibition of complement-mediated lysis of sensitized sheep red blood cells.     

Regulation of arginine metabolism in Saccharomyces cerevisiae. Association of arginase and ornithine transcarbamoylase

Association of arginase and ornithine transcarbamoylase (OTCase) has been proposed to play an essential role in the regulation of arginine metabolism in Saccharomyces cerevisiae (Wiame, J.-M. (1971) Curr. Top. Cell. Reg. 4, 1-39). In this report multienzyme complex formation is directly demonstrated in the presence of the active-site ligands for OTCase and arginase. Using equilibrium sedimentation, a dissociation constant for complex formation was determined to be 2.3 X 10(-8) M in the presence of ornithine and agmatine, active-site ligands for OTCase and arginase, respectively. A molecular stoichiometry in the complex of one molecule of OTCase to one molecule of arginase was verified using transmission electron microscopy. The dimensions of the complex were determined by negative staining and rotary and unidirectional shadowing techniques to be 102 A wide by 81 A high. These dimensions are quantitively consistent with dimensions of the individual enzymes (Duong, L. T., Eisenstein, E., Green, S. M., Ornberg, R. L., and Hensley, P. (1986) J. Biol. Chem. 261, 12807-12813). The enzymatic activity of OTCase is virtually completely inhibited when associated with arginase, reflecting the dramatic modulation of enzyme activity as a consequence of the acquisition of quaternary structure in this multienzyme complex.     

The purification and characterization of arginase from Saccharomyces cerevisiae

In Saccharomyces cerevisiae, ornithine transcarbamoylase and arginase form a regulatory multienzyme complex (Hensley, P. (1988) Curr. Top. Cell. Regul. 29, 35-75). In this complex, arginase acts as a negative allosteric effector for ornithine transcarbamoylase. Before an analysis of the factors which promote and stabilize complex formation, arginase was purified in milligram quantities from a plasmid-containing, enzyme-overproducing, protease-deficient yeast strain and its physical characterization undertaken. The purified enzyme has a specific activity of 885 mumol urea min-1 mg-1 and a Km for arginine of 15.7 mM. The ultraviolet spectrum has a maximum absorbance at 279 nm, and the steady-state fluorescence emission spectrum has a maximum intensity at 337 nm, suggesting that the 3 tryptophans/polypeptide chain are in a relatively hydrophobic environment. Arginase has a weakly bound manganese responsible for the maintenance of the catalytic activity and is known to be heat activated in the presence of manganese. This effect is half-maximal at 12.1 microM manganese. In addition to a catalytic requirement for manganese, the presence of a more tightly bound metal is suggested from sedimentation studies. The native trimeric enzyme has a sedimentation coefficient of 5.95 S. Removal of the weakly associated metal results in no change in the sedimentation coefficient. However, dialysis with EDTA causes the s-value to decrease to 4.65 S, suggesting that under these conditions, the trimeric enzyme may partially dissociate. An analysis of CD spectra shows that significant spectral changes result from the removal of both the weakly bound metal and dialysis against EDTA.     

The cytokineplast: purified, stable, and functional motile machinery from human blood polymorphonuclear leukocytes

We examined the formation of motile, chemotactically active, anucleate fragments from human blood polymorphonuclear leukocytes (PMN, granulocytes), induced by the brief application of heat. These granule-poor fragments are former protopods (leading fronts, lamellipodia) that become uncoupled from the main body of the cell and leave it, at first with a connecting filament that breaks and seals itself. The usual random orientation of such filaments can be controlled by preorientation of cells in a gradient of the chemotactic peptide, N-formylmethionylleucylphenylalanine (F-Met-Leu-Phe) (2x10(-9) M- 1x10(-8)). Cytochalsin B, 2.5-5 μg/ml, prevents fragment formation; colchicine, 10(-5) M, does not. In scanning electron micrographs, fragments are ruffled and the cell body rounded up and rather smooth. In transmission electron micrographs, fragments contain microfilaments but lack centrioles and microtubules. Like intact cells, both bound and free fragments can respond chemotactically to an erythrocyte destroyed by laser microirradiation (necrotaxis); the free, anucleate fragments may do so repeatedly, even after having been held overnight at ambient temperatures. We propse the name cytokineplast for the result of this self-purification of motile apparatus. The exodus of the motile machinery from the granulocyte requires anchoring of the bulk of the cell to glass and uncoupling, which may involve heat-induced dysfunction of the centrosome. In ultrastructural studies of the centrosomal region after heat, centriolar structure remains intact, but pericentriolar osmiophilic material appears condensed, and microtubules are sparse. These changes are found in all three blood cell types examined: PMN, eosinophil, and monocyte. Of these, the first two make fragments under our conditions; the more sluggish monocyte does not. Uncoupling is further linked to centrosomal dysfunction by the observation that colchicines-treated granulocytes (10(-5)M, to destroy the centrosome’s efferent arm) make fragments after less heat than controls. If motive force and orientation are specified mainly from the organelle-excluding leading front, then endoplasmic streaming in PMN is a catch-up phenomenon, and microtubules do not provide the vector of locomotion but rather stabilize and orient the “baggage” (nucleus, granuloplasm)—i.e., they prevent fishtailing. Moreover, constraints emanating from the centrosome may now be extended to include, maintenance of the motile machinery as an integral part of the cell.     

A derivative of the CRMP2 binding compound lanthionine ketimine provides neuroprotection in a mouse model of cerebral ischemia

Lanthionines are novel neurotrophic and neuroprotective small molecules that show promise for the treatment of neurodegenerative diseases. In particular, a recently developed, cell permeable lanthionine derivative known as LKE (lanthionine ketimine 5-ethyl ester) promotes neurite growth at low nanomolar concentrations. LKE also has neuroprotective, anti-apoptotic, and anti-inflammatory properties. Its therapeutic potential in cerebral ischemia and its mechanisms of neurotrophic action remain to be fully elucidated. Here, we hypothesize that the neuroprotective actions of LKE could result from induction or modulation of CRMP2. We found that treating primary cultured mouse neurons with LKE provided significant protection against t-butyl hydroperoxide-induced neuronal death possibly through CRMP2 upregulation. Similarly, in vivo studies showed that LKE pre and/or post-treatment protects mice against permanent distal middle cerebral artery occlusion (p-MCAO) as evidenced by lower stroke lesions and improved functional outcomes in terms of rotarod, grip strength and neurologic deficit scores in treated groups. Protein expression levels of CRMP2 were higher in brain cortices of LKE pretreated mice, suggesting that LKE’s neuroprotective activity may be CRMP2 dependent. Lower activity of cleaved PARP-1 and higher activity of SIRT-1 was also observed in LKE treated group suggesting its anti-apoptotic properties. Our results suggest that LKE has potential as a therapeutic intervention in cerebral ischemia and that part of its protective mechanism may be attributed to CRMP2 mediated action and PARP-1/SIRT-1 modulation.     

The sugarcane borer sex attractant

Investigations with the sugarcane borer sex pheromone have been conducted in the laboratory and field since 1964 to: (1) develop a suitable synthetic diet for rearing large numbers of larvae; (2) develop a reliable bioassay to aid chemical studies of the pheromone; (3) study male response in the field to the pheromone; (4) determine the effects on natural populations of trapping males in small field plots; and (5) identify the pheromone. More than 1,000,000D. saccharalis have been reared on synthetic diets during the past 4 years. Bioassay techniques for detecting active fractions separated during chemical studies have been developed. Statistically significant reductions in sugarcane borer infestations and damage to sugarcane have been attained in small replicated field plots. Male movement in the field has been studied utilizing marking and release techniques. About 1400 males of the bluegrass webworm,C. teterrellus, responded to cagedD. saccharalis females during two years of field work, thus providing evidence for a lack of species specificity of the pheromone.

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Résumé La découverte en 1964 d’une phéromone sexuelle efficace chez le foreur de la canne à sucre,Diatraea saccharalis (F.) a stimulé la recherche pour identifier et synthétiser la phéromone et faire l’étude de ses possibilités d’emploi dans la lutte contre ces ravageurs.D. saccharalis est le principal insecte nuisible à la canne à sucre en Louisiane où il cause environ 13% des pertes annuelles. Les femelles vierges commencent à émettre ce produit chimique après l’émergence et elles sont le plus attractives pendant les trois premiers jours de vie, ensuite l’attraction diminue avec l’age. Généralement les femelles cessent d’attirer les males après l’accouplement et la majorité des femelles s’accouplent seulement une fois dans la nature, où l’activité sexuelle a lieu entre une heure et quatre heures du matin. Plus d’un million de larves ont été élevées en laboratoire ces quatre dernières années. Environ 75% des abdomens des femelles obtenues ont été utilisés par les chimistes qui cherchent à identifier la phéromone. Des réductions significatives de l’infestation par le foreur et des préjudices à la canne à sucre ont été enregistrées dans de petites parcelles où étaient disposés 400 à 800 pièges par acre, chaque piège contenant une femelle vierge. Il n’y a pas eu de réductions significatives l’année suivante quand la densité des pièges a été réduite à 80 par acre, cependant les captures de males ont augmenté de presque 2,5 fois. Les nombres de males marqués recapturés au point de lacher ne diffèrent pas significativement si les lachers ont été faits à des distances de 40, 80, 160 et 320 pieds du piège renfermant les femelles vierges, et seulement 4,44 pour cent des males ont été recapturés. Ces phénomènes sont peut être dus à la dispersion des males à partir des points de lacher avant leur réponse à la phéromone. Le manque de spécificité de la phéromone a été déjà signalé. Pendant les deux ans d’essais dans la nature, les pièges avec les femelles vierges deD. saccharalis ont attiré beaucoup de males deCrambus teterrellus Zincken.

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Presented at the symposium OILB on borers of graminaceous plants. Paris, 24th sept. 1970.     

The arachidonic acid 5-lipoxygenase inhibitor nordihydroguaiaretic acid inhibits tumor necrosis factor alpha activation of microglia and extends survival of G93A-SOD1 transgenic mice

Familial forms of amyotrophic lateral sclerosis (ALS) can be caused by mutations in copper, zinc-superoxide dismutase (SOD1). Mice expressing SOD1 mutants demonstrate a robust neuroinflammatory reaction characterized, in part, by up-regulation of tumor necrosis factor alpha (TNFalpha) and its primary receptor TNF-RI. In an effort to identify small molecule inhibitors of neuroinflammation useful in treatment of ALS, a microglial culture system was established to identify TNFalpha antagonists. Walker EOC-20 microglia cells were stimulated with recombinant TNFalpha, with or without inhibitors, and the cell response was indexed by NO2- output. Three hundred and fifty-five rationally selected compounds were included in this bioassay. The arachidonic acid 5-lipoxygenase (5LOX) and tyrosine kinase inhibitor nordihydroguaiaretic acid (NDGA), a natural dicatechol, was one of the most potent non-cytotoxic antagonists tested (IC50 8 +/- 3 microm). Investigation of the G93A-SOD1 mouse model for ALS revealed increased message and protein levels of 5LOX at 120 days of age. Oral NDGA (2500 p.p.m.) significantly extended lifespan and slowed motor dysfunction in this mouse, when administration was begun relatively late in life (90 days). NDGA extended median total lifespan of G93A-SOD1 mice by 10%, and life expectancy following start of treatment was extended by 32%. Disease-associated gliosis and cleaved microtubule-associated tau protein, an indicator of axon damage, were likewise reduced by NDGA. Thus, TNFalpha antagonists and especially 5LOX inhibitors might offer new opportunities for treatment of ALS.