The limited proteolysis of tumor necrosis factor-α

The limited proteolysis of human recombinant TNF- by trypsin yields two stable products resulting from cleavage after Arg6 and Arg44. In solution these two products remain associated together in a trimer with a Stokes' radius slightly greater than the radius of intact TNF- and, therefore, could not be separated from each other under nondenaturing conditions. This limited digest retains at least 20% of the activity of the original TNF- sample, and has a tertiary structure that is similar to that of the native protein by circular dichroism. On the other hand, incorrectly folded, inactive TNF- undergoes extensive digestion following similar treatment with trypsin. These results indicate that the active form of TNF- has a tight core structure which is maintained afterN-terminal cleavage and removal.     

Specificity towards oligomannoside and hybrid type glycans of the endo-β-N-acetylglucosaminidase B from the basidiomy ceteSporotrichum dimorphosporum

We have previously shown that an endo--N-acetylglucosaminidase (EC 3.2.1.96) named Endo B, isolated from culture filtrates of the basidiomyceteSporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of theN-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43–49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)₁(GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₄(GlcNAc)₂ were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)₂(Man)₅(GlcNAc)₂ glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme.The oligomannoside-and theN-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule.Endo B represents a powerful tool for removing oligomannoside-andN-acetyllactosamine-type glycans fromN-glycopeptides andN-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.Abbreviations Gal d-galactose - Man d-mannose - GlcNAc N-acetyl-d-glucosamine - Con A concanavalin A - Asn asparagine - GLC gas liquid chromatography - TLC thin layer chromatography - Endo endo--N-acetylglucosaminidase - Endo B endo--N-acetylglucosaminidase isolated fromSporotrichum dimorphosporum - PBE polybuffer exchanger - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Rat ventral prostate as an independent source of inhibin-like material

Castration had no effect on prostatic inhibin-like activity as compared to normal controls. Consequent upon castration there is a significant reduction in the inhibin content of the ventral prostate on a per gland basis. However, when expressed as a function of protein content, there is no change. The data suggest that the very same epithelial cells which under the influence of androgen carry out the characteristic exocrine secretory activity are responsible for the synthesis and secretion of inhibin-like material.     

Cellular origin and distribution of transforming growth factor-β1 in the normal rat myocardium

Summary Transforming growth factor- (TGF-) is a biologically active polypeptide present in normal tissues as well as transformed cells. Two structurally related forms of this peptide are TGF- ₁ and TGF- ₂. Using freshly isolated cardiomyocytes and non-myocyte heart cells, and a [³²P]-labelled cDNA probe to human TGF- ₁, we demonstrated that mRNA for TGF- ₁ could be detected only in the nonmyocyte fraction of heart cells. In the present study, the distribution of TGF- ₁ in the heart was determined by immunofluorescence staining by use of a polyclonal antibody to porcine TGF- ₁ in cryostat sections of rat heart. Immunofluorescence staining was intense around the blood vessels and radially diffuse in the surrounding myocardium.     

Characterization of the follicles in the intermediate lobe of the pituitary gland of the Mongolian gerbil (Meriones unguiculatus)

Summary The Mongolian gerbil (Meriones unguiculatus) contains abundant follicles throughout the intermediate lobe (IL) of the pituitary gland in the adult animal. The mode of follicle formation, the nature of the follicle building cells and the distribution of follicles were investigated in semithin sections of the gerbil IL. The sections were stained conventionally, or immunohistochemically with antibodies directed against -melanocyte stimulating hormone (- MSH). Follicular cells were constantly -MSH-negative, and resembled the marginal cells lining the hypophyseal cleft with regard to their cytological and immunohistochemical properties. Moreover, follicular cells appeared to be derived from strands of marginal cells that regularly invaginated deep into the IL. Both follicular and marginal cells often made up cellular clusters. This process coincided with follicle formation and the generation or transport of the colloidal content found inside follicles and the hypophyseal cleft. Although the non-secretory cells of the IL obviously constituted one major source of pituitary colloid in the gerbil, -MSH-positive secretory cells, which occasionally were found to be discharged into the cleft cavity, might contribute to the colloidal contents.     

Distribution of δ-aminolevulinic acid biosynthetic pathways among phototrophic bacterial groups

Two biosynthetic pathways are known for the universal tetrapyrrole precursor, -aminolevulinic acid (ALA). In the ALA synthase pathway which was first described in animal and some bacterial cells, the pyridoxal phosphate-dependent enzyme ALA synthase catalyzes condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO₂. In the five-carbon pathway which was first described in plant and algal cells, the carbon skeleton of glutamate is converted intact to ALA in a proposed reaction sequence that requires three enzymes, tRNA^Glu, ATP, Mg²⁺, NADPH, and pyridoxal phosphate. We have examined the distribution of the two ALA biosynthetic pathways among various genera, using cell-free extracts obtained from representative organisms. Evidence for the operation of the five-carbon pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA, using 3,4-[³H]glutamate or 1-[¹⁴C]glutamate as substrate. ALA synthase activity was indicated by RNase-insensitive incorporation of label from 2-[¹⁴C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later. The five-carbon pathway is apparently the more widely utilized one among bacteria, while the ALA synthase pathway seems to be limited to the subgroup of purple bacteria.Abbreviations ALA -aminolevulinic acid - DTT dithiothreitol - PALP pyridoxal phosphate - SDS sodium dodecyl sulfate - tricine N-tris-(hydroxymethyl)methylglycine     

Les régulateurs de croissance d'insectes (RCI), mimiques de l'hormone juvénile,en tant que moyen de lutte morphogénétique et ovicide contre les tordeuses des vergers

Résumé La métamorphose des insectes est régie par un équilibre hormonal complexe dans lequel l'hormone juvénile (HJ) joue un rôle important. Au dernier stade larvaire, la teneur en HJ est particulièrement faible dans le corps de l'insecte. Si un régulateur de croissance d'insectes (RCI)-un mimétique de l'HJ-est appliqué à ce moment-là, la mue nymphale est pertubée provoquant des déformations morphogénétiques caractéristiques. La teneur en HJ est également très faible dans les ufs fraîchement pondus. Les traitements aux RCI peuvent par conséquent perturber le développement embryonnaire de certaines espèces et produire ainsi un effet ovicide. Depuis quelques années deux RCI-le fenoxycarb et le CGA 45 128-ont été testés pour leur activité morphogénétique sur le dernier stade larvaire de quelques ravageurs tels qu'Adoxophyes orana F.v.R., ainsi que pour leur activité ovicide sur les ufs frais de Cydia pomonella L. et Grapholita funebrana Tr. Après quelques années d'expérimentation et de commercialisation des RCI dans les vergers européens, il s'avère que l'utilisation de ces produits peu toxiques, sélectifs et peu nocifs pour la faune utile, constitue une amélioration considérable pour l'aménagement de la lutte intégrée.

---

Insect growth regulators (IGR), mimics of juvenile hormone, as morphological and ovicidal means of control against orchard tortricids

Summary Metamorphosis is regulated by a complex hormonal balance in which juvenile hormone (JH) plays an important part. At the last larval instar the content of JH is particularly low in the insect body. If an insect growth regulator (IGR) — a mimic of JH-is applied at this time, the pupal moult may be disturbed with the characteristic morphogenetical deformations. The JH content is also very low in freshly laid eggs. Therefore IGR treatments may disturb the embryonic development of some species and produce an ovicidal activity. During a few years two IGR-fenoxycarb and CGA 45128-were evaluated for their morphogenetical effect on the last larval instar of Adoxophyes orana F.v.R. and their ovicidal effect on freshly laid eggs of Cydia pomonella L. and Grapholita funebrana Tr. After a few years of experimentation with both compounds and of commercialisation of fenoxycarb in European orchards, IGR confirmed to present a considerable improvement in integrated pest management due to selectivity, and low mammal toxicity.

     

Abnormal expression ofα-L-fucosidase in lymphoid cell lines of fucosidosis patients

Fucosidosis is an autosomal recessive lysosomal storage disease due to a deficiency of-L-fucosidase activity in tissues and body fluids. Exponentially growing lymphoid cell cultures from four fucosidosis patients had 2.7-fold to 15.6-fold less extracellular-L-fucosidase protein and 28.8-fold to 144.0-fold less intracellular-L-fucosidase protein with negligible catalytic activity, compared to the mean of 19 control cultures. The percentage of total-L-fucosidase protein released extracellularly by cultures from the four patients was 64 to 85%, compared to 35±9% for control cultures. Intracellular and extracellular enzyme forms in fucosidosis and control cell lines were glycoproteins containing polypeptide chains ofM _r=52,000. During a 1.5-hr pulse-label with³⁵S-methionine,-L-fucosidase was synthesized by control cells and two fucosidosis cell lines as an intracellular form withM _r=58,000. During a subsequent 21-hr chase with unlabeled methionine, mutant enzyme was almost entirely processed to an extracellular form withM _r=62,000. In contrast, only 25–30% of control enzyme was processed to an extracellular form (M _r=62,000), with the remainder retained intracellularly (M _r=60,000). In the other two fucosidosis cell lines,-L-fucosidase was synthesized as an intracellular form withM _r=56,000 that was processed to an extracellular form withM _r=60,000. In summary, the fucosidosis mutation(s) affected the catalytic activity, quantity, and extracellular release of-L-fucosidase as expressed by lymphoid cells.This work was funded by NIH Grants DK 32161 to R. A. DiCioccio and GM 28428 to J. K. Darby.     

Genetic evidence for α2-adrenergic receptor subtypes in rat brain,heart and adrenal gland

Summary A complementary DNA (cDNA) clone - cA₂-47 - corresponding to a new ₂-adrenergic receptor subtype has been isolated from a rat brain cDNA library and used as a hybridization probe to scrutinize the ₂-receptor poly(A⁺) RNAs in rat brain, heart and adrenal gland. Hybridization of the 5 half of the coding region of this cDNA at 37°C to rat brain poly(A+) RNA revealed a single band at 5.8 kb as the size of its corresponding mRNA. Under identical hybridization conditions, a human platelet ₂-receptor genomic probe failed to hybridize to any rat brain mRNAs.Under lower stringency conditions, hybridization of the full-length cDNA, cA₂-47, to selected rat tissue poly(A⁺) RNA showed the presence of four different sized mRNAs in brain and three in both heart and adrenal gland. Messages of 1.3 kb and 2.1 kb were common in all three tissues (although the band at 2.1 kb was slightly higher in the heart and adrenal gland). A 5.8 kb mRNA was unique to the brain and a slightly higher band at 6.0 kb was consistently present in heart and adrenal gland but was absent in the brain. A fourth message at 3.4 kb was found predominantly in the brain and was either absent or present at very low levels in the other tissues examined. Under the same conditions, a human platelet ₂-receptor probe hybridized to similar sized messages of 2.1 and 5.8 kb in rat brain and 2.2 and 6.0 kb in rat heart and adrenal gland. This probe, however, failed to detect the abundant 1.3 kb mRNA common to all tissues or the 3.4 kb message in rat brain. The extent of homology of these messages with cA₂-47 is not confined to limited regions of the cDNA since similar hybridization patterns were observed using either 5-noncoding or 5-coding regions of the probe.These results provide the first direct evidence of a surprisingly large range of mRNA sizes for members of the ₂-receptor family in brain, heart, and adrenal gland. The unique nature of certain members of the family in each of the tissues examined raises the curious possibility that these members might contribute to some of the individualized functions of the brain, cardiovasculature and adrenal gland.