Preferential action of rat brain cathepsin B as a peptidyl dipeptidase converting pro-opioid oligopeptides

Purified rat brain cathepsin B (EC 3.4.22.1) converted prodynorphins or proenkephalins to shorter active forms by the preferential removal of C-terminal dipeptides. The substrate affinities for Met-enkephalin-Arg-Phe or -Arg-Gly-Leu were Km 46 and 117 microM, and kcat/Km ratios were 67 and 115 microM-1, min-1, respectively. Met-Enkephalin was inactivated by the same mechanism (Km-450 microM; kcat/Km = 0.12 microM-1 min-1). The comparison of cathepsin B hydrolysis for pro-opioids, a synthetic hexapeptide and its fragments, C-blocked peptides (pro-opioid amides, Met-enkephalin amide, substance P), and bovine myelin basic protein, provided information on the influence of the C-terminal residues on dipeptide release, the rates as correlated to peptide length, and the optimal arrangement of residues favoring scission at the P1-P'1 sites. The brain enzyme was stereospecific and did not act on peptides with C-terminal D-amino acid substituents. Arg hindered and Pro blocked the release of C-terminal dipeptides when in the P'2 positions. The suppression of dipeptide release by agents inhibiting endopeptidase actions such as E-64 and leupeptin, and the endogenous brain factor (cerebrocystatin) point to similar catalytic mechanisms for the exopeptidase action.     

Conservation of the DNA binding domain and other properties between porcine and rat glucocorticoid receptors

Activated glucocorticoid receptor protein (GCR) was partially purified from porcine liver cytosol by sequential chromatography on phosphocellulose and DNA-cellulose using a modification of a protocol developed for purification of rat GCR. This partially purified preparation, when separated by SDS-polyacrylamide gel electrophoresis and immunoblotted, indicated that a Mr = 94,000 protein band cross-reacts with a monoclonal antibody against rat GCR. A nitrocellulose filter binding assay showed that both the partially purified porcine and rat GCRs interact specifically with a cloned synthetic 24 base pair deoxyoligonucleotide containing the GCR binding sequence in the first intron of the human growth hormone (hGH) gene. This specific protein-DNA interaction is blocked by a single base pair change in the binding site. All three putative domains of the GCR molecule: the steroid binding, immunoreactive, and DNA binding have been conserved between two divergent species.     

Seed dispersal and seedling emergence in an old field community in central New York (USA)

Summary Seed dispersal and seedling emergence of common taxa growing in a Solidago-dominated old field in central New York (USA) were monitored from May 1982 to June 1984. Over 3.5x10⁴ seeds per m² were captured on seed traps in each of the two years, with peaks occuring in July (due to Hieracium) and in November (due to Solidago). About 4.0x10³ seedling/m² emerged beneath the intact community in each of the two years. Although seedlings emerged predominantly in the early spring, a secondary peak occurred in September and October when many seedlings of introduced grasses appeared. Two additional aspects of the reproductive biology of the major taxa were related to the seasonal timing of seed dispersal: As the date of peak seed dispersal (among taxa) became progressively later in the season, (1) the duration of dispersal increased from about one week to about one-half year, and (2) the delay between the peak of seed dispersal and the peak of seedling emergence increased from a few days to about one-half year.     

Production of native, correctly folded bovine pancreatic trypsin inhibitor by Escherichia coli

A gene for bovine pancreatic trypsin inhibitor (BPTI) was fused to the coding sequence for the Escherichia coli alkaline phosphatase signal peptide and expressed in E. coli under the control of the alkaline phosphatase promoter. When induced in phosphate-depleted medium such cells produced a trypsin inhibitor that was indistinguishable from native, properly folded BPTI. In particular, the BPTI produced by E. coli had three disulfide bonds that appeared to be identical to those found in native BPTI, as assayed by sensitivity to iodoacetate, dithiothreitol, and urea. This expression/secretion system will make possible the production of variant BPTI molecules, thus allowing the perturbing effects of amino acid substitutions on BPTI folding, structure, and function to be assessed.     

Use of diathermy for weeding heterogeneous tissue cultures

Summary Cultures generated from tissues consisting of multiple types of cells are often heterogeneous. Unless the cell type of interest has or can be given some selective growth advantage it may be overgrown by other cells. While developing techniques for the tissue culture of microvascular endothelial cells we evaluated an electrosurgical generator (diathermy) to selectively kill nonendothelail cells. Primary cell cultures were observed at ×100 magnification under phase contrast microscopy and a needle electrode apposed to the cell to be destroyed. A return electrode was constructed by placing a sterile clip in contact with the culture medium. The diathermy power setting controlled the area of lysis. Use of this technique allowed weeding of unwanted cells without damage to endothelial cells, which were able to grow to confluence in pure culture. Dr. Marks receives a Medical Postgraduate Research Scholarship from the National Health and Medical Research Council of Australia. Financial support was received from the Leo Leukaemia and Cancer Research Trust and the Scleroderma Association of New South Wales.     

A portable silicon photodiode luminometer

A simple, inexpensive, battery-powered, portable luminometer which is based on a silicon photodiode is described. The instrument is intended to measure the light produced by chemiluminescent and bioluminescent reactions. The devic shows a good detection limit and, in a bioluminescent reaction for adenosine 5′-triphosphate (ATP), detected 0.5 pmol in 1ml of aqueous solution. The instrument measures irradiance from 10^?13 to 10?¹¹ W cm^?2 at the sensor, within the range 300 to 900nm.     

Differential insulin responses to oral and intravenous glucose in the transplantable rat insulinoma--a role for GIP

We have previously demonstrated an impaired insulin response to intraperitoneal glucose and arginine by the transplantable NEDH rat insulinoma. The nature of this tumour B-cell defect has been further studied by investigating the response of insulinoma-bearing rats to intravenous and intragastric glucose. Intravenous glucose failed to stimulate plasma immunoreactive insulin (IRI) above high basal levels (14.5 +/- 1.1 micrograms/L). However, significant elevation of the plasma IRI concentration was observed following an intragastric glucose load (17.1 +/- 1.5 micrograms/L; P less than 0.02). In view of the different effects of oral and intravenous glucose on insulin secretion in the RIN, implicating an involvement of incretin factors from the gut, the response of the tumour to GIP was investigated. Plasma IRI concentrations rose significantly in these animals (20.6 +/- 2.5 micrograms/L at 5 min, P less than 0.02). We conclude that (a) the transplantable rat insulinoma is responsive to GIP, and (b) that whilst the tumour B-cell has lost its insulin responsiveness to hyperglycaemia produced by intraperitoneal or intravenous glucose, it retains its ability to respond to intragastric glucose. This could be due to incretin factors from the gut of which GIP is currently the strongest candidate.     

Synthesis of S100 Protein on Free and Membrane-Bound Polysomes of the Rabbit Brain

Abstract: Free and membrane-bound polysomes were isolated from the cerebral hemispheres and cerebellum of the young adult rabbit. The two polysomal populations were translated in an mRNA-dependent cell-free system derived from rabbit reticulocytes. Analysis of the [³⁵S]methionine-labeled translation products on two-dimensional polyacrylamide gels indicated an efficient separation of the two classes of brain polysomes. The relative synthesis of S100 protein by free and membrane- bound polysomes was determined by direct immuno-precipitation of the cell-free translation products in the presence of detergents to reduce nonspecific trapping. Synthesis of S100 protein was found to be twofold greater on membrane-bound polysomes compared with free polysomes isolated from either the cerebral hemispheres or the cerebellum. In addition, the proportion of poly- (A+)mRNA coding for SlOO protein was also twofold greater in membrane-bound polysomes compared with free polysomes isolated from the cerebral hemispheres. These results indicate that the cytoplasmic S100 protein is synthesized predominantly on membrane-bound polysomes in the rabbit brain. We suggest that the nascent S100 polypeptide chain translation complex is attached to the rough endoplasmic reticulum by an ionic interaction involving a sequence of 13 basic amino acids in S100 protein.     

Foraminifera from the Silberberg Formation («Lower Oligocene») at Silberberg,near Helmstedt (Germany)

From the Silberberg Quarry, type locality of the Silberberg Formation (Martini &Ritzkowski 1968), some planktonic foraminifera are described:Globigerina officinalis Subbotina, Globigerina ouachitaensisHowe &Wallace,G. praebulloides leroyi Blow &Banner, G.praebulloides occlusa Blow &Banner which taxa are here suggested to form intergrading morphotypes within the range of variation of a population dominated byG. officinalis. In addition,G. cf.danvillensis Howe &Wallace is found. The planktonic fauna, together with some elements in the benthonic fauna, suggests a possible correlation with the Danville Landing Member of the Jackson Formation, Louisiana, traditionally ascribed to the Upper Eocene. There is some similarity with European Uppermost Eocene — Lower Oligocene faunas described by various authors. The Silberberg Formation was suggested to be a Neostratotype for the Latdorfian Stage of the Lower Oligocene byMartini &Ritzkowski (1968). It is suggested that the Silberberg Formation may well be coeval with the Upper Priabonian as proposed byCita 1969.