Acid proteinase of hypothalamus

In a continuing study of the physiological role of protein breakdown in the hypothalamus, acid proteinase from bovine hypothalamus was purified about 1000-fold. The molecular weight of the enzyme was approximately 50,000. Maximal activity against hemoglobin was obtained at pH 3.2–3.5; serum albumin was split much more slowly. Hypothalamus acid proteinase was partially inhibited by -phenyl pyruvate, or benzethonium Cl, and was completely inhibited by low concentrations of pepstatin. This proteinase splits somatostatin, substance P, and analogs of substance P. The probable sites of enzyme action on these peptides were determined by the end group dansyl technique. The enzyme, most likely cathepsin D, may play an important role in the formation and breakdown of peptide hormones in the hypothalamus.     

PROLIDASE ACTIVITY IN BRAIN: COMPARISON WITH OTHER ORGANS

Prolidase (imidodipeptidase, EC 3.4.3.7) activity in brain was assayed spectrophotometrically, and the validity of the assay was confirmed by microchromatographic analysis of the dansyl derivatives of the reaction products. The ratio of the activity of prolidase from brain on Ala-Pro, Gly-Pro, and Pro-Pro was similar to that of the highly purified intestinal mucosal prolidase. The size and polarity of the amino acid side chain (R) of the N-terminal amino acid and whether the terminal amino and carboxy groups are free or blocked are important factors in determining prolidase substrate activity. Using Ala-Pro as substrate, we found prolidase activity in all rat tissues we investigated, with highest activity in kidney and lowest in plasma; 33 mmol of Ala-Pro was hydrolyzed per mg of protein per min by brain tissue, which is 20% of the activity for kidney and 23% of that for intestinal mucosa. The specific activity of prolidase was higher in the sciatic nerve than in brain or spinal cord, and activity was higher in the distal portion.     

THE EFFECTS OF RED BLOOD CELL EXTRACTS ON THE PROLIFERATION OF ERYTHROCYTE PRECURSOR CELLS, IN VIVO

Saline incubation extracts of mature erythrocytes were assayed in vivo by a variety of techniques in order to study their ability to modify the proliferation of maturing erythroid cells. Using comparable extracts from granulocytes and lymphocytes, the specificity of the effect of the red cell extract for erythroid cells was confirmed by measurement of autoradiographic labelling indices, radio-iron incorporation and spleen colony growth. The erythroid cells were found to be very sensitive to the effects of the extract, as little as 10 μg per mouse producing a maximum effect on iron incorporation. It was found that the extract does not block erythroid cell proliferation completely but simply lengthens the cell cycle, mainly by increasing the G₁ phase of the cycle. There was no effect on the committed erythroid precursor cells. The in vivo activity, specificity and non-toxicity to the cells, together with the cells' sensitivity to red cell extract suggest, therefore, that this inhibitor may play a physiological role in the control of red cell production.     

Developmental changes in the breakdown of brain tubulin by cerebral cathepsin D

The activity of cathepsin D on hemoglobin and on cytoplasmic tubulin was measured in brain preparations at different ages—in newborn, 10-and 21-day-old, and young adult rats. Enzyme activity increased after birth, reaching a maximum at around 21 days, and then declined. This increase was not parallel with decreased turnover of proteins during development, but was parallel with decreasing level and increasing microheterogeneity and rate of assembly of tubulin during development. The breakdown of tubulin was heterogeneous, with initial fast breakdown of a large portion, followed by breakdown at a lower rate. This heterogeneity in breakdown persisted throughout development. The breakdown of tubulin, unlike that of hemoglobin, was at all ages greater at pH 5.8 than at pH 3.2. The possible role of cathepsin D in tubulin metabolism and the developmental changes under physiological conditions need further exploration.     

Chromosomal localization of transfected genes by a combination of hot banding and fluorescence in situ hybridization.

We describe the combination of hot banding with fluorescence in situ hybridization as a rapid and efficient method to identify integration sites of transfected DNA sequences in chromosomes. As a test system we used SW480 EJ2, a clonal cell line obtained after transfection of SW480 with pSV2neoEJ, a plasmid containing a point-mutated, c-Ha-RAS oncogene. Nick-translated probes were compared with random primed-labeled probes to evaluate their relative efficiency in fluorescence in situ hybridization. The fluorescence signals were quantified in interphase nuclei by confocal scanning laser microscopy. Nick-translated probes were found to yield better results. Hot banding followed by fluorescence in situ hybridization localized the integration site of pSV2neoEJ in SW480 EJ2 at the site of a translocation on a marker chromosome Xp+. The combination of fluorescence in situ hybridization and hot banding can be used to (a) rapidly and efficiently analyze integration sites in large numbers of transfectants, (b) assess the clonality of transfected cell lines, and (c) localize the site of integration of transfected genes in the recipient genome.     

The intracellular distribution of adenylate kinase in the leaves of spinach,wheat and barley

Of the total adenylate-kinase activity in 10-d-old barley and wheat leaves, 40–50% is localised in the chloroplasts, while in mature spinach leaves 50–70% of the enzyme is chloroplastic. The extra-chloroplastic adenylate-kinase activity is associated with the mitochondria, very little, if any, is freely soluble in the cytoplasm. The adenylate pool of the cytoplasm could have access to adenylate-kinase activity in the intermitochondrial space because of the free permeation of adenylates across the outer mitochondrial membrane. Thus the adenylate pool of the cytoplasm could be subject to adenylate-kinase equilibrium. The mitochondrial adenylate kinase appeared to the localised exclusively in the intermembrane space.     

Endogenous peptide(s) inhibiting [3H]cocaine binding in mouse brain

The supernatant fraction of centrifuged homogenate of brain tissue contains material that inhibits the saturable binding of [³H]cocaine to crude mouse brain membranes. This material was subjected to heat treatment to remove protein; further purification was achieved by filtering through an Amicon UM-10 membrane ultrafilter and gel filtration of the ultrafiltrate on Sephadex G-25. Sensitivity to acid hydrolysis and peptidase action indicates that the inhibitory activity resides in peptide material with a low molecular weight. The partially purified inhibitor has similar effects to that of cocaine on the specific binding of various ligands to opiate and nonopiate receptors in mouse brain membranes.     

Mechanisms of haemopoietic stem cell proliferation control

The control of stem cell (CFU-S) proliferation is mediated by short-range acting factors which can be detected by the proliferation modifying activities present in media conditioned by haemopoietic cells. A specific inhibitor of stem cell proliferation is obtained from haemopoietic tissue containing minimally proliferating CFU-S, whilst stimulatory material is obtained from cell suspensions containing rapidly proliferating CFU-S. Used competitively, these factors, which are detected in different molecular weight range fractions, manipulate the rate of CFU-S proliferation in a manner compatible with a physiological control mechanism. In addition, a long-term bone marrow culture system has been shown to provide an in vitro model of stem cell control. Fractionation of cell populations from haemopoietic tissues reveals marked concentration differences of the CFU-S proliferation modifying activities depending on the proliferative state of the CFU-S. However, irrespective of whether the tissue contains stem cells that are actively or minimally proliferating, both stimulatory and inhibitory activities are detected. From dose-response studies it is concluded that stem cell proliferation is controlled by an appropriate balance of stimulatory and inhibitory factors which, however, are not produced by the stem cells themselves.