Separation and properties of a “Neutral” hexosaminidase from embryonic chicken brain

• 1.1. A “neutral” hexosaminidase has been separated from other hexosaminidase forms (I and II) by DEAE-cellulose chromatography and characterized in embryonic (16-days old) and 1-day old chicken brains.
• 2.2. Its properties differ from those of the forms I and II. It has optimum activity at about pH 6.0 and can be eluted from DEAE-cellulose with 0.25 M KCl only.
• 3.3. It has no N-acetylgalactosaminidase activity and cannot be successfully detected after isoelectric focusing since it is very acidic and completely unstable below pH 5.0.
• 4.4. “Neutral” hexosaminidase is heat-stable at pH 6.0 and is inhibited by chloride.
• 5.5. These properties, very different from those of forms I and II, suggest that this “neutral” form of hexosaminidase would be very similar to known hexosaminidase C separated from other materials.
• 6.6. We have found no significant differences for the above-mentioned three forms in chick embryos (16-days old) in comparison with those from 1-day old chicken.

     

Monoclonal antibodies prepared against heparin lyase I and their reactivity toward heparin lyase I,II and III

• 1.1. Six different monoclonal IgG mouse antibodies to heparin lyase I from Flavobacterium heparinum were prepared.
• 2.2. The monoclonal antibodies were used to detect heparin lyases I, II and III by dot-blotting immunoassay and by Western blotting.
• 3.3. Individual antibodies showed different reactivity toward the three heparin lyases.
• 4.4. The reactivity of two of the monoclonal antibodies was destroyed by exposing heparin lyases to sodium dodecyl sulfate.
• 5.5. The antibodies can be used to rapidly distinguish between the three heparin lyases.

     

The neural control of release of hyperlipaemic hormone from the corpus cardiacum of Locusta migratoria

• 1.1. The neural control of the release of hyperlipaemic hormone has been studied in isolated corpora cardiaca of Locusta migratoria using electrical stimulation of the nervi corporis cardiaci I and II, and assaying the bathing medium for hyperlipaemic activity.
• 2.2. Stimulation of nervus corpus cardiacum II (NCC II) results in the release of hormone which is dependent upon the arrival of compound action potentials within the glandular lobe of the corpus cardiacum.
• 3.3. Sodium deficient Ringer and Ringer containing 10⁻⁶ M tetrodotoxin abolish the compound action potential and with it the release of hormone.
• 4.4. The mechanism of release is dependent upon the presence of extracellular calcium-ions.
• 5.5. Stimulation of nervus corpus cardiacum I (NCC I) alone does not result in the release of hyperlipaemic hormone, but the presence of action potentials in NCC I potentiates the hyperlipaemic effect obtained by NCC II stimulation.

     

Regulatory role of δ-aminolevulic acid dehydratase in the dimorphic fungus Mucor rouxii

• 1.1. With the aim of finding a possible relationship between the known dimorphism phenomenon existing in the fungus Mucor rouxii and the biosynthesis of respiratory pigments, the activity of aminolevulic acid synthetase (ALA-S) and ALA-dehydratase (ALA-D) was studied in crude extracts and in 15,000 g supernatants of both mycelium and yeast-like cells.
• 2.2. The activity of ALA-S was unusually high (3 nmol ALA/hr/mg protein) compared with that reported for other tissues and did not vary with the fungus morphology.
• 3.3. Instead, ALA-D specific activity was found to be 16.5 nmol PBG/hr/mg protein in mycelium extracts, that is 7-fold greater than that measured in the yeast-like morphology (2.6 nmol PBG/hr/mg protein).
• 4.4. It was of importance to determine the activity levels of ALA-D along with the morphogenic transition from yeast to mycelium. It was observed that the greatest change and enhancing of specific activity occurred 2 hr before the emergence of the germ tubes and was held constant up to the complete development of mycelium.
• 5.5. Both hyphae formation and enhancement of ALA-D activity were diminished when cAMP was added to the culture shifted from the anaerobic atmosphere to air.
• 6.6. These findings and preliminary studies on the characterization of M. rouxii ALA-D indicate that this enzyme plays a regulatory role in porphyrin biosynthesis in this fungus as well as a key function in the characteristic morphogenic transition.

     

Dipeptidyl aminopeptidase activities of guinea-pig brain

• 1.1. The subcellular distribution ofdipeptidyl aminopeptidase activities in guinea-pig brain was investigated. Our studies show that DAP I (Gly-Arg-NH-Mec hydrolase) type activity was found to have an acidic optimum and was associated with the nuclear pellet.
• 2.2. No DAP II (Lys-Ala-NH-Mec hydrolase) type activity could be detected. Apparant hydrolysis was mainly due to aminopeptidase activity.
• 3.3. DAP III (Arg-Arg-NH-Mec hydrolase) type activity is largely cytoplasmic, but there was evidence of a membrane form associated with the synaptosomes.
• 4.4. DAP IV (Gly-Pro-NH-Mec hydrolase) type activity is present on the synaptosomal membrane, and also enriched in the microsomes. A soluble form of Gly-Pro-NH-Mec hydrolase activity is also present in the cytoplasm. Whether this activity is a DAP II or IV type activity is still yet to be determined.

     

The binding and degradation of 125I-labelled insulin by rat kidney brush-border membranes

• 1.1. The interaction of insulin with purified brush-border membranes from rat kidney was studied with the use of [¹²⁵I]insulin.
• 2.2. The specific binding of insulin by brush-borders could be demonstrated, and was time- and temperature-dependent.
• 3.3. [¹²⁵I]insulin was displaced by unlabelled insulin. A₁-B₂₉ dodecoyl insulin and insulin A- and B-chains in proportion to their relative bioactivity.
• 4.4. Brush-border membranes showed high insulin-degrading activity with an apparent K_m of 2.2 μM.
• 5.5. A number of proteinase inhibitors were effective in inhibiting insulin degradation but the greatest degree of inhibition was achieved by the use of thiol-blocking reagents.
• 6.6. No evidence was obtained for the involvement of the enzyme glutathione-insulin transhydrogenase.

     

Specific biochemical and immunological properties of some water-soluble glycoproteins produced by rat gastric mucosal scrapings in vitro

• 1.1. Two major glycoprotein components were released in vitro by rat gastric mucosal cells after 4 hr incubation with radioisotopes: a high molecular weight fraction with the characteristics of a fucomucin and a low molecular weight fraction, the latter having a higher specific radioactivity than the former.
• 2.2. Pulse chase experiments indicate that several low and high molecular weight glycoproteins are synthesized simultaneously with no precursor-product relationship between them.
• 3.3. Common antigenic determinants, specific to the stomach were found on the 2 fractions, using immunofluorescence, both fractions appeared to be present in the same cells.

     

Neurohypophysial hormones as molecular evolutionary tracers: investigations on the sturgeons Acipenser stellatus and Acipenser guldenstadti

• 1.1. Neurohypophysial hormones of two sturgeon species, Acipenser stellatus and Acipenser guldenstadti, have been purified through molecular sieving on Bio-Gel P4 and reverse-phase high pressure liquid chromatography on Nucleosil C18 columns.
• 2.2. Arginine vasotocin has been identified in both species by its retention time in partition chromatography, amino acid composition and, in the case of A. stellatus, by amino acid sequencing.
• 3.3. A second peptide has been purified and could be α-deamidated vasotocin.
• 4.4. Another peptide with oxytocic activity, distinct from the known oxytocin-like peptides, seems to be present in very small amounts.

     

Soluble cyclic amp-dependent protein kinases from chick liver: Effects of NaCl and heat-stable modulator

• 1.1. Two cyclic AMP-dependent protein kinases—Fraction I and II—have been isolated from chick liver soluble preparation on DEAE-cellulose.
• 2.2. Both fractions have an apparent K_m for ATP of 2 × 10⁻⁶M, are stimulated maximally by 5 × 10⁻⁸ M cyclic AMP and phosphorylate mainly basic proteins—histone and protamine.
• 3.3. They exhibit various pH values for optimal activity and show differences with respect to both sensitivity to NaCl and substrate specificity.
• 4.4. The heat-stable protein modulator inhibits the cyclic AMP-dependent protein kinase activity of both fractions, but with cyclic GMP one kinase is stimulated and the other inhibited.
• 5.5. Slight differences in histone triggered holoenzyme dissociation as well as the lack of difference between their ability for subunit reassociation do not allow to classify these isozymes as protein kinases of Type I and II, according to Corbin et al. (1975).

     

A comparative study of nuclear and chloroplast DNA dependent RNA polymerases from wheat leaves

• 1.1. Three DNA dependent RNA polymerases have been purified from chromatin and chloroplast fractions of wheat leaves.
• 2.2. The purified enzymes were completely dependent on exogenous DNA after purification by glycerol gradient, DEAE-Sephadex and phosphocellulose chromatography.
• 3.3. The nuclear enzymes, I and II, showed a strong preference for denatured nuclear DNA, whereas the chloroplast enzyme preferred denatured chloroplast DNA.
• 4.4. The three enzymes require either Mg²⁺ or Mn²⁺ for activity.
• 5.5. α-amanitin specifically inhibited RNA polymerase II but has no effect on polymerase I and chloroplast polymerase.
• 6.6. Enzyme I is most active at very low ionic strength (0.10 mM KC1), whereas enzyme II and chloroplast enzyme show maximum activity at 150mM and 50 mM KC1 respectively.

     

Immunochemical evidence for a gastrin-like peptide in the intestinal tissues of the earthworm Lumbricus terrestris

• 1.1. The presence of gastrin-like immunoreactive materials in intestinal tissues of the earthworm Lumbricus terrestris has been demonstrated by means of radioimmunoassay technique.
• 2.2. The greatest activity coincides with the areas of the intestines with high digestive enzyme activities. No gastrin-like activities were detected in nerve tissues.
• 3.3. The results also indicate that earthworm gut tissues may contain different forms of gastrin or gastrin-like peptides.
• 4.4. The absence of gastrin-like activity in earthworm nerve tissues may have significant phylogenetic implication for the proposed gastrin-cholecystokinin origin in neuronal elements of invertebrates.

     

A theoretical interpretation for M/F ratio index in electroantennogram of the American cockroach

• 1.1. In addition to the sexually active and inactive general odorous compounds for which behavioral activity and M/F I evaluations (male/female I value) have been done, several compounds were evaluated here for M/F I value.
• 2.2. To the reference M/F I value of camphor (1.0), the values were estimated to be 1.5–6.9 for sexually active compounds, and 0.5–1.4 for general odorous compounds having interaction with a sex pheromone receptor (SPR).
• 3.3. A theoretical interpretation was made for M/F I on the basis of the interaction of the compounds to general odor receptor (GOR) and SPR, and the resulting M/F I values.
• 4.4. In the M/F I interpretation, affinity and intrinsic activity of the compounds to SPR were newly introduced as factors.
• 5.5. It was assumed theoretically that EAG response due to the interaction of intrinsic activity of the compounds caused various electric responses (negative-, zero- and positive-signed responses), when the compounds had affinity to SPR.
• 6.6. Intrinsic activity of the sexually active compounds was expected to be a main factor to govern their pheromone activity, ie. M/F I values.

     

Light-induced effects of several enzymes of carbohydrate metabolism in Phycomyces blakesleeanus

• 1.1. The photoregulation shown by glyceraldehyde 3-phosphate dehydrogenase and glucose 6-phosphate dehydrogenase appears to be independent of the mad gene product(s) and also independent of carotene biosynthesis regulation.
• 2.2. The photoregulation of malate dehydrogenase appeared to be dependent on the mutation of the mad and car S genes.
• 3.3. Pyruvate kinase and lactate dehydrogenase may be classified as light-independent.
• 4.4. The action of ATP and fructose 1,6-bisphosphate on the enzymes studied was generally independent of light/dark grown conditions.
• 5.5. However, the effect of fructose 1,6-bisphosphate on Phycomyces pyruvate kinase appears to be light-dependent.

     

Larval Musca domestica lipophorin biosynthesis

• 1.1. Larval Musca domestica lipophorin biosynthesis was studied in vitro.
• 2.2. The newly synthesized lipophorin has a density a little lower than the circulating lipophorin after 1 hr of incubation. After 3 hr of incubation the fat body cells transfer lipids to the lipophorin that attains the density of circulating lipophorin.
• 3.3. The lipophorin synthesized in vitro is identical to circulating lipophorin in density and in electrophoretical behavior.
• 4.4. However these two molecules must have differences since the circulating lipophorin transfers lipids to fat body cells while the synthesized in vitro does not.
• 5.5. The biosynthesis of Musca lipophorin shows differences with the Manduca sexta lipophorin biosynthesis.

     

Light-induced transient increase of the activity of topoisomerase I in plasmodia of Physarum polycephalum

• 1.1. Activity of topoisomerase I and incorporation of [³H]uridine and [¹⁴C]thymidine were monitored during light-induced sporulation of the slime mold Physarum polycephalun.
• 2.2. A 4-fold transient increase of topoisomerase I activity but not of [³H]uridine or [¹⁴C]thymidine incorporation was observed after 42 hr of illumination with 6 hr impulses.
• 3.3. The activity of topoisomerase I did not increase in the absence of light impulses. However, ca 5-fold increase of the activity was observed in dark when 100 μ M dibutyryl-cAMP was administered 12 hr before harvesting of plasmodia.
• 4.4. Fluorodeoxyuridine and cycloheximide administered 36 hr after starting of the illumination cancelled the increase of the activity of topoisomerase I.
• 5.5. After 7 days of the illumination, when fruiting bodies appeared, the activity of topoisomerase I dropped to about 15% of the initial value.

     

The accumulation of age pigments during larval development of the spider crab,Hyas araneus (Decapoda,Majidae)

• 1.1. Lipofuscin, body carbon and respiration rates were measured in Hyas araneus from hatching to metamorphosis. Lipofuscin was measured spectrofluorometrically from the chloroform phase of chloroform/methanol extracts.
• 2.2. Excitation/emission spectra of both the chloroform and the methanol/aqueous phase showed one distinct fluorescence peak in the chloroform (410–415 nm emission/340–350 nm excitation) and the methanol/aqueous phase (405/350 nm) of zoea I (directly after hatching) and megalopa (0 and 24 days old).
• 3.3. Individual lipofuscin concentrations increased continuously during zoea I and halfway through zoea II, but remained constant through the entire megalopa despite high metabolic activity in this stage.
• 4.4. Individual lipofuscin concentrations were positively correlated with body carbon and carbonspecific lipofuscin was negatively correlated.
• 5.5. Moulting caused considerable loss of lipofuscin. During the first two larval ecdyses 17–18% were lost, with the shed moults containing only 3.4–4.5% of the lipofuscin found in late premoult individuals.
• 6.6. The different patterns of lipofuscin accumulation in respective larval stages is discussed in regard to mitotic activity of tissues. While in the zoea, growth is more related to lipid formation and biomass accumulation, in the megalopa morphogenetic processes require substantial epidermal growth, i.e. protein accumulation. However, the question why in the megalopa no increase in lipofuscin is found, remains unanswered.

     

Rat liver endoplasmic reticulum protein kinases

• 1.1. Rat liver microsomal membranes were studied for the presence of protein kinases. Microsomal proteins solubilized with Triton X-100 were analyzed by means of ion exchange chromatography.
• 2.2. Protein kinase activity was detected in the column fractions using specific assays for cAMP-dependent protein kinase, cGMP-dependent protein kinase, protein kinase C, Ca²⁺/calmodulin-dependent protein kinase and casein kinases.
• 3.3. Fractions with protein kinase activity were further analyzed by SDS-polyacrylamide gel electrophoresis.
• 4.4. The results indicate that cAMP-dependent protein kinase type I and II, casein kinases I and II, protein kinase C proenzymes I and II and Ca²⁺ /calmodulin kinase II are associated with the membranes of endoplasmic reticulum (ER).

     

Proteolysis post mortem in North Atlantic krill

• 1.1. The autoproteolytic processes in selected species of North Atlantic krill, Meganyctiphanes norvegica (M. Sars), Thysanoessa inermis (Krøyer) and T. raschii (M. Sars) have been examined at 0°C by following the release of peptides and free amino acids.
• 2.2. The krill contains high levels of peptide hydrolases, and autoproteolysis seems to be due mainly to digestive enzymes localized in the hepatopancreas and the intestinal tract of the animals.
• 3.3. During autoproteolysis the individual amino acids were generally released at rates corresponding to their proportion in the bulk protein of the krill. The major exceptions were alanine which accumulated in amounts larger than was to be expected from the composition of the krill protein, and glutamic acid/glutamine, aspartic acid/asparagine, arginine, and to some extent glycine, proline and serine, which accumulated to a lesser extent than was to be expected.
• 4.4. Storage of krill for 1 week resulted in only minor changes in the total content of amino acids as determined after acid hydrolysis, with the exception of alanine which increased in concentration.
• 5.5. The results suggest that the formation of free alanine is partly due to reactions other than proteolysis.
• 6.6. The release of free amino acids was accompanied by a considerable increase in the amount of small peptides, and glutamic acid/glutamine, aspartic acid/asparagine, glycine and proline tended to accumulate in these peptides.
• 7.7. The autoproteolytic activity of the Thysanoessa species showed seasonal variations, probably in response to food availability. In the case M. norvegica, the results suggest that there are smaller fluctuations in the level of proteolytic enzymes, probably indicating less pronounced variations in the food intake over the year.

     

Effect of methyl methacrylate on mitochondrial function and structure

• 1.1. Treatment of isolated rat liver mitochondria with methyl methacrylate (MM) produced membrane disruption as evidenced by the release of citrate synthase, and changes in the ultrastructure of mitochondria.
• 2.2. At concentration 0.1%, MM uncoupled oxidative phosphorylation as evidenced by stimulation of state 4 respiration supported either by pyruvate plus malate or succinate (+rotenone) and ATP-ase activity in intact mitochondria.
• 3.3. At concentration 1% MM stimulated ATP-ase activity in intact mitochondria and succinate (+rotenone) oxidation at state 4 and was without effect on this substrate oxidation at state 3.
• 4.4. MM inhibited pyruvate plus malate oxidation either at state 3 or in the presence of uncoupling agents.
• 5.5. MM inhibited the NADH oxidase of electron transport particles at a concentration which failed to inhibit either succinic oxidase or the NADH-ferricyanide reductase activity.
• 6.6. The data presented suggest that in the isolated mitochondria MM inhibits NADH oxidation in the vicinity of the rotenone sensitive site of complex I.
• 7.7. The general conclusion is that MM may block an electron transport and to uncouple oxidative phosphorylation in rat liver mitochondria. The overall in vitro effect would be to prevent ATP synthesis which could result in cell death under in vivo conditions.