Chromophoric peptide substrates for activity determination of animal aspartic proteinases in the presence of their zymogens: A novel assay

Solid-phase synthesis was used for the preparation of pyroglutamyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (I) and glycyl-glycyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (II), two water-soluble and sensitive chromophoric substrates of chicken pepsin, hog pepsin A, and bovine spleen cathepsin D. The kinetic constants of hydrolysis of the p-nitrophenylalanyl-phenylalanyl bond of the substrates were measured by difference spectrophotometry at 308 nm (Δ? = 860 m^?1 cm^?1) and by ninhydrin colorimetry (substrate I, ?₅₇₀ = 2.31 × 10⁴m^?1 cm^?1). The pH optimum of cleavage is 5 for the pepsins and 3.7 for cathepsin D. Since all three proteinases still have a significant activity at pH 5.5–6 a new, simple assay was designed for submicrogram quantities of pepsins in the presence of pepsinogens without interference of the latter. The method is particularly suitable for the analyses of the zymogen activation mixtures.     

Breakdown of Diazotized Proteins and Synthetic Substrates by Rumen Bacterial Proteases

Several different kinds of substrate were used to investigate the proteolytic activity of rumen bacteria and of proteases released from rumen bacteria by blending (“coat proteases”). These substrates included diazotized feed proteins and diazotized soluble and insoluble pure proteins. It was concluded that, while solubility was an important factor, the secondary and tertiary structure of a protein had a major influence on its rate of digestion. The resistance of elastin congo red to digestion indicated that similar fibrous proteins in plant material might resist proteolytic attack by rumen bacteria. Coat proteases had a broad specificity, including several exo- and endopeptidase activities, as determined by using synthetic peptide substrates.     

Differential recognition of the serologically defined HLA-A2 antigen by allogeneic cytotoxic T cells

A comprehensive analysis of human alloimmune cytotoxic T lymphocytes (CTLs) specific for the HLA-A2 antigen identified 11% of HLA-A2 positive cells as outliers. In total, 11 unrelated serologically indistinguishable, but distinguishable by cell-mediated lympholysis (CML) HLA-A2 positive outlier cells were identified. The outlier cells could be subdivided in two subgroups according to reactivity patterns obtained with CTLs directed against the HLA-A2 antigen of outlier cells and their inhibitory capacity in specific competitive inhibition experiments. Thus, the serologically defined HLA-A2 specificity can be divided into at least three subtypes using CTLs specific for the HLA-A2 antigen. Moreover, CTLs specific for an HLA-A2 subtype could be induced when responder cells expressed a different HLA-A2 subtype antigen. On the basis of several family studies, we conclude that the subtype HLA-A2 antigens are inherited in a codominant way.     

Protein kinase activity of purified components of the chicken oviduct progesterone receptor

Preparations of the 90K and 110K components of the chick oviduct progesterone receptor (PR) purified to near homogeneity were tested for protein kinase activity. The 90K component was shown to incorporate radioactive phosphate from [γ-³²P]-ATP in the presence of Ca²⁺ but not of Mg²⁺ ions, while the 110K component was phosphorylated in the presence of Mg²⁺, but not of Ca²⁺. The enzymatic activity of the 90K polypeptide appeared selective, since added proteins (histones) did not become phosphorylated. However, all proteins present in the 110K preparations were phosphorylated in the presence of Mg²⁺. These data suggest that components of the chick oviduct PR display protein kinase activity.     

Application of silver stains to gastric endocrine cells in plastic sections

Summary A simultaneous light and electron microscopic study of mouse gastric mucosa was made to determine whether the silver nitrate methenamine stain of Duk-Ho Lee could be used to stain gastric endocrine-like cells in plastic embedded tissue. Examination of consecutive thick and thin sections showed that this stain blackened the granules of the predominant type of endocrine-like cell present. Blackening of the granules with silver occured in tissue fixed in osmium tetroxide solution with or without dichromate salt or in tissue fixed in glutaraldehyde then treated with osmium. The intensity of staining was deepest in the osmium-dichromate fixed tissue, but the glutaraldehyde-osmium procedure gave less interference from diffuse silver impregnation and better preservation of detail for electron microscopy.     

Electron microscopic observations on iron-labelled secondary lysosomes in renal tubules in mice

Summary Labelling of renal tubule cytosomes with electron dense iron granules can be attained by daily intramuscular injections to mice of an iron sorbitol citric-acid compound in a total of approximately 50 mg Fe⁺⁺⁺/100 g of body weight. The labelled cytosomes correspond to secondary lysosomes and represent heterolysosomes or ambilysosomes. The evidence suggests that tubule and lysosome function are undisturbed by the labelling procedure. The use of this method for fine structural studies of the interaction between secondary lysosomes and other cytoplasmic organelles and elements is indicated.Microbodies do not incorporate administered Fe⁺⁺⁺. The morphological observations support the opinion that these bodies are formed in specialized portions of the smooth surfaced endoplasmic reticulum of proximal tubule cells.Supported in part by grants from the Swedish Medical Research Council (Projects No. K 67-12x-1006-2, B 67-12x-1006-02K, K 68-12x-1006-03, and B 69-12x-1006-04A). The assistance of Miss Silwa Mengarelli and Miss Britt-Marie Pettersson is gratefully acknowledged.     

Bronchial Reactivity to Cigarette and Cigar Smoke

The change in specific airway conductance produced by smoking a cigarette under standard conditions was measured in 91 heavy smokers. Subsequently 19 of the most reactive subjects smoked two cigarettes with different filters and another containing cigar tobacco. The results indicated that reactivity to cigarette smoke was reduced significantly by increasing the retention efficiency of the filter and that reactivity to inhaled cigar-tobacco smoke was no less than that to cigarette smoke.     

Changes in the anatomical structure of the shoot apex ofSenecio vulgaris L. during ontogenis in relation to the formation of leaves and inflorescence

An investigation was made of the anatomical structure of the shoot apex ofSenecio vulgaris L. a photoperiodically neutral plant, and compared with the formation of successive leaf primordia along the axis up to the initiation of the terminal inflorescence. In the shoot apex of a germinating plant a central zone can first be distinguished from the peripheral zone which is composed of small and intensely stained cells. Later, a rib meristem appears. At the time of the initiation of the middle (the largest) leaves, the shoot apex has a distinct small central zone and a well developed peripheral zone and rib meristem. Between these zones there is a group of cells dividing in all directions, the subcentral zone. At the time of initiation of the last leaves, the central zone extends to the flanks and gradually ceases to be distinguishable. At the same time, the subcentral zone increases in size. This is caused first by cell division and later, with the initiation of the last, most reduced leaves, by enlargement of the cells. Vacuolization in the inner part of the apex and the arrangement of the superficial cells in rows parallel to the surface of the apex, is a preparatory step to the initiation of the inflorescence.     

131I in the study of the fate of proteins in the organism

Авторы изучили исче зновения от su131 () Я марки ровкой бычьего сыво роточного альбумина из поток крови и ткан ей, и его распределен ие в subcellular печени дроби у кроликов, в ранний п ериод после администрации, в ход е наиболее заметным иммунологической ре акции и после 19 дней. Результаты этих эксп ериментов показыва ют, что в исследовани и судьбы белка антигенов в организ м белков йодированн ой пригодны для перв ого этапа только, i. д. распределение в тка ни, жидкости, белка половину жизни и имм унной фазы исчезнов ения от антигена. Так как в контрольны х группах, к которым K () su131 управление, йода со храняется в тканях деятельность по 19 ден ь, никто не может исключать возможно сть-даже в экспериме нтах в котором йод с рекламой белка была использована, то в настоящее время деятельность ткани в последующих этапов уже не доказ ательства присутст вия белка.