Purification and properties of cathepsin D from the mammary glands of lactating rabbits

Cathepsin D was purified from the lactating rabbit mammary gland by a rapid procedure, which included fractionation with (NH4)2SO4, acid precipitation, double affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100, resulting in approximately 360-fold purification of the enzyme over the homogenate and approximately 16% recovery. After isoelectric focusing, the enzyme dissociated into four (pI 5.8, 6.3, 6.5 and 7.2) multiple forms, but appeared homogeneous on polyacrylamide gel electrophoresis. Cathepsin D has a Mr of 45 kDa as determined by Sephadex G-100 column chromatography. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme gave a single protein band, corresponding to Mr of 45 kDa. The amino acid composition of the enzyme is similar to that of cathepsins D from other tissues. A single N-terminal amino acid was glycine. Cathepsin D contains 6.4% carbohydrates consisting of mannose, galactose, fucose and glucosamine at a ratio of 3:9:2:2. Cathepsin D is inhibited by pepstatin with Ki of 2.5 X 10(-9) M and irreversibly by N-diazoacetyl-N'-2.4-dinitrophenyl-ethylene diamine. The enzyme hydrolyzes bovine hemoglobin with the maximal activity at pH 3.0 with Km = 10(-5) M and HLeu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe with Km = 4 X 10(-5) M and Rcat = 0.95 s-1. The major cleavage sites were Leu15-Tyr16, Phe24-Phe25 and Phe25-Tyr26 during hydrolysis of the oxidized insulin B-chain by cathepsin D.     

Automated extraction of bank angles from bird‐borne video footage using open‐source software

The use of miniaturized video cameras to study the at‐sea behavior of flying seabirds has increased in recent years. These cameras allow researchers to record several behaviors that were not previously possible to observe. However, video recorders produce large amounts of data and videos can often be time‐consuming to analyze. We present a new technique using open‐source software to extract bank angles from bird‐borne video footage. Bank angle is a key facet of dynamic soaring, which allows albatrosses and petrels to efficiently search vast areas of ocean for food. Miniaturized video cameras were deployed on 28 Wandering Albatrosses (Diomedea exulans) on Marion Island (one of the two Prince Edward Islands) from 2016 to 2018. The OpenCV library for the Python programming language was used to extract the angle of the horizon relative to the bird’s body (= bank angle) from footage when the birds were flying using a series of steps focused on edge detection. The extracted angles were not significantly different from angles measured manually by three independent observers, thus being a valid method to measure bank angles. Image quality, high wind speeds, and sunlight all influenced the accuracy of angle estimates, but post‐processing eliminated most of these errors. Birds flew most often with cross‐winds (58%) and tailwinds (39%), resulting in skewed distributions of bank angles when birds turned into the wind more often. Higher wind speeds resulted in extreme bank angles (maximum observed was 94°). We present a novel method for measuring postural data from seabirds that can be used to describe the fine‐scale movements of the dynamic‐soaring cycle. Birds appeared to alter their bank angle in response to varying wind conditions to counter wind drift associated with the prevailing westerly winds in the Southern Ocean. These data, in combination with fine‐scale positional data, may lead to new insights into dynamic‐soaring flight.     

The uptake of liposome-entraped 125I-labelled poly(vinylpyrrolidone) by rat jejunum in vitro

The uptake of free and liposome-entrapped ¹²⁵I-labelled poly(vinylpyrrolidone) was measured in an intestinal sac preparation from adult rats. An an equal concentration of ¹²⁵I-labelled poly(vinylpyrrolidone), the rate of uptake of the liposome-entrapped material was four times that of the free macromolecule.     

In vitro study of lipid metabolism in the mealworm larval fat body

Lipid metabolism in Tenebrio larval fat body has been studied in vitro. Lipid release required the presence of diluted hemolymph in the incubation medium. This time-dependent release of lipid was strongly stimulated in a dose-dependent manner by Tenebrio corpora cardiaca (CC) extracts or synthetic adipokinetic hormone (AKH I). Furthermore, some glycerol was released when larval fat body was incubated without hemolymph, and this phenomenon was also dose dependent for added CC extracts. Lipid synthesis was estimated in vitro by following the incorporation of radioactivity from [6-¹⁴C] glucose into fatty acids. Lipogenesis occurred in the absence of added carbohydrates in the medium, but it was stimulated by the addition of glucose, and especially trehalose (10 mg ml^?1). Intestinal insulin-like peptide (ILP) also stimulated in vitro lipogenesis in a dose-dependent fashion. We conclude that lipolytic and lipogenetic activities of larval mealworm fat body in vitro are effectively under hormonal control.     

An ultrastructural study of centriolar complexes in adult and embryonic human aortic endothelial cells

Ultrastructural organization of centriolar complexes in 90 adult human aortic endothelial cells from uninvolved areas, fibrous and atheromatous plaques and 30 endothelial cells from human embryonic aorta were studied using serial sections. Primary cilia protruding from the abluminal cell surface were found on 28 of 30 endothelial cells from atheromatous plaques. Only five of 30 cells from either fibrous plaques or uninvolved areas developed primary cilia protruding to the lumen. Impaired primary cilia entirely immersed into the cytoplasm were found in embryonic endothelial cells. It was speculated that both the modes of formation and the functions of endothelial cilia in embryonic and adult aortas are different.     

Capacitive and ionic currents in BLM from phosphatidic acid in Ca2+-induced phase transition

The development of electric current with time in a bilayer lipid membrane (BLM) formed from dipalmitoylphosphatidic acid on introducing Ca2+ ions into the medium was studied at constant temperature and pH. The phase transition in the Ca2+-induced BLM is accompanied by the initial capacitive current followed by the occurrence of single ionic channels. The amount of transported charges in the capacitive current is 5 C/ microF. The conductivity of the single ionic channels ranges from 50 to 100 pSm.     

Ca2+ ions induce successive capacity and ion currents in BLM from phosphatidic acid as a result of phase transition

Time dependence of Ca2+-induced electric current in BLM formed from DPPA was studied at constant temperature and pH. The phase transition in BLM is accompanied by capacity current and following appearance of single ionic channels. It was shown that transferred charge was 5 nC/F, conductivity of single ionic channels--500-100 pSm.     

Study of the structural and functional properties of fibronectin--a high molecular weight plasma glycoproteins--by using monoclonal antibodies

Functional and structural properties of fibronectin--high molecular weight glyco-protein from human plasma--were studied by monoclonal antibodies against fibronectin. It was shown that monoclonal antibodies against human plasma fibronectin exhibit a certain species specificity. Antigenic determinant for our monoclonal antibody is located in the central part of the protein polypeptide chain--in the structural domain. The monoclonal antibodies studied do not inhibit any tested functions of fibronectin. In contrast, polyclonal antibodies are not species specific and inhibit all fibronectin functions.