The hydrolysis of some L-amino acid-p-nitranilides with the normal and pregnant's serum aminopeptidases

From 4 serum aminopeptidases (2 of pregnant and 2 of nonpregnant women's sera), the placental lysosomal (mol. wt 320,000) splits Lys-NAp only. B-Cys-NAp is hydrolyzed from the both placental enzymes, i.e. lysosomal and microsomal (mol. wt 145,000) AP. Ala-NAp is split by both nonpregnant serum AP more readily than Leu-NAp.     

Denaturation map of the ribosomal DNA of Lytechinus variegatus sperm.

Electron microscopy of the partially heat denatured ribosomal DNA (rDNA) from sea urchin (Lytechinus variegatus) sperm has demonstrated that it consists of repeating units of 3.6 +/- 0.2 micron, corresponding to a mol.wt of 7.2 +/- 0.4 x 10(6). Based on differential denaturability, each repeat unit is divided into 2 regions. The larger region of 2.47 +/- 0.11 micron (mol.wt 4.9 +/- 0.22 x 10(6)) corresponds in length to the ribosomal precursor RNA of sea urchins and the smaller, GC-rich, subunit of 1.16 +/- 0.09 micrometer (mol.wt 2.3 +/- 0.18 x 10(6)) is presumed to contain non-transcribed spacer sequences.     

The use of dipeptide-p-nitranilides for the study of aminopeptidase specificity.

The use of dipeptide-p-nitranilides for the study of 2 placental aminopeptidases separated on Sephadex G200 helped in establishing some regular features of their specifities. The high-molecular (320,000 daltons) one prefers Phe in position P'1 to Leu, whereas the lower-molecular aminopeptidase (145,000 daltons) prefers Leu. The high-molecular aminopeptidase splits very slowly the N-terminal Leu when Gly is in adjacent position. Leu-Gly-p-NA is therefore an inhibitor of this AP.     

Elastase digested urokinase (ED-UK)

Summary Elastase digested urokinase (ED-UK) was prepared from human high mol. wt urokinase (HMW-UK). It resembled low mol. wt urokinase (LMW-UK) in its mol. wt, specific activity, and active sites. The steady-state kinetic parameters of each enzyme for the activation of human Glu-plasminogen also resembled each other, as did their amidase parameters (with pyro-Glu-Gly-Arg-pNA).     

Elastase digested urokinase (ED-UK)

Elastase digested urokinase (ED-UK) was prepared from human high mol. wt urokinase (HMW-UK). It resembled low mol. wt urokinase (LMW-UK) in its mol. wt, specific activity, and active sites. The steady-state kinetic parameters of each enzyme for the activation of human Glu-plasminogen also resembled each other, as did their amidase parameters (with pyro-Glu-Gly-Arg-pNA).     

The use of dipeptide-p-nitranilides for the study of aminopeptidase specificity

Summary The use of dipeptide-p-nitranilides for the study of 2 placental aminopeptidases separated on Sephadex G200 helped in establishing some regular features of their specifities. The high-molecular (320,000 daltons) one prefers Phe in position P'₁ to Leu, whereas the lower-molecular aminopeptidase (145,000 daltons) prefers Leu. The high-molecular aminopeptidase splits very slowly the N-terminal Leu when Gly is in adjacent position. Leu-Gly-p-NA is therefore an inhibitor of this AP.     

Purification of an L-fucose binding lectin fromUlex europeus by affinity column chromatography

Summary An L-fucose binding lectin fromUlex europeus was purified by affinity column chromatography using an L-fucose-starch complex. The lectin thus purified had a mol.wt of 60,000, and consisted of 2 glycoprotein subunit with mol.wt 29,000 and 31,000, respectively.     

Tumor-derived angiogenesis factors from rat Walker 256 carcinoma: an experimental investigation and review

Angiogenesis, the process of developing a hemovascular network, is an essential feature of the growth of solid tumors, and is induced by factors secreted by tumor cells. Assay procedures suitable for the investigation of angiogenesis, and for the screening of angiogenesis factors during purification are reviewed; and a number of reports describing the purification of angiogenesis factors, primarily from the rat Walker 256 carcinoma as starting material, are discussed. Work from the authors' laboratory is also presented. Walker 256 cells grown in large-scale culture were the source of a reproducible and homogeneous source of angiogenic material. Factors secreted by these cells were isolated by a series of chromatographic steps. Ion exchange chromatography on carboxymethyl-Sephadex produced two active fractions, one of which was fractionated into several macromolecular species by lectin affinity and hydrophobic adsorption chromatography. The other gave a high mol.wt, active fraction that was resolved into a low mol.wt, active component and a non-angiogenic but possibly carrier molecule with a mol.wt of 140,000. While none of the angiogenic factors were identified chemically, the results demonstrate the existence of both high and low mol.wt tumor-secreted angiogenic substances, confirming the hypothesis for tumor-induced angiogenesis and predicting potential means to interfere with the process of tumor growth.     

EG-VEGF controls placental growth and survival in normal and pathological pregnancies: case of fetal growth restriction (FGR)

Identifiable causes of fetal growth restriction (FGR) account for 30 % of cases, but the remainders are idiopathic and are frequently associated with placental dysfunction. We have shown that the angiogenic factor endocrine gland-derived VEGF (EG-VEGF) and its receptors, prokineticin receptor 1 (PROKR1) and 2, (1) are abundantly expressed in human placenta, (2) are up-regulated by hypoxia, (3) control trophoblast invasion, and that EG-VEGF circulating levels are the highest during the first trimester of pregnancy, the period of important placental growth. These findings suggest that EG-VEGF/PROKR1 and 2 might be involved in normal and FGR placental development. To test this hypothesis, we used placental explants, primary trophoblast cultures, and placental and serum samples collected from FGR and age-matched control women. Our results show that (1) EG-VEGF increases trophoblast proliferation ([³H]-thymidine incorporation and Ki67-staining) via the homeobox-gene, HLX (2) the proliferative effect involves PROKR1 but not PROKR2, (3) EG-VEGF does not affect syncytium formation (measurement of syncytin 1 and 2 and β hCG production) (4) EG-VEGF increases the vascularization of the placental villi and insures their survival, (5) EG-VEGF, PROKR1, and PROKR2 mRNA and protein levels are significantly elevated in FGR placentas, and (6) EG-VEGF circulating levels are significantly higher in FGR patients. Altogether, our results identify EG-VEGF as a new placental growth factor acting during the first trimester of pregnancy, established its mechanism of action, and provide evidence for its deregulation in FGR. We propose that EG-VEGF/PROKR1 and 2 increases occur in FGR as a compensatory mechanism to insure proper pregnancy progress.     

Polypeptides of the lens fibre cell intracellular matrix

Summary The cytoskeletal proteins of the vertebrate lens fibre cell comprise polypeptides ranging in mol.wt from 43,000 to 250,000 daltons. The main intermediate filament polypeptide of the pickerel, Northern frog, chick, bovine, and human lens has a mol.wt of 54,000 daltons. Peptide analysis revealed that the chick 54,000 dalton protein was more similar to the bovine protein than to the pickerel protein.Supported by grants EY 01417 (H. Maisel) and HD-06390 (J.C. Brown, University of Virginia, Charlottesville) from the National Institutes of Health, U.S.P.H.S.     

Subcellular distribution and binding of heavy metals in the untreated liver of the squid; comparison with data from the livers of cadmium and silver-exposed rats

In natural squid liver, about 30% of the total Cd present was found in the cytosolic fraction. A large portion of this Cd was bound to high molecular weight species (mol. wt greater than 70,000). In contrast to Cd, about 60% of the total Ag occurred in the cytosolic fraction; Ag was bound mainly to low molecular weight species (mol. wt less than 20,000).     

A high molecular weight inhibitor of Ca2+ -dependent neutral protease in rat brain.

An endogenous, heat-stable inhibitor of high mol. wt (approximately 3x10(5)) was found to be present in rat brain, which inhibited Ca2+-dependent neutral protease specifically but not due to its binding of Ca2+ in the medium .     

Effects of thyroidectomy and thyroxine replacement on the responsiveness of the anterior pituitaries from male rats to thyrotropin-releasing hormone in vitro

Summary Thyroidectomy decreased prolactin concentrations in the anterior pituitary (AP) and serum of the male rat. The amount of basal and thyrotropin-releasing hormone (TRH)-stimulated release of prolactin by AP in vitro was lower in thyroidectomized (Tx) rats than in sham Tx rats. These results suggest that the inhibitory effects of thyroidectomy on pituitary and serum prolactin in male rats are mediated in part by the reduction of the production and spontaneous release of prolactin and the responsiveness of prolactin to TRH.     

Denaturation map of the ribosomal DNA ofLytechinus variegatus sperm

Summary Electron microscopy of the partially heat denatured ribosomal DNA (rDNA) from sea urchin (Lytechinus variegatus) sperm has demonstrated that it consists of repeating units of 3.6±0.2 m, corresponding to a mol.wt of 7.2±0.4×10⁶. Based on differential denaturability, each repeat unit is divided into 2 regions. The larger region of 2.47±0.11 m (mol.wt 4.9±0.22×10⁶) corresponds in length to the ribosomal precursor RNA of sea urchins and the smaller, GC-rich, subunit of 1.16±0.09 m (mol.wt 2.3±0.18×10⁶) is presumed to contain non-transcribed spacer sequences.Acknowledgments. This paper is dedicated to the memory of Dr D. P. Costello whose insprration made this work possible. I gratefully acknowledge the valuable advice of Drs D.W. Stafford and M.A. Bleyman.     

Tumor-derived angiogenesis factors from rat Walker 256 carcinoma: an experimental investigation and review

Summary Angiogenesis, the process of developing a hemovascular network, is an essential feature of the growth of solid tumors, and is induced by factors secreted by tumor cells. Assay procedures suitable for the investigation of angiogenesis, and for the screening of angiogenesis factors during purification are reviewed; and a number of reports describing the purification of angiogenesis factors, primarily from the rat Walker 256 carcinoma as starting material, are discussed. Work from the authors' laboratory is also presented. Walker 256 cells grown in large-scale culture were the source of a reproducible and homogeneous source of angiogenic material. Factors secreted by these cells were isolated by a series of chromatographic steps. Ion exchange chromatography on carboxymethyl-Sephadex produced two active fractions, one of which was fractionated into several macromolecular species by lectin affinity and hydrophobic adsorption chromatography. The other gave a high mol.wt, active fraction that was resolved into a low mol.wt, active component and a non-angiogenic but possibly carrier molecule with a mol.wt of 140,000. While none of the angiogenic factors were identified chemically, the results demonstrate the existence of both high and low mol.wt tumor-secreted angiogenic substances, confirming the hypothesis for tumor-induced angiogenesis and predicting potential means to interfere with the process of tumor growth.This work was supported by funds from the Monsanto Company under Agreements with Harward University and from the U.S. Public Health Service, Contract No. N01-CB-43942. We are particularly indebted to Bernard S. Wildi and Monte C. Throdahl for their advice, cooperation, and encouragement. We also thank Dr Judah Folkman for the initial supply of male mouse submaxillary glands and rat Walker carcinoma conditioned medium, for help with the CAM assays, assays in the rabbit cornea, and many helpful discussions; and R. Bretton, A. Ehrlich, B. Evans, F. Fu, N. Hay and B. Tsokanis for excellent technical assistance.Author to whom correspondence should be addressed.     

Purification of a nuclease from human serum

Summary The purification procedure for a nuclease from human serum is described. It includes ammonium sulfate precipitation, chromatography on DEAE-Sephadex and on Sephacryl-S 200, and preparative electrophoresis. The enzyme, purified about 2000-fold, is homogeneous in a sodium dodecyl sulfate electrophoretic system, where it has a mol. wt of 78,000. The pH optimum lies around pH 6.5; it is a sugar-nonspecific endonuclease.Acknowledgment. This work has been supported by a research grant from Stiftung Volkswagenwerk through the Akademie der Wissenschaften und der Literatur, Mainz. We thank Mrs R. Nehrbass and Mrs C. Wolpert for technical assistance.     

Satellite DNA (II) from sea urchin (Lytechinus variegatus) sperm

When DNA isolated from freshly collected sperm of sea urchin (Lytechinus variegatus) is centrifuged to equilibrium in CsCl, 2 heavy satellite bands appear beside the main band DNA. Satellite DNA (II) appears in between the main band DNA (rho = 1.695 g/cm3) and the rDNA satellite (rho = 1.722 g/cm3). Satellite DNA (II) has a buoyant density 1.710 g/cm3, corresponding to 50% GC content. It is speculated that the satellite DNA (II), which appears to be of high mol. wt, might contain the sequences complementary to histone mRNA.     

Neuronal ceroid lipofuscinosis protein CLN3 interacts with motor proteins and modifies location of late endosomal compartments

CLN3 is an endosomal/lysosomal transmembrane protein mutated in classical juvenile onset neuronal ceroid lipofuscinosis, a fatal inherited neurodegenerative lysosomal storage disorder. The function of CLN3 in endosomal/lysosomal events has remained elusive due to poor understanding of its interactions in these compartments. It has previously been shown that the localisation of late endosomal/lysosomal compartments is disturbed in cells expressing the most common disease-associated CLN3 mutant, CLN3?ex7-8 (c.462-677del). We report here that a protracted disease causing mutant, CLN3E295K, affects the properties of late endocytic compartments, since over-expression of the CLN3E295K mutant protein in HeLa cells induced relocalisation of Rab7 and a perinuclear clustering of late endosomes/lysosomes. In addition to the previously reported disturbances in the endocytic pathway, we now show that the anterograde transport of late endosomal/lysosomal compartments is affected in CLN3 deficiency. CLN3 interacted with motor components driving both plus and minus end microtubular trafficking: tubulin, dynactin, dynein and kinesin-2. Most importantly, CLN3 was found to interact directly with active, guanosine-5'-triphosphate (GTP)-bound Rab7 and with the Rab7-interacting lysosomal protein (RILP) that anchors the dynein motor. The data presented in this study provide novel insights into the role of CLN3 in late endosomal/lysosomal membrane transport.     

Placental origin of the progesterone binding protein in the pregnant guinea pig; immunohistoligical study]

The origin of the high affinity progesterone binding protein (PBP) occurring during pregnancy in the guinea pig serum has been investigated. An indirect immunofluorescence technique has been developed using a specific antiserum raised in rabbits. An accumulation of PBP-like immunofluorescent material was detected in the trophoblastic syncytium of the placenta tissue, whereas all other examined pregnant guinea pig organs gave negative reactions. It is concluded that this placental accumulation of immunoreactive PBP-like material may express a placental biosynthesis of this glycoprotein.     

Certain biochemical effects of garlic oil on rats maintained on high fat-high cholesterol diet

The feeding of a high fat-high cholesterol (HF-HC) diet to normal rats for 1 month increased the lipid components cholesterol and triglyceride in serum, liver and kidneys and decreased the serum albumin very significantly. Administration of garlic oil (100 mg/kg b. wt/day) for 1 month together with the HF-HC diet to another group almost nullified the lipid-increasing and albumin-decreasing effects of that diet. The reduction in total lipids, cholesterol and triglycerides and the restoration to normal level of serum albumin were highly significant in the garlic oil group. Adipose tissue triglyceride lipase activity was significantly increased in both the above groups with a much greater rise in the oil group.