Comparison of copper binding components in dog serum with those in other species.

The copper content of dog serum and its distribution to copper binding proteins was compared with that of rat and mouse. Total serum Cu concentrations of dogs and mice were one third those of the rat. Plasma ceruloplasmin, determined by azide-inhibitable oxidase activity with two substrates, was 8-fold less in the dog and 9- to 20-fold less in the mouse than in the rat, and, in both dogs and mice, there was 70-75% less ceruloplasmin Cu, determined by atomic absorption after gel filtration. In the dog, the largest proportion of total and exchangeable serum Cu was with the transcuprein fraction. Only one third as much Cu was with albumin in the dog (and mouse) versus the rat, and this was released much more readily through dialysis. In dogs and mice, the exchangeable (nonceruloplasmin) serum copper pool was half the size of that in rats and humans. Especially in the mouse (but also in rats and dogs), a small proportion of the exchangeable pool appeared bound to ferroxidase II. We conclude that the dog may rely more on transcuprein and low molecular weight complexes and less on albumin and ceruloplasmin for transport of copper to cells.     

Host-finding and host recognition of infective Ancylostoma caninum larvae

A. caninum larvae responded to environmental and host stimuli with four behavioral phases of host-finding. (1) Snake-like movement was stimulated by warmth and by defined vibrations of the substratum. (2) Waving behavior was a prerequisite for the passive change-over to dog hairs. It was stimulated by heat radiation and by the CO2 content, warmth, and humidity of an air stream. (3) Creeping direction: the larvae were attracted by heat in temperature gradients as weak as 0.04 degrees C mm-1 and by dog hydrophilic skin surface extracts, but not by skin lipids or serum. (4) Penetration into agar was stimulated by heat, dog hydrophilic skin fraction, and dog serum. The effective component of serum had a molecular weight of between 5000 and 30,000 and proved to be a protein, since it lost its effectiveness after digestion with proteinases. Dog saliva, urine, milk, and various pure dog serum components did not stimulate penetration. A. caninum larvae were able to penetrate mouse skin repeatedly, but they did not follow the tracks of previously penetrated larvae in agar.     

Effect of chloride and diamide on serum angiotensin i-converting enzyme activity from eight mammalian species

1. The effect of chloride on serum angiotensin I-converting enzyme (ACE) activity was characterized in eight mammalian species: dog, guinea pig, hamster, human, mouse, rabbit, rat, and sheep.2. Optimum chloride concentrations varied from 300 mM for rabbit to 1700 mM for hamster.3. The increments with these optimum concentrations with respect to 100 mM chloride concentration were from 1.4-fold in rabbit to 7.9-fold in hamster and dog.4. There was no correlation between serum chloride concentration or serum ACE activity and optimum chloride concentration.5. Serum ACE increased only in humans with diamide pretreatment suggesting the presence of endogenous inhibitors.     

Identification of cDNA encoding motilin related peptide/ghrelin precursor from dog fundus

A novel protein expressed by entero-endocrine cells of the mouse stomach was named prepromotilin Related Peptide (ppMTLRP) since it shares sequence similarities with the prepromotilin (Tomasetto et al.). The mouse ppMTLRP was found identical to the rat precursor of ghrelin (ppghrelin), an endogenous ligand specific for the Growth Hormone Secretagogue receptor identified from rat stomach (Kojima et al.). In the present study the cDNA encoding the dog counterpart of ppMTLRP/Ghrelin has been isolated and sequenced. The dog ppMTLRP/Ghrelin cDNA showed scores of respectively 80% and 75% homology with its human and mouse counterparts. By translation of the dog ppMTLRP/Ghrelin cDNA sequences, two ORFs could be deduced encoding either a 117 amino acid ppMTLRP/Ghrelin or the deleted Gln14 ppMTLRP/Ghrelin, as it was also known in mouse, rat and man. The dog ppMTLRP/Ghrelin shared 91% similarity and 78% identity, and 89% similarity and 78% identity with the human and mouse ppMTLRP/Ghrelin proteins respectively. The best score of homology was found in the MTLRP/Ghrelin sequence itself. Indeed the dog MTLRP/Ghrelin peptide shared 100% similarity and 93% identity, and 96% identity and similarity, with the human and mouse MTLRP/Ghrelin. Using Northern blot analysis to study dog ppMTLRP/Ghrelin gene expression on dog adult gut tissues, maximal expression level was found in the stomach fundus and corpus, and no expression could be detected in the stomach antrum nor in the duodenum, jejunum, ileum, colon or liver. In conclusion, we have identified ppMTLRP/Ghrelin from dog, and found that it is highly conserved with man, mouse or rat. The expression pattern along the gastro-intestinal tract is similar to the expression pattern previously described in mouse.     

Gas chromatographic determination of novel valproyl taurinamide derivatives in mouse and dog plasma

Valproyl taurinamides are a novel group of compounds that possess anticonvulsant activity. In this study a gas chromatographic micromethod was developed for the quantification of selected valproyl taurinamides and some of their metabolites in biological samples. Valproyl taurinamide (VTD), N-methyl valproyl taurinamide (M-VTD), N,N-dimethyl valproyl taurinamide (DM-VTD) and N-isopropyl valproyl taurinamide (I-VTD) were analyzed in mouse and dog plasma and in dog urine using gas chromatography. Flame ionization detection and mass spectrometric detection were compared. The plasma samples were prepared by solid-phase extraction using C(18) cartridges. The urine samples were prepared by liquid-liquid extraction. The sample volume used was 100 microl of dog plasma, 50 microl of mouse plasma and 20 microl of dog or mouse urine. The quantification range of the method was 1.5-50 mg/l in dog plasma (VTD only), 2.5-250 mg/l in mouse plasma (0.7-90 pmol injected) and 0.04-2 mg/ml in dog urine (VTD only). The inter-day precision in plasma and urine samples was around 10% for all quantified concentrations except LOQ (15-20%). The accuracy for all four compounds was between 90 and 110% within the entire concentration range. The developed method was suitable for quantification of a series of CNS-active valproyl taurineamide derivatives in biological samples at relevant in vivo concentrations.     

Diffusible amyloid oligomers trigger systemic amyloidosis in mice

AA (amyloid protein A) amyloidosis in mice is markedly accelerated when the animals are given, in addition to an inflammatory stimulus, an intravenous injection of protein extracted from AA-laden mouse tissue. Previous findings affirm that AA fibrils can enhance the in vivo amyloidogenic process by a nucleation seeding mechanism. Accumulating evidence suggests that globular aggregates rather than fibrils are the toxic entities responsible for cell death. In the present study we report on structural and morphological features of AEF (amyloid-enhancing factor), a compound extracted and partially purified from amyloid-laden spleen. Surprisingly, the chief amyloidogenic material identified in the active AEF was diffusible globular oligomers. This partially purified active extract triggered amyloid deposition in vital organs when injected intravenously into mice. This implies that such a phenomenon could have been inflicted through the nucleation seeding potential of toxic oligomers in association with altered cytokine induction. In the present study we report an apparent relationship between altered cytokine expression and AA accumulation in systemically inflamed tissues. The prevalence of serum AA monomers and proteolytic oligomers in spleen AEF is consistent to suggest that extrahepatic serum AA processing might lead to local accumulation of amyloidogenic proteins at the serum AA production site.     

Hydrophobic interactions of transcobalamin II (TC II) from mammalian sera

The hydrophobic properties of mammalian transcobalamin IIs (TC II) were studied by chromatography of radioactive cyanocobalamin (CN[57Co]Cbl)-labeled serum on phenyl-Sepharose CL-4B. Mammalian holo TC IIs (CN[57Co]Cbl-TC II) exhibited species variability in their affinity for the hydrophobic matrix in the order: dog greater than mouse greater than human greater than rat greater than rabbit. Phenyl-Sepharose chromatography of the isolated CN[57Co]Cbl-TC II peaks from gel filtration of dog and rat serum showed no hydrophobic change in dog TC II, but an increase in hydrophobicity of rat TC II. Phenyl-Sepharose chromatography of CN[57Co]Cbl-labeled rabbit serum (holo TC II) and the unlabeled serum (apo TC II) showed apo TC II to be more hydrophobic than holo TC II as has been shown for human TC II (Begley et al., Biochem Biophys Res Commun 103:434-441, 1981). Thus mammalian holo TC IIs differ in their hydrophobic properties and apo TC II, in man and rabbit, is more hydrophobic than holo TC II. In addition, isolation of the TC II in some animal sera by gel filtration may result in a TC II that is more hydrophobic than the native molecule.     

Adenosinetriphosphatase Activity in the Cell Membranes of Kidney Tubule Cells

This cytochemical study demonstrates high levels of apparent ATPase activity in the infolded cell membranes of the proximal tubules (dog, rat, human, mouse, monkey, and opossum) and ascending loops of Henle (dog, rat, human and, to a variable degree, mouse). Electron microscopy has shown (see Rhodin (1)) that these membranes separate adjacent cells where they interlock with one another by multiple cytoplasmic lamellae containing oriented mitochondria. The significance of the high ATPase activity is considered in relation to possible movements of the membranes and to "active transport" believed to occur there. In the rat, enzyme activity in the proximal tubule membranes does not survive formol-calcium fixation, and it is therefore necessary to use unfixed sections for its demonstration. However, in edematous rats with experimental nephrosis (induced by the injection of aminonucleoside or with antikidney serum) marked ATPase activity is exhibited in these membranes even after formol-calcium fixation. When proximal tubule or Henle loop cells of the dog acquire an altered metabolism, as indicated by accumulated lipide spheres or by "droplets," the infolded ATPase-rich membranes are no longer demonstrable.     

Immunochemical and autoantigenic properties of the globular domain of basement membrane collagen (type IV)

Polyclonal rabbit antibodies raised against the globular domain NC1 of collagen IV from human placenta and a mouse tumor react with conformational antigenic determinants present on the NC1 hexamers and also with the three major subunits obtained after dissociation. The antibodies recognized unique structures within basement membranes and showed a broad tissue reactivity but only limited species cross-reactivity. Using these antibodies, it was possible to detect small amounts of collagen IV antigens from cell cultures and in serum. Monoclonal rat antibodies against mouse NC1 revealed a similar reaction potential. Autoantibodies could be produced in mice against mouse NC1 which react with kidney and lung basement membranes in a pathological manner, mimicking Goodpasture syndrome.     

Adenosinetriphosphatase activity in the cell membranes of kidney tubule cells

This cytochemical study demonstrates high levels of apparent ATPase activity in the infolded cell membranes of the proximal tubules (dog, rat, human, mouse, monkey, and opossum) and ascending loops of Henle (dog, rat, human and, to a variable degree, mouse). Electron microscopy has shown (see Rhodin (1)) that these membranes separate adjacent cells where they interlock with one another by multiple cytoplasmic lamellae containing oriented mitochondria. The significance of the high ATPase activity is considered in relation to possible movements of the membranes and to "active transport" believed to occur there. In the rat, enzyme activity in the proximal tubule membranes does not survive formol-calcium fixation, and it is therefore necessary to use unfixed sections for its demonstration. However, in edematous rats with experimental nephrosis (induced by the injection of aminonucleoside or with antikidney serum) marked ATPase activity is exhibited in these membranes even after formol-calcium fixation. When proximal tubule or Henle loop cells of the dog acquire an altered metabolism, as indicated by accumulated lipide spheres or by "droplets," the infolded ATPase-rich membranes are no longer demonstrable.     

A comparison of the cellulose acetate electrophoretic serum lipoprotein-cholesterol profile of the adult male rat with other species

A comparison was made of the serum lipoprotein-cholesterol profile, obtained by cellulose acetate electrophoresis coupled with an enzymatic stain for total cholesterol, of the adult male rat, mouse, rabbit, dog, monkey and human. Four cholesterol-staining lipoprotein bands were detected in rat serum, while only three cholesterol-staining lipoprotein bands were present in the other species studied. The apparently unique lipoprotein-cholesterol band in the rat was found to electrophoretically migrate in the prealbumin region of rat serum, has been named prealbumin lipoprotein-cholesterol (PAL-C) and was shown to be a high density lipoprotein (HDL). Of the species studied those more susceptible to experimentally induced atherosclerosis had higher low density lipoprotein-cholesterol to HDL-cholesterol ratios compared to those species least susceptible to experimentally induced atherosclerosis.     

Interactions between globular proteins and F-actin in isotonic saline solution.

Solutions of each of three different globular proteins (cytochrome c, chromophorically labeled serum albumin, and chromophorically labeled aldolase), mixed with another unlabeled globular protein or with fibrous actin, were prepared in pH 8.0 Tris-HCl buffer containing 0.15 M NaCl. Each solution was centrifuged at low speed, at 5 degrees C, until unassociated globular protein in solution achieved sedimentation equilibrium. Individual absorbance gradients of both macrosolutes in the mixtures subsequent to centrifugation were obtained via optical scans of the centrifuge tubes at two wavelengths. The gradients of each macrosolute in mixtures of two globular proteins revealed no association of globular proteins under the conditions of these experiments, but perturbation of the gradients of serum albumin, aldolase, and cytochrome c in the presence of F-actin indicated association of all three globular proteins with F-actin. Perturbation of actin gradients in the presence of serum albumin and aldolase suggested partial depolymerization of the F-actin by the globular protein. Analysis of the data with a simple phenomenological model relating free globular protein, bound globular protein, and total actin concentration provided estimates of the respective equilibrium constants for association of serum albumin and aldolase with F-actin, under the conditions of these experiments, of the order of 0.1 microM-1.     

A simple one-step hemolytic assay for C2 with C2-deficient human serum.

A simple one-step procedure has been developed for the molecular titration of C2 by utilizing the ability of the test material to restore the hemolytic activity of human serum selectively deficient in C2 (C2D serum). In this assay, equal volumes of EA (10(8) cells/ml), C2D serum (1/20), and a suitable dilution of a source of C2 were incubated at 37 degrees C for 60 min and the fraction of cells lysed was used to calculate the effective molecules of C2/ml test material. The assay can be used to titrate C2 in human, guinea pig, rat, mouse and rabbit sera, but not C2 in dog serum. The assay is simple and reproducible, and comparable in sensitivity to the conventional two-step assay with EAC14 cells and Cgp-EDTA.     

Evidence from the anti-idiotypic network that the acetylcholine receptor is a rabies virus receptor.

We have developed idiotype-anti-idiotype monoclonal antibodies that provide evidence for rabies virus binding to the acetylcholine receptor (AChR). Hybridoma cell lines 7.12 and 7.25 resulted after fusion of NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with rabies virus strain CVS. Antibody 7.12 reacted with viral glycoprotein and neutralized virus infectivity in vivo. It also neutralized infectivity in vitro when PC12 cells, which express neuronal AChR, but not CER cells or neuroblastoma cells (clone N18), which have no AChR, were used. Antibody 7.25 reacted with nucleocapsid protein. Anti-idiotypic monoclonal antibody B9 was produced from fusion of NS-1 cells with spleen cells from a mouse immunized with 7.12 Fab. In an enzyme-linked immunosorbent assay and immunoprecipitation, B9 reacted with 7.12, polyclonal rabies virus immune dog serum, and purified AChR. The binding of B9 to 7.12 and immune dog serum was inhibited by AChR. B9 also inhibited the binding of 7.12 to rabies virus both in vitro and in vivo. Indirect immunofluorescence revealed that B9 reacted at neuromuscular junctions of mouse tissue. B9 also reacted in indirect immunofluorescence with distinct neurons in mouse and monkey brain tissue as well as with PC12 cells. B9 staining of neuronal elements in brain tissue of rabies virus-infected mice was greatly reduced. Rabies virus inhibited the binding of B9 to PC12 cells. Mice immunized with B9 developed low-titer rabies virus-neutralizing antibody. These mice were protected from lethal intramuscular rabies virus challenge. In contrast, anti-idiotypic antibody raised against nucleocapsid antibody 7.25 did not react with AChR.     

Maintenance of plasma-derived proteins at much lower concentrations in the uterine lumen of the rabbit: a kinetic study of passage.

Human serum albumin (HSA) and human gamma globulin (HGG) in serum and uterine fluid of nonpregnant rabbits at various times after an i.v. injection (100 mg/kg) were measured by a radial immunodiffusion test using specific antisera. The HSA concentration in uterine fluid rose to a peak at 12 hr when it was 11% of the serum concentration and then declined, whereas HGG reached a peak at 18 hr (3.2% of serum level) and decreased thereafter. The HSA passed 2 1/2 times faster than HGG, but both proteins equilibrated with uterine fluid in about 12-18 hr. Steady state levels of HSA and HGG indicated that uterine fluid: serum ratios were 1:10 and 1:20, respectively. Similar ratios were found for total protein and rabbit serum albumin (1:10) and rabbit gamma globulin (1:20). Therefore, except when there is a local immune response, the uterine lumen contains only about 5% of the serum antibody concentration. Available data in the mouse, rat and dog also indicate disparity between serum and uterine fluid protein levels.     

Protein binding of cortisol by means of competitive adsorption: application to cortisol binding by serum of sixteen eutherian mammals.

1. Binding of 3H-cortisol by serum proteins by means of competitive adsorption was relatively high by serum of the gerbil, human, rabbit, sheep, tree shrew, hamster, rhesus monkey and horse. 2. A somewhat lower binding was observed by serum proteins of the baboon, cattle, dog, rat and cat. 3. Serum taken from either the mouse, guinea pig or pig gave very flat binding curves, specific binding not exceeding 5% of added 3H-cortisol. 4. It is concluded that the measurement of protein-binding of 3H-cortisol by means of competitive adsorption is a reliable method for serum of most eutherian species but is unsuited if serum of the mouse, guinea pig or pig is used.     

Computational studies on prion proteins: effect of Ala(117)-->Val mutation

Molecular dynamics calculations demonstrated the conformational change in the prion protein due to Ala(117)-->Val mutation, which is related to Gerstmann-Str?ussler-Sheinker disease, one of the familial prion diseases. Three kinds of model structures of human and mouse prion proteins were examined: (model 1) nuclear magnetic resonance structures of human prion protein HuPrP (125-228) and mouse prion protein MoPrP (124-224), each having a globular domain consisting of three alpha-helices and an antiparallel beta-sheet; (model 2) extra peptides including Ala(117) (109-124 in HuPrP and 109-123 in MoPrP) plus the nuclear magnetic resonance structures of model 1; and (model 3) extra peptides including Val(117) (109-124 in HuPrP and 109-123 in MoPrP) plus the nuclear magnetic resonance structures of model 1. The results of molecular dynamics calculations indicated that the globular domains of models 1 and 2 were stable and that the extra peptide in model 2 tended to form a new alpha-helix. On the other hand, the globular domain of model 3 was unstable, and the beta-sheet region increased especially in HuPrP.     

Effects of temperature and glucose concentration on glycogen synthesis in Ancylostoma caninum

The glycogen content of starved worms incubated in Krebs-Ringer bicarbonate, ox serum or dog serum, increased with increasing glucose concentrations. The pattern of increase in the presence and absence of dog serum, however, differed. A maximum of worm glycogen content was reached at a much lower glucose concentration when dog serum was present than when it was absent. Studies on temperature effects on worms incubated at 27 and 37°C, in the presence and absence of dog serum showed that for worms incubated in dog serum, a temperature coefficient of 3.31 was obtained. For worms incubated in Krebs-Ringer bicarbonate, the temperature coefficient was 0.88, thus showing that the uptake of glucose for glycogen synthesis was different when dog serum was absent from the incubating medium.     

Chromosome preparations from microplate cultures of man,dog, rat,and mouse

Summary A simple method for making chromosomal preparations from 10⁵ leukocytes from man, dog, mouse, and rat and from 0.01 ml total human and dog blood is developed. The leukocytes and the peripheral blood are cultured in Cooke microtiter plates in a culture volume of 0.1 ml. The culture medium is R.P.M.I. 1640 supplemented with serum and phytohemagglutinin. The culture time is 72 or 96 h.