Distribution of 46Sc and 51Cr in tumor-bearing animals and the mechanism for accumulation in tumor and liver

Tumor uptake rates, concentrations in mitochondrial fraction (containing lysosome) of liver and tumors, avid accumulations in connective tissue (especially inflammatory tissue) and binding substances in these tissues for ⁴⁶Sc³⁺ and ⁵¹Cr³⁺ were essentially similar to those for ⁶⁷Ga³⁺, ¹¹¹In³⁺, ¹⁶⁹Yb³⁺, ¹⁶⁷Tm³⁺, ⁹⁵Zr⁴⁺ and ¹⁸¹Hf⁴⁺. However, the main binding substance of ⁴⁶Sc³⁺ and ⁵¹Cr³⁺ in tumor and liver was the acid mucopolysaccharide (as described concerning ⁹⁵Zr and ¹⁸¹Hf) whose molecular weight exceeded 40,000, although the main binding substance of ⁶⁷Ga³⁺, ¹¹¹In³⁺, ¹⁶⁹Yb³⁺ and ¹⁶⁷Tm³⁺ was the acid mucopolysaccharide with a molecular weight of about 10,000.     

Distribution of opiate-like substances in rat tissues

Rat tissues were tested for their ability to inhibit the binding of [³H]dihydromorphine or [³H]naloxone to membrane-bound opiate receptors. By this criterion, morphine-like substances were found in lung, heart, liver, and kidney as well as in brain. The relative activity of the extracts, based on initial tissue weight, differed with the radioactive ligand employed. With dihydromorphine, the order was as above. With naloxone, lung was most active, followed by heart, brain, liver, and kidney. The ability of all tissue extracts to inhibit opiate binding was reduced by 100 mM NaCl and slightly reduced by 1 mM MnCl₂. Gel filtration using Sephadex G-25 indicated that the inhibitory Substances were heterogeneous in molecular weight. Only with brain and kidney extracts was there significant activity at the elution volume where enkephalins would be expected. Fraction tion using Amberlite XAD-2, a resin which selectively absorbs hydrophobic materials, again indicated that the major portion of activit in all tissue extracts was due to substances other than enkephalins.     

Properties of a Solubilized Microsomal Auxin-binding Protein from Coleoptiles and Primary Leaves of Zea mays

An auxin-binding protein can be solubilized from microsomal membranes of Zea mays using either Triton X-100 extraction of the membranes or buffer extraction of the acetone-precipitated membranes. This paper describes the properties of the binding protein solubilized by these two methods. The binding is assayed by gel filtration chromatography in the presence of naphthalene [2-¹⁴C]acetic acid. Binding is rapid and reversible with an optimum at pH 5. Both preparations show similar molecular weights by gel filtration (80,000 daltons) at pH 7.6 and 0.1 molar NaCl, and both aggregate at low ionic strength. They appear to be the same active molecular species. The binding activity is destroyed by trypsin, pronase or para-chloromercuribenzoic acid, but not significantly reduced by phospholipase C, DNase, RNase, or dithioerythritol. Since saturating amounts of naphthalene acetic acid protect the molecule from inhibition by para-chloromercuribenzoic acid, it is concluded that the binding protein has a sulfhydryl group at the binding site, or protects such a group in its binding conformation. The dissociation constant of the protein for naphthalene acetic acid is 4.6 × 10⁻⁸ molar with 30 picomoles of sites per gram of tissue fresh weight. Binding constants were estimated for 13 other natural and synthetic auxins by competition with naphthalene[2-¹⁴C]acetic acid. Their dissociation constants are in general agreement with published values for their binding to intact membranes and their biological activity, although several exceptions were noted. A supernatant factor from the same tissue changes the apparent affinity of the protein for naphthalene acetic acid. This factor may be the same one as has been previously reported to alter the affinity of intact microsomes for auxin.     

The Effects of Hyaluronic Acid on Macrophage FC Receptor Binding and Phagocytosis Are Independent of the Mode of Depolymerization

In order to determine whether exposure of hyaluronic acid to oxygen radicals caused an alteration in its properties. independent of the change in molecular weight induced. we examined its effect upon macro-phage Fc receptor binding. High molecular weight hyaluronic acid (Healon-Pharmacia) caused a dose dependent inhibition of binding between the concentrations of 0.2–1 mg/ml. At a concentration of 0.3 mg/ml both oxygen radical depolymerized and enzymatically degraded hyaluronic acid caused an inhibition of Fc receptor binding at molecular weights of 1 × 10⁶. 1.5 × 10⁶ and 2 × 10⁶. Oxygen radical degraded hyaluronic acid caused a stimulation of Fc receptor binding at molecular weights of 2 × 10⁵ and 3.5 × 10⁵. and enzyme degraded hyaluronic acid causes stimulation at a molecular weight of 2.5 × 10⁶. Thus this “biological property” of hyaluronic acid is dependent upon molecular weight solely and not upon the mode of depolymerization.     

Association of microcystin-LR and its biotransformation product with a hepatic-cytosolic protein

Microcystin-LR (MCYST-LR), a cyclic peptide hepatotoxin, associates with high-molecular-weight, liver cytosolic components. Repetitive cycles of heat denaturation and pronase digestion released 80 ± 6% of the bound radiolabel from these components, parent toxin (22%), and two biotransformation products, with high-performance liquid chromatography (HPLC) retention times of 6.7 (52%) and 5.6 (13%) min. Both parent and the biotransformed (6.7 min) toxin appeared to be covalently bound to a monomeric protein of molecular weight 40,000 (protein plus radiolabeled toxin). Binding and biotransformation reactions were time- and temperature-dependent and did not require endogenous molecules <6,000 daltons. The binding appeared to be saturable with a maximum of 20 pmol MCYST-LR bound per mg protein. The binding protein(s) and biotransformation activity were present in rat liver, brain, kidney, heart, lung, small intestine, large intestine, testes, skeletal muscle, and to a lesser extent, in fat. Okadaic acid, a specific protein phosphatase inhibitor, showed a concentration-dependent inhibition of [³H]MCYST-LR binding to hepatic cytosol. The molecular weight and organ distribution of the binding protein(s), and inhibition of binding by okadaic acid were consistent with one of the binding sites being the catalytic subunit of protein phosphatase type 2A.     

Involvement of a low molecular weight component(s) in the mechanism of action of the glucocorticoid receptor.

[³H]Dexamethasone-receptor complexes from rat liver cytosol preincubated at 0° bind poorly to DNA-cellulose. However, if the steroid-receptor complex is subjected to gel filtration at 0–4° separating it from the low molecular weight components of cytosol, the steroid-receptor complex becomes “activated” enabling its binding to DNA-cellulose. This activation can be prevented if the gel filtration column is first equilibrated with the low molecular weight components of cytosol. In addition, if adrenalectomized rat liver cytosol, in the absence of exogeneous steroid, is subjected to gel filtration the macromolecular fractions separated from the “small molecules” of that cytosol have much reduced binding activity towards [³H]dexamethasone. These results suggest that rat liver cytosol contains a low molecular weight component(s) which maintains the glucocorticoid receptor in a conformational state that allows the binding of dexamethasone. Furthermore, this component must be removed from the steroid-receptor complex before binding to DNA can occur.     

Demonstration of an endogenous, competitive inhibitor(s) of [3H] diazepam binding in bovine brain.

An endogenous inhibitor(s) of [³H] diazepam binding to synaptosomes has been demonstrated in bovine brain. The inhibitory activity of crude extracts is heat stable, dialyzable, and not affected by ether extraction. Three distinct peaks of inhibitory activity were resolved using Sephadex G-25 chromatography. The lowest molecular weight peak (<700 daltons) had the highest specific inhibitory activity and its inhibition of [³H] diazepam binding was competitive. A similar low molecular weight fraction was not observed in either muscle or liver suggesting that it may be unique to brain. Thin layer chromatography of the Sephadex G-25 fractions revealed a discrete band of inhibitory activity in the two low molecular weight peaks.     

Binding of nonsuppressible insulinlike activity to human serum. Evidence for a carrier protein.

When ¹²⁵I-labeled nonsuppressible insulinlike activity—soluble in acid/ethanol (NSILA-S) is incubated with human serum between 10 and 20% of the radioactivity are bound to serum proteins and can be displaced specifically by cold NSILA-S. Chromatography of the incubation mixture on Sephadex G-200 at pH 7.5 reveals three peaks of radioactivity in the large molecular weight region and a fourth one corresponding to low molecular unbound labeled NSILA-S. An excess of cold NSILA-S during preincubation leads to the disappearance of the two major large molecular weight peaks and to a concomitant increase of the peak eluting in the low molecular weight range. Binding of ¹²⁵I-labeled NSILA-S is highly sensitive to small concentrations of cold NSILA-S, whereas insulin, ACTH and human growth hormone are completely ineffective in displacing bound ¹²⁵I-labeled NSILA-S. NSILA-S preparations of different purity show displacement according to their specific biological activities. Furthermore, binding of ¹²⁵I-labeled NSILA-S to serum pH- and time-dependent and displays saturation characteristics. Chromatography of serum on Sephadex G-200 with 0.15 m acetic acid/0.15 m NaCl localizes the binding fraction in the 50,000–70,000 molecular weight range. Boiling of serum for 5 min abolishes binding completely.These studies help explain why the molecular weight of NSILA varied considerably from one group of investigators to the other.     

Anti-obesity effect of Solidago virgaaurea extract in high-fat diet-fed SD rat

In our previous report, Solidago virgaurea var. gigantea (SV) extract was shown to exhibit anti-adipogenesis activity in 3T3-L1 adipocyte cells. In this study, anti-obesity activity of SV extract was investigated in in vivo animal model. Sprague-Dawley (SD) rats were administered with high-fat diet, and the effect of SV extract was tested. SD rats were treated orally with SV extract for eight weeks, and their body weight was measured every week. The oral treatment of SV extract decreased body weight, fat tissue weight, blood low-density lipoprotein-cholesterol level, and blood triglycerides level. The p-AMP-activated protein kinase (AMPK) (AMP kinase) protein level in the fat tissue of the SV extract-treated SD rats increased. The protein levels of AMPK-downstream proteins, c-AMP response element binding protein and acetyl-CoA carboxylase, fatty acid synthase, and FABP4 decreased, indicating that SV extract-activated AMPK induced inhibition of adipogenesis and lipid biosynthesis in fat tissue. ¹H-NMR measurements of the lipid soluble liver extract showed a decrease in the lipid metabolites, indicating that SV extract-activated fatty acid oxidation in the liver. Overall, our results suggest that orally treated SV extract has excellent anti-obesity effect against HFD-induced obesity of SD rat.     

Accessible hyaluronan receptors identical to ICAM-1 in mouse mast-cell tumours

Immunohistochemical studies of the hyaluronan (HA)-receptor (R), originally found on liver endothelial cells (LEC) and related to the intercellular adhesion molecule 1 (ICAM-1), showed that polyclonal antibodies against HARLEC (HA receptor on LEC) also stain structures in mouse mastocytomas, mainly vessels. To test if intravenously administered HA might target the tumour receptorsin vivo, mice carrying an inoculated mastocytoma in one hind leg muscle were injected in the tail vein with¹²⁵I-tyrosine (T)-labelled HA and killed 75 min after injection when organs and tissues were checked for radioactivity. When doses exceeding the binding capacity of the liver were injected, a significant increase in radioactivity (up to five-fold) within the tumour tissue was found. The weight adjusted difference between control and tumour tissue was greater for smaller tumours, probably due to necrosis in the larger. HA-staining of tumours from animals receiving¹²⁵I-T-HA, showed HA in areas that also stained weakly for ICAM-1 using monoclonal antibodies. ICAM-1 staining was dramatically increased after hyaluronidase treatment of the sections, indicating that the HA is bound to these receptors and thereby blocks antibody recognition.Abbreviations ICAM-1 intercellular adhesion molecule 1 - HA hyaluronan - HARLEC hyaluronan receptor on liver endothelial cells - MW molecular weight     

ONTOGENETIC CHANGES OF PROTEINS OF ENDOPLASMIC RETICULUM

The proteins of the smooth and rough endoplasmic reticulum from fetal, immature, and adult male rats were compared after incorporation of two radioactively labeled precursors, ¹⁴C-labeled amino acids and δ-aminolevulinic acid-³H by means of gel electrophoresis. The labeling patterns indicated that protein components present in two major electrophoretic bands underwent significant synthesis in fetal tissue while three actively incorporating protein bands were noted in adult tissue. Although the uptake of the amino acids-¹⁴C decreased for the smooth and rough elements of the endoplasmic reticulum as a whole during liver development, the qualitative patterns were not significantly different in adult and fetal livers. The over-all incorporation (disintegrations per minute per milligram protein) of the heme precursor into the smooth and rough elements also did not change with development. However, a change was noted in the distributional electrophoretic patterns with development. The estimation of molecular weight (by disc electrophoresis) and the incorporation of the heme precursor suggested the similarity of the two major protein bands to cytochrome P-450 and cytochrome b₅, components of the endoplasmic reticulum, thought to be involved in the mixed-function oxidase system. The evidence indicated that in fetal liver, at a time when the oxidase capability was low, the amino acid incorporation into these two protein groups was the same as in the adult. The incorporation of the heme moiety, however, was different, decreasing in the cytochrome b₅ region and increasing in the cytochrome P-450 region during development. These results correlate with the increase in oxidase activity associated with liver development.     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.     

Isolation of carcinogenic aminoazo dye-binding protein and its identification as alcohol dehydrogenase

A major carcinogenic aminoazo dye-binding protein having Ip of 9.7 (isoelectric focusing) was isolated from the liver cytosol of rats given 40 mg 3'MeDAB. The protein has the molecular weight of 6.8 × 10⁴ (gel-filtration) and two subunits of about 3.9 × 10⁴ molecular weight (SDS-polyacrylamide gel electrophoresis). The amino acid composition was similar to that reported for liver alcohol dehydrogenase of animals. The enzymatic activity was shown to be associated consistently with the dye-binding protein fractions throughout the purification steps suggesting identity of the dye-binding protein as liver alcohol dehydrogenase.     

Purification and properties of pig liver and muscle enolases

Enolases (2-phospho-d-glycerate hydrolase, EC 4.2.1.11) were purified from both pig liver and muscle. Graphs of InC vs.r ² from sedimentation equilibrium experiments are linear, which suggests homogeneous preparations of liver and muscle enolases. From these data the molecular weight of liver enolase is calculated to be approximately 92,000 D and that of muscle enolase to be approximately 85,000 D. SDS-PAGE experiments give a molecular weight value of 46,000 D for liver enolase and a value of 44,000 D for muscle enolase. These molecular weight values for liver and muscle enzymes are within the range for other enolases and show that both of these pig enolases are dimers. Amino acid composition data support the sedimentation equilibrium data and also give a smaller molecule weight (84,968 D) for muscle enolase compared to that of the liver enzyme (89,021 D). The two enzymes differ in their content of lysine [liver enolase (L)=94 residues, muscle enolase (M)=68 residues], histidine (L=13, M=21), serine (L=53, M=36), proline (L=52, M=34), and cysteine (L=4, M=21). Partial specific volumes of 0.737 ml/g for liver enolase and 0.735 ml/g for muscle enolase were calculated from the amino acid composition data. Pig liver and muscle enolases differ radically in their isoelectric points (pI=6.4–6.5 for liver enolase, and pI=8.8–9.0 for muscle enolase), and in their degree of inactivation by 750 mM LiCI (liver enolase is inactivated to a greater degree than the muscle enolase). Despite these physical and chemical differences, the kinetic constantsK _M values for Mg²⁺, 2-phosphoglyceric acid, and phospho(enol)pyruvate appear not to be significantly different for these two forms of enolase. The physical, chemical, and kinetic data for pig liver and muscle enolases are compared to similar data for pig kidney enolase.     

Regulation of buccal mucosal calcium channel activity by salivary mucins

• 1.1. The effect salivary mucins on the activity of calcium channel isolated from buccal mucosal cell membranes was investigated. The uptake of ⁴⁵Ca²⁺while only moderately (15%) affected by the intact low and high molecular weight mucin forms, was significantly inhibited, by the acidic low and high molecular weight salivary mucins which evoked 64 and 60% inhibition, respectively.
• 2.2. The inhibitory effect of salivary mucins was associated with the sialic acid and sulfate ester groups of the carbohydrate chains, as the removal of either group caused partial loss in the glycoproteins inhibition, and the complete loss in the inhibitory effect occurred following desialylation and desulfation.
• 3.3. The channel in the presence of epidermal growth factor (EGF) and ATP responded by an increase in tyrosine phosphorylation of 55 and 170 kDa proteins, and the phosphorylated channels showed a 46% increase in ⁴⁵Ca²⁺ uptake. The phosphorylation and the calcium uptake were susceptible to inhibition by a specific tyrosine kinase inhibitor, genistein.
• 4.4. The binding of EGF to calcium channel receptor protein was inhibited by the low and high molecular weight acidic mucins, causing 41.2 and 36.1% reduction, respectively. This reduction in binding was dependent upon the presence of sulfate ester and sialic acid groups, as evidenced by the loss of the glycoproteins' inhibitory capacity following removal of these groups.
• 5.5. The results for the first time demonstrate that salivary mucins actively participate in the modulation of the EGF-controlled buccal mucosal calcium channel activity expression, a process of importance to the preservation of oral tissue integrity.

     

Demonstration of a macromolecule cross-reacting with antibodies to luteinizing hormone releasing hormone and its tissue distribution

Hypothalamic cytosol contains a macromolecule which cross-reacts with antibodies to luteinizing hormone releasing hormone (LRH). This cross-reacting material (macro-CRM) is insoluble in methanol or acid ethanol, and its molecular weight is about 70,000. Macro-CRM is also found in cytosols of extra-hypothalamic regions of the brain, liver, kidney, spleen, and skeletal muscle. Plasma contains only marginal amounts of macro-CRM. This substance inhibits the binding of [¹²⁵I]LRH to LRH antibodies in a reversible, competitive manner.     

Distribution of 103Ru—chloride in tumor-bearing animals and the mechanism for accumulation in tumor and liver

Tumor uptake rates of ¹⁰³Ru—chloride were smaller than those for ⁶⁷Ga—citrate. In three tumors and liver, ¹⁰³Ru in the mitochondrial fraction containing lysosome increased with time after the administration of ¹⁰³Ru—chloride. The concentration of ¹⁰³Ru was more dominant in connective tissue (especially inflammatory tissue) than in viable tumor tissue or in necrotic tissue. Quite large amounts of ¹⁰³Ru in the tumor and liver were bound to the acid mucopolysaccharide whose molecular masses exceeded 40,000. Behavior of this nuclide was essentially similar to that of ⁶⁷Ga.