[125I]CGP 42112 reveals a non-angiotensin II binding site in 1-methyl-4-phenylpyridine (MPP+)-induced brain injury

Summary 1. Intracerebral injection of the oxidative metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridine (MPP⁺), into the substantia nigra of adult rats resulted in a lesion at the injection site.2. Using autoradiography, we localized specific [¹²⁵]CGP 42112 binding that was not recognized by angiostensin II or angiotensin II AT₁ or AT₂ receptorselective ligands.3. Our results suggest that [¹²⁵I]CGP 42112 may be binding to activated microglia that appear at the lesion site.     

Effects of natural complex carbohydrate (citrus pectin) on murine melanoma cell properties related to galectin-3 functions

Citrus pectin (CP) and pH-modified citrus pectin (MCP) are highly branched and non-branched complex polysaccharides, respectively, rich in galactoside residues, capable of combining with the carbohydrate-binding domain of galectin-3. We reported previously that intravenous injection of B16-F1 murine melanoma cells with CP or MCP into syngeneic mice resulted in a significant increase or decrease of lung colonization, respectively (Platt D, Raz A (1992)J Natl Cancer Inst 84:438–42). Here we studied the effects of these polysaccharides on cell-cell and cell-matrix interactions mediated by carbohydrate-recognition. MCP, but not CP, inhibited B16-F1 melanoma cells adhesion to laminin and asialofetuin-induced homotypic aggregation. Both polysaccharides inhibited anchorage-independent growth of B16-F1 cells in semisolid medium, i.e. agarose. These results indicate that carbohydrate-recognition by cell surface galectin-3 may be involved in cell-extracellular matrix interaction and play a role in anchorage-independent growth as well as thein vivo embolization of tumour cells.Abbreviations CP natural citrus pectin - MCP pH-modified CP - EHS Englebreth-Holm Swarm - CMF-PBS Ca²⁺-and Mg²⁺-free phosphate-buffered saline, pH 7.2 - HRP horseradish peroxidase - ABTS 2,2-azino-di(3-ethylbenzthiazoline sulfonic acid - DMEM Dulbecco's modified Eagle's minimal essential medium - BSA bovine serum albumin     

Human HE2 (μB) and μA motifs show the same function as whole IgH intronic enhancer in transgenic mice

In the murine IgH gene intronic enhancer (ENH_iH), two major functional domains were reported. One is the E4/octomer region and another includes the A and B motifs. In the human ENH_iH, it was reported that the HE2, which corresponds to the murine B, and E6 motifs play an important role in an enhancer activity and a tissue-specificity at cellular level. Here we examined thein vivo function of the E6, A and HE2 motifs within the human ENH_iH by using the transgenic mice technique. The A and HE2 motifs together revealed almost the same enhancer function as the whole human ENH_iH, but the E6 motif had lesser enhancer acitivty and tissue-specificity.     

Sequence homology and structure predictions of the creatine kinase isoenzymes

Comparisons of the protein sequences and gene structures of the known creatine kinase isoenzymes and other guanidino kinases revealed high homology and were used to determine the evolutionary relationships of the various guamidino kinases. A CK framework is defined, consisting of the most conserved sequence blocks, and diagnostic boxes are identified which are characteristic for anyone creatine kinase isoenzyme (e.g. for vertebrate B-CK) and which may serve to distinguish this isoenzyme from all others (e.g. from M-CKs and Mi-CKs). Comparison of the guanidino kinases by near-UV and far-UV circular dichroism further indicates pronounced conservation of secondary structure as well as of aromatic amino acids that are involved in catalysis.Abbreviations GuaK guanidino kinase - CK creatine kinase - B-and M-CK brain and muscle cytosolic CK isoenzyme - Mi-CK mitochondrial CK isoenzyme - ArgK arginine kinase - Cr creatine - PCr phosphorylcreatine - PArg phosphorylarginine     

Neurogenic peptides and the cardiomyopathy of magnesium-deficiency: Effects of substance P-receptor inhibition

Dietary deficiency of magnesium (Mg) in rodents results in cardiomyopathic lesion formation. In our rat model, these lesions develop after 3 weeks on the Mg-deficient diet; significant elevation of several cytokines, IL-1, IL-6 and TNF also occurs. In probing the mechanisms of lesion formation, we obtained data supporting the participation of free radicals (Freedman AMet al.: Bioch Biophys Res Commun 1990; 170: 1102). Recently, we identified an early elevation of circulating substance P and proposed a role of neurogenic peptides during Mg-deficiency (Weglicki WB, Phillips TM: Am J Phys 1992; 262: R734). The present study was designed to evaluate the contribution of neurogenic peptides to the pathogenesis of Mg-deficiency. In the blood, substance-P and calcitonin gene related peptide (CGRP) are elevated during the first week on the diet. During the second week, circulating histamine, PGE₂ and TBAR-materials were elevated and red cell glutathione was reduced, all prior to the elevation of the inflammatory cytokines during the third week. When the rats were treated with the substance P-receptor blocker [CP-96,345], the levels of substance P and CGRP remained elevated; however, increases in histamine, PGE₂, TBAR-materials, and the decrease in red cell glutathione were inhibited; also, the development of cardiac lesions was inhibited significantly. These data support a central role for neurogenic peptides, especially substance P, in the development of cardiomyopathic lesions during Mg-deficiency.     

A cell-based reporter assay for the identification of protein kinase C activators and inhibitors

Transmission of extra cellular signals across biological membranes results in the generation of lipid metabolites which in turn influence specific cellular events such as cell growth or differentiation. Many of these lipid messengers can activate protein kinase C (PKC) isozymes of which one function is to perpetuate the extracellular signals to the nucleus by phosphorylating other targets proteins. We have engineered mammalian cell lines to identify and evaluate activators and inhibitors of PKC-dependent and independent signal transduction pathways. The A31 mouse fibroblast cell line, has been stably transfected with a construct containing a triplet repeat of the TPA response element (TRE) upstream of a thymidine kinase promoter fused to the human growth hormone (hGH) gene. A31 cells containing this reporter construct exhibit significant increases in hGH secretion following stimulation by phorbol esters or other mitogens. The levels of hGH secretion are modulated in this system using different pharmacological agents. We demonstrate that this assay can be used to identify specific and general inhibitors as well as activators of the signal transduction pathway mediated by PKC isozymes. (Mol Cell Biochem141: 129–134, 1994)     

Purification and characterization of the high molecular weight microtubule associated proteins from neonatal rat brain

The changes in the levels of microtubule-associated proteins (MAPs) during advanced embryonic stages, neonatal and adult organisms reflect the importance of these cytoskeletal proteins in relation to the morphogenesis of the central nervous system. MAP-1B is found in prenatal brains and it appears to have the highests levels in neonatal rat brains, being a developmentally-regulated protein. In this research, a fast procedure to isolate MAP-1B, as well as MAP-2 and MAP-3 from neonatal rat brains was designed, based on the differential capacity of poly L-aspartic acid to release MAPs during temperature-dependent cycles of microtubule assembly in the absence of taxol. The high molecular weight MAP-1B was recovered in the warm supernatants after microtubular protein polymerization in the presence of low concentrations of polyaspartic acid. Instead, MAP-2 and a 180 kDa protein with characteristics of MAP-3 remained associated to the polymer after the assembly. Further purification of MAP-1B was attained after phosphocellulose chromatography. Isolation of MAP-2 isoforms together with MAP-3 was achieved on the basis of their selective interactions with calmodulin-agarose affinity columns. In addition, MAP-2 and MAP-3 were also purified on the basis of their capacities to interact with the tubulin peptide -II (422–434) derivatized on an Affigel matrix. However, MAP-1B did not interact with the -II tubulin fragment, but it showed interaction with the Affigel-conjugated -I (431–444) tubulin peptide. The different MAPs componentes were characterized by western blots using specific monoclonal antibodies. A salient feature of neonatal rat brain MAP-3 was its interactions with site-directed antibodies that recognize binding epitopes on the repetitive sequences of tau and MAP-2. However, these site-specific antibodies did not interact with MAP-1B from the neonatal rat brain tissue.Abbreviations PAA poly (L-aspartic acid) - HMW-MAPs high molecular weight microtubule associated proteins     

The impact of type I diabetes on rat liver γ-glutamyltranspeptidase

The impact of type 1 diabetes mellitus on liver -glutamyltranspeptidase, a premalignant marker, was studied. Diabetes was induced in male Sprague Dawley and Fischer 344 rats by administration of Streptozotocin, which produced a stable and moderately severe diabetic state. In liver homogenates, -glutamyltranspeptidase was increased over control levels: 1.2, 8.1 and 13,2 fold in Strague-Dawley rats; 4.8, 58.4 and 84.7 fold in Fischer 344 rats; at 1, 3 and 6 weeks following Streptozotocin treatment. In plasma membranes isolated from the livers of Fischer 344 rats, -glutamyltranspeptidase was increased over control levels: 5.6, 75 and 127 fold at weeks 1, 3 and 6 following Streptozotocin treatment. The relative specific activity of 5-nuleohdase was found to be similar: 9–14, indicating comparable degrees of plasma membrane purity. Plasma glutamate-pyruvate transaminase levels were minimally and similarly affected at all time points indicating lack of association of increasing -glutamyltranspeptidase activity with overt liver damage. Thyroid hormone replacement, with both T₃ (0.6 g/Kg) once a day and T₄ (6.0 g/kg) twice a day for three days elicited a further 30% increment in enzyme activity. Insulin replacement (20–40 units/200 g body weight) twice a day for five days reduced enzyme activity 51% at week 6. This was associated with an increase in -glutamyltranspeptidase in the plasma from 14 fold over control levels in the diabetic state at week 6 to 53 fold ever control levels after insulin replacement at week 6. It is proposed that the diabetes-induced increase in -glutamyltranspeptidase is reduced by an insulin-directed shedding of the enzyme into the plasma.     

3-Aminobenzamide,a potent inhibitor of poly (ADP-ribose) polymerase,causes a rapid death of HL-60 cells cultured in serum-free medium

HL-60 cells transferred from serum-supplemented to serum-free culture medium initially bound to culture plate tightly and then released from the plate on increasing the culture time and resumed exponential growth after about 8 h lag. At the initial stage of the culture, the cells became extremely sensitive to 3-aminobenzamide, a potent inhibitor of poly (ADP-ribose) polymerase, and, at 1 mM, 80 to 90% of the cells were lysed within 20 h, whereas the inhibitor was totally ineffective on the cell growth in serum-supplemented medium at the concentration. Non-inhibitory analogs of the inhibitor were ineffective. Assay of poly(ADP-ribose) polymerase activity in permeable cells indicated that a transient activation of the enzyme occurred during the culture in serum-free medium (the maximum activation was observed at 8 h of the culture). The cells conditioned in serum-free medium for 24 h acquired significant resistancy to the inhibitor. A low concentration of fibronectin (5 to 10/ml) and a relatively high concentration of bovine serum albumin (0.5 to 1 mg/ml) effectively blocked the cell attachment to plate and also the 3-aminobenzamide-induced cell lysis. These results suggest that poly(ADP-ribose) polymerase is involved in a process essential for HL-60 cells to adapt to a serumdeprived growth condition.