Binding characteristics of delta opioid receptors in different regions of the brain of young and old male rats as studied with the highly selective ligand [D-Pen2-D-Pen5] enkephalin.

The present experiments were performed to study the binding characteristics of delta opioid receptors in membrane preparations obtained from the brain of adult male rats, and to analyze whether aging modifies these binding parameters. The binding characteristics of delta opioid receptors were evaluated on membrane preparations derived from dissected brain regions (hypothalamus, amygdala, mesencephalon, corpus striatum, hippocampus, thalamus, frontal poles, anterior and posterior cortex) collected from male rats of 3 and 24 months of age; the highly selective ligand 3H-[D-Pen2-D-Pen5] enkephalin (3H-DPDPE) was used. The results obtained in young rats show that the distribution of delta opioid receptors is different in the various brain areas examined; these receptors appear to be maximally concentrated in the frontal poles, anterior and posterior cortex; lower concentrations were found in the other structures considered. Kd (dissociation constant) for the delta sites was found very similar in all areas. The distribution of delta opioid receptors in the brain of 24-month-old rats was similar to that observed in young animals; this result was surprising in view of the fact that aging modifies the number of other types of brain opioid receptors (mu and kappa).     

Increased muscular phospholipase A2 activity in diabetic rats.

Anomalies in prostaglandin (PG) synthesis have been suggested in both experimental and human diabetes mellitus; increased levels of plasma and tissue eicosanoids has been recently reported by several investigators. One step in prostaglandin synthesis is the enzymatic hydrolysis of membrane phospholipids by Phospholipase A2 (PLA2). Nevertheless the alternative pathway involving Phospholipase C must be considered. An evaluation of PLA2 activity is therefore a useful method for studying prostaglandin synthesis in the peripheral target tissues of insulin activity. We studied PLA2 activity in normal and diabetic rat muscle. Streptozotocin-induced diabetic rats showed significantly higher muscular PLA2 activity when compared with controls (3.04 x 10(-2) +/- 0.50 x 10(-2) versus 1.34 x 10(-2) +/- 0.35 x 10(-2) arachidonic acid pMol.mg protein-1.min-1 (p less than 0.01). This effect was not observed in diabetic animals successfully treated with insulin (1.78 x 10(-2) +/- 0.5 x 10(-2) versus 1.34 x 10(-2) +/- 0.35 x 10(-2) arachidonic acid pMol.mg protein-1.min-1), and a significant correlation was found between blood glucose and muscular PLA2 activity (r = 0.42; p less than 0.05). Our results clearly show that in streptozotocin-induced diabetic rats muscular PLA2 activity is significantly higher. The relationship between blood glucose levels and muscular PLA2 activity and the decrease of PLA2 activity after insulin treatment suggest that these changes may be related to a defect in insulin effect.     

Argon laser lesions of the retina; occurrence and origin of macrophages.

Macrophage infiltration is frequent in the early stages of various proliferative eye disorders, including subretinal neovascularization. In this study, we set out to establish the origin of macrophages found in an animal model of laser-induced subretinal neovascularization. One primate received several intravenous injections of a colloidal carbon suspension. We then applied standard argon laser lesions to the retina of both eyes, which were enucleated eight days later and sectioned serially for histological examination. A quantitative estimate of carbon-laden and non-laden leukocytes was made based on morphological criteria. Mononuclear leukocytes accumulated in the laser lesions and the percentage of carbon-laden mononuclear leukocytes in relation to the total leukocyte number was higher in the extravascular area of the laser sites than in the systemic circulation. These findings indicate that the majority of mononuclear leukocytes that accumulate at the sites of laser lesions are derived from the systemic circulation.     

Molecular biological and immunohistochemical analysis of tp53 in human ameloblastomas]

AIM: Ameloblastoma is the most frequent epithelial tumor of the jaws. The spread is locally invasive and it tends to recur. Malignant transformation and occurrence of metastases has been described. Immunohistochemical analysis shows an enhanced expression of P53 protein in ameloblastomas. Mutation of the tp53 tumor suppressor gene was verified in several human tumors. In this study histological sections were analyzed for the expression of P53 protein and the tp53 gene was examined for mutations. MATERIAL AND METHODS: Tumor DNA from 29 patients with an ameloblastoma was examined for mutations in exons 5 to 8 of the tp53 tumor suppressor gene using PCR, SSCP,- and sequential analysis. Histological sections of the tumors were analyzed immunohistochemically for an overexpression of P53 protein. RESULTS: Two tp53 mutations (6.9%) in ameloblastomas were verified for the first time. In all 58.6% of the tumors showed an immunoreactivity for P53 protein. There was a statistically significant positive correlation (Fisher's exact test p<0.0148) between an increased number of P53-positive tumor cells and the appearance of recurrence. DISCUSSION: In the face of the uncertain postoperative behavior of ameloblastomas, the immunohistochemical verification of more than 10% P53-positive tumor cells may give a prognostic indication for a tendency to recurrence.     

Ecological memory assessment in normal aging. A preliminary report on an Italian population

Forty-eight healthy volunteers were administered a computerized battery of "ecological" memory tests simulating real life everyday tasks and actions such as recalling people's names or telephone numbers. The group was dichotomized according to recent criteria proposed by a NIMH study group. Younger individuals (below 50 years of age) always showed better performances than those over 50. Although the learning curves were significantly lower for the older subgroup, the forgetting rate did not differ, suggesting that most of the memory complaints of the elderly might be attributable to the initial encoding phase of the memorizing activity rather than to a retrieval problem. It seems that easy distractability and inefficient strategic elaboration of incoming information are also, at least in part, responsible for the memory problems associated with normal aging. This article reports preliminary findings of a normative and representative Italian population sample.     

Transfer factor (TF) treatment of patients with HBs-Ag-positive chronic active hepatitis

Summary It is a clinically and experimentally well supported working hypothesis that infection with hepatitis B virus may result in chronic active hepatitis in patients with suspected immune deficiencies. On this basis, a pilot study was performed in order to evaluate the effect of specific transfer factor (TF) in the treatment of HB_s-Ag-positive chronic active hepatitis. From the leukocytes of 500 ml venous blood each of 40 volunteers that had completely recovered from acute virus hepatitis B within the last 6 months, a unique TF pool (40 units of TF) was prepared according to the method of Lawrence. Preexaminations indicated that this preparation was able to enhance cellular immune reactions in vitro. Thirteen patients with HB_s-antigenemia and chronic active hepatitis (i.e., two liver biopsies within the last 6 or more months with the histological criteria of chronic aggressive hepatitis according to de Groote, elevated serum levels of bilirubin, alkaline phosphatase, transaminase activities, and/or -globulines) were randomized: Seven received s.c. injections of two units of TF each on days 1 and 15, the other six saline. Conversion of skin reactions to some ubiquitous antigens occurred in the TF group, but no significant and constant drop of HB_s-Ag serum titers was observed. Although some of the biochemical parameters seemed to ameliorate in the TF group, the differences versus the control group did not prove to be significant within the limited number of patients under observation. The in vitro reactivity of patients' lymphocytes to HB_s-Ag, tested by means of the³H-thymidine uptake, was never found enhanced after TF application. In the used doses, specific TF was not effective in the treatment of HB_s-Ag-positive chronic active hepatitis; unfavorable side-effects were not observed.     

Primary osteoblasts response to shock wave therapy using different parameters

Over the past decade extracorporeal shock-wave therapy (ESWT) has been increasingly applied to orthopaedic and musculoskeletal pathologies, the aim of this study was to assess how the energy density of the shock waves and the number of impulses affect viability, differentiation and synthetic activity of osteoblasts. Primary sheep osteoblasts cultures were treated with ESWT with an electro-hydraulic shock wave generator by selecting three different energy levels (14-21-28 kV corresponding at 0.15-0.31-0.40 mJ/mm2) and two different total numbers of impulses (500, 1000) for each level. At the end of treatment, cell counts and viability were recorded. Cells were then cultivated for 48 hours starting from a concentration of 1 x 10(4) cells/ml. The biological activity and viability were evaluated at 24 and 48 hours after treatment. No cytodestructive effects were observed in Group A, while a cytodestructive effect of ESWT was seen in cultures receiving the highest energy treatments. The different shock wave treatment induced differences in MTT assays after 24 and 48 hours, in particular the highest level showed a detrimental effect on cell respiration at both experimental times as compared to the Control Group and the protein metabolism was generally depressed by ESWT with impulses at the highest energy level. After 24 hours such effect further increased with the growing number of impulses. The lowest energy level appeared to significantly improve the metabolic parameter in primary cell cultures as compared to controls when 500 impulses were selected. The current study has demonstrated that one of the most important aspects to be considered is not the total number of impulses used but the energy level of the shock waves, thus confirming that ESWT has a dose-dependent effect on cells.     

Splice variants of the relaxin and INSL3 receptors reveal unanticipated molecular complexity

LGR7 and LGR8 are G protein-coupled receptors that belong to the leucine-rich repeat-containing G-protein coupled receptor (LGR) family, including the thyroid-stimulating hormone (TSH), LH and FSH receptors. LGR7 and LGR8 stimulate cAMP production upon binding of the cognate ligands, relaxin and insulin-like peptide 3 (INSL3), respectively. We cloned several novel splice variants of both LGR7 and LGR8 and analysed the function of four variants. LGR7.1 is a truncated receptor, including only the N-terminal region of the receptor and two leucine rich repeats. In contrast, LGR7.2, LGR7.10 and LGR 8.1 all contain an intact seven transmembrane domain and most of the extracellular region, lacking only one or two exons in the ectodomain. Our analysis demonstrates that although LGR7.10 and LGR8.1 are expressed at the cell surface, LGR7.2 is predominantly retained within cells and LGR7.1 is partially secreted. mRNA expression analysis revealed that several variants are co-expressed in various tissues. None of these variants were able to stimulate cAMP production following relaxin or INSL3 treatment. Unexpectedly, we did not detect any direct specific relaxin or INSL3 binding on any of the splice variants. The large number of receptor splice variants identified suggests an unforeseen complexity in the physiology of this novel hormone-receptor system.     

Veränderte Aktivitäten der Enzyme des Energiestoffwechsels in Thrombocyten von Patienten mit Lebercirrhose und Splenomegalie

Zusammenfassung 1. Die Aktivitäten von 28 Enzymen aus verschiedenen Abschnitten des Energiestoffwechsels wurden in isolierten Thrombocyten von 17 gesunden Menschen und 15 Patienten mit gesicherter Lebercirrhose gemessen.2. Die für Gewebe bekannte Proportionskonstanz der Enzymaktivitäten des zentralen Segmentes der Glykolyse (TIM, GAPDH, PGK, GPM und EN) findet sich auch in den Thrombocyten als Ordnungsprinzip. Die mitochondrial lokalisierten Enzyme NAD-spezifische Isocitratdehydrogenase und Glycerophosphatoxydase wurden erstmalig in Thrombocyten mit relativ hoher Aktivität nachgewiesen.3. Die Plättchen von Patienten mit Lebercirrhose und Milzvergrößerung infolge portaler Hypertension zeigten signifikant erhöhte Enzymaktivitäten in allen untersuchten Stoffwechselwegen, insbesondere der mitochondrial lokalisierten Enzyme. Funktionell bedeutsam erscheinen der ausgeprägte Anstieg der Mg⁺⁺-aktivierbaren ATPase, die gleichbleibende Aktivität der Fructose-6-Phosphatkinase und eine Verminderung der Lactatdehydrogenase. Bei der Patientengruppe mit Cirrhose ohne Milzvergrößerung lagen fast alle gemessenen Aktivitäten der Thrombocyten im Normbereich.4. Es wird ein Patient mit Lebercirrhose ohne Milzvergrößerung beschrieben, dessen Thrombocyten um den Faktor 8 höhere Aktivitäten fast aller gemessenen Enzyme aufwiesen. Auch in diesen Plättchen zeigte die Lactatdehydrogenase eine Aktivitätsverminderung gegenüber der Norm.5. Ein Enzymdefekt der Plättchen wie bei Thrombasthenie wurde bei Lebercirrhose nicht gefunden. Nach unseren Kenntnissen über die Enzymausstattung von Blutzellen handelt es sich bei den Thrombocyten mit höherer Enzymaktivität um eine jugendliche Zellpopulation. Die Pathogenese wird besonders hinsichtlich der Rolle der vergrößerten Milz diskutiert.

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Summary 1. The activities of 28 enzymes from different pathways of energy producing metabolism were measured in the isolated platelets of 17 normal persons and 15 patients with proven cirrhosis of the liver.2. In mammalian tissues a constant proportion between the enzymes of the central segment of the glycolytic pathway (TIM, GAPDH, PGK, GPM, EN) has been described. This constant proportion has been demonstrated also in the platelets. The mitochondrially localized enzymes NAD-specific Isocitratdehydrogenase and Glycerophosphatoxidase have been measured for the first time in platelets with a high activity.3. The platelets of patients with liver cirrhosis and splenomegaly following portal hypertension showed significant higher enzyme activities in all investigated pathways, mainly in the citric acid cycle. Functionally important could be the marked increase of the Mg⁺⁺ activated ATPase, the unaltered activity of the Phosphofructokinase and a lowered activity of the Lactatdehydrogenase in these platelets. The patients with cirrhosis but without a large spleen had normal enzyme activities of the platelets.4. One patient is described with cirrhosis without splenomegaly who had an elevation of nearly all measured enzymes by a factor 8 in his platelets. The LDH showed a decreased activity.5. No enzyme defect in the platelets of cirrhotic patients as in thrombasthenia was found. Basing on our knowledge of the enzyme equipment of blood cells the conclusion is drawn that the platelets with higher enzyme content represent a young cell population. The pathogenesis is discussed with special reference to the role of the enlarged spleen.

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Teilergebnisse wurden vorgetragen auf dem 2. Symposium der European Association for the Study of the Liver, Göteborg, 1967 und der 24. Tagung der Deutschen Gesellschaft für Verdauungs- und Stoffwechselkrankheiten, Hamburg 1967.