Quantification of observable behaviors induced by typical and atypical kappa-opioid receptor agonists in male rhesus monkeys.

RATIONALE: Kappa-opioid receptor (KOR) agonists are antinociceptive but have side effects that limit their therapeutic utility. New KOR agonists have been developed that are fully efficacious at the KOR but may produce fewer or reduced side effects that are typical of KOR agonists. OBJECTIVES: We determined behavioral profiles for typical and atypical KOR agonists purported to differ in intracellular-signaling profiles as well as a mu-opioid receptor (MOR) agonist, oxycodone, using a behavioral scoring system based on Novak et al. (Am J Primatol 28:124-138, 1992, Am J Primatol 46:213-227, 1998) and modified to quantify drug-induced effects (e.g., Duke et al. J Pharmacol Exp Ther 366:145-157, 2018). METHODS: Six adult male rhesus monkeys were administered a range of doses of the typical KOR agonists, U50-488H (0.0032-0.1 mg/kg) and salvinorin A (0.00032-0.01 mg/kg); the atypical KOR agonists, nalfurafine (0.0001-0.001 mg/kg) and triazole 1.1 (0.01-0.32 mg/kg); the MOR agonist, oxycodone (0.0032-0.32 mg/kg); and as controls, cocaine (0.032-0.32 mg/kg) and ketamine (0.32-10 mg/kg). For time-course determinations, the largest dose of each KOR agonist or MOR agonist was administered across timepoints (10-320 min). In mixture conditions, oxycodone (0.1 mg/kg) was followed by KOR-agonist administration. RESULTS: Typical KOR agonists produced sedative-like and motor-impairing effects. Nalfurafine was similar to typical KOR agonists on most outcomes, and triazole 1.1 produced no effects on its own except for reducing scratch during time-course determinations. In the mixture, all KOR agonists reduced oxycodone-induced scratching, U50-488H and nalfurafine reduced species-typical activity, and U50-488H increased rest/sleep posture. CONCLUSIONS: Atypical "biased" KOR agonists produce side-effect profiles that are relatively benign (triazole 1.1) or reduced (nalfurafine) compared to typical KOR agonists.

Peroxynitrite mediates VEGF's angiogenic signal and function via a nitration-independent mechanism in endothelial cells.

The modulation of angiogenic signaling by reactive oxygen species (ROS) is an emerging area of interest in cellular and vascular biology research. We provide evidence here that peroxynitrite, the powerful oxidizing and nitrating free radical, is critically involved in transduction of the VEGF signal. We tested the hypothesis that VEGF induces peroxynitrite formation, which causes tyrosine phosphorylation and mediates endothelial cell migration and tube formation, by studies of vascular endothelial cells in vitro and in a model of hypoxia-induced neovascularization in vivo. The specific peroxynitrite decomposition catalyst FeTPPs blocked VEGF-induced phosphorylation of VEGFR2 and c-Src and inhibited endothelial cell migration and tube formation. Furthermore, exogenous peroxynitrite mimicked VEGF activity in causing phosphorylation of VEGFR2 and stimulating endothelial cell growth and tube formation in vitro and new blood vessel growth in vivo. The selective nitration inhibitor epicatechin enhanced VEGF's angiogenic function in activating VEGFR2, c-Src, and promoting endothelial cell growth, migration, and tube formation in vitro and retinal neovascularization in vivo. Decomposing peroxynitrite with FeTPPs or blocking oxidation using the thiol donor NAC blocked VEGF's angiogenic functions in vitro and in vivo. In conclusion, peroxynitrite is critically involved in transducing VEGF's angiogenic signal via nitration-independent and oxidation-mediated tyrosine phosphorylation.

Ancient mitogenomes of Phoenicians from Sardinia and Lebanon: A story of settlement, integration, and female mobility.

The Phoenicians emerged in the Northern Levant around 1800 BCE and by the 9th century BCE had spread their culture across the Mediterranean Basin, establishing trading posts, and settlements in various European Mediterranean and North African locations. Despite their widespread influence, what is known of the Phoenicians comes from what was written about them by the Greeks and Egyptians. In this study, we investigate the extent of Phoenician integration with the Sardinian communities they settled. We present 14 new ancient mitogenome sequences from pre-Phoenician (~1800 BCE) and Phoenician (~700-400 BCE) samples from Lebanon (n = 4) and Sardinia (n = 10) and compare these with 87 new complete mitogenomes from modern Lebanese and 21 recently published pre-Phoenician ancient mitogenomes from Sardinia to investigate the population dynamics of the Phoenician (Punic) site of Monte Sirai, in southern Sardinia. Our results indicate evidence of continuity of some lineages from pre-Phoenician populations suggesting integration of indigenous Sardinians in the Monte Sirai Phoenician community. We also find evidence of the arrival of new, unique mitochondrial lineages, indicating the movement of women from sites in the Near East or North Africa to Sardinia, but also possibly from non-Mediterranean populations and the likely movement of women from Europe to Phoenician sites in Lebanon. Combined, this evidence suggests female mobility and genetic diversity in Phoenician communities, reflecting the inclusive and multicultural nature of Phoenician society.

[New conjugates of antitumor antibiotic doxorubicin with water-soluble galactomannan: synthesis and biological activity].

New water-soluble conjugates in the form of Schiff bases (DGM-1 and DGM-2) were prepared by the interaction of water-soluble periodate-oxidized galactomannan with doxorubicin or N-(L-lysyl)doxorubicin, respectively. The water-soluble galactomannan (DAVANAT a commercial product of Pro-Pharmaceuticals company) was obtained by partial acidic hydrolysis of high-molecular-mass galactomannan from Cyamopsis tetragonoloba (guar gum) seeds. The conjugate stability was studied in aqueous solutions. The DGM-1 antiproliferative activity was comparable with that of doxorubicin on three models: cell lines of murine melanoma B 16-F1, human breast cancer MCF-7 (HTB-22), and human colon cancer HT-29 (HTB-38). DGM-2 was poorly active in all the three tests. DGM- 1 can thus be regarded as a high-molecular-mass depot form of doxorubicin.

Modulation of the lung colonization of B16-F1 melanoma cells by citrus pectin.

CONTEXT: Studies have shown that the galactoside-containing simple sugars and anti-galactoside-binding lectin antibodies may affect experimental tumor cell metastasis. However, the limited number of reagents used thus far necessitate further observations. PURPOSE: Natural citrus pectin (CP) and pH-modified CP (MCP), rich in galactose residues, were used to study the involvement of carbohydrates containing galactoside residues in cellular interaction in vitro and in lung colonization in vivo of B16-F1 melanoma cells. METHODS: B16-F1 melanoma cells were incubated with various concentrations of CP and MCP. Their ability to form homotypic aggregation in vitro and tumor lung colonization in vivo in 8-week-old female C57BL/6 mice was then analyzed. RESULTS: The CP binds to the surface of B16-F1 melanoma cells; this binding can be inhibited by lactose at a concentration of 0.15 M. Intravenous injection of the murine B16-F1 melanoma cells with the natural CP resulted in a significant increase (up to threefold) in the appearance of tumor colonies in the lung and in increased homotypic aggregation properties of the cells, while injection of MCP significantly decreased B16-F1 experimental metastasis (greater than 90%). CONCLUSIONS: Tumor galactoside-binding proteins mediate cellular recognition by linking oligosaccharides with terminal D-galactoside residues on adjacent cells. Successful interference with such a process with MCP may lead to a reduced ability to form tumor cell emboli and metastasis. IMPLICATIONS: These findings imply that the galactose-containing carbohydrate side chains of CP might mimic or compete with the natural ligand(s) of the tumor galactoside-binding protein (gal-lectin) and thus affect cellular interactions relevant for metastasis.

Naturally occurring cell-mediated cytotoxicity against simian virus 40-transformed 3T3 murine tumor cells.

The tumorigenicity and host protective mechanisms induced by simian virus 40 (SV40)-transformed 3T3 cells (SV403T3) were evaluated in syngeneic BALB/c mice. Tumors were regularly produced by sc inoculation of SV403T3 cells; the incidence, latent period, and survival were proportional to the size of the initial inoculum. With the use of an in vitro 18-hour 51Cr cytotoxicity assay, spleen cells from normal mice showed a dose-related killing activity against the SV403T3 cells. At an effector cell-to-target cell ratio of 200:1, the average lysis was 56 +/- 6%. This reaction appeared specific for the virally transformed targets; the mean lysis of parent 3T3 cells was 23 +/- 5%. Effectors were resistant to anti-theta serum and not removed by adherence to plastic or nylon wool. Tissue distribution studies indicated that these effectors were present in high concentrations in spleen, bone marrow, lymph nodes, and peritoneal cavity. Low levels of activity were associated with cells from the thymus. In the present studies specific T-cell cytotoxicity against the SV403T3 cells could not be demonstrated. Animals challenged with nonviable SV403T3 cells prior to tumor cell inoculation did not show increased in vivo resistance. In parallel, the in vitro cytotoxicity of animals inoculated with SV403T3 tumor cells showed no heightened cell killing compared to the cytotoxicity of normal controls.

Complement C1q and C3 mRNA expression in the frontal cortex of Alzheimer's patients.

The levels and cellular localization of mRNA for complement C1q and C3 were examined by RNA gel blot and nonradioactive in situ hybridization in the frontal cortex of patients with Alzheimer's disease (AD) and age-matched controls. We found that the hybridization signal for C1q mRNA was markedly increased (approx. 3.5-fold) in the frontal cortex of AD patients compared to that in age-matched controls. In contrast to previous reports we also found that the levels of C3 mRNA, although well expressed, did not differ significantly between AD cases and age-matched controls. Nonradioactive in situ hybridization using digoxigenin-labeled ribo-probes revealed that transcripts coding for both C1q and C3 were closely associated with neurons. These results support the hypothesis that complement could play a role in neuronal degeneration which has been observed in the brain of AD patients.

Osteoarthritis of the talocalcaneal joint in 18 horses.

REASONS FOR PERFORMING STUDY: Talocalcaneal osteoarthritis (TO) is an uncommon cause of moderate to severe hindlimb lameness, on which only isolated case reports have been published to date. OBJECTIVES: To review the clinical features of TO and determine optimal methods for diagnosis, management and prognosis. METHODS: The case records from 4 referral centres of 18 horses showing hindlimb lameness considered, as a result of clinical investigation, to be caused by TO, were reviewed. RESULTS: TO affected mature sports and pleasure horses (age 7-16 years) and caused moderate to severe lameness, usually of sudden onset with no obvious inciting cause. There were few localising signs, other than worsening of lameness by hock flexion. Tarsocrural joint analgesia produced improvement in lameness in 6/11 horses (55%) and perineural analgesia of the tibial and fibular nerves complete soundness in 6/14 horses (43%) in which it was performed; 7/14 horses (50%) showed a further substantial improvement. Radiological findings included subchondral bone lysis and sclerosis and irregular joint space width, seen most obviously in a lateromedial view. Nuclear scintigraphy revealed marked uptake of radiopharmaceutical predominantly plantaromedially in the region of the talus in the 7 horses in which it was performed. Fourteen horses were treated conservatively with box- or pasture-rest, with or without intra-articular corticosteroids, hyaluronic acid or polysulphated glycosaminoglycan, and all remained lame. Intra-articular corticosteroids appeared to have no effect in any horse. Of 10 horses receiving conservative management only, 6 were subjected to euthanasia, 3 were retired and 1 remained in light work, but was still lame. Two horses treated by either partial tibial and fibular neurectomy or subchondral forage failed to regain soundness and were retired. Six horses were treated by surgical arthrodesis of the talocalcaneal joint with 2 or three 5.5 mm AO screws introduced obliquely across the joint from the plantarolateral aspect of the calcaneus, which resulted in improvement in lameness in all cases. CONCLUSIONS: Osteoarthritis of the talocalcaneal joint causes acute onset severe lameness, but clinical findings and diagnostic analgesia often fail to identify precisely the site of pain. Consistent radiographic changes suggested TO was contributing to the lameness and this diagnosis was supported by nuclear scintigraphy. The poor success of conservative treatment (including intra-articular medication) suggests that surgical arthrodesis is the treatment of choice, although the prognosis is still poor for a return to full soundness. POTENTIAL RELEVANCE: The clinical features described should facilitate more accurate diagnosis and prognosis. A novel surgical treatment is described which appears to offer significant improvement in the lameness. Further work is necessary to determine the causes of this condition and more effective management.

Accelerated glial reactivity to stroke in aged rats correlates with reduced functional recovery.

Following cerebral ischemia, perilesional astrocytes and activated microglia form a glial scar that hinders the genesis of new axons and blood vessels in the infarcted region. Since glial reactivity is chronically augmented in the normal aging brain, the authors hypothesized that postischemic gliosis would be temporally abnormal in aged rats compared to young rats. Focal cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery in 3- and 20-month-old male Sprague Dawley rats. The functional outcome was assessed in neurobehavioral tests at 3, 7, 14, and 28 days after surgery. Brain tissue was immunostained for microglia, astrocytes, oligodendrocytes, and endothelial cells. Behaviorally, aged rats were more severely impaired by stroke and showed diminished functional recovery compared with young rats. Histologically, a gradual activation of both microglia and astrocytes that peaked by days 14 to 28 with the formation of a glial scar was observed in young rats, whereas aged rats showed an accelerated astrocytic and microglial reaction that peaked during the first week after stroke. Oligodendrocytes were strongly activated at early stages of infarct development in all rats, but this activation persisted in aged rats. Therefore, the development of the glial scar was abnormally accelerated in aged rats and coincided with the stagnation of recovery in these animals. These results suggest that a temporally anomalous gliotic reaction to cerebral ischemia in aged rats leads to the premature formation of scar tissue that impedes functional recovery after stroke.

Seizure induced C-Fos mRNA in the rat brain: comparison between young and aging animals.

The molecular mechanisms associated with age-related alterations in the pharmacological and physiological properties of hippocampal and cortical neurons in response to chemically induced seizure are largely unknown. Administration of pentylenetetrazole (PTZ) (50 mg/kg body weight) to rats of various ages evoked tonic-colonic seizures. Using RNA gel blot analysis we found that 1 h after the onset of seizure, the mRNA for the protooncogene c-fos was increased in the hippocampus and cortex of 3-month-old rats. The levels of c-fos mRNA in the hippocampus and cortex of 3-month-old rats returned to control levels by 3 h after PTZ administration. The levels of c-fos mRNA in the hippocampus and cortex of 20-month-old and 30-month-old rats peaked at 3 h and returned to basal levels by 15 h following PTZ treatment. These results suggest that the induction of immediate-early gene expression, as exemplified by c-fos, is not impaired in the aged rat brain. However, the aged rat brain responded more slowly to chemically induced seizure and the levels of c-fos mRNA induction are decreased by about 49% in the cortex and by 27% in the hippocampus of 30-month-old rats, as compared to the levels expressed by 3-month-old rats.

Interleukin-6 and selected plasma proteins in healthy persons of different ages.

In the present study, interleukin-6 (IL-6) and several acute phase proteins were measured in healthy participants (23-87 years of age). A linear correlation between IL-6 and age was established with an increase of 0.016 pg/ml (0.004) per year of life. Whereas CRP remained below 0.5 mg/dl in all participants, an increase with age for fibrinogen and an inverse relation for albumin as well as transferrin were obtained. However, the increase of IL-6 did not correlate with any of these changes. IL-6 associated diseases may therefore occur more often with advancing age, but in healthy participants IL-6 does not explain the changing plasma protein pattern resembling that of an acute phase reaction.

Biochemical studies of membrane glycoproteins during red cell aging.

(1) There is a significant decrease of ATPase activities in red cell membranes with increasing age of the donor. (2) A comparison of blood groups A and O relates the higher ATPase activities of young donors to blood group A. (3) Membrane ATPase activities reveal an age-dependent decrease in young and old donors with blood group A. (4) There is an age-dependent decrease of the N-acetylneuraminic acid concentration of glycophorin. (5) The galactose content of glycophorin only increases in blood group A. (6) There are no age-dependent changes of the exposed galactose. (7) The experiments so far reveal an increase of IgG absorption to old glycophorin.

Effect of essential phospholipids on the properties of ATPases of isolated rat liver plasma membranes of young and old animals.

"Essential" phospholipids (EPL), rich in dilinoleyl-phosphatidylcholine were given orally to young and 780-800 day-old rats. The double-labelled (14C/32P) dilinoleyl-phosphatidylcholine was incorporated in plasma membranes and the amounts incorporated into different organs investigated. Old animals incorporated higher radioactivity than young. The 14C/32P ratio however decreased in comparison with the substance administered. The amount of (Na+-K+)-ATPase in rat liver plasma membranes of old animals increased after pretreatment with EPL. The thermostability of ATPases was increased in plasma membranes of EPL treated old animals. In old animals EPL significantly lowered the cholesterol content. The membrane fluidity increased. The role of EPl in structural and functional properties of aged plasma membranes is discussed.

Lifespan of rabbit erythrocytes and activity of the reticulohistiocyte system.

The survival rate was measured of differently aged rabbit erythrocytes that had been taken from young and old animals, tagged with 51Cr and injected into young and old animals. The survival rate of the donor erythrocytes depends mostly on the age of the recipient animals. The life span of the injected cells found in old animals was much lower than that found in young animals. On the other hand, the age of the donor animal also has an influence, although only a small one, on the life span of the red blood cells. The cells from young animals live rather longer than those from old animals.

Phagocytic activity of the reticulohistiocyte system in rabbits after splenectomy and activation with ink.

The survival rate was measured of differently aged rabbit erythrocytes that had been taken from young animals, tagged with 51Cr and injected into young and old animals. The recipient animals had either been splenectomized, or had an ink-activated reticulohistiocyte system, or both. In young as well as in old ink-treated animals the injected cells lived for a much shorter time than in the non-treated animals. In splenectomized animals the red blood cells lived a little longer than those in the control group. In animals which had been both splenectomized and ink-treated the 51Cr-tagged cells again lived for a shorter time than those in the control group, but longer than in the ink-treated animals.

Age dependent kinetic studies of cytoplasmic and lysosomal enzymes of the normal and D-galactosamine injured rat liver.

Rats of two age groups (6 weeks and 30 months) received (1) a single dose of 600 mg D-galactosamine (GalN)/kg body weight by intraperitoneal (i.p.) injection, (2) a single dose of 600 mg GalN/kg body weight i.p. combined with 20 mg prednisolone/kg body weight subcutaneously at the beginning of the experiment. The kinetic studies disclose that GalN produces more severe changes in old than in young animals, represented by the activities of cytoplasmic (glutamic oxaloacetic transaminase, glutamic pyruvic transaminase) and lysosomal (beta-acetylglucosaminidase beta-glucuronidase, cathepsin D) enzymes. Prednisolone diminishes the morphological liver changes as well as the biochemical disturbances in young rats. There is only a protecting effect in morphological changes of old animals within the first 12 h. The prevention of cytoplasmic enzyme activity increase is limited to the first 12 h.

Diameters of erythrocytes of different ages measured by scanning electron-microscopy.

Contradictory reports exist about the age-dependent changes of erythrocyte diameters. All the investigations were carried out using Coulter Counter, by which the erythrocytes are sucked through a glass capillary. The contact with a glass surface causes the cell to deform instantly. This explains the lack of accuracy of the measurements subsequently taken. We measured the size of red cells of different ages by scanning electron-microscopy, thus avoiding deformations. We found that the average diameter decreases with the age of the red cell. The mean diameter was 7.77 microns in young cells, 7.34 microns in middle-aged and 7.00 microns in old cells.

Age-dependent physiochemical and biochemical studies of human red cell membranes.

Erythrocyte ghosts of male persons of two age groups, younger than thirty and older than seventy years, were analyzed to investigate the imfluence of age on lipid composition, the physical state of the lipid phase, Na+K+-ATPase activity and sialic acid content. The phospholipid content in red cell membranes of old donors is significantly lower than in young ones. Cholesterol and fatty acid compositions shows no difference between the two donor groups. The membrane fluidity in liposomes prepared from total lipids of old donor decreased. No significant difference in lipid composition and membrane fluidity reflected by the spin labels was observed between blood group A and O. The activity of Na+K+-ATPase of the young donors with blood group A is significantly higher than those of old donors. The results also demonstrate a decrease of sialic acid content of red cells of old donors.

Comparative molecular moment analysis (CoMMA): 3D-QSAR without molecular superposition.

3d-QSAR procedures utilize descriptors that characterize molecular shape and charge distributions responsible for the steric and electrostatic nonbonding interactions intimately involved in ligand-receptor binding. Comparative molecular moment analysis (CoMMA) utilizes moments of the molecular mass and charge distributions up to and including second order in the development of molecular similarity descriptors. As a consequence, two Cartesian reference frames are then defined with respect to each molecular structure. One frame is the principal inertial axes calculated with respect to the center-of-mass. For neutrally charged molecular species, the other reference frame is the principal quadrupolar axes calculated with respect to the molecular "center-of-dipole." QSAR descriptors include quantities that characterize shape and charge independently as well as quantities that characterize their relationship. 3D-QSAR partial least squares (PLS) cross-validation procedures are utilized to predict the activity of several training sets of molecules previously investigated. This is the first time that molecular electrostatic quadrupolar moments have been utilized in a 3D-QSAR analysis, and it is shown that descriptors involving the quadrupolar moments and related quantities are required for the significant cross-validated predictive r2's obtained. CoMMA requires no superposition step, i.e., no step requiring a comparison between two molecules at any stage of the 3D-QSAR calculation.

Anomalous expression of microtubule-associated protein 1B in the hippocampus and cortex of aged rats treated with pentylenetetrazole.

The aim of the present study was to assess the age-dependent response of microtubule-associated protein 1B, a plasticity-associated protein deriving from a late gene, following administration of an epileptogenic stimulus. The effect of a single administration of the convulsant pentylenetetrazole on microtubule-associated protein 1B expression in the hippocampal formation and cortex of three-, 18- and 28-month-old rats was assessed using northern blot analysis, in situ hybridization and immunohistochemistry. In three-month-old rats, we detected initial increases in microtubule-associated protein 1B messenger RNA at 15 h following pentylenetetrazole administration in the granule cells of the dentate gyrus, in the CA3 region of the hippocampus and in layers II/III of the entorhinal cortex, and these reached a maximum at 44 h. However, in the hippocampus and cortex of 18-month-old rats, the peak occurred at 15 h, and in the brains of 28-month-old rats a blunted peak was reached at 3 h. Pentylenetetrazole treatment in young rats resulted in a robust induction of microtubule-associated protein 1B immunoreactivity in the granule cells of the dentate gyrus and in layers II/III of the entorhinal cortex, but also produced a large decrease in the retrosplenial cortex. However, following pentylenetetrazole treatment in older rats, the granule cells of the dentate gyrus were nearly devoid of microtubule-associated protein 1B immunoreactivity, whereas the retrosplenial cortex showed no changes at all, and the entorhinal cortex had an expression pattern similar to that of young rats. Aberrant immunolabeling of microtubule-associated protein 1B occurred in cortical layer VI of the aged rats where, unlike in young rats, there was heavy staining of neuronal somata. These results suggest that the regulation of the plasticity-associated protein microtubule-associated protein 1B is altered in the ageing rat brain, with the peak of expression shifted to earlier times in 18-month-old rats and blunted, variable increases at even earlier times in 28-month-old rats.