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Chemically modified tetracycline (4-de-dimethylamino tetracycline), like commercially available tetracyclines, is known to inhibit experimentally induced pathologic collagen breakdown. A method for measurement of chemically modified tetracycline in small volumes (50 microliters) of rat serum was developed using reversed-phase HPLC; this was necessary because this tetracycline analog lacks antimicrobial activity and, therefore, cannot be measured with standard bioassays. This method uses the same solution for extraction and elution thus providing a simple and rapid assay for both drugs. Using this technique, the concentration of chemically modified tetracycline and tetracycline were determined in rat serum at different times after oral administration. The serum concentration of chemically modified tetracycline was much higher than that for tetracycline, and its serum half-life was greater. The IC50 of chemically modified tetracycline and tetracycline, as inhibitors of collagenase from rat polymorphonuclear leukocytes, was determined and found to be 4.1 x 10(-8) M (0.02 micrograms/ml) and 2.4 x 10(-4) M (120 micrograms/ml), respectively. Based on the serum levels of these drugs after oral administration, and their IC50 values, chemically modified tetracycline is potentially a far more potent inhibitor of excess collagenase activity than tetracycline, during pathologic conditions, and may have the added advantage of not producing some of the typical complications of long-term antibiotic therapy.

We review evidence that an immune and nervous system linkage exists in invertebrates similar to that shown in vertebrates. Immunoreactive cytokines appear to play a role in this interaction. We have demonstrated that M. edulis, a marine bivalve mollusc, reacts to the vertebrate monokines interleukin-1, -6 and TNF. We have also demonstrated endogenous immunoreactive interleukin-1, -6 and tumour necrosis factor in M. edulis hemolymph, immunocytes and pedal ganglia. Our findings suggest an important common signalling mechanism that has been conserved over 500 million years of evolution.

Recent reports have provided strong evidence indicating that Met-enkephalin is serving as a neuroimmune modulator. It acts as a bidirectional signal molecule in transmitting message between the endocrine system and the immune cells in the circulating fluid. In this study, we investigated peptidases which are capable of degrading Met-enkephalin in the hemolymph fluid and hemocyte membrane. Our results showed that aminopeptidase is present at a high level in the fluid and a low level in the membrane. Carboxypeptidase is not present in the fluid but it is present at a level higher than that of aminopeptidase in the membrane. Either ACE or neutral endopeptidase is also present in the hemolymph fluid and hemocyte membrane. Functional role of these peptidases in the overall scheme of the neuroimmune mechanism is currently under investigation.

Evidence for the presence of an enkephalinergic system in the ganglia of the pond snail, Lymnaea stagnalis, has been obtained with 3 experimental approaches. Scatchard analysis with [3H]etorphine reveals a monophasic high-affinity opiate binding site (Kd 2.3 nM) which is naloxone-sensitive. Immunocytochemical localization of Met- and Leu-enkephalin-like substances as well as alpha-MSH- and ACTH-like materials was demonstrated within specific populations of neurons. Substances with Met- and Leu-enkephalin and Met-enkephalin sulfoxide RIA reactivities were detected also in HPLC fractions corresponding to the retention times of authentic enkephalin standards. Together, the results provide strong evidence for the presence of enkephalinergic mechanisms in the nervous system of Lymnaea stagnalis. Additionally, the report provides indirect evidence for the existence of a macromolecular opioid precursor. This enkephalinergic system shows striking similarities to opioid mechanisms found in vertebrates and bespeaks a common evolutionary origin.

Mytilus edulis has been the subject of recent studies to determine whether the relationship between the immune and neuroendocrine systems seen in vertebrates also exists in invertebrates. The effects of mammalian monokines were studied in Mytilus immunocytes previously shown to produce and react to opioid peptides. These invertebrate cells respond to interleukin 1 (IL-1) and tumor necrosis factor (TNF), both in vitro and in vivo, in a manner similar to that of human granulocytes. As in the mammalian monokine network, the effect of IL-1 on the immunocytes is brought about, at least in part, by its stimulatory effect on the formation of TNF. In addition, the presence of immunoreactive IL-1 and TNF in Mytilus hemolymph was demonstrated.

1. This review article provides information on the evolutionary history of neuroendocrine and related regulatory mechanisms. It focuses on the presence, diverse roles, and modes of operation of one class of neuropeptides, the endogenous opioids, in insects. 2. Opioid peptides, closely resembling those of vertebrates, have been identified in the brain and related neuroendocrine structures by means of immunocytochemistry and high-pressure liquid chromatography. 3. The demonstration of naloxone-sensitive, high-affinity binding sites for Met-enkephalin-like neuropeptides in the brain and digestive tract of Leucophaea deserves special attention because it provides new insights into the functional significance of opiate receptors paralleling those known in vertebrates. 4. Possible roles of receptor-mediated opioid systems in the insects discussed are regulation of the cyclicity of the female reproductive system, maintenance of normal midgut function mediated by the recurrent nerve, and locomotor activity.

An acid extract of pedal ganglia of the mollusc Mytilus edulis was fractionated by high-pressure liquid chromatography with a reverse-phase column. Peak fractions with retention times of those of [Met]- and [Leu]enkephalin were subjected to binding assays in both invertebrate and vertebrate tissues. The results showed that these fractions have the same binding activities as authentic enkephalins. Peptides from these fractions were purified by high-pressure liquid chromatography under isocratic conditions. Sequential amino acid analyses showed that these peptides have the same primary structures as [Met]- and [Leu]enkephalin. These results with M. edulis suggest that invertebrates possess an enkephalinergic system similar to that of higher organisms.

As part of a programme to establish a panel of individuals with high titres of anticytomegalovirus (CMV) IgG for the production of CMV Ig, the CMV serostatus of 139 normal blood donors was determined by indirect haemagglutination assay, enzyme immunoassay, and complement fixation. The indirect haemagglutination assay was the most accurate and convenient method for the detection of CMV antibody negative donations. Plasma for production of hyperimmune CMV IgG could best be selected by an IgG-specific enzyme immunoassay procedure.

1. This report further demonstrates that etorphine influences presynaptic dopamine release, which in turn centrally modulates peripheral cilioinhibition. 2. In older animals cilioinhibition has become enhanced due to a lack of responsiveness to endogenous opioids which results in greater dopamine release, causing a higher level of cilioinhibition as demonstrated by challenging the visceral ganglia with etorphine or destroying the dopaminergic component with 6-hydroxydopamine. 3. Only the central cilioinhibitory, not the peripheral inhibitory response, mechanism appears to be altered in older animals. Thus, the alteration appears in the central integrative mechanisms involved with regulating ciliary activity. 4. The KCl-stimulated release of dopamine is unaltered in both young and old organisms, whereas the opiate inhibition of the KCl-stimulated release of dopamine is reduced in older organisms. Thus, the aging-associated alteration is associated with a specific process. 5. The reduction of opioid influence and the resulting enhanced cilioinhibitory activity may make the organisms more susceptible to environmental stress.

1. Computerized analysis of the crabs locomotor behavior revealed an initial increase in activity followed by a gradual decrease over a 12 min observation period. 2. Dopamine, in a dose-dependent manner, inhibits locomotor activity. The effect can be antagonized with the dopamine antagonist, haloperidol. This suggests that dopaminergic influences are involved with locomotor mechanisms. 3. FK 33,824, a stable opioid analog, significantly enhances the initial excitatory locomotor activity. Naloxone, a potent opiate antagonist, can block the excitatory action induced by FK 33 824. This suggests the presence of an opioid modulation mechanism in the regulation of locomotor activity. 4. Concomitant administration of the various agents results in the behavioral characteristics of the agonist appearing when the appropriate antagonist is not present. Thus, administration of dopamine + FK 33,824 + haloperidol results in enhanced locomotor activity. 5. Concomitant dopamine and FK 33,824 administration results in enhanced locomotor activity. This suggests that the opioid mechanism is closer to the last step in affecting the organism's locomotion or in initiating activity.

A survey (1964-1973) was conducted on naturally occurring western equine encephalomyelitis (WEE) virus infections of Richardson's ground squirrels (RGS) in agricultural areas of Saskatchewan. The survey included both serology and virus isolation attempts on live-trapped wild squirrels. Throughout the study, seropostive squirrels were found in the known enzootic regions of the province each summer. The infections rate was high (11.6% of 250) in the epidemic year of 1965 and low (less than 2% of 681) in the subsequent non-epidemic years. The maximal seasonal prevalence of seropositive RGS coincided with the time when the squirrel population and aedine mosquito abundance reached their seasonal peaks. Five virus isolations were obtained from the blood and brains of naturally infected squirrels. All isolations were from squirrels collected in June. The early seasonal infections in squirrels could provide a source of virus for Culex tarsalis, the epidemic vector. In view of their abundance in the enzootic agricultural areas and the high annual population turnover, the RGS may play an important role in the natural history of WEE virus in the Canadian prairies.

In Mytilus and Leucophaea the high-affinity binding site density is significantly lower in old animals than in young animals, whereas the low-affinity site density remains unchanged. In Mytilus the estimated met-enkephalin and met-enkephalin-Arg6-Phe7 levels are significantly higher in old than in young animals. In Leucophaea only the met-enkephalin level can be determined, and it is also higher in old animals. The decrease in the high-affinity binding site density and the corresponding increase in endogenous enkephalin levels suggest the existence of an opioid compensatory mechanism associated with the aging process. In Mytilus there is a demonstrated decrease with age in intraganglionic dopamine levels in response to applied opiates. In addition, the inhibition of dopamine-stimulated adenylate cyclase activity by opiates also decreases in older animals. In Leucophaea the sex difference in opioid binding densities diminishes with age.

The eyestalk of the land crab, Gecarcinus lateralis, is rich in Met-enkephalin-like materials. Leu-enkephalin, if present, is at a level which is below the sensitivity of our assay. The brain (cerebral ganglion) of this organism contains both Met- and Leu-enkephalin-like materials. Material which was reactive with our Met-enkephalin antibody was also detected in a peak that migrated after Leu-enkephalin. This biochemical study confirms earlier immunohistochemical reports which indicated that opioid substances are present in crustaceans. It also strongly supports the concept that opioid mechanisms are a product of early evolution and further demonstrates that the sequence of the smaller opioid compounds has been conserved.

Procollagen synthesized by freshly excised chick enbryo leg tendons is efficiently processed by proteolytic removal of first the amino propeptides and then the carboxyl propeptides. The same processes proceed in confluent short term cell cultures derived from such tendon explants; in sparse cultures cleavage of the amino propeptides predominates. Separate amino and carboxyl procollagen peptidase activities were demonstrated by specific assays in enzymes obtained from cell culture media by ammonium sulfate precipitation, ion exchange chromatography, and velocity sedimentation. Both enzymes are inhibited by EDTA and 1:10 phenanthroline but not by inhibitors of serine proteases. Evidence is provided that the proteolytic scissions are specific and similar to the physiologically occurring processes. The collagen telopeptides left after cutting by the enzymes can participate in lysyl oxidase-induced cross-linking. The enzymes can remove propeptides from cross-linked procollagens without destroying these links which occur through telopeptides. The enzymes act on the separated amino and carboxyl portions of procollagen fragmented by vertebrate collagenase and can act on procollagens which have been associated as well as on molecules in solution.

An extract of pedal ganglia of Mytilus edulis was fractioned by high-pressure liquid chromatography by use of a reverse-phase column. Peak fractions with the same retention time as Met-enkephalin-Arg6-Phe7 were subjected to binding assays in invertebrate neural tissue. The results showed that these fractions have the same binding activities as authentic Met-enkephalin-Arg6-Phe7. The heptapeptide from these fractions was purified by high-pressure liquid chromatography under isocratic conditions. Sequential amino acid analysis showed this peptide to have the same primary structure as Met-enkephalin-Arg6-Phe7. These results indicate that invertebrates such as Mytilus edulis possess enkephalinergic systems similar to those found in in higher organisms.

Eighteen isolations of Cache Valley virus (Bunyaviridae) were obtained from a total of 113,694 mosquitoes collected in Saskatchewan during the summers of 1972 to 1974. Most of the isolations were from mosquitoes collected during August. Culiseta inornata, the most abundant mosquito (38% of total collected), had the highest minimum vector-infection rate (0.83 isolations per 1000 mosquitoes). The virus was also isolated from Culex tarsalis and Aedes vexans. It is indicated in the isolations that the prairie grasslands of the province are enzootic for Cache Valley virus.

D'-Ala-2 met-5-enkephalinamide application to the cerebral ganglia of Leucophaea maderae results in a decrease in locomotor activity. The opiate antagonist, naloxone, can block this effect as well as the depressant effect of morphine on locomotor activity. D-Ala-2,leu-5-enkephalinamide and dynorphin enhance locomotor activity following their topical application to the cerebral ganglia. This effect also can be antagonized by concomitant naloxone treatment. Benzomorphans were the most potent ligands tested in their ability to displace [3H]D'-ala-2,met-5-enkephalinamide whereas mu and delta ligands were by comparison less potent. These results suggest the presence of kappa-like opioid receptors in Leucophaea cerebral ganglia. The kappa ligands also are potent in enhancing locomotor activity in addition to being weakly antagonized by naloxone. Again, these results indicate the presence of multiple-opiate receptor types in invertebrates.

Morphine stimulates food consumption in the land snail Helix aspersa. This stimulation of food consumption can be blocked by naloxone, the potent opiate antagonist. In addition, this opiate-induced food consumption exhibits tolerance by the sixth day of treatment. This study further highlights opioid mechanisms in relatively simple organisms and also suggests that these mechanisms are "ancient" signal systems.

Extract from the abdominal ganglia of Aplysia was fractionated by high performance liquid chromatography. Several large uv absorbing peaks were found. One of these peaks from the ganglionic extract was analyzed by displacement assay with the use of Mytilus edulis membranes. The results revealed the substance in this peak was able to displace 3H-D-Ala2, met5-enkephalinamide. In electrophysiological studies of abdominal and cerebral neurons, depolarizing and hyperpolarizing responses were obtained from some neurons, including neurons in the identified cerebral B cell cluster. A smaller population of cells exhibited biphasic responses. Some of the responses could be depressed by prior naloxone treatment. In conclusion, an endogenous opioid system, using substance related to but distinct from the enkephalins, may exist in Aplysia.

We have previously shown that serotonin (5-HT) suppresses interferon-gamma (IFN-gamma)-induced Ia expression. In the present report, we show that 5-HT as well as other monoamines, histamine and dopamine, modulate IFN-gamma-induced phagocytosis in murine bone marrow macrophages. The effect of 5-HT on IFN-gamma-induced phagocytosis varied according to the concentration of IFN-gamma to which the macrophages were exposed. At low concentrations of IFN-gamma, 5-HT augmented phagocytosis, whereas at high concentrations of IFN-gamma, 5-HT suppressed phagocytosis. At both low and high IFN-gamma concentrations the response to 5-HT was dose-related and occurred at physiologic concentrations; the half-maximal effect was 6 X 10(-7) M and 3 X 10(-7) M for low and high IFN-gamma concentrations, respectively. Both histamine and dopamine also augmented IFN-gamma (1 U/ml) induced phagocytosis, at half-maximal augmenting concentrations of 7 X 10(-8) M and 4 X 10(-7) M, respectively. The 5-HT effects were blocked by the 5-HT antagonists spiperone, ketanserin, LY53857, mCPP, and PAPP, but not by the histamine antagonists pyrilamine, chlorpheniramine, or cimetidine. Histamine augmentation of IFN-gamma-induced phagocytosis was blocked by the H1 antagonists pyrilamine and chlorpheniramine, but not by the H2 antagonist cimetidine. The dopamine effect was blocked by spiperone and pyrilamine, both of which have been shown to block dopaminergic effects in other systems. This data provides functional evidence that at least part of the modulation of IFN-gamma-induced phagocytosis by 5-HT occurs through a 5-HT receptor-mediated mechanism, and 5-HT, dopamine, and histamine modulate IFN-gamma-induced phagocytosis independently through their respective receptors.

To identify the mechanisms responsible for the paucity of recently synthesized collagen in connective tissues during diabetes, in vitro procollagen metabolism was studied in non-diabetic (control) and diabetic rats. Achilles tendons from the two groups were incubated for 1-8 h (35 degrees C) in medium containing [14C]proline and the radiolabeled collagen in the tissue, and that released into the media, were examined by SDS-polyacrylamide gel electrophoresis and fluorography. The bulk of the radiolabeled collagen in tendon from the diabetics was recovered as degradation products; these, but also procollagen and collagen components, were prominent in the control tissues. Moreover, the collagenous components synthesized by the diabetic rat tendons were more readily digested in vitro by trypsin than those produced by control tissues. We conclude that diabetes reduces collagen accretion in connective tissues in part due to increased intracellular degradation of procollagen.

The present report demonstrates that both mouse thymocytes and Mytilus edulis hemocytes contain a novel type of dopamine receptor. Scatchard analysis of these data revealed a single class of high-affinity binding sites with an affinity constant (Kd) of 6.6 nM, and a binding site density (Bmax) of 141 pmol/g protein in mice and a Kd of 7.6 nM and a Bmax of 66 pmol/g protein for the hemocytes. In older Mytilus the Kd was the same; however, there was a significant decrease in the Bmax (48 pmol/g protein; P greater than 0.05). Age changes were not evident for the mouse cells. The ability of a variety of other related drugs to displace specifically bound dopamine was investigated. The catecholamines (norepinephrine greater than epinephrine) and the dopamine agonist epinine were the most potent of the ligands tested and apomorphine and butaclamol were of moderate potency. This may serve to indicate that this dopamine binding site may be very different from the known D1 and D2 sites. This particular site may represent a third type of dopaminergic receptor. Furthermore, the hemocytes of Mytilus appear to be of two major types, namely, cells with a granular cytoplasm and those whose cytoplasm is agranular. Histofluorescence studies on the hemocytes reveal that a subpopulation of the granule-containing cells appears to contain serotonin.

Adult Richardson's ground squirrels were infected with western equine encephalomyelitis virus by intranasal instillation. Mortality followed the instillation of a minimum threshold of 4.7 logs of virus while infection was produced by a dosage of 2.3 logs. The incubation period was from four to seven days, being preceded by a viremic phase. Signs were depression, ataxia and paralysis of the limbs. Highest titres of virus were recovered from the brain and histopathological changes involving the central nervous system included meningitis, vasculitis, perivascular cuffing, gliosis, neuronophagia and neuronal degeneration. The virus was also found in a variety of extraneural tissues. Lesions in extraneural tissues included necrosis of brown fat and an apparent increase in number of Kupffer's cells in the liver. The lymphoid tissue was involved indicating a possible source for viremia. The duration and magnitude of viremia were ample enough to provide virus source for arthropods. The potential for transmission of the virus independent of arthropods was discussed in view of the pathogenesis demonstrated in the experimental infections.

Mytilus pedal ganglia extract was treated sequentially with TPCK-trypsin and carboxypeptidase B. The treated sample and an untreated sample were purified separately with Sep-Pak C18 and subjected to HPLC. Fractions with Rt's corresponding to the Met-, Leu-enkephalin and Met-enkephalin-Arg6-Phe7 were assayed for enkephalin activities by displacement studies using pedal ganglia membrane and 3H-DAMA. The data showed the activities in the regions of Met-, Leu-enkephalin and Met-enkephalin-Arg6-Phe7 of the treated sample increased over that of the untreated sample by 2.3, 2.5 and 1.6 folds respectively. These results provided strong evidence for the presence of enkephalin precursor material in Mytilus.

Evidence for the participation of opioid neuropeptides in immunoregulatory activities, especially cellular adherence and migration, has been obtained in representatives of two phyla of invertebrates, the mollusc Mytilus edulis and the insect Leucophaea maderae. The injection of a synthetic analog of [Met]enkephalin [( D-Ala2,Met5]enkephalinamide, DAMA; 10(-6) M) had a stimulatory, naloxone-reversible effect on the directed migration of immunocompetent hemocytes. Incubation of hemolymph in the presence of exogenous or endogenous opioid material significantly enhanced the adherence of hemocytes on albumin-coated slides as demonstrated by use of indirect Zeiss-Zonax reflectance computer analysis. Conversely, hemocyte adherence was markedly reduced by the addition of naloxone (10(-8) M) to the incubation medium, either alone or in combination with DAMA. The antagonistic effects of naloxone on the stimulatory activities of opioids indicate that, like those previously reported in mammals, they are receptor-mediated. The presence of an endogenous [Met]enkephalin-like material was demonstrated in cell-free hemolymph as well as sequestered hemocytes by use of high-pressure liquid chromatography and radioimmunoassay. These results demonstrate that the capacity of immunocytes to release and respond to opioid neuropeptide messengers is not restricted to mammalian organisms but was developed early in the course of evolution.

Both adult and suckling Richardson's ground squirrels (Spermophilus richardsonii) were susceptible to subcutaneous exposure with western equine encephalomyelitis (WEE) virus, but the virus was more virulent for sucklings than adults. In sucklings, the incubation period was from 4 to 5 days, followed by apparent signs of central nervous system (CNS) involvement. Death occurred 10 to 13 days postexposure. In adults, infections were inapparent or acute with typical signs of CNS involvement similar to those observed in sucklings. In both age groups, brain and lymph nodes were the most frequently involved tissues. The highest titres of virus were recovered from brain, sections of which also showed the most marked histological changes. Lesions in the brain included multifocal vasculitis, perivascular edema, perivascular cuffing, focal or diffuse gliosis, parenchymal hemorrhage, meningitis with infiltration of mononuclear cells, neuronal degeneration, and occasional demyelination. For both age groups, viremias were detected for 3 to 5 days with a maximum virus titre of 4 to 6 logs, a sufficient time and magnitude to infect numerous mosquitoes, further supporting the hypothesis that S. richardsonii may serve as an amplifying host of WEE virus in the prairie provinces.