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A typical Indonesian kretek cigarette brand and an experimental kretek reference cigarette were compared to the reference cigarette 2R4F in two 90-day inhalation studies. Male and female rats were exposed nose-only to mainstream smoke for 6 hours daily, for 90 consecutive days. Biological endpoints were assessed according to OECD guideline 413, with special emphasis on respiratory tract histopathology and on lung inflammation (broncho-alveolar lavage fluid levels of neutrophils, macrophages and lymphocytes). Histopathological alterations included: in the nose, hyperplasia and squamous metaplasia of the respiratory epithelium and squamous metaplasia and atrophy of the olfactory epithelium; in the larynx, epithelial squamous metaplasia and hyperplasia; in the lungs, accumulation of macrophages in alveoli and goblet cell hyperplasia in bronchial epithelium. The findings were qualitatively consistent with observations from previous similar studies on conventional cigarettes. Compared to 2R4F cigarette, however, kretek smoke exposure was associated with a pronounced attenuation of pulmonary inflammation and less severe histopathological changes in the respiratory tract. Neutrophilic inflammation was also significantly lower (>70%). These results are consistent with the observations made on smoke chemistry and in vitro toxicology. They do not support any increased toxicity of the smoke of kretek cigarettes compared to conventional American-blended cigarettes.

The biological activity of mainstream smoke from experimental kretek cigarettes with and without three mixes of ingredients was assessed in a 90-day rat inhalation study and in a 4-day in vivo micronucleus assay. 350 ingredients, commonly used in various combinations and in a limited number in a given brand in the manufacture of marketed kretek cigarettes, were applied at a low and a high target level to test cigarettes with a typical Indonesian blend of tobaccos and cloves. In the 90-day inhalation study, effects commonly seen in rat inhalation studies with mainstream smoke were observed. In general, no ingredients-related histopathological changes were found in the respiratory tract. In the 4-day micronucleus assay exposure of male rats to mainstream smoke from the test cigarettes containing any of the three mixes did not increase the proportions of micronucleated cells in peripheral blood and bone marrow over the proportion of micronucleated cells in the control group. Based on the results of these studies, it can be concluded that the addition of ingredients commonly used in the manufacture of kretek cigarettes did not change the overall in vivo toxicity profile of the mainstream smoke.

Cigarette smoking is the primary etiology of chronic obstructive pulmonary disease (COPD) and a risk factor for both lung and cardiovascular (CV) diseases, which are rarely investigated concomitantly. Although smoking cessation shows clear CV risk benefit, lung-related disease risk remains higher in former smokers than in never smokers. We sought to determine the differential molecular responses of murine respiratory tissues to better understand the toxicity pathways involved in smoking-related disease risk and those related to the benefits of smoking cessation. ApoE(-/-) mice were exposed to mainstream cigarette smoke (CS) or a smoking cessation-mimicking protocol for up to 6 months and transcriptomics analysis of nasal epithelium and lung parenchyma performed. We supported our gene expression profiling approach with standard lung histopathology and bronchoalveolar lavage fluid (BALF) analysis. Many BALF analytes involved in functions ranging from inflammation to cell proliferation and tissue remodeling were found elevated in BALF. Gene expression levels of these molecules were also increased in lung tissue, suggesting that the inflammatory response was the result of local tissue activation and the contribution of recruited inflammatory cells. Gene set enrichment analysis (GSEA) of expression data from murine lungs and nasal epithelium showed distinct activation patterns of inflammation, complement, and xenobiotic metabolism pathways during CS exposure that were deactivated upon smoking cessation. Pathways involved in cell proliferation and tissue remodeling were activated by CS and progressively deactivated upon smoke exposure cessation. Differential CS-mediated responses of pulmonary and nasal tissues reflect common mechanisms but also the varying degrees of epithelial functional specialization and exposure along the respiratory tract.

Cigarette smoking is the leading cause of lung cancer and chronic obstructive pulmonary disease, yet there is little mechanistic information available in the literature. To improve this, laboratory models for cigarette mainstream smoke (MS) inhalation-induced chronic disease development are needed. The current study investigated the effects of exposing male A/J mice to MS (6h/day, 5 days/week at 150 and 300 mg total particulate matter per cubic meter) for 2.5, 5, 10, and 18 months in selected combinations with postinhalation periods of 0, 4, 8, and 13 months. Histopathological examination of step-serial sections of the lungs revealed nodular hyperplasia of the alveolar epithelium and bronchioloalveolar adenoma and adenocarcinoma. At 18 months, lung tumors were found to be enhanced concentration dependently (up to threefold beyond sham exposure), irrespective of whether MS inhalation had been performed for the complete study duration or was interrupted after 5 or 10 months and followed by postinhalation periods. Morphometric analysis revealed an increase in the extent of emphysematous changes after 5 months of MS inhalation, which did not significantly change over the following 13 months of study duration, irrespective of whether MS exposure was continued or not. These changes were found to be accompanied by a complex pattern of transient and sustained pulmonary inflammatory changes that may contribute to the observed pathogeneses. Data from this study suggest that the A/J mouse model holds considerable promise as a relevant model for investigating smoking-related emphysema and adenocarcinoma development.

Cigarette smoke (CS) imposes a strong oxidative burden on exposed tissues resulting in a severely disturbed oxidant/antioxidant balance, which in the context of chronic exposure is assumed to be a key contributor to CS-related diseases. Because of its emerging central role in orchestrating the general cellular antioxidant response, the pathway leading to the activation of the transcription factor Nrf2 has received mounting attention over the past decade in investigations aimed at elucidating CS-induced pathophysiological mechanisms. To comprehensively characterize the impact of Nrf2 in acute and subchronic smoking scenarios, Nrf2(-/-) mice and their wild-type (wt) ICR littermates were exposed to either ambient air (sham exposure) or one of three doses of CS for up to 5 months, with two postexposure endpoints of 1 and 13 days. The lungs of the mice were monitored for transcriptomic changes on a genome-wide level, which confirmed an impaired expression of antioxidant and phase 2-related genes in CS-exposed Nrf2(-/-) mice. Importantly, in comparison to wt mice, an attenuated cell cycle/mitotic response and intensified stress gene expression pattern were observed in exposed Nrf2(-/-) mice, which was paralleled by clear dose-dependent effects on alveolar destruction and impaired lung function. In contrast, the inflammation-related transcriptional response and scores for various bronchioalveolar inflammation parameters were qualitatively and quantitatively similar in CS-exposed mice of both genotypes. Taken together, these results confirm the protective nature of Nrf2 in oxidative stress scenarios and suggest that the enhanced emphysematous phenotype exhibited by CS-exposed Nrf2(-/-) mice is more likely caused by an imbalance in cell loss and regeneration than by increased inflammation.

Since its discovery as a critical cofactor in the initiation of the protein C (PC) anticoagulant pathway [1,2], biochemical and structural investigations, combined with in vivo analyses of genetically engineered mice have revealed new, and in part PC- and thrombin-independent aspects of thrombomodulin (TM) function in fibrinolysis and inflammation, and in embryogenesis. This review summarizes more recent structural and functional investigations of TM, gives an overview of the association of TM gene polymorphisms with human disease, and provides a synopsis of what is know about TM function in disease states of thrombosis, stroke, arteriosclerosis, and cancer. Newly emerging aspects of TM function in inflammation and embryogenesis are presented and discussed in detail.

The biological effects of mainstream smoke (MS) from Indonesian-blended cigarettes with and without added cloves, cloves extracted with hot ethanol, and extracted cloves replenished with eugenol or clove oil were assessed in a 90-day inhalation study in rats. A separate 35-day inhalation study in rats was performed with MS from American-blended cigarettes with 0%, 2.5%, 5% or 10% added eugenol. Effects commonly seen in inhalation studies with MS were observed. These included histopathological changes indicative of irritation in the entire respiratory tract and inflammatory responses in the lung. Adding cloves to American- or Indonesian-blended cigarettes reduced the inflammatory response in the lung but with no difference between the two blend types. When the clove oil was extracted (∼75% reduction of eugenol achieved) from cloves, the inflammatory response in the lung was still reduced similarly to whole cloves but the severity of histopathological changes in the upper respiratory tract was less reduced. Add back of clove oil or pure eugenol reduced this response to a level similar to what was seen with whole cloves. When eugenol was added to American-blended cigarettes, similar findings of reduced lung inflammation and severity of histopathological changes in respiratory the tract was confirmed. These studies demonstrate a clear effect of cloves, and in particular eugenol, in explaining these findings.

Prostaglandin E 2 (PGE 2 )- and 6-keto-PGF 1α -like immunoactivity was measured in incubates of forestomach and gastric corpus mucosa in (a) unoperated rats, (b) rats with sham-operation of the kidneys and (c) rats with bilateral nephrectomy. In addition the mean ulcer area in the forestomach and gastric mucosa was assayed in all three groups of rats. The PGE 2 - and 6-keto-PGF 1α -like immunoactivity in gastric mucosa incubates and 6-keto-PGF 1α in the forestomach incubates was almost the same in unoperated and sham-operated rats. Increased 6-keto-PGF 1α in the forestomach and of PGE 2 and 6-keto-PGF 1α in the gastric mucosa was found in rats with bilateral nephrectomy before gastric lesions were seen. A higher mean ulcer area occurred in the forestomach and gastric mucosa 24–48 h after bilateral nephrectomy. We conclude, that the increased PGE 2 - and 6-keto-PGF 1α production by the gastric mucosa and forestomach was associated with the loss of normal renal tissue function. Despite the protection of PGE 2 and PGI 2 by gastric tissue, gastric lesions nevertheless occur in acute uraemic rats.

The possible role of dietary flax on pre-pubertal development of mammary glands and bone resorption was investigated in gilts. Fifty-seven gilts were fed 1 of 4 diets from 88 d of age until slaughter (d 212 +/- 1). Diets were control without flax (n = 14); 10% flaxseed supplementation (n = 13); 6.5% flaxseed meal supplementation (n = 15); and 3.5% flaxseed oil supplementation (n = 15). All diets were isonitrogenous and isocaloric. Jugular blood samples were obtained on d 78 and 210 to establish the fatty acid profile and to determine the concentrations of prolactin, estradiol, and cross-linked N-telopeptides of type I collagen. At slaughter, the mammary glands were excised, parenchymal and extraparenchymal tissues were dissected, and the composition of the parenchymal tissue (protein, fat, DM, and DNA) was determined. Histochemical analyses of the mammary parenchyma were performed, and fatty acid profiles in the extraparenchymal tissue were evaluated. Dietary flax increased (Por = 0.001) the concentrations of PUFA and decreased those of SFA (P0.01) and MUFA (Por = 0.001) in plasma and extraparenchymal tissues, which was largely due to the inclusion of 10% flaxseed or 3.5% flaxseed oil (Por = 0.01) but not 6.5% flaxseed meal. Circulating concentrations of prolactin and estradiol were unaltered by treatments (P0.1), but concentrations of cross-linked N-telopeptides of type I collagen tended to be greater (P0.1) in flax-supplemented gilts. The DM content of parenchymal tissue was the only mammary compositional value affected, showing an increase with flax addition (P0.05). No change (Por = 0.1) in the bromodeoxyuridine labeling index or estrogen receptor localization was observed with treatments. Dietary supplementation with flax as seed, meal, or oil, therefore, brought about the expected changes in the fatty acid profile but had no beneficial effects on mammary development or bone resorption.

An experiment was conducted to determine growth performance, carcass characteristics, and fat quality of growing-finishing pigs fed diets based on short-season corn hybrids. Twenty-four individually housed, Cotswold, growing pigs with an initial BW of 41.4 (SD = 1.4) kg were blocked by BW and sex and randomly allotted from within block to 1 of 3 diets to give 8 replicate pigs per diet. Experimental diets consisted of a control based on barley and 2 diets based on corn as the main energy sources. A 3-phase feeding program for 20 to 50 kg (phase I), 50 to 80 kg (phase II), and 80 to 110 kg (phase III) of BW was used. Diets for each phase contained approximately 3.5 Mcal/kg of DE, with total lysine of 0.95, 0.75, and 0.64% in phase I, II, and III diets, respectively. Average daily gain, ADFI, and G:F were monitored weekly during each phase. Pigs were slaughtered after reaching a minimum BW of 100 kg to determine carcass characteristics. There were no effects of diet on ADG, ADFI, and G:F (0.45 +/- 0.02, 0.34 +/- 0.02, and 0.31 +/- 0.02 for phase I, II, and III, respectively). Carcass length, dressing percent, LM area, loin depth, backfat thickness, belly firmness, and L*, b*, and a* fat color were not different across dietary treatments. Pigs fed one corn variety had no differences in fatty acid profile with barley-fed pigs, whereas those fed the other variety of corn had a greater (P0.05) concentration of PUFA in their backfat. The results indicate that growth performance, carcass characteristics, and fat quality of pigs fed diets based on short-season corn hybrids and those fed the barley-based diet were not different.

High resolution X-ray microtomography (micro-CT) was used for the detection of emphysema in live mice. Emphysema was induced in C57BL/6 J mice by intratracheal instillation of different amounts of porcine pancreatic elastase. This emphysema could be clearly detected by micro-CT seven weeks post-treatment: analysis of the whole data set of virtual cross-sections showed the presence of a dose-dependent level of emphysema.

ICC-1132 is a malaria vaccine candidate based on a modified hepatitis B virus core particle (HBc) bearing putative protective epitopes from the circumsporozoite protein (CS) of Plasmodium falciparum. While the epitope carrier itself is immunogenic, its potency can be increased by formulation with adjuvants. As a prelude to Phase I clinical trials, rhesus macaques were immunised twice with GMP grade ICC--1132 in saline or formulated with the adjuvants Alhydrogel (Alhydrogel) or Montanide((R)) ISA 720 (Montanide). Both adjuvant formulations gave significant humoral responses after the first injection, with titres increasing further after the second dose. The Montanide formulation was the most immunogenic, but undesirable reactogenicity in the form of sterile abscesses was associated with higher dosage levels of ICC--1132. These side effects could be avoided with lower antigen load, or by formulation of the second dose in Alhydrogel. Such measures also reduced peak titres and longevity of antibodies against CS, demonstrating the delicate balance between immunogenicity and reactogenicity of new vaccine formulations.

In the present study, the feasibility of applying highresolution microtomography (micro-CT) for the detection of lung tumors was investigated in live mice at an early and more advanced stage of tumor development. The chest area of anesthesized mice was scanned by X-ray micro-CT. In mice with a minor and heavy tumor load, micro-CT proved to be a fast and noninvasive imaging device for the detection of lung tumors. After validation of the CT data by histologic sectioning, it was shown that the majority of tumors could be distinguished in the reconstructed virtual slices obtained by micro-CT. The data from micro-CT were also confirmed by visual inspection of the inflated and excised lungs postmortem. In vivo micro-CT opens broad perspectives for imaging tumor development and its progression in a noninvasive way. Micro-CT also allows for longitudinal evaluation of the treatment of lung cancer by drugs.

The rate of dentinogenesis for the pig is quantified and the effects of dietary arachidonic acid supplementation and/or exogenous prostaglandin on dentine formation are defined. Thirty-six pigs were randomised to four groups, receiving either standard or supplemented formula and either prostaglandin E2 or placebo injections for fifteen days. Double tetracycline banding is used to measure rate of growth in the teeth. The average rate of dentinogenesis for all the study animals is 17.96 microm/day. Results show that the rate of dentinogenesis is not significantly affected by the interaction of hormone and dietary supplementation.

A male Asian elephant (Elephas maximus) died at the Berlin zoological gardens in August 1998 of systemic infection with the novel endotheliotropic elephant herpesvirus (ElHV-1). This virus causes a fatal haemorrhagic disease in Asian elephants, the so-called endothelial inclusion body disease, as reported from North American zoological gardens. In the present work, ElHV-1 was visualized ultrastructurally in affected organ material. Furthermore, a gene block comprising the complete glycoprotein B (gB) and DNA polymerase (DPOL) genes as well as two partial genes was amplified by PCR-based genome walking and sequenced. The gene content and arrangement were similar to those of members of the Betaherpesvirinae. However, phylogenetic analysis with gB and DPOL consistently revealed a very distant relationship to the betaherpesviruses. Therefore, ElHV-1 may be a member of a new genus or even a new herpesvirus subfamily. The sequence information generated was used to set up a nested-PCR assay for diagnosis of suspected cases of endothelial inclusion body disease. Furthermore, it will aid in the development of antibody-based detection methods and of vaccination strategies against this fatal herpesvirus infection in the endangered Asian elephant.

Herpesvirus infections which take a fatal turn on African elephants as well as on Asian elephants seem to occur increasingly not only in the USA but also in European stocks. The endotheliotropic herpesvirus causes a rapidly progressing and severe disease which makes any therapeutical effort unsuccessful and finally results in death of the animal, especially in young Asian elephants. As all attempts to culture the virus failed up to now, molecular biological procedures have to be used more often for diagnostical purpose together with the common methods of pathology, virology, and electronmicroscopical evaluation. This is a report on the case of 'KIBA', an eleven year old male elephant at the Zoological Garden Berlin, infected with the endotheliotropic elephants herpesvirus. 'KIBA' was born at the Zoo in Houston, Texas, and raised within his herd. Upon arriving in Berlin in November 1997 he adapted to the new premises and climate and new social circumstances without any problems. In June 1998 he already serviced three females of his new herd several times. In August 1998 he died after passing a peracute progression of the disease after residenting in Berlin for only 9 months. The dissection of the animal revealed some evidence on an agent damaging the endothelium. Major signs indicating this agent were bleedings in several serous membranes, mucosa and on the the right atrium, as well as other parts of the myocardium. Furthermore there have been ulcerations at various localisations of the whole digestive tract. Slightly basophilic intranuclear inclusion bodies have been found histologically in endothelial cells of different organ samples. An examination of altered organ-material by electronmicroscopy made some herpesvirus-like particles visible. A virological investigation first revealed evidence of giant cell formations with solitary basophilic intranuclear inclusion bodies in different cell cultures, however, without any distinct cytopathogenic effect. Supported by molecular biological procedures the infection of 'KIBA' could be verified as the elephants herpesvirus. By means of PCR and subsequent sequence analysis a DNA-sequence typical for the elephants herpesvirus could be obtained which showed an identity of 97% with the terminase sequence of the elephant herpesvirus described by American authors. The deduced amino acid-sequences were 100% identical. To the terminase of the human cytomegalovirus, the elephant sequence had an identity of 53% (similarity: 74%). Based on the cooperation of ILAT, Institute of Veterinary-Pathology/Free University Berlin, Robert-Koch-Institut Berlin, and Zoological Garden Berlin, the cause of 'KIBA's' death could be discovered immediately. Possible implications of this case especially on breeding and keeping elephants are discussed briefly.

Thrombomodulin (TM), recognized as an essential vessel wall cofactor of the antithrombotic mechanism, is also expressed by a wide range of tumor cells. Tumor cell lines subcloned from four patients with malignant melanoma displayed a negative correlation between TM expression and cell proliferation in vitro and in vivo. Overexpression of wild-type TM decreased cell proliferation in vitro and tumor growth in vivo. TM mutants with altered protein C activation capacity lead to a similar effect. In contrast, transfection of melanoma cells with mutant TM constructs, in which a portion of the cytoplasmic or lectin domain was deleted, abrogated the antiproliferative effect associated with overexpression of wild-type TM. Experiments performed with either peptide agonists/antagonists of the thrombin receptor, with hirudin, or with inhibitors of thrombin-TM interaction did not alter the growth inhibitory effect of TM overexpression. These data suggest that TM exerts an effect on cell proliferation independent of thrombin and the thrombin receptor, possibly related to the binding of novel ligands to determinants in the lectin domain which might trigger signal transduction pathways dependent on the cytoplasmic domain.

For four generations rats were fed a low selenium diet (2-7 micrograms Se kg-1) or the same diet with 250 or 300 micrograms Se kg-1 added as selenite. In male rats of the first generation that had been fed the diets from the age of 20 days onwards, selenium depletion led to slightly delayed testis growth during pubertal development that was compensated for in the later stages of maturation. In adult rats fed the low selenium diet for nearly a year no changes in testicular mass and morphology were observed. The serum concentration of testosterone of 6-month-old, selenium-depleted animals was, however, slightly lower than that of adequately supplied controls, and the stimulation of testosterone secretion by administration of GnRH or LH resulted in a significantly less marked rise in the serum concentration of testosterone. From the second generation onwards the testis mass, expressed as a percentage of the body mass, decreased and in the fourth generation was less than 50% of that of the controls. The male gonads of fourth generation animals showed a severe bilateral atrophy, in which the seminiferous tubules were considerably reduced in diameter and almost entirely lined by Sertoli cells and a few stem cells. Differentiated spermatozoa could not be detected. The alterations were reversible and spermatogenesis was restored by feeding the selenium-adequate diet. The findings indicate that testicular morphology and functions are affected by severe selenium deficiency and that the element is necessary for testosterone biosynthesis and the formation and normal development of spermatozoa.

In a 9-year-old male German Shepherd dog with a body weight of 37 kg which was euthanised because of a spinal meningioma, examination of the carcass revealed severe signs of degradation concerning the whole skeleton which were investigated by radiological, microradiological and pathologic-histological means. The skeletal alterations in form of systemic defects in corticalis and spongiosa in fact correspond to appearance and localisation of bone defects typical of fibrous osteodystrophia, but now are filled by conspicuous cell-poor matured collagenous connective tissue. In this connection it must be emphasised, that there are no osteoclasts detectable in the lacunae of degradation so that the disease process apparently has stopped.

The phosphoinositide cycle is an important second messenger system that may be involved in the therapeutic action of lithium. Elderly patients taking lithium often experience beneficial effects at lower doses and serum levels than their younger counterparts. To investigate age-related differences in lithium's effects on the phosphoinositide system, neostriatal slices from 3-, 10-, and 24- to 26-month-old Fischer 344 rats were labeled with [3H]-myoinositol and exposed for a 25-minute incubation period to oxotremorine-M and varying lithium chloride concentrations. Inositol phosphatase accumulation was significantly greater in 10- and 24- to 26-month-old than in 3-month-old rats. Further investigations may clarify the underlying mechanisms for such age-related differences.

Our approach to analyze molecular components of the blood-brain barrier led to the identification of additional transcripts which can be regarded as "BBB markers". Other candidates are presently analyzed in order to find hitherto unknown cell type-specific transcripts. We investigated the expression of these marker-genes in cell culture and found all genes still being transcribed after 10 days in primary cultures, although at a lower level. This is surprising, since other authors report the disappearance of BBB characteristics under such conditions. Moreover, the BBB marker gamma-GT is found to be not only expressed in BMEC, but also in the closely associated pericytes. The hitherto unknown physiological function of the enzyme, especially the abundance in pericytes is still under investigation. Since the method of subtractive cloning has been proven as a fruitful approach, we consider to establish further subtractive cDNA libraries, using different subtraction parameters. The PCR method is applicable for amplification of subtracted cDNA (Timblin et al., 1990) and we expect to find additional clones, mainly of lower abundance which are of functional importance for the BBB phenomenon. The described characterization of cultured BMEC now allows to proceed to study BBB-specific gene expression with special regard to regulatory elements. We will perform these experiments by use of enhancer trap vectors transfected into BMEC. The isolation of the corresponding genomic DNA fragments of the BBB markers is in progress.

The release of endogenous acetylcholine and dopamine and the appearance of their metabolites, choline and dihydroxyphenylacetic acid (DOPAC), from neostriatal slices prepared from Fischer 344 rats was examined under various experimental conditions. There was a dose-dependent increase in the amount of neurotransmitter or metabolite as the medium potassium concentration was increased from 5 to 50 mM. Over an eight minute period in Krebs Ringer bicarbonate buffer containing 25 mM potassium, the rate of release of acetylcholine was 6 to 13 times greater than that of dopamine. The dopamine endogenous to the slice preparation appeared to have little effect on the release of endogenous acetylcholine since manipulations that significantly altered dopamine release (depletion with 6-hydroxydopamine or uptake inhibition with nomifensine) had minimal effects on the cholinergic neurons. In contrast, increasing the endogenous acetylcholine in the preparation by inhibiting acetylcholinesterase resulted in a 1.2 to 12 fold increase in dopamine release depending upon the incubation time and the potassium concentration. These studies indicate that within the neostriatal slices there is minimal influence of the endogenous dopamine on the cholinergic neurons, whereas the extracellular acetylcholine can influence dopamine release when its concentration is increased by inhibition of acetylcholinesterase.

The purpose of this study was to investigate whether the muscarinic modulation of neostriatal acetylcholine release changes with senescence. Neostriatal slices from Fischer 344 rats aged 3, 10 and 28 months were prepared and incubated in Krebs-Ringer bicarbonate buffer oxygenated with 95% O2/5% CO2. Acetylcholine release from slices of each age group was monitored in the presence or absence of muscarinic agents, and the release in the presence of the drug was compared to the release from slices of age-matched controls in the absence of drug. The muscarinic agonist, oxotremorine, and two muscarinic antagonists, atropine and pirenzepine, were tested for their effects on acetylcholine release. Pirenzepine is selective in its interaction with the M1 muscarinic receptor subtype; atropine and oxotremorine are nonselective in their actions. Of the three drugs tested, pirenzepine displayed a significant age-related difference in its effects on acetylcholine release. Whereas the effects of pirenzepine (50 microM) on acetylcholine release modulation in slices from the 3-month rats were negligible, the M1-selective antagonist increased the release of acetylcholine from slices of 10- and 28-month rats by another 42 and 192% (P less than .05), respectively. Atropine (1 microM) was also tested, and an increase in acetylcholine release by another 64, 104 and 218% (all P less than .05) was observed in slices from the 3-, 10- and 28-month rats, respectively. In the presence of oxotremorine (50 microM), acetylcholine release decreased in slices from the 3-month rats by 35% (P less than .1), but changed by only 7 and 15% in the 10- and 28-month slices, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A double spinal cord trauma of an eight weeks old calf is reported, born on pasture from a heifer as a premature birth at the beginning of month 9. Observations made were rupture of intervertebral disk at the crossing of cervical to thoracic vertebrae followed by syndesmosis or synchondrosis resp., as well as a comminuted fracture of the 1st lumbar vertebra including both the adjoining vertebrae, with succeeding reactive callus formation. It is concluded that this traumatization has occurred during parturition. Motoric or sensory disturbances were observed neither.

In order to assess behavioral and neurochemical changes resulting from pesticide exposure, food‐restricted male weanling rats were exposed for 90 d to low doses (1 ppb‐10,000 ppb range) of individual pesticides (aldicarb, metribuzin, or methomyl) or mixtures of them. During exposure, rats were trained to run a T‐maze and tested for spatial discrimination reversal learning. At sacrifice, three brain regions (cortex, hippocampus, and neostriatum) were assayed for the neurotransmitters dopamine, acetyl‐choline, and serotonin. Animals treated with a mixture of two insecticides and one herbicide were found to have slower speeds in maze‐running (motor control) and also had altered levels of choline in their neostriatums. Rats treated with one herbicide compound (metribuzin) took longer to learn on two reversals; this group also had a significantly lower acetylcholine/choline ratio in their hippocampus.

The release of endogenous and newly synthesized acetylcholine (ACh) was examined in neostriatal slices prepared from young adult (10-month) and aged (28-month) Fischer 344 rats. Both spontaneous and potassium-stimulated release were tested after various in vitro incubation times (1, 3 or 5 hr). The potassium-stimulated release of ACh from slices of 28-month rats was decreased by 53% when tested after incubating the slices 1 hr. The age-related differences in ACh release lessened if the slices were incubated for longer times (3 or 5 hr) before monitoring release. The spontaneous release of ACh was similar among the slices from both age-groups and at all times points monitored. When the neostriatal slices were incubated in medium supplemented with deuterated choline, the release of both the endogenous and newly synthesized ACh from slices of 28-month rats was decreased by 33% when tested after a 1-hr incubation, but was again similar to that released from slices of 10-month rats when tested after a 3-hr incubation. Choline release from slices of the 28-month rats was similar to that released from the slices of the 10-month rats when acetylcholinesterase (AChE) was inhibited during the release incubation. In slices with intact AChE activity, however, the age-related difference in choline release was similar to that observed for ACh release when AChE was inhibited. That is, when AChE activity was intact, the potassium-stimulated choline release from slices of 28-month rats was less than that released from slices of 10-month rats when release was tested after a 1-hr incubation. This difference in choline release was not apparent when release was tested after longer incubation times. The results show that in vitro age-related differences in neostriatal ACh release are apparent in early periods of incubation and reverse with longer incubation times.

Intraperitoneal administration of methotrexate in a single dose of 40 mg/kg induces fluid accumulation in the small intestine of rats, significantly increasing jejunal PGE2 formation and simultaneously the amounts of PGE2 in the intestinal contents in vivo. Concomitantly, jejunal PGD2 and 6-keto-PGF1 alpha generation and the amounts of these prostaglandins in the intestinal contents were significantly lowered. However, PGD2 and 6-keto-PGF1 alpha jejunal release, and the amounts of these prostaglandins found in the intestinal contents, were already low after a sublethal dose (4 mg/kg) of methotrexate, whereas the jejunal release as well as the amounts in the intestinal contents of PGE2 were not altered. Fluid accumulation, the amounts of prostaglandins in the intestinal contents and jejunal release of prostaglandins are significantly inhibited by indomethacin. The increased jejunal synthesis of PGE2, with its enteropooling effect, may play a significant role in methotrexate-induced diarrhea in rats.

The in vitro preservation of neurons in 300 micron thin neostriatal slices, which are routinely used for electrophysiological studies, was examined by light and electron microscopy and was compared to 700 micron thick neostriatal slices. The thin slices displayed well-preserved cells after up to 5 h of incubation. This finding correlated well with whether electrical activity could be recorded. In cross-section, the thin slices consisted of three layers: the inner layer contained many intact cells (80%) and was sandwiched between the outer layers where deteriorating cells predominated. In contrast to the thin slices, the thick slices (700 micron) displayed no layering of intact cells in cross-section. Instead, the majority of cells throughout these thick slices was swollen (98%), with only small patches of intact cells. Two types of deteriorating cells were apparent: swollen cells and dark (pycnotic) cells. The proportion of swollen cells increased with incubation time. In the thin slices this swelling occurred in the outer layers with the middle layer of intact cells remaining relatively unchanged over long incubation periods, whereas all cells in the thick slices were swollen after 2 h of incubation. Dark cells were localized to the outer portion of both slices and the number of such dark cells did not change with incubation time.

— The high affinity transport of choline (Ch) and the synthesis of acetylcholine (ACh) were measured in synaptosomes by measuring the utilization of [2H4]Ch. The synthesis of ACh was reduced under several conditions which reduce the availability of acetyl coenzyme A (AcCoA) including no glucose added, replacement of glucose with succinate or impairment of glucose utilization by bromopyr-uvate, NaCN, or pentobarbital. These conditions did not reduce the amount of unacetylated [2H4]Ch in the synaptosomes indicating that the high affinity transport of Ch is not directly coupled to the synthesis of ACh.

Effects of 2,4-dithiobiuret (DTB) treatment in rats on neuromuscular transmission and the disposition of cholinergic substances, acetylcholine (ACh) and choline (Ch), were examined in a combined electrophysiological/biochemical study using an in vitro extensor digitorum longus (EDL) muscle-peroneal nerve preparation. EDL muscle preparations isolated from rats treated with DTB (1 mg/kg/day X 5 days, ip) displayed a 49% depression in the frequency of miniature end-plate potentials (MEPPs) and a 21% depression in mean MEPP amplitude. Statistical analysis of evoked end-plate potentials (EPPs) measured in curarized preparations indicated that the mean quantal content (m) was significantly depressed in EDL muscles from DTB-treated rats. At stimulation rates of 1, 10, 20, and 50 Hz the estimated values of m in EDL preparations from DTB-treated rats were, respectively, 21, 25, 45, and 51% of that in control preparations. Biochemical determinations of ACh and Ch revealed a significant DTB-induced increase in endogenous ACh and Ch content in EDL preparations fixed for extraction of ACh and Ch immediately after dissection from the treated rats. In vitro, however, there were negligible changes in overall ACh synthesis since the total (tissue and medium) tracer ACh (2H4-ACh) synthesized from tracer Ch (2H4-Ch; 10 microM) supplied in the perfusion medium was similar in EDL preparations from DTB-treated and control rats. Also, in EDL muscles from DTB-treated rats the resting release of ACh was not affected, but when exogenous Ch (2H4-Ch) was not supplemented in the medium the evoked release (via peroneal nerve stimulation) of ACh was depressed. Thus, decreases in spontaneous quantal ACh release, as detected in the electrophysiological experiments, were not reflected by changes in the biochemically determined ACh resting release. The biochemical determination of evoked ACh release, however, correlated with the decrease in quantal content detected in the electrophysiological analysis of evoked EPPs when exogenous Ch was not supplemented in the perfusion medium. Significant and consistent increases (two to three times) in both Ch content and efflux occurred in the EDL muscles from DTB-intoxicated rats. These results indicate that DTB induces a prejunctional impairment of neuromuscular transmission that is not specifically directed at ACh synthesis. Rather those processes by which ACh is incorporated into or released from vesicles appear to be altered.

Rat jejunal tissue in vitro synthesizes large amounts of prostaglandin (PG) D 2 and smaller amounts of 6-keto-PGF 1α and PGE 2 , whereas human small intestinal mucosa synthesizes much smaller amounts of the three PG determined with about equal amounts of PGE 2 and PGD 2 . Intraperitoneal administration of bacterial endotoxin to rats induces fluid accumulation in the small intestine and increases significantly the release of PGD 2 , PGE 2 and 6-keto-PGF 1α into the small intestinal lumen in vivo. Endotoxin-induced stimulation of PG release is particularly pronounced for PGD 2 . Fluid accumulation and PG output are inhibited by indomethacin. It seems possible that the different total amounts of PG synthesized by small intestinal tissue of man and rat as well as the different pattern of PG released might contribute to species-specific responses of the gastrointestinal tract to various pathophysiological stimuli.

Using sequential incubations in media of different K+ composition, we investigated the dynamics of choline (Ch) uptake and acetylcholine (ACh) synthesis in rat brain synaptosomal preparations, using two different deuterated variants of choline and a gas chromatographic-mass spectrometric (GC-MS) assay for ACh and Ch. Synaptosomes were preincubated for 10 min in a Krebs medium with or without high K+ and with 2 micrometer-[(2)H(9)]Ch. At the end of the preincubation al variants of ACh and Ch were measured in samples of the pellet and medium. In the second incubation (4 min) samples of synaptosomes were resuspended in normal or high K+ solutions containing [(2)H(4)]Ch (2 micrometer) and all variants of ACh and Ch were measured in the pellet and medium at the end of this period. This protocol allowed us to compare the effects of preincubation in normal or high K+ solution on the metabolism during a second low or high K+ incubation of a [(2)H(9)]Ch pool accumulated during the preincubation period. Moreover, we were able to compare and contrast the effects of this protocol on [(2)H(9)]Ch metabolism versus [(2)H(4)]Ch metabolism. The most striking result we obtained was that [(2)H(9)]Ch that had been retained by the synaptosomes after the preincubation was not acetylated during a subsequent incubation in normal or high K+ media. This result suggests that if an intraterminal pool of Ch is involved in ACh synthesis, the size of this pool is below the limits of detection of our assay. We have confirmed the observation that a prior depolarizing incubation results in an enhanced uptake of Ch during a second incubation in normal K+ Krebs. Moreover, Ch uptake is stimulated by prior incubation under depolarizing conditions relative to normal preincubation when the second incubation is in a high K+ solution. These results are discussed in terms of current models of the regulation of ACh synthesis in brain.

Choline (Ch) uptake and release and acetylcholine (ACh) synthesis and release have been studied by gas chromatography mass spectrometry (GCMS) in slices of rat neostriatum in vitro to assess the effects of depolarization by 25 mM K+ and the influence of elevated concentrations of Ch in the incubation medium. During the first 60 min after preparation, 25 mM K+ increased ACh release by 182% and reduced ACh levels by 40%. The rate of ACh synthesis was unchanged. After a 1-h equilibration period, the rate of ACh synthesis was considerably less (2.41 nmol mg-1 h-1, compared to 9.78 nmol mg-1 h-1). Exposure to 25 mM K+ during the second hour increased the rate to 6.47 nmol mg-1 h-1. During the first 10 min of exposure to 25 mM K+, ACh synthesis was reduced, regardless of incubation. Increasing concentrations of external [2H4]Ch apparently favored initial rates of net ACh synthesis, since the rank order of initial net ACh synthesis rates is the same as the rank order of external [2H4] Ch concentration under both normal and depolarized conditions. However, the only significant effect of external [2H4]Ch on ACh metabolism was that it increased ACh release during the initial 10 min, when the preparation was depolarized with K+. The efflux of endogenous [2H0]Ch was increased initially (10 min) and slowed over a 60-min period by 25 mM K+, and increased when [2H4]Ch in the medium was increased. Changes in ACh synthesis and release were dependent upon the time exposure of slices to high K+, and the results suggest that Ch favors initial rates of ACh synthesis, but that Ch influences ACh release primarily under conditions of stress (i.e., depolarization).

When brain synaptosomes are obtained from animals that have been injected intravenously with [2H4]choline 1 minute before being killed, their high affinity [3H] choline uptake is correlated inversely with their acetylcholine content and directly with the rate at which they synthesize [2H4]acetylcholine. The control of such choline uptake by the cytoplasmic acetylcholine concentration is proposed as a mechanism regulating acetylcholine synthesis in cholinergic nerve terminals.

1. The balance between acetylcholine (ACh) synthesis and degradation and the availability of choline were studied in the neuromuscular junction of rats aged 10 (mature adult) and 28 (aged) months. Endogenous and 2H4-labelled variants of ACh and choline were assayed during steady-state stimulated and resting conditions using gas chromatography-mass spectrometry. 2. Endogenous ACh levels were 34% less per nerve terminal in the older rats; in non-innervated tissue, the levels were 12-14% as large as in innervated tissue. Endogenous choline levels in innervated and non-innervated tissue were similar and were 28% higher in the older animals. 3. Accumulation of 2H4-labelled choline was significantly greater in the 28-month animals; the specific activity at equilibrium was 60% higher in the older rats. 4. Incorporation of [2H4]choline into [2H4]ACh was significantly faster in the aged rats; times to half-maximal values were 2.0 and 0.6 min in the 10- and the 28-month animals, respectively. There was no measurable synthesis of ACh in the non-innervated tissue. 5. There were no major age-related differences in the amount of ACh released during stimulation between 1 and 20 Hz. In contrast, choline efflux during stimulation was significantly greater in the aged animals; flux values (+/- S.E. of mean) were 0.48 (+/- 0.014) and 0.66 (+/- 0.038) nmol/min for the 10- and the 28-month rats, respectively. 6. Under resting conditions, endogenous ACh efflux was significantly greater in the older rats; the rates (+/- S.E. of mean) were 2.00 (+/- 0.21) and 3.05 (+/- 0.43) pmol/min for the younger and the older animals, respectively; similarly, choline efflux was greater in the aged tissue. 7. These results indicated that lower intracellular ACh levels can be attributed to enhanced leakage rather than to decreased synthesis. Greater ACh efflux is accompanied by correspondingly greater choline uptake in the 28-month rats; this is associated with greater choline efflux and higher steady-state choline levels in the aged tissue.

An in vitro slice technique was employed to study the receptors involved in intrinsic cholinergic excitation in the rat neostriatum. The locally evoked synaptic potentials were suppressed by antinicotinic agents, mecamylamine (10 muM), d-tubocurarine (3 muM) or hexamethonium (100 muM), but not by the antimuscarinic agent atropine (100 muM). If the slices were exposed to an acetylcholinesterase (AChE)-inhibitor (paraoxon 1--20 muM, physostigmine 0.1--0.5 muM), the synaptic potentials were potentiated. The amplitude of the orthodromic population spike increased, and it was further facilitated when the stimulus frequencies were raised from 1--3 Hz to 10--30 Hz. The frequency facilitation following exposure to an AChE-inhibitor was blocked by atropine (1--100 muM). Intracellular recording indicated that a slow depolarizing potential caused the frequency potentiation of the orthodromic discharges. Apparently rat neostriatum is similar to cholinergic systems in sympathetic ganglia and spinal Renshaw cells, in that nicotinic receptors mediate fast excitation and muscarinic receptors mediate slow excitation.

Chicken thymus, spleen, and bursa lymphocytes were isolated by different methods and incubated under differing conditions in order to obtain and characterize avian lymphokines. The biological activity of lymphokine-containing cell culture supernatants was measured by their antiviral activity (interferon(IFN)-units) and by their capacity to induce cytostatic effects in bone-marrow-derived macrophages (50% cytostasis-inducing dose, CID). Lymphokine production by thymus lymphocytes required concanavalin A (ConA)-stimulation, while spleen cells, when cultured at high density, released CID and IFN activities into the culture medium even without mitogen-stimulation. By way of comparison, the highest lymphokine content was found in the supernatant of lymphocyte cultures, which were incubated for 72 hours at 41 degrees C after stimulation with an optimal ConA dose. For stimulation of thymus lymphocytes 30 micrograms ConA/ml were found to be optimal, independent of serum content and cell density in the cultures. In contrast, the optimal ConA dose for spleen lymphocytes not only depended on the serum content but also on the cell density in the cultures and varied within a range of 2.5 micrograms and 45 micrograms ConA/ml.

Some classical and nonclassical muscarinic agents were tested for their effects on potassium-evoked acetylcholine (ACh) release from rat neostriatal slices. Release was monitored by measuring endogenous ACh when acetylcholinesterase (AChE) was inhibited with physostigmine (30 microM) or by measuring endogenous choline when AChE activity was left intact. The classical antagonist, atropine (0.1-2 microM), induced an increase in release whether AChE activity was inhibited or intact. The putative M-1 selective antagonist, pirenzepine, had minimal effects over a broad concentration range (2-200 microM) and induced an increase in ACh release only when AChE activity was inhibited. The classical agonist, oxotremorine (10-100 microM) decreased effectively ACh release (by 22-35%), but only when AChE activity was intact. The oxotremorine analog, oxotremorine-M, was apparently more potent than oxotremorine, but also decreased ACh release (by 24-41%) only when AChE activity was intact. Another oxotremorine analog, BM-5, behaved more like a muscarinic antagonist in its effects on neostriatal ACh release, and the highest concentration tested (100 microM) increased release (by 47%) when AChE activity was left intact. As in many other cholinergic systems, the agonists tested in this study were not selective in their action on ACh release modulation in the rat neostriatum. The antagonists, however, were more selective in their action, i.e., pirenzepine was relatively ineffective, and on the basis of this selective action, it can be concluded that modulation of endogenous ACh release in the rat neostriatum is mediated by a M-2 muscarinic receptor subtype.

Two classical and two nonclassical muscarinic agents were tested for their effects on neostriatal acetylcholine (ACh) release. The classical muscarinic antagonist, atropine (0.1-2 microM), increased ACh release in a dose-dependent fashion, whereas the 'nonclassical' antagonist, pirenzepine (2-200 microM), had no effect on ACh release. The muscarinic agonist, oxotremorine (1-100 microM), and its analog, oxotremorine-M (0.1-50 microM), both decreased ACh release in a dose-dependent fashion. The inhibitory potency of oxotremorine-M was approximately 20 times that of oxotremorine, and the inhibitory effects of both agonists were blocked by atropine. The results obtained with the muscarinic antagonists indicate that 'M-2 type' muscarinic receptors mediate ACh release in the neostriatum, but further pharmacological analyses are required to assign with certainty the receptor subtype involved in neostriatal ACh release.

The effects of different ionic environments were measured on the concentration of acetyl-choline (ACh) from synaptosomes and their effect on subsequent high affinity choline (Ch) transport and ACh synthesis after resuspension of the synaptosomes in the normal Krebs medium. KCl (40 mM) was used to induce ACh release and reduce synaptosomal ACh content. The effects of Na+ omission, Ca2+ omission, and high Mg2+ on spontaneous (KC1: 4.75 mM) and potassium induced (KC1: 40 mM) ACh release and other cholinergic parameters are presented. The high affinity transport of Ch was more highly correlated with the reciprocal of the ACh level (r= 0.934, P= 9.7 × 10-4) than with the ACh release rate during preincubation (r= 0.792, P= 3.4 × 10-2). The results are consistent with the view that the consequences of the various ionic conditions on Ch transport and ACh synthesis are dependent on their effects on intrasynaptosomal ACh levels and only secondarily on synaptosomal ACh release.