Screening of 64 tryptamines at NMDA, 5-HT1A, and 5-HT2A receptors: a comparative binding and modeling study.

Tryptamine (T) and several T derivatives (Ts) inhibit in a voltage-dependent manner the NMDA receptor (NR). This effect is influenced by substituents at various positions, but has not yet been subjected to a detailed SAR study. Here, 64 Ts have been tested as inhibitors of [3H]MK-801 binding to NRs on rat brain membranes. For comparison, they were also tested as inhibitors of [3H]8-OHDPAT binding to 5-HT1A and of [3H]ketanserin binding to 5-HT2A receptors. Since most of these Ts have not been tested before at any of these receptors, we start with a review of the effects of Ts on 5-HT1A and 5-HT2A binding sites. NRs were inhibited with IC50s from 2 to 7 µM by Ts with alkyl or halogen at positions 2, 5, and/or 7. Inhibition by some Ts was attenuated more than 10-fold by 30 µM spermine. The most potent inhibitors at 5-HT1A receptors were 5-carboxamido-T (IC50 0.00015 µM) and serotonin (0.0016 µM), at 5-HT2A receptors 2-Me-4,7-Cl2-T (1.2 µM) and 2,7-Me2-4-Cl-T (2.0 µM). Fujita-Ban modified Free-Wilson analyses pointed to the individual significance of particular substituents. Also QSARs based on molecular operating environment descriptors resulted in sound correlations at all 4 targets. No similarities between the NR and 5-HT receptors could be found. At the NR, only L-Trp-NH2 bound 10 times better than at both 5-HT receptors studied. L-Trp-NH2 may be a structural lead to endogenous non-competitive NR antagonists.

Clinicopathological associations of CD44 mRNA and protein expression in primary breast carcinomas.

AIMS: The purpose of this study was to examine the occurrence of CD44 isoforms in breast carcinomas and their role in predicting clinical outcome. METHODS AND RESULTS: Shock-frozen tumour tissues from 110 patients with breast carcinoma were examined by immunohistochemistry using antibodies directed against CD44s, v5, v6, v7 and v3-10. In addition, 80 of these tumours were available for quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of CD44s and CD44v6. Immunohistochemically, the positive tumours showed cytoplasmic and/or membranous staining with all antibodies. Staining results did not correlate with histological subtype, lymph node status, status of steroid receptors, tumour size or age. Neither was any correlation found for overall and disease-free survival. Quantitative real-time RT-PCR of CD44s and CD44v6, however, revealed that expression of CD44v6 mRNA was significantly associated with lower pathological grade (Pearson chi(2) test P = 0.009; linear-by-linear association P = 0.003). Linear-by-linear association between CD44s mRNA expression and lower pathological grade was also seen (P = 0.02). Survival analysis with the Kaplan-Meier method demonstrated that increased CD44s mRNA expression was significantly associated with both disease-free survival and overall survival (P = 0.0185 and P = 0.0344, respectively). A similar trend for CD44v6 mRNA expression was seen in these cases, but the difference was not significant. CONCLUSIONS: Quantitative real-time RT-PCR revealed clinical correlations of CD44s and CD44v6 mRNA expression in breast carcinomas while immunohistochemistry for the protein expression of CD44s and other CD44 variants did not. This contradictory result merits further studies concerning the clinical impact of CD44 molecules in breast carcinomas.

Estrogen receptor-alpha and C-ERBB-4 expression in breast carcinomas.

The presence of estrogen receptors (ERs) in breast carcinomas is important for clinical response to endocrine therapy. However, the cellular mechanisms following ER activation are not fully understood. It has been indicated that expression of the ER is associated with the expression of c-erbB-4. To address this question, 103 breast carcinoma samples were studied using reverse transcriptase polymerase chain reaction (RT-PCR) analysis after application of a microselection method for RNA isolation. Total RNA for RT-PCR was isolated from 20-microm-thick frozen sections, which were made from microselected areas. Paraffin blocks from 98 of these 103 tumors were also immunohistochemically examined. Significant associations between ER-alpha and c-erbB-4 mRNA and protein expressions were found in the present study with both methods. One-fourth of the tumors did not express ER-alpha (22%, 24%, and 26% with chemical binding, immunohistochemistry, and RT-PCR, respectively). About one-half of the ER-alpha negative tumors did not express c-erbB-4 on both mRNA and protein levels (48% with RT-PCR and 46% with immunohistochemistry, P=0.001 for both methods). The endocrine therapy responsive breast cancer cell lines MCF-7 and T47-D were positive for both ER-alpha and c-erbB-4 expression, while the endocrine therapy nonresponsive breast cancer cell lines MDA-MD-231 and SK-BR-3 were not. Thus, we confirm the association between the expression of ER-alpha and c-erbB-4 mRNA and protein in breast carcinomas, indicating a role for c-erbB-4 in estrogen signal transduction.

Expression of CD44 isoforms in infiltrating lobular carcinoma of the breast.

Expression of CD44 has been shown to correlate with the progression and prognosis of some malignant tumors. The aim of this study was to evaluate the expression of CD44 isoforms in infiltrating lobular carcinomas and analyse their potential as prognostic indicators. A panel of 39 tumors were examined for their expression of membranous and cytoplasmic CD44s, v3, v5, v6, v7 and v3-10 in the infiltrating cells, by immunohistochemical staining. The protein positive tumors showed membranous and/or cytoplasmic staining with all antibodies used except for CD44v7, which only displayed cytoplasmic staining. Cytoplasmic expression of CD44v3 (P = 0.014) and membranous expression of v6 (P = 0.039) were significantly associated with alveolar, classical/alveolar carcinomas and mucinous/alveolar carcinomas. Furthermore, in alveolar, classical/alveolar and mucinous/alveolar carcinomas, cytoplasmic staining of CD44v5 was correlated with lymph node negative patients (P = 0.048), whereas membranous v5 was correlated with lymph positive patients (P = 0.048). In classical, classical/trabecular and trabecular carcinomas expression of membranous CD44s was significantly correlated with lymph node status (P = 0.042).

E-cadherin and alpha-, beta-, and gamma-catenin protein expression is up-regulated in ovarian carcinoma cells in serous effusions.

The aims of this study were firstly, to investigate the expression of E-cadherin complex proteins in ovarian carcinoma cells in serous effusions and in primary and metastatic lesions; and secondly to study the value of these four proteins and calretinin, a mesothelial marker, in the differential diagnosis of ovarian carcinoma cells from reactive mesothelial cells in effusions. Sixty-seven malignant effusions and 97 corresponding primary (n=36) and metastatic (n=61) lesions were immunohistochemically stained for E-cadherin and alpha-, beta-, and gamma-catenin. Staining extent and intensity were scored. Effusion specimens were additionally analysed for calretinin immunoreactivity. Membrane immunoreactivity for E-cadherin and alpha-, beta-, and gamma-catenin was detected on carcinoma cells in the majority of the effusions, but rarely on reactive mesothelial cells (p<0.001 for all markers). Calretinin immunoreactivity was confined to mesothelial cells (p<0.001). An association was seen between E-cadherin and alpha-catenin expression, in both effusions and solid tumours, and for beta-catenin in solid tumours (range p<0. 001 to p=0.014). Up-regulation of all four cadherin complex proteins was seen in carcinoma cells in effusions, when compared with corresponding primary tumours (range p<0.001 to p=0.028). As with effusions, metastatic lesions showed up-regulation of alpha-, beta-, and gamma-catenin when compared with primary carcinomas (p=0.002-0. 015). Carcinoma cells in effusions showed in addition elevated levels of E-cadherin when compared with metastatic lesions (p<0.001). Staining results in effusions showed no association with effusion site, tumour type or histological grade. Immunoblotting on 29 malignant effusions confirmed the presence of all four proteins in the majority of samples and co-precipitation of E-cadherin and beta-catenin was seen in ten specimens examined. E-cadherin complex proteins are widely expressed in ovarian carcinoma cells. Together with calretinin, they form a powerful battery of markers for the cytological diagnosis of carcinoma cells in effusions. The up-regulation of E-cadherin complex proteins in serous effusions and metastatic lesions may mark an early metastatic phenotype and possibly mediates survival of tumour cells at these sites through the inhibition of apoptosis.

Endometrial brush biopsy (Tao brush). Histologic diagnosis of 200 cases with complementary cytology: an accurate sampling technique for the detection of endometrial abnormalities.

We examined 200 cases of endometrial brush biopsy (EBB) using the Tao brush and correlated findings with histologic findings from subsequent dilatation and curettage (D&C) or hysterectomy specimens. Diagnosis by EBB relied mainly on histologic evaluation of H&E-stained tissue sections and was complemented by additional cytologic smear examination. EBB correctly detected the following cases: endometrioid adenocarcinoma, 3; complex hyperplasia with atypia, 1; simple hyperplasia without atypia (SH), 2; and benign endometrium, 177. In 3 cases the diagnosis of atrophic endometrium was made by EBB; corresponding D&C specimens were nondiagnostic. Five cases of SH were interpreted by EBB as proliferative endometrium, and 13 endometrial polyps were not identified by EBB. Nine samples were nondiagnostic. Sensitivity and specificity were 100% for detecting atypical hyperplasia and carcinoma. However, it was difficult for EBB to distinguish SH from disordered proliferative endometrium or to diagnose endometrial polyps. We found that diagnosis by EBB is reproducible; a second pathologist blinded to histologic follow-up correctly identified all adenocarcinoma/atypical hyperplasia cases. EBB is an accurate, safe, and easy procedure that is well tolerated by patients and should be considered in the initial evaluation of high-risk outpatients.

Differential expression of CD44s and CD44v3-10 in adenocarcinoma cells and reactive mesothelial cells in effusions.

The detection of malignant cells in serous effusions obtained from patients diagnosed with cancer marks the presence of metastatic disease and is associated with a poor outcome. The purpose of this study was to evaluate the role of CD44s and CD44v isoforms in the distinction between mesothelial cells and malignant epithelial cells in effusions. Fifty-nine fresh pleural and peritoneal effusions were studied. These consisted of 41 specimens from patients with known gynecological neoplasms, 9 from patients diagnosed with breast adenocarcinoma, and 9 effusions from patients with various nongynecological malignancies or tumors of unknown origin. Forty-three effusions contained malignant/atypical epithelial cells, and 16 effusions were diagnosed as reactive. Three effusions contained exclusively malignant cells. Specimens were stained with anti-CD44s, v3, v5, v6, v7 and v3-10. The presence of staining in cancer cells, benign mesothelial cells and lymphocytes was evaluated. CD44s immunoreactivity was seen in 10 of 43 (23%) cases in malignant/atypical epithelial cells and in 53 of 56 (94%) cases in benign cells. In contrast, CD44v3-10 was seen in 23 of 43 (55%) cases in malignant/atypical epithelial cells and in 3 of 56 (6%) cases in benign cells. We advocate the use of CD44s and CD44v3-10 immunostaining in diagnostic evaluation of difficult serous effusions.

Expression of CD44 in effusions of patients diagnosed with serous ovarian carcinoma--diagnostic and prognostic implications.

CD44 is a family of cell adhesion molecules involved in a variety of cellular functions. The present study analysed the expression of two CD44 isoforms in serous effusions of patients diagnosed with ovarian carcinoma and corresponding primary and metastatic lesions. Fifty-eight effusions, 23 primary ovarian tumours, and 44 metastatic lesions were studied for protein expression of CD44s and v3-10 using immunohistochemistry. Results were correlated with clinical parameters. CD44v3-10 was seen in carcinoma cells in the majority of cases at all sites. Malignant effusions showed an up-regulation of CD44s compared to both primary tumours and metastatic solid lesions. Mesothelial cells frequently expressed CD44s, but were rarely immunoreactive for v3-10. CD44s immunoreactivity in cancer cells in effusions was significantly more often observed in patients with FIGO stage 3 than in stage 4 patients (P = 0.045). Staining results did not correlate with age, effusion site, metastatic site, tumour grade or residual tumour mass after initial surgery. Likewise, comparison of overall and disease-free survival with expression of the CD44 isoforms studied did not reveal any statistically significant associations. The up-regulation in CD44 levels in effusions, primarily in stage 3 disease, suggests that adhesion of ovarian carcinoma cells to mesothelium may be regulated at the level of CD44s expression, and provides further evidence of phenotypic alteration in the transition from primary tumour cell clones to effusions. The similar expression profile of CD44 in carcinoma cells in peritoneal and pleural effusions supports our previous observations and the hypothesis that carcinoma cells in peritoneal effusions are truly metastatic.

[Prevention of myopathies and cardiomyopathies in swine using magnesium aspartate hydrochloride]. / Prophylaxe von Myopathien und Kardiomyopathien beim Schwein durch den Einsatz von Magnesium-Aspartat-Hydrochlorid.

The efficiency of magnesium-aspartate-hydrochloride (MAH) in prophylaxis of porcine stress-induced myopathy was investigated. For the examination a conveyor belt was used. MAH was administered orally or intramuscularly. Clinical investigations, blood specimen collections and ECG were carried out before and after running. Stress-induced tachypnoea and tachycardia and hyperthermia were reduced after both forms of administration of MAH. The enzyme activity of CK, LDH and alpha-HBDH increased slighter after oral and i. m. application. The increase of serum glucose concentration as well as serum lactate level were reduced after oral and i. m. substitution with MAH. In the ECG apart from the relative diastolic period all time values were significantly prolonged after MAH-substitution. The results indicate that several stress-induced changes of clinical and metabolic parameters can be reduced by administration of MAH.

[The effectiveness of tilmicosin in respiratory diseases of swine]. / Zur Wirksamkeit von Tilmicosin bei respiratorischen Erkrankungen des Schweines.

Within the scope of the clinical evaluation of Tilmicosin in Enzootic Pneumonia of pigs, nasal swabs from 78 animals were taken, before and after oral medication of different doses (0, 100, 200, 300 mg Tilmicosin/kg dry food), and examined bacteriologically for Pasteurella multocida, Bordetella bronchiseptica und Haemophilus parasuis. The bacteria aforementioned were isolated from the nasal secretions of 83% of the pigs, 14 days after crowding without any prophylactic regime. It could be demonstrated, that pigs with clinical symptoms of Enzootic Pneumonia had a 50% higher prevalence-rate of multi-colonization with pneumotropic bacteria than healthy animals. Feeding 300 mg Tilmicosin/kg food for 9 and 14 days respectively, resulted in elimination of Pasteurella spp. and Haemophilus spp. The rate of newly Bordetella bronchiseptica infected pigs was lower than in the placebo-group. Parallel to these bacteriological results improvement of clinical signs and increased daily weight gain were observed.

[The protective effect of adsorbents against ochratoxin A in swine]. / Untersuchungen zur protektiven Wirkung von Adsorbentien gegenüber Ochratoxin A beim Schwein.

Adsorption of the mycotoxin ochratoxin A by activated charcoal, various bentonites (acid, alkaline, neutral), and hydrated sodium calcium aluminosilicate was tested in vitro as well as in feeding experiments with pigs. In vitro tests showed that the 1% addition of activated charcoal leads to complete adsorption of ochratoxin A from aqueous solutions. This effect was not influenced by pH-values ranging from 3-8. In contrast, adsorption by bentonite and hydrated sodium calcium aluminosilicate occurred primarily in the acid range (pH 3-4). Dietary addition of hydrated sodium calcium aluminosilicate (1%) and acid bentonite (1%, 10%) to ochratoxin A-contaminated feed (1.0 mg/kg) had no effect on the blood or tissue levels of the toxin in pigs. The addition of 1% activated charcoal caused a slight decrease of ochratoxin A in the blood, whereas a tenfold dosage resulted in a 50% to 80% reduction of ochratoxin A levels in both blood and tissue. Reduction of ochratoxin A absorption via the dietary administration of activated charcoal (5%) was confirmed in a 16 week feeding experiment. However, this experiment also showed the serum level of vitamin E to be lower than in the controls receiving adsorbent-free feed.

[Diseases of the extremities of swine in relationship to the floor condition with special reference to bursitis]. / Krankheiten der Extremitäten des Schweines in Abhängigkeit von der Bodenbeschaffenheit unter besonderer Berücksichtigung der Bursitiden.

In 2 pig farms, 102 sows kept in single ranging on plain concrete floor, 168 sows kept in single ranging on cast-iron grates and 27 fattening pigs kept in group penning on concrete slatted floors were repeatedly examined for the incidence of auxiliary synovial bursae (ASB). The clinical examination was completed by radiological, patho-anatomical and histological examinations of the rear legs. In sows, ASB were found on two prominences of the bones: on the latero-plantar area of the calcaneum and the fourth tarsal bone as well as on the plantar tarsal sesamoid bone. ASB on the rear legs were found in 41.2% of the sows kept on plain concrete floor and in 59.5% of the sows kept on cast-iron grates. The ASB of the sows kept on cast-iron grates were larger than those of the sows kept on plain concrete floor. In 49% of the sows kept on plain concrete floor the skin covering the ASB was reddened, in 45% of the sows kept on cast-iron grates it was covered with scab. Injury and necrosis of the skin as well as injury of the ASB themselves were found only in animals kept on cast-iron grates and made up 5% of the cases. In 30% of the cases, palpation of the ASB induced defensive movement. The palpation of ball-shaped, soft bursae more often caused defensive movement than did the palpation of flat-shaped, hard bursae. The occurrence of ASB was combined with kyphosis of the spine, false posture of the rear legs, dystasia, hyperextension of the fetlock, arthrosis of the ankle joint, and anomalies of the claws. In fattening pigs, the ASB were localized at the lateroplantar area of the calcaneum and the fourth tarsal bone as well as at the posterior surface of the calcaneum. At the end of the fattening period, ASB were detected in 26 out of 27 fattening pigs. All of the 48 ASB that were examined histologically showed signs of inflammation. In 30 cases the inflammation was chronic, in 18 cases it turned out to be florid. The results indicate that ASB cause pain during the developing phase and also in the chronic stage.

[Microbial changes as the cause of failures of the therapy of bacterial diseases of the urogenital organs of swine]. / Erregerwechsel als Ursache von Misserfolgen bei der Therapie bakteriell bedingter Krankheiten der Urogenitalorgane des Schweines.

In long-term clinical and bacteriological controlled breeding sows of 15 stocks with a high rate of urinary tract infections in 41 sows (= 8.97%) of 457 chronic infected sows a spontaneous change of the causative microorganism was seen. In clinical trials to test the effectiveness of new chemotherapeutics in sows suffering from urogenital infections changes of bacteria were noticed during and after therapy. In selected cases the proceeding of change using a quinoxaline-di-N-oxide-derivative and an aminoglycoside-antibiotic is demonstrated. After parenteral application of the quinoxaline-di-N-oxide-derivative in 3 of 6 cases a change of the causative agent happened. Treating the sows with the aminoglycoside-antibiotic there was a change in 18.2% of urinary tract infections and in 25.0% of genital tract infections. The causes of changes of the causative microorganisms are discussed and conclusions are drawn for diagnosis and therapy of urinary and genital tract infections in the sow.

[Pathological indicators in sows relevant to animal welfare in modern housing systems]. / Tierschutzrelevante pathologische Indikatoren bei Sauen in neuzeitlichen Haltungssystemen.

Methods and problems are discussed evaluating pathological indicators potentially related to animal welfare in modern husbandry systems of brood sows. A variety of interrelationships of diseases and genetic dispositions concerning the locomotive and cardiovascular system interferes with the required forensic evidence between individual sickness and housing and/or neglected care. There are no defined pathological processes indicating disproportionate husbandry as well as insufficient ability of physical exercise. Highly restricted possibilities to move caused by modern housing may result in pathologically manifest alterations. Technopathic diseases (induced by housing systems) may be prevented by enhanced care concerning the health control of animals and the functioning of the technical equipment as well as by appropriate prophylaxis. Farmers and veterinary surgeons should co-operate for the needs of animal welfare. One of the resulting conclusions is to plan for and to provide more labour for specific care necessary to maintain animal welfare. One should consider such management calculations with major precaution in which the size of working units relative to manpower gets increasingly maximized.

Estimation of frontal-plane cardiac vectors in heart disease.

Frontal-plane vector angle and magnitude values were clinically estimated in 100 patients with cardiac disease. Maximal deviations from isopotential in standard limb leads I and II were summed and mathematically transformed into estimated vector magnitudes (mV) and estimated angles (degrees). These values were then compared against reference vectors (previously shown to be excellent standards) defined from the same QRS complexes. The estimated magnitude values reached a degree of accuracy of +/- .25 mV in 78% of the cases, and the estimated angle values reached a degree of accuracy of +/- 15 degrees in 80% of the cases. The lowest estimate errors occurred in the infarct group, whereas the largest errors occurred in the chamber-enlargement and axis-deviation groups.