Determination of myoglobin concentration and oxidative capacity in cryostat sections of human and rat skeletal muscle fibres and rat cardiomyocytes

Myoglobin plays various roles in oxygen supply to muscle mitochondria. It is difficult, and in some cases impossible, to study the relationship between the myoglobin concentration and the oxidative capacity of individual muscle cells because myoglobin has to be fixed in situ whereas determination of oxidative capacity, for example, succinate dehydrogenase activity, requires unfixed cryostat sections. We have investigated whether a vapour-fixation technique allows the use of serial sections to study the relationship between myoglobin and succinate dehydrogenase activity. The technique is used to study a rat soleus muscle, two human skeletal muscle biopsies and biopsies of two patients with chronic heart failure, and in a control and hypertrophied rat heart. Staining intensities were quantified by microdensitometry. The absorbance values were calibrated using sections cut from gelatine blocks containing known amounts of myoglobin. The results show that it is possible to use serial sections for the determination of the myoglobin concentration and succinate dehydrogenase activity, and indicate that myoglobin can lead to a substantial reduction (18–60%) of the extracellular oxygen tension required to prevent an anoxic core in muscle cells.     

Developmental changes in rat brain monoamine metabolism and β-adrenoceptor subtypes after chronic prenatal exposure to propranolol

The aim of this study was to investigate whether a chronic prenatal β-blockade can alter the maturation of the noradrenergic system in the rat brain. Pregnant female and adult male rats were treated for 10 days with the β-antagonist propranolol dissolved in the drinking water (40–50 mg/kg/day). Direct and long-term effects on β-adrenoceptors and monoamine metabolism in various rat brain regions were determined.

After the prenatal treatment the propranolol level in the foetal brain was 0.9 μg/g, while in the adult brain 2.0 μg/g was present. The foetal β1-receptors were significantly up-regulated by propranolol (200%), whereas the β2-receptor number remained unaltered. On postnatal days 4 and 21 the number of both β-subtypes was the same as that of controls. Noradrenaline, its metabolite 3-methoxy-4-hydroxyphenylglycol and their ratio were unaltered directly after the prenatal treatment. In the PN 21 offspring, however, the metabolite level had increased in the frontal cortex (+17%) and hippocampus (+32%), and the ratio in the hippocampus (37%) and medulla pons (+34%). Prenatal treatment also induced a significant increase of the 5-hydroxyindolacetic acid·5-hydroxytryptamine ratio (+15%) in the medulla pons at GD 21. No direct or lasting effects were found on dopamine metabolism. Propranolol treatment of adult rats gave no direct changes in monoamine metabolism.

We concluded that chronic prenatal propranolol exposure (a) reversibly up-regulates foetal β1-adrenoceptors, and (b) increases the NA activity in the brain in later life.     

Selection, characterization and regeneration of hydroxyproline-resistant cell lines of Solanum tuberosum: Tolerance to NaCl and freezing stress

Sixty-seven hydroxyproline-resistant (hyp^r) cell lines were selected from cell suspensions of a diploid potato ( Solanum tuberosum L., clone H²578) after plating on 5 and 10 m M hydroxyproline (hyp). Resistant colonies were obtained with a spontaneous frequency of 2.9×10⁻⁶. No clear influence could be shown from treatment with N-ethyl-N-nitrosourea (10 or 50 μ M ). Ninety % of the variant lines contained more proline than the wild type when cells were grown away from hyp for 1 month. Total free amino acid content was increased 2.2 to 6.8 times. When the lines were grown for another 2–5 months on non-selective medium, the content of proline and other amino acids and hyp resistance decreased. After this period the values were, however, still substantially higher than in the wild type. When tested for growth on media with other amino acid analogues (azetidine-2-carboxylic acid and dehydroproline, analogues of proline; aminoethyl-cysteine, analogue of lysine and 3-fluorotyrosine, analogue of tyrosine) and on media with inhibitory concentrations of lysine + threonine. lines H4a and H4b4 were cross resistant to these compounds. When tested on media with inhibitory NaCl concentrations, variant lines H2a, H4a, and H6 showed better tolerance than the wild type. One variant cell line (H4a) was successfully regenerated into plants. Preliminary results showed an increased frost tolerance in the leaves of these plants (−4.5°C compared to −3°C for the wild type), accompanied by a higher leaf proline content. Callus initiated from leaves of the regenerated clones was more resistant to hyp than wild type callus, indicating that the variant trait might be due to a mutation.     

Isolation and characterization of nitrate reductase-deficient mutants of Arabidopsis thaliana

Summary Chlorate resistant mutants of Arabidopsis thaliana were isolated, of which 10 exhibited a lowered nitrate reductase activity and 51 were chlorate-resistant because of an impaired uptake of chlorate. The 51 mutants of this type are all affected in the same gene. The mutants with a lowered nitrate reductase activity fall into 7 different complementation groups. Three of these mutants grow poorly on media with nitrate as the sole nitrogen source, while the others apparently can reduce sufficient nitrate to bring about growth. In all cases a low nitrate reductase activity coincides with an enhanced nitrite reductase activity. After sucrose gradient centrifugation of wildtype extracts nitrate reductase is found at the 8S position, whereas cytochrome-c reductase is found both at 4 and 8S positions. It is suggested that the functional nitrate reductase is a complex consisting of 4S subunits showing cytochrome-c reductase activity and a Mo-bearing cofactor. All mutants except B25 are capable of assembling the 4S subunits into complexes which for most mutants have a lower S value and exhibit a lower nitrate reductase activity than the wildtype complexes. Since the mutants B25 and B73 exhibit a low xanthine dehydrogenase activity, the Mo-bearing cofactor is probably less available in these mutants than in the wildtype. B73 appears to be the only mutant which is partly repaired by excessive Mo. The possible role of several genes is discussed.     

Deletions in herpes simplex virus glycoprotein D define nonessential and essential domains.

Herpes simplex virus glycoprotein D (gD) is a major component of the virion envelope and infected cell membranes and is essential for virus entry into cells. We have recently shown that gD interacts with a limited number of cell surface receptors which are required for virus penetration into cells. To define domains of gD which are required for aspects of virus replication including receptor binding, deletion mutations of 5 to 14 amino acids were constructed by using oligonucleotide-directed mutagenesis. Plasmids containing mutant genes for gD were assayed for the ability to rescue a recombinant virus, F-gD beta, in which beta-galactosidase sequences replace gD-coding sequences. Effects on global folding and posttranslational processing of the molecules were assessed by using a panel of monoclonal antibodies which recognize both continuous and discontinuous epitopes. A region near the amino terminus (residues 7 to 21) of gD which is recognized by monoclonal antibodies able to neutralize herpes simplex virus in the absence of complement was not essential for function. In addition, virtually all of the cytoplasmic domain of gD and an extracellular domain close to the membrane were dispensable. In contrast, deletion mutations in the central region of the molecule, save for one exception, led to alterations in global folding of the molecule and maturation of the protein was inhibited.     

Potent dihydroquinolinone dopamine D2 partial agonist/serotonin reuptake inhibitors for the treatment of schizophrenia

A dihydroquinolinone moiety was found to be a potent serotonin reuptake inhibitor pharmacophore when combined with certain amines. This fragment was coupled with selected D₂ ligands to prepare a series of dual acting compounds with attractive in vitro profiles as dopamine D₂ partial agonists and serotonin reuptake inhibitors. Structure–activity studies revealed that the linker plays a key role in contributing to D₂ affinity, function, and SRI activity.     

Does the presence of ant nests matter for oviposition to a specialized myrmecophilous Maculinea butterfly?

More than 50% of the lycaenid butterflies have an ant-associated lifestyle (myrmecophily) which may vary from coexistence to specific mutualistic or even parasitic interactions. Ant-related host-plant selection and oviposition has been observed in some myrmecophilous lycaenids. Therefore, it is remarkable that there is no evidence for this behaviour in the highly specialized, obligate myrmecophilous butterflies of the genus Maculinea. In contrast with previous findings, our results provide evidence for ant-related oviposition patterns in Maculinea alcon in relation to the distribution of specific host-ant nests (i.e. Myrmica ruginodis) based on repeated egg counts during the flight period in two populations. We also show that ant-related oviposition can be counterbalanced by intraspecific competition and oviposition deterrency when host plants already carry several eggs. Therefore, the absence of a correlation between egg load and the presence of host-ant nests at the end of the flight period should be interpreted carefully Whether ovipositional cues are obtained either directly (from ants or their nests) or indirectly (from vegetation structure), and whether alternative explanations based on the phenology and growth form of host plants are possible, is discussed.     

PACAP mediates the neural proliferative pathway of Mastomys enterochromaffin-like cell transformation.

BACKGROUND AND AIM: Pituitary adenylate-cyclase activating peptide (PACAP) is a more potent proliferative agent than gastrin for rat enterochromaffin-like (ECL) cell proliferation in vitro. The role of this neurotransmitter during gastrin-mediated ECL cell tumor formation and gastrin-autonomous ECL cell neoplasia is unknown. METHODS AND RESULTS: ECL cell transformation was induced in the Mastomys using 16 wk H2 receptor blockade of acid inhibition. Examination of the epithelial fundic mucosa demonstrated that PACAP-immunoreactivity significantly increased in the tumor mucosa compared to the na?ve stomach, and was associated with ECL cells. Na?ve and tumor ECL cells were then purified (approximately 95%) from Mastomys and the presence of all three PACAP/VPAC receptor subtypes was demonstrated by polymerase chain-reaction amplification. Thereafter, cells were maintained in short-term (48 h) primary cultures. PACAP significantly (p<0.05) increased 24 h bromo-deoxyuridine uptake (approximately 4-fold) in both cell types with estimated EC(50) values of approximately 4x10(-16) M and approximately 2x10(-16) M, respectively. Specific receptor antagonists (PAC1/VPAC1) of PACAP competitively inhibited these proliferative effects in na?ve cells. Oligonucleotide antisense directed against PAC1 significantly inhibited PACAP-stimulated DNA synthesis by approximately 85% (p<0.05) in tumor cells. CONCLUSION: PACAP is a potent and effective modulator of ECL cell proliferation. The expression of this neuropeptide and its receptors, particularly PAC1, suggest the existence of a neural regulatory pathway of ECL cell proliferation and transformation.     

Effect of homoeopathic medicines on daily burden of symptoms in children with recurrent upper respiratory tract infections.

OBJECTIVE--To investigate the intrinsic effects of individually prescribed homoeopathic medicines. DESIGN--Randomised double blind placebo controlled study. SETTING--Paediatric outpatient department of university hospital. PATIENTS--175 children with frequently recurring upper respiratory tract infections. Of the 170 children evaluable, 86 were randomised to homoeopathic medicines (47 boys, 39 girls; median age at start 4.2 years; median number of episodes in past year 4) and 84 to placebo (43 boys, 41 girls; median age at start 3.6 years; median number of episodes in past year 4). MAIN OUTCOME MEASURES--Mean score for daily symptoms, number of antibiotic courses, and number of adenoidectomies and tonsillectomies over one year of follow up. RESULTS--The mean daily symptom score was 2.61 in the placebo group and 2.21 in the treatment group (difference 0.41; 95% confidence interval -0.02 to 0.83). In both groups the use of antibiotics was greatly reduced compared with that in the year before entering the trial (from 73 to 33 in the treatment group and from 69 to 43 in the placebo group). The proportion of children in the treatment group having adenoidectomies was lower in the treatment group (16%, 8/50) than in the placebo group (21%, 9/42). The proportion having tonsillectomies was the same in both groups (5%). CONCLUSION--Individually prescribed homoeopathic medicines seem to add little to careful counselling of children with recurrent upper respiratory tract infection in reducing the daily burden of symptoms, use of antibiotics, and need for adenoidectomy and tonsillectomy.