Myocardial calcium cycling defect in furazolidone cardiomyopathy.

We have previously demonstrated that in furazolidone-induced congestive heart failure in turkeys the specific Ca(2+)-ATPase activity of myocardial sarcoplasmic reticulum (SR) is 60% increased in compensation for a 50% depression in net Ca(2+)-sequestration activity. This study tested the hypothesis that SR Ca(2+)-uptake and Ca(2+)-ATPase activities were uncoupled in this cardiomyopathy because of increased Ca(2+)-release channel activity. A novel microassay was used to monitor Ca2+ transport by myocardial homogenates using the fluorescent Ca2+ dye indo 1 to indicate extravesicular ionized Ca2+. The method is applied to cyropreserved biopsy specimens of myocardium and requires only 50 mg tissue. Both SR Ca(2+)-pump and SR Ca(2+)-channel activity were estimated using the channel-inhibitor ruthenium red (RR) and the mitochondrial inhibitor sodium azide. The specificity of the RR inhibition was confirmed using ryanodine. Cardiomyopathy was induced in 2-week-old turkey poults by the addition of 0.07% furazolidone to their feed for 4 weeks. Compared with controls, myocardial maximal Ca(2+)-channel activity relative to maximal Ca(2+)-pump activity was 22% greater and duration of Ca(2+)-channel activity was 100% increased. However, the heart failure birds had 43 and 53% decreases in absolute maximal Ca(2+)-pumping and Ca(2+)-channel activities, respectively. The abnormal Ca(2+)-channel activity resulted in 200% greater time before initiation of net Ca2+ sequestration and 700% greater final myocardial Ca2+ concentrations. For all birds, the Ca(2+)-accumulating activity was highly correlated with Ca(2+)-release activity (all p less than 0.05). These data indicate that in this animal model of congestive heart failure there is defective SR Ca(2+)-channel function resulting in abnormal Ca2+ homeostasis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the patterns of inheritance of splenomegaly and serum IgM levels in the Watut of Papua New Guinea

Hyperreactive malarious splenomegaly (HMS) reflects abnormal immune responses to malarial infection. The central question is whether HMS results from unusual patterns of malarial infection or from immune incompetence in the host. Family distributions of two features of the syndrome, splenomegaly and excessively high IgM levels, have been examined in a Papau New Guinea population in which HMS is exceptionally common. Segregation analysis of spleen grade shows that a major sex-linked gene controls hyperresponsiveness to malaria. This finding is supported by additional segregation analysis, which shows that an autosomal locus cannot account for a significant proportion of variation in spleen grade, and by path analysis, which rejects a model that assumes that parents contribute equally to the child's genotype. The sex-linked gene contributing to HMS was not mediated through sex linkage of a major gene for IgM concentrations, as shown by segregation analysis. It has yet to be determined whether this pattern of inheritance also applies to HMS occurring sporadically in other less severely affected populations. The applicability of these findings to the general variability in "normal" IgM responses to malaria also remains to be established.     

Mechanisms of food selection in Daphnia

A conceptual behavioural and mechanistic Holling-type model of food selection in Daphnia pulicaria is derived from SEM observations with animals feeding on mixtures of spherical-cylindrical diatoms, oblongate green algae, and filamentous cyanobacteria, as well as ultrafine particles. The algae used were Stephanodiscus hantzschii (<- 6 µm length), Monoraphidium setiforme ( 20 µm), and Oscillatoria aghardii (strands, >- 80 µm). Cell (strand) selection can occur at any or all of three stages: (i) interception from the feeding currents, (ii) collection and channeling to the food groove, and (iii) compaction and transport to the mouth. During each stage, given equal initial cell densities, elongate cells are more likely to escape collection than spherical cells and are more likely to be rejected. In addition, filaments require increased handling time at stages (ii) and (iii) and promote entanglement with limb 5 and the postabdominal claw. Food is collected primarily with the aid of limbs 3 (and 4), but limbs 1 and 2 also intervene. Neither the leaky sieve hypothesis alone nor any other single-process hypothesis explains the observations on examined in corpore positions, morphology, and derived movements of the feeding limbs. Attachment and mucus appear to be important for the ingestion of bacteria and ultrafine particles.The model is consistent with many experimental results of differential feeding by Daphnia pulicaria on mixtures of variously shaped algae and other observations on Daphnia feeding behaviour. The paradigm of invariate, nonselective feeding by Daphnia is rejected.     

Cerebral Metabolic Effects of Monoamine Oxidase Inhibition in Normal and 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Acutely Treated Monkeys

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces dopaminergic cell death in the substantia nigra pars compacta (SNpc) and clinical parkinsonism in humans and experimental animals. Pretreatment with monoamine oxidase inhibitors prevents this cell death and associated parkinsonism by blocking the oxidation of MPTP to a toxic intermediate. The 2-deoxyglucose method was used to study the acute effects of MPTP in the monkey brain and the effects of monoamine oxidase inhibition on local cerebral glucose utilization in both normal and MPTP-treated monkeys. MPTP administration alone caused a major increase in glucose utilization in the SNpc and smaller increases in some subnuclei within the ventral tegmental area in which eventual dopaminergic cell loss also occurs. Pretreatment with pargyline abolished these metabolic increases, a finding suggesting both that the oxidized product of MPTP generates the metabolic increases and that the increased glucose consumption may contribute to cell toxicity. On the other hand, in most cortical, thalamic, striatal, brainstem, and cerebellar areas MPTP alone caused reductions in glucose utilization, and pargyline failed to prevent these effects. Pargyline alone depressed metabolism in the locus coeruleus and a few other monoaminergic structures.     

Cell Damage Associated with Changing the Medium of Mesencephalic Cultures in Serum-Free Medium Is Mediated via N-Methyl-D-Aspartate Receptors

Dopaminergic neurons from embryonic rat mesencephalon were grown in simple serum-free media. The cells develop over a period of several weeks in vitro, particularly between day 14 and day 23. Removing the culture medium and replacing it with fresh medium during this interval caused severe damage to the cultures; this damage is mediated by excitatory amino acids acting through glutamate receptors. Damage could be completely prevented by antagonists of the N-methyl-D-aspartate subtype of glutamate receptor. As expected, medium that contains glutamate (i.e., Ham's F-12 medium) caused damage; however, medium that contains no glutamate or aspartate (i.e., Dulbecco's modified Eagle medium) also caused severe damage, and most of the damage was dependent on the presence of glutamine in the medium. The presence of the antibiotics penicillin and streptomycin greatly enhanced damage caused by medium change.     

Identification of cell-surface heparin/heparan sulfate-binding proteins of a human uterine epithelial cell line (RL95).

The interaction of heparin (HP) with the cell-surface components of a human uterine epithelial carcinoma cell line (RL95) was studied. Binding of [3H]HP to cell surfaces was saturable in a dose- and time-dependent manner. HP and certain forms of heparan sulfate (HS) efficiently compete for [3H]HP binding. In contrast, other glycosaminoglycans, such as chondroitin sulfate, keratan sulfate, hyaluronic acid, and dermatan sulfate, do not compete for binding to these sites. Scatchard analysis revealed that [3H]HP bound to these sites with an apparent KD of 0.7-0.9 microM and a binding capacity of 9 x 10(6) sites/cell to attached cells. EDTA-detached cells displayed a similar apparent KD, but an approximately 2-fold increase in binding capacity. Protease digestion of cells on ice markedly reduced [3H]HP binding, indicating that these binding sites were associated with proteins. In contrast, heparinase treatment of cells stimulated binding by approximately 2-fold, indicating that a large fraction of these binding sites were occupied with endogenous ligand. We examined the structural features of HP/HS required for HP/HS binding. O-Sulfation, substitution of amino groups, and, to a lesser extent, the presence of carboxyl groups were important recognition features of HP/HS by cell-surface HP/HS-binding sites. N-Sulfation was not required. Photoaffinity labeling with 125I-sulfosuccinimidyl 2-(p-azidosalicylamido)-ethyl-1, 3-dithiopropionate-HP was used to identify HP/HS-binding proteins on RL95 cell surfaces. Proteins with M(r) values of 14,000-18,500 and 31,000 were photolabeled at the surfaces of attached cells. Photolabeling was blocked by the addition of excess HP, but not chondroitin sulfate. Additional proteins with M(r) values greater than 31,000 were photolabeled specifically on EDTA-detached cells. Moreover, the M(r) 14,000-18,500 and 31,000 proteins were retained on the EDTA-detached cells. These observations indicated that certain cell-surface HP/HS-binding proteins were not exposed when cells were attached to substrata. Proteins of similar M(r) values as the photolabeled components as well as many additional proteins were identified by heparin-agarose chromatographic selection of extracts of cells labeled metabolically with [35S]methionine or vectorially with Na125I at the cell surface. Fragments of cell-surface HP/HS-binding proteins were released from intact RL95 and mouse uterine epithelial cells by mild trypsinization and isolated by heparin-agarose affinity chromatography. Three peptides with M(r) values between 6000 and 14,000 required greater than 0.5 M salt for elution from heparin-agarose, retained HP binding activity in a 125I-HP gel overlay assay, and selectively bound [3H]HP in a solid-phase binding assay.(ABSTRACT TRUNCATED AT 400 WORDS)     

Right-side dominance for song control in the zebra finch.

Adult male zebra finches underwent unilateral denervation of the syrinx or unilateral lesion of the forebrain nucleus HVC known to be important for song control. Disruptive effects on song were greater after right-side than after left-side operations. After denervation of the right half of the syrinx, the fundamental frequencies of all syllables within a song converged on a value near 500 Hz, and nearly all syllables were altered in type. In contrast, the syllables produced after denervation of the left side of the syrinx largely maintained their preoperative frequencies, and fewer syllables changed in type. Unlike nerve sections, HVC lesions did not result in strikingly lateralized effects on syllable phonology; however, HVC lesions did affect the temporal patterning of a bird's song, whereas nerve sections did not, and changes in temporal patterning were more marked after right than after left HVC lesions. Right-side dominance for zebra finch song control is the reverse of that described in other songbird species with lateral asymmetry for vocal communication. We suggest that the need for a dominant side is more important than the side of dominance.     

Construction of a sequencedClostridium perfringens-Escherichia coli shuttle plasmid

A newClostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from theC. perfringens replicon pIP404 and theE. coli vector pUC18. The multiple cloning site and lacZ gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants inE. coli. Both chloramphenicol and erythromycin resistance can be selected inC. perfringens andE. coli since pJIR418 carries theC. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes fromC. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.     

Inhibition of transplasma membrane electron transport by monoclonal antibodies to the transferrin receptor

Reduction of iron in diferric transferrin is inhibited by monoclonal antibodies to the transferrin receptor which bind at sites other than the high affinity transferrin binding site. These antibodies include B3/25, GB16 and GB22. Two antibodies which bind at the high affinity site for transferrin, 42/6 and GB18, do not inhibit iron reduction by transplasma membrane electron transport. The results are consistent with the proposal that differric transferrin reduction or stimulation of transmembrane NADH oxidase activity involves a site different from the high affinity diferric transferrin binding site. A synergistic action of antibodies with epitopes at the tight binding site involved in iron uptake and the antibodies which inhibit electron transport, B3/25 and GB16, can explain the increased inhibition of growth observed when both 42/6 and B3/25 are added to proliferating cells.     

Identification and characterization of a new family of high-affinity receptors for Escherichia coli heat-stable enterotoxin in rat intestinal membranes.

Novel high-affinity, low-capacity binding sites in intestinal membranes for the heat-stable toxin produced by Escherichia coli have been defined. The appearance of these sites is observed in the presence of physiological concentrations of NaCl in binding reactions. Scatchard analyses of equilibrium binding in the absence of NaCl demonstrated a single class of binding sites with KD = 1.9 x 10(-9) M and Bmax = 0.75 pmol/mg of protein. In contrast, similar experiments in the presence of NaCl demonstrated, in addition to the previously described low-affinity site, a high-affinity site with a KD of 2.1 x 10(-11) M and a Bmax of 73 fmol/mg of protein. Confirmation of the presence of high- and low-affinity sites was obtained in studies of the kinetics of ST binding. These sites exhibited similar dissociation but markedly different association kinetics. Determination of the association and dissociation constants permitted calculation of the KD's for the high- and low-affinity sites, which were 1.15 x 10(-11) M and 1.89 x 10(-9) M, respectively. These data agree closely with those obtained in studies of equilibrium binding. Furthermore, similar values for the KD's of these sites were obtained in experiments of competitive displacement of labeled ST, confirming the presence of two receptors for this toxin. Binding of ST to high-affinity sites is completely reversible and does not appear to be coupled to activation of particulate guanylate cyclase. In contrast, binding of ST to low-affinity sites appears to be partially reversible and may be coupled to activation of guanylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)