The reactive serine residue of epidermolytic toxin A.

Comparison of amino acid sequence data suggested that there may be a functional relationship between the staphylococcal epidermolytic toxins and V8 proteinase. The hypothesis was tested by treating epidermolytic toxin with di-isopropyl phosphorofluoridate, which bound specifically at serine-195, the homologue of the active-site serine residue of V8 proteinase.     

Complete development of Caryospora bigenetica (Apicomplexa: Eimeriidae) in vitro

The development of Caryospora bigenetica in vitro is described by light microscopy. Sporozoites from snake-derived oocysts were purified and inoculated onto cultures of primary testicle cells of the cotton rat, cotton rat kidney cells, and human fetal lung cells. Intracellular sporozoites were observed one and two days postinoculation (DPI). Motile, extracellular first-generation merozoites were present 3 DPI, and second-generation merozoites were present 5 DPI. Mature gamonts were observed 9 DPI and developed into unsporulated oocysts by 10 DPI. Oocystes sporulated in vitro, and excystation was observed. Cells that were penetrated by in vitro-produced sporozoites formed caryocysts by 16 DPI. To test infectivity of in vitro-derived stages, merozoites were removed from cultured cells 5 DPI and inoculated intraperitoneally into a mouse; infection resulted. Sporulated oocysts removed from cell cultures 12 DPI produced facial swelling in an orally inoculated cotton rat.     

Point mutations in the 23 S rRNA genes of four lincomycin resistant Nicotiana plumbaginifolia mutants could provide new selectable markers for chloroplast transformation

Summary Experiments designed to establish stable chloroplast transformation require selectable marker genes encoded by the chloroplast genome. The antibiotic lincomycin is a specific inhibitor of chloroplast ribosomal activity and is known to bind to the large ribosomal subunit. We have investigated a defined region of the chloroplast 23 S rRNA genes from four lincomycin resistant Nicotiana plumbaginifolia mutants and from wild-type N. plumbaginifolia. The mutants LR415, LR421 and LR446 have A to G transitions at positions equivalent to the nucleotides 2058 and 2059 in the Escherichia coli 23 S rRNA. The mutant, LR400, possesses a G to A transition at a position corresponding to nucleotide 2032 of the E. coli 23 S rRNA.     

Inhibition by corticosteroids of epidermal growth factor-induced recovery of cyclooxygenase after aspirin inactivation

Cultures of vascular smooth muscle cells superfused with [14C]arachidonic acid synthesized the antiplatelet substance prostacyclin as the major cyclooxygenase product. Prostacyclin synthesis was inactivated by aspirin, which irreversibly acetylates cyclooxygenase. Aspirin-treated cells recovered within 2 h by a process that was blocked by cycloheximide but not by actinomycin D, and that required a serum component identified as epidermal growth factor (EGF). EGF-induced recovery of cyclooxygenase was greatly potentiated by type beta transforming growth factor (TGF-beta). Incubation with EGF and TGF-beta in the 0.1-1.0 nanomolar range stimulated cyclooxygenase recovery up to 20-fold without increasing [35S]methionine incorporation into other cell proteins. Induction of cyclooxygenase by EGF and TGF-beta also was prevented by cycloheximide but not by actinomycin D. EGF-dependent recovery was blocked by preincubation with dexamethasone (2 microM), an effect that was duplicated by pure lipocortin (2-4 micrograms/ml). Incubation of membrane preparations from these cells with EGF selectively activated phosphorylation of a 35-kDa cellular protein that comigrated with lipocortin. The results suggest that cyclooxygenase recovery in aspirin-inactivated vascular smooth muscle cells is mediated by an EGF-dependent translational control that is inhibited by corticosteroids. The findings also provide a new mechanism whereby corticosteroids suppress inflammatory prostaglandins.     

POMC-derived peptides in the neuromuscular system of streptozotocin-diabetic mice.

Immunoreactivity for beta-endorphin and alpha-MSH/ACTH was demonstrated in intramuscular nerves in soleus, extensor digitorum longus, and diaphragm muscles of normal and streptozotocin-diabetic mice. There was a higher incidence of immunoreactive nerves in the muscles of the diabetic mice. Specific binding for [125I]ACTH was detected in a proportion of the muscle fibers, using autoradiography. There were significantly more fibers with specific [125I]beta-endorphin sites and specific [125]ACTH sites in some muscles in the diabetic mice. The increased expression of POMC-derived peptides and their receptors in the neuromuscular system of streptozotocin-diabetic mice may indicate early neuropathic change.     

Transport regulation of recombinant gene expression in E. coli and B. subtilis

Expression kinetics of the lactose (lac) operon in Escherichia coli are reviewed for both wild-type and recombinant cell cultures under chemostatic conditions. A unified model which involves regulation of active inducer (lactose) transport, promoter-operator regulated expression of the lac operon, glucose-mediated inducer exclusion, and catabolite repression is summarized and supporting data is shown to verify its accuracy. The synthesis of alpha-amylase with a recombinant form of Bacillus subtilis is also reviewed to point out generic features in transport regulation, the lac operon model providing a point of departure. While there are many similarities in the influence of transport on both regulating models, there are also important differences. In a chemostat system, the synthesis of alpha-amylase is nongrowth associated, while beta-galactosidase is a growth-associated enzyme. Nevertheless, transport regulation is an important feature in both instances.     

Inhibitors of protein and RNA synthesis block structural changes that accompany long-term heterosynaptic plasticity in Aplysia.

Synaptic connections between the sensory and motor neurons of Aplysia in culture undergo long-term facilitation in response to serotonin (5-HT) and long-term depression in response to FMRFamide. These long-term functional changes are dependent on the synthesis of macromolecules during the period in which the transmitter is applied and are accompanied by structural changes. There is an increase and a decrease, respectively, in the number of sensory neuron varicosities in response to 5-HT and FMRFamide. To determine whether macromolecular synthesis is also required for the structural changes, we examined in parallel the effects of inhibitors of protein (anisomycin) or RNA (actinomycin D) synthesis on the structural and functional changes. We have found that anisomycin and actinomycin D block both the enduring alterations in varicosity number and the long-lasting changes in synaptic potential. These results indicate that macromolecular synthesis is required for expression of the long-lasting structural changes in the sensory cells and that this synthesis is correlated with the long-term functional modulation of sensorimotor synapses.     

Substance P, neurokinin A and calcitonin gene-related peptide during development of the rat gastrointestinal tract.

Substance P, neurokinin A and calcitonin gene-related peptide (CGRP) were determined in the stomach and small intestine of rats during late foetal development and up to 35 days postnatal life. Concentrations of substance P in stomach and intestine increased from 14 gestational days to 3 days postpartum, and declined thereafter. Concentrations of neurokinin A in stomach declined from 14 days gestation over the period 3-35 postnatal days. In the intestine, concentrations of neurokinin A increased steadily from 14 days gestation to 21-35 postnatal days. Concentrations of CGRP in stomach and intestine declined from 14 days gestation to 7 postnatal days. Thereafter, concentrations of CGRP increased in both stomach and intestine. Total contents of each of the three peptides increased progressively with gestational and postnatal age in parallel with increasing stomach and intestinal weights. The results demonstrate different patterns of change in the concentrations of substance P, neurokinin A and CGRP during the dynamic phases of growth and maturation of the gastrointestinal tract in the foetal and postnatal rat.