An affinity matrix for the purification of poly(ADP-ribose) glycohydrolase.

The preparation of quantities of poly(ADP-ribose) glycohydrolase sufficient for detailed structural and enzymatic characterizations has been difficult due to the very low tissue content of the enzyme and its lability in late stages of purification. To date, the only purification of this enzyme to apparent homogeneity has involved a procedure requiring 6 column chromatographic steps. Described here is the preparation of an affinity matrix which consists of ADP-ribose polymers bound to dihydroxyboronyl sepharose. An application is described for the purification of poly(ADP-ribose) glycohydrolase from calf thymus in which a single rapid affinity step was used to replace 3 column chromatographic steps yielding enzyme of greater than 90% purity with a 3 fold increase in yield. This matrix should also prove useful for other studies of ADP-ribose polymer metabolism and related clinical conditions.     

Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.     

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.     

Mechanism involved in the mobilization of neutrophil calcium by 5-hydroxyeicosatetraenoate

5-Hydroxyeicosatetraenoate (5-HETE), like leukotriene B4 and platelet-activating factor, stimulated human polymorphonuclear neutrophils to mobilize intracellular calcium. The three compounds acted through mechanisms that were inhibited by pertussis toxin, cholera toxin, and PMA. Each agonist, furthermore, desensitized (or down-regulated) the neutrophil's calcium mobilization response to a second challenge with the same agonist. However, 5-HETE and leukotriene B4 had little or no activity in cross-desensitizing neutrophil responses to each other or to platelet-activating factor. Furthermore, 5-HETE interfered minimally or not at all with the binding of radiolabeled leukotriene B4 and platelet-activating factor to their respective receptors on neutrophils. Thus, 5-HETE mobilizes neutrophil calcium by a mechanism different from those used by leukotriene B4 and platelet-activating factor. This mechanism appears to involve specific 5-HETE receptors that couple to pertussis toxin-inhibitable, GTP-binding proteins.     

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.     

Interaction of opioid peptides and other drugs with multiple kappa receptors in rat and human brain. Evidence for species differences.

Previous experiments resolved four kappa binding sites in guinea pig brain termed kappa 1a, kappa 1b, and kappa 2b. The present study was undertaken to examine the occurrence of kappa receptor subtypes in rat and human brain. [3H]U69,593 and [3H]bremazocine were used to label kappa 1 and kappa 2 binding sites, respectively, present in brain membranes depleted of mu and delta binding sites by pretreatment with the irreversible ligands, BIT and FIT. Low levels of [3H]U69,593 binding precluded a detailed quantitative study of kappa 1 binding sites in these species. Quantitative examination of [3H]bremazocine binding resolved two kappa 2 binding sites in both rat and human brain whose ligand selectivity patterns differed from that of the guinea pig. These observations suggest that there may be considerable variation in the ligand recognition site of kappa receptor subtypes among mammalian species.     

Transthyretin Pro55, a variant associated with early-onset,aggressive, diffuse amyloidosis with cardiac and neurologic involvement

Summary Mutations in the protein transthyretin cause amyloidosis involving the heart, peripheral nerves, and other organs. A family from West Virginia developed an unusually aggressive form of widespread transthyretin amyloidosis. Single-strand conformation polymorphism analysis revealed a variant in the transthyretin gene, which was found on sequencing to be a TC transversion at position 2 of codon 55, corresponding to a Leu Pro substitution. The variant sequence was confirmed by restriction analysis and polymerase chain reaction (PCR)-primer introduced restriction analysis.