Crystallization of the complex of actin with gelsolin segment 1.

Crystals of a 1:1 complex between human gelsolin segment 1 and actin have been grown from solutions containing polyethylene glycol 6000. The crystals are orthorhombic, space group P2(1)2(1)2(1); the axes are a = 57.4 A, b = 70.4 A, c = 184.5 A. They are moderately stable to X-rays and diffract to beyond 2.5 A. There is one molecule of complex in the asymmetric unit.     

Evidence for functional homology in the F-actin binding domains of gelsolin and alpha-actinin: implications for the requirements of severing and capping

The F-actin binding domains of gelsolin and alpha-actinin compete for the same site on actin filaments with similar binding affinities. Both contain tandem repeats of approximately 125 amino acids, the first of which is shown to contain the actin-binding site. We have replaced the F-actin binding domain in the NH2-terminal half of gelsolin by that of alpha-actinin. The hybrid severs filaments almost as efficiently as does gelsolin or its NH2-terminal half, but unlike the latter, requires calcium ions. The hybrid binds two actin monomers and caps the barbed ends of filaments in the presence or absence of calcium. The cap produced by the hybrid binds with lower affinity than that of gelsolin and is not stable: It dissociates from filament ends with a half life of approximately 15 min. Although there is no extended sequence homology between these two different F-actin binding domains, our experiments show that they are functionally equivalent and provide new insights into the mechanism of microfilament severing.     

Identification of a region in segment 1 of gelsolin critical for actin binding.

The actin severing and capping protein gelsolin contains three distinct actin binding sites. The smallest actin binding domain of approximately 15,000 Mr was originally obtained by limited proteolysis and it corresponds to the first of six repeating segments contained in the gelsolin sequence. We have expressed this domain (here termed segment 1 or N150 to define its amino acid length) in Escherichia coli, together with a series of smaller mutants truncated at either N- or C-terminal ends, in an attempt to localize residues critical of actin binding. Limited truncation of segment 1 by 11 residues at its N-terminal end has no observable effect on actin binding, but on removal of a further eight residues, actin binding is totally eliminated. Although this loss of actin binding may reflect ablation of critical residues, we cannot rule out the possibility that removal of these residues adversely affects the folding of the polypeptide chain during renaturation. Truncation at the C-terminus of segment 1 has a progressive effect on actin binding. Unlike intact segment 1, which shows no calcium sensitivity of actin binding within the resolution of our assays, a mutant with 19 residues deleted from its C-terminus shows unchanged affinity for actin in the presence of calcium, but approximately 100-fold weaker binding in its absence. Removal of an additional five residues from the C-terminus produces a mutant that binds actin only in calcium. Further limited truncation results in progressively weaker calcium dependent binding and all binding is eliminated when a total of 29 residues has been removed. Although none of the expressed proteins on their own binds calcium, 45Ca is trapped in the complexes, including the complex between actin and segment 1 itself. These results highlight a region close to the C-terminus of segment 1 that is essential for actin binding and demonstrate that calcium plays an important role in the high affinity actin binding by this domain of gelsolin.     

Opiate binding to cerebroside sulfate: a model system for opiate-receptor interaction.

Cerebroside sulfate was shown to bind etorphine and levorphanol with high affinity. The relative potency of narcotic analgesics in preventing the binding of levorphanol to cerebroside sulfate correlated well with their reported analgetic activity. The data indicate similarities between cerebroside sulfate and a purified opiate receptor from mouse brain which has been reported to be a proteolipid. Some preliminary animal data also imply the involvement of CS in opiate action We, therefore, propose that CS may serve as a useful “receptor” model for the study of opiate-receptor interaction $\text{in vitro}$.     

Failure of vasopressin and oxytocin to antagonize acute morphine antinociception or facilitate narcotic tolerance development

The effects of vasopressin and oxytocin on acute morphine antinociception and on tolerance development were examined in mice and rats. The studies failed to demonstrate any alteration of chronic morphine effects using two separate models of tolerance development in the mouse. Adrenalectomy enhanced the antinociceptive, hyperthermic, and cataleptic effects of acute morphine treatment, but vasopressin was without additional effect in either adrenalectomized or sham-control rats. Furthermore, neither vasopressin nor oxytocin pretreatment altered brain concentrations of acutely injected morphine. It is concluded that the role of vasopressin and oxytocin as endogenous mediators of opiate analgesia or tolerance/dependence is minimal at best.     

Opioid properties of beta-lipotropin fragment 60-65, H-Arg-Try-Gly-Gly-Phe-Met-OH.

The pharmacologic activity of the hexapeptide fragment corresponding to the amino acid fragment 60–65 in β-lipotropin, (β-LPH-(60–65)) was studied $\text{in vitro}$ and $\text{in vivo}$. In binding assays on synaptosomal plasma membrane the peptide was found to be equipotent to met-enkephalin, but behaved differently to cations; in contrast to met-enkephalin both Mn⁺² and Na⁺ enhanced the binding of β-LPH-(60–65) to synaptosomal plasma membrane. On both the quinea pig ileum and mouse vas deferens β-LPH-(60–65) inhibited contractions elicited by electrical stimulation and each effect was reversible by naloxone. On the guinea pig ileum β-LPH-(60–65) was equipotent to met-enkephalin and 0.5 as potent as normorphine but on the vas deferens it was 4.6 times more potent than normorphine. The activities of β-LPH-(60–65) appear to be due to the intact compound rather than to its conversion to met-enkephalin, since the peptide extracted from the ileum assay was found to behave identically as β-LPH-(60–65) with high pressure liquid chromatography. When β-LPH-(60–65) was administered centrally to mice and rats, no overt central actions were observed and an antinociceptive effect could not be demonstrated. Nor did β-LPH-(60–65) antagonize morphine action or precipitate the withdrawal syndrome in morphine dependent animals. It is concluded that the good agreement which generally exists between $\text{in vitro}$ and $\text{in vivo}$ assay procedures for opiate-like activity of morphine and its surrogates does not necessarily hold for the endogenous peptides with similar actions.     

Effects of morphine tolerance and dependence on Mg++ dependent ATPase activity of synaptic vesicles.

The effect of morphine on ATPase of synaptic plasma membranes (SPM) and synaptic vesicles isolated from the mouse brain was studied. The activity of synaptic vesicle Mg⁺⁺-dependent ATPase from mice rendered morphine tolerant and dependent by pellet implantation was 40% higher than that from placebo implanted mice. However, the activities of Mg⁺⁺-dependent ATPase and Na⁺, K⁺ activated ATPase of SPM of tolerant and nontolerant mice were not significantly different. The activity of synaptic vesicular Mg⁺⁺-dependet ATPase was dependent on the concentration of Mg⁺⁺ but not of Ca⁺⁺; maximum activity was obtained with 2 mM MgCl₂. On the other hand, Mg⁺⁺-dependent ATPase activity of SPM was dependent on both Mg⁺⁺ and Ca⁺⁺, activity being maximum using 2 mM MgCl₂ and 10^?5 M CaCl₂. It is suggested that this stimulation of ATPase activity may alter synaptic transmission and may thus be involved in some aspects of morphine tolerance and dependence.     

Reactivation of koi herpesvirus infections in common carp Cyprinus carpio

Two co-habitation studies with common carp were conducted to determine whether latent infections of koi herpesvirus (KHV) exist. Fish were exposed to KHV using 2 different temperature profiles, which induced low and high initial mortality. Subsequently, certain groups of fish were co-habited with naive fish while others were not. Koi herpesvirus was reactivated in fish from 3 of the 5 experimental tanks. Reactivation of the virus occurred regardless of the initial mortality associated with the virus or whether fish were co-habited with naive fish. The reactivation of the virus in our experiments occurred several months after the initial exposure to KHV and appeared to be temperature dependent.     

The design and implementation of the immune epitope database and analysis resource

Epitopes are defined as parts of antigens interacting with receptors of the immune system. Knowledge about their intrinsic structure and how they affect the immune response is required to continue development of techniques that detect, monitor, and fight diseases. Their scientific importance is reflected in the vast amount of epitope-related information gathered, ranging from interactions between epitopes and major histocompatibility complex molecules determined by X-ray crystallography to clinical studies analyzing correlates of protection for epitope based vaccines. Our goal is to provide a central resource capable of capturing this information, allowing users to access and connect realms of knowledge that are currently separated and difficult to access. Here, we portray a new initiative, The Immune Epitope Database and Analysis Resource. We describe how we plan to capture, structure, and store this information, what query interfaces we will make available to the public, and what additional predictive and analytical tools we will provide.