Mechanism of action of GTP in the induction of Ca2+ release from hepatic microsomes

The mechanism by which GTP induces Ca2+ release from Ca2(+)-preloaded rat hepatic microsomes was studied. In the same concentration range as that for Ca2+ release, GTP inhibited the initial rate of ATP-driven Ca2+ uptake. It also inhibited the formation by ATP of the phosphorylated intermediate of Ca2(+)-ATPase, which had previously been identified by us as a 97-116 kDa protein (Fleschner, C.R., et al. (1985) Biochem. J. 226, 839). Vanadate, an inhibitor of Ca2(+)-ATPase, also caused Ca2+ release in a similar fashion, but its effect was not additive to that of GTP. Although the non-metabolizable GTP analogues, GMPPNP and GTP gamma S, did not cause Ca2+ release by themselves, GTP gamma S completely and GMPPNP partially blocked the effect of GTP. Pretreatment of vesicles with either cholera or pertussis toxin did not alter the responsiveness to GTP. These results indicate that GTP inhibits microsomal Ca2(+)-ATPase, independently of the Gs and Gi proteins. Because a decrease in Ca2+ uptake results in a net increase in Ca+ release, this effect of GTP seems to account, at least partially, for the GTP-induced Ca2+ release from microsomes.     

Analysis of metal-induced DNA lesions and DNA-repair replication in mammalian cells

The potency of several metal compounds in causing lesions in DNA either directly or by exposure of intact cultured cells has been examined using the neutral conditions of nucleoid gradient sedimentation. HgCl2 was clearly the most potent inducer of single-strand breakage when added to isolated nucleoids or when nucleoids were prepared from cells treated with this compound. CaCrO4 , however, caused DNA-strand breaks in nucleoids isolated from cells treated with this agent but did not induce DNA strand breaks when added directly to nucleoids. Although less potent than HgCl2, NiCl2 also caused significant single strand breakage in isolated nucleoids or in nucleoids prepared from cells treated with this metal. Since strand breakage of DNA in intact cells may occur secondary to activation of DNA-dependent nucleases during repair replication, CsCl gradient density sedimentation was utilized to examine whether repair processes were induced by exposure of cells to NiCl2, HgCl2 and CaCrO4 . CaCrO4 and NiCl2 induced substantial DNA-repair activity at concentrations and exposure times where DNA lesions could not be detected whereas HgCl2 induced a 10-fold lower level of DNA-repair activity compared to CaCrO4 at optimal concentrations which again were below the concentrations of this metal that produced measurable DNA lesions. Both the induction of DNA-repair activity and DNA-strand breakage by these metals was concentration- and time-dependent. These results demonstrate some unique aspects of the interaction of HgCl2, NiCl2 and CaCrO4 with the DNA of intact cells and point to the possible important correlation of induction of DNA repair to carcinogenesis since nickel and chromate have clearly been implicated as carcinogens and induce considerable repair whereas HgCl2 is not considered a carcinogen and induces the least DNA repair despite its potency in producing DNA lesions.     

Behavioral syndromes and the evolution of correlated behavior in zebrafish

Studies of "behavioral syndromes" in different populations andspecies of animals can be used to shed light on the underlyingmechanisms of evolution. For example, some personality syndromessuggest the existence of an underlying hormonal link, whereasother relationships between boldness and aggression appear tobe the result of similar selective pressures. Here, we used1 wild-derived and 2 laboratory strains of zebrafish (Daniorerio) to examine relationships among 5 behavioral measures:shoaling, activity level, predator approaches, latency to feedafter a disturbance, and biting to a mirror stimulus. We foundevidence of an activity syndrome, as if underlying metaboliccosts influence variation in multiple forms of behavior. Evidencefor a relationship between boldness and aggression was alsoapparent but depended both on strain and which specific behaviorpatterns were identified as measures of "boldness." Althoughsome comparisons of laboratory and wild-derived strains wereconsistent with a domestication syndrome, others were not. Mostobserved relationships were relatively weak and occasionallyinconsistent, arguing against strong underlying genetic linkagesor pleiotropic effects relating any of the behavioral measures.Instead, it may be more important to study the details of selectivecontext or the long-term impact of linkages between some, butnot all, of a large set of genes influencing complex behavioraltraits. We found profound differences among strains in mostbehavior patterns, but few sex differences. One strain (TM1)was consistently different from the others (SH and Nadia) beingmore social, more likely to approach predators, and taking lesstime to recover from disturbance than were the other 2 strains.     

The effects of early and adult social environment on zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) behavior

Because early social experience can have a profound effect on later mate and social choices, the availability of options and decisions made early in development can have major effects on adult behavior. Herein, we use strain differences among zebrafish, Danio rerio, as an experimental tool to test the effects of social experience on behavior. By manipulating the strain composition of groups in which the subject fish are housed at different ages, we tested (1) whether mixing with dissimilar individuals influenced subsequent behavior, (2) whether prolonged mixing during the juvenile stage had a more pronounced effect than a shorter period of mixing during adulthood and (3) whether mixing had a lasting effect after animals were resorted into groups of same strain animals. We found that social experience had a profound impact on social behavior. Both Nadia and TM1 individuals engaged in more frequent biting after having been in mixed strain groups compared to pure strain groups. This was true of groups mixed as juveniles, as well as adults, indicating that this response was not dependent on exposure during a critical developmental period. Also, TM1 fish (but not Nadia) having recently been housed in mixed-strain groups were more willing to leave the immediate vicinity of a shoal than were TM1 fish raised in pure strain groups. This change was more pronounced in groups mixed as juveniles than as adults. In addition, the observed changes persisted after mixed groups were separated into pure strain groups for a month. Other behavioral measures including Activity Level, Predator Response and Stress Recovery were unaffected by previous social experience.     

Multivalent interactions of calcium/calmodulin-dependent protein kinase II with the postsynaptic density proteins NR2B, densin-180, and alpha-actinin-2

Dendritic calcium/calmodulin-dependent protein kinase II (CaMKII) is dynamically targeted to the synapse. We show that CaMKIIalpha is associated with the CaMKII-binding proteins densin-180, the N-methyl-D-aspartate receptor NR2B subunit, and alpha-actinin in postsynaptic density-enriched rat brain fractions. Residues 819-894 within the C-terminal domain of alpha-actinin-2 constitute the minimal CaMKII-binding domain. Similar amounts of Thr286-autophosphorylated CaMKIIalpha holoenzyme [P-T286]CaMKII bind to alpha-actinin-2 as bind to NR2B (residues 1260-1339) or to densin-180 (residues 1247-1495) in glutathione-agarose cosedimentation assays, even though the CaMKII-binding domains share no amino acid sequence similarity. Like NR2B, alpha-actinin-2 binds to representative splice variants of each CaMKII gene (alpha, beta, gamma, and delta), whereas densin-180 binds selectively to CaMKIIalpha. In addition, C-terminal truncated CaMKIIalpha monomers can interact with NR2B and alpha-actinin-2, but not with densin-180. Soluble alpha-actinin-2 does not compete for [P-T286]CaMKII binding to immobilized densin-180 or NR2B. However, soluble densin-180, but not soluble NR2B, increases CaMKII binding to immobilized alpha-actinin-2 by approximately 10-fold in a PDZ domain-dependent manner. A His6-tagged NR2B fragment associates with GST-densin or GST-actinin but only in the presence of [P-T286]CaMKII. Similarly, His6-tagged densin-180 or alpha-actinin fragments associate with GST-NR2B in a [P-T286]CaMKII-dependent manner. In addition, GST-NR2B and His6-tagged alpha-actinin can bind simultaneously to monomeric CaMKII subunits. In combination, these data support a model in which [P-T286]CaMKIIalpha can simultaneously interact with multiple dendritic spine proteins, possibly stabilizing the synaptic localization of CaMKII and/or nucleating a multiprotein synaptic signaling complex.     

Vampire squid: detritivores in the oxygen minimum zone

Vampire squid (Vampyroteuthis infernalis) are considered phylogenetic relics with cephalopod features of both octopods and squids. They lack feeding tentacles, but in addition to their eight arms, they have two retractile filaments, the exact functions of which have puzzled scientists for years. We present the results of investigations on the feeding ecology and behaviour of Vampyroteuthis, which include extensive in situ, deep-sea video recordings from MBARI''s remotely operated vehicles (ROVs), laboratory feeding experiments, diet studies and morphological examinations of the retractile filaments, the arm suckers and cirri. Vampire squid were found to feed on detrital matter of various sizes, from small particles to larger marine aggregates. Ingested items included the remains of gelatinous zooplankton, discarded larvacean houses, crustacean remains, diatoms and faecal pellets. Both ROV observations and laboratory experiments led to the conclusion that vampire squid use their retractile filaments for the capture of food, supporting the hypothesis that the filaments are homologous to cephalopod arms. Vampyroteuthis'' feeding behaviour is unlike any other cephalopod, and reveals a unique adaptation that allows these animals to spend most of their life at depths where oxygen concentrations are very low, but where predators are few and typical cephalopod food is scarce.     

Loss of lymphocyte cyclic AMP dependent protein kinase activity in malignant melanoma.

Cyclic AMP dependent protein kinase activity was depressed in whole thymus and spleen as well as isolated splenic lymphocytes from B16 melanoma bearing C57B1/6J mice as compared to control animals. A similar loss of enzyme activity was observed in human peripheral blood lymphocytes from melanoma bearing patients as compared to normal subjects. An unaltered level of activity in the heart of tumor bearing mice suggested some specificity for the lymphoid system. This depressed enzyme activity was the result of a diminished Vmax for cAMP stimulated calf histone phosphorylation. The tumor bearing state in the mouse was also accompanied by a depletion of small lymphocytes from both thymus and spleen and it is hypothesized that the losses of lymphocytes and cAMP dependent protein kinase activity are related.