Influence of the carbon source on glycerol kinase activity in Neurospora crassa.

The level of glycerol kinase activity in Neurospora crassa was shown to change in response to resuspension of sucrose-grown mycelia in fresh medium containing a new carbon source: the magnitude of the change depended on the new carbon source provided. Certain carbon sources, such as glucose and fructose, inhibited the small increase that occurred in the absence of any carbon source. Others, and in particular deoxyribose, galactose, glycerol and ribose, greatly enhanced this increase. The activity induced by deoxyribose and galactose had the same stability, both in vivo and in vitro, as that induced by glycerol, and as that induced by incubation of Neurospora cultures at low temperatures. The inhibitory carbon sources, such as glucose and fructose, also restricted the increases induced by deoxyribose, galactose and glycerol: they had more effect on the increases induced by glycerol and deoxyribose than on that induced by galactose. The increase in activity that occurs at low temperature was also inhibited by glucose and sucrose.     

Opiates, opioid peptides and single neurones.

The use of electrophysiological techniques to study the mechanisms of action of opiates has three distinct advantages. In the first place, they measure directly the important functional parameter of the nervous system - the electrical activity of single neurones. Second, they have a high resolution in time which facilitates arguing from effect to cause. Third, they have a high resolution in space because single neurones, or single loci on the same neurone, can be examined selectively. The present review deals exclusively with recordings at the level of the single neurone.     

Fish as proxies of ecological and environmental change

Anthropogenic impacts have shifted aquatic ecosystems far from prehistoric baseline states; yet, understanding these impacts is impeded by a lack of available long-term data that realistically reflects the organisms and their habitats prior to human disturbance. Fish are excellent, and largely underused, proxies for elucidating the degree, direction and scale of shifts in aquatic ecosystems. This paper highlights potential sources of qualitative and quantitative data derived from contemporary, archived and ancient fish samples, and then, using key examples, discusses the types of long-term temporal information that can be obtained. This paper identifies future research needs with a focus on the Southern Hemisphere, as baseline shifts are poorly described relative to the Northern Hemisphere. Temporal data sourced from fish can improve our understanding of how aquatic ecosystems have changed, particularly when multiple sources of data are used, enhancing our ability to interpret the current state of aquatic ecosystems and establish effective measures to safeguard against further adverse shifts. The range of biological, ecological and environmental data obtained from fish can be integrated to better define ecosystem baseline states on which to establish policy goals for future conservation and exploitation practices.     

Structural Based Analyses of the JC Virus T-Antigen F258L Mutant Provides Evidence for DNA Dependent Conformational Changes in the C-Termini of Polyomavirus Origin Binding Domains

The replication of human polyomavirus JCV, which causes Progressive Multifocal Leukoencephalopathy, is initiated by the virally encoded T-antigen (T-ag). The structure of the JC virus T-ag origin-binding domain (OBD) was recently solved by X-ray crystallography. This structure revealed that the OBD contains a C-terminal pocket, and that residues from the multifunctional A1 and B2 motifs situated on a neighboring OBD molecule dock into the pocket. Related studies established that a mutation in a pocket residue (F258L) rendered JCV T-ag unable to support JCV DNA replication. To establish why this mutation inactivated JCV T-ag, we have solved the structure of the F258L JCV T-ag OBD mutant. Based on this structure, it is concluded that the structural consequences of the F258L mutation are limited to the pocket region. Further analyses, utilizing the available polyomavirus OBD structures, indicate that the F258 region is highly dynamic and that the relative positions of F258 are governed by DNA binding. The possible functional consequences of the DNA dependent rearrangements, including promotion of OBD cycling at the replication fork, are discussed.     

Propagule pressure and environmental conditions interact to determine establishment success of an invasive plant species,glossy buckthorn (<Emphasis Type="Italic">Frangula alnus</Emphasis>), across five different wetland habitat types

Many invasive plant species are able to establish within a wide range of community types. This establishment success depends on high propagule pressure and successful recruitment of seedlings following propagule dispersal into receptive environments. This study aimed to investigate interactions between propagule pressure and environmental resistance to seedling recruitment of the invasive shrub, glossy buckthorn (Frangula alnus Mill.), over a range of wetland habitat types. We measured propagule deposition using seed traps and recruitment success using sown plots, while characterizing vegetation and abiotic environmental conditions in five adjacent wetland habitat types. Drier habitats, which included Cedar Swamp, Shrub Carr, and String, had lower resistance to buckthorn establishment than the wetter Flark and Cattail Marsh. The drier habitats supported more woody species and provided more raised hummock surfaces essential for successful buckthorn recruitment and establishment. Propagule pressure was also higher in dry habitats that supported higher densities of adult glossy buckthorn, while long-distance dispersal into areas with low adult density was uncommon. Natural recruitment was highest in sites with intense propagule pressure, but experimental sowing of seeds demonstrated that buckthorn establishes in wet sites with higher resistance if propagule pressure is increased and seeds are deposited on hummocks. This study demonstrates the affinity of glossy buckthorn for drier wetland sites, and provides empirical evidence that environmental resistance can be overcome by higher propagule pressure.     

γ-Irradiation facilitates the expression of adoptive immunity against established tumors by eliminating suppressor T cells

Summary It was found that sublethal (550 rad) whole-body -irradiation of mice bearing established immunogenic tumors enabled tumor-sensitized spleen cells infused intravenously 1 h later to cause complete tumor regression in all mice. In contrast, -irradiation alone caused only a temporary halt in tumor growth, and immune cells gave practically no therapeutic effect at all. This result was obtained with the SA1 sarcoma, Meth A fibrosarcoma, P815 mastocytoma, and P388 lymphoma. Additional experiments with the Meth A fibrosarcoma revealed that the spleen cells from tumor-immune donors that caused tumor regression in -irradiated recipients were T cells, as evidenced by their functional elimination by treatment with anti-Thy-1.2 antibody and complement. It was shown next that adoptive T-cell-mediated regression of tumors in -irradiated recipients was inhibited by an intravenous infusion of spleen cells from donors with established tumors, but not by spleen cells from normal donors. The spleen cells that suppressed the expression of adoptive immunity were functionally eliminated by treatment with anti-Thy-1.2 antibody and complement. Moreover, they were destroyed by exposing the tumor-bearing donors to 500 rad of -radiation 24 h before harvesting their spleen cells. The results are consistent with the interpretation that -radiation facilitates the expression of adoptive T-cell-mediated immunity against established tumors by eliminating a population of tumor-induced suppressor T cells from the tumor-bearing recipient.Supported by Grants CA-16642 and CA-27794 from the National Cancer Institute, a Grant-in-Aid from R. J. Reynolds Industries, Inc. and Grant RR-05705 from the Division of Research Resources, NIH     

Loop 2 of limulus myosin III is phosphorylated by protein kinase A and autophosphorylation

Little is known about the functions of class III unconventional myosins although, with an N-terminal kinase domain, they are potentially both signaling and motor proteins. Limulus myosin III is particularly interesting because it is a phosphoprotein abundant in photoreceptors that becomes more heavily phosphorylated at night by protein kinase A. This enhanced nighttime phosphorylation occurs in response to signals from an endogenous circadian clock and correlates with dramatic changes in photoreceptor structure and function. We seek to understand the role of Limulus myosin III and its phosphorylation in photoreceptors. Here we determined the sites that become phosphorylated in Limulus myosin III and investigated its kinase, actin binding, and myosin ATPase activities. We show that Limulus myosin III exhibits kinase activity and that a major site for both protein kinase A and autophosphorylation is located within loop 2 of the myosin domain, an important actin binding region. We also identify the phosphorylation of an additional protein kinase A and autophosphorylation site near loop 2, and a predicted phosphorylation site within loop 2. We show that the kinase domain of Limulus myosin III shares some pharmacological properties with protein kinase A, and that it is a potential opsin kinase. Finally, we demonstrate that Limulus myosin III binds actin but lacks ATPase activity. We conclude that Limulus myosin III is an actin-binding and signaling protein and speculate that interactions between actin and Limulus myosin III are regulated by both second messenger mediated phosphorylation and autophosphorylation of its myosin domain within and near loop 2.     

Translation factor IF2 at the interface of transposition and replication by the PriA-PriC pathway

Bacteriophage Mu DNA synthesis is initiated during transposition by replication restart proteins PriA, DnaT and either PriB or PriC. The PriA-PriC pathway requires PriA's helicase activity and other host factors that promote the orderly transition from transpososome to replisome on the Mu DNA template. The host factor MRFalpha-PR, which removes obstacles to PriA binding and promotes the PriA-PriC pathway, was identified to be the translation initiation factor IF2. Purified isoform IF2-2, which is truncated at the N-terminal end, had full MRFalpha-PR activity whereas full-length IF2-1 was inactive. IF2-2 was bound to the Mu DNA template specifically at the step for prereplisome assembly. Prior steps in the orderly transition from transpososome were essential to promote efficient IF2-2 binding. Moreover, PriA helicase activity was subsequently needed to displace IF2-2, remodelling the template to permit replisome assembly. IF2's role in the transition mechanism as well as its function as G protein and translation factor suggest its potential to regulate DNA synthesis by this pathway.     

Ring substituent effects on biological activity of vinyl sulfones as inhibitors of HIV-1

In a previous study, we prepared a small library of chicoric acid analogs that possessed both potent anti-integrase and antiviral activity. It was also shown that active compounds fell into one of two groups: those that inhibited an early stage in viral replication and those that inhibited at a later stage. In this study, a series of vinyl geminal disulfone-containing compounds possessing a range of ring substituents has been synthesized to probe the impact of structure on inhibitory mechanisms. Four active compounds were identified using HIV drug susceptibility assays. Three of the inhibitors possessing either no substituents or electron-withdrawing substituents on the aromatic rings led to high levels of cytotoxicity and antiviral activity. Intrigued by the potential implications of electronic effects on activity, we probed whether the active compounds could be nonspecifically reacting via 1,4-addition. To investigate this hypothesis, the compounds were incubated with glutathione and upon LC/MS analysis, molecular ion peaks corresponding to both mono and double addition adducts were identified. Second, we synthesized analogs lacking the ability to participate in 1,4-addition and tested them for antiviral activity and cytotoxicity, and found the compounds inactive for both activities. Taken together, the studies reported herein suggest that compounds lacking electron-donating substituents on the aromatic ring are promiscuous acceptors of biological nucleophiles, whereas compounds possessing electron-donating substituents seem to resist addition or at least be more selective and significantly less toxic.