The principal islet of the coho salmon (Oncorhyncus kisutch) contains the BB isoenzyme of creatine kinase

The importance of creatine kinase (E.C. 2.7.3.2) in endocrine tissues has been generally overlooked. Using a specific radiometric assay, we have demonstrated the existence of CK in the Brockmann body (principal islet) of the Coho salmon. We have purified this protein from insular tissue and concurrently purified CK from brain and muscle of the salmon. Purification characteristics, immunological cross-reactivity, and N-terminal sequence analysis have demonstrated that the predominant cytosolic CK from the Brockmann body is indistinguishable from the BB (brain) isoenzyme. Immunocytochemical studies indicated that the enzyme resides in the endocrine parenchyma. Phosphocreatine may serve as a reservoir of energy in the islet and augment its capacity to secrete hormones. The induction of CK-BB in the islet by other hormones could influence the secretion of insular hormones. Interorgan flux of the substrate creatine may be an undescribed mechanism of physiological regulation.     

Actions of Rho family small G proteins and p21-activated protein kinases on mitogen-activated protein kinase family members.

The mitogen-activated protein (MAP) kinases are a family of serine/threonine kinases that are regulated by distinct extracellular stimuli. The currently known members include extracellular signal-regulated protein kinase 1 (ERK1), ERK2, the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs), and p38 MAP kinases. We find that overexpression of the Ste20-related enzymes p21-activated kinase 1 (PAK1) and PAK2 in 293 cells is sufficient to activate JNK/SAPK and to a lesser extent p38 MAP kinase but not ERK2. Rat MAP/ERK kinase kinase 1 can stimulate the activity of each of these MAP kinases. Although neither activated Rac nor the PAKs stimulate ERK2 activity, overexpression of either dominant negative Rac2 or the N-terminal regulatory domain of PAK1 inhibits Ras-mediated activation of ERK2, suggesting a permissive role for Rac in the control of the ERK pathway. Furthermore, constitutively active Rac2, Cdc42hs, and RhoA synergize with an activated form of Raf to increase ERK2 activity. These findings reveal a previously unrecognized connection between Rho family small G proteins and the ERK pathway.     

Contrasting patterns of Andean diversification among three diverse clades of Neotropical clearwing butterflies

The Neotropical region is the most biodiverse on Earth, in a large part due to the highly diverse tropical Andean biota. The Andes are a potentially important driver of diversification within the mountains and for neighboring regions. We compared the role of the Andes in diversification among three subtribes of Ithomiini butterflies endemic to the Neotropics, Dircennina, Oleriina, and Godyridina. The diversification patterns of Godyridina have been studied previously. Here, we generate the first time‐calibrated phylogeny for the largest ithomiine subtribe, Dircennina, and we reanalyze a published phylogeny of Oleriina to test different biogeographic scenarios involving the Andes within an identical framework. We found common diversification patterns across the three subtribes, as well as major differences. In Dircennina and Oleriina, our results reveal a congruent pattern of diversification related to the Andes with an Andean origin, which contrasts with the Amazonian origin and multiple Andean colonizations of Godyridina. In each of the three subtribes, a clade diversified in the Northern Andes at a faster rate. Diversification within Amazonia occurred in Oleriina and Godyridina, while virtually no speciation occurred in Dircennina in this region. Dircennina was therefore characterized by higher diversification rates within the Andes compared to non‐Andean regions, while in Oleriina and Godyridina, we found no difference between these regions. Our results and discussion highlight the importance of comparative approaches in biogeographic studies.     

Lamin A/C binding protein LAP2alpha is required for nuclear anchorage of retinoblastoma protein

The phosphorylation-dependent anchorage of retinoblastoma protein Rb in the nucleus is essential for its function. We show that its pocket C domain is both necessary and sufficient for nuclear anchorage by transiently expressing green fluorescent protein (GFP) chimeras of Rb fragments in tissue culture cells and by extracting the cells with hypotonic solutions. Solid phase binding assays using glutathione S-transferase-fusion of Rb pockets A, B, and C revealed a direct association of lamin C exclusively to pocket C. Lamina-associated polypeptide (LAP) 2alpha, a binding partner of lamins A/C, bound strongly to pocket C and weakly to pocket B. When LAP2alpha was immunoprecipitated from soluble nuclear fractions, lamins A/C and hypophosphorylated Rb were coprecipitated efficiently. Similarly, immunoprecipitation of expressed GFP-Rb fragments by using anti-GFP antibodies coprecipitated LAP2alpha, provided that pocket C was present in the GFP chimeras. On redistribution of endogenous lamin A/C and LAP2alpha into nuclear aggregates by overexpressing dominant negative lamin mutants in tissue culture cells, Rb was also sequestered into these aggregates. In primary skin fibroblasts, LAP2alpha is expressed in a growth-dependent manner. Anchorage of hypophosphorylated Rb in the nucleus was weakened significantly in the absence of LAP2alpha. Together, these data suggest that hypophosphorylated Rb is anchored in the nucleus by the interaction of pocket C with LAP2alpha-lamin A/C complexes.     

P-selectin and CD63 use different mechanisms for delivery to Weibel-Palade bodies

The biogenesis of endothelial-specific Weibel-Palade bodies (WPB) is poorly understood, despite their key role in both haemostasis and inflammation. Biogenesis of specialized organelles of haemopoietic cells is often adaptor protein complex 3-dependent (AP-3-dependent), and AP-3 has previously been shown to play a role in the trafficking of both WPB membrane proteins, P-selectin and CD63. However, WPB are thought to form at the trans Golgi network (TGN), which is inconsistent with a role for AP-3, which operates in post-Golgi trafficking. We have therefore investigated in detail the mechanisms of delivery of these two membrane proteins to WPB. We find that P-selectin is recruited to forming WPB in the trans-Golgi by AP-3-independent mechanisms that use sorting information within both the cytoplasmic tail and the lumenal domain of the receptor. In contrast, CD63 is recruited to already-budded WPB by an AP-3-dependent route. These different mechanisms of recruitment lead to the presence of distinct immature and mature populations of WPB in human umbilical vein endothelial cells (HUVEC).     

Ocular and regional cerebral blood flow in aging Fischer-344 rats

Vascularremodeling and changes in vascular responsiveness occur in the ratcerebrum with old age. This includes reductions in cerebral arteriolarnumerical density, cross-sectional area, distensibility, the relativeproportion of distensible elements in the cerebral arteriolar wall, andreduced endothelium-dependent relaxation. The purpose of this study wasto test the hypothesis that old age results in an increase in vascularresistance and, correspondingly, a decrease in blood flow to ocular,regional cerebral, and spinal tissue in the rat. Blood flow wasmeasured in the eye, olfactory bulb, left and right cerebrum, pituitary gland, midbrain, pons, cerebellum, medulla, and spinal cord of juvenile(2-mo-old, n = 6), adult (6-mo-old,n = 7), and aged (24-mo-old,n = 7) male Fischer-344 rats. Arterialpressure and blood flow were used to calculate vascular resistance.Vascular resistance in the eye of aged rats (6.03 ± 1.08 mmHg · ml¹ · min · 100 g) was higher than that in juvenile (3.83 ± 0.38 mmHg · ml¹ · min · 100 g) and adult rats (3.12 ± 0.24 mmHg · ml¹ · min · 100 g). Similarly, resistance in the pons of older rats (2.24 ± 0.55 mmHg · ml¹ · min · 100 g) was greater than in juvenile (0.66 ± 0.06 mmHg ·ml¹ · min · 100 g) and adult rats (0.80 ± 0.11 mmHg · ml¹ · min · 100 g). In contrast, vascular resistance in the pituitary gland was lowerin the aged rats (juvenile, 3.09 ± 0.22; adult, 2.79 ± 0.42;aged, 1.73 ± 0.32 mmHg · ml¹ · min · 100 g, respectively). Vascular resistance was not different in othercerebral tissues or in the spinal cord in the aged rats. These datasuggest that regional cerebral and spinal blood flow and vascularresistance remain largely unchanged in conscious aged rats at rest butthat elevations in ocular vascular resistance and, correspondingly,decreases in ocular perfusion with advanced age could have seriousadverse effects on visual function.

     

Intracellular positioning of isoforms explains an unusually large adenylate kinase gene family in the parasite Trypanosoma brucei

Adenylate kinases occur classically as cytoplasmic and mitochondrial enzymes, but the expression of seven adenylate kinases in the flagellated protozoan parasite Trypanosoma brucei (order, Kinetoplastida; family, Trypanosomatidae) easily exceeds the number of isoforms previously observed within a single cell and raises questions as to their location and function. We show that a requirement to target adenylate kinase into glycosomes, which are unique kinetoplastid-specific microbodies of the peroxisome class in which many reactions of carbohydrate metabolism are compartmentalized, and two different flagellar structures as well as cytoplasm and mitochondrion explains the expansion of this gene family in trypanosomes. The three isoforms that are selectively built into either the flagellar axoneme or the extra-axonemal paraflagellar rod, which is essential for motility, all contain long N-terminal extensions. Biochemical analysis of the only short form trypanosome adenylate kinase revealed that this enzyme catalyzes phosphotransfer of gamma-phosphate from ATP to AMP, CMP, and UMP acceptors; its high activity and specificity toward CMP is likely to reflect an adaptation to very low intracellular cytidine nucleotide pools. Analysis of some of the phosphotransfer network using RNA interference suggests considerable complexity within the homeostasis of cellular energetics. The anchoring of specific adenylate kinases within two distinct flagellar structures provides a paradigm for metabolic organization and efficiency in other flagellates.     

Primary reactions of the LOV2 domain of phototropin,a plant blue-light photoreceptor

The phototropins constitute an important class of plant photoreceptor kinases that control a range of physiological responses, including phototropism, light-directed chloroplast movement, and light-induced stomatal opening. The LOV2 domain of phototropin binds a molecule of flavin mononucleotide (FMN) and undergoes a photocycle involving light-driven covalent adduct formation between a conserved cysteine residue and the C(4a) atom of FMN. This product state promotes C-terminal kinase activation and downstream signal transduction. Here, we report the primary photophysics and photochemistry of LOV2 domains of phototropin 1 of Avena sativa (oat) and of the phy3 photoreceptor of Adiantum capillus-veneris (maidenhair fern). In agreement with earlier reports [Swartz, T. E., et al. (2001) J. Biol. Chem. 276, 36493-36500], we find that the FMN triplet state is the reactive species from which the photoreaction occurs. We demonstrate that the triplet state is the primary photoproduct in the LOV2 photocycle, generated at 60% efficiency. No spectroscopically distinguishable intermediates precede the FMN triplet on the femtosecond to nanosecond time scale, indicating that it is formed directly via intersystem crossing (ISC) from the singlet state. Our results indicate that the majority of the FMN triplets in the LOV2 domain exist in the protonated form. We propose a reaction mechanism that involves excited-state proton transfer, on the nanosecond time scale or faster, from the sulfhydryl group of the conserved cysteine to the N5 atom of FMN. This event promotes adduct formation by increasing the electrophilicity of C(4a) and subsequent nucleophilic attack by the cysteine's thiolate anion. Comparison to free FMN in solution shows that the protein environment of LOV2 increases the ISC rate of FMN by a factor of 2.4, thus improving the yield of the cysteinyl-flavin adduct and the efficiency of phototropin-mediated signaling processes.     

A SAS-6-Like Protein Suggests that the Toxoplasma Conoid Complex Evolved from Flagellar Components

SAS-6 is required for centriole biogenesis in diverse eukaryotes. Here, we describe a novel family of SAS-6-like (SAS6L) proteins that share an N-terminal domain with SAS-6 but lack coiled-coil tails. SAS6L proteins are found in a subset of eukaryotes that contain SAS-6, including diverse protozoa and green algae. In the apicomplexan parasite Toxoplasma gondii, SAS-6 localizes to the centriole but SAS6L is found above the conoid, an enigmatic tubulin-containing structure found at the apex of a subset of alveolate organisms. Loss of SAS6L causes reduced fitness in Toxoplasma. The Trypanosoma brucei homolog of SAS6L localizes to the basal-plate region, the site in the axoneme where the central-pair microtubules are nucleated. When endogenous SAS6L is overexpressed in Toxoplasma tachyzoites or Trypanosoma trypomastigotes, it forms prominent filaments that extend through the cell cytoplasm, indicating that it retains a capacity to form higher-order structures despite lacking a coiled-coil domain. We conclude that although SAS6L proteins share a conserved domain with SAS-6, they are a functionally distinct family that predates the last common ancestor of eukaryotes. Moreover, the distinct localization of the SAS6L protein in Trypanosoma and Toxoplasma adds weight to the hypothesis that the conoid complex evolved from flagellar components.     

Do Low-Mercury Terrestrial Resources Subsidize Low-Mercury Growth of Stream Fish? Differences between Species along a Productivity Gradient

Low productivity in aquatic ecosystems is associated with reduced individual growth of fish and increased concentrations of methylmercury (MeHg) in fish and their prey. However, many stream-dwelling fish species can use terrestrially-derived food resources, potentially subsidizing growth at low-productivity sites, and, because terrestrial resources have lower MeHg concentrations than aquatic resources, preventing an increase in diet-borne MeHg accumulation. We used a large-scale field study to evaluate relationships among terrestrial subsidy use, growth, and MeHg concentrations in two stream-dwelling fish species across an in-stream productivity gradient. We sampled young-of-the-year brook trout (Salvelinus fontinalis) and Atlantic salmon (Salmo salar), potential competitors with similar foraging habits, from 20 study sites in streams in New Hampshire and Massachusetts that encompassed a wide range of aquatic prey biomass. Stable isotope analysis showed that brook trout used more terrestrial resources than Atlantic salmon. Over their first growing season, Atlantic salmon tended to grow larger than brook trout at sites with high aquatic prey biomass, but brook grew two-fold larger than Atlantic salmon at sites with low aquatic prey biomass. The MeHg concentrations of brook trout and Atlantic salmon were similar at sites with high aquatic prey biomass and the MeHg concentrations of both species increased at sites with low prey biomass and high MeHg in aquatic prey. However, brook trout had three-fold lower MeHg concentrations than Atlantic salmon at low-productivity, high-MeHg sites. These results suggest that differential use of terrestrial resource subsidies reversed the growth asymmetry between potential competitors across a productivity gradient and, for one species, moderated the effect of low in-stream productivity on MeHg accumulation.