Morphological and morphometric studies of the dysmyelinating mutant,the Long Evans shaker rat

The Long Evans shaker (les) rat is a recently identified CNS myelin mutant with an autosomal recessive mode of inheritance. Although scattered myelin sheaths are present in some areas of the CNS, most notably the ventral spinal cord in the young neonatal rat, this myelin is gradually lost, and 8-12 weeks little myelin is present throughout the CNS. Despite this severe myelin deficiency, some mutants may live beyond one year of age. Rare, thin myelin sheaths that are present early in development lack myelin basic protein (MBP) and on ultrastructural examination are poorly compacted and lack a major dense line. Many oligodendrocytes develop an accumulation of vesicles and membranous bodies, but no abnormal cell death is observed. In the optic nerve, cell kinetic studies show an increase in proliferation at early time points in les, while total glial cell counts are also increased in les from 2 months of age. In situ hybridization studies demonstrate that the numbers of mature oligodendrocytes are similar to controls early in life and increase with time compared to controls. There is both a progressive astrocyte hypertrophy and microgliosis. While les has a mutation in the myelin basic protein (mbp) gene, it is dissimilar in both genotype and phenotype to the previously described mbp mouse mutants, shiverer (shi) and shiverermld. Unlike shi and its allele, where myelin increases with time and oligodendrocytes become ultrastructurally normal, les oligodendrocytes are permanently disabled, continue to demonstrate cytoplasmic abnormalities, and fail to produce myelin beyond the first weeks of life.     

Molecular characterization and sequencing of antifreeze proteins from larvae of the beetle Dendroides canadensis

The deduced amino acid sequences of antifreeze proteins (AFPs) from larvae of the beetle Dendroides canadensis were determined from both complementary DNAs (cDNAs) and from peptide sequencing. These consisted of proteins with a 25-residue signal peptide and mature proteins 83 (Dendroides antifreeze protein; DAFP-1) or 84 (DAFP-2) amino acids in length which differed at only two positions. Peptide sequencing yielded sequences which overlapped exactly with those of the deduced cDNA sequences of DAFP-1 and DAFP-2, while the partial sequence of another AFP (DAFP-3) matched 21 of 28 residues. Seven 12- or 13-mer repeating units are present in these antifreeze proteins with a consensus sequence consisting of: Cys-Thr-X₃-Ser-X₅-X₆-Cys-X₈-X₉-Ala-X₁₁-Thr-X₁₃, where X₃ and X₁₁ tend toward charged residues, X₅ tends toward threonine or serine, X₆ toward asparagine or aspartate, X₉ toward asparagine or lysine, and X₁₃ toward alanine in the 13-mers. The most interesting feature of these proteins is that throughout the length of the mature antifreeze proteins every sixth residue is a cysteine. These sequences are not similar to any of the known fish AFPs, but they are similar to AFPs from the beetle Tenebrio molitor. Accepted: 14 November 1997     

Bioluminescent organs of two deep-sea arrow worms, Eukrohnia fowleri and Caecosagitta macrocephala, with further observations on Bioluminescence in chaetognaths

Bioluminescence in the deep-sea chaetognath Eukrohnia fowleri is reported for the first time, and behavioral, morphological, and chemical characteristics of bioluminescence in chaetognaths are examined. Until this study, the only known species of bioluminescent chaetognath was Caecosagitta macrocephala. The luminescent organ of that species is located on the ventral edge of each anterior lateral fin, whereas that of E. fowleri runs across the center of the tail fin on both dorsal and ventral sides. Scanning electron microscopy showed that the bioluminescent organs of both species consist of hexagonal chambers containing elongate ovoid particles-the organelles holding bioluminescent materials. No other luminous organism is known to use hexagonal packing to hold bioluminescent materials. Transmission electron microscopy of particles from C. macrocephala revealed a densely packed paracrystalline matrix punctuated by globular inclusions, which likely correspond to luciferin and luciferase, respectively. Both species use unique luciferases in conjunction with coelenterazine for light emission. Luciferase of C. macrocephala becomes inactive after 30 min, but luciferase of E. fowleri is highly stable. Although C. macrocephala has about 90 times fewer particles than E. fowleri, it has a similar bioluminescent capacity (total particle volume) due to its larger particle size. In situ observations of C. macrocephala from a remotely operated vehicle revealed that the luminous particles are released to form a cloud. The discovery of bioluminescence in a second chaetognath phylogenetically distant from the first highlights the importance of bioluminescence among deep-sea organisms.     

Substrate modulation of enzyme activity in the herpesvirus protease family

The herpesvirus proteases are an example in which allosteric regulation of an enzyme activity is achieved through the formation of quaternary structure. Here, we report a 1.7 A resolution structure of Kaposi's sarcoma-associated herpesvirus protease in complex with a hexapeptide transition state analogue that stabilizes the dimeric state of the enzyme. Extended substrate binding sites are induced upon peptide binding. In particular, 104 A2 of surface are buried in the newly formed S4 pocket when tyrosine binds at this site. The peptide inhibitor also induces a rearrangement of residues that stabilizes the oxyanion hole and the dimer interface. Concomitant with the structural changes, an increase in catalytic efficiency of the enzyme results upon extended substrate binding. A nearly 20-fold increase in kcat/KM results upon extending the peptide substrate from a tetrapeptide to a hexapeptide exclusively due to a KM effect. This suggests that the mechanism by which herpesvirus proteases achieve their high specificity is by using extended substrates to modulate both the structure and activity of the enzyme.     

Ventriculo-aortic junction in human root. A geometric approach

With advances in tissue engineering and improvement of surgical techniques, stentless biological valves and valve-sparing procedures have become alternatives to traditional aortic valve replacement with stented bioprostheses or mechanical valves. New surgical techniques preserve the advantages of native valves but require better understanding of the anatomical structure of the aortic root. Silicone rubber was injected in fresh aortic roots of nine human cadavers under the physiological closing pressure of 80 mmHg. The casts reproduced every detail of the aortic root anatomy and were used to digitize 27 leaflet attachment lines (LALs) of the aortic valves. LALs were normalized and described with a mathematical model. LALs were found to follow a pattern with the right coronary being the largest followed by the non-coronary and then the left coronary. During diastole, the aortic valve LAL can be described by an intersection between a created tube and an extruded parabolic surface. This geometrical definition of the LAL during end diastole gives a better understanding of the aortic root anatomy and could be useful for heart valve design and improvement of aortic valve reconstruction technique.     

Plasma membrane domain organization regulates EGFR signaling in tumor cells

Macromolecular complexes exhibit reduced diffusion in biological membranes; however, the physiological consequences of this characteristic of plasma membrane domain organization remain elusive. We report that competition between the galectin lattice and oligomerized caveolin-1 microdomains for epidermal growth factor (EGF) receptor (EGFR) recruitment regulates EGFR signaling in tumor cells. In mammary tumor cells deficient for Golgi beta1,6N-acetylglucosaminyltransferase V (Mgat5), a reduction in EGFR binding to the galectin lattice allows an increased association with stable caveolin-1 cell surface microdomains that suppresses EGFR signaling. Depletion of caveolin-1 enhances EGFR diffusion, responsiveness to EGF, and relieves Mgat5 deficiency-imposed restrictions on tumor cell growth. In Mgat5(+/+) tumor cells, EGFR association with the galectin lattice reduces first-order EGFR diffusion rates and promotes receptor interaction with the actin cytoskeleton. Importantly, EGFR association with the lattice opposes sequestration by caveolin-1, overriding its negative regulation of EGFR diffusion and signaling. Therefore, caveolin-1 is a conditional tumor suppressor whose loss is advantageous when beta1,6GlcNAc-branched N-glycans are below a threshold for optimal galectin lattice formation.     

High-level secretion of dipetarudin, a chimeric thrombin inhibitor, by Pichia pastoris

Dipetarudin is a potent direct thrombin inhibitor that was genetically engineered as a chimera between dipetalogastin II and hirudin. Dipetarudin was initially cloned and purified from Escherichia coli, but with a very low yield of about 0.3 mg/l of culture medium. In this study, we report the production of dipetarudin in the methylotrophic yeast Pichia pastoris using pPIC9 vector. The His+ transformants were screened for the best expression performances by prolongation of the ecarin clotting time. An optimal dipetarudin's expression was reached by addition of methanol in culture medium to a final concentration of 0.5%, every 8h during 4 days. Secreted dipetarudin was purified essentially using a two-step purification scheme: anion exchange chromatography in a Resource Q column, followed by C18-reversed phase HPLC. About 150 mg purified dipetarudin was obtained from 1l culture supernatant. This yield is 500-fold higher than the yield obtained with the E. coli system. The molecular mass of dipetarudin calculated by MALDI-TOF (7450 Da) was in agreement with the mass calculated by the amino acid composition (7454 Da), indicating correct processing of the signal sequence. The Ki value of dipetarudin was 399+/-83 fM, which is in agreement with that calculated for the inhibitor isolated from E. coli. This efficient and cost-effective expression system facilitates large-scale production and purification of dipetarudin for further structural, functional and pharmacological investigations.     

RAL-1 controls multivesicular body biogenesis and exosome secretion

Exosomes are secreted vesicles arising from the fusion of multivesicular bodies (MVBs) with the plasma membrane. Despite their importance in various processes, the molecular mechanisms controlling their formation and release remain unclear. Using nematodes and mammary tumor cells, we show that Ral GTPases are involved in exosome biogenesis. In Caenorhabditis elegans, RAL-1 localizes at the surface of secretory MVBs. A quantitative electron microscopy analysis of RAL-1–deficient animals revealed that RAL-1 is involved in both MVB formation and their fusion with the plasma membrane. These functions do not involve the exocyst complex, a common Ral guanosine triphosphatase (GTPase) effector. Furthermore, we show that the target membrane SNARE protein SYX-5 colocalizes with a constitutively active form of RAL-1 at the plasma membrane, and MVBs accumulate under the plasma membrane when SYX-5 is absent. In mammals, RalA and RalB are both required for the secretion of exosome-like vesicles in cultured cells. Therefore, Ral GTPases represent new regulators of MVB formation and exosome release.     

Greater shrub dominance alters breeding habitat and food resources for migratory songbirds in Alaskan arctic tundra

Climate warming is affecting the Arctic in multiple ways, including via increased dominance of deciduous shrubs. Although many studies have focused on how this vegetation shift is altering nutrient cycling and energy balance, few have explicitly considered effects on tundra fauna, such as the millions of migratory songbirds that breed in northern regions every year. To understand how increasing deciduous shrub dominance may alter breeding songbird habitat, we quantified vegetation and arthropod community characteristics in both graminoid and shrub dominated tundra. We combined measurements of preferred nest site characteristics for Lapland longspurs (Calcarius lapponicus) and Gambel's White‐crowned sparrows (Zonotrichia leucophrys gambelii) with modeled predictions for the distribution of plant community types in the Alaskan arctic foothills region for the year 2050. Lapland longspur nests were found in sedge‐dominated tussock tundra where shrub height does not exceed 20 cm, whereas White‐crowned sparrows nested only under shrubs between 20 cm and 1 m in height, with no preference for shrub species. Shrub canopies had higher canopy‐dwelling arthropod availability (i.e. small flies and spiders) but lower ground‐dwelling arthropod availability (i.e. large spiders and beetles). Since flies are the birds' preferred prey, increasing shrubs may result in a net enhancement in preferred prey availability. Acknowledging the coarse resolution of existing tundra vegetation models, we predict that by 2050 there will be a northward shift in current White‐crowned sparrow habitat range and a 20–60% increase in their preferred habitat extent, while Lapland longspur habitat extent will be equivalently reduced. Our findings can be used to make first approximations of future habitat change for species with similar nesting requirements. However, we contend that as exemplified by this study's findings, existing tundra modeling tools cannot yet simulate the fine‐scale habitat characteristics that are critical to accurately predicting future habitat extent for many wildlife species.