The Landscape of Intertwined Associations in Homooligomeric Proteins

We present an overview of the full repertoire of intertwined associations in homooligomeric proteins. This overview summarizes recent findings on the different categories of intertwined associations in known protein structures, their assembly modes, the properties of their interfaces, and their structural plasticity. Furthermore, the current body of knowledge on the so-called three-dimensional domain-swapped systems is reexamined in the context of the wider landscape of intertwined homooligomers, with a particular focus on the mechanistic aspects that underpin intertwined self-association processes in proteins. Insights gained from this integrated overview into the physical and biological roles of intertwining are highlighted.     

Insight into glucosidase II from the red marine microalga Porphyridium sp. (Rhodophyta)

N‐glycosylation of proteins is one of the most important post‐translational modifications that occur in various organisms, and is of utmost importance for protein function, stability, secretion, and loca‐lization. Although the N‐linked glycosylation pathway of proteins has been extensively characterized in mammals and plants, not much information is available regarding the N‐glycosylation pathway in algae. We studied the α 1,3‐glucosidase glucosidase II (GANAB) glycoenzyme in a red marine microalga Porphyridium sp. (Rhodophyta) using bioinformatic and biochemical approaches. The GANAB‐gene was found to be highly conserved evolutionarily (compo‐sed of all the common features of α and β subunits) and to exhibit similar motifs consistent with that of homolog eukaryotes GANAB genes. Phylogenetic analysis revealed its wide distribution across an evolutionarily vast range of organisms; while the α subunit is highly conserved and its phylogenic tree is similar to the taxon evolutionary tree, the β subunit is less conserved and its pattern somewhat differs from the taxon tree. In addition, the activity of the red microalgal GANAB enzyme was studied, including functional and biochemical characterization using a bioassay, indicating that the enzyme is similar to other eukaryotes ortholog GANAB enzymes. A correlation between polysaccharide production and GANAB activity, indicating its involvement in polysaccharide biosynthesis, is also demonstrated. This study represents a valuable contribution toward understanding the N‐glycosylation and polysaccharide biosynthesis pathways in red microalgae.     

Increased serum IGF-1 levels protect the musculoskeletal system but are associated with elevated oxidative stress markers and increased mortality independent of tissue igf1 gene expression

Although the literature suggests a protective (anabolic) effect of insulin-like growth factor-1 (IGF-1) on the musculoskeletal system during growth and aging, there is evidence that reductions in IGF-1 signaling are advantageous for promoting an increase in life span through reduction in oxidative stress-induced tissue damage. To better understand this paradox, we utilized the hepatocyte-specific IGF-1 transgenic (HIT) mice, which exhibit 3-fold increases in serum IGF-1, with normal IGF-1 expression in other tissues, and mice with an IGF-1 null background that exclusively express IGF-1 in the liver, which thereby deliver IGF-1 by the endocrine route only (KO-HIT mice). We found that in the total absence of tissue igf1 gene expression (KO-HIT), increases in serum IGF-1 levels were associated with increased levels of lipid peroxidation products in serum and increased mortality rate at 18 months of age in both genders. Surprisingly, however, we found that in female mice, tissue IGF-1 plays an important role in preserving trabecular bone architecture as KO-HIT mice show bone loss in the femoral distal metaphysis. Additionally, in male KO-HIT mice, increases in serum IGF-1 levels were insufficient to protect against age-related muscle loss.     

Nascent polypeptide sequences that influence ribosome function

Ribosomes catalyze protein synthesis using transfer RNAs and auxiliary proteins. Historically, ribosomes have been considered nonspecific translational machines, having no regulatory functions. However, a new class of regulatory mechanisms has been discovered that is based on interactions occurring within the ribosomal peptide exit tunnel that result in ribosome stalling during translation of an appropriate mRNA segment. These discoveries reveal an unexpectedly dynamic role ribosomes play in regulating their own activity. By using nascent leader peptides in combination with bound specific amino acids or antibiotics, ribosome functions can be altered significantly resulting in regulated expression of downstream coding regions. This review summarizes relevant findings in recent articles and outlines our current understanding of nascent peptide-induced ribosome stalling in regulating gene expression.     

A potential role for shed soluble major histocompatibility class I molecules as modulators of neurite outgrowth

The neurobiological activities of classical major histocompatibility class I (MHCI) molecules are just beginning to be explored. To further examine MHCI's actions during the formation of neuronal connections, we cultured embryonic mouse retina explants a short distance from wildtype thalamic explants, or thalami from transgenic mice (termed "NSE-Db") whose neurons express higher levels of MHCI. While retina neurites extended to form connections with wildtype thalami, we were surprised to find that retina neurite outgrowth was very stunted in regions proximal to NSE-Db thalamic explants, suggesting that a diffusible factor from these thalami inhibited retina neurite outgrowth. It has been long known that MHCI-expressing cells release soluble forms of MHCI (sMHCI) due to the shedding of intact MHCI molecules, as well as the alternative exon splicing of its heavy chain or the action proteases which cleave off it's transmembrane anchor. We show that the diffusible inhibitory factor from the NSE-Db thalami is sMHCI. We also show that COS cells programmed to express murine MHCI release sMHCI that inhibits neurite outgrowth from nearby neurons in vitro. The neuroinhibitory effect of sMHCI could be blocked by lowering cAMP levels, suggesting that the neuronal MHCI receptor's signaling mechanism involves a cyclic nucleotide-dependent pathway. Our results suggest that MHCI may not only have neurobiological activity in its membrane-bound form, it may also influence local neurons as a soluble molecule. We discuss the involvement of complement proteins in generating sMHCI and new theoretical models of MHCI's biological activities in the nervous system.     

RNAi screen reveals host cell kinases specifically involved in Listeria monocytogenes spread from cell to cell

Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereas the mechanisms supporting cytosolic actin-based motility are fairly well understood, it is unclear whether specific host factors may be required for supporting the formation and resolution of membrane protrusions. To address this gap in knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library covering the human kinome and identified 7 candidate kinases whose depletion led to severe spreading defects in cells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlated with the formation of protrusion that did not resolve into double-membrane vacuoles. Moreover, we developed sending and receiving cell-specific RNAi procedures and showed that CSNK1A was required in the sending cells, but was dispensable in the receiving cells, for protrusion resolution. Finally, we showed that the observed defects were specific to Listeria monocytogenes, as Rickettsia conorii displayed wild-type cell-to-cell spread in CSNK1A1- and CSNK2B-depleted cells. We conclude that, in addition to the specific host factors supporting cytosolic actin-based motility, such as CSNK2B, Listeria monocytogenes requires specific host factors, such as CSNK1A1 in order to form productive membrane protrusions and spread from cell to cell.     

ALGAL COMMUNITIES OF SPRINGS AND STREAMS IN THE MT. ST. HELENS REGION,WASHINGTON, U.S.A. FOLLOWING THE MAY 1980 ERUPTION1

Mt. St. Helens, a volcanic peak in the Cascade Range in southern Washington erupted violently on May 18, 1980, causing enormous damage to both terrestrial and aquatic ecosystems. The initial explosion evaporated, scoured or buried all springs and streams in the blast impact area. Ash fall and erosion from defoliated hillsides subsequently filled most of the lotic habitats with organic debris and volcanic ash. Recolonization of springs and streams by algae occurred quickly in areas where erosion through the ash progressed down to bedrock. Within 15 months or less of the eruption, algal communities were established throughout the blast impact area. However, as a result of the initial and continued disturbance these communities remained in an early successional stage. Floral assemblages were highly variable except that they were composed mostly of diatoms, with Achnanthes minutissima dominating most lotic sites. Springs showed the most rapid development toward stable floras.     

Comparison of in vitro laboratory analyses with the fertility of cryopreserved stallion spermatozoa

Assessing the fertilizing potential of a semen sample is important for effective stallion management and for rapid progress in evaluating new cryopreservation technologies. Unfortunately, sperm motility does not estimate fertility well. These experiments established assays to measure cell viability, acrosomal integrity and mitochondrial function for cryopreserved stallion spermatozoa, using flow cytometry, and determined the variability associated with these assays. Correlations between results for these laboratory assays and stallion fertility were also determined. The inter-assay variability for visual motility, computer assisted motility, and sperm velocity, sperm viability, percent viable-acrosome intact cells and mitochondrial function of cells were all similar, however, intra-assay variability was lower for flow cytometric assays than for motility assays. The reliability of all assays were >0.72, except for sperm velocity (0.32). Although visual motility and the straightness of sperm motility conducted 90 min after thawing were correlated with seasonal fertility (0.56 and 0.55, respectively), data from no single assay were correlated with first-cycle fertility rates (P > 0.05). Best models using data from multiple assays explained 66 to 73, 76 to 89 and 79 to 94% of the variability in fertilizing potential, when two, three and four variables were included, respectively. Caution is required in interpreting these data, as only a few stallions were evaluated and relatively few mares were bred to each stallion, however, they do indicate that using a few rapid and inexpensive sperm assays, we can begin to understand factors important in stallion sperm fertilizing capacity, and we can use these assays to more effectively evaluate new methods for cryopreserving stallion spermatozoa.     

Establishment of pregnancies after serial dilution or direct transfer by vitrified equine embryos

Experiments were conducted to determine viability of equine embryos in vivo after vitrification. In a preliminary study (Experiment 1), embryos were exposed in three steps to vitrification solutions containing increasing concentrations of ethylene glycol and glycerol (EG/G); the final vitrification solution was 3.4 M glycerol + 4.6 M ethylene glycol in a base medium of phosphate-buffered saline. Embryos were warmed in a two-step dilution and transferred into uteri of recipients. No pregnancies were observed after transfer of blastocysts >300 microm (n = 3). Transfer of morulae or blastocysts < or = 300 microm resulted in four embryonic vesicles (4/6, 67%). In a second experiment, embryo recovery per ovulation was similar for collections on Day 6(28/36, 78%) versus Days 7 and 8(30/48, 62%). Embryos < or = 300 and >300 microm were vitrified, thawed and transferred as in Experiment 1. Some embryos < or = 300 microm were also transferred using a direct-transfer procedure (DT). Embryo development rates to Day 16 were not different for embryos < or = 300 microm that were treated as in Experiment 1(10/22, 46%) or transferred by DT (16/26, 62%). Embryos > 300 microm (n = 19) did not produce embryonic vesicles.     

Factors affecting the success of oocyte transfer in a clinical program for subfertile mares

Oocyte transfer is a potential method to produce offspring from valuable mares that cannot carry a pregnancy or produce embryos. From 2000 through 2004, 86 mares, 19.2 +/- 0.4 yr of age (mean +/- S.E.M.), were used as oocyte donors in a clinical program at Colorado State University. Oocytes were collected from 77% (548/710) of preovulatory follicles and during 96% (548/570) of cycles. Oocytes were collected 21.0+/-0.1h after administration of hCG to estrous donors and cultured 16.4 +/- 0.2 h prior to transfer into recipients' oviducts. At 16 and 50 d after transfer, pregnancies were detected in 201 of 504 (40%) and 159 of 504 (32%) of recipients, respectively, with an embryo-loss rate of 21% (42/201). Pregnancy rates were similar (P > 0.05) for cyclic and noncyclic recipients and for recipients inseminated with cooled, fresh or frozen semen. One or more recipients were detected pregnant at 16 and 50 d, respectively, for 80% (69/86) and 71% (61/86) of donors. More donors <20 than > or = 20 yr (mean ages +/- S.E.M. of 15.5 +/- 0.4 and 23.0 +/- 0.3 yr, respectively) tended (P = 0.1) to have one or more pregnant recipients at 50 d (36/45, 80%; 28/45, 62%, respectively). Results of the program confirm that pregnancies can consistently be obtained from older, subfertile mares using oocyte transfer.