ULTRASTRUCTURAL ORGANIZATION OF CHLOROPLAST THYLAKOIDS OF THE GREEN ALGA "OOCYSTIS MARSSONII"

Intact cells of "Oocystis marssonii" were thin sectioned and freeze-etched, using conventional and double-recovery techniques. Thylakoids extend the length of the single chloroplast and occur in stacks of three to five. The peripheral thylakoids in a stack often alternate between adjacent stacks. Interpretation of double-recovery results suggests that membranes in unstacked regions are asymmetrical, with one face smooth and the matching face covered with closely packed 85–90 Å diameter particles. Adjacent membranes in stacked regions evidently share 170 Å diameter particles, and either membrane in a stacked region may fracture. The two fracture planes thus made possible may expose nearly entire 170 Å particles or only the upper portion of such particles, creating in the latter case images of 125–135 Å diameter particles. Fracture planes in all cases appear to occur through the interior of the membrane, in the plane between the hydrophobic ends of the lipid bilayer proposed in numerous membrane models.     

Exome sequencing reveals a <Emphasis Type="Italic">nebulin</Emphasis> nonsense mutation in a dog model of nemaline myopathy

Nemaline myopathy (NM) is a congenital muscle disorder associated with muscle weakness, hypotonia, and rod bodies in the skeletal muscle fibers. Mutations in 10 genes have been implicated in human NM, but spontaneous cases in dogs have not been genetically characterized. We identified a novel recessive myopathy in a family of line-bred American bulldogs (ABDs); rod bodies in muscle biopsies established this as NM. Using SNP profiles from the nuclear family, we evaluated inheritance patterns at candidate loci and prioritized TNNT1 and NEB for further investigation. Whole exome sequencing of the dam, two affected littermates, and an unaffected littermate revealed a nonsense mutation in NEB (g.52734272 C>A, S8042X). Whole tissue gel electrophoresis and western blots confirmed a lack of full-length NEB in affected tissues, suggesting nonsense-mediated decay. The pathogenic variant was absent from 120 dogs of 24 other breeds and 100 unrelated ABDs, suggesting that it occurred recently and may be private to the family. This study presents the first molecularly characterized large animal model of NM, which could provide new opportunities for therapeutic approaches.     

Sphingosine kinase 1 expression is regulated by signaling through PI3K, AKT2, and mTOR in human coronary artery smooth muscle cells

Sphingosine kinase 1 (SphK1) is a lipid kinase implicated in mitogenic signaling pathways in vascular smooth muscle cells. We demonstrate that human coronary artery smooth muscle (HCASM) cells require SphK1 for growth and that SphK1 mRNA and protein levels are elevated in PDGF stimulated HCASM cells. To determine the mechanism of PDGF-induced SphK1 expression, we used pharmacological inhibitors of the PI3K/AKT/mTOR signaling pathway. Wortmannin, SH-5, and rapamycin significantly blocked PDGF-stimulated induction of SphK1 mRNA and protein expression, indicating a regulatory role of the PI3K/AKT/mTOR pathway in SphK1 expression. To determine which isoform of AKT regulates SphK1 mRNA and protein levels, siRNAs specific for AKT1, AKT2, and AKT3 were used. We show that AKT2 siRNA significantly blocked PDGF-stimulated increases in SphK1 mRNA and protein expression levels as well as SphK1 enzymatic activity levels. In contrast, AKT1 or AKT3 siRNA did not have an effect. Together, these results demonstrate that the PI3K/AKT/mTOR signaling pathway is involved in regulation of SphK1, with AKT2 playing a key role in PDGF-induced SphK1 expression in HCASM cells.     

Increased growth rates of stream salamanders following forest harvesting

Timber harvesting can influence headwater streams by altering stream productivity, with cascading effects on the food web and predators within, including stream salamanders. Although studies have examined shifts in salamander occupancy or abundance following timber harvest, few examine sublethal effects such as changes in growth and demography. To examine the effect of upland harvesting on growth of the stream‐associated Ouachita dusky salamander (Desmognathus brimleyorum), we used capture–mark–recapture over three years at three headwater streams embedded in intensely managed pine forests in west‐central Arkansas. The pine stands surrounding two of the streams were harvested, with retention of a 14‐ and 21‐m‐wide forested stream buffer on each side of the stream, whereas the third stream was an unharvested control. At the two treatment sites, measurements of newly metamorphosed salamanders were on average 4.0 and 5.7 mm larger post‐harvest compared with pre‐harvest. We next assessed the influence of timber harvest on growth of post‐metamorphic salamanders with a hierarchical von Bertalanffy growth model that included an effect of harvest on growth rate. Using measurements from 839 individual D. brimleyorum recaptured between 1 and 6 times (total captures, n = 1229), we found growth rates to be 40% higher post‐harvest. Our study is among the first to examine responses of individual stream salamanders to timber harvesting, and we discuss mechanisms that may be responsible for observed shifts in growth. Our results suggest timber harvest that includes retention of a riparian buffer (i.e., streamside management zone) may have short‐term positive effects on juvenile stream salamander growth, potentially offsetting negative sublethal effects associated with harvest.     

An approach to functionally relevant clustering of the protein universe: Active site profile‐based clustering of protein structures and sequences

Protein function identification remains a significant problem. Solving this problem at the molecular functional level would allow mechanistic determinant identification—amino acids that distinguish details between functional families within a superfamily. Active site profiling was developed to identify mechanistic determinants. DASP and DASP2 were developed as tools to search sequence databases using active site profiling. Here, TuLIP (Two‐Level Iterative clustering Process) is introduced as an iterative, divisive clustering process that utilizes active site profiling to separate structurally characterized superfamily members into functionally relevant clusters. Underlying TuLIP is the observation that functionally relevant families (curated by Structure‐Function Linkage Database, SFLD) self‐identify in DASP2 searches; clusters containing multiple functional families do not. Each TuLIP iteration produces candidate clusters, each evaluated to determine if it self‐identifies using DASP2. If so, it is deemed a functionally relevant group. Divisive clustering continues until each structure is either a functionally relevant group member or a singlet. TuLIP is validated on enolase and glutathione transferase structures, superfamilies well‐curated by SFLD. Correlation is strong; small numbers of structures prevent statistically significant analysis. TuLIP‐identified enolase clusters are used in DASP2 GenBank searches to identify sequences sharing functional site features. Analysis shows a true positive rate of 96%, false negative rate of 4%, and maximum false positive rate of 4%. F‐measure and performance analysis on the enolase search results and comparison to GEMMA and SCI‐PHY demonstrate that TuLIP avoids the over‐division problem of these methods. Mechanistic determinants for enolase families are evaluated and shown to correlate well with literature results.     

JAM-A-independent, antibody-mediated uptake of reovirus into cells leads to apoptosis

Apoptosis plays a major role in the cytopathic effect induced by reovirus following infection of cultured cells and newborn mice. Strain-specific differences in the capacity of reovirus to induce apoptosis segregate with the S1 and M2 gene segments, which encode attachment protein σ1 and membrane penetration protein μ1, respectively. Virus strains that bind to both junctional adhesion molecule-A (JAM-A) and sialic acid are the most potent inducers of apoptosis. In addition to receptor binding, events in reovirus replication that occur during or after viral disassembly but prior to initiation of viral RNA synthesis also are required for reovirus-induced apoptosis. To determine whether reovirus infection initiated in the absence of JAM-A and sialic acid results in apoptosis, Chinese hamster ovary (CHO) cells engineered to express Fc receptors were infected with reovirus using antibodies directed against viral outer-capsid proteins. Fc-mediated infection of CHO cells induced apoptosis in a σ1-independent manner. Apoptosis following this uptake mechanism requires acid-dependent proteolytic disassembly, since treatment of cells with the weak base ammonium chloride diminished the apoptotic response. Analysis of T1L × T3D reassortant viruses revealed that the μ1-encoding M2 gene segment is the only viral determinant of the apoptosis-inducing capacity of reovirus when infection is initiated via Fc receptors. Additionally, a temperature-sensitive, membrane penetration-defective M2 mutant, tsA279.64, is an inefficient inducer of apoptosis. These data suggest that signaling pathways activated by binding of σ1 to JAM-A and sialic acid are dispensable for reovirus-mediated apoptosis and that the μ1 protein plays an essential role in stimulating proapoptotic signaling.     

Hormonal effects of maltreatment in Nazca booby nestlings: implications for the "cycle of violence"

Non-breeding Nazca booby adults exhibit an unusual and intense social attraction to non-familial conspecific nestlings. Non-parental Adult Visitors (NAVs) seek out and approach unguarded nestlings during daylight hours and display parental, aggressive, and/or sexual behavior. In a striking parallel to the “cycle of violence” of human biology, degree of victimization as a nestling is strongly correlated with frequency of future maltreatment behavior exhibited as an adult. Here, we investigate candidates for permanent organization of this behavior, including immediate and long-term changes in growth and circulating corticosterone and testosterone due to victimization, by protecting some nestlings with portable exclosures that prevented NAV visits and comparing them to controls. During maltreatment episodes, nestlings experience an approximately five-fold increase in corticosterone concentration, and corticosterone remains elevated approximately 2.8-fold until at least the following morning. Our results are consistent with the possibility that repeated activation of the hypothalamic-pituitary-adrenal axis permanently organizes future adult maltreatment behavior. No effect on growth, acute or chronic changes in testosterone, or chronic corticosterone elevation was detected or appeared to be components of an organizational effect. This unusual behavior presents an opportunity to investigate neural, endocrine, and behavioral organization resulting from early social trauma that may be conserved across vertebrate classes.