Light capture and pigment diversity in marine and freshwater cryptophytes

Phenotypic traits associated with light capture and phylogenetic relationships were characterized in 34 strains of diversely pigmented marine and freshwater cryptophytes. Nuclear SSU and partial LSU rDNA sequence data from 33 of these strains plus an additional 66 strains produced a concatenated rooted maximum likelihood tree that classified the strains into 7 distinct clades. Molecular and phenotypic data together support: (i) the reclassification of Cryptomonas irregularis NIES 698 to the genus Rhodomonas, (ii) revision of phycobiliprotein (PBP) diversity within the genus Hemiselmis to include cryptophyte phycocyanin (Cr‐PC) 569, (iii) the inclusion of previously unidentified strain CCMP 2293 into the genus Falcomonas, even though it contains cryptophyte phycoerythrin 545 (Cr‐PE 545), and (iv) the inclusion of previously unidentified strain CCMP 3175, which contains Cr‐PE 545, in a clade with PC‐containing Chroomonas species. A discriminant analysis‐based model of group membership correctly predicted 70.6% of the clades using three traits: PBP concentration · cell^?1, the wavelength of PBP maximal absorption, and habitat. Non‐PBP pigments (alloxanthin, chl‐a, chl‐c₂, α‐carotene) did not contribute significantly to group classification, indicating the potential plasticity of these pigments and the evolutionary conservation of the PBPs. Pigment data showed evidence of trade‐offs in investments in PBPs vs. chlorophylls (a +c₂).     

Transcriptomic analysis of IgG4 Fc-fusion protein degradation in a panel of clonally-derived CHO cell lines using RNASeq

In this study, we report an investigation of a panel of clonally-derived Chinese hamster ovary (CHO) cell lines exhibiting variability in the proportion of full-length IgG4 Fc-fusion protein produced. The recombinant protein was found to be degraded during cell culture into four shorter “clipped” species (three of the four cleavage sites occurred at arginine residues) and preliminary analyses suggested that a host cell enzyme was responsible for proteolysis. To identify the specific enzyme responsible, RNA sequencing was used to identify gene expression differences between the cell lines with a “high” and “low” clipping phenotype. From this analysis, six protease-encoding genes were found to be significantly upregulated in those cell lines yielding the lowest proportion of full-length IgG4 Fc-fusion protein. Four of these protease candidates were deprioritized after examination of their cleavage site specificity. The remaining enzymes, Adam19 and Furin, were found to be capable of cleavage at arginine residues, and inhibitors for both proteases were added to cell-free media to determine if the product degradation could be reduced. While the Adam19 inhibitor had no impact, Furin inhibitor I (specific for the proprotein convertase family of enzymes) was found to result in a 33–39% increase in complete IgG4 Fc-fusion protein when compared with untreated samples.     

The effect of hydration upon the conformation and dynamics of neocarrabiose,a repeat unit of β-carrageenan

Molecular mechanics calculations have been performed for the disaccharide neocarrabiose, one of the repeat units of β-carrageenan, as a general model for the (1 → 3)-linkage in the carrageenans. An adiabatic conformational energy map for this molecule has been prepared by constrained energy minimization and compared to previously reported relaxed maps. Neither the experimentally determined crystal structure of neocarrabiose nor the fiber diffraction conformation of β-carrageenan is a low energy conformation on the relaxed Ramachandran map. Molecular dynamics simulations in vacuum produced trajectories consistent with this relaxed vacuum surface. However, a simulation with explicitly included solvent water molecules produced a trajectory that remained in the region of the two experimental structures. This dramatic solvation effect is apparently the result of the breaking of an interring hydrogen bond between the O2 hydroxyl groups of neocarrabiose as both groups hydrogen bond to solvent. © 1996 John Wiley & Sons, Inc.     

Stability and folding rates of domains spanning the large A-band super-repeat of titin

Titin is a very large (>3 MDa) protein found in striated muscle where it is believed to participate in myogenesis and passive tension. A prominent feature in the A-band portion of titin is the presence of an 11-domain super-repeat of immunoglobulin superfamily and fibronectin-type-III-like domains. Seven overlapping constructs from human cardiac titin, each consisting of two or three domains and together spanning the entire 11-domain super-repeat, have been expressed in Escherichia coli. Fluorescence unfolding experiments and circular dichroism spectroscopy have been used to measure folding stabilities for each of the constructs and to assign unfolding rates for each super-repeat domain. Immunoglobulin superfamily domains were found to fold correctly only in the presence of their C-terminal fibronectin type II domain, suggesting close and possibly rigid association between these units. The domain stabilities, which range from 8.6 to 42 kJ mol(-1) under physiological conditions, correlate with previously reported mechanical forces required to unfold titin domains. Individual domains vary greatly in their rates of unfolding, with a range of unfolding rate constants between 2.6 x 10(-6) and 1.2 s(-1). This variation in folding behavior is likely to be an important determinant in ensuring independent folding of domains in multi-domain proteins such as titin.     

Involvement of hematopoietic progenitor kinase 1 in T cell receptor signaling

Hematopoietic progenitor kinase 1 (HPK1), a mammalian Ste20-related serine/threonine protein kinase, is a hematopoietic-specific upstream activator of the c-Jun N-terminal kinase. Here, we provide evidence to demonstrate the involvement of HPK1 in T cell receptor (TCR) signaling. HPK1 was activated and tyrosine-phosphorylated with similar kinetics following TCR/CD3 or pervanadate stimulation. Co-expression of protein-tyrosine kinases, Lck and Zap70, with HPK1 led to HPK1 activation and tyrosine phosphorylation in transfected mammalian cells. Upon TCR/CD3 stimulation, HPK1 formed inducible complexes with the adapters Nck and Crk with different kinetics, whereas it constitutively interacted with the adapters Grb2 and CrkL in Jurkat T cells. Interestingly, HPK1 also inducibly associated with linker for activation of T cells (LAT) through its proline-rich motif and translocated into glycolipid-enriched microdomains (also called lipid rafts) following TCR/CD3 stimulation, suggesting a critical role for LAT in the regulation of HPK1. Together, these results identify HPK1 as a new component of TCR signaling. T cell-specific signaling molecules Lck, Zap70, and LAT play roles in the regulation of HPK1 during TCR signaling. Differential complex formation between HPK1 and adapters highlights the possible involvement of HPK1 in multiple signaling pathways in T cells.     

Manganese-containing superoxide dismutase and its gene from Candida albicans

Mitochondrial manganese-containing superoxide dismutase was purified around 112-fold with an overall yield of 1.1% to apparent electrophoretic homogeneity from the dimorphic pathogenic fungus, Candida albicans. The molecular mass of the native enzyme was 106 kDa and the enzyme was composed of four identical subunits with a molecular mass of 26 kDa. The enzyme was not sensitive to either cyanide or hydrogen peroxide. The N-terminal amino acid sequence alignments (up to the 18th residue) showed that the enzyme has high similarity to the other eukaryotic manganese-containing superoxide dismutases. The gene sod2 encoding manganese-containing superoxide dismutase has been cloned using a product obtained from polymerase chain reaction. Sequence analysis of the sod2 predicted a manganese-containing superoxide dismutase that contains 234 amino acid residues with a molecular mass of 26173 Da, and displayed 57% sequence identity to the homologue of Saccharomyces cerevisiae. The deduced N-terminal 34 amino acid residues may serve as a signal peptide for mitochondrial translocation. Several regulatory elements such as stress responsive element and haem activator protein 2/3/4/5 complex binding sites were identified in the promoter region of sod2. Northern analysis with a probe derived from the cloned sod2 revealed a 0.94-kb band, which corresponds approximately to the expected size of mRNA deduced from sod2.     

Haem oxygenase-1 prevents cell death by regulating cellular iron

Haem oxygenase-1 (HO1) is a heat-shock protein that is induced by stressful stimuli. Here we demonstrate a cytoprotective role for HO1: cell death produced by serum deprivation, staurosporine or etoposide is markedly accentuated in cells from mice with a targeted deletion of the HO1 gene, and greatly reduced in cells that overexpress HO1. Iron efflux from cells is augmented by HO1 transfection and reduced in HO1-deficient fibroblasts. Iron accumulation in HO1-deficient cells explains their death: iron chelators protect HO1-deficient fibroblasts from cell death. Thus, cytoprotection by HO1 is attributable to its augmentation of iron efflux, reflecting a role for HO1 in modulating intracellular iron levels and regulating cell viability.     

Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells

Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor beta1 (TGF-beta1), and addition of TGF-beta1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-beta1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFbeta1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFbeta1-driven matrix production within a diabetic milieu.     

Recent intron gain in elongation factor-1alpha of colletid bees (Hymenoptera: Colletidae)

We discovered the presence of a unique spliceosomal intron in the F1 copy of elongation factor-1alpha (EF-1alpha) restricted to the bee family Colletidae (Hymenoptera: Apoidae). The intron ranges in size from 101 to 1044 bp and shows no positional sliding. Our data also demonstrate the complete absence of this intron from exemplars representing all other bee families, as well as from close hymenopteran relatives. A review of the literature finds that this intron is likewise absent from all other arthropods for which data are available. This provides unambiguous evidence for a relatively recent intron insertion event in the colletid common ancestor and, at least in this specific instance, lends support to the introns-late hypothesis. The comparative distribution of this novel intron also supports the monophyly of Colletidae and the exclusion of the Stenotritidae from this family, providing an example of the potential of some introns to act as robust markers of shared descent.