cDNA Sequence, Protein Structure, and Evolution of the Single Hemocyanin from Aplysia californica, an Opisthobranch Gastropod

By protein immunobiochemistry and cDNA sequencing, we have found only a single hemocyanin polypeptide in an opisthobranch gastropod, the sea hare Aplysia californica, which contrasts with previously studied prosobranch gastropods, which express two distinct isoforms of this extracellular respiratory protein. We have cloned and sequenced the cDNA encoding the complete polypeptide of Aplysia californica hemocyanin (AcH). The cDNA comprises 11,433 bp, encompassing a 5UTR of 77 bp, a 3UTR of 1057 bp, and an open reading frame for a signal peptide of 20 amino acids plus a polypeptide of 3412 amino acids (M_r ca. 387 kDa). This polypeptide is the subunit of the cylindrical native hemocyanin (M_r ca. 8 MDa). It comprises eight different functional units (FUs: a, b, c, d, e, f, g, h) that have been identified immunobiochemically after limited proteolysis of AcH purified from the hemolymph. Each FU shows a highly conserved copper-A and copper-B site for reversible oxygen binding. FU AcH-h carries a specific C-terminal extension of ca. 100 amino acids that include two cysteines that may be utilized for disulfide bridge formation. Potential N-glycosylation sites are present in six FUs but lacking in AcH-b and AcH-c. On the basis of multiple sequence alignments, phylogenetic trees and a statistically firm molecular clock were calculated. The latter suggests that the last common ancestor of Haliotis and Aplysia lived 373±47 million years ago, in convincing agreement with fossil records from the early Devonian. However, the gene duplication yielding the two distinct hemocyanin isoforms found today in Haliotis tuberculata occurred 343±43 million years ago.[Reviewing Editor: Dr. Axel Meyer]The sequence reported in this paper has been deposited in the GenBank database under accession number AJ556169.     

Prostaglandin E2 Induces Interleukin-6 Synthesis in Human Astrocytoma Cells

Abstract: Prostaglandins (PGs) and cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6), have been implicated in the etiopathology of various inflammatory and degenerative disorders, including Alzheimer's disease (AD) and prion diseases. Nonsteroidal antiinflammatory drugs (NSAIDs), potent inhibitors of PG synthesis, appear to be beneficial in the treatment of AD. To assess whether PGs are able to induce IL-6 synthesis in cells of the CNS, IL-6 mRNA and protein syntheses were measured in a human astrocytoma cell line after stimulation with different PGs. PGE₁ and PGE₂, but not PGD₂ and PGF_2α, led to a rapid and transient induction of IL-6 mRNA, followed by IL-6 protein synthesis. Furthermore, PGE₂ potentiated IL-1β-induced IL-6 mRNA synthesis. These results are discussed with respect to the participation of PGs in neurodegenerative diseases (and its inhibition by NSAIDs) by affecting cytokine expression.     

Heat-Sensitive Early Function in Induced λ Nsus Lysogens

Mutations in gene N of lambda prevent killing of the host bacterium after infection. However, derepression of Nsus prophages in nonpermissive (pm(-)) bacteria results in death of the lysogens. When prophages in pm(-)(lambdaCItsA-Nsus) lysogens are derepressed by raising the temperature to 45 C, the cells remain viable as long as they are at 45 C. However, they cannot form colonies at 33 C unless they have been superinfected, at the high temperature, by lambdaCI(+)-Nsus phage which produces repressor at 45 C. A large fraction of these "rescued," heat-inducible lysogens are lysogenized by the superinfecting phage, but lysogenization is not required for rescue. In pm(-)(lambdaCItsA-Nsus) lysogens growing at 45 C, the rate of deoxyribonucleic acid (DNA) synthesis shows a characteristic increase after the temperature is lowered. This increased DNA synthesis, which is correlated with loss of rescue potential, does not occur as long as the cultures are maintained at 45 C.     

λ Mutants Which Persist as Plasmids

Lambda phages mutated in gene N do not kill sensitive host bacteria, but persist as plasmids. Plasmids are formed by genomes containing cI(+), and also by sus, ts, or c mutants of cI. Bacteria infected with two or more phage particles give rise to clones in which most of the bacteria are carriers. The introduced lambda genomes replicate more than once per bacterial division until there are 10 to 20 lambda plasmids per host genome. In bacteria containing both F and lambda plasmids, both replicate independently, and elimination by growth in acridine orange is also independent. Carriers of lambda Nsus plasmids are not immune, and there is complementation between the plasmids and superinfecting lambda mutants.     

Very Short Patch Mismatch Repair in Phage Lambda: Repair Sites and Length of Repair Tracts

Five amber mutations in the repressor (cI) gene of bacteriophage lambda recombine anomalously with nearby cI mutations. When any of these markers is used in four-factor crosses, cI+ recombinants that are expected to require three cross-overs occur at high frequencies. These recombinants are attributable to very-short-patch (VSP) repair of specific mismatches in DNA heteroduplexes formed during recombination between the markers flanking cI. The sites of the repair-prone mutations and the lengths of repair tracts have now been determined. Amber mutations subject to VSP repair are C to T transitions in 5'CCATGG, the sequence methylated by the product of gene dcm, and also in the related 5'CAGG or 5'CCAG sequences. Ambers arising in CAG sequences found in other contexts, or in codons other than CAG, were not subject to VSP repair. Repair tracts rarely, if ever, exceed ten nucleotides in length, and can be as short as two nucleotides. A repair-prone mutation does not stimulate recombination between flanking cI markers.     

Asymmetric and Symmetric Dimethylarginine as Risk Markers for Total Mortality and Cardiovascular Outcomes: A Systematic Review and Meta-Analysis of Prospective Studies

BackgroundA growing number of studies linked elevated concentrations of circulating asymmetric (ADMA) and symmetric (SDMA) dimethylarginine to mortality and cardiovascular disease (CVD) events. To summarize the evidence, we conducted a systematic review and quantified associations of ADMA and SDMA with the risks of all-cause mortality and incident CVD in meta-analyses accounting for different populations and methodological approaches of the studies.MethodsRelevant studies were identified in PubMed until February 2015. We used random effect models to obtain summary relative risks (RR) and 95% confidence intervals (95%CIs), comparing top versus bottom tertiles. Dose-response relations were assessed by restricted cubic spline regression models and potential non-linearity was evaluated using a likelihood ratio test. Heterogeneity between subgroups was assessed by meta-regression analysis.ResultsFor ADMA, 34 studies (total n = 32,428) investigating associations with all-cause mortality (events = 5,035) and 30 studies (total n = 30,624) investigating the association with incident CVD (events = 3,396) were included. The summary RRs (95%CI) for all-cause mortality were 1.52 (1.37–1.68) and for CVD 1.33 (1.22–1.45), comparing high versus low ADMA concentrations. Slight differences were observed across study populations and methodological approaches, with the strongest association of ADMA being reported with all-cause mortality in critically ill patients. For SDMA, 17 studies (total n = 18,163) were included for all-cause mortality (events = 2,903), and 13 studies (total n = 16,807) for CVD (events = 1,534). High vs. low levels of SDMA, were associated with increased risk of all-cause mortality [summary RR (95%CI): 1.31 (1.18–1.46)] and CVD [summary RR (95%CI): 1.36 (1.10–1.68) Strongest associations were observed in general population samples.ConclusionsThe dimethylarginines ADMA and SDMA are independent risk markers for all-cause mortality and CVD across different populations and methodological approaches.     

C-Terminal Modulatory Domain Controls Coupling of Voltage-Sensing to Pore Opening in Cav1.3 L-type Ca Channels

Activity of voltage-gated Ca_v1.3 L-type Ca²⁺ channels is required for proper hearing as well as sinoatrial node and brain function. This critically depends on their negative activation voltage range, which is further fine-tuned by alternative splicing. Shorter variants miss a C-terminal regulatory domain (CTM), which allows them to activate at even more negative potentials than C-terminally long-splice variants. It is at present unclear whether this is due to an increased voltage sensitivity of the Ca_v1.3 voltage-sensing domain, or an enhanced coupling of voltage-sensor conformational changes to the subsequent opening of the activation gate. We studied the voltage-dependence of voltage-sensor charge movement (Q_ON-V) and of current activation (I_Ca-V) of the long (Ca_v1.3_L) and a short Ca_v1.3 splice variant (Ca_v1.3_42A) expressed in tsA-201 cells using whole cell patch-clamp. Charge movement (Q_ON) of Ca_v1.3_L displayed a much steeper voltage-dependence and a more negative half-maximal activation voltage than Ca_v1.2 and Ca_v3.1. However, a significantly higher fraction of the total charge had to move for activation of Ca_v1.3 half-maximal conductance (Ca_v1.3: 68%; Ca_v1.2: 52%; Ca_v3.1: 22%). This indicated a weaker coupling of Ca_v1.3 voltage-sensor charge movement to pore opening. However, the coupling efficiency was strengthened in the absence of the CTM in Ca_v1.3_42A, thereby shifting I_Ca-V by 7.2 mV to potentials that were more negative without changing Q_ON-V. We independently show that the presence of intracellular organic cations (such as n-methyl-D-glucamine) induces a pronounced negative shift of Q_ON-V and a more negative activation of I_Ca-V of all three channels. These findings illustrate that the voltage sensors of Ca_v1.3 channels respond more sensitively to depolarization than those of Ca_v1.2 or Ca_v3.1. Weak coupling of voltage sensing to pore opening is enhanced in the absence of the CTM, allowing short Ca_v1.3_42A splice variants to activate at lower voltages without affecting Q_ON-V.     

Fast evolving 18S rRNA sequences from Solenogastres (Mollusca) resist standard PCR amplification and give new insights into mollusk substitution rate heterogeneity

Background  

The 18S rRNA gene is one of the most important molecular markers, used in diverse applications such as molecular phylogenetic analyses and biodiversity screening. The Mollusca is the second largest phylum within the animal kingdom and mollusks show an outstanding high diversity in body plans and ecological adaptations. Although an enormous amount of 18S data is available for higher mollusks, data on some early branching lineages are still limited. Despite of some partial success in obtaining these data from Solenogastres, by some regarded to be the most "basal" mollusks, this taxon still remained problematic due to contamination with food organisms and general amplification difficulties.