The yeast cyclophilin multigene family: purification, cloning and characterization of a new isoform.

Cyclophilins (Cyps) constitute a highly conserved family of proteins present in a wide variety of organisms. Historically, Cyps were first identified by their ability to bind the immunosuppressive agent cyclosporin A (CsA) with high affinity; they later were found to have peptidyl-prolyl cis-trans isomerase (PPIase) activity, which catalyzes the folding of oligopeptides at proline-peptide bonds in vitro and may be important for protein folding in vivo. Cells of Saccharomyces cerevisiae contain at least two distinct Cyp-related PPIases encoded by the genes CYP1 and CYP2. A yeast strain (GL81) containing genomic disruptions of three known yeast PPIase-encoding genes [CYP1, CYP2 and RBP1 (for rapamycin-binding protein); Koltin et al., Mol. Cell. Biol. 11 (1991) 1718-1723] was previously constructed and found to be viable. Soluble fractions of these cells possess residual CsA-sensitive PPIase activity (2-5% of that present in wild-type cells as assayed in vitro). We have purified an approx. 18-kDa protein exhibiting PPIase activity from a soluble fraction of GL81 cells and determined that its N-terminal amino acid (aa) sequence exhibits significant homology (but nonidentity) to the Cyp1 and Cyp2 proteins. We designate the gene for this new protein, CYP3. Using a degenerate oligodeoxyribonucleotide (oligo) based on the N-terminal aa sequence, plus an internal oligo homologous to a conserved region within the portion of CYP1 and CYP2 that had been deleted in the genome, a CYP3-specific DNA fragment was generated by the polymerase chain reaction (PCR) using GL81 genomic DNA as a substrate. This PCR fragment was used as a probe to isolate CYP3 genomic and cDNA clones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Size and structure of 'footprints' produced by Daphnia: impact of animal size and density gradients

Daphnids with higher density than the surrounding water body have to push water downwards to hold their position in the water column. This swimming current is mainly the result of the movement of the second antennae and only to a small extent due to the filter current. The water structures under the Daphnia are very variable depending on the swimming behavior. We focused on the hop and sink behavior, where the downward-pushed water masses form a wake which can be described as a mushroom-shaped vortex. The volume of this structure increases linearly with time. The wake volume is much larger than the zooplankton itself which can be important for the perceptibility by mechanically sensing carnivorous predators. In water with a density gradient of 10 kg m^-4 the wake length WL can be described by WL = 1.64 (0.096) x L^1.58(±0.14) with the length of the Daphnia L in millimeters. The wake length, measured after 5 s, remained constant for gradients up to 1 kg m^-4. Above this value, the wake length declines in good agreement with the value expected from theory with WL = 10.66 (± 0.21) x (d/dz)^{-0.265 (± 0.010)}. Since the intra- and inter-specific 'communication' (e.g. mate seeking, nutrient partition between phytoplankton and bacteria, predator avoidance) can be expected to be bound to the hydrodynamic properties of the wakes, different wake forms and sizes most likely have an important impact on the information sent out.      

Early and late changes in Pax6 expression accompany eye degeneration during cavefish development

We have compared Pax6 expression during embryonic development in the eyed surface form (surface fish) and several different eyeless cave forms (cavefish) of the teleost Astyanax mexicanus. Despite lacking functional eyes as adults, cavefish embryos form small optic primordia, which later arrest in development and show various degrees of eye degeneration. The pattern of Pax6 mRNA expression was modified early and late during cavefish development. In early surface fish embryos, two bilateral Pax6 expression domains are present in the anterior neural plate, which extend across the midline and fuse to form the forebrain and optic primordia. In cavefish embryos, these Pax6 domains are diminished in size and remain separated, resulting in an anterior gap in Pax6 expression and presumably the formation of smaller optic primordia. The anterior gap in Pax6 expression was confirmed by double staining for Pax6 and distalless-3 mRNA, which marks the anterior margin of the neural plate and is unaltered in cavefish. Similar anterior gaps in Pax6 expression occurred in independently derived cavefish populations, suggesting that they are important in eye degeneration. Later during surface fish development, Pax6 protein is expressed in the cornea, lens, and ganglion and amacrine cells of the neural retina. Pax6 expression was gradually reduced during cavefish lens development, concomitant with lens arrest and degeneration, and was absent in the corneal epithelium, which does not differentiate in cavefish. In contrast, Pax6 expression in the retinal ganglion and amarcine cells is unmodified in cavefish, despite retarded retinal development. The results suggest that changes in Pax6 expression are involved in the evolution of cavefish eye degeneration.     

Synthesis and in vitro biological evaluation of aryl boronic acids as potential inhibitors of factor XIa

A series of functionalized aryl boronic acids were synthesized and evaluated as potential inhibitors of factor XIa. Crystal structures of the protein-inhibitor complexes led to the design and synthesis of second generation compounds showing single digit micromolar inhibition against FXIa and selectivity against thrombin, trypsin, and FXa.     

Intermolecular Overhauser effects in fluoroalcohol solutions of cyclo-alanylglycine

Interactions between the diketopiperazine cyclo-alanylglycine and four fluorinated alcohols in water-fluoroalcohol mixtures were examined by (1)H[(19)F] intermolecular nuclear Overhauser effects (NOE) experiments. The alcohols studied were trifluoroethanol, hexafluoroacetone trihydrate, 1,1,1,3,3,3-hexafluoroisopropanol and perfluoro-t-butanol. The experimental methods used permit detection of solvent-solute NOEs of 0.1% or less. Solute and solvent diffusion coefficients were determined and apparent molecular radii of the fluoroalcohols estimated. Using these data, it was shown that observed (1)H[(19)F] intermolecular NOEs are consistent with expectations based on theory. A method for extending conventional theory to take into account the shape of a solute and the exposure of its hydrogens to solvent is described. This approach gives reasonable agreement with experimental results, particularly if it is assumed that solute-solvent interactions take place in such a way that the fluorines of a fluoroalcohol are preferentially oriented toward the solute during solute-solvent encounters. The results support the suggestion that intermolecular (1)H[(19)F] NOEs may become a useful tool for studies of peptide and protein conformations in fluoroalcohol-water solvent mixtures.     

Percutaneous coronary intervention with adjunctive abciximab and clopidogrel in a patient with chronic idiopathic thrombocytopaenic purpura

The use of the antiplatelet agents abciximab and clopidogrel is now accepted therapy in percutaneous coronary intervention. We present a case in which these agents were used in a patient with idiopathic thrombocytopaenic purpura and a platelet count of 40x10(9)/l undergoing primary multivessel coronary stenting. This case shows that unstable coronary syndromes can occur in patients with thrombocytopaenia and that antiplatelet agents may be used safely in this context.     

Common germinal-center B-cell origin of the malignant cells in two composite lymphomas, involving classical Hodgkin's disease and either follicular lymphoma or B-CLL

BACKGROUND: Classical Hodgkin's disease (HD) and B-cell non-Hodgkin lymphoma (NHL) occasionally occur in the same patient. Such composite lymphomas represent interesting models to study the pathogenesis of B-cell lymphomas and the relationship between HD and B-cell NHL. MATERIALS AND METHODS: We analyzed two composite lymphomas (a combination of classical HD with follicular lymphoma [FL] and a combination of classical HD with B-cell chronic lymphocytic leukemia [B-CLL]) by micromanipulation of single cells from tissue sections and amplification of immunoglobulin V region genes for the clonal relationship of the tumor cells. RESULTS: In both cases, clonally related variable (V) genes with both shared as well as distinct somatic mutations were obtained from the two lymphomas, showing that in each of the cases the distinct tumor cells were members of a common germinal center (GC) B-cell clone. FL cells from two different lymph nodes of patient 1 showed a similar mutation pattern, suggesting that infiltration of these lymph nodes by tumor cells was not restricted to a particular FL cell or subclone. In the FL, a single cell was identified with a mutation signature indicating that premalignant cells can persist in the tissue. CONCLUSIONS: The cases presented here further underline the close relationship between HD and B-cell NHL and the role of the GC in lymphomagenesis. Whereas the latter was already suggested for FL and HD, the present study indicates that also in the B-CLL subset characterized by mutated Ig genes, important steps in malignant transformation happen in the GC, and that HRS cells can derive from CD5-positive B cells.     

Protein stability and surface electrostatics: a charged relationship

Engineering proteins to withstand a broad range of conditions continues to be a coveted objective, holding the potential to advance biomedicine, industry, and our understanding of disease. One way of achieving this goal lies in elucidating the underlying interactions that define protein stability. It has been shown that the hydrophobic effect, hydrogen bonding, and packing interactions between residues in the protein interior are dominant factors that define protein stability. The role of surface residues in protein stability has received much less attention. It has been believed that surface residues are not important for protein stability particularly because their interactions with the solvent should be similar in the native and unfolded states. In the case of surface charged residues, it was sometimes argued that solvent exposure meant that the high dielectric of the solvent will further decrease the strength of the charge-charge interactions. In this paper, we challenge the notion that the surface charged residues are not important for protein stability. We computationally redesigned sequences of five different proteins to optimize the surface charge-charge interactions. All redesigned proteins exhibited a significant increase in stability relative to their parent proteins, as experimentally determined by circular dichroism spectroscopy and differential scanning calorimetry. These results suggest that surface charge-charge interactions are important for protein stability and that rational optimization of charge-charge interactions on the protein surface can be a viable strategy for enhancing protein stability.     

Tadpole begging reveals high quality

Parents can benefit from allocating limited resources nonrandomly among offspring, and offspring solicitation (i.e. begging) is often hypothesized to evolve because it contains information valuable to choosy parents. We tested the predictions of three ‘honest begging’ hypotheses – Signal of Need, Signal of Quality and Signal of Hunger – in the tadpoles of a terrestrial frog (Oophaga pumilio). In this frog, mothers provision tadpoles with trophic eggs, and when mothers visit, tadpoles perform a putative begging signal by stiffening their bodies and vibrating rapidly. We assessed the information content of intense tadpole begging with an experimental manipulation of tadpole condition (need/quality) and food deprivation (hunger). This experiment revealed patterns consistent with the Signal of Quality hypothesis and directly counter to predictions of Signal of Need and Signal of Hunger. Begging effort and performance were higher in more developed and higher condition tadpoles and declined with food deprivation. Free‐living mothers were unlikely to feed tadpoles of a nonbegging species experimentally cross‐fostered with their own, and allocated larger meals to more developed tadpoles and those that vibrated at higher speed. Mother O. pumilio favour their high‐quality young, and because their concurrent offspring are reared in separate nurseries, must do so by making active allocation decisions. Our results suggest that these maternal choices are based at least in part on offspring signals, indicating that offspring solicitation can evolve to signal high quality.     

Hepatitis C Viremia Is Associated with Cytomegalovirus IgG Antibody Levels in HIV-Infected Women

Background

Individuals with HIV infection exhibit high cytomegalovirus (CMV) IgG levels, but there are few data regarding the association of hepatitis C virus (HCV) with the immune response against CMV.

Methods

Associations of HCV with CMV seropositivity and CMV IgG levels were studied in 635 HIV-infected women, 187 of whom were HCV-seropositive, with adjustment in multivariable models for age, race/ethnicity, and HIV disease characteristics. Eighty one percent of the women reported receipt of highly active antiretroviral therapy (HAART) prior to or at CMV testing.

Results

In adjusted models women with chronic HCV had higher CMV IgG levels than those without HCV RNA (β = 2.86, 95% CI:0.89 – 4.83; P = 0.004). The association of HCV RNA with CMV IgG differed by age (P _interaction = 0.0007), with a strong association observed among women in the low and middle age tertiles (≤45.3 years of age; β = 6.21, 95% CI:3.30 – 9.11, P<0.0001) but not among women in the high age tertile. CMV IgG levels were not associated with non-invasive measures of liver disease, APRI and FIB-4, or with HCV RNA level and adjustment for Epstein-Barr virus (EBV) IgG levels did not affect the association between HCV and CMV.

Conclusions

CMV IgG levels are higher in HCV/HIV co-infected women than in HIV mono-infected women. Further research on the association of HCV with CMV IgG is indicated because prior studies have found CMV IgG to be associated with morbidity and mortality in the general population and subclinical carotid artery disease in HIV-infected patients.