An evolutionary model for protein-coding regions with conserved RNA structure

Here we present a model of nucleotide substitution in protein-coding regions that also encode the formation of conserved RNA structures. In such regions, apparent evolutionary context dependencies exist, both between nucleotides occupying the same codon and between nucleotides forming a base pair in the RNA structure. The overlap of these fundamental dependencies is sufficient to cause "contagious" context dependencies which cascade across many nucleotide sites. Such large-scale dependencies challenge the use of traditional phylogenetic models in evolutionary inference because they explicitly assume evolutionary independence between short nucleotide tuples. In our model we address this by replacing context dependencies within codons by annotation-specific heterogeneity in the substitution process. Through a general procedure, we fragment the alignment into sets of short nucleotide tuples based on both the protein coding and the structural annotation. These individual tuples are assumed to evolve independently, and the different tuple sets are assigned different annotation-specific substitution models shared between their members. This allows us to build a composite model of the substitution process from components of traditional phylogenetic models. We applied this to a data set of full-genome sequences from the hepatitis C virus where five RNA structures are mapped within the coding region. This allowed us to partition the effects of selection on different structural elements and to test various hypotheses concerning the relation of these effects. Of particular interest, we found evidence of a functional role of loop and bulge regions, as these were shown to evolve according to a different and more constrained selective regime than the nonpairing regions outside the RNA structures. Other potential applications of the model include comparative RNA structure prediction in coding regions and RNA virus phylogenetics.     

Effect of ovary holding temperature and time on equine granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology

The effects of ovary holding time and temperature on granulosa cell apoptosis, oocyte chromatin configuration and cumulus morphology were investigated through a series of experiments. Three experiments were performed to determine the effect of ovary holding time and temperature on granulosa cell apoptosis. Ovaries were held (1) at 20, 30 or 35-37 degrees C for up to 2h, (2) at 30 degrees C for 0-1, 1-2, 2-3, 3-4, 4-6 or 6-10h, and (3) granulosa cells were held for 0, 1, 2, 3, 5, 12 or 24h in M199 with Hank's salts at room temperature (suboptimal incubation). Granulosa cell DNA was analysed by ethidium bromide staining or 3'-end labelling. Two experiments were performed to determine the effect of ovary holding time and temperature on oocyte chromatin configuration. Ovaries were held (1) at 20, 30 or 35-37 degrees C for up to 3h and (2) at 20-37 degrees C for 0-1, 1-2, 2-3, 3-4, 4-6, 6-8 or 8-12h. The oocytes were stained with Hoechst stain 33258 and the chromatin configuration was evaluated. Two experiments were performed to determine the effect of ovary holding time and temperature on cumulus oophorus morphology. Ovaries were held at (1) 20-30 or 35-37 degrees C for up to 2h and (2) for 0-2, 2-4, 4-6, and 6-10h at 35-37 degrees C. The cumulus oocyte complex (COC) were retrieved and the cumulus morphology was evaluated. There was no difference in proportion of follicles with non-apoptotic granulosa cells in the two groups below body temperature (20 and 30 degrees C), but more follicles had apoptotic granulosa cells when the ovaries were held at 35-37 degrees C (P < 0.001). Holding ovaries at 30 degrees C for more than 3h increased the proportion of follicles with apoptotic granulosa cells (P < 0.01). When follicles with non-apoptotic granulosa cells were incubated at room temperature, there was no granulosa cell apoptosis in any of the follicles within the first 3h, but at 5h apoptosis was present in the granulosa cells of 22% of the follicles, and 78% of the follicles contained apoptotic granulosa cells at 24h (P < 0.001). The temperature at which the ovaries were held did not influence oocyte chromatin, although there was a tendency towards more condensed chromatin configurations in the groups below body temperature. More denuded and expanded COCs were present in the lower temperature group (P < 0.001). Oocyte chromatin configuration changed after 6h of holding (P < 0.001), and numbers of compact COCs decreased after 2h (P < 0.05). The present studies suggest that equine follicles should be held for no more than 3h at 20-30 degrees C if granulosa cell apoptosis is to be avoided. To avoid changes in cumulus oophorus morphology, ovaries should be held at 35-37 degrees C and for less than 2h before processing, and to avoid oocyte chromatin configuration changes, ovaries should be stored for less than 6h. When ovaries are to be used in oocyte maturation studies, and assuming that (1) CC is the chromatin configuration of choice for oocyte maturation, (2) that presence of granulosa cell apoptosis promotes maturation of the oocyte and (3) that expanded cumulus oocytes are preferable, the present data suggests that ovaries should be stored for 4-6h before oocyte retrieval.     

Stimulation of PAI-1 and adipokines by glucose in human adipose tissue in vitro

Adipokines such as Plasminogen activator inhibitor-1 (PAI-1), interleukin (IL)-8, and tumor necrosis factor (TNF)-alpha are elevated in patients with obesity, insulin resistance, and type 2 diabetes. In the present study, we investigated whether glucose affected the production of these adipokines in human adipose tissue in vitro. Glucose (up to 35mM) increased secretion of PAI-1 (p<0.01) and IL-8 (p<0.01), but not TNF-alpha, in a dose- and time-dependent manner. Half-maximal stimulatory concentration of glucose was about 1mM. Glucosamine (5mM) decreased production of PAI-1 (p<0.05) and IL-8 (p<0.05), indicating that the hexosamine biosynthesis pathway is not involved in the glucose-induced increment in adipokine secretion. The present data demonstrate that glucose increases PAI-1 and IL-8 secretion. However, glucose concentrations above 5mM had no additional effects on adipokine secretion, suggesting that mechanisms other than diabetes/insulin resistance-related hyperglycemia may be involved in the observed elevation of these adipokines.     

Astroglial c-Myc overexpression predisposes mice to primary malignant gliomas

Malignant astrocytomas are common human primary brain tumors that result from neoplastic transformation of astroglia or their progenitors. Here we show that deregulation of the c-Myc pathway in developing astroglia predisposes mice to malignant astrocytomas within 2-3 weeks of age. The genetically engineered murine (GEM) gliomas harbor a molecular signature resembling that of human primary glioblastoma multiforme, including up-regulation of epidermal growth factor receptor and Mdm2. The GEM gliomas seem to originate in an abnormal population of glial fibrillary acidic protein-expressing cells in the ventricular zone and, analogous to human glioblastomas, exhibit molecular and morphological heterogeneity. Levels of connexin 43 in the majority of the tumors are unaltered from normal tissue, indicating that GEM tumors have retained the capacity to establish syncytial networks. In line with this, individual glioma foci are composed of a mixture of actively proliferating cells expressing c-Myc and proliferating cell nuclear antigen and less dividing bystander cells that express glial fibrillary acidic protein and the broad complex tramtrack bric-a-brac/poxvirus and zinc finger domain protein HOF. A subset of the transgenic mice harbored, in addition to brain tumors, vestigial cerebellums in which granule cell migration and radial Bergman glial cell differentiation were disturbed. These observations argue for a window of vulnerability during astrocyte development where c-Myc overexpression is sufficient to trigger the neoplastic process, presumably by inducing the sustained growth of early astroglial cells. This is in contrast to most other transgenic studies in which c-Myc overexpression requires co-operating transgenes for rapid tumor induction.     

Tumor tissue concentrations of the proteinase inhibitors tissue inhibitor of metalloproteinases-1 (TIMP-1) and plasminogen activator inhibitor type 1 (PAI-1) are complementary in determining prognosis in primary breast cancer

The purpose of this study was to investigate the association between tumor tissue levels of total tissue inhibitor of metalloproteinases-1 (TIMP-1) and prognosis in patients with primary breast cancer and to analyze whether measurement of TIMP-1 in tumor extracts added prognostic information to that obtained from measurements of urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 (PAI-1). An established sandwich enzyme-linked immunosorbent assay was thoroughly validated for the measurement of total TIMP-1 in tumor tissue extracts and used to determine levels of total TIMP-1 in 341 detergent-extracted tumor tissue samples from patients with primary breast cancer. The median age of the patients was 56 years (range, 29-75 years), and 164 were lymph node-negative, and 177 were lymph node-positive. The median follow-up time of the patients was 8.5 years (range, 7.3-11.3 years), and during follow-up 153 patients experienced recurrence of disease, and 136 patients died. In univariate survival analysis, we found a significant association between tumor tissue TIMP-1 level and both shorter recurrence-free survival (p = 0.0004) and shorter overall survival (p = 0.03). In multivariate survival analysis, higher tumor tissue TIMP-1 levels significantly and independently predicted shorter recurrence-free survival (p < 0.05, hazard ratios >1, comparing quartiles II-IV with I). In addition, we found that measurement of TIMP-1 levels added prognostic information to that obtained from measurement of PAI-1. In conclusion, high levels of TIMP-1 in tumor tissue extracts are significantly associated with a poor prognosis in patients with primary breast cancer. Furthermore TIMP-1 adds prognostic information to that obtained from PAI-1. However, further validation in independent data sets is needed.     

Cytokine response to eccentric exercise in young and elderly humans

To examine the plasma interleukin(IL)-6 response in elderly (E) and young (Y) humans, 10 E and 10 Ysubjects completed 60 min of eccentric lower limb exercise at the samerelative oxygen uptake. Plasma IL-6 was measured before, immediatelyafter, and 5 days into recovery from exercise, as were the biochemicalmarkers of muscle damage, creatine kinase (CK), and myoglobin. In both groups, IL-6 increased (P < 0.05) immediately afterexercise and peaked 4 h after exercise at 4.35 ± 1.7 vs.5.05 ± 3.17 pg/ml for E and Y subjects, respectively. However,the increase in IL-6 in both groups was modest relative to theincreases in CK peaking at 539 ± 413 vs. 10,301 ± 5,863 U/lfor E and Y subjects, respectively. In addition, the increase in IL-6was less pronounced (P < 0.05) in E subjects comparedwith Y subjects. These results suggest that IL-6 increasesprogressively after eccentric exercise, suggesting that this increaseis related to muscle damage. However, the modest increase in IL-6,despite large increases in CK, suggests that the IL-6 response tomuscle damage does not make an important contribution to the largeincrease in IL-6 observed during concentric exercise of long duration.Our data also suggest that aging may be associated with impaired repairmechanisms for exercise-induced muscle damage.

     

Sulfation in the Golgi lumen of Madin-Darby canine kidney cells is inhibited by brefeldin A and depends on a factor present in the cytoplasm and on Golgi membranes

Madin-Darby canine kidney cells are more resistant than most other cell types to the classical effects of brefeldin A (BFA) treatment, the induction of retrograde transport of Golgi cisternae components to the endoplasmic reticulum. Here we show that sulfation of heparan sulfate proteoglycans (HSPGs), chondroitin sulfate proteoglycans (CSPGs), and proteins in the Golgi apparatus is dramatically reduced by low concentrations of BFA in which Golgi morphology is unaffected and secretion still takes place. BFA treatment seems to reduce sulfation by inhibition of the uptake of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) into the Golgi lumen, and the inhibitory effect of BFA was similar for HSPGs, CSPGs, and proteins. This was different from the effect of chlorate, a well known inhibitor of PAPS synthesis in the cytoplasm. Low concentrations of chlorate (2-5 mm) inhibited sulfation of CSPGs and proteins only, whereas higher concentrations (15-30 mm) were required to inhibit sulfation of HSPGs. Golgi fractions pretreated with BFA had a reduced capacity for the synthesis of glycosaminoglycans (GAGs), but control level capacity could be restored by the addition of cytosol from various sources. This indicates that the PAPS pathway to the Golgi lumen depends on a BFA-sensitive factor that is present both on Golgi membranes and in the cytoplasm.     

Bacteriophage resistance of a deltathyA mutant of Lactococcus lactis blocked in DNA replication

The thyA gene, which encodes thymidylate synthase (TS), of Lactococcus lactis CHCC373 was sequenced, including the upstream and downstream regions. We then deleted part of thyA by gene replacement. The resulting strain, MBP71 deltathyA, was devoid of TS activity, and in media without thymidine, such as milk, there was no detectable dTTP pool in the cells. Hence, DNA replication was abolished, and acidification by MBP71 was completely unaffected by the presence of nine different phages tested at a multiplicity of infection (MOI) of 0.1. Nonreplicating MBP71 must be inoculated at a higher level than CHCC373 to achieve a certain pH within a specified time. For a pH of 5.2 to be reached in 6 h, the inoculation level of MBP71 must be 17-fold higher than for CHCC373. However, by adding a limiting amount of thymidine this could be lowered to just 5-fold the normal amount, while acidification was unaffected with MBP71 up to an MOI of 0.01. It was found that nonreplicating MBP71 produced largely the same products as CHCC373, though the acetaldehyde production of the former was higher.     

Sodium regulation during dehydration of a herbivorous and a carnivorous beetle from African dry savannah

The sodium regulation of carnivorous carabid beetles of the genus Cypholoba and herbivorous tenebrionid beetles of the species Phrynocolus petrosus from dry savannah in East Africa was investigated while the beetles went through dehydration in the laboratory. In both species the water loss took place mainly at the expense of the extracellular fluid, and in both species the loss of extracellular water was accompanied by a loss of extracellular sodium. In the carabid beetles the sodium removed from the extracellular fluid was excreted from the body, while in the tenebrionids sodium was kept within the body. It is proposed that the different manners in which the two species handle their sodium reflect differences in their access to dietary water and sodium.