Cell signaling: wingless and glypicans together again

The role of the Glypican proteoglycans in Wingless signaling has been controversial. New studies show that the Glypican Dally-like can have both positive and negative effects on Wingless signaling; moreover, signaling can be regulated by removing Dally-like from the cell surface.     

Characterization of proteins in human pancreatic cancer serum using differential gel electrophoresis and tandem mass spectrometry

The purpose of this study was to develop techniques for identifying cancer biomarkers in human serum using differential in-gel electrophoresis (DIGE), and characterizing the protein biomarkers using tandem mass spectrometry (MS/MS). A major problem in profiling protein expression by DIGE comes from the presence of high concentrations of a small number of proteins. Therefore, serum samples were first chromatographed using an immunoaffinity HPLC column (Agilent Technologies), to selectively remove albumin, immunoglobulins, transferrin, haptoglobin, and antitrypsin. Serum samples from three individuals with pancreatic cancer and three individuals without cancer were compared. Serum samples were processed using the immunoaffinity column. Differential protein analysis was performed using DIGE. A total of 56 protein spot-features were found to be significantly increased and 43 significantly decreased in cancer serum samples. These spot features were excised, trypsin digested, and analyzed by MALDI/TOF/TOF (4700 Proteomics Analyzer, Applied Biosystems). We identified 24 unique proteins that were increased and 17 unique proteins that were decreased in cancer serum samples. Western blot analysis confirmed increased levels of several of these proteins in the pancreatic cancer serum samples. In an independent series of serum samples from 20 patients with pancreatic cancer and 14 controls, increased levels of apolipoprotein E, alpha-1-antichymotrypsin, and inter-alpha-trypsin inhibitor were found to be associated with pancreatic cancer. These results suggest that affinity column enrichment and 2-D DIGE can be used to identify numerous proteins differentially expressed in serum from individuals with pancreatic cancer.     

Chronosequence predictions are robust in a Neotropical secondary forest,but plots miss the mark

Tropical secondary forests (TSF) are a global carbon sink of 1.6 Pg C/year. However, TSF carbon uptake is estimated using chronosequence studies that assume differently aged forests can be used to predict change in aboveground biomass density (AGBD) over time. We tested this assumption using two airborne lidar datasets separated by 11.5 years over a Neotropical landscape. Using data from 1998, we predicted canopy height and AGBD within 1.1 and 10.3% of observations in 2009, with higher accuracy for forest height than AGBD and for older TSFs in comparison to younger ones. This result indicates that the space‐for‐time assumption is robust at the landscape‐scale. However, since lidar measurements of secondary tropical forest are rare, we used the 1998 lidar dataset to test how well plot‐based studies quantify the mean TSF height and biomass in a landscape. We found that the sample area required to produce estimates of height or AGBD close to the landscape mean is larger than the typical area sampled in secondary forest chronosequence studies. For example, estimating AGBD within 10% of the landscape mean requires more than thirty 0.1 ha plots per age class, and more total area for larger plots. We conclude that under‐sampling in ground‐based studies may introduce error into estimations of the TSF carbon sink, and that this error can be reduced by more extensive use of lidar measurements.     

THE FINE STRUCTURE OF ACANTHAMOEBA CASTELLANII : I. The Trophozoite

The fine structure of the trophozoite of Acanthamoeba castellanii (Neff strain) has been studied. Locomotor pseudopods, spikelike "acanthopodia," and microprojections from the cell surface are all formed by hyaline cytoplasm, which excludes formed elements of the cell and contains a fine fibrillar material. Golgi complex, smooth and rough forms of endoplasmic reticulum, digestive vacuoles, mitochondria, and the water-expulsion vesicle (contractile vacuole) are described. A canicular system opening into the water-expulsion vesicle contains tubules about 600 A in diameter that are lined with a filamentous material. The tubules are continuous with unlined vesicles or ampullae of larger diameter. Centrioles were not observed, but cytoplasmic microtubules radiate from a dense material similar to centriolar satellites and are frequently centered in the Golgi complex. Cytoplasmic reserve materials include both lipid and glycogen, each of which amounts to about 10% of the dry weight.     

THE APPARENT DISTORTION OF BRIEF RECTANGULAR ELECTRICAL STIMULI IN NERVE

If it is assumed that the kinetics of the process of excitation in nerve is given by dp/dt = KI – kp, I being the actual exciting component of the current, p the state of excitation, and K and k constants, it is necessary to postulate that on application of a rectangular stimulus of voltage, V, the current, I, undergoes a transient exponential variation, usually a decrease, in order that the integral of the differential equation (above) may fit the strength-duration data in V and t. This hypothesis is substantiated by data by Sakamoto on single fibers of the sciatic nerve of the frog. The time constant of the postulated current transient is of the order of 10^–4 sec. for single fibers and of the order of 10^–5 sec. or less in the sciatic nerve trunk. The latter value is about the same as that found by Cole in the same tissue by purely physical measurements. Some criticisms by Rushton (1934) are discussed.     

Does restoration work? It depends on how we measure success

The restoration of 4 partial stream barriers was evaluated in watersheds of Terra Nova National Park, Newfoundland, Canada from 2009 to 2011. Brook trout (n = 462) were tagged and tracked moving through our study sites using passive‐integrated transponder telemetry and the restoration actions were assessed using 3 different measures: passage success rates; the range of passable flows; and the availability of passable flows. We considered the observed results within a before‐after‐control‐impact (BACI) design that included reference reaches and pre‐restoration observations. The conclusions of BACI analyses were also contrasted with those that would have been obtained from commonly used before‐after (B‐A) or control‐impact (C‐I) study designs. While the restoration actions changed hydrological conditions in a way that should facilitate fish passage, our biological measures indicated that success was variable across culverts and within culverts depending on the measure evaluated. Furthermore, the natural temporal and spatial variability of fish movements often resulted in different conclusions between the more robust BACI design and the more commonly used B‐A and C‐I designs. Our results demonstrate that restoration of partial barriers may not always yield dramatic improvements. Furthermore, without suitable controls, the chances of drawing false conclusions regarding restorations in temporally and spatially dynamic systems are substantial.     

Systemic RNAi in western corn rootworm,Diabrotica virgifera virgifera,does not involve transitive pathways

Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is highly sensitive to orally delivered double‐stranded RNA (dsRNA). RNAi in WCR is systemic and spreads throughout the insect body. This raises the question whether transitive RNAi is a mechanism that functions in WCR to amplify the RNAi response via production of secondary siRNA. Secondary siRNA production is achieved through RNA‐dependent RNA polymerase (RdRP) activity in other eukaryotic organisms, but RdRP has not been identified in WCR and any other insects. This study visualized the spread of the RNAi‐mediated knockdown of Dv v‐ATPase C mRNA throughout the WCR gut and other tissues using high‐sensitivity branched DNA in situ hybridization. Furthermore, we did not detect either secondary siRNA production or transitive RNAi in WCR through siRNA sequence profile analysis. Nucleotide mismatched sequences introduced into either the sense or antisense strand of v‐ATPase C dsRNAs were maintained in siRNAs derived from WCR fed with the mismatched dsRNAs in a strand specific manner. The distribution of all siRNAs was restricted to within the original target sequence regions, which may indicate the lack of new dsRNA synthesis leading to production of secondary siRNA. Thus, the systemic spread of RNAi in WCR may be derived from the original dsRNA molecules taken up from the gut lumen. These results indicate that the initial dsRNA dose is important for a lethal systemic RNAi response in WCR and have implications in developing effective dsRNA traits to control WCR and in resistance management to prolong the durability of RNAi trait technology.     

Identification of drm, a novel gene whose expression is suppressed in transformed cells and which can inhibit growth of normal but not transformed cells in culture.

Using differential display analysis, we compared the expression of RNA in v-mos-transformed cells and their flat revertant and isolated a novel gene, drm (down-regulated in mos-transformed cells), whose expression is down-regulated in parental v-mos-transformed cells but which is expressed at a high level in the revertant and normal rat fibroblasts (REF-1 cells). Analysis of different oncogene-transformed cells revealed that drm gene expression was also suppressed in REF-1 cells transformed by v-ras, v-src, v-raf, and v-fos. The drm cDNA contains a 184-amino-acid-protein-encoding open reading frame which shows no significant homologies to known genes in DNA databases. Polyclonal antibodies raised against drm peptide detect a protein with the predicted size of 20.7 kDa in normal cells and under nonpermissive conditions in cells conditionally transformed by v-mos but not in parental v-mos-transformed cells. Northern analysis of normal adult tissues shows that drm is expressed as a 4.4-kb message in a tissue-specific manner, with high expression in the brain, spleen, kidney, and testis and little or no expression in the heart, liver, and skeletal muscle. In situ hybridization analysis in adult rat tissue reveals good correlation with this pattern and indicates that drm mRNA is most highly expressed in nondividing and terminally differentiated cells, such as neurons, type 1 lung cells, and goblet cells. Transfection of a drug-selectable drm expression vector dramatically reduced the efficiency of colony formation in REF-1 and CHO cells, and the drm-transfected REF-1 survivors expressed low or nondetectable levels of exogenous drm mRNA. The toxic effects of drm can be overcome by cotransfection with constructs expressing oncogenic ras; furthermore, cells expressing high levels of drm and conditionally transformed with mos-expressing Moloney murine sarcoma virus rapidly undergo apoptosis when shifted to the nonpermissive temperature. Taken together, our data suggest that cells expressing high levels of drm undergo apoptotic death in the absence of oncogene-induced transformation and that drm represents a novel gene with potential roles in cell growth control or viability and tissue-specific differentiation.     

Snake relationships revealed by slow-evolving proteins: a preliminary survey

We present an initial evaluation of relationships among a diverse sample of 215 species of snakes (8% of the world snake fauna) representing nine of the 16 commonly-recognized families. Allelic variation at four slow-evolving. protein-coding loci, detected by starch-gel electrophoresis, was found to be informative for estimating relationships among these species at several levels. The numerous alleles detected at these loci [ Arp -2 (42 alleles). Ltlh -2 (43), Mdh -1 (29), Pgm (Z)] provided unexpected clarity in partitioning these taxa. Most congeneric species and several closely-related genera have the same allele at all four loci or differ at only a single locus. At thc other extreme are those species with three or four unique alleles; these taxa cannot be placed in this analysis. Species sharing two or three distinctive alleles are those most clearly separated into clades. Typhlopids, pythonids, viperids, and elapids were resolved into individual clades. whereas bods were separated into boincs and erycines, and colubrids appeared as scveral distinct clades (colubrines, natricines, psammophines, homalopsines, and xenodontines). Viperids were recognized as a major division containing three separate clades: Asian and American crotalines. Pabearctic and Oriental viperines, and Ethiopian causines. The typhlopids were found to be the basal clade, with the North American erycine boid Chrrrino and the West Indian woodsnakes Tropidophi, Y near the base. A number of species and some small clades were not allocated because of uninformative (common, unique, or conflicting) alleles. Of the 21 S species examined, five to eight appear to have been misplaced in the analysis of these electrophoretic data.     

Environment of infants during sleep and risk of the sudden infant death syndrome: results of 1993-5 case-control study for confidential inquiry into stillbirths and deaths in infancy. Confidential Enquiry into Stillbirths and Deaths Regional Coordinators and Researchers.

OBJECTIVE--To investigate the role of sleeping arrangements as risk factors for the sudden infant death syndrome after a national risk reduction campaign. DESIGN--Two year population based case-control study. Parental interviews were conducted for each infant who died and for four controls matched for age and date of interview. SETTING--Three regions in England with a total population of 17 million people. SUBJECTS--195 babies who died and 780 matched controls. RESULTS--Prone and side sleeping positions both carried increased risks of death compared with supine when adjusted for maternal age, parity, gestation, birth weight, exposure to smoke, and other relevant factors in the sleeping environment (multivariate odds ratio = 9.00 (95% confidence interval 2.84 to 28.47) and 1.84 (1.02 to 3.31), respectively). The higher incidence of side rather than prone sleeping led to a higher population attributable risk (side 18.4%, prone 14.2%). More of the infants who died were found with bed covers over their heads (21.58; 6.21 to 74.99). The use of a dummy had an apparent protective effect (0.38; 0.21 to 0.70). Bed sharing for the whole night was a significant risk factor for infants whose mothers smoked (9.25; 2.31 to 34.02). No protective effect of breast feeding could be identified on multivariate analysis. CONCLUSIONS--This study confirms the importance of certain risk factors for the sudden infant death syndrome and identifies others-for example, covers over the head, side sleeping position-which may be amenable to change by educating and informing parents and health care professionals.