Sap flow in wheat under free-air CO2 enrichment

The effects of elevated carbon dioxide (CO₂) concentration on plant water use are best evaluated on plants grown under field conditions and with measurement techniques that do not disturb the natural function of the plant. Heat balance sap flow gauges were used on individual main stems of wheat (Triticum aestivum L. cv Yecora rojo) grown under normal ambient conditions (control) and in a free-air CO₂ enrichment (FACE) system in Arizona with either high (control + high H₂O = CW; FACE + high H₂O = FW) or low (control + low H₂O = CD; FACE + low H₂O = FD) irrigation regimens. Over a 30d period (stem elongation to anthesis), combinations of treatments were monitored with,10–40 gauges per treatment. The effects of increased CO₂ on tiller water use were inconsistent in both the diurnal patterns of sap flow and the statistical analyses of daily sap flow (F_tot). Initial results suggested that the reductions in F_tot, from CO₂ enrichment were small (,0–10%) in relation to the H₂O treatment effect (,20–30%). For a 3d period, F_tot of FW was,19–26% less than that of CW (P = 0.10). Examination of the different sources of variation in the study revealed that the location of gauges within the experimental plots influenced the variance of the sap flow measurements. This variation was probably related to positional variation in subsurface drip lines used to irrigate plots. A sampling design was proposed for use of sap flow gauges in FACE systems with subsurface irrigation that takes into account the main treatment effects of CO₂ enrichment and the other sources of variation identified in this study. Despite the small and often statistically non-significant differences in F_tot between the CW and FW treatments, cumulative water use of the FW treatment at the end of the first three test periods ranged from 7 to 23% lower than that of the CW treatment. Differences in sap flow between FW and CW compared well with treatment differences in evapotranspiration. The results of the study, based on the first reported sap flow measurements of wheat, suggest that irrigation requirements for wheat production, in the present climatic regimen of the south-western US, may be predicted to decrease slightly because of increasing atmospheric CO₂.     

Growth and recession of aquatic macrophytes on a shaded section of the River Lambourn, England, from 1971 to 1980

SUMMARY. The growth and recession of macrophytes on a shaded section of the R. Lambourn were documented by a mapping procedure. With the exception of Ranunculus spp., the changes in total cover did not indicate directly the pattern of growth and recession of the macrophyles. Analysis of gross changes, expressed as gains and losses on cover, indicated that colonization of gravel and silt by the dominant macrophyte, Berula erecta, did not vary seasonally. Colonization was at a constant rate of about 8% of the site each month throughout the year and this accounted for 50% of the total number of gains by Berula. Gains of Berula from Ranunculus showed an annual cycle with a maximum during the summer when Ranunculus was in recession. Gains of Berula from Callitriche spp. also varied annually but the maximum was during the autumn. Total losses of Berula were at a constant rate throughout the year but were to gravel and silt during the winter, to Ranunculus in spring and early summer and to Callitriche in late summer and autumn. Analysis of loss of Berula with time indicated that the position of the Berula carpet was constantly changing. The growth and recession of Berula could not be linked in a meaningful way to environmental variables. Callitriche and Ranunculus both showed an annual pattern of growth and recession. There was temporal separation of the two macrophytes with Ranunculus growing mainly in spring and early summer and Callitriche showing maximum growth in late summer and autumn, and some evidence of spatial separation. The observed differences between years in the growth of Callitriche could not be attributed to any of the environmental variables measured. Discharge was thought to be an important variable controlling the growth of Ranunculus since increase of Ranunculus in the spring was positively correlated with the mean discharge at that time. In years when discharge was low, the growth of Rununculus appeared to be restricted by shading from epiphytic algae which accumulated on the plant surfaces under these conditions.     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Major morphological changes in a Lake Victoria cichlid fish within two decades

During the upsurge of the introduced predatory Nile perch in Lake Victoria in the 1980s, the zooplanktivorous Haplochromis ( Yssichromis ) pyrrhocephalus nearly vanished. The species recovered coincident with the intense fishing of Nile perch in the 1990s, when water clarity and dissolved oxygen levels had decreased dramatically due to increased eutrophication. In response to the hypoxic conditions, total gill surface in resurgent H. pyrrhocephalus increased by 64%. Remarkably, head length, eye length, and head volume decreased in size, whereas cheek depth increased. Reductions in eye size and depth of the rostral part of the musculus sternohyoideus, and reallocation of space between the opercular and suspensorial compartments of the head may have permitted accommodation of larger gills in a smaller head. By contrast, the musculus levator posterior, located dorsal to the gills, increased in depth. This probably reflects an adaptive response to the larger and tougher prey types in the diet of resurgent H. pyrrhocephalus . These striking morphological changes over a time span of only two decades could be the combined result of phenotypic plasticity and genetic change and may have fostered recovery of this species.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 41–52.     

Early evolution of metazoan serine/threonine and tyrosine kinases: identification of selected kinases in marine sponges

The phylum Porifera (sponges) was the first to diverge from the common ancestor of the Metazoa. In this study, six cDNAs coding for protein- serine/threonine kinases (PS/TKs) are presented; they have been isolated from libraries obtained from the demosponges Geodia cydonium and Suberites domuncula and from the calcareous sponge Sycon raphanus. Sequence alignments of the catalytic domains revealed that two major families of PS/TK, the "conventional" (Ca(2+)-dependent) protein kinase C (PKC), the cPKC subfamily, as well as the "novel" (Ca(2+)- independent) PKC (nPKC), form two separate clusters. In each cluster, the sequence from S. raphanus diverges first. To approach the question about the origin of protein-tyrosine kinases (PTK), which are found only in Metazoa, we analyzed two additional PS/TKs which have been cloned from S. domuncula: the stress-responsive protein kinase (KRSvSD) and the protein-kinase-C-related kinase (PRKvSD). The construction of the phylogenetic tree, comprising the eight PS/TKs and the PTK cloned previously from G. cydonium, revealed that the PTK derived from the branch including the KRSvSD kinase. These data facilitate the first molecular approach to elucidate the origin of metazoan PTK within the PS/TK superfamily.      

Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.     

Bovine mammary gland UDP-GalNAc:GlcNAcbeta-R beta1-->4-N- acetylgalactosaminyltransferase is glycoprotein hormone nonspecific and shows interaction with alpha-lactalbumin

We have identified a novel N -acetylgalactosaminyltransferase activity in lactating bovine mammary gland membranes. Acceptor specificity studies and analysis of products obtained in vitro by 400 MHz1H-NMR spectroscopy revealed that the enzyme catalyses the transfer of N - acetylgalactosamine (GalNAc) from UDP-GalNAc to acceptor substrates carrying a terminal, beta-linked N -acetylglucosamine (GlcNAc) residue and establishes a beta1-->4-linkage forming a GalNAcbeta1-->4GlcNAc ( N, N '-diacetyllactosediamine, lacdiNAc) unit. Therefore, the enzyme can be identified as a UDP-GalNAc:GlcNAcbeta-R beta1-->4-N- acetylgalactosaminyltransferase (beta4-GalNAcT). This enzyme resembles invertebrate beta4-GalNAcT as well as mammalian beta4- galactosyltransferase (beta4-GalT) in acceptor specificity. It can, however, be clearly distinguished from the pituitary hormone-specific beta4-GalNAcT by its incapability of acting with an elevated activity on a glycoprotein substrate carrying a hormone-specific peptide motif. Furthermore, the GalNAcT activity appeared not to be due to a promiscuous action of a beta4-GalT as could be demonstrated by comparing the beta4-GalNAcT and beta4-GalT activities of the mammary gland, bovine colostrum, and purified beta4-GalT, by competition studies with UDP-GalNAc and UDP-Gal, and by use of an anti-beta4-GalT polyclonal inhibiting antibody. Interestingly, under conditions where mammalian beta4-GalT forms with alpha-lactalbumin (alpha-LA) the lactose synthase complex, the mammary gland beta4-GalNAcT was similarly induced by alpha-LA to act on Glc with an increased efficiency yielding the lactose analog GalNAcbeta1-->4Glc. This enzyme thus forms the second example of a mammalian glycosyltransferase the specificity of which can be modified by this milk protein. It is proposed that the mammary gland beta4-GalNAcT functions in the synthesis of lacdiNAc- based, complex-type glycans frequently occurring on bovine milk glycoproteins. The action of this enzyme is to be considered when aiming at the production of properly glycosylated protein biopharmaceuticals in the milk of transgenic dairy animals.      

Factors that drive the increasing use of FFPE tissue in basic and translational cancer research

The decision to use 10% neutral buffered formalin fixed, paraffin embedded (FFPE) archival pathology material may be dictated by the cancer research question or analytical technique, or may be governed by national ethical, legal and social implications (ELSI), biobank, and sample availability and access policy. Biobanked samples of common tumors are likely to be available, but not all samples will be annotated with treatment and outcomes data and this may limit their application. Tumors that are rare or very small exist mostly in FFPE pathology archives. Pathology departments worldwide contain millions of FFPE archival samples, but there are challenges to availability. Pathology departments lack resources for retrieving materials for research or for having pathologists select precise areas in paraffin blocks, a critical quality control step. When samples must be sourced from several pathology departments, different fixation and tissue processing approaches create variability in quality. Researchers must decide what sample quality and quality tolerance fit their specific purpose and whether sample enrichment is required. Recent publications report variable success with techniques modified to examine all common species of molecular targets in FFPE samples. Rigorous quality management may be particularly important in sample preparation for next generation sequencing and for optimizing the quality of extracted proteins for proteomics studies. Unpredictable failures, including unpublished ones, likely are related to pre-analytical factors, unstable molecular targets, biological and clinical sampling factors associated with specific tissue types or suboptimal quality management of pathology archives. Reproducible results depend on adherence to pre-analytical phase standards for molecular in vitro diagnostic analyses for DNA, RNA and in particular, extracted proteins. With continuing adaptations of techniques for application to FFPE, the potential to acquire much larger numbers of FFPE samples and the greater convenience of using FFPE in assays for precision medicine, the choice of material in the future will become increasingly biased toward FFPE samples from pathology archives. Recognition that FFPE samples may harbor greater variation in quality than frozen samples for several reasons, including variations in fixation and tissue processing, requires that FFPE results be validated provided a cohort of frozen tissue samples is available.