bluuebunny/crossref_metadata_2025 huggingface dataset:
CELLULAR SIGNALLING AFTER IN VIVO HEAT SHOCK IN THE LIVER
Authors: Maroni Paola , Bendinelli Paola , Zuccorononno Cinzia , Schiaffonati Luisa , Piccoletti Roberta
Published: September 2002

In an experimental model of in vivo hyperthermia, we investigated the involvement of a number of signalling events in rat liver. We report that in vivo heat shock causes a powerful activation of c‐Jun N‐terminal kinase and p38 kinase but does not trigger poly(ADP‐ribose) polymerase cleavage, a signature event of apoptosis. Among the upstream regulators of the kinases, we show that stress‐activated protein kinase/extracellular signal‐regulated kinase/nitrogen‐activated protein kinase kinase 4 SEK1/MKK4 is not involved whereas MKK3 and/or MKK6 are activated. PAK activity displays a transient rise, whereas GCK does not change. PI3‐kinase activity increases in anti‐phosphotyrosine immunoprecipitates, suggesting a tyrosine kinase‐dependent induction mechanism, and the co‐immunoprecipitation of PI3‐kinase with p60 Src kinase supports the involvement of this latter. GSK3, which may act downstream to PI3‐kinase through AKT, undergoes hyperphosphorylation, thus playing a possible role in the protection from apoptosis and in the modulation of heat‐shock transcription factor activity.
EFFECT OF SERUM WITHDRAWAL ON THE PROLIFERATION OF B16F10 MELANOMA CELLS
Authors: Rodriguez‐Ayerbe Clara , Smith‐Zubiaga Isabel
Published: September 2002

B16F10 murine melanoma cell proliferation was inhibited after 48h in medium with serum in the range 0.1 to 0.5% by volume. Cell viability was mostly retained, whereas cells completely deprived of serum died. Growth‐arrested cultures showed serum‐dependent suppression of DNA synthesis. The response was typically that of a ‘cell cycle freeze’, verified by flow cytometric distribution of cells. Consequently, serum deprivation did not lead to synchony when serum was restored to arrested populations. Furthermore, there was no change in PCNA expression in arrested cells.
INFLUENCE OF INHIBITORS OF CYTOSKELETON PROTEINS ON WATER EXCHANGE OF WHEAT ROOTS UNDER THE AFTER‐ACTION OF WATER STRESS
Authors: Volobuyeva O. V. , Khokhlova L. P. , Ganiyeva G. R. , Velikanov G. A.
Published: September 2002

The dynamics of water molecular state and transport in winter wheat (Triticum aestivum L.) of roots different resistance cultivars was studied by a biophysical method, Nuclear Magnetic Resonance (NMR), and a physiological method, Water‐Holding Capacity (WHC). The effective coefficient of water self‐diffusion (Deff), spin—spin relaxation times (T2) and WHC were measured after structural modification of cytoskeleton by colchicine and cytochalasin B after the action of water stress. New information about molecular mechanisms of water state and water transport regulation determined by the influence of dynamic cytoskeleton structure has been obtained. This is very important for the development of a fundamental theory of water exchange in plants, and for the ways of its optimization under conditions of environmental stress.
HELICOBACTER PYLORI VACUOLATING CYTOTOXIN (VacA) ALTERS CYTOSKELETON‐ASSOCIATED PROTEINS AND INTERFERES WITH RE‐EPITHELIALIZATION OF WOUNDED GASTRIC EPITHELIAL MONOLAYERS
Authors: Pai Rama , Sasaki Eiji , Tarnawski Andrzej S.
Published: September 2002

In previous studies, we demonstrated that Helicobacter pylori vacuolating cytotoxin (VacA) inhibits gastric epithelial cell proliferation and inhibits epidermal growth factor (EGF)‐activated signal transduction. Cell proliferation and migration, both essential for mucosal healing are dependent on the cell cytoskeleton. Other investigators demonstrated that VacA induces vacuolation of eukaryotic cells. Since in some cells, control of actin cytoskeleton involves GTP‐binding proteins of Rho family, in this study we examined whether VacA affects wound re‐epithelialization, cell cytoskeleton‐associated proteins Rho, Rac1 in a gastric epithelial (RGM1) cell monolayer wound model, and whether these changes correlate with vacuolation. VacA treatment significantly inhibited wound re‐epithelialization, cell proliferation vs control. VacA‐induced cell vacuolation strongly correlated with inhibition of wound re‐epithelialization. Furthermore, VacA reduced Rac‐1 protein expression and distribution, and C3‐mediated ADP‐ribosylation of Rho. These findings suggest that VacA may interfere with repair of gastric mucosal injury and ulcer re‐epithelialization by altering cytoskeleton‐dependent cell functions and signaling.
REPLICATION PROTEIN A 32kDa SUBUNIT (RPA p32) BINDS THE SH2 DOMAIN OF STAT3 AND REGULATES ITS TRANSCRIPTIONAL ACTIVITY
Authors: Kim Jeonghoon , Kim Dohoon , Chung Jongkyeong
Published: September 2002

STATs (signal transducers and activators of transcription) are transcription factors that contain SH2 domains and are activated by tyrosine phosphorylation in response to cytokines and growth factors. Replication protein A (RPA) is a heterotrimeric complex that consists of three subunits, p70, p32 and p11, and has important functions in DNA replication and metabolism. Here, we present evidence that the RPA p32 subunit binds specifically to the SH2 domain of STAT3 in a phosphotyrosine‐independent manner. We confirm their protein—protein interactions by yeast 2‐hybrid analyses and in vitro binding assays using recombinant proteins generated from bacteria and in vitro translation. We also show that STAT3 binds to RPA p32 in vivo by conducting co‐precipitation experiments. As the SH2 domain is highly involved in the tyrosine phosphorylation and the transcriptional activity of STAT3, over‐expression of RPA p32 correspondingly augmented growth factor‐stimulated tyrosine phosphorylation and transcription activities of STAT3.
C/EBPα AND C/EBPβ ARE PERSISTENTLY ASSOCIATED WITH THE RAT LIVER NUCLEAR MATRIX THROUGHOUT DEVELOPMENT AND THE ACUTE PHASE RESPONSE
Authors: Dinic Svetlana , Ivanovic‐Matic Svetlana , Bogojevic Desanka , Mihailovic Mirjana , Poznanovic Goran
Published: September 2002

The partitioning of C/EBPα and C/EBPβ on the nuclear matrix structure was examined during the different transcriptional activities accompanying liver development and the acute phase (AP) response. The presence of C/EBPα and C/EBPβ was established on the nuclear matrix. Their relative concentrations on the matrix always reflected the developmental stage‐ and AP‐related fluctuations observed in the nuclear extract. Thus, they progressively increased as development proceeded, whereas during the AP response, C/EBPα decreased and C/EBPβ increased. In addition, the levels of both transcription factors were always notably higher in the nuclear matrix than in the extracts. We conclude that the observed changes and overall enrichment of the nuclear matrix with regulatory proteins is a reflection of the importance of such interactions for the in vivo functioning of C/EBP proteins.
COMPARISON BETWEEN THE IN VIVO AND IN VITRO EXPRESSION OF THREE ADHESION‐SIGNALING PROTEINS IN EMBRYONIC CELLS
Authors: Ridyard Marc S. , Sanders Esmond J.
Published: September 2002

We have examined the relationship between the in vivo and in vitro expression of three adhesion‐signaling proteins (FAK, PYK2 and Paxillin), using cells of the early chick embryo, where pure cell populations may be isolated and cultured, and in which epithelial‐to‐mesenchymal transformation is occurring. Focal Adhesion Kinase (FAK) and Proline‐rich Tyrosine Kinase‐2 (PYK2) are related in molecular structure, and may have some overlapping functions in signal transduction associated with cell‐substratum adhesion. Paxillin, a cytoskeletal protein, is also localized to focal adhesions. We show that the immunocytochemical detection of these molecules in vivo does not reflect their in vitro localization. Focal Adhesion Kinase showed a developmentally regulated localization to the cytoplasm, but not to sites of adhesion, in epithelial cells in vivo, while Paxillin was associated with migrating mesoderm cells. Proline‐rich Tyrosine Kinase‐2 was undetectable in vivo. The level of expression of these molecules was compared under in vivo and in vitro conditions. While the expression of Focal Adhesion Kinase showed a tissue‐specific regulation of expression with the change to in vitro conditions, Proline‐rich Tyrosine Kinase‐2 showed a more uniform and less tissue‐specific up‐regulation. Levels of Paxillin expression also showed an increase with this change in conditions. We conclude that despite the structural and functional relationships between these three molecules in the developing embryo, the expression and localization of each is independently regulated. We suggest that this provides these cells with the adaptability that they require in order to respond to the changing extracellular environment in the early embryo, and to undergo epithelial‐to‐mesenchymal transformation.
INFLUENCE OF MEMBRANE FLUIDITY MODIFIERS ON LYSOSOMAL OSMOTIC SENSITIVITY
Authors: Yang Lu , Zhang Guo‐Jiang , Zhong Yi‐Gang , Zheng Yan‐Zhen
Published: September 2002

Since lysosomes are prone to osmotic lysis, we have examined the correlation between their physical state and sensitivity to osmotic challenge, using agents which modify membrane fluidity. The latency loss of β‐hexosaminidase after an incubation in hypotonic sucrose medium was followed under different conditions of membrane fluidity, recorded by steady‐state fluorescence anisotropy of 1,6‐diphenyl‐1,3,5‐hexatriene. Increasing fluidity of the lysosomal membranes with benzyl alcohol (BA) and greater rigidity caused by cholesteryl hemisuccinate (CHS) increased and decreased the enzyme latency loss, respectively. The effects of BA and CHS treatments on osmotic sensitivity were reversible subsequently by reciprocal treatments of the lysosomes with CHS and BA, respectively. The results indicate that the physical state of the membrane does indeed affect lysosomal osmotic stability.
MULTIPLE RESPIRATORY BURSTS AS A RESPONSE TO VERATRATE STRESS IN RHODOCOCCUS ERYTHROPOLIS CELLS
Authors: Malarczyk Elzbieta , Paździoch‐Czochra Marzanna
Published: September 2002

Although Rhodococcus spp. strains are able to degrade methoxyphenols by enzymatic means, the contact with veratric acid (3,4‐dimethoxybenzoic acid, hereafter called veratrate) is very stressful for the cells ofRhodococcus erythropolis DSM 1069 (Rh). Within 5min of contact veratrate in phosphate buffer, the emergence of many vacuoles was observed in the cell body and respiratory bursts, with violent endogenous oxygen uptake, took place several times during the 24h incubation. During these peaks (where the cells were in their MAX states), increased activity of NADH oxidase was noted, accompanied by maximal accumulation of vanillic and isovanillic acids (3‐methoxy‐4‐hydroxybenzoic acid and 4‐hydroxy‐3‐methoxybenzoic acid respectively, hereafter called vanillates) in the incubation medium, which appeared to be products of veratrate demethylation. At the troughs (cell in their MIN state), the vacuoles disappeared from the cell body, oxygen uptake was normal, and the pool of vanillates decreased while the veratrate level in the medium increased. The cells from MAX and MIN states reacted in opposite ways in the presence of either formaldehyde and GSH, or paraquate and cAMP. The NADH oxidase activity, measured as oxygen uptake against NADH in the membrane pellets of MAX and MIN stage cells, differed in their response to the exogenous presence of FAD, ATP, cAMP, catalase, GSH, H2O2and methoxyphenolic substrates. The periodic character of these events is described here. Co‐operation between two multiprotein membrane complexes (NAD(P)H oxidase and 3‐O/4‐O‐demethylases) in Rhodococcus erythropolis cells and their competition for two common substrates—NAD(P)H and O2—is proposed as an explanation for rhythmical nature of these reactions.
RHYTHMIC PATTERNS IN THE EXPRESSION OF THE ras ONCOGENE IN PROLIFERATING AND DIFFERENTIATING ERYTHROLEUKAEMIA CELLS
Authors: Hammond K. D. , Savage N. , Littlewood M.
Published: September 2002

Temporal variations in the expression of the ras oncogene, and its protein product, were investigated during hexamethylene bisacetamide (HMBA)‐induced differentiation of murine erythroleukaemic (MEL) cells. We highlight the fact that when comparisons were made between untreated, proliferating cells and HMBA‐treated, differentiating cells using only one time‐point, differences, both for the expression of the gene and the protein, were in most cases insignificant; standard deviations were high and the interpretation could be made that HMBA had little effect. Such interpretation fails to take account of the dynamic nature of the system, with single time‐point studies giving incomplete information, which can be misleading. Multiple time analyses showed clearly that rhythmic patterns of expression were modulated by the differentiating agent. Time‐dependent changes in the expression of mRNA specific to H‐ ras and N‐ ras, as well as in the expression of the Ras protein, when measured over periods of minutes or hours, were apparent. HMBA affected frequency and phasing of the rhythms. Regulation of the dynamics in this way may be crucial to the control of cell function and transformation.
CYTOTOXICITY OF DAUNORUBICIN IN TRISOMIC (+21) HUMAN FIBROBLASTS: RELATION TO DRUG UPTAKE AND CELL MEMBRANE FLUIDITY
Authors: Przybylska Maria , Koceva‐Chyla Aneta , Rózga Blazej , Józwiak Zofia
Published: September 2002

The influence of daunorubicin (DNR) on survival of human normal (S‐126) and trisomic, with respect to chromosome 21 (T‐164; S‐240), skin fibroblasts and some parameters related to it, such as intracellular drug accumulation, distribution and interaction with cell membrane, were studied. The in vitro growth‐inhibition assay indicated that DNR was less cytotoxic for trisomic than for normal cells. Comparison of kinetic parameters and intracellular distribution of this compound showed that the uptake and the amount of intracellular free DNR were greater in normal than in trisomic cells. Contrary to this, there were no significant differences between the amount of DNA‐bound drug in both types of cells. TMA‐DPH and 12‐AS fluorescence anisotropy measurements demonstrated that DNR decreased lipid fluidity in the inner hydrophobic region of plasma membrane in both cell types, but did not influence the fluidity of the outer surface of membrane. We conclude that fibroblasts derived from individuals affected with Down's syndrome are better protected from the damage induced by DNR than normal cells.
CIRCADIAN RHYTHM OF DNA SYNTHESIS AND MITOTIC ACTIVITY IN TONGUE KERATINOCYTES
Authors: García M.N. , Barbeito C.G. , Andrini L.A. , Badrán A.F.
Published: September 2002

Tongue keratinocytes have a high mitotic index (MI) with an evident circadian variation. Our study set out to compare and contrast two phases of the cell cycle: DNA synthesis (S‐phase), with inmunocytochemical detection by bromodeoxyuridine (BrdU), and mitosis (M‐phase), by the colchicine‐arrest of metaphase method, exploring both the dorsal and ventral surfaces of the mouse tongue throughout a circadian period. Adult male mice standardized for light periodicity used for MI experiment were injected intraperitoneally with colchicine. Other animals were injected intraperitoneally with 5‐BrdU for S‐phase determination. Animals given both treatments were divided into six groups and killed at 4h intervals until 20:00h. Tongue samples were processed for histology and immuno‐histochemistry. S and M indices were expressed as labelled nuclei or colchicine metaphases, respectively, per 1000 nuclei. Peak MI occurred at 12:00, with the minimum value at 20:00 on dorsal and ventral tongue surfaces. Peak S‐phase was at 04:00, whereas the minimum value was at 16:00 for both surfaces. These results show that the proliferative activity of the tongue epithelium is of similar intensity and temporal distribution on both surfaces.
S100‐ANNEXIN COMPLEXES: SOME INSIGHTS FROM STRUCTURAL STUDIES
Authors: Lewit‐Bentley Anita , Réty Stéphane , Sopkova‐de Oliveira Santos Jana , Gerke Volker
Published: September 2002

Several annexins have been shown to bind proteins that belong to the S100 calcium‐binding protein family. The two best‐characterized complexes are annexin II with p11 and annexin I with S100C, the former of which has been implicated in membrane fusion processes. We have solved the crystal structures of the complexes of p11 with annexin II N‐terminus and of S100C with annexin I N‐terminus. Using these structural results, as well as electron microscopy observations of liposome junctions formed in the presence of such complexes (Lambert et al., 1997 J Mol Biol 272, 42–55), we propose a computer generated model for the entire annexin II/p11 complex.
THE t‐SNARE SYNTAXIN IS SUFFICIENT FOR SPONTANEOUS FUSION OF SYNAPTIC VESICLES TO PLANAR MEMBRANES
Authors: Woodbury Dixon J. , Rognlien Kathie
Published: September 2002

Vesicular trafficking and exocytosis are directed by the complementary interaction of membrane proteins that together form the SNARE complex. This complex is composed of proteins in the vesicle membrane (v‐SNAREs) that intertwine with proteins of the target membrane (t‐SNAREs). Here we show that modified synaptic vesicles (mSV), containing v‐SNAREs, spontaneously fuse to planar membranes containing the t‐SNARE, syntaxin 1A. Fusion was Ca2+‐independent and did not occur with vesicles lacking v‐SNAREs. Therefore, syntaxin alone forms a functional fusion complex with v‐SNAREs. Our functional fusion assay uses synaptic vesicles that are modified, so each fusion event results in an observable transient current. The mSV do not fuse with protein‐free membranes. Additionally, artificial vesicles lacking v‐SNAREs do not fuse with membranes containing syntaxin. This technique can be adapted to measure fusion in other SNARE systems and should enable the identification of proteins critical to vesicle—membrane fusion. This will further our understanding of exocytosis and may improve targeting and delivery of therapeutic agents packaged in vesicles.
THY‐1 ASSOCIATED PP85–90 IS A POTENTIAL DOCKING SITE FOR SH2 DOMAIN‐CONTAINING SIGNAL TRANSDUCTION MOLECULES
Authors: Durrheim Glenda A. , Garnett David , Dennehy Kevin M. , Beyers Albert D.
Published: September 2002

Thy‐1, a glycosylphosphatidylinositol (GPI)‐anchored glycoprotein expressed at high levels on thymocytes, has been implicated in positive and negative signal transduction. We show that Thy‐1 associates with a protein of 85–90kDa, which is prominently phosphorylated in vitro as well as in vivo following the stimulation of thymocytes with pervanadate. pp85–90 is not identical to known proteins that are phosphorylated following T cell activation. The SH2 domains of fyn, csk, phosphatidylinositol 3′‐kinase, rasGAP, vav and lck bind to pp85–90 with varying affinities. The SH2 domains of ZAP70, SHP‐1 and PLCγ1 and the SH3 domains of lck, vav and HS1 did not bind to pp85–90. The molecular weight, iso‐electric point, efficient phosphorylation by fyn and lck and preferential binding to the SH2 domain of fyn compared to that of lck indicate that Thy‐1‐associated pp85–90 may be identical to a recently cloned, fyn‐associated transmembrane adaptor protein, PAG‐85.
FLOW CYTOMETRIC ANALYSIS OF THE TH1–TH2 BALANCE IN HEALTHY INDIVIDUALS AND PATIENTS INFECTED WITH THE HUMAN IMMUNODEFICIENCY VIRUS (HIV) RECEIVING A PLANT STEROL/STEROLIN MIXTURE
Authors: Breytenbach Ulrike , Clark Anel , Lamprecht Johan , Bouic Patrick
Published: September 2002

The Th1–Th2 balance plays a pivotal role in determining the outcome of an immune response to an infectious organism. It is proposed that during HIV infection, disease progression is characterized by a loss of Th1 activity, a shift to a more ‘allergic’ Th2‐type response and hence loss of cytotoxic cell activity against infected host cells. This study was undertaken to investigate this balance in three groups of individuals: HIV‐negative volunteers (n=10), a group of HIV‐infected patients on no therapy (n=10) as well as a group of patients managed with a mixture of plant sterols/sterolins (n=9). In parallel, their response to mitogens and the subsequent expression of the activation antigen CD69 was measured. This study was conducted by three‐colour flow cytometry in order to obviate the less sensitive cytokine secretion assays that have yielded controversial results. The results indicate that HIV‐infected patients on no therapy exhibit a pre‐dominant Th2 response (IL‐4 secretion), whereas those on the sterol/sterolin mixture exhibit a beneficial Th1 response (IFN‐γ). Surprisingly, in both patient groups, the expression of CD69 was abnormally low when compared to the uninfected volunteers, implying that chronic activation is already present in vivo. It appears that the detrimental Th2 driven response might be swung to the more beneficial Th1 response with the immune modulatory sterols/sterolin mixture. Clinical use of this mixture in HIV infection has yielded results which corroborate the above observations in that patients using the plant sterol/sterolin mixture maintain their CD4 cell numbers over an extended period of time in the absence of any anti‐retroviral therapy.
CELL DEATH IN TETRAHYMENA THERMOPHILA: NEW OBSERVATIONS ON CULTURE CONDITIONS
Authors: Christensen Søren T. , Sørensen Heidi , Beyer Natascha H. , Kristiansen Karsten , Rasmussen Leif , Rasmussen Morten I.
Published: September 2002

We previously suggested that the cell fate of the protozoan ciliate, Tetrahymena thermophila, effectively relates to a quorum‐sensing mechanism where cell‐released factors support cell survival and proliferation. The cells have to be present above a critical initial density in a chemically defined nutrient medium in order to release a sufficient level of these factors to allow a new colony to flourish. At a relatively high rate of metabolism and/or macromolecular synthesis and below this critical density, cells began to die abruptly within 30min of inoculation, and this death took the form of an explosive disintegration lasting less than 50 milliseconds. The cells died at any location in the culture, and the frequency of cell death was always lower in well‐filled vials than those with medium/air interface. Cell death was inhibited by the addition of Actinomycin D or through modifications of the culture conditions either by reducing the oxygen tension or by decreasing the temperature of the growth medium. In addition, plastic caps in well‐filled vials release substances, which promote cell survival. The fate of low‐density cultures is related to certain ‘physical’ conditions, in addition to the availability of oxygen within closed culture systems.
LINOLEIC AND LINOLELAIDIC ACIDS DIFFERENTIALLY INFLUENCE PROLIFERATION AND APOPTOSIS OF MOLT‐4 LEUKAEMIA CELLS
Authors: Phoon M. C. , Desbordes Charles , Howe J. , Chow Vincent T. K.
Published: September 2002

The effects of varying concentrations of linoleic acid and its transisomer linolelaidic acid on the proliferation the ultrastructural morphology of MOLT‐4 T‐lymphoblastic leukaemia cells were investigated. At 2 and 4 days after exposure to the fatty acids, the cells were counted by flow cytometry and observed by electron microscopy. After 4 days of treatment, linoleic acid was growth stimulatory at concentrations of 200μm or less, but was markedly inhibitory at 400μm. In contrast, linolelaidic acid stimulated proliferation at concentrations of 100 and 200μm, but inhibited cell growth at 400μm. Cells treated with 400μm linoleic acid displayed dense accumulations of characteristic lipid globules and glycogen granules, and exhibited ultrastructural evidence of apoptosis including vacuolization, membrane blebbing and chromatin margination at the nuclear periphery. These results support the notion that geometrical isomerism and concentration of polyunsaturated fatty acids influence the proliferative destiny of cancer cells. Reverse transcription polymerase chain reaction (RT‐PCR) analysis revealed a previously documented larger alternatively spliced p53 gene transcript in MOLT‐4 cells cultured under reduced serum conditions. However, only wild‐type p53 transcripts were amplified by RT‐PCR of MOLT‐4 cells exposed to phytohaemagglutinin, linoleic acid or linolelaidic acid.
CHARACTERISATION OF MYOGENIC CELL MEMBRANE: II. DYNAMIC CHANGES IN MEMBRANE LIPIDS DURING THE DIFFERENTIATION OF MOUSE C2 MYOBLAST CELLS
Authors: Nakanishi Masami , Hirayama Etsuko , Kim Jeman
Published: September 2002

Proliferating mouse C2 myoblast cells resist haemagglutinating virus of Japan, Sendai virus (HVJ) mediated cell fusion. However, differentiating C2 cells can be induced to fuse by HVJ, suggesting that the rigid membrane of C2 cells changes during the differentiation. To investigate this phenomenon, changes in membrane lipids which affect fluidity were examined. Membrane cholesterol gradually decreased with the differentiation of C2 cells. However, spontaneous fusion to form myotubes and artificial fusion induced by HVJ were both inhibited when the level of cholesterol was prevented from falling in the cell membrane. The membranes of differentiating C2 cells contained more unsaturated fatty acids than those of proliferating cells. Thus, when differentiating C2 cells were treated with stearate (a saturated fatty acid), they failed to form myotubes and were insensitive to HVJ‐mediated fusion. Whereas, if proliferating C2 cells were given linolenate (an unsaturated fatty acid), they became capable of HVJ‐induced fusion. These results indicate that differentiating C2 cells change their fusion sensitivity by decreasing cholesterol, probably at the same time as they increase the unsaturated fatty acid content of the cell membrane.
IMMUNOGOLD LABELLING OF FIBROBLAST FOCAL ADHESION SITES VISUALISED IN FIXED MATERIAL USING SCANNING ELECTRON MICROSCOPY, AND LIVING, USING INTERNAL REFLECTION MICROSCOPY
Authors: Richards R. G. , Stiffanic M. , Owen G. Rh , Riehle M. , Gwynn Ap I. , Curtis A. S. G.
Published: September 2002

A new immunogold labelling method for the visualisation of vinculin, an integral protein in focal adhesions of cells, is reported. Quantification of vinculin is indicative of substrate cytocompatibility (cytocompatibility is one aspect of biocompatibility; it is the cellular response to a biomaterial). For efficient labelling, most of the cell body above the cell—substrate interface was removed with detergent. The antigen blocking procedure, size of label (5nm) and duration of silver‐enhancement (6min), for visualisation of the labelled sites on the whole cell by scanning electron microscopy (SEM), were determined. Imaging living cells with interference reflection light microscopy, followed by backscattered electron (BSE) imaging of the same fixed and immunolabelled cells confirmed the results. Collecting low voltage BSE images of embedded cells after the substrate had been removed provided ‘sectional’ views through the cell. This enabled visualisation of vinculin exclusively within the cell‐substrate contact zone; the focal adhesions. The method could be of general use in the imaging of protein distribution at biological tissue/substrate interfaces.
Viruses infecting canola (Bassica napus) in south-west Australia: incidence, distribution, spread, and infection reservoir in wild radish (Raphanus raphinistrum)
Authors: Coutts B. A. , Jones R. A. C.
Published: September 2002

Over 2 growing seasons, the incidences of infection with beet western yellows
(BWYV), cauliflower mosaic (CaMV), and turnip mosaic (TuMV) viruses were
determined in canola (Brassica napus) crops growing in
the agricultural area of south-west Australia. Tissue blot immunoassay was
used to detect BWYV and enzyme-linked immunosorbent assay to detect CaMV and
TuMV. In 1998, BWYV was detected in 59% of 159 crops surveyed, whereas
in 1999 it was found in 66% of 56 crops. Incidences within individual
infected crops were 1–65% (1998) and 1–61% (1999).
Infection occurred widely in high and medium rainfall zones, but was also
readily detected in the low rainfall zone. In addition, BWYV was found in
canola samples from 5 sites in New South Wales. Most cultivars tested (9 of
10) in the canola crop survey were infected with BWYV. No clear relationship
was found between BWYV infection and any particular type of disease symptom.
Overall, the incidence of BWYV at the crop edge was marginally greater than
that inside the crop. CaMV was detected in 27% of 143 crops in 1998 but
in only 2 of 47 in 1999. Incidences within individual infected crops were
1–17% in 1998 but only 1% in 1999. CaMV infected 6 of 10
cultivars and was present in high, medium, and low rainfall zones. Obvious
chlorotic ringspot symptoms were associated with CaMV infection. TuMV was
detected in 5% of 139 crops in 1998 but in only 1 of 47 from 1999.
Incidences within the individual infected crops were 1–5% in 1998
and 1% in 1999; 3 of 10 cultivars were infected and it was found in
high and medium rainfall zones.
BWYV, CaMV, and TuMV were all found infecting wild radish
(Raphanus raphinistrum). In general, incidences of BWYV
were greater in wild radish than in canola. In 1998, BWYV was detected in wild
radish at 9 of 12 sites sampled in 5 of 6 districts, with infection incidences
up to 48%. In 1999, it was detected at all 10 sites sampled in 7
districts, with incidences up to 96%. Infected samples came from all
rainfall zones, and from several different types of sites, some of which were
distant from canola crops. Despite the presence of possible viral symptoms in
wild radish, none was clearly associated with BWYV infection. In contrast,
TuMV caused obvious mottle and ‘oak leaf’ patterns in wild radish
plants.
The finding of widespread virus infection in canola crops and a substantial
virus reservoir in wild radish weeds is cause for concern to the canola
industry.
Degradability of crude protein from clover herbages used in irrigated dairy production systems in northern Victoria
Authors: Cohen D. C.
Published: September 2002

An in sacco (nylon bag) technique was used to estimate
the degradability of dry matter (DM) and crude protein, and to estimate the
effective rumen degradability of protein (ERDP), for 3 irrigated clover
herbages. Pasture characteristics (nutritive values and leaf: stem ratios)
were also described, and relationships to ERDP established. The nutritive
value characteristics and degradabilities of white clover
(Trifolium repens L.), Persian clover
(Trifolium resupinatum L.), and subterranean clover
(Trifolium subterraneum L.) were also compared for
various regrowth periods (of 3, 4, 6, and 12 weeks). Nutritive value
characteristics of clover herbages varied from 9.9 to 11.9 MJ/kg DM for
metabolisable energy, 153 to 304 g/kg DM for crude protein, and 209 to 377
g/kg DM for neutral detergent fibre. The leaf: stem ratio correlated well
with the crude protein content of the clovers, with herbages consisting of
more leaf generally having superior crude protein content
(R2 = 0.64,
P < 0.001).
The effective rumen degradability of protein for clover herbages ranged from
60 g/kg DM for mature (12 week regrowth) subterranean clover to 195
g/kg DM for vegetative (3 weeks regrowth) Persian clover. For clover
herbages with a 3-week regrowth period, after initial cutting to 5 cm, and at
ruminal outflow rates of 0.08/h, the effective rumen degradability of
protein varied from 136 to 195 g/kg DM. A positive relationship between
crude protein and ERDP (R2
= 0.82) suggested that ERDP could be estimated for clover herbages that
have been previously assessed for crude protein content, obviating the need
for in sacco studies. Calculated degradabilities were
lower for all clovers when higher ruminal outflow rates were assumed.
Using the metabolisable protein system, metabolisable protein supply and
metabolisable protein in excess of animal requirements were calculated.
Ruminal losses of nitrogen were also estimated for cows consuming white clover
of varying regrowth periods in both early and late lactation. It was concluded
that metabolisable protein supply is unlikely to limit production in these
examples. At regrowth periods of 3 weeks, metabolisable protein contributions
from microbial and dietary sources were similar, and twice that required by
the animal. Ruminal losses of nitrogen were substantial and amounted to
66—23% of nitrogen intake. In the experiments reported here,
if all of the energy required to excrete excess protein (as urea) could
instead be used for the production of milk, cows may have produced
0.5mp;mdash;2.0 kg more milk per day. Such losses could potentially be
reduced if the protein content and/or degradabilities of clover herbages
were reduced, and/or energy rich supplements were offered.
Increases in grain yield of wheat by breeding for an osmoregulation gene: relationship to water supply and evaporative demand
Authors: Morgan J. M.
Published: September 2002

The effect of water stress on yield increases produced by breeding for an
osmoregulation gene (or) was examined using both
backcross-bred lines (produced using allele identification in pollen grains)
and inbred lines (produced using leaf tests). Yields were measured in 39 field
experiments spanning 8 seasons. These included experiments where water was
supplied through drip irrigators and rain excluded with a mobile shelter.
Several approaches to the measurement of stress environment were examined. The
commonly used site mean yield, although most accessible and so utilising the
most data, was least effective in explaining variation in yield response.
Comparatively high efficiency (84%) could be achieved using
measurements of rainfall or evaporative demand for specific periods of crop
ontogeny, but this suffered the limitation of being season or treatment
specific. The results did, however, demonstrate the value of the pollen
selection method in increasing yield under conditions of reduced water supply.
In keeping with past climatic analyses, and with the theory underlying
variations in leaf water potential to which osmoregulation responds, an index
incorporating water supply and evaporative demand accounted well for the yield
increase (85%) over the wide range of seasons and treatments examined.
It requires inputs of available soil water at sowing, rainfall, and free-water
evaporation during the growing period, together with sowing and anthesis
dates. The index was used to assess potential yield responses in the various
climates covered by the Australian wheatbelt. Greatest potential lay at the
drier, inland, margins especially where soils are lighter and water-holding
capacities lower.
Genetic parameters for growth and feed efficiency in weaner versus yearling Charolais bulls
Authors: Arthur P. F. , Renand G. , Krauss D.
Published: September 2002

Data on young Charolais bulls were used to evaluate the effect of age of dam,
and provide genetic parameter estimates for growth and feed efficiency traits
in weaners compared with yearlings. Records on 510 performance test bulls born
from 1985 to 1989 and 792 progeny test bulls born from 1988 to 1997 were used,
plus available pedigree information (total n =
5493). After weaning (approximately 225 days of age), progeny test bulls were
housed in barns and tested until 15 months for all bulls and 19 months of age
for half of the bulls. From the records, the test period was artificially
partitioned into a weaner and a yearling test. Both tests were for 140 days,
with the weaner test commencing at approximately 274 days of age (6–8
weeks after weaning), and the yearling test commencing at approximately 430
days of age. For each test (weaner or yearling), average daily gain, daily
feed intake, feed conversion ratio (feed intake per unit weight gain), and
residual feed intake (feed intake net of the expected requirements for growth
and maintenance) were calculated. Liveweights at 12 and 18 months of age were
also studied.
Age of dam effect was significant (P < 0.05) for
weight at 12 and 18 months of age, and for all weaner traits except residual
feed intake. All the traits studied were moderately heritable
(0.20–0.46) except for feed conversion ratio in yearlings (0.10).
Genetic correlations for the same traits measured in weaners and later in
yearlings were less than unity (0.42–0.95), with the 3 highest
correlations obtained for weight (0.950.03), feed intake (0.900.07), and
residual feed intake (0.750.12). Therefore, for genetic improvement purposes,
traits measured in weaners should be considered different from those measured
in yearlings, except for weight and feed intake. However, the medium to very
high genetic correlations between weaner and yearling tests for most of the
traits studied indicate that selection based on weaners will have a positive
correlated response in yearlings and vice versa.
Effects of enzyme supplementation on the replacement value of canola meal for soybean meal in broiler diets
Authors: Kocher A. , Choct M. , Morrisroe L. , Broz J.
Published: September 2002

A broiler growth study was conducted to investigate the replacement value of
canola meal (CM) for soybean meal (SBM) in broiler diets with or without the
addition of two commercial enzyme products. A total of 3600 broilers were used
in this experiment. Four isoenergetic diets replicated 10 times with 90 birds
each were fed ad libitum as starter (Day 0–21) and
finisher (Day 21–37) crumbles. All diets included a high proportion of
sorghum as the main cereal component, and soybean meal (20% and
24.5%) or canola meal (35% and 28.9%) were included as
the sole protein concentrate. At Day 38 selected birds from each dietary
treatment were commercially processed and carcass yield and weight of selected
meat portions were measured. The inclusion of CM in place of SBM and the
addition of enzyme to CM-based diets did not affect feed intake, growth, and
feed conversion ratio (FCR). Mortality in birds fed the unsupplemented CM diet
was significantly reduced compared with the mortality in birds fed the SBM
control diet or the CM diets supplemented with enzymes. Birds fed the
unsupplemented CM had significantly (P < 0.05)
reduced eviscerated weight, breast meat, and thigh yield and a significantly
(P < 0.05) reduced water-holding capacity of muscle
tissue compared with the SBM control diet. The addition of Enzyme A to
CM-based diets increased breast meat and the addition of Enzyme B
significantly (P < 0.05) increased dress yield,
breast meat, and Marylands (thigh and drumstick portions). Carcass yield and
quality on this diet was comparable to the SBM diet.
Translocation from flowering to daughter tillers in perennial ryegrass (Lolium perenne L.)
Authors: Matthew C.
Published: September 2002

Recent New Zealand cultivars of Lolium perenne often have
a high rate of tiller death and replacement in late spring–early summer.
A majority of the new tillers are daughter tillers of flowering tillers.
Previous research has led to a hypothesis that defoliation may influence the
amount of assimilate exported by flowering tillers, and hence the rate of
daughter tiller formation.
To test this hypothesis, flowering tillers of
of nutrition that enabled the dam to gain 100
g/day of conceptus-free liveweight in addition to expected fetal growth)
during the mid pregnancy (post-shearing) period (from Day 70 of pregnancy
(P70) until P101). At the end of this period ewes were offered either
maintenance or a high level of nutrition during late pregnancy
(P102–P140).
Pregnancy shearing was found to increase lamb birthweight (average birthweight
5.4±0.1 v. 5.1± 0.1 kg) without differentially affecting
the birthweight of singles or twins. Dam feeding level post-shearing did not
affect the birthweight response to mid pregnancy shearing. Pregnancy shearing
treatment had no effect on the summit metabolic rate of twin lambs. However,
when results of this study are compiled with those of previous mid to late
pregnancy shearing studies under similar conditions, it appears that the birth
weight response from pregnancy shearing is greatest where lightweight lambs
are born to unshorn (control) ewes.
A comparison of the adaptation of yellow lupin (Lupinus luteus L.) and narrow-leafed lupin (L. angustifolius L.) to acid sandplain soils in low rainfall agricultural areas of Western Australia
Authors: French R. J. , French R. J. , Sweetingham M. W. , Sweetingham M. W. , Shea G. G. , Shea G. G.
Published: September 2002

Almost the entire lupin industry of Western Australia is based on the single
species Lupinus angustifolius L. (narrow-leafed lupin),
which is very well adapted to coarse-textured, mildly acid soils. However,
L. angustifolius is not well suited to the strongly acid
sand plain soils along the low rainfall fringe of Western Australia’s
agricultural areas, and alternative grain legume species may be preferable.
These soils, known locally as wodjil soils, have very low nutrient contents,
often high levels of extractable Al in the subsoil, and are common in areas
where severe brown spot and root rot disease is caused
byPleiochaeta setosa. Yellow lupin,
Lupinus luteus L., may be a better species on these
soils.
This paper describes a series of trials comparing the grain yields of
narrow-leafed lupin and yellow lupin on a range of soils in the agricultural
areas of Western Australia. These trials were sown on a range of dates and in
a range of rotational backgrounds between 1995 and 1998. With current
cultivars, narrow-leafed lupin clearly has higher yield potential than yellow
lupin when soil-extractable [Al] at a depth of 15–25 cm
(measured in a 1 :5 extract of soil in 0.01 M CaCl2) is
<10 mg/kg. When extractable [Al] at this depth is greater,
yellow lupin can produce greater yields than narrow-leafed lupin, depending on
other environmental characteristics, especially when extractable
[Al] exceeds 28 mg/kg, but its yield advantage is often small.
Yellow lupin is less sensitive to delayed sowing than narrow-leafed lupin, and
more tolerant of brown spot, but narrow-leafed lupin is more responsive to
good seasonal conditions and less sensitive to frost.
We conclude that yellow lupin has a place in Western Australian farming
systems on soils with >10 mg/kg extractable [Al] where these
soils are in close lupin rotations, in areas where brown spot is severe, or in
low rainfall areas where narrow-leafed lupin yield potential does not often
exceed 1 t/ha on these soils. However, yellow lupin has had relatively
little breeding effort in Australia and its place will remain precarious until
better adapted, higher yielding cultivars become available.
Induction of late maturity a-amylase in wheat by cool temperature
Authors: Mrva K. , Mares D. J.
Published: September 2002

Wheat genotypes prone to late maturity α-amylase (LMA) produced high
levels of germination-type (high pI isozymes) α-amylase following
exposure to cool temperature during grain development. Plants grown in the
glasshouse, plants grown in the field and transplanted to the glasshouse after
flowering, and tillers taken from field or glasshouse grown plants all
responded in a similar manner. Plants or detached tillers can therefore be
used in screening tests to identify germplasm with the LMA genotype. The cool
temperature treatment was effective when applied continuously from shortly
after flowering until near-ripeness, or when limited to the phase of grain
development (26–35 days after anthesis) in LMA-prone genotypes that
appears to be most sensitive to cool temperature. Based on these observations,
guidelines for screening wheat germplasm are proposed and the advantages of
using detached tillers discussed. The detached tiller method was successfully
applied to a range of genotypes, some of which were known to be prone to LMA,
and to a doubled haploid population derived from the cross Janz (low amylase)
BD159 (LMA genotype). The preliminary data from this population were
consistent with control by a single gene and similar therefore to the model
proposed previously for cv. Spica.
Effect of magnesium and nitrogenous fertilisers on the growth and alkaloid content in Lupinus angustifolius L.
Authors: Barlóg P. K.
Published: September 2002

The effect of kieserite (MgSO4.H2O) and nitrogenous fertilisers in various forms [control, Ca(NO3)2, (NH4)2SO4, NH4NO3, CO(NH2)2] on the growth and alkaloid content in L. angustifolius was investigated. Mineral fertilisers were applied at doses of 60 kg/ha of Mg and 75 kg/ha of N before sowing. Kieserite did not have a significant influence on the growth and dry matter production of lupin. Ca(NO3)2 and NH4NO3, although increasing the yield of straw, did not affect the yield of seeds. The application of (NH4)2SO4 decreased the yield of straw and seeds when compared with the effect of the above-mentioned fertilisers. The effect of Mg and nitrogenous fertilisers on alkaloid biosynthesis in vegetative organs depended, most of all, on the weather conditions in the vegetation period and on the developmental stage of lupin at which they were applied. At the stage of 6-7 leaves, the alkaloid content was negatively correlated with the content of Mg in plants (R2 = 67%). The influence of mineral forms of nitrogen on the alkaloid content in the dry matter increased in the following order: (NH4)2SO4 <�NH4NO3 <�Ca(NO3)2. The nitrogen fertilisers studied significantly modified the alkaloid content in the seeds, but the effect of nitrogen fertilisers also depended on Mg. In the treatments without Mg, nitrogenous fertilisers significantly increased the content of alkaloids by 9-17%. The greatest increase in alkaloid content was found when NH4NO3 was applied, followed by (NH4)2SO4. Simultaneous application of Mg and nitrogen decreased the content of alkaloids in seeds, depending on the year of study, by 2-8%.
Water stress and watertable depth influence yield, water use efficiency, and nitrogen recovery in bell pepper: lysimeter studies
Authors: Costa L. Dalla , Gianquinto G.
Published: September 2002

Lysimeters of size 1.5 by 1.5 by 1.5 m, containing a loamy soil, were used to
study the effects of both water stress during the reproductive stage and
underground water supply on bell pepper (Capsicum annuum
L.). Continuous water stress treatments were obtained providing irrigation
equal to 40%, 50%, 60%, 80%, and 120% of
maximum evapotranspiration (ET), evenly applied from flowering to end of the
season. Another set of treatments involved a transient drought stress imposed
by withholding 80 mm of irrigation, based on ET, beginning at 2 growth stages
(early fruit set, or mid fruit growth). A well-watered control (irrigated
restoring the entire ET) was included for comparison. The underground water
supply treatments involved 3 permanent watertable depths (0.40, 0.70, and 1.10
m below soil surface) and one variable depth increasing from 0.40 to 1.30 m.
Continuous water stress significantly reduced total fresh weight of fruit,
which was linearly related to the water consumption. The highest marketable
yield was found at irrigation of 120% ET; the lowest at 40% ET.
Marketable yield did not differ among 60%, 80%, and 100%
ET. Withholding water during early fruit set restricted fruit yield
significantly, whereas plants undergoing drought later in fruit growth yielded
as well as the well-watered control. Total fruit yield with a shallow
watertable (0.40 m depth) was comparable to above-ground irrigation restoring
100% ET, but marketable yield was significantly reduced. Highest
marketable yield was obtained with watertable at 0.7 m depth, but was reduced
by lowering watertable depth. The lower yield observed in water shortage
conditions was generally due to a reduced fruit number.
In most cases, water use efficiency decreased with increasing water
consumption, but was not affected by transient drought stress. Crop nitrogen
content was the highest when irrigation provided 100% and 120%
ET restoration. It was significantly reduced only by both 40% ET and
watertable irrigated treatments. Generally, a shallow watertable allowed less
nitrogen rescue than a deeper one.
The critical period for water was identified between the beginning of fruit
set and first maturing fruits when soil water potential should be maintained
above –0.02 MPa.