Hydrological science, society and the sustainable management of Scottish freshwaters resources in the 21st century.

Hydrology in Scotland has emerged as a diverse and maturing discipline in recent years following its origins in engineering and the environmental sciences. Despite significant progress in understanding the physical, chemical and biological aspects of the hydrological cycle in Scotland, hydrologists face a number of significant challenges. These include: improved basic process understanding and modelling of catchment functioning; increased understanding of climatic variability and change; the collection of more extensive and well-integrated data sets; improved understanding of the role of hydrology in maintaining good ecological status in managed rivers; and a rapidly evolving policy agenda both within Scotland and the EU. So far, the response of the scientific community to these challenges has been encouraging. However, it is concluded that in the future, hydrologists need to be increasingly engaged in interdisciplinary research projects and communicate better with environmental planners and various stakeholder groups if the discipline is going to make its full contribution to sustainable water resource management in Scotland.

Hydrology in Scotland: towards a scientific basis for the sustainable management of freshwater resources--foreword to thematic issue.

The physical background to hydrology in Scotland is briefly reviewed. The main scientific events associated with understanding the science of hydrology in Scotland are summarized and major contemporary research themes are outlined. A brief overview of each paper in the current volume is given.

Polyamine depletion in human melanoma cells leads to G1 arrest associated with induction of p21WAF1/CIP1/SDI1, changes in the expression of p21-regulated genes, and a senescence-like phenotype.

The cell cycle regulatory events that interface with polyamine requirements for cell growth have not yet been clearly identified. Here we use specific inhibitors of polyamine biosynthetic enzymes to investigate the effect of polyamine pool depletion on cell cycle regulation. Treatment of MALME-3M cells with either the ornithine decarboxylase inhibitor alpha-difluoromethylornithine or the S-adenosylmethionine decarboxylase inhibitor MDL-73811 lowered specific polyamine pools and slowed cell growth but did not induce cell cycle arrest. By contrast, treatment with the combination of inhibitors halted cell growth and caused a distinct G1 arrest. The latter was associated with marked reduction of all three polyamine pools, a strong increase in p21(WAF1/CIP1/SDI1) (p21), and hypophosphorylation of retinoblastoma protein. All effects were fully prevented by exogenous polyamines. p21 induction preceded p53 stabilization in MALME-3M cells and also occurred in a polyamine-depleted, p53-nonfunctional melanoma cell line, indicating that p21 is induced at least in part through p53-independent mechanisms. Conditional overexpression of p21 in a fibrosarcoma cell line was shown previously to inhibit the expression of multiple proliferation-associated genes and to induce the expression of genes associated with various aspects of cell senescence and organism aging. Polyamine depletion in MALME-3M cells was associated with inhibition of seven of seven tested p21-inhibited genes and with induction of 13 of 14 tested p21-induced genes. p21 expression is also known to induce a senescence-like phenotype, and phenotypic features of senescence were observed in polyamine-depleted MALME-3M cells. Cells increased in size, appeared more granular, and expressed senescence-associated beta-galactosidase. Cells released from the polyamine inhibition lost the ability to form colonies, failed to replicate their DNA, and approximately 25% became bi- or multinucleated. These events parallel the outcome of prolonged p21 induction in fibrosarcoma cells. The results of this study indicate that polyamine pool depletion achieved by specific biosynthetic enzyme inhibitors causes p21-mediated G1 cell cycle arrest followed by p21-mediated changes in gene expression, development of a senescence-like phenotype, and loss of cellular proliferative capacity.

Sp1 and krüppel-like factor family of transcription factors in cell growth regulation and cancer.

The Sp/KLF family contains at least twenty identified members which include Sp1-4 and numerous krüppel-like factors. Members of the family bind with varying affinities to sequences designated as 'Sp1 sites' (e.g., GC-boxes, CACCC-boxes, and basic transcription elements). Family members have different transcriptional properties and can modulate each other's activity by a variety of mechanisms. Since cells can express multiple family members, Sp/KLF factors are likely to make up a transcriptional network through which gene expression can be fine-tuned. 'Sp1 site'-dependent transcription can be growth-regulated, and the activity, expression, and/or post-translational modification of multiple family members is altered with cell growth. Furthermore, Sp/KLF factors are involved in many growth-related signal transduction pathways and their overexpression can have positive or negative effects on proliferation. In addition to growth control, Sp/KLF factors have been implicated in apoptosis and angiogenesis; thus, the family is involved in several aspects of tumorigenesis. Consistent with a role in cancer, Sp/KLF factors interact with oncogenes and tumor suppressors, they can be oncogenic themselves, and altered expression of family members has been detected in tumors. Effects of changes in Sp/KLF factors are context-dependent and can appear contradictory. Since these factors act within a network, this diversity of effects may arise from differences in the expression profile of family members in various cells. Thus, it is likely that the properties of the overall network of Sp/KLF factors play a determining role in regulation of cell growth and tumor progression.

Autologous endometrial coculture in patients with in vitro-fertilization (IVF) failure: correlations of outcome with leukemia inhibiting factor (LIF) production.

PROBLEM: To determine if LIF produced by autologous endometrial co-culture (ECC) was associated with outcome in 46 patients with a history of multiple IVF failures. METHOD OF STUDY: The conditioned media (CM) from ECC cells exposed or non-exposed to human embryos was analyzed for LIF. RESULTS: Exposure or non-exposure to an embryo did not result in differing levels of LIF in the CM. LIF levels were significantly greater in the CM than in the serum controls (LIF was not found in the serum controls). Embryos grown on ECC demonstrated a significant improvement in number of blastomeres and fragmentation when compared to embryos grown in conventional media without ECC (6.7 +/- 1.3 vs. 5.6 +/- 1.2 blastomeres and 17.6% +/- 9.3 vs. 26.4% +/- 9.8 fragmentation; P < 0.05). When LIF levels were detectable in the CM, the embryos grown in ECC were of improved quality as compared to the embryos grown only in conventional media and demonstrated a non-significant increase in pregnancy rates (60 vs. 48%, P = 0.50). CONCLUSIONS: We have demonstrated a significant improvement in embryo quality with ECC. The cells in the ECC express LIF. The presence of LIF in the CM was associated with embryonic development and clinical pregnancy.

Protein kinase C signaling mediates a program of cell cycle withdrawal in the intestinal epithelium.

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G(0). PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21(waf1/cip1) and p27(kip1), thus targeting all of the major G(1)/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G(0) as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCalpha alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt-villus axis revealed that PKCalpha activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit-specific events in situ. Together, these data point to PKCalpha as a key regulator of cell cycle withdrawal in the intestinal epithelium.

Regulation of E2F: a family of transcription factors involved in proliferation control.

Members of the E2F family of transcription factors are key participants in orchestration of the cell cycle, cell growth arrest and apoptosis. Therefore, an understanding of the regulation of E2F activity is essential for an understanding of the control of cellular proliferation. E2F activity is regulated by the retinoblastoma family of tumor suppressors and by multiple other mechanisms. This review will describe our current knowledge of these mechanisms which together constitute a highly complex network by which the cell cycle and cellular proliferation can be controlled.

Growth/cell cycle regulation of Sp1 phosphorylation.

Sp1 sites can mediate growth/cell cycle induction of dihydrofolate reductase in late G1 (Jensen, D. E., Black, A. R. Swick, A. G., and Azizkhan, J. C. (1997) J. Cell. Biochem. 67, 24-31). To investigate mechanisms underlying this induction, effects of serum stimulation on regulation of Sp1 were examined. In Balb/c 3T3 cells, serum stimulation did not affect Sp1 synthesis or the relative binding of Sp1 family members to DNA; however, it did result in a rapid, approximately 2-fold increase in Sp1 levels and an approximately 3-fold increase in specific Sp1 phosphorylation in mid-G1. In normal human diploid fibroblasts, serum stimulation also increased Sp1 phosphorylation in mid-G1 but did not affect Sp1 levels. Therefore, Sp1 phosphorylation is regulated in a growth/cell cycle-dependent manner which correlates temporally with induction of dihydrofolate reductase transcription. Further studies revealed a kinase activity specifically associated with Sp1 in a growth-regulated manner. This activity is distinct from purified kinases previously shown to phosphorylate Sp1 in vitro and phosphorylates Sp1 between amino acids 612 and 678 in its C terminus, a region also phosphorylated in mid-G1 in vivo. Therefore, this study indicates that phosphorylation of the C terminus of Sp1 may play a role in the cell cycle regulation of its transcriptional activity.

Distinct roles for Sp1 and E2F sites in the growth/cell cycle regulation of the DHFR promoter.

Dihydrofolate reductase activity is required for many biosynthetic pathways including nucleotide synthesis. Its expression is therefore central to cellular growth, and it has become a key target for cancer chemotherapy. Transcription of the dihydrofolate reductase gene is regulated with growth, being expressed maximally in late G1/early S phase following serum stimulation of quiescent cells. This regulation is directed by a promoter which contains binding sites for only the transcription factors Sp1 and E2F. In this study, the role of these promoter elements in growth/cell cycle regulation of dihydrofolate transcription was addressed directly by transient transfection of Balb/c 3T3 cells with mutant promoter-reporter gene constructs. The E2F sites were found to repress transcription in G0 and early G1 but did not contribute to the level of transcription in late G1/S phase. In contrast, Sp1 sites were able to mediate induction of transcription from the dihydrofolate reductase promoter, as well as a heterologous promoter, following serum stimulation of quiescent cells. These findings add dihydrofolate reductase to a growing list of genes at which E2F sites are primarily repressive elements and delineate a role for Sp1 sites in the growth/cell cycle regulation of transcription.

Identification of a viral kinase that phosphorylates specific E2Fs and pocket proteins.

The transcription factor E2F and its regulation by pRB and related pocket proteins are central to cell cycle control in higher eukaryotes. Much of our knowledge of this regulation has come from studies using immediate-early proteins of DNA tumor viruses. Previously, we reported that the 72-kDa immediate-early region 1 gene product of the human cytomegalovirus, IE72, transactivates the dihydrofolate reductase promoter through the E2F site and that it physically interacts with E2F1 (M. J. Margolis, S. Pajovic, E. L. Wong, M. Wade, R. Jupp, J. A. Nelson, and J. C. Azizkhan, J. Virol. 69:7759-7767, 1995). In this study, we further characterized the mechanism by which IE72 modulates E2F-dependent transcription. In vitro phosphorylation reactions using gel-purified bacterially expressed proteins revealed that IE72 is a kinase that autophosphorylates and phosphorylates E2F1, -2, and -3 (but not E2F4 or -5) and the RB-related pocket proteins p130 and p107 (but not pRB). The region of IE72 spanning amino acids 173 to 197 shows a high level of homology to the ATP binding sites in over 500 kinases. The kinase-negative protein IE72deltaATP, from which this region has been deleted, cannot activate E2F-dependent transcription. The kinase activity of IE72 is also required for its ability to reduce the association of E2F4 with p107 and p130. Taken together, these data suggest that the kinase activity of IE72 is required for E2F-dependent transcriptional activation and that this is likely to result from phosphorylation of specific members of the E2F and pocket protein families by IE72.

Alternate splicing of the rTS gene product and its overexpression in a 5-fluorouracil-resistant cell line.

rTS is a gene with convergent and overlapping transcription relative to thymidylate synthase (TS). Screening of a cDNA library has identified a second rTS RNA (rTSbeta) which is related to a previously described rTS RNA (rTSalpha). rTSbeta differs from rTSalpha by the insertion of an extra 116 nucleotides after base 128 of rTSalpha and an altered 3'-noncoding region such that rTSbeta RNA is not complementary to TS mRNA. rTSalpha and rTSbeta RNAs have open reading frames predicted to encode proteins of 361 and 416 amino acids, respectively. Antibody to rTSbeta protein detects two proteins of the predicted molecular weight in the H630 colon tumor cell line. rTSbeta is overexpressed 40-70-fold in a 5-fluorouracil-resistant H630 subline compared with 7-8-fold for TS and rTSalpha.

Cell cycle-regulated association of E2F1 and Sp1 is related to their functional interaction.

Because of the large number of growth-regulated genes containing binding sites for the transcription factors Sp1 and E2F and the reported ability of E2F to mediate cell cycle (growth) regulation, we studied interactions between E2F1 and Sp1. In transient transfection assays using Drosophila melanogaster SL2 cells, transfection with both Sp1 and E2F1 expression vectors resulted in greater than 85-fold activation of transcription from a hamster dihydrofolate reductase reporter construct, whereas cotransfection with either the Sp1 or E2F1 expression vector resulted in 30- or <2-fold activation, respectively. Therefore, these transcription factors act synergistically in activation of dihydrofolate reductase transcription. Transient transfection studies demonstrated that E2F1 could superactivate Sp1-dependent transcription in a promoter containing only Sp1 sites and that Sp1 could superactivate transcription of promoters through E2F sites, further demonstrating that these physically associated in Drosophila cells transfected with Sp1 and E2F1 expression vectors and in human cells, with maximal interaction detected in mid- to late G1. Additionally, E2F1 and Sp1 interact in vitro through specific domains of each protein, and the physical interaction and functional synergism appear to require the same regions. Taken together, these data demonstrate that E2F1 and Sp1 both functionally and physically interact; therefore this interaction, Sp1 and E2F1 may regulate transcription of genes containing binding sites for either or both factors.

Expression of rTS correlates with altered growth regulation of thymidylate synthase.

The recently discovered rTS gene is convergent with and overlaps the thymidylate synthase gene in the antisense orientation. K562 B1A and KB 1BT are two methotrexate-resistant cell lines that have amplified genes for dihydrofolate reductase. K562 B1A cells have increased levels of rTS mRNA and protein compared with their parental K562 cells, whereas KB 1BT cells show unaltered rTS expression compared with their parental (KB) cells. Altered levels and loss of growth-regulation of in situ thymidylate synthase activity were found in methotrexate-resistant K562 B1A cells but not in K562, KB, and KB 1BT cells. These data point to a link between rTS and the regulation of TS activity.

rTS gene expression is associated with altered cell sensitivity to thymidylate synthase inhibitors.

rTS is a recently discovered gene, phylogenetically conserved and found to be expressed in a wide variety of cell lines. rTS has also been found to be overexpressed in two cell lines resistant to FU and to MTX. The MTX-resistant cell line was found to have a high degree of cross resistance to several TS inhibitors. An apparent paradox to this correlation of rTS overexpression and resistance to TS inhibitors is the observation that expression of transfected rTS alpha results in enhanced sensitivity of cells to the TS inhibitor prodrug TFT and a modest increase in resistance to FUdR. Since immunoprecipitation of TS leads to the co-immunoprecipitation of two proteins within the expected molecular weight range of the two rTS proteins, it may be that both proteins bind to TS in vivo and modify its activity. Preliminary data substantiate this conclusion. It is conceivable that the ratio of the two rTS proteins associated with TS in vivo may differentially alter TS activity depending upon their stoichiometry or possibly posttranslational modification. Thus it may be possible for rTS to confer greater sensitivity to one pyrimidine analog (e.g., TFT) which is a product analog but to increase resistance or have a minor effect on a substrate analog (e.g., FdUMP) by stabilizing different conformations of TS. The structure of the rTS proteins suggests they are expected to have catalytic activity which involves proton abstraction from an alpha-carbon of a carboxyl group. Whether this enzyme activity is functional and related to pyrimidine metabolism awaits further study.

Involvement of rRNA synthesis in the enhanced survival and recovery of protein synthesis seen in thermotolerance.

Although acquired thermotolerance has been linked to the induction of heat shock proteins, the molecular mechanism(s) by which cells become resistant to heat is unknown. The present study shows a strong correlation between the survival of cells following heat shock and the rate of recovery of protein, total RNA, and rRNA synthesis. Increasing exposure of CHO cells to 45 degrees C was found to decrease survival and cause a lengthening delay in these synthetic processes. The same reciprocal correlation was seen in thermotolerant cells. As thermotolerance develops, more cells survive a heat challenge and the delay in synthesis decreases. These data argue that enhanced recovery of protein and RNA synthesis is one factor which plays a key role in thermotolerance. The involvement of rRNA synthesis was further investigated by using actinomycin D at 0.1 microgram m1(-1), a concentration at which rRNA synthesis is selectively inhibited. When the drug was present during the recovery from a challenge heat treatment, the survival of thermotolerant cells was approximately 3-fold lower than expected from the mild toxicity of the drug. As this could not be accounted for by an interaction of the drug with the response of cells to single heat treatments, it is concluded that the drug inhibits the expression of thermotolerance in cells which would otherwise express a full degree of thermotolerance. The time and concentration dependence of this effect indicates that the drug acts though inhibition of rRNA synthesis. Therefore, enhanced recovery of RNA synthesis, presumably rRNA synthesis, is identified as one of the mechanisms responsible for enhanced survival of thermotolerant cells following heat shock.

Solubilization and physical characterization of acceptors for dendrotoxin and beta-bungarotoxin from synaptic membranes of rat brain.

Dendrotoxin (DTX), an Mr 7000 convulsant polypeptide from the venom of Dendroaspis angusticeps, or its facilitatory homologues act through blockade of certain voltage-sensitive K+ currents in a variety of neurons. High-affinity acceptors for DTX have been demonstrated in synaptic plasma membranes of rat or chick brain, and a fraction of these avidly bind beta-bungarotoxin (beta-BuTX), a presynaptically active protein whose lighter B polypeptide is homologous to this toxin. Extraction of rat synaptic plasma membranes using Triton X-100 in K+-containing buffer yielded binding sites with KD values of approximately 0.5 and 0.7 nM for 125I-labeled DTX and beta-BuTX, respectively. The content of high-affinity sites obtained for beta-BuTX, including the contribution of a lower affinity component, approximates to the Bmax (approximately 1.3 pmol/mg of protein) obtained for the apparent single set of DTX acceptors. On solubilization, the pharmacological specificity of the acceptor for neurotoxic DTX congeners was retained. 125I-beta-BuTX binding (2.1 nM) was blocked efficaciously by DTX (IC50 = 1.6 nM) while the binding of 2.1 nM 125I-DTX was inhibited completely by beta-BuTX (IC50 = 25 nM); the lower potency of the latter could relate to the noncompetitive nature of the mutual competition and to the presence of high- and low-affinity sites for beta-BuTX. On gel filtration, or sedimentation analysis in H2O/sucrose and 2H2O/sucrose gradients, one peak of DTX binding activity was observed, and this was inhibitable by beta-BuTX. From the hydrodynamic properties of the acceptor/detergent/lipid complex (s20,w = 13.2 S; Stokes radius = 8.6 nm), a molecular weight of 405,000-465,000 was estimated.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of neuronal acceptors for dendrotoxin in its convulsive action in rat brain.

Dendrotoxin, a snake-venom polypeptide, is a potent convulsant that facilitates transmitter release apparently by inhibition of voltage-sensitive K+ channels responsible for A-currents. A biologically active 125I-iodinated derivative of this toxin was prepared and used to characterize kinetically homogeneous non-interacting high-affinity acceptors in synaptic membranes from rat cerebral cortex and hippocampus. Binding of radiolabelled toxin from Dendroaspis angusticeps to its membrane acceptor protein was inhibitable by homologous polypeptides from other mamba snakes; most importantly, their rank order of potency was identical with that for their central neurotoxicities in rats, furnishing evidence for involvement of this binding component in the convulsive symptoms observed. Beta-Bungarotoxin, a presynaptically acting neurotoxin whose action on neurotransmitter release at the neuromuscular junction and effects on brain synaptosomes are antagonized by dendrotoxin, was only able to inhibit the binding of the 125I-labelled toxin with low efficacy, although dendrotoxin apparently interacts avidly with the acceptor sites for beta-bungarotoxin. This weak interaction of beta-bungarotoxin with the acceptor was not attributable to its phospholipolytic action. Other neurotoxins and ion-channel antagonists failed to affect the binding of dendrotoxin. The findings presented here, together with recent electrophysiological data, favour the interpretation that dendrotoxin binds to a membrane protein comprising, or closely associated with, this one group of voltage-dependent K+ channels.

Two acceptor sub-types for dendrotoxin in chick synaptic membranes distinguishable by beta-bungarotoxin.

Using chick synaptic membranes, proteinaceous acceptors were characterized for dendrotoxin, a polypeptide from Dendroaspis angusticeps with convulsant activity due to its facilitation of transmitter release, resulting from inhibition of A-current K+ channels in brain. Both equilibrium and kinetic measurements of radioiodinated toxin binding showed that two populations of membraneous acceptors were discernible with different affinities (Kd approximately 0.5 nM and 15 nM; Bmax approximately 90 and 400 fmol/mg protein). Only the high-affinity component interacted avidly with beta-bungarotoxin, an inhibitory presynaptic neurotoxin whose lighter chain is homologous to dendrotoxin. Facilitatory homologues of dendrotoxin from Dendroaspis species antagonised its binding to both acceptor sub-types in proportion to their central neurotoxicities, whereas various other toxins (crotoxin, apamin), trypsin inhibitors and lectins proved ineffective. Cross-linking of toxin specifically bound to its membrane acceptors, using bis-imido esters followed by electrophoretic analysis in the presence of sodium dodecyl sulphate, revealed a polypeptide with Mr of 75,000 together with lesser amounts of a 69,000-Mr component. Notably, the covalent labelling of each of these bands was inhibited partially by low concentrations of beta-bungarotoxin, indicating that they are derived from both acceptor species. The demonstrated existence of an acceptor form shared by dendrotoxin and beta-bungarotoxin, together with another sub-type selective for dendrotoxin, is discussed in relation to the known pharmacological interactions of these toxins which exert opposite effects on transmitter release.

Botulinum neurotoxin and dendrotoxin as probes for studies on transmitter release.

Acceptors for BoNT have been detected autoradiographically on the terminal membrane of motor nerves at a density of approximately 150/micron2 and shown to mediate toxin internalization, a process deemed essential for its inhibition of transmitter release. DTX, a protein with pronounced central neurotoxicity, was shown to induce convulsive states in hippocampal slices from guinea-pig. Synaptic transmission was facilitated and spontaneous epileptiform activity produced in intact cell populations. Voltage clamp analysis of hippocampal neurones revealed that DTX specifically attenuated a transient voltage-dependent K+ conductance (A-current) and this could account for the excitatory effects observed. Proteinaceous acceptors with high affinity for DTX were identified on brain synaptosomal membranes and found to contain a 65 000 Mr polypeptide. Their location in rat brain regions was established and contrasted with that of the binding sites for beta-bungarotoxin. These findings indicate the usefulness of DTX as a probe for a protein associated with one variety of K+ channel while the larger subunit of BoNT was found to interact with a membraneous component that resides at cholinergic nerve terminals and, hence, is likely to have a unique role.