Changes in enzyme activities involved in formation and interconversion of UDP-sugars during the cell cycle in a synchronous culture of Catharanthus roseus

Changes in the activities of enzymes involved in UDP-sugar formation [UDP-glucose pyrophosphorylase (EC 2.7.7.9), sucrose synthase (EC 2.4.1.13) and UDP-glucuronic acid pyrophosphorylase (EC 2.7.7.44)], and interconversion [UDP-glucuse 4-epimerase (EC 5.1.3.2), UDP-glucose dehydrogenase (EC 1.1.1.22), UDP-glucuronic acid decarboxylase (EC 4.1.1.35) and UDP-xylose 4-epimerase (EC 5.1.3.5)] were investigated during the cell cycle in a synchronous culture of Catharanthus roseus (L.) G. Don. The specific activities of UDP-glucose pyrophosphorylase and UDP-glucose 4-epimerase increased in the G2 phase before the first cell division, and those of sucrose synthase, UDP-glucose dehydrogenase and UDP-glucuronic acid pyrophosphorylase increased in the G1 phase after the first cell division. However, during the cell cycle, UDP-glucuronic acid decarboxylase and UDP-xylose 4-epimerase did not change significantly in their specific activities. Changes in enzyme activities are discussed in relation to those reported previously for cell wall composition (S. Amino et al. 1984. Physiologia Plantarum 60: 326–332).     

Separation of membranes from semiprotoplasts of suspension-cultured sycamore maple (Acer pseudoplatanus) cells

Semiprotoplasts were produced from suspension-cultured Acer pseudoplatanus (sycamore maple) cells prior to cell disruption by passing them through a 60 μm nylon screen. Cell membranes from homogenates were separated by ultracentrifugation on linear sucrose density gradients. Samples were collected by gradient fractionation and subcellular fractions were assayed for membrane markers and glycosyl transferase activities. Results of standard marker assays (cytochrome c reductase for endoplas-mic reticulum. uridine and inosine diphosphatases for Golgi. and eosin-5'-maleimide binding for plasma membrane) showed partial separation of these three membrane types. Golgi and plasma membrane markers overlapped in most gradients. Incorporation of ¹⁴C-labeled sugars from UDP-glucose and UDP-xylose into ethanol precipitated polysaccharides was used to detect glucan synthases I & II (glucosyl transferases) and xylosyl transferase activities in Golgi membrane fractions. All three glycosyl transferases overlapped in fractions corresponding to both Golgi and plasma membrane markers, although peak activities for all three occurred in different fractions. More than one peak of glucan synthase I activity was found. Glucan synthase II, associated with ß-l.3 glucan (cullose) synthesis in plasma membranes, was also detected and exhibited a 10-fold stimulation in the presence of Ca²⁺.     

Changes in intracellular UDP-sugar levels during the cell cycle in a synchronous culture of Catharanthus roseus

UDP-sugar contents were measured using high performance liquid chromatography and gas chromatography during the cell cycle in a synchronous culture of Catharanthus roseus (L.) G. Don. UDP-glucose, UDP-galactose, UDP-glucuronic acid, UDP-xylose and UDP-arabinose could be determined, and 75–90% of the UDP-sugars were UDP-glucose. The contents of UDP-glucose and UDP-galactose increased in the late G2-M and the late S-M phases, respectively, whereas UDP-glucoronic acid and UDP-arabinose increased in amount in the G1 phase. These changes in the levels of UDP-sugars during the cell cycle generally correlated well with the changes in cell wall constituents and in the activities of the enzyme involved in synthesis and interconversion of UDP-sugars reported by S. Amino et al. (Physiol. Plant. 1985. 64: 111–117).     

Separation of membranes by flotation centrifugation for in vitro synthesis of plant cell wall polysaccharides

Summary Membranes from etiolated maize seedlings were isolated using sucrose gradients for in vitro studies of polysaccharide synthesis. Following downward centrifugation, flotation centrifugation improved the purity of membrane fractions, in particular the Golgi apparatus. Based on naphthylphthalamic acid binding to plasma membrane and inosine-5-diphosphatase activity in Golgi apparatus, flotation centrifugation removed about 70% of the plasma membrane which cosedimented with the Golgi apparatus in downward centrifugation. The addition of chelators during flotation centrifugation allowed separation of the Golgi apparatus from endoplasmic reticulum, as indicated by NADH cytochromec reductase activity. Glucan and xylan synthase activities were measured as the radioactivity incorporated from either UDP-¹⁴C-glucose or UDP-¹⁴C-xylose into 80% ethanol insoluble materials. Glucan synthase activity at a substrate concentration of 1 mM UDP-glucose without CaCl₂ was greatest in fractions enriched in Golgi apparatus, but in the presence of 3 mM CaCl₂ the activity was greatest in fractions enriched in plasma membrane. Glucan synthase activity at a substrate concentration of 10M UDP-glucose in the presence of 3 mM MnCl₂ was greatest in fractions enriched in plasma membrane, but was also high in fractions enriched in Golgi apparatus. Xylan synthase activity, at a substrate concentration of 1 M UDP-xylose in the presence of 3 mM MnCl₂, was greatest in fractions enriched in Golgi apparatus. To further characterize these synthase reactions, the glycosyl linkages of the products formed were analyzed with a gas chromatograph coupled to a radiogas proportional counter. With the substrate, UDP-¹⁴C-glucose, and fractions enriched in Golgi apparatus, both (13)- and (14)-radioactive glucosyl linkages were formed, whereas the main linkage formed by fractions enriched in plasma membrane was (13)-glucosyl. With the substrate, UDP-¹⁴C-xylose, mostly (14)-xylosyl and some terminal-xylosyl linkages were formed by fractions enriched in Golgi apparatus. Only xylan synthase activity copurified with Golgi apparatus and, because plasma membrane lacked this activity, xylan synthase may be used as a reasonable indicator of Golgi apparatus.Abbreviations ATP adenosine-5-triphosphate - CR crude fraction from downward centrifugation - FL purified fraction from flotation centrifugation - GC gas chromatography - GC-RPC gas chromatography-radiogas proportional counting - IDP inosine-5-disphosphate - NPA naphthylphthalamic acid - UDP uridine-5-diphosphate - TEM transmission electron microscopy     

Aluminum resistance in wheat (Triticum aestivum) is associated with rapid, Al-induced changes in activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in root apices

We have investigated the effect of aluminum (Al) on the activity of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) isolated from 5-mm root apices of 4-day-old wheat ( Triticum aestivum ) cultivars differing in resistance to Al. Rapid increases in G6PDH and 6PGDH activities were observed in Al-resistant cultivars (PT741 and Atlas 66) during the first 10 h of treatment with 100 μ M Al, while no change in the activity of either enzyme was observed in Al-sensitive cultivars (Katepwa and Neepawa) during a 24-h exposure to Al. The Al-induced increases in enzyme activities observed in the Al-resistant PT741 appear to reflect an induction of protein synthesis since the increases were completely abolished by 1 m M cycloheximide. No differences in G6PDH and 6PGDH activities were observed between the Al-sensitive and the Al-resistant genotypes when Al was supplied in vitro. Under these conditions, an increase in Al concentration from 0 to 1.4 m M caused a gradual decrease in activity of both enzymes, irrespective of the Al-resistance of whole seedlings. Aluminum-sensitive and aluminum-resistant cultivars also differed in the rate and extent of accumulation of slowly-exchanging Al in 5-mm root apices. During the first 6 h of Al treatment, Al accumulation was only 10% more rapid in Katepwa than in PT741. After 24-h exposure, accumulation in the Al-sensitive Katepwa, was two-fold higher. A decline in Al accumulation in a slowly-exchanging compartment as well as a decrease in activities of G6PDH and 6PGDH were found in the Al-resistant PT741, when seedlings were transferred to Al-free treatment solutions after 16-h exposure to 100 μ M Al. These results suggest that rapid induction of G6PDH and 6PGDH in the Al-resistant line PT741 by Al may play a role in the mechanism of Al resistance, possibly by regulation of the pentose phosphate pathway.     

Aluminum resistance in wheat (Triticum aestivum) is associated with rapid, Al-induced changes in activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in root apices

We have investigated the effect of aluminum (Al) on the activity of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) isolated from 5-mm root apices of 4-day-old wheat ( Triticum aestivum ) cultivars differing in resistance to Al. Rapid increases in G6PDH and 6PGDH activities were observed in Al-resistant cultivars (PT741 and Atlas 66) during the first 10 h of treatment with 100 μ M Al, while no change in the activity of either enzyme was observed in Al-sensitive cultivars (Katepwa and Neepawa) during a 24-h exposure to Al. The Al-induced increases in enzyme activities observed in the Al-resistant PT741 appear to reflect an induction of protein synthesis since the increases were completely abolished by 1 m M cycloheximide. No differences in G6PDH and 6PGDH activities were observed between the Al-sensitive and the Al-resistant genotypes when Al was supplied in vitro. Under these conditions, an increase in Al concentration from 0 to 1.4 m M caused a gradual decrease in activity of both enzymes, irrespective of the Al-resistance of whole seedlings. Aluminum-sensitive and aluminum-resistant cultivars also differed in the rate and extent of accumulation of slowly-exchanging Al in 5-mm root apices. During the first 6 h of Al treatment, Al accumulation was only 10% more rapid in Katepwa than in PT741. After 24-h exposure, accumulation in the Al-sensitive Katepwa, was two-fold higher. A decline in Al accumulation in a slowly-exchanging compartment as well as a decrease in activities of G6PDH and 6PGDH were found in the Al-resistant PT741, when seedlings were transferred to Al-free treatment solutions after 16-h exposure to 100 μ M Al. These results suggest that rapid induction of G6PDH and 6PGDH in the Al-resistant line PT741 by Al may play a role in the mechanism of Al resistance, possibly by regulation of the pentose phosphate pathway.     

In vitro biosynthesis of ether-type glycolipids in the methanoarchaeon Methanothermobacter thermautotrophicus

The biosynthesis of archaeal ether-type glycolipids was investigated in vitro using Methanothermobacter thermautotrophicus cell-free homogenates. The sole sugar moiety of glycolipids and phosphoglycolipids of the organism is the beta-D-glucosyl-(1-->6)-D-glucosyl (gentiobiosyl) unit. The enzyme activities of archaeol:UDP-glucose beta-glucosyltransferase (monoglucosylarchaeol [MGA] synthase) and MGA:UDP-glucose beta-1,6-glucosyltransferase (diglucosylarchaeol [DGA] synthase) were found in the methanoarchaeon. The synthesis of DGA is probably a two-step glucosylation: (i) archaeol + UDP-glucose --> MGA + UDP, and (ii) MGA + UDP-glucose --> DGA + UDP. Both enzymes required the addition of K(+) ions and archaetidylinositol for their activities. DGA synthase was stimulated by 10 mM MgCl(2), in contrast to MGA synthase, which did not require Mg(2+). It was likely that the activities of MGA synthesis and DGA synthesis were carried out by different proteins because of the Mg(2+) requirement and their cellular localization. MGA synthase and DGA synthase can be distinguished in cell extracts greatly enriched for each activity by demonstrating the differing Mg(2+) requirements of each enzyme. MGA synthase preferred a lipid substrate with the sn-2,3 stereostructure of the glycerol backbone on which two saturated isoprenoid chains are bound at the sn-2 and sn-3 positions. A lipid substrate with unsaturated isoprenoid chains or sn-1,2-dialkylglycerol configuration exhibited low activity. Tetraether-type caldarchaetidylinositol was also actively glucosylated by the homogenates to form monoglucosyl caldarchaetidylinositol and a small amount of diglucosyl caldarchaetidylinositol. The addition of Mg(2+) increased the formation of diglucosyl caldarchaetidylinositol. This suggested that the same enzyme set synthesized the sole sugar moiety of diether-type glycolipids and tetraether-type phosphoglycolipids.     

Calcium-Activated Neutral Protease Activity Is Decreased in PC12 Cells After Ethanol Exposure

Abstract: Calcium-activated neutral protease activity was determined in PC12 cells exposed to ethanol for 96 h using a fluorescence-based assay with N -succinyl-Leu-Tyr 7-amido-4-methylcoumarin as the substrate. Stimulated activity was measured at high (1,400 µ M ) or low (140 µ M ) Ca²⁺ concentrations in the presence of 20 µ M ionomycin. Kinetic parameters were derived by fitting a model relating fluorescence intensity to time: F_t = F _final*(1 − e ^{− k obs t} ). Cell extracts were subjected to nondenaturing gel electrophoresis and casein zymography with quantification of the activity of the two calpain isoforms. Exposure to ethanol significantly decreased whole cell calpain activity measured by k _obs beginning at 20 m M , to 27.8% of control at 1,400 µ M Ca²⁺ and 29.2% of control at 140 µ M Ca²⁺ in the presence of 20 µ M ionomycin. No changes in μ-calpain or m-calpain activities were found in cell extracts from cells exposed to 20 m M ethanol, whereas at 40 and 80 m M ethanol, significant decreases in both μ-calpain and m-calpain activities were discovered.     

Base excision repair of ionizing radiation-induced DNA damage in G1 and G2 cell cycle phases

Background

Major genomic surveillance mechanisms regulated in response to DNA damage exist at the G₁/S and G₂/M checkpoints. It is presumed that these delays provide time for the repair of damaged DNA. Cells have developed multiple DNA repair pathways to protect themselves from different types of DNA damage. Oxidative DNA damage is processed by the base excision repair (BER) pathway. Little is known about the BER of ionizing radiation-induced DNA damage and putative heterogeneity of BER in the cell cycle context. We measured the activities of three BER enzymes throughout the cell cycle to investigate the cell cycle-specific repair of ionizing radiation-induced DNA damage. We further examined BER activities in G2 arrested human cells after exposure to ionizing radiation.

Results

Using an in vitro incision assay involving radiolabeled oligonucleotides with specific DNA lesions, we examined the activities of several BER enzymes in the whole cell extracts prepared from synchronized human HeLa cells irradiated in G1 and G2 phase of the cell cycle. The activities of human endonuclease III (hNTH1), a glycosylase/lyase that removes several damaged bases from DNA including dihydrouracil (DHU), 8-oxoguanine-DNA glycosylase (hOGG1) that recognizes 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) lesion and apurinic/apyrimidinic endonuclease (hAPE1) that acts on abasic sites including synthetic analog furan were examined.

Conclusion

Overall the repair activities of hNTH1 and hAPE1 were higher in the G1 compared to G2 phase of the cell cycle. The percent cleavages of oligonucleotide substrate with furan were greater than substrate with DHU in both G1 and G2 phases. The irradiation of cells enhanced the cleavage of substrates with furan and DHU only in G1 phase. The activity of hOGG1 was much lower and did not vary within the cell cycle. These results demonstrate the cell cycle phase dependence on the BER of ionizing radiation-induced DNA damage. Interestingly no evidence of enhanced BER activities was found in irradiated cells arrested in G2 phase.     

Inhibition of ERK and JNK Decreases Both Osmosensitive Taurine Release and Cell Proliferation in Glioma Cells

Cell swelling is associated with the activation of an increase in the osmosensitive taurine release (OTR) rate, which serves to decrease cell volume as part of a process known as regulatory volume decrease. OTR, which is sensitive to many pharmacological agents including anion channel blockers and signalling pathway modulators, has also been suggested to play a role in cell cycle progression. At non-cytotoxic concentrations, the anion channel blocker NPPB (25 μM), the extra-cellular signal-regulated kinase inhibitor PD98059 (50 μM), and the c-Jun NH2-terminal kinase inhibitor SP 600125 (5 μM) each decreased the OTR rate by ≥50%, decreased cell proliferation, and increased G0/G1 cell cycle arrest.     

Biochemical controls of citrate synthase in chickpea bacteroids

Bacteroids formed by Mesorhizobium ciceri CC 1192 in symbiosis with chickpea plants (Cicer arietinum L.) contained a single form of citrate synthase [citrate oxaloacetate-lyase (CoA-acetylating) enzyme; EC 4.1.3.7], which had the same electrophoretic mobility as the enzyme from the free-living cells. The citrate synthase from CC 1192 bacteroids had a native molecular mass of 228 ± 32 kDa and was activated by KCl, which also enhanced stability. Double reciprocal plots of initial velocity against acetyl-CoA concentration were linear, whereas the corresponding plots with oxaloacetate were nonlinear. The K _m value for acetyl-CoA was 174 μM in the absence of added KCl, and 88 μM when the concentration of KCl in reaction mixtures was 100 mM. The concentrations of oxaloacetate for 50% of maximal activity were 27 μM without added KCl and 14 μM in the presence of 100 mM KCl. Activity of citrate synthase was inhibited 50% by 80 μM NADH and more than 90% by 200 μM NADH. Inhibition by NADH was linear competitive with respect to acetyl-CoA (K _is = 23.1 ± 3 μM) and linear noncompetitive with respect to oxaloacetate (K _is = 56 ± 3.8 μM and K _ii = 115 ± 15.4 μM). NADH inhibition was relieved by NAD⁺ and by micromolar concentrations of 5′-AMP. In the presence of 50 or 100 mM KCl, inhibition by NADH was apparent only when the proportion of NADH in the nicotinamide adenine dinucleotide pool was greater than 0.6. In the microaerobic environment of bacteroids, NADH may be at concentrations that are inhibitory for citrate synthase. However, this inhibition is likely to be relieved by NAD⁺ and 5′-AMP, allowing carbon to enter the tricarboxylic acid cycle. Received: 14 July 1999 / Accepted: 20 September 1999     

Mechanisms of α-Sialosyl Cholesterol Action to Suppress Both Cyclic AMP Production and DNA Synthesis of Rat Glial Cells

Abstract: α-Sialosyl cholesterol (α-SC) that elicited morphological differentiation of rat astrocytes not only lowered intracellular cyclic AMP (cAMP) levels but also inhibited cAMP production induced by either α-isoproterenol, cholera toxin, or forskolin. The targets of α-SC in the cAMP production system of rat astrocytes were investigated to understand the mechanism of the a-SC action on cAMP production. cAMP production evoked by a-isoproterenol (1 μ M ) was entirely canceled by β blockers such as propranolol and timolol (1 μ M ), but not by α-SC. Concentrations of α-SC greater than 15 μ M were required for 50% inhibition of the activation by a β agonist. Although α-SC inhibited in a dose-dependent manner the activities of membrane-associated adenylate cyclase that had been stimulated by either GTPγS or forskolin, α-SC inhibited neither GTP-binding activities nor GTPase activities of the membrane-associated G proteins. These findings suggest that α-SC suppresses adenylate cyclase directly, but not β receptors or G proteins, and that it promotes the morphological differentiation of rat astrocytes through a mechanism regulating directly the cytoskeletal organization, regardless of intracellular cAMP level. α-SC (30 μ M ) suppressed 40% of DNA synthesis in the cell-free system, which contained the cytosolic extracts and the nucleus fraction prepared from rat astrocytoma C6 cells. Approximately 25% of α-SC incorporated in the astrocyte cytoplasm was transferred to the nuclei by 10 min after the addition. Thus, it is likely that α-SC that had been incorporated in the cytosol suppressed adenylate cyclase by acting from the cytosolic side of the plasma membrane, and separately suppressed nuclear DNA synthesis.     

Nitric Oxide Synthase Activity Endogenously Modulates NMDA Receptors

Abstract: We tested the possibility that endogenous nitric oxide synthase activity regulated NMDA receptors in primary cultured striatal neurons. We monitored NMDA-induced increase in intra-cellular Ca²⁺ levels with fura-2 ratio imaging, while nitric oxide synthase activity was either increased with l -arginihe (the natural substrate of nitric oxide synthase) or inhibited using nitro- l -arginine (a specific inhibitor of nitric oxide synthase). We found that the NMDA receptor effect was slowly but strongly diminished after an l -arginine (1 m M , 15 min) treatment ( l -arginine preincubation reduced the 100 μM NMDA-induced maximal effect by 30–50%). The l -arginine blockade of NMDA receptors was long-lasting but could be partially reversed by hemoglobin (100 μM , 10 min), which binds nitric oxide. This was not observed when the neurons were treated with l -arginine together with nitro- l -arginine. Our data strongly suggest that physiological nitric oxide synthase activity could regulate NMDA receptors.     

IDENTIFICATION OF A CYCLIC AMP-DEPENDENT PROTEIN KINASE IN THE DINOFLAGELLATE AMPHIDINIUM OPERCULATUM

Single cell analysis by flow cytometry is a powerful tool that has been employed to identify many different characteristics of phytoplankton populations. Cell volume is an important physiological component of many cellular processes. We have used a Coulter EPICS XL flow cytometer to measure cell volume in the spheroid dinoflagellate Amphidinium operculatum as a function of forward scatter. Cell volume measurements of this alga were quantified as equivalent spherical diameters from a standard curve obtained with latex beads of known diameter. This parameter was used to monitor cell diameter throughout the cell division cycle. In log phase cultures, A. operculatum showed increasing cell volumes throughout the light phase and a maximum cell volume concurrent with the onset of cell division late in the light phase. The maximum equivalent spherical diameter measured 14 μm, while the minimum equivalent spherical diameter was 10 μm that occurred late in the dark phase. Stationary phase cultures of A. operculatum did not exhibit oscillating cell volumes throughout the diel cycle. Chemical inhibition of the cell cycle using 100 μM olomoucine diminished cell volume changes during the light phase. These results suggest a coupling of size control to the cell division cycle.     

Specific Muscarinic-Cholinergic Desensitization in the Neuroblastoma-Glioma Hybrid NG108-15

Abstract: The cholinergic agonist carbachol, epinephrine, and the opiate morphine all inhibit prostaglandin E₁ (PGE₁)-stimulated adenylate cyclase in homogenates from the neuroblastoma-glioma hybrid NG108-15. Pretreatment of the hybrid with 100 μ M carbachol resulted in the rapid loss (desensitization) of the carbachol inhibition of adenylate cyclase (tM_1/2< 3 min). The desensitization of the carbachol inhibition was blocked by 0.1 μ M atropine. Pretreatment with carbachol (1–24 h) did not significantly affect the inhibition of adenylate cyclase by either epinephrine or morphine, nor did it alter the PGE₁-stimulated activity, that is, no supersensitization was observed. Cholate extracts of the particulate fraction from either carbachol-desensitized or of control NGlOS-15 were able to reconstitute adenylate cyclase activities of the coupling proteins (G/F)-deficient cyclymphoma cell membranes with equal efficacy. These results suggested that the coupling proteins of the adenylate cyclase were not altered by the carbachol pretreatment and that desensitization occurs at the receptor or at a receptor-associated level. However, the possibility remained that specific domains of the G/F, which interact only with muscarinic receptors, were altered.     

Cyclin E-cdk2 activation is associated with cell cycle arrest and inhibition of DNA replication induced by the thymidylate synthase inhibitor Tomudex

Tomudex (ZD1694) is a specific antifolate-based thymidylate synthase inhibitor active in a variety of solid tumor malignancies. Studies were carried out in vitro to evaluate downstream molecular alterations induced as a consequence of the potent and sustained inhibition of thymidylate synthase by Tomudex. Twenty-four hours following the initial 2-h treatment with Tomudex, human A253 head and neck squamous carcinoma cells, not expressing p53 and p21(WAF1), were accumulated with DNA content characteristic of early S phase of the cell cycle with a concomitant reduction of cells in G1 and G2/M phases. The changes in cyclin and cdk protein expression and their kinase activities were examined in control and drug-treated A253 cells. Tomudex treatment resulted in the decrease in p27(kip1) expression, with an increase in cyclin E and cdk2 protein expression and kinase activities 24 h after a 2-h exposure. Although cyclin A protein expression was markedly increased, cyclin A kinase activity was only slightly increased. Cyclin D1, cyclin B, cdk4, and cdc2 protein expression and kinase activities remain constant. Lack of activation of cyclin A- and B-cdc2 was associated with a reduced proportion of cells in G2/M phases. Increased cyclin E-cdk2 protein expression was accompanied by the inhibition of DNA synthesis, with a decrease in E2F-1 expression. These results propose that cyclin E-cdk2 kinase can negatively regulate DNA replication. The studies with dThyd rescue from cyclin E-cdk2 protein overexpression and growth inhibition by Tomudex indicate that increased cyclin E-cdk2 protein expression is associated with effective inhibition of thymidylate synthase and resultant dNTP pool imbalance. Provision of dThyd more than 24 h after exposure to Tomudex allowed cells to replicate DNA for a single cycle back to G1, but did not prevent the profound growth-inhibitory effect manifested in the following 5 days. Tomudex treatment resulted in a time-dependent induction of the megabase DNA fragments, followed by secondary 50- to 300-kb DNA fragmentation. The 50- to 300-kb DNA fragmentation may be derived from the inhibition of DNA synthesis associated with cyclin E-cdk2 activation. These results suggest that the megabase DNA fragmentation is induced as a consequence of inhibition of thymidylate synthase by Tomudex and kilobase DNA fragmentation may correlate with the reduction of p27(kip1) expression and the increase in cyclin E and cdk2 kinase activities. Activation of cyclin E and cdk2 kinases allows cells to transit from G1 to S phase accompanied by the inhibition of DNA synthesis. The changes in cell cycle regulatory proteins associated with growth inhibition and DNA damage by Tomudex are not p53 dependent.     

CHANGES IN ACTIVITIES OF THYMIDYLATE SYNTHASE AND DIHYDROFOLATE REDUCTASE IN SEA URCHIN EGGS AFTER FERTILIZATION

Thymidylate synthase activity in sea urchin eggs increases just after fertilization and decreases 30 min later. Then, cyclic variation in the activity occurs in association with the cleavage cycle. Dihydrofolate reductase activity in fertilized eggs is almost the same as in unfertilized eggs and shows no marked change within 3 hr after fertilization. Aminopterin, an analogue of dihydrofolate, inhibits dihydrofolate reductase, and arrests cleavage. On incubation in sea water containing aminopterin (20-100μM) from the time of fertilization, the development of Clypeaster and Pseudocentrotus eggs was arrested at the 32–64 cell stage, and that of Anthocidaris eggs was arrested at the morula stage. Dihydrofolate (100μM) counteracts the inhibitory effect of aminopterin on egg cleavage. Thymidine at concentrations above 10μM also prevents inhibition by aminopterin. Other deoxyribonucleosides at concentrations of 10μM to 100μM do not affect inhibition of egg cleavage by aminopterin. Deoxyadenosine at concentrations above 5 mM inhibits egg cleavage, but other deoxyribonucleosides have no effect.     

Novel characteristics of UDP-glucose dehydrogenase activities in maize: non-involvement of alcohol dehydrogenases in cell wall polysaccharide biosynthesis

UDP-glucose dehydrogenase (UDPGDH) activity was detected in extracts of maize cell-cultures and developing leaves. The reaction product was confirmed as UDP-glucuronate. Leaf extracts from null mutants defective in one or both of the ethanol dehydrogenase genes, ADH1 and ADH2, had similar UDPGDH activities to wild-type, showing that UDPGDH activity is not primarily due to ADH proteins. The mutants showed no defect in their wall matrix pentose:galactose ratios, or matrix:cellulose ratio, showing that ADHs were not required for normal wall biosynthesis. The majority of maize leaf UDPGDH activity had K _m (for UDP-glucose) 0.5–1.0 mM; there was also a minor activity with an unusually high K _m of >50 mM. In extracts of cultured cells, kinetic data indicated at least three UDPGDHs, with K _m values (for UDP-glucose) of roughly 0.027, 2.8 and >50 mM (designated enzymes E_L, E_M and E_H respectively). E_M was the single major contributor to extractable UDPGDH activity when assayed at 0.6–9.0 mM UDP-Glc. Most studies, in other plant species, had reported only E_L-like isoforms. Ethanol (100 mM) partially inhibited UDPGDH activity assayed at low, but not high, UDP-glucose concentrations, supporting the conclusion that at least E_H activity is not due to ADH. At 30 μM UDP-glucose, 20–150 μM UDP-xylose inhibited UDPGDH activity, whereas 5–15 μM UDP-xylose promoted it. In conclusion, several very different UDPGDH isoenzymes contribute to UDP-glucuronate and hence wall matrix biosynthesis in maize, but ADHs are not responsible for these activities.     

Evidence for the involvement of calcium in the hatching of Globodera rostochiensis

Low concentrations of ruthenium red and lanthanum chloride inhibited hatching of juveniles from eggs in cysts of Globodera rostochiensis in potato root diffusate. Pro-bit analysis for 31 cysts at seven concentrations of ruthenium red showed that 50% inhibition with 95% fiducial limits occured at 47 ± 23 μm; a similar value of 59 ± 14 μm was obtained using eggs removed from cysts. Results for 10 to 20 cysts at six concentrations of lanthanum chloride suggested a somewhat higher value for 50% inhibition of 110 ± 83 μM. In contrast hatching of eggs in cysts of Heterodera schachtii in water was unaffected by 5 ITIM lanthanum chloride and 625 μM ruthenium red, concentrations which cause over 90% inhibition of hatch in G. rostochiensis.
Two calcium ionophores synergised hatching of a 1971 population of G. rostochiensis in dilute diffusate. Optimal concentrations of 2 μM for A23187 and 10 μM for BrX537A increased the hatch from 17 ± 3–6 juveniles/cyst to 114 ± 44 juveniles/cyst and 138 ± 40 juveniles/cyst respectively. Ionophores in the absence of diffusate hatched very few eggs of this population but caused a greater hatch in a second (1975) population which gave a high hatch in water of 43 ± 10 juveniles/cyst. This was increased by A23187 to 181 ± 41 juveniles/cyst. The results with both the inhibitors and the ionophores suggest that hatching in G. rostochiensis might be a calcium-mediated process.