Mass screening of rice mutant populations at low CO2 for identification of lowered photorespiration and respiration rates.

Introduction: Identifying rice (Oryza sativa) germplasm with improved efficiency of primary metabolism is of utmost importance in order to increase yields. One such approach can be attained through screening genetically diverse populations under altered environmental conditions. Growth or treatment under low carbon dioxide (CO2) concentrations can be used as a means of revealing altered leaf photorespiration, respiration and other metabolic variants. Methods: We developed a pipeline for very high throughput treatment of gamma- and ethyl methanesulfonate- (EMS) induced mutant populations of IR64 rice seedlings at very low CO2 for 7 days. 1050 seedlings per batch at 5th leaf stage were exposed to 60 ppm CO2 for the first day and 30 ppm for the remaining three days. Following this, putative candidates were identified by measuring chlorophyll depletion using SPAD. Screening results showed a distinct difference between the mutants and the WTs. Results and discussion: The mean chlorophyll loss in WTs ranged from 65% to 11% respectively, whereas in the mutant lines chlorophyll loss ranged from 0 to 100%, suggesting considerable phenotypic variation. Rice mutants with a reduced chlorophyll reduction (<10%) were identified as 'Chlorophyll retention mutants' (CRMs) under low CO2 stress. In total, 1909 mutant lines (14,000 seedlings) were screened for chlorophyll content under 30 ppm CO2, with 26 lines selected for detailed screening. These 26 putative candidates were self-seeded to produce an M5 generation, used to determine the genetic control of the altered response to low CO2. Gas exchange of light and CO2 response revealed that there were significant variations among photosynthetic properties in two selected rice mutants. The CO2 compensation points in the absence of photorespiration and leaf respiration rates were lower than the WTs and anatomical analyses showed that CRM 29 had improved mesophyll cell area. We propose that this approach is useful for generating new material for breeding rice with improved primary metabolism.

Up-regulation of p21(WAF1/CIP1) by histone deacetylase inhibitors reduces their cytotoxicity.

Histone deacetylase inhibitors show promise as chemotherapeutic agents and have been demonstrated to block proliferation in a wide range of tumor cell lines. Much of this antiproliferative effect has been ascribed to the up-regulated expression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1). In this article, we report that p21 expression was up-regulated by relatively low doses of the histone deacetylase inhibitor azelaic bishydroxamic acid (ABHA) and correlated with a proliferative arrest. Higher doses of ABHA were cytotoxic. Cells that did not up-regulate p21 expression were hypersensitive to killing by ABHA and died via apoptosis, whereas up-regulation of p21 correlated with reduced sensitivity and a block in the apoptotic mechanism, and these cells seemed to die by necrosis. Using isogenic p21(+/+) and p21(-/-) cell lines and direct inhibition of caspase activity, we demonstrate that the reduced sensitivity to killing by ABHA is a consequence of inhibition of apoptosis by up-regulated p21 expression. These data indicate the enormous potential of therapeutic strategies that bypass the cytoprotective effect of p21 and act on the same molecular targets as the histone deacetylase inhibitors.

Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts.

Use of specific histone deacetylase inhibitors has revealed critical roles for the histone deacetylases (HDAC) in controlling proliferation. Although many studies have correlated the function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed whether the accumulation of acetylated histones, caused by HDAC inhibitor treatment, is responsible for growth inhibition. In the present study we show that HDAC inhibitors cause growth inhibition in normal and transformed keratinocytes but not in normal dermal fibroblasts. This was despite the observation that the HDAC inhibitor, suberic bishydroxamate (SBHA), caused a kinetically similar accumulation of hyperacetylated histones. This cell type-specific response to SBHA was not due to the inactivation of SBHA by fibroblasts, nor was it due to differences in the expression of specific HDAC family members. Remarkably, overexpression of HDACs 1, 4, and 6 in normal human fibroblasts resulted in cells that could be growth-inhibited by SBHA. These data suggest that, although histone acetylation is a major target for HDAC inhibitors, the accumulation of hyperacetylated histones is not sufficient to cause growth inhibition in all cell types. This suggests that growth inhibition, caused by HDAC inhibitors, may be the culmination of histone hyperacetylation acting in concert with other growth regulatory pathways.

Loop diuretics inhibit detubulation and vacuolation in amphibian muscle fibres exposed to osmotic shock.

The effect of loop diuretics at concentrations known to influence cellular water entry coupled to Na-K-Cl co-transport, upon the vacuolation and detubulation following osmotic shock, was investigated in amphibian skeletal muscles. These were exposed to a glycerol-Ringer solution (18 min), an isotonic Ca2+/Mg2+ Ringer solution and cooling. Adding bumetanide (1.0 and 2.0 microM) to these solutions sharply reduced the incidence of detubulation, assessed by abolition or otherwise of action potential after-depolarisations, from 93.9 +/- 4.7% (n = 6) to 5.0 +/- 1.1% (n = 4: mean +/- SEM: 2.0 microM bumetanide). It dramatically reduced the number and fraction of muscle volume occupied by tubular vacuoles, measured using confocal microscopy, from 60.3 +/- 4.3% (n = 10) to 9.0 +/- 1.1% (n = 35). The incidence of large horseradish peroxidase-lined tubular vacuoles, viewed using electronmicroscopy, similarly was reduced with 2 microM bumetanide in the glycerol-Ringer solution. Bumetanide acted through cellular volume adjustments early in the detubulation protocol. Thus, it exerted its maximum effect when added to the glycerol-Ringer, rather than the Ca2+/Mg2+ Ringer solution. Furthermore, whereas fibre diameters measured using scanning electron microscopy returned to normal during glycerol treatment relative to those of control fibres left in isotonic Ringer, addition of 2.0 microM bumetanide in the glycerol Ringer left markedly smaller fibre diameters. Finally equipotent concentrations of the chemically distinct loop diuretics. furosemide and ethacrynic acid similarly influenced detubulation. These findings implicate Na-K-Cl co-transport in the water entry into muscle fibres that would be expected following introduction of extracellular glycerol. This might then enable the subsequent Na-K-ATPase dependent water extrusion that produces the tubular distension (vacuolation) and detachment (detubulation) following glycerol withdrawal, phenomena also observed in muscular dystrophy.

Reconstitution experiments provide no evidence for a role for the 53-kDa glycoprotein in coupling Ca2+ transport to ATP hydrolysis by the (Ca(2+)-Mg2+)-ATPase in sarcoplasmic reticulum.

The sarcoplasmic reticulum (SR) of skeletal muscle contains a 53 kDa glycoprotein of unknown function, as well as the (Ca(2+)-Mg2+)-ATPase. It has been suggested that the glycoprotein couples the hydrolysis of ATP by the ATPase to the transport of calcium. It has been shown that if SR vesicles are solubilized in cholate in media containing low K+ concentrations followed by reconstitution, then vesicles are formed containing the glycoprotein and with ATP hydrolysis coupled to Ca2+ accumulation, as shown by a large stimulation of ATPase activity by addition of A23187. In contrast, if SR vesicles are solubilized in media containing a high concentration of K+, then the vesicles that are produced following reconstitution lack the glycoprotein and show low stimulation by A23187 (Leonards, K.S. and Kutchai, H. (1985) Biochemistry 24, 4876-4884). We show that the effect of K+ on reconstitution does not follow from any changes in the amount of glycoprotein but rather from an effect of K+ on the detergent properties of cholate. In low K+ media, the cmc of cholate is high, cholate is a relatively poor detergent and incomplete solubilization results in 'reconstitution' of vesicles with the correct orientation of ATPase molecules. In high K+ media, the cmc of cholate is reduced and more complete solubilization of the SR leads to a true reconstitution with the formation of vesicles with a random orientation of ATPase molecules. The experiments provide no evidence for an effect of the glycoprotein on the (Ca(2+)-Mg2+)-ATPase.

Chemical crosslinking and enzyme kinetics provide no evidence for a regulatory role for the 53 kDa glycoprotein of sarcoplasmic reticulum in calcium transport.

m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) was used to cross-link the protein components of rabbit skeletal muscle sarcoplasmic reticulum. Analysis of cross-linked material by SDS-polyacrylamide gel electrophoresis showed that both the (Ca(2+)-Mg2+)-ATPase and the 53 kDa glycoprotein could be cross-linked, since the amount of protein at the locations on the gel corresponding to uncross-linked material was reduced in the presence of 1.0 mM MBS. Cross-linked products of 130 kDa, 200-260 kDa and approx. 300 kDa were identified. Probing the cross-linked products with monoclonal antibodies against ATPase, 53 kDa glycoprotein and calsequestrin revealed no cross-linked products containing the ATPase and either calsequestrin or the 53 kDa glycoprotein over the range of molecular weights examined here. Possible interactions between the ATPase and calsequestrin or the 53 kDa glycoprotein were also investigated by studying the ATPase activity for the purified ATPase and for the ATPase in sarcoplasmic reticulum vesicles made permeable to Ca2+ with A23187. Effects of Ca2+ and ATP on the two systems were indistinguishable, providing no evidence for a major modulatory role of calsequestrin or the 53 kDa glycoprotein on the ATPase.

Scintillation proximity assay: competitive binding studies with [125I]endothelin-1 in human placenta and porcine lung.

Scintillation proximity assay (SPA) technology has been used to investigate the competitive binding of [125I]endothelin-1 (ET-1) by ET-1, endothelin-2 (ET-2), endothelin-3 (ET-3), sarafotoxin 6b (SFTX-b), and big endothelin (big ET) to endothelin receptors in human placenta and porcine lung. Specific binding of [125I]ET-1 to high-affinity receptors was detected in membranes coupled to wheat germ agglutinin (WGA)-coated beads impregnated with scintillant (fluomicrospheres). The binding characteristics of ET-1 were similar in both of the tissues studied. In contrast, the receptor-binding properties of ET-2, ET-3, and SFTX-b were different in human placental and porcine lung membranes. This suggests that different endothelin receptor populations exist in these tissues. In addition, the binding characteristics of ET-3 and SFTX-b differed markedly to those of ET-1 and ET-2 in both tissues. This may be due to the four amino acid residue substitution in the N-terminal loop of both ET-3 and SFTX-b. We have also demonstrated that the ET-1 precursor, big ET, competitively inhibits [125I]ET-1 binding in these tissues, but is 100-fold less potent than ET-1.

The large glycoprotein subunit of the skeletal muscle voltage-sensitive calcium channel. Deglycosylation and development.

Deglycosylation was used to assess the size of the core polypeptide of the large alpha 2-glycoprotein subunit of the 1,4-dihydropyridine-sensitive calcium channel from rabbit skeletal muscle. The extent of glycosylation was assessed by measuring the shift in apparent molecular mass of the alpha 2 component following electrophoresis in sodium dodecyl sulphate/polyacrylamide gels, using anti-(alpha 2-subunit) monoclonal antibody staining of immunoblots. Chemical deglycosylation with trifluoromethanesulphonic acid produced a shift in apparent molecular mass of the alpha 2 component from Mr 140,000 to Mr 105,000, consistent with a carbohydrate content of approximately 25%. Enzymatic treatments were insufficient to deglycosylate the alpha 2 subunit fully, possibly due to the inaccessibility of glycosidic bonds to enzyme attack. Enzymatic deglycosylation procedures did, however, reduce the 1,4-dihydropyridine-binding activity of transverse-tubule membranes. Neuraminidase alone or together with endo-beta-N-acetylglucosaminidase (endoglycosidase F) reduced the number of sites for (+)[3H]PN 200-110 by 73 +/- 2% and 77 +/- 5% respectively, with no change in apparent dissociation constant, implying a possible role for the glycosylated subunits in the binding of 1,4-dihydropyridines to the calcium-channel complex. The development of the alpha 2 component in rat skeletal muscle was shown to be indistinguishable from the appearance of 1,4-dihydropyridine binding activity consistent with the involvement of the alpha 2 subunit in the calcium-channel complex at all stages of development.

Monoclonal antibodies against the 1,4-dihydropyridine receptor associated with voltage-sensitive Ca2+ channels detect similar polypeptides from a variety of tissues and species.

Four monoclonal antibodies have been raised against voltage-sensitive Ca2+ channel dihydropyridine receptors from rabbit skeletal muscle. When tested by immunoblot assay of denatured transverse tubule membranes in reducing polyacrylamide gels, each recognised a single polypeptide of Mr approximately 140,000 that co-migrated with the large glycoprotein subunit of the purified receptor. In blots of nonreducing gels, a larger protein of Mr approximately 170,000 was seen and three of the antibodies recognised additional components at Mr approximately 310,000 and approximately 330,000. Crossreactive material of similar molecular mass was also seen in rabbit heart and brain, and in the skeletal muscle of other species.

On the problem of counting and sizing mitochondria: a general reappraisal based on ultrastructural studies of mammalian lymphocytes.

The authors ask the question whether the parameters "numbers" and "volume" are suitable for the morphometric analysis of mitochondria. In several types of cell, irregularity of mitochondrial shape makes it technically difficult, if not impossible, to obtain reliable stereological estimates of mean organelle volume or number per unit volume. Of more fundamental concern is whether number of mitochondria per cell is of any real value as a structural correlate of respiratory potential and hence as a measure of cell function. Alternative parameters might serve better for this purpose. Though the problem is illustrated by reference to quantitative studies of lymphocytes, it is also pertinent to the investigation of many other cell types.