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Although the enucleate conducting cells of the phloem are incapable of protein synthesis, phloem exudates characteristically contain low concentrations of soluble proteins. The role of these proteins and their movement into and out of the sieve tubes poses important questions for phloem physiology and for cell-to-cell protein movement via plasmodesmata. The occurrence of protein turnover in sieve tubes was investigated by [(35)S]methionine labeling and by the use of aphid stylets to sample the sieve tube contents at three points along a source-to-sink pathway (flag leaf to grains) in wheat plants (Triticum aestivum L.). Protein concentration and composition were similar at all sampling sites. The kinetics of (35)S-labeling of protein suggested a basically source-to-sink pattern of movement for many proteins. However, an appreciable amount of protein synthesis and, presumably, removal also occurred along the path. This movement appeared to be protein specific and not based on passive molecular sieving. The results have important implications for the transport capacities of plasmodesmata between sieve tubes and companion cells. The observations considerably expand the possible basis for ongoing sieve tube-companion cell interactions and, perhaps, interaction between sources and sinks.

Castor bean (Ricinus communis L.) has a high photosynthetic capacity under high humidity and a pronounced sensitivity of photosynthesis to high water vapor pressure deficit (VPD). The sensitivity of photosynthesis to varying VPD was analyzed by measuring CO(2) assimilation, stomatal conductance (g(s)), quantum yield of photosystem II (phi(II)), and nonphotochemical quenching of chlorophyll fluorescence (q(N)) under different VPD. Under both medium (1000) and high (1800 micromoles quanta per square meter per second) light intensities, CO(2) assimilation decreased as the VPD between the leaf and the air around the leaf increased. The g(s) initially dropped rapidly with increasing VPD and then showed a slower decrease above a VPD of 10 to 20 millibars. Over a temperature range from 20 to 40 degrees C, CO(2) assimilation and g(s) were inhibited by high VPD (20 millibars). However, the rate of transpiration increased with increasing temperature at either low or high VPD due to an increase in g(s). The relative inhibition of photosynthesis under photorespiring (atmospheric levels of CO(2) and O(2)) versus nonphotorespiring (700 microbars CO(2) and 2% O(2)) conditions was greater under high VPD (30 millibars) than under low VPD (3 millibars). Also, with increasing light intensity the relative inhibition of photosynthesis by O(2) increased under high VPD, but decreased under low VPD. The effect of high VPD on photosynthesis under various conditions could not be totally accounted for by the decrease in the intercellular CO(2) in the leaf (C(i)) where C(i) was estimated from gas exchange measurements. However, estimates of C(i) from measurements of phi(II) and q(N) suggest that the decrease in photosynthesis and increase in photorespiration under high VPD can be totally accounted for by stomatal closure and a decrease in C(i). The results also suggest that nonuniform closure of stomata may occur in well-watered plants under high VPD, causing overestimates in the calculation of C(i) from gas exchange measurements. Under low VPD, 30 degrees C, high light, and saturating CO(2), castor bean (C(3) tropical shrub) has a rate of photosynthesis (61 micromoles CO(2) per square meter per second) that is about 50% higher than that of tobacco (C(3)) or maize (C(4)) under the same conditions. The chlorophyll content, total soluble protein, and ribulose-1,5-bisphosphate carboxylase/oxygenase level on a leaf area basis were much higher in castor bean than in maize or tobacco, which accounts for its high rates of photosynthesis under low VPD.

During the period from 1982-1990, a total of 1644 patients were admitted to our hospital with proximal femoral fractures. Trochanteric fractures were encountered in 54 per cent of the cases (887/1644) with a mean age of 68 years. Cervical fractures were encountered in 46 per cent of the cases (757/1644) with a mean age of 62 years. We found 28 patients who had been readmitted with a second hip fracture in this retrospective study, including twenty three males and five females. Two of these second hip fractures occurred on the same side as the initial fracture and twenty six occurred on the opposite side. The time interval between the first and the second hip fracture was shorter in males than that in females (1.7 years V.S. 2.7 years) and the interval also was shorter in those with a previous trochanteric fracture as compared to those with a previous cervical fracture (1.5 years V.S. 2.6 years). It appeared that a hip fracture reduced the risk of a subsequent hip fracture on the same side, and almost all second hip fractures were located on the opposite side of the patient (93 per cent). A possible reason for this "protective mechanism" is increased strength of the bone due to callus formation. A significant symmetry (trochanteric-trochanteric or cervical-cervical) was found between the first and the second hip fractures (69 per cent). We believe the symmetrical fracture pattern is due to senile osteoporosis, a kind of ongoing systemic weakening of the skeletal system among the elderly.

The expression of maize (Zea mays) phophoenolpyruvate carboxylase (PPC) gene constructions was studied in transgenic tobacco plants (Nicotiana tabacum). Where transcription was under the control of a maize PPC gene promoter, a low level of aberrantly large PPC transcript was detected. Analysis of this PPC transcript indicated that transcription initiation occurs upstream of the normal site. Despite the aberrant transcription initiation, expression of the PPC transcript was still light-regulated. Higher levels of maize PPC transcript of the correct size were obtained with a chimeric gene construction containing a tobacco (Nicotiana plumbaginifolia) chlorophyll a/b binding protein gene promoter. The PPC activities in the leaves of these transgenic plants were up to twofold higher than those of nontransformed plants. Two forms of PPC with different kinetic properties were identified in leaf extracts of the transgenic plants: one form with a high apparent K(m) for phosphoenolpyruvate (maize isozyme), and a second form exhibiting a low apparent K(m) (tobacco isozyme). Biochemical analyses of these plants indicated that the transgenic plants had significantly elevated levels of titratable acidity and malic acid. These biochemical differences did not produce any significant physiological changes with respect to photosynthetic rate or CO(2) compensation point.

Mesophyll and paraveinal mesophyll protoplasts (PVMP) were isolated from leaves of soybean (Glycine max) at various stages of physiological development, and protein compositions of the two protoplast types were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Polypeptides of 27, 29 (previously shown to be storage proteins), and 94 kilodaltons were found to be PVMP-specific proteins and were present in both nodulated and nonnodulated plants. The 27 and 94 kilodalton polypeptides were major PVMP constituents. All three polypeptides accumulate as early as one-quarter leaf expansion. Immunoblotting and immunocytochemical studies using antibodies against the 27/29 kilodalton proteins confirmed that they are specific to the paraveinal mesophyll (PVM) and that they are localized in the PVM vacuole. The 27 kilodalton polypeptide increased significantly by two weeks depodding, and this accumulation was restricted to the PVM vacuole. Radiolabeling experiments showed that the difference in relative amounts of the 27 and 29 kilodalton polypeptides was due to a greater rate of synthesis of the 27 kilodalton polypeptide. The 94 kilodalton polypeptide accumulated to a maximum at anthesis, but was absent at 2 weeks postanthesis in both depodded and podded nodulated plants, probably because they were nitrogen limited. In nonnodulated plants, it was present through 2 weeks postanthesis. The results confirm that the 27 and 29 kilodalton proteins of soybean leaf are stored in the PVM vacuole and show that they are accumulated early during leaf development while they are still strong sinks for nitrogen. The 94 kilodalton protein, previously found to accumulate in leaves after depodding, is also a PVM protein and is likely a third vegetative storage protein, although its accumulation appears to be more dependent on excess nitrogen availability. The results further support the hypothesis that the PVM is a specialized leaf tissue that functions in synthesis and compartmentation of storage proteins.

The genus Flaveria shows evidence of evolution in the mechanism of photosynthesis as its 21 species include C(3), C(3)-C(4), C(4)-like, and C(4) plants. In this study, several physiological and biochemical parameters of photosynthesis and photorespiration were measured in 18 Flaveria species representing all the photosynthetic types. The 10 species classified as C(3)-C(4) intermediates showed an inverse continuum in level of photorespiration and development of the C(4) syndrome. This ranges from F. sonorensis with relatively high apparent photorespiration and lacking C(4) photosynthesis to F. Among the intermediates, the photosynthetic CO(2) compensation points at 30 degrees C and 1150 micromoles quanta per square meter per second varied from 9 to 29 microbars. The values for the three C(4)-like species varied from 3 to 6 microbars, similar to those measured for the C(4) species. The activities of the photorespiratory enzymes glycolate oxidase, hydroxypyruvate reductase, and serine hydroxymethyltransferase decreased progressively from C(3) to C(3)-C(4) to C(4)-like and C(4) species. On the other hand, most intermediates had higher levels of phosphenolpyruvate carboxylase and NADP-malic enzyme than C(3) species, but generally lower activities compared to C(4)-like and C(4) species. The levels of these C(4) enzymes are correlated with the degree of C(4) photosynthesis, based on the initial products of photosynthesis. Another indication of development of the C(4) syndrome in C(3)-C(4)Flaveria species was their intermediate chlorophyll a/b ratios. The chlorophyll a/b ratios of the various Flaveria species are highly correlated with the degree of C(4) photosynthesis suggesting that the photochemical machinery is progressively altered during evolution in order to meet the specific energy requirements for operating the C(4) pathway. In the progression from C(3) to C(4) species in Flaveria, the CO(2) compensation point decreased more rapidly than did the decrease in O(2) inhibition of photosynthesis or the increase in the degree of C(4) photosynthesis. These results suggest that the reduction in photorespiration during evolution occurred initially by refixation of photorespired CO(2) and prior to substantive reduction in O(2) inhibition and development of the C(4) syndrome. However, further reduction in O(2) inhibition in some intermediates and C(4)-like species is considered primarily due to the development of the C(4) syndrome. Thus, the evolution of C(3)-C(4) intermediate photosynthesis likely occurred in response to environmental conditions which limit the intercellular CO(2) concentration first via refixation of photorespired CO(2), followed by development of the C(4) syndrome.

The facultative halophyte, Mesembryanthemum crystallinum, shifts its mode of carbon assimilation from the C(3) pathway to Crassulacean acid metabolism (CAM) in response to water stress. In this study, exogenously applied abscisic acid (ABA), at micromolar concentrations, could partially substitute for water stress in induction of CAM in this species. ABA at concentrations of 5 to 10 micromolar, when applied to leaves or to the roots in hydroponic culture or in soil, induced the expression of CAM within days (as indicated by the nocturnal accumulation of total titratable acidity and malate). After applying ABA there was also an increase in phosphoenolpyruvate carboxylase and NADP-malic enzyme activities. The degree and time course of induction by ABA were comparable to those induced by salt and water stress. Electrophoretic analyses of leaf soluble protein indicate that the increases in phosphoenolpyruvate carboxylase activity during the induction by ABA, salt, and water stress are due to an increase in the quantity of the enzyme protein. ABA may be a factor in the stress-induced expression of CAM in M. crystallinum, serving as a functional link between stress and biochemical adaptation.

Twenty cases of tardy ulnar palsy were treated by anterior subcutaneous transposition in the past 6 years at the Veterans General Hospital-Taichung. These cases were followed up for at least 9 months after operation. Mean duration of follow up was 39.5 months. Age distribution was from 40y/o to 72y/o, mean age was 54.5y/o. Duration of symptoms was from 2 months to 72 months, mean was 20.3 months. Eight cases were attributed to the idiopathic type. 16 cases (80%) improved after operation. Four factors were analyzed to evaluate their status as a prognostic guide: age, duration of syndrome, severity before operation and precipitating factors. Only severity before operation correlated with prognosis, but good recovery can be achieved in severe cases. Anterior subcutaneous transposition is a good method for treating tardy ulnar palsy.

The nature and sequence of metabolic events during phase II (early morning) Crassulacean acid metabolism in Opuntia erinacea var columbiana (Griffiths) L. Benson were characterized. Gas exchange measurements under 2 and 21% O(2) revealed increased O(2) inhibition of CO(2) fixation with progression of phase II. Malate and titratable acidity patterns indicated continued synthesis of C(4) acids for at least 30 minutes into the light period. Potential activities of phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme exhibited little change during phase II, while light activation of NADP-malate dehydrogenase, pyruvate, orthophosphate dikinase, and ribulose-1,5-bisphosphate carboxylase was apparent. Short-term (14)CO(2) fixation experiments showed that the per cent of (14)C incorporated into C(4) acids decreased while incorporation into other metabolites increased with time. PEPC exhibited increased sensitivity to 2 millimolar malate, and the K(i)(malate) for PEPC decreased markedly with time. Sensitivity of PEPC to malate inhibition was considerably greater at pH 7.5 than at 8.0. The results indicate that decarboxylation and synthesis of malate occur simultaneously during the early morning period, and that phase II acid metabolism is not limited by CO(2) diffusion through stomata. With progression of phase II, CO(2) fixation by PEPC decreases while fixation by ribulose-1,5-bisphosphate carboxylase increases.

The relationship between the state of oligomerization and activity of purified maize leaf phosphoenolpyruvate carboxylase using size exclusion high performance liquid chromatography was examined. Maximum activities of 35 to 38 micromoles per minute per milligram protein were found when 100% of the enzyme was in its tetrameric form. The effects of the sulfhydryl group modifiers CuCl(2) and p-chloromercuribenzoate on enzyme inhibition and the state of aggregation of the protein complex were examined. Aggregation of the enzyme is temperature and pH sensitive with low temperature and high pH favoring depolymerization. Stability of the tetrameric form is largely dependent upon histidyl residues, and to some extent this explains the biphasic response of enzyme activity to changes in MgCl(2) concentrations. Modification of the tetramer's histidyl residues by the inhibitor diethylpyrocarbonate (0.125 millimolar) results in its dissociation to the dimeric form and loss of activity. Subsequent treatment with 0.4 molar hydroxylamine results in reassociation to the tetramer and restoration of enzymic activity.

During the early morning period, light and temperature exert distinctively different influences on the gas exchange patterns of the Crassulacean acid metabolism plant Opuntia erinacea through their effects on acid metabolism. An initial decrease in CO(2) uptake was triggered by illumination and was apparently due to a decreased CO(2) diffusion gradient through light-mediated decarboxylation of malate. In contrast, the morning burst of CO(2) uptake occurred at high temperature presumably in response to increases in both stomatal conductance and the CO(2) diffusion gradient, resulting from the temperature-regulated fixation of endogenous CO(2), primarily into malate. Subsequent stomatal closure, apparently due to elevated levels of internal CO(2) through rapid decarboxylation of malate at high temperature, was primarily responsible for the final termination of early morning Crassulacean acid metabolism.

Light microscopic examination of leaf cross-sections showed that Flaveria brownii A. M. Powell exhibits Kranz anatomy, in which distinct, chloroplast-containing bundle sheath cells are surrounded by two types of mesophyll cells. Smaller mesophyll cells containing many chloroplasts are arranged around the bundle sheath cells. Larger, spongy mesophyll cells, having fewer chloroplasts, are located between the smaller mesophyll cells and the epidermis. F. brownii has very low CO(2) compensation points at different O(2) levels, which is typical of C(4) plants, yet it does show about 4% inhibition of net photosynthesis by 21% O(2) at 30 degrees C. Protoplasts of the three photosynthetic leaf cell types were isolated according to relative differences in their buoyant densities. On a chlorophyll basis, the activities of phosphoenolpyruvate carboxylase and pyruvate, Pi dikinase (carboxylation phase of C(4) pathway) were highest in the larger mesophyll protoplasts, intermediate in the smaller mesophyll protoplasts, and lowest, but still present, in the bundle sheath protoplasts. In contrast, activities of ribulose 1,5-bisphosphate carboxylase, other C(3) cycle enzymes, and NADP-malic enzyme showed a reverse gradation, although there were significant activities of these enzymes in mesophyll cells. As indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the banding pattern of certain polypeptides of the total soluble proteins from the three cell types also supported the distribution pattern obtained by activity assays of these enzymes. Analysis of initial (14)C products in whole leaves and extrapolation of pulse-labeling curves to zero time indicated that about 80% of the CO(2) is fixed into C(4) acids (malate and aspartate), whereas about 20% of the CO(2) directly enters the C(3) cycle. This is consistent with the high activity of enzymes for CO(2) fixation by the C(4) pathway and the substantial activity of enzymes of the C(3) cycle in the mesophyll cells. Therefore, F. brownii appears to have some capacity for C(3) photosynthesis in the mesophyll cells and should be considered a C(4)-like species.

(Benzamidooxy)acetic acid (common name benzadox) which has herbicidal properties was evaluated as a potential inhibitor of photosynthesis in C4 plants. Among enzymes of the C4 pathway, it was a relatively strong inhibitor of alanine aminotransferase in in vitro experiments at concentrations of 5mM. In benzadox treated leaves of Panicum miliaceum, a NAD-malic enzyme type C4 species, there was strong inhibition of both alanine and aspartate aminotransferase and of photosynthetic O2 evolution within one hour. Consistent with the inhibition of these enzymes of the C4 cycle, the pool sizes of metabolites of the cycle was altered: the aspartate level was increased two fold, while the levels of other metabolites such as pyruvate, alanine, oxalacetate and malate were decreased. Kinetic studies with partially purified alanine aminotransferase showed that benzadox is a competitive inhibitor with respect to alanine and a noncompetitive inhibitor with respect to 2-oxoglutarate. Comparisons between the structures and inhibitory actions of benzadox and (aminooxy)acetic acid, the latter a potent inhibitor of alanine and aspartate aminotransferases, suggest that in vivo, benzadox may exert its effect through metabolism to (aminooxy)acetic acid.

The initial products of photosynthesis by the C(3) species Flaveria cronquistii, the C(4) species F. trinervia, and the C(3)-C(4) intermediate species F. ramosissima were determined using a pulse-chase technique with (14)CO(2)-(12)CO(2). The intermediate species F. ramosissima incorporated at least 42% of the total soluble (14)C fixed into malate and aspartate after 10 seconds of photosynthesis in (14)CO(2), as compared with 90% for the C(4) species F. trinervia and 5% for the C(3) species F. cronquistii. In both F. ramosissima and F. trinervia, turnover of labeled malate and aspartate occurred during a chase period in (12)CO(2), although the rate of turnover was slower in the intermediate species. Relative to F. cronquistii, F. ramosissima showed a reduced incorporation of radioactivity into serine and glycine during the pulse period. These results indicate that a functional C(4) pathway of photosynthesis is operating in F. ramosissima which can account for its reduced level of photorespiration, and that this species is a true biochemical intermediate between C(3) and C(4) plants.

Photosynthesis was examined in leaves of Flaveria brownii A. M. Powell, grown under either 14% or 100% full sunlight. In leaves of high light grown plants, the CO(2) compensation point and the inhibition of photosynthesis by 21% O(2) were significantly lower, while activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and various C(4) cycle enzymes were considerably higher than those in leaves grown in low light. Both the CO(2) compensation point and the degree of O(2) inhibition of apparent photosynthesis were relatively insensitive to the light intensity used during measurements with plants from either growth conditions. Partitioning of atmospheric CO(2) between Rubisco of the C(3) pathway and phosphoenolpyruvate carboxylase of the C(4) cycle was determined by exposing leaves to (14)CO(2) for 3 to 16 seconds, and extrapolating the labeling curves of initial products to zero time. Results indicated that approximately 94% of the CO(2) was fixed by the C(4) cycle in high light grown plants, versus approximately 78% in low light grown plants. Thus, growth of F. brownii in high light increased the expressed level of C(4) photosynthesis. Consistent with the carbon partitioning patterns, photosynthetic enzyme activities (on a chlorophyll basis) in protoplasts from leaves of high light grown plants showed a more C(4)-like pattern of compartmentation. Pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase were more enriched in the mesophyll cells, while NADP-malic enzyme and ribulose 1,5-bisphosphate carboxylase/oxygenase were relatively more abundant in the bundle sheath cells of high light than of low light grown plants. Thus, these results indicate that F. brownii has plasticity in its utilization of different pathways of carbon assimilation, depending on the light conditions during growth.

The activity and extent of light activation of three photosynthetic enzymes, pyruvate,Pi dikinase, NADP-malate dehydrogenase (NADP-MDH), and fructose 1,6-bisphosphatase (FBPase), were examined in maize (Zea mays var Royal Crest) leaves relative to the rate of photosynthesis during induction and under varying light intensities. There was a strong light activation of NADP-MDH and pyruvate,Pi dikinase, and light also activated FBPase 2- to 4-fold. During the induction period for whole leaf photosynthesis at 30 degrees C under high light, the time required to reach half-maximum activation for all three enzymes was only 1 minute or less. After 2.5 minutes of illumination the enzymes were fully activated, while the photosynthetic rate was only at half-maximum activity, indicating that factors other than enzyme activation limit photosynthesis during the induction period in C(4) plants.Under steady state conditions, the light intensity required to reach half-maximum activation of the three enzymes was similar (300-400 microEinsteins per square meter per second), while the light intensity required for half-maximum rates of photosynthesis was about 550 microEinsteins per square meter per second. The light activated levels of NADP-MDH and FBPase were well in excess of the in vivo activities which would be required during photosynthesis, while maximum activities of pyruvate,Pi dikinase were generally just sufficient to accommodate photosynthesis, suggesting the latter may be a rate limiting enzyme.There was a large (5-fold) light activation of FBPase in isolated bundle sheath strands of maize, whereas there was little light activation of the enzyme in isolated mesophyll protoplasts. In mesophyll protoplasts the enzyme was largely located in the cytoplasm, although there was a low amount of light-activated enzyme in the mesophyll chloroplasts. The results suggest the chloroplastic FBPase in maize is primarily located in the bundle sheath cells.

Carbon-isotope ratios were examined as δ(13)C values in several C3, C4, and C3-C4 Flaveria species, and compared to predicted δ(13)C, values generated from theoretical models. The measured δ(13)C values were within 4‰ of those predicted from the models. The models were used to identify factors that contribute to C3-like δ(13)C values in C3-C4 species that exhibit considerable C4-cycle activity. Two of the factors contributing to C3-like δ(13)C values are high CO2 leakiness from the C4 pathway and pi/pa values that were higher than C4 congeners. A marked break occurred in the relationship between the percentage of atmospheric CO2 assimilated through the C4 cycle and the δ(13)C value. Below 50% C4-cycle assimialtion there was no significant relationship between the variables, but above 50% the δ(13)C values became less negative. These results demonstrate that the level of C4-cycle expression can increase from, 0 to 50% with little integration of carbon transfer from the C4 to the C3 cycle. As expression increaces above 50%, however, increased integration of C3- and C4-cycle co-function occurs.

Our previous two articles have shown that glutaraldehyde-fixed bovine pericardium is nearly isotropic, whether fixed without constraints, with tethering, or with pressure. In this study, we have used uniaxial stress during fixation to produce bovine pericardial material with marked tensile anisotropy. Rectangular and cruciate pericardial samples have been mechanically examined after one of four treatments: (i) fixation under 88-kPa uniaxial stress, (ii) fixation under 176-kPa uniaxial stress, (iii) 3 h of 176-kPa uniaxial stress in saline followed by 24-h fixation under the same stress, (iv) fixation under 176-kPa uniaxial stress followed by a second fixation under 176-kPa stress in a direction normal to the first. Strips of material were cut at 0 degree, 30 degrees, 60 degrees, and 90 degrees to direction of the initial stress, and tested for response to cyclic loading, stress relaxation, plastic deformation, and fracture properties. Fixation under uniaxial stress produced anistropy similar to that seen in porcine aortic valve leaflets; however, the overall extensibility of the material depended on the applied stress and the aspect ratio of the stressed sample. While loading in saline produced no change, the sequential biaxial stressing produced a reduction in anisotropy, suggesting exposure of additional crosslinking sites. Uniaxial stress during fixation may be a useful method for construction of anisotropic heart valve leaflets.

Mesophyll cells and bundle sheath strands isolated from leaves of the C(4) plant Digitaria sanguinalis (L.) Scop. are capable of utilizing aspartate as a Hill oxidant. The resulting O(2) evolution upon illumination depends on the presence of 2-oxoglutarate, is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and is stimulated by methylamine. The rate of aspartate-dependent O(2) evolution with mesophyll cells was similar to those with phosphoenolpyruvate + CO(2) or with oxalacetate. Amino-oxyacetate, an inhibitor of aspartate aminotransferase, inhibited the aspartate-dependent O(2) evolution. Aspartate aminotransferase and NADP(+) -malate dehydrogenase are located in the mesophyll chloroplasts. These data suggest that aspartate is converted to oxalacetate via aspartate aminotransferase in the chloroplasts of mesophyll cells and that oxalacetate is subsequently reduced to malate, which is coupled to the photochemical evolution of O(2). This suggestion is further verified by the inhibition of phosphoenolpyruvate-dependent (14)CO(2) fixation by aspartate + 2-oxoglutarate, which presumably acts as oxalacetate and competes with phosphoenolpyruvate + CO(2) for NADPH. dl-Glyceraldehyde inhibited aspartate-dependent O(2) evolution in the bundle sheath strands but not in the mesophyll cells. The data indicate that aspartate may be converted to malate in both mesophyll and bundle sheath cells. In NADP(+) -malic enzyme species, aspartate may exist as a C(4)-dicarboxylic acid reservoir which can contribute to the C(4) cycle through its conversion to malate.

Acetaminophen (Tylenol) is a widely used analgesic/antipyretic drug which is enzymatically bioactivated, or toxified, by the cytochromes P-450 to a hepatotoxic reactive intermediary metabolite. Brief general anesthesia with diethyl ether has been shown to inhibit both the toxifying cytochromes P-450 and enzymatic glucuronidation, the latter constituting up to 60% of acetaminophen elimination via a nontoxifying pathway. Thus ether potentially could produce a temporally differentiated inhibition of bioactivating and "detoxifying" pathways, resulting in an enhancement of acetaminophen hepatotoxicity if the balance favored bioactivation. To evaluate this possibility, separate groups of male NIH strain mice were treated with acetaminophen at different times after 5 min of anesthesia with ether. Ether produced a 40-fold enhancement in acetaminophen hepatotoxicity as determined by plasma glutamic-pyruvic transaminase (GPT) concentrations. This toxicologic enhancement was observed only if acetaminophen administration was delayed, with a maximal enhancement when acetaminophen was given 6 hr after ether, and no effect with a delay of 16 hr. Similar studies in male CD-1 mice were carried out using halothane (Fluothane) as the general anesthetic given either over 5 min or over 1 hr. While halothane given over 5 min had no effect, a 1 hr anesthetic duration produced a 10-fold increase in acetaminophen hepatotoxicity as determined by peak GPT concentration, with no observed hepatotoxicity in the halothane controls. Toxicologic enhancement occurred only with delayed administration of acetaminophen; however, the maximal enhancement observed with a 6-hr delay was still evident with a 12-hr delay. Conversely, inhibition of acetaminophen hepatotoxicity was observed if acetaminophen was given either 2 hr or 18 hr after halothane.(ABSTRACT TRUNCATED AT 250 WORDS)

Leaves of Flaveria brownii exhibited slightly higher amounts of oxygen inhibition of photosynthesis than the C(4) species, Flaveria trinervia, but considerably less than the C(3) species, Flaveria cronquistii. The photosynthetic responses to intercellular CO(2), light and leaf temperature were much more C(4)-like than C(3)-like, although 21% oxygen inhibited the photosynthetic rate, depending on conditions, up to 17% of the photosynthesis rate observed in 2% O(2). The quantum yield for CO(2) uptake in F. brownii was slightly higher than that for the C(4) species F. trinervia in 2% O(2), but not significantly different in 21% O(2). The quantum yield was inhibited 10% in the presence of 21% O(2) in F. brownii, yet no significant inhibition was observed in F. trinervia. An inhibition of 27% was observed for the quantum yield of F. cronquistii in the presence of 21% O(2). The photosynthetic response to very low intercellular CO(2) partial pressures exhibited a unique pattern in F. brownii, with a break in the linear slope observed at intercellular CO(2) partial pressure values between 15 and 20 mubar when analyzed in 21% O(2). No significant break was observed when analyzed in 2% O(2). When taken collectively, the gas-exchange results reported here are consistent with previous biochemical studies that report incomplete intercellular compartmentation of the C(3) and C(4) enzymes in this species, and suggest that F. brownii is an advanced, C(4)-like C(3)-C(4) intermediate.

Photosynthetic gas exchange, activities of six key C(4) cycle enzymes, amounts of soluble protein, chlorophyll, and DNA, and various leaf anatomical and structural features were measured in naturally occurring tetraploid and octaploid plants of the NAD-malic enzyme type C(4) grass Panicum virgatum L. On a leaf area basis, the photosynthetic rate and concentrations of DNA, soluble protein, and chlorophyll were 40 to 50% higher, and enzyme activities 20 to 70% higher in the octaploid than in the tetraploid. Photosynthetic cells in the octaploid were only 17 to 19% larger in volume, yet contained 33 to 38% more chloroplasts than cells in the tetraploid. On a per cell basis the contents of DNA, soluble protein, and chlorophyll, activities of carboxylating photosynthetic enzymes, and carbon assimilation rate were all doubled in octaploid compared with tetraploid cells. Since cellular volume did not double with genome doubling, cellular constituents were more concentrated in the cells of the octaploid. The influences of polyploidy were balanced between mesophyll and bundle sheath cells since the changes in physical and biochemical parameters with ploidy level were similar in both cell types. We conclude that photosynthetic activity in these two polyploid genotypes of P. virgatum is determined by enzyme activities and concentrations of biochemical constituents, and that selection for smaller cell volume has led to higher photosynthetic rates per unit leaf area in the octaploid. The ratio of DNA content to cellular volume is a major factor determining the concentrations of gene products in cells. The number of chloroplasts, however, is controlled more by cellular volume than by the number of nuclear chromosomes.

Four species of the genus Flaveria, namely F. anomala, F. linearis, F. pubescens, and F. ramosissima, were identified as intermediate C(3)-C(4) plants based on leaf anatomy, photosynthetic CO(2) compensation point, O(2) inhibition of photosynthesis, and activities of C(4) enzymes. F. anomala and F. ramosissima exhibit a distinct Kranz-like leaf anatomy, similar to that of the C(4) species F. trinervia, while the other C(3)-C(4) intermediate Flaveria species possess a less differentiated Kranz-like leaf anatomy. Photosynthetic CO(2) compensation points of these intermediates at 30 degrees C were very low relative to those of C(3) plants, ranging from 7 to 14 microliters per liter. In contrast to C(3) plants, net photosynthesis by the intermediates was not sensitive to O(2) concentrations below 5% and decreased relatively slowly with increasing O(2) concentration. Under similar conditions, the percentage inhibition of photosynthesis by 21% O(2) varied from 20% to 25% in the intermediates compared with 28% in Lycopersicon esculentum, a typical C(3) species. The inhibition of carboxylation efficiency by 21% O(2) varied from 17% for F. ramosissima to 46% for F. anomala and were intermediate between the C(4) (2% for F. trinervia) and C(3) (53% for L. esculentum) values. The intermediate Flaveria species, especially F. ramosissima, have substantial activities of the C(4) enzymes, phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, NADP-malic enzyme, and NADP-malate dehydrogenase, indicating potential for C(4) photosynthesis. It appears that these Flaveria species may be true biochemical C(3)-C(4) intermediates.

The keto form of oxaloacetate (OAA), a product of phosphoenolpyruvate carboxylase (PEPC) activity, can undergo various nonenzymatic conversions which make conventional methods of assaying the enzyme difficult, because the products may either act as inhibitors or go undetected. In studies with PEPC isolated from leaves of maize, an assay coupled with reduction of OAA to malate was compared with product analysis using high-performance liquid chromatography and an assay based on Pi release. The results show that activity of the enzyme in the assay coupled to malate dehydrogenase is underestimated, to varying extents, depending on magnesium concentration, buffer, and pH. In the assay coupled to malate dehydrogenase, inaccuracies occur due to conversion of the keto form of OAA to the enol form, which is not utilized as a substrate, and due to loss of OAA by decarboxylation to pyruvate. The assay based on Pi formation is considered to give the true rate of catalysis. With this assay the pH optimum is 7.8, compared to 8.3-8.5 for the assay coupled to malate dehydrogenase. The metal enol complex of oxaloacetate (M-OAAenol) is an inhibitor of PEPC and conditions which are favorable for forming this tautomer, high pH with divalent metal ions or high concentrations of Tris buffer at a pH below its pKa value, limit catalysis. Glycine stimulates enzyme activity, and it may have its effect by preventing the formation of the hydrated M-OAAenol complex and maintaining more of the OAA in the keto form. This interpretation is consistent with glycine stimulation of malate synthesis in the assay of PEPC coupled to malate dehydrogenase, with glycine stimulation of the decarboxylation of OAA, and with a reduction in the level of the M-OAAenol complex in the presence of glycine.

The potential for C4 photosynthesis was investigated in five C3-C4 intermediate species, one C3 species, and one C4 species in the genus Flaveria, using (14)CO2 pulse-(12)CO2 chase techniques and quantum-yield measurements. All five intermediate species were capable of incorporating (14)CO2 into the C4 acids malate and aspartate, following an 8-s pulse. The proportion of (14)C label in these C4 products ranged from 50-55% to 20-26% in the C3-C4 intermediates F. floridana Johnston and F. linearis Lag. respectively. All of the intermediate species incorporated as much, or more, (14)CO2 into aspartate as into malate. Generally, about 5-15% of the initial label in these species appeared as other organic acids. There was variation in the capacity for C4 photosynthesis among the intermediate species based on the apparent rate of conversion of (14)C label from the C4 cycle to the C3 cycle. In intermediate species such as F. pubescens Rydb., F. ramosissima Klatt., and F. floridana we observed a substantial decrease in label of C4-cycle products and an increase in percentage label in C3-cycle products during chase periods with (12)CO2, although the rate of change was slower than in the C4 species, F. palmeri. In these C3-C4 intermediates both sucrose and fumarate were predominant products after a 20-min chase period. In the C3-C4 intermediates, F. anomala Robinson and f. linearis we observed no significant decrease in the label of C4-cycle products during a 3-min chase period and a slow turnover during a 20-min chase, indicating a lower level of functional integration between the C4 and C3 cycles in these species, relative to the other intermediates. Although F. cronquistii Powell was previously identified as a C3 species, 7-18% of the initial label was in malate+aspartate. However, only 40-50% of this label was in the C-4 position, indicating C4-acid formation as secondary products of photosynthesis in F. cronquistii. In 21% O2, the absorbed quantum yields for CO2 uptake (in mol CO2·[mol quanta](-1)) averaged 0.053 in F. cronquistii (C3), 0.051 in F. trinervia (Spreng.) Mohr (C4), 0.052 in F. ramosissima (C3-C4), 0.051 in F. anomala (C3-C4), 0.050 in F. linearis (C3-C4), 0.046 in F. floridana (C3-C4), and 0.044 in F. pubescens (C3-C4). In 2% O2 an enhancement of the quantum yield was observed in all of the C3-C4 intermediate species, ranging from 21% in F. ramosissima to 43% in F. pubescens. In all intermediates the quantum yields in 2% O2 were intermediate in value to the C3 and C4 species, indicating a co-function of the C3 and C4 cycles in CO2 assimilation. The low quantum-yield values for F. pubescens and F. floridana in 21% O2 presumably reflect an ineffcient transfer of carbon from the C4 to the C3 cycle. The response of the quantum yield to four increasing O2 concentrations (2-35%) showed lower levels of O2 inhibition in the C3-C4 intermediate F. ramosissima, relative to the C3 species. This indicates that the co-function of the C3 and C4 cycles in this intermediate species leads to an increased CO2 concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase and a concomitant decrease in the competitive inhibition by O2.

The weedy species Parthenium hysterophorus (Asteraceae) possesses a Kranz-like leaf anatomy. The bundle sheath cells are thick-walled and contain numerous granal chloroplasts, prominent mitochondria, and peroxisomes, all largely arranged in a centripetal position. Both mesophyll and bundle sheath chloroplasts accumulate starch. P. hysterophorus exhibits reduced photorespiration as indicated by a moderately low CO(2) compensation concentration (20-25 microliters per liter at 30 degrees C and 21% O(2)) and by a reduced sensitivity of net photosynthesis to 21% O(2). In contrast, the related C(3) species P. incanum and P. argentatum (guayule) lack Kranz anatomy, have higher CO(2) compensation concentrations (about 55 microliters per liter), and show a greater inhibition of photosynthesis by 21% O(2). Furthermore, in P. hysterophorus the CO(2) compensation concentration is relatively less sensitive to changes in O(2) concentrations and shows a biphasic response to changing O(2), with a transition point at about 11% O(2). Based on these results, P. hysterophorus is classified as a C(3)-C(4) intermediate. The activities of diagnostic enzymes of C(4) photosynthesis in P. hysterophorus were very low, comparable to those observed in the C(3) species P. incanum (e.g. phosphoenolpyruvate carboxylase activity of 10-29 micromoles per milligram of chlorophyll per hour). Exposures of leaves of each species to (14)CO(2) (for 8 seconds) in the light resulted in 3-phosphoglycerate and sugar phosphates being the predominant initial (14)C products (77-84%), with

The rate of photosynthesis under high light (1000 micromole quanta per square meter per second) and at 25 degrees C was measured during development of the third leaf on wheat plants and compared with the activity of several photosynthetic enzymes and the level of metabolites. The rate of photosynthesis reached a maximum the 7th day after leaf emergence and declined thereafter. There was a high and significant correlation between the rate of photosynthesis per leaf area and the activities of the enzymes ribulose 5-phosphate kinase (r = 0.91), ribulose 1,5-bisphosphate (RuBP) carboxylase (r = 0.94), 3-phosphoglycerate (PGA) kinase (r = 0.82), and fructose 1,6-bisphosphatase (r = 0.80) per leaf area. There was not a significant correlation of photosynthesis rate with chlorophyll content. The rate of photosynthesis was strongly correlated with the level of PGA (r = 0.85) and inversely correlated with the level of triose phosphate (dihydroxyacetone phosphate and glyceraldehyde 3-phosphate) (r = 0.92). RuBP levels did not change much during leaf development; therefore photosynthesis rate was not correlated with the level of RuBP. The rate of photosynthesis was at a maximum when the ratio of PGA/triose phosphate was high, and when the ratio of RuBP/PGA was low. Although several enzymes change in parallel with leaf development, the metabolite changes suggest the greatest degree of control may be through RuBP carboxylase. The sucrose content of the leaf was highest under high rates of photosynthesis. There was no evidence that later in leaf development, photosynthesis (measured under high light and at 25 degrees C) was limited by utilization of photosynthate.