Biomedical subjects

R L Heath

Publications and source records attributed to R L Heath.

At least 19 recordsLinked to original sources

PubMed2006

Comparison of calculated and measured foliar O3 flux in crop and forest species.

We designed a new gas exchange system that concurrently measures foliar H2O, O3, and CO2 flux (HOC flux system) while delivering known O3 concentrations. Stomatal responses of three species were tested: snapbean, and seedlings of California black oak (deciduous broadleaf) and blue oak (evergreen broadleaf). Acute O3 exposure (120-250 ppb over an hour) was applied under moderate light and low vapor pressure deficits during near steady state conditions. The rate of stomatal closure was measured when the whole plant was placed in the dark. An adjacent leaf on each plant was also concurrently measured in an O3-free cuvette. Under some conditions, direct measurements and calculated foliar O3 flux were within the same order of magnitude; however, endogenously low gs or O3 exposure-induced depression of gs resulted in an overestimation of calculated O3 fluxes compared with measured O3 fluxes. Sluggish stomata in response to light extinction with concurrent O3 exposure, and incomplete stomatal closure likewise underestimated measured O3 flux.

Carbon Dioxide↗

PubMed1995

Structures of a series of 6-kDa trypsin inhibitors isolated from the stigma of Nicotiana alata.

The three-dimensional structures of a series of 6-kDa trypsin inhibitors isolated from the stigma of the ornamental tobacco Nicotiana alata have been determined by 1H NMR spectroscopy combined with simulated annealing calculations. The proteins, T1-T4, are proteolytically cleaved from a 40.3-kDa precursor protein, NA-proPI, together with a chymotrypsin inhibitor, C1, the structure of which was reported recently [Nielsen, K.J., Heath, R.L., Anderson, M.A., & Craik, D.J. (1994) J. Mol. Biol. 242, 231-243]. Each of the proteinase inhibitors comprises 53 amino acids, including 8 cysteine residues which are linked to form 4 disulfide bridges. The proteins have a high degree of sequence identity and differ mainly in residues around the putative reactive sites. The structure of T1 was determined using a set of 533 interproton distance restraints derived from NOESY spectra, combined with 33 dihedral restraints derived from 3JNH-H alpha coupling constants and 16 hydrogen bonds. The structures of the remaining inhibitors (T2-T4) were deduced to be almost identical to T1, on the basis of their similar chemical shifts and 2D spectra. The current study demonstrates that the structures of the trypsin inhibitors (T1-T4) are similar to that previously found for the chymotrypsin inhibitor, C1. Despite differences in sequence, there is conservation in backbone geometry between the reactive site loops of the two classes of inhibitors. From this, it is clear that the nature of the side chain on the primary binding residue, rather than the backbone fold, is the main determinant of the enzyme specificities of these proteinase inhibitors.

Amino Acid Sequence↗

PubMed1995

Characterization of the protease processing sites in a multidomain proteinase inhibitor precursor from Nicotiana alata.

A gene encoding a 40.3-kDa serine proteinase inhibitor (PI) precursor is expressed at high levels in the stigma of the ornamental tobacco, Nicotiana alata. The precursor is processed proteolytically in vivo to release five homologous proteinase inhibitors of approximately 6 kDa, as well as two flanking peptides. The five PIs have been purified from stigmas and identified by N-terminal sequencing, electrospray mass spectrometry and inhibition activity against chymotrypsin or trypsin. One of the PIs inhibits chymotrypsin and the other four are most active on trypsin. Cleavage occurs in a linker region (EEKKND) that is repeated six times in the precursor molecule. In the plant, the initial cleavage probably occurs between asparagine and the aspartate residues and ragged ends are formed by subsequent trimming. In vitro, the protease-sensitive linker region is selectively cleaved by the endoproteinases Asp-N, Glu-C and Lys-C to release fully active approximately 6-kDa PIs that are resistant to further proteolytic digestion. The precursor, produced by a recombinant baculovirus, inhibits chymotrypsin more effectively than trypsin. The stoichiometry of 2.6 trypsin molecules/1 precursor molecule indicates that processing is required to activate or expose all of the four trypsin inhibitory sites.

Amino Acid Sequence↗

PubMed1995

Prosodic characteristics of speech pre- and post-right hemisphere stroke.

Case-control studies have shown right hemisphere specialization in the production of intonation in speech. We examined spontaneous prosody in audiotapes of interviews with a 77-year-old right-handed woman recorded 6 months before and 6 weeks after she suffered a stroke affecting the right frontotemporo-parietal regions and the right basal ganglia. Post-stroke, the patient had a normal Mini-Mental Status Examination Score of 29, hemispatial neglect, and impairments in the comprehension of facial expression and prosody. Self-rated mood was within normal limits. We compared beginning, peak, and ending fundamental frequencies (fo) in breath groups, the timing of these fo changes, rate of speech, pause duration, and breath-group duration. We found that post-stroke, the patient had a more restricted fo contour, no changes in the timing of peak fo, an increased rate of speech, less variability in pause duration, and no changes in breath-group duration.

Affect↗

PubMed1994

The three-dimensional solution structure by 1H NMR of a 6-kDa proteinase inhibitor isolated from the stigma of Nicotiana alata.

The three-dimensional structure and disulfide connectivities of a 6-kDa protein isolated from the stigma of the ornamental tobacco Nicotiana alata has been determined by 1H NMR spectroscopy combined with simulated annealing calculations. The protein, termed C1, is a chymotrypsin inhibitor and is one of five homologous proteinase inhibitors that are proteolytically cleaved from a 40.3-kDa precursor protein. The other four proteinase inhibitors (T1 to T4) contain reactive sites for trypsin. The three-dimensional structure of C1 is generally well defined and contains a triple stranded beta-sheet as the dominant secondary structural feature. Several turns and a short region of 3(10) helix are also present. The putative chymotrypsin reactive site is present on an exposed loop which is less defined than the rest of the protein. The overall shape of C1 is disc-like and the N and C termini are exposed, supporting the proposal that this protein results from post-translational processing of the 40.3-kDa precursor protein.

Amino Acid Sequence↗

PubMed1993

Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors.

A cDNA clone, NA-PI-II, encoding a protein with partial identity to proteinase inhibitor (PI) II of potato and tomato has been isolated from a cDNA library constructed from Nicotiana alata stigma and style mRNA. The cDNA encodes a polypeptide of 397 amino acids with a putative signal peptide of 29 amino acids and six repeated domains, each with a potential reactive site. Domains 1 and 2 have chymotrypsin-specific sites and domains 3, 4, 5, and 6 have sites specific for trypsin. In situ hybridization experiments demonstrated that expression of the gene is restricted to the stigma of both immature and mature pistils. Peptides with inhibitory activity toward chymotrypsin and trypsin have been isolated from stigmas of N. alata. The N-terminal amino acid sequence obtained from this protein preparation corresponds to six regions in the cDNA clone NA-PI-II. The purified PI protein preparation is likely to be composed of a mixture of up to five similar peptides of approximately 6 kD, produced in vivo by proteolytic processing of a 42-kD precursor. The PI may function to protect the reproductive tissue against potential pathogens.

Amino Acid Sequence↗

PubMed1992

Fluorescence Quenching in the Varied Photosynthetic Modes of Portulacaria afra (L.) Jacq.

The kinetics of chlorophyll fluorescence were measured in Portulacaria afra (L.) Jacq. when the plants were functioning in either Crassulacean acid metabolism (CAM) or C(3)/CAM cycling (called cycling) modes, as determined by fluctuation in titratable acidity and gas exchange properties. Cycling plants showed primarily daytime CO(2) uptake typical of C(3) plants, but with a slight diurnal acid fluctuation, whereas CAM plants showed nocturnal CO(2) uptake, daytime stomatal closure, and a large diurnal acid fluctuation. Results from fluorescence measurements indicated no significant differences in photochemical quenching between cycling and CAM plants; however, sizable differences were detected in nonphoto-chemical quenching (q(n)), with the largest differences being observed during the middle of the day. Cycling plants had lower q(n) than CAM plants, indicating altered photosynthetic regulation processes. This q(n) difference was believed to be related to reduced internal CO(2) concentration in the CAM plants because of daytime stomatal closure and reduced deacidification rates in the late afternoon when most of the malic acid has been utilized. Experimentally, higher external CO(2) given to plants in the CAM mode resulted in a decline in q(n) in comparison to that measured in plants in the cycling mode. No changes were observed in photochemical quenching when CO(2) was added.

Journal Article↗

PubMed1990

Ca transport in membrane vesicles from pinto bean leaves and its alteration after ozone exposure.

The influence of ozone on Ca(2+) transport in plant membranes from pinto bean (Phaseolus vulgaris L. var Pinto) leaves was investigated in vitro by means of a filtration method using purified vesicles. Two transport mechanisms located at the plasma membrane are involved in a response to ozone: (a) passive Ca(2+) influx into the cell and (b) active Ca(2+) efflux driven by an ATP-dependent system, which has two components: a primary Ca(2+) transport directly linked to ATP which is partially activated by calmodulin and a H(+)/Ca(2+) antiport coupled to activity of a H(+)-ATPase. The passive Ca(2+) permeability is increased by ozone. A triangular pulse of ozone stimulates a higher influx of Ca(2+) than does a square wave, even though the total dose was the same (0.6 microliter per liter x hour). Leaves exposed to a square wave did not exhibit visible injury and were still able to recover from oxidant stress by activation of calmodulin-dependent Ca(2+) extrusion mechanisms. On the other hand, leaves exposed to a triangular wave of ozone, exhibit visible injury and lost the ability of extruding Ca(2+) out of the cell.

Journal Article↗

PubMed1989

Alteration of Extracellular Enzymes in Pinto Bean Leaves upon Exposure to Air Pollutants, Ozone and Sulfur Dioxide.

Diamine oxidase and peroxidase, associated with the wall in pinto bean (Phaseolus vulgaris L. var Pinto) leaves, can be washed out by vacuum infiltration and assayed without grinding the leaf. The diamine oxidase activity is inhibited in vivo by exposure of the plants to ozone (dose of 0.6 microliters per liter x hour), whereas the peroxidase activity associated with the wall space is stimulated. This dose does not cause obvious necrosis or chlorosis of the leaf. These alterations are greater when the dose of ozone exposure is given as a triangular pulse (a slow rise to a peak of 0.24 microliters per liter followed by a slow fall) compared to that given as a constant square wave pulse of 0.15 microliters per liter for the same 4 hour period. Exposure of the plants to sulfur dioxide (at a concentration of 0.4 microliters per liter for 4 hours) does not result in any change in the diamine oxidase or peroxidase activities, yet the total sulfhydryl content of the leaf is increased, demonstrating the entry of sulfur dioxide. These two pollutants, with different chemical reactivities, affect the activities of the extracellular enzymes in different manners. In the case of ozone exposure, the inhibition of extracellular diamine oxidase could profoundly alter the movements of polyamines from cell to cell.

Journal Article↗

PubMed1988

Use of serotype-specific monoclonal antibodies to study the epidemiology of rotavirus infection.

The development of an enzyme immunoassay (EIA) capable of serotyping human rotavirus (HRV) in faecal extracts has enabled us to retrospectively study the epidemiology of rotavirus infection in Melbourne. Of 552 stored specimens obtained from individuals with rotavirus-associated gastroenteritis between 1975 and 1986, the serotype could be determined in 62%. Infection was most prevalent in two groups, neonates and children aged between 12 and 24 months. In these groups infection was due to different serotypes, type 1 in older children and an untypable virus in infants. Serotype 1 strains were detected in greater numbers than the other serotypes and circulated in each year of the study. Serotype 2 rotaviruses were associated with a large epidemic in 1978, but have been detected only rarely since.

Age Factors↗

PubMed1988

Synthesis of human rotavirus polypeptides in cell culture.

The replication strategy of a human serotype 1 rotavirus, adapted to rapid growth in CV-1 cells, was investigated. A single cycle growth curve revealed eclipse and latent periods of 3 and 4 hours, respectively. Although the extent of reduction of host cell protein synthesis was directly related to the multiplicity of infection of the virus, incorporation of actinomycin D and excess NaCl into the medium resulted in significant reduction in host cell background and enabled observation of viral polypeptides as early as 2 hours post infection. Five polypeptides were found to be structural components of the virion, and a further eight appeared to be nonstructural proteins or intermediates. Five polypeptides were glycosylated during virus replication, but only one of these, VP7, was a definite structural glycoprotein. Pulse-chase experiments revealed that four low molecular weight polypeptides underwent post-translational modifications.

Animals↗

PubMed1985

Photosynthetic water oxidation. A new chemical model.

A sequential four-step chemical model for the water oxidation process in photosystem II is presented, based on the observation that a peroxide-linked biquinone complex can be chemically formed as a result of hydroxide ion addition to quinone. In our model, the hydroxide ion intermediate is generated in photosystem II as a result of proton abstraction from water. In the model, the first two flashes of light raise the oxidation state of the bimanganese center, while the third and fourth flashes of light sequentially generate the peroxide-linked biquinone which is then directly oxidized by the bimanganese center to produce oxygen and regenerate quinone.

Benzoquinones↗

PubMed1985

Isolation of feline rotaviruses and their relationship to human and simian isolates by electropherotype and serotype.

Rotaviruses were detected by electron microscopy in the faecal specimens of six clinically well cats and virus was subsequently isolated from four of them. Analysis of the RNA of the isolates showed the existence of three electrophoretic types characteristic of the 'long' RNA electrophoretic pattern exhibited by rotaviruses. All feline isolates were neutralized only by antiserum to SA11 rotavirus, indicating that these isolates were serotype 3 rotaviruses. Antiserum prepared against a feline strain neutralized all the feline isolates as well as SA11 but showed no neutralizing activity against human isolates of serotype 1, 2 or 4.

Animals↗

PubMed1985

Inhibition of the k-stimulated ATPase of the plasmalemma of pinto bean leaves by ozone.

Three varieties of Phaseolus vulgaris which differ in their sensitivity to ozone were examined for changes in some physiological and structural plasma membrane characteristics. Plasma membrane vesicles were prepared from control and ozone-treated (0.2 to 0.5 microliters per liter ozone for 5 hours) leaf tissue, and the (K(+) + Mg(2+))-ATPase activity determined and compared. No major changes were observed in the resistant varieties. The sensitive variety showed a severe inhibition of ATPase activity which was largely due to a decrease in the K(+)-stimulated component. This inhibition was completely reversed by the addition of sulfhydryl compounds.Ozone-induced plasma membrane permeability changes may be effected by damage to membrane proteins, perhaps by oxidation of amino acid sulfhydryl groups to disulfide and sulfenic moieties.

Journal Article↗

PubMed1985

Effects of polyethylene-glycol-induced osmotic stress on transpiration and photosynthesis in pinto bean leaf discs.

A new leaf disc chamber allows measurements of chlorophyll fluorescence and CO(2) and H(2)O vapor exchanges during infusion of solution into the cut edge of the disc. Polyethylene glycol (molecular weight, 6000) was used to apply a mild external osmotic stress to the leaf disc within this chamber. This stress rapidly caused a temporary increase in transpiration. This increase was reversed (5-6 minutes later) and after 20 to 25 min, the stomates nearly completely closed. Internal CO(2) (calculated) and leaf temperature followed the transpiration measurements. However, chlorophyll fluorescence (small rise) followed internal CO(2) (small rise). This complete sequence of events resembles those caused by exposure of leaves to certain air pollutants which have been seen to cause such a transient increase followed by a decrease in stomatal closure.

Journal Article↗

PubMed1984

A New Type of Hexose Monophosphate Shunt in Chlorella sorokiniana: CO(2) Release from Differentially Labeled Glucose.

Using differentially labeled glucose as a substrate to probe the operation of the hexose monophosphate shunt (pentose cycle) in Chlorella sorokiniana, we found that the labeling patterns for the release of (14)CO(2) over the first 5 minutes are compatible with the operation of the recently described L-type pentose shunt. Experimentally, this L-type differs from the F-type or ;textbook' variety in that no radioactivity is obtained from C-2 labeled glucose, and the small amount derived from C-6 labeled glucose is due to a second pass of the glucose molecule (derived from the pentose cycle) through the pentose cycle.

Journal Article↗

PubMed1984

Decline in Energy Reserves of Chlorella sorokiniana upon Exposure to Ozone.

Ozone exposure has been shown to increase the loss of K from Chlorella cells due to an increase in passive permeability and a depolarization of membrane potential. One factor which likely influences or can be influenced by these changes is the energy state of the cell. To study this relationship, various indicators of cell energy status were examined in the presence and absence of O(3).The active uptake of chloride and deoxyglucose is nearly completely inhibited by O(3) at a dose at which cellular death, measured by plating efficiency, is minimal. Glucose-stimulated respiration, dependent upon ATP/ADP balance, is depressed to a greater degree than endogenous respiration in ozonated cells. Total ATP and glucose-6-phosphate levels also decrease but not as rapidly, and labeled intermediates of glucose metabolism are lost.Thus, exposure to O(3) results in a depletion of the cell's energy reserves as substantiated by changes observed in processes which both utilize and generate ATP. This loss in energy reserves occurs at the same exposure level of O(3) as do the changes in passive transport properties. Thus, we cannot tell which occurs first; and the processes seem to be linked with respect to O(3) injury.

Journal Article↗

PubMed1982

Ozone Inhibition of Photosynthesis in Chlorella sorokiniana.

Exposure of Chlorella sorokiniana (07-11-05) to ozone inhibits photosynthesis. In this study, the effects of ozone on O(2) evolution and fluorescence yields are used to characterize this inhibition. At an ozone dose of about 3 micromoles delivered to 2 x 10(9) cells, the photosynthetic rate of the cells is inhibited 50%, as indicated by a decrease in bicarbonate-stimulated O(2) evolution (control rate, 1.4 +/- 0.3 x 10(-15) moles per cell per minute).Normal patterns of chlorophyll fluorescence are also altered. Upon continuous exposure to ozone (3.5 x 10(-7) moles O(3) per minute), three stages of change in relative fluorescence yields are observed: (a) a rise in variable yield with no corresponding change in nonvariable yield (after 1-2 minutes), which was interpreted to be a shift in the energy flow pathway; (b) a decline in variable yield with a slight rise in nonvariable yield (requiring 3-5 minutes), interpreted to be a blockage in the CO(2) fixation pathways; and (c) complete blockage of variable yield with a concurrent decline in nonvariable yield (8-10 minutes), interpreted to be a destruction of the pigment system. The timing of each stage depended upon the ozone concentration and its delivery rate to the cell suspension. These results are compared with ozone-induced decline in photosynthesis and leaf water potential changes reported for other plant systems.Evidence is also presented to suggest that ozone effects on the photosynthetic processes are attributable to ionic imbalances brought about by ozone interaction with the plasmalemma rather than a direct effect on the chloroplast.

Journal Article↗