Utilization of (E)-2-butenoate (Crotonate) by Clostridium kluyveri and some other Clostridium species

Clostridium La 1 obtained from a Clostridium kluyveri culture was compared with a typical C. kluyvery strain (DSM 555). The former grows on crotonate and is unable to use ethanol-acetate as carbon sources. The latter grows on crotonate only after long adaptation periods. Resting cells of both strains show also pronounced differences in the fermentation of crotonate. This holds even for C. kluyveri grown on crotonate. Besides several other differences the most striking is that there is no hybridization between the DNA of both strains.Crotonate seems not to be a very special carbon source since C. butyricum and C. pasteurianum grow on crotonate medium supplemented by peptone and yeast extract.Non Standard Abbreviations EA-medium ethanol and acetate as carbon source - C-medium crotonate as carbon source - DSM Deutsche Sammlung von Mikroorganismen     

Carbon isotope composition of intermediates of the starch-malate sequence and level of the crassulacean acid metabolism in leaves of Kalanchoe blossfeldiana Tom Thumb

Isotype analyses were performed on biochemical fractions isolated from leaves of Kalanchoe blossfeldiana Tom Thumb. during aging under long days or short days. Irrespective of the age or photoperiodic conditions, the intermediates of the starch-malate sequence (starch, phosphorylated compounds and organic acids) have a level of ¹³C higher than that of soluble sugars, cellulose and hemicellulose. In short days, the activity of the crassulacean acid metabolism pathway is predominant as compared to that of C₃ pathway: leaves accumulate organic acids, rich in ¹³C. In long days, the activity of the crassulacean acid metabolism pathway increases as the leaves age, remaining, however, relatively low as compared to that of C₃ pathway: leaves accumulate soluble sugars, poor in ¹³C. After photoperiodic change (long daysshort days), isotopic modifications of starch and organic acids suggest evidence for a lag phase in the establishment of the crassulacean acid metabolism pathway specific to short days. The relative proportions of carbon from a C₃-origin (RuBPC acitivity as strong discriminating step, isotope discrimination in vivo=20) or C₄-origin (PEPC activity as weak discriminating step, isotope discrimination in vivo=4) present in the biochemical fractions were calculated from their ¹³C values. Under long days, 30 to 70% versus 80 to 100% under short days, of the carbon of the intermediates linked to the starch-malate sequence, or CAM pathway (starch, phosphorylated compounds and organic acids), have a C₄-origin. Products connected to the C₃ pathway (free sugars, cellulose, hemicellulose) have 0 to 50% of their carbon, arising from reuptake of the C₄ from malate, under long days versus 30 to 70% under short days.Abbreviations CAM crassulacean acid metabolism - CAM pathway pathway with malate accumulation by -carboxylation of PEP, arising from glycolysis of starch (starch-malate sequence) - C₃-metabolism metabolism with primary carbon fixed by the Calvin and Benson pathway (C₃-origin) - C₄-metabolism metabolism with primary carbon fixed by the Hatch and Slack pathway (C₄-origin) - C₃-pathway pathway with RuBPC activity and the Calvin and Benson pathway, irrespective of the CO₂-source, atmospheric or reuptake of the C₄ from malate - ¹³C()=(R_sample-R_PDR)10³/R_PDB where PDB Pee Dee belemnite (belemnite from the Pee Dee formation, South Carolina) and R=¹³C/¹²C - D isotope discrimination - PEP phosphoenolpyruvate - PEPC (EC 4.1.1.31) PEP carboxylase - PGA phosphoglyceric acid - Py.di-PK (EC 2.7.9.1) pyruvate, Pi-dikinase - RuBP ribulose bisphosphate - RuBPC (EC 4.1.1.39) RuBP carboxylase - SD short days - LD long days     

Drug testing data from the 2007 Pan American Games: δ C values of urinary androsterone, etiocholanolone and androstanediols determined by GC/C/IRMS

The main purpose of this article is to show the application of the CG/C/IRMS in real time during competition in the steroid confirmation analysis. For this reason, this paper summarizes the results obtained from the doping control analysis during the period of the 2007 Pan American Games held in Rio de Janeiro, Brazil. Approximately 5600 athletes from 42 different countries competed in the games. Testing was performed in accordance to World Anti-Doping Agency (WADA) technical note for prohibited substances. This paper reports data where abnormal urinary steroid profiles, have been found with the screening procedures. One 8 mL urine sample was used for the analysis of five steroid metabolites with two separate analyses by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Urine samples were submitted to GC/C/IRMS for confirmation analysis to determine the ¹³C/¹²C ratio of selected steroids. Fifty-seven urine samples were analyzed by GC/C/IRMS and the δ ¹³C values (‰) of androsterone, etiocholanolone, 5β-androstane-3α, 17β-diol (5β-diol), 5α-androstane-3α, 17β-diol (5α-diol) and 5β-pregnane-3α, 20α-diol (5β-pdiol), the endogenous reference compound are presented. One urine sample with a testosterone/epitestosterone (T/E) ratio of 4.7 was confirmed to be positive of doping by GC/C/IRMS analysis. The δ values of 5β-diol and 5α-diol were 3.8 and 10.8, respectively, compared to the endogenous reference compound 5β-pdiol, which exceeded the WADA limit of 3‰. The results obtained by CG/C/IRMS confirmation analyses, in suspicious samples, were conclusive in deciding whether or not a doping steroid violation had occurred.     

The activities of hydrogenase and enoate reductase in two Clostridium species,their interrelationship and dependence on growth conditions

The enzyme activities of Clostridium La 1 and Clostridium kluyveri involved in the stereospecific hydrogenation of ,-unsaturated carbonyl compounds with hydrogen gas were measured. In C. La 1 the specific activities of hydrogenase and enoate reductase depended heavily on the growth phase and the composition of the medium. During growth in batch cultures on 70 mM crotonate the specific activity of hydrogenase increased and then dropped to about 10% of its maximum value, whereas the activity of enoate reductase reached its maximum in cells of the stationary phase. Under certain conditions during growth the activity ratio hydrogenase: enoate reductase changed from 120 to 1. Thus, the rate limiting enzyme for the hydrogenation can be either the hydrogenase or the enoate reductase, depending on the growth conditions of the cells.The specific activities of ferredoxin-NAD reductase and butyryl-CoA dehydrogenase increased 3-4-fold during growth on crotonate. By turbidostatic experiments it was shown that at constant input of high crotonate concentrations (200 mM) the enoate reductase activity was almost completely suppressed; it increased steadily with decreasing crotonate down to an input concentration of 35 mM.Glucose as carbon source led to high hydrogenase and negligible enoate reductase activities. The latter could be induced by changing the carbon source of the medium from glucose to crotonate. Tetracycline inhibited the formation of enoate reductase.A series of other carbon sources was tested. They can be divided into ones which result in high hydrogenase and rather low enoate reductase activities and others which cause the reverse effect.When the Fe²⁺ concentration in crotonate medium was growth limiting, cells with relatively high hydrogenase activity and very low enoate reductase activity in the stationary phase were obtained. At Fe²⁺ concentrations above 3·10^-7 M enoate reductase increased and hydrogenase activity reached its minimum. The ratio of activities changes by a factor of about 200. In a similar way the dependence of enzyme activities on the concentration of sulfate was studied.In batch cultures of Clostridium kluyveri a similar opposite time course of enoate reductase and hydrogenase was found.The possible physiological significance of this behavior is discussed.Non Standard Abbreviations O.D.₅₇₈ Optical density at 578 nm Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

Variation in stable isotope signatures of seston and a zooplanktivorous fish in a eutrophic Chinese lake

Temporal and spatial changes in ¹³C and ¹⁵N of seston (mainly phytoplankton) and isotopic relationship between seston and the lake anchovy (Coilia ectenes) were studied in the large eutrophic freshwater Lake Chaohu in China. Much of the spatial and temporal variation in ¹³C of lake anchovies was explained by variation in seston, indicating a strong link between pelagic primary production and higher order consumers. Because the lake is shallow, there were no significant differences in ¹³C and ¹⁵N of seston between surface and overlying waters. Spatially, the relatively high ¹³C and ¹⁵N of seston in the western part of the lake might be due to high levels of anthropogenically derived N and C introduced from the surrounding cities through sewage drainage systems. The trophic position of the lake anchovy in the food web of Lake Chaohu was estimated to be 2.9–4.1 (3.5 ± 0.4), which agrees well with the previous stomach content analysis suggesting that the lake anchovy fed both on zooplankton and small planktivorous fishes.     

Sources of organic and inorganic carbon in a headwater stream: Evidence from carbon isotope studies

A combination of stable isotope studies and ¹⁴Cdating were used to identify the main sources andprocesses controlling streamwater DOC and TIC in atemperate non-forested watershed. ¹³Cvalues for terrestrial (–24.9 to –29.1) and aquatic(–30.5 to –33.5) plants were similar to valuesreported in the literature for similar ecosystems.¹³C values for DOC in soil solution andstreamwater were consistent with soil and terrestrialvegetation, indicating that the terrestrial ecosystemis the dominant source of aquatic DOC in thiswatershed. ¹³C values of soil atmosphereCO₂ (–17.2 to –25.2) were slightly lessnegative than would be expected for production viaaerobic soil microbial decomposition and rootrespiration. There was a close correspondence between¹³C values (–15.5 to –21.5) forstreamwater TIC and soil atmospheric CO₂ in thecentral part of the catchment where the stream drainsCO₂-rich peats. ¹⁴C dating showed thatalthough peat has been accumulating in the watershedfor at least 2700 years, DOC in soil pore water andstreamwater contains carbon of predominantly recentorigin (post-AD 1955).     

Changes in soil organic matter with cropping as measured by organic carbon fractions and 13C natural isotope abundance

The decline in soil organic matter with cropping is a major factor affecting the sustainability of cropping systems. Changes in total C levels are relativelyinsensitive as a sustainability measure. Oxidation with different strength KMnO₄ has been shown to be a more sensitive indicator of change. The relative size of soil C fractions oxidised by 333 mM KMnO₄ declined with cropping, whilst the relative size of the unoxidised fraction increased. Changes in ¹³C ratio have been used to measure C turnover in systems which include C3 and C4 species.     

Carbon isotope discrimination by plants follows latitudinal and altitudinal trends

Summary In an earlier paper we provided evidence that carbon isotope discrimination during photosynthesis of terrestrial C3 plants decreases with altitude, and it was found that this was associated with greater carboxylation efficiency at high altitudes. Changing partial pressures of CO₂ and O₂ and changing temperature are possible explanations, since influences of moisture and light were reduced to a minimum by selective sampling. Here we analyse plants sampled using the same criteria, but from high and low altitudes along latitudinal gradients from the equator to the polar ends of plant distribution. These data should permit separation of the pressure and temperature components (Fig. 1). Only leaves of fully sunlit, non-water-stressed, herbaceous C3 plants are compared. The survey covers pressure differences of 400 mbar (ca. 5000 m) and 78 degrees of latitude (ca 25 K of mean temperature of growth period). When habitats of similar low temperature (i.e. high altitude at low latitude and low altitude at polar latitude) are compared, discrimination increases towards the pole (with decreasing altitude and thus increasing atmospheric pressure). Latitudinally decreasing temperature at almost constant atmospheric pressure (samples from low altitude) is associated with a decrease in discrimination. So, polar low-altitude plants have ¹³C values half way between humid tropical lowland and tropical alpine plants. It is unlikely that latitudinal changes of the light regime had an effect, since low and high altitude plants show contrasting latitudinal trends in ¹³C although local altitudinal differences in overall light consumption were small. These results suggest that both temperature and atmospheric pressure are responsible for the altitudinal trends in ¹³C discrimination. Temperature effects may partly be related to increased leaf thickness (within the same leaf type) in cold environments. Theoretical considerations and laboratory experiments suggest that it is the oxygen partial pressure that is responsible for the pressure related change in discrimination. The study also provided results of practical significance for the use of carbon isotope data. Within a community of C3 plants, discrimination in species of similar life form, exposed to similar light, water and ambient CO₂ conditions ranges over 4, with standard deviations for 10–30 species of ±0.6 to 1.2. This natural variation has to be taken into account by using a sufficient sample size and standardization of sampling in any attempt at ecological site characterization using carbon isotope data. Evidence of a pronounced genotypic component of this variation in ¹³C discrimination in wild C3 plant species is provided. Correlations with dry matter partitioning, mesophyll thickness and nitrogen content are also present.     

Role of Sodium Ions and Their Uptake by Cells of Cultured Blue-Green Algae, Spirulina platensis and Spirulina maxima

The growth of the blue-green algae Spirulina platensis and Spirulina maxima, cultured in complete mineral Zarouk medium containing Na⁺ or Na⁺-deficient medium, was studied over a period of 24 h. The optical densities of S. platensis and S. maxima cells, determined during the last hour of exposure to sodium deficiency, amounted to 55.6 and 32.6%, respectively, of the optical densities of the same cells grown in complete Zarouk medium. Moreover, the cultures grown in Na⁺-deficient medium exhibited increased ability to take up sodium (which was low in S. platensis and S. maxima cells cultured in complete mineral medium). It is concluded that the two species studied are characterized by periodic, on the order of minutes, changes in the cellular uptake and release of sodium.     

A global survey of carbon isotope discrimination in plants from high altitude

Summary Carbon 13/12 isotope ratios have been determined from leaves of a hundred C₃ plant species (or ecotypes) from all major mountain ranges of the globe, avoiding drought stressed areas. A general increase in ¹³C content was found with increasing altitude, i.e. overall discrimination against the heavy isotope is reduced at high elevation. The steepest decline of discrimination is observed in taxa typically ranging to highest elevations (e.g. the genus Ranunculus). Mean ¹³C for all samples collected between 2500 and 5600 m altitude is-26.15 compared to the lowland average of-28.80 (P<0.001). Forbs from highest elevations reach-24. According to theory of ¹³C discrimination this indicates decreasing relative limitation of carbon uptake by carboxylation. In other words, we estimate that the ratio of internal to external partial pressure of CO₂ (p _i /p _a )in leaves of high elevation plants is lower than in leaves of low altitude. These results confirm recent gas exchange analyses in high and low elevation plants.     

Carbon and nitrogen isotope ratios in different compartments of a healthy and a declining Picea abies forest in the Fichtelgebirge,NE Bavaria

Summary Natural carbon and nitrogen isotope ratios were measured in different compartments (needles and twigs of different ages and crown positions, litter, understorey vegetation, roots and soils of different horizons) on 5 plots of a healthy and on 8 plots of a declining Norway spruce (Picea abies (L.) Karst.) forest in the Fichtelgebirge (NE Bavaria, Germany), which has recently been described in detail (Oren et al. 1988a; Schulze et al. 1989). The ¹³C values of needles did not differ between sites or change consistently with needle age, but did decrease from the sun-to the shade-crown. This result confirms earlier conclusions from gas exchange measurements that gaseous air pollutants did no long-lasting damage in an area where such damage was expected. Twigs (¹³C between-25.3 and-27.8) were significantly less depleted in ¹³C than needles (¹³C between-27.3 and-29.1), and ¹³C in twigs increased consistently with age. The ¹⁵N values of needles ranged between-2.5 and-4.1 and varied according to stand and age. In young needles ¹⁵N decreased with needle age, but remained constant or increased in needles that were 2 or 3 years old. Needles from the healthy site were more depleted in ¹⁵N than those from the declining site. The difference between sites was greater in old needles than in young ones. This differentiation presumably reflects an earlier onset of nitrogen reallocation in needles of the declining stand. ¹⁵N values in twigs were more negative than in needles (-3.5 to-5.2) and showed age- and stand-dependent trends that were similar to the needles. ¹⁵N values of roots and soil samples increased at both stands with soil depth from-3.5 in the organic layer to +4 in the mineral soil. The ¹⁵N values of roots from the mineral soil were different from those of twigs and needles. Roots from the shallower organic layer had values similar to twigs and needles. Thus, the bulk of the assimilated nitrogen was presumably taken up by the roots from the organic layer. The problem of separation of ammonium or nitrate use by roots from different soil horizons is discussed.     

Separation and characterization of the complexes constituting the cellulolytic enzyme system of Clostridium thermocellum

Clostridium thermocellum, strain JW20 (ATCC 31449) when growing in cellulose produces a cellulolytic enzyme system, that at the early stage of the fermentation is largely bound to the substrate. As cellulose is consumed the bound enzyme is released as free enzyme to the culture fluid. The bound enzyme fraction extracted with distilled water from the cellulose contains two major components, a large complex (M_r100×10⁶) and a small complex M_r4.5×10⁶) which were separated by gel filtration and sucrose solved by affinity chromatography into a complex that binds to the column and into a non-bindable mixture of proteins. All four fractions have endo--glucanase activity but only the two bound complexes and the free bindable complex hydrolyze crystalline cellulose with cellobiose as the main product. These three complexes are qualitatively similar in that they each contain about 20 different polypeptides (M_r values from 45,000 to 200,000) of which about ten are major components. However, the relative amounts of some of the peptides in the complexes differ. At least four polypeptides of the complexes have endo--glucanase activity.Abbreviations CM cellulose, carboxymethyl cellulose - CMCase carboxymethyl cellulase cosidered endo--1,4-glucanase - SDS sodium dodecyl sulfate - YAS yellow affinity substance - YAS-cellulose yellow affinity substance-cellulose complex     

Stable carbon isotope analysis of soil organic matter illustrates vegetation change at the grassland/woodland boundary in southeastern Arizona,USA

In southeastern Arizona, Prosopis juliflora (Swartz) DC. and Quercus emoryi Torr. are the dominant woody species at grassland/woodland boundaries. The stability of the grassland/woodland boundary in this region has been questioned, although there is no direct evidence to confirm that woodland is encroaching into grassland or vice versa. We used stable carbon isotope analysis of soil organic matter to investigate the direction and magnitude of vegetation change along this ecotone. ¹³C values of soil organic matter and roots along the ecotone indicated that both dominant woody species (C₃) are recent components of former grasslands (C₄), consistent with other reports of recent increases in woody plant abundance in grasslands and savannas throughout the world. Data on root biomass and soil organic matter suggest that this increase in woody plant abundance in grasslands and savannas may increase carbon storage in these ecosystems, with implications for the global carbon cycle.     

The importance of seed reserves for seedling performance: an integrated approach using morphological,physiological, and stable isotope techniques

To investigate how seed reserves affect early seedling performance, we conducted a factorial greenhouse experiment using Lithocarpus densiflora (Tanoak). Seedlings were grown from large (5.8±0.7 g) and small (3.2±0.4 g) seeds and, following shoot emergence, seeds were either removed or left attached. Seedlings were harvested for quantification of biomass and ¹³C at seven time periods following seed removal (2, 4, 8, 16, 32, 64, 128 days) and seedling photosynthesis was measured three separate time periods (2–4, 49–82, 95–128 days after seed removal). Biomass increased for all seedlings, but the increase was significantly larger for seedlings with attached seeds than with removed seeds. Seed removal just after shoot emergence significantly decreased seedling biomass, but seed removal 64 days after shoot emergence had no effect on seedling biomass. Seedling photosynthesis per unit leaf area varied by time and seed presence, but not by seed size. At the first period, seedlings with attached seeds had significantly higher photosynthetic rates than seedlings with removed seeds, at the second period there was no effect of seed removal, and at the third time period seedlings with attached seeds had significantly lower photosynthetic rates than seedlings with removed seeds. Despite temporal variation in photosynthesis per unit leaf area, seedlings with attached seeds always had significantly greater leaf area than seedlings with removed seeds, resulting in significantly higher total plant photosynthesis at all three time periods. The ¹³C values of both the leaves and roots were more similar to that of the seed for seedlings with attached seeds than for seedlings with removed seeds, however, seed removal and seed size strongly affected root ¹³C. This study demonstrates that seed reserves have important effects on the early growth, physiology, and ¹³C of L. densiflora seedlings.     

Polar bears make little use of terrestrial food webs: evidence from stable-carbon isotope analysis

Summary The mean stable-carbon isotope ratios (¹³C) for polar bear (Ursus maritimus) tissues (bone collagen –15.7, muscle –17.7, fat –24.7) were close to those of the same tissues from ringed seals (Phoca hispida) (–16.2, –18.1, and –26.1, respectively), which feed exclusively from the marine food chain. The ¹³C values for 4 species of fruits to which polar bears have access when on land in summer ranged from –27.8 to –26.2, typical of terrestrial plants in the Arctic. An animal's ¹³C signature reflects closely the ¹³C signature of it's food. Accordingly, the amount of food that polar bears consume from terrestrial food webs appears negligible, even though some bears spend 1/3 or more of each year on land during the seasons of greatest primary productivity.     

Keeling plots for hummingbirds: a method to estimate carbon isotope ratios of respired CO<Subscript>2</Subscript> in small vertebrates

The carbon isotope composition of an animals breath reveals the composition of the nutrients that it catabolizes for energy. Here we describe the use of Keeling plots, a method widely applied in ecosystem ecology, to measure the ¹³C of respired CO₂ of small vertebrates. We measured the ¹³C of Rufous Hummingbirds (Selasphorus rufus) in the laboratory and of Mourning (Zenaida macroura) and White-winged (Z. asiatica) Doves in the field. In the laboratory, when hummingbirds were fed a sucrose based C3 diet, the ¹³C of respired CO₂ was not significantly different from that of their diet (¹³C_{C3 diet}). The ¹³C of respired CO₂ for C3 fasted birds was slightly, albeit significantly, depleted in ¹³C relative to ¹³C_{C3 diet}. Six hours after birds were shifted to a sucrose based C4 diet, the isotopic composition of their breath revealed that birds were catabolizing a mixture of nutrients derived from both the C3 and the C4 diet. In the field, the ¹³C of respired CO₂ from Mourning and White-winged Doves reflected that of their diets: the CAM saguaro cactus (Carnegeia gigantea) and C3 seeds, respectively. Keeling plots are an easy, effective and inexpensive method to measure ¹³C of respired CO₂ in the lab and the field.     

Introduction of genes for leucine biosynthesis from Clostridium pasteurianum into C. acetobutylicum by cointegrate conjugal transfer

Summary A Clostridium pasteurianum gene bank was constructed in Escherichia coli, using plasmid pAT153, and several chromosomal fragments found which complemented both leuB and leuC mutations in auxotrophic E. coli K12 strains. No fragments capable of complementing leuA or leuD mutations were identified. Conjugal transfer of the LeuB/leuC genes from Bacillus subtilis into two different Leu^- C. acetobutylicum auxotrophic strains was elicited by their incorporation into a large plasmid cointegrate composed of the conjugal plasmid pAM1 and a specially constructed gram-positive, replication-deficient plasmid, pMTL21 EC. Inheritance of the cointegrate plasmid restored one of the auxotrophic C. acetobutylicum strains to prototrophy. The second strain remained Leu^-.     

Growth and Endogenous Cytokinins of Juniper Shoots as Affected by High Metal Concentrations

The growth and the content of endogenous cytokinins (CKs) of current-year-old shoots from juniper plants (Juniperus communis L.) growing over and off ore site were compared. The juniper shoots from ore site (M plants) had higher metal content and exhibited delayed growth. Less bases and nucleosides of Z- and iP- type CK and more iP-conjugates were present in the M shoots. These changes were probably due to inhibited CK export from the roots and/or altered CK metabolism forming less biologically active CKs.     

Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)     

Common and form-specific cell wall antigens of Candida albicans as released by chemical and enzymatic treatments

In order to investigate the antigenic properties of the proteins and mannoproteins present in the cell surface of Candida albicans, and to identify individual antigenic moieties and their distribution, a number of polyclonal antisera were obtained by immunizing rabbits with chemical and enzymatic cell wall extracts obtained from intact cells from both growth forms (yeast and mycelium) of the fungus. Prior to injection, wall moieties present in the extracts were subjected to different treatments and/or purification procedures such as adsorption onto polystyrenelatex microbeads or electrophoretic separation. When used as probes in indirect immunofluorescence assays, the different antisera gave rise to different fluorescence patterns varying in intensity and topological localization of the reactivity in C. albicans cells. When the different antisera were used as probes in Western blots of wall proteinaceous materials solubilized from both blastospores and germ tubes, differences in reactivity and specificity were readily discernible, allowing to identify a number of common and form-specific cell wall components. Of special interest was the fact that one of the antisera raised, after adsorption onto heat-killed blastospores, specifically recognized medium to low molecular weight moieties present only in the cell wall extracts from germ tubes. When this antiserum was used as probe in immunofluorescence assays, reactivity was confined to the hyphal extensions. Together, these observations seem to indicate that the different antibody preparations described in this report could represent important tools in the study of different aspects of the cell wall biology in C. albicans, including the identification and study of the distribution of common and form-specific cell wall-bound antigens.Abbreviations IIF Indirect immunofluorescence - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - PAb polyclonal antibody