Chemical and histochemical studies of normal and diseased human gastrointestinal tract. III. Changes in the histochemical and chemical properties of the epithelial glycoproteins in the mucosa close to colonic tumours

Summary Histochemical, chemical and histological studies were performed on 26 specimens of human colonic tumours and 62 specimens of mucosa taken at distances of 0.5–5.0 cm from the tumour. The tumour glycoproteins were divided almost equally between three anionic types, sulphomucin, sialomucin and mixed sialomucin and sulphomucin. All showed a reduction in staining for side chainO-acylated sialic acid. In 56% of the tumours, this was accompanied by loss of glycoprotein while, in 44%, abundant mucin was still present.Histochemical examination of the mucosal specimens indicated that in 24.2% the side chainO-acylated sialic acids did not differ from normal. In 41.9% there was a focal change and in 33.9% there was a generalized field reduction in the proportion of side chainO-acyl sialic acids. The latter were subdivided into moderate and severe. Chemical analyses correlated well with the histochemical classification of the mucosal specimens and showed that, on average, the classifications focal and severe field change were not due to sampling error. Forty-five per cent of the cases showed only focal change and 40% only field change. Mucosal specimens associated with 60% of the moderately differentiated tumours showed only focal change while those associated with 75% of well-differentiated tumours showed only field change. Abnormal patterns of staining for side chainO-acylated sialic acids (a) were largely independent of the distance from the tumour, (b) occurred in the presence of a normal pattern of staining for sialomucins and sulphomucins and (c) were associated with 61.4% of the specimens that showed no discernible evidence of histological abnormality. In contrast, only one specimen showed evidence of histological change without a corresponding change inO-acylated sialic acids. The data suggest that abnormal patterns of staining forO-acylated sialic acids may represent premalignant change but their precise significance and specificity requires further studies of non-neoplastic diseases of the colon.     

Chemical and histochemical studies of normal and diseased human gastrointestinal tract. V. A differential diagnostic method for the histochemical classification of glycoproteins

Summary A differential diagnostic scheme is described for the division of colonic epithelial glycoproteins into eleven histochemically distinct classes. The scheme depends upon the use of seven histochemical techniques which, collectively, permit the differential staining ofO-sulphate ester, sialic acid and its side chainO-acyl variants and vicinal diols located on carbohydrate residues other than sialic acids. Elements of the scheme also provide a general approach to the classification of epithelial glycoproteins in anatomic sites other than the colon.Application of the scheme permitted the classification of the epithelial glycoproteins in the mucosa 0.5–5.0 cm from human colonic tumours and provided direct confirmation of previous observations that changes from normal in the relative proportions of either side chainO-acylated sialic acids or sialic acids andO-sulphate esters can occur independently of one another.     

Chemical and histochemical studies of normal and diseased human gastrointestinal tract. II. A comparison between histologically normal small intestine and Crohn's disease of the small intestine

Summary Comparative chemical and histochemical studies were performed on formalin-fixed, surgical specimens of human small intestine from cases of Crohn's disease and normal controls. The sialic acids of the crude glycoproteins isolated from normal ileum were significantly less neuraminidase-susceptible and more C₄ substituted (P<0.01) than those of the glycoproteins isolated either from normal upper small intestine (duodenum and jejunum) or from cases of Crohn's disease of the ileum. Fractionation yielded two major sialic acid-containing fractions, eluting from DEAE-cellulose with 0.2m or 0.3m sodium chloride. Both fractions contained fucose, galactose, glucosamine and galactosamine in addition to sialic acids both with and withoutO-acyl substituents at position C₄ and/or in the side-chain (side-chainO-acylated sialic acids were also detected by histochemical procedures). The fractions differed significantly from one another with respect to the neuraminidase susceptibility of their sialic acids (P<0.01), the percentage of C₄ (P<0.01) and side-chain substituted sialic acids (P<0.05), and the molar fucose-sialic acid ratio (P<0.05). TheO-acyl substitution patterns of the sialic acids of both the 0.2m and 0.3m fractions of the upper small intestine glycoproteins differed significantly from those of the corresponding fractions from normal ileum, while the sialic acids of the 0.2m fractions from Crohn's disease of the ileum differed significantly from normal with respect to neuraminidase susceptibility (P<0.01) and percentage C₄ substitution (P<0.01); the 0.3m fractions differed only in the percentage of sialic acids substituted at C₄. The differences between the sialic acids from the normal and Crohn's disease specimens were shown to be independent of either the anatomical origin of the specimen or the histopathological sub-group of the Crohn's disease specimens; no significant differences were noted between the sub-groups but all the sub-groups differed from normal.     

Binding specificity of influenza C-virus to variablyO-acetylated glycoconjugates and its use for histochemical detection ofN-acetyl-9-O-acetylneuraminic acid in mammalian tissues

The specificity of influenza C-virus binding to sialoglycoconjugates was tested with various naturallyO-acetylated gangliosides or syntheticallyO-acetylated sialic acid thioketosides, which revealed binding to 9-O-acetylatedN-acetylneuraminic acid. Binding was also observed with a sample of Neu5,7Ac₂-GD3, however at a lower degree. Sialic acids with two or threeO-acetyl groups in the side chain of synthetic sialic acid derivatives are not recognized by the virus. In these experiments, bound viruses were detected with esterase substrates. Influenza C-virus was also used for the histological identification of mono-O-acetylated sialic acids in combination with an immunological visualization of the virus bound to thin-sections. The occurrence of these sialic acids was demonstrated in bovine submandibular gland, rat liver, human normal adult and fetal colon and diseased colon, as well as in human sweat gland. Submandibular gland and colon also contain significant amounts of glycoconjugates with two or three acetyl esters in the sialic acid side chain, demonstrating the value of the virus in discriminating between mono- and higherO-acetylation at the same site. The patterns of staining showed differences between healthy persons and patients with colon carcinoma, ulcerative colitis or Crohn's disease. Remarkably, some human colon samples did not showO-acetyl sialic acid-specific staining. The histochemical observations were controlled by chemical analysis of tissue sialic acids.Abbreviations BSA bovine serum albumin - BSM bovine submandibular gland mucin - HAU haemagglutination units - HPLC high-performance liquid chromatography - HPTLC high-performance thin-layer chromatography - Neu5Ac N-acetylneuraminic acid - Neu5,9Ac₂ N-acetyl-9-O-acetylneuraminic acid - Neu5,7,9Ac₃ N-acetyl-7,9-di-O-acetylneuraminic acid - Neu5,7,8,9Ac₄ N-acetyl-7,8,9-tri-O-acetylneuraminic acid - PBS phosphate-buffered saline - TLC thin-layer chromatography Dedicated to Prof. Dr Nathan Sharon on the occasion of his 70th birthday.     

Chemical and histochemical studies of normal and diseased human gastrointestinal tract. I. A comparison between histologically normal colon, colonic tumours, ulcerative colitis and diverticular disease of the colon

Summary Chemical and histochemical methods were used to compare the epithelial glycoproteins from formalin-fixed surgical specimens of normal human large intestine, colonic tumours, ulcerative colitis and diverticular disease. All the epithelial glycoproteins contained fucose, galactose, glucosamine, galactosamine and, in addition, sialic acids both with and withoutO-acyl substituents in the side chain and/or at position C₄. The glycoproteins of the normal ascending and descending colons differed significantly with respect to the percentage of the sialic acids released following digestion of the de-O-acylated glycoprotein withVibrio cholera neuraminidase and to the molar fucose-sialic acid ratio. Statistical analysis of the chemical data showed that (a) compared to normal, the sialic acids of the tumour and ulcerative colitis glycoproteins from the descending colon were significantly less substituted in the side chain and at position C₄; (b) theO-acetyl substitution pattern of the sialic acids of the ulcerative colitis glycoproteins from the ascending colon and the quantitative composition of the carbohydrate prosthetic groups of the ulcerative colitis glycoproteins from both ascending and descending colons differed from normal; (c) it was not always possible to distinguish between the ulcerative colitis and tumour glycoproteins on the basis of theO-acetyl substitution pattern of their sialic acids; and (d), there were minor differences between normal glycoproteins and those from cases of diverticular disease.     

A new method for the histochemical demonstration of O-acyl sugars in human colonic epithelial glycoproteins

Summary A new general method has been developed for the specific histochemical identification ofO-acyl sugars in any epithelial glycoprotein. These sugars include hexose, 6-deoxyhexose andN-acetylhexosamine with an ester substituenent(s) located on a potentialvicinal diol(s). In the procedure reported [the periodic acid-borohydride reduction-saponification-selective periodate oxidation-borohydride reduction-periodic acid-Schiff (PA-Bh-KOH-PA-Bh*-Bh-PAS) method] the initial PA-Bh treatment rendersvicinal diols located on either sialic acid or neutral sugars PAS unreactive. In the subsequent steps ester substituents are removed from bothO-acyl sugars andO-acyl sialic acids by saponification (KOH), sialic acidvicinal diols are selectively removed by the PA^*-Bh sequence andO-acyl sugars are stained with the PAS technique. This method has the advantage that the results are obtained with a single section and the results are either positive or negative. Consequently, it is superior to the three indirect methods investigated because it does not require an observer to compare the intensity or the shade of the staining obtained with serial sections.Using the PA-Bh-KOH-PA^*-Bh-PAS method we have demonstrated, for the first time, thatO-acyl sugars occur in the epithelial goblet cell glycoproteins of adult human colon. The effect of the presence ofO-acyl sugars on the interpretation of a number of other methods for the histochemical investigation of glycoproteins is discussed. It is recommended that the results obtained with the PA-Bh-KOH-PA^*-Bh-PAS method be evaluated before histochemical procedures for the investigation of neutral sugars andO-acyl sialic acids are selected.     

Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its O-acyl variants or O-sulphate ester. II. Methods based upon the periodic acid-phenylhydrazine-Schiff reaction

Summary Four methods based upon the periodic acid—phenylhydrazine—Schiff reaction have been developed for the simultaneous visualization of neutral sugars with periodate oxidizablevicinal diols (hexose, 6-deoxyhexose,N-acetylhexosamine) and either sialic acids or side chainO-acyl sialic acids. In the first of these procedures, the saponification—periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A—Schiff—saponification (KOH—PA—DNPH—Az—KOH) method, all sialic acids stain Azure blue, neutral sugars with oxidizablevicinal diols stain yellow and mixtures of such components stain in various shades of green. In the second technique, periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A Schiff—saponification (PA—DNPH—Az—KOH), Azure Blue staining is confined to sialic acids without side chain substituents or which have anO-acyl substituent at position C7, while in the third method, the selective periodate oxidation—borohydride reduction—saponification—periodic acid oxidation—2,4-dinitrophenyl hydrazine—Azure A—Schiff—saponification (PA^*—Bh—KOH—PA—DNPH—Az—KOH) technique, only sialic acids withO-acyl substituents at positions C7, C8 or C9 (or which have two or threeO-acyl side chain substituents) stain Azure blue. Finally in the fourth procedure, periodic acid oxidation—2,4-dinitrophenylhydrazine—Azure A—Schiff—saponification—borohydride reduction—periodic acid oxidation—Schiff (PA—DNPH—Az—KOH—Bh—PAS), sialic acids without side chain substituents or which haveO-acyl substituents at C7 stain Azure blue, sialic acids substituted at position C8 or C9 (or which are di- or tri-substituted) stain magenta and neutral sugars stain yellow. Where mixtures of these components are present, a wide range of colours is obtained.     

Histochemical identification of side chain substituted O-acylated sialic acids: the PAT-KOH-Bh-PAS and the PAPT-KOH-Bh-PAS procedures

Summary Two new methods, based on the original periodic acid-Thionin Schiff-saponification-periodic acid-Basic Fuchsin Schiff (PAT-KOH-PAS) technique of Cullinget al. (1976), have been devised for the histochemical identificantion of side-chainO-acylated sialic acids. In the first of these, the periodic acid-Thionin Schiff-saponification-borohydride reduction-periodic acid-Basic Fuchsin Schiff (PAT-KOH-Bh-PAS) procedure, the specificity of the original PAT-KOH-PAS technique was improved by: (a) extending, when necessary, the initial period of periodate oxidation, (b) increasing the period of exposure to Thionin Schiff reagent from 30 min to 4 h, (c) using a Thionin Schiff reagent prepared by a different method, (d) interposing a borohydride reduction step between the saponification and PAS steps and, (e) extending the period of oxidation in the final PAS step from 10 to 30 min. In the second procedure, the periodic acid-phenylhydrazine-Thionin Schiffborohydride reduction-periodic acid-Basic Fuchsin Schiff (PAPT-KOH-Bh-PAS), based on the periodic acid-phenylhydrazine-Schiff (PAPS) technique of Spicer (1961), blue Thionin Schiff staining was confined to sialic acid residues with oxidizable side chainvicinal diols by interposing a treatment with 0.5% (w/v) aqueous phenylhydrazine hydrochloride for 2 h at room temperature between the initial periodic acid oxidation and the Thionin Schiff steps of the PAT-KOH-Bh-PAS procedure. These procedures are discussed within the general framework of the methods available for the histochemical identification of side-chainO-acylated sialic acids.     

Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its side chain O-acyl variants or O-sulphate ester. I. Methods based upon the selective periodate oxidation of sialic acids

Summary Five new methods, based upon the selective oxidation of sialic acid residues with 0.4mm periodic acid in approximately 1m hydrochloric acid at 4°C for 1 h (PA^*), have been devised for the simultaneous visualization of neutral sugars and either sialic acid and its side chainO-acyl variants orO-sulphate ester. In the first of these, the selective periodate oxidation—borohydride reduction—saponification—selective periodate oxidation—Thionin Schiff—saponification—borohydride reduction—periodic acid—Schiff (PA^*—Bh—KOH—PA^*—T—KOH—Bh—PAS) technique, sialic acids withO-acyl substituents at C7, C8 or C9 (or which have two of three side chainO-acyl substituents) stain blue while neutral sugars with periodate-sensitivevicinal diols (hexose, 6-deoxyhexose, andN-acetylhexosamine) stain magenta. The second method, the saponification—selective periodate oxidation—Thionin Schiff—saponification—borohydride reduction—periodic acid—Schiff (KOH—PA^*—T—KOH—Bh—PAS), stains all sialic acids blue and neutral sugars magenta. In the third procedure, the selective periodate oxidation—Thionin Schiff—borohydride reduction—periodic acid—Schiff—saponification (PA^*—T—Bh—PAS—KOH) method, sialic acids without side chain substituents (or which have anO-acyl substituent at C7) stain blue and neutral sugars stain magenta. In the fourth method, the saponification-selective periodate oxidation—borohydride reduction—Alcian Blue pH 1.0—periodic acid—Schiff (KOH—PA^*—Bh—AB1.0—PAS) technique,O-sulphate esters stain aquamarine blue and neutral sugars stain magenta. In all of these techniques mixtures of the components stain in various shades of purple. Performance of the KOH—PA^*—Bh—AB1.0—PAS technique without the Alcian Blue pH 1.0 step provides a method for the selective identification of neutral sugars in macromolecules that also contain sialic acids.     

Metapopulation structure,population trends,and status of Florida Grasshopper Sparrows

ABSTRACT The federally Endangered Florida Grasshopper Sparrow (FGSP, Ammodramus savannarum floridanus) is endemic to dry prairie habitat in central Florida and is currently only found at three public management areas (Avon Park Air Force Range, APAFR; Kissimmee Prairie Preserve State Park, KPPSP; and Three Lakes Wildlife Management Area, TLWMA). We analyzed long‐term (1991–2008) point‐count data to compare population trends of FGSPs at these management areas to determine if they function as independent populations by using Spearman's rank correlation to test for independence between annual trends. We also examined banding and resighting data to infer metapopulation structure. Populations fluctuated across years at all three sites, declining significantly at APAFR (r_s=?0.89, P < 0.001, N= 13) and KPPSP (r_s=?0.78, P= 0.004, N= 11) and remaining stable at TLWMA (r_s= 0.19, P= 0.46, N= 17). Population trends among the three management areas appeared independent (absolute value of r_s≤ 0.50, P≥ 0.12). Previous studies indicated that sparrows in the three areas were not genetically differentiated, and two cases of dispersal between APAFR and KPPSP have been documented. However, dispersal rates among areas appear to be too low to influence demographic dynamics within individual areas. Within APAFR, FGSPs are aggregated into three spatially distinct habitat patches previously considered separate populations, but dispersal among these patches is more frequent than previously reported and population trends among these patches are correlated (r_s≥ 0.91, P < 0.001). These patterns suggest that a single metapopulation of FGSPs exists consisting of three distinct populations (APAFR, KPPSP, and TLWMA), and the spatially distinct aggregations at APAFR constitute a single population using several habitat patches. The population at APAFR is at risk of extirpation, and immediate action is needed if that population is to recover. Taking broader metapopulation dynamics into account will be useful for guiding management efforts aimed at conserving the FGSP in the broader central Florida landscape.     

Successional changes in soil nitrogen availability,non-symbiotic nitrogen fixation and carbon/nitrogen ratios in southern Chilean forest ecosystems

Vast areas of southern Chile are now covered by second-growth forests because of fire and logging. To study successional patterns after moderate-intensity, anthropogenic fire disturbance, we assessed differences in soil properties and N fluxes across a chronosequence of seven successional stands (2–130 years old). We examined current predictions of successional theory concerning changes in the N cycle in forest ecosystems. Seasonal fluctuations of net N mineralization (N_min) in surface soil and N availability (N_a; N_a=NH ₄ ⁺ –N+NO ₃ ^– –N) in upper and deep soil horizons were positively correlated with monthly precipitation. In accordance with theoretical predictions, stand age was positively, but weakly related to both N_a (r ²=0.282, P<0.001) and total N (N_tot; r ²=0.192, P<0.01), and negatively related to soil C/N ratios (r ²=0.187, P<0.01) in surface soils. A weak linear increase in soil N_min (upper plus deep soil horizons) was found across the chronosequence (r ²=0.124, P<0.022). N_min occurred at modest rates in early successional stands, suggesting that soil disturbance did not impair microbial processes. The relationship between N fixation (N_fix) in the litter layer and stand age best fitted a quadratic model (r ²=0.228, P<0.01). In contrast to documented successional trends for most temperate, tropical and Mediterranean forests, non-symbiotic N_fix in the litter layer is a steady N input to unpolluted southern temperate forests during mid and late succession, which may compensate for hydrological losses of organic N from old-growth ecosystems.     

Habitual physical activity and peak anaerobic power in elderly women

The aim of this study was to investigate the relationship between maximal anaerobic power (P _max) and corresponding optimal velocity (V _opt) and habitual physical activity (PA) on the one hand and with maximal oxygen consumption (V˙O_2max) on the other hand, in elderly women. Twenty-nine community dwelling, healthy women aged 66–82 years participated in the study. PA was evaluated using the Questionnaire d'Activite Physique Saint-Etienne (QAPSE) and expressed using two QAPSE activity indices: mean habitual daily energy expenditure (MHDEE) and daily energy expenditure corresponding to leisure time sports activities (sports activity). The subjects' P _max and V _opt were measured while they cycled on a friction-loaded non-isokinetic cycle ergometer. P _max was expressed relative to body mass [P _max/kg(W · kg⁻¹)], and relative to the mass of two quadriceps muscles [P _{max /Quadr}(W·kg_Quadr ⁻¹)]. A negative relationship between P _max/kg (Spearman's r = −0.56; P < 0.01), P _max/Quadr (r = −0.53; P < 0.01) and V _opt (r = −0.45; P < 0.05) and age was found. P _max/kg was positively associated with MHDEE (r = 0.51; P < 0.01) and sports activity (r = 0.58; P < 0.01), as were P _max/Quadr and V _opt (r = 0.55; P < 0.01 and r = 0.54; P < 0.01, respectively). P _max/kg, P _max/Quadr and V _opt correlated positively with V˙O_2max. The positive relationship between ergometer measurements and PA indices was similar to that between V˙O_2max and PA. P _max/kg was, moreover, closely related to V _opt (r = 0.77; P < 0.001). When a multiple stepwise regression analysis was used to select the variables influencing ergometer measurements, MHDEE contributed significantly to P _max/kg variance, whereas sports activity contributed to P _max/Quadr and V _opt variances. In conclusion, the data from this cross-sectional study suggest that in healthy elderly women habitual PA, and especially leisure time PA, alleviates the decline of the P _max of the quadriceps muscles. Accepted: 30 January 1997     

Enantioselective autocatalysis. III. Configurational and conformational studies on a 1,4-benzodiazepinooxazole derivative

We report here on configurational and conformational studies undertaken on the bromofluoro-1,4-benzodiazepinooxazole derivative I, which has previously been found capable of undergoing total spontaneous resolution under racemizing conditions. Due to its bridgehead N-atom I may exist in any of four diastereomeric forms,C _r N _r (orC _s N _s ) andC _r N _s (orC _s N _r ). Molecular mechanics calculations revealed that in their lowest energy conformations theC _r N _r (orC _s N _s ) diastereomers were favored over theC _r N _s (orC _s N _r ) diastereomers by some 3.42 kcal/mol, while semi-empirical quantum mechanical calculations indicated heats of formation also favoring theC _r N (orC _s N _s ) diastereomers by 3.83 kcal/mol. The¹H NMR spectra of the three crystalline modifications (, and) of I were examined in acetone, and found to be identical. These data, combined with the results of the above calculations, suggest that I exists in methanol or methanol-acetone solutions as theC _r N (orC _s N _s ) enantiomer prior to its racemization.     

Inter- and intra-habitat relationships between woodland cryptostigmata species diversity and the diversity of soil and litter microhabitats

Summary Soil animal communities contain a large number of species exhibiting a low degree of trophic specialization. Competition between animal species with similar food requirements is frequently reduced by partitioning habitat space and this ecological principle is demonstrated for woodland mite communities. Microhabitat diversity was determined for the litter (L), fermentation (F) and humus (H) sub-horizons using gelatine embedded soil sections and compared with mite species diversity for the same layers of the soil and litter profile. Cryptostigmata species diversity was correlated with microhabitat diversity (r=0.67, P<0.01) in six woodland soils with a range of humus forms. Intra-habitat relationships were determined for one site on two sampling occasions: in November an exceptionally high correlation was obtained (r=0.91, P<0.001) but samples collected in February showed a lower correlation (r=0.63, P<0.01). Within sub-horizon relationships showed significant correlations for the L (r=0.59, P<0.01) and H sub-horizons (r=0.72, P=0.001). The F sub-horizon data were more variable than the other two sub-horizons but a correlation of 0.60 (P=0.05) was obtained for the intrahabitat study.     

Genetic variability in photosynthetic rate and leaf characters in Brassicaceae coenospecies

Thirty-nine Brassica coenospecies grown in pot cultures during 1993 and 1994 were screened for variability in photosynthetic rate (P _N ) and leaf characters. There were significant differences among the species in P _N per unit leaf area, chlorophyll (Chl) content, specific leaf mass (SLM), stomatal resistance (r _s ) and individual leaf size. The interactions species x year and species x date of measurement were small compared to the species effect. There was a significant negative correlation between P _N and r _s and a significant positive one between P _N and both Chl content and SLM. This revised version was published online in August 2006 with corrections to the Cover Date.     

Interannual variation in soil CO2 efflux and the response of root respiration to climate and canopy gas exchange in mature ponderosa pine

We examined a 6‐year record of automated chamber‐based soil CO₂ efflux (F_s) and the underlying processes in relation to climate and canopy gas exchange at an AmeriFlux site in a seasonally drought‐stressed pine forest. Interannual variability of F_s was large (CV=17%) with a range of 427 g C m^?2 yr^?1 around a mean annual F_s of 811 g C m^?2 yr^?1. On average, 76% of the variation of daily mean F_s could be quantified using an empirical model with year‐specific basal respiration rate that was a linear function of tree basal area increment (BAI) and modulated by a common response to soil temperature and moisture. Interannual variability in F_s could be attributed almost equally to interannual variability in BAI (a proxy for above‐ground productivity) and interannual variability in soil climate. Seasonal total F_s was twice as sensitive to soil moisture variability during the summer months compared with temperature variability during the same period and almost insensitive to the natural range of interannual variability in spring temperatures. A strong seasonality in both root respiration (R_r) and heterotrophic respiration (R_h) was observed with the fraction attributed to R_r steadily increasing from 18% in mid‐March to 50% in early June through early July before dropping rapidly to 10% of F_s by mid‐August. The seasonal pattern in R_r (10‐day averages) was strongly linearly correlated with tree transpiration (r²=0.90, P<0.01) as measured using sap flux techniques and gross ecosystem productivity (GEP, r²=0.83, P<0.01) measured by the eddy‐covariance approach. R_r increased by 0.43 g C m^?2 day^?1 for every 1 g C m^?2 day^?1 increase in GEP. The strong linear correlation of R_r to seasonal changes in GEP and transpiration combined with longer‐term interannual variability in the base rate of F_s, as a linear function of BAI (r²=0.64, P=0.06), provides compelling justification for including canopy processes in future models of F_s.     

Isolation and properties of two sialate-<Emphasis Type="Italic">O</Emphasis>-acetylesterases from horse liver with 4- and 9-<Emphasis Type="Italic">O</Emphasis>-acetyl specificities

Sialate-O-acetylesterase was purified almost 900-fold from particle-free supernatants of horse liver by gel filtration, ion-exchange chromatography and isoelectric focussing. The native enzyme on gel filtration exhibits a molecular weight of 54,000 Da. It was separated by isoelectric focussing into two forms with pI values of 4.8 and 5.7, respectively. The esterase with a lower pI hydrolyses only 9-O-acetyl groups from sialic acids (K_M 1.1 mM), while that with the higher pI esterifies both 4- and 9-O-acetylated monosaccharides at similar rates (K_M 0.3 M and 1.3 mM, respectively). Both forms are inactive with 7-O-acetylated N-acetylneuraminic acid. Enzyme assays were carried out at the pH optimum (pH 8.4–8.6) using free O-acetylated sialic acids followed by direct analysis of the reaction products by isocratic anion-exchange HPLC. Glycosidically bound sialic acids can also be de-O-acetylated. Horse liver esterase seems to be an essential enzyme for the catabolism of 4-O-acetylated sialoglycoconjugates, since sialidase from this tissue cannot act on 4-O-acetylated sialic acids.     

Photosynthetic characteristics of diploid honeysuckle (<Emphasis Type="Italic">Lonicera japonica</Emphasis> Thunb.) and its autotetraploid cultivar subjected to elevated ozone exposure

In order to investigate the effect of chromosome doubling on ozone tolerance, we compared the physiological responses of a diploid honeysuckle (Lonicera japonica Thunb.) and its autotetraploid cultivar to elevated ozone (O₃) exposure (70 ng g⁻¹, 7 h d⁻¹ for 31 d). Net photosynthetic rate (P _N) of both cultivars were drastically (P<0.01) impaired by O₃. Although there were significantly positive correlation between P _N and stomatal conductance (g _s) in both cultivars under each treatment, the decreased g _s in O₃ might be the result rather than the cause of decreased P _N as indicated by stable or increasing the ratio of intercellular to ambient CO₂ concentration(C _i/C _a). P _N under saturating CO₂ concentration (P _Nsat) and carboxylation efficiency (CE) significantly decreased under O₃ fumigation, which indicated the Calvin cycle was impaired. O₃ also inhibited the maximum efficiency of photosystem II (PSII) photochemistry in the dark-adapted state (F_v/F_m), actual quantum yield of PSII photochemistry (Φ_PSII), electron transport rate (ETR), photochemical quenching coefficient (qP), non-photochemical quenching (NPQ), the maximum in vivo rate of Rubisco carboxylation (V_cmax) and the maximal photosynthetic electron transport rate (J_max) which demonstrated that the decrease in P _N of the honeysuckle exposed to elevated O₃ was probably not only due to impairment of Calvin cycle but also with respect to the light-harvesting and electron transport processes. Compared to the diploid, the tetraploid had higher relative loss in transpiration rate (E), (g _s), (P _Nsat), V_cmax and J_max. This result indicated that the Calvin cycle and electron transport in tetraploid was damaged more seriously than in diploid. A barely nonsignificant (P=0.086) interaction between O₃ and cultivar on P _N suggested a higher photosynthetic sensitivity of the tetraploid cultivar.     

Histochemical studies of the colonic epithelial glycoproteins of the normal rabbit

Summary Two general classes of glycoproteins have been identified in the colonic epithelial cells of New Zealand white rabbits. Each is associated with an ultrastructurally distinct secretory cell. The first of these classes is found in cells, termed vesiculated columnar cells, characterized by electron-translucent vesicles, a small rough endoplasmic reticulum-Golgi complex and prominent microvilli. The glycoproteins of the vesiculated cells contain abundantO-sulphate ester, sialic acids with ester substituents at positions C-8 or C-9 (or with two or three side chain substituents) and neutral sugars withvicinal diols whose periodate oxidation is prevented by anO-acyl ester substituent(s). The second class of glycoproteins occurs in goblet cells characterized by electron-dense vesicles, an abundant rough endoplasmic reticulum, a well-developed Golgi apparatus and few, if any, microvilli. Goblet cells along the entire length of the crypts contain neutral sugars with periodate-oxidisablevicinal diols and a ferriferricyanide-reactive component. Cells in the upper halves of the crypts also contain components that are sulphated, Schiff-reactive and acid-fast. In the lower halves of the crypts, the goblet cells contain smaller quantities of the above components plus sialic acids, some of which possibly have anO-acyl substituent located at position C-8 or C-9 (or which have two or three side chainO-acyl substituents). It is suggested that the function of the glycoproteins from the vesiculated columnar cells is protective and that from the goblet cells is lubricative.     

Leaf growth, stomatal diffusion resistances and photosynthesis during droughting of Lolium perenne populations selected for contrasting stomatal length and frequency

Lolium perenne selection lines with high calculated stomatal resistances to diffusion (r_s) as a result of either few or short stomata, maintained leaf extension rates and photosynthetic rates longer than selection lines with low resistances when deprived of water. There were no significant differences between high and low r_s plants in light saturated CO₂ uptake of turgid attached leaves. When grown in soil drying to 21% moisture, plants with low calculated r_s exhibited lower minimum leaf resistances (r_l) than those with high, measured with a diffusion porometer, on all except the last day. The daily maximum r_l (1.5 h after the start of the light period) became greater among low than high r_s plants as the difference in rate of leaf extension between the two groups of plants increased with drying soil. Rate of leaf extension was negatively correlated with daily maximum r_l and started to decline when relative leaf water content (RLWC), at 5 h after the start of the light period, fell below about 88%. Transpiration rate of plants grown in different soil moisture regimes was correlated (r=+ 0.83, P < 0.01) with mean maximum adaxial leaf conductance (reciprocal of resistance). There was a highly significant correlation (r=+ 0.62, P < 0.01) between calculated adaxial r_s and mean minimum measured r_l among plants growing at high or intermediate soil moisture, but not at low. Therefore, some random variation in minimum r_l, even with adequate moisture, seemed to be unaccounted for by variation in stomatal numbers or size. Selection for increased numbers of adaxial stomata also resulted in more on the abaxial surface, but mean adaxial/abaxial ratio in the ‘frequent’ stomata plants was still only about 9:1.