Tree-ring analysis and conifer growth responses to increased atmospheric CO2 levels

Summary Tree-ring data of naturally grown connifers were analyzed to evaluate the possibility of enhanced tree growth due to increased atmospheric CO₂. Tree cores were obtained from 34 sites in four different climatic regions in the northern hemisphere. In each of the four regions, the sampling sites were located along ecological gradients between the subalpine treeline and low elevations and, sometimes, the arid forest border. Growth trends after 1950, when the atmospheric CO₂ concentration increased by more than 30 l·l^-1 indicate an increase in ring-widths at eight of the 34 sites. These chronologies were from sites which moderate temperature or water stress. In four cases the growth increase in the post-1950 period coincided with favorable climatic conditions. In the remaining four cases, the growth increase exceeded the upper bound response expected from CO₂ enrichment experiments with seedling conifer species. Therefore, increased growth in any of the tree-ring chronologies examined could not be solely attributed to higher atmospheric CO₂ concentrations.Major financial supporters: Swiss National Science Foundation (application no. 1.869-0.83); Swiss Federal Institute of Forestry Research, 8903 Birmensdorf, Switzerland; other financial supporters: Carbon Dioxide Research Division, U.S. Department of Energy under subcontract no. 11X-57507V with Martin Marietta Energy Systems, IncOperated by Martin Marietta Energy Systems, Inc., under contract DE-AC05-840R21400 with U.S. Department of Energy     

Genome size in wild Pisum species

Genome size was measured in 75 samples of the wild pea species Pisum abyssinicum, P. elatius, P. fulvum and P. humile by ethidium-bromide (EB) flow cytometry (internal standard: Triticum monococcum) and Feulgen densitometry (internal standard: Pisum sativum Kleine Rheinländerin). Total variation of EB-DNA between samples covered 97.7% to 114.9% of the P. sativum value, and Feulgen DNA values were strongly correlated with EB-DNA values (r=0.9317, P < 0.001). Only P. fulvum was homogeneous in genome size (108.9% of P. sativum). Wide variation was observed between samples in P. abyssinicum (100.9–109.7%), P. elatius (97.7–114.9%) and P. humile (98.3–111.1% of P. sativum). In view of the world-wide genome size constancy in P. sativum, the present data are interpreted to show that the pea taxa with variable genome size are genetically inhomogeneous and that the current classification is not sufficient to describe the biological species groups adequately.     

Comparison of methods for the demarcation between earlywood and latewood in tree rings of Norway spruce

The precise demarcation between earlywood and latewood is important for the detailed analysis of intra-annual tree ring features. Different techniques based on visual assessment, wood anatomy analysis and X-ray densitometry have been developed and are currently used for this purpose. Depending on the chosen method, tree species and environmental conditions, the results can significantly vary. Thus, it is important to determine the technique optimal for a particular research. Here, we investigated Norway spruce (Picea abies) tree rings to examine the agreement among the following demarcation methods: (1) direct visual assessment, (2) Mork’s index (anatomical definition of the transition from earlywood to latewood based on cell wall-lumen ratio) and (3) fixed and floating density thresholds applied to intra-ring density profiles. The aim was to modify both the Mork’s criterion and density thresholds on the basis of reference values given by visual identification of earlywood/latewood transition. A total of 231 tree rings were analysed by all methods. Our results showed that the usage of floating threshold (defined for each ring separately based on density profiles) is more reliable in comparison with fixed threshold (the same threshold value used for all tree rings and samples). Statistical analysis revealed the best correspondence between visual identification of earlywood/latewood transition and demarcation based on the standard Mork’s index and the floating density threshold derived as 80 % of maximum latewood density. In terms of Mork’s index calibration, the results showed that to determine latewood cells in Norway spruce trees growing in temperate conditions, it is sufficient to use an index value equal to 0.83. The results are applicable for the studied spruce population growing in a temperate climate. The methodology itself, however, is universal and can help to calibrate criteria for earlywood-latewood demarcation under specific conditions.     

Affinity Chromatography of B-Glucosidase and Endo-B-Glucanase from Aspergillus Niger on Concanavalin A-Sepharose: Implications for Cellulase Component Purification and Immobilization

Affinity chromatography of a commercial preparation of 3-glu-cosidase from Aspergillus niger using concanavalin A-Sepharose (CAS) was employed as a means of purifying this glycoprotein. However, mannose (up to 1.08 M) was ineffective as an eluent of this enzyme from CAS, as were several other sugars and their derivatives, including 0.5 M glucose. Also, washing the CAS:8-glucosidase complex with buffer at pH 3.5 in the absence of MnCl₂ and CaCl₂ (required to preserve the binding activity of concanavalin A below pH 5.0) did not result in elution of this enzyme. On the contrary, endo-glucanase activity present in a crude cellulase complex (A. niger) which bound to CAS could be eluted by mannose (0.5–0.7 M) and was fractionated Into at least two components. The CAS:β-glucosidase complex hydrolyzed cellobiose to glucose and possessed an activity of 2, 158 units/g dry CAS. It could be used, therefore, for continuous cellobiose hydrolysis without leakage of enzyme from the support.     

Bioelectrical impedance vs. four-compartment model to assess body fat change in overweight adults

Objective: The Tanita TBF‐305 body fat analyzer is marketed for home and clinical use and is based on the principles of leg‐to‐leg bioelectrical impedance analysis (BIA). Few studies have investigated the ability of leg‐to‐leg BIA to detect change in percentage fat mass (%FM) over time. Our objective was to determine the ability of leg‐to‐leg BIA vs. the four‐compartment (4C) model to detect small changes in %FM in overweight adults. Research Methods and Procedures: Thirty‐eight overweight adults (BMI, 25.0 to 29.9 kg/m²; age, 18 to 44 years; 31 women) participated in a 6‐month, randomized, double‐blind, placebo‐controlled study of a nutritional supplement. Body composition was measured at 0 and 6 months using the Tanita TBF‐305 body fat analyzer [using equations derived by the manufacturer (%FM_T‐Man) and by Jebb et al. (%FM_T‐Jebb)] and the 4C model (%FM_4C). Results: Subjects in the experimental group lost 0.9%FM_4C (p = 0.03), a loss that did not reach significance using leg‐to‐leg BIA (0.6%FM_T‐Man, p = 0.151; 0.6%FM_T‐Jebb, p = 0.144). We observed large standard deviations (SDs) in the mean difference in %FM between the 4C model and the Tanita_Manufacturer (2.5%) and Tanita_Jebb (2.2%). Ten subjects fell outside ±1 SD of the mean differences at 0 and 6 months; those individuals were younger and shorter than those within ±1 SD. Discussion: Leg‐to‐leg BIA performed reasonably well in predicting decreases in %FM in this group of overweight adults but resulted in wide SDs vs. %FM_4C in individuals. Cross‐sectional determinations of %FM of overweight individuals using leg‐to‐leg BIA should be interpreted with caution.     

Chemical fingerprinting of Andrographis paniculata using HPLC, HPTLC and densitometry

An attempt has been made to develop a method by which to determine the chemical fingerprint of Andrographis paniculata (Acanthaceae). High-performance thin layer chromatography (HPTLC) was used to analyse hexane, chloroform, methanol and water extracts of leaves of A. paniculata. A computerised densitometer was applied to the two-dimensional spectrographic image analysis of the HPTLC plates. An HPLC equipped with a photodiode array detector was used for the analyses of these different extracts. The analyses showed that andrographolide and neoandrographolide are absent in the hexane extract but are present in greater amounts in the methanol extract as compared with the other extracts. These chromatograms may serve as a chemical fingerprint of the drug A. paniculata for quality control purposes and in the preparation of formulations based on the drug.     

Immunohistochemical, Histochemical and Radioassay Analysis of Nitric Oxide Synthase Immunoreactivity in the Lumbar and Sacral Dorsal Root Ganglia of the Dog

Summary In this study, immunohistochemistry for neuronal nitric oxide synthase (bNOS-IR), nicotinamide adenine dinucleotide phosphate diaphorase histochemistry (NADPHd) and nitric oxide synthase radioassay were used to study the occurrence, number and distribution pattern of nitric oxide synthesizing neurons in the lumbar (L1–L7) and sacral (S1–S3) dorsal root ganglia of the dog. Nitric oxide synthase immunolabelling was present in a large number of small- (area <1000 μm²) and medium-sized (area 1000–2000 μm²) as well as in a limited number of large-sized (area >2000 μm²) neurons. Although neuronal nitric oxide synthase immunolabelling and histochemical staining provided intense staining of multiple small- and medium-sized neurons in all lumbar and sacral dorsal root ganglia, immunolabelled or histochemically stained somata exhibited little topographic distribution in individual dorsal root ganglia. Great heterogeneity was noticed in the immunolabelling of medium-sized nitric oxide synthase immunopositive neurons ranging from lightly immunolabelled somata to heavily immunoreactive ones with completely obscured nuclei. Both staining procedures proved to be highly effective in visualizing intraganglionic fibers of various diameters. In general, the largest fibers revealed at the peripheral end of lumbar and sacral dorsal root ganglia were larger, 6.49–9.35 μm in diameter, while those running centrally and proceeding into the dorsal roots were about 30% reduced, ranging between 5.32 and 8.67 μm in diameter. Peripherally, the occurrence of nitric oxide synthase detected in axonal profiles, and confirmed histochemically, in the specimens of the femoral and sciatic nerves, is the first indication of the presence of nitric oxide synthase in the peripheral processes of somata located in L4–S2 dorsal root ganglia. Large and thin central nitric oxide synthase immunoreactive processes of L1–S3 dorsal root ganglion neurons segregate shortly before entering the spinal cord, the former making a massive medial bundle in the dorsal root accompanied by a slim lateral bundle penetrating Lissauer's tract. Quantitative assessment of the distribution of bNOS-IR and/or NADPHd-stained neurons showed a peculiar pattern in relation to spinal levels. Apparent incongruity was found in the total number of NADPHd-stained versus bNOS-IR neurons, demonstrating a clear prevalence of small bNOS-IR somata in all lumbar ganglia, while medium-sized NADPHd-stained somata clearly prevailed all along the rostrocaudal axis with a peak in L5 ganglion. While the number of small bNOS-IR neurons clearly outnumbered NADPHd-stained and NADPHd-unstained somata in S1–S3 ganglia, an inverse relation appeared comparing the total number of medium-sized NADPHd-stained and NADPHd-unstained somata compared with the number of moderate and intense bNOS-IR neurons. Densitometry of bNOS-IR and NADPHd-stained neurons in lumbar and sacral ganglia revealed two distinct subsets of densitometric profiles, one relating to more often found medium-sized bNOS immunolabelled and the other, characteristic for moderately bNOS immunoreactive somata of the same cell size. Considerable differences in catalytic nitric oxide synthase activity, determined by conversion of [³H]arginine to [³H]citrulline were obtained in lumbosacral dorsal root ganglia all along the lumbosacral intumescence, the lowest (0.898± 0.2 dpm/min/μg protein) being in the L4 dorsal root ganglion and the highest (4.194± 0.2 dpm/min/μg protein) in the S2 dorsal root ganglion.     

X-ray microdensitometry applied to subfossil tree-rings: growth characteristics of ancient pines from the southern boreal forest zone in Finland at intra-annual to centennial time-scales

A collection of subfossil wood of Pinus sylvestris (Scots pine) was exposed to X-ray densitometry. The collection of 64 samples from the southern boreal forest zone was dendrochronologically cross-dated to a.d. 673-1788. Growth characteristics were determined by performing density profiles including the following parameters: minimum density, earlywood and latewood boundary density, maximum density, earlywood width, earlywood density, latewood width, latewood density, annual ring width and annual ring density. Seven out of the nine parameters were found to contain non-climatic growth trends and six were found to be heteroscedastic in their variance. Tree-specific records were indexed, to remove the non-climatic growth trends and stabilize the variance, and combined into nine parameter-specific tree-ring chronologies. Growth characteristics of the pines changed in parallel with the generally agreed climatic cooling from the Medieval Warm Period to the Little Ice Age: pine tree-rings showed decreasing maximum densities from the period a.d. 975-1150 to a.d. 1450–1625. A concomitant change in the intra-annual growth characteristics was detected between these periods. The findings indicate that not only the trees growing near the species’ distributional limits are sensitive to large-scale climatic variations but also the trees growing in habitats remote from the timberline have noticeably responded to past climate changes.     

Stability of Adiposity Phenotypes from Childhood and Adolescence into Young Adulthood with Contribution of Parental Measures

Objective: The stability of several indicators of body composition and adipose tissue distribution over 12 years was quantified. Research Methods and Procedures: The participants were 77 boys and 76 girls who were evaluated along with their parents at baseline as children and adolescents (8 to 18 years of age) and remeasured as young adults 12 years later. Indicators of body composition included the body mass index, fat mass, fat free mass, percentage of body fat, sum of six skinfolds (SF6), and the first principal component of six age‐adjusted skinfold residuals. Relative adipose tissue distribution was represented by the second principal component of skinfold residuals and a trunk‐to‐extremity skinfold ratio, adjusted for SF6. Results: Partial interage correlations, controlling for initial age and length of follow‐up, were 0.65 and 0.59 for the body mass index, 0.59 and 0.64 for fat mass, 0.65 and 0.57 for fat free mass, 0.50 and 0.57 for percentage of body fat, 0.66 and 0.44 for SF6, 0.64 and 0.42 for the first principal component of six age‐adjusted skinfold residuals, 0.19 and 0.31 for the second principal component of skinfold residuals, and 0.41 and 0.47 for trunk‐to‐extremity skinfold ratio, adjusted for SF6, in men and women, respectively. Multiple regression analyses indicated that the significant partial R² values of parental measurements on the prediction of their offspring in young adulthood ranged from 2% to 9%. Discussion: The results indicate moderately high stability of indicators of body composition and somewhat lower stability of measures of adipose tissue distribution. Overall, parental measures offer less predictive value than do measures of childhood and adolescent body composition and adipose tissue distribution.     

Cryptopolyploidy revisited: the case of Vinca (Apocynaceae)

This investigation presents a look back to ancient times of karyology with modern optical instruments. `Cryptopolyploidy', i.e. an intrinsically polyploid but numerically non-polyploid structure of chromosome complements, today is an obsolete concept of chromosome architecture and evolution, but was actively discussed up to the mid-seventies of the past century. We focus here at a hypothesis of cryptooctoploidy in Vinca difformis (2n = 46), which was based on a measured four-fold chromosome volume compared with V. minor (2n = 46), the proposed diploid. We used DNA flow cytometry and Feulgen densitometry to see, if the postulate of cryptooctoploidy in V. difformis in the retrospect could be justified. It was found not defendable, because V. difformis differed only about 1.55-fold in C-value from V. minor, which is far from a regular multiple and much less than the 4-fold. C-values are given also for V. major, V. herbacea and V. rosea.