Biodiversity and bamboo genetic resources in Asia:in situ, community-based and ex situ approaches to conservation

亚洲的生物多样性及竹类遗传资源：就地保护、社区保护和易地保护“Ｔｈｅｉｒｓｔｒｅｎｇｔｈ，ｌｉｇｈｔｎｅｓｓ，ｓｍｏｏｔｈｎｅｓ，ｓｔｒａｉｇｈｔｎｅｓ，ｒｏｕｎｄｎｅｓｓ，ａｎｄｈｏｌｏｗｎｅｓ，ｔｈｅｆａｃｉｌｉｔｙａｎｄｒｅｇｕｌａｒｉｔｙｗ...     

In situ Protection Management and Conservation Study of Some Medicinal Plants

In situ study on eleven commercially important species viz;Adiantum capillus veneris L,Bergenia ciliata (Haw) Sternb,Colchicum luteum Baker,Polygonum amplexicaule D.Den,Cuminum cyminum L,Dioscorea deltoidea Wall Kunth,L Morchella esculenta L,Paeonia emodi Wall ex H Kf,Podophyllum hexandrum (Royle) Chatt & Mukh,Valeriana wallichii DC and Viola serpens Wall ex Roxb was conducted in four locations viz; Malam (1 400 to 2 000?m),Bargin (1 700 to 2 300?m),Biakand (1 500 to 2 100?m) and Shinko (2 100 to 2 700?m).The data was recorded from both protected and unprotected sites of each site.Each site had 3 altitudinal sampling point.The density, herbage coverage and fresh biomass were determined in each locations for every species.All the investigated parameters generally showed an increase of 3 to 6 times over unprotected sites in each locations.Morchella esculenta,Dioscorea deltoidea,Colchicum luteum and Podophyllum hexandrum were absent in all unprotected sites while other species had low values in these stes.The investigated parameters of Colchicum luteum,Bergenia ciliata,Paeonioa emodi,Dioscorea deltoidea and Podophyllum hexandrum generally increased with the increasing elevation.Soil analysis,soil and air temperatures were recorded for each site.The air and soil temperature were slightly higher in open areas than in the protected site and showed decrease with increasing elevation.While the soil fertility was relatively high in protected sites as compared to unprotected area.The study shows that protection promotes the growth, distribution and occurrence of medicinal plants.It is possible with the participation of local communities to conserve these resources.     

A convenient method for distinguishing human and mouse cells in situ

Animal models play critical roles in the field of biomedical research.Mouse models with transplanted human cells or tissues,such as cell line-derived xenografts(CDX)and patient-derived xenografts(PDX),have been widely used[1].Tracking transplanted cells and understanding the spatial relationship between donor and host are extremely important for the study of these models.To distinguish cell sources in mouse xenograft models at the morphological level,antibody recognition methods(immunohistochemistry and immunofluorescence),transgenic reporter methods,and chromosome-specific probe labeling methods are widely used[2–5].However,these methods all need to consider the issues of label specificity,signal intensity,in situ applicability,and complexity of experimental operations,which increase the difficulty in use.In this report,we described a simple method for accurately distinguishing human and mouse nuclei based on 4′,6-diamidino-2-phenylindole(DAPI)staining.This method can accurately identify species not only in cultured cells,but also in mouse xenograft models.Using short-wavelength excitation and high brightness of DAPI stains,this easy-to-use method is expected to be widely used in the field of biomedical research.     

Are in vivo and in situ brain tissues mechanically similar?

Brain tissue mechanical properties have been well-characterized in vitro, and were found to be inhomogeneous, nonlinear anisotropic and influenced by neurological development and postmortem time interval prior to testing. However, brain in vivo is a vascularized tissue, and there is a paucity of information regarding the effect of perfusion on brain mechanical properties. Furthermore, mechanical properties are often extracted from preconditioned tissue, and it remains unclear if these properties are representative of non-preconditioned tissue. We present non-preconditioned (NPC) and preconditioned (PC) relaxation responses of porcine brain (N = 10) obtained in vivo, in situ and in vitro, at anterior, mid and posterior regions of the cerebral cortex during 4mm indentations at either 3 or 1 mm/s. Material property characteristics showed no dependency on the site tested, thus revealing that cortical gray matter on the parietal and frontal lobes can be considered homogenous. In most cases, preconditioning decreased the shear moduli, with a more pronounced effect in the dead (in situ and in vitro) brain. For most conditions, it was found that only the long-term time constant of relaxation (tau > 20 s) significantly decreased from in vivo to in situ modes (p < 0.02), and perfusion had no effect on any other property. These findings support the concept that perfusion does not affect the stiffness of living cortical tissue.     

Chemostat and in‐situ colonization kinetics of thermothrix thiopara on calcite and pyrite surfaces

Growth and attachment rates of Thermothrix thiopara on calcite and pyrite were quantitated in a thiosulfate‐limited chemostat and in the thermal spring where the organism is found in nature. Surface growth rates were quantitated by using the surface colonization and exponential growth equations. These two models were compared as means of determining surface growth rates. In the chemostat, T. thiopara cells colonizing calcite and pyrite surfaces grew at approximately one‐third the rate of suspended cells. However, T. thiopara attached to pyrite faster than to calcite. In the thermal spring, growth and attachment rates were equal on calcite and pyrite. It was concluded that the exponential growth equation overestimates in‐situ surface growth rates and that T. thiopara grows more slowly when colonizing mineral surfaces than when growing in suspension. Lower growth rates on surfaces may be due to a reduced cell surface area for nutrient uptake or an increased specific maintenance rate.     

Quantifying RNA–protein interactions in situ using modified-MTRIPs and proximity ligation

The stabilization, translation and degradation of RNA are regulated by interactions between trans-acting factors, such as microRNA and RNA-binding proteins (RBP). In order to investigate the relationships between these events and their significance, a method that detects the localization of these interactions within a single cell, as well as their variability across a cell population, is needed. To visualize and quantify RNA–protein interactions in situ, we developed a proximity ligation assay (PLA) that combined peptide-modified, multiply-labelled tetravalent RNA imaging probes (MTRIPs), targeted to sequences near RBP binding sites, with proximity ligation and rolling circle amplification (RCA). Using this method, we detected and quantified, with single-interaction sensitivity, the localization and frequency of interactions of the human respiratory syncytial virus (hRSV) nucleocapsid protein (N) with viral genomic RNA (gRNA). We also described the effects of actinomycin D (actD) on the interactions of HuR with β-actin mRNA and with poly(A)+ mRNA at both native and increased HuR expression levels.     

Magnitude and modulation of pancreatic β-cell gap junction electrical conductance in situ

The parallel gap junction electrical conductance between a -cell and its nearest neighbors was measured by using an intracellular microelectrode to clamp the voltage of a -cell within a bursting islet of Langerhans. The holding current records consisted of bursts of inward current due to the synchronized oscillations in membrane potential of the surrounding cells. The membrane potential record of the impaled cell, obtained in current clamp mode, was used to estimate the behavior of the surrounding cells during voltage clamp, and the coupling conductance was calculated by dividing the magnitude of the current bursts by that of the voltage bursts. The histogram of coupling conductance magnitude from 26 cells was bimodal with peaks at 2.5 and 3.5 nS, indicating heterogeneity in extent of electrical communication within the islet of Langerhans. Gap junction conductance reversibly decreased when the temperature was lowered from 37 to 30°C and when the extracellular calcium concentration was raised from 2.56 to 7.56 mm. The coupling conductance decreased slightly during the active phase of the burst. Activation of adenylate cyclase with forskolin (10 m) resulted in an increase in cell-to-cell electrical coupling. We conclude that -cell gap junction conductance can be measured in situ under near physiological conditions. Furthermore, the magnitude and physiological regulation of -cell gap junction conductance suggest that intercellular electrical communication plays an important role in the function of the endocrine pancreas.The authors thank Dr. Arthur Sherman for sharing his insights on in situ coupling measurements, and for helpful discussions throughout the work. DM acknowledges partial support by a National Institutes of Health training grant to the Johns Hopkins University, Department of Biomedical Engineering (5 T32 GM7057). This work was supported in part by a National Science Foundation PYI award (ECS-9058419) to NFS.     

What is the relevance of smallholders’ agroforestry systems for conserving tropical tree species and genetic diversity in circa situm, in situ and ex situ settings? A review

Smallholders’ agroforests may be valuable for conserving tropical trees through three main mechanisms. First, trees planted and/or retained by farmers in agricultural landscapes where wild stands were once found may be circa situm reservoirs of biodiversity. Second, farmland trees may support conservation in situ by providing an alternative source of product to reduce extraction from forest, and by acting as ‘corridors’ or ‘stepping stones’ that connect fragmented wild stands. Third, the additional value that planting assigns to trees may result in greater interest in including them in seed collections, field trials and field ‘genebanks’ that support ex situ conservation. Here, we critically review the evidence for these mechanisms, and highlight areas for research and for intervention so that agroforestry practices can better support conservation in each setting, with an emphasis on often neglected genetic-level considerations. Based on current global challenges to diversity, conservation will need to rely increasingly on a smallholder-farm circa situm approach, but concerns on long-term effectiveness need to be properly quantified and addressed. Connectivity between widely dispersed, low density trees in agricultural landscapes is an important factor determining the success of the circa situm approach, while improving farmers’ access to a diversity of tree germplasm that they are interested in planting is required. The circumstances in which agroforestry plantings can support in situ conservation need to be better defined, and research on the stability of active tree seed collections (how long are species and populations retained in them?) as ex situ reservoirs of biodiversity is needed.     

In situ activation of β-galactosidase of Kluyveromyces bulgaricus resting cells by sodium and potassium phosphates and chlorides

Summary Incubation of Kluyveromyces bulgaricus in sodium and potassium salts led to in vivo activation of -galactosidase. The activation reaction was relatively slow since, at 37°C, it took 30 min to come to completion. The reaction was irreversible and was favoured by high salt concentrations with chlorides proving to be more efficient than phosphates. After incubation in KCl, the final activity obtained was 1.49 U/mg dry yeast and this represented a 10-fold increase in activity compared with the control value measured in ammonium phosphate. Hydrolysis of onitrophenyl--galactoside (ONPG) was insensitive to inhibitors of the transport systems and energy metabolism. There results suggest that K. bulgaricus resting cells take up substrates and ions that readily influence -galactosidase activity.     

‘In vitro’ and ‘in situ’ decomposition of nuisance macroalgae Cladophora glomerata and Pilayella littoralis

The decomposition of two macroalgal species Cladophora glomerata (CHLOROPHYTA) and Pilayella littoralis (PHAEOPHYTA) was studied in the laboratory and field conditions. These species are known to cause the extensive macroalgal blooms in the whole coastal range of the Baltic Sea. The objective of the experiments was to determine decomposition rates of the macroalgae, follow the changes in tissue nutrient content and validate the role of benthic invertebrates in this process. In the laboratory conditions, the differences in the decomposition rates of the algae were mainly due to the oxygen conditions. The weight loss of C. glomerata was slightly higher in anaerobic conditions than in aerobic conditions. If 99% of initial dry weight of P. littoralis was lost in aerobic conditions then only 20% was lost in anaerobic conditions. In general, the loss of phosphorus and nitrogen in algal tissues followed the weight loss. As an exception, the amount of nitrogen changed very little during the decomposition of C. glomerata. In field conditions, the photosynthetic activity exceeded the decomposition rate of C. glomerata at lower temperatures in spring. The decomposition of P. littoralis was estimated at 49% of its initial dry weight. The addition of benthic invertebrates had no effect on the decomposition process. In summer, the decomposition rates were estimated at 65% for C. glomerata and 68% for P. littoralis being in the same order of magnitude as observed in laboratory conditions. If the decomposition of C. glomerata was faster at the end of the experiment, the most significant losses of weight of P. littoralis took place during the first 2 weeks of deployment. Idotea baltica significantly contributed to the loss of C. glomerata. The decomposition rate of P. littoralis was reduced by the presence of Mytilus edulis and increased by Gammarus oceanicus.     

Comparison of 3′ and 5′ biotin labelled oligonucleotides for in situ hybridisation

Oligonucleotide probes enzymatically labelled at the 3-end with biotin have been used successfully to detect target RNA and DNA in combination with in situ hybridisation. Addition of multiple biotin residues to the 3-end increases the hybridisation signals, but it is not known whether the same principle is applicable to the 5-end. We have labelled a 35-base oligonucleotide during synthesis with 1, 5 and 12 biotin molecules at the 5-end and compared it to conventional 3-labelling. In additional experiments the probes were labelled at both ends. Probes were applied to histological sections obtained from paraffin-embedded cell-clot-complexes that contain uninfected and Rhinoviral-infected cells, using a standard in situ hybridisation protocol with appropriate controls. Hybridisation signals were compared for intensity of cytoplasmic signal and sensitivity as number of positive cells. Both parameters increased in parallel with higher numbers of biotin residues attached to the 5-end and 12 biotin residues were almost as effective as 3-enzymatic tailing. The sensitivity could be increased above that of either 3- or 5-labelling by the addition of residues at both ends of the probe. The 5-attachment of biotin residues can extend the value of oligonucleotide probes employed for in situ hybridisation and yield increased sensitivity when combined with 3-enzymatic labelling.     

In situ quantification of fish‐cage fouling by underwater photography and image analysis

Close‐up underwater photography and image analysis were used to quantify mesh occlusion by biofouling of salmon‐cage netting. This technique allows fast, non‐destructive sampling of cages in situ for the determination of temporal and spatial changes in fouling. The area of net blockage can be easily determined, allowing rapid evaluation of cleaning or antifouling performance.     

In situ observation of a cell adhesion and metabolism using surface infrared spectroscopy

In this study, we report on an in situ monitoring system of living cultured cells using infrared absorption spectroscopy in the geometry of multiple internal reflections (MIR-IRAS). In order to observe living cultured cells, the temperature in the sample chamber of a FT-IR spectrometer was maintained at 37 °C and a humidified gas mixture containing 5% CO₂ was introduced into the sample chamber. Human breast cell line MCF-7 cultured on Si MIR prisms were placed in the sample chamber and infrared spectra of MCF-7 cells were collected for 5 h. It was found that the adhesion and metabolism of MCF-7 cells could be monitored by the absorption intensity of amide-II protein band (1,545 cm⁻¹) and also by the absorption intensities of CH _x bands (2,700–3,100 cm⁻¹). These results suggest that our system is useful for a nondestructive and non-label monitoring of cell viability. Our method based on infrared absorption spectroscopy has a potential for bioscreening application.     

Tricholoma matsutake– an assessment of in situ and in vitro infection by observing cleared and stained whole roots

 Structures present within field-collected Tricholoma matsutake/Pinus densiflora ectomycorrhizas and in vitro infections of P. densiflora roots by T. matsutake were observed by clearing, bleaching and staining whole lateral roots and mycorrhizas. Field mycorrhizas were characterized by a lack of root hairs, by the presence of a sparse discontinuous mantle composed of irregularly darkly staining hyphae over the root surface, primarily behind the root cap, and by the presence of Hartig net mycelium within the root cortex. Hartig net 'palmettis' were classified into three basic structures, each with distinctive morphologies. Aerial hyphae, bearing terminal swellings, were observed emanating from the mantle. Cleared, bleached and stained in vitro-infected roots possessed multibranched hyphal structures within the host root cortex and aerial hyphae bearing terminal swellings were observed arising from the mycelium colonizing the root surface. T. matsutake on P. densiflora conforms to the accepted morphology of an ectomycorrhiza. This staining protocol is particularly suited to the study of Matsutake mycorrhizal roots and gives rapid, clear, high-contrast images using standard light microscopy while conserving spatial relationships between hyphal elements and host tissues. Accepted: 26 August 1999     

In situ Localization and Isolation of Actin Filaments from Pollen Tubes of Amaryllis vittata Ait

ＩｎｓｉｔｕＬｏｃａｌｉｚａｔｉｏｎａｎｄＩｓｏｌａｔｉｏｎｏｆＡｃｔｉｎＦｉｌａｍｅｎｔｓｆｒｏｍＰｏｌｌｅｎＴｕｂｅｓｏｆＡｍａｒｙｌｌｉｓｖｉｔｔａｔａＡｉｔＣＡｌＸｕｅ（蔡雪）;ＤＯＮＧＹｕｎ－ｚｈｏｕ（董云洲）（ＣｏｌｌｅｇｅｏｆＬｉｆｅＳ...     

Expression and localization of ionotropic glutamate receptor subunits in the goldfish retina—an in situ hybridization and immunocytochemical study

The expression and distribution of AMPA, kainate and NMDA glutamate receptor subunits was studied in the goldfish retina. For the immunocytochemical localization of the AMPA receptor antisera against GluR2, GluR2/3 and GluR4 were used, and for in situ hybridization rat specific probes for GluR1 and GluR2 and goldfish specific probes for GluR3 and GluR4 were used. The localization of the low affinity kainate receptor and NMDA receptor was studied using antisera against GluR5-7 and NR1. All AMPA receptor subtypes were demonstrated to be present in the goldfish retina both by immunocytochemistry and in situ hybridization. In situ hybridization revealed expression of all AMPA receptors subunit at the inner border of the INL. Only GluR3 was also strongly expressed in the outer border of the INL. Some of the ganglion cells displayed a strong signal for GluR1, GluR3 and GluR4. GluR1-immunoreactivity was present in subsets of bipolar, amacrine, and ganglion cells. GluR2 and GluR2/3-immunoreactivity was mainly localized in the outer plexiform layer. GluR2 and GluR2/3-immunoreactivity are associated with the photoreceptor synaptic terminals. GluR4-immunoreactivity is present on Müller cells in the inner retina and on dendrites of bipolar cells in the OPL, whereas GluR5-7-immunoreactivity was prominently present on horizontal cell axon terminals. Finally, NR1-immunoreactivity was confined to amacrine cells, the inner plexiform layer and ganglion cells. This study shows that there is a strong heterogeneity of glutamate receptor subunit expression in the various layers of the retina. Of the AMPA receptor subunits GluR3 seems to be expressed the most widely in all layers with strong glutamatergic synaptic interactions whereas all the other subunits seem to have a more restricted expressed pattern.