Ultrastructural distribution of alpha-bungarotoxin binding sites in the suprachiasmatic nucleus of the rat hypothalamus

Summary The distribution of (¹²⁵I) alpha bungarotoxin (-BTX) binding sites in the suprachiasmatic nucleus (SCN) of the adult female rat was examined by electron-microscopic autoradiography. The ultrastructural distribution of silver grains was analysed by line source, direct point count, and 50% probability circle methods. Real grain distribution was significantly different from that of randomly generated hypothetical grains. Line source analysis demonstrated two populations of sources: one associated with membranes, and one inside neuronal structures. Probability circle analysis of shared grains indicated that membrane-bound-radioactive sources were mainly asssociated with axo-dendritic appositions. Only a small proportion of labeled neuronal interfaces exhibited synaptic differentiations in the plane of section. However, the compartment containing synaptic terminals was the most enriched when comparing real to hypothetical grains. Probability circle analysis of exclusive grains demonstrated that sources that were not associated with neuronal plasma membranes were likely to be within nerve cell bodies and dendrites. It is concluded that the majority of specifically labeled -BTX binding sites in the SCN is membrane bound, and may be associated with axodendritic synaptic transmission. The presence of a significant proportion of the label in the soma and dendrites of suprachiasmatic neurons 24 h after ventricular infusion suggests that some of the labeled binding sites (junctional or nonjunctional) may be internalized within these two compartments.     

A NEW METHOD FOR ANALYZING ELECTRON MICROSCOPE AUTORADIOGRAPHS USING HYPOTHETICAL GRAIN DISTRIBUTIONS

A new method for the analysis of electron microscope autoradiographs is described which has two advantages over other methods. This method provides estimates for the activity in different regions of the section and takes into account the actual geometrical shape and position of different regions within the section. Use is made of the measured distribution of grains about a thin line source to construct a hypothetical distribution of autoradiographic grains which is then compared with the actual distribution of grains. Different activities are then ascribed to various regions until a satisfactory agreement with the autoradiographic grains is obtained using the χ² significance. In the next paper, an example of the use of this method is given for the distribution of grains in the nucleus of erythroid bone marrow cells labeled with radioactive iron, ⁵⁵Fe.     

Measurement of the <Superscript>15</Superscript>N/<Superscript>14</Superscript>N ratio of phenylalanine in fermentation matrix by isotope ratio mass spectrometry

Objectives

To determine the origin of ¹⁵N-labeled phenylalanine in microbial metabolic flux analysis using ¹⁵N as a tracer, a method for measuring phenylalanine δ¹⁵N using HPLC coupled with elemental analysis-isotope ratio mass spectrometry (EA-IRMS) was developed.

Results

The original source of the ¹⁵N-labeled phenylalanine was determined using this new method that consists of three steps: optimization of the HPLC conditions, evaluation of the isotope fractionation effects, and evaluation of the effect of pre-processing on the phenylalanine nitrogen stable isotope. In addition, the use of a ¹⁵N-labeled inorganic nitrogen source, rather than ¹⁵N-labeled amino acids, was explored using this method.

Conclusions

The method described here can also be applied to the analysis of metabolic flux.

     

Autoradiographic study on the localization of estradiol-17β in neonatal mouse uterus and cervix

Summary The uptake of ³H-estradiol-17 in the neonatal mouse uterus and cervix has been studied by an autoradiographic method. When the radio-active hormone is administered in vivo and in vitro, grains are found to be concentrated above the nuclei both in the uterine and cervical epithelium and stroma. Grain counts revealed that the nuclear concentration of grains is higher at 4 h than at 2 h after isotope injection. The cervical epithelium has a higher nuclear concentration than the uterine epithelium both in vivo and in vitro. In the stroma, this situation is reversed except after in vitro treatment of the tissues.In the cervix, more of the hormone seems to be located within the nucleus while in the uterus a higher proportion of the grains are found in the vicinity of the nuclear periphery.Although the nuclear concentration of grains is higher at 4 h than at 2 h, the number of grains above the sections is lower at 4 h. Both in vivo and in vitro, the number of grains is higher above the stromal than above the epithelial compartments of the uterus and cervix.Five days old animals showed the same labeling pattern. The differences in uptake and distribution of ³H-estradiol are discussed in relation to other known differences in the hormone responsiveness in these tissues.We are greatly indebted to Professor W.E. Stumpf and the Laboratories for Reproductive Biology, University of North Carolina Medical School for the opportunity to study the method of dry mount autoradiography. The work has been supported by the Norwegian Research Council for Science and the Humanities and by the Norwegian Cancer Society (Landsforeningen mot Kreft)     

13C标记代谢流分析中天然稳定性同位素修正矩阵构建方法及应用

稳定性同位素¹³C标记实验是分析细胞代谢流的一种重要手段,主要通过质谱检测胞内代谢物中¹³C标记的同位素分布,并作为胞内代谢流计算时的约束条件,进而通过代谢流分析算法得到相应代谢网络中的通量分布。然而在自然界中,并非只有C元素存在天然稳定性同位素¹³C,其他元素如O元素也有其天然稳定性同位素¹⁷O、¹⁸O等,这使得质谱方法所测得的同位素分布中会夹杂除¹³C标记之外的其他元素的同位素信息,特别是分子中含有较多其他元素的分子,这将导致很大的实验误差,因此需要在进行代谢流计算前进行质谱数据的矫正。本研究提出了一种基于Python语言的天然同位素修正矩阵的构建方法,用于修正同位素分布测量值中由于天然同位素分布引起的测定误差。文中提出的基本修正矩阵幂方法用于构建各元素修正矩阵,结构简单、易于编码实现,可直接应用于¹³C代谢流分析软件数据前处理。将该修正方法应用于¹³C标记的黑曲霉（Aspergillus niger）胞内代谢流分析,结果表明本研究提出的方法准确有效,为准确获取微生物胞内代谢流分析提供了可靠的数据修正方法。     

Pathway of Photosynthetic Malate Formation in Vitis vinifera, a C(3) Plant

The time course of intramolecular isotope distribution in phosphoglyceric acid and serine was determined after exposure of grape leaf discs (Vitis vinifera L.) to ¹⁴CO₂ (1000 microliters per liter) for variable metabolic periods, and the labeling patterns were compared with the respective isotope distribution in the C_1-3 fragment of malic acid. The results clearly support the classical concept of a close precursor-product relationship between photosynthetic phosphoglycerate and malic acid. Under the assimilatory conditions used in this study, there was no indication of an immediate carbon transfer from serine to malate as has been suggested for C₃ plants (Kent et al. 1974 Plant Physiol 53: 491-495) because of a coincident labeling of these compounds in Vicia faba. According to our data, previous evidence in favor of this hypothetical pathway is based largely on an unusual ¹⁴C distribution in serine, due to an extreme suppression of photorespiration, as well as on arbitrary comparisons between compounds of divergent kinetic characteristics and consequently different degrees of metabolic label randomization.     

Environmental analysis of airborne pollen occurrence,pollen source distribution and phenology of <Emphasis Type="Italic">Fraxinus angustifolia</Emphasis>

Narrow-leafed ash (Fraxinus angustifolia) is a common polygamous tree growing on the banks of rivers in the western Mediterranean region. Pollination occurs during winter, and the tree’s pollen is among the most abundant during that season. This work aims to relate the phenology of pollen shedding, source tree distribution, meteorology and airborne pollen occurrence for the species. Aerobiological sampling was conducted in Badajoz (south-western Spain) using a Hirst volumetric sampler over 24 years (1993–2016). Trees were geo-localized in a circle 500 m in diameter surrounding the pollen sampler. During the last two periods, pollination phenology was studied in 10 specimens, five in the surroundings of the pollen station and five 3 km apart, at a frequency of 3–4 days on average. Moreover, a detailed analysis of pollen occurrence was conducted for these two periods. Daily data for the whole period and hourly data over the last 2 years were used, including pollen monitoring and meteorology. A comparison was made between pollen occurrence and source distribution. The main pollen season lasted on average 53 (28–75) days. Average values were less than 10 grains m^?3, except for two periods of 23–24 grains m^?3. Daily data and hourly data correlation with meteorology showed different signs in correlation analysis. Hourly analysis showed that the maximum concentration occurred just after noon. Most pollen was recorded at an average temperature of 9 °C. Analysis of pollen sources and pollen occurrence showed a close relationship between predominant wind directions and tree distribution. Peaks of phenology were not coincident with pollen peaks. No trends in pollination were found. Non-homogeneous distribution of pollen sources for Fraxinus angustifolia provided a suitable tool to demonstrate that wind direction plays a relevant role when aerobiological data are interpreted according to source distribution. A limitation in phenology analysis and aerobiological data was noted in the narrow-leafed ash species.     

Microsampling of Fish Otoliths: A Comparison Between DM 2800 and Dremel in Stable Isotope Analysis

This paper describes and compares two microsampling methods, DM 2800 and Dremel, which were used in taking aragonite samples from otolith zones of cod, Gadus morhua, for stable isotope analysis. There were no significant differences between the two sampling methods both in carbon and oxygen isotope ratios (¹³C/¹²C, ¹⁸O/¹⁶O) and in annual and seasonal otolith zones. The possible finite effect with Dremel could be minimized or negligible by selecting otoliths of younger fishes. Thus, Dremel may be accepted as a convenient microsampling method in stable isotope analysis of otoliths.     

Incorporation of 3H-oleic acid by the proximal convoluted tubule cells of the chick (Gallus domesticus)

Summary Lipid metabolism in the cells of the renal proximal convoluted tubules (PCT) was investigated in healthy fowls and in fowls with the Fatty Liver and Kidney Syndrome (FLKS). The tissue was fixed at 10–25 min intervals after intravenous injection of ³H-oleic acid. The distribution of autoradiographic grains was analysed by the circle method. In normal cells most of the silver grains were associated with the cytoplasmic organelles. Lipid droplets and Golgi elements had the highest specific activity relative to the nuclear activity, which was little above background level. Lysosome-like bodies and mitochondria had lower values. In the cells of the FLKS-affected birds a large proportion of the grains was located over the lipid droplets, which are abundant in this condition. The specific activity of the cytoplasmic organelles was barely 2-fold higher than the nuclear activity. The results suggest that there is a diminished incorporation of esterified fatty acids by the organelles of these cells and that the excess is transferred to the lipid droplets. The identity of low electron density particles observed in the PCT cells of severely affected birds is discussed.     

Stability of elastin in the developing mouse aorta: a quantitative radioautographic study

Elastic lamina growth during development and the ultimate stability of elastin in the mouse aortic media was investigated by light and electron microscopic radioautography. Following a single subcutaneous injection of l-[3,4-³H]valine at 3 days of age, animals were killed at 9 subsequent time intervals up to 4 months of age. One day after injection, radioautographic silver grains were primarily observed over the elastic laminae; however, silver grains were also seen over the smooth muscle cells and extracellular matrix. By 21 to 28 days of age, the silver grains were almost exclusively located over the elastic laminae. From 28 days to 4 months of age, the distribution of silver grains appeared relatively unchanged. Quantitation of silver grain number/m² of elastin showed a steady decrease in the concentration of silver grains associated with the elastic laminae from 4 to 21 days of age. After this time, no significant difference in silver grain concentration was observed. Since the initial decrease in grains/m² of elastin corresponds to a period of rapid post-natal growth, the decrease is likely to be a result of dilution of the radiolabel due to new elastin synthesis. With the assumption that little or no significant turnover occurs during this time, a constant growth rate of 4.3% per day was predicted by linear regression analysis. Since no significant difference in the concentration of silver grains was observed from 28 to 118 days of age, no new growth or turnover of elastin can be said to occur during this time period. This is supported by the observation that animals injected with radiolabeled valine at 28 days and 8 months of age showed no significant incorporation of radiolabel into the elastic laminae. The results from this study present the first long-term radioautographic evidence of the stability of aortic elastin and emphasize that initial deposition of elastin and proper assembly of elastic laminae is a critical event in vessel development.     

Zinc fluxes into developing barley grains: use of stable Zn isotopes to separate root uptake from remobilization in plants with contrasting Zn status

Background and Aims

Zn imported into developing cereal grains originates from either de novo Zn uptake by the roots or remobilization of Zn from vegetative tissues. The present study was focused on revealing the quantitative importance of the two pathways for grain Zn loading and how their relative contribution varies with the overall plant Zn status.

Methods

The stable isotope ⁶⁷Zn was used to trace Zn uptake and remobilization fluxes in barley (Hordeum vulgare L.) plants growing in hydroponics at 0.1?μM (low Zn), 1.5?μM (medium Zn) or 5?μM Zn (high Zn). When grain development reached 15?days after pollination the Zn source was changed to an enriched ⁶⁷Zn isotope and plants were harvested after 6 to 48?h. Zn concentrations and isotope ratios were determined using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).

Results

Plants with low Zn status absorbed 3-fold more Zn than plants with medium or high Zn status when roots were exposed to an external concentration of 1.5?μM ⁶⁷Zn. Stems and ears were the primary recipients of the de novo incorporated Zn with preferential allocation to the developing grains over time. The leaves received in all cases a very small proportion (<5?%) of the newly absorbed Zn and the proportion did not increase over time. Zn fluxes derived from uptake and remobilization were almost equal in plants with low Zn status, while at high Zn status remobilization delivered 4 times more Zn to the developing grains than did root Zn uptake.

Conclusions

Stable isotopes in combination with ICP-MS provided a strong tool for quantification of Zn fluxes in intact plants. The importance of Zn remobilization compared to de novo root absorption of Zn increased with increasing plant Zn status. Very little de novo absorbed Zn was translocated to the leaves during generative growth stages.     

An indirect method for estimating 15N isotope fractionation during nitrogen fixation by a legume under field conditions

A new technique is proposed for measuring ¹⁵N isotope fractionation during N fixation that obviates some of the possible disadvantages of existing methods. Accurate calculation of N fixation by legumes using the ¹⁵N natural abundance technique requires a value for the isotopic composition of fixed N as an input. Isotopic fractionation in fixed N in legumes has usually been measured using N- free solution culture but results can vary with Rhizobium strain and growth conditions. The proposed method avoids these problems and can be used as an integral part of a field experiment for evaluating N fixation.The technique is essentially a process of adjusting values of ¹⁵ N for fixed N until % N fixation calculated by the ¹⁵N natural abundance method best matches % N fixation estimated by the ¹⁵N enrichment method. The use of high % N fixation values improves the sensitivity and reliability of the method.A field evaluation of this comparison technique using chickpea (Cicer arietinum L.) provided a ¹⁵N isotope fractionation factor (–2.37) for fixed N close to that obtained by N-free solution culture methods (–2.10). The availability of these two independent techniques allowed mutual corroboration of estimates of ¹⁵N isotope fractionation during N fixation.

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Stable carbon isotope analysis as a crop management indicator at Arslantepe (Malatya,Turkey) during the Late Chalcolithic and Early Bronze Age

In Near Eastern archaeology, studies on crop management during the early stages of civilisation have been based on cuneiform texts and only recently have geoarchaeological surveys and archaeobotanical studies tried to identify agricultural practices. Nevertheless, direct evidence for irrigation and water management is very rare and difficult to interpret. New possibilities for analysing these ways of management have been provided by stable carbon isotope analysis of ancient crops. In this study carbon isotope discrimination (Δ¹³C) was performed to assess the growing conditions of fossil cereal grains. Charred grains of emmer and barley recovered from the same site, Arslantepe in eastern Anatolia, over a 1,000 years long continuous sequence during the third millennium b.c. have been analysed and compared. The variation in water availability during grain filling, estimated by Δ¹³C, can be attributed to climate change and/or human practices. Distinguishing exactly which is to blame is complex but essential in reconstructing specific management practices. Variations in the ¹³C/¹²C ratio suggest that barley and emmer grew under different water regimes. In particular, a different management system was adopted between 3000 and 2800 cal. b.c. when emmer was sustained by human-induced water supply, whereas the more resistant barley was rain-fed and possibly assigned to more marginal terrains. Our results are compared with the stable carbon isotope data available for other Near Eastern sites. Emmer and barley records are also compared with deciduous oak Δ¹³C from Arslantepe in order to better understand the influence of environmental factors, climate and human impact.     

Decimal Place Slope,A Fast and Precise Method for Quantifying 13C Incorporation Levels for Detecting the Metabolic Activity of Microbial Species

The metabolic incorporation of stable isotopes such as ¹³C or ¹⁵N into proteins has become a powerful tool for qualitative and quantitative proteome studies. We recently introduced a method that monitors heavy isotope incorporation into proteins and presented data revealing the metabolic activity of various species in a microbial consortium using this technique. To further develop our method using an liquid chromatography (LC)-mass spectrometry (MS)-based approach, we present here a novel approach for calculating the incorporation level of ¹³C into peptides by using the information given in the decimal places of peptide masses obtained by modern high-resolution MS. In the present study, the applicability of this approach is demonstrated using Pseudomonas putida ML2 proteins uniformly labeled via the consumption of [¹³C₆]benzene present in the medium at concentrations of 0, 10, 25, 50, and 100 atom %. The incorporation of ¹³C was calculated on the basis of several labeled peptides derived from one band on an SDS-PAGE gel. The accuracy of the calculated incorporation level depended upon the number of peptide masses included in the analysis, and it was observed that at least 100 peptide masses were required to reduce the deviation below 4 atom %. This accuracy was comparable with calculations of incorporation based on the isotope envelope. Furthermore, this method can be extended to the calculation of the labeling efficiency for a wide range of biomolecules, including RNA and DNA. The technique will therefore allow a highly accurate determination of the carbon flux in microbial consortia with a direct approach based solely on LC-MS.The metabolic incorporation of stable isotopes such as ¹³C or ¹⁵N into proteins has become a powerful component of qualitative and quantitative proteome studies (1). Incorporation of heavy isotopes can be used to analyze microbial processes such as turnover rates and also to help to establish structure-function relationships within microbial communities. Stable isotope probing (SIP¹) techniques based on DNA-SIP (2) and RNA-SIP (3) have been used for this purpose previously. With the introduction of protein-SIP (4), the need for an accurate alternative method for calculating label incorporation into biomolecules arose. Protein-SIP has several advantages compared with DNA/RNA-SIP, the most important being its capacity to detect dynamic levels of incorporation, whereas only labeled or unlabeled states can be categorized by means of DNA/RNA-SIP because of the need to separate ¹³C-DNA/RNA by density gradient centrifugation. Quantitative analysis of ¹³C incorporation levels is of the utmost importance, especially when unraveling carbon fluxes through either microbial communities or food webs with different trophic levels.In contrast to the incorporation of isotopically labeled amino acids, which is often used in quantitative proteomics (5), metabolic labeling by growth substrates and nutrients (e.g. salts) is often imperfect and makes the processing of mass spectrometry (MS) data difficult. For example, when the incorporation of ¹³C exceeds ∼2 atom %, common database search algorithms fail to identify peptides and proteins. The problem can only be managed successfully if a stable, known degree of ¹³C incorporation can be achieved during the experiment (6). Using a low labeling efficiency of roughly 5 atom %, Huttlin et al. (6) chose the altered envelope chain for calculating the incorporation and simultaneously used the signal intensity for a quantitative comparison with the sample that had a natural abundance of ¹³C. Database approaches for peptide identification can cope only with the natural abundance of carbon isotopes; they fail if the incorporation of ¹³C significantly exceeds the natural isotope abundance or if incorporation patterns occur in unpredictable ways (7).The simplest method for determining the incorporation level is to compare the unlabeled average mass of the monoisotopic peptide with the mass of the labeled protein, as estimated by matrix-assisted laser desorption/ionization or electrospray ionization MS (8, 9). A more advanced approach for determining the isotopic mass distribution of peptides is based on the isotopic distribution of the peaks of a peptide envelope (10, 11). Here, for a given isotopomer, the incorporation efficiency is defined as the percentage of incorporated ¹³C atoms with relation to the total number of carbon atoms with the natural isotope abundance (approximately 1.01 atom % ¹³C). As a reference, the theoretical isotopic distribution of a peptide is calculated based upon an algorithm described elsewhere (12). The isotope distribution of both unlabeled and labeled peptides can subsequently be used to calculate the incorporation level. For this method, an Excel spreadsheet (ProSIPQuant.xls) was developed (4). A similar approach, also based on the calculation of isotopic distributions, has been used in other studies (7). In these studies, however, the identification of the peptides is limited to those that have unlabeled counterparts; in addition, an exact calculation can be hampered by overlapping signals coming from additional peaks with similar masses.In the present study, we describe a new way of determining the isotope incorporation level. Our method makes use of characteristic patterns in the digits after the decimal point of the peptide masses generated by high-accuracy instruments such as the linear ion trap LTQ-Orbitrap (Thermo Fisher Scientific, Bremen, Germany). For tryptic peptides, typical regularities in the decimal places of the monoisotopic masses have been observed (13, 14). These observations have been explored in detail for theoretical and experimental data of proteins originating from Helicobacter pylori (15). As a result, a rule called the “half decimal place rule” (HDPR) was defined; it states that the decimal place is nearly half of the first digit for tryptic peptides with masses in the range of 500–1,000 Da. In other words, the exact mass of a peptide is equal to its nominal mass times ∼1.005. Because the difference between ¹²C and ¹³C is slightly greater than 1 Da, exactly 1.0033548378, the decimal places of a tryptic peptide''s mass are shifted in a regular manner by the incorporation level and lead to a significantly increased slope for the digits in the third and fourth place after the decimal point. This shift can be used to estimate the incorporation level of heavy isotopes into the protein. Detecting such shifts requires the highly accurate measurement possible with modern mass spectrometers such as the LTQ-Orbitrap, the Fourier transform ion cyclotron resonance, or the quadrupole time of flight. In this communication, we demonstrate the applicability of this approach using Pseudomonas putida ML2 proteins labeled uniformly via the consumption of [¹³C₆]benzene with five different substrate concentrations (0, 10, 25, 50, and 100 atom % of ¹³C). The ¹³C incorporation was calculated based on several labeled peptides derived from different proteins in one SDS-PAGE band. By these means, we have established a method that allows the determination of ¹³C incorporation into proteins and can be used to assess the metabolic activity of a given species within a mixed community.     

INCORPORATION OF H3-THYMIDINE IN LEAF NUCLEI OF XANTHIUM PENNSYLVANICUM

The basic kinetics and the pattern of incorporation of H³-thymidine was studied in the leaf lamina of Xanthium pennsylvanicum. A method of foliar absorption was used to incorporate the radioisotope into leaf nuclei. The autoradiographic techniques employed provided data on the amount of the isotope incorporated. It was determined that 10 μc/ml (sp. act. 6.7 c/mmole) of H³-thymidine with 1–8 hr of isotopic growth and 4 hr of postisotopic growth gave the most satisfactory results. The percent of labelled nuclei and the number of grains per nucleus were presented as functions of isotopic and postisotopic growth periods. Distribution of grains in the nuclei approximated the Poisson distribution at 1 hr of isotopic growth. Increased time of isotopic growth changed the pattern of grain distribution. No deleterious effects were observed using an 8-hr period of isotopic growth, but prolonged incubation time significantly decreased the proportion of mitotic figures in the lamina. The amount of incorporation of the DNA precursor expressed as percent of labelled nuclei was linear to about 16 hr of isotopic growth and thereafter decreased gradually. As indicated by the average number of grains per nucleus, H³-thymidine incorporation increased to about 16 hr, and soon after reached a saturation level. The percent of labelled nuclei and the number of grains per nucleus decreased as a function of the postisotopic growth period. However, they were significantly greater in the lamina near the vein than in the lamina region at some distance from the vein. The radioactive precursor was initially absorbed by the cells of the lamina and was subsequently translocated into the vascular system. There it was circulated and made available to the dividing cells near veins of the lamina. This region may be a metabolically distinct part of the lamina with significantly higher rates of incorporation and mitotic turnover.     

Amino-acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells useful for NMR studies

Culture conditions for successful amino–acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells are described. The method was applied to the selective labeling of the catalytic domain of c-Abl kinase with ¹⁵N-phenylalanine, ¹⁵N-glycine, ¹⁵N-tyrosine or ¹⁵N-valine. For the essential amino acids phenylalanine, tyrosine and valine high ¹⁵N-label incorporation rates of 90% and approximately the expected number of resonances in the HSQC spectra were observed, which was not the case for the non-essential amino acid glycine. The method should be applicable to amino-acid-type selective isotope labeling of other recombinant proteins which have not been amenable to NMR analysis.     

Ultrastructural relationship between monoamine- and TRH-containing axons in the rat median eminence as revealed by combined autoradiography and immunocytochemistry in the same tissue section

Summary The correlation of dopamine (DA)-, noradrenaline (NA)- or serotonin (5HT)-containing neurons and thyrotropin releasing hormone (TRH)-containing neurons in the median eminence of the rat, as well as the coexistence of monoamines (MA) and TRH in the neurons, were examined by subjecting ultrathin sections to a technique that combines MA autoradiography and TRH immunocytochemistry. The distribution and localization of silver grains after ³H-MA injection were examined by application of circle analysis on the autoradiographs.TRH-like immunoreactive nerve terminals containing the immunoreactive dense granular vesicles were found to have an intimate contact with monoaminergic terminals labeled after ³H-DA, ³H-NA or ³H-5HT infusion in the vicinity of the primary portal capillaries in the median eminence. Synapses between TRH-like immunoreactive axons and MA axons labeled with silver grains, however, have not been observed to date. Findings suggesting the coexistence of TRH and MA in the same nerve terminals or the uptake of ³H-MA into TRH-like immunoreactive nerve terminals, where silver grains after ³H-MA injection were concurrently localized in TRH-like immunoreactive nerve terminals, were rarely observed in the median eminence. Percentages of the nerve terminals containing both immunoreactive granular vesicles and silver grains after ³H-MA injection to total nerve terminals labeled after ³H-MA infusion silver grains were equally very low in ³H-DA, ³H-NA or ³H-5HT, amounting to less than 6.1%.This work was supported in part by grant-in-aid for scientific research from the Japan Ministry of Education (No. 557018).     

Mercury determination in blood by gas chromatography-mass spectrometry

A stable isotope dilution gas chromatography-mass spectrometry method using¹⁹⁶Hg as an internal standard is described for determining Hg in blood. In this method, the blood samples are not subjected to any digestion to avoid the loss of Hg. A solution of 0.6M HCl is used to free Hg present in blood from proteins. The pH of the solution is adjusted to 9 using borate buffer and Hg chelated using lithiumbis(trifluoroethyl)dithiocarbamate. All isotope ratio measurements are made using an organic mass spectrometer. Overall precision values for the five major Hg isotopes relative to²⁰²Hg are 1.6–2.3% when 10 ng samples of chelated Hg are analyzed. No appreciable memory or carryover effect is observed when two synthetic mixtures differing in¹⁹⁶Hg/²⁰²Hg ratios by a factor of 30 are sequentially analyzed. The method is validated by determining Hg in blood samples using isotope dilution GC-MS.     

Trophic partitioning in tropical rain forest birds: insights from stable isotope analysis

Bird communities reach their highest taxonomic and trophic diversity in tropical rain forest, but the use of different foraging strategies to meet food requirements in such competitive environments is poorly understood. Conventional dietary analyses are poorly suited to investigate dietary patterns in complex systems. We used stable carbon (¹³C/¹²C) and nitrogen (¹⁵N/¹⁴N) isotope analysis of whole blood to examine avian trophic patterns and sources of diet in the tropical rain forest of Los Tuxtlas, Veracruz, Mexico. We used stable nitrogen isotope analysis to delineate trophic levels, and stable carbon isotope analysis to distinguish the relative contribution of C-3 and CAM/C-4 ultimate sources of proteins to diets. There was large inter- and intraspecific variation in whole blood ¹³C and ¹⁵N values in 23 species of birds. Stable nitrogen isotope analysis separated birds into several trophic levels, including species that obtained their dietary protein mostly from plants, insects or a combination of both food sources. Stable carbon isotope analysis showed that most birds fed on C3-based foods but Stub-tailed Spadebills (Platyrinchus cancrominus) included C-3- and C-4/CAM-specialist individuals. Our analyses provided insights into the nutritional contribution of plant and animal sources of protein and distinguish their photosynthetic origin over relatively long average time periods.     

An approach to the theory of quantitative and double label autoradiography

The distributions of grains and tracks per cell, as functions of the isotope content in the cell, are basic to all quantitative autoradiography. In this paper a formulation of these distributions in terms of the distribution of grains per disintegration is suggested. An empirical method for evaluating this distribution for various isotopes and various geometrical relationships between source and emulsion is described. The results are used to examine the usefulness of different techniques for double labelling autoradiography.