Isolation, purification and characterization of glycosaminoglycans in the fluids of the mollusc Anodonta cygnea

The isolation, purification and characterization of Anodonta cygnea haemolymph, and extrapallial fluid glycosaminoglycans (Gags), which have high calcium affinity, were carried out in order to better understand the process of nacreous shell biomineralization. Our results show the existence of two different Gags with similar contents in the two fluids, throughout the year, but with significant seasonal variations for both. In the winter months, we identified by electrophoresis, only one kind of Gag chain (hyaluronic acid) while two different Gags (hyaluronic acid and heparan sulphate-like) from both fluids of A. cygnea were found in summer months. Quantification showed the total Gag fractions of both fluids in average, at their highest concentration (79.8 mg/L) in the highest calcification summer period. In contrast, the period of the year with the lowest concentration of total Gags (54.6 mg/L) occurred during the winter when calcification is reduced. This significant decrease between seasons is correlated mainly with the sulphated fraction, being 37.1 mg/L in the summer while only 9.2 mg/L in the winter haemolymph. The present data suggest that a heparan sulphate-like Gag has a relevant role in the biomineralization mechanisms acting as the calcium carbonate nucleator in the shell.     

The action of Cd, Cu, Cr, Zn, and Pb on fluid composition of Anodonta cygnea (L.): organic components

The heavy metals, Cd, Cu, Cr, Zn, and Pb, were used to incubate healthy specimens of the freshwater mussel species, Anodonta cygnea. Afterwards, their biological fluids, either haemolymph or extrapallial fluid were analyzed for the presence of several organic constituents, known to be important for biomineralization, such as proteins, glycosaminoglycans (GAGs) and glucosamine. Proteins were subjected to further study, namely through the total amino acid determination after acid hydrolysis. The most disturbing pollutants tested seem to be Pb, Zn, and Cr, which caused highly decreased overall compositions, namely with respect to protein, and glucosamine, in comparison to the control group. This suggests that this group contributes to a decrease of the metabolic activity, and thus mineralization, in the exposed animals.     

Influence of the yeast strain on the changes of the amino acids,peptides and proteins during sparkling wine production by the traditional method

The influence of five yeast strains on the nitrogen fractions, amino acids, peptides and proteins, during 12 months of aging of sparkling wines produced by the traditional or Champenoise method, was studied. High-performance liquid chromatography (HPLC) techniques were used for analysis of the amino acid and peptide fractions. Proteins plus polypeptides were determined by the colorimetric Bradford method. Four main stages were detected in the aging of wines with yeast. In the first stage, a second fermentation took place; amino acids and proteins plus polypeptides diminished, and peptides were liberated. In the second stage, there was a release of amino acids and proteins, and peptides were degraded. In the third stage, the release of proteins and peptides predominated. In the fourth stage, the amino acid concentration diminished. The yeast strain used influenced the content of free amino acids and peptides and the aging time in all the nitrogen fractions. Received 25 March 2002/ Accepted in revised form 31 July 2002     

Certain biochemical characteristics of an insulin-like substance from the bivalve mollusk Anodonta cygnea

An insulin-like substance (ILS) was isolated from the visceral organs of the bivalve mollusc Anodonta cygnea by chromatography on a sulfocationite CU-23 and purified by reverse phase liquid chromatography. ILS was shown to be made up to several fractions with Mr ranging from 9 to 20 kDa which have identical amino acid composition but different hydrophobicity and N-terminal amino acids. It was supposed that the heterogeneity of ILS fractions is due to its genetical or posttranslational polymorphism. ILS has a low (0.02%) affinity for the mammalian insulin receptor and a low immune affinity for mammalian insulin and possesses a mitogenic activity which is commensurate with that of the epidermal growth factor. The data obtained suggest that Anodonta cygnea ILS represents a separate branch of a relatively ancient family of insulin-like hormones and growth factors responsible for metabolism and proliferation of invertebrate tissues.     

Annual variations in plasma sex steroid-binding protein and testosterone concentrations in the adult male little brown bat: relation to the asynchronous recrudescence of the testis and accessory reproductive organs

The annual reproductive cycle of the male little brown bat, in contrast to seasonal reproductive patterns of other mammals, is differentiated by an asynchronous recrudescence of the testis and the accessory reproductive glands. Spermatogenesis occurs during the summer, whereas fully stimulated accessory organs, stored epididymal spermatozoa, and sexual behavior are expressed later during a mating period that extends, albeit interrupted by hibernation, from late summer until early spring. To investigate whether changes in high affinity androgen-binding activity in the circulation are related to the delayed renewal of the accessory organs, plasma sex steroid-binding protein (SBP) and total testosterone (T) levels were measured throughout the year. From these data and determinations of association constants for T binding to SBP and albumin at both hibernating (4 degrees C) and active (40 degrees C) temperatures, estimates of the unbound ("free") and albumin-bound T fractions were made and correlated with changes in the accessory reproductive organs. Plasma SBP concentrations (mean +/- SEM) exhibited wide seasonal fluctuations: they were baseline in May (10 +/- 2 nM) following spring arousal, increased dramatically in June (184 +/- 24 nM), and reached peak levels in early July (262 +/- 29 nM), where they remained until August. In late August they began to fall (104 +/- 23 nM) and then returned to baseline during the hibernation period (October-April). Although total T levels were also elevated in June, it appeared that the unbound ("free") and the unbound plus albumin-bound T fractions did not increase until late July. Since the accessory gland weights did not begin to increase until late July as well, it was concluded that increases in the unbound and albumin-bound T fractions may be an important factor in the recrudescence of the accessories and that increased SBP activity in early summer may play a role in the regression and delayed renewal of these organs. However, what factor(s) maintain the accessory glands, epididymal spermatozoa, and sexual behavior during the breeding and hibernation periods when all T fractions were low are, as yet, undetermined.     

Patterns in the Chemical Fractionation of Organic Nitrogen in Rocky Mountain Streams

The intraannual dynamics of particulate organic nitrogen (PON) and two fractions of dissolved organic nitrogen (DON) were investigated in two Rocky Mountain streams draining watersheds with low rates of N deposition. Organic nitrogen accounted for over 60% of the total annual nitrogen export and consisted mostly of DON. Nitrate peaked during winter months and declined considerably during the growing season (less than 10 µg/L) suggesting the importance of biotic uptake. Concentrations of PON, total DON, and two DON fractions (humic and non-humic) peaked during spring runoff and were positively related to discharge, indicating hydrologic influence. Total DON and its two fractions showed significant inverse relationships to nitrate, indicating that DON and nitrate followed different intraannual patterns. Despite its seasonal fluctuations in concentration, PON showed a consistent carbon–nitrogen (C:N) ratio suggesting that it was relatively uniform in composition. Fractionation studies indicated that DON was primarily of non-humic origin, whereas dissolved organic carbon (DOC) was mainly derived from humic sources. The two DON fractions differed from each other in seasonal patterns of concentration and C:N ratio. The proportion of humic DON increased during snowmelt, and there were diverging seasonal patterns in the C:N ratio of the two fractions implying variations in bioavailability. Although organic nitrogen is commonly treated as a single pool in ecological studies, our results indicated that DON consists of fractions that undergo large intraannual changes in proportions and chemical composition. Treatment of DON as a single pool may be misleading from the viewpoint of understanding ecosystem processes directly related to changes in its sources and biological reactivity.     

Seasonal seawater temperature as the major determinant for populations of culturable bacteria in the sediments of an intact mangrove in an arid region

Mangroves are highly productive marine ecosystems where bacteria (culturable and non-culturable) actively participate in biomineralization of organic matter and biotransformation of minerals. This study explores spatial and seasonal fluctuations of culturable heterotrophic bacteria and Vibrio spp. in the sediments of an intact mangrove ecosystem and determines the dominant environmental factors that govern these fluctuations. Sediment samples were collected monthly from three stations in the mangroves of Laguna de Balandra, Baja California Sur, Mexico, through an annual cycle. Physicochemical parameters included seawater temperature, salinity, and concentration of dissolved oxygen. Viable counts of culturable heterotrophic bacteria and Vibrio spp. were made. In one sample (March 2003), nutrient concentrations (ammonium, nitrites, nitrates, and phosphates), organic matter, pH and sediment texture were also determined. General cluster analyses, analysis of variance of specific variables, and several principal component analyses demonstrated that seawater temperature is the principal determinant of seasonal distribution of culturable heterotrophic bacteria and Vibrio spp. in mangrove sediments. Soil texture, concentration of nutrients, and organic matter concentration contribute to heterogenicity to a lesser extent. A large spatial variation in bacterial populations was observed over short distances ( approximately 1 m) in sampling areas within the same site. These analyses show that the culturable bacterial distribution in sediments of mangroves has high spatial and temporal heterogeneity.     

Enzymes of Nitrogen Assimilation Undergo Seasonal Fluctuations in the Roots of the Persistent Weedy Perennial Cichorium intybus

Chicory (Cichorium intybus), a deep rooted weed, grows in regions with temperate climates. Seasonal partitioning of compounds between the root and shoot results in fluctuations in the soluble carbohydrate, nitrate, amino acid, and protein pools within the roots. The activities of nitrate reductase (NR) (EC 1.6.6.1), glutamine synthetase (EC 6.3.1.2), NADH (EC 1.4.1.14), ferrodoxin glutamate synthase (EC 1.4.7.1), and glutamate dehydrogenase (GDH) (EC 1.4.1.2-4) vary throughout the year and coincide with seasonal alterations in nitrate, fructose, and sucrose. During the winter, NR, glutamine synthetase and ferrodoxin glutamate synthase activities increase in the root, while GDH displays the opposite trend with elevated activity in the summer months. All of these enzymes exhibit seasonal alterations in abundance as detected by Western blot analysis, increasing during the winter and, therefore, contributing to the seasonally dynamic protein pool. Extensive fluctuations in abundance and activity of these enzymes in the root occur during the spring and fall and coincide with shoot growth and senescence, respectively. Several observations indicate that posttranslational modifications of NR and GDH are taking place throughout the year; for example, NR is particularly unstable during the spring and fall, and seasonal GDH activity does not correlate with protein abundance.     

Lysosomal enzymes in relation to the isoproterenol-induced secretory cycle in rat parotid gland

Two lysosomal enzymes, cathepsin D and acid phosphatase, were detected in significant amounts in the lysosome-containing subcellular fractions of rat parotid tissue and found to have dissimilar distributions in these fractions. The total levels of these enzymes were measured at various times throughout a complete secretory cycle induced synchronously by fasting rats overnight and administering isoproterenol at time zero. The results showed a 30% increase in cathepsin D activity in the glands by 10 h post-stimulation, and a 20% decrease in acid phosphatase activity 7 h after stimulation. These results suggest that there are cyclic changes in lysosomal enzymes during the secretory cycle of this gland, but that these changes are complex ones and cannot be related to specific cellular processes at this time.     

Seasonal and within-plant gradients in the intramolecular carbon isotopic composition of amino acids of Spartina alterniflora

Autotrophy and heterotrophy create different patterns of carbon flux through the central metabolic pathway. One consequence of these different fluxes is that the α-carboxyl carbon of amino acids is derived from different carbon sources and has a different isotopic composition under autotrophic and heterotrophic conditions. In Spartina alterniflora, a C4 grass and a common and ecologically important component of coastal ecosystems, the isotopic composition of bulk acid hydrolyzable carbon and total amino acid carboxyl carbon were compared over a seasonal cycle. The isotopic composition of plants varied significantly between aboveground and below ground tissues, and the δ¹³C of both hydrolyzable organic carbon and total amino acid carboxyl carbon showed significant variation among seasons. The isotopic heterogeneity within amino acids was used to infer seasonal changes in source/sink relationships for amino acid carbon among plant organs. Comparison of the intramolecular isotope data for Spartina and C3 freshwater marsh plants indicates that the patterns and processes inferred for Spartina are not unique to this taxon.     

An improved large scale fractionation of high mobility group non-histone chromatin proteins.

1. Methodology is presented for the large scale preparation and fractionation of high mobility group proteins from calf thymus chromatin. The total high mobility group protein from approx. 1 kg calf thymus tissue can be separated into five fractions by CM-Sephadex C25 ion-exchange chromatography. High mobility group proteins 1 and 2 comprise two fo the fractions. From a third fraction two more chromatin proteins, protein 3 and 17, can be isolated by trichloroacetic acid precipitation and CM-cellulose chromatography at pH 5.5. 2. The four proteins thus purified are lysine-rich proteins. Proteins 1 and 2 are additionally characterised by their high contents of acidic amino acids, as described previously (Goodwin, G. H. and Johns, E. W. (1973) Eur. J. Biochem. 40, 215-219). Proteins 3 and 17, having lower contents of acidic amino acids, are basic proteins similar to the histones. All four proteins exhibit single N-terminal amino acids; glycine is the N-terminal group of proteins 1, 2 and 3; protein 17 has a proline N-terminal amino acid. The proteins are not highly phosphorylated nor are they associated with appreciable quantities of nucleic acid.     

Separation and determination of 14C-labelled intermediates of the citric acid cycle and related compounds

A new systematic procedure is presented which permits a complete chromatographic resolution of the intermediates of the citric acid cycle as well as the related amino and keto acids. The adsorption onto ion-exchange columns followed by the subsequent elution therefrom forms the first step of the present procedure. Meanwhile, unstable keto acids are enzymatically converted into the respective amino acids. This preliminary process is very effective in desalting the biological specimen and also results in a broad division of a large number of intermediates into three subgroups, i.e., amino, keto and other organic acid fractions. Each of these subgroups is then readily resolved into individual acids by means of one-dimensional thinlayer chromatography. ¹⁴C-Compounds added to the reaction mixture of rat liver mitochondria were recovered with a good reproducibility throughout the entire course of the present procedure.     

Complementary DNA Cloning and Characterization of Pearlin, a New Class of Matrix Protein in the Nacreous Layer of Oyster Pearls

: Calcified shell layer is composed of two polymorphs of CaCO3, aragonite or calcite, and an organic matrix. The organic matrix consists of EDTA-soluble and insoluble fractions. These fractions are thought to regulate the formation of the elaborate shell structure. After decalcification of powdered pearl with 0.3 M EDTA, an EDTA-insoluble fraction was extracted with 0.3 M EDTA/8 M urea. This extraction step enabled us to purify a new class of EDTA-insoluble protein, Pearlin, almost homogeneously. Pearlin has a molecular weight of about 15 kDa and contains a sulfated mucopolysaccharide. We cloned the complementary DNA coding for Pearlin and deduced its complete amino acid sequence. Sequence analysis reveals that Pearlin is composed of 129 amino acids with a high proportion of Gly (10.8%), Tyr (10.0%), Cys (8.5%), Asn (7.7%), Asp (7.7%), and Arg (7.7%). Northern blot analysis showed that Pearlin messenger RNA was expressed specifically in mantle epithelium. From the sequencing data, Pearlin was shown to be quite different from the fibrous protein rich in Ala and Gly. The function of this protein in biomineralization is discussed.     

黑河下游湿地土壤有机氮组分剖面的分布特征

结合野外调查,用Bremner法研究了黑河下游湿地不同土壤类型的有机氮组分,结果表明:在0—50 cm土层,5种土壤有机氮均以酸解性氮为主,占全氮的71.04%—81.79%。泥炭土、沼泽土、草甸土、亚高山草甸土所含的酸解氮、非酸解氮和酸解氮组分氨态氮、氨基酸态氮、氨基糖态氮含量的剖面分布总体上均随土层深度的增加而呈降低趋势,而风沙土却相反,上述有机氮组分呈升高趋势。5种土壤酸解氮及其组分氨态氮、氨基酸态氮、氨基糖态氮占全氮比例的剖面分布总体上均随土层深度的增加而呈降低趋势,而非酸解氮却呈升高趋势。5种土壤酸解未知态氮含量及占全氮比例均在剖面分布上无明显特征。在0—30 cm各相同土层内,5种土壤酸解氮各组分含量及占全氮比例的大小顺序均为氨基酸态氮氨态氮未知态氮氨基糖态氮;而在30—50 cm土层,5种土壤酸解氮各组分含量及占全氮比例的大小顺序均无明显特征。此外,黑河下游湿地土壤干化、沙化过程中,表层0—10 cm土壤有机氮组分含量变化明显,其中土壤氨态氮对生态环境变化最为敏感。     

Seasonal distribution and changes in the nutritive quality of living,dead and detrital fractions of Zostera marina L.

Samples of eelgrass, Zostera marina L., were collected monthly from December 1974 through December 1975 in a shallow embayment near Beaufort, N.C., and separated into green leaves, dead leaves, and leaf detrital material. Each component was analysed for dry weight, organic matter, inorganic and organic carbon, nitrogen and amino compounds.The standing crop of green and dead blades reached a maximum in April through June, while detrital matter had peaks in December, April and July–September. Inorganic carbon in the three grass fractions showed seasonal variations similar to those observed for epiphytic biomass in previous years, and represented 14, 24, and 30% of the total carbon associated with the green and dead leaves and detrital fragments, respectively. Organic carbon represented a decreasing proportion of the dry weight of these three fractions on a dry weight basis while there was a significant increase in organic carbon on an ash-free dry weight basis in the detrital fragments relative to the head dead blades. During senescence there was a loss of nitrogen from the leaves and an increase in the nitrogen content of the organic matter of the detritus relative to the dead leaves. The latter suggests that there was microbial growth on the detritus and subsequent nitrogen immobilization from the surrounding medium. There were significant decreases of lysine, histidine, arginine, glycine, tyrosine, and glucosamine in the dead leaves relative to the detritus. The glucosamine, derived from N-acetyl-glucosamine, a product of murein which is a component of microbial cell walls, had a seasonal distribution similar to that of the epiphytic community and available inorganic nitrogen in the surrounding water. The relative proportions of N-acetyl-glucosamine, nitrogen and organic carbon were all higher in the fall and winter.     

滇西太保山森林公园子囊菌门虫生真菌物种多样性及其消长动态

云南及青藏高原存在着丰富的虫生真菌资源。本文选择云南省保山市省级城市森林公园太保山森林公园为研究区域, 对子囊菌门虫生真菌物种多样性及其季节消长动态开展了系统研究。在2016年每月采集土样和罹病昆虫, 分离虫生真菌菌株, 采用多基因(nrSSU, nrLSU, EF-1α, RPB1和RPB2)系统发育分析进行物种鉴定, 通过α多样性分析来研究虫生真菌的数量特征和种群消长动态。太保山森林公园子囊菌门虫生真菌全年均有分布, 共获得395个菌株, 包括3科9属24种; 优势属为虫草属(Cordyceps) (6个种199个菌株), 白僵菌属(Beauveria) (5个种80个菌株), 优势种依次为Akanthomyces sp.、Cordyceps tenuipes、C. cicadae、C. fumosorosea和Beauveria bassiana。7月菌株检出率最高(85株, 占总数的21.4%), 8月物种丰富度(15种)和多样性指数(2.35)最高。在5个优势种中, 只有Cordyceps cicadae受季节变化影响较大, 集中分布于5-9月, 7月分布最丰富(35株, 占该月菌株总数的41.2%); 其他4个种一年大部分时间(11或12个月)都能检测到。结果表明, 太保山森林公园子囊菌门虫生真菌资源丰富, 多数优势种对季节变化适应能力较强。     

Seasonal variation of nitrogenous compounds in the xylem sap of Citrus

Samples of tracheal sap of Citrus sinensis (L.) Osbeck cv. Washington Navel were taken from field trees throughout the year and the nitrogen composition of the sap was determined. The nitrogenous fraction of the sap was composed mainly of free amino acids (92–97% of total nitrogen) and nitrates throughout the year. Proline was the most abundant amino acid during almost the entire cycle, and its concentration was especially high during the autumn and winter period. Nevertheless, a significant part (40–60%) of the total organic nitrogen was transported as arginine. Total nitrogen as well as amino acids and nitrates were maximal at spring flush. At spring flush and summer flush there was also a diversification of α-amino nitrogen among different amino acids. During the spring flush, nitrates, asparagine and γ-aminobutyric acid in the xylem sap seemed to have a radicular origin, whereas glutamic acid and arginine were released from the surrounding parenchyma. The results suggest a metabolic transformation in the wood parenchyma of nitrogenous compounds coming from the roots (including reduction of nitrates) and a turnover of different nitrogen metabolites between the xylem and surrounding cells.     

Fractionation of plant extracts by thin-layer electrophoretic and chromatographic procedures

Methods are described for separating plant tissue extracts into amino acid, organic acid, and neutral fractions by means of thin-layer electrophoresis. The electrophoresis also provides the first-dimensional oeparation for both amino acid and organic acid fractions. Separation of amino acids and organic acids in the second dimension, and of sugars in both dimensions, is achieved by thin-layer chromatography. Suitable procedures for including the study of phospholipids and phosphate esters are suggested. Extracts from 20 mg tissue can be studied easily in this way.     

Free amino acids and protein concentrations in reproductive tract fluids of the mare

Uterine tubal fluids (UTF) were collected daily over a 214-day period (March through August) from three mares. Individual UTF samples identified by day of estrous cycle for five complete cycles within this six-month span were analyzed for free amino acids and total protein. Biochemical comparisons were made to blood plasma by drawing samples daily from each mare. Free amino acids and total protein were determined also on follicular fluids collected from three different mares on days 5 and 6 of standing estrus.The free amino acid level of UTF was significantly greater than was the amino acid concentration in blood plasma or follicular fluid. The highest concentration of amino acids in UTF was on day 13. Cyclic trends were observed for the amino acids, histidine, methionine, half-cystine, serine, proline, glycine, alanine, isolecine, and leucine. Glycine and alanine were found in the highest concentrations in UTF, peaking on day 17 of the estrous cycle. Protein concentration in UTF was highest on day 13 and lowest on days 7 and 19. Protein values for diestrus (33.1 mg/ml) were significantly greater (p<0.05) than for estrus (28.0 mg/ml).     

The two-step mode of growth in the scleractinian coral skeletons from the micrometre to the overall scale

It has been known since the 19th century that coral skeletons are built of aragonite crystals with taxonomy-linked arrangements, but the way by which each coral species controls this crystallization process remains an unsolved question. The problem became still more intriguing when it was shown that isotopic compositions of coral aragonite were subject to taxonomy-linked influences (the "vital effect"). On the other hand, presence of an organic component in coral skeletons is also long known, but localization of these compounds is admittedly restricted to particular structures called "centres of calcifications." Fibres, the largely predominant part of the coral skeletons, are usually considered as purely mineral units. In this paper, it is shown that in both "centres of calcification" and fibres, organic compounds are associated with the mineral material at a deep structural level. A series of variously scaled observations and localized measurements allow recognition of the presence of an organic component at the nanometre scale. Far from being a freely operating process, crystallization of coral fibres is thus permanently controlled by the polyp basal ectoderm through a cyclic two-step process acting at the micrometre-scale. The biomineralization cycle begins by secretion of a proteoglycan matrix. As the composition of these sugars-proteins assemblages has been shown taxonomy dependent, the hypothesis can be made that multiple and long recognized specificities of coral skeletons are linked to this biochemically driven crystallization process. Additionally, this new concept of the biomineralization process in coral skeletons provides us with an access to the long term evolution of the Scleractinia. Remarkably, results of a skeleton-based approach using microstructural criteria (i.e., the spatial relationships of "centres of calcification" and the three-dimensional arrangements of fibres), are consistent with a molecular phylogenetic analysis carried out on the same species. Clearly, at the overall ontogenic level, the two-step growth mode of coral skeletons is also a valuable tool to reconstruct the evolutionary history of Scleractinia.