Morphological and immunological confirmation of the presence of chlamydiae in the gut tissues of the chesapeake bay clam,Mercenaria mercenaria

The original electron microscopic identification by other investigators in 1977 of chlamydiae in the gut tissues of the Chesapeake Bay hard clam (Mercenaria mercenaria) is corroborated and further supported by evidence ofChlamydia-specific immunofluorescence (IF). Our electron microscopy demonstrated that gut tissue cells were heavily infected with chlamydiae in all stages of development but the intrachlamydial phage-like particles reported in 1977 were not seen. Tissue sections stained with IF reagents were strongly positive, and IF was blocked in varying degrees with chlamydial antisera produced in a goat and a turkey. The IF-positive tissue sections contained intracytoplasmic inclusions that stained darkly with Lugol's iodine (indicating the presence of glycogen) while IF-negative tissues had little if any iodinestaining material. Furthermore, electron micrographs of chlamydiae-containing phagosomes showed numerous rosettes of electron-dense particles typical of glycogen. The presence of iodine-positive phagosomes with electron dense rosettes suggests that the organisms are glycogen-producing chlamydiae biochemically related toChlamydia trachomatis. Repeated attempts to cultivate chlamydiae from the clam tissues in cell cultures and laboratory animals failed.     

Lifespan and patterns of accumulation and mobilization of nutrients in the sugar-fed phorid fly, Pseudacteon tricuspis

Abstract. The effect of sugar feeding on the survival of adult phorid fly Pseudacteon tricuspis is investigated. Flies fed 25% sucrose in aqueous solution continuously throughout their lifespan have greater longevity (mean ± SE longevity: female = 7.9 ± 0.8 days, male = 8.9 ± 0.9 days) than completely starved (provided no water and no sugar solution) flies, sugar-starved (provided water only) flies, or flies fed sugar solution only on their first day of adult life. Completely starved flies rarely lived beyond one day. Provision of water increases longevity by 2 days, and one full day of sugar feeding further increases longevity by an additional 1–2 days. Flies fed 50% sucrose have similar survivorship as those fed 25% sucrose. The temporal patterns of nutrient accumulation and utilization are also compared in P. tricuspis fed different diets: sugar-starved, sucrose-fed on the first day of adult life only, and sucrose-fed continuously. Adult P. tricuspis emerge with no gut sugars, and only minimal amounts of body sugars and glycogen. Although the levels of body sugars and glycogen decline gradually in sugar-starved flies, a single day of sugar feeding results in the accumulation of maximum amounts of gut sugars, body sugars and glycogen. High levels of these nutrients are maintained in female and male phorid flies fed on sucrose continuously over the observation period, whereas nutrient levels decline in flies fed only on the first day of life, beginning 1 day postfeeding. Female and male P. tricuspis emerge with an estimated 12.3 ± 2.3 and 7.2 ± 1 g of lipid reserves per fly, respectively. These teneral amounts represent the highest lipid levels detected in adult flies, irrespective of their diet, and are maintained over the life times of sucrose-fed female and male flies, but declined steadily in sugar-starved females. These data suggest that adult P. tricuspis are capable of converting dietary sucrose to body sugars and glycogen, but not lipids.     

Dietary polysaccharides: fermentation potentials of a primitive gut ecosystem

The alimentary canal of the earthworm is representative of primitive gut ecosystems, and gut fermenters capable of degrading ingested biomass-derived polysaccharides might contribute to the environmental impact and survival of this terrestrial invertebrate. Thus, this study evaluated the postulation that gut microbiota of the model earthworm Lumbricus terrestris ferment diverse biomass-derived polysaccharides. Structural polysaccharides (e.g. cellulose, chitin) had marginal impact on fermentation in anoxic gut content treatments. In contrast, nonstructural polysaccharides (e.g. starch, glycogen) greatly stimulated (a) the formation of diverse fermentation products (e.g. H₂, ethanol, fatty acids) and (b) the facultatively fermentative families Aeromonadaceae and Enterobacteriaceae. Despite these contrasting results with different polysaccharides, most saccharides derived from these biopolymers (e.g. glucose, N-acetylglucosamine) greatly stimulated fermentation, yielding 16S rRNA gene-based signatures of Aeromonadaceae-, Enterobacteriaceae- and Fusobacteriaceae-affiliated phylotypes. Roots and litter are dietary substrates of the earthworm, and as proof-of-principle, gut-associated fermenters responded rapidly to root- and litter-derived nutrients including saccharides. These findings suggest that (a) hydrolysis of certain ingested structural polysaccharides may be a limiting factor in the ability of gut fermenters to utilize them and (b) nonstructural polysaccharides of disrupted biomass are subject to rapid fermentation by gut microbes and yield fatty acids that can be utilized by the earthworm.     

Regulation of fat body glycogen phosphorylase activity during refeeding in Manduca sexta larvae

In 12-h-starved larvae of the tobacco hornworm, Manduca sexta, fat body glycogen phosphorylase was quickly inactivated when insects were refed with normal diet and agar which contained 3% sucrose. Only the first 2 min of refeeding were necessary to induce enzyme inactivation. During this short period, larvae did not ingest enough sucrose to increase the hemolymph glucose concentration. This may indicate that the gut released a hormone(s) which directly or indirectly led to the inactivation of fat body glycogen phosphorylase. Inactivation of the enzyme could also be induced by injection of glucose (30 mg) into the hemolymph of starving M. sexta larvae suggesting that there may be separate control from a neuroendocrine site such as the brain or the corpora cardiaca. Trehalose was less effective. Bovine insulin (2 and 4 μg/starved larva) did not induce phosphorylase inactivation over 20 min or decrease hemolymph carbohydrate or lipid concentrations within 60 min. It is, therefore, necessary to screen insect tissues for substances which could bring about inactivation of fat body glycogen phosphorylase. © 1992 Wiley-Liss, Inc.     

Liver glycogen metabolism during intestinal perfusion in the rat.

The effects of metabolic constituents on glycogen metabolism in the liver can be studied by perfusing the small intestine with those constituents capable of being absorbed across the gut mucosa. Perfusion of the gut with glucose to give concentrations of approx. 20 mM in the portal vein produced a 5-fold increase in liver glycogen after 60 min. The rate of synthesis over the 15–30 min period of perfusion of 1.24 μmol/min/g liver fell to 0.34 in the 30–60 min period. This stimulation was accompanied by a transient activation of glycogen synthase which occurred in the first 30 min period of perfusion but which had disappeared by 60 min. Glucose perfusion had no effect on total and active phosphorylase at the time intervals studied. While the pattern of insulin secretion during perfusion could not be ascertained, the activation of glycogen synthase and increase in glycogen synthesis correlated with the production of gastric inhibitory polypeptide (GIP), a potent stimulator of insulin secretion. Perfusion of the gut with fructose did not stimulate glycogen synthesis or activate glycogen synthase or stimulate GIP secretion, but instead it produced a marked activation of phosphorylase after 30 min of perfusion.     

Fine structure and function of the digestive tract of Cyathura carinata (Krøyer) (Crustacea,Isopoda)

Summary The entire gut of Cyathura carinata is lined by a cuticle indicating its completely ectodermal origin. By flattening of the epithelial folds and possibly also of reserve-folds of the plasma membrane the intestine is highly dilatable, an adaptation towards a rapid uptake of the food which is sucked in by means of specialized mouthparts, which pierce the body wall of its main prey, the polychaete Nereis diversicolor. Bundles of microtubules within the intestinal cells presumably represent cytoskeletal structures providing protection against mechanical stress. Spirally arranged muscle fibres, which form peculiar contact areas with the gut, can easily follow any dilatation. A few indications of the metabolic functions of the anterior gut epithelium have been found: Basally and apically located labyrinthine structures of the plasma membrane, apically located clear vesicles, positive reactions for lysosomal, mitochondrial and membraneous enzymes, a strikingly thin and loosely arranged cuticle through which food substances of low molecular weight may diffuse. The cells of the gut and also of the digestive caeca are interconnected by desmosomes, extensive pleated septate junctions, and gap junctions. In the pleon the gut is less dilatable and devoid of plasma membrane specializations. In this area tendon cells, particularly rich in microtubules, serve as attachment sites for the dilating muscles of the rectum. The digestive caeca synthetize and secrete digestive enzymes, mix food and enzymes in their lumen, resorb food molecules, store lipids and glycogen. In the glandular epithelium small cells, rich in rough ER, and a majority of large cells, rich in lipid droplets, occur which, however, are interconnected by a series of morphologically intermediate cells. All cells bear an apical brush border, form a basal labyrinth and contain high to medium activities of acid phosphatase, nonspecific esterases, ATPase, and succinic dehydrogenase. The ER-rich cells are far less frequent than in the omnivorous or herbivorous isopods (Sphaeroma, Idothea, Asellidae, Oniscoidea).     

Role of glucose and insulin in regulating glycogen synthase and phosphorylase activities in rainbow trout hepatocytes

This study, using ¹³C nuclear magnetic resonance spectroscopy showed enrichment of glycogen carbon (C1) from ¹³C-labelled (C1) glucose indicating a direct pathway for glycogen synthesis from glucose in rainbow trout (Oncorhynchus mykiss) hepatocytes. There was a direct relationship between hepatocyte glycogen content and total glycogen synthase, total glycogen phosphorylase and glycogen phosphorylase a activities, whereas the relationship was inverse between glycogen content and % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio. Incubation of hepatocytes with glucose (3 or 10 mmol·1^-1) did not modify either glycogen synthase or glycogen phosphorylase activities. Insulin (porcine, 10^-8 mol·1^-1) in the medium significantly decreased total glycogen phosphorylase and glycogen phosphorylase a activities, but had no significant effect on glycogen synthase activities when compared to the controls (absence of insulin). In the presence of 10 mmol·1^-1 glucose, insulin increased % glycogen synthase a and decreased % glycogen phosphorylase a activities in trout hepatocytes. Also, the effect of insulin on the activities of % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio were more pronounced at low than at high hepatocyte glycogen content. The results indicate that in trout hepatocytes both the glycogen synthetic and breakdown pathways are active concurrently in vitro and any subtle alterations in the phosphorylase to synthase ratio may determine the hepatic glycogen content. Insulin plays an important role in the regulation of glycogen metabolism in rainbow trout hepatocytes. The effect of insulin on hepatocyte glycogen content may be under the control of several factors, including plasma glucose concentration and hepatocyte glycogen content.     

Evidence for the periplasmic location of glycogen in Saccharomyces

Treatment of yeast cells with hot alkali fails to solubilize a significant amount of glycogen. The insoluble glycogen is readily hydrolyzed by insolubilized α-amylase indicating that the apparent insolubility of this glycogen fraction does not result from its physical entrapment within an insoluble glucan membrane. The alkali-insoluble glycogen fraction of glutaraldehyde treated-cells is rapidly degraded by a mixture of snail gut enzymes during the formation of spheroplasts but the alkali-soluble glycogen fraction is unaffected. These results indicate that a major fraction of yeast glycogen is located outside the cellular membrane.     

Physiological reactions of aquatic oligochaetes to environmental anoxia

Aquatic oligochaetes are well known for their ability to resist prolonged periods of anoxia. In fact, the observed mortality is more likely to result from laboratory stress (unnatural sediment, starvation, accumulation of toxic substances) than from lack of oxygen per se. Lumbriculus variegatus feeds under anoxia at 6°C at a low rate and survives more than 40 days. A sudden transfer into anoxic water, however, results in a cessation of defaecation before the gut is half emptied, whereas the gut is completely emptied under aerobic conditions within 8–10 hours (11°C).Anoxic heat dissipation as measured by direct calorimetry is reduced by up to 80% relative to aerobic rates. The basal rate of oxygen uptake is independent of PO₂ above 3 kPa (15% air saturation), but the active rate shows a high degree of oxygen conformity. Whereas the theoretical oxycaloric equivalent yields an accurate estimation of aerobic heat dissipation in Lumbriculus, anoxic catabolism of glycogen explains only up to 60% of the directly measured rates of anoxic heat dissipation in Lumbriculus and Tubifex. Since unknown bioenergetic processes may be important under anoxia, direct calorimetry is required to assess total rates of energy expenditure in anoxic oligochaetes.     

A functional glycogen biosynthesis pathway in Lactobacillus acidophilus: expression and analysis of the glg operon

Glycogen metabolism contributes to energy storage and various physiological functions in some prokaryotes, including colonization persistence. A role for glycogen metabolism is proposed on the survival and fitness of Lactobacillus acidophilus, a probiotic microbe, in the human gastrointestinal environment. L. acidophilus NCFM possesses a glycogen metabolism (glg) operon consisting of glgBCDAP‐amy‐pgm genes. Expression of the glg operon and glycogen accumulation were carbon source‐ and growth phase‐dependent, and were repressed by glucose. The highest intracellular glycogen content was observed in early log‐phase cells grown on trehalose, which was followed by a drastic decrease of glycogen content prior to entering stationary phase. In raffinose‐grown cells, however, glycogen accumulation gradually declined following early log phase and was maintained at stable levels throughout stationary phase. Raffinose also induced an overall higher temporal glg expression throughout growth compared with trehalose. Isogenic ΔglgA (glycogen synthase) and ΔglgB (glycogen‐branching enzyme) mutants are glycogen‐deficient and exhibited growth defects on raffinose. The latter observation suggests a reciprocal relationship between glycogen synthesis and raffinose metabolism. Deletion of glgB or glgP (glycogen phosphorylase) resulted in defective growth and increased bile sensitivity. The data indicate that glycogen metabolism is involved in growth maintenance, bile tolerance and complex carbohydrate utilization in L. acidophilus.     

Progressive redistribution of alcohol dehydrogenase during vitellogenesis in Drosophila melanogaster: characterization of ADH-positive bodies in mature oocytes

Summary The use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH) provides a powerful tool in the analysis of the tissue and temporal patterns of Adh gene expression. Immunocytochemical techniques at the light- and electron-microscopic levels have been used to determine the distribution of ADH in the ovarian follicles of D. melanogaster during oogenesis. In the early stages of oogenesis, small amounts of ADH are detectable in the cystocytes. At the beginning of vitellogenesis (S₇), ADH appears to be located mainly in the nurse cells. From stage S₉ onwards, the ADH protein is evenly distributed in the ooplasm until the later stages of oogenesis (S_13–14), when multiple ADH-positive bodies of varying size appear in the ooplasm. This change in distribution is a result of the compartmentalization of the ADH protein within the glycogen yolk or -spheres. Yolk becomes enclosed within the lumen of the primitive gut during embryonic development, and thus our results suggest a mechanism for the transfer of maternally-inherited enzymes to the gut lumen via yolk spheres.     

A modified PAS stain combined with immunofluorescence for quantitative analyses of glycogen in muscle sections

Simultaneous analyses of glycogen in sections with other subcellular constituents within the same section will provide detailed information on glycogen deposition and the processes involved. To date, staining protocols for quantitative glycogen analyses together with immunofluorescence in the same section are lacking. We aimed to: (1) optimise PAS staining for combination with immunofluorescence, (2) perform quantitative glycogen analyses in tissue sections, (3) evaluate the effect of section thickness on PAS-derived data and (4) examine if semiquantitative glycogen data were convertible to genuine glycogen values. Conventional PAS was successfully modified for combined use with immunofluorescence. Transmitted light microscopic examination of glycogen was successfully followed by semiquantification of glycogen using microdensitometry. Semiquantitative data correlated perfectly with glycogen content measured biochemically in the same sample (r²=0.993, P<0.001). Using a calibration curve (r²=0.945, P<0.001) derived from a custom-made external standard with incremental glycogen content, we converted the semiquantitative data to genuine glycogen values. The converted semiquantitative data were comparable with the glycogen values assessed biochemically (P=0.786). In addition we showed that for valid comparison of glycogen content between sections, thickness should remain constant. In conclusion, the novel protocol permits the combined use of PAS with immunofluorescence and shows valid conversion of data obtained by microdensitometry to genuine glycogen data.     

Utilization of α-starch in ayu, Plecoglossus altivelis, relating to growth and body composition

To examine the possibility of dietary α‐starch in reducing feed costs in a practical diet, α‐starch was supplemented at 10, 20, 30 and 40% in a composed diet having the same protein level. The four diets were fed to ayu, Plecoglossus altivelis (initial weight 9.1 g) for 43 days. Growth and feed efficiency increased with the supplement, with values highest in the 30–40%α‐starch diet. The level of dietary α‐starch did not affect the proximate muscle composition; although the hepatosomatic index was not affected, liver glycogen increased with increasing dietary α‐starch. The dietary α‐starch did not influence evacuation time from the gut, and was well digested through passage in the gut, mainly between the stomach and the anterior part of the intestine. Ayu have an ability to adapt their metabolism to high dietary α‐starch, and can digest 40% or more in a composed diet. Although the muscle lipid content did not change, the fatty acid composition was influenced by dietary starch. With the elevation of dietary starch, a decrease of C18:2n‐6 and an increase of C22:6n‐3 occurred. These results indicate that at least 40%α‐starch can be used in practical diets for ayu.     

Mode of Loss of Phosphatidyl-ethanolamine in Autoxidation

This report identifies and describes the chemical structure of granular material that accumulates in the cytoplasm of Selenomonas ruminantium grown in glucose or lactate medium. The granular material was identified to be glycogen. Its molecular weight was about 2 x 10⁷ daltons. Conversion into maltose with α-amylase, β-amylase, and isoamylase was 61%, 37%, and 15%, respectively. The glycogen was digested completely by joint action of β-amylase and isoamylase, and its conversion into maltose was 103%. The average chain length of the glycogen was 23.5. The maximum absorption of the iodine complex of the glycogen was at 520 nm. These results led us to conclude that the chemical structure of this glycogen was similar to that of plant amylopectin, unlike normal Microbiol or animal glycogen so far known. When S. ruminantium was grown in glucose medium, the amount of glycogen in cells reached about 260 µg/mg dry weight of cells during late exponential phase and early stationary phase.     

Feeding behaviour, digestive physiology and lipid content in macropterous females of Pyrrhocoris apterus (L.) (Heteroptera: Pyrrhocoridae)

Abstract. Macropterous females of Pyrrhocoris apterus (L.) reared under short-day conditions (LD 12:12 h) were analysed for temporal patterns of feeding and drinking behaviour, activities of digestive enzymes in the gut, and lipid and glycogen content in the haemolymph and fat body. Peaks of drinking activity were recorded at days 3, 7 and 10 during the first 14 days after imaginal ecdysis. Feeding activity peaked on the third day, ceasing completely after the fourth day of adult life. Esterase, protease, amylase and aminopeptidase activities exhibited the highest overall activity in the first days after imaginal emergence; then enzyme activities decreased. In the fat body, the content of lipids was highest on day 5, then a decrease of about 40% was observed at day 14; the amount of glycogen was highest on day 1 at 11 μg of glucose equivalents/mg of fat body, then decreased to 2 μg at day 14 after the imaginal moult. In the haemolymph, the lipid content rose until day 8 when it reached almost 0.3 μmol/μl; at day 14 the value was slightly lower. The association of fasting with reproductive arrest in macropterous females of P. apterus, accompanied by a decrease in digestive enzyme activities and a mobilization of lipid reserves from the fat body, was demonstrated.     

Non‐invasive quantification of brain glycogen absolute concentration

The only currently available method to measure brain glycogen in vivo is ¹³C NMR spectroscopy. Incorporation of ¹³C‐labeled glucose (Glc) is necessary to allow glycogen measurement, but might be affected by turnover changes. Our aim was to measure glycogen absolute concentration in the rat brain by eliminating label turnover as variable. The approach is based on establishing an increased, constant ¹³C isotopic enrichment (IE). ¹³C‐Glc infusion is then performed at the IE of brain glycogen. As glycogen IE cannot be assessed in vivo, we validated that it can be inferred from that of N‐acetyl‐aspartate IE in vivo: After [1‐¹³C]‐Glc ingestion, glycogen IE was 2.2 ± 0.1 fold that of N‐acetyl‐aspartate (n = 11, R² = 0.77). After subsequent Glc infusion, glycogen IE equaled brain Glc IE (n = 6, paired t‐test, p = 0.37), implying isotopic steady‐state achievement and complete turnover of the glycogen molecule. Glycogen concentration measured in vivo by ¹³C NMR (mean ± SD: 5.8 ± 0.7 μmol/g) was in excellent agreement with that in vitro (6.4 ± 0.6 μmol/g, n = 5). When insulin was administered, the stability of glycogen concentration was analogous to previous biochemical measurements implying that glycogen turnover is activated by insulin. We conclude that the entire glycogen molecule is turned over and that insulin activates glycogen turnover.     

外源糖原调控猪链球菌基因表达的转录组分析

【背景】碳水化合物的利用与猪链球菌在宿主体内的定殖和致病性密切相关。感染期间,宿主细胞释放的糖原可能是猪链球菌重要的碳源。【目的】从转录组学角度解析猪链球菌全基因转录水平对外源糖原诱导的响应,特别是毒力基因。【方法】将猪链球菌2型强毒株分别用糖原和葡萄糖进行液体培养,通过高通量转录组测序,比较分析糖原对猪链球菌代谢通路和毒力基因差异表达的影响,并通过体外试验和攻毒试验进行验证。【结果】猪链球菌在糖原培养基中生长良好。转录组数据显示,糖原培养条件下的猪链球菌共有908个基因差异表达,基因组占比46.07%,其中501个基因上调表达,407个基因下调表达。富集分析结果表明,糖原影响了猪链球菌广泛的基础代谢过程,但糖酵解途径保持稳定。30个毒力基因的表达水平发生变化,重要的毒力因子SLY、ApuA、ArcABC等的基因转录水平大幅度升高(倍数>20)。糖原培养后的猪链球菌的溶血活性、黏附和侵入能力显著上升,对受试动物的毒力增强,证实猪链球菌能够响应糖原诱导,糖原能调控猪链球菌的致病性。【结论】外源糖原的利用显著影响了猪链球菌的基因表达谱,这种对碳源的响应是细菌对不断变化的生存环境的适应...     

Microphotometric determination of glycogen in single fibres of human quadriceps muscle

Synopsis A technique for the quantitation of glycogen in single fibres of human skeletal muscle is described. By using microphotometry the loss of glycogen from cryostat sections during a PAS-staining procedure was shown to be negligible. Further, it was found that nearly all the PAS-positive material (98.5%) inside a muscle fibre is glycogen. A significantly higher mean glycogen concentration (P<0.001) was found in type II fibres than in type I fibres in the resting quadriceps muscle of sedentary young males. The coefficient of variation for the glycogen concentration within each fibre type was found to be 17% and 15% for type I and type II respectively. The specificity of the PAS-staining technique for glycogen was confirmed by a statistically significant correlation (r=0.78,P<0.001) between the glycogen concentration measured biochemically and that calculated from microphotometry and area and thickness measurements. With the technique described, it seems possible to measure the glycogen concentration of single muscle fibres in serial sections and to calculate this in standard biochemical terms.     

Carbohydrate Composition during Germination and Outgrowth of Ascospores of Saccharomyces cerevisiae

Single spores of Saccharomyces cerevisiae were examined to distinguish changes in the synthesis and degradation of intracellular and wall carbohydrates during germination and outgrowth. Intracellular carbohydrate was fractionated into trehalose and glycogen. Trehalose degradation occurred during germination and outgrowth. The intracellular glycogen was degraded during germination and then synthesized during outgrowth. Wall carbohydrate was fractionated into glycogen, glucan and mannan. The wall glycogen and the KOH-soluble glucan were degraded during germination and then synthesized prior to and during outgrowth, respectively. The major component of the KOH-insoluble glucan in the wall is β-1,3-glucan. The glucan and mannan were synthesized during outgrowth.

The study revealed that the development of a vegetative cell from a spore follows rapid decreases in the amounts of trehalose, glycogen and KOH-soluble glucan during germination, and great increases in the amounts of glycogen, β-1,3-glucan and mannan during outgrowth.