An agarose slide method to follow the fate of bacteria within digestive vacuoles of protozoa

Abstract A method to follow the fate of ingested bacteria within digestive vacuoles of protozoa is presented. Tetrahymena pyriformis , previously fed with bacteria, is deposited onto glass microscope slides covered with a film of nutritive agarose. The protozoa lyse and the digestive vacuoles containing the bacteria stay undamaged and can be observed microscopically. After incubation, microcolonies reveal those vacuoles which contained living bacteria. The method can be used to study the survival ability of the ingested bacteria. It is a potentially valuable technique for studies on digestion efficacy, virulence ability, or escape mechanisms of bacteria from digestion.     

Untersuchungen zum Einfluß der Lagerungsdauer auf den Futterwert von Strohpellets

Während der Tränkperiode (ca. 55. Lebenstag) und 2 Wochen nach dem vollständigen Tränkmilchentzug (ca. 80. Lebenstag) wurde an Kälbern, welche mit Duodenalbrückenfisteln ausgerüstet waren, der Einfluß eines Heuanteiles in der Ration in Höhe von 6, 10, 14 bzw. 18 % in der Tränkperiode und von 16,23, 31 bzw. 37% (auf TS‐Basis) in der Periode nach dem Milchentzug auf den ruminalen N‐Umsatz geprüft.

Mit der Steigerung des Heuanteiles in der Ration für ruminerende Kälber reduzierte sich in der Tendenz die duodenale NAN‐Passage, deren Bakterien‐N‐Anteil, die postruminale NAN‐Verdaulichkeit sowie, durch eine schlechtere Verwertung des für die Bakterien‐N‐Synthese verfūgbaren N bedingt, die Bakterien‐N‐Syntheserate. Die scheinbare Fermentation der organischen Substanz war in der Tendenz mit wachsendem Heuanteil in der Ration gesteigert worden.     

Variation of ADM1 by using temperature-phased anaerobic digestion (TPAD) operation

The objective of the study was to examine the application of the Anaerobic Digestion Model No. 1 (ADM1) developed by the IWA task group for mathematical modelling of anaerobic process. Lab-scale temperature-phased anaerobic digestion (TPAD) process were operated continuously, and were fed with co-substrate composed of dog food and flour. The model platform implemented in the simulation was a derivative of the ADM1. Sensitivity analysis showed that k_m.process (maximum specific uptake rate) and K_S.process (half saturation value) had high sensitivities to model components. Important parameters including maximum uptake rate for propionate utilisers (k_m.pro) and half saturation constant for acetate utilisers (K_S.ac) in the thermophilic digester and maximum uptake rate for acetate utilisers (k_m.ac) in the mesophilic digester were estimated using iterative methods, which optimized the parameters with experimental results. Simulation with estimated parameters showed good agreement with experimental results in the case of methane production, uptake of acetate, soluble chemical oxygen demand (SCOD) and total chemical oxygen demand (TCOD). Under these conditions, the model predicted reasonably well the dynamic behavior of the TPAD process for verifying the model.     

Evaluation of cell surface-displayed protein stability against simulated gastric fluid

A molecular display technology that uses the displayed proteins on cell surfaces has many applications in microbiology and molecular biology. Here, we describe the resistance of displayed proteins to proteases using simulated gastric fluid (SGF), which included pepsin at pH 2. The displayed β-glucosidase resisted pepsin digestion compared with secreted, free β-glucosidase. In SDS-PAGE and Western blotting analysis, the secreted β-glucosidase was immediately digested within 1 min following SGF treatment, although the displayed β-glucosidase was stable for more than 60 min following SGF treatment. In addition, the residual activity of secreted β-glucosidase was completely destroyed after 10 min SGF treatment. However, displayed β-glucosidase retained 14% of its residual activity following the same treatment. These results clearly show that cell surface display technology using enzymes can reveal the protease resistance of a protein of interest under various conditions.     

Connective tissue growth factor is a substrate of ADAM28

ADAM28, a member of the ADAM (a disintegrin and metalloproteinase) gene family, is over-expressed by carcinoma cells and the expression correlates with carcinoma cell proliferation and progression in human lung and breast carcinomas. However, information about substrates of ADAM28 is limited. We screened interacting molecules of ADAM28 in human lung cDNA library by yeast two-hybrid system and identified connective tissue growth factor (CTGF). Binding of CTGF to proADAM28 was demonstrated by yeast two-hybrid assay and protein binding assay. ADAM28 cleaved CTGF in dose- and time-dependent manners at the Ala¹⁸¹-Tyr¹⁸² and Asp¹⁹¹-Pro¹⁹² bonds in the hinge region of the molecule. ADAM28 selectively digested CTGF in the complex of CTGF and vascular endothelial growth factor₁₆₅ (VEGF₁₆₅), releasing biologically active VEGF₁₆₅ from the complex. RT-PCR and immunohistochemical analyses demonstrated that ADAM28, CTGF and VEGF are commonly co-expressed in the breast carcinoma tissues. These data provide the first evidence that CTGF is a novel substrate of ADAM28 and suggest that ADAM28 may promote VEGF₁₆₅-induced angiogenesis in the breast carcinomas by the CTGF digestion in the CTGF/VEGF₁₆₅ complex.     

磷酸化对人肝癌细胞株SMMC-7721转醛醇酶活性的影响

目的:研究人肝癌细胞株SMMC-7721中转醛醇酶(Transaldolase,TAL)活性变化与磷酸化的关系。方法:SMMC-7721细胞培养,细胞周期同步化后分组加不同浓度(10%、1%)胎牛血清孵育,分别测定不同孵育时间(1、2、4、8h)TAL酶活性;Western blot检测TAL蛋白表达水平。磷酸酶消化法检测TAL活性变化与磷酸化的关系。结果:①人肝癌细胞株SMMC-7721中TAL活性在10%胎牛血清培养时明显高于1%胎牛血清,2、4、8h显著性差异(P<0.05)。②不同浓度(10%.1%)胎牛血清孵育2、4、8hTAL蛋白表达水平无显著性差异。③磷酸化消化后TAL活性,与消化前相比呈显著性下降。结论:人肝癌细胞株SMMC-7721中增加血清浓度明显提高TAL活性,酶活性变化与TAL蛋白表达水平无关,提示活性调节发生在蛋白翻译后修饰,其中磷酸化起重要作用。     

Pre-hydrolysis improves utilisation of dietary protein in the larval teleost Atlantic halibut (Hippoglossus hippoglossus L.)

A protein preparation labelled by incorporation of [U]¹⁴C-AA was hydrolysed to various degrees and administered to a teleost fish larva (Atlantic halibut, Hippoglossus hippoglossus L.) by tube-feeding, and its post-administration utilisation was studied. Three treatments were prepared: IntP—intact protein, PHP—pepsin-hydrolysed protein, and HHP—highly hydrolysed protein (using pepsin, trypsin, endoproteinase Glu-C, Asp-N, and Pro-C). At small doses (11.4±1.5 μg larvae⁻¹), the intact protein (IntP) was digested and absorbed to 36±5.5%. However, the relative absorption efficiency of the intact protein was reduced as the dose increased. Absorption efficiency was higher when the protein was hydrolysed prior to feeding the larvae and was constant at 63% (R²=98) independent of degree of proteolysis and dose (ranging from 3.5 to 35 μg larvae⁻¹). The initial absorption rate increased with the degree of hydrolysis. Calculations based on data collected during the first 30-120 min show that the absorption of PHP and HHP into extra-intestinal body tissues was 2.2 and 3 times as fast, respectively, as that of intact protein. However, the rates of absorption did not influence the distribution of absorbed AA into either catabolism or anabolism, as all larvae, independent of which protein solution they were given, catabolised 42±7% of the absorbed AA, and accumulated 49±6% into the body tissue, during the 20 h post-feeding incubation period.Larval age and size did not influence the absorption and utilisation of the hydrolysed protein preparations. This was different from the intact protein, as significantly higher fractions of the intact protein were absorbed by the larvae at 31 days past first feeding (dpff) than by larvae at 25 dpff. Analysis of the faecal evacuation suggested that the poor protein utilisation in the younger larvae was due to enhanced faecal evacuation, which in some larvae was more than 50% only 4 h post diet administration, at a time when the process of digestion and absorption was far from complete. This indicated that faecal evacuation is a critical factor in the utilisation of slowly digested and absorbed feed components, such as intact proteins, by fish larvae.     

Changes in the digestive gland of the loliginid squid Sepioteuthis lessoniana (Lesson 1830) associated with feeding

Changes associated with feeding in the histological and cytological structure of the digestive gland of the loliginid squid Sepioteuthis lessoniana were examined, along with the nature of both the intracellular and extracellular enzymes produced by the gland. The timing of the release of the extracellular enzymes during the digestive cycle was also determined using a quantitative experimental program. Like that of all coleoid cephalopods, the digestive gland was characterised by one type of cell with several functional stages. As is the case for other loliginid squids, however, the digestive cells did not contain the large enzyme-carrying boules that characterise the digestive glands of most cephalopods. Instead, smaller secretory granules were found in the digestive cells and these may be the enzyme carriers. The prominent rough endoplasmic reticulum, large mitochondria and active Golgi complexes present in the digestive cells are characteristic of cephalopods and indicate a high metabolic activity. Like that of other cephalopods, endocytotic absorption of nutrients and intracellular digestion occurs in the digestive gland of this squid. From quantitative and qualitative examinations of structural changes in the digestive gland of S. lessoniana after feeding, a schedule of its function during the course of digestion was proposed. This indicated that digestion was very rapid, being completed in as little as 4 h in S. lessoniana. Extracellular digestive enzymes were only released after the first hour following feeding, which implies that they are stored in the stomach between meals to increase digestive efficiency.     

Ultrasound-assisted digestion: a useful alternative in sample preparation

Ultrasound-assisted digestion is a promising alternative in the analysis of solid samples when either simple dissolution or direct analysis is not applicable. However, the field of application of ultrasonic sample digestion is still small in comparison with classical digestion alternatives and, particularly, with microwave-assisted digestion. This fact can be justified by the scant knowledge analytical chemists have about the advantages ultrasonic energy provides to digestion. Among these, the strict control at low temperatures of ultrasound applications allows the implementation of ultrasonic-assisted steps in biochemical analyses. In this connection, two specific biological applications, ultrasonic enzymatic digestion and assistance of ultrasound for cell disruption, are also reviewed.     

Digestive responses of two omnivorous rodents (<Emphasis Type="Italic">Peromyscus maniculatus</Emphasis> and <Emphasis Type="Italic">P. alstoni</Emphasis>) feeding on epigeous fungus (<Emphasis Type="Italic">Russula occidentalis</Emphasis>)

The sporocarps of hypogeous and epigeous fungi are important dietary items for forest dwelling rodents in temperate and tropical forests throughout the world. However, results of some pioneering works have demonstrated that fungi cannot be considered as nutritionally high-quality food items for some mycophagous small rodents. According to these studies, when mycophagous rodents feed on fungus, they showed a minimal digestibility, but whether this applies to most rodent species that include fungi in their diets is unknown. In this study, we experimentally evaluated body mass changes and feed preferences in captive deer (Peromyscus maniculatus) and volcano (P. alstoni) mice when fed on epigeous fungus (Russula occidentalis). In experiment 1, the animals were fed with fungus as the only feedstuff in comparison to regular rodent chow and oat. In experiment 2, the animals were fed with fungus in a free-choice arrangement together with equal amounts of rodent chow and oat. Both species lost ∼15% of their body mass within 4 days when fed on fungus alone, but gained 5–10% body mass during the same time period when ingesting oat and rodent chow, respectively, as the only feedstuff. However, in contrast, in the free-choice arrangement with all three feedstuffs, both species gained 20–30% body mass, and showed the highest feed preference for fungus followed by oat and rodent chow. In addition, apparent digestibility of energy and nitrogen were analyzed in both rodent species, which were 50–60% for fungus, whereas approximately 90–94% for rodent chow and oat. According to our results, animals need to supplement their diets with alternative high-quality food items in order to maintain and increase their body mass, suggesting that epigeous fungi are only of moderate nutritional value for small rodents. Futures studies should focus on exploring the importance of a mixture of fungal species in the diet of small mycophagous rodents.