Survival, Physiology, and Lysis of Lactococcus lactis in the Digestive Tract

The survival and the physiology of lactococcal cells in the different compartments of the digestive tracts of rats were studied in order to know better the fate of ingested lactic acid bacteria after oral administration. For this purpose, we used strains marked with reporter genes, the luxA-luxB gene of Vibrio harveyi and the gfp gene of Aequora victoria, that allowed us to differentiate the inoculated bacteria from food and the other intestinal bacteria. Luciferase was chosen to measure the metabolic activity of Lactococcus lactis in the digestive tract because it requires NADH, which is available only in metabolically active cells. The green fluorescent protein was used to assess the bacterial lysis independently of death. We report not only that specific factors affect the cell viability and integrity in some digestive tract compartments but also that the way bacteria are administrated has a dramatic impact. Lactococci which transit with the diet are quite resistant to gastric acidity (90 to 98% survival). In contrast, only 10 to 30% of bacteria survive in the duodenum. Viable cells are metabolically active in each compartment of the digestive tract, whereas most dead cells appear to be subject to rapid lysis. This property suggests that lactococci could be used as a vector to deliver specifically into the duodenum the proteins produced in the cytoplasm. This type of delivery vector would be particularly appropriate for targeting digestive enzymes such as lipase to treat pancreatic deficiencies.     

Genetically engineered yeasts as a new delivery vehicle of active compounds to the digestive tract: In vivo validation of the concept in the rat

An innovative “biodrug” concept based on oral administration of living recombinant microorganisms as a vehicle to deliver active compounds directly into the digestive tract has recently been developed. To validate this concept, we studied a recombinant Saccharomyces cerevisiae strain in order to investigate its viability and its ability to produce a protein (glutathione-S-transferase (GST)-V₅H₆) in the rat. Following oral administration, the recombinant yeast showed a survival rate of around 40% in the upper parts of the digestive tract, but was more sensitive to the conditions in the large intestinal, where viability dropped to 1%. Western blot analysis was able to detect the model protein throughout the digestive tract, including stomach, duodenum, jejunum (proximal, median and distal), ileum, cecum and colon. The gastrointestinal sac technique was employed to quantify GST-V₅H₆ in all the digestive compartments. These results suggest that S. cerevisiae may represent a useful host for producing compounds of interest directly in the digestive tract.     

hGH消化道途径转基因的研究

消化道途径转基因过程方便、快捷、易适应,可为基因治疗提供全新的模式。为了研究人 生长激素(bGH)基因的经消化道途径转基因过程,实验首先应用ECHO克隆系统。在供载体 pUni-hGH和宿主载体pcDNA4／TO-E的基础上,构建出hGH的哺乳动物表达载体pcDNA4-hGH;然 后结合酿酒酵母表达载体pESC-URA,构建出hGH的酵母-哺乳动物穿梭栽体pESC-CMV-hGH,测 序鉴定后转化酿酒酵母。用阳性重组酵母对小鼠进行灌胃免疫实验,间接ELISA方法在实验组 小鼠的血清中检测到抗hGH抗体的存在。结果证实hGH基因可通过消化道途径转进小鼠体细 胞并进行表达,初步证明了hGH的消化道基因治疗的可行性。     

凹耳蛙消化道组织学和嗜银细胞形态观察

为了揭示凹耳蛙(Odorrana tormota)消化道的基本特征,运用石蜡切片法和龙桂开银浸法对凹耳蛙消化道组织学结构及嗜银细胞的形态与分布密度进行了观察。结果显示:①凹耳蛙的胃壁具明显的纵行皱襞和胃小凹,胃腺发达,小肠可分为十二指肠和回肠,杯状细胞分散在十二指肠上皮细胞之间,十二指肠中未见十二指肠腺分布。②凹耳蛙嗜银细胞见于消化道全长,呈毛笔头样、锥体形、梭形、椭圆形和长条形等;幽门腺上皮和十二指肠绒毛上皮中的嗜银细胞具指向腺泡腔或肠腔的突起,提示其可能具有腔分泌的功能。嗜银细胞的分布密度胃幽门部最高,十二指肠和胃体其次,食道最低。据此认为胃既是凹耳蛙的主要消化器官,也是消化道中主要的内分泌器官;十二指肠是凹耳蛙消化道中的主要吸收部位,同时也具有内分泌功能;消化道嗜银细胞具有内分泌的功能,还可能具有腔分泌的功能。     

八肽胆囊收缩素在猪,大鼠和豚鼠肠道的免疫组织化学定位和分布

实验采用ＡＢＣ免疫组织化学方法研究了八肽胆囊收缩素样免疫反应物质在猪、大鼠和豚鼠肠道的定位与分布,并用相邻切片免疫双标记法观察了它与５－羟色胺的关系,结果表明,ＣＣＫ－８－ＩＲ细胞主要位于肠腺的底部,少数位于绒毛上皮。节段性分布上,在猪可见于从十二指肠到结肠全长的粘膜,大鼠和豚鼠ＣＣＫ－８－ＩＲ细胞则见于从十二指肠至回肠的粘膜,但均以十二指肠密度最高;免疫双标记法证实,在三种动物肠道中均有ＣＣＫ＝     

Distribution of endocrine cells in the digestive tract of Alligator sinensis during the active and hibernating period

The digestive tract is the largest endocrine organ in the body; the distribution pattern of endocrine cells varies with different pathological and physiological states. The aim of the present study was to investigate the distributed density of 5-hydroxytryptamine (5-HT), gastrin (GAS), somatostatin (SS) and vasoactive intestinal peptide (VIP) immunoreactive (IR) cells in the digestive tract of Alligator sinensis during the active and hibernating period by immunohistochemical (IHC) method. The results indicated that 5-HT-IR cells were distributed throughout the entire digestive tract, which were most predominant in duodenum and jejunum. The density increased significantly in stomach and duodenum during hibernation. GAS-IR cells were limited in small stomach and small intestine. The density decreased significantly in small stomach during hibernation, while increased in duodenum. What's more, most of the endocrine cells in duodenum were generally spindle shaped with long cytoplasmic processes ending in the lumen during hibernation. SS-IR cells were limited in stomach and small stomach. The density increased in stomach while decreased in small stomach during hibernation, meanwhile, fewer IR cells occurred in small intestine. VIP-IR cells occurred in stomach and small stomach. The density decreased in small stomach, while increased in stomach during hibernation. These results indicated that the endocrine cells in different parts of digestive tract varied differently during hibernation, their changes were adaptive response to the hibernation.     

Recombinant Saccharomyces cerevisiae expressing P450 in artificial digestive systems: a model for biodetoxication in the human digestive environment

The use of genetically engineered microorganisms such as bacteria or yeasts as live vehicles to carry out bioconversion directly in the digestive environment is an important challenge for the development of innovative biodrugs. A system that mimics the human gastrointestinal tract was combined with a computer simulation to evaluate the survival rate and cinnamate 4-hydroxylase activity of a recombinant model of Saccharomyces cerevisiae expressing the plant P450 73A1. The yeasts showed a high level of resistance to gastric and small intestinal secretions (survival rate after 4 h of digestion, 95.6% +/- 10.1% [n = 4]) but were more sensitive to the colonic conditions (survival rate after 4 h of incubation, 35.9% +/- 2.7% [n = 3]). For the first time, the ability of recombinant S. cerevisiae to carry out a bioconversion reaction has been demonstrated throughout the gastrointestinal tract. In the gastric-small intestinal system, 41.0% +/- 5.8% (n = 3) of the ingested trans-cinnamic acid was converted into p-coumaric acid after 4 h of digestion, as well as 8.9% +/- 1.6% (n = 3) in the stomach, 13.8% +/- 3.3% (n = 3) in the duodenum, 11.8% +/- 3.4% (n = 3) in the jejunum, and 6.5% +/- 1.0% (n = 3) in the ileum. In the large intestinal system, cinnamate 4-hydroxylase activity was detected but was too weak to be quantified. These results suggest that S. cerevisiae may afford a useful host for the development of biodrugs and may provide an innovative system for the prevention or treatment of diseases that escape classical drug action. In particular, yeasts may provide a suitable vector for biodetoxication in the digestive environment.     

黑龙江林蛙冬眠和非冬眠消化道内分泌细胞的比较研究

目的应用5-羟色胺(5-HT)、生长抑素(SS)、胃泌素(GAS)、胰高血糖素(Glu)、胰多肽(PP)和P-物质(SP)6种特异性抗血清,对黑龙江林蛙冬眠期和非冬眠期消化道内分泌细胞进行检测。方法应用卵白素-生物素-过氧化物酶复合物(avidin-biotin-peroxidase complex,ABC)免疫组织化学法。结果在冬眠期和非冬眠期黑龙江林蛙消化道内检测到5-HT细胞、SS细胞和Glu细胞,而GAS细胞、PP细胞和SP细胞均未检测到。5-HT细胞分布广泛,存在于黑龙江林蛙冬眠期和非冬眠期从食管到直肠的整个消化道中,在冬眠期分布密度高峰为胃幽门部和十二指肠,而非冬眠期则为胃幽门部;SS细胞分布也较为广泛,在黑龙江林蛙冬眠期和非冬眠期消化道中,除了直肠以外,均有分布,且这两个时期SS细胞的分布密度高峰均在胃幽门部。Glu细胞在冬眠期仅在胃贲门部、胃体、空肠和回肠检测到,而在非冬眠期除以上四个部位外直肠部也检测到了Glu细胞,并且Glu细胞分布密度高峰都在胃体部。结论黑龙江林蛙在冬眠期和非冬眠期消化道中内分泌细胞分布型的不同,可能与机体适应不同时期的生理功能有关。     

扬子鳄消化道内分泌细胞的免疫组织化学研究

应用７种特异性胃肠激素抗血清对扬子鳄消化道内分泌细胞进行了免疫组织化学定位。５－羟色胺细胞在消化道各段都有分布,以十二指肠密度最高,食道、直肠其次。生长抑素细胞在胃幽门部非常密集,胃体中等,胃贲门部较少,十二指肠偶见。胃泌素细胞主要分布于十二指肠前段,空肠、回肠和直肠偶见。许多血管活性肠肽细胞分布于胃贲门部,胃体和胃幽门部少数。胰高血糖素、胰多肽和Ｐ－物质在消化道各段均未检出阳性细胞。结合扬子鳄的     

Distribution and morphology of argyrophilic cells in the digestive tract of the African ostrich

We used light microscopy to elucidate the morphological features of argyrophilic cells in the digestive tract of the African ostrich (Struthio camelus). The results indicated that argyrophilic cells were found to be distributed among the epithelial cells of the mucosa or glands throughout the digestive tract, except for the esophagus; two types of argyrophilic cells were found; i.e., closed-type cells and cells with triangular or elongated shapes and with their apical cytoplasmic process in contact with the lumen (open-type cells); the greatest number of argyrophilic cells was found in the proventriculus, and the argyrophilic cell density gradually decreased from the proventriculus to the rectum; Furthermore, the number of argyrophilic cells in the duodenum and ileum was higher than that in the jejunum. This text still combined the characteristics that the argyrophilic cells in digestive tract of ostrich maybe related to different digestive function of different region and the basis of their morphology to carry on a discussion. It was speculated that argyrophilic cells in the digestive tract may have both endocrine and exocrine functions.     

Passage of the intravenously administered 15N urea into the digestive tract and its excretion in the sheep.

The experiments performed on two wethers provided with simple rumen cannulas and reentrant cannulas, inserted into the proximal duodenum and ileum, showed a passage of 15N from labelled urea, injected intravenously, from the blood to the digestive tract. The amount of the 15N in the digesta was the highest in duodenum, slightly lower in the rumen and slightly lower in ileum. Approximately 50% of the injected 15N was excreted in urine. The amount of the 15N eliminated with feces was very small; 0.6 to 2.8% of the dose injected per day. About 73--84% of the 15N which passed the duodenum was absorbed in further parts of the digestive tract. It can be concluded that all parts of the digestive tract take part in utilization of the endogenous urea.     

Large differences in the fraction of active bacteria in plankton,sediments, and biofilm

Generally, only a small fraction of free-living pelagic bacteria are metabolically active, while particle-associated bacteria usually exhibit a larger proportion of active bacteria. Most previous studies on the active fraction of bacteria focus on planktonic communities, and there are only a few studies on sediment and epiphytic biofilm bacteria. We compared the active fraction of the total number of bacteria in three different habitats of the littoral zone of Lake Erken, Sweden, including the sediments, the epiphytic biofilm on the submerged macrophyte Ranunculus circinatus, and the water column. Active bacteria were detected as those with an active electron transport system, identified by the capacity to reduce the tetrazolium salt CTC (5-cyano-2,3-ditolyltetrazolium chloride) into its fluorescent, water insoluble state. There were large differences between habitats. The active fraction of the total number of bacteria detected by fluorescence microscopy (annual mean +/- SD) in the sediments was 46 +/- 10%, on R. circinatus 37 +/- 18%, and in the water column 4 +/- 4%. The abundance of CTC-reducing cells was correlated with total bacterial abundance, and the fraction of CTC-reducing bacteria generally increased with total bacterial abundance, for all the habitats. Consequently, the difference in the fraction of CTC-reducing bacteria between the habitats could be attributed to different densities of bacteria, with a larger proportion of active bacteria at higher bacterial densities.     

Biodiversity of active and inactive bacteria in the gut flora of wood-feeding huhu beetle larvae (Prionoplus reticularis)

Huhu grubs (Prionoplus reticularis) are wood-feeding beetle larvae endemic to New Zealand and belonging to the family Cerambycidae. Compared to the wood-feeding lower termites, very little is known about the diversity and activity of microorganisms associated with xylophagous cerambycid larvae. To address this, we used pyrosequencing to evaluate the diversity of metabolically active and inactive bacteria in the huhu larval gut. Our estimate, that the gut harbors at least 1,800 phylotypes, is based on 33,420 sequences amplified from genomic DNA and reverse-transcribed RNA. Analysis of genomic DNA- and RNA-derived data sets revealed that 71% of all phylotypes (representing 95% of all sequences) were metabolically active. Rare phylotypes contributed considerably to the richness of the community and were also largely metabolically active, indicating their participation in digestive processes in the gut. The dominant families in the active community (RNA data set) included Acidobacteriaceae (24.3%), Xanthomonadaceae (16.7%), Acetobacteraceae (15.8%), Burkholderiaceae (8.7%), and Enterobacteriaceae (4.1%). The most abundant phylotype comprised 14% of the active community and affiliated with Dyella ginsengisoli (Gammaproteobacteria), suggesting that a Dyella-related organism is a likely symbiont. This study provides new information on the diversity and activity of gut-associated microorganisms that are essential for the digestion of the nutritionally poor diet consumed by wood-feeding larvae. Many huhu gut phylotypes affiliated with insect symbionts or with bacteria present in acidic environments or associated with fungi.     

无蹼壁虎消化道5-羟色胺细胞的免疫组织化学定位

目的研究无蹼壁虎(Gekkoswinhonis)消化道内5-HT免疫反应阳性内分泌细胞的分布以及形态。方法应用显示5-HT的免疫组织化学方法。结果5-HT阳性细胞在无蹼壁虎消化道各段均有分布,其中以十二指肠部位分布密度最多,食道和直肠其次,回肠部位分布密度最低。消化道各段5-HT细胞形态多样,有圆形、椭圆形、梭形、不规则形,其中梭形细胞多具有胞突。结论无蹼壁虎的消化道5-HT细胞的分布、形态与其功能相适应。     

Chitinolytic enzymes and the bacterial microflora in the digestive tract of cod, Gadus morhua

The levels of chitinolytic enzymes and chitinolytic bacteria in the digestive tract of feeding and fasting cod were compared. Enzyme activities within a given tissue were of a similar order irrespective of the presence of chitinolytic bacteria and/or chitin. Cod chitinase and chitobiase are therefore endogenous and constitutive enzymes. Fasting cod had similar numbers of bacteria within the gastro-intestinal compartments as feeding fish. Representative bacterial isolates from fasting fish were characterized.     

东方铃蟾消化道组织学的初步研究

采用组织学方法对东方铃蟾的消化道进行了研究。结果表明:肠分为十二指肠、空肠和大肠。消化道管壁由粘膜层、粘膜下层、肌层和浆膜层构成。食道、胃和肠均为单层柱状上皮。胃和十二指肠的粘膜皱褶最丰富。食道腺为复泡状腺,胃腺属于单管状腺,肠的各段无多细胞腺体,但空肠和大肠有丰富的杯状细胞。肌层均为平滑肌,内层环肌较厚,外侧纵肌较薄,其中大肠的外侧纵肌最发达。     

Proteomic investigation of the adaptation of Lactococcus lactis to the mouse digestive tract

Lactic acid bacteria are used on an industrial scale for the manufacturing of dairy products. It is now intended to develop novel applications of lactic acid bacteria that could be used as living vehicles for the targeting of antigens or therapeutics to the digestive mucosa. The aim of this study was to analyze the adaptations of Lactococcus lactis, a model lactic acid bacteria to the digestive tract and to identify functions required for colonization of the intestine. For this purpose, we combined gnotobiology with proteomics: axenic mice were colonized with a dairy L. lactis strain and the bacterial proteome was examined by 2-DE. As compared to cultures in broth, the proteome profile of bacteria grown in the intestine indicates the activation of metabolic pathways involved in various carbon sources assimilation and suggests the adoption of a mixed acids fermentative metabolism. We identified the product of the ywcC gene as essential for the colonization of the digestive tract and demonstrated that the corresponding gene product (YwcC) possesses a phosphogluconolactonase activity, suggesting an important role of the pentose phosphate pathway for the development of L. lactis in the digestive environment.     

Postnatal development of intra-epithelial leukocytes in the chicken digestive tract: phenotypical characterization in situ

In the present study, we characterized intra-epithelial leukocytes in the digestive tract of chickens during postnatal development. Their phenotype was characterized by monoclonal antibodies in cryostat sections and the numbers of the different cell-types were counted in the epithelium of the esophagus, proventriculus, duodenum, jejunum, cecum, and colon. All intra-epithelial leukocytes bore the leukocyte-common antigen CD45; 35% were T lymphocytes, and 50% bore a B-cell marker. However, no immunoglobulin-bearing cells were detected in the epithelium. Monocytes and macrophages were found only in the epithelium of the esophagus. A remaining population of non-B, non-T, non-monocyte cells (15%) was present in all parts of the digestive tract. The number of intra-epithelial leukocytes was greatest in the duodenum and jejunum, and decreased in the proximal part of the cecum and in the colon. Intra-epithelial leukocytes were only sporadically detected in the proventriculus. The total number of intra-epithelial leukocytes increased until 8 weeks after hatching and then decreased at 18 months. In the esophagus, the total number of intra-epithelial leukocytes changed little during aging. We found that the intra-epithelial leukocytes of chickens and rodents are distinct in that chicken intra-epithelial leukocytes comprise a cell population that bears a B-cell antigen but that lacks surface immunoglobulins.     

东方蝾螈消化道5-羟色胺免疫活性细胞的分布与形态学观察,前插2

采用ABC免疫组织化学方法对东方蝾螈Cynops orientalis消化道内5-羟色胺免疫活性内分泌细胞的分布及形态进行观察.结果显示,5-羟色胺(5-HT)细胞从食管到直肠各段均有分布,其中十二指肠的分布密度最高,空肠次之,幽门和直肠部最低.5-HT细胞形态多样呈圆形、锥体形和梭形等,根据其形态认为东方蝾螈消化道5-HT免疫活性细胞具有内分泌、外分泌两种功能.