In vivo activity of nifurzide and nifuroxazide in intestinal bacteria in man and gnotobiotic mice

Although Gram-negative enteropathogenic bacteria are the target strains of nifurzide and nifuroxazide treatments, neither drug affected faecal counts of in vitro-susceptible Enterobacteriaceae in healthy volunteers. This absence of activity was shown to be due to the poor solubility of the drugs tested. Therefore, effect of high doses of nifurzide was investigated in gnotobiotic mice. Activity against in vitro susceptible enteropathogens was then observed. Normal bacterial cells were replaced in the faeces by elongated, nonseptate and unflagellated mutants. Moreover, the resistance to colonization by enterotoxigenic Escherichia coli and Shigella flexeri of an anaerobic flora of human origin was sharply decreased.     

Inhibition of conjugal transfer of R plasmids by nitrofurans

Nifurzide is a nitrofuran with antibacterial activity. As nitrofurans have been reported to interact with DNA, we tested the ability of nifurzide to inhibit plasmid transfer. Inhibition of plasmid transfer between Escherichia coli strains was obtained for ten plasmids belonging to nine incompatibility groups. The same effect was observed when plasmid RP4 was harboured in six different members of the Enterobacteriaceae. Inhibition depended on the reduction of the -NO2 group of nifurzide and was obtained with four other nitrofuran derivatives.     

Effects of cryopreservation on glucose metabolism and survival of bovine morulae and blastocysts derived in vitro or in vivo

Bovine morulae and blastocysts were either produced in vitro through maturation, fertilization and culture of immature oocytes recovered from slaughterhouse-derived ovaries, collected in vivo or obtained after 24 h in vitro culture of in vivo collected embryos. The morulae and blastocysts were classified into four categories of embryo quality and two stages of embryonic development. Embryos were frozen by a controlled freezing method using 10% glycerol as a cryoprotectant. The ability of individual embryos to withstand freeze/thawing was measured immediately before and after cryopreservation by changes in CO2 production from (U-14C)glucose during a 2 h incubation period in a non-invasive closed system immediately before and after cryopreservation. Post-thaw survival was assessed by development in vitro during a 48 h culture period. Survival rates and oxidative metabolism after freeze/thawing were similar in embryos of the two developmental stages. However, after freeze/thawing, the rate of CO2 production of in vitro produced embryos was reduced to one half of their pre-freeze levels and was associated with poor survival rates. In vivo collected embryos had a significantly better tolerance to freezing and higher survival rates. However, when in vivo embryos were exposed to in vitro culture conditions, the rates of CO2 production and survival were significantly reduced. Pre-freeze embryo quality affected post-thaw in vitro development and metabolic activity markedly in embryos produced in vitro or pre-exposed to in vitro culture conditions. While there was no relationship between pre-freeze levels of CO2 production and post-thaw in vitro embryo development, all embryos which developed in vitro after freezing/thawing retained at least 58% of the pre-freeze levels of CO2 production regardless of their origin. Results of the present study indicate that embryos produced in vitro or pre-exposed to in vitro culture conditions are more sensitive to cryo-injury. This sensitivity is affected by embryo quality and is similarly reflected at the biochemical level. Determination of oxidative metabolism offers a feasibility for selection of viable morulae/blastocysts after freezing/thawing.     

In vivo and in vitro correlation of trachealis muscle contraction in dogs.

Maximal trachealis muscle shortening in vivo was compared with that in vitro in seven anesthetized dogs. In addition, the effect of graded elastic loads on the muscle was evaluated in vitro. In vivo trachealis muscle shortening, as measured using sonomicrometry, revealed maximal active shortening to be 28.8 +/- 11.7% (SD) of initial length. Trachealis muscle preparations from the same animals were studied in vitro to evaluate isometric force generation, isotonic shortening, and the effect of applying linear elastic loads to the trachealis muscle during contraction from optimal length. Maximal isotonic shortening was 66.8 +/- 8.4% of optimal length in vitro. Increasing elastic loads decreased active shortening and velocity of shortening in vitro in a hyperbolic manner. The elastic load required to decrease in vitro shortening to the extent of the shortening observed in vivo was similar to the estimated load provided by the tracheal cartilage. We conclude that decreased active shortening in vivo is primarily due to the elastic afterload provided by cartilage.     

Comparison of the frequencies of sister chromatid exchanges induced by thiophosphamide in vivo and in vitro

The effects of thiophosphamide on rabbit lymphocytes were studied and compared in vivo and in vitro. In the course of in vitro action the dose was calculated by multiplying the thiophosphamide concentration by the time of in vitro treatment. In vivo the dose was calculated as integral of concentration variation function of thiophosphamide from the time of injection till the time of blood collection. It was demonstrated that as the dose was raised, the rate of sister chromatid exchanges increased linearly in vivo and in vitro. The coefficients of linear regressions were found to coincide. The data point to equal efficacy of thiophosphamide in vivo and in vitro.     

Frog oocyte glycogen synthase: enzyme regulation under in vitro and in vivo conditions

Frog oocyte glycogen synthase properties differ significantly under in vitro or in vivo conditions. The K(mapp) for UDP-glucose in vivo was 1.4mM (in the presence or absence of glucose-6-P). The in vitro value was 6mM and was reduced by glucose-6-P to 0.8mM. Under both conditions (in vitro and in vivo) V(max) was 0.2 m Units per oocyte in the absence of glucose-6-P. V(max) in vivo was stimulated 2-fold by glucose-6-P, whereas, in vitro, a 10-fold increase was obtained. Glucose-6-P required for 50% activation in vivo was 15 microM and, depending on substrate concentrations, 50-100 microM in vitro. The prevailing enzyme obtained in vitro was the glucose-6-P-dependent form, which may be converted to the independent species by dephosphorylation. This transformation could not be observed in vivo. We suggest that enzyme activation by glucose-6-P in vivo is due to allosteric effects rather than to dephosphorylation of the enzyme. Regulatory mechanisms other than allosteric activation and covalent phosphorylation are discussed.     

Detection of activated O2 species in vitro and in rat lungs by chemiluminescence

This study used chemiluminescence, an "on-line" photon-counting technique, to detect and characterize activated O2 species in vitro and in isolated rat lungs. The sensitivity and specificity of enhanced chemiluminescence for superoxide anion (O2-.) and hydrogen peroxide (H2O2) was evaluated in vitro. The effect of media conditions (such as O2 tension, albumin concentration, and sulfhydryl group availability) on luminescence was assessed in vitro. Xanthine-xanthine oxidase (X-XO) primarily produced superoxide anion in vitro. Enhanced chemiluminescence varied directly with the dose of luminescent probe used and the quantity of activated O2 species administered. The strength of the luminescent signal was also dependent on the concentration of albumin and O2 in the media. Lucigenin was more sensitive than luminol to the presence of O2-. and, unlike luminol, lucigenin did not alter radical production by XO. However, neither luminescent probe was specific for O2-., as both detected H2O2 and O2 in vitro. H2O2-induced chemiluminescence was inhibited by catalase but not superoxide dismutase (SOD), while X-XO-induced luminescence was inhibited by SOD but not catalase. SOD-inhibitable chemiluminescence was a sensitive and specific marker for O2-. production in vitro. Once the sensitivity-specificity of enhanced chemiluminescence was defined in vitro, this technique was used to explore the mechanism by which exogenous X-XO reduced hypoxic vasoconstriction in isolated rat lungs. The vascular paresis, caused by administration of X-XO to the rat lung, resulted from a brief burst of O2-. production rather than a sustained alteration of lung radical levels.     

Effects of thyrotropin and thyroid hormones in vivo on thyroid responsiveness to thyrotropin in vitro.

The thyroid gland of rats fed propylthiouracil is known to be unresponsive in vitro to thyrotropin; to investigate further the underlying mechanism groups of rats were variously treated with propylthiouracil and thyroid hormone or subjected to hypophysectomy. In vitro responsiveness of the thyroids was tested by measuring an increase in the concentration of c AMP when thyrotropin or prostaglandin E1 was added to the medium. Results showed that responsiveness to thyrotropin partially returned with rats fed prophylthiouracil and hypophysectomized 5, but not 2, days before death; hypophysectomy of normal rats led to increased in vitro responsiveness to thyrotropin and this was partially reversed by injections of thyrotropin for a week before death. Administration of thyroid hormone had little effect in these investigations and in vitro responsiveness to prostaglanding E1 was not consistently influenced by any of the in vivo regimens. From this experience we conclude that, at least as studied in vitro, circulating thyrotropin has a significant role in modulating responsiveness of the thyroid to thyrotropin.     

Collagen in developing chick muscle in vivo and in vitro

Because of the known role of collagen in chick skeletal muscle differentiation the collagen synthesized by embryonic chick muscle was studied. The major collagen synthesized by this muscle was found to be type I collagen. In addition, the effectiveness of types I, II, III and IV collagens in promoting myoblast fusion in vitro was compared. These collagens were found to be equally effective as in vitro substrates.     

Palmitate oxidation by in vivo and in vitro grown Mycobacterium lepraemurium.

Oxidation of palmitate by Mycobacterium lepraemurium isolated from C3H mice lepromata (in vivo) and also grown on Ogawa egg-yolk medium (in vitro) was investigated. Palmitate was found to be oxidized, after a lag period of about 8 h, by both the in vivo and in vitro grown bacilli. Cell-free extracts prepared from in vivo and in vitro grown cells catalysed an active oxidation of palmitate after a lag period of 3-4 h. The amount of ATP increased, with the increase in time during oxidation of palmitate by the cell-free extracts. The generation of ATP was strongly inhibited by the inhibitors rotenone, antimycin A and cyanide as well as by the uncouplers 2,4-dinitrophenol and 2,6-dibromophenol. These results indicated that oxidation of palmitate by the in vivo and in vitro grown M. lepraemurium is mediated through the respiratory chain using oxygen as the terminal electron acceptor.     

体外受精和孤雌活化过程中小鼠胚胎细胞骨架的动态变化

为研究微管在体外受精与孤雌活化过程中的动态变化,本实验比较了体外受精胚胎、SrCl2激活的孤雌胚胎和体内受精的原核期胚胎在体外发育的情况,采用免疫荧光化学与激光共聚焦显微术检测卵母细胞孤雌活化过程中及体外受精后微管及核的动态变化,以分析微管在减数分裂过程中的作用及其对早期发育的影响.结果显示,体内受精胚胎的发育率显著高于体外受精和孤雌激活胚胎体外发育率(P<0.05),而体外受精与孤雌激活胚胎在各阶段发育率差异均不显著.在体外受精中,精子入卵,激活卵母细胞,减数分裂恢复,纺锤丝牵拉赤道板卜致密排列的母源染色体向纺锤体两侧迁移;后期将染色体拉向两极;末期时,微管分布于两组已去凝集的母源染色体之间,卵母细胞排出第二极体(the second polarbody,Pb2),解聚的母源染色体形成雌原核.同时,在受精后5～8 h精子染色质发生去浓缩与再浓缩,形成雄原核.在原核形成的同时,胞质星体在雌、雄原核的周围重组形成长的微管,负责雌、雄原核的迁移靠近.孤雌活化过程中,卵母细胞恢复减数分裂,姐妹染色单体分离,被拉向两极,经细胞松弛素B处理后,活化4～6 h,卵周隙中未见Pb2,而在胞质中出现两个混合的单倍体原核,之间由微管相连接,负责两个单倍体原核的迁移靠近.与体外受精相比较,孤雌活化时卵母细胞更容易被激活,减数分裂期间微管的发育早且更完善.     

Development and comparison of in vivo and in vitro models for endometritis in cows and mares

In order to investigate pathogenic mechanisms of acute endometritis in cows and mares, we established an in vivo model in both species. Based on the results of an in vitro transmigration system, human recombinant interleukin-8 (rhIL-8; 1.25 microg per mare and 5 microg per cow in 50 ml phosphate-buffered saline) was used to attract polymorphonuclear neutrophil granulocytes (PMNs) into the uteri. Peak numbers of uterine neutrophils were attracted after 6h, in both cows and mares. On average, mares responded more sensitively than cows, with 15 times higher numbers of rhIL-8-attracted uterine neutrophils (72+/-8 x 10(7)cells). In contrast to in vitro studies, in vivo migrated neutrophils (uterine neutrophils) of both species displayed a significantly reduced MHC class I expression. Expression of the CD11a molecule was significantly enhanced on equine uterine neutrophils but downregulated on bovine cells. Compared with untreated autologous peripheral neutrophils, both uterine and in vitro migrated neutrophils showed no alteration of phagocytic capacity. The ability to generate reactive oxygen species (ROS) was significantly upregulated in bovine and equine uterine neutrophils. This was also observed after in vitro migration of equine neutrophils, whereas ROS generation by bovine neutrophils was significantly depressed. In summary, the concept of inducing endometritis directly by local application of human interleukin-8 has been reliably successful in cows and mares. The model permits the analysis of PMN migration into the uterus under defined and controlled conditions. The observed differences between cows and mares with respect to phenotypical and functional characteristics of in vivo attracted uterine cells point to species-related features of neutrophil migration. In vitro transmigrated bovine and equine cells partially differ in phenotype and function from uterine neutrophils. Therefore, the in vitro transmigration assay cannot completely represent the in vivo endometritis model described here.     

Uptake and degradation of formaldehyde-treated 125I-labelled human serum albumin in rat liver cells in vivo and in vitro

The uptake of formaldehyde-treated 125I-labelled human serum albumin in rat hepatocytes and nonparenchymal liver cells was measured in vivo and in vitro. Isolated liver cells were prepared by treating the perfused liver with collagenase. Purified hepatocytes and nonparenchymal cells were obtained by differential centrifugation. Human serum albumin was found to be taken up exclusively or almost exclusively by nonparenchymal cells in vitro and in vivo (after intravenous injection). The maximal rate of human serum albumin-uptake in vitro was comparable to that in vivo. Nonparenchymal cells degraded human serum albumin in vitro as indicated by release of trichloroacetic acid-soluble radioactivity. Degradation started about 20-30 min after addition of human serum albumin to cells and rate of degradation was proportional to rate of uptake. Human serum albumin-degradation could be studied without interference of concurrent uptake by separating cells that had been preincubated with human serum albumin from the medium and then reincubating them with human serum albumin-free medium. The lag phase before human serum albumin-degradation starts and the inhibitory effect of chloroquine on degradation indicate that human serum albumin is degraded in lysosomes. The data obtained show that enzymatically prepared nonparenchymal liver cells retain their endocytic activity in vitro. Denatured human serum albumin should be useful both as a marker for rat liver macrophages and for the study of intracellular proteolysis in these cells.     

Comparative studies between in vivo and in vitro spermatogenesis of Japanese eel (Anguilla japonica)

In order to check the quality of in vitro spermatogenesis of Japanese eel, in vitrol 1-ketotestosterone (11-KT) induced spermatogenesis was compared with in vivo spermatogenesis induced by a single injection of human chorionic gonadotropin (hCG) in detail. DNA contents of germ cells from in vitro and in vivo testicular fragments were compared using flow cytometry. Since the in vitro result of flow cytometry showed prominent 1C peak including spermatozoa and spermatids, the reduction of DNA by meiosis was assumed to progress normally, (i.e., haploid spermatozoa were produced in this in vitro system). In the testes of in vitro culture, however, spermatozoa were not released into lumen. Furthermore, the number of mitotic divisions of the in vitro experiment (6 divisions) was fewer than that of in vivo (10 divisions). In electron microscopy observations, both of in vivo and in vitro spermatozoon had a crescent-shaped nucleus with a flagellum, and a single large spherical mitochondrion. However, the elongation of the sperm head was not sufficient and the mitochondrion was not always located at the anterior end as is observed for the spermatozoa obtained from hCG injected eels. Eel spermatogenesis related substance-11 (eSRS11) is homologue of histone H1 which is up-regulated during spermatogenesis. Using this probe, in vitro spermatogenesis was also evaluated in molecular levels. In Northern blot analysis, eSRS11 mRNA was detected in both in vivo and in vitro testes. However, the expression of in vitro was much weaker than that of in vivo. These differences indicate that the stimulation of 11-KT is not sufficient, and another factors are needed to induce complete spermatogenesis in vitro.     

Asynchronism of thymocyte development in vivo and in vitro

Although fetal thymus organ culture (FTOC) has become widely used to investigate T-cell development, the differences between thymocyte development in vivo and in vitro (in FTOC) remain largely unknown. In this study, the viability and numbers of thymocytes recovered from embryonic thymus lobes in different gestation days (gd) mice or from 15 day embryonic thymus lobes cultured for different days in FTOC system were evaluated. The expression of CD3, CD4, CD8, CD95 ligand (CD95L), and CD69 on thymocytes were analyzed by FACS. The results showed that thymocytes, either in vivo or in vitro, could differentiate from double negative (DN) cells to double positive (DP) cells and to single positive (SP) cells. But the number of total thymocytes and the percentage of DP cells in vitro were less than that in vivo, and the expression of CD95L and CD69 on thymocytes in vitro was higher than that in vivo. Our results suggested that although thymocyte development in vitro could recapitulate thymic development in vivo, the proliferation of thymocytes in vitro was less intensive than that in vivo; the differentiation of thymocytes in vitro was delayed compared with that in vivo; and the apoptosis and activation of thymocytes in vitro were higher than that in vivo. In conclusion, FTOC is a useful system for the study of T cell differentiation, but it is necessary to interpret the results from in vitro studies carefully since the thymocyte development in vitro is asynchronous from that in vivo.     

Chronology of apoptosis in bovine embryos produced in vivo and in vitro

The postimplantation developmental potential of embryos can be affected by various forms of cell death, such as apoptosis, at preimplantation stages. However, correct assessment of apoptosis is needed for adequate inference of the developmental significance of this process. This study is the first to investigate the independent chronological occurrence of apoptotic changes in nuclear morphology and DNA degradation (detected by the TUNEL reaction) and incidences of nuclei displaying these features at various preimplantation stages of bovine embryos produced both in vivo and in vitro. Different elements of apoptosis were observed at various developmental stages and appeared to be differentially affected by in vitro production. Nuclear condensation was observed from the 6-cell stage in vitro and the 8-cell stage in vivo, whereas the TUNEL reaction was first observed at the 6-cell stage in vitro and the 21-cell stage in vivo. Morphological signs of other forms of cell death were also observed in normally developing embryos produced both in vivo and in vitro. The onset of apoptosis seems to be developmentally regulated in a stage-specific manner, but discrete features of the apoptotic process may be differentially regulated and independently modulated by the mode of embryo production. Significant differences in indices of various apoptotic features were not evident between in vivo- and in vitro-produced embryos at the morula stage, but such differences could be observed at the blastocyst stage, where in vitro production was associated with a higher degree of apoptosis in the inner cell mass.     

Macrophage activation in vivo and in vitro

Morphology, lysosomal enzyme activity and phagocytic ability were tested in peritoneal macrophage cultures after stimulation in vivo or in vitro with endotoxin, mineral oil or latex particles, and compared to the same parameters in normal peritoneal macrophages. Treatment with latex did not give changes in the parameters tested after in vivo or in vitro stimulation. In all other types of stimulation the cells displayed varying degrees of spreading and changes in granule content. Extensive ruffling of cell membrane was obvious in endotoxin-stimulated cells. The pattern of lysosomal enzyme activity was complex and depended on the means of stimulation. Acid phosphatase showed the greatest increase after both in vivo and in vitro stimulation, N-acetyl-glucosaminidase could not be increased in vitro. Internalization of opsonized red cells mediated by the Fc receptor increased after in vivo stimulation. No such change was observed after in vitro stimulation. Normal peritoneal macrophages do not internalize significantly via the C₃ receptor. In vivo stimulation triggered the capacity to internalize up to 45% of the attached red cells. A similar reaction was obtained in vitro when both endotoxin and FCS were added to the culture medium, but not when endotoxin or FCS were used alone. We conclude that the use of the term activation of macrophages should always be based on quantitative changes in well defined parameters. Changes in one parameter will not necessarily be accompanied by the whole range of biochemical and morphological perturbations. The capacity to ingest via the C₃ receptor may be the most useful parameter.     

Initiation and maintenance of in vitro decidualization are independent of hormonal sensitization in vivo.

The effects of in vivo hormonal sensitization on the competence of uterine stromal (US) cells to decidualize in vitro were assessed. In vitro differentiation of uterine stroma isolated from Day 4 pregnant rats, sensitized to respond to a decidual stimulus, was compared to that in nonsensitized immature, castrated or cycling rats. The initiation of in vitro decidualization--as monitored by the expression of the decidual markers desmin and laminin in rat US cells--was independent of the hormonal status of the animal from which the cells were isolated and occurred in the absence of serum in the medium. Differentiation was accelerated in high-density cultures where contact inhibition suppressed proliferation and decreased the extent of cell growth. The extent to which in vitro decidualization imitates in vivo stromal cell differentiation was assessed by comparing decidualization in the rabbit, a species with only a limited decidual cell response, and in the rat. US cells isolated from nonpregnant rabbits differentiated in vitro by expressing laminin, but not desmin. Indirect immunofluorescence of frozen uterine sections from pregnant and nonpregnant rabbits validated in vitro differentiation as a faithful reflection of the in vivo program of decidualization. Although the program of US cell differentiation may vary between the species, initiation of differentiation in vitro appeared to be independent of hormonal preparation in vivo for both the species examined.     

Different lipolytic effects of theophylline and dibutyryl cyclic AMP in vivo and in vitro.

Both dcAMP and theophylline are known to promote lipolysis in vitro by increasing intracellular cAMP. Although theophylline stimulates FFA mobilization in vivo as well, a report of low circulating FFA levels in the rat given dcAMP suggested that dcAMP may inhibit lipolysis in the intact animal. To explore this possibility, a comparison of the in vitro and in vivo lipolytic effects of theophylline and dcAMP was made in the young dog. Circulating glycerol and FFA levels rose following the administration of theophylline. While glycerol and FFA fell slightly in puppies given dcAMP, only the FFA change was significant. Epinephrine infusions given alone produced sustained elevations of glycerol and FFA. When theophylline was given in conjunction with ongoing epinephrine infusions, plasma glycerol and FFA levels remained high. On the other hand, epinephrine-stimulated lipolysis was markedly inhibited by dcAMP, as shown by pronounced falls of glycerol and FFA from the elevated levels found with epinephrine alone. In vitro studies involving fragments of puppy adipose tissue reveal that epinephrine, theophylline, and dcAMP promoted glycerol release. In contrast to the in vivo observations, lipolysis was also stimulated by combinations of both epinephrine and theophylline as well as by epinephrine and dcAMP. Thus, theophylline stimulates lipolysis in vitro and in vivo in the puppy. In contrast, dcAMP stimulates lipolysis in vitro but inhibits this action in the intact animal. This important difference in the two pharmacologic agents suggests the need for caution when using them in in vivo studies involving the action of cAMP.     

Successful capacitation and homologous fertilization in vitro in Calomys musculinus and Calomys laucha (Rodentia - sigmodontinae)

Small South American rodents of the genus Calomys have been used extensively for virology and ecological research. Previous studies have demonstrated that Calomys musculinus and Calomys laucha have a relatively short oestrous cycle and that superovulation and parthenogenetic activation can be induced. The purpose of this study was to determine the requirements for in vitro manipulation of the male gamete and in vitro fertilization. Two culture media and different concentrations of spermatozoa were tested for their ability to support sperm motility, hyperactivation and the acrosome reaction. The ability of capacitated Calomys spermatozoa to penetrate zona-free hamster eggs was also evaluated. In vitro fertilization was assessed by examining attachment and binding to the zona pellucida, second polar body extrusion, pronucleus formation and the fertilizing sperm tail. The results of the study showed that: (i) Tyrode's albumin lactate pyruvate (TALP) medium was more effective than T6 medium for maintaining sperm motility in vitro; (ii) hyperactivation was achieved with TALP but not with T6; (iii) the acrosome reaction was easily distinguished by light microscopy and depends on time and sperm concentration; (iv) capacitated spermatozoa are able to penetrate zona-free hamster eggs; and (v) superovulated oocytes can be fertilized in vitro. This is the first report of capacitation and in vitro fertilization for Calomys sp. These results provide opportunities to use C. musculinus and C. laucha as new laboratory animals for research into reproductive biology.