Influence of splenomegaly on aortic and liver parenchymal CT numbers during contrast-enhance CT in patients with cirrhosis.

INTRODUCTION: To compare CT (computed tomography) values for enhancement of the abdominal aorta and liver parenchyma during dynamic contrast enhancement (CE) CT in cirrhotic patients with and without splenomegaly (SM). METHODS: We considered 258 patients (83 males and 46 females for the splenomegaly group, and 83 males and 46 females for the control group) for this retrospective study. We measured CT values in the abdominal aorta and hepatic parenchyma during the hepatic arterial (HAP) and portal venous (PVP) phases. The aortic CE at HAP and the hepatic parenchymal CE at PVP were compared between the two groups. For success rate of scans, we also calculated the optimal CE rates (>280 HU in the abdominal aorta and >50 HU in the hepatic parenchyma) for each group. RESULTS: In the SM group, the CE for abdominal aorta was decreased during the aortic phase for a dynamic CE-CT (p < 0.05). When evaluating the success rates, they were found to be 65.1 % and 58.9 % in the SM group and 81.4 % and 72.3 % in the non-SM group (p < 0.05). CONCLUSION: The success rate of scans and CE for the abdominal aorta during the aortic phase exhibited a significant decrease during dynamic CE-CT scans on patients with SM. Patients with SM may have reduced diagnostic ability with typical contrast injection protocols. IMPLICATIONS FOR PRACTICE: It may be necessary to change the injection rates and contrast medium volume during CE-CT depending on the presence or absence of SM.

Comparison of formation of thiolactones and active metabolites of prasugrel and clopidogrel in rats and dogs.

Prasugrel and clopidogrel are antiplatelet prodrugs that are converted to their respective active metabolites through thiolactone intermediates. Prasugrel is rapidly hydrolysed by esterases to its thiolactone intermediate, while clopidogrel is oxidized by cytochrome P450 (CYP) isoforms to its thiolactone. The conversion of both thiolactones to the active metabolites is CYP mediated. This study compared the efficiency, in vivo, of the formation of prasugrel and clopidogrel thiolactones and their active metabolites. The areas under the plasma concentration versus time curve (AUC) of the thiolactone intermediates in the portal vein plasma after an oral dose of prasugrel (1 mg kg(-1)) and clopidogrel (0.77 mg kg(-1)) were 15.8 +/- 15.9 ng h ml(-1) and 0.113 +/- 0.226 ng h ml(-1), respectively, in rats, and 454 +/- 104 ng h ml(-1) and 23.3 +/- 4.3 ng h ml(-1), respectively, in dogs, indicating efficient hydrolysis of prasugrel and little metabolism of clopidogrel to their thiolactones in the intestine. The relative bioavailability of the active metabolites of prasugrel and clopidogrel calculated by the ratio of active metabolite AUC (prodrug oral administration/active metabolite intravenous administration) were 25% and 7%, respectively, in rats, and 25% and 10%, respectively, in dogs. Single intraduodenal administration of prasugrel showed complete conversion of prasugrel, resulting in high concentrations of the thiolactone and active metabolite of prasugrel in rat portal vein plasma, which demonstrates that these products are generated in the intestine during the absorption process. In conclusion, the extent of in vivo formation of the thiolactone and the active metabolite of prasugrel was greater than for clopidogrel's thiolactone and active metabolite.

Phage diversity in a methanogenic digester.

It has been shown that phages are present in natural and engineered ecosystems and influence the structure and performance of prokaryotic communities. However, little has been known about phages occurring in anaerobic ecosystems, including those in methanogenic digesters for waste treatment. This study investigated phages produced in an upflow anaerobic sludge blanket methanogenic digester treating brewery wastes. Phage-like particles (PLPs) in the influent and effluent of the digester were concentrated and purified by sequential filtration and quantified and characterized by transmission electron microscopy (TEM), fluorescence assay, and field inversion gel electrophoresis (FIGE). Results indicate that numbers of PLPs in the effluent of the digester exceeded 1 x 10(9) L-1 and at least 10 times greater than those in the influent, suggesting that substantial amounts of PLPs were produced in the digester. A production rate of the PLPs was estimated at least 5.2 x 10(7) PLPs day-1 L-1. TEM and FIGE showed that a variety of phages were produced in the digester, including those affiliated with Siphoviridae, Myoviridae, and Cystoviridae.

Multiple laryngeal foreign bodies composed of dried fish vertebral bones: a case report.

We present a rare case of tracheostomy for removal of laryngeal foreign bodies consisting of three connected fish vertebral bones in a 15-month-old girl. Recent endoscopic techniques have made it possible to extract nearly all tracheobronchial foreign bodies with rigid bronchoscopes. However, the three connected foreign bodies in this report could not be extracted entirely by single endoscopy because the glottis as an exit was narrow due to severe oedema. Accordingly, tracheostomy was required to assist ventilation, prevent prolonged post-operative endotracheal intubation, remove the secondary tracheal foreign bodies and to provide a conduit for the introduction of a bronchoscope. This suggests that tracheostomy should be considered to avoid the potential dangers of severe laryngeal oedema and to secure the route for removal of foreign bodies from the trachea when treating patients with multiple laryngeal foreign bodies and laryngeal oedema.

Effects of renal sorbitol accumulation on urinary excretion of enzymes in hyperglycaemic rats.

We studied the effects of epalrestat, a specific inhibitor of aldose reductase, on renal sorbitol accumulation and the resulting urinary enzyme excretion in hyperglycaemic rats. The activities of proximal tubule-derived enzymes such as N-acetyl-beta-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), gamma-glutamyltranspeptidase (GGT) and dipeptidyl aminopeptidase IV (DAPIV) in urine were determined in five groups of male Wistar rats (each n = 7): (a) 0.9% saline-loaded, (b) 10% glucose-loaded, (c) 10% glucose-loaded with epalrestat pretreatment, (d) 10% mannitol-loaded and (e) 10% mannitol-loaded with epalrestat pretreatment. Epalrestat was given mixed in chow at a dose of 50 mg/kg body weight. Urinary NAG, AAP, GGT and DAPIV activities were significantly increased (P<0.005, P<0.05, P<0.01, P<0.01, respectively) by the induction of hyperglycaemia. In contrast, enzyme excretion was not increased in the mannitol- or saline-loaded groups. Pre-treatment with epalrestat completely prevented the increased urinary excretion of NAG, AAP and GGT. At the end of the infusion study, renal cortical glucose concentrations of the glucose-loaded groups with and without epalrestat pretreatment were approximately fivefold higher than those of the mannitol- or saline-loaded groups (P<0.005 each). Renal cortical sorbitol concentrations of the glucose-loaded group was also approximately twofold higher than those of the mannitol- or saline-loaded groups (P<0.01 each). However, in the group that received both glucose and epalrestat, renal cortical sorbitol concentration was not increased. These results suggest that accumulation of intracellular sorbitol leads to proximal tubular cell dysfunction and abnormal enzymuria.

Development-inhibiting activity of some tropical plants against Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae).

Thirteen tropical plants were evaluated for development-inhibiting activity against Sitophilus zeamais. The bioassays were carried out by incorporating seeds or leaves at various dose levels into an artificial diet for the test insect. It was found that seeds of Basella alba and leaves of Operculina turpethum and Calotropis gigantea were potent in delaying development and in reducing adult emergence, and hence the capacity for population increase. At 0.5% concentration, adult emergence in tests with B. alba, O. turpethum and C. gigantea was reduced by 62, 95 and 70%, respectively. In B. alba and C. gigantea, the development periods were 2.2 and 1.8 times those in the control and the capacities for increase/day were only 0.0324 and 0.0328 compared with 0.1004 in the control. B. alba, O. turpethum and C. gigantea were active at concentrations as low as 0.01, 0.05 and 0.1%. The potential of these materials in insect pest management is discussed.

Tissue specific expression and chromosomal mapping of a human UDP-N-acetylglucosamine: alpha1,3-d-mannoside beta1, 4-N-acetylglucosaminyltransferase.

A human cDNA for UDP- N -acetylglucosamine:alpha1,3-d-mannoside beta1,4- N- acetylglucosaminyltransferase (GnT-IV) was isolated from a liver cDNA library using a probe based on a partial cDNA sequence of the bovine GnT-IV. The cDNA encoded a complete sequence of a type II membrane protein of 535 amino acids which is 96% identical to the bovine GnT-IV. Transient expression of the human cDNA in COS7 cells increased total cellular GnT-IV activity 25-fold, demonstrating that this cDNA encodes a functional human GnT-IV. Northern blot analysis of normal tissues indicated that at least five different sizes of mRNA (9.7, 7.6, 5.1, 3.8, and 2.4 kb) forGnT-IV are expressed in vivo. Furthermore, these mRNAs are expressed at different levels between tissues. Large amounts of mRNA were detected in tissues harboring T lineage cells. Also, the promyelocytic leukemia cell line HL-60 and the lymphoblastic leukemia cell line MOLT-4 revealed abundant mRNA. Lastly, the gene was mapped at the locus on human chromosome 2, band q12 by fluorescent in situ hybridization.

Suppressed impact of nitric oxide on renal arteriolar function in rats with chronic heart failure.

We performed experiments to test the hypothesis that experimental heart failure (HF) is associated with altered nitric oxide (NO)-dependent influences on the renal microvasculature, including diminished modulation of constrictor responses to ANG II. Eight to ten weeks after inducing HF in rats by coronary artery ligation, we administered enalaprilat to suppress ANG II synthesis and studied renal arteriolar function using the in vitro blood-perfused juxtamedullary nephron technique. In kidneys from sham-operated rats, NO synthase inhibition [100 microM Nomega-nitro-L-arginine (L-NNA)] reduced afferent arteriolar diameter by 4.1 +/- 0.6 microm and enhanced ANG II responsiveness (10 nM ANG II decreased afferent diameter by 10.1 +/- 1.4 micrometer before and 12.8 +/- 1.6 micrometer during L-NNA treatment; P < 0.05). In kidneys from HF rats, L-NNA did not alter afferent arteriolar baseline diameter or ANG II responsiveness (10 nM ANG II decreased diameter by 12.5 +/- 1.5 micrometer before and 12.5 +/- 2.3 micrometer during L-NNA). The effects of L-NNA on efferent arteriolar function were also abated in HF rats. In renal cortex of HF rats, NO synthase activity was decreased by 63% and superoxide dismutase activity was diminished by 39% relative to tissue from sham-operated rats. Urinary nitrate/nitrite excretion was also reduced in HF rats. Thus both diminished synthesis and augmented degradation are likely to contribute to a decreased renal microvascular impact of endogenous NO during chronic HF, the consequences of which include loss of NO-dependent modulation of ANG II-induced vasoconstriction.

Expression and characterization of rat UDP-N-acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is a useful host for the production of heterologous proteins through the secretory pathway. However, because of the potential antigenicity of mannan-type sugar chains in humans, yeast cannot be used as a host for the production of glycoprotein therapeutics. To overcome this problem, we are trying to breed a yeast which can produce hybrid- or complex-type carbohydrates. UDP- N- acetylglucosamine:alpha-3-d-mannoside beta-1, 2- N- acetylglucosaminyltransferase I (GnT-I) is essential for the conversion of high mannose-type N- glycans to hybrid- and complex-type ones. As yeast lacks this enzyme, we have introduced the rat GnT-I cDNA into yeast cells. The transformed yeast cells expressed GnT-I activity in vitro. The expressed GnT-I was localized in all organella, including the endoplasmic reticulum (ER), Golgi apparatus, and vacuole, suggesting that the mammalian Golgi retention signal of GnT-I did not function in yeast cells. Analysis of the GnT-I gene product with a c-Myc epitope tag at the C-terminus elucidates that the N - terminal region of GnT-I, including the mammalian Golgi retention signal, should be removed in the yeast ER.

Production of human compatible high mannose-type (Man5GlcNAc2) sugar chains in Saccharomyces cerevisiae.

A yeast mutant capable of producing Man5GlcNAc2 human compatible sugar chains on glycoproteins was constructed. An expression vector for alpha-1,2-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. An in vitro alpha-1,2-mannosidase assay and Western blot analysis showed that it was successfully localized in the endoplasmic reticulum. A triple mutant yeast lacking three glycosyltransferase activities was then transformed with an alpha-1, 2-mannosidase expression vector. The oligosaccharide structures of carboxypeptidase Y as well as cell surface glycoproteins were analyzed, and the recombinant yeast was shown to produce a series of high mannose-type sugar chains including Man5GlcNAc2. This is the first report of a recombinant S. cerevisiae able to produce Man5GlcNAc2-oligosaccharides, the intermediate for hybrid-type and complex-type sugar chains.

High density culture of immortalized liver endothelial cells in the radial-flow bioreactor in the development of an artificial liver.

Liver endothelial cells are important components of the tissue along the hepatic sinusoid. They are responsible for microcirculation in the liver and scavenger functions. It would therefore be important to include these cells in any hybrid type of artificial liver in addition to hepatocytes. However, it is difficult to culture these cells in vitro. The development of a liver endothelial cell line, which maintains the characteristics of the primary culture, would thus be of great benefit in the development of an artificial liver. In the present study we established immortalized liver endothelial cells from the liver of an H-2Kb-tsA58 transgenic mouse, which harbors the SV40 TAg gene. Hepatic sinusoidal cells isolated from H-2Kb-tsA58 mouse proliferated in the presence of gamma-interferon at 33 degrees C. Four clones were established, out of which clone M1 had the highest amounts of PGI2 production, as well as plasminogen activator activity and internalized acetylated low density lipoprotein. On culture dishes the M1 cells grew individually and spread. Sieve plates on the cell surface were not readily visible, but small pores were detected under electron microscopic observation. These results suggest that M1 clone cells originated from liver endothelial cells. Moreover it was possible to culture the immortalized liver endothelial cells in a radial-flow bioreactor for 5 days, with a maximum 6-keto prostaglandin F1alpha production of 25 microg per day. This suggests that immortalized liver endothelial cells and a radial-flow bioreactor can prove useful tools in the development an artificial liver.

cDNA cloning and expression of bovine UDP-N-acetylglucosamine: alpha1, 3-D-mannoside beta1,4-N-acetylglucosaminyltransferase IV.

UDP-N-acetylglucosamine:alpha1,3-D-mannoside beta1, 4-N-acetylglucosaminyltransferase (GnT-IV) is one of the essential enzymes in the production of tri- and tetra-antennary Asn-linked sugar chains. Recently, we have successfully purified GnT-IV from bovine small intestine. Based on the partial amino acid sequence of the purified bovine GnT-IV enzyme, its cDNA has been cloned from bovine small intestine. The open reading frame is 1,605 base pairs long, and this sequence produced GnT-IV activity on transient expression in COS-7 cells. Although the deduced amino acid sequence does not have any significant homology with other known N-acetylglucosaminyltransferases (GnTs), the hydrophobicity profile showed a typical type II transmembrane protein structure, which is common to many glycosyltransferases. N-terminal amino acid sequencing of the purified GnT-IV revealed that 92 amino acids, including a transmembrane region, were truncated during purification. Of the three potential N-glycosylation sites Asn-458 was actually glycosylated in the purified enzyme, although this N-glycosylation site could be abolished without any reduction in GnT-IV activity. Serial deletions at both the N and C termini proved that the catalytic domain of GnT-IV is located in the central region of the enzyme. The GnT-IV mRNA level correlated with enzymatic activity in the various bovine tissues tested.

Massive culture of human liver cancer cells in a newly developed radial flow bioreactor system: ultrafine structure of functionally enhanced hepatocarcinoma cell lines.

With a view to initiating clinical trials, cell morphology and function for a newly developed artificial liver support system employing highly functional human liver cell line, FLC-7, cultured in a radial flow bioreactor were compared to cells grown in a conventional monolayer culture. The radial flow bioreactor consists of a vertically extended cylindrical matrix comprised of porous glass bead microcarriers through which liquid medium flows from the periphery in toward the central axis generating a beneficial concentration gradient of oxygen and nutrients, while preventing excessive shear stresses or buildup of waste products. The three-dimensional culture system supports high-density (1.1 x 10(8) cells/ml-matrix), large scale cultures (4.4 x 10(10) cells/400 ml-bioreactor) with long-term viability. Scanning and transmission electron microscopy (SEM and TEM) revealed that cells cultured in a monolayer system were flattened and extended with numerous cytoplasmic projections. Cells in the three-dimensional culture were spherical and covered with microvillilike processes resembling liver cells in vivo. The cells were solidly attached on the surfaces and within the pores of the microcarriers in highly dense colonies. The spherical cells remained in close contact with adjacent cells, while circulation of liquid medium flowed freely through spaces between cells. FLC-7 cells produced albumin at a rate of 6.41 micrograms/24 h/10(6) cells. Alpha-fetoprotein (AFP) production dropped nearly threefold in comparison to monolayer cultures. Results demonstrated that the new artificial liver support systems (ALSS) provides a superior three-dimensional culture environment that allows cells to perform at naturally functioning levels.

Functional evidence for UDP-galactose transporter in Saccharomyces cerevisiae through the in vivo galactosylation and in vitro transport assay.

The oligosaccharide profiles in glycoproteins are determined by a series of processing reactions catalyzed by Golgi glycosyltransferases and glycosidases. Recently in vivo galactose incorporation in Saccharomyces cerevisiae has been demonstrated through the expression of human beta-1,4-galactosyltransferase in an alg1 mutant, suggesting the presence of a UDP-galactose transporter in S. cerevisiae (Schwientek, T., Narimatsu, H., and Ernst, J. F. (1996) J. Biol. Chem. 271, 3398-3405). However, this is quite unexpected, because S. cerevisiae does not have galactose residues in its glycoproteins. To address this question we have constructed S. cerevisiae mnn1 mutant strains expressing Schizosaccharomyces pombe alpha-1,2-galactosyltransferase. The mnn1 mutant of S. cerevisiae provides endogenous acceptors for galactose transfer by the expressed alpha-1,2-galactosyltransferase. We present here three lines of evidences for the existence of UDP-galactose transporter in S. cerevisiae. (i) About 15-20% of the total transformed mnn1 cells grown in a galactose medium were stained with fluorescein isothiocyanate-conjugated alpha-galactose-specific lectin, indicating the presence of alpha-galactose residues on the cell surface. (ii) Galactomannan proteins can be precipitated with agarose-immobilized alpha-galactose-specific lectin from a whole cell lysate prepared from transformed mnn1 cells grown in a galactose medium. (iii) The presence of UDP-galactose transporter was demonstrated by direct transport assay. This transport in S. cerevisiae is dependent on time, temperature, and protein concentration and is inhibited by nucleotide monophosphate and Triton X-100. The overall UDP-galactose transport in S. cerevisiae is comparable with that in S. pombe, indicating a more or less similar reaction velocity, while the rate of GDP-mannose transport is higher in S. pombe than in S. cerevisiae.

A novel second isoenzyme of the human UDP-N-acetylglucosamine:alpha1,3-D-mannoside beta1,4-N-acetylglucosaminyltransferase family: cDNA cloning, expression, and chromosomal assignment.

We isolated a novel cDNA encoding a second isoenzyme of UDP-N-acetylglucosamine:alpha1,3-D-mannoside beta1,4-N-acetylglucosaminyltransferase (GnT-IV; EC 2.4.1.145). The nucleotide and deduced amino acid sequences of the cDNA were homologous to those of the previously cloned human GnT-IV cDNA (63% and 62% identity, respectively). The new cDNA is also confirmed to express GnT-IV activity, suggesting that two isoenzymes of human GnT-IV exist. Although genomic Southern analysis suggested that both genes exist in many mammalian species and the chicken, northern analysis revealed that both genes are expressed in different ways in human tissues. This is the first report concerning the gene family of an N-acetylglucosaminyltransferase in mammals.

Enhanced constrictor responses of skeletal muscle arterioles during chronic myocardial infarction.

The goal of this study was to test the hypothesis that chronic myocardial infarction potentiates agonist-induced constrictor responses of rat skeletal muscle arterioles in vivo. Eight weeks after we performed coronary artery ligation or sham (control) surgery, the spinotrapezius muscle was prepared for direct visualization of the microcirculation. Diameter of third-order arterioles (40.7 +/- 0.5 microns) to topical suffusion of angiotensin II (ANG II; 0.1-10 nM), arginine vasopressin (AVP; 0.1-10 nM), endothelin-1 (ET-1; 1.0-100 pM), and the thromboxane analog U-46619 (1.0-100 nM) was measured in both groups. Myocardial-infarcted rats exhibited enhanced arteriolar constrictor responses to ANG II and AVP compared with the responses in controls. In contrast, ET-1- and U-46619-induced constrictor responses were similar in control and myocardial-infarcted rats. Additional experiments explored the impact of NG-monomethyl-L-arginine (L-NMMA; 0.1 mM) on arteriolar reactivity. In control animals, L-NMMA potentiated ANG II- and AVP-induced vasoconstriction, achieving values similar to those observed in myocardial-infarcted rats. L-NMMA did not alter vasoconstrictor responses in rats with chronic myocardial infarction. These observations suggest that enhanced agonist-induced vasoconstriction during heart failure may reflect a loss of nitric oxide-mediated modulation of arteriolar tone.

Purification and characterization of UDP-N-acetylglucosamine: alpha1,3-D-mannoside beta1,4-N-acetylglucosaminyltransferase (N-acetylglucosaminyltransferase-IV) from bovine small intestine.

A new beta1,4-N-acetylglucosaminyltransferase (GnT) which involves in branch formation of Asn-linked complex-type sugar chains has been purified 224,000-fold from bovine small intestine. This enzyme requires divalent cations, such as Mn2+, and catalyzes the transfer of GlcNAc from UDP-GlcNAc to biantennary oligosaccharide and produces triantennary oligosaccharide with the beta1-4-linked GlcNAc residue on the Manalpha1-3 arm. The purified enzyme shows a single band of Mr 58,000 and behaves as a monomer. The substrate specificity demonstrated that the beta1-2-linked GlcNAc residue on the Manalpha1-3 arm (GnT-I product) is essential for the enzyme activity. beta1-4-Galactosylaion to this essential beta1-2-linked GlcNAc residue or N-acetylglucosaminylation to the beta-linked Man residue (bisecting GlcNAc, GnT-III product) blocks the enzyme action, while beta1-6-N-acetylglucosaminylation to the Manalpha1-6 arm (GnT-V product) increases the transfer. Based on these findings, we conclude that the purified enzyme is UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,4-N-acetylglucosaminyltransferase IV (GnT-IV), that has been a missing link on biosynthesis of complex-type sugar chains.

Discovery of the shortest sequence motif for high level mucin-type O-glycosylation.

The consensus primary amino acid sequence for mucin-type O-glycosylation sites has not been identified. To determine the shortest motif sequence required for high level mucin-type O-glycosylation, we prepared more than 100 synthetic peptides and assayed in vitro O-GalNAc transfer to serine or threonine in these peptides using a bovine colostrum UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyl transferase (O-GalNAcT). We chose the sequence PDAASAAP from human erythropoietin (hEPO) for further systematic substitutions because it accepted GalNAc and was a fairly simple sequence consisting only of four kinds of amino acids. Several substitutions showed that threonine is approximately 40-fold better than serine as the glycosylated amino acid and a proline at position +3 on the C-terminal side is very important. To define the effect of proline residues around the glycosylation site, we analyzed a series of peptides containing one to three proline residues in a parent peptide AAATAAA. The results clearly indicated that prolines at positions +1 and +3 had a positive effect. The O-GalNAc transfer level of AAATPAP was increased approximately 90-fold from AAATAAA. The deletion of amino acids from the N-terminal side of the glycosylation site suggested that five amino acids from position -1 to +3 were especially important for glycosylation. Moreover, the influence of all 20 amino acids at positions -1, +2, and +4 was analyzed. Uncharged amino acids were preferred at position -1, and small or positively charged amino acids were preferred at position +2. No preference was observed at position +4. We propose a mucin-type O-glycosylation motif, XTPXP, which may be suitable as a signal for protein O-glycosylation. The features observed in this study also appear to be very useful for prediction of mucin-type O-glycosylation sites in glycoproteins.

[Quality control and cytogenetic analysis of human pre-embryos fertilized and cultured in vitro].

For the purpose to evaluate in vitro culture conditions of human preembryos, the efficacy of conventional culture and co-culture systems on embryonic development and genetic disorders was studied. Firstly, the development of cultured mouse embryos grown in standard media (Whitten's, GPM, HFT and Ham F10) or in HFT medium with different helper cell layers was compared. Embryonic growth was substantially reduced during in vitro culture, demonstrably by impaired cell proliferation, compared with in vivo controls. In in vitro fertilization and culture condition, SCEs of blastocysts were significantly increased. Development in co-culture with the feeder layers was notably better than in standard media. These results suggest that human preembryos could be rescued by the use of helper cells. Increased developmental rates and the cell numbers of blastocysts were the most evident morphological features of human preembryos that developed in co-culture with uterine luminal epithelial cells. However mosaicism may be caused by in vitro culture conditions and its onset may indicate when a disturbance in the embryonic development has occurred. It is advisable to perform further research into the mechanism of feeder cell-embryo interaction for understanding the optimal conditions of embryonic development in vitro.