Embryonic body culturing in an all-glass microfluidic device with laser-processed 4 µm thick ultra-thin glass sheet filter.

In this paper, we report the development and demonstration of a method to fabricate an all-glass microfluidic cell culturing device without circulation flow. On-chip microfluidic cell culturing is an indispensable technique for cellular replacement therapies and experimental cell biology. Polydimethylsiloxane (PDMS) have become a popular material for fabricating microfluidic cell culture devices because it is a transparent, biocompatible, deformable, easy-to-mold, and gas-permeable. However, PDMS is also a chemically and physically unstable material. For example, PDMS undergoes aging easily even in room temperature conditions. Therefore, it is difficult to control long term experimental culturing conditions. On the other hand, glass is expected to be stable not only in physically but also chemically even in the presence of organic solvents. However, cell culturing still requires substance exchanges such as gases and nutrients, and so on, which cannot be done in a closed space of a glass device without circulation flow that may influence cell behavior. Thus, we introduce a filter structure with micropores onto a glass device to improve permeability to the cell culture space. Normally, it is extremely difficult to fabricate a filter structure on a normal glass plate by using a conventional fabrication method. Here, we demonstrated a method for fabricating an all-glass microfluidic cell culturing device having filters structure. The function of this all-glass culturing device was confirmed by culturing HeLa, fibroblast and ES cells. Compared with the closed glass devices without a filter structure, the numbers of cells in our device increased and embryonic bodies (EBs) were formed. This method offers a new tool in microfluidic cell culture technology for biological analysis and it expands the field of microfluidic cell culture.

Health benefits of fermented milk containing Bifidobacterium bifidum YIT 10347 on gastric symptoms in adults.

We conducted a preliminary open trial (trial 1) and a double-blind, placebo-controlled, crossover trial (trial 2) to examine how fermented milk containing the probiotic Bifidobacterium bifidum YIT 10347 affects gastric and lower abdominal symptoms in adults taking no medication. In trial 1, subjects with or without gastric and lower abdominal symptoms ingested fermented milk containing B. bifidum YIT 10347 daily for 2 wk. In trial 2, subjects with gastric symptoms ingested fermented milk containing B. bifidum YIT 10347 (active preparation) or placebo daily for 2 wk, followed by crossover for 3 wk after a washout period. Before (baseline) and 1 and 2 wk after ingestion, subjects completed a questionnaire. In trial 1 (305 subjects), the prevalence of gastric and lower abdominal symptoms was 46 and 58%, respectively, at baseline. Ingestion of B. bifidum YIT 10347 significantly decreased the prevalence of gastric and lower abdominal symptoms from 45 to 33% at 1 wk and to 28% at 2 wk, and from 57 to 40% at 2 wk, respectively. In subjects with gastric symptoms at baseline, the average gastric symptom score per subject significantly decreased by 0.9 at 1 wk and 1.2 at 2 wk. In trial 2 (27 subjects), ingestion of the active preparation significantly decreased the average gastric symptoms score per subject by 1.0 at 1 wk and 1.1 at 2 wk, but ingestion of placebo milk had no effect. No side effects were reported by any subjects in either trial. We conclude that fermented milk containing B. bifidum YIT 10347 has the potential to provide health benefits by alleviating gastric symptoms in subjects taking no medication.

Increased expression of cell adhesion molecule 1 by mast cells as a cause of enhanced nerve-mast cell interaction in a hapten-induced mouse model of atopic dermatitis.

BACKGROUND: Neuroimmunological disorders are involved in the pathogenesis of atopic dermatitis (AD), partly through enhanced sensory nerve-skin mast cell interaction. Cell adhesion molecule 1 (CADM1) is a mast-cell adhesion molecule that mediates the adhesion to, and communication with, sympathetic nerves. OBJECTIVES: To investigate the role of mast cell CADM1 in the pathogenesis of AD, CADM1 expression levels by comparing between lesional and nonlesional skin mast cells of an AD mouse model, which was developed by repeated application of trinitrochlorobenzene, and to examine, in cocultures, how the alterations in CADM1 detected in lesional mast cells might affect the sensory nerve-mast cell interaction. METHODS: AD-like lesional and nonlesional skin mast cells were collected separately by laser capture microdissection. CADM1 expression was examined by reverse transcription-polymerase chain reaction and CADM1 immunohistochemistry. In cocultures, adhesion between dorsal root ganglion (DRG) neurites and IC2 mast cells was analysed by loading a femtosecond laser-induced impulsive force on neurite-attendant IC2 cells, while cellular communication was monitored as the IC2 cellular response ([Ca(2+)]i increase) after nerve-specific stimulant-induced DRG activation. RESULTS: AD-like lesional mast cells expressed three-fold more CADM1 transcripts than nonlesional cells. This was supported at the protein level, shown by immunohistochemistry. In coculture, CADM1 overexpression in IC2 cells strengthened DRG neurite-IC2 cell adhesion and doubled the population of IC2 cells responding to DRG activation. A function-blocking anti-CADM1 antibody abolished these effects in a dose-dependent manner. CONCLUSIONS: Increased expression of CADM1 in mast cells appeared to be a cause of enhanced sensory nerve-mast cell interaction in a hapten-induced mouse model of AD.

Deficiency of tenascin-C delays articular cartilage repair in mice.

OBJECTIVE: In human articular cartilage, tenascin-C (TN-C) expression decreases during maturation of chondrocytes, and almost disappears in adults; however, it reappears in damaged cartilage. To examine the effects of TN-C on cartilage degeneration and repair, we compared articular cartilage degeneration between wild-type (WT) and tenascin-C knockout mouse (TNKO) mice using a spontaneous osteoarthritis (OA) in aged joints and surgical OA model. In addition, we made full-thickness cartilage defects and compared the cartilage repair process between the two groups. METHODS: The surgical procedure to create degenerative OA model was performed by transecting the anterior cruciate ligament and medial collateral ligament. Full-thickness defects were created in the center of the femoral trochlea to evaluate cartilage repair. Sections of cartilage were stained with hematoxylin and eosin or safranin-O, and immunostaining for TN-C. The degrees of degeneration and repair were graded. RESULTS: In the WT surgical OA model, the articular cartilage was almost normal at 2 weeks, but safranin-O decreased staining at 4 weeks. In TNKO mice, safranin-O decreased staining at 2 weeks, and cartilage was injured intensely at 4 weeks. In the cartilage repair model, TN-C was expressed after 1 week, was strongly expressed in the upper layer of regenerated tissue after 3 weeks, and disappeared after 6 weeks. The defects were restored until 6 weeks in WT mice; however, defects in TNKO mice were filled with fibrous tissue with no cartilage-like tissue. CONCLUSIONS: This study revealed that cartilage repair in TNKO mice was significantly slower than that in WT mice and that the deficiency of TN-C progressed during cartilage degeneration.

Bifidobacterium bifidum BF-1 suppresses Helicobacter pylori-induced genes in human epithelial cells.

Helicobacter pylori infection alters gene expression in host cells. Specifically, inflammatory chemokines such as IL-8 are upregulated in the gastric mucosa during H. pylori infection. Although the mechanism by which H. pylori causes inflammation of the gastric mucosa is not yet understood, many studies have suggested that nuclear factor kappa B (NF-κB) plays a key regulatory role in host cells. We have shown that preincubation with Bifidobacterium bifidum strain BF-1, a probiotic strain known to improve H. pylori-associated gastritis, suppresses induction of IL-8 by the pathogen. To investigate how how BF-1 affects gene expression in H. pylori-infected cells, we performed microarray analysis to assess gene expression in epithelial cells, which had been preincubated with BF-1 and infected with H. pylori. We found that preincubation with BF-1 suppresses the expression of H. pylori-induced genes in human cells and that most of the affected genes are related to the NF-κB signaling pathways. These results suggest that BF-1 can affect the regulatory mechanism of the NF-κB signaling pathways.

Effect of Bifidobacterium bifidum fermented milk on Helicobacter pylori and serum pepsinogen levels in humans.

Helicobacter pylori infection is an important risk factor for gastric diseases. Some probiotics are useful for suppressing H. pylori infection. Bifidobacterium bifidum YIT 4007 can improve the experimental gastric injury in rats and the disease stages on the gastric mucosa in peptic ulcer patients. We evaluated the fermented milk using a clone (BF-1) having the stronger ability to survive in the product than this parent strain to clarify the in vitro suppressive effect of BF-1 on H. pylori and the in vivo efficacy of BF-1 fermented milk on H. pylori and gastric health. In the mixed culture assay of BF-1 and H. pylori, the number of pathogens was decreased such that it was not detected after 48 h in the Brucella broth with a decrease in pH values. In the cell culture experiment with human gastric cells, the H. pylori infection-induced IL-8 secretion was suppressed by the preincubation of BF-1. In a human study of 12-wk ingestion (BF-1 group, n = 40; placebo group, n = 39) with a randomized double-blind placebo-control design, the H. pylori urease activity and gastric situation were evaluated using a urea breath test (UBT) and the serum pepsinogen (PG) levels as biomarkers for inflammation or atrophy, respectively. In the H. pylori-positive subjects, the difference (DeltaUBT) of the UBT value from the baseline value in the BF-1 group (n = 34) was lower than that in the placebo group (n = 35) at 8 wk. The baseline UBT values showed a negative correlation with DeltaUBT values at 8 and 12 wk in the BF-1 group but not in the placebo. In the PG-positive subjects classified by the PG test method, the BF-1 group was lower in DeltaUBT values than the placebo group at 8 and 12 wk. In the active gastritis class by PG levels, the BF-1 group was lower in their DeltaUBT values than the placebo at 8 and 12 wk. The PG I levels in the BF-1 group were lower than the placebo at 12 wk. The PG II levels in the BF-1 group did not change during the ingestion period, but the placebo was increased. The PG I/II ratios slightly decreased from baseline at 12 and 20 wk in the BF-1 and placebo groups. These patterns were also observed in the H. pylori-positive subjects. The improving rates of upper gastrointestinal symptomatic subjects and total symptom numbers in the BF-1 group were higher than those in the placebo. These results indicate that BF-1 fermented milk may affect H. pylori infection or its activity, gastric mucosal situation, and the emergence of upper gastrointestinal symptoms.

A novel RNA helicase-like protein during early embryonic development in silkworm Bombyx mori: molecular characterization and intracellular localization.

In order to understand the molecular mechanism of development during early embryogenesis in diapause and non-diapause of the silkworm, mRNA from diapause and non-diapause eggs was compared using the differential display technique. We cloned the full length of a cDNA encoding a novel RNA helicase-like (RHL) protein by the RACE method using a cDNA fragment which was one of the specific cDNAs in the non-diapause eggs. A BLAST search using the predicted amino acid sequence of RHL revealed a low homology (21-25% identity of its partial length) with that of the DEAD-box RNA helicase. Gene expression of the RHL gene of the diapause and non-diapause eggs was investigated by RT-PCR until 60h after oviposition. Amplified RHL cDNA was observed through all the stages in the non-diapause eggs, while in the diapause eggs, cDNA was found in eggs 0-12h after oviposition but disappeared 24-60h after oviposition. When the diapause eggs were activated by HCl treatment after chilling at 4 degrees C for 6 days from 48h after oviposition (artificial diapause termination), cDNA was observed from 12h after HCl treatment. We also investigated the immunohistochemical distribution and localization of RHL in non-diapause eggs using anti-recombinant His-tag RHL antiserum. RHL was distributed in blastoderm cells and yolk cells and was localized in the nucleus and the cytosol of yolk cells. These data suggest that RHL has an important role in the early embryo of the silkworm.

Fer tyrosine kinase oligomer mediates and amplifies Src-induced tumor progression.

c-Src is upregulated in various human cancers, suggesting its role in malignant progression. However, the molecular circuits of c-Src oncogenic signaling remain elusive. Here we show that Fer tyrosine kinase oligomer mediates and amplifies Src-induced tumor progression. Previously, we showed that transformation of fibroblasts is promoted by the relocation of c-Src to non-raft membranes. In this study, we identified Fer and ezrin as non-raft c-Src targets. c-Src directly activated Fer by initiating its autophosphorylation, which was further amplified by Fer oligomerization. Fer interacted with active c-Src at focal adhesion membranes and activated Fer-phosphorylated ezrin to induce cell transformation. Fer was also crucial for cell transformation induced by v-Src or epidermal growth-factor receptor activation. Furthermore, Fer activation was required for tumorigenesis and invasiveness in some cancer cells in which c-Src is upregulated. We propose that the Src-Fer axis represents a new therapeutic target for treatment of a subset of human cancers.

Living cells of probiotic Bifidobacterium bifidum YIT 10347 detected on gastric mucosa in humans.

The probiotic strain Bifidobacterium bifidum YIT 10347 has been demonstrated to inhibit Helicobacter pylori activity, prevent injury to the gastric mucosa, and improve general gastric malaise symptoms in H. pylori positive patients. This study aimed to investigate the adhering activity and localisation of B. bifidum YIT 10347 to gastric cells and tissue in vitro, and in human in vivo to clarify the mechanism of its beneficial effects on the stomach. The in vitro study found the adhesion rate of B. bifidum YIT 10347 to human gastric epithelial cells was about 10 times higher than that of lactic acid bacteria and other bifidobacteria. In the human study, 5 H. pylori negative and 12 H. pylori positive subjects ingested milk fermented with B. bifidum YIT 10347. B. bifidum YIT 10347 cells were measured by RT-qPCR for in gastric biopsy samples. Living B. bifidum YIT 10347 cells were detected in the biopsy samples in H. pylori negative subjects (105 cells/g and 104 cells/g at 1 h and 2 h after ingestion, respectively) and H. pylori positive subjects (104 cells/g at 1 h after the ingestion). Moreover, immunostaining analysis of tissue sections found that B. bifidum YIT 10347 cells were located at the interstitial mucin layer of the stomach. These results suggest that cells of probiotic B. bifidum YIT 10347 adhered to the human gastric mucosa in a live state, and that the higher adhering activity of B. bifidum YIT 10347 to the gastric mucosa may be involved in its beneficial effects on the human stomach.

Discovery of a new tetrahydrobiopterin-synthesizing enzyme in the lemon mutant of the silkworm Bombyx mori.

A new tetrahydrobiopterin-synthesizing enzyme, which is different from sepiapterin reductase (EC 1.1.1.153), was discovered in the integument of the lemon mutant of the silkworm Bombyx mori. This enzyme converted 6-pyruvoyltetrahydropterin to tetrahydrobiopterin, an essential cofactor in the hydroxylation of aromatic amino acids, in the presence of NADPH. The reaction proceeded via 6-lactoyltetrahydropterin and 1'-hydroxy-2'-oxopropyltetrahydropterin as intermediates. The molecular mass of this enzyme was estimated to be 40 kDa. N-Acetylserotonin, a potent inhibitor of sepiapterin reductase, slightly inhibited the enzymatic reaction. In the presence of 0.5 mM N-acetylserotonin, the formation of tetrahydrobiopterin by sepiapterin reductase purified from the normal strain silkworm was completely inhibited. However, the formation of tetrahydrobiopterin by the enzyme purified from the lemon mutant was inhibited by only about 50%. These results suggest an alternative biosynthetic pathway to tetrahydrobiopterin.

FlgB, FlgC, FlgF and FlgG. A family of structurally related proteins in the flagellar basal body of Salmonella typhimurium.

The flagellar basal body of Salmonella typhimurium consists of four rings surrounding a rod. The rod, which is believed to transmit motor rotation to the filament, is not well characterized in terms of its structure and composition. FlgG is known to lie within the distal portion of the rod, in the region where it is surrounded by the L and P rings, just before the rod-hook junction. The FlgC and FlgF proteins are also known to be flagellar basal-body components; by comparison of deduced and experimental N-terminal amino acid sequences we show here that FlgB is a basal-body protein. The flgB, flgC, flgF and flgG gene sequences and the deduced protein sequences are presented. The four proteins are clearly related to each other in primary sequence, especially toward the N and C termini, supporting the hypothesis (based on examination of basal-body subfractions) that FlgB, FlgC and FlgF are, like FlgG, rod proteins. From this and other information we suggest that the rod is the cell-proximal part of a segmented axial structure of the flagellum, with FlgB, FlgC and FlgF located (in unknown order) in successive segments of the proximal rod, followed by FlgG located in the distal rod; the axial structure then continues with the hook, HAPs and filament. Although the rod is external to the cell membrane, none of the four rod proteins contains a consensus signal sequence for the primary export pathway; comparison with the experimentally determined N-terminal amino acid sequence indicates that FlgB has had its N-terminal methionine removed, while the other three are not processed at all. This demonstrates that these proteins are not exported by the primary cellular pathway, and suggests that they are exported by the same flagellum-specific pathway as the flagellar filament protein flagellin. The observed sequence similarities among the rod proteins, especially a six-residue consensus motif about 30 residues in from the N terminus, may constitute a recognition signal for this pathway or they may reflect higher-order structural similarities within the rod.

Selective abortion of two nonsister nuclei in a developing ascus of the hfd-1 mutant in Saccharomyces cerevisiae.

A recessive mutation, hfd1-1, in strain SOS4 of Saccharomyces cerevisiae leads the mutant cells to produce predominantly two-spored asci. Light microscopical examination of Giemsa-stained cells revealed no significant differences in the meiotic figures between mutant and wild-type strains. However, only two of the four meiotic products in a developing ascus matured to ascospores in SOS4. Dyad analysis was carried out on an hfd1-1 mutant strain heterozygous for three markers, asp5, gal1, and arg4, which are closely linked to their centromeres, and for his4, which is loosely linked to its centromere. The two-spored asci produced by the hfd1-1 mutant segregated dominant (+) and recessive (-) alleles of each marker in a 1:1 ratio; they generally contained one + and one - spore for any given marker. The occurrence of rare dyads with two + or two - spores can be explained quantitatively by recombination between the marker and its centromere. From the results of these cytological and genetical analyses, we infer that, in the mutant strain, one genome set is partitioned to each of the four second-meiotic division poles, but only two nonsister genomes are incorporated into mature spores. Thus, the hfd1-1 mutation in SOS4 blocks incorporation of two nonsister nuclei into mature ascospores, but does not block enclosure of the remaining two nonsister nuclei.

Definition of additional flagellar genes in Escherichia coli K12.

Twenty-nine flagellar genes in Escherichia coli K12 have previously been assigned to three regions of the genome. Flagellar region I is located between pyrC and ptsG, region II between aroD and uvrC, and region III between uvrC and his. In this study, flagellar mutants in Escherichia coli K12 were obtained by selection for resistance to the flagellotropic phage, chi. They were analyzed in complementation tests using P1 phage-mediated transduction. In addition to the fla genes already described, eight more flagellar genes were identified. This analysis defined six more fla genes in region I (flaU, etc.), one more in region II (flbB) and one more in region III (flbC). Region I was shown to include at least 12 fla cistrons. Complementation analysis with polar Mu phage-induced Fla- mutants and with lambda fla phage defined four transcriptional units in region I. These were: flaU, flbA-flaW-flaV-flaK-flaX-flaL-flaY-flaM, flaZ and flaS- flaT, with transcription proceeding from left to right. The flB gene was found to be part of an operon: flB-flaI in region II. In region III, a previously unidentified gene flbC was located between hag and flaN.

Chondroprotective effects of Salubrinal in a mouse model of osteoarthritis.

OBJECTIVES: Salubrinal is a synthetic agent that elevates phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) and alleviates stress to the endoplasmic reticulum. Previously, we reported that in chondrocytes, Salubrinal attenuates expression and activity of matrix metalloproteinase 13 (MMP13) through downregulating nuclear factor kappa B (NFκB) signalling. We herein examine whether Salubrinal prevents the degradation of articular cartilage in a mouse model of osteoarthritis (OA). METHODS: OA was surgically induced in the left knee of female mice. Animal groups included age-matched sham control, OA placebo, and OA treated with Salubrinal or Guanabenz. Three weeks after the induction of OA, immunoblotting was performed for NFκB p65 and p-NFκB p65. At three and six weeks, the femora and tibiae were isolated and the sagittal sections were stained with Safranin O. RESULTS: Salubrinal suppressed the progression of OA by downregulating p-NFκB p65 and MMP13. Although Guanabenz elevates the phosphorylation level of eIF2α, it did not suppress the progression of OA. CONCLUSIONS: Administration of Salubrinal has chondroprotective effects in arthritic joints. Salubrinal can be considered as a potential therapeutic agent for alleviating symptoms of OA. Cite this article: Bone Joint Res 2015;4:84-92.

A fluoroquinolone (DR-3355) protects human lymphocyte cell lines from HIV-1-induced cytotoxicity.

HIV-1 infection of human CD4+ lymphocyte cell lines results in cell death. Treatment, but not pretreatment, of infected cells, with a fluoroquinolone antibiotic, DR-3355, protects a significant subfraction of cells from HIV-1-mediated cytolysis. All surviving cells have lost expression of the CD4 antigen, but do (MT-4) or do not (CEM) express viral antigens and produce infective virus. The rescued CEM and MT-4 cells are phenotypically stable and do not require continuous exposure to the drug for survival.

A gene for DNA invertase and an invertible DNA in Escherichia coli K-12.

An assay system for the pin gene function, which suppresses the vh2 mutation of Salmonella, was developed and used to show that most strains of Escherichia coli K-12 are Pin+, whereas all the strains of E. coli C examined are Pin-. An E. coli host strain was constructed and used for detection of DNA fragments carrying the E. coli K-12 pin gene cloned in the plasmid vector pBR322. Restriction analysis of the cloned fragments showed that the invertible DNA (designated P region) is adjacent to the pin gene and that its inversion is mediated by the pin gene product. The pin gene was found to be functionally homologous to the gin gene of Mu phage and the cin gene of P1 phage. The P region most probably resides within the cryptic prophage e14, and the Pin- phenotype is likely to be associated with the loss of e14.

Sequence analysis of the flgA gene and its adjacent region in Salmonella typhimurium, and identification of another flagellar gene, flgN.

The flagellar genes flgA and flgM are located at the terminus of the region-I flagellar gene cluster on the chromosome of Salmonella typhimurium. The flgA gene is involved in P-ring formation of the flagellar basal body, whereas flgM encodes the anti-sigma factor which acts as a negative regulator of the flagellar regulon. The nucleotide sequence of the DNA fragment containing these flagellar genes and the adjacent region was determined. The flgA gene was found to encode a 219-amino-acid (aa) protein of 23,556 Da. The N-terminal region of FlgA has the characteristics of a typical signal sequence, suggesting that FlgA may function in the periplasmic space where P-ring assembly takes place. The flgM gene was found to constitute an operon together with an ORF which encodes a 140-aa protein of 15,899 Da. A gene disruption mutant was constructed by inserting a cat gene cartridge into the ORF on the chromosome. This mutant showed only weak motility, indicating that the product of the ORF is involved in flagellar formation. Therefore, this ORF was designated as flgN. Electron microscopic observation revealed that most of the flagellar structures produced by the flgN mutant are hook-basal body complexes lacking the filament portions. Based on these results, we concluded that the flgN product is required for the efficient initiation of filament assembly.

Sequence analysis of operator mutants of the phase-1 flagellin-encoding gene, fliC, in Salmonella typhimurium.

In phase-2 cells of diphasic Salmonella strains, expression of the phase-1 flagellin-encoding gene, fliC, is repressed by the repressor encoded by the fljA gene. Nine operator-constitutive (Oc) mutants of fliC were isolated from S. typhimurium by selecting those which could express fliC in the presence of the repressor. Among them, eight mutants could express fliC both in the presence and the absence of the repressor, whereas the ninth one could express only in the presence of the repressor. Nucleotide sequence analysis revealed that the Oc mutations of the former type were all located between bp 7 and 20 upstream from the coding region of fliC, which suggests that this region may correspond to the operator for fliC. The latter mutant was found to have a tandem duplication of 28 bp which contains a part of the operator sequence, and seems to require the repressor to activate fliC expression.

Molecular cloning and characterization of cellular genes whose expression is repressed by the adenovirus E1a gene products and growth factors in quiescent rat cells.

Several cDNA clones of cellular genes, whose expression is repressed by the adenovirus type-12 E1a gene products, were isolated from a rat 3Y1 cell cDNA library by differential plaque hybridization with labeled cDNA probes prepared from 3Y1 and the derivative cell line expressing the E1a gene constitutively. The changes in the levels of these gene transcripts during cell-cycle progression from G0 to G1 to S phase were analyzed with 3Y1 cells and gMA cell lines, derived from 3Y1 cells, in which the expression of the E1a gene or its 13S, 12S cDNA can be switched on by the addition of dexamethasone. Quantitation of the transcripts by Northern-blot hybridizations and by nuclear run-on experiments revealed the following. (i) The level of clone-53 mRNA (which turned out to be the fibronectin (FN)-coding mRNA) is very high in resting gMA cells and decreased rapidly after switching on of the E1a gene or its 13S, or 12S cDNA. (ii) The addition of serum or platelet-derived growth factor to resting 3Y1 cells also resulted in a rapid decrease in the FN mRNA level, but the addition of epidermal growth factor (EGF) had little or no effect. (iii) The level of clone-56 mRNA in gMA cells was not affected by the induction of the E1a gene expression; however, the addition of EGF to resting gMA or 3Y1 cells resulted in a decrease of this mRNA after a 12- to 16-h lag period. Induction of the E1a gene expression in gMA cells treated with EGF shortened the lag period. The addition of serum to resting 3Y1 cells decreased the clone-56 mRNA level without a significant lag period.

An immunization method using antigen entrapped in erythrocyte ghosts.

Intraperitoneal injection into mice of dinitrophenol-conjugated ovalbumin entrapped in autologous erythrocyte ghosts gave rise to an increase of anti-dinitrophenol antibody production without use of artificial adjuvants. This technique could be useful for vaccination or other immuno-therapeutic purposes.