Search PubMed⌕ Search

Biomedical subjects

J Toy

Publications and source records attributed to J Toy.

16 recordsLinked to original sources
PubMed2000

Lipid-mediated gene transfection into chick embryo retinal cells in ovo and in vitro.

Several lipofection reagents were tested on chick embryo retinal cultures using green fluorescent protein (GFP) as a reporter gene; best results were obtained with the GenePORTER (GP) reagent, which yielded approximately 4.4% of the cells with intense GFP fluorescence. Cell survival and structural differentiation appeared normal, but one of the immunocytochemical markers studied (visinin) was less frequently observed in GP-treated cultures. When similar plasmid-GP mixtures were injected into chick embryo eyes in ovo, bright GFP-fluorescent cells were observed in different retinal layers, without detectable detrimental effects on retinal morphology. Particularly extensive reporter gene expression was obtained upon intraocular injection of GP plus naked DNA from a RCAS retrovirus, which resulted in the development of abundant radial columns of alkaline phosphatase-positive cells, separated by columns of negative cells. We conclude that lipid-based transfection offers a quick, simple and fairly innocuous means for gene delivery into proliferating and postmitotic retinal cells, in vitro as well as in the developing eye in ovo, and that transfection of naked retroviral DNA can lead to extensive expression of foreign genes by retinal cells, bypassing the time-consuming steps required for the generation of high-titer virion stocks.

Animals↗
PubMed1999

Expression of the optx2 homeobox gene during mouse development.

Optx2, a member of the sine oculis-Six family of homeobox genes, is first expressed in the anterior neural plate of the mouse embryo, and subsequently in the optic vesicle and ventral forebrain. During later development, expression is further restricted to precursors of the neural retina, optic chiasm, adenohypophysis and neurohypophysis. In the adult mouse retina, Optx2 mRNA is found in cells within the ganglion cell layer and inner nuclear layer.

Animals↗
PubMed1998

The optx2 homeobox gene is expressed in early precursors of the eye and activates retina-specific genes.

Vertebrate eye development begins at the gastrula stage, when a region known as the eye field acquires the capacity to generate retina and lens. Optx2, a homeobox gene of the sine oculis-Six family, is selectively expressed in this early eye field and later in the lens placode and optic vesicle. The distal and ventral portion of the optic vesicle are fated to become the retina and optic nerve, whereas the dorsal portion eventually loses its neural characteristics and activates the synthesis of melanin, forming the retinal pigment epithelium. Optx2 expression is turned off in the future pigment epithelium but remains expressed in the proliferating neuroblasts and differentiating cells of the neural retina. When an Optx2-expressing plasmid is transfected into embryonic or mature chicken pigment epithelial cells, these cells adopt a neuronal morphology and express markers characteristic of developing neural retina and photoreceptors. One explanation of these results is that Optx2 functions as a determinant of retinal precursors and that it has induced the transdifferentiation of pigment epithelium into retinal neurons and photoreceptors. We also have isolated optix, a Drosophila gene that is the closest insect homologue of Optx2 and Six3. Optix is expressed during early development of the fly head and eye primordia.

Amino Acid Sequence↗
PubMed1994

Mutagenesis of the Lactobacillus casei folylpolyglutamate synthetase gene at essential residues resembling an ATP binding site.

Site-directed mutagenesis studies were performed on a region of the Lactobacillus casei folylpoly-gamma-glutamate synthetase (FPGS) protein (residues 49 to 52), which is highly conserved when compared to the Escherichia coli and human FPGS proteins. The amino acid sequence of this region, GKGS/T, is similar to the consensus sequence for the A region of a nucleotide binding site, a motif which encodes a phosphate-binding loop. Mutation G49A or K50R, with substitution to amino acids of similar size and charge, resulted in decreases in Vmax/Km of 40- to over 100-fold, depending on the variable substrate. Alteration of G51 to S or T resulted in a large increase in the Km for glutamate. The Km for ATP was not affected more than 4-fold by any of the mutations. Our studies indicate that the conserved region is essential for FPGS function, since many of the mutations resulting in functionally conservative substitutions produced inactive enzymes. However, the mutations affected binding of all three substrates, so there is no direct evidence for involvement of the region in ATP binding.

Adenosine Triphosphate↗
PubMed1993

Cloning and sequence determination of the valS gene, encoding valyl-tRNA synthetase in Lactobacillus casei.

The DNA sequence of the valS gene from Lactobacillus casei and the predicted amino acid sequence of its valyl-tRNA synthetase product have been determined. An open reading frame coding for a protein of 901 amino acids was found. A clone containing the intact L. casei valS gene functionally complemented the temperature-sensitive growth of the valS mutant strain 236c of Escherichia coli. The valS gene and the downstream folylpolyglutamate synthetase gene are transcribed in the same direction but are separated by a putative transcription terminator.

Amino Acid Sequence↗
PubMed1992

Purification and crystallization of Lactobacillus casei folylpolyglutamate synthetase expressed in Escherichia coli.

Folylpolyglutamate synthetase (FPGS) from Lactobacillus casei has been crystallized with polyethylene glycol and acetate buffer at pH 5.0. The enzyme was obtained from Escherichia coli strain SF4 harboring the L. casei FPGS chromosomal gene on a pEMBL vector (pGT3-8.1). Crystals of the enzyme were obtained which diffract to 2.6 A resolution. The crystals are monoclinic, space group P2(1), with unit cell dimensions of a = 54.07 A, b = 45.83 A, c = 84.37 A and beta = 107.92 degrees. A unit cell contains one molecule of the 43,000 Da enzyme per asymmetric unit. A complete X-ray data set on the native crystals has been collected.

Escherichia coli↗
PubMed1990

Cloning and expression of the gene encoding Lactobacillus casei folylpoly-gamma-glutamate synthetase in Escherichia coli and determination of its primary structure.

A genomic library of Lactobacillus casei DNA containing 10,000 individual clones was constructed in the plasmid pUC13. The gene encoding the L. casei folylpolyglutamate synthetase was isolated from the library by complementation of a folC mutant of Escherichia coli. The gene was expressed in E. coli from its own promoter and produced amplified folylpolyglutamate synthetase activity with properties identical with those of the purified L. casei enzyme. The absence of dihydrofolate synthetase activity and the preferential utilization of 5,10-methylenetetrahydrofolate, rather than 10-formyltetrahydrofolate as folate substrate, distinguishes this activity from the E. coli folylpolyglutamate synthetase-dihydrofolate synthetase. A protein of Mr = 43,000, identical with that of purified L. casei folylpolyglutamate synthetase, was expressed in maxicells containing the complementing plasmid. The nucleotide sequence of the folylpolyglutamate synthetase gene was determined. An open reading frame of 1,284 bases was found predicting a protein product of 428 amino acids with Mr = 44,169. The predicted amino acid sequence of the gene is 33% homologous to that of the E. coli folylpolyglutamate synthetase. Primer extension studies indicate that the transcription initiation site is at -59 base pairs, relative to the initiation ATG codon of the folylpolyglutamate synthetase gene, suggesting that the gene is transcribed independently of upstream genes. A second open reading frame was found downstream of the folylpolyglutamate synthetase open reading frame, overlapping the final codon by 1 base pair. This downstream gene may be co-transcribed with the folylpolyglutamate synthetase gene.

Amino Acid Sequence↗
PubMed1985

Preliminary clinical observations with recombinant interleukin-2 in patients with AIDS or LAS.

The toxicity of recombinant Interleukin-2 (IL-2) was studied in patients with acquired immunodeficiency syndrome (AIDS) or persistent lymphadenopathy syndrome (LAS). Increasing doses of the drug from 10(3) Units/m2 to 10(6) U/m2 were given as an intravenous bolus injection. At the high-dose levels some minor effects, such as fever up to 39.5 degrees C, chills, malaise or vomiting, were observed. The administration of 10(6) U/m2 as a 4-hour infusion showed identical results. No particular alterations of laboratory parameters were found. At the high-dose level the serum concentration of neopterin, which is released from macrophages after interferon gamma stimulation, was significantly (p less than 0.001) elevated above pretreatment levels. The clinical observation of daily infusions of 10(6)/m2 for 14 days revealed the same side effects. All patients developed lymphocytosis and eosinophilia. Two patients had suffered from severe diarrhoea for several weeks presumably due to cryptosporidiosis. In both cases diarrhoea ceased under the treatment with IL-2 and did not occur in the following two months.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Acquired immunological tolerance of foreign cells is impaired by recombinant interleukin 2 or vitamin A acetate.

The susceptibility of newborn mice to the inception of tolerance after exposure to antigen is associated with their deficiency in the production of endogenous interleukin 2 (IL-2). As further evidence of the complicity of IL-2 in the inception and maintenance of tolerance, it is shown here that a solid and long-lasting state of tolerance induced by the intravenous injection into newborn CBA mice of lymphoid cells from (CBA X C57BL/10ScSn)F1 hybrids can be brought to an end by the administration of exogenous IL-2 or by supplementing an otherwise normal diet with vitamin A acetate, the effect of which is to increase the proportion of the moiety of the T-cell population that produces IL-2. These results indicate that certain nonspecific stimuli can influence whether immunological tolerance is maintained.

Animals↗
PubMed1977

A combination of chlorambucil, vinblastine, procarbazine and prednisolone for treatment of Hodgkin's disease.

Seventy patients with Hodgkin's disease have been treated with a combination of chlorambucil, vinblastine, procarbazine and prednisolone (Ch1VPP). The complete remission rate of 75-7% compares well with that produced by other combinations. The combination is non-toxic, easily administered and can be given safely to outpatients. Its main advantage is that it is far less upsetting to patients than combinations containing nitrogen mustard.

Adolescent↗