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Biomedical subjects

J Brumbaugh

Publications and source records attributed to J Brumbaugh.

16 recordsLinked to original sources
PubMed2000

Autosomal albino chicken mutation (ca/ca) deletes hexanucleotide (-deltaGACTGG817) at a copper-binding site of the tyrosinase gene.

We compared tyrosinase cDNA sequences from a line of autosomal albino and Black Silky chickens isolated from cultured melanocytes by reverse transcription-polymerase chain reaction (RT-PCR). Both sources produce a single DNA fragment of predicted normal tyrosinase size. Direct sequencing of the PCR product showed three mutated sites in the tyrosinase gene of the albino chicken. Two silent point mutations and a deletion of six nucleotides (-deltaGACTGG) at 817 bp in the tyrosinase cDNA sequence were observed when compared with the White Leghorn and Black Silky cDNA sequences. The deduced albino chicken tyrosinase protein lacks two amino acids, aspartic acid and tryptophan. The position of these amino acids is consistent with one of the potential copper-binding sites that should be indispensable for function of the enzyme. We speculate that the six-base deletion is responsible for the inactive tyrosinase in this line of albino chickens.

Amino Acid Sequence↗
PubMed1994

Tissue-specific expression of mouse tyrosinase gene in cultured chicken cells.

A mouse tyrosinase cDNA has been combined with different promoters and inserted into several replication-competent avian leukosis proviruses and the viruses were transferred into cultured albino chick cells by viral infection. Expression of the tyrosinase gene depended on one of four promoter sequences: the resident constitutive promoter (Rous sarcoma virus long-terminal repeat; RSV-LTR), 471 bp from the mouse tyrosinase gene-associated promoter, 519 bp from the Japanese quail tyrosinase gene associated promoter, or 369 bp from the quail tyrosinase promoter. The infected cells expressed tyrosinase and produced pigment which could be seen with the light microscope. Immunofluorescence microscopy, using an anti mouse tyrosinase T1-specific antibody, also showed the presence of mouse tyrosinase. When infected with the same viral titer, gene expression was highest with the constitutive LTR promoter. The quail tyrosinase promoter, while less efficient than the LTR, was more efficient than the other tyrosinase promoter. Fibroblasts and hepatocytes infected with the construct carrying the constitutive promoter or the truncated quail promoter expressed tyrosinase. The mouse and quail promoters appeared to show tissue-specific expression since fibroblasts and hepatocytes infected with viruses carrying these promoters did not express mouse tyrosinase. Toxicity is associated with constitutive expression of tyrosinase in nonmelanocytes. Therefore the viruses that carry the tissue specific promoters should be useful for in vivo studies.

Animals↗
PubMed1992

The expression of mouse tyrosinase in chick cells in vitro and in vivo when controlled by a constitutive promoter.

Virally introduced mouse tyrosinase expression was checked both in vitro and in vivo in chicken cells and tissues. The results indicate that a constitutive promoter is able to express mouse tyrosinase in a variety of cells and tissues both in vitro and in vivo. Tyrosinase expression is marked by pigment production in situ, which is visible at macroscopic as well as microscopic levels without the use of substrates. It is concluded that tyrosinase can be a valuable marker for tracking gene insertion since it is spontaneously expressed. The expression of tyrosinase in some cells and tissues has a detrimental effect, however, and should be controlled by tissue-specific promoters.

Animals↗
PubMed1990

Transcription and sequence studies of a 4.3-kbp fragment from a ds-DNA eukaryotic algal virus.

A 4.3-kbp portion of the genome from the Chlorella virus, PBCV-1, has been cloned and sequenced. Minimally, five open reading frames (ORFs) were identified on this fragment. Transcriptional analysis indicates that each ORF encodes complex patterns of RNA. The total length of transcribed RNA exceeds that of the ORF indicating either post-transcriptional modification or multiple transcriptional start/stop sites. The sequence TTTTTNT, previously described as the transcriptional stop site for the early genes of vaccinia virus, is also found downstream of each of our ORFs. The regions 5' to each ORF were very A + T-rich (approx 80%) but distinct promoter sequences were not unambiguously identified.

Base Sequence↗
PubMed1988

Function of c-mos proto-oncogene product in meiotic maturation in Xenopus oocytes.

The c-mos proto-oncogene is expressed as a maternal mRNA in oocytes and early embryos of Xenopus laevis, but its translation product pp39mos is detectable only during progesterone-induced oocyte maturation. Microinjection of mos-specific antisense oligonucleotides into oocytes not only prevents expression of pp39mos, but also blocks germinal vesicle breakdown, indicating that it functions during reinitiation of meiotic division.

Animals↗
PubMed1986

Minimal pigment: a new type of oculocutaneous albinism.

Minimal pigment, a new type of oculocutaneous albinism (OCA), is described. At birth, affected individuals had no skin or eye pigment, and white hair and blue irides, but minimal amounts of pigment developed in the iris during the first decade of life. They had no measurable hairbulb tyrosinase activity. A characteristic and unusual pattern of parental activity was found in each of three families studied, with one parent having normal and the other parent having abnormally low tyrosinase activity. The melanocyte ultrastructure was normal and variations in premelanosomal pigmentation correlated with tyrosinase activity. This clinical and biochemical pattern has not been seen in any of the previously described types of OCA. The biochemical defect in minimal pigment OCA is unknown.

Albinism↗
PubMed1985

Brown oculocutaneous albinism. Clinical, ophthalmological, and biochemical characterization.

The clinical, ophthalmological, and biochemical characteristics of a 28-year-old black woman with brown oculocutaneous albinism were determined. Hair color was medium brown and skin color was light brown, and a faint tan developed with sun exposure. The irides were light brown in the central one-third, blue-gray in the peripheral two-thirds, and showed punctate and radial translucency. Visual acuity was 20/60 in the right eye and 20/100 in the left eye. There was a moderate pendular nystagmus, and previous surgeries had corrected an exotropia. The foveal reflex was muted, and the retinal pigment was reduced. Hairbulb tyrosinase activity was 1.75 pmoles/120 min/hairbulb, hairbulb glutathione content 0.83 nmoles/hairbulb, and urine excretion of 5-S-cysteinyldopa 174.9 ng/mg creatinine. Electron microscopy of hairbulb and skin melanocytes showed arrested melanosomal development. These findings suggest that there is a partial block in the distal eumelanin pathway in this form of albinism. The ophthalmological characteristics of six additional cases of this form of albinism are also presented.

Adult↗
PubMed1985

Quantitative analysis of immunosuppression in cyclosporine-treated heart transplant patients with lymphoma.

Lymphoma remains an important complication after heart transplantation in the era of cyclosporine immunosuppression. It has been generally assumed that the occurrence of lymphoma related to quantitative degree of immunosuppression, and this assumption is exemplified in the treatment of some such lymphomas with reduction of immunosuppression. Seventy-five consecutive survivors of heart and heart-lung transplantation between December 1980 and July 1983 were treated with cyclosporine and steroids; some received rabbit anti-thymocyte globulin for prophylaxis or treatment of rejection. Measured quantitative parameters of immunosuppression during the first three months after transplantation included mean cyclosporine level, total rabbit anti-thymocyte globulin dosage, number of days of T-cell suppression, and mean cyclosporine level during T-cell suppression. Serial Epstein-Barr virus antibody titers were measured. Lymphoma was diagnosed in six patients. The mean number of episodes of rejection did not differ between the lymphoma and the non-lymphoma groups. All quantitative measures of immunosuppression were higher in the lymphoma group, but this difference achieved statistical significance only in the case of total dosage of rabbit anti-thymocyte globulin (p less than 0.02). Four of the six lymphoma patients received some or all of their rabbit anti-thymocyte globulin dosage as prophylaxis against rejection; one received rabbit anti-thymocyte globulin solely for rejection; and the sixth received no rabbit anti-thymocyte globulin. Ebstein-Barr virus titer conversion (four-fold rise in titer) alone was not significantly associated with occurrence of lymphoma. However, logistic regression analysis suggested that Epstein-Barr virus conversion in concurrence with high mean cyclosporine levels predicted a higher risk of lymphoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Viral↗
PubMed1982

Genetic comparisons of eumelanosome ultrastructure.

We recently reported the existence of several new ultrastructural components of the eumelanosome matrix from developing chick pigment epithelium. The material used in the original study was obtained from a random sample of heterogeneous chicken. We have now extended this analysis to four genetically well-characterized chicken types: jungle fowl, blue, dominant white, and recessive wheaten. In these four genotypes there are specific ultrastructural alterations in the eumelanosome matrix. These alterations are different for each of the genotypes studied and are probably reflective of differences in the functional nature of the genes involved. The data thus provide evidence as to which genes are structural and which regulatory in action. Furthermore, since the material analyzed was all derived from pigmented pigment epithelium, it is clear that these melanosomes are affected by genes previously thought to leave them untouched. Finally, data are presented that these new ultrastructural elements are also found in a different class of animals, the C57BL/10 mouse.

Animals↗
PubMed1982

A method for culturing chick melanocytes: the effect of BRL-3A cell conditioning and related additives.

A method for growing chick embryo melanocytes is described that utilizes medium conditioned by Buffalo Rat liver (BRL-3A) cells. The dissected trunk region of each 72 h (Stages 14 to 19) embryo produces approximately 200,000 melanocytes (purity, 80%) when processed and cultured for 8 d. Thus, a typical experiment involving 20 embryos would produce a total of 4 x 10(6) melanocytes. Choice of serum, serum concentration, and cell density were determined experimentally. Partially purified multiplication stimulating activity (MSA) from BRL-3A cells and insulin were also tested as medium additives. MSA was not stimulatory, whereas insulin gave a positive response in 2% but not 10 or 0% serum. The final protocol used a modified F12 medium with 10% bovine calf serum conditioned by BRL-3A cells. Cultures were fed every other day. Small colonies of cells became evident by culture Day 3 and increased rapidly to Day 5 when pigmentation became obvious. Colony size continued to increase but more slowly from Days 5 to 8, whereas pigmentation increased rapidly and maximized on Day 8. There is a factor, or factors, present in BRL-3A conditioned medium that stimulates embryonic chick melanocytes to divide preferentially over contaminating cell types. This results in cultures that can provide adequate numbers and purity for biochemical studies.

Animals↗
PubMed1977

Transformation of chicken embryo retinal melanoblasts by a temperature-sensitive mutant of Rous sarcoma virus.

Retinal melanoblasts were transformed by a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV). At the permissive temperature for transformation, the cells cease melanin synthesis, degrade their melanosomes and release much of their accumulated melanin into the medium. At the nonpermissive temperature, the cells assume an epithelioid morphology, actively synthesize melanin and become difficult to distinguish from normal uninfected control cultures. Both the transformed phenotype and the differentiated cell phenotype are temperature-dependent. Infected retinal melanoblasts which are incubated at the nonpermissive temperature and which accumulate a large amount of melanin are unable to transform in response to a temperature shift; instead, the cells degenerate and die. Retinal melanoblasts can be infected by subgroups A, B, C and D of RSV; however, their level of susceptibility to infection is about 1/40 compared to fibroblasts. Cultures infected by ts-RSV produce virus at both temperatures, suggesting that cell phenotype does not regulate virus synthesis.

Animals↗