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Biomedical subjects

J Speck

Publications and source records attributed to J Speck.

At least 19 recordsLinked to original sources
PubMed1997

Age-related distribution of cones and ON-bipolar cells in the rd mouse retina.

PURPOSE: Retinal dystrophic (rd) mice lose most of their rod photoreceptors within the first three weeks after birth. We determined the age-related distribution of peanut agglutinin lectin (PNA)-labeled cones during the first 12 months of age. We also investigated whether the density of ON-bipolar cells expressing L7 protein was affected by their loss of photoreceptor inputs. METHODS: rd mice were selected from a transgenic strain which expresses an L7-beta-galactosidase fusion gene localized to ON-bipolar cells. Cones were stained with PNA and ON-bipolar cells with bluo-gal (halogenated indolyl-beta-D-galactoside). Retinas were flat-mounted and observed at 1, 2, 3, 6 and 12 months of age. RESULTS: PNA-labeled cones are distributed unevenly across the retina at 1 month postnatal. Their concentration decreases first in the central and far peripheral retina, leaving a ring of labeled cells in the midperipheral region. At 3 months, a larger patch of cones remains in the supero-temporal midperipheral region and a smaller patch in the infero-nasal retina. By 6 months, few cones remain in the infero-nasal retina; by 1 year approximately 100 cones remain in the entire retina, localized to the superior midperipheral region. ON-bipolar cells appear evenly distributed at 1 month. By 2-3 months, relatively more bluo-gal staining is seen in the midperipheral regions underlying dense cone populations. At 6-12 months, bluo-gal label is distributed in a spotty pattern with little or no staining seen in areas of apparent neovascularization. CONCLUSIONS: (1) PNA-labeling of cones in the rd retina deteriorates in a distinct spatial pattern with the longest cone survival in the midperipheral superior retina. (2) ON-bipolar cells are more densely labeled in regions of high cone density during the early months of cone degeneration and, in later stages, show relative decreases in regions of apparent neovascularization.

Age Factors↗
PubMed1996

4-Oxoretinol, a new natural ligand and transactivator of the retinoic acid receptors.

All-trans-retinoic acid (at-RA) induces cell differentiation in a wide variety of cell types, including F9 embryonic teratocarcinoma cells, and can influence axial pattern formation during embryonic development. We now identify a novel retinoid synthetic pathway in differentiating F9 cells that results in the intracellular production of 4-oxoretinol (4-oxo-ROL) from retinol (vitamin A). Approximately 10-15% of the total retinol in the culture is metabolized to 4-hydroxyretinol and 4-oxo-ROL by the at-RA-treated, differentiating F9 cells over an 18-hr period, but no detectable metabolism of all-trans-retinol to at-RA or 9-cis-retinoic acid is observed in these cells. Remarkably, we show that 4-oxo-ROL can bind and activate transcription of the retinoic acid receptors whereas all-trans-retinol shows neither activity. Low doses of 4-oxo-ROL (e.g., 10(-9) or 10(-10 M) can activate the retinoic acid receptors even though, unlike at-RA, 4-oxo-ROL does not contain an acid moiety at the carbon 15 position. 4-oxo-ROL does not bind or transcriptionally activate the retinoid X receptors. Treatment of F9 cells with 4-oxo-ROL induces differentiation without conversion to the acid and 4-oxo-ROL is active in causing axial truncation when administered to Xenopus embryos at the blastula stage. Thus, 4-oxo-ROL is a natural, biologically active retinoid that is present in differentiated F9 cells. Our data suggest that 4-oxo-ROL may be a novel signaling molecule and regulator of cell differentiation.

Animals↗
PubMed1996

[Avian yolk antibodies in diagnosis and research].

Hens were immunized with bacterial polysaccharide (alginate), Hepatitis B surface antigen (HBsAg), and potato viruses (PVA, PVS, PVM, PVX, and PVY). The antibodies were isolated noninvasively from the yolks of laid eggs. The purified yolk immunoglobulins (IgY) were tested in an array of various assays and diagnostic techniques. The methods employed were precipitation reactions, immun-electrophoresis, ELISA (after biotinylation of IgY), immuno-gold electron microscopy, and western and immuno blotting. Some of these methods had to be modified according to the special requirements of avian antibodies. The special handling of this animal system is described in regard to antibody production. The results demonstrate that IgY derived from hens can replace IgG produced by traditional methods in mammals. The advantages of this alternate animal system are emphasized in respect to animal care, high productivity, and special suitability of avian antibodies for certain diagnostic purposes.

Animals↗
PubMed1995

[Comparison of different methods for the diagnosis of freemartinism--blood group serology, cytology and polymerase chain reaction].

The percentage of freemartins among blood samples tested by chromosome analysis amounted to 83.9%, by blood group serologie 71.4%. 126 blood samples have been tested by blood group serology and PCR. Employing blood group serology, 71.3% and using PCR with BOV97M primers 85.8% of the animals proved to be freemartins. 40 blood samples were additionally analysed using PCR with zinc-finger-gene primers. 36 animals (90%) were identified as being freemartins by means of BOV97M and 34 animals (85%) by means of the zinc-finger-gene primer. The PCR method proved to be a rapid and very sensitive method for the diagnosis of freemartins and also suitable for routine testing. The BOV97M primer showed to have a higher Y chromosome specificity than the zinc-finger-gene primer.

Animals↗
PubMed1994

Binding of 9-cis-retinoic acid and all-trans-retinoic acid to retinoic acid receptors alpha, beta, and gamma. Retinoic acid receptor gamma binds all-trans-retinoic acid preferentially over 9-cis-retinoic acid.

Both 9-cis-retinoic acid (RA) and all-trans-RA (t-RA) compete for [3H]9-cis-RA binding to RA receptors (RAR alpha, beta, and gamma) in nucleosol fractions from transiently transfected COS-1 cells with IC50 values of approximately 12 and 5 nM, respectively. Curiously, 9-cis-RA competes for [3H]t-RA binding to mouse RAR alpha, beta, and gamma with IC50 values of 31, 8, and 60 nM, respectively, while t-RA itself does not exhibit such differential competition (IC50 values for RARs, 5 nM). A similar pattern is observed with human retinoic acid receptors (RARs). Differential binding of 9-cis-RA to the RAR beta and gamma receptors is also found following in vitro transcription and translation of these receptors. Displacement assays demonstrate that t-RA exhibits similar off-rates for RAR alpha, beta, and gamma. However, 9-cis-RA is 6-fold more rapidly displaced from RAR gamma than from RAR beta. When RAR-transfected COS-1 cells are incubated with [3H]t-RA, [3H]-9-cis-RA or various mixtures of these two radioligands, high performance liquid chromatography analysis demonstrates that the ligands bound in nucleosol fractions from RAR beta-transfected cells reflect the isomer content of the media. However, in identical whole cell assays, nucleosol fractions from RAR gamma-transfected cells preferentially bind t-RA over 9-cis-RA, consistent with the in vitro data. These binding kinetics in vitro and in whole cells suggest that there could be differences in the interactions of the receptor subtypes with the endogenous retinoic acids under physiologic conditions.

Animals↗
PubMed1994

Distinct binding determinants for 9-cis retinoic acid are located within AF-2 of retinoic acid receptor alpha.

Retinoids exert their physiological action by interacting with two families of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), which regulate gene expression by forming transcriptionally active heterodimeric RAR/RXR or homodimeric RXR/RXR complexes on DNA. Retinoid receptor activity resides in several regions, including the DNA and ligand binding domains, a dimerization interface, and both a ligand-independent (AF-1) and a ligand-dependent (AF-2) transactivation function. While 9-cis retinoic acid (RA) alone is the cognate ligand for the RXRs, both 9-cis RA and all-trans RA (t-RA) compete for binding with high affinity to the RARs. This latter observation suggested to us that the two isomers may interact with a common binding site. Here we report that RAR alpha has two distinct but overlapping binding sites for 9-cis RA and t-RA. Truncation of a human RAR alpha to 419 amino acids yields a receptor that binds both t-RA and 9-cis RA with high affinity, but truncation to amino acid 404 yields a mutant receptor that binds only t-RA with high affinity. Remarkably, this region also defines a C-terminal boundary for AF-2, as addition of amino acids 405 to 419 restores receptor-mediated gene activity to a truncated human RAR alpha lacking this region. It is interesting to speculate that binding of retinoid stereoisomers to unique sites within an RAR may function with AF-2 to cause differential activation of retinoid-responsive gene pathways.

Amino Acid Sequence↗
PubMed1993

Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids.

The binding of endogenous retinoids and stereoisomers of retinoic acid (RA) to the retinoid nuclear receptors, RA receptor (RARs) and retinoid X receptors (RXRs), was characterized using nucleosol preparations from transiently transfected COS-1 cells. Among several stereoisomers of RA tested, including 7-cis-, 9-cis-, 11-cis-, 13-cis-, and all-trans-RA, only 9-cis-RA effectively competes with 9-cis-[3H]RA binding to the RXRs. Additionally, the endogenous retinoid trans-didehydro-RA (t-ddRA) does not interact with RXRs, whereas the 9-cis form of ddRA competes effectively. RXRs (alpha, beta, and gamma) bind 9-cis-RA with dissociation constants (Kd) of 15.7, 18.3, and 14.1 nM, respectively. In contrast to the selectivity of RXRs for 9-cis-RA, RARs bind both t-RA and 9-cis-RA with high affinity, exhibiting Kd values in the 0.2-0.7 nM range for both ligands. Unlike RARs, the cellular RA binding proteins CRABPI or CRABPII bind t-RA but do not bind 9-cis-RA. Consistent with the binding data, 9-cis-RA and 9-cis-ddRA transcriptionally activate both GAL4-RXR and GAL4-RAR chimeric receptors with EC50 values of 3-20 nM for 9-cis-RA and 9-cis-ddRA, whereas t-RA and t-ddRA efficiently activate only GAL4-RAR chimeric receptors. Thus, 9-cis forms of endogenous retinoids can contribute to the pleiotropic effects of retinoids by interacting with both the RARs and RXRs.

Animals↗
PubMed1992

9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha.

Vitamin A (retinol) and its natural derivatives are required for many physiological processes. The activity of retinoids is thought to be mediated by interactions with two subfamilies of nuclear retinoic acid receptors, RAR and RXR. The RARs bind all-trans retinoic acid (t-RA) with high affinity and alter gene expression as a consequence of this direct ligand interaction. RXR alpha is activated by t-RA, yet has little binding affinity for this ligand. t-RA may be converted to a more proximate ligand that directly binds and activates RXR alpha, and we have developed a method of nuclear receptor-dependent ligand trapping to test this hypothesis. Here we report the identification of a stereoisomer of retinoic acid, 9-cis retinoic acid, which directly binds and activates RXR alpha. These results suggest a new role for isomerization in the physiology of natural retinoids.

Animals↗
PubMed1992

The M RNA of impatiens necrotic spot Tospovirus (Bunyaviridae) has an ambisense genomic organization.

The nucleotide sequence of Impatiens necrotic spot virus (INSV) M RNA was determined from cDNA clones. The INSV M RNA was 4972 nucleotides in length with two open reading frames (ORFs) in an ambisense genomic organization. The larger ORF near the 3' end of the viral RNA, coding for a protein with a predicted molecular weight of 124.9 kDa, was in the viral complementary sense and produced the G2 and G1 proteins. A smaller ORF in the viral sense was capable of coding for a 34.1-kDa polypeptide, designated the NSm protein. Two subgenomic RNA species were detected in INSV-infected tissue that corresponded to the predicted sizes (3.3 and 1.0 kb) of the G2-G1 and NSm mRNAs. The ORFs were separated by a 478 nucleotide A-U-rich intergenic region similar to the regions found in other viral RNAs with ambisense ORFs. The intergenic region was predicted to form a stable stem-loop structure (-81.2 kcal/mole). The ambisense genomic organization is characteristic of the S RNA for members of the Phlebovirus, Uukuvirus, and Tospovirus genera in the Bunyaviridae family. This is the first report of an ambisense Bunyaviridae M RNA.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequence of the 3' non-coding region and N gene of the S RNA of a serologically distinct tospovirus.

A tomato spotted wilt-like virus (TSWV-I) is a distinct member of the Tospovirus genus of the Bunyaviridae and is distinguished from the typical TSWV by having a serologically distinct nucleoprotein (N). A cDNA clone extending from the 3' terminus of the viral RNA through the entire N open reading frame (ORF) was sequenced. The TSWV-I N ORF is capable of encoding a polypeptide of 262 amino acids with a predicted Mr of 28.8K. In vitro transcription and translation of the clone produced a protein which comigrated with TSWV-I N and was immunoprecipitated by TSWV-I antibodies. Hybridization analysis of lithium chloride-precipitated RNA from healthy and TSWV-I-infected tissue detected a virus-specific 1.2 kb subgenomic RNA. The TSWV-I S RNA terminal consensus sequence (8 nucleotides) was identical to that of TSWV; the remaining TSWV-I untranslated region showed only 51% identity with that of TSWV. Comparison of the TSWV-I and TSWV N proteins showed 67% identity at the amino acid level. The degree of similarity in the terminal sequence, untranslated region and N ORF is similar to that expected between distinct serogroups within certain genera of the Bunyaviridae.

Amino Acid Sequence↗
PubMed1989

[In vitro parameters for understanding the immunocompetence of chickens of different lineage. 1. The cytotoxicity of avian whole blood against Saccharomyces cells].

A cytotoxicity test was performed as a parameter for the nonspecific defense capacity in chickens using blood and saccharomyces cells (SCT). The test has the special merit of frequent repetitions of the results by two parallel test arrangements. The results of individual animals were not altered during a period of 14 days, as well as relatively not being affected by actual day fluctuation as long as environmental changes or antigenic changes do not appear. The cytotoxic activity is due to cellular defense mechanisms, especially concerning the activity of granulocytes, thrombocytes and monocytes but not lymphocytes and erythrocytes. The lysozyme contents of plasma show a very space cytotoxic activity against saccharomyces cells.

Animals↗
PubMed1989

[In vitro parameters for the comprehension of the immunocompetence of chickens of different lines. 2. Testing an immunocompetent parameter as a selection criterion in the chicken].

A cytotoxicity test using blood and saccharomyces cells (SCT) was experimentally applied in a sample of pedigreed commercial chickens from different lines over 3 generations to test its usefulness as a parameter for the nonspecific defense capacity. The results clearly show a significant difference between the SC rates in the different lines. Positive heritabilities exist between the SC rates of the 3 generations. The correlations between SC rates of males in the first generation and parameters of layer productivity of their daughters are nonsignificant. The possibility to integrate the parameter SC into the selection index is critically discussed.

Animals↗
PubMed1985

Ursodeoxycholic acid treatment of bile reflux gastritis.

Intractable epigastric pain associated with nausea and bilious vomiting often follows gastric surgery and has been attributed to reflux of bile and the irritating effects of endogenous bile acids on the gastric remnant. To test the effect of changing bile acid composition of the refluxed material on the symptoms and gastric mucosal histology, 12 patients with symptomatic alkaline reflux gastritis were treated for 1 mo with placebo and for 1 mo with ursodeoxycholic acid, 1000 mg/day. Before treatment, all patients were symptomatic and manifested epigastric pain, nausea, and bilious vomiting. The gastric mucosa was erythematous, friable, and bile stained, and the histology revealed chronic inflammation. No significant change in symptoms was noted during administration of placebo. In contrast, ursodeoxycholic acid treatment resulted in a profound decrease in the intensity and frequency of pain and almost abolished nausea and vomiting. During bile acid therapy the proportion of ursodeoxycholic acid in gastric bile rose to 50% of total bile acids, whereas cholic and deoxycholic acids decreased and chenodeoxycholic acid remained unchanged. The macroscopic and microscopic appearance of the gastric mucosa, however, did not change after 1 mo of ursodeoxycholic acid treatment. These results suggest that increasing the proportion of ursodeoxycholic acid in refluxed gastric bile reduces the pain and frequency of symptoms associated with bile reflux.

Adult↗
PubMed1985

Lethal atherosclerosis associated with abnormal plasma and tissue sterol composition in sitosterolemia with xanthomatosis.

Tissue sterol composition was determined in an 18-year-old male with sitosterolemia with xanthomatosis who died suddenly and whose coronary and aortic vessels showed extensive atherosclerosis and, for comparison, in an 18-year-old male with minimal atherosclerosis who died accidently. Sterols in the control tissues (plasma, erythrocytes, cardiac muscle, lung, liver, aorta, and brain) contained cholesterol with only trace amounts of cholestanol. In contrast, sterols in corresponding tissues of the sitosterolemic subject (except brain) were composed of cholesterol, increased amounts of plant sterols, campesterol and sitosterol, and 5 alpha-saturated stanols, cholestanol, 5 alpha-campestanol, and 5 alpha-sitostanol, that were deposited in approximately the same ratio as present in plasma. However, sitosterolemic brain sterol composition resembled that of the control brain with cholesterol and only trace amounts (less than 1%) of cholestanol and phytosterols. The sitosterolemic aorta was extensively atherosclerotic and contained more than twice the quantity of sterols as the control aorta (5.6 mg/g versus 2.6 mg/g) with increased amounts of cholesterol, plant sterols, and 5 alpha-saturated stanols. These results indicate that cholesterol, plant sterols, and 5 alpha-stanols are deposited prematurely and are associated with accelerated atherosclerosis in subjects with sitosterolemia with xanthomatosis.

Adolescent↗
PubMed1982

Identification of 5 alpha-stanols in patients with sitosterolemia and xanthomatosis: stereochemistry of the protonolysis of steroidal organoboranes.

Plasma and fecal sterols of patients who exhibited tendon xanthomas with normal plasma cholesterol levels were studied. Plasma levels were (mg/dl mean +/- SD): cholesterol 232 +/- 36; cholestanol 5.9 +/- 2.1 (normal less than 0.6); sitosterol 18 +/- 5.6 (normal less than 1.0); campesterol 11 +/- 3.3 (normal less than 1.0). Other sterols such as sitostanol and campestanol (the 5 alpha-dihydro derivatives of sitosterol and campesterol) were also present in large amounts. Examination of the feces from these subjects showed cholesterol, sitosterol, campesterol and their 5 beta-saturated derivatives. Presence of 5 alpha-stanols in the plasma, and their absence in the feces indicates that the 5 alpha-stanols are synthesized endogenously within the body rather than in the intestine by colonic bacteria. The absolute identification of the structures of these 5 alpha-stanols was elucidated via hydroboration of their corresponding unsaturated sterols coupled with protonolysis of the resultant organoboranes.

Boranes↗
PubMed1982

Ursodeoxycholic acid: a safe and effective agent for dissolving cholesterol gallstones.

Ursodeoxycholic acid, 250 to 300, 500 to 600, or 900 to 1000 mg/d, was given orally for 6 to 38 months to 53 patients with cholesterol gallstones and functioning gallbladders. Forty-two patients had greater than 50% reduction in gallstone volume, number, or both, without apparent dose dependence and 27 of these patients had complete gallstone dissolution. Results of laboratory studies including liver function tests were not affected adversely and biliary lithocholic acid concentration did not increase during therapy. Most biliary symptoms seemed to disappear within 3 months and no patient developed diarrhea. Large diameter and increased number of gallstones were found to hinder dissolution. The percentage of biliary ursodeoxycholic acid increased with increasing dose and reached a maximum of 50% to 60% of total bile acids at a dose of about 10 to 12 mg/kg body weight. d. Biliary lithogenic index was reduced significantly during treatment with ursodeoxycholic acid, 500 to 600 and 900 to 1000 mg/d. Thus, ursodeoxycholic acid appears to be a safe and effective alternative to surgery in selected patients with gallstones.

Bile Acids and Salts↗