Showing papers by "DeWitt S. Goodman published in 1990"

Distribution of transthyretin in the rat eye.

Abstract: We reported previously synthesis of transthyretin (TTR), or prealbumin, a transport protein for thyroxine and retinol, in the eyes of rats and cows and showed that in the rat eye, TTR mRNA is localized exclusively in the retinal pigment epithelium (RPE). We now demonstrate by immunohistochemistry that TTR has a more widespread distribution in the rat eye than does its mRNA. Intense immunoreactivity for TTR was found in the RPE, ciliary epithelium, iris epithelium, corneal endothelium, optic nerve fiber layer of the retina, and lens capsule. Depending on the method of processing, immunoreactivity of varying intensity was found also in other ocular structures. In particular, the retinal ganglion cells were strongly immunoreactive on frozen sections but not on paraffin sections. Although vitreous humor was not included in the sections of adult rat eye, sections of a 25-mm rat embryo showed intense immunoreactivity in the vitreous humor. Since plasma TTR does not cross Bruch's membrane into the retina, our findings suggest that ocular TTR is synthesized, at least in part, in the RPE and is transported to specific locations within the eye. Although the physiologic role of ocular TTR is unknown, it is possible that it participates in retinol cycling within the eye. The widespread ocular distribution of TTR may account for the occurrence of various forms of ocular amyloidosis in the familial amyloidotic polyneuropathies, a group of dominantly inherited disorders caused by point mutations in the TTR gene.

Lack of effect of lovastatin therapy on the parameters of whole-body cholesterol metabolism.

Abstract: The effects of lovastatin therapy on the parameters of body cholesterol metabolism were explored in nine hypercholesterolemic patients. Long-term cholesterol turnover studies were performed before therapy, and were repeated after 15 mo of lovastatin therapy (40 mg/d) while continuing on therapy. The major question addressed was whether a reduction in plasma cholesterol level with lovastatin would be associated with a reduction in the whole-body production rate of cholesterol or with the sizes of exchangeable body cholesterol pools as determined by the three-pool model of cholesterol turnover. The mean plasma cholesterol level decreased 19.4% (from 294 to 237 mg/dl), and low-density lipoprotein cholesterol decreased 23.8% (from 210 to 159 mg/dl) with lovastatin therapy. Changes in high-density lipoprotein cholesterol level were not significant. The cholesterol production rate did not change significantly with therapy (1.09 +/- 0.10 [mean +/- S.D.] vs. 1.17 +/- 0.09 g/d). By comparison, colestipol and niacin treatment in three other subjects more than doubled the cholesterol production rate (1.14 +/- 0.28 vs. 2.42 +/- 0.34 g/d). Thus, hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibition by lovastatin at the therapeutic dose used here did not change the steady-state rate of whole-body cholesterol synthesis. Despite the changes in plasma cholesterol levels, no significant changes were seen in the values of M1, of M3 or of Mtot, the sizes of the pools of rapidly, of slowly, and of total body exchangeable cholesterol. Conclusion: lovastatin therapy to lower plasma cholesterol does not lead to corresponding reductions in body cholesterol pools or to a reduction in the rate of whole-body cholesterol synthesis. In the new steady state that exists during long-term lovastatin therapy, along with increased expression of the genes for HMG-CoA reductase and the LDL receptor, the body compensates for the effects of the drug so that cholesterol production rate and tissue pool sizes are not changed from pretreatment values.

Localization of cellular retinol-binding protein mRNA in rat testis and epididymis and its stage-dependent expression during the cycle of the seminiferous epithelium.

Abstract: Anatomical localization of cellular retinol-binding protein (CRBP) mRNA was examined in normal rat testis and epididymis and also in retinoid-deficient rat testis. In situ hybridization was performed with 35S-labeled rat CRBP cRNA probes on frozen tissue sections. In normal testis, CRBP mRNA was mainly localized in the Sertoli cells and to some extent in peritubular cells. A distinct cyclic variation of the relative levels of hybridizable CRBP mRNA was observed during the spermatogenic cycle. The peak of CRBP mRNA content was seen in the stages of the cycle that preceded those in which peak CRBP protein content had been observed previously in our laboratory by immunohistochemistry. No appreciable amount of CRBP mRNA was observed in the interstitial space or in the lumen of the tubules. CRBP mRNA displayed the same anatomical localization in the retinoid-deficient testis, but the level of hybridizable CRBP mRNA was substantially reduced. A strong hybridization signal for CRBP mRNA was seen in proximal epididymis and was strikingly localized in the ductular epithelium. CRBP mRNA was not detectable in the distal portion of the epididymis. These studies provide information about the cell-specific expression of CRBP synthesis within the testis and epididymis and about its cyclic variation and regulation.

Purification and properties of plasma retinol-binding protein.

Abstract: Publisher Summary This chapter describes some of the physicochemical properties of retinol-binding protein (RBP) with an emphasis on those properties that are important for the purification of RBP. The chapter describes, in detail, procedures that employ conventional column chromatography for the purification of rat RBP and human RBP from plasma. The procedures for the purification of RBP from these two species are similar. Both procedures initially isolate RBP as a complex with transthyretin (TTR) and separate the RBP from the TTR in a final gel-filtration step. For the purification of rat and human RBP, some representative column elution profiles have been provided. The chapter concludes with a brief discussion on RBP gene structure and expression. It has been hypothesized that RBP synthesized in extrahepatic tissues may play an important role in the transport and recycling of retinol in the body.

Monoclonal antibody studies of the antigenic determinants of human plasma retinol-binding protein.

Abstract: A battery of monoclonal antibodies (MoAbs) against human retinol-binding protein (RBP) was produced to obtain useful probes for the study of the antigenic determinants of RBP. The 12 antibodies all reacted with human RBP by immunoblotting. Based on antibody cross-competition radioimmunoassays, four distinct and different groups of antibodies were identified: group I, 1A4 and 2F4; group II, 1G10, 5C5, 6F4, and 7G3; group III, 5H6, 6C7, 10G5, and 14E3; and group IV, 5H9 and 13A1. Information about the epitopes of RBP recognized by these MoAbs was obtained by testing the reactivity of each antibody with human, rabbit, and rat RBPs by immunoblotting. Group I and group IV antibodies reacted to a similar extent with human, rabbit, and rat RBPs. Group II antibodies reacted strongly with human and rabbit RBPs, but reacted very weakly with rat RBP. Group III antibodies reacted strongly with human RBP, but did not react with rabbit or rat RBP. Thus, the epitopes for group I and group IV antibodies appear to be regions of the RBP molecule that are conserved across the three species, whereas group III antibodies recognized only human RBP. In a preliminary study, the reactivity of each antibody with purified cyanogen bromide fragments of RBP was tested by slot immunoblotting. None of the MoAbs reacted with any of the cyanogen bromide fragments. This study shows that MoAbs specific for at least four different regions of the RBP molecule can be produced; hence, RBP contains at least four major antigenic domains.

In situ hybridization of retinoid-binding protein messenger RNA.

Abstract: Publisher Summary In situ hybridization is a very powerful technique to localize specific mRNA molecules directly within a cell. This technique provides a combination of molecular and morphological information about individual cells in a tissue, in contrast to the information that can be obtained from the analysis of a pool of RNA isolated from a tissue or organ. Because it combines both molecular and morphological analyses, in situ hybridization is a valuable technique in several areas of biomedical research, including developmental biology, cell biology, genetics, and pathology. The technique of in situ hybridization has been used to localize retinol binding protein (RBP) mRNA and transthyretin (TTR) mRNA in fetuses of a wide range of gestational ages, brain, kidney, adipose tissue, and F9 teratocarcinoma cells. This chapter discusses the technique of in situ hybridization that is divided into four parts: (1) preparation of tissue sections, (2) probe preparation, (3) hybridization of tissue sections, and (4) autoradiographic analysis and staining of tissue sections.