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M S Brown

Publications and source records attributed to M S Brown.

At least 91 records · Page 5Linked to original sources
PubMed1997

Sphingomyelin depletion in cultured cells blocks proteolysis of sterol regulatory element binding proteins at site 1.

The current studies explore the mechanism by which the sphingomyelin content of mammalian cells regulates transcription of genes encoding enzymes of cholesterol synthesis. Previous studies by others have shown that depletion of sphingomyelin by treatment with neutral sphingomyelinase causes a fraction of cellular cholesterol to translocate from the plasma membrane to the endoplasmic reticulum where it expands a regulatory pool that leads to down-regulation of cholesterol synthesis and up-regulation of cholesterol esterification. Here we show that sphingomyelinase treatment of cultured Chinese hamster ovary cells prevents the nuclear entry of sterol regulatory element binding protein-2 (SREBP-2), a membrane-bound transcription factor required for transcription of several genes involved in the biosynthesis and uptake of cholesterol. Nuclear entry is blocked because sphingomyelinase treatment inhibits the proteolytic cleavage of SREBP-2 at site 1, thereby preventing release of the active NH2-terminal fragments from cell membranes. Sphingomyelinase treatment thus mimics the inhibitory effect on SREBP processing that occurs when exogenous sterols are added to cells. Sphingomyelinase treatment did not block site 1 proteolysis of SREBP-2 in 25-RA cells, a line of Chinese hamster ovary cells that is resistant to the suppressive effects of sterols, owing to an activating point mutation in the gene encoding SREBP cleavage-activating protein. In 25-RA cells, sphingomyelinase treatment also failed to down-regulate the mRNA for 3-hydroxy-3-methylglutaryl CoA synthase, a cholesterol biosynthetic enzyme whose transcription depends on the cleavage of SREBPs. Considered together with previous data, the current results indicate that cells regulate the balance between cholesterol and sphingomyelin content by regulating the proteolytic cleavage of SREBPs.

Animals↗
PubMed1997

Identification of complexes between the COOH-terminal domains of sterol regulatory element-binding proteins (SREBPs) and SREBP cleavage-activating protein.

SREBP cleavage-activating protein (SCAP) stimulates the proteolytic cleavage of membrane-bound SREBPs, thereby initiating the release of NH2-terminal fragments from cell membranes. The liberated fragments enter the nucleus and stimulate transcription of genes involved in synthesis and uptake of cholesterol and fatty acids. Sterols repress cleavage of SREBPs, apparently by interacting with the membrane attachment domain of SCAP. In the present studies we show that SCAP, like the SREBPs, is located in membranes of the endoplasmic reticulum and nuclear envelope. The COOH-terminal domain of SCAP, like that of the SREBPs, is located on the cytosolic face of the membranes. Co-immunoprecipitation experiments show that SCAP and SREBP-2 form a complex that can be precipitated with antibodies to either component. Complex formation occurs when cells express only the COOH-terminal domain of either SREBP-2 or SCAP, indicating that the complex forms between the two COOH-terminal domains. Truncation of SREBP-2 at its COOH terminus prevents the formation of complexes with SCAP and simultaneously reduces proteolytic cleavage. We conclude that proteolytic cleavage of SREBPs requires the formation of a complex with the COOH-terminal domain of SCAP and that SCAP is therefore a required element in the regulation of sterol and fatty acid metabolism in animal cells.

Animals↗
PubMed1997

Cleavage site for sterol-regulated protease localized to a leu-Ser bond in the lumenal loop of sterol regulatory element-binding protein-2.

A sterol-regulated protease initiates release of the NH2-terminal segments of sterol regulatory element-binding proteins (SREBPs) from cell membranes, thereby allowing them to enter the nucleus and to stimulate transcription of genes involved in the uptake and synthesis of cholesterol and fatty acids. Using SREBP-2 as a prototype, we here identify the site of sterol-regulated cleavage as the Leu522-Ser523 bond in the middle of the 31-residue hydrophilic loop that projects into the lumen of the endoplasmic reticulum and nuclear envelope. This site was identified through use of a vector encoding an SREBP-2/Ras fusion protein with a triple epitope tag that allowed immunoprecipitation of the cleaved COOH-terminal fragment. The NH2 terminus of this fragment was pinpointed by radiochemical sequencing after replacement of selected codons with methionine codons and labeling the cells with [35S]methionine. Alanine scanning mutagenesis revealed that only two amino acids are necessary for recognition by the sterol-regulated protease: 1) the leucine at the cleavage site (leucine 522), and 2) the arginine at the P4 position (arginine 519). These define a tetrapeptide sequence, RXXL, that is necessary for cleavage. Cleavage was not affected when the second transmembrane helix of SREBP-2 was replaced with the membrane-spanning region of the low density lipoprotein receptor, indicating that this sequence is not required for regulation. Glycosylation-site insertion experiments confirmed that leucine 522 is located in the lumen of the endoplasmic reticulum. We conclude that the sterol-regulated protease is a novel enzyme whose active site faces the lumen of the nuclear envelope, endoplasmic reticulum, or another membrane organelle to which the SREBPs may be transported before cleavage.

Amino Acid Sequence↗
PubMed1997

Differential expression of exons 1a and 1c in mRNAs for sterol regulatory element binding protein-1 in human and mouse organs and cultured cells.

The 5' end of the mRNA-encoding sterol regulatory element binding protein-1 (SREBP-1) exists in two forms, designated 1a and 1c. The divergence results from the use of two transcription start sites that produce two separate 5' exons, each of which is spliced to a common exon 2. Here we show that the ratio of SREBP-1c to 1a transcripts varies markedly among organs of the adult mouse. At one extreme is the liver, in which the 1c transcript predominates by a 9:1 ratio. High 1c:1a ratios are also found in mouse adrenal gland and adipose tissue and in human liver and adrenal gland. At the other extreme is the spleen, which shows a reversed 1c:1a ratio (1:10). In five different lines of cultured cells, including the HepG2 line derived from human hepatocytes, the 1a transcript predominated (1c:1a ratio < 1:2). In mouse 3T3-L1 preadipocytes, the 1a transcript was present, but the 1c transcript was not detectable. When these cells were differentiated into adipocytes by hormone treatment in culture, the amount of 1a transcript rose markedly (8.2-fold), and the 1c transcript remained virtually undetectable. We conclude that the SREBP-1a and 1c transcripts are controlled independently by regulatory regions that respond differentially to organ-specific and metabolic factors.

Adipose Tissue↗
PubMed1997

Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured cells.

We have produced transgenic mice whose livers express a dominant positive NH2-terminal fragment of sterol regulatory element binding protein-1c (SREBP-1c). Unlike full-length SREBP-1c, the NH2-terminal fragment enters the nucleus without a requirement for proteolytic release from cell membranes, and hence it is immune to downregulation by sterols. We compared SREBP-1c transgenic mice with a line of transgenic mice that produces an equal amount of the NH2-terminal fragment of SREBP-1a. SREBP-1a and -1c are alternate transcripts from a single gene that differ in the first exon, which encodes part of an acidic activation domain. The 1a protein contains a long activation domain with 12 negatively charged amino acids, whereas the 1c protein contains a short activation domain with only 6 such amino acids. As previously reported, livers of the SREBP-1a transgenic mice were massively enlarged, owing to accumulation of triglycerides and cholesterol. SREBP-1c transgenic livers were only slightly enlarged with only a moderate increase in triglycerides, but not cholesterol. The mRNAs for the LDL receptor and several cholesterol biosynthetic enzymes were elevated in SREBP-la transgenic mice, but not in 1c transgenic mice. The mRNAs for fatty acid synthase and acetyl CoA carboxylase were elevated 9- and 16-fold in la animals, but only 2- and 4-fold in 1c animals. Experiments with transfected cells confirmed that SREBP-1c is a much weaker activator of transcription than SREBP-1a when both are expressed at levels approximating those found in nontransfected cells. SREBP-1c became a strong activator only when expressed at supraphysiologic levels. We conclude that SREBP-1a is the most active form of SREBP-1 and that SREBP-1c may be produced when cells require a lower rate of transcription of genes regulating cholesterol and fatty acid metabolism.

Acetyl-CoA Carboxylase↗
PubMed1997

Complementation cloning of S2P, a gene encoding a putative metalloprotease required for intramembrane cleavage of SREBPs.

We report the cloning of a gene, S2P, that encodes a putative metalloprotease required for intramembrane proteolysis of sterol-regulatory element-binding proteins (SREBPs) at Site-2. SREBPs are membrane-bound transcription factors that activate genes regulating cholesterol metabolism. The active NH2-terminal domains of SREBPs are released from membranes by sequential cleavage at two sites: Site-1, within the lumen of the endoplasmic reticulum; and Site-2, within a transmembrane segment. The human S2P gene was cloned by complementation of mutant CHO cells that cannot cleave SREBPs at Site-2 and are cholesterol auxotrophs. S2P defines a new family of polytopic membrane proteins that contain an HEXXH sequence characteristic of zinc metalloproteases. Mutation of the putative zinc-binding residues abolishes S2P activity. S2P encodes an unusual metalloprotease that cleaves proteins within transmembrane segments.

Animals↗
PubMed1997

Method for segmenting chest CT image data using an anatomical model: preliminary results.

We present an automated, knowledge-based method for segmenting chest computed tomography (CT) datasets. Anatomical knowledge including expected volume, shape, relative position, and X-ray attenuation of organs provides feature constraints that guide the segmentation process. Knowledge is represented at a high level using an explicit anatomical model. The model is stored in a frame-based semantic network and anatomical variability is incorporated using fuzzy sets. A blackboard architecture permits the data representation and processing algorithms in the model domain to be independent of those in the image domain. Knowledge-constrained segmentation routines extract contiguous three-dimensional (3-D) sets of voxels, and their feature-space representations are posted on the blackboard. An inference engine uses fuzzy logic to match image to model objects based on the feature constraints. Strict separation of model and image domains allows for systematic extension of the knowledge base. In preliminary experiments, the method has been applied to a small number of thoracic CT datasets. Based on subjective visual assessment by experienced thoracic radiologists, basic anatomic structures such as the lungs, central tracheobronchial tree, chest wall, and mediastinum were successfully segmented. To demonstrate the extensibility of the system, knowledge was added to represent the more complex anatomy of lung lesions in contact with vessels or the chest wall. Visual inspection of these segmented lesions was also favorable. These preliminary results suggest that use of expert knowledge provides an increased level of automation compared with low-level segmentation techniques. Moreover, the knowledge-based approach may better discriminate between structures of similar attenuation and anatomic contiguity. Further validation is required.

Computer Simulation↗
PubMed1997

Industry information technology trends.

The coming year will be full of critical challenges for home care and hospice industry executives and senior managers. Information technology (IT), although posing its own challenges, will be a key solution to surviving and thriving through 1998 and beyond. These are the results of a ground-breaking survey conducted during the 16th Annual Meeting of the National Association for Home Care (NAHC) and the World Congress on Homecare and Hospice.

Computer Communication Networks↗
PubMed1997

Cornea-contact lens interaction in the aquatic environment.

PURPOSE: A large population of ametropic scuba divers wear contact lenses. We discuss optics and corneal physiology, as well as the types of contact lenses that are appropriate for underwater activities. METHODS: We reviewed an extensive body of literature to formulate guidelines to aid the contact lens fitter in satisfying individual sport diver's needs. RESULTS: Optical factors such as image displacement and light wave-length shifts require that contact lenses for underwater use be suitably modified. Underwater images appear nearer and larger (requiring greater accommodation) and are made up almost exclusively of the short wavelength end of the spectrum. Correction of presbyopia, in particular, is influenced by these factors. For example, presbyopic contact lens-corrected myopes require greater near adds underwater than when viewing the same objects in air. In general, presbyopes should consider monovision correction to facilitate underwater visual tasks. Although divers wearing rigid gas permeable contact lenses run the risk of more corneal problems than soft lens wearers if conservative ascents are not adhered to, there are no compelling reasons to change lens types in patients who are already fully adapted. Soft contacts, while very stable on the eye during diving, present a greater risk of lens contamination by sea or fresh water exposure. However, the latter problems are easily overcome by using disposable soft lenses. CONCLUSION: In this paper, we present several suggestions for lens material, modifications required for underwater ametropia correction, and wearing modalities for the sport divers. An understanding of the dramatic changes that impact the properties of light, corneal physiology, and visual perception which accompany the diver below the surface will enable the contact lens fitter to design a lens appropriate to the needs of the individual patient.

Contact Lenses, Hydrophilic↗
PubMed1996

Recurrent G-to-A substitution in a single codon of SREBP cleavage-activating protein causes sterol resistance in three mutant Chinese hamster ovary cell lines.

Oxygenated sterols such as 25-hydroxycholesterol kill Chinese hamster ovary cells because they inhibit the proteolytic processing of sterol regulatory element binding proteins (SREBPs), a pair of membrane-bound transcription factors that activate genes controlling cholesterol synthesis and uptake from lipoproteins. The unprocessed SREBPs remain membrane-bound, they cannot activate the cholesterol biosynthetic pathway, and the cells die of cholesterol deprivation. Several sterol-resistant hamster cell lines have been isolated previously by chemical mutagenesis and selection for resistance to killing by 25-hydroxycholesterol. We recently identified the defect in one such cell line (25-RA cells) as a point mutation in a newly discovered membrane protein of 1276 amino acids, designated SREBP cleavage-activating protein (SCAP). The mutation in the 25-RA cells resulted from a G-to-A transition in codon 443 of the SCAP gene, changing aspartic acid to asparagine. Wild-type SCAP, when overexpressed by transfection, stimulates the proteolytic processing of both SREBPs. The D443N substitution is an activating mutation that increases the activity of SCAP and renders it resistant to inhibition by 25-hydroxycholesterol. We here report the identical G-to-A transition in two additional lines of Chinese hamster ovary cells that were mutagenized and isolated by a similar protocol. The three mutations occurred independently as indicated by haplotype analysis of the mutant genes using two intragenic sequence polymorphisms. All three cell lines were mutagenized with alkylating agents (nitrosoethylurea or ethylmethane sulfonate) that favor G-to-A transitions. Nevertheless, the finding of the same nucleotide substitution at the same location in all three cell lines indicates that SCAP may be unique in its ability to stimulate SREBP cleavage, and residue 443 is a crucial determinant of the protein's ability to be inhibited by 25-hydroxycholesterol.

Adenine↗