Biomedical subjects
Publications and source records attributed to D W Russell.
The second article in this three-part series on researching and evaluating geriatric mental health programs focuses on measurement of outcomes. Five issues are discussed: (1) specifying relevant outcome variables, (2) reliability of measures, (3) systematic biases in assessment, (4) validity of measures, and (5) evaluation of program costs.
Effects of social support, negative life events, and daily hassles on depressive symptoms were assessed in 301 adults aged 65 or older, in person 3 times at 6-month intervals and by mail questionnaires every month over a 12-month period. Initial social support predicted severity of depressive symptoms 12 months later. Social support and initial levels of depressive symptomatology predicted number of daily hassles but not number of major life events. Effects of social support, depression, and major life events on the incidence of daily hassles remained significant without the inclusion of hassles reflecting depressive symptomatology or problems in relationships or whose content overlapped with major life events. Daily hassles mediated the effects of major life events on subsequent depression. Results did not differ for men and women. Implications for models of the relations among social support, stress, and depression are discussed.
DNA methylation is known to regulate several prokaryotic replication origins. In particular, the Escherichia coli chromosomal origin oriC and the pMB1 plasmid origin (which is homologous to the ColE1 origin) replicate poorly when hemimethylated at dam (GATC) sites. Because the mismatch repair protein MutH is known to recognize hemimethylated dam sites, its role in the replication of these origins was investigated. The results presented here show that the mutH gene product is partially responsible for the poor replication of the pMB1 origin when hemimethylated but has no effect on the replication of oriC. Methylation levels at individual dam sites suggest that the MutH protein binds to an inverted repeat in the pMB1 replication primer promoter. These findings suggest a mechanism for the coordinated control of DNA repair and replication.
Cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase) is a microsomal cytochrome P-450 that catalyzes the first and rate-limiting step in bile acid biosynthesis, the major catabolic pathway in cholesterol homeostasis. The gene encoding the rat 7 alpha-hydroxylase has been isolated and characterized. Southern blotting experiments demonstrated that the gene is present in a single copy in the rat genome. DNA sequence analysis showed that the 7 alpha-hydroxylase gene is unique among the characterized cytochrome P-450s in that it contains only six exons. Nuclease S1 and primer-extension mapping experiments positioned the 5'-ends of the 7 alpha-hydroxylase mRNA approximately 20-25 nucleotides downstream of a consensus TATAAA sequence. RNA blotting experiments demonstrated the presence of multiple 7 alpha-hydroxylase mRNAs that differ in the lengths of their 3'-untranslated regions.
The rate-limiting step in bile acid biosynthesis is catalyzed by the microsomal cytochrome P-450 cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase). The expression of this enzyme is subject to feedback regulation by sterols and is thought to be coordinately regulated with enzymes in the cholesterol supply pathways, including the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase and synthase. Here we report the purification of rat 7 alpha-hydroxylase and the determination of a partial amino acid sequence. Oligonucleotides derived from peptide sequence were used to clone a full-length cDNA encoding 7 alpha-hydroxylase. DNA sequence analysis of the cDNA revealed a 7 alpha-hydroxylase protein of 503 amino acids with a predicted molecular weight of 56,890 which represents a novel family of cytochrome P-450 enzymes. Transfection of a 7 alpha-hydroxylase cDNA into simian COS cells resulted in the synthesis of a functional enzyme whose activity was stimulated in vitro by the addition of rat microsomal cytochrome P-450 reductase protein. RNA blot hybridization experiments indicated that the mRNA for 7 alpha-hydroxylase is found only in the liver. The levels of this mRNA increased when bile acids were depleted by dietary cholestyramine and decreased when bile acids were consumed. Dietary cholesterol led to an increase in 7 alpha-hydroxylase mRNA levels. The enzymatic activity of 7 alpha-hydroxylase paralleled the observed changes in mRNA levels. These results suggest that bile acids and sterols are able to alter the transcription of the 7 alpha-hydroxylase gene and that this control explains the previously observed feedback regulation of bile acid synthesis.
This study investigated whether social support was related to immune function among spouses of cancer patients. Effects of depression and negative life events were examined as potential mediators. Results showed evidence of greater immunocompetence on 2 of 3 dynamic measures: natural killer cytotoxicity and proliferation response to phytohemagglutinin among spouses who reported high levels of social support. All six components of social support assessed by the Social Provisions Scale (Cutrona & Russell, 1987) were strongly related to these indices of immune function. No evidence was found for mediation by either life events or depression.
The microsomal enzyme steroid 5 alpha-reductase is responsible for the conversion of testosterone into the more potent androgen dihydrotestosterone. In man, this steroid acts on a variety of androgen-responsive target tissues to mediate such diverse endocrine processes as male sexual differentiation in the fetus and prostatic growth in men. Here we describe the isolation, structure, and expression of a cDNA encoding the human steroid 5 alpha-reductase. A rat cDNA was used as a hybridization probe to screen a human prostate cDNA library. A 2.1-kilobase cDNA was identified and DNA sequence analysis indicated that the human steroid 5 alpha-reductase was a hydrophobic protein of 259 amino acids with a predicted molecular weight of 29,462. A comparison of the human and rat protein sequences revealed a 60% identity. Transfection of expression vectors containing the human and rat cDNAs into simian COS cells resulted in the synthesis of high levels of steroid 5 alpha-reductase enzyme activity. Both enzymes expressed in COS cells showed similar substrate specificities for naturally occurring steroid hormones. However, synthetic 4-azasteroids demonstrated marked differences in their abilities to inhibit the human and rat steroid 5 alpha-reductases.
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Reviewing the medical literature since 1981 show that patients with acquired immunodeficiency syndrome (AIDS) usually suffer from poor ingestion and absorption, increased excretion of nutrients, metabolic and endocrine abnormalities and immunologic abnormalities which impair nutrition. Human immunodeficiency virus (HIV) and associated opportunistic infections stimulate a broad and complex array of responses which include fever, hypermetabolism, leukocytosis, proteolysis of skeletal muscle, and synthesis, by the liver, of acute-phase reactant proteins and various intracellular enzymes. These responses increase gluconeogenesis and lipogenesis, decrease albumin synthesis, and redistribute and/or sequester various trace elements. Infection-induced depletion of body nutrients serves to weaken host resistance. The deterioration of the nutritional status of these patients is likely to have an important effect on the course of the disease. It is thus evident that comprehensive management of HIV infection must include nutritional evaluation and treatment. When oral nutrition proves to be insufficient to maintain adequate intake, alternative routes must be considered. Selection of an appropriate nutritional support formula requires an evaluation of the absorptive capacity of the gastrointestinal (GI) tract, the length of time required for nutritional repletion, costs, patient acceptance, and feasibility of at-home use. Nutritional support maximizes the ability of the AIDS patient to resist infection, may favor the response to medication by decreasing the incidence of adverse drug reactions, and may prolong the quality and productivity of life.
Seven imperfect repeats of a 40-amino acid cysteine-rich sequence constitute the ligand binding domain of the low density lipoprotein (LDL) receptor. To assess the contribution of each repeat, three site-directed mutations were made individually in each repeat: 1) deletion of the repeat, 2) substitution of a conserved isoleucine with aspartic acid, and 3) substitution of a conserved aspartic acid with tyrosine. cDNAs containing these mutations were transfected into simian COS cells and assayed for their ability to bind LDL, which contains a 500-kDa protein ligand (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains multiple copies of a 33-kDa ligand (apoE). The results showed that binding of the two ligands required different combinations of repeats. LDL binding required repeats 3-7; deletion of any one of these repeats markedly reduced LDL binding. In contrast, beta-migrating very low density lipoprotein binding was insensitive to the loss of any single repeat with the important exception of repeat 5, whose loss reduced binding by 60%. The same effects were obtained when each of the repeats was altered by either of the two substitution mutations. The current findings suggest that a multiplicity of cysteine-rich repeats may allow a single protein to bind several different protein ligands by employing different combinations of repeats.
The conversion of testosterone into the more potent androgen, dihydrotestosterone, catalyzed by the enzyme steroid 5 alpha-reductase, is required for the differentiation of male external genitalia. Here, we report the isolation of cDNA clones encoding the rat steroid 5 alpha-reductase using expression cloning in Xenopus oocytes. DNA sequence analysis demonstrates that the liver and ventral prostate forms of steroid 5 alpha-reductases are identical hydrophobic proteins of 29 kDa. The amount of steroid 5 alpha-reductase mRNA in liver increased in response to castration, but remained unchanged in the prostate. Testosterone administration to castrates induced expression of mRNA in the prostate but had no effect on liver. The data suggest that the steroid 5 alpha-reductase gene is differentially regulated by testosterone in androgen-responsive versus non-responsive tissues.
DNA methylation in Escherichia coli plays a role in many key cellular processes, including DNA replication, repair, restriction, and transcription. However, several mutant bacterial strains exist which are deficient in DNA methylase activities. Thus, it has been suggested that methylation produced by the dam (DNA adenine methylase) gene is required for the viability of E. coli and that dam- strains still produce low levels of methylation. Current experimental methods are not sensitive enough to detect a few potentially essential methylated sites per genome. Here we describe a method for the detection of N6-methyladenine at specific sites with a sensitivity of one site in more than 10 megabases. We show that methylation produced by both the dam and hsd (EcoK) genes is not required for the growth of E. coli and identify the site of EcoK modification. Minor adaptations of the technique should enable the identification of other rare DNA modifications.
The conversion of cholesterol into bile acids in the liver represents the major catabolic pathway for the removal of cholesterol from the body. In this complex biosynthetic pathway, at least 10 enzymes modify both the ring structure and side chain of cholesterol, resulting in the formation of the primary bile acids, cholic acid, and chenodeoxycholic acid. To gain insight into the details and regulation of this pathway, we have used protein sequencing and molecular cloning techniques to isolate and characterize a cDNA encoding the rabbit mitochondrial sterol 26-hydroxylase. This enzyme catalyzes the first step in the oxidation of the side chain of sterol intermediates in the biosynthesis of bile acids. The structure of the sterol 26-hydroxylase, as deduced by both DNA sequence analysis of the cDNA and protein sequence analysis, reveals it to be a mitochondrial cytochrome P-450. A signal sequence of 36 residues precedes a coding region of 499 amino acids, predicting a molecular weight of 56,657 for the mature protein. The identity of the 26-hydroxylase cDNA was further confirmed by expression in monkey COS cells employing a versatile eukaryotic expression vector. Blotting experiments revealed that the mRNA for this enzyme is expressed in many tissues and that it is encoded by a low copy number gene in the rabbit genome.
Cholesterol homeostasis is maintained by coordinate regulation of endogenous synthesis and exogenous uptake of lipoprotein cholesterol by low density lipoprotein (LDL) receptors. In the lymphocyte, limiting the availability of exogenous cholesterol is known to increase the rate of endogenous sterol biosynthesis. However, the effect of cholesterol deprivation on the expression and regulation of the LDL receptor gene has not been delineated in lymphocytes. Here, LDL receptor mRNA was detected in freshly isolated human peripheral mononuclear cells. LDL receptor mRNA levels increased by 3-fold during a one-h in vitro culture in lipoprotein-deficient medium and by 6-fold during a 2-h incubation. Actinomycin D blocked the synthesis of LDL receptor mRNA in these cultures. However, neither cycloheximide nor LDL or oxygenated sterols suppressed the increase in LDL receptor mRNA levels observed after a 2-h incubation. The increase in LDL receptor mRNA was maintained for 24 h of culture in the absence of LDL. Ongoing gene transcription and not mRNA stabilization accounted for this expression. Inhibition of protein synthesis with cycloheximide completely prevented the sustained increase in LDL receptor mRNA levels measured after 24 h. Low concentrations of LDL (5 micrograms of cholesterol/ml) and oxygenated sterols also suppressed the level of LDL receptor mRNA measured after a 24-h incubation. These data show that the initial upregulation of LDL receptor gene expression is independent of protein synthesis and not suppressed by either LDL or oxygenated sterols. In contrast, the continued transcription necessary for the maintenance of steady-state levels of LDL receptor mRNA requires synthesis of new protein and is regulated by LDL and oxygenated sterols.
The Iowa 65+ Rural Health Study gathered health status information on all elderly persons living in two rural Iowa counties. In this report these data are used to determine the ratio of persons with activities of daily living (ADL) dependencies living in the community to those in institutions. Results indicated that the "community/institutional dependency ratio" is about 1 to 1 for these counties, which is about half the ratio representing conventional wisdom. Possible explanations for this difference are discussed. In addition, it was found that the level of ADL dependency (need) can serve alone as an almost certain predictor of institutionalization for some elderly. For others, ADL dependency (need) is only one factor. The likely variability of the community/institutional dependency ratio across different geographic areas has implications for government funding of home health care, for long-term care insurance, and for eliminating excess demand. These implications are discussed.
Certain individuals with familial hypercholesterolemia (FH) produce mutant forms of the low density lipoprotein (LDL) receptor that fail to move from the endoplasmic reticulum to the Golgi complex. Here, we describe the cloning and expression of one such mutant allele, FH 429. The mutation causes a substitution of a Val for a Gly at residue 544. When recreated in an expressible cDNA, this substitution gives rise to an LDL receptor that is not transported to the cell surface and is rapidly degraded. Three previously mapped transport-deficient alleles of the LDL receptor were traced to the cysteine-rich repeats of the protein, suggesting that the generation of non-disulfide-bonded (free) cysteines might cause the block in transport. The FH 429 mutation is not located in a cysteine-rich region, however. We have attempted to test the role of cysteine by expressing mutant cDNAs that encode proteins blocked in transport and predicted to contain free cysteines. The results suggest that free cysteines are not obligatory for the blocked intracellular movement of mutant LDL receptors.
The ligand binding domain of the low density lipoprotein (LDL) receptor contains seven imperfect repeats of a 40-amino acid cysteine-rich sequence. Each repeat contains clustered negative charges that have been postulated as ligand-binding sites. The adjacent region of the protein, the growth factor homology region, contains three cysteine-rich repeats (A-C) whose sequence differs from those in the ligand binding domain. To dissect the contribution of these different cysteine-rich repeats to ligand binding, we used oligonucleotide-directed mutagenesis to alter expressible cDNAs for the human LDL receptor which were then introduced into monkey COS cells by transfection. We measured the ability of the mutant receptors to bind LDL, which contains a single protein ligand for the receptor (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains apoB-100 plus multiple copies of another ligand (apoE). The results show that repeat 1 is not required for binding of either ligand. Repeats 2 plus 3 and repeats 6 plus 7 are required for maximal binding of LDL, but not beta-VLDL. Repeat 5 is required for binding of both ligands. Repeat A in the growth factor homology region is required for binding of LDL, but not beta-VLDL. Repeat B is not required for ligand binding. These results support a model for the LDL receptor in which various repeats play additive roles in ligand binding, each repeat making a separate contribution to the binding event.
The human low density lipoprotein (LDL) receptor is shown to carry out efficient receptor-mediated endocytosis in Xenopus laevis oocytes. Microinjection of mRNAs encoding the human receptor led to synthesis of a 120-kDa precursor possessing high mannose N-linked sugars and core O-linked sugars. During its transport to the cell surface, the protein increased in apparent size to 160 kDa, which is similar to the change that occurs in human cells. This increase was not seen when the receptor lacked the serine/threonine-rich region that undergoes O-linked glycosylation. The surface receptors bound 125I-LDL at 0 degrees C and internalized it with a half-time of 2 min when the cells were warmed to 19 degrees C. The rate of internalization was slowed by 7-fold when a single residue in the cytoplasmic domain (Tyr807) was changed to a cysteine, an alteration that slows incorporation into coated pits in mammalian cells. Deletion of the cytoplasmic domain abolished rapid internalization. We conclude that the signals for O-linked glycosylation and receptor-mediated endocytosis of the LDL receptor have been conserved throughout vertebrate evolution.