A candidate gene approach to searching for low-penetrance breast and prostate cancer genes.

Most cases of breast and prostate cancer are not associated with mutations in known high-penetrance genes, indicating the involvement of multiple low-penetrance risk alleles. Studies that have attempted to identify these genes have met with limited success. The National Cancer Institute Breast and Prostate Cancer Cohort Consortium--a pooled analysis of multiple large cohort studies with a total of more than 5,000 cases of breast cancer and 8,000 cases of prostate cancer--was therefore initiated. The goal of this consortium is to characterize variations in approximately 50 genes that mediate two pathways that are associated with these cancers--the steroid-hormone metabolism pathway and the insulin-like growth factor signalling pathway--and to associate these variations with cancer risk.

Lessons from postgenome-wide association studies: functional analysis of cancer predisposition loci.

In the last few years, genome-wide association studies (GWASs) have identified hundreds of predisposition loci for several types of human cancers. Recent progress has been made in determining the underlying mechanisms through which different single-nucleotide polymorphisms (SNPs) affect predisposition to cancer. Although there has been much debate about the clinical utility of GWASs, less attention has been paid to how GWASs and post-GWASs functional analysis have contributed to understanding the aetiology of cancer. Most common variants associated with cancer risk are localized in nonprotein-coding regions highlighting transcriptional regulation as a common theme in the mechanism of cancer predisposition. Here, we outline strategies to functionally dissect predisposition loci and discuss their limitations as well as challenges for future studies.

Membranes in polyribosome formation by rabbit reticulocytes.

When rabbit reticulocytes are incubated with n-butanol, an agent disruptive to the structure and function of cellular membranes, there is a rapid disaggregation of the polyribosomes. Reaggregation is promoted when the n-butanol is diluted below a critical concentration or when the cells are washed free of the alcohol and the incubation is continued. Neither disaggregation nor regeneration will occur in the absence of protein synthesis. These observations suggest that integrity of the reticulocyte membrane is necessary for the attachment of ribosomes to messenger RNA and for the formation of polyribosomes.

A rabbit reticulocyte model for the role of hemin-controlled repressor in hypochromic anemias.

Hemin allows maximal protein synthesis in intact rabbit reticulocytes and their cell-free lysate preparations by retarding the formation of a translational repressor (HCR) found in the postribosomal supernate. In order to evaluate the role of HCR in the pathogenesis of hypochromic anemias, HCR was isolated and partially purified from intact rabbit reticulocytes incubated in vitro with either 0.1 mM alpha,alpha-dipyridyl (an iron-chelating agent) or 0.1 M ethanol. Both of these agents inhibit reticulocyte protein synthesis. Hemin (50 muM) protects against the inhibition by both agents. A ferrous iron-transferrin mixture, however, protects only against alpha,alpha-dipyridyl. Both alpha,alpha-dipyridyl and ethanol inhibit heme synthesis before the time that protein synthesis is affected, while neither lowers either ATP or GSH levels. These results indicate that while both agents inhibit heme synthesis, alpha,alpha-dipyridyl does so by inducing iron deficiency while ethanol works at a non-iron-requiring step. When HCR was isolated from intact cells and assayed in the reticulocyte cell-free systems, plus and minus hemin, premature appearance of HCR was found in cells incubated in vitro with alpha,alpha-dipyridyl or ethanol. When hemin was present in the intact cell incubation, the appearance of HCR was retarded. The HCR from alpha,alpha-dipyridyl ethanol-treated cells was partially purified and eluted at the same location on a Sephadex G-200 column (molecular weight approximately 3 x 10(5)) as that from postribosomal supernates incubated minus hemin. In addition rabbits with phenylhydrazine-induced hemolytic anemia were given intravenous ethanol in vivo at a dose of 0.4 ml/kg. This concentration of alcohol resulted in an inhibition of the rate of heme synthesis and protein synthesis as well as an acceleration of HCR formation in reticulocytes. The HCR from these in vivo treated rabbits was isolated, partially purified, and assayed in an identical fashion as the in vitro experiments. These in vivo experiments further support the physiological and pathophysiological role of HCR in reticulocytes. On the basis of these results a model for a role of HCR in some of the hypochromic anemias is proposed. In iron deficiency or chronic disease (where iron is not available to the erythroblast for heme synthesis) HCR appears prematurely and inhibits protein synthesis. When heme synthesis is inhibited by ethanol but there is sufficient intracellular iron, HCR appears prematurely and inhibits protein synthesis, iron accumulates in the erythroblast, and the end result is sideroblastic anemia.

Heat inhibition of reticulocyte protein synthesis. Evidence for a mechanism independent of the hemin-controlled repressor.

Incubation of rabbit reticulocytes at 45 degrees C results in a prompt but reversible decrease in protein synthesis and a concomitant conversion of polyribosomes to smaller aggregates. These effects occur even in the presence of 100 micrometer hemin in the incubation medium. There is also inhibition of heme synthesis but this occurs at a later time than the effect on protein synthesis. The inhibtion of heme synthesis results from a decrease in activity of beta-aminolevulinic acid synthetase. This decrease of heme synthesis appears to be secondary to the inhibition of protein synthesis with resultant accumulation of intramitochondrial heme (which will decrease beta-aminolevulinic acid synthetase activity). An inhibitor of reticulocyte cell-free protein synthesis formed in the postribosomal supernatants of cells incubated at both 45 and 37 degrees C but not at 0 degrees C. No temporal or quantitative differences in the amount of this inhibitor from cells treated at either 37 or 45 degrees C was apparent. The inhibitor was not found in the fraction where the hemin-controlled repressor is isolated. It is concluded that heat inactivation of intact reticulocyte protein synthesis does not depend upon a decrease in heme synthesis, heme concentration or generation of the hemin-controlled repressor. Furthermore, it appears that the inhibitor formed in the post-ribosomal supernatant cannot be the sole cause of the heat inhibition of protein synthesis.

Iron requirement for isolated rat liver mitochondrial protein synthesis.

Isolated rat liver mitochondrial protein synthesis was severly inhibited by alpha, alpha-dipyridyl (a ferrous iron-chelating agent), chloramphenicol and hemin (10(-7) M or greater). In contrast, gamma, gamma-dipyridyl (a non-iron-chelating analogue of alpha, alpha-dipyridyl), cycloheximide and lower concentrations of hemin were non-inhibitory. The inhibitory action of alpha, alpha-dipyridyl was reversed by addition of Fe(NH4)2(SO4)2 while ZnCl2, CuCl2 and CoCl2 were ineffective. Hemin, however, did not protect against the alpha, alpha-dipyridyl inhibition of mitochondrial protein synthesis. These results indicate that ferrous iron is required for mitochondrial protein synthesis and suggests that it is through a mechanism independent of hemin concentration.

Red cell distribution width, mean corpuscular volume, and transferrin saturation in the diagnosis of iron deficiency.

The usefulness of the red cell distribution width, mean corpuscular volume, and the transferrin saturation in diagnosing iron deficiency anemia were evaluated in a retrospective study of 247 anemic hospitalized patients, many of whom had chronic liver disease. A red cell distribution width greater than 15% had a sensitivity of 71% and a specificity of 54% for iron deficiency as diagnosed by a low serum ferritin or bone marrow examination. A mean corpuscular volume less than 80 femtoliters had a sensitivity of 53% and a specificity of 84%. Transferrin saturation less than 16% had a sensitivity of 61% and a specificity of 86%. Because the sensitivities and specificities of these tests are less than reported in studies of healthier populations, they cannot be relied on for screening for iron deficiency in sick hospitalized patients.

Quantitative neuronal c-fos and c-jun expression in Alzheimer's disease.

Apoptosis, or programmed cell death, has been proposed as a mechanism of neuropathology in Alzheimer's disease (AD). Activation of immediate early genes (IEG) c-jun and c-fos appears to be required for the initiation of apoptosis. Furthermore, the expression of c-jun is induced in cultured neurons that undergo beta-amyloid-mediated apoptosis suggesting a direct role for c-jun in the apoptosis of AD neurons. Using immunohistochemical methods, we calculated the average number of neuronal profiles per unit area expressing c-Jun and c-Fos within hippocampal regions CA1, CA2/3, and CA4 in postmortem brain samples from AD patients and age-matched non-AD patients. There was an increase in c-Jun-positive and c-Fos-positive neuronal profile density in nearly all AD hippocampal regions examined. In cerebellum there was no evidence of apoptosis as determined by using TUNEL technique, and negligible c-Jun labeling.

Delayed gastric emptying and improvement with domperidone in a patient with anorexia nervosa.

A woman with anorexia nervosa who displayed severe bloating after eating was treated with domperidone, a novel compound with prokinetic properties. Both subjective ratings of satiety and assessment of gastric emptying documented improvement.

Age-related decline in the biosynthesis of mitochondrial inner membrane proteins.

Isolated mitochondria from rat livers of various ages show a gradual decline in the rate of inner membrane-matrix protein synthesis with advancing age of the animal. Rats at 112-120 weeks synthesize these proteins at only 40% of the rate of 2-8-week-old animals. The initial rates of incorporation of label were 145 cpm/mg/minute for the "old" animals, and 320 cpm/mg/minute for the "young" animal. No difference in either amino acid pool size or leakage of label through the mitochondrial membrane was detected in the two age groups. Treatment of the mitochondria with ferrous ammonium sulphate produced a 1.62 fold increase in mitochondrial protein synthesis in the young animal but not in the old. Hemin treatment produced a similar effect. These results suggest that the decrease in delta-aminolevulinic acid synthase activity seen with age (Paterniti et al., 1978) may be due to a decrease in the synthesis of mitochondrial inner membrane proteins.

Effect of inhibitors and stimulators on isolated liver cell mitochondrial protein synthesis from young and old rats.

Mitochondria isolated from the livers of old rats (26-30 months) were found to incorporate 41% less leucine into mitochondrial proteins as compared to those from young rats (2-3 months). The initial rates of incorporation of label were 145 cpm/mg/min for the "old" animals, and 320 cpm/mg/min for the young animal. No difference in either amino acid pool size or leakage of label through the mitochondrial membrane was detected in the two age groups. Young rats were treated in vivo with cycloheximide (10 mg/kg) followed by isolation and incubation of their mitochondria in vitro two hours later. There was a two-fold increase in incorporation of leucine into mitochondrial proteins. In contrast, mitochondria isolated from old rats showed a markedly blunted response to cycloheximide pre-treatment. When mitochondria isolated from young and old rats were exposed to inhibitors of mitochondrial protein synthesis, alpha-alpha-dipyridyl (2 x 10(-4)M) and ethanol (0.15M), the old mitochondria showed greater susceptibility to inhibition. These results suggest that the control of the biosynthesis of mitochondrial proteins is altered in the old animals.

Effect of age on rat liver heme and drug metabolism.

Old (24-months) rats have lower activities of hepatic delta-aminolevulinic synthase and the microsomal cytochrome P-450 monooxygenase activities--aminopyrine N-demethylase and aniline hydroxylase--as compared to young (2-months) animals. In contrast, the activity of the heme degradative enzyme, heme oxygenase, is higher in the old rats. Cytochrome P-450 and microsomal heme contents were maintained in the old. When inducibility and inhibition of these enzymes were studied, the old rats responded to the same degree as the young. These results indicate that the ability of the heme synthetic and degradative enzymes to respond to decreasing cellular heme levels is not significantly altered with age. The observations that there is a lower baseline activity of ALA-synthase and good maintenance of microsomal heme and cytochrome P-450 content, in spite of elevated heme oxygenase activity in the old, suggest that, at least in the senescent rat, hepatic heme utilization and degradation are only loosely coupled to heme production. It appears, therefore, that alternate sources of heme for cytochrome P-450 are available in the old animals. Furthermore, it is suggested that the old rat has a baseline change in ALA-synthase, heme oxygenase, and cytochrome P-450 that may be overcome under the appropriate conditions.

Effect of cimetidine on delta-aminolevulinic acid synthase and microsomal heme oxygenase in rat liver.

Cimetidine is a well known inhibitor of the heme-containing enzyme cytochrome P-450. We have found that it also inhibits delta-aminolevulinic acid synthase (ALA-S) and microsomal heme oxygenase, the rate-limiting enzymes for heme synthesis and heme degradation respectively. Cytochrome P-450 content was decreased but microsomal heme concentration remained unaltered for a period of 30 min after in vivo cimetidine administration to rats. In vitro incubation of cimetidine with each of the above enzymes revealed no direct effect of cimetidine on ALA-S but about 50% inhibition of heme oxygenase and 20% reduction in cytochrome P-450 content. This suggests that a metabolite of cimetidine inhibits ALA-S activity in vivo, while the drug itself or a metabolite inhibits heme oxygenase both in vivo and in vitro. A rise in ALA-S activity seen after its early inhibition and its return to approximate control values after 60 min suggest a reversible inhibition of ALA-S by a metabolite of cimetidine and may correspond to its clearance from the animal. An elevation in microsomal heme content paralleled the rise in ALA-S activity while microsomal heme oxygenase activity returned to only 65% of control value 60 min after cimetidine treatment. Cytochrome P-450 content did not change after its initial decrease, suggesting that irreversible alteration had occurred.

Cervical cancer screening in women aged 65 and over.

Pap smear screening is an accepted method of detecting cervical dysplasia or malignancy, and use of this test has been shown to reduce the incidence of invasive squamous cervical cancer in young and middle aged women. Conflicting recommendations exist concerning the screening of women over the age of 65, and little information is available concerning the results of screening in this population. Elderly women are routinely omitted from screening, as it is generally believed that they usually refuse testing. This paper reports a study attempting to evaluate the feasibility of screening in women over the age of 64 attending a large urban outpatient geriatric clinic. It demonstrates that pap smear screening is acceptable to this population, particularly if performed by the usual primary care provider. It also confirms previous reports that women in this age group have had very little screening in the past, and that screening uncovers a large number of abnormalities. Of note is the high rate of false positive smears in this population.

Folate and vitamin B12 levels in an urban elderly population with chronic diseases. Assessment of two laboratory folate assays: microbiologic and radioassay.

Folate levels of serum and red blood cells (RBC) and vitamin B12 serum levels were investigated in 326 urban chronically ill elderly ambulatory patients and 41 healthy young control subjects. Two laboratory methods were used for investigating the folate levels, the microbiologic assay (MBA) with Lactobacillus casei and radioassay (RA). Serum and RBC samples of 326 patients were tested by the folate MBA and 270 of the same samples by the RIA methods. In the MBA 6.8% of the patients and 12.2% of controls had low levels of folate RBC (less than 200 ng/mL) and 1.8% of patients and 4.8% of controls had low serum folate levels (less than 5 ng/mL). All of the patients with the low folate levels had normal hematologic findings and no clinical symptoms of folate deficiency. In the RIA method, all of the patients and almost all of the control subjects (except one) had normal folate levels. Ten of the patients (3%) had low levels of serum vitamin B12 (less than 200 pg/mL). They were hematologically normal. They had normal Schilling tests and normal vitamin B12 dietary intake. Their RBC folate levels were normal and even somewhat higher. Forty percent of these patients had macular degeneration of the eyes. The data indicate the superiority of the RA method over the MBA and bring into question the accuracy of previous studies. The data furnish further evidence that a low vitamin B12 level in the elderly is not necessarily a true vitamin B12 deficiency and raise the possibility of an association between low B12 serum levels and macular degeneration of the eyes.

Erythrocyte parameters in the elderly: an argument against new geriatric normal values.

Erythrocyte parameters in 292 unselected geriatric patients were studied retrospectively. Statistically significant mean decreases compared to the laboratories' normal mean values were found in the red blood cell count (RBC), hemoglobin level, hematocrit, and mean corpuscular hemoglobin concentration; there also was a slight increase in mean corpuscular volume and mean corpuscular hemoglobin. However, when 17 patients with a hemoglobin level less than 10 gm/dl or a hematocrit reading less than 35 percent were excluded, all of the mean values for erythrocyte parameters fell within the normal range. In 71 percent of these 17 patients the etiology of the anemia was documented. Although one cannot definitely exclude a slight change in erythrocyte parameters with aging, it is concluded that the establishment of so-called new geriatric norms is premature, as the population studied included patients with various diseases. Good medical practice dictates continued evaluation and monitoring of patients in whom erythrocyte values are outside the established normal ranges.

Low serum B12 levels in a hematologically normal elderly subpopulation.

Serum Vitamin B12 levels were determined on 378 patients ranging in age from 56 to 104 years with a median age of 77 for both males and females. A radiodilution method was employed for these determinations. This screening procedure identified 26 patients with low serum B12 levels. Nineteen of these patients had no other symptoms and were hematologically normal. The B12 binding proteins, transcobalamins (TC) I, II, and III were quantitated employing QUSO and DEAE cellulose batch separations. The total number of TC II binding/affinity sites for B12 were elevated in both the normal and low B12 elderly groups. About 17% of the total serum B12 was carried by TC II in the control group while only 4% of the total was carried on TC II in both the normal and low B12 elderly. This was accompanied by an increase in unsaturation in TC II for these two groups. The findings suggest that an alteration in the TC II-B12 delivery system has occurred in the elderly.

Pneumococcal bacteremia in adults: age-dependent differences in presentation and in outcome.

In order to evaluate the effect of age on the presentation of and response to acute bacterial infection, the hospital charts of 187 adult patients with community-acquired pneumococcal bacteremia admitted to Bellevue Hospital over a nine-and-a-half year period were reviewed. Compared with younger patients, older patients (aged 65 or older) more frequently had (1) a lower fever in response to the infection, (2) an unclear history of illness, (3) a delay in diagnosis and/or therapy, and (4) a higher risk of dying. On admission, their leukocyte counts and heart rates were similar to those in a group of younger patients, which was composed largely of alcoholic patients and those addicted to intravenous drugs. Response to therapy was also similar in surviving older patients. Lower temperature and an unclear history were features most commonly associated with both delayed diagnosis and higher mortality. When patients with a history of alcohol abuse and those dying shortly after admission (i.e., presenting in a moribund state) were eliminated from the analysis, many of these age-related differences in presentation and outcome became even more evident.

Mutation spectrum in gamma-irradiated shuttle vector replicated in ataxia-telangiectasia lymphoblasts.

Cells from ataxia-telangiectasia (AT) patients are hypersensitive to the lethal effects of ionizing radiation. To assess radiation mutagenesis in these cells, the SV40-based shuttle vector, pZ189, was used to analyze gamma-ray-induced mutations following the plasmid's replication in AT lymphoblasts. Progenies from the AT line GM2783 exposed to 50 Gy showed a mutation frequency of 7.6 x 10(-3), 63-fold over background; surviving plasmids were 3.4% of control. Both values were essentially the same as those of irradiated plasmids replicated in a normal lymphoblast line, GM606. In addition, pZ189 exposed to 25 Gy of gamma radiation and replicated in another normal lymphoblast line and in cells of two additional AT lymphoblast lines showed similar mutation frequencies and percentages of surviving plasmids. Qualitative comparison of plasmid mutations from AT and normal cells showed no significant differences, indicating that the damaged DNA was repaired with similar fidelity in AT and normal cells. These studies suggest that there is no correlation between the enhanced sensitivity of AT cells to killing by ionizing radiation and gamma-radiation-induced mutagenesis of plasmid DNA processed in these cells.

Decreased brain glucose metabolism in microvessels from patients with Alzheimer's disease.

We studied brain glucose metabolism in patients with Alzheimer's disease and age-matched controls in vivo by PET and assessed brain glucose utilization and the phosphorylation constant K3 for hexokinase. In addition we determined in vitro the binding of 2DG and measured its phosphorylation to 2DG-phosphate in cerebral microvessels obtained at autopsy from subjects with Alzheimer's disease and age-matched controls. In patients with Alzheimer's disease we found a marked decrease in the kinetic constant K3 for the hexokinase, and a marked decrease in the overall metabolism of glucose in our PET studies; in microvessels there was a marked decrease in the affinity of 2DG and a decrease in hexokinase activity. Alzheimer's disease may be related to a complex alteration in brain glucose metabolism.