A genomewide suppressor and enhancer analysis of cdc13-1 reveals varied cellular processes influencing telomere capping in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Cdc13 binds telomeric DNA to recruit telomerase and to "cap" chromosome ends. In temperature-sensitive cdc13-1 mutants telomeric DNA is degraded and cell-cycle progression is inhibited. To identify novel proteins and pathways that cap telomeres, or that respond to uncapped telomeres, we combined cdc13-1 with the yeast gene deletion collection and used high-throughput spot-test assays to measure growth. We identified 369 gene deletions, in eight different phenotypic classes, that reproducibly demonstrated subtle genetic interactions with the cdc13-1 mutation. As expected, we identified DNA damage checkpoint, nonsense-mediated decay and telomerase components in our screen. However, we also identified genes affecting casein kinase II activity, cell polarity, mRNA degradation, mitochondrial function, phosphate transport, iron transport, protein degradation, and other functions. We also identified a number of genes of previously unknown function that we term RTC, for restriction of telomere capping, or MTC, for maintenance of telomere capping. It seems likely that many of the newly identified pathways/processes that affect growth of budding yeast cdc13-1 mutants will play evolutionarily conserved roles at telomeres. The high-throughput spot-testing approach that we describe is generally applicable and could aid in understanding other aspects of eukaryotic cell biology.

Cortical concentrations of corticotropin-releasing hormone and its receptor in Alzheimer type dementia and major depression.

Corticotropin-releasing hormone-immunoreactivity (CRH-IR) and CRH receptors (binding capacity and affinity) were measured in postmortem cortical areas from depressed subjects, two groups of senile dementia of the Alzheimer type (SDAT), and age-, sex-, and postmortem-delay-matched controls. No difference in CRH-IR and CRH receptor status between depressed subjects and controls was noted. CRH-IR was decreased in all cortical areas in SDAT, with a corresponding increase in CRH receptor binding capacity (with no change in affinity) in occipital cortex. No effects of postmortem delay were seen. It is suggested that the increase in CRH receptor numbers in SDAT is related to the degree of distribution of pathological involvement in specific regions.

5HT2 receptor changes in major depression.

The 5HT2 receptor has been studied using quantitative tritium film autoradiography in the postmortem frontal cortex of 15 cases suffering from major depression and controls, matched for age, gender, postmortem delay, and storage time. In unmedicated depressives there was a significant increase in 5HT2 receptor binding over matched control values. Antidepressant-treated depressives dying while depressed had 5HT2 receptor densities not significantly different from control values. Depressives dying euthymic, (i.e., recovered) showed a marked reduction in 5HT2 receptor binding when compared with controls. A tentative hierarchy of 5HT2 receptors in affective states is proposed.

Neocortical concentrations of neuropeptides in senile dementia of the Alzheimer and Lewy body type: comparison with Parkinson's disease and severity correlations.

Corticotropin releasing hormone (CRH), somatostatin (SRIF), and arginine vasopressin (AVP) concentrations were estimated using radioimmunoassay in the temporal and occipital cortices in postmortem brain from patients clinically and neuropathologically diagnosed as senile dementia of the Lewy body type (SDLT), senile dementia of the Alzheimer type (SDAT), and Parkinson's disease (PD) and from neurologically normal controls. The concentration of temporal and occipital neocortical CRH was diminished in both SDAT and SDLT compared to control values, whereas SRIF was reduced only in temporal cortex in both these conditions. In contrast, the concentrations of both CRH and SRIF were unaltered in PD. The concentrations of AVP in SDLT, SDAT, and PD were similar to those found in the control groups. The decrement in SRIF, but not CRH, was found to be correlated with some indices of severity of illness in SDAT; a similar but nonsignificant trend for SRIF was observed in SDLT.

Cortisol, ACTH, and dexamethasone concentrations in a psychogeriatric population.

Cortisol and adrenocorticotrophic hormone (ACTH) were measured at 2 time points before the administration of 1 mg of dexamethasone (day 1) and 1 time point on the following day (day 2). Thirteen severely depressed elderly patients, 15 patients with Alzheimer-type dementia (ATD), and 16 normal controls were studied. Cortisol was markedly elevated in depressed patients compared with the other subjects in day 1 samples. Following dexamethasone, both the depressed and ATD patients showed a similar elevation of cortisol compared with controls. ACTH concentrations were not significantly different between the groups before dexamethasone, but were significantly higher in both depressed and ATD patients after dexamethasone. More depressed patients than ATD patients exhibited hypersecretion of ACTH after dexamethasone. This implies that ACTH is less responsive to glucocorticoid feedback in elderly depressed patients, which may be a factor in causing hypercortisolemia.

Apolipoprotein E and alpha-1 antichymotrypsin polymorphism genotyping in Alzheimer's disease and in dementia with Lewy bodies. Distinctions between diseases.

The possibility of gene interactions in Alzheimer's disease (AD) has been suggested by the finding of an association of the AA genotype of the alpha-1 antichymotrypsin (AACT) gene and the apolipoprotein E (apoE) epsilon 4/4 genotype in AD. We tested this possibility by genotyping a large series of clinically and neuropathologically confirmed cases of AD and a series of cases with dementia with Lewy bodies (DLB) with a matched control group for the AACT locus and apoE. ApoE genotyping showed the established finding of an increased frequency of the apoE epsilon 4 allele in AD and in DLB. The AD and DLB groups differed between each other with a higher epsilon 2 allele frequency and a reduced incidence of the epsilon 4/4 genotype in DLB. Differences in the apoE frequencies may account for some of the differences between the two diseases. No association was found for the AACT A allele in AD or DLB in the groups as a whole or when stratified with respect to apoE, with the exception of a trend showing an increased incidence of the apoE epsilon 4/4 AACT AA genotype combination in AD patients (chi 2 = 3.18, p = 0.07), although in DLB this was not apparent (chi 2 = 0.0, p = 1.0). The AACT A allele is not a major risk factor for late-onset AD or DLB.

The biological activity of a single dose of tamoxifen in the adult ovariectomized rat.

1 The peripheral and central activities of tamoxifen were studied in the ovariectomized adult rat, up to 16 d after a single dose of 7.0 or 0.7 mg/kg. 2 Food consumption and body weight were decreased; only food consumption returned to control values after 16 d. 3 The weights of the uterus and of the uterine luminal fluid were increased for up to 8 d. 4 Serum follicle-stimulating hormone (FSH) concentrations decreased, but only significantly 1 and 8 d after 7.0 mg tamoxifen/kg. Serum luteinizing hormone (LH) and prolactin concentrations were elevated for up to 4 d. 5 Lordosis behaviour was absent throughout the period studied. 6 Within 3 d of administration, tamoxifen partially antagonized the oestrogen-induced changes in uterine luminal fluid, prolactin and LH secretion and lordosis behaviour. 7 Tamoxifen did not alter oestrogen-induced changes in uterine weight, food consumption and body weight. 8 The experiments demonstrate that tamoxifen is active for up to 16 d after a single intraperitoneal dose; oestrogen agonist, partial agonist and antagonist activities were demonstrated. The duration and type of activity depends upon the dose of tamoxifen and the target tissue response examined.

Characterization of the prolactin receptor in human prostate.

The uptake of 125I-labelled human prolactin by subcellular fractions obtained from the human hyperplastic prostate was investigated and the specific binding sites were characterized. The binding was both time- and temperature-dependent with maximum specific uptake achieved after incubation for 20 h at 4 degrees C (dissociation constant = 2.4 x 10(-10) mol/l). The present studies also indicate that the bulk of the specific prolactin binding was confined to the 105 000 and 15 000 g membrane fractions whilst the cytosol and nuclear pellet exhibited a lower capacity for the peptide hormone. Attempts to optimize the binding revealed that pretreatment of the subcellular fractions with 4 mol MgCl2/l quadrupled the binding sites; a similar effect was produced after pretreatment with dextran-coated charcoal, but the changes were less pronounced. Furthermore, our studies suggest that the binding was specific for the human prolactin, with other hormones such as ovine prolactin, human LH, human FSH and human GH showing little or no competition. The addition of magnesium and copper ions to the incubation medium also markedly increased the specific binding, whereas calcium, manganese and EDTA inhibited the prolactin uptake. Freezing and storage of tissue at -70 degrees C did not greatly affect the binding sites. The results suggest that we are clearly dealing with a specific prolactin-binding protein.

Time-related effects of en-clomiphene upon central and peripheral oestrogen target tissues and cytoplasmic receptors.

The temporal effects of the oestrogen antagonist en-clomiphene were studied in the 3 week ovariectomized rat for up to 24 days after a single dose of 0.5 or 50 mg/kg. Cytosol binding of [3H]oestradiol was measured in central and peripheral target tissues. One day after treatment both doses of en-clomiphene decreased binding of [3H]oestradiol to similar levels within tissues. In brain cytosols binding of [3H]oestradiol returned to expected control values by 16 days with the exception of the hypothalamic cytosol after treatment with 50 mg/kg which, paradoxically, achieved control levels by 8 days. However, a single-point saturation assay was inadequate for the measurement of high-affinity oestrogen receptors in brain tissue. Uterine binding of [3H]oestradiol returned to control levels by 2 and 16 days after treatment with 0.5 and 50 mg/kg respectively. Replenishment of [3H]oestradiol binding was slower in pituitary cytosol and was achieved by 8 and 24 days after treatment with 0.5 and 50 mg/kg respectively. En-clomiphene was uterotrophic; the maximal increase was at 4 days and 1 day after 0.5 and 50 mg/kg and was sustained, albeit at a lower level, for 4 and 8 days respectively. En-clomiphene at 50 mg/kg increased total uterine DNA 2 days after treatment. Small dose-related sustained increases in weight of uterine luminal fluid were also observed. En-clomiphene had no effect upon serum FSH. Serum LH was decreased only on day 8 after treatment. Serum prolactin was increased to a similar level by both doses of en-clomiphene; however, the maximum was achieved 2 and 16 days after treatment with 0.5 and 50 mg/kg respectively. Rats were tested for sexual receptivity after 50 mg/kg en-clomiphene and increases were detected between 7 and 9 days of treatment. Progesterone facilitated this effect only on days 8 and 9. En-clomiphene produced dose-related falls in food intake and body weight, the latter remaining below the weights of control rats throughout the period studied. It is apparent that the temporal effects of en-clomiphene upon cytoplasmic [3H]oestradiol binding are diverse, replenishment of binding being dependent upon the dose and the tissue examined. In addition, the changes in biological activity as measured by uterine and neuroendocrine effects also appear temporally unrelated to each other and to the binding of [3H]oestradiol. Thus further studies are necessary to elucidate possible differences in tissue oestrogen receptor mechanisms and the mode of action of the oestrogen antagonists.

Hypothalamic, pituitary and uterine cytoplasmic and nuclear oestrogen receptors and their relationship to the serum concentration of tamoxifen and its metabolite, 4-hydroxytamoxifen, in the ovariectomized rat.

High-affinity oestrogen receptors were measured in the cytosol and nuclei prepared from the hypothalamus, pituitary gland and uterus of the ovariectomized rat up to 16 days after a single dose of tamoxifen or 4-hydroxytamoxifen. Tamoxifen produced a dose-related fall in the concentration of cytosol receptors in the hypothalamus, pituitary gland and uterus. Minimum values were observed after 24 h; cytosol receptor concentrations were restored slowly and only reached expected control values between 4 and 8 days and 2 and 4 days for 7.0 mg/kg and 0.7 mg/kg doses of tamoxifen respectively. The nuclear receptor changes were inversely related to the cytosol receptor changes, except that hypothalamic nuclear receptor concentrations after 0.7 mg tamoxifen/kg were not changed. 4-Hydroxytamoxifen (0.14 mg/kg) depleted cytosol and raised nuclear oestrogen receptors in the pituitary gland and uterus. Receptor concentrations had returned to the expected control values by day 4. Oestrogen receptor concentrations in the hypothalamus were unchanged. The apparent resistance of the receptor system in the hypothalamus to translocation by tamoxifen and 4-hydroxytamoxifen was probably due to the blood-brain barrier since the apparent affinity of tamoxifen for the cytosol receptor was similar for all three tissues (dissociation constant 4 nmol/l). Serum concentrations of tamoxifen and 4-hydroxytamoxifen measured after a single dose of 7.0 mg tamoxifen/kg were maximal after 24 h and undetectable by 4 days, at which time nuclear and cytosol receptor levels were still changed. Concentrations of 4-hydroxytamoxifen were approximately one-third those of tamoxifen. The results suggest that the receptor changes after tamoxifen are not simply related to the serum concentration of tamoxifen and its metabolites and that the retention of ligand within the target tissue may be an important determinant.

Interaction between prolactin and zinc in the human prostate gland.

The interaction between prolactin and zinc was examined in vitro in the human prostate gland. The results indicated that prolactin did not modulate the acute uptake of zinc into benign prostatic hypertrophy tissue whereas zinc, in contrast, increased the uptake of prolactin into the prostate gland. Our study further showed that the augmented uptake of prolactin by zinc was partly due to an increase in the non-specific binding properties of the peptide hormone. We were also able to demonstrate that the specific binding of 125I-labelled human prolactin to the receptor was reduced in the presence of zinc by a competitive mechanism.

Agonist and antagonist activity of en-clomiphene upon oestrogen-mediated events in the uterus, pituitary gland and brain of the rat.

The oestrogen agonist and antagonist activity of a single dose of en-clomiphene (0.5--50 mg/kg) was studied in peripheral and brain tissue in the 3 week ovariectomized rat. 17 beta-Oestradiol benzoate (100 microgram/kg) or vehicle was injected 24 h after en-clomiphene administration and data collected at 72 h. En-clomiphene produced a dose-related (agonist) fall in body weight and food intake. Agonist action was not observed upon sexual receptivity and prolactin secretion; oestrogen antagonism of these parameters was only seen at the higher doses. The effects of en-clomiphene upon serum LH and FSH were complex, both agonist and antagonist activity being demonstrated in the absence and presence of oestrogen. En-clomiphene was uterotrophic at all doses tested; however, oestrogen antagonism was only seen at the higher doses. Inhibition of the accumulation of uterine luminal fluid at the higher doses of en-clomiphene was a sensitive index of oestrogen antagonism. The results are discussed in relation to previous studies of the effects of en-clomiphene on oestrogen receptors. With the exception of the antagonism of oestrogen-induced sexual receptivity no correlation could be made between biological activity and the status of oestrogen receptors in tissue.

Peptides in the neocortex in Alzheimer's disease and ageing.

Studies which have examined neuropeptides in Alzheimer's disease (AD) and normal ageing are reviewed. A marked specificity and selectivity is noted: most neuropeptides are normal, and the only two peptides consistently altered are somatostatin (SRIF) and corticotropin releasing hormone (CRH). Binding sites for CRH are increased in number in a reciprocal fashion to the reduction in CRH. These findings (1) provide evidence for selective vulnerability within the cortex in AD, (2) suggest that the primary site of pathology in AD may be cortical, and (3) indicate that the pathological process of AD is distinct from that of normal ageing.

No association between a polymorphism in the presenilin 1 gene and dementia with Lewy bodies.

The discovery of mis-sense mutations linking the presenilin-1 (PS-1) gene on chromosome 14 to Alzheimer's disease (AD) has lead to a thorough investigation of this locus. The PS-1 gene contains a polymorphism creating two alleles. The most common allele, allele 1, has been linked with late-onset AD. Given the clinical and pathological overlaps between AD and dementia with Lewy bodies we genotyped 46 pathologically confirmed cases of dementia with Lewy bodies for the PS-1 polymorphism and compared the allelic frequencies with 87 age-matched control cases and 103 age-matched AD cases. No association between dementia with Lewy bodies and PS-1 allele 1 was found either in the group as a whole, or in the group stratified according to dosage of the epsilon4 allele of the apolipoprotein E gene. We suggest that either the presenilin polymorphism has no effect on dementia with Lewy bodies or that any linkage is precluded by another, more influential, locus.

Brain matrix metalloproteinase 1 levels are elevated in Alzheimer's disease.

Several lines of evidence indicate that there may be an inflammatory component to the pathology of Alzheimer's disease (AD), the major form of degenerative dementia in the elderly. Activity of inflammatory cells, and the elaboration of toxic molecules by such cells may be a significant factor in disease progression. In peripheral inflammatory states, the increased activity of matrix metalloproteinase (MMP) enzymes are a major cause of tissue breakdown and secondary damage in diseases such as rheumatoid arthritis. The activity of such enzymes in the normal or diseased central nervous system is, however, not well characterized. We have therefore determined the levels of MMP 1 (collagenase) in the normal human brain and in AD. MMP1 levels were relatively low though were significantly elevated by approximately 50% in AD in all cortical areas examined. Given the activity towards collagen of MMP1, it is possible that enhanced MMP1 activity in AD, may contribute to the blood-brain barrier dysfunction seen in AD.

No association between an intronic polymorphism in the presenilin-1 gene and Alzheimer's disease.

Mutations in the presenilin-1 (PS-1) gene are believed to be responsible for the majority of familial early-onset Alzheimer's disease (AD). The finding of an intronic polymorphism in the PS-1 gene prompted an investigation into its relevance in AD. An association between homozygosity for the most common allele (allele 1) in this intronic polymorphism and late-onset AD has been shown and has been confirmed by others though some studies do not support these findings. We genotyped a large series of sporadic AD cases (n = 120) and age-matched controls (n = 108) for this intronic polymorphism. We then compared both the frequency of allele 1 and allele 1 homozygosity between the AD group as a whole and in early-onset (n = 26) and late-onset (n = 94) groups with age-matched control groups (n = 29 and n = 79, respectively). No increase in the frequency or homozygosity of allele 1 in either the AD group as a whole, or when divided into late- and early-onset cases was found. Increases in the frequency of allele 1 homozygotes and in the number of non-apolipoprotein E epsilon4 carrying allele 1 homozygotes/heterozygotes was demonstrated in the early-onset AD cases although these values did not reach significance. We conclude that there is no relationship between this intronic polymorphism in the PS-1 gene and AD in the homogenous population genotyped in this study.

Effect of apolipoprotein E genotype on Alzheimer's disease neuropathology in a cohort of elderly Norwegians.

A cohort of elderly Norwegians dying in nursing homes in the Oslo region have been genotyped for the Apolipoprotein E (ApoE) gene. Alzheimer's disease (AD) cortical neuropathology and clinical evidence of dementia were used to assign cases without evidence of other confounding neuropathology. Senile plaque (SP) and neurofibrillary tangle (NFT) densities in frontal, temporal and parietal cortex were then correlated with ApoE genotype to determine any relationship between ApoE genotype and AD pathology. Comparisons with ApoE epsilon 3, epsilon 4 and epsilon 2 allele dosage failed to show any significant effect on cortical SP densities in any cortical area. NFT densities were increased by epsilon 4 allele dosage in the frontal cortex but not in other cortical regions. A reduction was seen in cortical NFT densities with epsilon 2 allele, though again this was not consistently significant in any of the groups. The epsilon 3 allele failed to show any consistent effect on cortical NFT densities. Assessment by individual genotypes showed epsilon 2/3 < epsilon 2/4 < epsilon 3/3 < epsilon 3/4 < epsilon 4/4 which had highest cortical NFT densities in all areas. By genotype, SP densities were generally of the order epsilon 2/4 < epsilon 2/3 < epsilon 3/3 < epsilon 4/4 < epsilon 3/4 though in none of the groups was this significant. Duration of disease showed no consistent effect on neuropathological burden. ApoE genotype may have an effect on determining whether individuals suffer from AD and the age at onset of disease but may only have a minimal effect on pathology burden.

Molecular biology of APO E alleles in Alzheimer's and non-Alzheimer's dementias.

Current research into the aetiology of the dementias is focused upon genetic factors which give rise to the disease process. Recently the Apolipoprotein E gene (APO E) and in particular the epsilon 4 allele has been shown to be a risk factor for late onset Alzheimer's disease (AD) where there is an increased frequency of the epsilon 4 allele. The epsilon 4 allele has also been shown to reduce the age at onset of dementia in AD in a dose dependent manner, with the epsilon 2 allele having an opposing effect. We have genotyped a large series of clinically and neuropathologically confirmed cases of AD and found the expected increase in the Apolipoprotein epsilon 4 allele frequency when compared to a control population. Similarly, in Lewy Body Dementia (LBD) an increased epsilon 4 frequency is also found though a normal epsilon 2 frequency exists, unlike in AD where the epsilon 2 frequency is reduced. No changes in APO E allele frequencies were found in presenile AD, Parkinson's disease with or without dementia, or in Down's syndrome. No association was found between any of the APO E alleles and the histopathological indices of AD, cortical senile plaques and neurofibrillary tangles, in any disease category. Neurochemical indicators of AD, loss of choline acetyltransferase activity was also unaffected by APO E genotype. Whilst their appears to be a strong association between the APO E allele and AD and also in LBD, other related neurodegenerative disorders associated with dementia do not show such a linkage. Changes in the epsilon 2 allele frequency may indicate a genetic difference between AD and LBD. The epsilon 4 allele does not appear to influence the burden of AD type pathology and this is particularly relevant given the relative lack of NFT in LBD indicating that factors other than SP or NFT may govern the onset of dementia.

Plasma N-POMC, ACTH and cortisol following hCRH administration in major depression and dysthymia.

The concentrations of plasma N-terminal pro-opiomelanocortin, adrenocorticotropic hormone and cortisol in response to a 14:30 h human corticotrophin releasing hormone (hCRH) stimulation test (1 microgram/kg) were measured in control, major depression and dysthymic subjects. The increases in all three hormones were similar in the depressed groups when compared to the control values. The elevation in cortisol after hCRH was significantly greater in major depression when compared to the dysthymic subjects. The significance of these findings is discussed.

Alterations in neuropeptides in aging and disease. Pathophysiology and potential for clinical intervention.

Marked specific and selective changes in the levels of some neuropeptides in age-related diseases, such as senile dementia of the Alzheimer (SDAT) or Lewy body (SDLT) types, Parkinson's disease, Huntington's disease and major depressive disorder, versus normal aging have been noted. However, the levels of most neuropeptides are normal. The only 2 peptides consistently altered in SDAT are somatostatin and corticotrophin-releasing hormone both of which are reduced. In Huntington's disease, the level of substance P in the basal ganglia is reduced suggesting a preferential vulnerability of spiny neurones in this disease. In Parkinson's disease, substance P is attenuated in the basal ganglia while somatostatin is reduced in the neocortex. These and other results suggest that substance P deficits are related to movement disorders while somatostatin deficits are related to cognitive impairment. SDLT is a type of dementia with features common to both SDAT and Parkinson's disease, although the changes in neuropeptides suggest that neurochemically the disease is more closely related to SDAT. In major depressive disorder, the level of corticotrophin-releasing hormone is reduced while there is a reciprocal increase in corticotrophin-releasing hormone receptors suggesting that the neurones remain functional. Potential clinical intervention has been limited by problems such as poor penetration of agents into the brain and the short half-lives of neuropeptide agonists and antagonists. However, some currently available agents may act, at least in part, through modulation of neuropeptide pathways, e.g. carbamazepine and alprazolam both modulate the corticotrophin-releasing hormone system in animals, and both have clinically proven antidepressant activity.