A preliminary study of cerebral blood flow, aging and dementia in people with Down syndrome.

BACKGROUND: People with Down syndrome (DS) develop Alzheimer's disease (AD) at an earlier age of onset than those with sporadic AD. AD neuropathology is typically present in DS by 40 years of age with an onset of dementia approximately 10 years later. This early onset is due to the overexpression of amyloid precursor protein from the third copy of chromosome 21. Cerebrovascular neuropathology is thought to contribute in 40-60% of cases sporadic AD. However, the vascular contribution to dementia in people with DS has been relatively unexplored. We hypothesised that vascular perfusion is compromised in older adults with DS relative to younger individuals and is further exacerbated in those with dementia. METHOD: Cerebral blood flow (CBF) was measured using pulsed arterial spin labelling in 35 cognitively characterised adults with DS (26-65 years). DS participants were also compared with 15 control subjects without DS or dementia (26-65 years). Linear regression evaluated the difference in CBF across groups and diagnosis along with assessing the association between CBF and cognitive measures within the DS cohort. RESULTS: Cerebral blood flow was significantly lower among DS participants with probable AD compared with controls (P = 0.02) and DS participants with no dementia (P = 0.01). Within the DS cohort, CBF was significantly associated with the Severe Impairment Battery (SIB) measure and the Dementia Questionnaire for People with Learning Disabilities (DLD) rating (F3,25  = 5.13; P = 0.007). Both the SIB (ß = 0.74; t = 2.71; P = 0.01) and DLD (ß = -0.96; t = -3.87; P < 0.001) indicated greater impairment as global CBF decreased. Age was significantly associated with CBF among participants with DS. There was a non-linear effect of age, whereby CBF declined more rapidly after 45 years of age. CONCLUSIONS: This preliminary study of CBF in DS indicates that cerebrovascular pathology may be a significant contributor to dementia in DS. CBF was associated with diagnosis, cognition and age. Notably, CBF decreases at a greater rate after age 45 and may represent a significant prodromal event in AD progression.

[Effect of transient ischemic attack on early neurological deterioration in patients with ipsilateral ischemic stroke].

Objective: To explore the effect of anterior circulation transient ischemic attack (TIA) on early neurological deterioration (END) in patients with ipsilateral ischemic stroke and its mechanism. Methods: (1) One hundred and thirteen patients with ipsilateral ischemic stroke and 36 healthy volunteers (healthy control group) in Neurology Department of Tianjin Medical University General Hospital from November 2014 to July 2015 were recruited into this study. According to whether got TIA before ischemic stroke, patients were divided into simple ischemic stroke group (CI group, n=87) and TIA-CI group (n=26). Their END, NIHSS score, 3-month mRS score, infarct size, serum hs-CRP and other risk factors were compared. (2) The peripheral blood mononuclear cells (PBMCs) were extracted from peripheral blood of TIA-CI group and CI group at 24 h, the 3 th day, the 7 th day and 12th day after ischemic stroke onset. At the same time, PBMCs of control group were collected. Western blot was carried out to evaluate the expression of nuclear factor-κB (NF-κB). Immunofluorescence was used to detect the cytoplasmic-to-nuclear shuttling of NF-κB. Results: (1) The incidence of END, NIHSS score at discharge, 3-month mRS score and serum hs-CRP level were significantly lower whereas the infarct size was significantly smaller of TIA-CI group than CI group (11.5% vs 31.0%, 1.9±2.3 vs 3.3±3.7, 0.9±0.8 vs 1.8±1.8, 1.1(0.3, 2.5) cm(3) vs 2.4(0.5, 22.8) cm(3,) (2.5±3.2) mg/L vs (6.2±3.2) mg/L, all P<0.05) . hs-CRP was positively correlated with END (r=0.311, P<0.05). (2) Expression of NF-κB: ① Compared with control group, the NF-κB expression increased first and then decreased in both of the two patient groups, and it decreased earlier in TIA-CI group than CI group.②In each time point, NF-κB expression of TIA-CI group was lower than CI group(t=1.754, P<0.05; t=1.858, P<0.05; t=0.609, P<0.05; t=0.519, P<0.05). (3) Activity of NF-κB: Most of NF-κB were inactivation and located in cytoplasm of control group. NF-κB of TIA-CI group and CI group was activated and translocated from cytoplasm into nuclear at the 24 h and 3 th day after ischemic stroke. At the 7 th day and 12th day, the accumulation of NF-κB in nuclear decreased and most of them located in cytoplasm in TIA-CI group, whereas most of NF-κB still located in nuclear in CI group. The activated station of NF-κB lasted shorter in TIA-CI group than CI group. Conclusions: TIA can reduce the incidence of END in patients of ipsilateral ischemic stroke. Its protective effect may relate with the inhibition of inflammation which induced by NF-κB.

Early gene expression in salivary gland after isoproterenol treatment.

Isoproterenol, a ß-adrenergic agonist, has been shown to induce salivary gland hyperplasia. However, the mechanism involved in this pharmacological phenomenon is not well understood. To gain a better understanding of the underlying changes, including genes, networks and pathways altered by isoproterenol, microarray-based gene expression analysis was conducted on rat parotid glands at 10, 30, and 60 min after isoproterenol injection. After isoproterenol treatment, the number of differentially expressed genes was increased in a time-dependent manner. Pathway analysis showed that cell hyperplasia, p38(MAPK), and IGF-1 were the most altered function, network and pathway, respectively. The balanced regulation of up- and down-expression of genes related to cell proliferation/survival may provide a better understanding of the mechanism of isoproterenol-induced parotid gland enlargement without tumor transformation.

Association of polymorphism in ICAM-1 (K469E) and cytology parameters in patients' initial blood test with acute ischemic stroke.

Acute ischemic stroke (AIS) has become a serious health problem in many countries because of its poor outcome and worsening epidemic trend. Early identification of genetic risk factors and physiological indicators for stroke occurrence may help to reduce the incidence of stroke. Therefore, we conducted a case-control study including 50 AIS patients and 50 healthy individuals from a Chinese population to explore the association between AIS and patient complete blood profiles and the association between AIS and the genetic polymorphism K469E in intercellular adhesion molecule-1 (ICAM-1). Compared to the control group, AIS patients showed a high percentage of mononuclear cells, low platelet count, low ratio of platelet to lymphocyte count, high frequency of the 469K allele, and low frequency of the 469E allele. White blood cell count, percentage of neutrophils, percentage of lymphatic cells, platelet distribution width, mean platelet volume, and platelet hematocrit levels showed no significant differences between the 2 groups and between different genotypes. Our results suggested an association of elevated levels of mononuclear cells and reduced platelet count with higher AIS risk. Our results also supported the hypothesis that the KK genotype at the K469E locus in ICAM-1 is a risk factor for AIS.

Anticandidal activity and biocompatibility of a rechargeable antifungal denture material.

OBJECTIVES: Candida-associated denture stomatitis is a recurrent and debilitating oral mucosal disease. Development of anticandidal denture materials represents a promising strategy to manage this condition. We have previously shown that miconazole incorporated in methacrylic acid (MAA) copolymerized diurethane dimethacrylate (UDMA) denture materials has long-term anticandidal activity. In this study, we examined the ability of culture medium conditioned with drug-free- or miconazole-MAA-UDMA discs to prevent Candida infection in an in vitro oral epithelial cell/Candida albicans coculture system. MATERIALS AND METHODS: Candida albicans (C. albicans)-induced OKF6/TERT-2 cell damage was quantified by the release of lactate dehydrogenase from epithelial cells, cytokine production was quantified using protein cytokine arrays, and the expression of C. albicans genes was measured by RT-qPCR. RESULTS: Candida albicans had limited growth with altered expression levels of secreted aspartyl proteinase-2 and -5 in culture medium conditioned by miconazole-MAA-UDMA discs. Significantly, the ability of C. albicans to induce oral epithelial cell damage and trigger epithelial proinflammatory cytokine production was also inhibited by miconazole disc conditioned media. CONCLUSION: Miconazole released from MAA-UDMA denture materials effectively prevents the development of candidal infection in an in vitro oral epithelial system. Further characterization of this drug-rechargeable denture material is warranted.

Cellular signals underlying ß-adrenergic receptor mediated salivary gland enlargement.

We examined the cellular signaling pathways involved in parotid gland enlargement induced by repeated isoproterenol administration in rats. Immunoblot analysis revealed early (1h) activation of the mitogen activated protein kinase (MAPK) ERK1/2, and progressive activation of epidermal growth factor receptor (EGFR), p38MAPK and p70S6 kinase (p70S6K) during 72h of isoproterenol treatment. Expression of ß-adrenergic receptors (ARs) of the ß2, but not ß1, subtype increased over time in parallel with increases in the proliferation marker PCNA and parotid gland weight. Levels of ß2-AR mRNA, assessed by quantitative RT-PCR and Northern blot analysis, were upregulated in parotid glands of isoproterenol treated rats. cAMP response element binding protein (CREB), a positive regulator of ß2-AR transcription, was activated at 1h after isoproterenol administration, as evidenced by increased nuclear translocation and DNA binding using immunohistochemical staining and electrophoretic mobility shift assay. ELISA of NF-κB, also a ß2-AR transcriptional regulator, revealed an increase in p65 and p50 subunits in nuclear protein extracts from parotid glands of isoproterenol treated rats. Together, these results demonstrate that ß-adrenergic stimulation activates diverse cell survival and progrowth signaling pathways, including cAMP and EGFR linked activation of ERK1/2, p38MAPK, and p70S6K, and also induction of ß2-ARs, possibly mediated by CREB and NF-κB, resulting in salivary gland enlargement. We propose that during isoproterenol treatment activation of the ß1-AR, the predominant ß-AR subtype in unstimulated salivary glands, initiates proliferative signaling cascades, and that upregulation of the ß2-AR plays an essential role in later stages of salivary gland growth.

Dendritic cells transfected with lentiviral vector-encoding human granulocyte-macrophage colony-stimulating factor augment anti-tumour T-cell response in vitro.

Dendritic cells (DC) are professional antigen-presenting cells that can actively taken up and present tumour-derived proteins to induce a tumour-specific immune response. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a pivotal role in the generation, sensitization, maturation and survival of DC. We charged the peripheral blood monocyte cell-derived DC with tumour lysate, and then transfected the DC with lentiviral vector-encoding human GM-CSF (hGM-CSF). The antigen-presenting capacity of the hGM-CSF-transfected DC was tested by means of the mixed lymphocyte reaction and cytotoxic T-lymphocyte assay using wild-type DC as the control. The Lenti-hGM-CSF-transfected DC was able to stimulate the proliferation of naive allogeneic T lymphocytes and to generate tumour-specific cytotoxic T lymphocytes more efficiently than the wild-type DC. This data indicates that Lenti-hGM-CSF-transfected DC could potentially be used as an effective clinical approach for cancer immunotherapy.

Salivary secretion, mucin concentrations and Candida carriage in HIV-infected patients.

OBJECTIVES: To test whether the submandibular/sublingual (SMSL) salivary secretion, mucin concentration and candida carriage status were altered in human immunodeficiency virus-positive (HIV+) patients. SUBJECTS AND METHODS: SMSL saliva collected from 48 HIV-infected and 31 HIV-negative men were analyzed for flow rates, total protein and mucin concentrations. Salivary cultures were performed for Candida assessment. RESULTS: The salivary flow rate and protein secretion of the HIV+ patients was 37% and 32% less than that of the controls (P < 0.0001, P = 0.0087). The mucin concentrations (MG1 and MG2) were higher in the HIV+ subjects compared with controls (P = 0.0186, P = 0.0014); however, the mucin secretions were not different. The frequency of Candida-positive cultures was higher in the HIV+ subjects than in the controls (61.4%vs 24.1%, P = 0.0018). In the HIV-infected group, the unstimulated SMSL flow rates were lower in Candida-positive than in Candida-negative patients (P = 0.0158). CONCLUSION: The salivary secretion of the SMSL glands was reduced in HIV infection. Although the mucin concentration increased in HIV+ subjects, mucin secretion was not altered. Highly active antiviral therapy had no effect on salivary function. We found an association between the level of candida carriage and salivary flow rate in HIV-infected patients.

(1)H-MRS metabolites in adults with Down syndrome: Effects of dementia.

To determine if proton magnetic resonance spectroscopy ((1)H-MRS) detect differences in dementia status in adults with Down syndrome (DS), we used (1)H-MRS to measure neuronal and glial metabolites in the posterior cingulate cortex in 22 adults with DS and in 15 age- and gender-matched healthy controls. We evaluated associations between (1)H-MRS results and cognition among DS participants. Neuronal biomarkers, including N-acetylaspartate (NAA) and glutamate-glutamine complex (Glx), were significantly lower in DS patients with Alzheimer's should probably be changed to Alzheimer (without ' or s) through ms as per the new naming standard disease (DSAD) when compared to non-demented DS (DS) and healthy controls (CTL). Neuronal biomarkers therefore appear to reflect dementia status in DS. In contrast, all DS participants had significantly higher myo-inositol (MI), a putative glial biomarker, compared to CTL. Our data indicate that there may be an overall higher glial inflammatory component in DS compared to CTL prior to and possibly independent of developing dementia. When computing the NAA to MI ratio, we found that presence or absence of dementia could be distinguished in DS. NAA, Glx, and NAA/MI in all DS participants were correlated with scores from the Brief Praxis Test and the Severe Impairment Battery. (1)H-MRS may be a useful diagnostic tool in future longitudinal studies to measure AD progression in persons with DS. In particular, NAA and the NAA/MI ratio is sensitive to the functional status of adults with DS, including prior to dementia.

Sexual dimorphism characterizes steroid hormone modulation of rat aortic steroid hormone receptors.

Quantification of aortic androgen and estrogen receptor content and distribution in AXC/SSh rats established that the total androgen receptor content in intact young mature males (mean +/- SD, 55 +/- 13 fmol/mg DNA) was indistinguishable (P greater than 0.05) from that in proestrous females (50 +/- 3 fmol/mg DNA). However, 60% of male aortic androgen receptors were in the nuclear fraction, whereas all proestrous female aortic androgen receptors were in the cytoplasmic fraction. The total aortic estrogen receptor content of intact young mature males (70 +/- 16 fmol/mg DNA) was indistinguishable (P greater than 0.05) from that of proestrous (92 +/- 12) or diestrous (77 +/- 4) females. However, 50% of proestrous female aortic estrogen receptors were in the nuclear fraction, whereas male or diestrous female aortic estrogen receptors were restricted to the cytoplasmic fraction. To assess estrogen receptor function, we characterized aortic cytoplasmic progesterone receptors and established that the receptor content of intact male aortae (101 +/- 3 fmol/mg DNA) was not significantly different (P greater than 0.05) from that of diestrous female aortae (100 +/- 11). 17 beta-Estradiol injection of intact males failed to affect aortic progesterone receptor content (93 +/- 17 fmol/mg DNA). However, injection of orchiectomized males with 17 beta-estradiol significantly (P less than 0.05) increased progesterone receptor content to 208 +/- 24 fmol/mg DNA. This value is twice that of intact males and is not significantly different (P greater than 0.05) from the aortic cytoplasmic progesterone receptor content (190 +/- 32 fmol/mg DNA) of 17 beta-estradiol-injected oophorectomized females. These studies establish that intracellular distribution of aortic androgen and estrogen receptors of male or female AXC/SSh rats is regulated by endogenous hormones. The observation that 17 beta-estradiol modulates aortic progesterone receptor content indicates that rat aortic estrogen receptors are physiologically functional. Our data imply that steroid hormones directly regulate aspects of rat cardiovascular cell function and that sexually dimorphic differential regulation may characterize male and female aortic metabolism.

Rat prostate cancer cells contain functional receptors for transforming growth factor-beta.

Clonally derived C-, D-, and T-family AXC/SSh rat prostate cancer cell lines contain transforming growth factor-beta (TGF beta) receptors. The content in C3, D1, T1, and T5 cells, respectively, was 8,560 +/- 1,450, 13,160 +/- 1,240, 2,425 +/- 490, and 10,540 +/- 1,025 sites/cell (mean +/- SEM). Respective Kd values were 160 +/- 48, 200 +/- 53, 24 +/- 3, and 115 +/- 15 pM (mean +/- SEM). T1 cell TGF beta receptor site content and Kd differed significantly from those of other prostate cancer cell lines (P less than 0.05). TGF beta is a bifunctional concentration-dependent modulator of T1 and T5 cell thymidine incorporation. At low concentrations, thymidine incorporation was inhibited, whereas as the medium TGF beta content was increased, T1 and T5 cell thymidine incorporation was stimulated. The concentrations of TGF beta causing half-maximum inhibition of T1 or T5 cell thymidine incorporation, respectively, were 0.11 and 0.24 pM, whereas the respective TGF beta concentrations causing half-maximum stimulation of thymidine incorporation were 14.4 and 134 pM. These findings establish that rat prostate cancer cell sensitivity to TGF beta inhibition of function is at least 2 orders of magnitude greater than that of most other mammalian cells. In contrast, the sensitivity of rat prostate cancer cells to TGF beta enhancement of function is comparable to that of other mammalian cells. TGF beta inhibited basic fibroblast growth factor (bFGF) stimulation of T1 and T5 cell thymidine incorporation. Because the concentration of bFGF required for half-maximum increase of T5 cell thymidine incorporation was independent of medium TGF beta content, the effect of TGF beta is distal to the T5 cell bFGF receptor. In contrast, the concentration of bFGF required for half-maximum increase in T1 cell thymidine incorporation increased 5-fold as the medium TGF beta content was increased; suggesting that the effect of TGF beta in T1 cells is proximal to the T1 cell bFGF receptor. Our studies establish that rat prostate cancer cells contain functional TGF beta receptors, imply the presence of functional bFGF receptors, and demonstrate that mitogen modulation of prostate cancer cell function is multifactorial. The finding that TGF beta is a bifunctional effector of prostate cancer cell DNA synthesis provides some insight into the potential complexity of mitogen modulation of prostate cancer cell proliferation. The mechanism by which these mitogens interact is unknown; however, our studies suggest that some interactive effects may be cell line specific.

Androgen modulation of prostate cancer cell androgen receptor content is cell line specific.

Using methods for cell lysis and fractionation which yield essentially quantitative recovery of rat prostate cancer cell cytosolic and nuclear androgen receptors, we examined androgen modulation of androgen receptor content of clonally derived prostate cancer cell lines. We showed that testosterone elicited a concentration-dependent 2.3-fold increase in T5 cell androgen receptor content which was maximum after 48 h and was maintained through at least 72 h of culture. Testosterone caused only a 1.4-fold elevation in D2 cell androgen receptor content which was maximum between 6 and 12 h of culture and was maintained through at least 72 h culture. In contrast, testosterone did not cause a change in C3 cell androgen receptor content. Cycloheximide inhibition showed that both the testosterone-mediated increase in and maintenance of basal prostate cancer cell androgen receptor content required protein synthesis. Because testosterone and the nonmetabolizable androgen R1881 were essentially equipotent as effectors of the increase in T5 cell androgen receptor content, findings using testosterone appear to represent maximum effects. RU 23908 antagonized both R1881 and testosterone promoted elevations of prostate cancer cell androgen receptor content. Effectiveness of RU 23908 was comparable to the relative binding affinity of R1881, testosterone and RU 23908 for androgen receptors. This implies that at least part of the androgen-promoted increase in prostate cancer cell androgen receptor content is mediated through the action of androgen receptors and suggests that androgen receptors may act as both cis and trans regulatory elements. The mechanisms which determine basal or androgen-modulated prostate cancer cell androgen receptor content remain to be elucidated.

Sexual dimorphism characterizes baboon myocardial androgen receptors but not myocardial estrogen and progesterone receptors.

Using biochemical methods we established that estrogen receptor content and distribution and progesterone receptor content in female and male baboon myocardium did not differ between sexes. In contrast, myocardial androgen receptor distribution between cytosolic and nuclear compartments was sexually dimorphic. Female baboon myocardial androgen receptors were restricted to the cytosolic compartment, whereas male myocardial androgen receptors were distributed between the cytosolic and nuclear compartments. Using human estrogen receptor cDNA we showed that baboon aorta, myocardium and uterus contain a 6.3 kb estrogen receptor transcript. Analyses performed with human progesterone receptor cDNA established that baboon aorta and uterus contain an 8 kb progesterone receptor transcript; however, progesterone receptor transcripts were not demonstrable in baboon myocardial RNA preparations. Because relative hybridization signal intensity reflected known uterine and aortic progesterone receptor content, failure to detect progesterone receptor transcripts in myocardial preparations may reflect sensitivity limitations and the fact that aortic progesterone receptor content is 5-fold greater than that of myocardium. Immunocytochemical analyses demonstrated that baboon myocardial progesterone receptors were present in greater than 25% of myocytes and generally absent from other myocardial cells. Our studies establish that: (1) gonadal steroid hormone receptor gene transcription occurs in cells of the baboon cardiovasculature, (2) these steroid hormone receptors may be physiologically functional, and (3) gonadal steroid hormone receptors may be restricted to specialized cells of the cardiovasculature.

Alteration in salivary function in early HIV infection.

The etiology of salivary gland hypofunction in HIV(+) patients is unclear. This study was designed to determine the effect of early-stage HIV(+) infection (CD4(+) > 200 cells/ micro L; n = 139) on salivary gland function and the relationship of this dysfunction to the taking of xerostomic medications. Salivary flow rates and the content of electrolytes and antimicrobial proteins in stimulated parotid and submandibular/sublingual saliva were determined. Compared with healthy controls (n = 50), the HIV(+) group showed significant reductions in flow rates of unstimulated whole (35%), stimulated parotid (47%), unstimulated submandibular/sublingual (23%), and stimulated submandibular/sublingual (39%) saliva. The flow rates for the HIV(+) patients taking xerostomic medications did not differ from those of patients who did not. Concentrations of some salivary gland components were altered in the HIV(+) group. Analysis of these data suggests that salivary gland function is adversely affected early in HIV infection and that these changes do not appear to be compounded by the taking of xerostomic medications.

Measuring short-term gamma-irradiation effects on mouse salivary gland function using a new saliva collection device.

A restraining device was designed specifically for the collection of whole saliva from mice without using anesthesia. As the procedure does not involve surgical cannulation of the salivary glands, saliva can be collected from the same mouse at different times. The time between the injection of a secretory stimulant (pilocarpine) and the appearance of saliva in the mouth (lag time) was 100.5 +/-8.5 s (mean+/-S.E.M., n=10) for control mice. The volume of saliva collected in the first 5 min was three times greater than that collected between 15 and 20 min. The average flow rate for a collection period of 15 min was 16.7 +/-1.8 microl/min (n=10). The flow rate was decreased 50% (P<0.005) whereas the lag time was increased more than 300% (P<0.05) at 24 h after irradiation. The concentrations of a 23.5-kDa protein and a mucin were decreased after irradiation whereas there was no significant effect on the concentration of amylase or peroxidase.

Estrogen-mediated cytoplasmic and nuclear distribution of rat cardiovascular estrogen receptors.

We used either the synthetic estrogen R2858 (moxestrol) or estradiol-17 beta to characterize estrogen receptors in cytoplasmic (R2858) and nuclear (estradiol-17 beta) preparations from rat aorta and myocardium. Relative steroid specificity studies showed that only estrogens were effective inhibitors of R2858 or estradiol-17 beta binding to aortic and myocardial estrogen receptors, whereas androgens, progestins, and cortisol were ineffective inhibitors. Low ionic strength sucrose density gradient analyses showed that myocardial estrogen receptors that localized in the cytoplasmic fraction migrated as macromolecules with sedimentation coefficients of 8S to 9S. In contrast, two binding components of sedimentation coefficients 8S to 9S and 10S to 11S were characteristic of the estrogen receptors localized in aortic cytoplasmic preparations. High ionic strength sucrose density gradient analysis showed that aortic and myocardial estrogen receptors localized in the nuclear fraction migrated as macromolecules with sedimentation coefficients of 4S to 6S. Saturation analyses showed that aortic and myocardial cytoplasmic preparations from intact young mature male rats contained 50.6 +/- 12.9 (mean +/- SD) and 51.0 +/- 14.1 fmol receptor/mg DNA, respectively. The respective R2858 dissociation constants were 0.42 and 0.15 nM. Estrogen receptors could not be demonstrated in nuclear preparations from cardiovasculature of intact males. Estradiol-17 beta injection of intact young mature male rats caused "depletion" of aortic and myocardial cytoplasmic fraction estrogen receptors and resulted in the appearance of 51.9 +/- 21.0 and 36.9 +/- 9.5 fmol receptor/mg DNA in the corresponding nuclear fractions. The respective estradiol-17 beta dissociation constants were 1.56 and 0.71 nM. Increased estrogen receptor content of cardiovascular nuclear fractions of estradiol-17 beta injected male rats correlated well with the concomitant decreased cytoplasmic fraction receptor content. The ability of estradiol-17 beta to affect localization of cardiovascular estrogen receptors between cytoplasmic and nuclear fractions suggests these estrogen receptors are physiologically functional and indicates that estrogen may directly regulate cardiovascular cell function.

Adenosine deaminase impairment and ribonucleotide reductase activity and levels in HeLa cells.

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4)-deficient patients recently were found to have abnormally high levels of dATP, a negative allosteric effector of ribonucleotide reductase (ribonucleoside-diphosphate reductase, 2'-deoxyribonucleoside-diphosphate:oxidized thioredoxin 2'-oxidoreductase, EC 1.17.4.1). Therefore it was proposed that the immunodeficiency associated with adenosine deaminase deficiency is mediated through inhibition of ribonucleotide reductase and hence DNA replication. HeLa cells, treated with an adenosine deaminase inhibitor, erythro-9(2-hydroxy-3-nonyl)adenine, and deoxyadenosine to mimic the adenosine deaminase-deficient state, were monitored to determine directly the effects on ribonucleotide reductase activity and levels. A low concentration of erythro-9-(2-hydroxy-3-nonyl)adenine, which did not inhibit cell growth, nevertheless retarded the cells in G2 + M phase of the cell cycle and increased reductase activity. Reductase activity was also elevated in cells treated with a low level of deoxyadenosine which did not affect the cell cycle or cell growth. However, ribonucleotide reductase activity was reduced to one-half of the control value in cells treated with either enough deoxyadenosine to inhibit cell growth or with a combination of erythro-9(2-hydroxy-3-nonyl)adenine and deoxyadenosine, each at concentrations which individually do not inhibit cell growth. Removal of deoxynucleotides, particularly dATP, from these extracts increased ribonucleotide reductase activity to several-fold higher than control values. The reduced activity of ribonucleotide reductase in the simulated adenosine deaminase-deficient HeLa cells provides direct evidence for the thesis that adenosine deaminase deficiency disease is mediated through elevated levels of dATP which inhibit ribonucleotide reductase. In addition, the cell cycle patterns and ribonucleotide reductase levels suggest that the regulatory substance(s) that controls the level of ribonucleotide reductase is not operative until the late S or G2 phase of the cell cycle.