An epigenome-wide association study meta-analysis of educational attainment.

The epigenome is associated with biological factors, such as disease status, and environmental factors, such as smoking, alcohol consumption and body mass index. Although there is a widespread perception that environmental influences on the epigenome are pervasive and profound, there has been little evidence to date in humans with respect to environmental factors that are biologically distal. Here we provide evidence on the associations between epigenetic modifications-in our case, CpG methylation-and educational attainment (EA), a biologically distal environmental factor that is arguably among the most important life-shaping experiences for individuals. Specifically, we report the results of an epigenome-wide association study meta-analysis of EA based on data from 27 cohort studies with a total of 10 767 individuals. We find nine CpG probes significantly associated with EA. However, robustness analyses show that all nine probes have previously been found to be associated with smoking. Only two associations remain when we perform a sensitivity analysis in the subset of never-smokers, and these two probes are known to be strongly associated with maternal smoking during pregnancy, and thus their association with EA could be due to correlation between EA and maternal smoking. Moreover, the effect sizes of the associations with EA are far smaller than the known associations with the biologically proximal environmental factors alcohol consumption, body mass index, smoking and maternal smoking during pregnancy. Follow-up analyses that combine the effects of many probes also point to small methylation associations with EA that are highly correlated with the combined effects of smoking. If our findings regarding EA can be generalized to other biologically distal environmental factors, then they cast doubt on the hypothesis that such factors have large effects on the epigenome.

Genomic profiles and predictors of early allograft dysfunction after human liver transplantation.

Early hepatic allograft dysfunction (EAD) manifests posttransplantation with high serum transaminases, persistent cholestasis, and coagulopathy. The biological mechanisms are poorly understood. This study investigates the molecular mechanisms involved in EAD and defines a gene expression signature revealing different biological pathways in subjects with EAD from those without EAD, a potential first step in developing a molecular classifier as a potential clinical diagnostic. Global gene expression profiles of 30 liver transplant recipients of deceased donor grafts with EAD and 26 recipients without graft dysfunction were investigated using microarrays of liver biopsies performed at the end of cold storage and after graft reperfusion prior to closure. Results reveal a shift in inflammatory and metabolic responses between the two time points and differences between EAD and non-EAD. We identified relevant pathways (PPARα and NF-κB) and targets (such as CXCL1, IL1, TRAF6, TIPARP, and TNFRSF1B) associated with the phenotype of EAD. Preliminary proof of concept gene expression classifiers that distinguish EAD from non-EAD patients, with Area Under the Curve (AUC) >0.80 were also identified. This data may have mechanistic and diagnostic implications for EAD.

Examination of protective effect of ischemic postconditioning after small bowel autotransplantation.

Ischemia/reperfusion (I/R) injury is a serious condition that results from some surgical procedures, including intestinal transplantation. Ischemic postconditioning is defined as brief periods of reperfusion alternating with reocclusion applied during the early minutes after reperfusion. The objective of this study was to investigate the effect of ischemic postconditioning before small bowel autotransplantation. Total orthotopic intestinal autotransplantation was performed in 30 white domestic pigs. Grafts were stored in cold University of Wisconsin solution for 1, 3, or 6 hours. Duration of reperfusion was 3 hours in all grafts. Before reperfusion, the intestine was postconditioned via 3 cycles of ischemia for 30 seconds and reperfusion for 30 seconds (ischemic postconditioning protocol). Tissue from the small intestine was obtained after laparotomy (control group) and at the end of reperfusion periods. To monitor oxidative stress, tissue concentrations of malondialdehyde and reduced glutathione, and activity of superoxide dismutase were determined at spectrophotometry. Tissue damage on sections stained with hematoxylin- eosin was evaluated using a quantitative method (Scion Image software; Scion Corp, Frederick, Maryland). Our results demonstrated that ischemic postconditioning significantly decreased the reperfusion-ended lipid peroxidation value (mean +/- SEM, 142.0 +/- 7.1 micromol/g vs 125.0 +/- 2.1 micromol/g; P < .05). Moreover, the capacity and activity of endogenous antioxidant protective systems (glutathione 789+/-8.0 micromol/g vs 934 +/- 5.7 micromol/g, and superoxide dismutase 110 +/- 9 IU/g vs 126 +/- 4 IU/g; P < .05) remained higher in the ischemic postconditioning groups compared with tissues without ischemic postconditioning. At quantitative analysis, tissue injury was increased by the duration of cold preservation. The greatest injury was observed in the mucosal and submucosal layers and in the depth of crypts after 6 hours of preservation. Ischemic postconditioning significantly decreased intestinal wall injury in each group (P < .05). It was concluded that ischemic postconditioning before reperfusion mitigated oxidative stress and histologic damage during small bowel autotransplantation.

Computer-aided detection (CAD) as a second reader using perspective filet view at CT colonography: effect on performance of inexperienced readers.

AIM: To evaluate whether computer-aided detection (CAD) as a second reader using perspective filet view [three-dimensional (3D) filet] improves the performance of inexperienced readers at computed tomography colonography (CTC) compared with unassisted 3D filet and unassisted two-dimensional (2D) CTC. MATERIAL AND METHODS: Fifty symptomatic patients underwent CTC and same-day colonoscopy with segmental unblinding. Two inexperienced readers read the CTC studies on 3D filet and 2D several weeks apart. Four months later, readers re-read the cases only evaluating CAD marks using 3D filet. Suspicious CAD marks not previously described on 3D filet were recorded. Jackknife free-response receiver operating characteristic (JAFROC-1) analysis was used to compare the observers' performances in detecting lesions with 3D filet, 2D and 3D filet with CAD. RESULTS: One hundred and three lesions > or =3mm were detected at colonoscopy with segmental unblinding. CAD alone had a sensitivity of 73% (75/103) at a mean false-positive rate per patient of 12.8 in supine and 11.4 in prone. For inexperienced readers sensitivities with 3D filet with CAD were 58% (60/103) and 48% (50/103) with an improvement of 14-16 percentage points (p<0.05) compared with 2D and of 10-11 percentage points (p<0.05) compared with 3D filet. For inexperienced readers, the false-positive rate was 25-41% and 71-200% higher with 3D filet with CAD compared with 3D filet and 2D, respectively. JAFROC-1 analysis showed no significant differences in per-lesion overall performance among reading modes (p=0.8). CONCLUSION: CAD applied as a second reader using 3D filet increased both sensitivity and the number of false positives by inexperienced readers compared with 3D filet and 2D, thus not improving overall performance, i.e., the ability to distinguish between lesions and non-lesions.

Primary three-dimensional analysis with perspective-filet view versus primary two-dimensional analysis: evaluation of lesion detection by inexperienced readers at computed tomographic colonography in symptomatic patients.

BACKGROUND: "Perspective-filet view" is a novel three-dimensional (3D) viewing technique for computed tomography colonography (CTC). Studies with experienced readers have shown a sensitivity for perspective-filet view similar to that of 2D or 3D endoluminal fly-through in detection of colorectal lesions. It is not known whether perspective-filet view, compared to axial images, improves lesion detection by inexperienced readers. PURPOSE: To compare primary 3D analysis using perspective-filet view (3D Filet) with primary 2D analysis, as used by inexperienced CTC readers. Secondary aims were to compare lesion detection by 3D Filet when used by experienced and inexperienced readers, and to evaluate the effect of combined 3D Filet + 2D analysis. MATERIAL AND METHODS: Fifty symptomatic patients were prospectively enrolled. An experienced reader performed 3D Filet analysis followed by complete 2D analysis (3D Filet + 2D), before colonoscopy with segmental unblinding. Two inexperienced readers (readers 2 and 3), blinded to CTC and colonoscopy findings, retrospectively performed 3D Filet analysis and, after 5 weeks, 2D analysis. True positives >or=6 mm detected by the inexperienced readers with 3D Filet and/or 2D were combined to obtain 3D Filet + 2D. RESULTS: Colonoscopy revealed 116 lesions: 16 lesions >or=10 mm, 19 lesions 6-9 mm, and 81 lesions or=6 mm with 3D Filet and 3D Filet + 2D were 77% and 83%, respectively. For the inexperienced readers, sensitivities for lesions >or=6 mm with 3D Filet and 2D were 51% and 57% (reader 2) and 40% and 43% (reader 3), respectively. There was no significant difference between 3D Filet and 2D regarding sensitivity and reading time. For lesions >or=6 mm, 3D Filet + 2D improved the sensitivity of reader 2 to 63% and of reader 3 to 51%. CONCLUSION: Lesion detection by inexperienced readers using perspective-filet view is comparable to that obtained by 2D. Lesion detection improves by combining 3D Filet + 2D, but not to the level of an experienced reader.

Reperfusion injury and inflammatory responses following acute lower limb revascularization surgery.

After revascularization of an acute arterial occlusion the development of a serious ischaemic-reperfusion injury is a menacing challenge and a hard task in peripheral vascular surgery. A whale of evidences point to oxidative stress, as an important trigger, in the complex chain of events leading to reperfusion injury. In the present study authors aimed to examine oxidative stress parameters, antioxidant-prooxidant state and leukocyte adhesion molecules (CD11a and CD18) expression following acute revascularization surgery of lower limb.10 patients were examined in the prospective randomized study. Peripheral blood sample was collected in ischaemic period, and after reperfusion in the 2nd and 24th hours, and on 7th day. Superoxide-dismutase activity, reduced glutathion concentration and leukocytes free radical production were measured. The degree of lipidperoxidation was marked with the quantity of malondialdehyde. The expressions of adhesion molecules were measured with flowcytometry.The speed and rate of free radical production significantly increased in the early reperfusion (p<0.05). The level of antioxidant enzymes decreased after revascularization. The CD11a and CD18 expression of the granulocytes significantly (p<0.05) decreased right after the revascularization, but with a gradual elevation until the 7th day they exceed the ischaemic value. Our results showed a time specific turnover of the sensitive antioxidant-prooxidant balance after revascularization operation.

Insulin growth factor-binding protein 2 is a candidate biomarker for PTEN status and PI3K/Akt pathway activation in glioblastoma and prostate cancer.

PTEN is an important tumor-suppressor gene associated with many cancers. Through expression profiling of glioblastoma tissue samples and prostate cancer xenografts, we identified a molecular signature for loss of the PTEN tumor suppressor in glioblastoma and prostate tumors. The PTEN signature consists of a minimum of nine genes, several of which are involved in various pathways already implicated in tumor formation. Among these signature genes, the most significant was an increase in insulin growth factor-binding protein 2 (IGFBP-2) mRNA. Up-regulation of IGFBP-2 was confirmed at the protein level by Western blot analysis and validated in samples not included in the microarray analysis. The link between IGFBP-2 and PTEN was of particular interest because elevated serum IGFBP-2 levels have been reported in patients with prostate and brain tumors. To further investigate this link, we determined that IGFBP-2 expression is negatively regulated by PTEN and positively regulated by phosphatidylinositol 3-kinase (PI3K) and Akt activation. In addition, Akt-driven transformation is impaired in IGFBP2(-/-) mouse embryo fibroblasts, implicating a functional role for IGFBP-2 in PTEN signaling. Collectively, these studies establish that PTEN and IGFBP-2 expression are inversely correlated in human brain and prostate cancers and implicate serum IGFBP-2 levels as a potential serum biomarker of PTEN status and PI3K Akt pathway activation in cancer patients.

Analysis of oncogenic signaling networks in glioblastoma identifies ASPM as a molecular target.

Glioblastoma is the most common primary malignant brain tumor of adults and one of the most lethal of all cancers. Patients with this disease have a median survival of 15 months from the time of diagnosis despite surgery, radiation, and chemotherapy. New treatment approaches are needed. Recent works suggest that glioblastoma patients may benefit from molecularly targeted therapies. Here, we address the compelling need for identification of new molecular targets. Leveraging global gene expression data from two independent sets of clinical tumor samples (n = 55 and n = 65), we identify a gene coexpression module in glioblastoma that is also present in breast cancer and significantly overlaps with the "metasignature" for undifferentiated cancer. Studies in an isogenic model system demonstrate that this module is downstream of the mutant epidermal growth factor receptor, EGFRvIII, and that it can be inhibited by the epidermal growth factor receptor tyrosine kinase inhibitor Erlotinib. We identify ASPM (abnormal spindle-like microcephaly associated) as a key gene within this module and demonstrate its overexpression in glioblastoma relative to normal brain (or body tissues). Finally, we show that ASPM inhibition by siRNA-mediated knockdown inhibits tumor cell proliferation and neural stem cell proliferation, supporting ASPM as a potential molecular target in glioblastoma. Our weighted gene coexpression network analysis provides a blueprint for leveraging genomic data to identify key control networks and molecular targets for glioblastoma, and the principle eluted from our work can be applied to other cancers.

The modulatory effect of estrogen on the neuronal activity in the barrel cortex of the rat. An electrophysiological study.

In acute experiments, the effects of iontophoretically applied 17 beta-estradiol hemisuccinate on the activity of the primary somatosensory cortical neurons were studied in ovariectomized rats by extracellular single-unit recording. 17 beta-Estradiol increased both the spontaneous and the vibrissa deflection-evoked responses, with an average latency of 24 min. It is suggested that this relatively long latency of the 17 beta-estradiol effect is based not so much on membrane mechanisms as on genomic mechanisms.

Two MHC surface amino acid differences distinguish foreign peptide recognition from autoantigen specificity.

KRN T cells can recognize two self MHC alleles with differing biological consequences. They respond to the foreign peptide RN(42--56) bound to I-A(k) or alternatively initiate autoimmune arthritis by interacting with a self Ag, GPI(282--294), on I-A(g7). Five surface amino acid differences between the two MHC molecules collectively alter which peptide side chains are recognized by the KRN TCR. In this study, it is shown that mutation of only two of these residues, alpha 65 and beta 78, in I-A(k) to their I-A(g7) counterparts is sufficient to allow recognition of the TCR contacts from GPI(282--294). To provide a detailed mechanism for the specificity change, the distinct contributions of each of these two mutations to the global effect on peptide specificity were analyzed. The alpha65 mutation is shown to broaden the spectrum of amino acids permissible at P8 of the peptide. In contrast, the beta 78 mutation alone blocks KRN TCR interaction with I-A(k) and requires the simultaneous presence of the alpha 65 mutation to preserve recognition. In the presence of the alpha 65 mutation, the beta 78 residue broadens peptide recognition at P3 and prevents recognition of the P8 L in RN(42--56), thus producing the observed specificity shift. These results localize the functionally relevant differences between the surfaces of two self-restricted MHC molecules to two residues that have counterbalanced positive and negative contributions to interaction with a single TCR. They highlight how subtle structural distinctions attributable to single amino acids can stand at the interface between foreign Ag responsiveness and pathogenic autoreactivity.

Structural and functional consequences of altering a peptide MHC anchor residue.

To better understand TCR discrimination of multiple ligands, we have analyzed the crystal structures of two Hb peptide/I-E(k) complexes that differ by only a single amino acid substitution at the P6 anchor position within the peptide (E73D). Detailed comparison of multiple independently determined structures at 1.9 A resolution reveals that removal of a single buried methylene group can alter a critical portion of the TCR recognition surface. Significant variance was observed in the peptide P5-P8 main chain as well as a rotamer difference at LeuP8, approximately 10 A distal from the substitution. No significant variations were observed in the conformation of the two MHC class II molecules. The ligand alteration results in two peptide/MHC complexes that generate bulk T cell responses that are distinct and essentially nonoverlapping. For the Hb-specific T cell 3.L2, substitution reduces the potency of the ligand 1000-fold. Soluble 3.L2 TCR binds the two peptide/MHC complexes with similar affinity, although with faster kinetics. These results highlight the role of subtle variations in MHC Ag presentation on T cell activation and signaling.

Nitric oxide improves cisplatin cytotoxicity in head and neck squamous cell carcinoma.

OBJECTIVE: To test whether nitric oxide (NO) enhances the cytotoxicity of cisplatin in a head and neck squamous cell carcinoma (HNSCC) cell line. BACKGROUND: Cisplatin is one of the most frequently used chemotherapeutic agents in the treatment of HNSCC. NO has been shown to play an important role in regulating tumor growth. Previous studies demonstrate that NO can enhance the cytotoxicity of cisplatin in Chinese hamster lung fibroblasts. In this report, we examined the in vitro interaction of NO and cisplatin in a HNSCC cell line. MATERIALS AND METHODS: CCL23 cells were pretreated with three different NO donors: PAPA/NO (t 1/2 = 15 min), DPTA/NO (t 1/2 = 3 h), and DETA/NO (t 1/2 = 20 h). The cells were rinsed and exposed for 6 hours to a culture medium containing cisplatin. Cell survival and LD50 of cisplatin were calculated with and without NO pretreatment. RESULTS: PAPA/NO and DPTA/NO did not show any cytotoxic activity and did not change the LD50 of cisplatin. DETA/NO when used alone resulted in 25.6% cell death at its peak dose (100 microM). Pretreatment with DETA/NO resulted in almost a threefold reduction of the LD50 of cisplatin (6.8 vs. 2.4 microg/mL). Pretreatment with DETA/NO sensitized the HNSCC cells to subsequent cisplatin activity (two-sided P =.00016). CONCLUSION: Pretreatment of HNSCC cells with long-acting NO donors enhances cisplatin activity. Short- and medium-acting NO donors do not exert a toxic effect and do not augment the activity of cisplatin. NO agonists should be considered in the future as a possible adjunct to cisplatin in the treatment of HNSCC. Further studies with animal models are necessary to further clarify this relationship.

Trimidox, an inhibitor of ribonucleotide reductase, induces apoptosis and activates caspases in HL-60 promyelocytic leukemia cells.

Ribonucleotide reductase (RR) is the rate-limiting enzyme for the de novo synthesis of deoxyribonucleotides. Its activity is significantly increased in tumor cells related to the proliferation rate. Therefore, the enzyme is considered to be an excellent target for cancer chemotherapy. In the present study, we investigated whether the antineoplastic effects of trimidox (3,4, 5-trihydroxybenzamidoxime), a novel inhibitor of RR, were due to induction of apoptosis.HL-60 cells were incubated with various concentrations of trimidox. Consequently, cell morphology, DNA condensation, annexin binding, DNA fragmentation, and signature type cleavage of poly(ADP-ribose)polymerase and gelsolin were determined. We also tested the involvement of CD95 and CD95 ligand in apoptosis induction. Furthermore, we examined the c-myc expression of HL-60 cells after incubation with trimidox in order to elucidate a possible association between c-myc expression and induction of apoptosis in the case of trimidox. Trimidox incubation caused a time-dependent increase of c-myc RNA expression and this was accompanied by the induction of apoptosis. Apoptosis was triggered independently of CD95 by the activation of caspases and PARP cleavage. We conclude that trimidox is able to induce programmed cell death. The induction of apoptosis was demonstrated by various biochemical and morphological methods and seems to be associated with the induction of c-myc. Apoptosis was induced by the activation of caspases and without change of the CD95 and CD95 ligand expression.

Molecular basis for recognition of an arthritic peptide and a foreign epitope on distinct MHC molecules by a single TCR.

KRN TCR transgenic T cells recognize two self-MHC molecules: a foreign peptide, bovine RNase 42-56, on I-Ak and an autoantigen, glucose-6-phosphate isomerase 282-294, on I-Ag7. Because the latter recognition event initiates a disease closely resembling human rheumatoid arthritis, we investigated the structural basis of this pathogenic TCR's dual specificity. While peptide recognition is altered to a minor degree between the MHC molecules, we show that the receptor's cross-reactivity critically depends upon a TCR contact residue completely conserved in the foreign and self peptides. Further, the altered recognition of peptide derives from discrete differences on the MHC recognition surfaces and not the disparate binding grooves. This work provides a detailed structural comparison of an autoreactive TCR's interactions with naturally occurring peptides on distinct MHC molecules. The capacity to interact with multiple self-MHCs in this manner increases the number of potentially pathogenic self-interactions available to a T cell.

Estrogen effects on arcuate neurons in rat. An in situ electrophysiological study.

In acute experiments, the effects of i.p. 17beta-estradiol on the activity of arcuate neurons were studied in ovariectomized rats. 17Beta-estradiol (100 microg/100g, i.p.) increased the spontaneous activity of the observed arcuate neurons with a latency of 20-25 min. In some neurons spontaneous activity could be influenced by stimulation of the olfactory and somatosensory systems. Activation of the trigeminal system significantly increased the spontaneous activity of the studied units, while stimulation of the accessory olfactory bulb decreased it, both with and without 17beta-estradiol treatment. It is suggested that the 20-25 min latency of the 17beta-estradiol effect is based not so much on membrane as on genomic mechanisms. This suggestion is supported by immunocytochemical studies: 17beta-estradiol treatment significantly decreased the number of GABA-positive axo-somatic synapses in the arcuate nucleus.

A modified method of intracytoplasmic sperm injection without the use of polyvinylpyrrolidone.

In a controlled study we compared the outcome of intracytoplasmic sperm injection (ICSI) performed by two different methods. The oocytes from 20 patients were equally divided into two groups and injected either by conventional ICSI using polyvinylpyrrolidone (PVP) or by a modified PVP-free ICSI procedure. While in the conventional ICSI method the spermatozoon is aspirated into the injection pipette, in the modified ICSI procedure the spermatozoon is attached to the end of the narrow micropipette by aspirating its tail. The sperm head is never drawn into the pipette. Accordingly, even a fast-moving spermatozoon can be 'caught' easily. As a result of such an aspiration the spermatozoon loses its motility. Therefore, PVP is required neither to slow down the movement of the spermatozoon nor to facilitate the movement of the solution in the injection pipette. A total of 230 mature oocytes were injected by both methods and the results were analysed. No differences were observed in survival rate between the two ICSI procedures (89% and 91%, respectively). However, the proportion of normally fertilized oocytes was significantly higher after microfertilization by modified ICSI (74%) when compared with the outcome of the conventional ICSI method (62%). The frequency of abnormal fertilization was not influenced by the method of ICSI used. The cleavage rate and quality of resulting embryos were also comparable. In conclusion, we have demonstrated a modified ICSI method which does not require the use of PVP. When compared with the conventional ICSI procedure, even better fertilization rates can be achieved. The proposed ICSI modification may provide an alternative procedure for elimination of the potentially harmful effects which may be associated with conventional ICSI.

Biochemical evidence for a novel low molecular weight 2-5A-dependent RNase L in chronic fatigue syndrome.

Previous studies from this laboratory have demonstrated a statistically significant dysregulation in several key components of the 2',5'-oligoadenylate (2-5A) synthetase/RNase L and PKR antiviral pathways in chronic fatigue syndrome (CFS) (Suhadolnik et al. Clin Infect Dis 18, S96-104, 1994; Suhadolnik et al. In Vivo 8, 599-604, 1994). Two methodologies have been developed to further examine the upregulated RNase L activity in CFS. First, photoaffinity labeling of extracts of peripheral blood mononuclear cells (PBMC) with the azido 2-5A photoaffinity probe, [32P]pApAp(8-azidoA), followed by immunoprecipitation with a polyclonal antibody against recombinant, human 80-kDa RNase L and analysis under denaturing conditions. A subset of individuals with CFS was identified with only one 2-5A binding protein at 37 kDa, whereas in extracts of PBMC from a second subset of CFS PBMC and from healthy controls, photolabeled/immunoreactive 2-5A binding proteins were detected at 80, 42, and 37 kDa. Second, analytic gel permeation HPLC was completed under native conditions. Extracts of healthy control PBMC revealed 2-5A binding and 2-5A-dependent RNase L enzyme activity at 80 and 42 kDa as determined by hydrolysis of poly(U)-3'-[32P]pCp. A subset of CFS PBMC contained 2-5A binding proteins with 2-5A-dependent RNase L enzyme activity at 80, 42, and 30 kDa. However, a second subset of CFS PBMC contained 2-5A binding and 2-5A-dependent RNase L enzyme activity only at 30 kDa. Evidence is provided indicating that the RNase L enzyme dysfunction in CFS is more complex than previously reported.

Inhibition of growth of estrogen receptor positive and estrogen receptor negative breast cancer cells in culture by AA-etherA, a stable 2-5A derivative.

The design, chemical synthesis and biological activities of a nuclease-resistant, nontoxic bioactive 2-5A derivative, AA-etherA [i.e., adenylyl-(2'-5')-adenylyl-(2'-2")-9-[(2'-hydroxyethoxy)-methyl]adenine], are described as a new approach to the inhibition of breast cancer cell growth. AA-etherA inhibits DNA replication and cell division of both estrogen receptor positive (MCF-7) and estrogen receptor negative (BT-20) breast cancer cells in culture in a dose-dependent manner. Maximal inhibition in MCF-7 and BT-20 cells was obtained with 100 microM AA-etherA after four days of treatment, with an GI50 of 58 and 37 microM, respectively. AA-etherA is stable in the cytoplasm. Treated cells accumulate within the late G1/early S phase of the cell cycle and then progress only very slowly through S phase. AA-etherA does not activate RNase L, as do 2-5A and other 2-5A derivatives, nor does it increase p68 kinase (PKR) content of the cells. High resolution, two-dimensional protein gel electrophoresis reveals twofold or greater inhibition of synthesis of 92 proteins out of 682 proteins that were reproducibly detected as high quality spots with average rates of synthesis of > or = 20 p.p.m. in untreated cells. The specificity of the effects of AA-etherA on select proteins and its failure to activate RNase L indicate that AA-etherA does not act through a general effect on mRNA translation or stability, but rather inhibits cell proliferation through a block to DNA replication, with a concommitant reduction in the synthesis of specific proteins, some of which may be required for cell cycle transit. Two likely targets to account for the AA-etherA inhibition of DNA replication are DNA topoisomerase I, which is inhibited by AA-etherA in other cell lines, and thymidine kinase, which could be inhibited in a manner similar to the effect of acyclovir. These data indicate that 2-5A analogs, particularly bifunctional 2-5A analogs like AA-etherA, will be useful for controlling cancer cell growth. Further development of such 2-5A analogs may provide highly specific compounds for chemotherapy and chemoprevention.

Structure of a histidine-X4-histidine zinc finger domain: insights into ADR1-UAS1 protein-DNA recognition.

The solution structure for a mutant zinc finger peptide based on the sequence of the C-terminal ADR1 finger has been determined by two-dimensional NMR spectroscopy. The mutant peptide, called PAPA, has both proline residues from the wild-type sequence replaced with alanines. A nonessential cysteine was also replaced with alanine. The behavior of PAPA in solution implicates the prolines in the conformational heterogeneity reported earlier for the wild-type peptide [Xu, R. X., Horvath, S. J., & Klevit, R. E. (1991) Biochemistry 30, 3365-3371]. The solution structure of PAPA reveals several interesting features of the zinc finger motif. The residue immediately following the second cysteine ligand adopts a positive phi angle, which we propose is a common feature of this class of zinc fingers, regardless of whether this residue is a glycine. The NMR spectrum and resulting solution structure of PAPA suggest that a side-chain to side-chain hydrogen bond involving an arginine and an aspartic acid analogous to one observed in the Zif268 protein-DNA cocrystal structure exists in solution in the absence of DNA [Pavletich, N. P., & Pabo, C. O. (1991) Science 252, 809-817]. A model for the interaction between the two ADR1 zinc fingers and their DNA binding sites was built by superpositioning the refined solution structures of PAPA and ADR1b onto the Zif268 structure. This model offers structural explanations for a variety of mutations to the ADR1 zinc finger domains that have been shown to affect DNA-binding affinity or specificity.

Galanin immunoreactive neurons in the medulla oblongata of rats.

The distribution of galanin-immunoreactive (-ir) neurons in the medulla oblongata was mapped with light microscopic immunohistochemistry. No immunopositive perikarya were seen in untreated rats. Two days after colchicine treatment, galanin immuno-positive neurons were localized in the following areas: 1) raphe nuclei (magnus, pallidus and obscurus); 2) in various parts of the reticular formation, mainly in the territory of the catecholaminergic groups and in the peritrigeminal subdivision of the lateral reticular nucleus; 3) vagal nuclei (nucleus of the solitary tract, nucleus ambiguous); 4) two cell groups at the ventral surface of the rostro-caudal middle portion of the medulla oblongata (they do not correspond to any known demarkated anatomical nuclei, but related to the chemosensitive medullary area); 5) in the gelatinous part of the spinal trigeminal nucleus. The wide distribution of galanin neurons in the medulla support data that had been reported on the role of this peptide in various viscerosensory and autonomic mechanisms. In addition to these, galanin seems to be an important factor in the restoration of lesioned neurons (nerve growth factor-like activity). An increased galanin mRNA expression can be seen in dorsal vagal or hypoglossal motor neurons after intracranial transections of vagal or hypoglossal nerves, respectively. Transections of the olivocerebellar tract induced galanin gene expression in neurons of the contralateral inferior olive. After brainstem hemisection, galanin immunopositivity was seen in cells of the nucleus of the solitary tract due to the transection of ascending projections of this primary autonomic center in the medulla oblongata.