Characterization of Mg2+ binding to the DNA repair protein apurinic/apyrimidic endonuclease 1 via solid-state 25Mg NMR spectroscopy.

Apurinic/apyrimidinic endonuclease 1 (APE1), a member of the divalent cation-dependent phosphoesterase superfamily of proteins that retain the conserved four-layered alpha/beta-sandwich structural core, is an essential protein that functions as part of base excision repair to remove mutagenic and cytotoxic abasic sites from DNA. Using low-temperature solid-state (25)Mg NMR spectroscopy and various mutants of APE1, we demonstrate that Mg(2+) binds to APE1 and a functional APE1-substrate DNA complex with an overall stoichiometry of one Mg(2+) per mole of APE1 as predicted by the X-ray work of Tainer and co-workers (Mol, C. D.; Kuo, C. F.; Thayer, M. M.; Cunningham, R. P.; Tainer, J. A. Nature 1995, 374 , 381-386). However, the NMR spectra show that the single Mg(2+) site is disordered. We discuss the probable reasons for the disorder at the Mg(2+) binding site. The most likely source of this disorder is arrangement of the protein-ligands about the Mg(2+) (cis and trans isomers). The existence of these isomers reinforces the notion of the plasticity of the metal binding site within APE1.

A 4-mm probe for (13)C CP/MAS NMR of solids at 21.15 T.

The design of a broadband 4-mm magic-angle spinning (MAS) X-(1)H/(19)F double resonance probe for cross-polarization (CP)/MAS NMR studies at 21.15 T ((1)H at 900 MHz) is described. The high-frequency (1)H/(19)F channel employs a new and efficient transmission line tuning design. The first (13)C CP/MAS NMR spectra recorded at 21.15 T have been obtained with this probe and exhibit the best S/N per milligram sample of hexamethylbenzene achieved so far for a 4-mm rotor.

A general strategy for the NMR observation of half-integer quadrupolar nuclei in dilute environments.

A general strategy for the observation of low gamma half-integer quadrupolar nuclides in biological systems is presented. The methodology combines low-temperature (4-100 K) techniques with cross-polarization (CP) experiments while employing a so-called Carr-Purcell-Meiboom-Gill spin-echo sequence (CPMG). This combined approach is termed CP/QCPMG. Also discussed are data processing issues that are unique to the induced signals that result from the QCPMG pulse sequence. Central to this strategy is the development of a stable low-temperature (4 to 250 K) NMR double-resonance probe. The probe is robust enough to handle multiple contact experiments and long acquisition periods with 1H decoupling. This approach is illustrated with low-temperature solid-state 67Zn and 25Mg NMR CP/QCPMG experiments on model compounds. The conclusion reached is that the strategy affords sufficient sensitivity to examine Zn2+ and/or Mg2+ binding sites in metalloproteins.

Heparin-binding epidermal-growth-factor-like growth factor gene expression is induced by scrape-wounding epithelial cell monolayers: involvement of mitogen-activated protein kinase cascades.

Peptide growth factors can promote the cell migration and proliferation that is needed to repair epithelia after mechanical or chemical injury. We report here that scrape-wounding rat intestinal epithelial (RIE-1) cell monolayers caused a rapid increase in levels of heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) mRNA, with a maximal response at approx. 1 h. Hybridization in situ showed that transcript induction occurred primarily in cells at or near wound borders. The increase in HB-EGF mRNA was preceded by activation of the p42 mitogen-activated protein kinase (MAPK) in the wounded cell cultures. Moreover, the induction of HB-EGF mRNA was blocked by PD098059 and U0126, inhibitors that prevent the activation of p42/p44 MAPKs and extracellular signal-regulated protein kinase 5 (ERK5). Both p42 MAPK activation and HB-EGF mRNA induction were inhibited by genistein, indicating a requirement for an upstream tyrosine kinase activity. In contrast, neither response was affected by inhibition of phosphoinositide 3-kinase activity, down-regulation of protein kinase C, or disruption of the actin cytoskeleton with cytochalasin B. We conclude that scrape-wounding epithelial cell monolayers induces HB-EGF mRNA expression by a mechanism that most probably requires p42/p44 MAPK activation, although we cannot exclude a role for ERK5. Our results suggest a physiological role for locally synthesized HB-EGF in promoting epithelial repair after injury.

An integrated confocal and magnetic resonance microscope for cellular research.

Complementary data acquired with different microscopy techniques provide a basis for establishing a more comprehensive understanding of health and disease at a cellular level, particularly when data acquired with different methodologies can be correlated in both time and space. In this Communication, a brief description of a novel instrument capable of simultaneously performing confocal optical and magnetic resonance microscopy is presented, and the first combined images of live Xenopus laevis oocytes are shown. Also, the potential benefits of combined microscopy are discussed, and it is shown that the a priori knowledge of the high-resolution optical images can be used to enhance the boundary resolution and contrast of the MR images.

Cyclic AMP inhibits agonist-induced heparin-binding EGF gene expression independently of effects on p42/p44 MAPK activation.

Heparin-binding EGF-like growth factor (HB-EGF) mRNA levels are increased up to 20-fold in RIE-1 cells by two agonists that act through distinct receptor types. We demonstrated a common requirement for p42/p44 mitogen-activated protein kinase (MAPK) in this response using the selective MAPK kinase (MEK) inhibitor, PD 098059. Agonist-mediated induction of HB-EGF mRNA was markedly suppressed in cells that had been treated with cyclic AMP-elevating agents. In contrast, the activation of p42 MAPK in response to agonists was not affected by raising cellular cyclic AMP levels. We conclude that cyclic AMP negatively regulates the HB-EGF gene, but that the inhibitory action is either independent of the p42/p44 MAPK pathway or the site of action is distal to MAPK activation.

Characterisation of the rat heparin-binding epidermal growth factor-like growth factor gene promoter.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene expression is strongly activated by a variety of extracellular stimuli, acting through the Raf/MEK/MAP kinase pathway. To study the elements that respond to this pathway, we have isolated and sequenced a fragment of the rat HB-EGF gene promoter. By transfection of a series of promoter/reporter constructs into cells, a minimal promoter element was demonstrated to lie between 448 bp upstream of the transcriptional start site and 103 bp into the first exon of the gene. However co-transfection of the promoter constructs with a plasmid directing expression of RafCAAX, an activated c-Raf-1 protein, gave a fold-stimulation of activity no greater than that seen for the parental pGL3-Basic plasmid alone. In addition, agonist stimulation of cell lines stably transfected with a HB-EGF promoter/luciferase construct produced little or no increase in reporter enzyme activity. These results suggest that the c-Raf-1 responsive elements lie outside the tested region of the rat HB-EGF gene. However, it has been reported that a c-Raf-1 responsive element is present within the equivalent region of the mouse gene. A comparison of the 5'-flanking regions of the mouse, rat and human HB-EGF genes indicated that the mouse sequence diverges abruptly from that of the other two species approximately 260 bp upstream of the transcriptional start site. PCR analysis of mouse genomic DNA suggests that this sequence divergence is due to DNA rearrangement during the cloning of the mouse gene. Additional studies are therefore required to identify Raf/MAP kinase responsive elements in the HB-EGF gene.

Management of neck lumps--a triage model.

We report our experience in the out-patient triage of 100 patients presenting with a lump in the neck. The out-patient visit consisted of a general history and examination, assessment of the upper air and food passages and, where indicated, ultrasound and core needle biopsy of the lump. Other investigations were performed as appropriate. One hundred neck lump patients were seen in a 9 month period. Ninety-six of these lumps were diagnosed on an out-patient basis, only four requiring admission for formal excision biopsy. Among the diagnoses were eleven lymphomas, nine parotid neoplasms, nine lymph node metastases, five thyroglossal cysts, and four branchial cysts. Almost half the patients seen had either a reactive lymphadenopathy, or no abnormality. The establishment of a tissue diagnosis on an out-patient basis allowed appropriate referrals to be made and management plans to be formulated. The theoretical risk of seeding of malignant cells in the needle tract is acknowledged and discussed.

Nov gene encodes adhesion factor for vascular smooth muscle cells and is dynamically regulated in response to vascular injury.

Nephroblastoma overexpressed (NOV) is a member of the CCN family (connective tissue growth factor, CYR61, and NOV) of proteins that are involved in regulating the proliferation, differentiation, and adhesion of a variety of cell types. We have examined the expression of the NOV: gene and NOV protein by vascular smooth muscle cells (VSMCs), in vitro and in vivo, and the effects of recombinant NOV on VSMCs. Rat aortic VSMCs were found to express NOV: mRNA and NOV protein in vitro and in vivo. NOV: expression in adult rat tissues was very high in the aorta and was detected only weakly in the brain and lung by Northern analysis (relative levels 33:3:1). During postnatal development (3 days to 12 weeks), the expression of NOV: was correlated with markers of the differentiated smooth muscle cell phenotype (smooth muscle myosin heavy chain and SM22 alpha). In the rat carotid artery balloon injury model, NOV: was detectable by in situ hybridization and was downregulated in the media of the injured artery compared with the uninjured artery at 7 and 14 days after injury. Expression in the developing intima was barely detectable at 7 days after injury except for strong expression at the luminal surface. At 14 days after injury, NOV: expression was substantially increased throughout the intima. In vitro studies of the function of NOV protein showed that it promoted VSMC adhesion via a mechanism that was divalent cation and Arg-Gly-Asp independent but that it did not modulate VSMC proliferation or phenotype. The strong expression and dynamic regulation of NOV: in the arterial wall, together with its ability to promote VSMC adhesion, suggest that it may be involved in homeostasis and repair.

Development and use of a virtual NMR facility.

We have developed a "virtual NMR facility" (VNMRF) to enhance access to the NMR spectrometers in Pacific Northwest National Laboratory's Environmental Molecular Sciences Laboratory (EMSL). We use the term virtual facility to describe a real NMR facility made accessible via the Internet. The VNMRF combines secure remote operation of the EMSL's NMR spectrometers over the Internet with real-time videoconferencing, remotely controlled laboratory cameras, real-time computer display sharing, a Web-based electronic laboratory notebook, and other capabilities. Remote VNMRF users can see and converse with EMSL researchers, directly and securely control the EMSL spectrometers, and collaboratively analyze results. A customized Electronic Laboratory Notebook allows interactive Web-based access to group notes, experimental parameters, proposed molecular structures, and other aspects of a research project. This paper describes our experience developing a VNMRF and details the specific capabilities available through the EMSL VNMRF. We show how the VNMRF has evolved during a test project and present an evaluation of its impact in the EMSL and its potential as a model for other scientific facilities. All Collaboratory software used in the VNMRF is freely available from www.emsl.pnl.gov:2080/docs/collab.

1H dynamic nuclear polarization in supercritical ethylene at 1.4 T.

(1)H dynamic nuclear polarization (DNP) has been measured in supercritical ethylene in the pressure range 60-300 bar in an external field of 1.4 T. A single-cell sapphire tube was used as a high-pressure cell, and powdered 1,3-bisdiphenylene-2-phenyl allyl (BDPA) free radicals were added and distributed at the wall of the cell. At all pressures the dominant DNP mechanism was a positive Overhauser enhancement, caused by proton-electron contact interactions at the fluid/solid radical interface. The observed enhancements varied from 12 at 60 bar to 17 at 300 bar. Besides the Overhauser enhancement, small solid state and thermal mixing enhancements also were observed, indicating that part of the ethylene was adsorbed at the radical surface for a prolonged time. The impacts of the experimental conditions on the Overhauser enhancement factors are discussed, and enhancements of at least 40-60 are estimated when the EPR saturation factor and the leakage factor become maximal. These data indicate that DNP-enhanced NMR has the potential of extending the impact of NMR in research areas involving supercritical fluids.

Identification of proteins in the equine embryonic capsule.

An acellular embryonic capsule envelops equine conceptuses between day 6 and day 23 after ovulation. As all of the factors mediating embryo-mother signalling must pass through the capsule, it acts like a 'mailbox'. Therefore, we have started to map the proteins in this special extracellular matrix at the interface between mother and embryo. In the present study, one- and two-dimensional gel electrophoresis were used to examine a range of proteins. Use of western blotting identified three specific proteins in the capsules of equine conceptuses recovered on day 16 after ovulation: insulin-like growth factor binding protein 3 (IGFBP-3), a 19 kDa uterine lipocalin (P19) and heparin-binding epidermal growth factor (HB-EGF). Western blotting of two-dimensional SDS-PAGE gels revealed the isoelectric points (pI values) of these proteins: IGFBP-3 was detected as the non-glycosylated 32 kDa form with two isoforms at about pI value 5.8; P19 had a pI value of 9.1; and several isoforms of HB-EGF were detected with molecular masses of approximately 28 kDa and a pI value range of 5.8-6.2. The origin of HB-EGF is not known, but IGFBP-3 is embryonic and P19 is maternal in origin and is thought to be a transport protein. In addition to playing a protective role, and probably also contributing to the mobility of the young conceptus within the uterus, the capsule may be thought of as the extracellular matrix of the embryo, which modulates the complex embryo-maternal signalling processes that take place during early pregnancy in mares.

A palatal haemangioma in a child.

Haemangiomas are the commonest benign tumours of the head and neck in childhood, but they are extremely rate on the palate. We report a case of a palatal haemangioma presenting with feeding difficulties in a young infant. Clinical diagnosis and pathological features are discussed and we suggest a classification which may help in the diagnosis of these cases.

An unusual complication of chemotherapy: an abscess in the pterygopalatine fossa.

Abscesses of the pterygopalatine fossa are extremely rare. The cases reported in the literature are all secondary to dental or periodontal disease. We present a case of an abscess in this site which was a complication of chemotherapy and was the result of haematogenous spread.

QCPMG-MAS NMR of half-integer quadrupolar nuclei.

By combination of fast magic-angle spinning (MAS) and detection of the free-induction decay during a rotor-synchronized quadrupolar Carr-Purcell-Meiboom-Gill (QCPMG) train of refocusing pulses, the sensitivity of quadrupolar-echo MAS NMR spectra for the central transition of half-integer quadrupolar nuclei exhibiting large quadrupolar couplings may be significantly enhanced. Enhancements by an order of magnitude may easily be realized while maintaining information about the anisotropic interactions. In the present study the so-called QCPMG-MAS experiment is demonstrated experimentally and by numerical simulations for the two 87Rb sites in Rb2SO4.

A multiphase 113Cd NMR investigation of metalloporphyrin reorientation in cadmium-substituted myoglobin.

113Cd NMR spectroscopy in both the solution and solid state has been used to investigate the role of the metal ion and the proximal histidine on metalloporphyrin reorientation in myoglobin. Heme disorder has been known to exist for many years but understanding its mechanism has proved difficult due to the short-lived nature of the minor porphyrin isomer in native myoglobin. Cadmium-substituted myoglobin can be generated in one form which contains different insertion isomers or in another form which contains predominantly only one of these species. This allows for direct investigation of heme disorder in metal-substituted myoglobin.

The differentiation of snoring mechanisms using sound analysis.

Ten subjects known to suffer from heavy snoring but not obstructive sleep apnoea were studied using the technique of sleep nasendoscopy. The mechanism of snoring was noted for each and sound recordings of the snoring noise were made. Six subjects were observed to snore using their soft palate only, three snored using only their tongue base and one snored using a combination of palate and tongue base. The sound recordings were subjected to computer analysis of waveform and frequency. Palatal flutter snoring and tongue base snoring appear to have distinct waveform and frequency patterns which allows them to be differentiated from each other.

Applicability of factor analysis in solid state NMR.

Factor analysis has been used to deduce the composition of multicomponent magic angle spinning NMR spectra comprised of overlapping isotropic peaks. The technique has been used to determine the number of constituents present and was combined with a target transformation minimization procedure to identify the component MAS spectra. The new analysis procedure is compared to the conventional least-squares approach and is found to be superior in all cases.