Plakophilins 2a and 2b: constitutive proteins of dual location in the karyoplasm and the desmosomal plaque

Using antibodies and recombinant DNA techniques, we have identified plakophilin 2, a novel desmosomal plaque protein of M(r) 100,000 (estimated from SDS-PAGE), which is a member of the arm-repeat family of proteins and can occur in two splice forms (2a and 2b) because of the insertion of a 44 amino acid (aa)-encoding exon. In its aa sequence (837 and 881 aa, calculated pIs: 9.33 and 9.38, mol wts 92,750 and 97,410 kD), it is conspicuously related to the 80-kD plakophilin 1, with which it shares a central region of 9 repeats of the arm-motif, preceeded by a long head region and followed by a very short (11 aa) carboxy-terminal sequence. Plakophilin 2 and its mRNA have been detected in a wide range of tissues and cell types, including cells devoid of desmosomes. By light and electron microscopical immunolocalization, plakophilin 2 has been localized to plaques of desmosomes of one-layered ("simple") and complex epithelia, carcinomas, diverse epithelium-derived cell culture lines, as well as cardiac tissue and the dendritic reticulum cells of lymphatic germinal centers, i.e., desmosomes in which plakophilin 1 is not detected. However, plakophilin 2 has also been localized in the desmosomes of certain but not all stratified epithelia where it coexists with plakophilin 1. Remarkably, plakophilin 2 is also enriched in the karyoplasm of a wide range of cell types, including many that lack desmosomes and in which, therefore, the nuclear state is the only locally enriched form of plakophilin 2 present. We conclude that plakophilins 2a and 2b are basic nuclear proteins that in certain cell types additionally assemble with other proteins to form the desmosomal plaque and serve general nuclear functions as well as a function specific to many but not all desmosomes.     

The pKa of the general acid/base carboxyl group of a glycosidase cycles during catalysis: a 13C-NMR study of bacillus circulans xylanase

The 20 kDa xylanase from Bacillus circulans carries out hydrolysis of xylan via a two-step mechanism involving a covalent glycosyl-enzyme intermediate. In this double-displacement reaction, Glu78 functions as a nucleophile to form the intermediate, while Glu172 acts as a general acid catalyst during glycosylation, protonating the departing aglycone, and then as a general base during deglycosylation, deprotonating the attacking water. The dual role of Glu172 places specific demands upon its ionization states and hence pKa values. 13C-NMR titrations of xylanase, labeled with [delta-13C]glutamic acid, have revealed pKa values of 4.6 and 6.7 for Glu78 and Glu172, respectively. These agree well with the apparent pKa values obtained from a study of the pH dependence of kcat/Km and demonstrate that, at the enzyme's pH optimum of 5.7, the nucleophile Glu78 is deprotonated and the general acid Glu172 initially protonated. Remarkably, the pKa for Glu172 drops to 4.2 in a trapped covalent glycosyl-enzyme intermediate, formed by reaction with 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside [Miao et al. (1994) Biochemistry 33, 7027-7032]. A similar pKa is measured for Glu172 when a glutamine is present at position 78. This large decrease in pKa of approximately 2.5 units is consistent with the role of Glu172 as a general base catalyst in the deglycosylation step and appears to be a consequence of both reduced electrostatic repulsion due to neutralization of Glu78 and a conformational change in the protein. Such "pKa cycling" during catalysis is likely to be a common phenomenon in glycosidases.     

Epidemiology and primary health care

University departments of epidemiology and primary medical care have tended to develop separately because they require very different skills. The Department of Community Medicine at St. Thomas's Hospital Medical School, London, is an example of a harmonious, combined development of the two disciplines. Working as a multidisciplinary team, the department has conducted studies initiated by epidemiologists, primary physicians and the interaction of the two. Examples are given from past and current research of the department. Through mutual cooperation the scientific knowledge of the epidemiologist and the down-to-earth practical knowledge of the clinician have complemented each other to produce valid research of practical relevance. The association has also provided educational facilities for medical students for whom, as physicians, protocol thinking, analysis and questioning of accepted "facts" will become increasingly important.     

Construction and characterization of a triple-recombinant vaccinia virus encoding B7-1, interleukin 12, and a model tumor antigen

BACKGROUND: Construction of recombinant viruses that can serve as vaccines for the treatment of experimental murine tumors has recently been achieved. The cooperative effects of immune system modulators, including cytokines such as interleukin 12 (IL-12) and costimulatory molecules such as B7-1, may be necessary for activation of cytotoxic T lymphocytes. Thus, we have explored the feasibility and the efficacy of inclusion of these immunomodulatory molecules in recombinant virus vaccines in an experimental antitumor model in mice that uses Escherichia coli beta-galactosidase as a target antigen. METHODS: We developed a "cassette" system in which three loci of the vaccinia virus genome were used for homologous recombination. A variety of recombinant vaccinia viruses were constructed, including one virus, vB7/beta/IL-12, that contains the following five transgenes: murine B7-1, murine IL-12 subunit p35, murine IL-12 subunit p40, E. coli lacZ (encodes beta-galactosidase, the model antigen), and E. coli gpt (xanthine-guanine phosphoribosyltransferase, a selection gene). The effects of the recombinant viruses on lung metastases and survival were tested in animals that had been given an intravenous injection of beta-galactosidase-expressing murine colon carcinoma cells 3 days before they received the recombinant virus by intravenous inoculation. RESULTS: Expression of functional B7-1 and IL-12 by virally infected cells was demonstrated in vitro. Lung tumor nodules (i.e., metastases) were reduced in mice by more than 95% after treatment with the virus vB7/beta/IL-12; a further reduction in lung tumor nodules was observed when exogenous IL-12 was also given. Greatest survival of tumor-bearing mice was observed in those treated with viruses encoding beta-galactosidase and B7-1 plus exogenous IL-12. CONCLUSION: This study shows the feasibility of constructing vaccinia viruses that express tumor antigens and multiple immune cofactors to create unique immunologic microenvironments that can modulate immune responses to cancer.     

Decrease in high density lipoprotein cholesterol (HDL-C) levels following gemfibrozil therapy

A matter of debate is whether the use of a video-endoscope impairs visual orientation and manual precision in endonasal surgery. We investigated the influence of video-endoscopy compared to endoscopy on stereoacuity in a model of the nasal cavity. Twenty medical staff members were asked to touch defined points in a spatial model of the nasal cavity as quickly as possible and in correct order using 0 degree and 30 degrees endoscopes, looking directly through the endoscope or looking at a video monitor connected to a CCD camera on the endoscope. Time, number of omissions of points and faults in point sequence were recorded. Manipulations were significantly quicker when the "operative field" was seen directly through the endoscope compared to orientation from the monitor for both 0 degree endoscope 96 +/- 4.7 s. vs. 108 +/- 5.6 s. and 30 degrees endoscope 84 +/- 3.9 s. vs. 96 +/- 5.5 s. (+/- SEM). There was no difference in number of omissions and faults in sequence between "endoscope" and "video-endoscope." The fact that the use of a video-endoscope did not increase the number of faults in our experiment does not support the notion that performing endoscopic sinus surgery using a monitor is unsafe. In the hands of the participants who were experienced with the endoscope, however, the use of a video-endoscope slowed down manipulations to a significant degree. To which extent this may be due to the effect of training or to superiority of the endoscope per se will remain a matter of discussion until a group of experienced video-endoscopists will have repeated the study.     

Comparison of an agonist, urocortin, and an antagonist, astressin, as radioligands for characterization of corticotropin-releasing factor receptors

The characteristics of a high-affinity antagonist radioligand are compared with those a high-affinity agonist in binding to the cloned corticotropin-releasing factor receptor type 1 (CRF-R1) and type 2 (CRF-R2) and to the native receptors that exist in rat cerebellum and brain stem. The relative potencies of CRF antagonists and agonists to the two types of cloned CRF receptors overexpressed stably in Chinese hamster ovary cells are determined using the antagonist radioligand 125I- [DTyr1]astressin (Ast*), and the agonist radioligand, 125I -[Tyr0]rat urocortin (Ucn*). The inhibitory binding constants (Ki) of astressin and urocortin are 1 to 2 nM for all receptors and are independent of which radioligand is employed. Astressin binds with high affinity to the native cerebellar/brain stem receptor and relative potencies of selected CRF analogs determined with Ast* on the native receptor are similar to those obtained for the cloned CRF-R1. The specific binding of Ast* to endogenous brain receptors is greater than that of Ucn*, resulting in more sites being detected by the antagonist than by the agonist. In contrast to another CRF agonist, the binding of Ucn* to the cloned receptors is relatively insensitive to guanyl nucleotides at both 20 degreesC and 37 degreesC; however, its binding to the native receptor is displaced by guanyl nucleotides at 37 degreesC and, to a lesser degree, at 20 degreesC. As expected, the binding of the antagonist Ast* is not affected by guanyl nucleotides. Because it is a high-affinity, specific CRF antagonist, astressin is eminently suitable as a ligand for detection and characterization of both endogenous and cloned CRF receptors.     

Toward earlier diagnosis of amyotrophic lateral sclerosis: revised criteria. rhCNTF ALS Study Group

We modified the World Federation of Neurology (WFN) diagnostic criteria for ALS to facilitate early diagnosis and used these criteria for enrollment of ALS patients in a clinical trial. The criteria developed required lower motor neuron (LMN) involvement in at least two limbs and upper motor neuron involvement in at least one region (bulbar, cervical, or lumbosacral). The EMG finding of fibrillation potentials was required for evidence of LMN involvement. Electrodiagnostic studies, neuroimaging, and laboratory studies were also used to exclude disorders that might mimic ALS. Using these criteria, the diagnosis of ALS was made at a mean time of 9.7 months from onset of symptoms, which compares favorably with the 12-month period cited in the literature. Using clinical assessment at completion of the trial, the diagnosis of ALS was believed to be accurate in those patients entered in the trial. However, pathologic confirmation of the diagnosis of ALS was not obtained. Based on our preliminary experience, we propose that these ALS diagnostic criteria will facilitate early diagnosis of ALS. Future studies should prospectively compare these criteria with the WFN criteria currently in use.     

Clinical pharmacokinetics of camptothecin topoisomerase I inhibitors

In this review the clinical pharmacokinetics of camptothecin topoisomerase I inhibitors, an important new class of anticancer drugs, is discussed. Two prototypes, topotecan and irinotecan, are currently marketed in many European countries and the USA for the treatment of patients with ovarian and colorectal cancer, respectively. Other camptothecin derivatives, including lurtotecan, 9-aminocamptothecin (9-AC) and 9-nitrocamptothecin (9-NC), are at different stages of clinical development. The common property of camptothecin analogues is their action against DNA topoisomerase I, but beyond this similarity the compounds differ widely in terms of antitumour efficacy, pharmacology, pharmacokinetics and metabolism. We review chemistry, mechanism of action, stability and bioanalysis of the camptothecins. Dosage and administration, status of clinical application, pharmacokinetics, pharmacodynamics and drug interactions are discussed.     

A comparison of empirical and neural network approaches for estimating corn and soybean leaf area index from Landsat ETM+ imagery

Plant foliage density expressed as leaf area index (LAI) is used in many ecological, meteorological, and agronomic models, and as a means of quantifying crop spatial variability for precision farming. LAI retrieval using spectral vegetation indices (SVI) from optical remotely sensed data usually requires site-specific calibration values from the surface or the use of within-scene image information without surface calibrations to invert radiative transfer models. An evaluation of LAI retrieval methods was conducted using (1) empirical methods employing the normalized difference vegetation index (NDVI) and a new SVI that uses green wavelength reflectance, (2) a scaled NDVI approach that uses no calibration measurements, and (3) a hybrid approach that uses a neural network (NN) and a radiative transfer model without site-specific calibration measurements. While research has shown that under a variety of conditions NDVI is not optimal for LAI retrieval, its continued use for remote sensing applications and in analysis seeking to develop improved parameter retrieval algorithms based on NDVI suggests its value as a “benchmark” or standard against which other methods can be compared. Landsat-7 ETM+ data for July 1 and July 8 from the Soil Moisture EXperiment 2002 (SMEX02) field campaign in the Walnut Creek watershed south of Ames, IA, were used for the analysis. Sun photometer data collected from a site within the watershed were used to atmospherically correct the imagery to surface reflectance. LAI validation measurements of corn and soybeans were collected close to the dates of the Landsat-7 overpasses. Comparable results were obtained with the empirical SVI methods and the scaled SVI method within each date. The hybrid method, although promising, did not account for as much of the variability as the SVI methods. Higher atmospheric optical depths for July 8 leading to surface reflectance errors are believed to have resulted in overall poorer performance for this date. Use of SVIs employing green wavelengths, improved method for the definition of image minimum and maximum clusters used by the scaled NDVI method, and further development of a soil reflectance index used by the hybrid NN approach are warranted. More importantly, the results demonstrate that reasonable LAI estimates are possible using optical remote sensing methods without in situ, site-specific calibration measurements.