Activation of adenovirus promoters by the adenovirus E1A protein in cell-free extracts.

The primary product of the adenovirus E1A gene is a protein that is sufficient for controlling host-cell proliferation and immortalizing primary rodent cells. The mechanism by which the protein induces these cellular effects is poorly understood, but might be linked to its ability to regulate RNA transcription from a number of viral and cellular genes. The mechanism of E1A's transcriptional-activation (trans-activation) was studied here by monitoring the protein's effect on specific adenovirus promoters in two types of transcriptional systems in vitro. One of these systems consisted of extracts from transformed cells constitutively expressing E1A, and the other consisted of extracts of HeLa cells supplemented with a plasmid-encoded E1A protein purified from Escherichia coli. The results show that the E1A protein specifically stimulates transcription from adenovirus promoters; thus, the induction of cellular transcription factors is not necessary to explain the stimulation of transcription by E1A.

X-ray Photographs of a Solar Active Region with a Multilayer Telescope at Normal Incidence.

An astronomical photograph was obtained with a multilayer x-ray telescope. A 4-centimeter tungsten-carbon multilayer mirror was flown as part of an experimental solar rocket payload, and successful images were taken of the sun at normal incidence at a wavelength of 44 angstroms. Coronal Si-XII emission from an active region was recorded on film; as expected, the structure is very similar to that observed at O-VIII wavelengths by the Solar Maximum Mission flat crystal spectrometer at the same time. The small, simple optical system used in this experiment appears to have achieved a resolution of 5 to 10 arc seconds.

The yohkoh mission for high-energy solar physics.

The Japanese Yohkoh satellite is now in orbit observing the sun with a set of x-ray imagers and x-ray and gamma-ray spectrometers. The data from this successful mission provide new information on solar flares and the sun's corona. This paper discusses the Yohkoh observations and presents a sample of the first scientific results from the mission.

The DNA strand of chimeric RNA/DNA oligonucleotides can direct gene repair/conversion activity in mammalian and plant cell-free extracts.

Chimeric oligonucleotides (chimeras), consisting of RNA and DNA bases folded by complementarity into a double hairpin conformation, have been shown to alter or repair single bases in plant and animal genomes. An uninterrupted stretch of DNA bases within the chimera is known to be active in the sequence alteration while RNA residues aid in complex stability. In this study, the two strands were separated in the hope of defining the role each plays in conversion. Using a series of single-stranded oligonucleotides, comprised of all RNA or DNA residues and various mixtures, several new structures have emerged as viable molecules in nucleotide conversion. When extracts from mammalian and plant cells and a genetic readout assay in bacteria are used, single-stranded oligonucleotides, containing a defined number of thioate backbone modifications, were found to be more active than the original chimera structure in the process of gene repair. Single-stranded oligonucleotides containing fully modified backbones were found to have low repair activity and in fact induce mutation. Molecules containing various lengths of modified RNA bases (2'-O-methyl) were also found to possess low activity. Taken together, these results confirm the directionality of nucleotide conversion by the DNA strand of the chimera and further present a novel, modified single-stranded DNA molecule that directs conversion in plant and animal cell-free extracts.

Evaluation of degradation pathways for plasmid DNA in pharmaceutical formulations via accelerated stability studies.

The stability of highly purified supercoiled plasmid DNA formulated in simple phosphate or Tris-buffered saline solutions has been characterized to establish the overall degradation processes that occur during storage in aqueous solution. Plasmid DNA stability was monitored during accelerated stability studies (at 50 degrees C) by measurements of supercoiled, open-circle, and linear DNA content, as well as the accumulation of apurinic sites and 8-hydroxydeoxyguanosine residues over time. The effects of formulation pH, demetalation, metal ion chelators, and ethanol (hydroxyl radical scavenger) on the supercoiled content of plasmid DNA during storage at 50 degrees C were also determined. The results indicate that free radical oxidation may be a major degradative process for plasmid DNA in pharmaceutical formulations unless specific measures are taken to control it by the addition of free radical scavengers, specific metal ion chelators, or both. The generation of hydroxyl radicals in phosphate-buffered saline was confirmed by examining the hydroxylation of phenylalanine over time by reverse phase high-performance liquid chromatography. Ethanol was found to enhance plasmid DNA stability and to inhibit the hydroxylation of phenylalanine; both observations are consistent with the known ability of ethanol to serve as a hydroxyl radical scavenger. Moreover, the combination of ethylenediamine tetraacetic acid (EDTA) and ethanol had a synergistic enhancing effect on DNA stability. However, the metal ion chelator diethylenetriaminepentaacetic acid (DTPA) was as potent as the combination of EDTA and ethanol for enhancing the stability of plasmid DNA. By controlling free radical oxidation with EDTA and ethanol, the rate constants of plasmid DNA degradation by means of depurination and beta-elimination were then determined, allowing accurate predictions of DNA storage stability as a function of formulation pH and temperature. The ability to predict plasmid DNA storage stability in the absence of free radical oxidation should prove to be a valuable tool for the design of stable pharmaceutical formulations of plasmid DNA.

Deamidation of polyanion-stabilized acidic fibroblast growth factor.

The deamidation of polyanion-stabilized acidic fibroblast growth factor (aFGF; FGF-1) can be induced by prolonged storage under accelerated conditions of elevated pH and temperature. A urea-isoelectric focusing (urea-IEF) method has been developed to monitor aFGF deamidation in the presence of highly negatively charged polyanions which are required to maintain the conformational stability of the protein. The kinetics of aFGF deamidation have been established by a combination of urea-IEF and an enzymatic ammonia assay. Native, non-deamidated aFGF (complexed with heparin) has a half-life of 16 weeks at pH 7, 30 degrees C, and 4 weeks at pH 8, 40 degrees C. The mitogenic activity and biophysical properties of deamidated aFGF were compared to the non-deamidated protein. These initial deamidation events have no significant effect on the protein's overall conformation, thermal stability, interaction with heparin, or bioactivity. At longer times, however, limited aggregation of the protein was observed after prolonged storage under some conditions. N-terminal protein sequencing of the protein's first 21 amino acid residues have identified one of the deamidation sites in a flexible, peptide-like region of the protein (Asn8-Tyr9).

Enhancing the avidity of a human recombinant anti-HIV-1 monoclonal antibody through oligomerization.

The oligomerization by chemical cross-linking of a recombinant human antiviral monoclonal antibody (MAb), r447-1, and its characterization are described. This MAb binds to an epitope residing in the hypervariable V3 region of the envelope protein (gp120/160) of HIV-1. A dimeric form of this MAb displays enhanced avidity and was found to be capable of neutralizing a greater variety of lymphoid cell culture-adapted HIV-1 variants and HIV-1 primary isolates than its monomeric form. The superior binding and breadth of reactivity of this antibody suggests it may have utility as a therapeutic and/or prophylactic agent, if it possesses an appropriate safety and immunogenicity profile.

Size exclusion HPLC method for the determination of acidic fibroblast growth factor in viscous formulations.

A size exclusion HPLC method has been developed to determine the protein concentration of pharmaceutical formulations of recombinant acidic fibroblast growth factor (aFGF). These topical aFGF formulations not only contain low levels of protein mass (50 micrograms ml-1), but also include buffer ions, polysaccharide polyanions to conformationally stabilize aFGF and 1% hydroxyethylcellulose to increase the solution's viscosity. A cesium chloride mobile phase is utilized during SEC-HPLC to dissociate aFGF from the pharmaceutical excipients and to minimize nonspecific interaction of the protein with the column matrix. The protein content of a viscous aFGF formulation is determined by comparison of aFGF peak areas to standards of known concentration. Fluorescence spectroscopy was utilized to directly demonstrate that the protein remains in its native conformation during sample preparation and analysis.

Biomonitoring environmental contaminants near a municipal solid-waste combustor.

Tetrachlorodibenzo-p-dioxin (TCDD), tetrachlorodibenzofuran (TCDF) and selected metal concentrations were measured in eggs and nestlings of anhingas (Anhinga anhinga) and white ibises (Eudocimus albus) collected from a colony next to a municipal solid-waste (MSW) combustor and ash landfill. Most of the measured residues, including TCDD, TCDF, arsenic, beryllium, cadmium and nickel, remained at pre-operational levels during the first five years of facility operation. Selenium (in anhingas) and mercury (in both anhingas and ibises) occurred at their lowest concentrations in samples collected during the fifth year of facility operation (Year-5). Alternatively, concentrations of lead in ibis nestlings were highest in Year-1 and Year-5 compared to Year-0. The MSW combustor could neither be ruled out nor confirmed as the source of this lead.

Function of submaxillary gland following iatrogenic damage to chorda tympani nerve.

Flow rate, protein and amylase content of submaxillary saliva were determined before and after iatrogenic damage to the chorda tympani nerve during otosurgery. Parallel to these investigations, gustatory function was checked. Chronic otopathy itself did not lead to a disturbance of either the sense of taste or the function of the submaxillary gland in any of the 14 patients. On the other hand, the flow rate of the affected gland dropped after chorda division (8 patients) and after chorda stretching (6 patients), and the protein concentration of the saliva was increased while the concentration of amylase remained unchanged. Dysgeusia was always observed after chorda division but only once among 6 cases of chorda stretching.

[A review of spontaneous cerebrospinal fluid rhinorrhoea (author's transl)]. / Ein Beitrag zur Definition der spontanen Rhinoliquorrhoe.

Thirty-two patients described in the literature between 1947 and 1977, together with a personal one, with spontaneous cerebrospinal fluid rhinorrhoea are presented. These 32 patients are assessed and the different aspects of their problems discussed. There is a striking female to male preponderance of 5:1.

[Early diagnosis of chordoma by fine-needle aspiration biopsy (author's transl)]. / Frühe Diagnose des Chordoms durch Feinnadelpunktion.

Successful surgical therapy or radiation of chordoma depends on an early morphologic diagnosis. Two cases of chordoma in the sphenooccipital region are reported, in which fine-needle aspiration biopsy was performed. This method is controlled by X-ray. For gaining sufficient material it is superior to surgical biopsy. The cytomorphology of chordoma and its differential diagnosis are discussed.

Use of an altered sugar-nucleotide to unmask the transition state for alpha(2-->6) sialyltransferase.

Rat liver alpha(2-->6) sialyltransferase catalyzes the formation of a glycosidic bond between N-acetylneuraminic acid and the 6-hydroxyl group of a galactose residue at the nonreducing terminus of an oligosaccharide. This reaction has been investigated through the use of the novel sugar-nucleotide donor substrate UMP-NeuAc. A series of UMP-NeuAc radioisotopomers were prepared by chemical deamination of the corresponding CMP-NeuAc precursors. Kinetic isotope effects (KIEs) on V/K were measured using mixtures of radiolabeled UMP-NeuAc's as the donor substrate and N-acetyllactosamine as the acceptor. The secondary beta-(2)H KIE was 1.218 +/- 0.010, and the primary (14)C KIE was 1.030 +/- 0.010. A large inverse (3)H binding isotope effect of 0.944 +/- 0.010 was measured at the terminal carbon of the NeuAc glycerol side chain. These KIEs observed using UMP-NeuAc are much larger than those previously measured with CMP-NeuAc [Bruner, M., and Horenstein, B. A. (1998) Biochemistry 37, 289-297]. Solvent deuterium isotope effects of 1.3 and 2.6 on V/K and V(max) were observed with CMP-NeuAc as the donor, and it is revealing that these isotope effects vanished with use of the slow donor substrate UMP-NeuAc. Bell-shaped pH versus rate profiles were observed for V(max) (pK(a) values = 5.5, 9.0) and V/K(UMP)(-)(NeuAc) (pK(a)values = 6.2, 9.0). The results are considered in terms of a mechanism involving an isotopically sensitive conformational change which is independent of the glycosyl transfer step. The isotope effects reveal that the enzyme-bound transition state bears considerable charge on the N-acetylneuraminic acid residue, and this and other features of this mechanism provide new directions for sialyltransferase inhibitor design.

[Tumors of the testis in children]. / Les tumeurs du testicule de l'enfant.

Tumours of the testicle are rare in children (about one case in 100,000 male children). Their histological type usually leads to favourable outcome. We report here our series of 12 cases together with an analysis of the large published series in order to review the prognosis and therapeutic indications which depend on the histology. The most frequent intratesticular tumours in the child are nonseminoma malignant germ cell tumours. At birth the tumour is usually a mature or mixed teratoma. Between the ages of 1 and 3 years the most frequently observed tumour arises from the vitelline sac representing about 50% of all tumours of the testicle in the child. From 3 years to puberty, paratesticular rhabdomyocarsomas and Leydig cell tumours are the most frequently encountered. Finally, at puberty, tumours of the testicle are the same as those observed in the adult, notably seminomas which are not observed before puberty. Other tumours occur only exceptionally. Orchiectomy after ligature of the spermatic cord is the usual treatment. Complementary chemotherapy and node dissection are not required as first intention therapy when the tumour is well localized (complete exeresis, no lymph node invasion, no pulmonary metastasis), but are reserved for relapse or residual tumours revealed by a secondary rise in alpha foetoprotein levels or by extension from the initial site. A present, in 80% of the patients with localized tumours, outcome is favourable without lymph node dissection or chemotherapy. The three-year survival rate is approximately 90%.

Isotope trapping and kinetic isotope effect studies of rat liver alpha-(2-->6)-sialyltransferase.

A mechanistic study of rat liver alpha-(2-->6) sialyltransferase (ST) is presented that includes isotope trapping experiments and kinetic isotope effects on V/K for the ST-catalyzed reaction of isotopically labeled CMP-N-acetylneuraminate and N-acetyllactosamine. The isotope trapping experiments confirmed that the kinetic mechanism is steady-state random, and further analysis indicated that for this sialyltransferase the experimentally observed isotope trapping ratio (product trapped/substrate released) was equivalent to the commitment to catalysis, Cf, the quantity required to correct the kinetic isotope effects. Cf was found to range from 1.0 (at 1.6 mM LacNAc) to 1.7 (at 100 mM LacNAc). After correction for Cf, the isotope effects were as follows: secondary beta-dideuterium, 1.04-1. 05; anomeric carbon primary 14C, 1.000 +/- 0.004; a small 3H binding effect of 1.016 +/- 0.007 at C9; and a carboxylate carbon secondary 14C isotope effect of 0.998 +/- 0.004. This pattern of KIEs is quite different than observed for solvolysis of CMP-NeuAc [Horenstein, B. A., and Bruner, M. (1996) J. Am. Chem. Soc. 118, 10371-10379]. Based on the results of ab-initio modeling of isotope effects, a hypothesis is presented which reconciles the unusual pattern of KIEs on the basis of binding interactions at the carboxylate carbon.

[Evoked response audiometry and vestibular examinations in patients with multiple sclerosis (author's transl)]. / EEG-Audiometrie und Vestibularisuntersuchungen bei MS-Patienten

A possible involvement of the VIII cranial nerve in Multiple Sklerosis was investigated. 15 of the 135 MS-patients were tested by evoked response audiometry (ERA) and vestibular examinations. Only a central nystagmus, central vertigo and coordination disorder were seen. Lesions of the peripheral vestibular system and pathological data of the evoked response audiometry were not found.

[Treatment of acute idiopathic vestibular paresis (author's transl)]. / Zur Therapie des isolierten idiopathischen Vestibularis-ausfalles.

In 31 patients with sudden isolated unilateral idiopathic lesion of the vestibular function the effect of therapeutic application of low molecular weight dextran and cortison was examined. 15 out of 31 patients received no drugs, 16 patients were treated with dextran-infusion and cortison for 10 days. Without drug application the vestibular function was restored in only 4 out of 15 patients after about 9 months, but after the therapeutic application of low molecular weight dextran and cortison a normal reaction of the vestibular system was seen in 11 out of 16 patients.

[Trigemino-facial reflex as a diagnostic aid in Recklinghausen's neurofibromatosis]. / Der trigemino-faciale Reflex als diagnostiches Hilfsmittel bei der Neurofibromatose Recklinghausen.

The blink reflex evoked by unilateral electrical stimulation was studied in 7 patients with Neurofibromatosis and 50 normal persons. Studing data of the early R1 reflex, there was no difference between normal persons and patients with von Recklinhausen disease. A slightly, but significantly increased reflex latency of the direct R2 was recognized in the majority of the patients with Neurofibromatosis.

Purification and biological characterization of an adenovirus type 2 E1A protein expressed in Escherichia coli.

The adenovirus 2 E1A gene encodes a multifunctional protein of 289 amino acids that can immortalize primary rodent cells and transcriptionally activate a number of viral and cellular genes. To facilitate an understanding of the molecular basis for the various actions of E1A, we have redesigned our bacterial expression vector (Ko, J.-L., and Harter, M. L. (1984) Mol. Cell. Biol. 4, 1427-1439) containing the cloned E1A gene such that a soluble authentic E1A protein now constitutes approximately 1.5% of the total Escherichia coli cellular protein. Further, we have developed a simple rapid purification scheme without the use of detergents or denaturants and show a purity of greater than 98% with a yield of approximately 53%. The E1A so purified is biologically active, stimulating cellular DNA synthesis following microinjection into quiescent NIH 3T3 and REF52 cells. In another report (Spangler, R., Bruner, M., Dalie, B., and Harter, M. L. (1987) Science 237, 1044-1046) we have also shown that our purified E1A protein activates transcription from appropriate promoters in an in vitro system.

[The facial nerve as a carrier of secretory fibres for the parotid gland (author's transl)]. / Der N. Facialis als Träger sekretorischer Parotisfasern.

In patients with unilateral facial nerve paresis the parotid reflex secretions of both sides were measured and chemically analyzed to investigate whether an alteration of composition and volume of parotid secretion could be demonstrated in facial nerve paresis, i.e., whether a functional influence of the facial nerve on the parotid gland could be recognized. Flow rate as well as protein and alpha-amylase concentrations were determined in the parotid secretions. A highly significant decrease of flow rates (ml/min) was observed on the paretic side whereas no differences could be found in the concentrations of protein and amylase. These results can be explained by assuming intratemporal anastomoses between the facial and glossopharyngeal nerves. An additional analysis by topodiagnostic criteria makes it likely that this anastomosing takes place only distal to the geniculate ganglion.