Use of Density Functional Based Tight Binding Methods in Vibrational Circular Dichroism.

Vibrational circular dichroism (VCD) is a spectroscopic technique used to resolve the absolute configuration of chiral systems. Obtaining a theoretical VCD spectrum requires computing atomic polar and axial tensors on top of the computationally demanding construction of the force constant matrix. In this study we evaluated a VCD model in which all necessary quantities are obtained with density functional based tight binding (DFTB) theory. The analyzed DFTB parametrizations fail at providing accurate vibrational frequencies and electric dipole gradients but yield reasonable normal modes at a fraction of the computational cost of density functional theory (DFT). Thus, by applying DFTB in composite methods along with DFT, we show that it is possible to obtain accurate VCD spectra at a much lower computational demand.

[Severe type 1-allergy to raw bell pepper]. / Schwere Soforttypallergie nach Verzehr roher Paprika.

We report on a patient with rare anaphylaxis after ingestion of raw bell pepper. A complex cluster of sensitization including grass and birch pointed out a possible pollen-associated food allergy. We suggest that the severe reaction is due to cross-reactivity towards Bet v 1. Western blot showed binding of the patient's serum to an 11 kDa protein, which has not been described yet and might be a new allergenic structure of the bell pepper plant or a fragment of the Bet v 1-homologous bell pepper protein.

[Linear ulcers with central keratinous plug]. / Linear angeordnete Ulzerationen mit zentralem Pfropf.

Reactive perforating dermatosis (RPD) is a primary perforating dermatosis. Histologically, it presents with transepidermal discharge of basophile material and vertical arrangement of collagen fibers. RPD is treated using external keratolytic agents, topical and systemic glucocorticosteroids, retinoids and antihistamines. Good results have also been reported using photo(chemo)therapy. In the case study presented here, the patient responded very well to treatment with allopurinol.

Selective immunoaffinity-based enrichment of CD34+ cells transduced with retroviral vectors containing an intracytoplasmatically truncated version of the human low-affinity nerve growth factor receptor (deltaLNGFR) gene.

Human hematopoietic stem cells remain one of the most promising target cells for gene therapeutic approaches to treat malignant and nonmalignant diseases. To rapidly characterize transduced cells and to isolate these from residual nontransduced, but biologically equivalent, cells, we have used a Moloney murine leukemia virus (Mo-MuLV)-based retroviral vector containing the intracytoplasmatically truncated human low-affinity nerve growth factor receptor (deltaLNGFR) cDNA as a marker gene. Supernatant transduction of CD34+ cells (mean purity 97%) in fibronectin-coated tissue culture flasks resulted in 5.5-45% (mean 26%) transduced cells expressing deltaLNGFR (LNGFR+ cells). After transduction, more than 65% of the transduced cells remained CD34+. Compared with control (mock- and nontransduced) CD34+ cells, transduction did not decrease the cloning efficiency of CD34+ cells. Immunomagnetic selection of the transduced cells with a monoclonal anti-LNGFR antibody resulted in >90% LNGFR+ cells. Further phenotypic characterization of these highly enriched LNGFR+ cells indicated that the majority co-expressed the CD34 and CD38 antigens. These results show that transduced cells expressing an ectopic cell-surface protein can be rapidly and conveniently quantitated and characterized by fluorescence-activated cell sorting (FACS) analysis and fast and efficiently enriched by immunoadhesion using magnetic beads. The use of cell-surface reporters should facilitate optimization of methods of gene transfer into more primitive hematopoietic progenitors.

Rapid filter assay for the detection of DNA polymerase activity: direct identification of the gene for the DNA polymerase from Thermus aquaticus.

A method for rapid identification of DNA polymerase activity employing an activated DNA substrate covalently bound to nitrocellulose membranes is described. Samples containing DNA polymerase are spotted and the membranes are incubated in an appropriate polymerization buffer containing radioactively labelled dNTPs. By autoradiography of the dried filters, DNA polymerase activity can be directly identified. The method can be used for fast and large-scale screening of chromosomal expression libraries for heterologous DNA polymerases characterized by activity optima different from those of the host organisms. We have identified the gene of the thermostable DNA polymerase from Thermus aquaticus in an expression library of Escherichia coli.

Nonradioactive labeling and high-sensitive detection of PCR products.

The polymerase chain reaction (PCR) represents the most common and widespread method for the direct amplification of specific sequences of nucleic acid target molecules. Incorporation of nonradioactive labeled nucleotides during PCR by Taq DNA polymerase results in directly detectable amplification products or generates nonradioactively labeled probes for nucleic acid hybridization. Here we provide a reliable and easy to follow protocol for direct incorporation of digoxigenin-(DIG) or biotin-labeled nucleotides during PCR. The combination of high-efficient PCR amplification and high-sensitive digoxigenin technology is leading to the detection of single DNA molecules by applying digoxigenin-specific antibodies in an ELISA-type detection reaction. Following a transfer to nylon membranes, the detection of digoxigenin-labeled amplification products can also be accomplished either with a colorimetric or a chemiluminescent reaction. Using the digoxigenin-labeled amplification products as hybridization probes, sensitivities in the 0.1-pg range are obtained in Southern blot procedures.

Preoperative localization of the central sulcus by dipole source analysis of early somatosensory evoked potentials and three-dimensional magnetic resonance imaging.

Surgery of lesions within or close to the central area of the brain always carries the risk of iatrogenic motor or sensory deficits. Functional localization by means of intraoperative direct stimulation of the motor area or by recording somatosensory evoked potentials (SSEP's) from the surface of the somatosensory cortex is believed to reduce the operative risk. The authors introduce the combination of dipole source analysis of scalp-recorded SSEP's with three-dimensional (3-D) magnetic resonance (MR) imaging as a tool for preoperative localization of the central sulcus. This provides information on both functional and structural localization for preoperative planning. Four repeated measurements of right and left median nerve SSEP's were obtained from 20 subjects. Dipole source analysis showed a retest reliability of the 3-D localization error of 2.9 +/- 2.0 mm. Compared to the MR evaluation, dipole source analysis was found to mark the central sulcus within 3 mm for 15 conditions (subjects x side of stimulation), while the 3-D MR measurement was accurate to within 6 mm for 10 conditions and 9 mm for 14 conditions. Dipole locations were confirmed in six patients who underwent surgery of the central region. With respect to this application, dipole source analysis combined with 3-D MR imaging appears to be a valuable tool for preoperative functional localization. The accuracy in localization will be further improved when realistic head models become available that can take into account individual head geometry. Further development of the proposed new method holds promise that evoked potentials and electroencephalography will gain greater use in presurgical functional localization.

Nonradioactive labeling of polymerase chain reaction products.

Polymerase chain reaction (PCR) was originally introduced to amplify in vitro particular DNA sequences by the application of temperature cycles (1). In a modification, RNA molecules also may serve as templates by an additional reverse transcription step converting RNA in complementary DNA sequences (2).

[Vaccination against hepatitis. Present and future]. / Impfung gegen Hepatitis. Gegenwart und Zukunft.

Hepatitis A virus, a member of the picornavirus group, has been adapted to growth in cell culture. Recently developed inactivated vaccines and attenuated hepatitis A strains are presently used in first field advance. Hepatitis B virus is prototype of the hepadna virus group. Based on purification of viral surface antigene (HBs Ag) from human sera, a safe and effective vaccine has been developed, which is now available for general use. Future vaccines against hepatitis B are presently developed with methods of modern genetic engineering. These vaccines will be based on the in vitro synthesis of oligopeptides or on expression of cloned viral DNA in bacteria, yeast cells or tissue cultures. The nature of the agents causing non-A-non-B hepatitis is still unknown. Thus there is no method available now to develop a procedure for immunoprevention of non-A-non-B hepatitis.

Cytomegalovirus DNA in colorectal carcinoma tissues.

Numerous studies had linked human cytomegalovirus (HCMV) infections with neoplasia. Among various other malignant tumors, colonic carcinoma tissues were reported to contain DNA sequences hybridizing with DNA extracted from virus particles. Gene technology allowed us to use a cloned viral DNA library to measure HCMV in colorectal tumors more specifically. Four of 38 tissue specimens did contain DNA sequences homologous to cloned viral DNA probes; however, in each of those cases, identical hybridization patterns were seen with specimens from non-infiltrated surrounding intestinal wall. The amount of HCMV DNA in normal tissues was at least as much as in tumor biopsies. We conclude that nucleic acid hybridizations at high sensitivity with moleculary cloned HCMV DNA did not support the notion of a correlation between colorectal carcinomas and human cytomegalovirus.

The role of a repetitive palindromic sequence element in the human cytomegalovirus major immediate early enhancer.

The major enhancer, extending from nucleotides -530 to -120 upstream of the transcription initiation site of immediate early (IE) genes 1 and 2 in human cytomegalovirus (HCMV), contains four groups of repeated sequence motifs that consist of 17, 18, 19 or 21 bp, respectively. One of these elements, the 19 bp repeat, is a symmetrical palindrome that is also part of IE regulatory sequences of other cytomegalovirus-type herpesviruses, but not of unrelated members of the herpesvirus group. Synthetic oligonucleotides representing the 19 bp repeat unit strongly reduced the activity of the IE1/2 enhancer/promoter in cotransfection assays after transient expression. The HCMV enhancer can substitute for the 72 bp repeats of simian virus 40 (SV40). Replication-competent deletion mutants of SV40/HCMV enhancer recombinants were constructed that contained a single palindromic 19 bp repeat with a central cleavage site for AhaII. If deletions were introduced into the single remaining 19 bp repeat most of the mutant viruses were still replication-competent in CV-1 monkey kidney cells. Insertion of two nucleotides into the single AhaII site did not significantly alter transient SV40 T antigen expression. Deletion of four nucleotides or more from the single 19 bp palindrome reduced the stimulation of T antigen synthesis by the HCMV enhancer/SV40 promoter unit down to about 50%. More extended deletions (28 to 80 bp) did not further reduce T antigen expression. All mutants without an intact 19 bp repeat contained the 18 bp and/or the 21 bp sequence motif. DNase I footprinting and gel retardation assays indicated sequence-specific protein binding by the 19 bp palindrome. Altered palindromes, correlating with reduced enhancer activity, lost most of their protein-binding properties. Thus, the 19 bp repeat element is one of several protein-binding sites that contribute to enhancer strength. However, the 19 bp sequence motif can be deleted entirely to leave reduced activity. The HCMV IE1/2 upstream sequence appears to be the perfect model of an enhancer as a complex of multiple binding sites for trans-activating proteins in a modular fashion.

Human cytomegalovirus DNA sequences with homologies to the cellular genome.

DNA from a cosmid-cloned gene library of human cytomegalovirus (HCMV) strain AD169 was found to share sequence homologies with cellular DNA of various origins. Viral DNA fragments subcloned in plasmid vectors enabled localization of the homologous sequences to five regions of the HCMV genome. Four of these regions were found in the long unique (UL) segment of the viral genome (EcoRI fragments R, I and b, and HindIII fragment S); one region with virus--cell homology was located in the terminal inverted repeats (EcoRI fragments O and H). All hybridization reactions were carried out under stringent reannealing conditions (18 degrees C to 21.5 degrees C below average Tm of HCMV DNA). Fragments from these five HCMV DNA regions did not cross-hybridize with each other. The sequence homologies were seen in DNA from normal human placental tissue, liver autopsy material, intestinal tissue, white blood cells, and colon carcinoma cells, and in a series of haematopoietic cell lines. Sequence homologies were also detected in DNA from owl monkeys and Chinese hamsters, but not in mouse DNA. They were not related to human Alu repeats or cloned human c-myc sequences.

Genome organization of herpesvirus aotus type 2.

Herpesvirus aotus type 2, a virus commonly found in owl monkeys without overt disease, has a similar genome structure to the oncogenic herpesviruses of nonhuman primates (herpesvirus saimiri, herpesvirus ateles). Virion DNA of herpesvirus aotus type 2 (M-DNA) has an unique 110-kilobase-pair region of low G + C content (40.2%, L-DNA), inserted between stretches of repetitive H-DNA (68.7% G + C, about 41 kilobase pairs per molecule) that are variable in length. A minority of virions contain defective genomes that consist of repetitive H-DNA only. The H-DNA is composed of various types of repeat units that are related in sequence with each other. The two dominant types of repeats (2.3 and 2.7 kilobase pairs) were cloned and compared by restriction enzyme cleavages and partial nucleotide sequencing. They are homologous in at least 1.3 kilobase pairs. The two forms of repeat units are randomly arranged and oriented in tandem. Reassociation kinetics did not allow detection of sequence homologies between H- and L-DNA of herpesvirus aotus type 2 and the respective sequences of oncogenic primate herpesviruses.

Reactivation of the methylation-inhibited late E2A promoter of adenovirus type 2 by a strong enhancer of human cytomegalovirus.

Promoter inactivation by sequence-specific methylation was demonstrated by using a construct which contained the late E2A promoter of adenovirus type 2 (Ad2) DNA and the prokaryotic gene for chloramphenicol acetyltransferase (CAT) as indicator. After the in vitro methylation of 5'-CCGG-3' sequences at positions -215, +6, and +24 relative to the cap site of the promoter, the construct was inactive upon transfection into mammalian cells. The same pAd2E2AL-CAT construct was active in the unmethylated form. Promoter inactivation could be overcome when the strong immediate early enhancer of human cytomegalovirus DNA, which lacked 5'-CCGG-3' sites, was inserted into the construct either in a position immediately antecedent to the promoter or in a location several thousand nucleotides remote from it. Reactivation of the 5'-CCGG-3' methylated pAd2E2AL-CAT construct entailed initiation of transcription at the authentic cap site of the late E2A promoter and maintenance of methylation at least during the duration of the transient expression experiment. Reactivation of the methylated late E2A promoter had also been demonstrated by the trans-activating 289 amino acid protein which was encoded in the E1A region of adenoviruses (B. Weisshaar et al., 1988, J. Mol. Biol. 202, 255-270). Thus there are several ways in which a methylated and silenced promoter can be reactivated in mammalian cells.

Human cytomegalovirus DNA in the temporal cortex of a schizophrenic patient.

Using highly sensitive nucleic acids hybridization techniques, which allow the detection of 0.1-0.5 single copy gene equivalents per cell, DNA from the temporal cortex of seven definite schizophrenics, five persons with schizophrenia-like psychoses, three patients with Huntington's chorea and nine mentally normal individuals were probed with human cytomegalovirus (HCMV) DNA. A clear hybridization signal was obtained with DNA from the temporal lobe of a young schizophrenic patient, whereas DNA from the temporal cortex of controls did not hybridize to the HCMV probe. This finding is in agreement with the cytomegalovirus hypothesis of schizophrenia and hints at the possibility that viral infection of the temporal cortex may in some sporadic cases be a contributing factor to the development of schizophrenic psychoses. There is no indication, however, that infection of the central nervous system with HCMV is an aetiological factor in the great majority of schizophrenic disorders. Clearly further studies, preferably in situ hybridizations of whole brains, are needed to prove or disprove the cytomegalovirus hypothesis of schizophrenia.

Non-radioactive labeling and detection of nucleic acids. IV. Synthesis and properties of digoxigenin-modified 2'-deoxyuridine-5'-triphosphates and a photoactivatable analog of digoxigenin (photodigoxigenin).

The chemical syntheses of novel digoxigenin-derivatized compounds are described which are modified substrates for enzymatically or photochemically non-radioactive digoxigenin labeling of nucleic acids. Various activated digoxigenin-haptens are coupled to 5-aminoallyl-substituted 2'-deoxyuridine-5'-triphosphate. This results in digoxigenin-modified nucleoside triphosphates of variable spacer lengths (Dig-[4]-dUTP/Dig-[11]-dUTP/Dig-[16]-dUTP) which can be used as substrates for enzymatic labeling of DNA with digoxigenin-haptens by Klenow enzyme-catalysed random-primed synthesis. In addition the synthesis of N-[4-azidobenzoyl]-N'-[(3-O-digoxigeninyl)methylcarbonyl)]-1 ,8-diamino- 3,6-dioxaoctane (photodigoxigenin), a photoactivatable analog of digoxigenin, is described which can be applied for photolabeling of DNA and RNA with digoxigenin-haptens leaving the nucleic acid molecules intact.

Expansion and fibronectin-enhanced retroviral transduction of primary human T lymphocytes for adoptive immunotherapy.

Human lymphocytes remain among the most promising target cells for gene therapy. Gene-modified lymphocytes have been used successfully to treat adenosine deaminase (ADA)-deficient patients and to control GvHD after allogeneic BMT. Because activation and proliferation of T cells are necessary for efficient retrovirus-mediated gene transfer and subsequent selection of transduced cells, mononuclear cells (MNC) from steady-state and G-CSF-stimulated peripheral blood were activated by short exposure to the mitogen PHA, the anti-CD3 antibody OKT3, or both in the presence of different concentrations of recombinant IL-2. Using OKT3 (10 or 30 ng/ml) and IL-2 (100 U/ml), T cells expanded efficiently during a 14-day culture period. Cell expansion was similar under serum-free conditions. The immunophenotypic profile over time showed a marked increase in CD8+ cells, leading to a reversed CD4/CD8 ratio of 1:2 and a slight increase in CD56+ cells. Supernatant-based centrifugal transduction of primary human T lymphocytes was compared with supernatant transduction on the extracellular matrix protein fibronectin. Transduction with cell-free retrovirus-containing supernatant in tissue culture flasks coated with human plasma fibronectin led to significantly higher transduction efficiencies (20% +/- 7.5%) than centrifugal transduction in uncoated culture flasks (13.6% +/- 5.1%)(p = 0.041). To both rapidly characterize transduced cells and isolate these from residual nontransduced but biologically equivalent cells, an amphotropic Moloney murine leukemia virus (MoMuLV)-based retroviral vector containing the intracytoplasmically truncated human low-affinity nerve growth factor receptor (deltaLNGFR) cDNA as a marker gene was used. FACS sorting of T cells after transduction resulted in >90% LNGFR+ cells and was much faster than enrichment of transduced cells through growth in G418-selection medium. These results show that supernatant-based retroviral gene transfer into primary human T lymphocytes can be enhanced by fibronectin. Ectopic expression of a cell surface protein can be used to rapidly and conveniently quantitate transduction efficiency through FACS analysis and to efficiently enrich transduced cells through FACS sorting.