Biomedical subjects
Publications and source records attributed to B Kofler.
Broad-range PCR has proven to be useful for the detection of bacteria. A set of broad-range PCR primers directed against conserved regions in the 16S rRNA gene was designed to specifically amplify either gram-positive or gram-negative bacteria. The gram type-specific broad-range PCR correctly classified all 62 pathogenic species tested.
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Surface solid dispersions were prepared via physical mixture and were either heated in a vacuum dryer or in a microwave oven for different periods of time. The physical state of felodipine in solid dispersions was studied using differential scanning calorimetry and x-ray powder diffractometry. USP paddle method was used for felodipine dissolution studies. The use of vacuum or microwave energy led to a significant improvement of felodipine dissolution which was caused partly by the amorphous state of felodipine and a large surface area of amorphous silicon dioxide.
The neuropeptide galanin elicits a range of biological effects by interaction with specific G-protein-coupled receptors. Human and rat GALR1 galanin receptor cDNA clones have previously been isolated using expression cloning. We have used the human GALR1 cDNA in hybridization screening to isolate the gene encoding GALR1 in both human (GALNR) and mouse (Galnr). The gene spans approximately 15-20 kb in both species; its structural organization is conserved and is unique among G-protein-coupled receptors. The coding sequence is contained on three exons, with exon 1 encoding the N-terminal end of the receptor and the first five transmembrane domains. Exon 2 encodes the third intracellular loop, while exon 3 encodes the remainder of the receptor, from transmembrane domain 6 to the C-terminus of the receptor protein. The mouse and human GALR1 receptor proteins are 348 and 349 amino acids long, respectively, and display 93% identity at the amino acid level. The mouse Galnr gene has been localized to Chromosome 18E4, homoeologous with the previously reported localization of the human GALNR gene to 18q23 in the same syntenic group as the genes encoding nuclear factor of activated T-cells, cytoplasmic 1, and myelin basic protein.
Using a probe obtained by PCR amplification from mouse genomic DNA, a genomic clone was isolated covering the entire mouse preprogalanin gene. The mouse gene has an exon:intron organisation very similar to that of the rat and human genes. The first exon is noncoding while exons 2-5 carry the coding region. Exon 6 also encodes the stop codon and a polyadenylation signal. The deduced amino-acid sequence of mouse preprogalanin is 94% and 68% identical to the rat and human peptide, respectively. The amino-acid sequence of mouse galanin was confirmed by RT-PCR amplification of mouse brain RNA. The cloning of the mouse galanin gene should allow elucidation of the regulatory characteristics of its promoter and facilitate transgenic approaches to the analysis of galanin gene function in this species.
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Two approaches have been used to elucidate the role of the nuclear polymerizing NAD+:protein(ADP-ribosyl)-transferase (ADPRT): i) comparison of the primary structure of Dictyostelium discoideum ADPRT derived from a 2 kb, partial cDNA sequence with the mammalian, fish, amphibian and insect counterparts revealed an overall homology of 25%. Whereas the automodification domain was not conserved at all, the NAD+ binding domain (aa 859-908) showed more than 70% identical amino acids in all species. Together with the similar enzymatic properties of the ADPRTs the genetic conservation underlined the notion that ADPRT plays a major role in various cellular processes; and ii) inactivation of the ADPRT gene in murine embryonic stem cells by homologous recombination led to mouse strains with a complete lack of nuclear poly(ADP-ribosyl)ation. These ADPRT mutant mice were viable and fertile indicating that ADPRT is dispensable in mouse development. Moreover, repair of UV and MNNG induced DNA damage was not affected in ADPRT/3T3 like fibroblasts, as measured by reactivation of in vitro damaged reporter plasmids and unscheduled DNA synthesis. However, about 30% of the ADPRT mutant mice developed pathological skin aberrations on a mixed 129/Sv x C57B1/6 genetic background. These mice will be extremely useful to define the precise biological role of poly(ADP-ribosyl)ation.
Expression of the human galanin gene was analysed using a 3.5-kb DNA fragment comprising the 5'-flanking sequence of the gene. This sequence contains a TATA box (ATATATA) preceded by numerous potential binding sites for transcription factors such as SP1, AP2, and NF kappa B. Three half-palindromic estrogen response elements (EREs, GGTCA) are also found at positions -1,162, -361, and -122 bp relative to the transcription start site. To localize functionally important portions of the promoter region, several shorter fragments of the galanin 5'-flanking region were placed upstream from the chloramphenicol acetyltransferase (CAT) reporter gene. In transient transfection assays, all constructs demonstrated substantial transcriptional activity in both rat glioma/mouse neuroblastoma hybrid cells (NG108-15) and Chinese hamster ovary (CHO-K1) cells. Comparison of the basal expression levels of the different constructs suggests the presence of a negative modulator between positions -1,891 and -207. When cotransfected into NG108-15 cells with the human estrogen receptor cDNA, estrogen did not induce transcription of the human galanin gene at physiological levels of estrogen receptor, although transcription was induced up to 30-fold in the presence of high levels of receptor.
Ubiquitin-conjugating enzymes catalyze the covalent attachment of ubiquitin to cellular substrates. Here we describe the isolation of a novel ubiquitin-conjugating enzyme from human placenta and the cloning of the corresponding cDNA. DNA sequencing revealed that this gene, UbcH2, encodes a protein with significant sequence similarity to yeast UBC8. In contrast to a previous report (Qin, S., Nakajima, B., Nomura, M., and Arfin, S. M. (1991) J. Biol. Chem. 266, 15549-15554), we discovered that UBC8 is interrupted by a single intron bearing an unusual branch point sequence. The revised amino acid sequence of yeast UBC8 exhibits 54% amino acid sequence identity to human UbcH2. Moreover, full-length UbcH2 and UBC8 enzymes expressed from their cDNAs show similar enzymatic activities in vitro by catalyzing the ubiquitination of histones, suggesting that the two enzymes may fulfill similar functions in vivo. Interestingly, comparison of the enzymatic activities of a truncated UBC8 (Qin, S., Nakajima, B., Nomura, M. and Arfin, S. M. (1991) J. Biol. Chem. 266, 15549-15554) and of the full-length enzyme (this report) suggests, that the first 12 amino-terminal residues of UBC8 are required for ubiquitination of histones in vitro but not for thiolester formation with ubiquitin. This suggests that the NH2 terminus of UBC8 may be necessary either for substrate recognition or for the transfer of ubiquitin onto substrates. The UbcH2 gene is located on chromosome 7 and shows a complex expression pattern with at least five different mRNAs.
A novel affinity-purification scheme based on the tight binding of NAD+:ADP-ribosyltransferase (polymerizing) [pADPRT; poly(ADP-ribose) polymerase; EC 2.4.2.30] to single-strand nicks in DNA, single-stranded patches and DNA ends has been developed to facilitate the purification of this enzyme from the lower eukaryote Dictyostelium discoideum. Two homogeneous forms of the enzyme, with M(r) values of 116,000 and 90,000, were prepared from D. discoideum by using poly(A) hybridized to oligo(dT)-cellulose as affinity material. The Km is 20 microM NAD+ for the 90,000-M(r) protein and 77 microM NAD+ for the 116,000-M(r) protein. The optimum conditions for the enzyme activity in vitro are 6-10 degrees C and pH 8. The time course is linear during the first 10 min of the reaction only. As in enzymes of higher eukaryotes, the activity is dependent on DNA and histone H1 and is inhibited by 3-methoxybenzamide, nicotinamide, theophylline, caffeine and thymidine.
In the legal judgment of suicidal cases following brain trauma the emphasis is put on the causality between trauma and suicide on the one hand, and on the question of free determination of intent, on the other hand. These problems are investigated empirically in the present study and an explanatory model based on act theory is proposed. At first, an annual sample of suicides in the region of Salzburg is put through a multidimensional diagnosis (suicidal axis syndrome-Mitterauer 1981). The suicidal axis syndrome consists of the following three components: 1. Suicide attempts in the history 2. Diagnosis of exogenous or (and) endogenous brain dysfunction 3. Suicide-positive history. Of a total of 130 exactly interpretable suicide cases there were 16 cases with a brain trauma followed by an organic brain syndrome in the history. Of these, 11 had announced their suicidal attempts previously and 4 presented with a suicide-positive family history. Although the assessment of suicide in the family history is difficult due to insufficient information, the suicidal axis syndrome was definitely confirmed in 4 suicide cases. We attempt to show that at least in those cases with a suicidal axis syndrome, the free determination of intent is to be negated at the moment of suicide, and that due to the existing chronic brain syndrome a causality between trauma and suicide seems to be given in a neuropsychiatric sense.
Differential scanning calorimetry (DSC), Fourier transform infra-red spectroscopy (FT-IR), HPLC and TLC were used to investigate the interactions between the mucolytic drug acetylcysteine and a number of commonly used tablet and capsule excipients. Acetylcysteine was found to be compatible with microcrystalline cellulose (Avicel PH 101), sodium carboxymethylcellulose, amorphous silicon dioxide (Aerosil), PVP, cross-linked PVP (Polyplasdone XL), corn starch, saccharose and magnesium stearate. Acetylcysteine thermal stability (onset degradation temperature) was decreased in mixtures with corn starch, magnesium stearate, saccharose and lactose. Interactions of acetylcysteine with lactose, PEG 4000 and 6000, glycine, adipic acid and saccharin sodium were found using DSC and studied in detail with FT-IR, HPLC and TLC. The results suggest that acetylcysteine in mixtures with PEG 4000, glycine or saccharin sodium is degraded during storage at conditions of high temperature and humidity.
This study investigated the usefulness of the Contingent Negative Variation (CNV) in the assessment of therapeutic efficacy of a nootropic substance, Cerebrolysin. A total of 41 geriatric patients with the diagnosis of moderate organic brain syndrome of different etiology were included in this randomized placebo-controlled double-blind study. After ten Cerebrolysin- plus multi-vitamins-Infusions, the treatment group of 27 patients showed a significant CNV-amplitude increase as compared to pre-treatment values. In the placebo group of 14 patients CNV-amplitudes even decreased after ten Infusions with multi-vitamins alone. These findings were interpreted in line with the vigilance model of nootropic drug effects as a vigilance-increasing effect of Cerebrolysin, i.e. as an increased neuronal performance readiness. The results confirmed the usefulness of the CNV method by providing an EEG-index of nootropic drug effects.
The method given by the slimming institute "Figurella International", to lose weight without applying chemistry or dieting, is scrutinized critically. The ozone treatment, one of the compounds of the method, proves to be useless. To overcome the general problem caused by photochemical oxidants the company should be set under pressure to stand back from producing ozone, which proves to be a major air pollutant but not to have any effect on the result of the slimming programme.
This study investigated the clinical usefulness of the contingent negative variation (CNV) as a neurophysiological index of cognitive dysfunction associated with cerebrovascular disease. A total of 31 patients ranging in age from 45 to 88 years with the diagnosis of a stroke were included. Nineteen patients were classified as demented according to clinical assessment and the Mental Deterioration Index (MDI) based on the WAIS, the other 12 patients as non-demented. In a discriminative CNV-paradigm two different tones served as warning stimuli for a flash of light presented 1.5 s later that could be turned off by a press on a button only after the relevant tone. According to our hypothesis, CNV amplitudes of demented patients were significantly smaller than those of the non-demented group. Moreover, a significant relationship was observed between severity of dementia (MDI) and degree of amplitude reduction. Thus, it was concluded that the CNV method could make an important clinical contribution to the assessment and course of dementia.
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A central topic of recent research on multiple sclerosis (MS) concerns the development of sensitive neurophysiological techniques with the ability to detect lesions at the earliest possible stage. Specifically, the recording of evoked potentials has during the past decade demonstrated its diagnostic value by disclosing clinically silent lesions especially of the optic nerve and brainstem region. The visual evoked potential (VEP) was registered in 177 patients with definite, probable and possible MS (McAlpine). Abnormal latencies were obtained in 86/46/34% of these three patient groups, respectively. 44% of all MS-patients without clinical or anamnestic evidence of optic nerve dysfunction showed delayed VEPs. The brainstem auditory evoked potential (BAEP) enables topodiagnostic statements to be made regarding brainstem lesion site. This is very valuable, since the brainstem region is especially prone to demyelination, which clinically as well as morphologically often evades detection. 74/60/48% of our definite, probable and possible MS-patients had pathological BAEPs. 50% of asymptomatic patients had an abnormal BAEP-result. Computer tomography yielded brainstem pathology in only 10% of all MS-patients.