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Biomedical subjects

B Sauer

Publications and source records attributed to B Sauer.

At least 55 records · Page 3Linked to original sources
PubMed1996

In situ color detection of alpha-L-arabinofuranosidase, a "no-background" reporter gene, with 5-bromo-3-indolyl-alpha-L-arabinofuranoside.

We describe the synthesis and use of 5-bromo-3-indolyl-alpha-L-arabinofuranoside (5-BI-ara) for the detection of alpha-L-arabinofuranosidase in bacterial colonies. Since the product of 5-BI-ara hydrolysis is an intensely colored indigo precipitate, it is a useful substrate for in situ detection of alpha-L-arabinofuranosidase activity. Here we show that colonies of an Escherichia coli strain expressing a recombinant alpha-L-arabinofuranosidase gene from Streptomyces lividans are readily identified by visual inspection on bacterial plates containing 5-BI-ara. Use of 5-BI-ara should facilitate the detection of endogenous alpha-L-arabinofuranosidase activity in a variety of microorganisms. In addition, since alpha-L-arabinofuranosidase activity is not commonly found in a large number of bacteria, yeast, and animal cells, 5-BI-ara will be useful in exploring the use of genes coding for alpha-L-arabinofuranosidase as gene expression reporters in heterologous systems.

Arabinonucleosides↗
PubMed1996

Multiplex Cre/lox recombination permits selective site-specific DNA targeting to both a natural and an engineered site in the yeast genome.

Variant lox sites having an altered spacer region (heterospecific lox sites) are not proficient for Cre-mediated recombination with the canonical 34 bp loxP site, but can recombine with each other. By placing different heterospecific lox sites at different genomic locations, Cre can catalyze independent DNA recombination events at multiple loci in the same cell without concern that unwanted inter-locus recombination events will be generated. Such heterospecific lox sites also allow Cre to specifically target efficient integration of exogenous DNA to endogenous lox-like sequences that naturally occur in the genome. Specific targeting occurs only with a DNA vector carrying a heterospecific lox site in which the spacer region has been redesigned to match the 'spacer' region of the targeted chromosomal element. Moreover, in cells expressing a catalytically active Cre recombinase, naturally occurring lox-like sequences can exhibit almost 20% mitotic recombination. Thus, in the same cell, heterospecific lox sites can be used independently at multiple loci for integration, for deletion and for enhanced mitotic recombination, thereby increasing the repertoire of genomic manipulations catalyzed by the Cre recombinase.

Base Sequence↗
PubMed1996

Efficient in vivo manipulation of mouse genomic sequences at the zygote stage.

We describe a transgenic mouse line carrying the cre transgene under the control of the adenovirus EIIa promoter that targets expression of the Cre recombinase to the early mouse embryo. To assess the ability of this recombinase to excise loxP-flanked DNA sequences at early stages of development, we bred EIIa-cre transgenic mice to two different mouse lines carrying loxP-flanked target sequences: (i) a strain with a single gene-targeted neomycin resistance gene flanked by 1oxP sites and (ii) a transgenic line carrying multiple transgene copies with internal loxP sites. Mating either of these loxP-carrying mouse lines to EIIa-cre mice resulted in first generation progeny in which the loxP-flanked sequences had been efficiently deleted from all tissues tested, including the germ cells. Interbreeding of these first generation progeny resulted in efficient germ-line transmission of the deletion to subsequent generations. These results demonstrate a method by which loxP-flanked DNA sequences can be efficiently deleted in the early mouse embryo. Potential applications of this approach are discussed, including reduction of multicopy transgene loci to produce single-copy transgenic lines and introduction of a variety of subtle mutations into the line.

Animals↗
PubMed1996

Escherichia coli rpoC397 encodes a temperature-sensitive C-terminal frameshift in the beta' subunit of RNA polymerase that blocks growth of bacteriophage P2.

Escherichia coli 397c is temperature sensitive for growth at 43.5 degrees C and unable to plate bacteriophage P2 at 33 degrees C. The mutation conferring these phenotypes was mapped to the rpoC gene. RNA synthesis is temperature sensitive in the mutant strain, and the beta' subunit of RNA polymerase isolated from this strain exhibits increased electrophoretic mobility. DNA sequence analysis revealed that the mutation is a deletion of 16 bp, resulting in a frameshift that leads to truncation of the beta' subunit at the carboxy terminus.

Amino Acid Sequence↗
PubMed1995

[The risk assessment of the need for help and care as well as the risk of mortality among older people. The results of an 18-month pilot study in a family practice].

OBJECTIVE: To investigate prospectively the prognostic power of various demographic and diagnostic parameters, as well as of medical history, for evaluating in elderly patients the probability of their requiring care and of their risk of dying soon. PATIENTS AND METHOD: All the elderly patients (> or = 70 years) of one general practitioner were included. Of 178 patients who fulfilled the inclusion criteria, 144 participated (93 women, 49 men; mean age 78.7 +/- 5.8 years). Demographic, basic diagnostic and medical history data of the kind that can be easily collected in general practice were recorded. The general practitioner was also asked to assess the probability of each patient requiring care and the likelihood of their dying soon. Several functional tests that record or measure daily activity were conducted. After 18 months all admissions to care, homes for the elderly or hospital and deaths were recorded. The different groups were compared. RESULTS: The probability of requiring care and of dying within the observation period were predicted more accurately by functional tests and questions than by conventional diagnostic methods. This was particularly so for functional investigations of basic daily activity, cognitive ability and manual skills. There were significant differences between the groups in the results of tests (P < 0.01). CONCLUSION: Functional tests can be rapidly performed and have high diagnostic and prognostic power. They are therefore recommended for use by general practitioners for assessing the probability of a given patient requiring care and for estimating the likelihood of early death.

Activities of Daily Living↗
PubMed1995

[Complications of endoscopic sclerotherapy of esophageal varices].

Within the framework of a retrospective study complications of endoscopic variceal sclerotherapy were analyzed. From April, 1, 1988 till August, 31, 1994 267 consecutive patients (158 male, 109 female, mean age 43 [27-78] years) with esophageal variceal hemorrhage due to liver cirrhosis and portal hypertension underwent endoscopic variceal injection treatment. Sclerotherapy was performed with 24.5 ml (12-34 ml) 1% of polydocanole on average per treatment. Each patient had 4.5 (2-7) therapy sessions on average. Local complications were: Transient dysphagia (73%), chest pain (65%), esophageal ulcerations (63%), ulerogenic bleeding (14%), posttherapeutic hemorrhage (13%), esophageal strictures (10%), pleural effusions (9%), subfebrile temperatures (6.4%), pericarditis (0.4%) and esophageal perforation (0.4%). No patient died from sclerotherapy-induced side effects. In conclusion, endoscopic injection therapy is an efficient treatment of acute variceal hemorrhage. Not severe local complications often occur, severe side effects are extremely rare, however.

Adult↗
PubMed1994

Purification and DNA-binding activity of the PacA subunit of the bacteriophage P1 pacase enzyme.

The bacteriophage P1 packaging site (pac) cleavage enzyme (pacase) consists of two phage encoded proteins, PacA and PacB. Both proteins are necessary for the recognition and cleavage of pac and for subsequent packaging of cleaved DNA into phage particles. We have purified PacA to homogeneity from a bacterial strain that overproduces the protein. Purified PacA complements an Escherichia coli extract containing the PacB protein for DNA cleavage at the pac site and recognizes and binds to methylated pac DNA independently of PacB in gel retardation experiments. The latter property of PacA is absolutely dependent on the presence of a wildtype E. coli extract, suggesting that E. coli host proteins play a role in the pac cleavage reaction.

Bacterial Proteins↗
PubMed1994

Faithful cleavage of the P1 packaging site (pac) requires two phage proteins, PacA and PacB, and two Escherichia coli proteins, IHF and HU.

The PacA and PacB subunits of the bacteriophage P1 DNA packaging enzyme (pacase) are necessary for cleavage of the phage packaging site (pac). In the accompanying paper, we show that the PacA subunit of the enzyme specifically binds to pac in the absence of PacB, but requires factors present in an Escherichia coli extract to do so. We show here that either of two E. coli DNA binding proteins, integration host factor (IHF) or HU, can replace this extract and promote the binding of PacA to pac. IHF binds to pac independently of PacA and DNase I footprinting experiments show that IHF protects approximately 40 bp of DNA around an IHF consensus sequence adjacent to the cleavage site. DNase I footprinting experiments also show that in the presence of either IHF or HU, PacA binds to the hexanucleotide sequences (5'-TGATCA/G) that flank the cleavage site and that have been previously shown to be essential for pac cleavage. The importance of IHF and HU in pac cleavage is further demonstrated by the severe reduction in both the fidelity and efficiency of pac cleavage in vitro with extracts lacking both IHF and HU. Addition of either IHF or HU to the deficient extracts renders them fully proficient for pac cleavage. Finally, we show that IHF bends DNA at the IHF site within pac. Based on these results, we propose a model that can account for the role of the various phage and host proteins, and for DNA bending in the pac cleavage reaction.

Bacterial Proteins↗
PubMed1994

High-performance liquid chromatographic separations of nystatin and their influence on the antifungal activity.

Several efficient analytical HPLC methods for the separation and comparison of the nystatin complex have been developed. These separations have been extended to the preparative scale in order to gain sufficient quantities of the purified nystatin components for testing antifungal activity. For the first time we describe a new HPLC method which does not affect the antifungal activity of nystatin.

Animals↗
PubMed1994

Site-specific recombination: developments and applications.

Site-specific DNA recombination systems have considerable utility in manipulating DNA and can facilitate many cloning and gene transfer techniques. The ability of a number of recombinases to catalyze efficient DNA recombination in higher eukaryotes has important and exciting consequences for precise chromosome and transgene engineering. Exploitation of these recombinases will facilitate the generation of transgenic animal disease models and help elucidate the function of developmental genes.

Animals↗
PubMed1994

Detection of Septata intestinalis in stool specimens and coprodiagnostic monitoring of successful treatment with albendazole.

We describe two patients with AIDS and chronic diarrhea in whom the microsporidian Septata intestinalis was detected with use of light and electron microscopic coprodiagnostic techniques. The ultrastructure of the microsporidian spores found in their stool specimens was distinctly different from that of Enterocytozoon bieneusi, another intestinal microsporidian found in patients infected with human immunodeficiency virus. Electron microscopic examination of duodenal biopsy specimens available from one of the patients enabled identification of S. intestinalis and confirmed the similarity of spores found in feces and in duodenal tissue. Both patients' diarrhea stopped when they were treated with albendazole. Coprodiagnostic monitoring indicated disappearance of the parasites and allowed the diagnosis of a relapse in one patient, who responded well to a second course of treatment.

AIDS-Related Opportunistic Infections↗
PubMed1994

Assessment of vitamin B2 status in performance athletes of various types of sports.

To determine the vitamin B2 supply, 62 athletes in various types of sports and 16 non-athletes were examined, using a weighed food record (7-day), alpha-EGR determination, microbiological blood, and HPLC urinary measurements. If the guidelines of 1.5 or 1.7 mg/day vitamin B2 or 0.6 mg/4,184 kJ (GRDA) are defined for adequate status, all sports groups (M +/- SEM) are above this level. The vitamin B2 blood concentrations show a range (mean) from 303-372 nmol/liter, which indicates that more than 90% of the athletes are above the reference value of 327 nmol/liter (mean). There is a correlation of r = 0.49 between the vitamin B2 intake (mean over 7 days) and the vitamin B2 blood level. Determination of vitamin B2 blood concentration is characterized by good precision (C.V. +/- 3.2%) and recovery (95.7%). The enzyme activation measurement (alpha-EGR) shows that all of the athletes are below the alpha-EGR reference value of < 1.50 (good supply). No significant correlations could be determined between alpha-EGR and vitamin B2 intake, blood concentrations, and urinary excretion. The measurement also shows high precision (C.V. 1.7%). The Vitamin B2 excretion in urine (mumol/g Cr.) was higher than the reference values for non-athletes in more than 90% of the athletes. There is a correlation of r = 0.89 between the vitamin B2 intake on the last day (Day 7) of the food record and the riboflavin excretion, and between vitamin B2 in blood and urinary excretion of r = 0.52. The C.V. of vitamin B2 urinary measurement using HPLC is 2.5%, with recovery of 98.2%. Combined measurement of vitamin B2 intake, blood concentration, and urine excretion appears to be a suitable procedure for determining the vitamin B2 status. Most of the performance athletes were sufficiently supplied with vitamin B2, compared to the reference values for non-athletes.

Adolescent↗
PubMed1994

Recycling selectable markers in yeast.

A series of excisable marker cassettes has been constructed to facilitate recycling of selectable markers in yeast. These cassettes exploit the use of the Cre DNA recombinase to precisely excise the marker gene when desired. They are especially useful for making gene disruptions and then removing the marker gene to allow subsequent genetic manipulations with that same marker. Also described are a number of cre expression vectors that allow galactose-induced expression of the recombinase in yeast. The procedure is simple and allows rapid processing of large numbers of transformants.

DNA Nucleotidyltransferases↗
PubMed1993

Genomic targeting with purified Cre recombinase.

Purified Cre recombinase protein introduced directly into cultured mammalian cells by lipofection catalyzes both site-specific chromosomal integration of a co-transfected lox targeting vector and precise excision of genomic DNA flanked by directly repeated lox sites. This procedure eliminates the need to transfect cre expression plasmids to activate recombination at lox sites. We used this simplified procedure to investigate the effect on targeting efficiency of both lox vector design and chromosomal position of the lox target. We show that such chromosomal position effects can exert at least a 50-fold per lox target difference in targeting efficiency in a human osteosarcoma cell line.

Animals↗
PubMed1993

Characterization of angiotensin AT1A receptor isoform by its ligand binding signature.

The objective of this study was to determine whether the binding signature of the cloned rat AT1A receptor transfected into Chinese hamster ovary cells could be distinguished from that of the endogenous AT1B receptor expressed in rat adrenal cortex. An extensive series of peptide and nonpeptide Ang II analogs was used for the characterization. The binding of [125I]Ang II to the recombinant AT1A receptors was quite sensitive to inhibition by GTP gamma S. Scatchard analysis of the competition of Ang II revealed two populations of binding sites (site 1: KD = 3.05 +/- 0.27 nM and a maximum binding (Bmax) of 134 +/- 26 fmol/mg protein; site 2: KD = 253 +/- 77 nM and Bmax = 1.05 +/- 0.19 pmol/mg protein). The ligand binding signature of the AT1A receptor is defined by the affinity (Ki = nM) and order of potency of the following ligands: saralasin (2.07) > Ang II (3.35) > losartan (14) > Ang III (20) > GR 117289C (28) > EXP6803 (160) > Ang I (281) > PD123177 (> 10,000). This binding signature of the cloned AT1A receptors appears to be similar to that displayed by rat adrenal cortical cells where AT1B is predominantly expressed. These findings suggest that AT1A and AT1B receptors may not be easily distinguishable by the currently available ligand agonists or antagonists. Consequently, AT1A or AT1B may be considered as isoforms rather than subtypes of the AT1 receptors.

Adrenal Cortex↗
PubMed1993

Intestinal coinfection with Enterocytozoon bieneusi and Cryptosporidium in a human immunodeficiency virus-infected child with chronic diarrhea.

The microsporidian Enterocytozoon bieneusi has been recognized as an important cause of chronic diarrhea in severely immunodeficient adults infected with human immunodeficiency virus (HIV). We report the first case of intestinal E. bieneusi infection in a child. The 9-year-old boy with connatal HIV infection presented with failure to thrive, chronic diarrhea, and intermittent abdominal pain. His CD4 lymphocyte count was 0.05 x 10(9)/L and dropped to 0.01 x 10(9)/L. No HIV-associated opportunistic infection other than oral hairy leukoplakia and oral candidiasis had been found before microsporidia were detected. Treatment of microsporidiosis with albendazole was of no benefit. During follow-up, the boy also developed intestinal cryptosporidiosis. Evaluation of chronic diarrhea in severely immunodeficient HIV-infected children should include examination for intestinal microsporidia. We recommend the use of a new coprodiagnostic technique that allows detection of microsporidial spores in stool specimens. Furthermore, consideration of dual or even multiple parasitic infections in the differential diagnosis of chronic diarrhea may have both important clinical and epidemiological implications.

AIDS-Related Opportunistic Infections↗
PubMed1992

Stoichiometry of the Cre recombinase bound to the lox recombining site.

The site-specific recombinase Cre from bacteriophage P1 binds and carries out recombination at a 34 bp lox site. The lox site consists of two 13 bp inverted repeats, separated by an 8 bp spacer region. Both the palindromic nature of the site and the results of footprinting and band shift experiments suggest that a minimum of two Cre molecules bind to a lox site. We report here experiments that demonstrate the absolute stoichiometry of the Cre-lox complex to be one molecule of Cre bound per inverted repeat, or two molecules per lox site.

Base Sequence↗