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Biomedical subjects

B Sauer

Publications and source records attributed to B Sauer.

At least 37 records · Page 2Linked to original sources
PubMed1998

Insulin-like growth factor-I affects perinatal lethality and postnatal development in a gene dosage-dependent manner: manipulation using the Cre/loxP system in transgenic mice.

Insulin-like growth factor-I (IGF-I) is essential for cell growth, differentiation and postnatal development. A null mutation in igf-1 causes intrauterine growth retardation and perinatal lethality. The present study was designed to test the lower limit of igf-1 gene dosage that ensures survival and postnatal growth by using the Cre/loxP system. Mice with variable reductions in IGF-I levels were generated by crossing EIIa-cre transgenic mice and mice with loxP-flanked igf-1 locus (igf-1/flox). EIIa-cre mice express bacteriophage P1 Cre (causes recombination) recombinase under the adenovirus promoter EIIa, during early embryonic development before implantation, and cause genomic recombination of the igf-1/flox locus. Mice with the most extensive recombination die immediately after birth, while the survivors have significant growth retardation in proportion to the reduction in their igf-1 gene. Interestingly, this gene dosage effect on body weight was not very significant before weaning. However, when the young animals were weaned at 3 weeks, the igf-1 gene dosage was the only independent predictor of the weight gain between 3 and 6 weeks among the parameters tested. Although growth retarded, mice with Cre-induced partial igf-1 deficiency were fertile and gave birth to null mice. Thus Cre-induced genomic recombination using the EIIa promoter occurs during development and creates distinct phenotypes compared with the conventional null mutation. This variability allows for postnatal survival and will enable one to begin to explore the role of the endocrine vs. paracrine effects of IGF-I.

Animals↗
PubMed1997

Generation of Cre recombinase-specific monoclonal antibodies, able to characterize the pattern of Cre expression in cre-transgenic mouse strains.

Transgene-encoded Cre recombinase can target alteration of loxP-tagged genes to specific cell types and developmental stages in mice, depending on the pattern of transgene expression. To facilitate determination of the latter, we have generated monoclonal anti-Cre antibodies which are specific for distinct epitopes on the recombinase and detect Cre both on immunoblots and intracellularly by immunofluorescence. We demonstrate the usefulness of these antibodies by an analysis of Cre expression in mice carrying a cre-transgene under B cell-specific control.

Animals↗
PubMed1997

Brief expression of a GFP cre fusion gene in embryonic stem cells allows rapid retrieval of site-specific genomic deletions.

The Cre DNA recombinase of bacteriophage P1 has become a useful tool for precise genomic manipulation in embryonic stem (ES) cells that have been gene modified by homologous recombination. We have re-engineered the cre gene to allow ready identification of living Cre+cells by constructing a functional fusion between Cre and an enhanced green fluorescent protein from Aequorea victoria (GFPS65T). The GFP cre fusion gene product rapidly targeted the nucleus in the absence of any exogenous nuclear localization signal. Moreover, GFPCre catalyzed efficient DNA recombination in both a mouse 3T3 derivative cell line and in murine ES cells. Fluorescence- activated cell sorting (FACS) of transiently GFP cre -transfected ES cells not only allowed rapid and efficient isolation of Cre+cells after DNA transfection but also demonstrated that a burst of Cre expression is sufficient to commit cells to Cre-mediated 'pop-out' of loxP -tagged DNA from the genome. Thus, GFP cre allows rapid identification of living cells in which loxP - flanked DNA sequences are destined to be removed from the genome by Cre-mediated recombination without reliance on recombinational activation or inactivation of a marker gene at the target locus. In addition, the GFP cre fusion gene will prove useful in tracing tissue-specific Cre expression in transgenic animals, thereby facilitating the generation and analysis of conditional gene knockout mice.

Animals↗
PubMed1997

Segmental genomic replacement by Cre-mediated recombination: genotoxic stress activation of the p53 promoter in single-copy transformants.

Genotoxic stress results in transcriptional activation of the p53 promoter. To gain more detailed information on genotoxic induction of the p53 promoter at a uniform genomic locus, we have developed an efficient strategy for replacing a defined genomic segment in mouse NIH 3T3 cells with exogenous transfected DNA using a 'double lox' targeting strategy mediated by Cre DNA recombinase. The strategy utilizes a pair of heterospecific lox sites engineered both into the genome and onto the targeting DNA. This allows direct replacement of genomic DNA by a Cre-catalyzed double crossover event. p53-CAT reporter constructs were site-specifically placed into the genomic target 20-fold more efficiently by double lox recombination than by Cre-mediated single crossover insertional recombination, and the absolute frequency of site-specific double lox targeting exceeded the frequency of transformation due to random illegitimate recombination of transfected DNA into the genome. Resulting targeted single-copy integrants of the p53-CAT reporter show strong genotoxic induction by mitomycin C, and a dynamic range of induction that exceeds that seen in transient transfection assays. The double lox strategy is generally applicable to Cre-mediated genomic targeting in any cell and should be of particular utility in the site-specific targeting of DNA into embryonic stem (ES) cells for the production of gene-modified mice.

3T3 Cells↗
PubMed1997

Disruption of the c/ebp alpha gene in adult mouse liver.

The liver-enriched transcription factor C/EBP alpha has been implicated in the regulation of numerous liver-specific genes. It was previously reported that mice carrying a homozygous null mutation at the c/ebp alpha locus died as neonates due to the absence of hepatic glycogen and the resulting hypoglycemia. However, the lethal phenotype precluded further analysis of the role of C/EBP alpha in hepatic gene regulation in adult mice. To circumvent this problem, we constructed a conditional knockout allele of c/ebp alpha by using the Cre/loxP recombination system. Homozygous c/ebp-loxP mice, (c/ebp alpha(fl/fl);fl, flanked by loxP sites) were found to be indistinguishable from their wild-type counterparts. However, when Cre recombinase was delivered to hepatocytes of adult c/ebp alpha(fl/fl) mice by infusion of a recombinant adenovirus carrying the cre gene, more than 80% of the c/ebp alpha(fl/fl) genes were deleted specifically in liver and C/EBP alpha expression was reduced by 90%. This condition resulted in a reduced level of bilirubin UDP-glucuronosyltransferase expression in the liver. After several days, the knockout mice developed severe jaundice due to an increase in unconjugated serum bilirubin. The expression of genes encoding phosphoenolpyruvate carboxykinase, glycogen synthase, and factor IX was also strongly reduced in adult conditional-knockout animals, while the expression of transferrin, apolipoprotein B, and insulin-like growth factor I genes was not affected. These results establish C/EBP alpha as an essential transcriptional regulator of genes encoding enzymes involved in bilirubin detoxification and gluconeogenesis in adult mouse liver.

Adenoviruses, Human↗
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↗