Strong structural effect of the position of a single acetylaminofluorene adduct within a mutation hot spot.

The NarI restriction enzyme recognition site, G1G2CG3CC, has been identified as a hotspot for -2 frameshift mutations induced by N-2-acetylaminofluorene (AAF) on the basis of a forward mutation assay in plasmid pBR322 in the bacterium Escherichia coli. AAF binds primarily to the C-8 position of guanine residues, and the three guanines of the NarI site are similarly reactive. Despite this similar chemical reactivity, only binding of AAF to the G3 residue causes the -2 frameshift mutations.

Intratracheal exposure of rats to Aspergillus fumigatus spores isolated from sawmills in Sweden.

Five strains of Aspergillus fumigatus (A, B, D, H, and K) isolated from sawmills were used to expose groups of three rats by intratracheal intubation. The dose was 10(9) spores per rat. At 48 h after administration, two rats from the D group and all rats from the K group died with symptoms of strong dyspnea and tachypnea.

Unusual helical packing in crystals of DNA bearing a mutation hot spot.

The target sequence of the restriction enzyme NarI (GGCGCC) is a hot spot for the -2 frameshift mutagenesis (GGCGCC----GGCC) induced by the chemical carcinogens such as N-2-acetyl-aminofluorene. Of the guanine residues, all of which show equal reactivity towards the carcinogen, only binding to the 3'-most proximal guanine within the NarI site is able to trigger the frameshift event. We selected the non-palindromic dodecamer d(ACCGGCGCCACA), whose sequence corresponds to the most mutagenic NarI site in pBR322 DNA; for X-ray structure analysis.

Excision of the imidazole ring-opened form of N-2-aminofluorene-C(8)-guanine adduct in poly(dG-dC) by Escherichia coli formamidopyrimidine-DNA glycosylase.

A polynucleotide containing N-(deoxyguanosine-8-yl)-2-aminofluorene residues (dGuo-C8-AF) was obtained by treatment of poly(dG-dC) with [3H]ring-N-hydroxy-2-amino-fluorene. This substrate was further treated under alkaline conditions to convert dGuo-C8-AF residues into their imidazole ring-opened derivative or 1-[6-(2,5-diamino-4-oxo-pyrimidinyl-N-6-deoxyribose]-3-(2-fluorenyl++ +)urea (iro-dGuo-C8-AF). The ring-opening of 50% of the dGuo-C8-AF residues occurs in 24 h at 37 degrees C in the presence of 0.

Z-DNA-forming sequences are spontaneous deletion hot spots.

Z-DNA-forming sequences are shown to elicit a biological response in Escherichia coli. Plasmids containing sequences capable of adopting the Z conformation (GC and CA/GT) are shown to be hot spots for spontaneous deletions. All the deletions involve an even number of base pairs.

Practical considerations in the production, purification, and formulation of monoclonal antibodies for immunoscintigraphy and immunotherapy.

Issues associated with the large-scale production of monoclonal antibodies for pharmaceutical applications are examined. The development of a commercial monoclonal antibody production process involves much more than just scaling-up the laboratory process and making it cost-effective. It involves establishing the hybridoma cell bank with cells that are free of adventitious agents such as viruses and mycoplasma, that have stability in continuous culture for antibody-production rate and cell viability, and that do not have unusual or expensive media requirements.

Single adduct mutagenesis: strong effect of the position of a single acetylaminofluorene adduct within a mutation hot spot.

2-Acetylaminofluorene (AAF), a potent rat liver carcinogen that binds covalently to the C-8 position of guanine residues in DNA, is an effective frameshift mutagen. The mutations are distributed nonrandomly, in that most are located at a few specific DNA sequences (i.e.

Construction of plasmids containing a unique acetylaminofluorene adduct located within a mutation hot spot. A new probe for frameshift mutagenesis.

N-2-acetylaminofluorene (AAF), a potent rat liver carcinogen, binds primarily to the C-8 position of guanine residues. In a bacterial forward mutation assay, more than 90% of the mutations induced by -AAF adducts are frameshift mutations located at specific sites: the so-called mutation hot spots. We are particularly interested in a class of -2 frameshift mutations occurring within a specific sequence, the NarI sequence.

Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants.

During endocytosis in Chinese hamster ovary (CHO) cells, Semliki Forest virus (SFV) passes through two distinct subpopulations of endosomes before reaching lysosomes. One subpopulation, defined by cell fractionation using free flow electrophoresis as "early endosomes," constitutes the major site of membrane and receptor recycling; while "late endosomes," an electrophoretically distinct endosome subpopulation, are involved in the delivery of endosomal content to lysosomes. In this paper, the pH-sensitive conformational changes of the SFV E1 spike glycoprotein were used to study the acidification of these defined endosome subpopulations in intact wild-type and acidification-defective CHO cells.

Toxicokinetics of ochratoxin A in several species and its plasma-binding properties.

The toxicokinetic profile of ochratoxin A was studied after the oral or intravenous administration of 50 ng/g b.w. to fish, quail, mouse, rat and monkey.

Acidification and ion permeabilities of highly purified rat liver endosomes.

While it is well established that acidic pH in endosomes plays a critical role in mediating the orderly traffic of receptors and ligands during endocytosis, little is known about the bioenergetics or regulation of endosome acidification. Using highly enriched fractions of rat liver endosomes prepared by free flow electrophoresis and sucrose density gradient centrifugation, we have analyzed the mechanism of ATP-dependent acidification and ion permeability properties of the endosomal membrane. This procedure permitted the isolation of endosome fractions which were up to 200-fold enriched as indicated by the increased specific activity of ATP-dependent proton transport.

Optimization of batch fermentor sterilization.

This article presents a calculation procedure useful for the optimization and scale up of batch sterilization cycles in large-scale fermentors. This technique determines the sterilization temperature and hold-time necessary to minimize nutrient damage in a specific fermentor. The method can also be used for "scaledown" experiments to eliminate sterilization conditions as a scale up parameter.

Molecular analysis of mutagenesis in E. coli.

In this paper we present a general strategy that is suitable for the analysis of the forward mutation spectrum induced by any physical or chemical mutagen or carcinogen. The assay is based upon the inactivation of the tetracycline resistance gene located on plasmid pBR322. Plasmid DNA is treated in vitro with the mutagen and transformed into the host bacteria of choice.

Genetic control of AAF-induced mutagenesis at alternating GC sequences: an additional role for RecA.

In a previous study, the forward mutation spectrum induced by the chemical carcinogen N-acetoxy-N-2-acetylaminofluorene was determined (Koffel-Schwartz et al. 1984). It was found that 90% of the induced mutations are frameshift mutations located within specific sequences (mutation hot spots).

A possible role for Na+,K+-ATPase in regulating ATP-dependent endosome acidification.

Endosomes maintain a slightly acidic internal pH, which is directly responsible for their ability to ensure proper sorting of incoming receptors and ligands during endocytosis. At least two distinct subpopulations of endosomes can be distinguished, designated "early" and "late" on the basis of their kinetics of labeling with endocytic tracers. The subpopulations differ not only in their functions (rapid receptor recycling and transport to lysosomes, respectively) but also in their capacities for acidification in intact cells and in vitro.

Patterns of physical activity among German adolescents: the Berlin-Bremen Study.

Patterns of leisure-time physical activity among 932 West German boys and girls from two distinct socioeconomic groups were examined longitudinally over a 2-year period beginning with seventh to eighth grade students. Activity indices reflect the weekly time spent in activities, the weekly frequency of participation, and the average duration per activity episode. The indices refer to all activities, to moderate or vigorous activities, or to each individual activity.

Distribution of 14C-ochratoxin A in the mouse monitored by whole-body autoradiography.

The tissue distribution of 14C-labeled ochratoxin A was studied in mouse using whole-body autoradiography. The distribution was followed for 18 days after one single intravenous injection of 5 microCi/animal, corresponding to 160-230 ng toxin/g body weight. Very long persistence of 14C-ochratoxin A in the circulation was noticed and the toxin was detected in the blood even after 18 days when the experiment was finished.

Frameshift mutagenesis in Escherichia coli by reversible DNA intercalators: sequence specificity.

The mutagenic potency of the simple reversible intercalators isopropyl-OPC (iPr-OPC) and 9-aminoacridine (9-AA) is assessed in E. coli using reversion assays based on plasmids derived from pBR322 carrying various frameshift mutations within the tetracycline resistance gene in repetitive sequences: +/- 2 frameshift mutations within alternating GC sequences; +/- 1 frameshift mutation at runs of guanines. The results obtained show that iPr-OPC and 9-AA have a sequence specificity for mutagenesis: they revert +1 and -1 frameshift mutations within runs of monotonous G:C base pairs.

Reliability of goniometric measurements of children with cerebral palsy.

The reliability of five lower-extremity goniometric measurements of 20 patients with moderate to severe hypertonicity was assessed by three pediatric physical therapists. The measurements were repeated on five of the children within one week. Intra-rater variation was less than inter-rater variation for all measurements, but variations of 10 to 15 degrees were found in inter-rater measurements.

Molecular cloning and sequencing of a cDNA coding for mature human kidney cathepsin H.

A cDNA library was established from human kidney RNA and screened with an extended oligonucleotide probe derived from the amino-acid sequence of human cathepsin H. A recombinant clone, pRF15, was isolated and characterized. DNA sequence analysis of its 1106-nucleotide-long insert revealed that pRF15 encodes the complete protein sequence of mature cathepsin H plus 28 amino acids of a propeptide, thus confirming that cathepsin H is synthesized as a larger precursor molecule and posttranslationally processed.