Response of dermal fibroblast cultures from patients with unusually severe responses to radiotherapy and from ataxia telangiectasia heterozygotes to fractionated radiation

We examined the cytotoxic effects of radiation delivered in daily fractions at clinically relevant doses in plateau phase cultures of skin fibroblast cell strains derived from ataxia telangiectasia (AT) heterozygotes, patients with unusually sensitive responses to radiotherapy, apparently normal patients, and cell bank controls. A gradual linear reduction in surviving fraction versus total dose was observed in the control group, comprised of apparently normal individuals and one patient with a normal clinical response to radiotherapy, after exposure to daily fractions of 2.0 Gy. There was a much steeper decline in surviving fraction among the AT heterozygotes and the group with sensitive responses to radiotherapy, such that after six daily fractions of 2.0 Gy (12 Gy total dose), the mean surviving fraction of the control group was significantly different from that of the AT heterozygotes (P = 0.0009) and that of the patients with unusually sensitive responses to radiotherapy (P = 0.0002). We propose that this assay may be a useful means of identifying cell strains from AT heterozygotes. Based on these results, the hypothesis is discussed that patients who suffer unusually sensitive clinical reactions to radiotherapy may be AT heterozygotes.     

secG and temperature modulate expression of azide-resistant and signal sequence suppressor phenotypes of Escherichia coli secA mutants

SecA is a dynamic protein that undergoes ATP-dependent membrane cycling to drive protein translocation across the Escherichia coli inner membrane. To understand more about this process, azide-resistant (azi) and signal sequence suppressor (prlD) alleles of secA were studied. We found that azide resistance is cold sensitive because of a direct effect on protein export, suggesting that SecA-membrane interaction is regulated by an endothermic step that is azide inhibitable. secG function is required for expression of azide-resistant and signal sequence suppressor activities of azi and prlD alleles, and in turn, these alleles suppress cold-sensitive and export-defective phenotypes of a secG null mutant. These remarkable genetic observations support biochemical data indicating that SecG promotes SecA membrane cycling and that this process is dependent on an endothermic change in SecA conformation.     

Identification and disruption of a plant shaker-like outward channel involved in K+ release into the xylem sap

SKOR, a K+ channel identified in Arabidopsis, displays the typical hydrophobic core of the Shaker channel superfamily, a cyclic nucleotide-binding domain, and an ankyrin domain. Expression in Xenopus oocytes identified SKOR as the first member of the Shaker family in plants to be endowed with outwardly rectifying properties. SKOR expression is localized in root stelar tissues. A knockout mutant shows both lower shoot K+ content and lower xylem sap K+ concentration, indicating that SKOR is involved in K+ release into the xylem sap toward the shoots. SKOR expression is strongly inhibited by the stress phytohormone abscisic acid, supporting the hypothesis that control of K+ translocation toward the shoots is part of the plant response to water stress.     

A comparative cerebral blood flow study in a baboon model with acetazolamide provocation: 99mTc-HMPAO vs 123I(IMP)

Pharmacological interactions are important when nuclear medical procedures are applied to patients under drug therapy, or drug provocation. This study compares in baboon models (regional) cerebral blood flow [(r)CBF] results from 99mTc-HMPAO and 123I-iodoamphetamine [123I(IMP)] each with and without acetazolamide, the latter a suggested drug for testing cerebrovascular reserve. Expected differences in cerebral uptake were observed between the two radio-tracers without acetazolamide. The increase in tracer uptake resulting from acetazolamide is significantly enhanced for 123I(IMP), which could have diagnostic implications.     

Postembryonic neurogenesis in the central nervous system of the tobacco hornworm, Manduca sexta. III. Spatial and temporal patterns of proliferation

Postembryonic neurogenesis leads to a dramatic increase in the number of functional neurons within the segmental ganglia of the moth, Manduca sexta. These adult-specific neurons are generated during larval life by segment-specific arrays of individually identifiable stem cells, or neuroblasts (Nbs). By the end of the feeding larval stage, each Nb has generated a discrete nest of progeny, which ranges in size from less than 10 to more than 70 progeny. The sizes of these identifiable nests of progeny vary in a segment-specific manner, with the thoracic nests containing a greater number of progeny compared with their homologues in the simpler abdominal ganglia. In order to describe those factors that influence the size of the post-embryonic neuronal lineages, we examined the spatial and temporal pattern of postembryonic neurogenesis in the segmental ganglia of Manduca. The rates at which the identifiable nests accumulated progeny were estimated by counting the number of progeny within the nests, using sectioned material isolated from animals at stages ranging from embryonic hatching until the end of the feeding larval stage. All of the postembryonic Nbs began to generate progeny at around the time of the molt to the third larval instar. Each nest added progeny at a rate that was a characteristic of its identity and segment of origin. Although all of the nests within the thorax continued to accumulate progeny throughout the feeding larval stage, several of the abdominal nests showed little or no growth following the molt to the fifth larval instar. The thymidine analog 5-bromo 2-deoxyuridine (5-BrdU) was used to estimate the mitotic rates of the identifiable Nbs. The number of labeled progeny within a nest 24 h after application of 5-BrdU ranged from a low of 1 to 2 to a high of 11 to 13 labeled cells. In some instances there was a good correlation between the estimated mitotic rate of an identified Nb and the rate of growth of its associated nest of progeny. However, several of the identifiable nests accumulated progeny at a slower rate than predicted based on the estimated mitotic rate of the Nb. Cell death appears to be responsible for slowing the growth of the nests during the feeding larval stage. We estimate that 10% to 70% of the neurons generated during the feeding larval stage degenerate within 24 h of their birth. The level of cell death observed within a nest was dependent on both its identity and its segment of origin.     

The OKT3 Antibody Response Study: a multicentre study of human anti-mouse antibody (HAMA) production following OKT3 use in solid organ transplantation

Human anti-murine antibody titres following patient exposure to the monoclonal antibody Orthoclone OKT3 (muromonab-CD3) are determined by laboratories using diverse analytical methods which are not standardized and whose concordance is not established. A multicentre study group therefore compared testing for IgG anti-OKT3 antibody among seven laboratories. A set of 270 sera was obtained from 30 heart, 30 kidney and 30 liver transplant recipients with no previous exposure to OKT3 who were receiving OKT3 for induction immunosuppression. Sera were collected from each patient prior to and at 24 +/- 2 days and 31 +/- 2 days following initial OKT3 exposure. Identical aliquots of all 270 sera were tested for IgG anti-OKT3 antibody by each laboratory. In addition, the limit of detection of each laboratory's method was estimated by titration of an affinity-purified IgG anti-OKT3 reference material of known concentration. Anti-OKT3 antibody formation differed greatly among the three organ groups. Cardiac patients demonstrated the least sensitization and almost exclusively lower titres, while kidney recipients had more frequent and higher titre antibody formation. Liver recipients yielded the highest sensitization rate and the most frequent high titre sera. Importantly, the seven laboratories differed widely in the number of pretreatment sera reported as positive (ranging from 0% to 41% among laboratories), the number of post-OKT3 sera reported as positive (17-63%), the number of post-OKT3 samples with titre > or = 1000 (2-31%), and the number of patients sensitized 19-69%). Concordance among laboratories was highly variable, with interlaboratory agreement ranging from 38% to 83% on the sample titres assigned to 180 post-OKT3 sera. Many of the discordant results were consistent with differences in the limit of detection of the analytical methods, which ranged from 0.19 microgram/ml to > or = 15 micrograms/ml, a nearly 100-fold difference among laboratories. This study demonstrated the presence of both good concordance and significant discordance among laboratories in determining human anti-mouse antibody titres, and demonstrated that common titre categories (100, 1000, 10,000) were not equivalent among laboratories. The level of concordance among methods should be considered when comparing anti-OKT3 antibody results from different centres and their correlation with clinical events. Universal comparative testing, patterned after proficiency testing programmes, is needed to assess differences among laboratories and to bring uniformity and a sound interpretative basis to this field of testing.