From segment to somite: segmentation to epithelialization analyzed within quantitative frameworks.

One of the most visually striking patterns in the early developing embryo is somite segmentation. Somites form as repeated, periodic structures in pairs along nearly the entire caudal vertebrate axis. The morphological process involves short- and long-range signals that drive cell rearrangements and cell shaping to create discrete, epithelialized segments. Key to developing novel strategies to prevent somite birth defects that involve axial bone and skeletal muscle development is understanding how the molecular choreography is coordinated across multiple spatial scales and in a repeating temporal manner. Mathematical models have emerged as useful tools to integrate spatiotemporal data and simulate model mechanisms to provide unique insights into somite pattern formation. In this short review, we present two quantitative frameworks that address the morphogenesis from segment to somite and discuss recent data of segmentation and epithelialization.     

Summary of complementation groups of UV-sensitive CHO cell mutants isolated by large-scale screening

A summary is given for the lineage and complementation groupassignments of 153 UV-sensitive mutants of the CHO AA8 cellline. The distribution of mutants among six complementationgroups was highly non-random, with the great majority of theisolates belonging to groups 1 and 2. This asymmetry is consistentwith the known hemizygosity of these two linked loci in CHOcells. The relative numbers of mutants induced in group 2 wasfound to depend greatly on the type of mutagen used. Mutagenesiswith UV radiation, ethyl methanesulfonate (EMS), N-methyl-N'-nitro-N-nitroso-guanidine and 7-bromomethylbenz[a]anthraceneproduced high frequencies of group 2 mutants. In contrast, ICR170and ICR191, which are thought to produce mostly frameshift mutations,yielded very few mutants in group 2. These results are of particularimportance in light of the recent finding that the human ERCC2gene, which corrects group 2 mutants, has very strong homologywith the yeast gene RAD3. RAD3 is an essential gene for viabilityin yeast, and the low recovery of group 2 mutants using theframeshift agents strongly suggests that frameshift mutationstend to be lethal in the hamster ERCC2 locus. Several mutagen-sensitivedouble mutants were isolated in two-step selections from EMS-,mitomycin C- or UV-sensitive parental cells, including the lineUVU1, the first mammalian line with two mutations that affectUV sensitivity. The first mutation inactivated excision repair,and the second mutation appears to have affected some otherrecovery process. UVU1 should be useful for studying recoveryprocesses that are separate from nucleotide excision repair. ¹To whom correspondence should be addressed     

Some notes on uncertainty; Federal policy and innovation

The legislative background and current responsibilities of the new National Center for Health Care Technology (NCHCT) are discussed. The NCHCT's charge is to consider the stage of development, the actual or potential risks, costs and rate of use of health care technology. The role of Government in supporting new technologies, stimulating innovation and encouraging application of research products is considered. These issues are discussed in the context of the uncertainty inherent in planning and the formulation of public policy.     

Hydrogen peroxide induces DNA single strand breaks in respiratory epithelial cells

The respiratory epithelium is often exposed to oxidant gases, including ozone from photochemical smog and toxic oxygen metabolites released from neutrophils recruited in conditions of airway inflammation. We evaluated DNA single strand break formation by alkaline elution as a measure of oxidant-induced DNA damage to bronchial epithelial cells. Human AdenoSV-40-transformed bronchial epithelial cells (BEAS), subclone R1.4 or nonhuman primate bronchial epithelial cells were cultured in growth factor supplemented Ham's F12 medium on polycarbonate filters. DNA was labeled by incubation with [³H]thymidine. Cells were incubated for 1 h in HBSS or HBSS and increasing concentrations of hydrogen peroxide (H₂O₂). Cells incubated in H₂O₂ demonstrated dose-dependent increases in strand break formation, and BEAS cells were more sensitive to H₂O₂-induced injury than primary bronchial epithelial cells. The addition of catalase or preincubation of cells with the iron chelator desferoxamine prevented H₂O₂-induced strand breakage. DNA strand break formation may be an important mechanism of oxidant injury in respiratory epithelial cells.This work was supported by NIEHS grant ES-00628 and California Primate Research Center Base grant. Portions of this work were presented at the American Thoracic Society annual meeting, May 1992, Miami, Florida.     

Duchenne muscular dystrophy and idiopathic hyperCKemia segregating in a family

A 7-month-old boy with gross motor delay and failure to thrive presented with rhabdomyolysis following an acute asthmatic episode. During hospitalization an electrocardiographic conversion to a Wolff-Parkinson-White type 1 (WPW) pattern took place. Duchenne muscular dystrophy (DMD) was suspected based on elevated creatine kinase (CK) serum levels, muscle biopsy, and family history. The diagnosis was confirmed by molecular analysis, which documented a deletion corresponding to cDNA probe 1-2a in the dystrophin gene, in the propositus and in an affected male cousin of his mother. “Idiopathic” hyperCKemia was found in the propositus, his father, and 5 of his relatives. We suggest that the unusually early and severe manifestations of DMD in this patient may be related to the coincidental inheritance of the maternal DMD gene and of a paternal gene, causing hyperCKemia. © 1995 Wiley-Liss, Inc.     

Genetic linkage analysis identifies new proximal and distal flanking markers for the X-linked agammaglobulinemia gene locus, refining its localization in Xq22

Genetic linkage analysis has been instrumental in mapping thegene for X-linked agammaglobulinemia (XLA) to the proximal longarm of the human X chromosome, to Xq22. Due to the relativerarity of this disease the localization of the gene within Xq22has remained imprecise. We have investigated twenty-nine familiesaffected by XLA and have found no recombinants with the DXS178locus in over 30 informative meioses. DXS178 is now the mostreliable and informative locus for use in pre-natal diagnosisand carrier detection of XLA. In addition, we have identifiednew closely linked proximal and distal flanking markers forXLA, DXS442 and DXS101, respectively. These loci are separatedby 2cM, considerably reducing the extent of DNA within whichthe XLA locus can be contained. This will open up the way formore directed positional cloning efforts for the isolation ofthe XLA gene.     

Improved contrast by a simplified post-staining procedure for ultrathin sections of resin-embedded bacterial cells: Application of ruthenium red

High contrast can be obtained in ultrathin sections of bacterial cells embedded in the low-temperature resin Lowieryl by post-staining of the sections on an aqueous ruthenium red solution. Post-staining with lead citrate can be omitted. Combination with post-staining with uranyl acetate results in further improvement of contrast. This is shown for Gram-positive, Gram-negative, and methanogenic bacteria. Improved visibility is demonstrated for wall layers including slime and capsule, cytoplasmic membrane, nucleoid, envelope of PHB inclusion bodies, polyphosphate, and intracellular defective bacteriophages. The procedure is suited as post-staining for immunocytochemical analyses.     

Rats fed only during the light period are resistant to stress-induced weight loss

Repeated restraint stress (3 h/day for 3 days) causes a chronic down-regulation of body weight in rats. This study determined whether weight loss was influenced by the time of day that rats had access to food or that stress was applied. Groups of male Sprague-Dawley rats were fed a 40% kcal fat diet with food given ad libitum, only during the light phase or only during the dark phase. After 2 weeks of adaptation, rats within each feeding treatment were divided into four groups. One was exposed to repeated restraint at the start of the light phase, another was restrained at the start of the dark phase and the remaining groups were nonstressed controls for restrained rats. Body weight was significantly reduced in ad libitum- and dark-fed restrained rats, compared with nonstressed controls, from Day 2 of restraint, regardless of the time of day that they were stressed. There was no significant effect of restraint on weight change of light-fed rats. Food intake was inhibited by stress in ad libitum- and dark-fed rats, but it was not changed in light-fed rats. Serum corticosterone was increased by restraint in all rats irrespective of feeding schedule. This study demonstrates that stress-induced weight loss only occurs when rats have food available during their normal feeding period (dark phase) and is not determined by increased corticosterone release.     

Cell-mediated and humoral immune responses induced by scarification vaccination of human volunteers with a new lot of the live vaccine strain of Francisella tularensis.

Tularemia is a disease caused by the facultative intracellular bacterium Francisella tularensis. We evaluated a new lot of live F. tularensis vaccine for its immunogenicity in human volunteers. Scarification vaccination induced humoral and cell-mediated immune responses. Indications of a positive immune response after vaccination included an increase in specific antibody levels, which were measured by enzyme-linked immunosorbent and immunoblot assays, and the ability of peripheral blood lymphocytes to respond to whole F. tularensis bacteria as recall antigens. Vaccination caused a significant rise (P less than 0.05) in immunoglobulin A (IgA), IgG, and IgM titers. Lymphocyte stimulation indices were significantly increased (P less than 0.01) in vaccinees 14 days after vaccination. These data verify that this new lot of live F. tularensis vaccine is immunogenic.