An efficient method to generate chromosomal rearrangements by targeted DNA double-strand breaks in Drosophila melanogaster

Homologous recombination (HR) is an indispensable tool to modify the genome of yeast and mammals. More recently HR is also being used for gene targeting in Drosophila. Here we show that HR can be used efficiently to engineer chromosomal rearrangements such as pericentric and paracentric inversions and translocations in Drosophila. Two chromosomal double-strand breaks (DSBs), introduced by the rare-cutting I-SceI endonuclease on two different mobile elements sharing homologous sequences, are sufficient to promote rearrangements at a frequency of 1% to 4%. Such rearrangements, once generated by HR, can be reverted by Cre recombinase. However, Cre-mediated recombination efficiency drops with increasing distance between recombination sites, unlike HR. We therefore speculate that physical constraints on chromosomal movement are modulated during DSB repair, to facilitate the homology search throughout the genome.     

Noise-induced cell death in the mouse medial geniculate body and primary auditory cortex

Noise-induced effects within the inner ear have been well investigated for several years. However, this peripheral damage cannot fully explain the audiological symptoms in noise-induced hearing loss (NIHL), e.g. tinnitus, recruitment, reduced speech intelligibility, hyperacusis. There are few reports on central noise effects. Noise can induce an apoptosis of neuronal tissue within the lower auditory pathway. Higher auditory structures (e.g. medial geniculate body, auditory cortex) are characterized by metabolic changes after noise exposure. However, little is known about the microstructural changes of the higher auditory pathway after noise exposure. The present paper was therefore aimed at investigating the cell density in the medial geniculate body (MGB) and the primary auditory cortex (AI) after noise exposure. Normal hearing mice were exposed to noise (10 kHz center frequency at 115 dB SPL for 3 h) at the age of 21 days under anesthesia (Ketamin/Rompun, 10:1). After 1 week, auditory brainstem response recordings (ABR) were performed in noise exposed and normal hearing animals. After fixation, the brain was microdissected and stained (Kluever-Barrera). The cell density in the MGB subdivisions and the AI were determined by counting the cells within a grid. Noise-exposed animals showed a significant ABR threshold shift over the whole frequency range. Cell density was significantly reduced in all subdivisions of the MGB and in layers IV-VI of AI. The present findings demonstrate a significant noise-induced change of the neuronal cytoarchitecture in central key areas of auditory processing. These changes could contribute to the complex psychoacoustic symptoms after NIHL.     

A Golgi deimpregnation study of neurons in the rhesus monkey visual cortex (Areas 17 and 18)

Summary The morhological features of 298 neurons impregnated according to Golgi-Kopsch in areas 17 and 18 of Macaca mulatta were analyzed, and the same neurons were deimpregnated to visualize structural details of the somata in different types of neurons. The following cell types were investigated: Pyramidal and pyramid-like cells, spiny stellate cells, double bouquet cells, bipolar cells, chandelier cells, neurogliaform cells, basket and related cells. This procedure allows the evaluation of the nuclear-cytoplasmic proportion and the position of the nucleus besides shape and size of the cell body. Pyramidal and pyramid-like cells (N=43), spiny stellate cells (N=26), basket and related cells (N=126) are variable in these features. A positive correlation between soma size and width of the cytoplasm is found in pyramidal, pyramid-like cells and spiny stellate cells. With the exception of some large somata in both these types of neurons the nucleus is found in a central position. Double bouquet cells (N=6), bipolar cells (N=13) and chandelier cells (N=11) exhibit small cytoplasmic rims and centrally located nuclei. The small somata of neurogliaform cells (N=37), however, and the small to very large somata of basket and related cells show broad cytoplasmic portions surrounding the eccentrically located nuclei. These findings allow the identification of different neuronal types in Nisslstained sections on the basis of these soma features. This is a prerequisite for further detailed quantitative studies on the laminar distribution of different neuronal types in the visual cortex of the monkey.     

Giant ragweed specific immunotherapy is not effective in a proportion of patients sensitized to short ragweed: analysis of the allergenic differences between short and giant ragweed

BACKGROUND: Short ragweed and giant ragweed pollen allergens are considered largely cross-reactive, and it is generally believed that 1 species is sufficient for skin testing and immunotherapy. However, in the area north of Milan (a zone widely invaded only by short ragweed), about 50% of patients submitted to injection specific immunotherapy with giant ragweed showed little or no clinical response, but showed an excellent outcome if they were shifted to short ragweed specific immunotherapy. OBJECTIVE: To investigate allergenic differences between short and giant ragweed. METHODS: IgE reactivity to short ragweed of sera from 16 patients allergic to ragweed was assessed by immunoblot before and after absorption with short and giant ragweed. Moreover, 41 ragweed-monosensitive patients underwent skin prick test with both ragweed species. RESULTS: In several cases, preabsorption of sera with giant ragweed extract was unable to inhibit IgE reactivity fully against both a 43-kd allergen and other allergens at different molecular weights in short ragweed. On skin prick test, short ragweed induced larger wheals than giant ragweed in the majority of patients, and 6 of 41 (15%) patients were strongly short ragweed-positive but giant ragweed-negative. The immunoblot with the serum from 1 of these subjects showed a strong IgE reactivity to short ragweed at about 43 kd in the absence of any reactivity to giant ragweed. CONCLUSION: Short and giant ragweed are not allergenically equivalent. Allergenic differences involve both the major allergens Amb a 1-2/Amb t 1-2 and some minor allergens. In patients allergic to ragweed, both diagnosis in vivo and immunotherapy should always be performed by using the ragweed species present in that specific geographic area.     

Abdominal pain in children and symptoms of somatization disorder

Children seen in a multispecialty medical clinic for abdominalpain were divided into three groups: 21 with confirmed organicfindings related to the abdominal pain, 14 with confirmed organicfindings unrelated to the pain, and 108 whose physical examinationswere negative (the functional pain group). For children withfunctional abdominal pain (but not for the others) the numberof symptoms of somatization disorder (Briquet's syndrome) wassignificantly related to the chronicity of the child's condition.Children with functional pain and no prior complaint had a meanof 1.95 symptoms; those with complaints of less than 1 year'sduration, 2.21 symptoms; those with complaints of more thana year since age 6, 4.04 symptoms; and those with complaintsfor more than a year with onset prior to age 6 years, 4.55 symptomsfrom the Somatization Disorder list. Findings were interpretedas preliminary evidence for a distinct, chronic, polysymptomatichysterical disorder beginning in childhood.     

The PAR1 (YAP1/SNQ3) gene of Saccharomyces cerevisiae,ac-jun homologue,is involved in oxygen metabolism

Summary The PAR1/SNQ3 gene of S. cerevisiae, which increases resistance to iron chelators in multi-copy transformants, is identical to the YAP1 gene, a yeast activator protein isolated as a functional homologue of the human c-jun oncogene by binding specifically to the AP-1 consensus box. The observed H₂O₂-sensitivity of par1 mutants has been attributed to an increased sensitivity to reduced oxygen intermediates. Accordingly, par1 mutants did not survive an elevated oxygen pressure and were very sensitive to menadione and methylviologene, two chemicals enhancing the deleterious effects of oxygen. The specific activities of enzymes involved in oxygen detoxification, such as superoxide dismutase, glucose 6-phosphate dehydrogenase and glutathione reductase, were decreased in par1 mutants and increased after PAR1 over-expression. As in the case of oxygen detoxification enzymes, the cellular levels of glutathione were similarly affected. These observations indicate that PAR1/YAP1/SNQ3 is involved in the gene regulation of certain oxygen detoxification enzymes. The finding that H₂O₂ promotes DNA-binding of human c-jun is consistent with a similar function for PAR1/YAP1/SNQ3 and c-jun in cellular metabolism.Dedicated to Professor Dr. R. W. Kaplan on the occasion of his 80th birthday     

Inhibitory interaction in the cat's lateral geniculate nucleus

Summary Spike activities of optic tract fibers and corresponding relay cells were recorded simultaneously in layers A and A₁ of the dorsal lateral geniculate nucleus of the cat. Light stimuli of various diameters were shone into the receptive field center of these unit pairs and their input/output ratios were determined. An increase of the stimulus size leads to an impairment of the input/output ratio in on-center and off-center relay cells. This suppressive effect has approximately the same latency as the excitatory response.Intracellular recordings suggest that the inhibitory effect of the surround is due to a postsynaptic process. Inhibitory postsynaptic potentials occur during and — under certain stimulus conditions —before the excitatory response. The short latency of these IPSPs suggests that they result from the activity of adjacent units with the same RF characteristics as the recorded neuron. This inhibitory input is not restricted to the RF periphery but may also be activated by stimulation within the RF center. Most neurons are also inhibited by units with antagonistic center responses.During the period of this research Ernst Pöppel held a training grant of the Stiftung Volkswagenwerk, Az. 11 1015.     

Production of stable anti-digoxin Fv in Escherichia coli.

We have created a bacterial expression-export system and have used it to express (14 mg l-1) the variable region fragment (Fv) of an anti-digoxin antibody (26-10) in Escherichia coli. The expression-export plasmid contains a T7 promoter and the E. coli signal sequences ompA [Movva et al., J. biol. Chem. 255, 27-29 (1980)] and phoA [Inouye et al., J. Bacteriol. 149, 434-439 (1982)] fused to heavy chain (VH) and light chain (VL) variable region sequences to generate an artificial cistron. The 26-10 Fv protein made using this system was soluble, unlike many other expression systems which produce insoluble proteins in the form of inclusion bodies. The 26-10 VH and VL proteins were cleaved at their mature N-termini and exported into the bacterial periplasm where they could be easily extracted and affinity purified on ouabain-Sepharose. 26-10 Fv bound to digoxin with similar affinity and specificity as the whole 26-10 antibody (Ka for Fv, 1.3 x 10(9) M-1, Ka for IgG, 7 x 10(9) M-1). 26-10 Fv appears to be remarkably stable in comparison with other Fv fragments. The half-life for chain dissociation of 26-10 Fv was 48 hr compared to the reported 1.5 hr half-life of McPC603 Fv. We present the proton NMR spectra of the 26-10 Fv as preliminary evidence that this expression-export system can be used to facilitate the analysis of the solution structure of 26-10 Fv by NMR.     

Differences in airway remodeling between subjects with severe and moderate asthma

BACKGROUND: Airway remodeling in asthma comprises a range of structural changes. Several studies have suggested an association between these changes and disease severity. The relationship between the extent of remodeling and lung function is not well defined. OBJECTIVE: We sought to contrast the structural changes in the airways of well-defined groups of subjects with severe and moderate asthma and to correlate the extent of remodeling with disease severity. METHODS: Endobronchial biopsy specimens were obtained from 15 subjects with severe and 13 subjects with moderate asthma. Epithelial integrity, cell-layer areas, subepithelial fibrosis, and the distance between epithelial and airway smooth muscle (ASM) layers were measured by means of image analysis. Collagen was identified by using Van Giesen stain, and ASM was defined by using smooth muscle alpha-actin immunostaining. Specific immunostains were performed for the evaluation of RANTES, IL-8, and eotaxin expression as markers of ASM phenotype. RESULTS: ASM area was greater in subjects with severe (0.24+/- 0.03 mm(2)) than in subjects with moderate (0.05+/- 0.01 mm(2)) asthma (P<.001). The distance between the epithelial and ASM layers was less in the severe group (0.12+/- 0.01 mm) than in the moderate group (0.24+/- 0.02, P<.001). A trend toward greater subepithelial fibrosis in subjects with severe asthma did not reach statistical significance. IL-8 and eotaxin expression, but not RANTES expression, were increased in the ASM of subjects with severe asthma compared with in subjects with moderate asthma. CONCLUSION: Smooth muscle alteration is the key structural change that distinguishes severe from moderate asthma, and phenotypic change in ASM might contribute to the difficulty in obtaining adequate control in some subjects with severe asthma.