Accurate measures of changes in regional lung air volumes from chest x-rays of small animals.

Objective. To develop a robust technique for calculating regional volume changes within the lung from x-ray radiograph sequences captured during ventilation, without the use of computed tomography (CT).Approach. This technique is based on the change in transmitted x-ray intensity that occurs for each lung region as air displaces the attenuating lung tissue.Main results. Lung air volumes calculated from x-ray intensity changes showed a strong correlation (R2= 0.98) against the true volumes, measured from high-resolution CT. This correlation enables us to accurately convert projected intensity data into relative changes in lung air volume. We have applied this technique to measure changes in regional lung volumes from x-ray image sequences of mechanically ventilated, recently-deceased newborn rabbits, without the use of CT.Significance. This method is suitable for biomedical research studies,enabling quantitative regional measurement of relative lung air volumes at high temporal resolution, and shows great potential for future clinical application.

Accuracy and precision of thickness determination from position-averaged convergent beam electron diffraction patterns using a single-parameter metric.

Position-averaged convergent beam electron diffraction patterns are formed by averaging the transmission diffraction pattern while scanning an atomically-fine electron probe across a sample. Visual comparison between experimental and simulated patterns is increasingly being used for sample thickness determination. We explore automating the comparison via a simple sum square difference metric. The thickness determination is shown to be accurate (i.e. the best-guess deduced thickness generally concurs with the true thickness), though factors such as noise, mistilt and inelastic scattering reduce the precision (i.e. increase the uncertainty range). Notably, the precision tends to be higher for smaller probe-forming aperture angles.

Characterization of a hypermutable strain of Drosophila simulans.

A hypermutable strain of Drosophila simulans that originated from a single spontaneous mutant male was characterized. Seven different mutations were isolated from roughly 100 generations of offspring. The genetic analysis of the viable mutants showed two mutations on the X chromosome, one in the lozenge locus and the other in the ruby gene. The autosomic mutations characterized were a dpp-heldout-like, a blistered-like and a homoeotic dominant mutant with an antenna-to-leg transformation and ectopic eyes that we called Zoinho-napata.

Subcellular localization of mRNA in neuronal cells. Contributions of high-resolution in situ hybridization techniques.

The development of technologies for high-resolution nucleic acid localization in cells and tissues has contributed significantly to our understanding of transcriptional and translational regulation in eukaryotic cells. These methods include nonisotopic in situ hybridization methods for light and electron microscopy, and fluorescent tagging for the study of nucleic acid behavior in living cells. In situ hybridization to detect messenger RNA has led to the discovery that individual transcripts may be selectively targeted to particular subcellular domains. In the nervous system, certain species of mRNA have been localized in distal processes in nerve cells and glia. Direct visualization of mRNA and its interactions with subcellular features, such as synaptic specializations, cytoskeletal elements, and nuclear pores, have been achieved. Of particular interest is the presence of mRNA and ribosomes in dendrites, beneath synaptic contacts, suggesting the possibility of synaptic regulation of protein synthesis. The following article will describe the application of high-resolution in situ hybridization and live imaging techniques to the study of mRNA targeting in neurons.

Developing compound eye in lozenge mutants of Drosophila: lozenge expression in the R7 equivalence group.

The lozenge locus is genetically complex, containing two functionally distinct units, cistrons A and B, that influence the structure of the compound eye. Extreme mutations of either cistron produce adult phenotypes that share similarities and that have striking differences. We have analyzed the expression of several developmentally important eye genes including boss, scabrous, rhomboid, seven-up, and Bar in lozenge mutant backgrounds representing both cistrons. This analysis follows the progressive recruitment of photoreceptor neurons during eye development and has confirmed that the initial development of photoreceptors is normal up to the five cell precluster stage (R8, R2/5 and R3/4). However, when lozenge is mutant, further eye development is perturbed. As cells R1, R6 and R7 are recruited, patterns of gene expression for seven-up and Bar become abnormal. We have also characterized the expression of two different enhancer trap alleles of lozenge. The lozenge product(s) appear to be first expressed in the eye disc in undifferentiated cells shortly after the five cell precluster forms. Then, as distinct cells are recruited to a fate, lozenge expression persists and is refined in those cells. Our data suggests that lozenge functions in cone cells and pigment cells as well as in specific glia. With respect to photoreceptor neurons, lozenge biases the developmental potential of cells R1, R6 and R7, by directly influencing the expression of genes important for establishing cell fate.

Ultrastructural localization of dendritic messenger RNA in adult rat hippocampus.

An ultrastructural examination of mRNA within adult rat CA1 hippocampal dendrites was conducted using two different methods. The messages for the alpha and beta forms of the calcium-calmodulin-dependent protein kinase II were localized in ultracryosections using silver-intensified gold detection of isoform-specific oligonucleotide probes. Labeling for both isoforms was observed within the cell bodies and proximal dendrites of pyramidal neurons, but only the alpha form was observed in more distal dendrites. Unfortunately, the morphological preservation of the tissue was not sufficient to determine the localization of labeling relative to subcellular features such as dendritic spines. To address this issue, a preembedding peroxidase-based method was developed, resulting in better preservation of the neuropil. The total population of polyadenylated [poly(A)] mRNA was localized in hippocampus using a biotinylated poly(dT) probe. Poly(A) mRNA was present in the nucleus and throughout the cell body of all hippocampal cells and within isolated dendrites and glial processes within the neuropil. Within pyramidal neurons, labeling was distributed in a longitudinal pattern in proximal apical dendrites. More distally, the amount of labeling diminished, and smaller foci of labeling were observed, particularly near the plasma membrane. Concentrated labeling was present at the base of dendritic spines and, less frequently, near synapses onto the dendritic shaft. These results suggest that dendritic mRNA is found in the vicinity of postsynaptic sites and provide additional evidence that local protein synthesis may play an important role in establishing and maintaining synaptic specializations.

Coexpression of Drosophila TRP and TRP-like proteins in Xenopus oocytes reconstitutes capacitative Ca2+ entry.

Capacitative Ca2+ entry is a component of the inositol-lipid signaling in which depletion of inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ stores activates Ca2+ influx by a mechanism that is still unknown. This pathway plays a central role in cellular signaling, which is mediated by many hormones, neurotransmitters, and growth factors. Studies of Drosophila photoreceptors provided the first putative capacitative Ca2+ entry mutant designated transient receptor potential (trp) and a Drosophila gene encoding TRP-like protein (trpl). It is not clear how the Ca2+ store depletion signal is relayed to the plasma membrane and whether both TRP and TRPL participate in this process. We report here that coexpressing Drosophila TRP and TRPL in Xenopus oocytes synergistically enhances the endogenous Ca(2+)-activated Cl- current and produces a divalent inward current. Both of these currents are activated by Ca2+ store depletion. In the absence of Ca2+, Mg2+ is the main charge carrier of the divalent current. This current is characterized by lanthanum sensitivity and a voltage-dependent blocking effect of Mg2+, which is relieved at both hyperpolarizing (inward rectification) and depolarizing (outward rectification) potentials. The store-operated divalent current is neither observed in native oocytes nor in oocytes expressing either TRP or TRPL alone. The production of this current implicates a cooperative action of TRP and TRPL in the depletion-activated current.

Genetic analysis of the lozenge gene complex in Drosophila melanogaster: adult visual system phenotypes.

Mutations at the lozenge (lz) locus are pleiotropic, primarily affecting the sense organs for sight, smell and taste. To better understand the role that lz plays in the visual system, we investigated its complex genetics and the effect mutations have on the structure of the compound eye. Complementation analysis within the lz locus reveals two functional units necessary for a normal eye, cistrons A and B. Previous recombination studies identified four subloci spanning 0.14 m.u. Cistron A mutations map to the distal-most spectacle sub-locus, which has been identified as an insertion point for P-elements. Southern blotting and chromosomal in situ hybridization show that P-allele lzmu2 contains a single P-element; a cosmid clone derived from lzmu2 confirms that the P-element is defective. Mutants of both cistrons perturb lens structure and eye pigmentation. However, the extent of the defects differs between the most severe mutations of the two cistrons. Within the eye, failure to form the fenestrated membrane permits photoreceptor neurons to "fall" into the brain disrupting neural structure. Our analysis shows that lz exerts control over the identity of cone cells, pigment cells and photoreceptor neurons.

TRP, a protein essential for inositide-mediated Ca2+ influx is localized adjacent to the calcium stores in Drosophila photoreceptors.

The Drosophila transient receptor potential (trp) gene product (TRP) shows some structural similarity to vertebrate voltage-gated Ca2+ channels. It appears to function as a novel Ca2+ channel responsible for light stimulated, inositol trisphosphate (InsP3)-mediated Ca2+ entry in the fly retina. The subcellular localization of TRP protein was determined in this study using immunohistochemical staining with anti-TRP antibody (MAb83F6). TRP was localized to the base of the microvilli in a region adjacent to the presumed InsP3-sensitive Ca2+ stores. This specific localization was supported by measuring the magnitude of a TRP-dependent inward current that results from spontaneous activation of the light-sensitive channels during whole-cell recordings (the rundown current, RDC). We found that reduction of the microvilli area through genetic dissection with the opsin null mutant, ninaEora, was correlated with a pronounced enhancement of the TRP-dependent inward current relative to wild type, suggesting that the TRP-dependent current was not produced along the length of the microvilli. We suggest that the functional localization of the TRP protein is on the plasma membrane loop found along the base of the rhabdomeric microvillus. Thus, the TRP channel may function in concert with the InsP3-sensitive Ca2+ stores.

Ca2+ limits the development of the light response in Drosophila photoreceptors.

The development of the light response was followed in Drosophila photoreceptors at 25 degrees C. In whole-cell recordings from dissociated ommatidia, responses to light were first detected at 82 h post-puparium formation; over the next 8 h sensitivity to light increased exponentially by 5 or 6 orders of magnitude. The end of this phase coincided with the maturation of the rhabdomere as measured by whole-cell capacitance. There was a modest 5-10fold further increase in sensitivity over the final 10 h of pupal development (90-100 h). During a narrow developmental time window (82-87 h) no responses could be detected using non-invasive recording techniques (electroretinogram or suction electrode), and responses to light could only be elicited in whole-cell recordings when micromolar concentrations of Ca2+ are included in the pipette. It seems unlikely that cytosolic Ca2+ per se is the limiting factor, and we suggest instead that the failure to respond to light is due to the lack of Ca2+ in the InsP3-sensitive intracellular stores and that the presence of Ca2+ in these stores is an absolute requirement for phototransduction in Drosophila.

Region-specific expression of a K+ channel gene in brain.

Northern blot analysis and in situ hybridization studies reveal the highly localized expression in rat brain of transcripts from a gene (KShIIIA) encoding components for voltage-gated K+ channels. KShIIIA expression is particularly prominent throughout the dorsal thalamus. The expression of KShIIIA is compared to that of a closely related gene, here called NGK2-KV4. These two genes encode transcripts that induce currents in Xenopus oocytes that are as of yet indistinguishable, but they show very different patterns of expression in rat brain. NGK2-KV4 transcripts are particularly abundant in the cerebellar cortex, where KShIIIA expression is very weak. These results demonstrate the existence of cell-type-specific K+ channel components and suggest that one reason for the unusually large diversity of K+ channel proteins is the presence of subtypes that participate in specific brain functions.

Expression of ion channel genes in Drosophila.

Ion channel transcripts from three genes were localized by the method of tissue in situ hybridization. The genes examined were the Drosophila Na+ channel genes, paralytic (para) and Drosophila Sodium Channel (DSC), and the K+ channel gene, Shaker( Sh). All three of the genes were expressed in cell bodies of the fly central nervous system, including optic lobes, central brain, suboesophageal ganglion, and thoracico-abdominal ganglion. Sh was additionally expressed in the photoreceptor cells of the retina and pupal flight muscle, while para and DSC were not. The temporal expression pattern of Sh in muscle was different from that in the central nervous system: muscle expression was transient and limited to mid-pupal stage while nervous system expression was observed throughout pupation, apparently peaking at the late-pupal stage. Only one class of 5' end was found in pupal muscle, possibly indicating regulation of splicing pathways.

Subcellular localization of transcripts in Drosophila photoreceptor neurons: chaoptic mutants have an aberrant distribution.

Photoreceptor neurons in the Drosophila retina are long (100 mu) and narrow, providing a system for the study of the intracellular distribution of transcripts and proteins. The chaoptic gene is expressed exclusively in photoreceptor neurons, and mutations of the gene result in reduced developmental competence of cells to generate normal rhabdomeric membranes. The mutant protein exhibited altered distribution both in developing and adult photoreceptor neurons. Furthermore, the transcript distribution in mutants was altered, decreasing with distance from the nucleus, instead of the normal uniform distribution throughout the cell soma. The deficit of transcript concentration correlated with the severity of developmental defect in rhabdomere formation along the cell. In contrast, the distribution of the opsin transcript was not affected by the chaoptic mutation. To observe RNA localization at the ultrastructural level, a high-resolution, electron microscopic in situ hybridization protocol was developed. The results indicate that the normal chaoptic transcript is present on the rough endoplasmic reticulum, which may be a vehicle for specific transcript distribution.

Twenty Drosophila visual system cDNA clones: one is a homolog of human arrestin.

From a group of 436 Drosophila melanogaster cDNA clones, we selected 39 that are expressed exclusively or predominantly in the adult visual system. By sequence analysis, 20 of the clones appear to represent previously unreported distinct cDNAs. The corresponding transcripts are detected in the retina and optic lobes. The genes are scattered throughout the genome, some near mutations known to affect eye function. One of these clones has been identified, by sequence analysis, as the structural gene (Arr) for a Drosophila homolog of human arrestin. Vertebrate arrestin interacts with rhodopsin in phototransduction and has been associated with an autoimmune form of uveitis in primates. The presence of an arrestin homolog in Drosophila suggests that both the vertebrate and invertebrate phototransduction cascades are regulated in a similar manner.

Transcript localization of four opsin genes in the three visual organs of Drosophila; RH2 is ocellus specific.

Drosophila and other Dipteran flies have three different kinds of visual organs; in the adult a pair of compound eyes and three dorsal ocelli; and in the larva a pair of internal photoreceptor organs. They develop in distinct ways, yet have certain features in common. All three organs use retinal-derived chromophores, coupled to distinct opsins, to provide a diversity of spectral sensitivities. Four opsin genes have been identified thus far in Drosophila; Rh1, Rh2, Rh3 and Rh4 (refs 6-11). We have used in situ hybridization to study the messenger RNAs expressed by these four opsin genes in all three visual organs. Rh1, Rh3 and Rh4 are already known to be expressed in different subsets of cells in the compound eye. We found that, in contrast, opsin Rh2 is the predominant opsin expressed in the ocelli. Opsin Rh1 is known to be expressed in the larval photoreceptor. We found that Rh3 and Rh4 are as well, but not Rh2. The ocellar-specific gene expression of Rh2 is of particular interest for its possible bearing on the function of the ocellus.

Molecular characterization and expression of sevenless, a gene involved in neuronal pattern formation in the Drosophila eye.

The Drosophila sevenless mutation results in lack of a single neuron (photoreceptor cell R7) in every ommatidium of the compound eye; the developmental defect occurs in the larval eye disc. We created P-element-induced alleles and used them to isolate the sev gene. An 8.2 kb transcript is expressed in the eye disc, behind the morphogenetic furrow, coincident with recruitment and differentiation of photoreceptor clusters. The transcript becomes localized at the apical surface, persists in the prepupa, and fades out at pupation. It is again detected in the adult head. In some alleles the 8.2 kb transcript is absent. In others, the transcript is expressed, in spite of the absence of cell R7. Localization of the gene product in the eye disc was obtained with antibody raised against sev protein.

Electrophoretic analysis of proteins from night-blind mutants of Phycomyces.

Using two-dimensional gel electrophoresis, we have analyzed proteins from a plasma membrane-enriched fraction from Phycomyces sporangiophores. Specifically, we have compared gels for night-blind mutants and a wild-type strain to find proteins involved in the early steps of the sensory transduction chain for phototropism. In the gels for a mutant affected in the gene madA, a protein spot [51 kilodaltons (kdal) and pI 6.35] appears that is absent from the wild-type and the other mad mutants. Mutants affect in either of two madB alleles lack a protein spot (57 kdal and pI 6.6) that is present in the wild-type and all other mad strains; this spot probably represents the madB gene product. In some madC mutants, two spots (59 kdal, pI 6.5, with a covalently linked flavin; and 50 kdal, pI 6.4) are absent; however, in other madC strains, one or both of these spots are present. These four protein spots that are altered in madA, madB, and madC mutants may represent component of the photoreceptor complex responsible for phototropism in Phycomyces.

Analysis of microsomal flavoproteins from Phycomyces sporangiophores: Candidates for the blue-light photoreceptor.

To help identify components of the blue-light photoreceptor system for phototropism in Phycomyces blakesleeanus Bgff., proteins from a microsomal fraction obtained from synchronous sporangiophores were studied. By two-dimensional gel electrophoresis, two proteins (FP1, FP2) with covalently bound flavins were found. FP1 has a molecular weight of 71 000 and an isoelectric point of 6.6; FP2 has a molecular weight of 59 000 and an isoelectric point of 6.5. These flavoproteins were purified by column chromatography and gel filtration while assaying for flavins by fluorescence. The relative concentrations of FP1 and FP2 were affected by light applied during growth. These flavoproteins are likely components of the blue-light photoreceptor complex mediating phototropism in Phycomyces.

Complications of catheter cerebral arteriography: analysis of 5,000 procedures. I. Criteria and incidence.

Computer analysis of complications of 5,000 catheter cerebral arteriograms performed at four hospitals (two training and two nontraining) revealed a total of 68 complications (1.4%). Eight (0.16%) of these complications were major: five patients required thrombectomy or embolectomy, two had permanent neurologic deficits, and one patient died. The complication rate for the training hospitals (3.9%) was significantly higher than that for the nontraining hospitals (0.9%), but the rates of permanent complications were identical in both groups (0.1%). Reporting criteria and complication types are described.