Relationship between Coating-Induced Soot Aggregate Restructuring and Primary Particle Number.

The restructuring of monodisperse soot aggregates due to coatings of secondary organic aerosol (SOA) was investigated in a series of photo-oxidation chamber experiments. Soot aggregates were generated by one of three sources (an ethylene premixed burner, a methane inverted diffusion burner, or a diesel generator), treated by denuding, size-selected by a differential mobility analyzer, and injected into a smog chamber, where they were exposed to the photo-oxidation products of p-xylene, which partitioned to form SOA coatings. The evolution of aggregates from their initial to final morphologies was investigated in situ by mobility and mass measurements and ex situ by transmission electron microscopy. At a given initial aggregate mobility diameter, diesel aggregates are less dense and composed of smaller primary particles than those generated by the two burners, and they restructure to a smaller final mobility diameter. Remarkably, the final degrees of restructuring of aggregates from all three sources exhibit the same linear dependence on the number of primary particles per aggregate. The observed linear relationship, valid for the atmospherically relevant SOA coating investigated here, could allow modelers to predict the evolution of aggregate morphology based on a single property of the aggregates.

Identification of transcripts overexpressed during airway epithelium differentiation.

Human airway epithelium, the defence at the forefront of protecting the respiratory tract, evacuates inhaled particles by a permanent beating of epithelial cell cilia. When deficient, this organelle causes primary ciliary dyskinesia, and, despite numerous studies, data regarding ciliated cell gene expression remain incomplete. The aim of the present study was to identify genes specifically expressed in human ciliated respiratory cells via transcriptional analysis. The transcriptome of dedifferentiated epithelial cells was subtracted from that of fully redifferentiated cells using complementary DNA representational difference analysis. In order to validate the results, gene overexpression in ciliated cells was confirmed by real-time PCR, and by comparing the present list of genes overexpressed in ciliated cells to lists obtained in previous studies. A total of 53 known and 12 unknown genes overexpressed in ciliated cells were identified. The majority (66%) of known genes had never previously been reported as being involved in ciliogenesis, and the unknown genes represent hypothetical novel transcript isoforms or new genes not yet reported in databases. Finally, several genes identified here were located in genomic regions involved in primary ciliary dyskinesia by linkage analysis. In conclusion, the present study revealed sequences of new cilia-related genes, new transcript isoforms and novel genes which should be further characterised to aid understanding of their function(s) and their probable disorder-related involvement.

[Confronting the practice of surgery on differentiated thyroid cancer with current guidelines in Germany. A multicenter trial]. / Chirurgische Praxis und Leitlinien beim differenzierten Schilddrüsenkarzinom in Deutschland: ein multizentrischer Vergleich.

AIM: For the surgical therapy of differentiated thyroid cancer precise guidelines are applied by the German medical societies. In a retrospective multicenter study, we investigated the following issues: Are the current guidelines respected? Is there a difference concerning the surgical radicalism and the outcome? Does the perioperative morbidity increase with the higher radicalism of the procedure? PATIENTS, METHODS: Data gained from 102 patients from 17 regional referral hospitals who underwent surgery for thyroid cancer and a following rodioiodine treatment (mean follow up: 42.7 [24-79] months) were analyzed. At least 71 criterias were analyzed in a SPSS file. RESULTS: 46.1% of carcinomas were incidentally detected during goiter surgery. The thyroid cancer (papillary n = 78; follicular n = 24) occurred in 87% unilateral and in 13% bilateral. Papillary carcinomas < 1 cm were detected in 25 cases; in five of these cases (20%) contralateral carcinomas < 1 cm were found. There were significant differences concerning the surgical radicalism: a range from hemithyroidectomy to radical thyroidectomy with lateral neck dissection. Analysis of the histopathologic reports revealed that lymph node dissection was not performed according to guidelines in 55% of all patients. The perioperative morbidity was lower in departments with a high case load. The postoperative dysfunction of the recurrent laryngeal nerve (mean: 7.9% total / 4.9% nerves at risk) variated highly, depending on differences in radicalism and hospitals. Up to now these variations in surgical treatment have shown no differences in their outcome and survival rates, when followed by radioiodine therapy. CONCLUSION: Current surgical regimes did not follow the guidelines in more than 50% of all cases. This low acceptance has to be discussed. The actual discussion about principles of treatment regarding, the so-called papillary microcarcinomas (old term) has to be respected within the current guidelines.

Virus inactivation and protein recovery in a novel ultraviolet-C reactor.

BACKGROUND AND OBJECTIVES: Ultraviolet-C (UVC) irradiation is a viral-inactivation method that was dismissed by many plasma fractionators as a result of the potential for protein damage and the difficulty in delivering uniform doses. A reactor with novel spiral flow hydraulic mixing was recently designed for uniform and controlled UVC treatment. The objective of this study was to investigate virus inactivation and protein recovery after treatment through the new reactor. MATERIALS AND METHODS: Virus- and mock-spiked Alpha1-proteinase inhibitor (Alpha1-PI) solutions were treated with UVC. The virus samples were assayed for residual infectivity and amplified by the polymerase chain reaction (PCR). The mock-spiked samples were assayed for protein integrity. RESULTS: Greater than 4 log10 of all test viruses were inactivated, regardless of the type of nucleic acid or presence of an envelope. Unlike previous studies, viruses with the smallest genomes were found to be those most sensitive to UVC irradiation, and detection of PCR amplicons > or = 2.0 kb was correlated to viral infectivity. Doses that achieved significant virus inactivation yielded recovery of > 90% protein activity, even in the absence of quenchers. CONCLUSIONS: The results demonstrate the effectiveness of UVC treatment, in the novel reactor, to inactivate viruses without causing significant protein damage, and confirm the utility of large PCR amplicons as markers for infectious virus.

Observation of optical Smith-Purcell radiation at an electron beam energy of 855 MeV.

Smith-Purcell radiation, generated when a beam of charged particles passes close to the surface of a diffraction grating, has been studied in the visible spectral range at wavelengths of 360 and 546 nm with the low emittance 855 MeV electron beam of the Mainz Microtron MAMI. The beam focused to a spot size of 4 microm (full width at half maximum) passed over optical diffraction gratings of echelle profiles with blaze angles of 0.8 degrees, 17.27 degrees, and 41.12 degrees and grating periods of 0.833 and 9.09 microm. Taking advantage of the specific emission characteristics of Smith-Purcell radiation a clear separation from background components, such as diffracted synchrotron radiation from upstream beam optical elements and transition radiation, was possible. The intensity scales with a modified Bessel function of the first kind as a function of the distance between electron beam and grating surface. Experimental radiation factors have been determined and compared with calculations on the basis of Van den Berg's theory [P.M. Van den Berg, J. Opt. Soc. Am. 63, 689 (1973)]. Fair agreement has been found for gratings with large blaze angles while the measurement with the shallow grating (blaze angle 0.8 degrees ) is at variance with this theory. Finally, the optimal operational parameters of a Smith-Purcell radiation source in view of already existing powerful undulator sources are discussed.

Cost-effectiveness of peritoneal dialysis catheter implantation by laparoscopy versus by open dissection.

This study evaluates the cost-effectiveness of laparoscopic implantation of peritoneal dialysis (PD) catheters as compared with insertion by open dissection. The cost analysis was based on clinical experience with 232 consecutive implants and 23 procedures to rescue catheters from flow dysfunction. Institutional expenses were calculated from the costs of labor and of disposable and reusable materials. Payer costs were taken from Medicare reimbursement schedules for outpatient, inpatient, professional, and ancillary services. A break-even percentage was calculated, representing the point at which the laparoscopic procedure became cost-effective because of a lower incidence of costly catheter rescue procedures. An observed difference in the incidence of catheter obstruction between laparoscopic and open procedures exceeding this percentage would indicate that the laparoscopic approach was cost effective. The calculated break-even value varied between 1.5% and 26% depending on whether the procedures were performed exclusively on an inpatient or outpatient basis. Given our inpatient/outpatient case mix, a weighted calculation of the break-even value was 9.4%. The observed difference in incidence between the two implant methods was 10.8% overall and 16.4% for the last 91 consecutive laparoscopic procedures. The analysis demonstrates that our laparoscopic implantation procedure is a cost-effective means of establishing PD access as compared with the open dissection technique.

The amnesiac gene product is expressed in two neurons in the Drosophila brain that are critical for memory.

Mutations in the amnesiac gene in Drosophila affect both memory retention and ethanol sensitivity. The predicted amnesiac gene product, AMN, is an apparent preproneuropeptide, and previous studies suggest that it stimulates cAMP synthesis. Here we show that, unlike other learning-related Drosophila proteins, AMN is not preferentially expressed in mushroom bodies. Instead, it is strongly expressed in two large neurons that project over all the lobes of the mushroom bodies, a finding that suggests a modulatory role for AMN in memory formation. Genetically engineered blockade of vesicle recycling in these cells abbreviates memory as in the amnesiac mutant. Moreover, restoration of amn gene expression to these cells reestablishes normal olfactory memory in an amn deletion background. These results indicate that AMN neuropeptide release onto the mushroom bodies is critical for normal olfactory memory.

Increased postwar symptoms and psychological morbidity among U.S. Navy Gulf War veterans.

To investigate reports on war-related morbidity, 527 active-duty Gulf War veterans and 970 nondeployed veterans from 14 Seabee commands were studied in 1994 with a questionnaire, sera collection, handgrip strength, and pulmonary function testing. The questionnaire assessed postwar symptoms, war exposures, and screened for chronic fatigue syndrome, post-traumatic stress disorder, and psychological symptoms suggesting neurosis (Hopkins Symptom Checklist). Sera were tested with four nonspecific reactant assays: C-reactive protein, transferrin, ferritin, and haptoglobin. Gulf War veterans reported a higher prevalence for 35 of 41 symptoms, scored higher on psychological symptom scales, were more likely to screen for post-traumatic stress disorder, had lower handgrip strength, and had higher serum ferritin assay results. Numerous comparisons of these morbidity outcomes with 30 self-reported exposures demonstrated many associations, but no unique exposure or group of exposures were implicated. Morbidity data are consistent with other postwar observations, but the etiology for morbidity findings remains uncertain.

Detection of Pneumocystis carinii DNA in blood specimens from human immunodeficiency virus-infected patients by nested PCR.

The detection of Pneumocystis carinii DNA in blood by PCR could be useful for studying the natural history of pneumocystosis and could also be a noninvasive diagnostic method. The results of previous studies are nevertheless conflicting. In our study, we compared three commercially available DNA extraction kits (GeneReleaser, QIAamp Tissue Kit, and ReadyAmp Genomic DNA Purification System) and proteinase K and proteinase K-phenol-chloroform treatments for the extraction of P. carinii DNA from dilutions of a P. carinii f. sp. hominis cyst suspension mixed with human whole blood. A rapid and simple nested PCR protocol which amplifies a portion of the mitochondrial large-subunit rRNA gene was applied to all the extraction products. The QIAmp Tissue Kit was the most effective kit for the isolation of amplification-ready P. carinii DNA and was used with nested PCR for the testing of whole-blood specimens from 35 immunocompetent control patients and 84 human immunodeficiency virus (HIV)-infected patients investigated for pulmonary disease and/or fever. In HIV-infected patients, P. carinii DNA was detected by nested PCR in blood samples from 3 of 14 patients with microscopically proven P. carinii pneumonia, 7 of 22 patients who were considered to be colonized with P. carinii, and 9 of 48 patients who were neither infected nor colonized with P. carinii. P. carinii DNA was not detected in blood specimens from the 35 immunocompetent patients. P. carinii DNA in blood might represent viable P. carinii organisms or DNA complexes released from pulmonary phagocytes. In conclusion, P. carinii DNA may be detected in whole blood from HIV-infected patients, but the nature and the meaning of the circulating form of P. carinii remain to be established.

Defective learning in mutants of the Drosophila gene for a regulatory subunit of cAMP-dependent protein kinase.

Disruptions of a Drosophila gene encoding a regulatory subunit of cAMP-dependent protein kinase homologous to mammalian RIbeta (dPKA-RI) were targeted to the first (noncoding) exon of dPKA-RI via site-selected P element mutagenesis. Flies homozygous for either of two mutant alleles showed specific defects in olfactory learning but not in subsequent memory decay. In contrast, olfactory acuity and shock reactivity, component behaviors required for normal odor avoidance learning, were normal in these mutants. Northern and Western blot analyses of mRNA and protein extracted from adult heads have revealed a complex lesion of the PKA-RI locus, including expression of a novel product and over- or underexpression of wild-type products in mutants. Western blot analysis revealed reductions in RI protein in mutants. PKA activity in the absence of exogenous cAMP also was significantly higher than normal in homogenates from mutant adult heads. These two mutant alleles failed to complement each other for each of these phenotypic defects, eliminating second-site mutations as a possible explanation. These results establish a role for an RI regulatory subunit of PKA in Pavlovian olfactory conditioning.

Cell-type specific calcium signalling in a Drosophila epithelium.

Calcium is a ubiquitous second messenger that plays a critical role in both excitable and non-excitable cells. Calcium mobilisation in identified cell types within an intact renal epithelium, the Drosophila melanogaster Malpighian tubule, was studied by GAL4-directed expression of an aequorin transgene. CAP2b, a cardioactive neuropeptide that stimulates fluid secretion by a mechanism involving nitric oxide, causes a rapid, dose-dependent rise in cytosolic calcium in only a single, genetically-defined, set of 77 principal cells in the main (secretory) segment of the tubule. In the absence of external calcium, the CAP2b-induced calcium response is abolished. In Ca2+-free medium, the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin, elevates [Ca2+]i only in the smaller stellate cells, suggesting that principal cells do not contain a thapsigargin-sensitive intracellular pool. Assays for epithelial function confirm that calcium entry is essential for CAP2b to induce a physiological response in the whole organ. Furthermore, the data suggest a role for calcium signalling in the modulation of the nitric oxide signalling pathway in this epithelium. The GAL4-targeting system allows general application to studies of cell-signalling and pharmacology that does not rely on invasive or cytotoxic techniques.

Biotin-pyrene conjugates with poly(ethylene glycol) spacers are convenient fluorescent probes for avidin and streptavidin.

Conventional biotin-fluorophore conjugates with approximately 14 atom spacers are strongly quenched when bound to avidin or streptavidin, whereas fluorescence becomes insensitive to receptor binding if typical fluorophores are linked to biotin via poly(ethylene glycol) (PEG) chains (Gruber et al., see the second of three papers in this issue). In the present study the antagonism between PEG-PEG repulsion and fluorophore interaction was examined more closely, using biotin-PEG-pyrene conjugates as model compounds. The antagonistic tendencies between hydrophilic PEG chains and hydrophobic pyrene labels were about balanced in the PEG1900 derivative since quenching was approximately 50% in 4:1 complexes with avidin or streptavidin. In contrast, strong quenching and concomitant excimer fluorescence was seen with the biotin-PEG800-pyrene conjugate, providing for a new fluorescence assay to accurately measure avidin and streptavidin concentrations at > or = 40 and > or = 10 nM, respectively. Association/ dissociation kinetics were analyzed from pyrene fluorescence changes, and dissociation constants were deduced. About 3-fold affinities were observed for streptavidin as compared to avidin, and little influence of PEG chain length was seen. All affinities were increased by a factor of approximately 3 when biotin-PEG-tetramethylrhodamine conjugates were used. The observed effect of fluorophore variation upon biotin binding is unexpectedly small; thus, the kinetic/thermodynamic data appear to be representative for biotin-PEG conjugates in general.

Biotin-fluorophore conjugates with poly(ethylene glycol) spacers retain intense fluorescence after binding to avidin and streptavidin.

Conventional biotin-fluorophore conjugates with approximately 14 atom spacers lose most of their fluorescence when binding to avidin or streptavidin, as is demonstrated in the present study. This explains the unusual fact that only biotinylated marker enzymes, but not fluorescent biotins, are regularly used in bioanalytic assays. Novel biotin-spacer-fluorophore conjugates are presented that retain intense fluorescence when binding to avidin or streptavidin. Preservation of fluorescence depends upon the use of poly(ethylene glycol) (PEG) spacers, which are shown not to interfere with biotin function. The observed absence of nonspecific interactions may also be attributed to the PEG chain. These novel fluorescent biotins are expected to be excellent new tools in fluorescence microscopy and related techniques.

Functional domains are specified to single-cell resolution in a Drosophila epithelium.

Specification of pattern is fundamental to the development of a multicellular organism. The Malpighian (renal) tubule of Drosophila melanogaster is a simple epithelium that proliferates under the direction of a single tip cell into three morphologically distinct domains. However, systematic analysis of a panel of over 700 P[GAL4] enhancer trap lines reveals unexpected richness for such an apparently simple tissue. Using numerical analysis, it was possible formally to reconcile apparently similar or complementary expression domains and thus to define at least five genetically defined domains and multiple cell types. Remarkably, the positions of domain boundaries and the numbers of both principal and secondary ("stellate") cell types within each domain are reproducible to near single-cell precision between individual animals. Domains of physiological function were also mapped using transport or expression assays. Invariably, they respect the boundaries defined by enhancer activity. These genetic domains can also be visualized in vivo, both in transgenic and wild-type flies, providing an "identified cell" system for epithelial physiology. Building upon recent advances in Drosophila Malpighian tubule physiology, the present study confirms this tissue as a singular model for integrative physiology.

Ectopic expression of sex-peptide in a variety of tissues in Drosophila females using the P[GAL4] enhancer-trap system.

Sex-peptide (SP), which is secreted by the accessory gland of Drosophila males, is transferred to the female during copulation, thereby reducing her sexual appetite (receptivity to males) and stimulating ovulation/oviposition. SP is known to be taken up into the hemolymph of mated females, but it is not clear whether there are two separate target tissues, for behavioral changes and ovulation or only one target for both responses. We have employed the GAL4-UAS system to express SP transgene constructs, both in different tissues and in different cellular components of virgin females. A cytoplasmic form of SP lacking a signal sequence did not evoke any responses, even when expressed ubiquitously. In contrast, a membrane-bound form of SP induced typical post-mating behavior, indicating that SP must be outside the cell in order to exert its biological effects. A total of 204 randomly selected P[GAL4] enhancer-trap lines were screened for their ability to induce SP responses in combination with the membrane-bound SP expressed under GAL4 control. Thirty-three lines were associated with both behavioral change and stimulated ovulation. No line was associated with only one of the two responses, implying that the SP target(s) mediating the two responses are either identical, very closely located, or present in two distinct tissues with a common set of genetic determinants. Western blot analysis of head, thorax, and abdominal extracts revealed that the biological activity was correlated with expression in the head fraction.

Molecular genetic analysis of V-ATPase function in Drosophila melanogaster.

V-ATPases are phylogenetically widespread, highly conserved, multisubunit proton pumps. Originally characterised in endomembranes, they have been found to energise transport across plasma membranes in a range of animal cells and particularly in certain epithelia. While yeast is the model of choice for the rapid generation and identification of V-ATPase mutants, it does not allow their analysis in a plasma membrane context. For such purposes, Drosophila melanogaster is a uniquely suitable model. Accordingly, we have cloned and characterised genes encoding several V-ATPase subunits in D. melanogaster and, using P-element technology, we have succeeded in generating multiple new alleles. Reporter gene constructs reveal ubiquitous expression, but at particularly high levels in those epithelial thought to be energised by V-ATPases, and several of the alleles have lethal recessive phenotypes characterised by epithelial dysfunction. These results, while providing the first gene knockouts of V-ATPases in animals, also illustrate the general utility of D. melanogaster as a model for the genetic analysis of ion transport and its control in epithelia.

[Value of color-coded Doppler sonography in the differential diagnosis of nodular thyroid gland changes]. / Stellenwert der farbcodierten Doppler-Sonographie in der Differentialdiagnostik nodulärer Schilddrüsenveränderungen.

AIM: The incidence of functional autonomous adenomas, detected in every second nodular goiter by scintigraphic methods is very high in an area of iodine deficiency. The color-coded Doppler sonography (CCDS) as a diagnostic tool in differentiating thyroid nodules is discussed controversially. METHODS: In this prospective study we investigated the value of CCDS in 200 patients with nodular thyroid alterations compared with 99m-Technetium (Tc) scintigraphy. RESULTS: Focal maximas of Tc-uptake were detected in 22.5% of all patients, and 44.5% of the thyroid nodules showed increased vascularity. There was no correlation between nodular vascularity and thyroid 99m-Tc uptake (TcTU). In contrast to this we could demonstrate a significant relation between vascularity and the diameter of the nodule (p < 0.0001). The results are discussed in the context of method specific limitations of ultrasound examinations. CONCLUSION: Our results confirm that CCDS has no great importance in the differentiation of thyroid nodules. Scintigraphy remains the diagnostic method of choice to assess the topographic thyroid function.

Associative learning disrupted by impaired Gs signaling in Drosophila mushroom bodies.

Disruptions in mushroom body (MB) or central complex (CC) brain structures impair Drosophila associative olfactory learning. Perturbations in adenosine 3',5' monophosphate signaling also disrupt learning. To integrate these observations, expression of a constitutively activated stimulatory heterotrimeric guanosine triphosphate-binding protein alpha subunit (Galphas*) was targeted to these brain structures. The ability to associate odors with electroshock was abolished when Galphas* was targeted to MB, but not CC, structures, whereas sensorimotor responses to these stimuli remained normal. Expression of Galphas* did not affect gross MB morphology, and wild-type Galphas expression did not affect learning. Thus, olfactory learning depends on regulated Gs signaling in Drosophila MBs.

Analysis and inactivation of vha55, the gene encoding the vacuolar ATPase B-subunit in Drosophila melanogaster reveals a larval lethal phenotype.

Vacuolar ATPases play major roles in endomembrane and plasma membrane proton transport in eukaryotes. A Drosophila melanogaster cDNA encoding vha55, the 55-kDa vacuolar ATPase (V-ATPase) regulatory B-subunit, was characterized and mapped to 87C2-4 on chromosome 3R. A fly line was identified that carried a single lethal P-element insertion within the coding portion of gene, and its LacZ reporter gene revealed elevated expression in Malpighian tubules, rectum, antennal palps, and oviduct, regions where V-ATPases are believed to play a plasma membrane, rather than an endomembrane, role. The P-element vha55 insertion was shown to be allelic to a known lethal complementation group l(3)SzA (= l(3)87Ca) at 87C, for which many alleles have been described previously. Deletions of the locus have been shown to be larval lethal, whereas point mutations show a range of phenotypes from subvital to embryonic lethal, implying that severe alleles confer a partial dominant negative phenotype. The P-element null allele of vha55 was shown also to suppress ectopic sex combs in Polycomb males, suggesting that transcriptional silencing may be modulated by genes other than those with known homeotic or DNA binding functions.