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An acoustic sensor system was used to detect three types of PD: point-point, point-plane and surface discharges. A wavelet-based de-noise method was carried out in signal extraction. The result of the extraction was provided with its high signal-to-noise ratio and excellent preservation of the energy and pulse amplitude. A group of frequency-domain indexes, which were generated through a spectral analysis describing the de-noised AE pulses of the three PD types, were defined and compared. The selected PD types were associated with AE pulses of characteristic shapes of frequency spectra and characteristic values of the associated descriptors. Then various types of PD can be identified by comparative analysis of the associated frequency spectra and bandwidths of dominant frequencies. Finally, using mean values and maximum values for AE pulses can be compared for different PD types when the lengths of the analyzed time intervals were same. For a specific PD type, the above values was depend on the time intervals.

In a differential screen for genes that might be important in the regulation of epithelial morphogenesis, we identified a novel gene, Debt91 (differentially expressed in branching tubulogenesis), which is up-regulated in an in vitro model of renal tubulogenesis and branching. Debt91 appears to encode a 381 amino acid molecule with high Ser and Thr composition and is highly conserved at its N-terminus across species. Sequence analysis suggests that it is a coiled-coil nuclear phosphoprotein with zinc finger motifs at the N-terminal conserved region, which is rich in cysteine and histidine. Debt91 is located on mouse chromosome 6 at a region that has conserved synteny with human chromosome 2p11.2, and appears to express two transcripts in several mouse cell lines and adult tissues. On whole murine embryo blots Debt91 expresses primarily its small transcript and is differentially regulated during development. Analysis of expression in in vitro cell culture models suggests that Debt91 is an immediate early gene up-regulated during growth factor-induced branching tubulogenesis.

Mouse pancreatic development is critically dependent on epithelial–mesenchymal interactions. The pancreas differs from other epithelial–mesenchymal organs in that the epithelium gives rise to both epithelial exocrine cells and non-epithelial endocrine cells. We studied the nature of the interactions between the epithelium and mesenchyme with respect to the decision between exocrine and endocrine lineages. We show here a tripartite influence of mesenchyme on the developing epithelium. First, close proximity or contact of mesenchyme with the epithelium induces exocrine differentiation. Second, this mesenchymal proximity to the epithelium suppresses endocrine differentiation. Third, mesenchyme has an overall enhancing effect on the degree of insulin differentiation, suggesting a pro-endocrine effect in those epithelial cells at a distance from the mesenchyme.Proximity or contact between the mesenchyme and epithelium appeared to be necessary for the pro-exocrine effects of mesenchyme. We found that, in a co-culture system, NIH3T3 cells were able to substitute for mesenchyme in exocrine induction as well as in both the endocrine induction and endocrine inhibition, implying that the responsible molecules are not unique to pancreatic mesenchyme. Laminin appears to be a key molecule mediating the epithelial–mesenchymal interactions that lead to exocrine differentiation, since inhibition of laminin expression resulted in blockage of the pro-exocrine induction of mesenchyme.

A screen for genes differentially regulated in a model of kidney development identified the novel gene embryonic epithelia gene 1 (EEG1). EEG1 exists as two transcripts of 2.4 and 3.5 kb that are most highly expressed at embryonic day 7 and later in the fetal liver, lung, placenta, and kidney. The EEG1 gene is composed of 14 exons spanning a 20-kb region at human chromosome 11p12 and the syntenic region of mouse chromosome 2. Six EEG1 exons have previously been assigned to a longer isoform of eosinophil major basic protein termed proteoglycan 2. Another gene distantly related to EEG1, POV1/PB39, is located 88 kb upstream from the EEG1 gene on chromosome 11. Temporal expression of 65 members of the solute carrier (SLC)-class of transport proteins was followed during kidney development using DNA arrays. POV-1 and EEG1, like glucose transporters, displayed very early maximal gene expression. In contrast, other SLC genes, such as organic anion and cation transporters, amino acid permeases, and nucleoside transporters, had maximal expression later in development. Thus, although the bulk of transporters are expressed late in kidney development, a fraction are expressed near the onset of nephrogenesis. The data raise the possibility that EEG1 and POV1 may define a new family of transport proteins involved in the transport of nutrients or metabolites in rapidly growing and/or developing tissues.

Central to the process of epithelial organogenesis is branching morphogenesis into tubules and ducts. In the kidney, this can be modeled by a very simple system consisting of isolated ureteric bud (UB) cells, which undergo branching morphogenesis in response to soluble factors present in the conditioned medium of a metanephric mesenchyme cell line. By employing a targeted screen to identify transcription factors involved early in the morphogenetic program leading to UB branching, we identified the mammalian ortholog of Timeless (mTim) as a potential immediate early gene (IEG) important in this process. In the embryo, mTim was found to be expressed in patterns very suggestive of a role in epithelial organogenesis with high levels of expression in the developing lung, liver, and kidney, as well as neuroepithelium. In the embryonic kidney, the expression of mTim was maximal in regions of active UB branching, and a shift from the large isoform of mTim to a smaller isoform occurred as the kidney developed. Selective down-regulation of mTim resulted in profound inhibition of embryonic kidney growth and UB morphogenesis in organ culture. A direct effect on the branching UB was supported by the observation that down-regulation of mTim in the isolated UB (cultured in the absence of mesenchyme) resulted in marked inhibition of morphogenesis, suggesting a key role for Tim in the epithelial cell morphogenetic pathway leading to the formation of branching tubules.

Introduction: Blood vessels have been recently found to be necessary for initiation of embryonic organ formation and subsequent differentiation. As a heparin-binding extracellular cytokine important in angiogenesis, neural and renal morphogenesis, and carcinogenesis, we hypothesized that pleiotrophin (PTN) plays a role in blood vessel-induced pancreatic organogenesis and differentiation. Methods: Mouse embryonic day (E) 8.25, nascent blood vessel (dorsal aorta), foregut endoderm, or E11.5 pancreas were either harvested fresh or cultured for 6 days in different in vitro systems under antisense conditions, and then subjected to RT-PCR and immunohistochemical analysis for markers including PTN, PDX-1, neurogenin 3, CD31, insulin, glucagon and amylase. Results: Prior to pancreatic evagination, PTN appears to be secreted from nascent dorsal aorta in the vicinity of the foregut endoderm. The budding pancreatic anlagen display PTN precisely in the areas of vasculogenesis adjacent to differentiating epithelium. E8.25 endoderm alone did not express PTN nor insulin in a 6-Day matrigel culture. When dorsal aorta was included, both PTN and insulin were detected by immunostaining. The apparent aortic induction of insulin expression was inhibited by PTN antisense. RT-PCR analysis confirmed that pancreatic specification (PDX-1 and Neurogenin 3 positive) still occurred without aorta, but failed to differentiate further (insulin, glucagon or amylase negative), whereas addition of dorsal aorta induced endocrine differentiation (insulin and glucagon positive). Subsequently, at E11-E13 expression of PTN is restricted to the pancreatic epithelium, especially in its basement membranes. As differentiation progresses, PTN becomes localized only to the undifferentiated “cord” region. By E18, PTN staining mainly overlapped with CD31, and thus was localized only to blood vessels. In 3D culture of E11 pancreas with PTN antisense, amylase and glucagon stainings were nearly undetectable, and insulin staining was very low. Interestingly, the low-level insulin expression became localized to ductal epithelium, suggesting that PTN may be necessary for formation of extra-epithelial islets. Conclusion: Together, these data strongly suggest that PTN plays a role in blood vessel-mediated mesenchymal induction of normal pancreatic specification, organogenesis, and subsequent endocrine and exocrine differentiation.

The effect of myocardial infarction upon the frequency content of the QRS complex was analyzed. Three bipolar signal-averaged surface electrograms, recorded during the early (10-15 days) and late (6 months) chronic phases of myocardial infarction, were analyzed in 61 patients and 11 healthy subjects. All patients were free of ventricular arrhythmia during 6 months of follow-up examinations. Time-frequency analysis of the QRS complex was based on the modified Wigner distribution, which is well suited to examine nonstationary character of data. Standard time-domain analysis for the presence of late potentials was used for comparison. High-frequency (> or = 90 Hz) components, separable from the dominant low-frequency components (< 90 Hz), were found in all groups. They were present throughout the QRS complex and were peaking in its middle portion. The high-frequency components were found significantly higher in postinfarction patients in both early (P < .007) and late chronic stage myocardial infarction (P < .05) compared to healthy subjects. Patients who tested positive for late potentials (24%) also had elevated high-frequency components; however, a comparable increase was also observed in late potential negative patients. Furthermore, the high-frequency component increase occurred in all patients earlier in the QRS than in its terminal 40 ms, where late potentials are traditionally evaluated. It is concluded that high-frequency components are an integral part of the QRS complex under physiologic conditions and persist in variable amount throughout its duration. The high-frequency components are increased in patients after myocardial infarction not associated with ventricular arrhythmia, and their elevation is not limited to the terminal QRS complex.

Introduction: TGF-beta isoforms are a multifunctional set of protein that regulates cell proliferation, differentiation and extracellular matrix synthesis. A few studies have suggested a role for TGF-beta in pancreatic organogenesis. We studied TGF beta signaling in the developing pancreas using ontogenic and inhibitory studies. Methods: Dorsal embryonic pancreatic rudiments were either harvested fresh for immunohistochemistry at serial ages, or placed in organ culture at E (embryonic day) 11.5 with TGF-beta specific pan-neutralizing antibody. After 6 days, tissues were processed for histological and immunohistochemical analysis of exocrine and endocrine markers. Results: The endogenous TGF-beta type II receptor (TBRII) was localized mainly to the epithelium at day 11, but with increasing age localized to the pancreatic islets and ducts. Blocking TGF-beta by pan-neutralizing antibody led to an increased differentiation of insulin-positive cells. Quantitative analysis showed nearly a 250% increase in insulin-positive cells. These insulin cells appeared to be mature, with co-expression of the beta cell marker, PDX-1. Amylase positive cells decreased by a small margin. Conclusions: Absence of TGF-beta signaling in embryonic period may direct pancreatic epithelial cells towards endocrine lineage, leading to growth and differentiation of endocrine cells. TBRII in the ducts and islets may normally serve to downregulate the production of beta cells.

We used resonance energy transfer and site-directed photo-cross-linking to probe the Ca(2+)-dependent proximity relationships between residue 117 next to the C-terminus of the inhibitory region in rabbit skeletal troponin-I (TnI) and residues in troponin-C (TnC) and in actin. A mutant TnI that contains a single cysteine at position 117 (I117) was constructed, and the distance between TnI residue 117 and TnC residue 98 was measured with the following results: for both the binary TnC-TnI complex and the ternary troponin complex, this distance was 30 and 41 A in the presence and absence of Ca(2+), respectively. The distance between TnI residue 117 and Cys374 of actin was 48 and 41 A in the presence and absence of Ca(2+), respectively. Six additional distances from this TnI residue to cysteines in TnC mutants were measured and used to localize this residue with respect to the crystal structure of TnC. The results show that in the presence of Ca(2+) it is localized near the B and C helices of TnC's N-terminal domain. In the absence of Ca(2+) this residue moves away from this location by approximately 8 A. Photo-cross-linking experiments show that I117 labeled with 4-maleimidobenzophenone photo-cross-linked to TnC but not to actin in both the presence and absence of Ca(2+). Taken together these results provide independent experimental support for the proposal (Y. Luo, J. L. Wu, B. Li, K. Langsetmo, J. Gergely, and T. Tao, 2000, J. Mol. Biol. 296:899-910) that upon Ca(2+) removal the region comprising TnI residues 114-125 triggers the movements of residues 89-113 and 130-150 toward actin, but does not itself interact with actin.