Dynamics of the N-terminal alpha-helix unfolding in the photoreversion reaction of phytochrome A

Time-resolved circular dichroism spectroscopy in the far-UV spectral region was used to examine the intermediates of the phytochrome photoreversion reaction (Pfr --> Pr). Three intermediates, lumi-F (tau = 320 ns), meta-Fa (tau = 265 micros) and meta-Fb (tau = 5.5 ms), have been identified in a simple sequential kinetic photoreversion mechanism by absorption spectroscopy [Linschitz, H., Kasche, V., Butler, W. L., & Siegelman, H. W. (1966) J. Biol. Chem. 241, 3395-3403; Pratt, L. H., & Butler, W. L. (1968) Photochem. Photobiol. 8, 477-485; Burke, M., Pratt, D. C., & Moscowitz, A. (1972) Biochemistry 11, 4025-4031; Spruit, C. J. P., Kendrick, R. E., & Cooke, R. J. (1975) Planta (Berlin) 127, 121-132; Eilfeld, P., & Rüdiger, W. (1985) Z. Naturforsch. 40c, 109-114; Chen, E., Lapko, V. N., Lewis, J. W., Song, P.-S., & Kliger, D. S. (1996) Biochemistry 35, 843-850]. In order to correlate the unfolding of the N-terminal alpha-helical segment with one or more of the intermediate species, time-resolved methods were coupled with the structurally sensitive probe of CD in the far-UV spectral region. Analysis of the TRCD data associates the decrease in alpha-helical content that occurs upon formation of Pr with decay of the meta-Fa intermediate. This unfolding process occurs with a time constant of 310 +/- 125 micros, which is consistent with the 265-micros lifetime for meta-Fa.     

Enantioselective analysis of praziquantel in plasma samples

In prokaryotes, DnaK-DnaJ chaperon is involved in the protein degradation catalyzed by proteases La and ClpA/B complex as shown in E. coli. To extend this into eukaryotic cells, we examined the effects of hsp70 genes, SSA1 and SSB1, and DnaJ genes, SIS1 and YDJ1, on the growth of proteasome subunit mutants of the yeast S. cerevisiae. The results identified SSB1 and SIS1 as a pair of chaperon genes specifically involved in efficient protein turnover in the yeast, whose overexpression suppressed the growth defects caused by the proteasome mutations. Moreover, a single amino acid substitution in the putative peptide-binding site of SSB1 protein profoundly enhanced the suppression activity, indicating that the activity is mediated by the peptide-binding activity of this chaperon. Thus SSB1, with its partner DnaJ, SIS1, modulates the efficiency of protein turnover through its chaperon activity.     

Seismic response and fragility analysis of a water storage structure

Stress analysis of a water storage structure has been carried out for static and seismic loading. Based on the stress analyses results, assessment of most likely failure modes for the structure caused by seismic event has been carried out. An attempt has been made to quantify the initial leakage rate and average emptying time for the structure during seismic event after evaluating the various crack parameters, viz., crack-width and crack-spacing at the locations of interest. Finally, the seismic fragility of the structure is developed as families of conditional probability curves plotted against peak ground acceleration (PGA) parameter at the location of interest considering the randomness and uncertainty associated with various parameters that could affect the seismic structural response.     

Riboflavin-mediated delivery of a macromolecule into cultured human cells

The effects of perivascular nerve stimulation and phenylephrine on osmolyte release were studied in the intact perfused rat liver and isolated liver parenchymal cells (PC) and nonparenchymal cells. In the perfused liver, electrical stimulation of perivascular nerves (20 Hz/2 ms/20 V) led to a phentolamine-sensitive increase of cell hydration by 6.5% +/- 1.2% (n = 3) and a transient phentolamine-sensitive stimulation of taurine and inositol, but not betaine, release. These nerve effects were mimicked by phenylephrine, but not prostaglandin F2alpha, and were not affected by sodium nitroprusside (SNP) or ibuprofen. Nerve stimulation-induced taurine, but not inositol, release was inhibited by 4, 4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS) (50 micromol/L). Single-cell fluorescence studies with isolated liver PC, Kupffer cells (KC), sinusoidal endothelial cells (SEC), and hepatic stellate cells (HSC) revealed that phenylephrine induced an increase in cytosolic free Ca2+ only in PC and HSC, but not in KC and SEC, whereas extracellular uridine triphosphate (UTP) produced Ca2+ transients/oscillations in all liver cell types studied. Phenylephrine had no effect on osmolyte release from isolated KC and SEC, but increased taurine (but not inositol) release from PC and inositol (but not taurine) efflux from HSC. The data suggest that: 1) liver cell hydration and-consecutively-osmolyte content are modulated by hepatic nerves via an alpha-adrenergic mechanism, which does not involve eicosanoids or hemodynamic changes; 2) that PC and HSC are the primary targets for nerve-dependent alpha-adrenergic activation, whereas 3) KC and SEC probably do not express alpha-adrenoceptors coupled to Ca2+ mobilization or osmolyte efflux.     

Evaluation of biochemical changes during in vivo erythrocyte senescence in the dog

One hypothesis to explain the age-dependent clearance of red blood cells (RBCs) from circulation proposes that denatured/oxidized hemoglobin (hemichromes) arising late during an RBC's life span induces clustering of the integral membrane protein, band 3. In turn, band 3 clustering generates an epitope on the senescent cell surface leading to autologous IgG binding and consequent phagocytosis. Because dog RBCs have survival characteristics that closely resemble those of human RBCs (ie, low random RBC loss, approximately 115-day life span), we decided to test several aspects of the above hypothesis in the canine model, where in vivo aged cells of defined age could be evaluated for biochemical changes. For this purpose, dog RBCs were biotinylated in vivo and retrieved for biochemical analysis at various later dates using avidin-coated magnetic beads. Consistent with the above hypothesis, senescent dog RBCs were found to contain measurably elevated membrane-bound (denatured) globin and a sevenfold enhancement of surface-associated autologous IgG. Interestingly, dog RBCs that were allowed to senesce for 115 days in vivo also suffered from compromised intracellular reducing power, containing only 30% of the reduced glutathione found in unfractionated cells. Although the small quantity of cells of age >/=110 days did not allow direct quantitation of band 3 clustering, it was nevertheless possible to exploit single-cell microdeformation methods to evaluate the fraction of band 3 molecules that had lost their normal skeletal linkages and were free to cluster in response to hemichrome binding. Importantly, band 3 in RBCs >/=112 days old was found to be 25% less restrained by skeletal interactions than band 3 in control cells, indicating that the normal linkages between band 3 and the membrane skeleton had been substantially disrupted. Interestingly, the protein 4.1a/protein 4.1b ratio, commonly assumed to reflect RBC age, was found to be maximal in RBCs isolated only 58 days after labeling, implying that while this marker is useful for identifying very young populations of RBCs, it is not a very sensitive marker for canine senescent RBCs. Taken together, these data argue that several of the readily testable elements of the above hypothesis implicating band 3 in human RBC senescence can be validated in an appropriate canine model.     

Differential reactivity of the rat S100A4(p9Ka) gene to sodium bisulfite is associated with differential levels of the S100A4 (p9Ka) mRNA in rat mammary epithelial cells

Elevated intracellular levels of S100A4, an S100-related calcium-binding protein, induce metastatic capability in benign mammary tumor-derived epithelial cells and in transgenic mice bearing oncogene-induced benign mammary tumors. The S100A4(p9Ka) gene in rat mammary epithelial cells expressing low levels of S100A4 yields a reduced number of fragments upon digestion with the methylation-sensitive restriction enzyme, HpaII, compared with the gene from high S100A4-expressing cells. Genomic sequencing of two potential regulatory elements in the S100A4 gene, an intronic enhancer and TATA box region, revealed that in low S100A4-expressing cells, most cytosine bases exhibited high levels of resistance to conversion to thymine by sodium bisulfite. In derivative cell lines, which express high levels of S100A4, only a small number of cytosine bases were resistant to treatment with sodium bisulfite. In contrast, cytosine bases in the DNA surrounding an upstream regulatory region, which binds inhibitory GC factor in the low-expressing cell lines, are sensitive to conversion to thymine by sodium bisulfite in both low- and high-expressing cell lines. The results suggest that the rat S100A4 gene is maintained in a different state in the low-expressing cell lines and that this state might be a consequence of the pattern of methylation in this regulated gene that does not contain a CpG island.     

Intestinal tuberculosis

Since the symptoms and clinical presentation of intestinal tuberculosis is nonspecific, the diagnosis is frequently delayed and may be achieved at autopsy only. Intestinal tuberculosis is very rare in Denmark, but may now be seen more often because of increasing numbers of immigrants from countries of the third world with a high prevalence of tuberculosis. A case of intestinal tuberculosis in a 28 year old Somalian female is reported. Methods of diagnosing intestinal tuberculosis are commented, and the frequent necessity of starting medical treatment before a bacteriological diagnosis is reached is emphasized.     

Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium

Estradiol-17beta (E2) acts through the estrogen receptor (ER) to regulate uterine growth and functional differentiation. To determine whether E2 elicits epithelial mitogenesis through epithelial ER versus indirectly via ER-positive stromal cells, uteri from adult ER-deficient ER knockout (ko) mice and neonatal ER-positive wild-type (wt) BALB/c mice were used to produce the following tissue recombinants containing ER in epithelium (E) and/or stroma (S), or lacking ER altogether: wt-S + wt-E, wt-S + ko-E, ko-S + ko-E, and ko-S + wt-E. Tissue recombinants were grown for 4 weeks as subrenal capsule grafts in intact female nude mice, then the hosts were treated with either E2 or oil a week after ovariectomy. Epithelial labeling index and ER expression were determined by [3H]thymidine autoradiography and immunohistochemistry, respectively. In tissue recombinants containing wt-S (wt-S + wt-E, wt-S + ko-E), E2 induced a similar large increase in epithelial labeling index compared with oil-treated controls in both types of tissue recombinants despite the absence of epithelial ER in wt-S + ko-E tissue recombinants. This proliferative effect was blocked by an ER antagonist, indicating it was mediated through ER. In contrast, in tissue recombinants prepared with ko-S (ko-S + ko-E and ko-S + wt-E), epithelial labeling index was low and not stimulated by E2 despite epithelial ER expression in ko-S + wt-E grafts. In conclusion, these data demonstrate that epithelial ER is neither necessary nor sufficient for E2-induced uterine epithelial proliferation. Instead, E2 induction of epithelial proliferation appears to be a paracrine event mediated by ER-positive stroma. These data in the uterus and similar studies in the prostate suggest that epithelial mitogenesis in both estrogen and androgen target organs are stromally mediated events.     

Contemporary percutaneous treatment of unprotected left main coronary stenoses: initial results from a multicenter registry analysis 1994-1996

BACKGROUND: Coronary artery bypass surgery (CABG) has been considered the therapy of choice for patients with unprotected left main (ULMT) coronary stenoses. Selected single-center reports suggest that the results of percutaneous intervention may now approach those of CABG. METHODS AND RESULTS: To assess the results of percutaneous ULMT treatment from a wide variety of experienced interventional centers, we requested data on consecutive patients treated after January 1, 1994, from 25 centers. One hundred seven patients were identified who were treated either electively (n=91) or for acute myocardial infarction (n=16). Of patients treated electively, 25% were considered inoperable, and 27% were considered high risk for bypass surgery. Primary treatment included stents (50%), directional atherectomy (24%), and balloon angioplasty (20%). Follow-up was 98.8% complete at 15+/-8 months. Results varied considerably, depending on presentation and treatment. For patients with acute myocardial infarction, technical success was achieved in 75%, and survival to hospital discharge was 31%. For elective patients, technical success was achieved in 98.9%, and in-hospital survival was strongly correlated with left ventricular ejection fraction (P=.003). Longer-term event (death, infarction, or bypass surgery) -free survival was correlated with ejection fraction (P<.001) and was inversely related to presentation with progressive or rest angina (P<.001). Surgical candidates with ejection fractions > or = 40% had an in-hospital survival of 98% and a 9-month event-free survival of 86+/-5%, whereas patients with ejection fractions < 40% had 67% and 22+/-12% in-hospital and 9-month event-free survivals, respectively. Nine hospital survivors (10.6%) experienced cardiac death within 6 months of hospital discharge. CONCLUSIONS: While results for selected patients appear promising, until early post-hospital discharge cardiac death can be better understood and minimized, percutaneous revascularization of ULMT stenosis should not be considered an alternative to bypass surgery for most patients. When percutaneous revascularization of ULMT is required, directional atherectomy and stenting appear to be the preferred techniques, and follow-up angiography 6 to 8 weeks after treatment is probably advisable.