A recurrent glycine substitution mutation, G2043R, in the type VII collagen gene (COL7A1) in dominant dystrophic epidermolysis bullosa

OBJECTIVE: To evaluate the effect of hemofilter pore size on the efficacy of continuous arteriovenous hemofiltration (CAVH) in improving morbidity and mortality in an immature swine model of Staphylococcus aureus-induced septicemia. DESIGN: Prospective, randomized study with age-matched controls. SETTING: Biomedical research facility. SUBJECTS: Fourteen 4 to 8-wk-old, weaned Poland-China swine, weighing 5 to 10 kg. INTERVENTIONS: Spontaneously breathing, ketamine-sedated swine (4 to 8 wks of age) were given an intravenous lethal dose of live S. aureus. Animals were then filtered with either a 50-kilodalton (kD) pore size filter (control) or a 100-kD pore size filter (experimental). No animals received antibiotics. MEASUREMENTS AND MAIN RESULTS: Physiologic, biochemical, and hematologic parameters were measured in all animals every 1 to 3 hrs. Animals were monitored continuously and survival time (hr) was recorded (permanent survival = 168 hrs/7 days). Animals filtered with the 100-kD filter survived significantly longer than control animals (103 +/- 18 [SEM] vs. 56 +/- 9 hrs). The 100-kD-filtered group had one permanent survivor (168 hrs). Protein concentration of the ultrafiltrate obtained from the 100-kD-filtered animals was eight-fold higher than control ultrafiltrate. The protein removed did not contain a high percentage of albumin (as determined by autoanalyzer methods). No significant differences were seen in any of the other measured parameters. CONCLUSIONS: CAVH significantly improved survival in swine with S. aureus-induced sepsis. The superior performance of the 100-kD filter vs. the 50-kD filter suggests that higher molecular weight mediators that are not removed efficiently by the 50-kD filter may be responsible for the morbidity and mortality seen in this model of sepsis. These mediators may be removed in greater proportion by our customized (100-kD pore size) filter.     

Plasma angiotensin(1-7) immunoreactivity is increased by salt load, water deprivation, and hemorrhage

In this study we investigated the effects of dehydration and hemorrhage on circulating levels of the heptapeptide, angiotensin(1-7). In water-deprived rats, a twofold increase in plasma angiotensin(1-7) was associated with similar increases in plasma renin activity, and angiotensin I and angiotensin II levels. In salt-loaded rats, plasma angiotensin(1-7) levels increased fourfold; however, other components of the renin-angiotensin system were suppressed or unchanged. In salt-loaded rats, increases in plasma angiotensin II levels in response to hemorrhage in normal rats were severely blunted, whereas angiotensin(1-7) plasma levels increased proportionately to the loss of blood volume. These results suggest that angiotensin(1-7) plasma concentration can be selectively regulated during dehydration and hemorrhage.     

Chiral separation of amines by high-performance liquid chromatography after tagging with 4-(N,N-dimethylaminosulphonyl)-7-(2-chloroformylpyrrolidin-1-yl)- 2,1,3-benzoxadiazole

Chiral tagging reagents, 4-(N,N-dimethylaminosulphonyl)-7-(2-chloroformylpyrrolidin-1 -yl)-2,1,3- benzoxadiazole (R(+)-DBD-Pro-COCl and S(-)-DBD-Pro-COCl), react with mirror image enantiomers of amines to produce corresponding diastereomers in the presence of pyridine as a catalyst. The maximal excitation and emission wavelengths of the resulting diastereomers were ca. 450 nm and 560 nm, respectively. The diastereomers derived from some aliphatic amines were resolved by a reversed-phase chromatography with water-acetonitrile or normal-phase chromatography with n-hexane-ethyl acetate as the eluent. The reactivities of both enantiomers of DBD-Pro-COCl to chiral amines were almost comparable, whereas a slight difference of fluorescence intensity was observed with S(-)-DBD-Pro-COCl. When (S-)-DBD-Pro-COCl was used as the derivatization reagent, amines corresponding to S-configuration were eluted faster than R-configuration. The opposite elution order was obtained with the use of R(+)-DBD-Pro-COCl, instead of S(-)-DBD-Pro-COCl. The Rs values obtained from 1-cyclohexylethylamine (CEA) having aliphatic ring structure was larger than those of amines (1-(1-naphthyl)ethylamine (NEA) and 1-phenylethylamine (PEA)) having aromatic ring structures.     

Interferon-gamma and interleukin-10 inhibit antigen presentation by Langerhans cells for T helper type 1 cells by suppressing their CD80 (B7-1) expression

CD80(B7-1) and CD86(B7-2) co-stimulatory molecules have been reported to activate Th1/Th2 development pathways differentially. It is well known that Langerhans cells (LC), potent antigen-presenting dendritic cells in the epidermis, express several co-stimulatory molecules and that this expression is modulated by several cytokines. Based on the recently reported effect of interferon (IFN)-gamma and interleukin (IL-)-10 on the expression of CD80 and CD86 by LC, we examined the effects of these cytokines on the expression of CD54 (intercellular adhesion molecule-1) and CD40 in addition to CD80 and CD86 on LC, and correlated the expression of each co-stimulatory molecule with antigen presentation for a Th1 clone by cultured LC (cLC) treated with these cytokines. LC cultured for 72 h significantly up-regulated MHC class II antigen expression and all the co-stimulatory molecules were examined. As previously reported, IL-10 or IFN-gamma inhibited the up-regulation of CD80 expression. Granulocyte/macrophage-colony-stimulating factor (GM-CSF) partially restored the suppression of CD80 expression induced by IFN-gamma on cultured LC, while it had virtually no effect on the inhibition induced by IL-10. Antigen presentation for the myoglobin-specific syngeneic Th1 clone by cLC, which were pre-incubated with these cytokines, correlated well with their CD80 expression. In addition, among the antibodies for CD80, CD86, CD28 or CD40, the suppression of the Th1 clone stimulation by LC was found to occur only with anti-CD80 and anti-CD28 antibodies. Finally, we studied the effects of IFN-gamma and IL-10 on GM-CSF production by epidermal keratinocytes (KC). We could show that only IFN-gamma, but not IL-10, suppressed GM-CSF production by KC. These findings suggest that both IFN-gamma and IL-10 suppress antigen presentation by LC for Th1 cells by suppressing their CD80 expression. The inhibitory effect of IFN-gamma on CD80 expression on LC appears to be partially mediated through the suppression of GM-CSF production by KC.     

Synthesis and evaluation of 4-amino-substituted 7-methoxy (and 7-hydroxy)- 11-methyl (and 5,11-dimethyl)-10H-pyrido [2,3-b] carbazoles and 4-amino- substituted 11-methyl (and 5,11-dimethyl)-10H-pyrido[3',4':4,5] pyrrolo [3,2-g] quinolines, two new series related to antitumor ellipticine derivatives

2-Acetyl-4-chloro-3-lithiopyridine ethylene glycol ketal (6b) was reacted with 3-formyl-5-methoxy-1-methyl-indole (9) and 3-formyl-1-methyl-1H-pyrrolo [3,2-c] pyridine (12), giving the corresponding expected alcohols. Reduction of these intermediates with triethylsilane trifluoroacetic acid and subsequent cyclodehydration then led to 4-chloro-7-methoxy-10,11-dimethyl-10H-pyrido [2,3-b] carbazole (8a) and the corresponding 7-aza-analog (8b). The synthesis of 4-chloro-11-methyl (and 5,11-dimethyl)-10-unsubstituted derivatives of these two series was performed through an independent pathway, involving condensation of conveniently substituted 2-amino carbazoles (17) and 7-amino-5H-pyrido [4,3-b] indoles (18) with 5-(ethoxymethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione, thermal cyclization of the resulting compounds with concomitant decarboxylation to the corresponding tetracyclic fused-4-quinolone systems and final chlorination with phosphorus oxychloride. Nucleophilic substitution of various 4-chloro derivatives was then easily performed in an excess of the required dialkylamino alkylamines at reflux and 4-amino substituted-7-hydroxy-10H- pyrido [2,3-b] carbazoles (25d-e) were obtained from 7-methoxy precursors (25a-b), by demethylation with boron tribromide in methylene chloride at -65 degrees C or with boiling 47% hydrobromic acid. Cytotoxicity determination of all new aminosubstituted derivatives and in vivo antitumor evaluation of the most active compounds clearly show that these two series of ellipticine analogs closely related to highly active products are devoid of antitumor properties in two experimental models shown to be sensitive to ellipticines. The place of the pyridinic nitrogen atom in these series has thus been demonstrated to play a crucial role in antitumor activity.     

Detection of two loci involved in (1-->3)-beta-glucan (curdlan) biosynthesis by Agrobacterium sp. ATCC31749, and comparative sequence analysis of the putative curdlan synthase gene

A 28-item questionnaire was returned by 291 psychiatrists who had completed training between 1962 and 1992. There were positive correlations between the amount of couple and family therapy training (CFTT) they received and the following: the extent to which graduate psychiatrists practice CFT; their involvement as supervisors, teachers, teaching program directors, or researchers; the extent to which they seek continuing education in CFT; their positive attitude toward CFT; and the extent to which they feel that their attitude to and interest in CFT has had a positive effect on the milieu in which they practice and on their personal lives.     

Structural and Electrical Properties of Layered Pervoskite Manganites Nd2-2x Sr1＋2x Mn2O7 （x=0.25, 0.3, 0.4）

Tetragonally layered perovskite manganites of Nd2-2xSr1＋2xMn2O7（x =0.25, 0.3, 0.4） were fabricated by using solid-state reaction technique. Structural characterization of the compounds was investigated by X-ray diffraction （XRD） and FT-IR absorption spectra. The XRD patterns revealed that all the samples were single phase. X-ray photoemission spectroscopy （XPS） was used to investigate their electronic structures. It was found that manganese was in mixed states of Mn^3＋ and Mn^4＋ whereas lattice oxygen and chemical absorbed oxygen were existed in all the samples. The high temperature electrical properties of Nd2-2xSr1＋2xMn2O7 （x = 0.3, 0.4） were measured by standard four-probe technique. The results showed that both compounds had semi-conductivity behavior in the temperature range of 300 - 1073 K, and the electrical conduction was dominated by thermally activated behavior above 500 K.     

In vitro cytotoxicity and DNA damage production in Chinese hamster ovary cells and topoisomerase II inhibition by 2-[2''-(dimethylamino)ethyl]-1, 2-dihydro-3H-dibenz[de,h]isoquinoline-1,3-diones with substitutions at the 6 and 7 positions (azonafides)

The p53 tumor suppressor gene encodes a phosphoprotein which when overexpressed can induce growth arrest at the G1 and G2/M phases of the cell cycle, promote differentiation and apoptosis. This paper demonstrates that p53 can associate with trk tyrosine kinase. Expression of a murine temperature-sensitive (ts) p53 mutant in PC12 cells overexpressing trk (a model system to analyse cellular differentiation and signal transduction induced by NGF) induces morphological changes in the absence of NGF stimulation at 32 degrees C but not at 37 degrees C. In cells differentiated by p53, trk, but not EGFr, was hyperphosphorylated on tyrosine. Furthermore trk was not phosphorylated when expressed in Saos-2 cells (human osteosarcoma cells that lack expression of both endogenous trk and p53) at either temperature. However, transfection of ts p53 into these cells induces trk phosphorylation at 32 degrees C in the absence of NGF stimulation. Association of trk and p53 can be detected in NIH3T3 and PC12 cells co-expressing trk and the ts p53 mutant, in NIH3T3 and PC12 cells transfected with trk alone, and in untransfected PC12 cells, showing that overexpressed and/or endogenous trk associates with endogenous, low levels of p53. These data suggest a novel function for p53 which involves the stimulation of signal transduction pathways (mediating morphological properties of cells), possibly through association with and hyperphosphorylation of trk.     

DNA adducts of 2,3-epoxy-4-hydroxynonanal: detection of 7-(1', 2'-dihydroxyheptyl)-3H-imidazo[2,1-i]purine and 1,N6-ethenoadenine by gas chromatography/negative ion chemical ionization/mass spectrometry

2,3-Epoxy-4-hydroxynonanal (EH) is a bifunctional aldehyde formed by epoxidation of trans-4-hydroxy-2-nonenal, a peroxidation product of omega-6 polyunsaturated fatty acids. EH is mutagenic and tumorigenic and capable of modifying DNA bases forming etheno adducts in vitro. Recent studies showed that etheno adducts are present in tissue DNA of humans and untreated rodents, suggesting a potential endogenous role of EH in their formation. A sensitive assay is needed so we can determine whether EH is involved in etheno adduct formation in vivo and study the biological significance of the etheno adducts in DNA. In this study, we developed a gas chromatography/negative ion chemical ionization/mass spectrometry assay for the analysis of 1, N6-ethenoadenine (epsilonAde) and 7-(1', 2'-dihydroxyheptyl)-3H-imidazo[2,1-i]purine (DHH-epsilonAde) in DNA; both are products from the reaction of adenine with EH. The assay entails the following sequence of steps: (1) addition of [15N5]epsilonAde and [15N5]DHH-epsilonAde to DNA as internal standards, (2) acid hydrolysis of DNA, (3) adduct enrichment by C18 solid phase extraction (SPE), (4) derivatization by pentafluorobenzylation (PFB), (5) separation of PFB-epsilonAde and PFB-DHH-epsilonAde on a Si SPE column, (6) acetonide (ACT) formation of PFB-DHH-epsilonAde, and (7) GC/MS analysis with selective ion monitoring (SIM). The limit of detection by on-column injection for PFB-epsilonAde monitoring of the (M - PFB)- ion at m/z 158 was 30 amol and for ACT-PFB-DHH-epsilonAde monitoring of the (M - PFB)- ion at m/z 328 was 0.4 fmol; the detection limits for the entire assay were 6.3 fmol for epsilonAde and 36 fmol for DHH-epsilonAde. In calf thymus DNA modified with EH at 37 degreesC for 50 h, both epsilonAde and DHH-epsilonAde were detected at high levels by this method, 4.5 +/- 0.7 and 90.8 +/- 8.7 adducts/10(3) adenine, respectively. These levels were also verified by HPLC fluorescence analysis, indicating that EH extensively reacts with adenine in DNA, forming etheno adducts. The high sensitivity of the assay suggests that it may be used in the analysis of ethenoadenine adducts in vivo.