FIST/HIPK3: a Fas/FADD-interacting serine/threonine kinase that induces FADD phosphorylation and inhibits fas-mediated Jun NH(2)-terminal kinase activation.

Fas is a cell surface death receptor that signals apoptosis. Several proteins have been identified that bind to the cytoplasmic death domain of Fas. Fas-associated death domain (FADD), which couples Fas to procaspase-8, and Daxx, which couples Fas to the Jun NH(2)-terminal kinase pathway, bind independently to the Fas death domain. We have identified a 130-kD kinase designated Fas-interacting serine/threonine kinase/homeodomain-interacting protein kinase (FIST/HIPK3) as a novel Fas-interacting protein. Binding to Fas is mediated by a conserved sequence in the COOH terminus of the protein. FIST/HIPK3 is widely expressed in mammalian tissues and is localized both in the nucleus and in the cytoplasm. In transfected cell lines, FIST/HIPK3 causes FADD phosphorylation, thereby promoting FIST/HIPK3-FADD-Fas interaction. Although Fas ligand-induced activation of Jun NH(2)-terminal kinase is impaired by overexpressed active FIST/HIPK3, cell death is not affected. These results suggest that Fas-associated FIST/HIPK3 modulates one of the two major signaling pathways of Fas.

BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth.

Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family, designated BAFF (for B cell activating factor belonging to the TNF family), which is expressed by T cells and dendritic cells. Human BAFF was mapped to chromosome 13q32-34. Membrane-bound BAFF was processed and secreted through the action of a protease whose specificity matches that of the furin family of proprotein convertases. The expression of BAFF receptor appeared to be restricted to B cells. Both membrane-bound and soluble BAFF induced proliferation of anti-immunoglobulin M-stimulated peripheral blood B lymphocytes. Moreover, increased amounts of immunoglobulins were found in supernatants of germinal center-like B cells costimulated with BAFF. These results suggest that BAFF plays an important role as costimulator of B cell proliferation and function.

Detection of testosterone propionate administration in horse hair samples.

A sensitive and specific method has been developed to detect semi-quantitatively testosterone in horse hair samples. The method involved a washing step with sodium dodecylsulfate aqueous solution. The mane and tail hair samples (100mg) were dissolved in 1 mL of sodium hydroxide for 15 min at 95 degrees C in the presence of d3-boldenone used as internal standard. The next three steps involved diethyl ether extraction and a solid phase extraction on Isolute C18 (EC) cartridges eluted with methanol. The residue was derivatized by adding 100 microL of acetonitrile and 30 microL of PFPA then incubating for 15 min at 60 degrees C. After evaporation, 30 microL of hexane was added and 2.5 microL was injected into the column (a bonded phase fused silica capillary column DB5MS, 30 m x 0.25 mm i.d. x 0.25 microm film thickness) of a Trace GC chromatograph. In order to improve the sensitivity of the method, damping gas flow has been optimized. Testosterone was identified in MS(2) full scan mode on the Polaris Q instrument. The assay was capable of detecting less than 1 pg mg(-1). The recovery was close to 90%. The analysis of tail and mane samples collected from a gelding horse having received a single dose of testosterone propionate (1 mg kg(-1)) showed the presence of testosterone in the range of 1-6 pg mg(-1) in hair collected during 5 months after administration.

Radon exhalation of cementitious materials made with coal fly ash: Part 1--scientific background and testing of the cement and fly ash emanation.

Increased interest in measuring radionuclides and radon concentrations in fly ash, cement and other components of building products is due to the concern of health hazards of naturally occurring radioactive materials (NORM). The current work focuses on studying the influence of fly ash (FA) on radon-exhalation rate (radon flux) from cementitious materials. The tests were carried out on cement paste specimens with different FA contents. The first part of the paper presents the scientific background and describes the experiments, which we designed for testing the radon emanation of the raw materials used in the preparation of the cement-FA pastes. It is found that despite the higher (226)Ra content in FA (more than 3 times, compared with Portland cement) the radon emanation is significantly lower in FA (7.65% for cement vs. 0.52% only for FA).

Radon exhalation of cementitious materials made with coal fly ash: Part 2--testing hardened cement-fly ash pastes.

Increased interest in measuring radionuclides and radon concentrations in fly ash (FA), cement and other components of building products is due to the concern about health hazards of naturally occurring radioactive materials (NORM). The paper focuses on studying the influence of FA on radon exhalation rate (radon flux) from cementitious materials. In the previous part of the paper the state of the art was presented, and the experiments for testing raw materials, Portland cement and coal fly ash, were described. Since the cement and FA have the most critical role in the radon release process relative to other concrete constituents (sand and gravel), and their contribution is dominant in the overall radium content of concrete, tests were carried out on cement paste specimens with different FA contents, 0-60% by weight of the binder (cement+FA). It is found that the dosage of FA in cement paste has a limited influence on radon exhalation rate, if the hardened material is relatively dense. The radon flux of cement-FA pastes is lower than that of pure cement paste: it is about approximately 3 mBq m(-2) s(-1) for cement-FA pastes with FA content as high as 960 kg m(-3).

Chemical modification of the carboxyl groups of protein substrates enhances their thrombin susceptibility.

Native or denatured protein substrates which are hardly digested by thrombin can become much more efficiently cleaved by the enzyme after chemical modification of their carboxyl groups. Five antibody kappa-chains were used to demonstrate this effect. The selective cleavage sites were determined by quantitative N-terminal analysis and N-terminal sequencing. All five kappa-chains share the same cleavage sites at Arg-Thr (residues 108-109), Arg-Glu (residues 142-143, Glu side chain modified with glycine amide), Lys-Ser (residues 207-208) and Arg-Gly (residues 211-212). One of the major cleavage sites (Arg-Thr) is located at the joint of the variable/constant region. The amino acids adjacent to these cleavage sites underline the proposed structural requirements for a potential thrombin substrate [(1985) Eur. J. Biochem. 151, 217-224]. This approach can facilitate the application of thrombin in generating large polypeptide fragments of proteins.

Caspase-induced inactivation of the anti-apoptotic TRAF1 during Fas ligand-mediated apoptosis.

The activation of the transcription factor NF-kappaB often results in protection against apoptosis. In particular, pro-apoptotic tumor necrosis factor (TNF) signals are blocked by proteins that are induced by NF-kappaB such as TNFR-associated factor 1 (TRAF1). Here we show that TRAF1 is cleaved after Asp-163 when cells are induced to undergo apoptosis by Fas ligand (FasL). The C-terminal cleavage product blocks the induction of NF-kappaB by TNF and therefore functions as a dominant negative (DN) form of TRAF1. Our results suggest that the generation of DN-TRAF1 is part of a pro-apoptotic amplification system to assure rapid cell death.

Observation of the cabibbo-suppressed decay xi(+)(c) --> pK(-)pi(+)

We report the first observation of the Cabibbo-suppressed charm baryon decay Xi(+)(c)-->pK(-)pi(+). We observe 150+/-22+/-5 events for the signal. The data were accumulated using the SELEX spectrometer during the 1996-1997 fixed target run at Fermilab, chiefly from a 600 GeV/c Sigma(-) beam. The branching fractions of the decay relative to the Cabibbo-favored Xi(+)(c)-->Sigma+K-pi(+) and Xi(+)(c)-->Xi(-)pi(+)pi(+) are measured to be B(Xi(+)(c)-->pK(-)pi(+))/B(Xi(+)(c)-->Sigma+K-pi(+)) = 0.22+/-0. 06+/-0.03 and B(Xi(+)(c)-->pK(-)pi(+))/B(Xi(+)(c)-->Xi(-)pi(+)pi(+)) = 0.20+/-0.04+/-0.02, respectively.

Family adaptation to institutionalised mentally retarded patients. A preliminary report on an Italian population.

Family interactive patterns were investigated in the relatives of 20 mentally retarded patients institutionalised in two centres in Northern Italy. Expressed emotion (EE) was used as evaluation instrument. The results show a surprisingly high rate (45%) of high EE, even in relatives of patients who did not live inside the family. High EE was positively correlated to the presence of behavioral disorders in the patients, as already shown for children with conduct disorders. Treatment implications are discussed; there is an opportunity for the implementation of a family psychoeducational approach, aimed both at managing the behavioural consequences of the disorder and at enhancing rehabilitation programmes.

Sexual dimorphism in body composition, weight status and growth in prepubertal school chiildren from rural areas of eastern Austria.

Sexual dimorphism in stature, weight status and body composition were analyzed in a sample of 398 prepubertal children (213 girls, 185 boys) ageing between 7 and 10. Furthermore the prevalence of overweight was tested. Body composition parameters were determined using TBF 105 Body composition analyzer according to BIA-method. Highly significant sex differences in body composition were observed (p < 0.001). In contrast, stature, weight and BMI showed no significant differences between the two sexes. Nevertheless, a significant higher portion (p < 0.05) of girls (29%) corresponded to the definition overweight according to ASNS (Austrian Survey of Nutritional Status), while only 20% of the boys felt into the category overweight. The results of the present study showed not only significant sex differences in body composition, especially in fat mass, long before puberty onset, but also a significantly higher prevalence of overweight among prepubertal girls in comparison to prepubertal boys.

Measurement of radon diffusion length in thin membranes.

Building regulations in Israel require the insulating of buildings against radon (222)Rn penetration from soil. In radon-prone areas membranes stretched between the soil and the building foundation are used, together with sealing other possible penetration routes. Designing the radon mitigation procedure requires checking that all sealing materials are practically, radon tight, having a thickness of at least three times the radon diffusion length. In this work, a very simple technique to evaluate the radon diffusion length in thin membranes, using a radon source of known activity and an activated charcoal canister as radon detector is presented. The theoretical formalism and measurement results for polyethylene membranes of different densities obtained in a recent comparison exercise are presented.

Termination of pregnancy in the second trimester with the aid of laminaria tents.

PIP: A rather old technique for terminating pregnancies in the second trimester is laminaria tents which this study, using 390 women, found very useful and safe. Laminaria tents are dried and sterilized seaweed which the authors used in 7-8 cm lengths. 2-3 laminaria tents are inserted into the uterus for 24 hrs and as they expand they dilate the cervical os. New ones can be inserted 2 to 3 times and left longer than the 24 hrs until the cervix is able to be dilated to 40 mm with a Hegar dilator. After evacuation of the fetus, suction curettage is used to further empty the uterus. Mortality was nil. 1 patient lost more than 1 pint of blood, 1 perforation occurred, and 1 patient required antibiotics for a spreading infection.^ieng

Interocular transfer of expansion, rotation, and translation motion aftereffects.

The motion aftereffect demonstrates the existence of direction-selective mechanisms in the visual system. However, direction-selective cells exist within many visual areas, including V1 and MT/V5. Can motion aftereffects be generated within each of these areas? In visual cortical areas beyond V1 almost all cells are binocular, whereas a smaller percentage are binocular in V1. The degree of binocularity can be revealed psychophysically by assessing interocular transfer. Interocular transfer of motion aftereffects generated from expanding rotating, and translating dynamic random-dot patterns were therefore compared, since these stimuli should activate cells in higher visual areas selectively. Partial interocular transfer was found that was greater for expansion and rotation than for translation. The results support the involvement of higher visual areas in motion aftereffects to complex animation sequences.

Retention behaviour of a template-assembled synthetic protein and its amphiphilic building blocks on reversed-phase columns.

The retention behaviour of a six-helix bundle template-assembled synthetic protein (TASP) molecule and its amphiphilic building blocks was investigated. The TASP consists of a circular template, cyclo(1-12)[KG]6, and six identical potentially alpha-helical peptides of the sequence KLALKLALKALKLALKLA. As an alpha-helix, this peptide is amphiliphilic along the axis of its helix. Based on this sequence, the retention times of a set of acetylated peptides containing from seven to twenty amino acids on a Nucleosil C18 column were compared with another set of peptides with the same amino acid composition but a non-amphiphilic structure. Peptide elution was effected with linear trifluoroacetic acid (TFA)-water to TFA-acetonitrile gradients. The difference in retention times increased with peptide length; the 9-mers eluted at the same time, but there was a difference of 3.5 min for the 13-mers and 22.3 min for the 20-mer, indicating the induction of secondary structure on binding to the stationary phase. The same pair of 20-mers on Vydac C18, C4 and biphenyl columns gave differences in retention times of 23.2, 16.7 and 12.3 min respectively. The TASP molecule was irreversibly adsorbed to C18 stationary phases, whereas it was eluted from C4 and biphenyl columns as a single sharp peak. Several side-products resulting from the synthesis of the TASP molecule were identified by matrix-assisted laser desorption ionization mass spectroscopy. A comparison of the retention times of these side-products and the results of pre-column denaturation experiments indicated that the tertiary structure of the TASP molecule is maintained on binding to biphenyl and C4 columns.

Population pharmacokinetic parameters in patients treated with oral mexiletine.

A new data analysis approach, NON-MEM, proposed by Sheiner and Beal, has been employed to estimate the population pharmacokinetic parameters of oral mexiletine in patients treated for arrhythmias. 452 serum concentration measurements in 58 patients were available for analysis. 27 patients had congestive heart failure and 8 had abnormal liver function tests at the time of the study. The population averages of the pharmacokinetic parameters and their interindividual variability were: oral total body clearance (Cl) 0.38 l/h/kg +/- 43% (C.V.), apparent volume of distribution (Vd) 5.3 l/kg +/- 40%, absorption rate constant 3.1 h-1 +/- 205%, absorption time-lag 0.3 h. Congestive heart failure and sex did not show a significant effect on Cl and Vd; the number of patients with severe liver function impairment was too small for a definite conclusion. Normalizing Cl and Vd for body weight significantly decreased their interindividual variability. Based on these results, a dosage regimen is recommended which is expected to produce a "therapeutic" serum concentration (0.8-2 mg/l) in over 60% of patients. Because of its unique features, which allow estimation of pharmacokinetic parameters and their variability from fragmentary patient data, the NONMEM system has great potential applicability to clinical pharmacokinetic studies.

Isolation and purification of novel hirudins from the leech Hirudinaria manillensis by high-performance liquid chromatography.

The isolation and purification of novel hirudins from a crude extract of the leech Hirudinaria manillensis and their analytical characterization are reported. Initial purification by gel permeation chromatography on Sephadex G50 and anion-exchange chromatography on Q Sepharose fast-flow removed most contaminants and yielded a highly active extract. Two isohirudins (designated hirudin P6 and P18) were isolated and purified by successive reversed-phase high-performance liquid chromatography on silica-based stationary phases and anion-exchange chromatography on Mono Q. The final products were characterized by reversed-phase high-performance liquid chromatography, 252Cf plasma desorption time-of-flight mass spectrometry and capillary zone electrophoresis. The molecular masses determined by 252Cf plasma desorption mass spectrometry were 7416 dalton for hirudin P6 and 7199 dalton for hirudin P18.

Analysis of synthetic peptides using matrix-assisted laser desorption ionization mass spectrometry.

Matrix-assisted laser desorption ionization mass spectroscopy (LDI MS), a novel method for analysis of large molecules, has been used for characterization of synthetic peptides and their by-products. The potential of LDI MS is demonstrated by analyzing crude synthetic peptides representing typical members of newly designed peptides and proteins. In the first case, a fragment condensation reaction yielding a highly hydrophobic six-helic bundle template-assembled synthetic protein (TASP) is monitored. Then, a crude 19-mer peptide designed to adopt an amphiphilic alpha-helical structure and its by-products from SPPS are identified. Finally, analysis of crude hirulog-1, a 20-mer peptide designed as a thrombin inhibitor, using C18 reversed phase high performance liquid chromatography (RP HPLC), capillary electrophoresis (CE) and LDI MS, manifests the potential of the latter method.