Induction of chromatid-type aberrations in peripheral lymphocytes of hospital workers exposed to very low doses of radiation

Radiological personnel represent workers exposed to low cumulative doses of radiation. As their surveillance is generally based on physical dosimetry, there is little or inconclusive information on biological effects due to radiation exposure at these doses. We aimed to explore the extent of chromosomal damage in circulating lymphocytes of hospital workers (technicians, nurses and physicians) chronically exposed to a very low level of radiation using conventional and molecular cytogenetic analyses (chromosome painting with chromosomes #2, #3 and #10 as probe cocktail). Compared with controls, exposed workers displayed a significant increase in the frequency of aberrant lymphocytes (1.26+/-0.11/100 cells versus 1.63+/-0.17/100 cells). In particular, exposed technicians showed significantly higher mean values than nurses or physicians (3.68+/-1.17/100 cells versus 1.36+/-0.18/100 cells and 1.36+/-0.09/100 cells, respectively). Interestingly, we found that the chromosomal damage was prevalently expressed as chromatid-type aberrations. Chromosome painting indicated that the frequency of chromosome rearrangements (CR; translocations and dicentrics pooled together) was approximately comparable between radiological workers and the control group. Moreover, we did not detect any significant difference due to radiation exposure when CR rates were considered separately for each of the three chromosomes in the probe cocktail.     

Relative and absolute configuration of antitumor agent SW-163D

Our interest on engineering non-ribosomal synthetase responsible for SW-163 biosynthesis prompted us to determine the relative and absolute configuration of antitumor cyclic depsipeptide SW-163s. We first isolated and identified SW-163 homologs D, F and G as known compounds UK-63598, UK-65662 and UK-63052, respectively. Both enantiomers of the unusual constitutive amino acid, N-methylnorcoromic acid, were synthesized in chiral forms starting from (R)- and (S)-1,2-propanediol. The hydrolyzate of SW-163D, a major constituent of this family, was converted with Marfey's reagent, 1-fluoro-2,4-dinitrophenyl-5-L-alanine-amide (L-FDAA), and the resulting mixture of amino acid derivatives was subjected to an LC/MS analysis. Compared with authentic samples, the analytical data unambiguously show that SW-163D consisted of L-Ala, D-Ser and (1S, 2S)-N-methylnorcoronamic acid. The remaining stereochemistry of the N-methylcysteine moieties was determined from NOE data.     

The effect of the chain length distribution of free fatty acids on the mixing properties of stratum corneum model membranes

The stratum corneum (SC) plays a fundamental role in the barrier function of the skin. The SC consists of corneocytes embedded in a lipid matrix. The main lipid classes in the lipid matrix are ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). The aim of this study was to examine the effect of the chain length of FFAs on the thermotropic phase behavior and mixing properties of SC lipids. Fourier transform infrared spectroscopy and Raman imaging spectroscopy were used to study the mixing properties using either protonated or deuterated FFAs. We selected SC model lipid mixtures containing only a single CER, CHOL and either a single FFA or a mixture of FFAs mimicking the FFA SC composition. The single CER consists of a sphingoid base with 18 carbon atoms and an acyl chain with a chain length of 24 carbon atoms. When using lignoceric acid (24 carbon atoms) or a mixture of FFAs, the CER and FFAs participated in mixed crystals, but hydration of the mixtures induced a slight phase separation between CER and FFA. The mixed crystalline structures did not phase separate during storage even up to a time period of 3 months. When using palmitic acid (16 carbon atoms), a slight phase separation was observed between FFA and CER. This phase separation was clearly enhanced during hydration and storage. In conclusion, the thermotropic phase behavior and the mixing properties of the SC lipid mixtures were shown to strongly depend on the chain length and chain length distribution of FFAs, while hydration enhanced the phase separation.     

Stem-loop binding protein is required for retinal cell proliferation,neurogenesis, and intraretinal axon pathfinding in zebrafish

In the developing retina, neurogenesis and cell differentiation are coupled with cell proliferation. However, molecular mechanisms that coordinate cell proliferation and differentiation are not fully understood. In this study, we found that retinal neurogenesis is severely delayed in the zebrafish stem-loop binding protein (slbp) mutant. SLBP binds to a stem-loop structure at the 3′-end of histone mRNAs, and regulates a replication-dependent synthesis and degradation of histone proteins. Retinal cell proliferation becomes slower in the slbp1 mutant, resulting in cessation of retinal stem cell proliferation. Although retinal stem cells cease proliferation by 2 days postfertilization (dpf) in the slbp mutant, retinal progenitor cells in the central retina continue to proliferate and generate neurons until at least 5 dpf. We found that this progenitor proliferation depends on Notch signaling, suggesting that Notch signaling maintains retinal progenitor proliferation when faced with reduced SLBP activity. Thus, SLBP is required for retinal stem cell maintenance. SLBP and Notch signaling are required for retinal progenitor cell proliferation and subsequent neurogenesis. We also show that SLBP1 is required for intraretinal axon pathfinding, probably through morphogenesis of the optic stalk, which expresses attractant cues. Taken together, these data indicate important roles of SLBP in retinal development.     

Involvement of highly sulfated chondroitin sulfate in the metastasis of the Lewis lung carcinoma cells

The altered expression of cell surface chondroitin sulfate (CS) and dermatan sulfate (DS) in cancer cells has been demonstrated to play a key role in malignant transformation and tumor metastasis. However, the functional highly sulfated structures in CS/DS chains and their involvement in the process have not been well documented. In the present study, a structural analysis of CS/DS from two mouse Lewis lung carcinoma (3LL)-derived cell lines with different metastatic potentials revealed a higher proportion of Delta(4,5)HexUA-GalNAc(4,6-O-disulfate) generated from E-units (GlcUA-GalNAc(4, 6-O-disulfate)) in highly metastatic LM66-H11 cells than in low metastatic P29 cells, although much less CS/DS is expressed by LM66-H11 than P29 cells. This key finding prompted us to study the role of CS-E-like structures in experimental lung metastasis. The metastasis of LM66-H11 cells to lungs was effectively inhibited by enzymatic removal of tumor cell surface CS or by preadministration of CS-E rich in E-units in a dose-dependent manner. In addition, immunocytochemical analysis showed that LM66-H11 rather than P29 cells expressed more strongly the CS-E epitope, which was specifically recognized by the phage display antibody GD3G7. More importantly, this antibody and a CS-E decasaccharide fraction, the minimal structure recognized by GD3G7, strongly inhibited the metastasis of LM66-H11 cells probably by modifying the proliferative and invading behavior of the metastatic tumor cells. These results suggest that the E-unit-containing epitopes are involved in the metastatic process and a potential target for the diagnosis and treatment of malignant tumors.     

Critical contribution of aromatic rings to specific recognition of polyether rings. The case of ciguatoxin CTX3C-ABC and its specific antibody 1C49

To address how proteins recognize polyether toxin compounds, we focused on the interaction between the ABC ring compound of ciguatoxin 3C and its specific antibody, 1C49. Surface plasmon resonance analyses indicated that Escherichia coli-expressed variable domain fragments (Fv) of 1C49 had the high affinity constants and slow dissociation constants typical of antigen-antibody interactions. Linear van't Hoff analyses suggested that the interaction is enthalpy-driven. We resolved the crystal structure of 1C49 Fv bound to ABC ring compound of ciguatoxin 3C at a resolution of 1.7A. The binding pocket of the antibody had many aromatic rings and bound the antigen by shape complementarity typical of hapten-antibody interactions. Three hydrogen bonds and many van der Waals interactions were present. We mutated several residues of the antibody to Ala, and we used surface plasmon resonance to analyze the interactions between the mutated antibodies and the antigen. This analysis identified Tyr-91 and Trp-96 in the light chain as hot spots for the interaction, and other residues made incremental contributions by conferring enthalpic advantages and reducing the dissociation rate constant. Systematic mutation of Tyr-91 indicated that CH-pi and pi-pi interactions between the aromatic ring at this site and the antigen made substantial contributions to the association, and van der Waals interactions inhibited dissociation, suggesting that aromaticity and bulkiness are critical for the specific recognition of polyether compounds by proteins.     

Spatial and temporal variability of surface water in the Kuroshio source region, Pacific Ocean, over the past 21,000 years: evidence from planktonic foraminifera

The Kuroshio Current is the major western boundary current of the North Pacific Ocean and has had a large impact on surface water character and climate change in the northwestern Pacific region. The Kuroshio Current becomes a distinctive surface flow in the Ryukyu Arc region after diverging from the North Equatorial Current and passing through the Okinawa Trough. Therefore, the Ryukyu Arc area can be called the Kuroshio source region. We reconstructed post-21-ka time–space changes in surface water masses in the Ryukyu Arc region using 15 piston cores which were dated by planktonic δ¹⁸O stratigraphy and AMS ¹⁴C ages. Our analysis utilized spatial and temporal changes in planktonic foraminiferal assemblages which were classified into the Kuroshio, Subtropical, Coastal, and Cold water groups on the basis of modern faunal distributions in the study region. These results indicate that the Kuroshio Current and adjacent surface water masses experienced major changes during: (1) the Last Glacial Maximum (LGM), and (2) the so-called Pulleniatina minimum event (PME) from 4,500 to 3,000 yr BP. The Kuroshio LGM event corresponds to severe global cooling and is marked by decreases in planktonic δ¹⁸O values and estimated sea-surface temperature (SST) with the dominance of the Cold water group of planktonic foraminifera. Cooling within the Kuroshio source region was enhanced during the LGM event because the Kuroshio Current was forced eastward due to the formation of a land bridge between Taiwan and the southern Ryukyu Arc which prohibited its flow into the Okinawa Trough. Except for the severe reduction and disappearance of the Pulleniatina group, no clear cooling signal was identified during the PME based on δ¹⁸O values, estimated SST values and variations in the composition of planktonic foraminiferal faunas. The PME assemblages are marked by high abundances of Neogloboquadrina dutertrei, a distinctive Kuroshio type species, along with other species assigned to the Coastal and Central water groups. Subtle ecological differences exist between Pulleniatina obliquiloculata and N. dutertrei; i.e. P. obliquiloculata exhibits lower rates of reproduction under conditions of lower primary productivity in the central Equatorial Pacific Ocean. El Niño-like conditions in the Equatorial Pacific Ocean result in lower rates of surface transport in the Kuroshio Current. In turn, this response triggers lower rates of primary productivity in central equatorial surface waters as well as in the upstream Kuroshio source region, ultimately resulting in a lower abundance of P. obliquiloculata. Thus, we interpret the PME as a possible proxy signal of El Niño-like conditions and enhancement of the El Niño Southern Oscillation climate system after the PME in the tropical and sub-tropical Pacific Ocean.     

Bacteriological studies on the sulfur cycle in the anaerobic part of the hypolimnion and in the surface sediments of rotsee in Switzerland scientific report of the advanced course of microbial ecology, sponsored by FEMS and the Swiss society for Microbiology, held at Kastanienbaum, Switzerland, 12 September–9 October 1982

Abstract The microbial ecology of the sulfur cycle in the anaerobic part of Rotsee (Switzerland) was studied. Almost all the sulfate reduction took place at the sediment surface at a rate of 2 mmol SO²⁻₄ reduced m⁻² day⁻¹. Approx. 10⁴ sulfate reducers per ml were present in the surface sediments. The sulfide produced was phototrophically consumed mainly by Thiopedia rosea, Lamprocystis roseopersicina and ' Pelochromatium roseum ' consortia. Thiopedia rosea migrated diurnally about one meter. Bacterial photosynthesis was limited by light and sulfide rather than by temperature.     

Multiple Enzymatic Activities Associated with Recombinant NS3 Protein of Hepatitis C Virus

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) contains at least two domains associated with multiple enzymatic activities; a serine protease activity resides in the N-terminal one-third of the protein, whereas RNA helicase activity and RNA-stimulated nucleoside triphosphatase activity are associated with the C-terminal portion. To study the possible mutual influence of these enzymatic activities, a full-length NS3 polypeptide of 67 kDa was expressed as a nonfusion protein in Escherichia coli, purified to homogeneity, and shown to retain all three enzymatic activities. The protease activity of the full-length NS3 was strongly dependent on the activation by a synthetic peptide spanning the central hydrophobic core of the NS4A cofactor. Once complexed with the NS4A-derived peptide, the full-length NS3 protein and the isolated N-terminal protease domain cleaved synthetic peptide substrates with comparable efficiency. We show that, as in the case of the isolated protease domain, the protease activity of full-length NS3 undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B and NS5A-NS5B. We have also characterized and quantified the NS3 ATPase, RNA helicase, and RNA-binding activities under optimized reaction conditions. Compared with the isolated N-terminal and C-terminal domains, recombinant full-length NS3 did not show significant differences in the three enzymatic activities analyzed in independent in vitro assays. We have further explored the possible interdependence of the NS3 N-terminal and C-terminal domains by analyzing the effect of polynucleotides on the modulation of all NS3 enzymatic functions. Our results demonstrated that the observed inhibition of the NS3 proteolytic activity by single-stranded RNA is mediated by direct interaction with the protease domain rather than with the helicase RNA-binding domain.