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PubMed2004

Mechanisms of DNA separation in entropic trap arrays: a Brownian dynamics simulation.

Using Brownian dynamics simulations, we study the migration of long charged chains in an electrophoretic microchannel device consisting of an array of microscopic entropic traps with alternating deep regions and narrow constrictions. Such a device has been designed and fabricated recently by Han and Craighead [Science 288 (2000) 1026] for the separation of DNA molecules. Our simulation reproduces the experimental observation that the mobility increases with the length of the DNA. A detailed data analysis allows to identify the reasons for this behavior. Two distinct mechanisms contribute to slowing down shorter chains. One has been described earlier by Han and Craighead [Science 288 (2000) 1026]: the chains are delayed at the entrance of the constriction and escape with a rate that increases with chain length. The other, actually dominating mechanism is here reported for the first time: some chains diffuse out of their main path into the corners of the box, where they remain trapped for a long time. The probability that this happens increases with the diffusion constant, i.e., the inverse chain length.

Computer Simulation↗

PubMed2005

Soyasaponin-I-modified invasive behavior of cancer by changing cell surface sialic acids.

OBJECTIVE: Sialylation involving tumor formation and invasive behavior goes along with altered sialyltransferase (ST) activity. A potent ST inhibitor, soyasaponin I (SsaI), was discovered to selectively inhibit the cellular alpha2,3-sialyltranserase activity. In this study, we further test the effects of SsaI on modifying the metastatic and invasive behaviors of cancer cell lines. METHODS: Nonmetastatic breast cancer cell line, MCF-7, and highly metastastic breast cancer cell line, MDA-MB-231, were used to investigate the effects of SsaI on tumor cells. RESULTS: SsaI did not affect cell growth cycle and also failed to inhibit cell growth in this study (the concentration of SsaI < or=100 muM). SsaI was as predicted to successfully inhibit cellular alpha2,3-ST activity and depressed the dose-dependent tumor cell surface alpha2,3-sialic acid expression. In addition, different concentrations of SsaI did stimulate MCF-7 cell adhesion to collagen type I linearly and significantly enhanced cell adhesion to the Matrigel-matrix. Furthermore, SsaI significantly decreased MDA-MB-231 cell migration. Reverse transcriptase polymerase chain reaction for evaluating mRNA expression of ST3Gal I, III and IV showed that SsaI also down-regulated the expression of ST3Gal IV but did not affect the other two. CONCLUSIONS: The results showed that SsaI was implicated in the invasive behavior of tumor cells, suggesting that altered alpha2,3-sialylation pathway played a crucial role in the adhesion and tumor metastases. SsaI is a good candidate for studying the biological roles of ST, and might provide a new preventive strategy in tumor metastasis.

Breast Neoplasms↗

PubMed2003

Geochemical speciation of heavy metals in semiarid soils of the central Ebro Valley (Spain).

This study describes the chemical speciation of Fe, Mn, Zn and Cr in four selected soil types representative of the more abundant agricultural soils in the central Ebro river valley in Aragón. Gypsisols, Calcisols, Solonchaks and Solonetzs represent around 20% of the total soil surface in the region. A total of 12 selected sites have been sampled and five subsamples were taken in each site to create a composite sample. The four elements have been analysed by the sequential extraction procedure of Tessier et al. [Anal. Chem. 51 (1979) 844.] by emission atomic spectrometry of solid state (ICP OES). Very little amounts of Fe and Mn were retrieved from the exchangeable phase, the ready available for biogeochemistry cycles in the ecosystems. Therefore, low quantities of Fe and Mn can be taken up by plants in these alkaline soils. Cr was not detected in the bioavailable forms as well as Zn that was only present in negligible amounts in very few samples. The absence of mobile forms of Cr in all soils eliminates the toxic risk both in the trophic chain and from its migration downwards the soil profile. The largest contents of Fe, Zn and Cr were retrieved from the residual phase where metals are strongly bound to minerals, whereas Mn from the carbonate and oxide phases amounts 80% of its total content. Gypsisols and Calcisols have the lowest metal contents while the highest are found in Solonetzs and Solonchaks.

Biological Availability↗

PubMed2002

Survey of natural and anthropogenic radioactivity in environmental samples from Yugoslavia.

The radioactivity of 238U, 226Ra, 232Th, 40K and 137Cs in sediments, soil, turf and honey from Serbia and Kosovo (Yugoslavia) was measured using gamma and alpha spectrometry in order to estimate the radiation hazard from natural and man-made sources, as well as to compile a database for radioactivity levels in those regions. One sample, collected in the vicinity of a "depleted uranium" (DU) shell of the recent Balkan war, revealed a high 238U activity and a non-natural 235U/238U activity ratio, confirming therefore its anthropogenic origin. However, some other soil samples coming from characteristic DU craters did not show any characteristic level of radioactivity. The other sediment and turf samples taken all around the country show low radioactivity levels for all the isotopes here considered. With the aim of obtaining some indication about radioactivity migration in the food chain, several honey samples have been examined too. All samples show very low radioactivity content, often indistinguishable from natural background.

Environmental Monitoring↗

PubMed1983

Carboxyl-terminal proteolytic processing during biosynthesis of the lysosomal enzymes beta-glucuronidase and cathepsin D.

During pulse-chase experiments in cultured porcine kidney cells, an early 75-kilodalton (kDa) form of beta-glucuronidase is converted to a late 72-kDa form. The relative molecular weight difference between the two forms is maintained on removal of high-mannose carbohydrate with endoglycosidase H. Both forms have the same partial NH2-terminal sequence, and both migrate as single polypeptide chains following reduction, alkylation, and electrophoresis under denaturing conditions. On treatment with carboxypeptidase Y, the early form released [35S]Met faster than the late form. Thus, the late form of beta-glucuronidase is generated by COOH-terminal proteolytic processing of the early form. During similar experiments, the mass of the 30-kDa heavy chain of porcine cathepsin D decreased by about 1 kDa. The heavy chain of the two-chain enzyme is derived from the COOH terminus of a 44-kDa single-chain enzyme. On treatment with carboxypeptidase Y, the early single-chain enzyme released COOH-terminal [35S]Met and [3H]Lys faster than the later 29-kDa heavy chain. Like beta-glucuronidase, cathepsin D evidently undergoes COOH-terminal proteolytic processing during biosynthesis.

Animals↗

PubMed1976

A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Partial characterization of the purified enzyme.

The purified Ca2+-activated protease (CAF) isolated from porcine skeletal muscle and capable of removing Z-disks from intact myofibrils is optimally active on either myofibril or casein substrates at pH 7.5 and in the presence of 1 mM Ca2+ and at least 2 mM 2-mercaptoethanol. No CAF activity is detected when 1 mM Mg2+, Mn2+, Ba2+, Co2+, Ni2+, and Fe2+ are added singly. When added with 1 mM Ca2+, Co2+, Cu2+, Ni2+, and Fe2+ inhibit, whereas Mg2+, Mn2+, and Ba2+ have no effect on CAF activity. CAF is irreversibly inhibited by iodoacetate but is unaffected by soybean trypsin inhibitor. S0/20,W=5.90 S, and sedimentation equilibrium molecular weight - 112 000 for purified CAF. Because purified CAF migrates as two polypeptide chains with molecular weights of 80 000 and 30 000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the CAF molecule must consist of one each of these two polypeptide chains. Approximate molecular dimensions of 38 X 220 A can be calculated for CAF from calibrated gel permeation column data or from S0/20,W and the molecular weight. Amino acid composition and physical properties of purified CAF distinguish it from the known catheptic enzymes and from other proteases found in blood or in granulocytes. Purified CAF removes Z-disks the 400-A periodicity associated with troponin in the I band and partly degrades M lines but causes no other ultrastructurally detectable effects when incubated with myofibrils. These results agree with the earlier finding that purified CAF degrades troponin, tropomyosin, and C-protein but has no effect on myosin, actin, or alpha-actinin, and suggest that CAF may have a physiological role in disassembly of intact myofibrils during metabolic turnover of myofibrillar proteins.

Amino Acids↗

PubMed1999

Palladium-Catalyzed Cross-Coupling of 2,5-Cyclohexadienyl-Substituted Aryl or Vinylic Iodides and Carbon or Heteroatom Nucleophiles.

2,5-Cyclohexadienyl-substituted aryl or vinylic iodides have been reacted with carbon nucleophiles (diethyl malonate and 2-methyl-1,3-cyclohexanedione), nitrogen nucleophiles (morpholine, potassium phthalimide, N-benzyl tosylamide, di-tert-butyl iminodicarboxylate, lithium azide, and anilines), a sulfur nucleophile (sodium benzenesulfinate), and oxygen nucleophiles (lithium acetate and phenols) to afford products of cyclization and subsequent cross-coupling in good to excellent yields. In most cases, this process is highly diastereoselective. The reaction is believed to proceed via (1) oxidative addition of the aryl or vinylic iodide to Pd(0), (2) organopalladium addition to one of the carbon-carbon double bonds, (3) palladium migration along the carbon chain on the same face of the ring to form a pi-allylpalladium intermediate, and (4) nucleophilic displacement of the palladium.

Journal Article↗

PubMed1983

Recruitment of cytosolic proteins to a secretory granule membrane depends on Ca2+-calmodulin.

An increase in free calcium triggers catecholamine secretion from chromaffin cells and calmodulin is strongly implicated as the intracellular Ca2+ receptor. In our recent studies of calmodulin action in the chromaffin cell, micromolar Ca2+ concentrations resulted in calmodulin and cytosolic proteins becoming bound to the chromaffin granule membranes. We now report that calmodulin is bound with high affinity to granule membrane proteins of molecular weights (Mrs) 25,000 and 22,000 (25K and 22K) at low Ca2+ (less than 10(-8) M) and to proteins with Mrs 69K and 50K at high Ca2+ (greater than 1 microM). Other cytosolic components (Mrs 70K, 36K, 34K and 32K) require calmodulin for their interfraction with membrane. These proteins separately bound to calmodulin-Sepharose at high Ca2+ concentrations. Although the functions of these adrenal proteins have not been established, the 34K and 32K Mr components co-migrate with clathrin light chains isolated from medullary coated vesicles and the Mr 34K components from both sources share the same one-dimensional peptide map. These interactions were observed at micromolar Ca2+ levels at 'intracellular' conditions of pH and ionic strength and would be expected to occur during secretion from the chromaffin cell.

Calcium↗

PubMed1978

Actin in Xenopus oocytes.

It has been found that a high-speed supernatant fraction from Xenopus oocytes extracted in the cold will form a clear, solid gel upon warming. Gel formation occurs within 60 min at 18 degrees-40 degrees C, and is, at least initially, temperature reversible. Gelation is strictly dependent upon the addition of sucrose to the extraction medium. When isolated in the presence of ATP, the gel consists principally of a 43,000-dalton protein which co-migrates with Xenopus skeletal muscle actin on SDS-polyacrylamide gels, and a prominent high molecular weight component of approx. 250,000 daltons. At least two minor components of intermediate molecular weight are also found associated with the gel in variable quantities. Actin has been identified as the major consituent of the gel by ultrastructural and immunological techniques, and comprises roughly 47% of protein in the complex. With time, the gel spontaneously contracts to form a small dense aggregate. Contraction requires ATP. In the absence of exogenous ATP, a polypeptide which co-migrates with the heavy chain of Xenopus skeletal muscle myosin becomes a prominent component of the gel. This polypeptide is virtually absent from gels which have contracted in ATP-containing extracts. It has also been found that Ca++ is required for gelation in oocyte extracts. At both low and high concentrations of Ca++ (defined as a ratio of Ca++/EGTA in the extraction medium), gelation is inhibited.

Actins↗

PubMed1999

Proton glass freezing in hydrated lysozyme powders.

At room temperature, the dielectric relaxation of hydrated powder of the protein lysozyme is known to be due to protons migrating between ionized side chains. A recent study of this relaxation at lower temperatures suggested a behavior typical of proton glasses. An analysis of the complex dielectric susceptibility by a temperature-frequency plot presented here has revealed that ergodicity is broken due to the divergence of the longest relaxation time at 266 K, indicating specifically that this hydrated protein is a proton glass. A change in the temperature behavior of the static dielectric constant and the average relaxation frequency at 273 K indicates a further transition occurring at this temperature, whose nature remains to be investigated.

Freezing↗

PubMed1980

Immunochemical characterization of a human B lymphocyte differentiation antigen (p65).

A human B-cell differentiation antigen (BDA-1) with a molecular weight of 65,000 was identified by use of an anti-B-cell xenoantiserum. BDA-1 was isolated by immunoprecipitation of cell lysates from a B-lymphoblastoid cell line (SB) and from blood B lymphocytes (sIg+ER-) of four normal individuals. It was not detectable in cell membrane lysates from two T-lymphoblastoid cell lines (HSB-2 and MOLT-3) or from enriched normal T cells (sIg-ER+). BDA is single-chain molecule since its migration in SDS-PAGE gels was not altered by heating the protein to 100 degree C or by treatment with 2-mercaptoethanol. Immunodepletion experiments demonstrated that the antigen recognized by anti-BDA xenoantiserum has neither structural nor antigenic relationships with Ia-like antigen or beta 2-microglobulin.

Antibodies↗

PubMed1990

Substance P contracts bovine tracheal smooth muscle via activation of myosin light chain kinase.

At near-threshold substance P concentrations, the isometric tension response of bovine tracheal strips is almost completely abolished by atropine, indicating mediation of contraction via substance P-stimulated release of acetylcholine from prejunctional nerve terminals. At near-maximal concentrations, the atropine-inhibited component of the tension response is less than 25%, indicating mainly direct activation. Under conditions in which activation by substance P is direct, peak tension is reached in approximately 11 min. Immunoblot analysis of the time course of phosphorylation of the 20-kDa myosin light chain (LC20) reveals incorporation of approximately 0.5 mol phosphate/mol light chain at 10 min. Two-dimensional tryptic phosphopeptide analysis of phosphorylated light chain reveals a single major phosphopeptide. The peptide migrates identically with that produced by myosin light chain kinase phosphorylation of purified tracheal myosin in vitro. Contraction stimulated by acetylcholine is more rapid, with attainment of peak tension in 2.5 min and a peak LC20 phosphorylation of 0.65 mol/mol. These results indicate that 1) substance P mediates contraction of bovine trachea both directly and indirectly, and 2) under conditions in which activation is direct, the tension and phosphorylation responses qualitatively resemble those observed with acetylcholine.

Animals↗

PubMed1999

A novel frameshift mutation in the first exon of the 21-OH gene found in homozygosity in an apparently nonconsanguineous family.

Congenital adrenal hyperplasia is most frequently due to steroid 21-hydroxylase (21-OH) deficiency. Due to the existence of a pseudogene in tandem duplicated with the 21-OH gene, asymmetric recombination causes the majority of the molecular defects underlying this deficiency: gene conversions and deletions of the functional gene. Screening for a small array of mutations, those existing in the pseudogene together with deletions, allows the characterization of most mutated alleles, 91% in the Spanish population. We report the case of a boy from a nonconsanguineous family, diagnosed during the neonatal period of a salt-wasting form of the deficiency, in which this screening did not allow the characterization of the paternal or the maternal allele. This infrequent finding in a nonconsanguineous family was further investigated. Single-strand conformation polymorphism screening for new mutations revealed an abnormally migrating pattern when polymerase chain reaction fragments from 21-OH gene exon 1 of the patient and relatives were analyzed. Upon direct sequencing, the insertion of a T at position 64 (64insT, frameshift generating a stop codon at exon 2) was found in homozygosity in the patient. Microsatellite typing in the HLA region revealed the patient to be homozygous for five markers (heterozygosities 0.62 to 0.74). Apparently this new mutation was generated several generations ago and has been preserved for years. Consanguinity had been discarded for several generations, although both families could be traced back to a small rural area in Navarra (Spain).

Amino Acid Sequence↗

PubMed2004

Frequent loss of heterozygosity but rare microsatellite instability in oesophageal cancer in Japanese and Chinese patients.

Reported frequencies for microsatellite instability (MSI) in oesophageal cancer differ widely in the literature, perhaps due to the high incidence of loss of heterozygosity (LOH) in this cancer. Using high-resolution fluorescent microsatellite analysis (HRFMA), we analysed microsatellite alterations in detail in 50 Japanese and 50 Chinese patients with squamous cell carcinoma in the oesophagus. In HRFMA, several devices have been developed to improve the detection characteristics, reproducibility of polymerase chain reaction and the migration accuracy of electrophoresis. All the alterations observed were separable into MSI, LOH and alterations ambiguous for both. MSI was rare in these panels of oesophageal carcinomas. The frequencies of MSI in the Japanese and Chinese subjects were 8 and 4%, respectively. All the alterations were mild (within 2 base pairs) and were observed in a limited number of markers. More drastic types of MSI, such as those typical in colorectal cancer, were not observed. On the other hand, the incidence of LOH was high, reaching 50% for the Japanese and 70% for the Chinese subjects. In many of these cases, LOH was observed in multiple microsatellite markers. The frequency of LOH in each marker was not apparently biased. Although in many cases MSI and LOH were clearly distinguished with use of the sensitive and quantitative fluorescent assay, theoretically indistinguishable patterns were noted in some cases. In conclusion, MSI is rare and LOH predominates in squamous cell carcinoma in the oesophagus.

Adult↗

PubMed2004

Human platelets contain forms of factor V in disulfide-linkage with multimerin.

Factor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factor V is stored complexed with the polymeric alpha-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains,were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Since unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that unusually large factor V was associated with multimerin and it remained associated in 0.5 M salt. Moreover, platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage. The disulfide-linked complexes of multimerin and factor V in platelets, which are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Factor Va generation and function from unusually large platelet factor V is only speculative at this time.

Blood Platelets↗

PubMed2007

Wading into the genomic pool to unravel acute lung injury genetics.

Acute lung injury (ALI) is a common and often devastating illness characterized by acute hypoxemia, alveolar flooding, and an unacceptably high morbidity and mortality. Because only a fraction of the patients exposed to ALI-inciting events progress to development of the syndrome, there is significant interest in the identification of genetic factors potentially contributing to ALI susceptibility or prognosis. Two complementary strategies used to elucidate ALI genetics formulate the "candidate gene approach," whereby genes are identified by either global gene expression profiling in humans or animal models of ALI, often yielding highly conserved candidates across multiple species, or by related literature searches. Relevant variants or single nucleotide polymorphisms (single base pair substitutions) in these ALI candidate genes are tested for differences in allelic frequency for both ALI susceptibility and outcome between ALI cases and control patients at risk for ALI. This approach has yielded important variants in a number of genes (angiotensin converting enzyme, surfactant protein B, heat shock protein 70, pre-B-cell colony enhancing factor, myosin light chain kinase, and macrophage migration inhibitory factor) contributing toward an ALI phenotype. An alternative strategy not yet used in ALI genetic studies includes genomewide analyses to locate "hot" genomic segments harboring several hundred genes, with potential ALI candidate genes embedded within these segments. Overall, the detailing of specific ALI-associated polymorphisms will continue to provide new insights in the understanding of ALI pathogenesis, reveal novel molecular targets, and promote the development of individualized therapies to reduce morbidity and mortality from this devastating disease.

Gene Expression Profiling↗

PubMed1989

Osteoporosis and familial idiopathic scoliosis: association with an abnormal alpha 2(I) collagen.

A positive family history is considered a risk factor for osteoporosis (OP) although the genetic or biochemical basis for this relationship remains undefined. Various mutations affecting normal synthesis of type I collagen have been reported in osteogenesis imperfecta (OI), a heritable disorder of connective tissue. Family A, in which the proband and a daughter are afflicted with OP and idiopathic scoliosis was examined for defects in collagen metabolism. Dermal fibroblast cultures were established to investigate de novo collagen synthesis. SDS-PAGE revealed an abnormally migrating alpha 2(I) chain and procollagen in two generations. Examination of the kinetics of type I collagen pC & N-propeptide processing demonstrated a rate 2x control in the proband. The phenotype family A is not OI. It shares features with families B & C, having familial clustering of OP. However, collagen synthesis was not abnormal in family B & C. These data suggest that in family A the alpha 2(I) structural defect may be related to defective skeletal matrix formation.

Adolescent↗

PubMed2000

[Studies of the genetic polymorphism of Schistosoma japonicum (Chinese mainland strains)].

In order to analyze the interspecies diversity and the extent of diversity among Schistosoma japonicum Chinese mainland strains, the genetic variation on gene level among 6 isolates collected from Jiangxi, Hunan, Hubei, Anhui, Sichuan, Yunnan and a cultured isolate from a laboratory were studied using molecular biological technique. There were only two different bases at position 112 and 143 in 536 bp sequence of 28S rDNA-D2 domain between Anhui and Yunnan isolates, the homology was 99.6%. The result could be explained the reason of why the migration rates of single chain 28S rDNA-D2 domain among the above 7 isolates were the same completely in PCR-SSCP. However, comparing with the sequences of the Philippine isolate of S. japonicum, S. mansoni and S. aematobium, there were 6, 94 and 93 different bases and the homologies were 98.9, 82.5 and 82.7 per cent separately. With 8 restriction endonucleases to analyze the ITS of rDNA obtained by PCR from the 7 isolates, the results showed that only 3 minor bands were different, e.g. 5.3% of total 58 fragments. It was suggested that the ITS of rDNA among 7 isolates were highly conserved. Using randomly amplified polymorphic DNA (RAPD) to analyze the genetic diversity of the genomes of the 7 isolates, the average genetic distance (D) calculated from total 284 amplified fragments was 0.22. The maximum D was 0.30 and existed between Anhui and Yunnan isolates. The minimum D was 0.13 and existed between Sichuan and Yunnan isolates. The clustering analysis of genetic distances showed that the 7 isolates could be gathered in one group. From above three results, it could be considered that the genetic diversity on gene level among S. japonicum Chinese mainland strains was very low.

Animals↗