Performance of BAC for DBPs precursors' removal for one year with micro-polluted lake water in East-China.

Effectiveness of biological activated carbon (BAC) filter in removing disinfection byproducts (DBPs) precursors of micro-polluted lake water for one year was conducted. The formation potential (FP) of DBPs (trihalomethanes (THMs), haloacetic acids (HAAs) and Nitrosamines (NAs)), dissolved organic carbon (DOC), molecular weight (MW) distribution and excitation emission matrix fluorescence (EEM) of dissolved organic material (DOM) in the influent and effluent of BAC were determined. The results indicated that the removal efficiency (RE) of DOC ranged from 42.9-28.3%. Neither virgin GAC nor long-term operated BAC could efficiently dispose of THMs and HAAs precursors (RE from 35.2-18.8%, from 42 to 8.4%, respectively), however, BAC still showed good ability in removal of NAs precursors after a year operation, of which RE just dropped from 81.7-69.6%. There was strong correlation between RE of NAs precursors and DOC with small MW (<0.5 kDa). The removal of HAAs precursors showed relatively close relation to aromatic protein-like components and soluble microbial pollutants (SMPs). Weak direct relationship was found between the water quality parameters and THMs precursors.

[Clinical and genetic investigation of families with Waardenburg syndrome type 2].

Objective:To investigate the clinical chacteration and molecular pathology of Waardenburg syndrome type 2 in seven families, and provide genetic diagnosis and hereditary counseling for family members. Method:Clinical data of seven families with WS2（14 patients）were collected. Peripheral blood samples of the probands and related family members were collected and genomic DNA was extracted. The coding sequences of microphthalmia associated transcription factor (MITF), sex-determining region Y-box 10(SOX10), snail family zinc finger 2 (SNAI2) and endothelin receptor type B（EDNRB）were analyzed by polymerase chain reaction and DNA sequencing. Then the raw data was analyzed. Result:The most common manifestations of WS2 are sensorineural hearing loss(10/14,71.4%), freckle(7/14, 50.0%)，heterochromia iridis(6/14, 42.9%) and premature greying(5/14,35.7%). All the deafness phenotype is congenital, bilateral profound sensorineural hearing loss. Freckles phenotype is different from cutaneous pigment abnormalities of WS in Westerners. The heterozygous mutation, c.328C>T in exon 3 of the MITF gene was detected in the proband and all patients of pedigree 2. However, no pathological mutation of the relevant genes (SOX10,SNAI2 and EDNRB) was detected in other pedigrees. Conclusion:There are obvious variations in clinical features of WS, while freckles may be a special subtype of cutaneous pigment disturbances. The MITF gene mutation, R110X,is therefore considered the disease causing mutation in pedigree WS02.However, there are novel disease causing genes or copy number variations in Waardenburg syndrome type 2, which require further research.

Taurine transporter is expressed in vascular smooth muscle cells.

The regulation of vascular smooth muscle cells (VSMCs) function by taurine has been a subject of increasing interest and investigation, and taurine is taken up into cells through a specific transporter system, the taurine transporter (TAUT). In the present study, we examined the expression of TAUT in VSMCs and the kinetic parameters of the uptake process of TAUT in VSMCs. RT-PCR and western blot demonstrated that the mRNA and protein of TAUT was expressed in VSMCs in vitro. Immunohistochemistry using antibody for TAUT revealed the expression of this protein in rat thoracic aorta. The maximal [(3)H]taurine uptake rate in VSMCs was 37.75 +/- 3.13 pmol/min per mg of protein, with a K ( m ) value of 5.42 +/- 0.81 microM. Thus, VSMCs are able to express a functional taurine transporter. The regulation and detailed function of taurine and TAUT in VSMCs remain unclear, but our findings suggest a functional role for them in VSMCs metabolism.

GCR1-dependent transcriptional activation of yeast retrotransposon Ty2-917.

Transcription of Saccharomyces cerevisiae Ty2-917 retrotransposon depends on regulatory elements both upstream and downstream of the transcription initiation site. An upstream activation sequence (UAS) and a downstream enhancer stimulate transcription synergistically. Here we show that activation by both of these sites depends on the GCR1 product, a transcription factor which also regulates the genes encoding yeast glycolytic enzymes. Eliminating GCR1 causes a 100-fold decrease in transcription of Ty2-917. Activation by the isolated Ty2-917 UAS also strongly depends on GCR1. Unexpectedly, GCR1-dependent activation by the Ty2-917 enhancer is strongly position-dependent. Activation by the enhancer in its normal position within the transcription unit depended strongly on GCR1, but eliminating GCR1 reduced activation only three-fold when the enhancer was moved upstream of the transcribed region. Gel mobility shift and DNaseI protection assays indicated that GCR1 binds specifically to multiple sites within the Ty2-917 UAS and enhancer regions.

Morphological analysis of lymph vessels and capillaries in gastric carcinoma.

AIM: To investigate the morphology of lymph vessel capillaries in both human gastric carcinomas and their peritumoral tissues, as well as the relation of this morphology to lymphatic metastasis. METHODS: The morphology and fine distribution of both lymph vessels and capillaries in and around the primary foci of gastric carcinoma were studied using the 5'Nase Alpase double staining method. The total amount of opened vessels and the opening rate of lymph vessels and capillaries were counted using a light microscope (100 × magnification), and the maximal luminal area, perimeter and diameter were measured using an image analysis technique. RESULTS: Lymph vessels and capillaries displayed strong 5'Nase-positive staining (brown and dark brown), while blood vessels and capillaries revealed significant Alpase activity (blue). There were many lymph vessels, capillaries and solid strip-like tissues found in the gastric carcinoma samples analyzed. The total amount of lymphatics in the metastatic group (gastric carcinoma vs peritumoral tissue) and non-metastatic group was 26.9 ± 14.2 vs 10.4 ± 4.0, 11.4 ± 3.4 and 9.7 ± 3.2, P < 0.01, respectively. Their opening rates were 21.2 vs 47.5 and 40.4 vs 46.0, P < 0.01, respectively. Their maximal luminal areas were 1502.98 ± 1236.91 vs 5526.80 ± 4853.42; 1918.14 ± 2299.24 vs 3836.16 ± 3549.16; 5526.80 ± 4853.42 vs 3836.16 ± 3549.16, P < 0.05, their perimeters were 220.33 ± 130.25 vs 441.43 ± 276.51; 241.79 ± 171.13 vs 333.80 ± 199.66; 441.43 ± 276.51 vs 333.80 ± 199.60, P < 0.05, and their diameters were 28.80 ± 14.98 vs 59.39 ± 28.53; 25.37 ± 15.79 vs 46.22 ± 20.85; 59.39 ± 28.53 vs 46.22 ± 20.85, P < 0.05, respectively. In summary, the lymphatics found in gastric carcinoma samples from the metastatic group were significantly lower than those of the other groups. CONCLUSION: There are newly formed lymph capillaries found in gastric carcinoma. Dilation of lymph capillaries may be related to edema found in peritumoral connective tissues. The observed lymph node metastases from gastric carcinoma occur through mature lymph capillaries that invade in and around primary gastric carcinoma foci.

The rhombotin gene family encode related LIM-domain proteins whose differing expression suggests multiple roles in mouse development.

The rhombotin (RBTN1 or Ttg-1) gene was first identified at a chromosome translocation in a T-cell acute leukaemia and later used to isolate two related genes (RBTN2 or Ttg-2 and RBTN3). Complete characterization of these genes in man and mouse shows that all three encode cysteine-rich proteins with typical LIM domains. RBTN1 and RBTN3-derived proteins have 98% identity in the LIM domains but are located on separate chromosomes in man and in mouse while RBTN1 and RBTN2, both located on human chromosome 11p but are on separate chromosomes in mouse, are only 48% identical in this part of the protein. The exon organization of RBTN1 and RBTN3 genes are similar, both having an intron, absent from the RBTN2 gene, in the LIM2-encoding region. The remarkable similarity between rbtn-1 and rbtn-3 proteins is parallelled in their expression patterns in mouse development, since both genes show high expression in restricted areas of the brain, but little lymphoid expression. rbtn-2 expression, however, is more ubiquitous. This gene shows a low level of thymus expression but high expression in fetal liver, adult spleen and B-cell lines, consistent with a role in B-cell development. These results suggest multiple cellular targets for the action of these proteins during development.

Specific interaction of the first three zinc fingers of TFIIIA with the internal control region of the Xenopus 5 S RNA gene.

A DNA plasmid encoding the first 101 amino acid residues of the Xenopus 5 S RNA gene-specific transcription factor IIIA (TFIIIA) was derived by polymerase chain reaction amplification of this region from the cDNA for TFIIIA. This polypeptide includes the first three zinc fingers of the TFIIIA DNA binding domain. The polypeptide was expressed in Escherichia coli and purified to greater than 95% homogeneity. The three finger polypeptide binds the internal control region of the 5 S RNA gene with sequence specificity and high affinity. Binding is metal-dependent and treatment of the polypeptide with EDTA abolishes binding. Polypeptide-DNA complexes exhibit a dissociation constant of 5.6(+/- 0.9) nM, while that for full-length Xenopus TFIIIA is 2.2(+/- 0.4) nM, measured under the same conditions. This suggests that the majority of the free energy of TFIIIA binding resides in these amino-terminal zinc fingers. The polypeptide protects 21 base-pairs of the internal control region from attack by DNase I, with protection from nucleotides +75 to +95 of the 120 base-pair gene. This region includes the C-block promoter element and several guanine residues that are essential for TFIIIA binding. Methylation interference experiments suggest that the mode of binding of the polypeptide and TFIIIA are similar. The minimal DNA sequences required for polypeptide binding were determined using a series of synthetic double-stranded deoxyribo-oligonucleotides. A 13 base-pair oligonucleotide spanning nucleotides +80 to +92 of the 5 S RNA gene retained specific and high-affinity binding, although the latter was reduced sixfold relative to longer DNA fragments. Polypeptides containing fingers 1 and 2, or fingers 2, 3 and 4 of TFIIIA do not exhibit sequence-specific DNA binding. Overall, these studies provide strong support for a model in which the first three zinc fingers of TFIIIA bind with high affinity between nucleotides +80 and +92 of the internal control region of the 5 S RNA gene.

A simple high-efficiency method for random mutagenesis of cloned genes using forced nucleotide misincorporation.

We describe a simple protocol for introducing random point mutations into cloned DNA fragments via forced misincorporation of deoxynucleoside triphosphates (dNTPs) by either of two polymerases which lack proofreading activity, reverse transcriptase or a mutant T7 DNA polymerase. A high ratio of one nucleotide to the other three is used to enhance the error rate. Mutagenesis is initiated from a specific primer and restricted to a relatively short segment by limiting the amounts of the nonmutagenic dNTPs. Our method is highly efficient, resulting in the isolation of greater than 50% mutant plasmids with most polymerase/forcing dNTP combinations. A wide spectrum of mutations can be obtained, in contrast to commonly used methods for random mutagenesis.

Enhancer and silencerlike sites within the transcribed portion of a Ty2 transposable element of Saccharomyces cerevisiae.

The Ty2-917 element is a member of the Ty2 class of retroviruslike transposable elements of Saccharomyces cerevisiae. We showed that regions downstream of the Ty2-917 transcription start site modulate its transcription. One region was located downstream of the transcription initiation site (position 240) and within the first 559 base pairs of the element. This region had a dramatic effect, causing an approximately 1,000-fold increase in steady-state levels of RNA. The region stimulated transcription when placed in either orientation upstream of a heterologous gene, HIS4, lacking its own upstream activation sequence (UAS). We termed this positively acting region an enhancer, by analogy to sites described in higher cells, to distinguish it from yeast UASs which do not function when placed within the transcribed portion of the gene. Though, like some higher eucaryotic enhancers, the Ty2-917 enhancer is located within the transcribed region, it is unlike them in that it occurs within a coding region rather than in an intron. The Ty2-917 enhancer and the Ty2-917 UAS had a synergistic effect on transcription, together stimulating transcription 15-fold over the predicted additive effect. We also identified a site which decreases RNA accumulation, located about 750 base pairs into the element. This site functioned in only one orientation when inserted upstream of the UAS-less heterologous gene. The site was similar to silencers, or negative enhancers, in that it acted to repress transcription from outside the transcribed region, but was distinct in that the function of a canonical silencer was independent of orientation.

Genetic analysis of Schizosaccharomyces pombe 7SL RNA: a structural motif that includes a conserved tetranucleotide loop is important for function.

We have studied the effects of mutations in a 6-base segment of Schizosaccharomyces pombe 7SL RNA, which lies within a 35-nucleotide domain whose sequence and secondary structure are conserved in RNAs from many divergent organisms, including the 7SL component of human signal recognition particle (SRP). Surprisingly, many changes in this region can be tolerated under normal growth conditions. An exception is the lethality of several mutations at positions 159 and 160, 2 nucleotides previously shown to be protected from RNase digestion by the 19-kDa canine SRP protein. Nucleotide 160 is, in addition, the most highly conserved base in a consensus sequence for the most common tetranucleotide loop in ribosomal RNAs. Mutations that are likely to affect the stability and/or conformation of the RNA give rise to a conditional phenotype: when osmolarity of the medium is raised, the RNAs become partially or completely defective in function at high temperature.

The upstream activation site of a Ty2 element of yeast is necessary but not sufficient to promote maximal transcription of the element.

The Ty elements of yeast are a family of retrovirus-like transposons that are highly transcribed, accounting for about 10% of total mRNA. We have mapped two sites to the nontranscribed region of the element upstream of the transcription start site that are required for maximal gene expression and are similar to sites previously defined in other genes. One, the TATA site, is located 74 base pairs upstream of the transcription start site and has the canonical sequence TATAAAA. This site is required for normal rates of initiation; deletion of the site greatly reduces the amount of Ty917 mRNA without changing its 5' end. A second site is located in a region from 140 to 110 base pairs upstream of the start site. Unlike other upstream activation site (UAS) elements previously defined, the Ty917 UAS is not sufficient to promote any transcription in the absence of downstream transcription regulatory sites. Thus the UAS is necessary but not sufficient for maximal transcription. Comparison of constructs lacking either the UAS or the downstream enhancer or both shows evidence of synergistic interaction between the sites since the effect of the sites on the rate of transcription initiation is more than additive.