The zinc finger repressor, ZBP-89, binds to the silencer element of the human vimentin gene and complexes with the transcriptional activator, Sp1

Vimentin is a component of the eukaryotic cytoskeleton belonging to the family of intermediate filament proteins. It exhibits a complex pattern of tissue- and development-specific expression. It is also a marker of the metastatic potential of many tumor cells. Previously, the human vimentin promoter was shown to contain several regions for the binding of positive and negative acting regulatory factors. Until now, the silencer element, which shuts down vimentin synthesis in selected tissues during development, was not precisely localized; nor was its binding protein known. In vivo DMS footprinting by ligation-mediated PCR delineated the position of guanine residues important to vimentin expression. Transient transfection assays in HeLa cells of various vimentin 5'-end promoter sequences and mutants thereof precisely defined two regulatory elements, a negative element and an adjoining positive acting element. Band shift assays, UV cross-linking, and Southwestern blot analysis confirm that the silencer element specifically binds a protein. Several lines of evidence show that ZBP-89, a zinc finger, Kruppel-like repressor protein is vimentin's silencer element binding factor. Co-immunoprecipitation and DNA affinity chromatography prove that Sp1 heterodimerizes with ZBP-89 when bound to the silencer element to yield a DNA-protein complex whose mobility is indistinguishable from that displayed by HeLa nuclear extract in band shift assays.     

Diverse reactions catalyzed by naphthalene dioxygenase fromPseudomonas sp strain NCIB 9816

Naphthalene dioxygenase (NDO) fromPseudomonas sp strain NCIB 9816 is a multicomponent enzyme system which initiates naphthalene catabolism by catalyzing the addition of both atoms of molecular oxygen and two hydrogen atoms to the substrate to yield enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. NDO has a relaxed substrate specificity and catalyzes the dioxygenation of many related 2- and 3-ring aromatic and hydroaromatic (benzocyclic) compounds to their respectivecis-diols. Biotransformations with a diol-accumulating mutant, recombinant strains and purified enzyme components have established that in addition tocis-dihydroxylation, NDO also catalyzes a variety of other oxidations which include monohydroxylation, desaturation (dehydrogenation),O-andN-dealkylation and sulfoxidation reactions. In several cases, the absolute stereochemistry of the oxidation products formed by NDO are opposite to those formed by toluene dioxygenase (TDO). The reactions catalyzed by NDO and other microbial dioxygenases can yield specific hydroxylated compounds which can serve as chiral synthons in the preparation of a variety of compounds of interest to pharmaceutical and specialty chemical industries. We present here recent work documenting the diverse array of oxidation reactions catalyzed by NDO. The trends observed in the oxidation of a series of benzocyclic aromatic compounds are compared to those observed with TDO and provide the basis for prediction of regio- and stereospecificity in the oxidation of related substrates. Based on the types of reactions catalyzed and the biochemical characteristics of NDO, a mechanism for oxygen activation by NDO is proposed.     

Detection of Cervical Cancer Biomarker Patterns in Blood Plasma and Urine by Differential Scanning Calorimetry and Mass Spectrometry

Improved methods for the accurate identification of both the presence and severity of cervical intraepithelial neoplasia (CIN) and extent of spread of invasive carcinomas of the cervix (IC) are needed. Differential scanning calorimetry (DSC) has recently been shown to detect specific changes in the thermal behavior of blood plasma proteins in several diseases. This methodology is being explored to provide a complementary approach for screening of cervical disease. The present study evaluated the utility of DSC in differentiating between healthy controls, increasing severity of CIN and early and advanced IC. Significant discrimination was apparent relative to the extent of disease with no clear effect of demographic factors such as age, ethnicity, smoking status and parity. Of most clinical relevance, there was strong differentiation of CIN from healthy controls and IC, and amongst patients with IC between FIGO Stage I and advanced cancer. The observed disease-specific changes in DSC profiles (thermograms) were hypothesized to reflect differential expression of disease biomarkers that subsequently bound to and affected the thermal behavior of the most abundant plasma proteins. The effect of interacting biomarkers can be inferred from the modulation of thermograms but cannot be directly identified by DSC. To investigate the nature of the proposed interactions, mass spectrometry (MS) analyses were employed. Quantitative assessment of the low molecular weight protein fragments of plasma and urine samples revealed a small list of peptides whose abundance was correlated with the extent of cervical disease, with the most striking plasma peptidome data supporting the interactome theory of peptide portioning to abundant plasma proteins. The combined DSC and MS approach in this study was successful in identifying unique biomarker signatures for cervical cancer and demonstrated the utility of DSC plasma profiles as a complementary diagnostic tool to evaluate cervical cancer health.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

mRNA stability and antibody production in CHO cells: Improvement through gene optimization

The productivity of stably transfected cell lines is of critical importance for the manufacturing of therapeutic proteins. Various methods have been successfully implemented to increase the production output of mammalian cell cultures. Increasing evidence suggests that optimization of the gene coding sequences of an expression vector can improve specific cell line yield of the recombinant protein. Here we demonstrate that gene optimization substantially enhances antibody production in Chinese hamster ovary cells. When gene optimization was applied to the heavy and light chain genes of a therapeutic antibody, we observed increased antibody production in transient transfection. Elevated heavy chain mRNA level was associated with the increase of antibody production. Further analysis suggested that the increased antibody expression is attributable to enhanced mRNA stability resulting from gene optimization. Gene optimization also led to increased antibody production in stable clones.     

Efficient method for the proteomic analysis of fixed and embedded tissues

Formalin-fixed and paraffin-embedded (FFPE) tissues present a particular challenge for proteomic analysis. Yet, most of the archived tissues in hospitals and tissue banks worldwide are only available in this form. We have developed conditions for removal of the embedding medium and protein digestion, such that informative tryptic peptides are released from fixed proteins which are suitable for analysis by liquid chromatography-mass spectrometry (LC-MS). We demonstrate that the peptide identifications made by this approach compare favorably to those made from matched fresh frozen tissue. Moreover, we demonstrate that a high level of sequence coverage can be observed for proteins of interest.     

PGRL1 Participates in Iron-induced Remodeling of the Photosynthetic Apparatus and in Energy Metabolism in Chlamydomonas reinhardtii

PGRL1 RNA and protein levels are increased in iron-deficient Chlamydomonas reinhardtii cells. In an RNAi strain, which accumulates lower PGRL1 levels in both iron-replete and -starved conditions, the photosynthetic electron transfer rate is decreased, respiratory capacity in iron-sufficient conditions is increased, and the efficiency of cyclic electron transfer under iron-deprivation is diminished. Pgrl1-kd cells exhibit iron deficiency symptoms at higher iron concentrations than wild-type cells, although the cells are not more depleted in cellular iron relative to wild-type cells as measured by mass spectrometry. Thiol-trapping experiments indicate iron-dependent and redox-induced conformational changes in PGRL1 that may provide a link between iron metabolism and the partitioning of photosynthetic electron transfer between linear and cyclic flow. We propose, therefore, that PGRL1 in C. reinhardtii may possess a dual function in the chloroplast; that is, iron sensing and modulation of electron transfer.