Glutamylsulfamoyladenosine and pyroglutamylsulfamoyladenosine are competitive inhibitors of E. coli glutamyl-tRNA synthetase

5'-O-[N-(L-glutamyl)-sulfamoyl] adenosine is a potent competitive inhibitor of E. coli glutamyl-tRNA synthetase with respect to glutamic acid (K(i) = 2.8 nM) and is the best inhibitor of this enzyme. It is a weaker inhibitor of mammalian glutamyl-tRNA synthetase (K(i) = 70 nM). The corresponding 5'-O-[N-(L-pyroglutamyl)-sulfamoyl] adenosine is a weak inhibitor (K(i) = 15 microM) of the E. coli enzyme.     

Adenovirus-mediated overexpression of tissue inhibitor of metalloproteinases-1 in the liver: efficient protection against T-cell lymphoma and colon carcinoma metastasis

BACKGROUND: Matrix metalloproteinases (MMPs) are critical for metastasis of tumor cells. Tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural MMP inhibitor, was shown to reduce metastasis in different models. Here, we investigated whether increased TIMP-1 levels in the liver achieved by adenoviral gene transfer will effectively inhibit liver metastasis of two independent tumor cell lines. METHOD: TIMP-1 was transferred with adenoviral vectors into the livers of DBA/2 and Balb/c mice, which were subsequently challenged by hematogenous experimental metastases of the T-cell lymphoma cell line L-CI.5s or the colorectal carcinoma cell line CT-26, respectively. RESULTS: MMP-9 expression in the liver was induced upon metastasis in both tumor types. Adenoviral gene transfer led to high transduction efficacy as indicated by lacZ expression in 60% of hepatocytes. TIMP-1, a key inhibitor of MMP-9, was expressed at 10(5)-fold higher levels by adenoviral gene transfer as compared with levels achieved in TIMP-1 transgenic mice, previously shown to be inefficient to reduce T-cell lymphoma metastasis. High local and systemic (serum) levels of TIMP-1 led to substantial (94%) reduction of T-cell lymphoma and colorectal carcinoma (73%) experimental liver metastasis. CONCLUSIONS: Adenoviral gene transfer led to systemic and local TIMP-1 levels sufficient to inhibit metastasis of a highly aggressive T-cell lymphoma, pointing at the requirement of threshold levels for effective anti-metastatic efficacy. This approach was also efficient in a colon carcinoma solid tumor model. We propose that viral gene transfer of TIMP-1 can provide a suitable defense strategy to prevent metastatic spread to the liver.     

Follow-up survey of parents of children with major birth defects in New York State

BACKGROUND: One of the objectives of the New York State birth defect surveillance system, the New York State Congenital Malformations Registry (CMR), is to identify children in need of special programs and assist prevention/intervention programs and services with evaluation. In 1999, the CMR started a statewide mass-mailing program to inform parents of children with major birth defects about programs and support groups that might be helpful to them. A follow-up survey by mail was conducted in 2002 to evaluate the usefulness of the mailing program. METHODS: The survey was mailed between September and December of 2001 to parents of children born in 2001 and reported to the CMR with selected major birth defects. Non-respondents were followed-up by telephone. RESULTS: Of the 226 eligible families, 54% (122) responded to the survey. Approximately 66% of responding parents stated that the information provided by the mailing program was helpful, and 48% had contacted and/or used the programs and services listed in the information sheets. Parents with children younger than 6 months of age at the time of mailing were more likely to find the information helpful, compared to parents with children 6 months of age or older. The majority of responding parents had contacted and/or used the Early Intervention Program. CONCLUSION: The findings from the current survey show that the statewide information-mailing program administered by CMR is helpful and useful to the families of children with major birth defects in New York State.     

Protease degradomics: mass spectrometry discovery of protease substrates and the CLIP-CHIP, a dedicated DNA microarray of all human proteases and inhibitors

The biological role of most proteases in vivo is largely unknown. Therefore, to develop robust techniques to analyze the protease degradome in cells and tissues and to elucidate their substrate degradomes we have developed a dedicated and complete human protease and inhibitor microarray that we have called the CLIP-CHIP Oligonucleotides (70-mers) for identifying all 715 human proteases, inactive homologs and inhibitors were spotted in triplicate onto glass slides with a dedicated subarray containing oligonucleotides for specific human breast carcinoma genes. Initial analyses revealed the elevated expression of a number of proteases in invasive ductal cell carcinoma including ADAMTS17, carboxypeptidases A5 and M, tryptase-gamma and matriptase-2. Matrix metalloproteinases (MMPs) showed a restricted expression pattern in both normal and cancerous breast tissues with most expressed at low levels. However, of the several MMPs expressed in significant quantities, the carcinoma samples showed only slightly elevated amounts other than for MMP-28 which was strongly elevated. To discover new protease substrates we developed a novel yeast two-hybrid approach we term 'inactive catalytic domain capture' (ICDC). Here, an inactive mutant protease catalytic domain lacking the propeptide was used as a yeast two hybrid bait to screen a human fibroblast cDNA library for interactor proteins as a substrate trap. Wnt-induced signaling protein-2 (WISP-2) was identified by ICDC and was biochemically confirmed as a new MMP substrate. In another approach we used isotope-coded affinity tag (ICAT) labeling with tandem mass spectrometry to quantitate the levels of secreted or shed extracellular proteins in MDA-MB-231 breast carcinoma cell cultures in the presence or absence of membrane type 1-MMP (MT1-MMP) overexpression. By this proteomic approach we identified and biochemically confirmed that IL-8, the serine protease inhibitor SLPI, the death receptor-6, pro-TNF-alpha and CTGF are novel substrates of MT1-MMP. The utility and quantitative nature of ICAT with MS/MS analysis as a new screen for protease substrate discovery based on detection of cleaved or shed substrate products should be readily adaptable to other classes of protease for assessing proteolytic function in a cellular context.     

A genome annotation-driven approach to cloning the human ORFeome

We have developed a systematic approach to generating cDNA clones containing full-length open reading frames (ORFs), exploiting knowledge of gene structure from genomic sequence. Each ORF was amplified by PCR from a pool of primary cDNAs, cloned and confirmed by sequencing. We obtained clones representing 70% of genes on human chromosome 22, whereas searching available cDNA clone collections found at best 48% from a single collection and 60% for all collections combined.     

Phosphorylation of CREB Ser142 regulates light-induced phase shifts of the circadian clock

Biological rhythms are driven in mammals by a central circadian clock located in the suprachiasmatic nucleus (SCN). Light-induced phase shifting of this clock is correlated with phosphorylation of CREB at Ser133 in the SCN. Here, we characterize phosphorylation of CREB at Ser142 and describe its contribution to the entrainment of the clock. In the SCN, light and glutamate strongly induce CREB Ser142 phosphorylation. To determine the physiological relevance of phosphorylation at Ser142, we generated a mouse mutant, CREB(S142A), lacking this phosphorylation site. Light-induced phase shifts of locomotion and expression of c-Fos and mPer1 in the SCN are significantly attenuated in CREB(S142A) mutants. Our findings provide genetic evidence that CREB Ser142 phosphorylation is involved in the entrainment of the mammalian clock and reveal a novel phosphorylation-dependent regulation of CREB activity.