Identification and Characterization of Two Labeled Intermediates in the Biosynthesis of Rat Thyroglobulin

Abstract Two protein components related to 19 S thyroglobulin and labeled at early times during its biosynthesis have been isolated and characterized. A 3–8 S fraction was prepared from the soluble extract of rat thyroid glands incubated in vitro with radioactive amino acids ([3H]leucine and [14C]isoleucine) or carbohydrates ([3H]- or [14C]mannose and [3H]- or [14C]galactose) or both. From this fraction three components were purified. Two of them, homogeneous by ultracentrifugal and electrophoretic criteria, had sedimentation rates of 6 S and 7 S and molecular weights close to 112,000 and 186,000, respectively. The molar ratios of labeled leucine to isoleucine incorporated into the 6 S and 7 S fractions were the same as those found in 19 S thyroglobulin and in its 12 S subunit. Both the 6 S and 7 S protein units were quantitatively precipitated by anti-rat thyroglobulin antibodies. Double labeling with [3H]leucine and 14C-carbohydrates demonstrated that mannose is present in the fully assembled 19 S and 12 S proteins as well as on the slower sedimenting 6 S and 7 S units, whereas galactose is incorporated only after the formation of the fully assembled stable thyroglobulin molecule. It is concluded that the 6 S and 7 S labeled proteins, which contain only part of the carbohydrate moiety of thyroglobulin, participate in the structure of newly formed unstable 19 S molecules and may represent intermediates during the assembly process of the elementary polypeptide chains of thyroglobulin

The Platelet-derived Growth Factor Controls c-myc Expression through a JNK- and AP-1-dependent Signaling Pathway *

Pro-inflammatory cytokines, environmental stresses, as well as receptor tyrosine kinases regulate the activity of JNK. In turn, JNK phosphorylates Jun members of the AP-1 family of transcription factors, thereby controlling processes as different as cell growth, differentiation, and apoptosis. Still, very few targets of the JNK-Jun pathway have been identified. Here we show that JNK is required for the induction of c-myc expression by PDGF. Furthermore, we identify a phylogenetically conserved AP-1-responsive element in the promoter of the c-myc proto-oncogene that recruits in vivo the c-Jun and JunD AP-1 family members and controls the PDGF-dependent transactivation of the c-myc promoter. These findings suggest the existence of a novel biochemical route linking tyrosine kinase receptors, such as those for PDGF, and c-myc expression through JNK activation of AP-1 transcription factors. They also provide a novel potential mechanism by which both JNK and Jun proteins may exert either their proliferative or apoptotic potential by stimulating the expression of the c-myc proto-oncogene

Activation of the Erk8 mitogen-activated protein (MAP) kinase by RET/PTC3, a constitutively active form of the RET proto-oncogene.

Mitogen-activated protein (MAP) kinases have a central role in several biological functions, including cell adhesion and spreading, chemotaxis, cell cycle progression, differentiation, and apoptosis. Extracellular signal-regulated kinase 8 (Erk8) is a large MAP kinase whose activity is controlled by serum and the c-Src non-receptor tyrosine kinase. Here, we show that RET/PTC3, an activated form of the RET proto-oncogene, was able to activate Erk8, and we demonstrate that such MAP kinase participated in RET/PTC3-dependent stimulation of the c-jun promoter. By using RET/PTC3 molecules mutated in specific tyrosine autophosphorylation sites, we characterized Tyr(981), a known binding site for c-Src, as a major determinant of RET/PTC3-induced Erk8 activation, although, surprisingly, the underlying mechanism did not strictly depend on the activity of Src. In contrast, we present evidence that RET/PTC3 acts on Erk8 through Tyr(981)-mediated activation of c-Abl. Furthermore, we localized the region responsible for the modulation of Erk8 activity by the RET/PTC3 and Abl oncogenes in the Erk8 C-terminal domain. Altogether, these results support a role for Erk8 as a novel effector of RET/PTC3 and, therefore, RET biological functions

Ribonuclease III-mediated processing of specific Neisseria meningitidis mRNAs.

Approx. 2% of the Neisseria meningitidis genome consists of small DNA insertion sequences known as Correia or nemis elements, which feature TIRs (terminal inverted repeats) of 26-27 bp in length. Elements interspersed with coding regions are co-transcribed with flanking genes into mRNAs, processed at double-stranded RNA structures formed by TIRs. N. meningitidis RNase III (endoribonuclease III) is sufficient to process nemis+ RNAs. RNA hairpins formed by nemis with the same termini (26/26 and 27/27 repeats) are cleaved. By contrast, bulged hairpins formed by 26/27 repeats inhibit cleavage, both in vitro and in vivo. In electrophoretic mobility shift assays, all hairpin types formed similar retarded complexes upon incubation with RNase III. The levels of corresponding nemis+ and nemis- mRNAs, and the relative stabilities of RNA segments processed from nemis+ transcripts in vitro, may both vary significantly

Asymmetrical Distribution of Neisseria Miniature Insertion Sequence DNA Repeats among Pathogenic and Nonpathogenic Neisseria Strains

Neisseria miniature insertion sequences (nemis) are miniature DNA insertion sequences found in Neisseria species. Out of 57 elements closely flanking cellular genes analyzed by PCR, most were conserved in Neisseria meningitidis but not in N. lactamica strains. Since mRNAs spanning nemis are processed by RNase III at hairpins formed by element termini, gene sets could selectively be regulated in meningococci at the posttranscriptional level

PCR-based rapid genotyping of Stenotrophomonas maltophilia isolates

BACKGROUND: All bacterial genomes contain repetitive sequences which are members of specific DNA families. Such repeats may occur as single units, or found clustered in multiple copies in a head-to-tail configuration at specific loci. The number of clustered units per locus is a strain-defining parameter. Assessing the length variability of clusters of repeats is a versatile typing methodology known as multilocus variable number of tandem repeat analysis (MLVA). RESULTS: Stenotrophomonas maltophilia is an environmental bacterium increasingly involved in nosocomial infections and resistant to most antibiotics. The availability of the whole DNA sequence of the S. maltophilia strain K279a allowed us to set up fast and accurate PCR-based diagnostic protocols based on the measurement of length variations of loci carrying a variable number of short palindromic repeats marking the S. maltophilia genome. On the basis of the amplimers size, it was possible to deduce the number of repeats present at 12 different loci in a collection of S. maltophilia isolates, and therefore label each of them with a digit. PCR-negative regions were labelled 0. Co-amplification of two pairs of loci provided a 4-digit code sufficient for immediate subtyping. By increasing the number of loci analyzed, it should be possible to assign a more specific digit profile to isolates. In general, MLVA data match genotyping data obtained by PFGE (pulsed-field gel electrophoresis). However, some isolates exhibiting the same PCR profiles at all loci display distinct PFGE patterns. CONCLUSION: The utilization of the present protocol allows to type several S. maltophilia isolates in hours. The results are immediately interpretable without the need for sophisticated softwares. The data can be easily reproducible, and compared among different laboratories