Initial activation of cyclin-B1-cdc2 kinase requires phosphorylation of cyclin B1

At the G₂/M transition of the cell cycle, the cdc25c phosphatase dephosphorylates inhibitory residues of cdc2, and cyclin-B–cdc2 kinase (MPF) is activated. Phosphorylation of cyclin B1 induces its nuclear accumulation, and, since cdc25c is also believed to accumulate and activate shortly before G₂/M in the nucleus, it has been proposed that this induces cyclin-B1–cdc2 kinase activation. We demonstrate that cyclin B1 phosphorylation has another essential function in vivo: it is required for cdc25c and MPF activation, which does not require nuclear accumulation of cyclin B1, and occurs in the cytoplasm.     

Identification of tyrosine residues in constitutively activated fibroblast growth factor receptor 3 involved in mitogenesis, Stat activation, and phosphatidylinositol 3-kinase activation

Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine rendered this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restored its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3.     

Membrane-anchored form of v-sis/PDGF-B induces mitogenesis without detectable PDGF receptor autophosphorylation

The v-sis protein is structurally and functionally related to PDGF. Forms of the v-sis protein which are anchored to the cell membrane via the transmembrane domain of the vesicular stomatitis virus G protein have been previously described (Hannink, M., and D.J. Donoghue. 1986. J. Cell Biol. 103:2311-2322). Several of these fusion proteins were shown to interact productively with the PDGF receptor (PDGFR) based on their ability to transform NIH 3T3 cells. In this report, we further characterized one of these membrane-anchored v-sis proteins, designated v-sis239-G. The gene encoding v-sis239-G was placed under control of the Drosophila melanogaster hsp70 promotor and synthesis of this protein was shown to induce a mitogenic response in NIH 3T3 cells. Unexpectedly, v-sis239-G did not induce detectable autophosphorylation of the PDGFR, in contrast to a similarly expressed secreted form of the v-sis protein. Thus, it appears that a PDGFR-mediated mitogenic response may be dissociated from detectable receptor autophosphorylation. Furthermore, induced synthesis of v-sis239-G was shown to lead to c-fos expression even in the absence of detectable receptor autophosphorylation. Interestingly, a nonmitogenic membrane-anchored form of the v-sis protein, designated v-sis239-G338, also induced c-fos without receptor autophosphorylation. These results raise interesting questions regarding the roles of autophosphorylation and c-fos induction in PDGFR-mediated signal transduction and suggest the possibility of an autophosphorylation-independent signal transduction pathway.     

Stability ofAlternaria toxins in fruit juices and wine

Alternaria alternata is a common fungal parasite on fruits and other plants and produces a number of mycotoxins, including alternariol (3,7,9-trihydroxy-1-methyl-6H-dibenzo [b,d]pyran-6-one), alternariol monomethyl ether (3,7-dihydroxy-9-methoxy-1-methyl-6H-dibenzo[b,d]pyran-6-one), and the mutagen altertoxin I {[1S-(1α,12aβ,12bα)] 1,2,11,12,12a, 12b-hexahydro-1,4,9,12a-tetrahydroxy-3,10-perylenedione}. Alternariol and alternariol monomethyl ether have previously been detected in some samples of fruit beverages. Stability studies of these toxins as well as altertoxin I added to fruit juices and wine (10–100 ng/mL) were carried out. To include altertoxin I in the analysis, cleanup with a polymer-based Varian Abselut solid phase extraction column was used, as recoveries from C-18 columns were low. The stabilities of alternariol and alternariol monomethyl ether in a low acid apple juice containing no declared vitamin C were compared with those in the same juice containing added vitamin C (60 mg/175 ml); there were no apparent losses at room temperature over 20 days or at 80°C after 20 min. in either juice. Altertoxin I was moderately stable in pH 3 buffer (75% remaining after a two week period). Furthermore, altertoxin I was stable or moderately stable in three brands of apple juice tested over 1–27 day periods and in a sample of red grape juice over 7 days. It is concluded that altertoxin I is sufficiently stable to be found in fruit juices and should be included in methods for alternariol and alternariol monomethyl ether.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

Histology of “placoderm” dermal skeletons: Implications for the nature of the ancestral gnathostome

The vertebrate dermal skeleton has long been interpreted to have evolved from a primitive condition exemplified by chondrichthyans. However, chondrichthyans and osteichthyans evolved from an ancestral gnathostome stem‐lineage in which the dermal skeleton was more extensively developed. To elucidate the histology and skeletal structure of the gnathostome crown‐ancestor we conducted a histological survey of the diversity of the dermal skeleton among the placoderms, a diverse clade or grade of early jawed vertebrates. The dermal skeleton of all placoderms is composed largely of a cancellar architecture of cellular dermal bone, surmounted by dermal tubercles in the most ancestral clades, including antiarchs. Acanthothoracids retain an ancestral condition for the dermal skeleton, and we record its secondary reduction in antiarchs. We also find that mechanisms for remodeling bone and facilitating different growth rates between adjoining plates are widespread throughout the placoderms. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

Multiplex PCR assay for the detection and quantification of Campylobacter spp., Escherichia coli O157:H7, and Salmonella serotypes in water samples

Three pathogens, Campylobacter, Salmonella, and Shiga-toxin-producing Escherichia coli, are leading causes of bacterial gastroenteritis in the United States and worldwide. Although these three bacteria are typically considered food-borne pathogens, outbreaks have been reported due to contaminated drinking water and irrigation water. The aim of this research was to develop two types of PCR assays that could detect and quantify three pathogens, Campylobacter spp., E. coli O157:H7, and Salmonella spp., in watershed samples. In conventional PCR, three target strains were detected by multiplex PCR (m-PCR) using each specific primer pair simultaneously. Under optimized m-PCR conditions, the assay produced a 90-bp product for Campylobacter jejuni, a 150-bp product for E. coli O157:H7, and a 262-bp product for Salmonella Typhimurium, and the limitation of detection was approximately 700 copies for all three bacteria. In addition, real-time PCR was performed to quantify the three pathogens using SYBR green fluorescence. The assay was designed so that each target had a different melting temperature [C. jejuni (80.1 °C), E. coli O157:H7 (83.3 °C), and S. Typhimurium (85.9 °C)]. Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction.