The Drosophila EGF receptor gene homolog: conservation of both hormone binding and kinase domains

Chicken v-erB probe was used to isolate a unique clone of Drosophila melanogaster DNA. It maps by in situ hybridization to position 57F on chromosome 2. A complete nucleotide sequence of the coding region has been obtained. The putative Drosophila EGF receptor protein is similar in overall organization to the human homolog. It shows three distinct domains: an extracellular putative EGF binding domain, a hydrophobic transmembrane region, and a cytoplasmic kinase domain. The overall amino acid homology is 41% in the extracellular domain and 55% in the kinase domain. Two cysteine-rich regions, a hallmark of the human ligand-binding domain, have also been conserved. Fusion of the coding sequences of the kinase and extracellular domains generating the receptor gene must have occurred over 800 million years ago.     

3H-oestradiol-17β counter current transfer from the ovarian vein into the ovarian artery in cows

During laparatomy the ovary in the luteal phase with the ovarian pedicle was isolated and transferred under stereomicroscope. The ovary was supplied with blood flowing out of the facial artery through a cannula. ³H-oestradiol-17β or ⁵¹Cr-labelled red blood cells (⁵¹Cr-RBC) were infused for 30 min through the cannula inserted into the ovarian vein 3 cm below the ovary. During and 30 min after the ³H-oestradiol-17β infusion, radioactivity was found both in the ovarian arterial blood near the ovary and in ovarian tissue. When ⁵¹Cr-RBC were infused in the same way as ³H-oestradiol, there was no radioactivity in the arterial blood or ovarian tissue. These experiments indicate the existence of a counter current transfer of ³H-oestradiol-17β from the ovarian vein into the ovarian artery in a cow's ovarian pedicle.     

Ultra-diffuse hydrothermal venting supports Fe-oxidizing bacteria and massive umber deposition at 5000?m off Hawaii

A novel hydrothermal field has been discovered at the base of Lōihi Seamount, Hawaii, at 5000 mbsl. Geochemical analyses demonstrate that ‘FeMO Deep'', while only 0.2 °C above ambient seawater temperature, derives from a distal, ultra-diffuse hydrothermal source. FeMO Deep is expressed as regional seafloor seepage of gelatinous iron- and silica-rich deposits, pooling between and over basalt pillows, in places over a meter thick. The system is capped by mm to cm thick hydrothermally derived iron-oxyhydroxide- and manganese-oxide-layered crusts. We use molecular analyses (16S rDNA-based) of extant communities combined with fluorescent in situ hybridizations to demonstrate that FeMO Deep deposits contain living iron-oxidizing Zetaproteobacteria related to the recently isolated strain Mariprofundus ferroxydans. Bioenergetic calculations, based on in-situ electrochemical measurements and cell counts, indicate that reactions between iron and oxygen are important in supporting chemosynthesis in the mats, which we infer forms a trophic base of the mat ecosystem. We suggest that the biogenic FeMO Deep hydrothermal deposit represents a modern analog for one class of geological iron deposits known as ‘umbers'' (for example, Troodos ophilolites, Cyprus) because of striking similarities in size, setting and internal structures.     

Three clusters of conformational states in p450cam reveal a multistep pathway for closing of the substrate access channel

Conformational changes in the substrate access channel have been observed for several forms of cytochrome P450, but the extent of conformational plasticity exhibited by a given isozyme has not been completely characterized. Here we present crystal structures of P450cam bound to a library of 12 active site probes containing a substrate analogue tethered to a variable linker. The structures provide a unique view of the range of protein conformations accessible during substrate binding. Principal component analysis of a total of 30 structures reveals three discrete clusters of conformations: closed (P450cam-C), intermediate (P450cam-I), and fully open (P450cam-O). Relative to P450cam-C, the P450cam-I state results predominantly from a retraction of helix F, while both helices F and G move in concert to reach the fully open P450cam-O state. Both P450cam-C and P450cam-I are well-defined states, while P450cam-O shows evidence of a somewhat broader distribution of conformations and includes the open form recently seen in the absence of substrate. The observed clustering of protein conformations over a wide range of ligand variants suggests a multistep closure of the enzyme around the substrate that begins by conformational selection from an ensemble of open conformations and proceeds through a well-defined intermediate, P450cam-I, before full closure to the P450cam-C state in the presence of small substrates. This multistep pathway may have significant implications for a full understanding of substrate specificity, kinetics, and coupling of substrate binding to P450 function.     

Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids

Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable to form pcPNA–pcPNA duplexes but can bind to complementary DNA sequences by Watson–Crick pairing via double duplex-invasion complex formation. We show that psoralen-conjugated pcPNAs can deliver site-specific photoadducts and mediate targeted gene modification within both episomal and chromosomal DNA in mammalian cells without detectable off-target effects. Most of the induced psoralen-pcPNA mutations were single-base substitutions and deletions at the predicted pcPNA-binding sites. The pcPNA-directed mutagenesis was found to be dependent on PNA concentration and UVA dose and required matched pairs of pcPNAs. Neither of the individual pcPNAs alone had any effect nor did complementary PNA pairs of the same sequence. These results identify pcPNAs as new tools for site-specific gene modification in mammalian cells without purine sequence restriction, thereby providing a general strategy for designing gene targeting molecules.     

MAGEB2 is Activated by Promoter Demethylation in Head and Neck Squamous Cell Carcinoma

Purpose

Although promoter hypermethylation has been an accepted means of tumor suppressor gene inactivation, activation of otherwise normally repressed proto-oncogenes by promoter demethylation has been infrequently documented.

Experimental Design

In this study we performed an integrative, whole-genome analysis for discovery of epigenetically activated proto-oncogenes in head and neck cancer tumors. We used the 47K GeneChip U133 Plus 2.0 Affymetrix expression microarray platform to obtain re-expression data from 5-aza treated normal cell line and expression data from primary head and neck squamous cell carcinoma (HNSCC) tumor tissues and normal mucosa tissues. We then investigated candidate genes by screening promoter regions for CpG islands and bisulfite sequencing followed by QUMSP and RT PCR for the best candidate genes. Finally, functional studies were performed on the top candidate gene.

Results

From the top 178 screened candidates 96 had CpG islands in their promoter region. Seven candidate genes showed promoter region methylation in normal mucosa samples and promoter demethylation in a small cohort of primary HNSCC tissues. We then studied the demethylation of the top 3 candidate genes in an expanded cohort of 76 HNSCC tissue samples and 17 normal mucosa samples. We identified MAGEB2 as having significant promoter demethylation in primary head and neck squamous cell carcinoma tissues. We then found significantly higher expression of MAGEB2 in tumors in a separate cohort of 73 primary HNSCC tissues and 31 normal tissues. Finally, we found that MAGEB2 has growth promoting effects on minimally transformed oral keratinocyte cell lines but not a definite effect on HNSCC cell lines.

Conclusion

In conclusion, we identified MAGEB2 as activated by promoter demethylation in HNSCCand demonstrates growth promoting effects in a minimally transformed oral keratinocyte cell line. More studies are needed to evaluate MAGBE2''s exact role in HNSCC.     

Egg size and composition in an ageing capital breeder – consequences for offspring performance

1. Egg size is often used as a proxy of egg quality although size and composition may vary, e.g. in insects egg size usually decreases as female ages. Whether this decrease in size reflects reduced concentrations of essential nutrients such as lipids and proteins of eggs laid by ageing females, or does reduced size per se explain often observed lower fitness of later laid eggs is poorly explored. 2. Egg properties were compared with fitness parameters of offspring laid on the first and fourth night during the oviposition period of a capital breeding moth, Cleorodes lichenaria (Hufnagel). The study aim was to explore whether decreased egg size is caused by decreased provisioning into later laid eggs measured as egg protein and lipid concentration, and whether it results in lower fitness of later laid offspring. 3. The fresh and dry weight of eggs decreased over the oviposition period, but the protein and lipid concentration remained constant. Survival of larvae was lower among the fourth night laid offspring on a low quality host Parmelia sulcata Taylor compared to a high quality host Ramalina fraxinea (L.) Ach. No differences were observed in egg fertility or hatchability, neonate survival without food and pupal mass between the offspring produced on different nights. 4. Decreased survival of offspring produced later was rather attributable to absolute provisioning (i.e. lower weight of eggs) than relative provisioning (i.e. decreased concentrations of nutrients in eggs). It is argued that lower survival of later laid smaller eggs on low quality diet is likely attributable to physical and chemical characteristics of host lichens and/or physical properties of tiny neonate larvae.     

CHK2-dependent phosphorylation of BRCA1 in hypoxia

Hypoxia induces a diverse spectrum of changes in the expression and activity of numerous DNA repair factors within the tumor microenvironment. In particular, we and others have shown that hypoxia induces phosphorylation and activation of the checkpoint kinase, CHK2, in an ATM-dependent manner. One downstream target of CHK2, the BRCA1 protein, plays a critical role in both DNA repair and cell cycle checkpoint regulation in mammalian cells. Here we report that BRCA1 is specifically phosphorylated on Serine 988 in response to hypoxic stress, and phosphorylation at this site is dependent on CHK2 expression. These findings enhance our understanding of ATM-CHK2 pathway activation in hypoxia, and they identify a novel role for BRCA1 in the response to hypoxic stress.