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Ovine 6-phosphogluconate dehydrogenase (6PGD), an enzyme of the pentose phosphate pathway, provides the necessary compounds of NADPH for the synthesis of fatty acids. Much of research has been conducted not only on the enzymatic level, but also on molecular level elucidating its cDNA sequence. Herein, we tried to elucidate if any correlation between enzymatic activity and expression of ovine 6PGD gene exists, in respect to two different weights from weaning to 4 months old. 18 male and 16 female lambs of Chios breed were randomly selected after weaning and assigned to two groups based on sex in a different experimental open-plan shed. Two subgroups were defined in each sex and they were slaughtered at 25 kg and 30 kg, respectively. Samples of adipose tissue (tail, perirenal and shoulder site) were collected and 6PGD enzymatic activity, gene expression, and characteristics of adipocytes were determined. According to the determined data, tail subcutaneous adipose tissue matures later than the others examined tissues and has a diminished lipogenic activity. A 6PGD gene expression pattern was not followed by analogous changes of its enzymatic activity, suggesting that other mechanisms such as post transcription or/and post translation regulation may be involved. [ABSTRACT FROM AUTHOR]

Abstract: The steroid/sterol sulfotransferase gene (SULT2B1) encodes for two isozymes of which one (SULT2B1b) sulfonates cholesterol and is selectively expressed in skin. The human SULT2B1 gene contains neither a TATAAA nor a CCAAT motif upstream of the coding region for SULT2B1b; however, this area is GC-rich. Of five Sp1 elements identified two had regulatory activity utilizing immortalized human keratinocytes: one element is located above the ostensible transcription initiation site, whereas the other is located within the 5′-untranslated region of the SULT2B1b mRNA. Sp1 and Sp2 transcription factors identified by supershift analyses induced reporter gene activity, an effect markedly augmented by histone deacetylase inhibition. [Copyright &y& Elsevier]

Excessive exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues including the breast. 4-Hydroxyequilenin (4-OHEN), a major catechol metabolite of equine estrogens present in Premarin®, an estrogen replacement formulation, has been shown to induce apoptosis and DNA damage in human breast cancer cells. It also has the potential to be a tumor initiator or promoter and complete carcinogen. To further understand the effects and mechanisms of equine catechol estrogen metabolite 4-OHEN action in vitro, human non-tumorigenic mammary epithelial MCF 10A cell line was used to study the toxic effects of 4-OHEN. In this study, we observed that 4-OHEN caused dose-dependent increases in apoptosis and DNA damage as measured by the DAPI nuclear screening assay and the Comet assay, respectively. Interestingly, cells treated with 100nM 4-OHEN biweekly for 4 weeks became resistant to cisplatin-induced apoptosis. The resistance to apoptosis of the 100nM 4-OHEN-treated cells was through multiple regulatory mechanisms. Compared to the DMSO-treated cells, the 100nM 4-OHEN-treated cells had higher GSH levels and total SOD activity, and a stronger GSH response after cisplatin treatment. Expression levels of several genes involved in cell growth, DNA repair, and apoptosis were either up- or down-regulated. These data indicate that long-term low-level equine estrogen metabolite exposure could induce DNA damage and initiate cells to become resistant to apoptosis. [Copyright &y& Elsevier]

Abstract: Calcitonin inhibits bone resorption by acting on osteoclasts via a specific receptor. The calcitonin receptor (CTR) is also found in many other normal and malignant tissues and cell lines. It has been cloned and sequenced in several species including humans. It belongs to a subclass of seven-transmembrane G protein-coupled receptors. Four human CTR (H-CTR) isoforms generated by alternatively spliced mRNA have previously been described. Two H-CTR encoding DNAs containing an unidentified 50-bp insert are now reported from T47D cells. The 50-bp insert corresponds to a DNA region located between exon 9 and exon 10, and appears to originate from an alternative splicing process. The two H-CTR cDNAs encode 274 and 290 aa long isoforms. Both are deleted from the putative fourth transmembrane domain to C-tail. They differ by the presence (H-CTR5) or absence (H-CTR6) of a previously known 16-aa insert in the putative first intracellular loop. Cell- and tissue-distribution analysis using RT-PCR demonstrates that the shorter one, HCTR6, is more prevalent. The mRNA of both isoforms was detected in giant cell tumor, whereas only H-CTR6 mRNA was detected in TT cells and kidney tissue. Neither H-CTR5 nor H-CTR6 could be detected in peripheral blood mononuclear cells cultured in the presence of RANKL, in MCF7 cells, and in cortical brain and ovarian tissues. When H-CTR6 was transiently expressed in HEK293 cells, CT failed to induce production of cAMP or to bind to the receptor. These suggest either an intrinsic loss of ligand binding function, or an altered intracellular trafficking. Our findings therefore indicate the existence of two novel splice variants of the H-CTR and confirm that multiple splicing patterns could be involved in the post-transcriptional regulation of the gene. [Copyright &y& Elsevier]

There is a growing family of novel GTPases conserved among higher plants and vertebrates, abbreviated as AIG1, IAP, IMAP, and IAN, respectively. Here, we comparatively analyze the human gene family encoding GTPases of the immunity-associated protein family recently re-termed GIMAP. Chromosome 7q36.1 contains, within 300 kb, a gimap gene cluster with seven functional genes and one pseudogene (hgimap3). The six genes hgimap1, hgimap2, hgimap4, hgimap5, hgimap6, and hgimap7 encode 33–46 kDa proteins with one GTP-binding domain, whereas hgimap8 encodes a very unusual 75-kDa protein with three GTP-binding domains. All hgimap genes except hgimap2 have orthologs in the mouse. Major expression sites of hgimap mRNAs are the spleen and lymph nodes, but also other organs such as muscle, heart, placenta, and digestive tract display detectable hgimap mRNA levels. The proteins hGIMAP4 and hGIMAP7 can be localized at ER and Golgi apparatus, but not in mitochondria, lysosomes and nuclei. All hgimap genes were expressed at very low levels—if at all—in diverse cancer cell lines. Our data support the view that the GIMAP proteins are involved in the control of cell survival not only in cells of the immune system as commonly anticipated. [Copyright &y& Elsevier]

During the complex life cycle of Plasmodium falciparum, through mosquito and human, the erythrocytic cycle is responsible for malarial disease and transmission. The regulation of events that occur during parasite development, such as proliferation and differentiation, implies a fine control of transcriptional activities that in turn governs the expression profiles of sets of genes. Pathways that underline gametocyte commitment are yet poorly understood even though kinases and transcription factors have been assumed to play a crucial role in this event. In order to understand the molecular mechanisms controlling the variation of gene expression profiles that might participate in early gametocytogenesis, the transcriptome of two clones, 3D7 and its gametocyte-less derivative F12, was compared at five time points of the erythrocytic asexual development. We have used a thematic DNA microarray containing 150 PCR fragments, representative of P. falciparum genes involved in signal transduction, cell cycle and transcriptional regulation. We identified several genes eliciting different expression profiles among which some implicated in gene regulation or encoding putative transcription factors. The differential expression of transcription factor and kinase transcripts observed in the two clones may enlighten genes that might have a role in impairment of the early gametocytogenesis of the F12 clone. [Copyright &y& Elsevier]

Small interfering RNAs (siRNAs) are a key component of RNA silencing, including cosuppression. Here, we show an example in which siRNA does not serve in the downregulation of target genes. A tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) catalyzes the formation of α-linolenate (18:3). Introduction of the NtFAD3 gene into tobacco plants caused strong reduction of 18:3 content in leaf tissues, which is associated with the production of the NtFAD3 siRNAs. However, this silencing effect was lacking in the root tissues. Both the introduced NtFAD3 and endogenous NtFAD3 genes were expressed successfully, and the roots showed increased 18:3 phenotype. Surprisingly, the NtFAD3 siRNAs were produced even in the root tissues. Expression of a hairpin double-stranded RNA against the NtFAD3 gene caused efficient reduction of 18:3 content in root tissues. Therefore, cosuppression of the NtFAD3 gene in tobacco appears to include an as yet unidentified developmental stage and tissue-specific mechanism of regulation of siRNA function. [Copyright &y& Elsevier]

All plants examined to date possess non-symbiotic hemoglobin whose physiological role remains unclear. The present study explored the catalytic function of three representative classes of the plant hemoglobin from Arabidopsis thaliana: AtGLB1, AtGLB2, and AtGLB3. Purified recombinant proteins of these hemoglobins displayed hydrogen peroxide-dependent oxidation of several peroxidase substrates that was sensitive to cyanide, revealing intrinsic peroxidase-like activity. In the presence of nitrite and hydrogen peroxide, AtGLB1 was the most efficient at mediating tyrosine nitration of its own and other proteins via the formation of reactive nitrogen species as a result of nitrite oxidation. AtGLB1 mRNA significantly accumulated in Arabidopsis seedlings exposed to nitrite, supporting the physiological relevance of its function to nitrite and nitrite-derived reactive nitrogen species. [Copyright &y& Elsevier]

SelS is a newly identified selenoprotein and its gene expression is up-regulated in the liver of Psammomys obesus after fasting. We have examined whether SelS is regulated by glucose deprivation and endoplasmic reticulum (ER) stress in HepG2 cells. Glucose deprivation and the ER stress inducers tunicamycin and thapsigargin increased SelS gene expression and protein content several-fold in parallel with glucose-regulated protein 78. The overexpression of SelS increased Min6 cell resistance to oxidative stress-induced toxicity. These results indicate that SelS is a novel member of the glucose-regulated protein family and its function is related to the regulation of cellular redox balance. [Copyright &y& Elsevier]

A set of genomic DNA sequences putatively encoding front-end desaturases were identified by in silico analysis of the draft genome of the marine microalga Thalassiosira pseudonana. Among these candidate genes, an open reading frame named TpdesN was found to be full-length, intronless, and constitutively expressed during cell cultivation. The predicted amino acid sequence of the corresponding protein, TpDESN, exhibited typical features of desaturases involved in the production of polyunsaturated fatty acids (PUFAs) in algae, i.e. a cytochrome b5-like domain at the N-terminus and three conserved histidine-rich motifs in the desaturase domain. Expression of TpDESN in Saccharomyces cerevisiae revealed that this enzyme was not involved in PUFA synthesis, but specifically desaturated palmitic acid 16:0 to 16:1Δ11. To our knowledge, until this report, Δ11-desaturase activity had only been detected in insect cells. [Copyright &y& Elsevier]

The genes for heat shock proteins (Hsps) can be upregulated in response to cellular trauma, resulting in enhanced cell survival and protection. Hsp32, also known as heme oxygenase 1, catalyzes the degradation of heme to produce carbon monoxide and bilirubin, which play a variety of cytoprotective functions at physiological concentrations, and iron, which is rapidly sequestered by the iron-binding protein ferritin. In the present study we examined the expression and localization of Hsp32 in the rat cochlea after heat shock using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry. Low levels of constitutive Hsp32 expression were observed in the normal rat cochlea by RT-PCR and Western blot. Hsp32 mRNA (messenger RNA) was present at higher levels in a subfraction containing sensorineural epithelium and lateral wall than in a subfraction containing modiolus. Western blot revealed that Hsp32 protein levels increase in the rat cochlea following heat shock. Immunocytochemistry showed scattered staining of outer hair cells in the organ of Corti of normal untreated rats. Following heat shock Hsp32 is upregulated in outer hair cells and the cells of the stria vascularis. These results suggest a potential role for Hsp32 as a component of the oxidative stress response pathway in the rat cochlea. [Copyright &y& Elsevier]

Differential mRNA display revealed that a cDNA encoding the major urinary protein 2 (MUP2) that belongs to the lipocalin superfamily was absent in livers of mice treated with 3-methylcholanthrene (MC). The expression of MUP2 is known to be stimulated by growth hormone (GH), through the GH receptor (GHR), Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) signal transduction pathway. Since MC is an aryl hydrocarbon receptor (AhR) ligand, the effects of MC treatment on the expression of GHR, JAK2 or STAT5 in the livers of wild-type or AhR-null mice were examined. The result indicated that the expression of GHR and JAK2 mRNA was greatly decreased by MC in wild-type mice but not in AhR-null mice. In addition, the binding activity of STAT5 bound to STAT5-binding element was reduced after MC treatment in wild-type mice but not in AhR-null mice. Based on these results, we conclude that the suppression of MUP2 mRNA expression by MC is caused by the AhR-mediated disruption of the GH signaling pathway. Possible mechanism(s) by which exposure to aromatic hydrocarbons causes a decrease in the body weight of mice, which has been referred to as wasting syndrome, will also be discussed. [Copyright &y& Elsevier]

Interleukin 4 (IL-4) is expected to play a dominant role in the development of T helper (Th) 2 cells. Th2 immune responses with expression of relatively large amounts of interleukin 4 (IL-4) but little interferon γ (IFN-γ) are characteristic for chronic helminth infections. But no information is available about IL4 expression during early Fasciola hepatica (F. hepatica) infections in cattle. Therefore, we investigated F. hepatica specific IL-4 and IFN-γ mRNA expression in peripheral blood mononuclear cells (PBMCs) from calves experimentally infected with F. hepatica. Cells were collected prior to infection and on post-inoculation days (PIDs) 10, 28 and 70. Interestingly, PBMCs responded to stimulation with F. hepatica secretory–excretory products (FhSEP) already on PID 10 and expressed high amounts of IL-4 but not of IFN-γ mRNA suggesting that F. hepatica induced a Th2 biased early immune response which was not restricted to the site of infection. Later in infection IL-4 mRNA expression decreased whereas IFN-γ mRNA expression increased slightly. Isolated lymph node cells (LNCs) stimulated with FhSEP and, even more importantly, non-stimulated LN tissue samples indicated highly polarized Th2 type immune responses in the draining (hepatic) lymph node, but not in the retropharyngeal lymph node. During preliminary experiments, two splice variants of bovine IL-4 mRNA, boIL-4δ2 and boIL-4δ3, were detected. Since a human IL-4δ2 was assumed to act as competitive inhibitor of IL-4, it was important to know whether expression of these splice variants of bovine IL-4 have a regulatory function during an immune response to infection with F. hepatica. Indeed, IL-4 splice variants could be detected in a number of samples, but quantitative analysis did not yield any clue to their function. Therefore, the significance of bovine IL-4 splice variants remains to be determined. [Copyright &y& Elsevier]

Pavlova lutheri, a marine microalga, is rich in the very long chain polyunsaturated fatty acids (VLCPUFAs) eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids. Using an expressed sequence tag approach, we isolated a cDNA designated Pldes1, and encoding an amino acid sequence showing high similarity with polyunsaturated fatty acid front-end desaturases. Heterologous expression in yeast demonstrated that PlDES1 desaturated 22:5n-3 and 22:4n-6 into 22:6n-3 and 22:5n-6 respectively, and was equally active on both substrates. Thus, PlDES1 is a novel VLCPUFA Δ4-desaturase. Pldes1 expression is four-fold higher during the mid-exponential phase of growth compared to late exponential and stationary phases. [Copyright &y& Elsevier]

Development of methods based on determining expression of individual genes resulted in the need for large amounts of high quality RNA preparations. It is widely accepted that in intact rRNA the 28S and 18S band ratio must be 2:1. It is not quite clear what is the main cause of lower rRNA bands intensity ratio. It is difficult to isolate RNA with 2:1 28S/18S ratio from RNase-rich and some tumor tissues. At the same time this requirement may be excessive and RNA preparations with lower 28S/18S rRNA ratio may be quite adequate for most techniques of determining gene expression. As demonstrated in this study, the level of a particular RNA may be reliably determined by RT-PCR even in a total RNA that is usually considered as degraded (28S to 18S ratio as low as 0.4), provided that random primer is used in RT. In contrast, the use of the oligo(dT) primer in RT-PCR may lead to underestimation of specific mRNA level in the degraded RNA samples, depending on the distance of amplified fragment from the poly(A) end. A criterion based on average degradation level of a number of reference genes is suggested to discriminate specific RNA degradation from random and unspecific ones. [Copyright &y& Elsevier]

Tumor necrosis factor (TNF) is a key mediator in regulating the inflammatory response. Previously two TNF genes have been cloned and sequenced from rainbow trout, Oncorhynchus mykiss. In this study, the mature peptides of the two TNF molecules were produced in bacteria, purified under native conditions and their bioactivities evaluated in vitro. Both trout rTNF1 and rTNF2 induced gene expression of a number of proinflammatory factors including IL1β, TNF1, TNF2, IL8 and COX2 in freshly isolated head kidney leucocytes and the macrophage cell line RTS11. The stimulatory doses of both rTNFs were ≥10 ng/ml. Moreover, leucocyte migration and phagocytic activity were enhanced in vitro by the rTNFs in a dose dependent manner. Western blot analysis revealed the presence of multiple forms of rTNF structures including monomeric, dimeric and trimeric forms, suggesting that formation of a homotrimeric structure may be essential for the TNF bioactivities. [Copyright &y& Elsevier]

Sequence of a tick gut lysozyme (TGL) from the soft tick Ornithodoros moubata was determined by cloning and sequencing of overlapping polymerase chain reaction (PCR) and RACE PCR products. It is the first lysozyme sequence representing the subphylum Chelicerata. The resulting open reading frame codes for a putative signal peptide of 22 amino-acid residues and a mature protein composed of 124 amino-acids. Calculated mass of the protein is 14037.75 Da and a theoretical isoelectric point is 8.16. The phylogenetic analysis revealed that the TGL belongs to the c-type lysozymes. It forms a distinct monophyletic group together with multiple lysozyme-like sequences found in the gene products agCP6542 from Anopheles gambiae strain PEST and CG8492-PA from Drosophila melanogaster. This group is referred to as an H-branch due to a unique histidine residue at position 52 which replaces the highly conserved tyrosine present in the vast majority of c-type lysozymes. TGL seems to be an interesting case in which the features of lysozymes with anti-bacterial and digestive function are combined. Semi-quantitative RT-PCR and Northern blotting analysis demonstrated that TGL is strongly up-regulated at the transcriptional level after a bloodmeal. The maximum lysozyme mRNA level was detected 16 h post bloodmeal and the message remained stable for 5 days and then it slowly dropped down to the level of non-fed ticks within 2 weeks. [Copyright &y& Elsevier]

Using specific proteinase inhibitors, we demonstrated that serine proteinases in the tarnished plant bug, Lygus lineolaris, are major proteinases in both salivary glands and gut tissues. Gut proteinases were less sensitive to inhibition than proteinases from the salivary glands. Up to 80% azocaseinase and 90% of BApNAse activities in the salivary glands were inhibited by aprotinin, benzamidine, and PMSF, whereas only 46% azocaseinase and 60% BApNAse activities in the gut were suppressed by benzamidine, leupeptin, and TLCK. The pH optima for azocaseinase activity in salivary glands ranged from 6.2 to 10.6, whereas the pH optima for gut proteinases was acidic for general and alkaline for tryptic proteinases. Zymogram analysis demonstrated that ~26-kDa proteinases from salivary glands were active against both gelatin and casein substrates. Three trypsin-like cDNAs, LlSgP2–4, and one trypsin-like cDNA, L1GtP1, were cloned from salivary glands and gut, respectively. Putative trypsin precursors from all cloned cDNAs contained a signal peptide, activation peptide, and conserved N-termini (IVGG). Other structural features included His, Asp, and Ser residues for the catalytic amino acid triad of serine proteinase active sites, residues for the binding pocket, and four pairs of cysteine residues for disulfide bridges. Deduced trypsin-like proteins from LlSgP2, LlSgP3, and LlGtP1 cDNAs shared 98–99% sequence identity with a previously reported trypsin-like precursor, whereas the trypsin-like protein of LlSgP4 shared only 44% sequence identity with all other trypsin-like proteins, indicating multi-trypsin forms are present in L. lineolaris. [Copyright &y& Elsevier]

The temperate Yersinia phage PY54 belongs to the unusual group of phages that replicate as linear plasmids with covalently closed ends. Besides Escherichia coli phage N15, PY54 is the only member of this group to be identified. We have determined the complete sequence (46,339 bp) of the PY54 genome. Bioinformatic analyses revealed 67 open reading frames (ORFs) with good coding potential located on both DNA strands. The comparison of the deduced PY54 gene products with known proteins encoded by other phages and bacteria along with functional studies have enabled us to assign the possible functions of 25 ORFs. In the left arm of the PY54 genome, we identified a number of ORFs that obviously code for head and tail proteins. Furthermore, this part of the phage genome contains genes probably involved in plasmid partitioning. Regarding the predicted gene functions and gene order, the PY54 and N15 left arms are similar. However, there are only weak DNA homologies and, in contrast to N15, the Yersinia phage harbours only a few ORFs related to genes found in lambdoid phages. The PY54 right arm comprises mainly regulatory genes as well as genes important for plasmid replication, DNA methylation, and host cell lysis. Out of 36 deduced products of the right arm, 13 revealed strongest database homologies to N15 proteins, of which the protelomerase and the Rep protein are exclusively homologous to their N15 counterparts. A number of PY54 genes essential for the lytic or lysogenic cycle were identified by functional analysis and characterization of phage mutants. In order to study transcription during the lytic and lysogenic stage, we analysed 34 PY54 ORFs by reverse transcriptase (RT)-PCR. The phage transcription patterns in lysogenic bacteria and at the late lytic stage of infection are nearly identical. The reasons for this finding are spontaneous release of phages during lysogeny and a high rate of phages that lysogenize their Yersinia host upon infection. [Copyright &y& Elsevier]

The sequence of a α2-macroglobulin (α2M) from the soft tick Ornithodoros moubata (TAM) was determined by cloning and sequencing of overlapping polymerase chain reaction (PCR) and rapid amplification of cDNA ends PCR products. The TAM cDNA sequence is 4944 bp long and contains one open reading frame coding for a protein precursor composed of 1494 amino-acid residues, including a 24-residue signal sequence. The mature protein is cleaved into two subunits similarly to the C3 and C4 components of complement and fish α2Ms. Phylogeny analysis revealed that TAM is closely related to Limulus α2M and displays the highest similarity to the partial sequence of α2M from hard tick Ixodes scapularis. The comparison of conserved cysteine residues between TAM and human and Limulus α2Ms made it possible to predict the pattern of disulfide bridges and explain the atypical molecular arrangement of TAM. Four variants of the TAM bait region differing only in a short central segment were found; our data indicate that TAM exists as a single-copy gene in the tick genome and its bait region variants likely arise by alternative splicing. TAM is produced by tick hemocytes and it is also significantly expressed in salivary glands. TAM mRNA levels were shown to be up-regulated upon blood meal. [Copyright &y& Elsevier]

We conducted differential screening to obtain cDNAs showing that gene expression is highly associated with the transformation from immature pollen to embryogenic cell, so-called embryogenic dedifferentiation of pollen, in a Nicotiana tabacum pollen culture system and analyzed their expression and sequences. Seventy-seven cDNA clones were independently isolated and distinguished into 16 groups based on their sequences. The groups were further categorized into two classes, Class I and II, based on the gene expression pattern of the representative clone of each group under various pollen culture conditions arranged for examining the coincidence with the dedifferentiation. The 13 groups in Class I showed prominent expression under the conditions allowing or facilitating pollen dedifferentiation and the expression level increased earlier than A-type cyclin genes, but they were not markedly expressed in the cell populations rich in S-phase cells, i.e. young anthers with pollen mother cells, BY-2 cells at the growth phase and early phase embryos derived from immature pollen. The other three groups in Class II encoded homologs to H1 histone, H2A histone and minichromosome maintenance (MCM) protein, respectively. The level of their transcripts increased during dedifferentiation, but it was also high in anthers containing pollen mother cells and in the proliferating BY-2 cells indicating that their expression is coincident with the S phase but not with dedifferentiation. These findings suggest that pollen dedifferentiation is a complex process accompanied with the reentrance of cell cycle and unknown events probably caused by specific expression of many genes, at least, listed in Class I. These genes should be used as reliable markers and important clues for further studies on the molecular mechanism of dedifferentiation. [Copyright &y& Elsevier]

The carnitine palmitoyltransferase I (EC.2.3.1.21; CPT I) mediates the transport of fatty acids across the outer mitochondrial membrane. In mammals, there are two different proteins CPT I in the skeletal muscle (M) and liver (L) encoded by two genes. The carnitine palmitoyltransferase system of lower vertebrates received little attention. With the aim of improving knowledge on the CPT family in fish, we examined CPT I cDNA and CPT activity in different tissues of rainbow trout (Oncorhynchus mykiss). Using RT-PCR, we successfully cloned a partial CPT I cDNA sequence (1650 bp). The predicted protein sequence revealed identities of 63% and 61% with human L-CPT I and M-CPT I, respectively. This mRNA is expressed in liver, white and red skeletal muscles, heart, intestine, kidney and adipose tissue of trout. This is in good agreement with the measurement of the CPT activity in the same tissues. The [IC50] that reflects the sensitivity to malonyl-CoA inhibition was 0.116±0.004 μM for the liver and 0.426±0.041 μM for the white muscle. These results demonstrate for the first time the existence of at least one gene encoding for CPT I present in both the liver and the muscle of rainbow trout. [Copyright &y& Elsevier]

We have identified a family of expansin transcripts that include seven α-expansins (MjExp1 through MjExp7) and three β-expansins (MjExpB1 through MjExpB3) from Mirabilis jalapa (Nyctaginaceae) that show dramatic changes in transcript abundance during the rapid expansion and subsequent senescence of the ephemeral flowers. In general, α-expansin expression was low in small buds, high during maximal elongation of the floral tube, reduced during floral display, and upregulated during calyx infolding and collapse. Transcripts encoding auxin responsive proteins (Aux/IAA) showed a similar pattern of expression. Northern analysis using a set of overlapping probes designed to the MjExp2 transcript demonstrated a gradient of sequence conservation along its length (high to low, from the 5′ to the 3′ end of the coding region), and identified the presence of floral senescence-specific expansins. Beta expansin transcripts were found to be preferentially expressed during early floral development and sharply downregulated coincident with rapid growth. All three β-expansin transcripts are highly related, and ψMjExpB2 is an intronless pseudogene derived from MjExpB1. MjExpB3 appears to have derived from MjExpB1 in a separate gene duplication event, and is predicted to encode a truncated protein. [Copyright &y& Elsevier]

The genomic organization of cbl genes from a variety of mammalian and non-mammalian species was determined by a combination of cloning and database searches. Humans and mice have three cbl genes (c-cbl,11The three human cbl genes are named cbl, cblb, and cblc by the HUGO Gene Nomenclature Committee. To avoid confusion when discussing the cbl genes in general and those, in particular, which are not clearly orthologues of mammalian cbl, c-cbl will be used to refer to human cbl and its orthologues. cblb, and cblc) which show remarkable conservation of the intron/exon structure over the region of the genes which encode the highly conserved N-terminal region of the proteins including the RING finger. Searches of genomic, cDNA, and EST databases revealed that one or more cbl genes exist in chordates, insects, and worms. Comparison of the complexity and genomic organization of the cbl gene family and the predicted Cbl proteins from various species suggests that the three mammalian cbl genes arose by two duplications of an ancestral gene. The genomic organization of the cbl genes from various species provides insight into the evolution of the cbl gene family. [Copyright &y& Elsevier]

Ephrins and Eph receptors are a family of molecules that have been implicated in many developmental processes including neuronal network formation, guidance of cell migration, and axonal pathfinding. These molecules exhibit the ability to send bidirectional signals following ligand–receptor interactions resulting from cell–cell contacts. Gene-targeted knockout mice of B-class ephrins and Eph receptors have been shown to display phenotypic responses that correlate with anatomical defects. For example, disruption of the EphB2 receptor leads to defects of the vestibular system, including pathfinding abnormalities in efferent axons and reduced endolymph production. Such developmental distortions lead to deficiencies in ionic homeostasis and repetitive circling behaviors. The present study demonstrates that B-class ephrins and Eph receptors are expressed in cochlear tissues, suggesting that they may play some role in auditory function. To determine whether ephrins and Eph receptors have a functional role in the peripheral auditory system, distortion-product otoacoustic emission (DPOAE) levels, collected across a broad frequency range, were compared between groups of mice expressing different Eph receptor genotypes. In particular, EphB1 and EphB3 receptor knockout mice exhibited significantly diminished DPOAE levels as compared to wild-type littermates, indicating that these specific Eph receptors are necessary for normal cochlear function. [Copyright &y& Elsevier]

Genetic polymorphisms that encode a tryptophan (Trp) residue in the triple-helical domain of the α2 (Trp2) or α3 chain (Trp3) of human type IX collagen have been linked to risk of degenerative intervertebral disc disease. To determine whether these two allelic variants express protein that may affect the extracellular matrix of cartilage in vivo, we examined the properties of resident type IX collagen in an anonymous collection of embryonic and fetal human cartilage samples screened for Trp genotypes. No difference was found in the yield and electrophoretic properties of pepsin-solubilized type IX collagen between Trp2, Trp3 and non-Trp cartilage samples. On Western blot analysis, a polyclonal antiserum raised against a synthetic peptide matching the immediate Trp-containing sequence of the Trp3 allele reacted specifically with the α3(IX) chain prepared from Trp3 cartilage samples. Two-dimensional peptide mapping of type IX collagen in CNBr-digests of whole tissue gave indistinguishable fingerprints for Trp2, Trp3 and control tissues, including the yield of cross-linked peptides. Analysis of one cartilage sample that was homozygous for the Trp2 allele also gave a normal yield of collagen IX, including its α2 chain and a normal profile of cross-linked peptides. Together, the findings indicate that both Trp2 and Trp3 allelic products are incorporated into the cross-linked fibrillar network of developing human cartilage apparently normally. Any pathological consequences are likely, therefore, to be long-term and indirect rather than from overt misassembly of matrix. [Copyright &y& Elsevier]

Ribosome display is a powerful tool for selecting and evolving protein functions through ligand-binding. Here, this in vitro system was used to perform selection based on the folding properties of proteins, independent of specific ligand-binding. The selection is based on two properties of misfolded proteins: (1) increased sensitivity to proteolysis and (2) greater exposure of hydrophobic area. By targeting these properties, we show that compactly folded and soluble proteins can be enriched over insoluble and random coil proteins. This approach may be especially useful for selection and evolution of folded proteins from random sequence libraries. [Copyright &y& Elsevier]

Human CD99 (MIC2) is a 32 kDa cell surface protein and its encoding gene is localized to the pseudoautosomal regions of both Xp and Yp chromosomes. Although sequences of several genes such as human PBDX and MIC2R are known to be related to that of CD99, the murine counterpart of CD99 has not been reported. Here we have identified a novel CD99 mouse paralog, named as CD99L2 (CD99 antigen-like 2), and its human, rat and zebrafish genes. Unlike the rapidly evolved CD99 gene, these CD99L2 genes were highly conserved among those species. However, the genomic organization of human and mouse CD99L2 genes showed a difference in their exon numbers possibly due to exon duplication during evolution. In addition, comparative analysis of the cDNA sequences identified the presence of variants in the region around the exons 3 and 4 even within a species due to a differential splicing event, resulting in species-specific patterns in their transcripts. As determined by in situ hybridization analysis, the CD99L2 gene appeared to be expressed particularly high in neuronal cells despite its ubiquitous distribution. The highly expression on neuronal cells without any variations between species reflects a dominant role of this molecule during neural development. Amino acid sequence alignment revealed five putative functional regions highly conserved between CD99L2 and CD99, indicating a close relationship between the two genes. Moreover, human and mouse CD99L2 were located on their X chromosomes, respectively, whereas the zebrafish mic2l1 gene was in the LG7 chromosome. These observations support the inference that the evolutionary conserved gene, CD99L2, originated from a common ancestor gene of CD99, and its high conservation among species implies at least some essential function. [Copyright &y& Elsevier]

The gene encoding the outer membrane adhesin/invasin protein OpcA was previously described in the genomes of two pathogenic Neisseria species, N. meningitidis (Nm) and N. gonorrhoeae (Ng). In order to understand the presence or absence of opcA in nonpathogenic Neisseria species, 13 strains of N. polysaccharea (Np), four strains of N. lactamica, three strains of N. subflava and nine strains of other species were examined by DNA hybridization, polymerase chain reaction (PCR) and nucleotide sequencing. The opcA gene was found in two Np strains (85322 and 89357). The Np-opcA gene is a novel member of this gene family with 93% homology to Ng-opcA. Comparison of opcA-surrounding regions among eight Neisseria strains revealed five types of genetic organization at the opcA locus in Neisseria, which result from insertion or deletion of genetic elements at the upstream region of opcA. Comparison of the deduced peptide sequences from two Np strains, two representative Ng strains, two representative Nm strains and 13 Nm sequence variants demonstrates interspecies diversity of the OpcA protein family with conserved transmembrane regions and species-specific polymorphism at the surface-exposed loops and periplasmic turns. Reverse transcription–PCR analysis and Northern blotting showed that Np-opcA was transcribable. From an alignment of the Np-OpcA and Ng-OpcA sequences against the three-dimensional crystal structure of Nm-OpcA we conclude that there is no obvious structural reason why these proteins would not be able to form stable, folded, outer membrane proteins. The data presented here provide additional information for understanding the distribution, variation and expression of opcA in Neisseria. [Copyright &y& Elsevier]

Screening of a flounder ovary cDNA library with a rainbow trout p53 probe led to the isolation of a p53-unrelated cDNA encoding an unknown 161 amino acid protein. In view of its apparent molecular weight and yet unknown function, the deduced protein was named Xp18. Corresponding orthologous cDNAs or expressed sequence tags have been identified in several species including human, rodents, bovine, chicken and zebrafish and a related cDNA has also been isolated in the fruit fly. Deduced amino acid sequences appeared to be extremely well conserved throughout vertebrate evolution. Structure predictions suggested that Xp18 may correspond to an integral protein comprising four transmembrane domains. The charged C-termini of all known vertebrate Xp18-like proteins displayed a characteristic KXKXX motif which is considered as an endoplasmic reticulum targeting sequence. Gene expression, as shown by Northern blot and quantitative reverse transcription-polymerase chain reaction analysis, was significantly higher in the ovary and to a lesser extent in the brain. Xp18 transcripts were also detected by in situ hybridization in most of the circumventricular regions of the brain of adult flounders. The gene encoding the human protein is located on chromosome Xq22.1, a genome region involved in numerous genetic diseases including premature ovarian failure. [Copyright &y& Elsevier]

Human enteroviruses and rhinoviruses are respiratory pathogens whose role in acute respiratory infection is underestimated due to the use of diagnostic procedures with poor sensitivity. To determine the prevalence of these two pathogens in the upper respiratory tract infections, a multiplex procedure was developed that both detect and differentiate rhinoviruses and enteroviruses. This sensitive procedure allowed the detection of both pathogens from archival material (nasal swabs) collected during the previous winters and differentiated rhinoviruses from enteroviruses. This procedure can be used to determine the role of these pathogens in mild or severe upper respiratory tract infections. [Copyright &y& Elsevier]

Rab GTPases are key regulators of vesicular traffic in eukaryotic cells. Here we sought a global characterization and description of the Plasmodium falciparum family of Rab GTPases. We used a combination of bioinformatic analyses, experimental testing of predictions, structure modelling and phylogenetics. These analyses led to the identification of seven new parasite Rabs. Accordingly we estimate that the P. falciparum family is made up of 11 genes. We show that ten members of this family are transcribed in infected erythrocytes. Concerning the various members of the family, a series of specific as well as global conclusions can be drawn. Rabs predicted to be compartment-specific show different subcellular distributions. This is demonstrated for PfRab1A and PfRab11A, with the generation of specific antisera. The sequence analyses reveal several peculiarities, with possible functional implications. One of the transcribed genes, Pfrab5b, does not encode a classical C-terminus, suggestive of a novel regulatory role for this GTPase. Another, Pfrab5a, previously identified as a rab gene located on chromosome 2, possesses a 30-amino-acid insertion in its GTP-binding domain. Structural considerations suggest that this insertion could represent a novel interaction interface. We used conserved RabF and RabSF motifs to discriminate between specific parasite Rabs, and followed their predicted change in position on the structure of PfRab6, as GTP is hydrolysed to GDP. This allowed us to propose their involvement in potential interaction surfaces, that we extended to human Rab6 and the motifs known to mediate Rabkinesine-6 binding. Finally, we compared the P. falciparum Rab family to those of Saccharomyces cerevisiae and Schizosaccharomyces pombe and found that parasite Rabs segregate into possible functional clads. Such grouping into clads may give clues to parasite Rab function, and may shed light on P. falciparum secretory/endocytic pathways. [Copyright &y& Elsevier]

Human ENX-2 is a homologue of Drosophila Enhancer of zeste, which is a member of Polycomb-group proteins regulating the expression of homeotic genes as chromatin-associated proteins. In this study, we demonstrate that ENX-2 plays an important role as a signaling molecule involved in T cell receptor-mediated signaling pathway. In immunoprecipitation experiments, ENX-2 and zeta associated protein-70 (ZAP-70) were co-precipitated from T cell lysate. When probed with an anti-phospho-tyrosine antibody, ENX-2 was found to be phosphorylated on tyrosine. On the other hand, ENX-2 was not phosphorylated on tyrosine in the mutant Jurkat cell, J.Cam1.6 lacking the activity of lymphocyte protein tyrosine kinase p56lck. The interaction between ENX-2 and ZAP-70 was abolished in the mutant cell. Furthermore, in-vitro kinase assay using purified p56lck demonstrated that ENX-2 became tyrosine phosphorylated by this kinase. These findings show that the phosphorylation of ENX-2 is responsible for the interaction between ENX-2 and ZAP-70. [Copyright &y& Elsevier]

The human Cx40 gene (NT_004434.5) was sorted out from the GenBank database and as a result of a BLAST homology search, two ESTs (BE784549 from a human lung database, and BE732411 from a human placenta database) overlapping with the coding exon 2 sequence and upstream regions of the gene were identified. These ESTs correspond to two transcripts 1A and 1B, which diverge from each other in their 5′ regions. The transcript 1A corresponds to the only transcript previously identified for the mouse and rat Cx40 genes; whereas the transcript 1B is a new transcript. The human Cx40 gene therefore comprises three exons: exon 1A (100 bp), exon 1B (132 bp) and coding exon 2, with the exons 1A and 1B at 14 and 1.3 kb of the exon 2, respectively. The expression of these transcripts is cell-type specific. Transcript 1A is expressed in endothelial cells. Its expression was demonstrated in human umbilical vein endothelial cells (HUVEC). Transcript 1B is expressed in placental cytotrophoblasts. Its expression was demonstrated in malignant trophoblastic cells, BeWo, JAR and JEG-3, and purified cytotrophoblasts from human first trimester placental tissues. Interestingly, both transcripts 1A and 1B are expressed in the right atrial appendages (RAA), although the cell-type expression of the two transcripts in this particular tissue has not yet been determined. Both transcripts were found to be expressed in the various heart regions investigated, where transcript 1B was found to always occur rarely in comparison with transcript 1A. Transcripts 1A and 1B are both more abundant in the atria than in the ventricles. Luciferase reporter gene assays demonstrated that two genomic regions containing the exons 1A and 1B induced a cell-type specific expression. The 1.2 kb sequence, containing the exon 1A, induced an increase of the luciferase activity in HUVEC; whereas the 1.9 kb sequence, containing the exon 1B, induces an increase of expression of the luciferase activity in BeWo cells. The DNA sequence upstream of the exon 1A contains SP1 binding sites, but no TATA- or CAAT-box; whereas the region upstream of the exon 1B is preceded by three CAAT-boxes. Thus, in contrast to the mouse and rat Cx40 genes, the human Cx40 gene organized in three exons and generates two transcripts, which are cell-type specific. [Copyright &y& Elsevier]

Plants detoxify and accumulate several compounds as glucosides. In this work, detoxification of the exogenously added harmful compound naphthol in tobacco cells (Nicotiana tabacum L. Bright Yellow) was studied. When 250 μM of 1-naphthol or 2-naphthol was added to the tobacco cells, most of the naphthol was accumulated in the cell as glucosides and in further modified forms. The glucosylation activities against naphthols were increased in proportion to the concentration of naphthols in the culture medium. Addition of 1 mM naphthols caused cell death. Three glucosyltransferase genes, namely NtGT1a, NtGT1b and NtGT3 were isolated and characterized. The recombinant enzymes encoded by these genes showed glucosylation activity against naphthols and other phenolic compounds. It was also shown that these genes were induced following the addition of naphthols to the tobacco cells. These results suggest that naphthols are metabolized by glucosyltransferases whose production is inducible by naphthol itself. [Copyright &y& Elsevier]

Members of the ADAM (A Disintegrin And Metalloprotease) family of cell surface proteins have been discovered in a variety of tissues and species. We have cloned and sequenced a mouse cDNA encoding a novel member of the ADAM family. This mouse ADAM gene was named ADAM32 after an human homologue previously sequenced but not characterized. The cDNA sequence of ADAM32 has an open reading frame which encodes 750 (mouse ADAM32) and 787 (human ADAM32) amino acids, sharing 69% homology between the two species. The deduced amino acid sequences of mouse and human ADAM32 cDNAs contain all of the domains common to ADAM family members. Comparison of amino acid sequences showed that ADAM32 is highly homologous to ADAM2 and ADAM3, sperm surface proteins required for fertilization. We found that the ADAM32 gene is expressed predominantly in the testis. Examination of transcription in pre-pubertal and adult mouse testes revealed that expression of ADAM32 begins during meiotic prophase, in pachytene spermatocytes. These results suggest a potential role for ADAM32 in sperm development or fertilization. Genome database searches revealed that the mouse and human ADAM32 genes are located on chromosomes 8 and 8q11, respectively. The ADAM32 gene covers a relatively large region of the genome (120 kb for mouse ADAM32 and 180 kb for human ADAM32) and consists of multiple small exons (at least 22 exons in mouse ADAM32 and 25 exons in human ADAM32). [Copyright &y& Elsevier]