Expression of Pax258 in the gastropod statocyst: insights into the antiquity of metazoan geosensory organs

Most animals have sensory systems that allow them to balance and orient relative to the pull of gravity. Structures responsible for these functions range from very simple statocysts found in many aquatic invertebrates to the complex inner ear of mammals. Previous studies suggest that the specialized mechanosensory structures responsible for balance in vertebrates and insects may be homologous based on the requirement and expression of group II Pax genes (i.e., Pax-2/5/8 genes). Here we report the expression of a Pax-258 gene in the statocysts and other chemosensory and mechanosensory cells during the development of the gastropod mollusk Haliotis asinina, a member of the Lophotrochozoa. Based on the phylogenetic distribution of geo-sensory systems and the consistent expression of Pax-258 in the cells that form these systems, we propose that Pax-258, along with POU-III and -IV genes, has an ancient and conserved role in the formation of structures responsible for balance and geotaxis in eumetazoans.

Expression of ephs and ephrins in developing mouse inner ear

Levels of expression of mRNAs encoding the different Ephs and ephrins were measured by semi-quantitative reverse-transcription polymerase chain reaction in developing mouse whole inner ears, and in dissected fractions of the neonatal mouse inner ear. Nineteen of the 24 known Ephs and ephrins were surveyed. The results showed that between embryonic age (E) 11.5 days and E12.5, levels increased 10-300 times per unit of tissue. In neonatal mice, the fraction containing combined organ of Corti and spiral ganglion showed relatively strong expression of EphA4, EphB3, ephrin-A3, ephrin-B2 and ephrin-B3. In the lateral wall, EphA4, ephrin-A3 and ephrin-B2 were strongly expressed, while ephrin-A3 was particularly strongly expressed in utricular and saccular sensory epithelia. The results suggest that the Ephs and ephrins are likely to play a part in the differentiation of the structures of the inner ear, and show which Ephs and ephrins are most likely to play important roles in the different structures. (C) 2003 Elsevier Science B.V. All rights reserved.

Suppressor of cytokine signalling gene expression is elevated in breast carcinoma

Cytokines are important for breast cell function, both as trophic hormones and as mediators of host defense mechanisms against breast cancer. Recently, inducible feedback suppressors of cytokine signalling (SOCS/JAB/SSI) have been identified, which decrease cell sensitivity to cytokines. We examined the expression of SOCS genes in 17 breast carcinomas and 10 breast cancer lines, in comparison with normal tissue and breast lines. We report elevated expression of SOCS-1-3 and CIS immunoreactive proteins within in situ ductal carcinomas and infiltrating ductal carcinomas relative to normal breast tissue. Significantly increased expression of SOCS-1-3 and CIS transcripts was also shown by quantitative in situ hybridisation within both tumour tissue and reactive stroma. CIS transcript expression was elevated in all 10 cancer lines, but not in control lines. However, there was no consistent elevation of other SOCS transcripts. CIS protein was shown by immunoblot to be present in all cancer lines at increased levels, mainly as the 47 kDa ubiquitinylated form. A potential proliferative role for CIS overexpression is supported by reports that CIS activates ERK kinases, and by strong induction in transient reporter assays with an ERK-responsive promoter. The in vivo elevation of SOCS gene expression may be part of the host/tumour response or a response to autocrine/paracrine GH and prolactin. However, increased CIS expression in breast cancer lines appears to be a specific lesion, and could simultaneously shut down STAT 5 signalling by trophic hormones, confer resistance to host cytokines and increase proliferation through ERK kinases.

Human melanoblasts in culture: Expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3

The BRN2 transcription factor (POU3F2, N-Oct-3) has been implicated in development of the melanocytic lineage and in melanoma. Using a low calcium medium supplemented with stem cell factor, fibroblast growth factor-2, endothelin-3 and cholera toxin, we have established and partially characterised human melanocyte precursor cells, which are unpigmented, contain immature melanosomes and lack L-dihydroxyphenylalanine reactivity. Melanoblast cultures expressed high levels of BRN2 compared to melanocytes, which decreased to a level similar to that of melanocytes when cultured in medium that contained phorbol ester but lacked endothelin-3, stem cell factor and fibroblast growth factor-2. This decrease in BRN2 accompanied a positive L-dihydroxyphenylalanine reaction and induction of melanosome maturation consistent with melanoblast differentiation seen during development. Culture of primary melanocytes in low calcium medium supplemented with stem cell factor, fibroblast growth factor-2 and endothelin-3 caused an increase in BRN2 protein levels with a concomitant change to a melanoblast-like morphology. Synergism between any two of these growth factors was required for BRN2 protein induction, whereas all three factors were required to alter melanocyte morphology and for maximal BRN2 protein expression. These finding implicate BRN2 as an early marker of melanoblasts that may contribute to the hierarchy of melanocytic gene control.

Electropalatographic assessment of tongue-to-palate contacts exhibited in dysarthria following traumatic brain injury: Spatial characteristics

Consonant imprecision has been reported to be a common feature of the dysarthric speech disturbances exhibited by individuals who have sustained a traumatic brain injury (TBI). Inaccurate tongue placements against the hard palate during consonant articulation may be one factor underlying the imprecision. To investigate this hypothesis, electropalatography (EPG) was used to assess the spatial characteristics of the tongue-to-palate contacts exhibited by three males (aged 23-29 years) with dysarthria following severe TBI. Five nonneurologically impaired adults served as control subjects. Twelve single-syllable words of CV or CVC construction (where initial C = /t, d, S, z, k, g/, V=/i, a/) were read aloud three times by each subject while wearing an EPG palate. Spatial characteristics were analyzed in terms of the location, pattern, and amount of tongue-to-palate contact at the frame of maximum contact during production of each consonant. The results revealed that for the majority of consonants, the patterns and locations of contacts exhibited by the TBI subjects were consistent with the contacts generated by the group of control subjects. One notable exception was one subject's production of the alveolar fricatives in which complete closure across the palate was demonstrated, rather than the characteristic groove configuration. Major discrepancies were also noted in relation to the amount of tongue-to-palate contact exhibited, with two TBI subjects consistently demonstrating increased contacts compared to the control subjects. The implications of these findings for the development of treatment programs for dysarthric speech disorders subsequent to TBI are highlighted.

The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells

Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyltransferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARgamma/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.

OxyR acts as a repressor of catalase expression in Neisseria gonorrhoeae

It has been reported that Neisseria gonorrhoeae possesses a very high level of catalase activity, but the regulation of catalase expression has not been investigated extensively. In Escherichia coli and Salmonella enterica serovar Typhimurium, it has been demonstrated that OxyR is a positive regulator of hydrogen peroxide-inducible genes, including the gene encoding catalase. The oxyR gene from N. gonorrhoeae was cloned and used to complement an E. coli oxyR mutant, confirming its identity and function. The gene was inactivated by inserting a kanamycin resistance cassette and used to make a knockout allele on the chromosome of N. gonorrhoeae strain 1291. In contrast to E. coli, the N. gonorrhoeae oxyR::kan mutant expressed ninefold-more catalase activity and was more resistant to hydrogen peroxide killing than the wild type. These data are consistent with OxyR in N. gonorrhoeae acting as a repressor of catalase expression.

Expression of hNP22 is altered in the frontal cortex and hippocampus of the alcoholic human brain

Background: Human neuronal protein (hNP22) is a gene with elevated messenger RNA expression in the prefrontal cortex of the human alcoholic brain. hNP22 has high homology with a rat protein (rNP22). These proteins also share homology with a number of cytoskeleton-interacting proteins. Methods: A rabbit polyclonal antibody to an 18-amino acid epitope was produced for use in Western and immunohistochemical analysis. Samples from the human frontal and motor cortices were used for Western blots (n = 10), whereas a different group of frontal cortex and hippocampal samples were obtained for immunohistochemistry (n = 12). Results: The hNP22 antibody detected a single protein in both rat and human brain. Western blots revealed a significant increase in hNP22 protein levels in the frontal cortex but not the motor cortex of alcoholic cases. Immunohistochemical studies confirmed the increased hNP22 protein expression in all cortical layers. This is consistent with results previously obtained using Northern analysis. Immunohistochemical analysis also revealed a significant increase of hNP22 immunoreactivity in the CA3 and CA4 but not other regions of the hippocampus. Conclusions: It is possible that this protein may play a role in the morphological or plastic changes observed after chronic alcohol exposure and withdrawal, either as a cytoskeleton-interacting protein or as a signaling molecule.

Decreased coumarin 7-hydroxylase activities and CYP2A6 expression levels in humans caused by genetic polymorphism in CYP2A6 promoter region (CYP2A6*9)

One of seven poor metabolizers of coumarin found in Thai subjects was previously genotyped as heterozygote for the CYP2A6*4 (whole deletion) and CYP2A6*9. Thus, we aimed to investigate the relationship between the genetic polymorphism in the TATA box of the CYP2A6 gene (CYP2A6*9), expression levels of CYP2A6 mRNA and coumarin 7-hydroxylase activities in human livers. Levels of CYP2A6 mRNA were quantified by real-time quantitative reverse transcriptase-polymerase chain reaction. The mean expression levels of CYP2A6 mRNA in individuals with CYP2A6*1/*4, CYP2A6*1/*9 and CYP2A6*4/*9 were 58%, 71% and 21% of the individuals genotyped as CYP2A6*1/*1, respectively. The mean in-vitro coumarin 7-hydroxylase activities in subjects carrying CYP2A6*1/*4, CYP2A6*1/*9 and CYP2A6*4/*9 were 41%, 71% and 12%, respectively, compared to those of the subjects judged as wild-type. Vmax values for coumarin 7-hydroxylation in the liver microsomes from human subjects with genotypes of CYP2A6*1/*1, CYP2A6*1/*4, CYP2A6*1/*9 and CYP2A6*4/*9 were 0.58, 0.26, 0.44 and 0.13 nmol/min/nmol total P450, respectively. CYP2A6 protein levels in human liver microsomes with the CYP2A6*4 and the CYP2A6*9 alleles were markedly decreased. These results suggest that the genetic polymorphism in the promoter region of the CYP2A6 gene (CYP2A6*9) reduced the expression levels of CYP2A6 mRNA and protein in human livers, resulting in the decrease of coumarin 7-hydroxylase activities. Individuals judged as CYP2A6*4/*9 were expected to be poor metabolizers, having extremely low activity of CYP2A6.

Purification of post-translationally modified proteins from bacteria: homologous expression and purification of histidine-tagged pilin from Neisseria meningitidis

Until recently, glycosylation of proteins in prokaryotes was regarded as uncommon and thought to be limited to special cases such as S-layer proteins and some archeal outer membrane proteins. Now, there are an increasing number of reports of bacterial proteins that are glycosylated. Pilin of pathogenic Neisseria is one of the best characterised post-translation ally modified bacterial proteins, with four different types of modifications reported, including a novel glycosylation. Pilin monomers assemble to form pilus fibres, which are long protein filaments that protrude from the surface of bacterial cells and are key virulence factors. To aid in the investigation of these modifications, pure pilin is required. A number of pilin purification methods have been published, but none are appropriate for the routine purification of pilin from many different isolates. This study describes a novel, rapid, and simple method of pilin purification from Neisseria meningitidis C311#3, which facilitates the production of consistent quantities of pure, native pilin. A 6 x histidine tag was fused to the C-terminus of the pilin subunit structural gene, pilE, via homologous recombination placing the 6 x histidine-tagged allele in the chromosome of N. meningitidis C311#3. Pilin was purified under non-denaturing conditions via a two-step process using immobilised metal affinity chromatography (IMAC), followed by dye affinity chromatography. Analysis of the purified pilin confirmed that it retained both of the post-translational modifications examined. This novel approach may prove to be a generally applicable method for purification and analysis of post-translationally modified proteins in bacteria. (C) 2003 Elsevier Science (USA). All rights reserved.

Identification and characterization of pptA: a gene involved in the phase-variable expression of phosphorylcholine on pili of Neisseria meningitidis

Pili of pathogenic Neisseria are major virulence factors associated with adhesion, cytotoxicity, twitching motility, autoaggregation, and DNA transformation. Pili are modified posttranslationally by the addition of phosphorylcholine. However, no genes involved in either the biosynthesis or the transfer of phosphorylcholine in Neisseria meningitidis have been identified. In this study, we identified five candidate open reading frames (ORFs) potentially involved in the biosynthesis or transfer of phosphorylcholine to pilin in N. meningitidis. Insertional mutants were constructed for each ORF in N. meningitidis strain C311#3 to determine their effect on phosphorylcholine expression. The effect of the mutant ORFs on the modification by phosphorylcholine was analyzed by Western analysis with phosphorylcholine-specific monoclonal antibody TEPC-15. Analysis of the mutants showed that ORF NMB0415, now defined as pptA (pilin phosphorylcholine transferase A), is involved in the addition of phosphorylcholine to pilin in N. meningitidis. Additionally, the phase variation (high frequency on-off switching of expression) of phosphorylcholine on pilin is due to changes in a homopolymeric guanosine tract in pptA.

Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4

Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1. families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1 L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c-both tagged and detagged-from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies. Crown copyright (C) 2003 Published by Elsevier Inc. All rights reserved.

Endogenous angiotensin II modulates nNOS expression in renovascular hypertension

Nitric oxide (NO) influences renal blood flow mainly as a result of neuronal nitric oxide synthase (nNOS). Nevertheless, it is unclear how nNOS expression is modulated by endogenous angiotensin II, an inhibitor of NO function. We tested the hypothesis that the angiotensin II AT1 receptor and oxidative stress mediated by NADPH oxidase contribute to the modulation of renal nNOS expression in two-kidney, one-clip (2K1C) hypertensive rats. Experiments were performed on male Wistar rats (150 to 170 g body weight) divided into 2K1C (N = 19) and sham-operated (N = 19) groups. nNOS expression in kidneys of 2K1C hypertensive rats (N = 9) was compared by Western blotting to that of 2K1C rats treated with low doses of the AT1 antagonist losartan (10 mg·kg-1·day-1; N = 5) or the superoxide scavenger tempol (0.2 mmol·kg-1·day-1; N = 5), which still remain hypertensive. After 28 days, nNOS expression was significantly increased by 1.7-fold in the clipped kidneys of 2K1C rats and by 3-fold in the non-clipped kidneys of 2K1C rats compared with sham rats, but was normalized by losartan. With tempol treatment, nNOS expression increased 2-fold in the clipped kidneys and 1.4-fold in the non-clipped kidneys compared with sham rats. The changes in nNOS expression were not followed by changes in the enzyme activity, as measured indirectly by the cGMP method. In conclusion, AT1 receptors and oxidative stress seem to be primary stimuli for increased nNOS expression, but this up-regulation does not result in higher enzyme activity.

Spatial variability in nutritional status of arabic coffee based on dris index

The combined use of precision agriculture and the Diagnosis and Recommendation Integrated System (DRIS) allows the spatial monitoring of coffee nutrient balance to provide more balanced and cost-effective fertilizer recommendations. The objective of this work was to evaluate the spatial variability in the nutritional status of two coffee varieties using the Mean Nutritional Balance Index (NBIm) and its relationship with their respective yields. The experiment was conducted in eastern Minas Gerais in two areas, one planted with variety Catucaí and another with variety Catuaí. The NBIm of the two varieties and their yields were analyzed through geostatistics and, based on the models and parameters of the variograms, were interpolated to obtain their spatial distribution in the studied areas. Variety Catucai, with grater spatial variability, was more nutritional unbalanced than variety Catuai, and consequently produced lower yields. Excess of Fe and Mn makes these elements limiting yield factors.

Spatial variability of particle size fractions of an Oxisol cultivated with conilon coffee

Information on the spatial distribution of particle size fractions is essential for use planning and management of soils. The aim of this work to was to study the spatial variability of particle size fractions of a Typic Hapludox cultivated with conilon coffee. The soil samples were collected at depths of 0-0.20 and 0.20-0.40 m in the coffee canopy projection, totaling 109 georeferentiated points. At the depth of 0.2-0.4 m the clay fraction showed average value significantly higher, while the sand fraction showed was higher in the depth of 0-0.20 m. The silt showed no significant difference between the two depths. The particle size fractions showed medium and high spatial variability. The levels of total sand and clay have positive and negative correlation, respectively, with the altitude of the sampling points, indicating the influence of landscape configuration.