Carlos Reguenga

Characterization of peroxisomal Pex5p from rat liver. Pex5p in the Pex5p-Pex14p membrane complex is a transmembrane protein.

Pex5p is the receptor for the vast majority of peroxisomal matrix proteins. Here, we show that about 15% of rat liver Pex5p is found in the peroxisomal fraction representing 0.06% of total peroxisomal protein. This population of Pex5p displays all the characteristics of an intrinsic membrane protein. Protease protection assays indicate that this pool of Pex5p has domains exposed on both sides of the peroxisomal membrane. The strong interaction of Pex5p with the membrane of the organelle is not affected by mild protease treatment of intact organelles, conditions that result in the partial degradation of Pex13p. Cytosolic Pex5p is a monomeric protein. In contrast, virtually all peroxisomal Pex5p was found to be part of a stable 250-kDa protein assembly. This complex was isolated and shown to comprise just two subunits, Pex5p and Pex14p.

Functional Transient Receptor Potential Vanilloid 1 is Expressed in Human Urothelial Cells

PURPOSE We investigated the expression and functional status of TRPV1 receptor in human urothelial cells. MATERIAL AND METHODS Human urothelium was cultured and TRPV1 receptor expression was studied by immunocytochemistry and reverse transcriptase-polymerase chain reaction. The influence of inflammatory mediators on TRPV1 mRNA levels was also studied. Functional assays (cobalt uptake measurements and whole cell voltage clamp records) were used to study the response of urothelial cells to capsaicin, temperature, low pH and inflammatory mediators. Capsaicin induced adenosine triphosphate release from urothelial cells was assessed by bioluminescence. RESULTS TRPV1 protein and mRNA were detected in human urothelial cells and mRNA more than tripled in the presence of inflammatory mediators. Nerve growth factor treatment alone did not affect TRPV1 mRNA expression. Capsaicin (100 nM and 1 microM) and heat (41C and 45C) evoked cobalt uptake and inflammatory mediators lowered the temperature threshold for TRPV1 activation to 37C. Capsaicin (1 microM) induced TRPV1 desensitization to further applications of the agonist. In whole cell patch clamp experiments 1 microM capsaicin and a heat ramp from 37C to 50C caused inward currents. The same concentration of capsaicin induced the release of about 7 fmol adenosine triphosphate per mg. CONCLUSIONS TRPV1 receptors expressed by human urothelial cells respond to capsaicin and thermal stimuli. Capsaicin evoked release of adenosine triphosphate suggests that human urothelial TRPV1 is involved in the afferent branch of the micturition reflex. Inflammatory mediators decrease the TRPV1 thermal threshold of activation to body temperature and increase its expression. This finding may be relevant for symptoms associated with cystitis.

Mammalian Pex14p: membrane topology and characterisation of the Pex14p-Pex14p interaction

Peroxisomal biogenesis is a complex process requiring the action of numerous peroxins. One central component of this machinery is Pex14p, an intrinsic peroxisomal membrane protein probably involved in the docking of Pex5p, the receptor for PTS1-containing proteins (peroxisomal targeting signal 1-containing proteins). In this work the membrane topology of mammalian Pex14p was studied. Using a combination of protease protection assays and CNBr cleavage, we show that the first 130 amino acid residues of Pex14p are highly protected from exogenously added proteases by the peroxisomal membrane itself. Data indicating that this domain is responsible for the strong interaction of Pex14p with the organelle membrane are presented. All the other Pex14p amino acid residues are exposed to the cytosol. The properties of recombinant human Pex14p were also characterised. Heterologous expressed Pex14p was found to be a homopolymer of variable stoichiometry. Finally, in vitro binding assays indicate that homopolymerisation of Pex14p involves a domain comprising amino acid residues 147-278 of this peroxin.

DRG11 immunohistochemical expression during embryonic development in the mouse

DRG11 is a paired domain transcription factor that is necessary for the assembly of the nociceptive circuitry in the spinal cord dorsal horn. It is expressed in small dorsal root ganglion (DRG) neurons and in their projection area in the spinal cord. Drg11 knockout mice exhibit structural and neurochemical defects both at the DRG and spinal superficial dorsal horn and present reduced nociceptive responses. In this study, a polyclonal antibody against DRG11 was generated and used for a detailed systematic spatio‐temporal analysis of DRG11 expression during development. DRG11 is first detected at E10.5 in the spinal dorsal horn, DRG and trigeminal ganglion, where it persists until P14‐21. At the cranial level, DRG11 expression is observed from E10.5 up to the same early post‐natal ages in several cranial sensory ganglia and brain nuclei. These results suggest that DRG11 is required for the establishment of the first neuronal sensory relay along development. Developmental Dynamics 236:2653–2660, 2007.

Selective detection of UCP 3 expression in skeletal muscle: effect of thyroid status and temperature acclimation

A novel peptide antibody to UCP 3 is characterized which is sensitive and discriminatory for UCP 3 over UCP 2, UCP 1 and other mitochondrial transporters. The peptide antibody detects UCP 3 expression in E. coli, COS cells and yeast expression systems. The peptide antibody detects a single approximately 33 kDa protein band in mitochondria from isolated rat skeletal muscle, mouse and rat brown adipose tissue, and in whole muscle groups (soleus and extensor digitorum longus) from mice. No 33 kDa band is detectable in isolated mitochondria from liver, heart, brain, kidney and lungs of rats, or gastrocnemius mitochondria from UCP 3 knock-out mice. From our data, we conclude that the peptide antibody is detecting UCP 3 in skeletal muscle, skeletal muscle mitochondria and brown adipose tissue mitochondria. It is also noteworthy that the peptide antibody can detect human, mouse and rat forms of UCP 3. Using the UCP 3 peptide antibody, we confirm and quantify the increased (2.8-fold) UCP 3 expression observed in skeletal muscle mitochondria isolated from 48-h-starved rats. We show that UCP 3 expression is increased (1.6-fold) in skeletal muscle of rats acclimated over 8 weeks to 8 degrees C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 degrees C. Furthermore, UCP 3 expression is increased (2.3-fold) in skeletal muscle from hyperthyroid rats compared to euthyroid controls. In addition, we show that UCP 3 expression is only coincident with the mitochondrial fraction of skeletal muscle homogenates and not peroxisomal, nuclear or cytosolic and microsomal fractions.

Prrxl1 is Required for the Generation of a Subset of Nociceptive Glutamatergic Superficial Spinal Dorsal Horn Neurons

Perception of noxious events relies on activation of complex central neuronal circuits. The spinal cord dorsal horn plays a pivotal role in the process relaying to the brain various types of somatosensory input. These functions are accomplished by distinct sensory neurons specifically organized in different laminae. They differentiate during development in a spatial‐temporal order due to the expression of combinatorial sets of homeodomain transcription factors. Here we demonstrate that the differential expression of the homeodomain transcription factors Prrxl1 (DRG11), Tlx3, and Lmx1b defines various subpopulations of spinal cord dorsal horn glutamatergic early born and late born neurons. Accordingly, in the superficial dorsal horn of Prrxl1−/− mice, the number of glutamatergic neurons is reduced by 70%, while the number of Golgi‐impregnated and noxious‐induced Fos immunoreactive neurons is reduced by 85%. These results suggest a crucial role for Prrxl1 in the generation of various subpopulations of nociceptive glutamatergic superficial dorsal horn neurons. Developmental Dynamics 239:1684–1694, 2010.

Identification of a 24 kDa intrinsic membrane protein from mammalian peroxisomes

A 24 kDa protein from rat liver peroxisomal membrane was isolated and subjected to Edman degradation. Using the N-terminal sequence of this polypeptide we have identified several rat and human expressed sequence tags in the GenBank Database. The complete sequence of a human cDNA clone was determined. The open reading frame encodes an extremely basic protein 212 amino acid residues long. A high similarity between this mammalian protein and hypothetical proteins from Caenorhabditis elegans and Neurospora crassa was found. Hydropathy analysis reveals the existence of two putative membrane-spanning domains in conserved regions of the three homologous proteins.

Characterization of the Peroxisomal Cycling Receptor Pex5p Import Pathway

Pex5p, the receptor for most peroxisomal matrix proteins, is thought to mediate the transport of newly synthesised proteins from the cytosol to the peroxisomal compartment. However, little is known about the mechanism of this pathway. Much of the data regarding this matter derives from steady-state level analysis of peroxisomal Pex5p on several mutant cell lines (Collins et al, 2000) or from studies aiming at the characterisation of peroxin-peroxin interactions (Fransen et al, 2002).

The g.1170C>T polymorphism of the 5′ untranslated region of the human alpha-galactosidase gene is associated with decreased enzyme expression—Evidence from a family study

SummarySubnormal leukocyte α-galactosidase (α-Gal) activity was found during evaluation of an adolescent male with cryptogenic cerebrovascular small-vessel disease. The only molecular abnormality found was the g.1170C>T single-nucleotide polymorphism (SNP) in the 5′ untranslated region of exon 1 in the α-Gal gene (GLA). Historically, this polymorphism has been considered to be biologically neutral. To test the hypothesis that the g.1170T allele might be associated with lower α-Gal expression, we genotyped GLA exon 1 and measured leukocyte and plasma α-Gal in the parents, brother and sister of the index case. The g.1170T allele co-segregated with a subnormal leukocyte α-Gal activity in the three siblings. Although plasma enzyme activities were within the normal range in all five relatives, the ranking of their values suggested a dosage effect of the g.1170T allele. Western blotting assays of leukocyte protein extracts showed that the relative expression of α-Gal in both the patient and his sister was significantly lower than in sex-matched hemizygous or homozygous controls for the g.1170C allele, either normalized to the β-actin immunoblot expression or standardized to a known amount of recombinant human α-Gal. These family data, in combination with results from a recent GLA SNP screening study among healthy Portuguese individuals, suggest that the g.1170C>T SNP may be co-dominantly associated with a relatively decreased GLA expression at the transcription and/or translation level. Larger population studies are needed to confirm these findings and to test the hypothesis that the GLA g.1170C>T may contribute to the multifactorial risk of ischaemic small-vessel cerebrovascular disease.

Cystitis is associated with TRPV1b-downregulation in rat dorsal root ganglia.

The recently identified splice variant of the transient receptor vanilloid type 1 (TRPV1) molecule, TRPV1b, produces a negative-dominant effect on the responsiveness of the TRPV1 channel, which is increased by peripheral inflammatory processes. Here, we studied, using real-time polymerase chain reaction, whether cyclophosphamide injection-evoked cystitis is associated with altered TRPV1/TRPV1b expression in the L5-L6 dorsal root ganglia, which innervate the urinary bladder. We found that while TRPV1 mRNA expression was unchanged, the amount of TRPV1b transcript was significantly reduced in L5-L6 dorsal root ganglia during cystitis. These data indicate that peripheral inflammatory events induce changes in TRPV1b expression in primary sensory neurons, which may result in increased responsiveness of the TRPV1 channel.