Search

Thioredoxins (Trx) are a class of small multifunctional redox-active proteins found in all organisms. Recently, we reported the cloning of a mitochondrial thioredoxin, Trx2, from rat heart. To investigate the biological role of Trx2 we have isolated the human homologue, hTrx2, and generated HEK-293 cells overexpressing Trx2 (HEK-Trx2). Here, we show that HEK-Trx2 cells are more resistant toward etoposide. In addition, HEK-Trx2 are more sensitive toward rotenone, an inhibitor of complex I of the respiratory chain. Finally, overexpression of Trx2 confers an increase in mitochondrial membrane potential, ΔΨm. Treatment with oligomycin could both reverse the effect of rotenone and decrease the membrane potential suggesting that Trx2 interferes with the activity of ATP synthase. Taken together, these results suggest that Trx2 interacts with specific components of the mitochondrial respiratory chain and plays an important role in the regulation of the mitochondrial membrane potential.

BackgroundA rat line developed by selective breeding for high alcohol sensitivity has blunted corticosterone responses to alcohol and stress. In the present study, we determined possible differences in adrenal activation after alcohol and motor performance testing between the alcohol‐sensitive alcohol‐nontolerant and alcohol‐insensitive alcohol‐tolerant rats.

Basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) proteins form dimeric transcription factors to mediate diverse biological functions including xenobiotic metabolism, hypoxic response, circadian rhythm and central nervous system midline development. The Ah receptor nuclear translocator protein (ARNT) plays a central role as a common heterodimerization partner. Herein, we describe a novel, embryonically expressed, ARNT interacting protein (AINT) that may be a member of a larger coiled-coil PAS interacting protein family. The AINT C-terminus mediates interaction with the PAS domain of ARNT in yeast and interacts in vitro with ARNT and ARNT2 specifically. AINT localizes to the cytoplasm and overexpression leads to non-nuclear localization of ARNT. A dynamic pattern of AINT mRNA expression during embryogenesis and cerebellum ontogeny supports a role for AINT in development.

ADAM family of metalloprotease-disintegrins, including enzymes that process TNF-α and β-amyloid precursor protein, has been indicated in neuronal development, but the role of these protease/adhesion/signaling proteins in adult nervous system remains poorly understood. Present study provides a systematic examination of ADAM gene expression in rodent CNS, showing the first quantitative characterization of ADAM mRNA distribution therein. At least 17 ADAM mRNAs were expressed. Individual ADAM mRNAs and their isoforms showed strikingly different expression patterns. Expression of mRNAs for ADAM10, the putative α-secretase, and ADAM17 (TACE), also indicated in APP processing, was further characterized using in situhybridization. Expression of ADAM10 mRNA was widespread, while ADAM17 showed a more restricted pattern. Altogether, the wide and differential expression of ADAM mRNAs suggests versatile roles for ADAMs in the adult CNS.

Ligand-induced transcriptional activation of gene expression by nuclear receptors is dependent on recruitment of coactivators as intermediary factors. The present work describes the cloning and characterization of RAP250, a novel human nuclear receptor coactivator. The results of in vitroand invivoexperiments indicate that the interaction of RAP250 with nuclear receptors is ligand-dependent or ligand-enhanced depending on the nuclear receptor and involves only one short LXXLL motif called nuclear receptor box. Transient transfection assays further demonstrate that RAP250 has a large intrinsic glutamine-rich activation domain and can significantly enhance the transcriptional activity of several nuclear receptors, acting as a coactivator. Interestingly, Northern blot and in situhybridization analyses reveal that RAP250 is widely expressed with the highest expression in reproductive organs (testis, prostate and ovary) and brain. Together, our data suggest that RAP250 may play an important role in mammalian gene expression mediated by nuclear receptor.

The role of estrogen (ER) and progesterone receptors (PR) in breast cancer is well established. Identification of the second human estrogen receptor, the estrogen receptor β (ERβ), prompted us to evaluate its role in breast cancer. We studied the expression of ERβ by immunohistochemistry and mRNA in situhybridization in 92 primary breast cancers and studied its association with ERα, PR, and various other clinicopathological factors. Sixty percent of tumors were defined as ERβ-positive (nuclear staining in 20% of the cancer cells). Normal ductal epithelium and 5 of 7 intraductal cancers were also found to express ERβ. Three-fourths of the ERα- and PR-positive tumors were positive for ERβ, whereas ERα and PR were positive in 87% and 67. of ERβ-positive tumors, respectively. ERβ was associated with negative axillary node status (P< 0.0001), low grade (P= 0.0003), low S-phase fraction (P= 0.0003), and premenopausal status (P= 0.04). In conclusion, the coexpression of ERβ with ERα and PR as well as its association with the other indicators of low biological aggressiveness of breast cancer suggest that ERβ-positive tumors are likely to respond to hormonal therapy. The independent predictive value of ERβ remains to be established.

The forkhead transcription factor FREAC-1 is a potent transcriptional activator. We have localized a transcriptional activation domain in the C-terminus of FREAC-1 and another one to a stretch of approximately 60 amino acids in the central part of the protein. While the C-terminal activation domain activates in all cell lines tested, the activation domain in the central part of the protein is functional only in cell lines derived from lung. This cell-type-specific activity is retained when the activation domain is fused to the heterologous DNA binding domain of Gal4. The humanFREAC-1gene was found to consist of two exons separated by an intron of 1.2 kb. Exon 1 encodes the forkhead DNA binding domain and the cell-type-specific activation domain. Exon 2 encodes the general activation domain. The distribution ofFREAC-1expression during embryogenesis was investigated byin situhybridization.FREAC-1mRNA was found in mesenchyme in immediate proximity to endodermal epithelia throughout the digestive, urinary, and respiratory tracts. Mesenchyme surrounding the notochord and adjacent to the ectodermal epithelia of the oral cavity and developing teeth also expressesFREAC-1.The pattern ofFREAC-1expression, with highest levels in the mesenchyme next to the epithelium and gradually diminishing as the distance from the epithelium increases, suggests thatFREAC-1expression is a response to epithelial paracrine signaling and thatFREAC-1may play a role in epitheliomesenchymal interactions.

Heat shock transcription factors (HSFs) are generally known as regulators of cellular stress response. The mammalian HSF1 functions as a classical stress factor, whereas HSF2 is active during certain developmental processes, including embryogenesis and spermatogenesis. In the present study, we examined HSF2 expression at specific stages of the rat seminiferous epithelial cycle. We found that expression of the alternatively spliced HSF2-α and HSF2-β isoforms is developmentally regulated in a stage-specific manner. Studies on cellular localization demonstrated that HSF2 is present in the nuclei of early pachytene spermatocytes at stages I–IV and in the nuclei of round spermatids at stages V–VIIab. In contrast a strong HSF2 immunoreactivity was detected in small distinct cytoplasmic regions from zygotene spermatocytes to maturation phase spermatids. Immunoelectron microscopic analysis revealed that these structures are mainly cytoplasmic bridges between germ cells. Our results on cellular localization of HSF2 and stage-specific expression of the HSF2 isoforms indicate that HSF2, in addition to its function as a nuclear transcription factor, may be involved in other cellular processes during spermatogenesis, possibly in the sharing process of gene products between the germ cells.

THE present paper describes the effect of capsaicin-induced stressful stimulus on the expression of immediate early genes (IEGs) c-fos, c-jun, junB and junD in the hypothalamic paraventricular nucleus (PVN) and the central amygdaloid nucleus (ACe) using in situhybridization. Stress caused an intense expression of c-fos,c-junand junB especially in the PVN and ACe and also a clear induction of junD was observed in the PVN. This suggests that the PVN and the ACe are two major targets of stress in the brain. The intense expression of the IEGs in the ACe and PVN suggests that stress may affect neurotransmitter gene expression through Fos and Jun proteins in both these nuclei.

We studied the effect of ageing on the mRNA levels of neurotrophins and their receptors in the rat pituitary gland using in situhybridization. In 2-month-old rats, mRNA for brain derived neurotrophic factor (BDNF) was observed in the anterior and intermediate lobes of the pituitary. TrkB and trkC, the putative receptors for BDNF and neurotrophin-3, were also expressed in the pituitary. In contrast, trk mRNA could not be detected in any of the pituitaries studied. Ageing did not affect BDNF mRNA levels. In 2-year-old rats, trkB and trkC mRNA levels were decreased in the anterior lobe. TrkB mRNA levels decreased also in the neural lobe. These results show that although the expression of BDNF does not change during ageing in the pituitary, expression of trkB and trkC mRNAs are altered in senescence. These changes may reflect overall impairments in pituitary endocrine functions in old age or they may be associated with morphological alterations in pituitary innervation.

DIOXINS are environmental pollutants, whose detrimental effects on health are the cause of wide public concern due to their accumulation in the food chain and resistance to metabolism. The most well known dioxin is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dioxins exert their effects through a ligand activated transcription factor termed the dioxin or aryl hydrocarbon receptor (Ahr), which acts in concert with another structurally related protein: the aryl hydrocarbon nuclear translocator (Arnt). In the present study, we have employed in situ hybridization to study the localization of the mRNAs for these two proteins in the rat brain. We found mRNAs for both Ahr and Arnt predominantly in the same neuronal populations: in the olfactory bulb, the hippocampus, and the cerebral and cerebellar cortices. Arnt, however, had a more widespread expression than Ahr in the brain. The present results demonstrate that dioxins may act directly in the brain and that the effects of dioxin may occur in discrete neuronal populations. However, in some parts of the brain, e.g. the hypothalamus, that are thought to be targets of the toxic effects of dioxins, we did not observe detectable levels of Ahr mRNA. Furthermore, it appears that Arnt may have additional functions in the brain, apart from being the heterodimerization partner of Ahr, possibly through heterodimerizing with other transcription factors.

USING in situhybridization histochemistry and immunocytochemistry the induction of immediate early genes (IEGs) was studied in the rat superior cervical ganglion (SCG) after deafferentation, axotomy and sialectomy (removal of the submandibular gland). Control SCGs showed very low levels of IEGs, whereby Fos proteins were found in 1 and Jun protein in 6 of neurons, but not in non-neuronal cells. Denervation and axotomy induced C-fos, NGFI-A, c-jun, junB and junD mRNA expression for up to 6 days in non-neuronal cells, whereas in sympathetic neurons the expression of only c-junmRNA was induced after axotomy and sialectomy. The induction of Fos and Jun proteins by neuronal injury was demonstrated by immunocytochemistry. The results indicate that neuronal injury induces IEGs mainly in non-neuronal cells and that in neurons only c-junis induced after axotomy. It is hypothesized that the induction of IEG other than c-junin neurons after brain injury is an indirect event unrelated to the neuronal response to injury.

THIS paper describes the localization of the a-type peroxisome proliferator-activated receptor (PPARa) in the rat brain using immunocytochemistry and in situhybridization. Expression of PPARa mRNA was highest in the granular cells of the cerebellar cortex and in the dentate gyrus, with a somewhat lower expresssion in areas CA1-CA4 of the hippocampus. PPARa mRNA was also found in some neurones of the cerebral cortex (layers II-IV) and the molecular layer of the cerebellar cortex, and in the olfactory tubercle. Immunocytochemistry revealed nuclear PPARα-immunoreactivity (-IR) in the same areas as seen with the in situhybridization. Furthermore, PPARa-IR was also localized in oligodendrocytes, whereas the other glial cell types appeared to lack PPARα. These results suggest that peroxisome proliferators and chemicals acting similarly have effects on discrete populations of neurones. The presence of PPARa in oligodendrocytes lends further support to the suggestion that peroxisomes are imprortant in the assembly and degradation of myelin.

In this study we investigated the expression of the immediate early genes (IEGs) c-fos, c-jun, and junD on mRNA and protein levels during the spermatogenic cycle of the rat using Northern blotting, in situ hybridization, and immunocytochemistry. The expression of these IEG mRNAs and proteins exhibited stage-specific variations. The results suggest that IEGs take part in transcriptional events that are involved in regulating the proliferation and differentiation of spermatogenic cells during specific stages of the cycle of the seminiferous epithelium.

The localization of somatostatin-like immunore-activity (SOM-LI) was examined in human lumbar sympathetic ganglia using the peroxidase-antiperoxidase method. Few of the principal neurons showed immunolabelling for somatostatin and sparse networks of nerve terminals were unevenly associated with ganglion cells. Using light microscopy, the most intense SOM-LI was seen in the perinuclear zone of the neurons. Electron-microscopically, the staining was localized on the membranes of the Golgi apparatuses. In the nerve terminals, SOM-LI was seen inside the small vesicles (40–60 nm diameter). All neurons with SOM-LI were also found to be tyrosine-hydroxylase immunoreactive when excamined with a double-staining technique. These results provide evidence that somatostatin and noradrenaline co-exist in human sympathetic neurons.

Calbindin D-28k-immunoreactivity has been demonstrated in some of the intrafusal muscle fibres and in the capsule of adult rat muscle spindles. In this study, the immunocytochemical localization of calbindin D-28k in the muscle spindles of triceps surae muscle was studied during postnatal maturation and after denervation. In young rats calbindin D-28k-immunoreactivity was seen in a few intrafusal fibres, first at the age of 4 days. At the 7th day, three calbindin D-28k-immunoreactive fibres and one unlabelled fibre were seen in most muscle spindles, as in adult rats. The spindle capsule and perineurial sheath of nerves were first seen to exhibit calbindin D-28k immunoreactivity at the age of 14 days, and thereafter the localization of calbinding D-28k-like immunoreactivity was similar to that in adult rats. After denervation, calbindin D-28k-immunoreactivity remained in intrafusal muscle fibres and the spindle capsule for a long period. After two months of denervation, calbindin D-28k immunoreactivity could still be seen in the spindle capsule, but the intrafusal fibres were not labelled. The innervation is known to have trophic effects on the intrafusal fibres. The present findings suggest that the expression of calbindin D-28k-immunoreactivity in maturating muscle spindles may be induced by the developing innervation. The decrease of calbindin D-28k-immunoreactivity in intrafusal fibres after denervation may be due to the loss of trophic factors released by the nerves.

The de novo synthesis and expression of sulfo sLex glycan on vascular endothelial glycoproteins has a central role in the initiation of inflammatory reactions, serving as a putative ZIP code for organ-specific trafficking of leukocytes into sites of inflammation. The synthesis of sulfo sLex requires energy carrying donors, CMP-sialic acid (CMP-SA), GDP-fucose (GDP-Fuc), and adenosine 3′-phosphate 5′-phosphosulphate (PAPS) for donation of SA, Fuc, and sulfate, respectively. These donors are synthesized in the nucleus or cytosol and translocated into Golgi by specific transporters where corresponding transferase and proteins as well as enzymatic activities increase on inflammatory stimuli. Here we analyze the transcriptional coregulation of CMP-SA, GDP-Fuc, and PAPS transporters with in situ hybridization and real-time PCR in acute inflammation using kidney and heart allografts as model systems. Our results indicate that these three transporters display coordinated transcriptional regulation during the induction of the sulfo sLex glycan biosynthesis. With in silico analysis, the data generated with 230 human Affymetrix U133A gene chips indicated that the coregulated expression of CMP-SA and GDP-Fuc transporters was not common. Taken together our results suggest that inflammation-induced transcriptional regulation exists for Golgi membrane transporters required for the synthesis of the inflammation-inducible ZIP code sulfo sLex glycans.