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Background/Aims: Calbindin D28k has been reported to be involved in transcellular calcium transport along the rat distal convoluted tubule (DCT). It has also been shown that administration of bumetanide is associated with hypercalciuria. The experiments reported here were designed to verify whether chronic infusion of this diuretic affects the gene expression and protein abundance of calbindin D28k along rat kidney DCT. Methods: Bumetanide was subcutaneously infused by an osmotic minipump for 7 days at a rate of 1.5 mg·h–1·kg–1. cDNA was synthesized from total RNA extracted from DCT microdissected from collagenase-treated kidneys. Results: Calbindin D28k mRNA abundance, quantified by competitive PCR, was found to be 13.7 ± 1.9 amol·ng–1 total RNA in DCT of control rats (n = 4) as compared to 24.2 ± 2.4 amol·ng–1 total RNA in DCT of bumetanide- treated rats (n = 5) (p < 0.01). This effect was associated with a 52% increase (p < 0.005) in calbindin D28k protein abundance, as detected by Western blot performed on tissue slices from renal cortex (n = 4). Conclusion: These data not only demonstrate that bumetanide upregulates the mRNA and protein abundance of calbindin D28k in rat DCT, but also suggest that DCT calcium reabsorption is increased following the administration of this loop diuretic.

In this study we show that SV-IV, a major immunomodulatory, anti-inflammatory, and sperm immunoprotective protein secreted from the rat seminal vesicle epithelium, acts in vitro as a substrate of protein kinase C (PKC) competing efficiently with H1 histone, a very well known PKC substrate. Electrospray mass spectrometry (ES-MS) analysis demonstrated that approximately 10% of the native SV-IV molecules were phosphorylated by PKC and that such a modification involved only a single serine residue (Ser58) out of the 22 occurring in the protein. Interestingly, this modification produced a substantial enhancement (approximately 50%) of the native SV-IV's ability to stimulate the activity of both horseradish peroxidase (POD) and selenium-dependent glutathione peroxidase (GPX), an enzyme that is known to protect the mammalian spermatozoa from oxidative stress and loss of motility in the female genital tract following ejaculation. In contrast, the phosphorylation of SV-IV on Ser58 did not produce any effect on the anti-inflammatory properties of SV-IV, as measured by its ability to inhibit the phospholipase A2.

SV-IV is a basic, thermostable, secretory protein of low Mr (9758) that is synthesized by rat seminal vesicle (SV) epithelium under strict androgen transcriptional control. This protein is of obvious pharmacological interest because it has potent nonspecies-specific immunomodulatory, anti-inflammatory, and pro-coagulant activities. In evaluating the clinical relevance and the possible use in medicine of SV-IV, we became interested in the study of its structure–function relationships and aimed to identify in its polypeptide chain specific peptide fragments possessing the marked anti-inflammatory properties of the protein not associated with other biological activities (pro-coagulation and immunomodulation) typical of this molecule. By using two different experimental approaches (the fragmentation of the protein into peptide derivatives by chemical methods and the organic synthesis on solid phase of selected peptide fragments), data were obtained showing that in this protein: (a) the immunomodulatory activity is related to the structural integrity of the whole molecule; (b) the anti-inflammatory activity is located in the N-terminal region of the molecule, the 8–16 peptide fragment being the most active; (c) the identified anti-inflammatory peptide derivatives do not seem to possess pro-coagulant activity, even though this particular function has been located in the 1–70 segment of the molecule.

The effect of nontoxic, low concentrations (10 −8 M) of retinoic acid (RA) for a relatively long time (28 days) on a Kirsten ras-virus transformed cell line (Ki-SVC1), derived from the rat seminal vesicle epithelium, was investigated. In these experimental conditions, the cell treatment with RA induced a decrease of the proliferation rate, apoptosis and a marked reduction of both anchorage-independent growth and tumorigenicity. These biological responses were either preceded or associated with important changes in adenylate cyclase/protein kinase C signaling pathways, the activation of important apoptosis-linked genes and a marked decrease of the v-Ki-ras p21 protein. The significance of these findings is discussed.

The sequential use of reverse transcriptase and the polymerase chain reaction (RT-PCR) has provided molecular biology research with an exquisitely sensitive and fast technique for studying gene expression. This method is particularly useful to study transcripts in the nervous system, which are on average present at low levels and the amount of tissue or cells to be analyzed is often limited. Here, we describe a RT-PCR assay which allows the simultaneous detection and semi-quantitation of several transcripts (multiplex). Multiple PCR primer pairs are used to detect different target transcripts in a single reaction, together with a pair of primers able to amplify the hypoxantine-phosphoribosyl-transferase (HPRT), a gene constitutively expressed at low levels throughout the nervous system. HPRT levels remain constant also during neurogenesis and it is thus apt to be used in developmental neurobiology. This internal standard is the mRNA of reference to evaluate sample variation in RT and PCR reactions and to monitor the degradation and recovery of RNAs. Normalization with respect to HPRT cDNA allows to estimate the relative abundance of each target mRNA.

Organisms exposed to ionizing radiation are mainly damaged by free radicals, which are generated by the radiolysis of water contained in the cells. Recently a significant reduction of tissue injury from irradiation damage was demonstrated by using MnSOD-plasmid/liposome treatments in the protection of murine lung. In this study we show that a new active recombinant human MnSOD (rMnSOD), easily administered in vivo, not only exerts the same radioprotective effect on normal cells and organisms as any MnSOD, but it is also radiosensitizing for tumor cells. In addition, we show how healthy animals, exposed to lethal doses of ionizing radiation and daily injections with rMnSOD, were protected from radiodamage and were still alive 30 days after the irradiation, while animals treated with only PBS solution, in the absence of rMnSOD, died after 7-8 days from the radiotreatments. The molecular analysis of all irradiated tissues revealed that the antiapoptotic AVEN gene appeared activated only in the animals treated in the presence of rMnSOD. The data suggest that rMnSOD deserves to be considered as a pharmaceutical tool for making radiotherapy more selective on cancer cells and to prevent and/or cure the accidental damage derived from exposure to ionizing radiation.

Seminal vesicle protein IV (SV-IV) is a secretory anti-inflammatory, procoagulant, and immunomodulatory protein produced in large amounts by the seminal vesicle epithelium of the rat under the strict transcriptional control of androgen. In particular, this protein was shown to possess the ability to markedly inhibit in vivo the humoral and cell-mediated immune responses of mice to nonbacterial cellular antigens (sheep erythrocytes and spermatozoa). We report data that demonstrate that in mice treated with SV-IV and infected withSalmonella entericaserovar Typhimurium, SV-IV is able to downregulate some important immunological and biochemical parameters that serovar Typhimurium normally upregulates in these animals. This event did not correlate with a lower bacterial burden but was associated with a markedly increased one (300%). Furthermore, the treatment of mice with SV-IV alone also produced a significant increase in the rate of mortality among serovar Typhimurium-infected animals. The mechanism underlying these phenomena was investigated, and the strong immunosuppression produced by SV-IV in serovar Typhimurium-infected mice was suggested to be the basis for the increased rate of mortality. The SV-IV-mediated immunosuppression was characterized by a decrease in the humoral and cell-mediated immune responses, altered lymphocyte-macrophage interaction, downregulation of cytokine and inducible nitric oxide synthase gene expression, inhibition of macrophage phagocytosis and intracellular killing activities, and absence of apoptosis in the splenocyte population of SV-IV- and serovar Typhimurium-treated mice. The immunosuppressive activity of SV-IV was specific and was not due to aspecific cytotoxic effects. SV-IV-specific receptors (Kd= 10−8M) occurring on the macrophage and lymphocyte plasma membranes may be involved in the molecular mechanism underlying the SV-IV-mediated immunosuppression. Some results obtained by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay also revealed a functional impairment of mitochondria (a decrease in mitochondrial dehydrogenase activity), thus indicating the possible implication of these organelles in the immunosuppressive process.

Retinoic acid (RA) and sodium butyrate (NaB) are regulators of cell growth and differentiation. We studied their effect on normal (SVC1) or v-Ki-ras-transformed (Ki-SVC1) rat seminal vesicle (SV) epithelial cell lines. The treatment of these cells with 10(-((7( M RA did not produce significant changes in the morphological and biochemical parameters analyzed. When RA was used in combination with 2 mM NaB, the treatment induced substantial morphological changes, apoptosis-independent growth arrest, up-regulation of tissue transglutaminase (tTGase), and down-regulation of beta and gamma RA receptor (RAR) mRNA expression. The same cells did not express RAR alpha either before or after NaB/RA treatment. A similar treatment did not change the amount of mRNA coding for the protein SV-IV (a typical differentiation marker of the SV epithelium) in normal or ras-transformed cells nor the level of v-Ki-ras mRNA in Ki-SVC1 cells. These findings suggest that a defective RA/RARs signaling pathway is probably the biochemical condition that underlies the unresponsiveness to RA of our in vitro culture system, and indirectly points to the possibility that the NaB/RA-induced effects were brought about by a cooperation at the transcription level between the histone deacetylase inhibitory activity of NaB and the ability of RA/RAR to modulate the expression of various genes involved in the control of cell growth and differentiation.

Homozygous wobbler mouse mutants develop a progressive paralysis due to spinal motoneuron degeneration. To understand the molecular aspect underlying the genetic defect we have studied the embroyonic (from E13) and postnatal expression of the three neurofilament and choline acetyltransferase genes in each member from several wild-type (wt) and wobbler (wr) progenies. There are no variations among wt littermates at all ages studied. In contrast, analyses of neurofilament mRNA reveals a 3–4-fold increase of medium neurofilament (NFM) mRNA in wobbler mice ( wr wr ). The pattern of increased NFM mRNA during development, prior to the appearance of the wobbler phenotype, among littermates (from heterozygous carriers) conforms to a mendelian inheritance of a single gene defect 1:2:1: (wr/wr:wr/ +:+/+). Light and heavy neurofilament mRNA levels are also increased later in development exclusively in those individuals with high NFM mRNA values indicating that increase of the latter is associated with increase of the light and heavy subunit expression. Also NF proteins are increased. Expression of choline acetyltransferase gene is instead always comparable to normal control. Our study provides novel insights into the nature of the wobbler defect, strengthening the hypothesis that neurofilament accumulation plays a pivotal role in the etiopathogenesis of motoneuron degeneration.

The amino acid glutamate is a widespread excitatory neurotransmitter in the brain. It activates cation-selective channels expressed by nearly every neuron and by glial cells; also various second messenger cascades. Little is known about the ontogeny of glutamate neurotransmission during neurogenesis. We have analyzed the development and differentiation of excitatory amino acid responses and Na+ channels in cells dissociated from embryonic rat ventral mesencephalon and striatum as well as cortex and cerebellum using fluorescent voltage-sensitive oxonol dyes and flow cytometry. Analysis of fluorescence distribution revealed complex profiles under resting conditions which changed in a characteristic manner over the period studied (Embryonic (E) Days 12-20). The response to the Na+ channel agonist veratridine appeared at E12/13 in the mesencephalon. At E13 L-glutamate and kainate evoked changes in membrane potential interpreted as cellular hyperpolarization. At E15 some cells still responded by hyperpolarizing but an equal number began to depolarize. By E18 most cells depolarized. Both hyper- and depolarizations were eliminated by a specific antagonist at kainate receptors (6-cyano-7-nitroquinoxyline-2,3-dione) and by resuspending the cells in Na(+)-free medium. Both responses exhibited a concentration dependency with higher doses evoking stronger effects. In contrast, there was little effect of veratridine in the striatum at E15-E16, and the response to kainate or L-glutamate was predominantly depolarizing during the same embryonic period, with little or no effect until E18. These data show that in the developing CNS, sodium channel responses as well as excitatory aminoacid neurotransmitter responses first become functional in the mesencephalon and subsequently in the striatum, thus suggesting an anatomical gradient of expression. Our results also show that glutamate receptor-coupled functions vary with embryonic age and with regional distribution, suggesting possible roles of glutamate in early CNS embryogenesis, as morphogens or modulators of synaptic plasticity.

Summary SV-IV (seminal vesicle protein no. 4) is a potent immunomodulatory and anti-inflammatory secretory protein (Mr 9758) produced in large amounts by the rat seminal vesicle epithelium. Here we show that this protein possesses the ability to upregulate in J774 macrophages the expression of the gene coding for the inducible nitric oxide synthase (iNOS). The increase in NO production consequent on the marked enhancement of iNOS activity was not associated with apoptotic damage of the SV-IV-treated cells. In the same experimental model, however, LPS induced upregulation of iNOS coupled with an increase in NO production and marked apoptotic death. Differences in the ability of SV-IV and LPS to control the life/death signal balance in target cells via trans-membrane activation of apoptotic (mediated by TNF-α and NO/iNOS system) and anti-apoptotic (mediated by bcl-2, c-myc, etc.) pathways are suggested to be the basis of the apoptotic fate of the experimentally treated cells. In addition, considering the important role played by NO in the process of mammalian reproduction, SV-IV may be involved in the fine tuning of NO concentration in the female genital tract mucosa via an SV-IV-mediated control of iNOS gene expression in local macrophages.

The major toxic components in the outer membranes of gram-negative bacteria are lipopolysaccharides (LPS) (endotoxins) and porins. The latter are hydrophobic proteins (mass, about 35,000 Da) that account for more than 50% (18, 35) of the total membrane proteins and that are named porins for their ability to form transmembrane channels for the passive diffusion of small molecules across the cell membrane (26, 27). Purified porins possess immunomodulatory and procoagulant activities and are considered pathogenicity determinants. Depending on the dose, LPS and porins are either frankly toxic to a number of target cells or significantly alter normal cell functions (10, 45). By acting on human polymorphonuclear leukocytes (PMNs), subtoxic concentrations of porins, for example, inhibit phagocytosis and intracellular killing of Salmonella typhimurium (45), decrease oxidative burst and cell hydrophobicity, and cause significant morphological changes in the target cells (46). Subcutaneous injection of porins in rat hind paws causes local inflammation without complement activation (12, 13), a process that is known to be triggered in vitro by these proteins. Finally, it has recently been demonstrated that nontoxic concentrations of porins stimulate the synthesis and release of platelet-activating factor from different types of human cells (PMNs and mesangial and endothelial cells) (6, 41, 42) and promote proinflammatory and immunomodulatory cytokine release from immunocompetent cells or other cellular sources (11). Porins from different microorganisms have similar effects (8, 23, 43, 44). The cell damage from severe infections by gram-negative bacteria comes mainly from exotoxins and enterotoxins, even though the occurrence of LPS and porins in the bacterial microenvironment contributes significantly. Exposure of target cells to high concentrations of these substances leads to a rapid lytic death of the cells (1, 5, 10, 34, 45). In contrast, lower concentrations of LPS produce a less dramatic type of death: programmed cell death (PCD), or apoptosis (49). Classic morphological and biochemical signs of apoptosis in the toxin-injured cells are cell shrinkage, extensive blebbing of the cell surface, disappearance of microvilli, chromatin condensation, internucleosomal DNA fragmentation, presence of large intracytoplasmic vacuoles, increase of intracellular calcium ion and free radical levels, enhancement of endonuclease and protease activities, cell cycle arrest, and modified transcriptional activity of different apoptosis-linked genes (p53, bcl-2, c-myc, etc.) (2, 7). An increase in the expression of type II transglutaminase (tissue transglutaminase [tTGase]) has also been reported to be associated with apoptosis in several cell types, and an important role for tTGase in this process has been proposed (15). The ability of bacterial endotoxin to kill cells by necrosis and/or apoptosis has been found to play a key role in the pathogenesis of many alterations occurring in subjects with infections by gram-negative bacteria, including disseminated intravascular coagulation, septic shock, degenerative processes in different tissues, and reduced fertility or sterility (4, 10, 24, 48). The tissue lesions produced by Pseudomonas aeruginosa are of particular interest. P. aeruginosa is an opportunistic pathogen that commonly invades immunocompromised patients. The main targets of the LPS and porin released by this gram-negative rod during its lysis or active growth are the endothelial and epithelial cells present in many anatomical regions, including the skin, eyes, genitourinary tract, and heart valves. In particular, it has been reported that low levels of natural porin or LPS in patients with mild chronic infections severely damage the human spermatozoa in vitro and lead to reduced fertility or sterility (29). On the basis of these data and considerations, and taking into account the scarce information available on the cytotoxic effects of very low concentrations of LPS or porin on specific target cells, we were prompted to investigate the possible involvement of these substances in the induction of apoptosis in a cell line (SVC1) derived from the rat seminal vesicle secretory epithelium (36, 37, 49).

Human telomerase reverse transcriptase (hTERT) gene expression in resected specimens of oral squamous cell carcinoma (OSCC) and their surrounding tissue, either apparently normal or clearly histologically dysplastic, was evaluated by both real-time RT-PCR and immunohisto-chemical protein analyses. The expression level of hTERT in oral dysplasia and in OSCC was markedly higher than in normal tissues. The correlation between hTERT expression in OSCC and clinico-pathological parameters or survival of OSCC patients was statistically analyzed. Our study demonstrates that there is no significant relationship between hTERT expression and classical clinico-pathological parameters. Interestingly, survival analysis showed both overexpressing cases and lower survival rate in the early stage of OSCC (p=0.03 for immunohistochemistry; p=0.04 for RT real-time PCR). The histological location of hTERT in these tumors has been discussed in the context of the cancer stem cell theory.