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F Sierra

Publications and source records attributed to F Sierra.

At least 37 records · Page 2Linked to original sources
PubMed1997

T-kininogen present in the liver of old rats is biologically active and readily forms complexes with endogenous cysteine proteinases.

We have previously reported an increase in T-kininogen mRNA levels in the liver of ageing Sprague-Dawley rats. T-Kininogen functions both as a precursor to the vasoactive peptide T-kinin, and as a potent and specific inhibitor of cysteine proteinases. Under normal physiological conditions, the majority of cysteine proteinases are found intracellularly and we have shown that a significant proportion of T-kininogen also accumulates intracellularly in the liver of old rats. Therefore, our aim was to determine whether or not this T-kininogen is biologically active as an inhibitor of cysteine proteases. Titration of whole liver extracts indicates that old rats do indeed contain a 4-fold higher level of cysteine proteinase inhibitory activity than younger counterparts. Using gel permeation chromatography in conjunction with an enzyme inhibitor assay, we show that this difference is mainly due to the presence of a low level of free biologically active T-kininogen. However, Western blot analysis of the gel permeation chromatography fractions demonstrate that most of the intrahepatic T-kininogen is found as enzyme-inhibitor complexes. Alkaline inactivation of the cysteine proteinase component of these complexes leads to the release of biologically competent free T-kininogen. These findings are discussed with regard to the possible mechanisms responsible for the accumulation of T-kininogen within the aged rat liver.

Aging↗
PubMed1997

Differential expression of voltage-gated Ca2+ conductances in human neuroblastoma NB69 cells cultured in defined serum-free and astrocyte-conditioned media.

Voltage-gated Ca2+ conductances were investigated with the whole-cell patch-clamp technique-either using Ca2+ or Ba2+ as charge carriers-in NB69 human neuroblastoma cells plated in "defined" serum-free (DM) and in "astroglial-conditioned" media (CM). Cells expressed the microtubule associated protein 1A when plated in both media, indicating neuronlike differentiation. Cells of similar sizes and shapes were selected for recordings. Different sets of voltage-gated Ca2+ current types were usually expressed in DM- and CM-plated cells. DM-plated cells exhibited a high-voltage-activated current (HVAC) in isolation, whereas 43% of the CM-plated cells also displayed the low-voltage-activated current (LVAC). The membrane surface density of the HVAC was about twofold higher in CM than in DM-plated cells and increased with plating time from 10 and 16pA/pF (days 1-4) to 24 and 37 pA/pF (days 5-10) in DM- and CM-plated cells, respectively. However, the amplitude of the LVAC did not change significantly with culture age. In conclusion, NB69 cells expressed HVAC in isolation when plated in DM, whereas both HVAC and LVAC were present in many CM-plated cells, suggesting that the CM contained diffusible factors secreted by astroglial cells which: (1) could induce the appearance of the LVAC and (2) increased HVAC current expression.

Astrocytes↗
PubMed1996

Altered expression of the twist gene in young versus senescent human diploid fibroblasts.

We have recently reported the cloning and characterization of the human twist gene (H-twist), which encodes an evolutionarily conserved helix-loop-helix transcription factor. In several species, twist has been shown to play a role in mesoderm differentiation. We now report on the differential expression of H-twist as a function of in vitro life span in human diploid fibroblasts. H-twist was found to be expressed at a relatively high level in quiescent young cells. Addition of fresh serum to young G0 cultures results in a rapid repression of H-twist mRNA expression. This repression depends on the presence of growth factors, specifically EGF, and requires protein synthesis. mRNA stability studies using either actinomycin D or alpha-amanitin indicate a similar decay rate of H-twist mRNA in both young and senescent cells, suggesting that the differential expression of H-twist in young versus old is not primarily regulated at the level of mRNA turnover.

Cell Line↗
PubMed1996

Preparation of giant myelin vesicles and proteoliposomes to register ionic channels.

Myelin vesicles, reconstituted liposomes with proteolipid protein (PLP), the main protein component of myelin, and electrophysiological patch-clamp are potentially powerful tools to study the role of myelin in functional ionic channels. However, technical difficulties in the vesiculation of myelin and the small size of the vesicles obtained do not permit the application of micropipettes for current recordings. From a suspension of purified myelin we have prepared oligolamellar vesicles (mean diameter of 144 nm) using the so-called French pressure system. From this preparation we obtained giant myelin vesicles approximately 10 microns in mean diameter, using a dehydration-rehydration procedure. Qualitative analysis of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed no significant loss of any component in these vesicles due to pressure, in comparison with non-vesiculated myelin. A way of preparing giant liposomes of approximately 80-100 microns and proteoliposomes of approximately 30 microns in mean diameter, using the same dehydration-rehydration procedure, is also reported. Reconstitution of purified PLP in giant liposomes was confirmed by fluorescent labeling of PLP and by fluorescence microscopy. The current recordings from these vesicles prove the validity of these methods and provide significant evidence of the existence of ionic channels in myelin membranes and the possibility that PLP functions as a channel. The physiological significance and characterization of these channels remain yet unresolved. These results have a special significance for elucidating the molecular role of myelin in the regulation of neural activity and in the brain ion microenvironment.

Animals↗
PubMed1996

Cerebral tissue oxygenation during hypoxia and hyperoxia using artificial placentation in lamb.

Aiming at a better understanding of the pathophysiologic basis of perinatal encephalopathy, we evaluated patterns of tissue oxygenation during hypoxia and hyperoxia. We utilized both laserspectroscopy and invasive tissue-Po2 microneed measurements synchronously in five newborn lambs (141-143 days of gestation). The model of artificial placentation provided defined changes of the blood gases, using a extracorporeal circuit with interposition of membrane lung. During hyperoxia, the Po2 at the blood outlet port of the lung was raised to > 300 mmHg for five minutes. During hypoxia, Po2 was diminished as oxygen at the gas phasis was replaced by nitrogen. After the induction of hyperoxia, a rise of tissue-Po2 was observed. The synchronously recorded data of the laserspectroscopy showed adequately rising HbO2 values in concordance (r = 0.97, p < 0.001). As a constant finding we did not observe Cyt-aa3 changes during induced hyperoxia with tissue-Po2 values of < 40 mmHg. Furthermore, no changes in blood volume occurred in this case. A different pattern of the laserspectroscopic parameters was found when the tissue-Po2 rose above a value of > 40 mmHg and Cyt-aa3 rose after a lag-time occurred. During induced hypoxia an immediate fall of tissue-Po2 corresponding with a fall of HbO2 in the spectroscopic tracing occurred (r = 0.87, p < 0.001). A fall of the Cyt-aa3 level was seen with a lag-time when the tissue-Po2 had reached values of below 10 mmHg. In addition, a rise of blood volume was recorded in all cases of induced hypoxia. In conclusion, the results indicated that cellular redoxe state remains stable over a large range of oxygen partial pressure changes.

Animals↗
PubMed1995

Both T- and K-kininogens increase in the serum of old rats but by different mechanisms.

In the rat, kininogens are produced from two distinctly regulated gene families, T- and K-kininogens. We have previously established that T-kininogen (T-KG, thiostatin) gene expression is increased in the liver of old rats, as compared to their younger counterparts. In this report, we have used Northern blot analysis to show that the steady state mRNA levels for either the LMW (Low Molecular Weight) or HMW (High Molecular Weight) forms of K-KG do not change significantly between 15 and 25 months of age. On the other hand, probing of Western blots containing total serum proteins showed that, as for T-KG, old animals have consistently elevated levels of HMW-KG. These results indicate that old rats contain elevated serum levels of both T- and K-KG. Nuclear run on experiments have shown that the increase in T-KG gene expression is controlled primarily at the transcriptional level. Our present results with K-KG, on the other hand, indicate that its expression is controlled post-transcriptionally.

Adolescent↗
PubMed1995

Identification of several isoforms of T-kininogen expressed in the liver of aging rats.

We have previously shown that senescent Sprague-Dawley rats have significantly increased levels of kininogen (KG) mRNA and protein, when compared with younger counterparts. In the rat, five different isoforms of kininogen have been identified: high-molecular-weight K-kininogen, low-molecular-weight K-kininogen, and T1 alpha, T1 beta, and T2 kininogens. Several of these isoforms have been shown to differ in their biological properties. It was therefore considered relevant to establish which of these isoforms are expressed in the liver of old rats. To this end, we have isolated and sequenced nine independent cDNA clones from a library constructed using liver mRNA from healthy senescent rats. Predicted protein sequences indicate the presence of T-kininogens only. The relative lack of induction of K-kininogens during aging was further confirmed by RNA hybridization experiments. The nucleotide sequences reveal a microheterogeneity of silent polymorphisms, suggesting the presence and expression of several different alleles of the genes. From our data we conclude that (i) Several isoforms of T-KG are expressed in the liver of senescent Sprague-Dawley rats and (ii) T1 kininogens appear to be the most highly represented T-KG mRNA species in old rat livers.

Aging↗
PubMed1995

N-type Ca2+ channels mediate transmitter release at the electromotoneuron-electrocyte synapses of the weakly electric fish Gymnotus carapo.

The effects of omega-conotoxin-GVIA (omega-CgTX) on synaptic transmission were studied in the electromotoneuron-electrocyte synapses of the electric organ (EO) of the weakly electric fish Gymnotus carapo. omega-CgTX selectively and irreversibly blocked excitatory postsynaptic potentials (EPSPs) in a dose dependent-manner. The toxin had no effect on: (a) resting postsynaptic membrane potential and conductance; (b) postsynaptic action potentials elicited by depolarizing transmembrane current pulses; (c) the action potential conduction in the presynaptic fiber; (d) acetylcholine (ACh)-induced postsynaptic responses. Nifedipine - a selective dihydropyridine antagonist of the L-type voltage-dependent Ca2+ channels (VDCCs) - did not affect synaptic transmission. Transmission was also undisturbed by the peptide omega-Agatoxin (omega-Aga-IVA), the low molecular weight polyamine, funnel-web toxin (FTX) - both included in the venom of the spider Agelenopsis aperta - and its synthetic analog sFTX, all selective blockers of P-type VDCCs. Since omega-CgTX irreversibly blocks the N-type VDCCs, we conclude that presynaptic N-type VDCCs mediate transmitter release at electromotoneuron terminals. The VDCCs involved in fish peripheral electromotoneuron-electrocyte presynaptic transmitter release are therefore similar to those in amphibian, reptilian and avian peripheral synapses, but differ from mammalian and invertebrate motoneuron terminals.

Animals↗
PubMed1993

Type 1 plasminogen activator inhibitor is not an acute phase reactant in rats. Lack of IL-6- and hepatocyte-dependent synthesis.

The contributing role of hepatocytes and IL-6 in the acute phase-like elevation of plasma type 1 plasminogen activator inhibitor (PAI-1) in vivo is not known. We addressed this question by comparing the effects of two inflammatory stimuli known to increase plasma concentrations of IL-6 on the plasma concentrations and site of synthesis of PAI-1 and cysteine proteinase inhibitor (CPI) in rats. Subcutaneous injection of turpentine results in a sustained increase in plasma IL-6 and CPI Ag levels over a 24-h period. In contrast, plasma PAI-1 activity was not altered by turpentine treatment. Northern blot analysis of poly(A)+ mRNA extracted from freshly isolated hepatocytes of saline- or turpentine-treated animals demonstrated induction of CPI mRNA expression but failed to detect basal or induced PAI-1 or IL-6 mRNA expression. However, PAI-1 mRNA was detected in rat hepatocytes in primary culture for 24 h and was induced by dexamethasone. Intravenous infusion of bacterial LPS (4 mg/kg) induced a sustained increase in plasma CPI Ag and transient increases in plasma IL-6 and PAI-1. Northern blot analysis of freshly isolated, fractionated liver cells indicated that LPS treatment (3 h) induced PAI-1 mRNA expression most significantly in the endothelial and Kupffer cell fractions. IL-6 mRNA expression was induced in Kupffer cells and CPI mRNA was induced in hepatocytes. Immunocytochemical analysis revealed LPS-induced accumulation of PAI-1 Ag associated with the vascular endothelium, subendothelial matrix, and sinusoidal lining cells. Our results indicate that PAI-1 mRNA is not significantly expressed by rat hepatocytes in vivo and that plasma PAI-1 levels are not influenced by increased IL-6 expression in Kupffer cells or in plasma.

Acute-Phase Proteins↗
PubMed1992

Serum T-kininogen levels increase two to four months before death.

We have reported an accumulation of T-kininogen mRNA in the liver of aging Sprague-Dawley rats. T-kininogen is a cysteine proteinase inhibitor. Since a disruption of the intracellular protein degradation machinery is known to occur during senescence, we wished to further define the role of this protein in the aging process. As a first step, we have measured T-kininogen levels both in serum and within the liver. We have found that serum protein levels are indeed augmented during senescence, although not as dramatically as the mRNA (2.5-fold versus 8.3-fold). Immunocytochemistry, as well as Western blot analysis suggests that this is due to the presence of T-kininogen within hepatic cells in aged rats. Life-long dietary restriction, a known age-prolonging treatment, decreases the overexpression of the protein in 24-month-old rats. Later, diet-restricted animals still show an increased expression from the gene, the effect being delayed but not abolished by dietary manipulation. Interestingly, a longitudinal study indicated the existence of a positive correlation between the time of increase of serum T-kininogen and the time of death of the animal. Serum T-kininogen was found to increase 2.5-4 months before death.

Aging↗
PubMed1990

Differential in vitro transcription from the promoter of a rat alpha 2u globulin gene in liver and spleen nuclear extracts.

When used in an in vitro transcription assay, the promoter of a cloned alpha 2u globulin gene is much more active in liver than in spleen nuclear extracts. Promoter deletion experiments suggest that both positive and negative regulatory mechanisms may be involved in the differential in vitro transcription from the alpha 2u globulin promoter in these two nuclear extracts. Interestingly, removal of promoter elements upstream from position -74 results in a significant increase of in vitro transcription in spleen but not in liver nuclear extracts, and thus reduces the difference in transcription observed with longer alpha 2u promoters in these two extracts. Deletion of additional nucleotides to position -43 strongly reduces the in vitro transcription efficiency of the promoter in extracts from both tissues. None of the examined promoters containing between 3000 and 22 nucleotides of 5' flanking regions are differentially transcribed in liver nuclear extracts from either male or female rats. Thus, in contrast to cell-type specificity, sex-specificity could not be observed in our in vitro transcription experiments. DNase I protection experiments with crude nuclear extracts and partially or highly purified nuclear proteins suggests the presence of six recognition sites for DNA-binding factors between the TATA element and position -210. Some of these factors could be identified as proteins that also bind to elements within the albumin gene promoter.

Alpha-Globulins↗
PubMed1989

T-kininogen gene expression is induced during aging.

We have constructed a cDNA library from senescent (24-month-old) rat liver mRNA and, by differential screening, have selected clones corresponding to mRNA species with increased abundance in aging rats. Direct sequencing of the inserts indicated that most of the clones (9 of 10) contained sequences coding for T-kininogen, also called major acute-phase protein, cysteine protease inhibitor, or thiostatin. Nuclear elongation experiments showed that the increase in mRNA concentration was controlled at the transcriptional level. RNase mapping and S1 analysis indicated that the age-dependent induction operated preferentially at one of the three transcriptional start sites of the gene(s). The acute-phase reaction (inflammation) is known to also induce these genes at the level of transcription; however, two of the three start sites are induced by inflammation. Transcription from one of these sites was induced by both phenomena, aging and inflammation.

Acute-Phase Proteins↗
PubMed1986

Different tissue-specific expression of the amylase gene Amy-1 in mice and rats.

We cloned the rat alpha-amylase gene Amy-1 and compared its structure and expression with its mouse counterpart. The results showed that the general organization of the transcriptionally active rat Amy-1 gene was similar to that of its mouse counterpart; i.e., the rat gene also contained two independent transcriptional promoters. The distance between the two promoters in the rat gene was, however, more than double (6 kilobases) that measured in the mouse gene (2.8 kilobases). In addition, the rat genome also contained an independent, orphonlike version of the weaker Amy-1 promoter, which was transcriptionally silent. In spite of the similar overall organization of the Amy-1 genes in mouse and rat cells, an interesting difference was observed in the expression of the weak promoter in these two closely related rodents. In rats this promoter was significantly active only in liver cells, while in mice it was utilized with similar efficiencies in parotid, liver, and pancrease cells. Moreover, the transcripts produced in rat liver had a very heterogeneous population of 5' ends, located between 180 and 220 nucleotides upstream of the two homologous start sites observed for this promoter in mouse liver, even though the sequences around this region were strongly conserved between the two species.

Animals↗
PubMed1983

Structure and in vitro transcription of a human H4 histone gene.

A human H4 histone gene was isolated and the nucleotide sequences of the mRNA coding as well as the 5' and 3' flanking regions were determined. No intervening sequences were found in this gene. A series of sequences which have been assigned putative regulatory roles in histone genes and/or in other genes were identified both upstream and downstream from the H4 histone protein coding region. Deletion mutants were constructed by BAL-31 nuclease digestion of sequences in the 5' flanking region of this H4 histone gene and were assayed in an in vitro transcription system. No regions upstream from the TATA box were required for site specific initiation in vitro. Data are presented which suggest that sequences located downstream from the 3' end of the coding region may influence the in vitro transcription of this human H4 histone gene.

Amino Acid Sequence↗