Biomedical subjects

J Marcinkiewicz

Publications and source records attributed to J Marcinkiewicz.

At least 19 recordsLinked to original sources

PubMed2006

Comparison of taurine chloramine and taurine bromamine effects on rheumatoid arthritis synoviocytes.

Fibroblast-like synoviocytes (FLS) participate in rheumatoid arthritis (RA) chronic synovitis by producing pro-inflammatory cytokines (IL-6, IL-8), growth factors (VEGF) and other inflammatory mediators (PGE2, NO). We have previously reported that Tau-Cl, generated by neutrophils, inhibits in vitro some of these pathogenic RA FLS functions. Taurine bromamine (Tau-Br) originates from eosinophils and neutrophils, and its immunoregulatory activities are poorly known. Therefore, we investigated the effects of Tau-Br on RA FLS functions and compared it to Tau-Cl anti-inflammatory action. When applied at noncytotoxic concentrations: (i) Tau-Br inhibited IL-6 and PGE2 production with potency similar to Tau-Cl (IC50 approximately 250 microM), (ii) Tau-Br failed to affect VEGF and IL-8 synthesis, while Tau-Cl exerted inhibitory effect (IC50 approximately 400 microM), (iii) none of these compounds affected NO generation and iNOS expression. Thus, Tau-Cl is more effective than Tau-Br in normalization of pro-inflammatory RA FLS functions.

Arthritis, Rheumatoid↗

PubMed2006

Taurine chloramine inhibits proliferation of rheumatoid arthritis synoviocytes by triggering a p53-dependent pathway.

OBJECTIVE AND DESIGN: Taurine chloramine (Tau-Cl), originating from activated neutrophils, possesses antiinflammatory activities. Fibroblast-like synoviocytes (FLS) participate in the chronic synovitis and synovial membrane hyperplasia that are characteristic pathological features of rheumatoid arthritis (RA). The present study was conducted to investigate the mechanism of the Tau-Cl effect on the proliferation of these cells in culture. MATERIALS AND METHODS: FLS were stimulated in vitro with platelet derived growth factor (PDGF) alone or together with Tau-Cl. Cell proliferation was evaluated by counting the total and dividing cell numbers and by measurement of (3)H-thymidine incorporation. Expression of the key cell-cycle regulators was evaluated at the protein (Western blotting) and/or mRNA (RT-PCR) levels. RESULTS: Treatment of RA FLS with Tau-Cl (200-500 microM) resulted in an early nuclear accumulation of p53 tumor suppressor protein. Moreover, Tau-Cl inhibited PDGF-triggered cell proliferation (IC(50) value approximately 250-300 microM), accompanied by characteristic modulation of p53 transcriptional targets: down-regulation of proliferating cell nuclear antigen (PCNA) and survivin, and concomitant up-regulation of p21 mitotic inhibitor. CONCLUSION: We propose that Tau-Cl inhibits proliferation of RA FLS by triggering a p53-dependent cell-cycle arrest and conclude that this compound suppresses pathways in FLS that are known to contribute to the pathology of RA.

Anti-Inflammatory Agents↗

PubMed2005

Is there a role of taurine bromamine in inflammation? Interactive effects with nitrite and hydrogen peroxide.

OBJECTIVE AND DESIGN: The myeloperoxidase system of neutrophils generates chlorinating and brominating oxidants in vivo. The major haloamines of the system are taurine chloramine (TauCl) and taurine bromamine (TauBr). It has been demonstrated in vitro that TauCl exerts both antiinflammatory and anti-bacterial properties. Much less is known about TauBr. The present study was conducted to compare bactericidal and immunoregulatory capacity of TauBr with that of the major chlorinating oxidants: HOCl and TauCl. Moreover, the effect of nitrites and H(2)O(2) on TauBr activity was investigated. MATERIALS: TauBr was prepared by reaction of HOBr with taurine. The reaction was monitored by UV absorption spectra. METHODS: Bactericidal activity of TauBr, TauCl and HOCl was tested by incubation of E. coli with the compounds and determined by the pour-plate method. To test the anti-inflammatory activity the compounds were incubated with LPS and IFN-gamma stimulated murine peritoneal macrophages. The production of following mediators was measured: nitrites by Griess reaction; TNF-alpha, IL-6, IL-10, IL-12p40 using capture ELISA. In some experiments the compounds were incubated with either nitrites or H(2)O(2). RESULTS: In our experimental set-up TauBr and HOCl exerted strong bactericidal effects on E. coli (MBC = 110 microM and 8 microM, respectively), while TauCl (< 1000 microM) did not kill test bacteria. However, both, TauBr and TauCl, at noncytotoxic concentrations (< 300 microM) inhibited the cytokine and nitric oxide production by macrophages. H(2)O(2) completely abolished the biological activities of TauBr but not those of TauCl. Nitrites did not affect any activity of TauBr or TauCl while they diminished the HOCl(-) mediated bacterial killing. CONCLUSION: TauBr, despite very low concentration of Br(-) in body fluids, may support TauCl and HOCl in the regulation of inflammatory response and in killing of bacteria by neutrophils. However, TauBr activity in vivo will depend on the presence of H(2)O(2) and possible other mediators of inflammation which can compete with target molecules for TauBr.

Animals↗

PubMed2004

Taurine chloramine and taurine bromamine induce heme oxygenase-1 in resting and LPS-stimulated J774.2 macrophages.

Taurine chloramine (TauCl) and taurine bromamine (TauBr) are products of activated neutrophils and eosinophils, respectively. It has been reported that TauCl, has strong anti-inflammatory properties. In a number of separate studies it has been shown that heme oxygenase-1 (HO-1), a stress inducible protein, exerts similar anti-inflammatory effects. In this study we investigated the influence of HO-1 on TauCl/TauBr mediated suppression of NO generation in J774.2 macrophages. Expression of HO-1 and inducible nitric oxide synthase (NOS-2) in LPS stimulated J774.2 cells provides an opportunity for determining these interactions. TauCl and TauBr, at non-cytotoxic concentrations, in a similar, dose-dependent manner, inhibited the expression of NOS-2, as evidenced by western blotting technique. Surprisingly, TauCl and TauBr induced expression of HO-1 in both non-activated and LPS-activated macrophages. Importantly, the fall in NOS-2 protein level was associated with a concomitant, dose-dependent induction of HO-1. In addition, an inhibitor of HO-1 activity, chromium III mesoporhyrin (CrMP), attenuated the inhibitory activity of TauBr but not that of TauCl, as measured by nitrite accumulation. These results suggest that at a site of inflammation, TauCl and TauBr may provide a link between taurine-dependent and HO-1-dependent cytoprotective mechanisms.

Animals↗

PubMed2002

Taurine chloramine modulates cytokine production by human peripheral blood mononuclear cells.

The effect of taurine (Tau) and taurine chloramine (Tau-Cl) on the production of TNF- alpha, IL-1 beta, and IL-6 by peripheral blood mononuclear cells of healthy volunteers was examined. Cells were stimulated with bacterial lipopolysaccharide (LPS) in the presence of either Tau or Tau-Cl. After 24 h culture the cytokine concentrations were measured in both culture supernatants (secreted) and cell lysates (cell-associated) using ELISA. In LPS-stimulated cells Tau-Cl inhibited both the secreted and cell-associated IL-1 beta and IL-6, while exerted dual effect on TNF- alpha production: raising it slightly at low and reducing at higher concentration. By contrast, Tau had no significant effect on the cytokine production. These results indicate that Tau-Cl modulates synthesis of pro-inflammatory cytokines, and therefore it may play a role in the initiation and propagation of immune response.

Adult↗

PubMed2002

Effect of taurine chloramine, the product of activated neutrophils, on the development of collagen-induced arthritis in DBA 1/J mice.

Taurine chloramine (TauCl), a product of neutrophil myeloperoxidase - halide system, formed by a reaction of taurine with HOCl, is known as an anti-microbial and anti-inflammatory long-lived oxidant. We previously reported that TauCl inhibits in vitro the production of proinflammatory cytokines (IL-6, IL-8) by RA synoviocytes. Therefore we performed this study to investigate the effect of TauCl treatment on the development of collagen-induced arthritis (CIA) in DBA1/J mice. Early administration of TauCl (after primary immunization) resulted in the delay of the onset of CIA, but had no effect on severity of arthritis. TauCl, given daily for 21 days after booster immunization, did not reduce the symptoms of arthritis in those mice, which already developed CIA, but significantly diminished incidence of the disease (55% vs. 90% of placebo mice). The mechanism of this effect is unknown. This is the first in vivo study suggesting that TauCl may be used for immune intervention in chronic inflammatory diseases.

Animals↗

PubMed2001

A defect in bone marrow derived dendritic cell maturation in the nonobesediabetic mouse.

The pathogenesis of diabetes in the nonobese diabetic (NOD) mouse is characterized by a selective destruction of the insulin-producing beta-cells in the islets of Langerhans mediated by autoreactive T cells. The function of T cells is controlled by dendritic cells (DC), which are not only the most potent activators of naïve T cells, but also contribute significantly to the establishment of central and peripheral tolerance. In this study, we demonstrate that the NOD mouse (H2: K(d), Ag(7), E*, D(b)) shows selective phenotypic and functional abnormalities in DC derived from bone marrow progeny cells in response to GM-CSF (DC(NOD)). NOD DC, in contrast to CBA DC, have very low levels of intracellular I-A molecules and cell surface expression of MHC class II, CD80, CD86 and CD40 but normal beta 2-microglobulin expression. Incubation with the strong inflammatory stimulus of LPS and IFN-gamma does not increase class II MHC, CD80 or CD86, but upregulates the level of CD40. The genetic defect observed in the DC(NOD) does not map to the MHC, because the DC from the MHC congenic NOD.H2(h4) mouse (H2: K(k), A(k), E(k), D(k)) shares the cell surface phenotype of the DC(NOD). DC from these NOD.H2(h4) also fail to present HEL or the appropriate HEL-peptide to an antigen-specific T cell hybridoma. However all the DC irrespective of origin were able to produce TNF-alpha, IL-6, low levels of IL-12(p70) and NO in response to LPS plus IFN-gamma. A gene or genes specific to the NOD strain, but outside the MHC region, therefore must regulate the differentiation of DC in response to GM-CSF. This defect may contribute to the complex genetic aetiology of the multifactorial autoimmune phenotype of the NOD strain.

Animals↗

PubMed2001

Brain-derived neurotrophic factor superinduction parallels anti-epileptic--neuroprotective treatment in the pilocarpine epilepsy model.

Antiepileptic drugs provide neuroprotection in several animal models of brain damage, including those induced by status epilepticus (SE). The mechanisms involved in this action are unknown, but neurotrophic factors such as brain-derived neurotrophic factor (BDNF) may play a role. In this study we investigated the changes in BDNF levels in rats in which SE had been induced by pilocarpine injection (400 mg/kg i.p.) and continued for several hours (unprotected group). In other animals (protected groups), SE was suppressed after 30 min by intraperitoneal injection of either diazepam (10 mg/kg) + pentobarbital (30 mg/kg) or paraldehyde (0.3 mg/kg). In diazepam + pentobarbital-treated rats the hippocampal damage caused by SE was significantly lower (p < 0.05) than in unprotected animals. In addition, 2 and 24 h after pilocarpine injection, the levels of BDNF mRNA were moderately increased in the unprotected group, but 'superinduced' in protected animals, especially in the neocortex and hippocampus. A time-dependent increase in BDNF immunoreactivity was also found by western blot analysis in rats treated with diazepam + pentobarbital. In contrast, a decrease of BDNF immunoreactivity occurred in the unprotected group. In conclusion, these results show that neuroprotection induced by anti-epileptic drugs in pilocarpine-treated rats is accompanied by strong potentiation of BDNF synthesis in brain regions involved in SE.

Animals↗

PubMed2001

Selective expression of the proprotein convertases furin, pc5, and pc7 in proliferating vascular smooth muscle cells of the rat aorta in vitro.

The aim of this study was to investigate whether transformation of quiescent vascular smooth muscle cells (VSMCs) into proliferating secretory cells is accompanied by an expression of processing enzymes that activate de novo-synthesized growth factors. Three enzymes belonging to the family of the kexin/subtilisin-like mammalian proprotein convertases (PCs), furin, PC5, and PC7, were found to be upregulated after balloon denudation in vivo. To determine their importance in these cell processes, we investigated their gene regulation using a short-term organ culture system. After incubation of rat aorta for 4 and 24 hr in serum-free medium, we demonstrated a significant induction of VSMC proliferation. The affected subset of VSMCs, positive for alpha-smooth muscle actin, also expressed proliferating cell nuclear antigen (PCNA). Our results revealed a parallel upregulation of furin, PC5, and PC7 in PCNA-immunolabeled cells. As a substrate model for comparison with PCs we used nerve growth factor (NGF). NGF is known to be activated by PCs. As shown by Northern blotting analysis, NGF mRNA concentration was significantly increased in cultured explants. NGF was released into the culture medium. In conclusion, both PCs and NGF are coordinately modulated on induction of VSMC proliferation.

Animals↗

PubMed2000

The mechanism of taurine chloramine inhibition of cytokine (interleukin-6, interleukin-8) production by rheumatoid arthritis fibroblast-like synoviocytes.

OBJECTIVE: Taurine chloramine (Tau-Cl) has been shown to inhibit the production of proinflammatory cytokines (interleukin-6 [IL-6] and IL-8) by fibroblast-like synoviocytes (FLS) isolated from rheumatoid arthritis (RA) patients. The present study was conducted to elucidate the mechanism of inhibitory action exerted by Tau-Cl. METHODS: The effects of Tau-Cl on 1) the transcription of genes coding for IL-6 and IL-8, and 2) the activity of nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) transcription factors, which are crucial for the transcription of these cytokine genes, were investigated in FLS isolated from the synovial tissue of RA patients. FLS were cultured in vitro for 3-6 passages and stimulated with recombinant human IL-1beta (1 ng/ml) in the presence of either Tau or Tau-Cl, which were added simultaneously with the stimulus at concentrations of 250 microM or 500 microM. The relative expression of IL-6 and IL-8 messenger RNA (mRNA) was evaluated after 4 hours of stimulation, using competitive reverse transcriptase-polymerase chain reaction. The DNA binding activity of NF-kappaB and AP-1 was examined 30 minutes and 2 hours after cell stimulation, respectively, using electromobility gel shift assay. RESULTS: IL-1beta triggered a significant rise in the activity of transcription factors NF-kappaB and AP-1, followed by an elevation of cytokine IL-6 and IL-8 mRNA expression. Tau-Cl, but not Tau, reduced IL-1beta-triggered cytokine mRNA expression, exerting stronger inhibitory activity on the levels of IL-6 than on those of IL-8. Importantly, Tau-Cl also diminished the activity of NF-kappaB and, to a lesser extent, that of AP-1 transcription factor. Neither IL-1beta nor Tau-Cl affected the activity of octamer transcription factor 1. CONCLUSION: Tau-Cl inhibition of IL-6 and IL-8 synthesis in FLS from RA patients results from the ability of this compound to diminish the activity of the major transcriptional regulators (NF-kappaB and AP-1), which subsequently reduces the transcription of these cytokine genes.

Aged↗

PubMed2000

Antimicrobial and cytotoxic activity of hypochlorous acid: interactions with taurine and nitrite.

OBJECTIVE: HOCl, a major bactericidal product of neutrophil MPO-halide system reacts with taurine to form taurine chloramine (TauCl), a less toxic anti-inflammatory mediator. Recently, it has been reported that HOCl may also react with nitrite (NO2-), a major end-product of nitric oxide (NO) metabolism, to form very active oxidant, nitryl chloride (NO2Cl). The present study was conducted to elucidate the effect of nitrite on bactericidal and some immunoregulatory properties of HOCl and TauCl. MATERIALS: TauCl was prepared from NaOCl and taurine. The reaction was carried out at pH 5.0 and pH 7.4, in the presence or absence of nitrite. All reactions were monitored by UV absorption spectra. METHODS: Bactericidal activity of HOCl and TauCl in the presence of nitrite was tested by incubation of E. coli with the compounds and determined by the pour-plate method. To test the effect of the compounds on activity of inflammatory cells, murine peritoneal neutrophils (PMN) and macrophages were used. The cells were activated in vitro with either LPS, IFN-gamma or zymosan and the production of following mediators was measured: reactive oxygen species using luminol-dependent chemiluminescence; nitric oxide by Griess reaction; TNF-alpha using capture ELISA. In addition, we tested the effect of HOCl and TauCl on activity of myeloperoxidase (MPO). RESULTS: At physiological pH nitrite reacts with HOCl but not with TauCl. This reaction was abolished in the presence of taurine. Nitrite prevented HOCl-mediated bacterial killing, inhibition of MPO activity, cellular cytotoxicity and inhibition of TNF-alpha production. Nitrite did not affect any activity of TauCl. CONCLUSION: We have shown that nitrite may react in vitro with HOCl but not with TauCl, to form new biologically active product(s). We did not confirm the hypothesis that a product of HOCl reaction with nitrite is more toxic than HOCl. To the contrary, we found that nitrite diminished bactericidal and immunoregulatory properties of HOCl. In vivo, nitrite will also compete with taurine for reaction with PMN-released HOCl. Nevertheless, due to high concentration of taurine in PMN cytosol, formation of TauCl will be a major regulatory mechanism of MPO-halide-system.

Animals↗

PubMed2000

Differential effects of pentoxifylline, a non-specific phosphodiesterase inhibitor, on the production of IL-10, IL-12 p40 and p35 subunits by murine peritoneal macrophages.

Pentoxifylline (PTX), a methylxanthine derivative, has been reported to be an effective drug in inhibiting TNF-alpha responses during septic shock. The inhibition of TNF-alpha production seems to be correlated with increased intracellular cAMP levels. PTX also affects the production of other cytokines such as IL-1, IL-6, IL-10, IL-12, and IFN-gamma. However, inhibition, as well as enhancement of cytokine production, has been observed in vitro, depending on the PTX concentration and cell type used.IL-12 is a heterodimeric cytokine that plays an important role in the development of Th1-mediated inflammatory responses. IL-12 along with TNF-alpha and other proinflammatory cytokines has shown to be responsible for the pathological reaction, which may lead to septic shock. For biological activity, the expression of both subunits of IL-12, p35 and p40, is required. Moreover, the p40 chain of IL-12 specifically inhibits the effects of the IL-12 heterodimer. In this study, we investigated the effects of PTX on the production of both proinflammatory (TNF-alpha, IL-6, IL-12) and anti-inflammatory (IL-10) cytokines by murine macrophages (Mφ). We have found that PTX, at concentrations below 100 microg/ml, selectively inhibited the production of TNF-alpha. Forskolin, a cAMP-elevating agent, similarly affected the production of the cytokines tested. However, at higher concentrations, PTX inhibited the production of TNF-alpha, IL-10, and IL-12 p35, but surprisingly, PTX enhanced the production of IL-12 p40. Concentrations of IL-10 were negatively correlated with the concentrations of IL-12 p40 subunit. These results further confirm the relevance of the use of PTX in clinical trials of immunological disorders characterised by inappropriate Th1 type immune responses.

Animals↗

PubMed2000

The effects of gender and menstrual phase on carbohydrate utilization during acute cold exposure.

OBJECTIVE: The purpose of this study was to evaluate the effects of gender and menstrual cycle on the percent of carbohydrate (CHO) utilized during cold water immersion (20 degrees C). Previous research has suggested that males and females utilize CHO differently during submaximal exercise. This study examined whether this differential response is replicated during a submaximal elevation in metabolism, as demonstrated during thermogenesis (i.e., shivering during acute cold exposure). METHODS: Male and female subjects between the ages of 18 and 30 years were recruited for this study. Female subjects underwent the experimental trial once during the follicular phase and once during the luteal phase of their menstrual cycle. Subjects were immersed to the first thoracic vertebra until esophageal temperature reached 36.5 degrees C or for a maximum preocclusion period of 40 minutes. Peripheral temperature homeostasis via cuff occlusion (right arm and left leg) took place for 10 minutes, after which the pressure cuffs were released (postocclusion) and the subjects remained in the water for an additional 10 minutes. The following variables were measured: respiratory exchange ratio, percent of CHO utilization, and oxygen consumption (Vo2). RESULTS: Analysis of variance demonstrated no significant difference between genders or phases of the menstrual cycle in respiratory exchange ratio, percent CHO utilization, or Vo2 during cold water immersion. A significant difference was observed between men and women for absolute Vo2. CONCLUSIONS: These data suggest that although men and women differ with respect to absolute aerobic metabolism, this distinction does not cause a differential response with respect to substrate utilization during acute cold exposure.

Adolescent↗

PubMed2000

The influence of gender and menstrual phase on thermosensitivity during cold water immersion.

BACKGROUND: This investigation evaluated the influence of gender and phase of menstrual cycle [follicular (FOL: days 2-6) and luteal (LUT: days 19-24) phases] on thermosensitivity and metabolic heat production (HP) during cold water immersion (20 degrees C) in 10 females (22.4 +/- 2.8 yr) and 16 males (22.4 +/- 2.9 yr). METHODS: Following a 20-min baseline period (BASE), subjects were immersed until esophageal temperature (Tes) reached 36.5 degrees C or for a maximum pre-occlusion (Pre-OCC) time of 40 min. An arm and thigh cuff were then inflated to 180 and 220 mmHg, respectively, for 10 min (OCC). Following release of the inflated cuffs (Post-OCC), the slope (beta) of the relationship between the decrease in Tes and the increase in HP was used to quantify thermosensitivity. RESULTS: ANOVA revealed no significant difference in thermosensitivity between phases of the menstrual cycle or between men and women (FOL = -2.76, LUT = -3.05, Males = -3.24 W x kg(-1) x degrees C(-1)). A significant (p < 0.05) main effect for gender for HP, and a significant (p < 0.05) main effect for menstrual phase for mean skin temperature (Tsk) were observed. CONCLUSIONS: These data suggest, despite gender differences in HP, that the thermosensitivity of HP during cold water immersion is similar between males and females and is not influenced by menstrual cycle phase. Therefore, these data indicate that when faced with a cold challenge, women respond similarly to men in both phases of their menstrual cycle.

Adolescent↗

PubMed2000

Influence of gender and menstrual cycle on a cold air tolerance test and its relationship to thermosensitivity.

This investigation evaluated the influence of gender and phase of menstrual cycle [follicular (FOL): Days 2-6) and luteal (LUT: Days 19-24)] on a cold air tolerance test (CATT: 90-min of exposure to 5 degrees C air) in 8 females (22.7 +/- 3.0 yr) and 15 males (22.3 +/- 2.9 yr). In addition, central thermosensitivity (beta; W x kg(-1) x degrees C(-1) [i.e., the slope of the relationship between the decrease in esophageal temperature (Tes) and the increase in heat production (HP)], gathered during a separate water trial in 20 degrees C water, was correlated to the change (delta) in Tes and HP across the 90 min of resting exposure during the CATT. Analysis of variance revealed no significant differences between phase of menstrual cycle or gender for HP, mean skin temperature (Tsk), and insulation; however, a main effect for time for these parameters was demonstrated. Despite these similarities, Tes differed (P < 0.05) between males and females. Additionally, no relationship was found between beta and deltaHP and deltaTes in the males and females. Also, there was no relationship between beta and thermoregulation during the CATT in these subjects. These data suggest that menstrual cycle phase did not cause a differential response in Tes, Tsk, and HP during a CATT. Furthermore, women maintained a higher Tes than men during the CATT despite similarities in HP and Tsk.

Adolescent↗

PubMed1999

The subtilisin/kexin family of precursor convertases. Emphasis on PC1, PC2/7B2, POMC and the novel enzyme SKI-1.

Proopiomelanocortin (POMC) is a precursor to various, bioactive peptides including ACTH, beta LPH, alpha MSH, and beta endorphin (beta END). Processing of POMC at dibasic residues is tissue-specific and is performed by either PC1 alone (resulting in ACTH and beta LPH, anterior pituitary corticotrophes) or by a combination of PC1 and PC2 (yielding alpha MSH and beta END, pituitary neurointermediate lobe and hypothalamus). The PC2-specific binding protein 7B2 is intimately involved in the zymogen activation of proPC2 into PC2. Structure-function studies of these enzymes demonstrated the presence of N- and C-terminal domains, as well as specific amino acids within the catalytic segment that influence the degree of activity of each enzyme and the interaction of PC2 with 7B2. The tissue distribution, plasticity of expression, and the multiple precursors that are differentially cleaved by PC1 and/or PC2, predict a wide array of combinatorial activities of these convertases within the endocrine and neuroendocrine system. The phenotypic consequences of the absence of genetic expression of either PC1 or PC2 are now explored using knockout mice and in human patients suffering from obesity and diabetes.

Animals↗

PubMed1999

Mammalian subtilisin/kexin isozyme SKI-1: A widely expressed proprotein convertase with a unique cleavage specificity and cellular localization.

Using reverse transcriptase-PCR and degenerate oligonucleotides derived from the active-site residues of subtilisin/kexin-like serine proteinases, we have identified a highly conserved and phylogenetically ancestral human, rat, and mouse type I membrane-bound proteinase called subtilisin/kexin-isozyme-1 (SKI-1). Computer databank searches reveal that human SKI-1 was cloned previously but with no identified function. In situ hybridization demonstrates that SKI-1 mRNA is present in most tissues and cells. Cleavage specificity studies show that SKI-1 generates a 28-kDa product from the 32-kDa brain-derived neurotrophic factor precursor, cleaving at an RGLT downward arrowSL bond. In the endoplasmic reticulum of either LoVo or HK293 cells, proSKI-1 is processed into two membrane-bound forms of SKI-1 (120 and 106 kDa) differing by the nature of their N-glycosylation. Late along the secretory pathway some of the membrane-bound enzyme is shed into the medium as a 98-kDa form. Immunocytochemical analysis of stably transfected HK293 cells shows that SKI-1 is present in the Golgi apparatus and within small punctate structures reminiscent of endosomes. In vitro studies suggest that SKI-1 is a Ca2+-dependent serine proteinase exhibiting a wide pH optimum for cleavage of pro-brain-derived neurotrophic factor.

Amino Acid Sequence↗

PubMed1999

Nerve growth factor and proprotein convertases furin and PC7 in transected sciatic nerves and in nerve segments cultured in conditioned media: their presence in Schwann cells, macrophages, and smooth muscle cells.

Synthesis of proteins such as nerve growth factor (NGF) is induced after nerve lesion. The NGF precursor (pro-NGF) requires a posttranslational processing by proprotein convertases to become active. In this report, we re-examine the localization of NGF protein and mRNA in injured nerve and show that the candidate pro-NGF convertases furin and PC 7 colocalize with NGF in non-neuronal cells in nerve. By Northern blot analysis, 1.5-kb and 1.3-kb NGF mRNAs were shown to be increased in distal and immediately proximal nerve segments on days 1, 4, and 14 after lesion; by Western blot analysis, NGF proteins of high molecular weight were detected after injury. In vivo, two phases of NGF immunopositivity were observed, in macrophages and perivascular cells shortly after lesion and in endoneurial cells on day 1 and 4. To identify the cells containing NGF, nerve segments were incubated in serum-containing medium with or without conditioning by white blood cells isolated from the circulation. Both hybridization and immunoreactivity signals for NGF were elevated after incubation of nerve segments for 4 hours in conditioned media, so that cells with NGF immunoreactivity could be identified by antibodies to specific cell markers. In these nerve fragments, Schwann cells, perivascular smooth muscle cells, and macrophages contained NGF immunoreactivity. The concentration of furin and PC7 mRNA also increased in lesioned nerves. By immunocytochemical investigation of nerve explants, furin and PC7 were detected in endoneurial cells, macrophages and perivascular cells and were colocalized with NGF. These in vitro and in vivo findings suggest that both furin and PC7 are associated with NGF in several cell types of the sciatic nerve and, hence, may be implicated in intracellular processing of pro-NGF.

Animals↗