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J Krupinski

Publications and source records attributed to J Krupinski.

At least 37 records · Page 2Linked to original sources
PubMed1998

Inducible nitric oxide production and expression of transforming growth factor-beta1 in serum and CSF after cerebral ischaemic stroke in man.

A residual blood supply to the ischaemic brain is a crucial determinant for tissue survival. Early changes in the vascular network and subsequent angiogenesis may be mediated by short-lived molecules like nitric oxide (NO) or growth factors such as transforming growth factor-beta1 (TGF-beta1). Although TGF-beta1 can inhibit NO production, this interaction has not been studied after ischaemia in humans. Serum samples were taken from patients at 24 h and 6 months and cerebrospinal fluid (CSF) samples at 24 h and 1 week later for possible correlation between the two factors. Tissue expression of TGF-beta1 and of the inducible isoform of NO synthase (NOS2) was assessed by immunohistochemistry. CSF levels of NO2-/NO3- as well as total (active + latent) TGF-beta1 were higher in stroke patients as compared to controls 24 h after the stroke. Both NO2-/NO3- and TGF-beta1 were lower 6 months after the stroke compared to 24 h. Levels of NO2-/NO3- correlated with levels of TGF-beta1 within the time points (P = 0.041, Kendall correlation coefficient). There was a strong staining for NOS2 in brain tissue sections in neurones, reactive astrocytes, infiltrating white blood cells, and endothelial cells of larger microvessels. TGF-beta1 expression was mainly limited to neurones and reactive astrocytes. These findings suggest that the interaction between TGF-beta1 and NOS2 might be important for angiogenesis after cerebral ischaemia and may indicate that TGF-beta1 is upregulated as a negative feedback response to elevated levels of NO.

Adult↗
PubMed1998

Genetic resistance to gastrointestinal nematode parasites in Polish long-wool sheep.

A study was undertaken from 1991 to 1994 on a farm in southern Poland to evaluate the genetic parameters of resistance to gastrointestinal nematodes. The predominant species were Teladorsagia circumcincta and Haemonchus contortus. A total of 32 sires were evaluated, around 15 per year. Faecal egg counts were measured twice during the 4-month grazing season for lambs (total 659 lambs) and three times for their mothers (total 327 ewes). Infection levels were high during the first 2 years and low during the last 2 years. Using an animal model, the heritability of log10(epg+25) increased from 0.20 in August to 0.33 in September for lambs, and from 0.18 in May to 0.25 in September for ewes. The repeatability of ewe faecal egg count between years was 0.25. A genetic correlation of 0.58 was found between faecal egg count in ewes and in 6-7-month-old lambs. A negative genetic correlation (-0.61) was estimated between faecal egg count in September and daily weight gain of lambs from 70 days of age to the end of grazing season (7 months of age). The results confirm the feasibility of genetic selection of sheep for resistance to nematode parasites in an environment where T. circumcincta and H. contortus are the dominant species.

Animals↗
PubMed1998

Angiogenic oligosaccharides of hyaluronan induce protein tyrosine kinase activity in endothelial cells and activate a cytoplasmic signal transduction pathway resulting in proliferation.

We have recently shown that the degradation products of hyaluronan of 3 to 10 disaccharides (o-HA), but not native high molecular weight hyaluronan, can induce angiogenesis in vivo and, as such, o-HA is an important regulator of the neovascularization process. As a continuation of this work, we have studied the cytoplasmic signal transduction pathways responsible for o-HA-activated endothelial cell proliferation. We show that the addition of o-HA (1 microg/ml) to bovine aortic endothelial cells induces tyrosine phosphorylation of multiple proteins within 1 minute and that the activity remains above basal levels for at least 24 hours. Increased phosphorylation of the CD44 receptor was also observed. Pretreatment of cells with an anti-CD44-receptor antibody (5 microg/ml) or the tyrosine kinase inhibitor genistein (10 microM) inhibited both o-HA-induced proliferation (p < 0.05) and protein tyrosine phosphorylation. In comparison, native hyaluronan had little effect on tyrosine phosphorylation across the same time period. Protein kinase C (PKC) activity was increased 2- to 3-fold in the membranes of cells treated with o-HA, and a pretreatment with phorbol 12,13-dibutyrate (PDBu) to down-regulate PKC significantly inhibited o-HA-induced cell proliferation (p < 0.05). Examination by Western blotting showed that only the betaI and epsilon isoforms remained translocated to the membrane for at least 24 hours. These isoforms seem to be involved in modulating the proliferative effects of o-HA, because the transient translocation of PKC isoforms by PDBu was not sufficient to induce mitogenesis. Furthermore, we show that PKC activation of the cytoplasmic kinase cascade (Raf-1 kinase, MAP kinase kinase [MEK-1], and extracellular signal-regulated kinase [ERK-1]) by o-HA culminated in the nuclear translocation of ERK-1. This pathway is essentially linear, as shown by the ability of specific enzyme inhibitors (PDBu and PD98059) to prevent both activation of ERK-1- and o-HA-induced proliferation. We conclude that phosphorylation of the CD44 receptor results in an increase in tyrosine phosphorylation, leading to the activation of a cytoplasmic cascade and cell proliferation; this concurs with previous work, which showed that o-HA-induced proliferation of endothelial cells is CD44-receptor-mediated and accompanied by early response gene activation.

Animals↗
PubMed1997

CREB (cAMP response element-binding protein) in the locus coeruleus: biochemical, physiological, and behavioral evidence for a role in opiate dependence.

Chronic morphine administration increases levels of adenylyl cyclase and cAMP-dependent protein kinase (PKA) activity in the locus coeruleus (LC), which contributes to the severalfold activation of LC neurons that occurs during opiate withdrawal. A role for the transcription factor cAMP response element-binding protein (CREB) in mediating the opiate-induced upregulation of the cAMP pathway has been suggested, but direct evidence is lacking. In the present study, we first demonstrated that the morphine-induced increases in adenylyl cyclase and PKA activity in the LC are associated with selective increases in levels of immunoreactivity of types I and VIII adenylyl cyclase and of the catalytic and type II regulatory subunits of PKA. We next used antisense oligonucleotides directed against CREB to study the role of this transcription factor in mediating these effects. Infusion (5 d) of CREB antisense oligonucleotide directly into the LC significantly reduced levels of CREB immunoreactivity. This effect was sequence-specific and not associated with detectable toxicity. CREB antisense oligonucleotide infusions completely blocked the morphine-induced upregulation of type VIII adenylyl cyclase but not of PKA. The infusions also blocked the morphine-induced upregulation of tyrosine hydroxylase but not of Gialpha, two other proteins induced in the LC by chronic morphine treatment. Electrophysiological studies revealed that intra-LC antisense oligonucleotide infusions completely prevented the morphine-induced increase in spontaneous firing rates of LC neurons in brain slices. This blockade was completely reversed by addition of 8-bromo-cAMP (which activates PKA) but not by addition of forskolin (which activates adenylyl cyclase). Intra-LC infusions of CREB antisense oligonucleotide also reduced the development of physical dependence to opiates, based on attenuation of opiate withdrawal. Together, these findings provide the first direct evidence that CREB mediates the morphine-induced upregulation of specific components of the cAMP pathway in the LC that contribute to physical opiate dependence.

Adenylyl Cyclases↗
PubMed1997

Isozyme-dependent sensitivity of adenylyl cyclases to P-site-mediated inhibition by adenine nucleosides and nucleoside 3'-polyphosphates.

Recombinant adenylyl cyclase isozyme Types I, II, VI, VII, and three splice variants of Type VIII were compared for their sensitivity to P-site-mediated inhibition by several adenine nucleoside derivatives and by the family of recently synthesized adenine nucleoside 3'-polyphosphates (Désaubry, L., Shoshani, I., and Johnson, R. A. (1996) J. Biol. Chem. 271, 14028-14034). Inhibitory potencies were dependent on isozyme type, the mode of activation of the respective isozymes, and on P-site ligand. For the nucleoside derivatives potency typically followed the order 2',5'-dideoxyadenosine (2',5'-ddAdo) > beta-adenosine > 9-(cyclopentyl)-adenine (9-CP-Ade) >/= 9-(tetrahydrofuryl)-adenine (9-THF-Ade; SQ 22,536), with the exception of Type II adenylyl cyclase, which was essentially insensitive to inhibition by 9-CP-Ade. For the adenine nucleoside 3'-polyphosphates inhibitory potency followed the order Ado < 2'-dAdo < 2',5'-ddAdo and 3'-mono- < 3'-di- < 3'-triphosphate. Differences in potency of these ligands were noted between isozymes. The most potent ligand was 2',5'-dd-3'-ATP with IC50 values of 40-300 nM. The data demonstrate isozyme selectivity for some ligands, suggesting the possibility of isozyme-selective inhibitors to take advantage of differences in P-site domains among adenylyl cyclase isozymes. Differential expression of adenylyl cyclase isozymes may dictate the physiological sensitivity and hence importance of this regulatory mechanism in different cells or tissues.

Adenosine↗
PubMed1997

A putative role for platelet-derived growth factor in angiogenesis and neuroprotection after ischemic stroke in humans.

BACKGROUND AND PURPOSE: Growth factors control two important processes in infarcted tissue, ie, angiogenesis and gliosis. We recently reported that transforming growth factor-beta1 (TGF-beta1) might be involved in angiogenesis after ischemic stroke in humans; here we present data of an extensive study on platelet-derived growth factor (PDGF) and its receptors. METHODS: We studied brain samples from patients who suffered from ischemic stroke for the expression of mRNA encoding PDGF-A, PDGF-B, and PDGF receptors (PDGF-R). Proteins were examined by Western blotting and immunohistochemistry using the antibodies to PDGF-AB, PDGF-BB, PDGF-R alpha, and PDGF-R beta. RESULTS: At the mRNA level, PDGF-A and PDGF-B were expressed mainly in neurons in penumbra. PDGF-R mRNA was strongly expressed in some astrocytes but mainly in type III/IV neurons in infarct and penumbra. The least expression was seen in the contralateral hemisphere (P<.001). In contrast, both PDGF-AB and PDGF-BB immunoreactive products were present in most cell types: PDGF-R alpha and PDGF-R beta mainly on neurons, and PDGF-R beta on some endothelial cells, with less staining of all the isoforms in the contralateral hemisphere. On Western blots, PDGF-AB and -BB were expressed more within white matter than gray matter of infarct/penumbra, whereas both isoforms of receptor were expressed mainly in gray matter compared with contralateral hemisphere. There was no or very weak expression of the receptor in white matter. CONCLUSIONS: PDGF proteins are highly expressed in white matter, suggesting that PDGF may exert its function in white matter participating either in regeneration of damaged axons or in glial scar formation. PDGF-BB and its receptor expressed on microvessel endothelial cells might be involved in angiogenesis after stroke. Thus, PDGF is likely to be angiogenic and neuroprotective in stroke.

Aged↗
PubMed1996

Splice variants of type VIII adenylyl cyclase. Differences in glycosylation and regulation by Ca2+/calmodulin.

Three alternatively spliced type VIII adenylyl cyclase messages have been identified by cDNA cloning and amplification from rat brain cDNA. Type VIII-A was previously referred to simply as type VIII (Cali, J. J., Zwaagstra, J. C., Mons, N., Cooper, D. M. F., and Krupinski, J. (1994) J. Biol. Chem. 269, 12190-12195). The types VIII-B and -C cDNAs differ from that of type VIII-A by deletion of 90 and 198 base pair exons, respectively, which encode a 30-amino acid extracellular domain with two consensus sites for N-linked glycosylation and a 66-amino acid cytoplasmic domain. Stable expression of types VIII-A, -B, and -C cDNAs in human embryonal kidney 293 (HEK-293) cells leads to the appearance of novel proteins, which are recognized by type VIII-specific antibodies and which co-migrate with immunoreactive species detected on immunoblots of rat brain membranes. Types VIII-A and -C are modified by N-linked glycosylation, while type VIII-B is insensitive to treatment with N-glycosidase F. An influx of extracellular Ca2+ stimulates cAMP accumulation in HEK-293 cells stably expressing type VIII-A, -B, or -C, but not in control cells. Adenylyl cyclase activity of each of the variants is stimulated by Ca2+/calmodulin and the EC50 for activation of type VIII-C is one fourth of that for either type VIII-A or -B. Type VIII-C also has a distinct Km for substrate, which is approximately 4-12-fold higher than that for types VIII-A or -B depending on whether Mn2+ or Mg2+ is the counterion for ATP. The differences in the structural and enzymatic properties of these three variants are discussed.

Adenylyl Cyclases↗
PubMed1996

Increased expression of TGF-beta 1 in brain tissue after ischemic stroke in humans.

BACKGROUND AND PURPOSE: Occlusion in cerebral vessels results in ischemic stroke and is followed by proliferation of microvessels, ie, angiogenesis. The process is particularly marked in the border zone of the infarct, known as the ischemic penumbra. This increase in vascularization is likely to be caused by the action of angiogenic factors, such as TGF-beta 1, which is a powerful regulator of angiogenesis. METHODS: In this study we examined 10 brain samples from patients who suffered from ischemic stroke for the expression of mRNA encoding TGF-beta 1. RESULTS: The ischemic penumbra contained the highest levels of TGF-beta 1 mRNA, whereas the normal contralateral hemispheres had the least (P < .001, Mann-Whitney U test). Unlike those from normal brain, protein extracts from infarcted tissue contained active TGF-beta 1 as a 25-kD band in Western blot analysis. Extracts from the penumbra also contained a 12.5-kD isoform of TGF-beta 1. Both penumbra and infarct contained TGF-beta 1 immunoreactive products as assessed with immunohistochemistry, whereas very weak staining was observed in the contralateral hemisphere. CONCLUSIONS: These results suggest that TGF-beta 1 is important in the pathogenesis of the angiogenic response in ischemic brain tissue and its modulation may be used for therapeutic purposes.

Aged↗
PubMed1996

The use of factor analysis for ascertaining patterns of psychopathology in children with intellectual disability.

The Developmental Behaviour Checklist (DBC) was developed to assess psychopathology in children with intellectual disabilities. A cut-off point between "non-cases' and "cases' was determined by comparison of the total scores with psychiatric clinical assessment. This paper describes a method aimed at determining the types of psychiatric problems in those regarded as "cases'. Factor analysis with varimax rotation carried out on a sample of 1093 subjects extracted six factors. Standardized factor scores were calculated for each subject in a community sample (n = 450), and the only or the dominant positive score was determined for each of the "cases'. Over 80% of "cases' could be allotted to one condition. Only a small number had none, or three or more conditions. The same procedure was replicated on a validation sample (n = 448) with even more satisfactory results. Differences in the prevalence of the six conditions by sex, age and level of mental retardation were ascertained. The validity and clinical relevance of this method are discussed.

Adolescent↗
PubMed1996

Immunocytochemical studies of cellular reaction in human ischemic brain stroke. MAB anti-CD68 stains macrophages, astrocytes and microglial cells in infarcted area.

Studies on human brains with ischemic insult were performed with immunohistochemical methods to identify antigenic properties of cells participating in that pathology. The 10 patients of whom the brains were obtained at autopsy aged from 51 to 81 years had survived from 3 to 17 days from the onset of stroke to death. Cells involved in the process of decomposition of infarcted tissue were analysed with immunocytochemical methods and CD68, EMB11 (Dako) monoclonal antibody (MAB) was used. Three cell types were visualized e.g. macrophages, astrocytes and microglial cells. Cells identified were numerous in the boundary zone of infarcts and only rarely distributed in the surrounding tissue at a distance from the necrotic tissue. A few cells only were stained in the contralateral hemisphere and none were astrocytes. Our results provide evidence that morphologically different cell types can be marked with the same antibody, which may be in agreement with their similar function in the pathogenesis of ischemic brain stroke.

Aged↗
PubMed1995

A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells.

G-protein coupled Angiotensin II receptors (AT1A), mediate cellular responses through multiple signal transduction pathways. In AT1A receptor-transfected CHO-K1 cells (T3CHO/AT1A), angiotensin II (AII) stimulated a dose-dependent EC50 = 3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT1, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca2+ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or ca2+/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [3H]thymidine incorporation in T3CHOA/AT1A cells. AII (1 microM) significantly inhibited cell number (51% at 96 h) and [3H]thymidine incorporation of 68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT1 receptor. Forskolin (10 microM) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [3H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity an tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT1A cells, in particular at 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.

Angiotensin II↗
PubMed1994

Molecular cloning and characterization of the type VII isoform of mammalian adenylyl cyclase expressed widely in mouse tissues and in S49 mouse lymphoma cells.

We have isolated a 5199-nucleotide cDNA from a mouse library containing an open reading frame encoding the 1099-amino acid type VII adenylyl cyclase protein. The type VII protein is most closely related in primary structure to an unpublished human adenylyl cyclase clone (GenBank accession no. D25538) and type II adenylyl cyclase. Northern blot analysis demonstrates that the type VII mRNA is most abundant in mouse heart, spleen, and lung. cAMP content rises rapidly in HEK 293 cells overexpressing type VII adenylyl cyclase following treatment with phorbol ester, peaking by 4 min, while cells expressing the type II adenylyl cyclase reach peak accumulation only after 20 min. Increases in intracellular calcium through treatment of type VII-293 cells with either ATP or A23187 alone failed to increase intracellular cAMP content. Phorbol ester treatment acted synergistically with beta-adrenergic stimulation to increase cAMP content in type VII-transformed cells. Pretreatment of type VII-transformed cells with pertussis toxin fails to prevent phorbol ester potentiation of isoproterenol stimulation. Thus the ability of phorbol ester to increase basal and isoproterenol-stimulated type VII activity appears to be a direct effect on this adenylyl cyclase isoform and not the result of modification of the inhibitory G protein, Gi.

Adenylyl Cyclases↗
PubMed1994

Type VIII adenylyl cyclase. A Ca2+/calmodulin-stimulated enzyme expressed in discrete regions of rat brain.

A cDNA that encodes type VIII adenylyl cyclase has been isolated from two rat brain libraries. The open reading frame encodes a 1248-amino acid protein predicted to have two sets of six transmembrane spans and two putative nucleotide binding domains as is characteristic of other mammalian adenylyl cyclases. Two type VIII messages are detected in rat brain with estimated sizes of 5.5 and 4.4 kilobases. In situ hybridization indicates that the type VIII messages are most abundantly expressed in the granule cells of the dentate gyrus, the pyramidal cells of hippocampal fields CA1-CA3, the entorhinal cortex, and the piriform cortex. Hybridization is also detected in the neocortex, the amygdaloid complex, and regions of the thalamus and hypothalamus. Stable expression of the type VIII cDNA in human embryonal kidney cells leads to the appearance of a novel 165-kDa glycoprotein in the membrane fraction. Stimulation of these cells with agents that increase intracellular Ca2+ results in up to 43-fold increases in cAMP accumulation over that of control cells transfected with the expression vector. Addition of isoproterenol alone does not lead to type VIII-specific effects in intact cells. Adenylyl cyclase activity in membranes prepared from type VIII-transformed cells is stimulated up to 40-fold by the addition of Ca2+/calmodulin (EC50 = 53 nM calmodulin). The addition of activated recombinant alpha subunit of Gs synergistically increases the Ca2+/calmodulin-stimulated activity. A possible role for type VIII adenylyl cyclase in long-term potentiation is discussed.

Adenylyl Cyclases↗
PubMed1994

Youth suicide in Victoria: a retrospective study.

OBJECTIVE: To determine the trends in youth suicide in Victoria and Australia as a whole, and their relation to youth unemployment. DESIGN: We used Australian Bureau of Statistics data to analyse suicide trends between 1907 and 1990 in young people aged 15-24 years and made an in-depth study of youth suicides between 1980 and 1990, for which computerised data are available. RESULTS: There has been a steady increase in youth suicide both in Victoria and Australia as a whole since 1960 in males but not females. There were significant differences in age, sex and area of residence in both the rate and the method of suicide. The increase in youth suicide was not associated with the rise in unemployment. Male (not female) suicide rates were higher in non-metropolitan areas and areas of high youth unemployment. The reasons for the increase in youth suicide remain obscure. CONCLUSIONS: There is a need for a prospective in-depth study to determine factors in the aetiology of youth suicide, with particular reference to possible areas for prevention.

Adolescent↗
PubMed1994

Role of angiogenesis in patients with cerebral ischemic stroke.

BACKGROUND AND PURPOSE: Stroke is one of the most common causes of mortality and morbidity in the Western world. It results from the occlusion of a cerebral artery followed by severe disturbances in blood supply through microvessels to brain tissue. Despite an extensive literature its pathophysiology is poorly understood, and this has severely impeded the logical development of therapy. METHODS: Brains were obtained from 10 patients aged 46 to 85 years with survival times of 5 to 92 days after their stroke. Infarcted areas and representative control tissues from the contralateral uninvolved brain hemisphere were collected. Microvessel density was measured microscopically. A total of 6520 microvessels were scored in 10,801 areas. The level of activation of the endothelial cells was studied by immunohistochemistry using three monoclonal antibodies, viz, E-9, raised against activated endothelial cells; IG11, recognizing vascular cell adhesion molecule-1; and anti-proliferating cell nuclear antigen. Angiogenic activity in tissue extracts was examined using an in vivo chicken chorioallantoic membrane assay. RESULTS: There was a statistically significant increase in the number of microvessels (Wilcoxon log-rank test; P < or = .01) in 9 of 10 infarcted brain tissues when compared with their contralateral normal hemisphere. In these patients the higher blood vessel counts correlated with longer survival, as ascertained by Spearman's p analysis (P < .02). The number of microvessels filled with blood cells was significantly lower in the infarcted hemispheres (P < .01). In contrast, statistically significant increased numbers of empty microvessels occurred in infarcted tissues compared with the contralateral hemisphere. Monoclonal antibody E-9 reacted weakly with normal-brain vascular endothelial cells; anti-proliferating cell nuclear antigen and IG11 were virtually negative. All three antibodies strongly stained the blood vessels of stroke tissues. The stroke tissues contained angiogenic activity, as shown by the induction of new blood vessels in a chorioallantoic membrane assay. CONCLUSIONS: We have shown that stroke causes active angiogenesis that is more developed in the penumbra. Further experiments are needed to determine if this angiogenesis has beneficial effect.

Aged↗