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M Shakibaei

Publications and source records attributed to M Shakibaei.

At least 55 records · Page 3Linked to original sources
PubMed1997

The malaria circumsporozoite protein: interaction of the conserved regions I and II-plus with heparin-like oligosaccharides in heparan sulfate.

The malaria circumsporozoite (CS) protein binds to glycosaminoglycans from heparan sulfate proteoglycans on the cell surface of hepatocytes and is specifically cleared from the bloodstream by the liver. We show here that the two conserved regions, I and II-plus, of the CS protein, in a concerted action, preferentially bind to highly sulfated heparin-like oligosaccharides in heparan sulfate. In a concentration-dependent manner, peptides representing region I and region II-plus inhibited the binding of recombinant CS protein to HepG2 cells by 62 and 84%, respectively. Furthermore, the action of endoproteinase Arg-C, which cleaves the recombinant CS constructs CS27IVC and CSFZ(Cys) predominantly at the conserved region I, was inhibited by heparin in a concentration-dependent fashion. CSFZ(Cys), which has a higher affinity to HSPGs than CS27IVC, was stabilized by heparin at a w/w ratio (CS protein:glycosaminoglycan) of 20/1, whereas full protection of CS27IVC required more heparin (5/1). Heparan sulfate provided full protection of CSFZ(Cys) only at a ratio of 1/10. Native fucoidan as well as normally sulfated fuco-oligosaccharides (0.76 mol sulfate/mol fucose) inhibited Plasmodium berghei development in HepG2 cells by 84 and 66%, respectively, in a concentration-dependent manner and sporozoite invasion into CHO cells by 80%. Desulfated fucoidan oligosaccharides were inactive. These results may explain the selective interaction between the CS protein and the unique heparan sulfate from liver, which is noted for its unusually high degree of sulfation, and may provide a plausible explanation for the selective targeting of the malaria CS protein to the liver.

Amino Acid Sequence↗
PubMed1997

Fibroblast growth factor-2 (FGF-2) and FGF-receptor (FGFR-1) immunoreactivity in embryonic spinal autonomic neurons.

The development of the nervous system appears to be under the control of multiple growth factors, neurotrophins and cytokines, which may be expressed either continuously or transiently throughout defined stages of cellular generation, proliferation or differentiation. Fibroblast growth factor (FGF) cytokines and their receptors are abundantly expressed in the embryonic nervous system but their localization at autonomic levels in the fetal spinal cord has not yet been detailed. Immunoreactivity to FGF-2, probably the best characterized member of the FGF family (FGF-1 to FGF-10) and of one of its high affinity receptors, FGFR-1, was found in autonomic neurons at embryonic day E14, the peak day of generation and proliferation in the common ventral motoneuron pool. It was also continuously present throughout the investigated subsequent stages (E15 to postnatal day P30). Immunogold electron microscopy revealed the cytoplasmic localization of FGF-2 and FGFR-1 in intermediolateral neurons, the major group of sympathetic preganglionic neurons in the spinal cord. In these neurons, immunocytochemistry from E14 onwards showed the co-distribution of both markers at the period of axonal outgrowth to peripheral targets, e.g. the adrenal medulla. Our findings suggest autocrine and/or paracrine actions of FGF-2 for sympathetic preganglionic development but do not support its role as a target-derived neurotrophic factor for autonomic neuron development.

Animals↗
PubMed1997

Molecular anatomy of the perivascular sheath in human placental stem villi: the contractile apparatus and its association to the extracellular matrix.

In previous studies, we have shown that smooth muscle cells and myofibroblast subpopulations of the perivascular stem villous sheath of the human placenta contain focal adhesion plaques and talin immunoreactivity. The close association of these cells to elastic and collagen fibres have led to the assumption of a functional myofibroelastic unit within the perivascular stem villous sheath. Interactions between the extracellular matrix and smooth muscle cells depend on a variety of structural protein assemblies. In the present study, we examined, by immunocytochemistry, whether the molecular assembly of extracellular matrix proteins and molecules of focal adhesions, known to be essential for signal transduction in smooth muscle cells, are also found in smooth muscle cells of the perivascular stem villous sheath of the human placenta. Vascular and extravascular smooth muscle cells were immunoreactive for alpha-actinin, vinculin, paxillin and tensin, the integrin chains alpha1 and beta1, and the basement membrane components laminin and heparan/-chondroitin sulfate proteoglycan perlecan. pp125(FAK) did not react. In the extracellular matrix of blood vessel walls and the perivascular stem villous sheath, we found immunoreactivity of fibronectin and collagen types I, VI and undulin (collagen type XIV). From our data we conclude that within the perivascular stem villous sheath, there exists a system of signal transduction molecules, indicating a cross talk between the smooth muscle cells of this sheath and their surrounding extracellular matrix.

Actins↗
PubMed1996

Dual interaction of the malaria circumsporozoite protein with the low density lipoprotein receptor-related protein (LRP) and heparan sulfate proteoglycans.

Speed and selectivity of hepatocyte invasion by malaria sporozoites have suggested a receptor-mediated mechanism and the specific interaction of the circumsporozoite (CS) protein with liver-specific heparan sulfate proteoglycans (HSPGs) has been implicated in the targeting to the liver. Here we show that the CS protein interacts not only with cell surface heparan sulfate, but also with the low density lipoprotein receptor-related protein (LRP). Binding of 125I-CS protein to purified LRP occurs with a Kd of 4.9 nM and can be inhibited by the receptor-associated protein (RAP). Blockage of LRP by RAP or anti-LRP antibodies on heparan sulfate-deficient CHO cells results in more than 90% inhibition of binding and endocytosis of recombinant CS protein. Conversely, blockage or enzymatic removal of the cell surface heparan sulfate from LRP-deficient embryonic mouse fibroblasts yields the same degree of inhibition. Heparinase-pretreatment of LRP-deficient fibroblasts or blockage of LRP on heparan sulfate-deficient CHO cells by RAP, lactoferrin, or anti-LRP antibodies reduces Plasmodium berghei invasion by 60-70%. Parasite development in heparinase-pretreated HepG2 cells is inhibited by 65% when RAP is present during sporozoite invasion. These findings suggest that malaria sporozoites utilize the interaction of the CS protein with HSPGs and LRP as the major mechanism for host cell invasion.

Animals↗
PubMed1996

Integrins on joint cartilage chondrocytes and alterations by ofloxacin or magnesium deficiency in immature rats.

Recently, we showed that magnesium deficiency induces lesions in knee joint cartilage from 5-week-old rats that are very similar to ofloxacin-induced cartilage defects. We concluded that quinolone-induced arthropathy is probably due to chelation of magnesium and thus a deficit in functionally available magnesium in joint cartilage (Stahlmann et al. 1995). As magnesium deficiency in joint cartilage could impair chondrocyte-matrix interaction which is mediated by cation-dependent integrin receptors of the beta 1-subfamily, we investigated integrin expression in joint cartilage from untreated, ofloxacin-treated and magnesium-deficient Wistar rats. With immunohistochemical methods using monoclonal and polyclonal antibodies, we showed that the integrin pattern in joint cartilage from rats corresponded largely to integrin expression described for human cartilage tissue: beta 1, alpha 1, alpha 3 and alpha v subunits and the alpha 5 beta 1 and alpha v beta 3 heterodimers were consistently expressed. Joint cartilage lesions were detected in ofloxacin-treated and magnesium-deficient rats. Lesions were more pronounced in the quinolone-treated group. Expression of several integrins was reduced in the vicinity of lesions after oral treatment with 2 x 600 mg ofloxacin/kg for 1 day. Gross-structural lesions (e.g., cleft formation, unmasked collagen fibres) in magnesium-deficient rats were very similar but changes in integrin expression were less pronounced. On the other hand, changes in cartilage matrix composition showed similar alterations in ofloxacin-treated and magnesium-deficient rats: fibronectin deposition in the cartilage matrix increased in both groups while glycosaminoglycan content decreased. In summary, similar defects occur in ofloxacin-treated and magnesium-deficient rats and with immunohistochemical methods subtle differences are demonstrable.

Animals↗
PubMed1996

Quinolone-induced cartilage lesions are not reversible in rats.

The reversibility of quinolone-induced cartilage lesions has not been studied in detail. We treated five groups of five to seven juvenile Wistar rats (male and female; age: 5 weeks) with 2 x 600 mg ofloxacin/kg by gastric intubation on 1 day only (9:00 a.m. and 5:00 p.m.) and studied the knee joints histologically 3 days, 1, 3, 8 and 17 weeks later. In addition, joint cartilage specimens from vehicle-treated control rats (n = 21) at corresponding age were examined. Cartilage lesions such as matrix swelling, loss of proteoglycans and horizontal clefts were found in nearly all knee joints (26 of 27 joints; incidence: 96%) of the ofloxacin-treated rats. Within the observation period of 4 months the size of these lesions in knee joint cartilage did not decrease significantly. The diameter of the lesions at the time points of evaluation was 1146 +/- 535, 1713 +/- 309, 1250 +/- 585, 1406 +/- 356, and 1542 +/- 467 microns, respectively (mean values +/- sd). Chondrocyte clusters producing glycosaminoglycans were observed 3 weeks after dosing and at later time points. They are considered to reflect the onset of repair but chondrocyte organization did not normalize during the study period, thus indicating the irreversibility of the effect under the experimental conditions. In principle, long-term joint cartilage damage has to be taken into account when the use of quinolones in children is considered. More detailed pharmacokinetic data are necessary for a reasonable risk assessment approach.

Animals↗
PubMed1995

The nuclear pore complex protein p62 is one of several sialic acid-containing proteins of the nuclear envelope.

While investigating the glycosylation of nuclear envelope proteins of neuroblastoma cells, we found several proteins that bound the sialic acid-specific Sambucus nigra agglutinin. The strongest signals were obtained for proteins with apparent molecular masses of 66 and 180 kDa. The specificity of the lectin binding was checked by acylneuraminyl hydrolase treatment of nuclear envelope proteins, which prohibited S. nigra agglutinin binding. Digestion of nuclear envelope proteins with the N-glycosidase F revealed that sialic acid was N-glycosidically linked to the 180-kDa protein and very probably O-glycosidically linked to the 66-kDa protein. Upon extraction, the latter behaved like the nucleoporin p62 in that it was partly extracted by high ionic strength buffers, could not be solubilized by nonionic detergent, and was completely removed from the nuclear envelope with urea. Two-dimensional gel electrophoretic comparison showed that the S. nigra agglutinin-binding protein and p62 have an identical isoelectric point of about 5.0 and an identical apparent molecular mass of 66 kDa. This, together with the binding of the anti-nucleoporin antibody, demonstrated the identity of the 66-kDa sialoprotein and p62. S. nigra agglutinin inhibits nuclear protein transport in neuroblastoma cells, strongly suggesting a functional significance of sialylation of p62.

Agglutinins↗
PubMed1995

Localization of non-conventional protein kinase C isoforms in bovine brain cell nuclei.

Using Western blotting and immunofluorescence microscopy we detected the protein kinase C isoforms delta, epsilon and zeta in isolated cell nuclei from bovine cerebral cortex. Both protein kinase C (PKC) delta and PKC epsilon are present in higher concentrations in neuronal than in glial nuclei and are located inside the nucleus and at the nuclear envelope. There they give a punctate staining in immunofluorescence microscopy. PKC zeta is also present both in the nucleoplasm and at the nuclear envelope. PKC eta could not be detected in the cell nuclei and, even in the homogenate of cerebral cortex, this isoform is present only in very low concentrations. The antibody against PKC eta bound strongly to a nucleoplasmic protein with an apparent molecular mass of 99 kDa. The localization of non-conventional PKC isoforms at the cell nucleus strongly indicates that these isoforms are directly involved in the regulation of nuclear processes.

Animals↗
PubMed1995

Citric acid extracts a specific set of proteins from isolated cell nuclei.

The treatment of isolated cell nuclei with citric acid was described as a method for separating inner and outer nuclear membrane. Using cell nuclei from bovine cerebral cortex, we can show that citric acid does not cause a separation of the two nuclear membranes, but extracts a specific set of proteins from the nuclei. The extraction of proteins is not just an effect of damaging the nuclear membrane or destructing the cytoskeleton, but rather a specific effect of citric acid treatment. One of the extracted proteins, chosen as a marker for the putative outer nuclear membrane fraction, has an apparent molecular weight of 145 kDa and is located in the nucleoplasm as shown by immunofluorescence microscopy. By sequencing tryptic peptides it was identified as RNA helicase A, an abundant nuclear protein assumed to participate in the processing of mRNA.

Amino Acid Sequence↗
PubMed1995

Co-existence of NADPH-diaphorase, fibroblast growth factor-2 and fibroblast growth factor receptor in spinal autonomic system suggests target-specific actions.

In the rat spinal cord, we found substantial co-existence of fibroblast growth factor-2, fibroblast growth factor receptor (type-1 or flg) immunoreactivity and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity (a histochemical marker for neuronal nitric oxide synthase) in preganglionic autonomic cell groups of intermediate layers VI, VII and X. Anti-fibroblast growth factor-2 and anti-nitric oxide synthase binding sites were confined to the cytoplasm of reactive neurons as judged by immunogold electron microscopy. Within the major autonomic nucleus, i.e. intermediolateral column, three different populations were identified: (i) fibroblast growth factor and fibroblast growth factor receptor, (ii) fibroblast growth factor/NADPH-diaphorase and (iii) NADPH-diaphorase-only stained cell groups. Sympathoadrenal neurons were prelabelled with fluorescent tracer Fast Blue and co-stained for fibroblast growth factor-like protein and NADPH-diaphorase, suggesting heterologous diversification of neuronal phenotypes and functional organization in the spinal autonomic system. Our findings suggest intriguing roles for nitric oxide and fibroblast growth factor-2 cytokine in the preganglionic sympathetic spinal cord system: The "short-term" diffusible messenger nitric oxide may act as "tonic" and/or "phasic" signal within rostrocaudally oriented function-specific preganglionic units necessary for integrated target control. The "long-term" messenger fibroblast growth factor-2 may be involved in, for example, cytokine-dependent regulation of neuronal NADPH-diaphorase/nitric oxide synthase. Furthermore, co-existence of NADPH-diaphorase, fibroblast growth factor-2 and receptor in sympathoadrenal neurons suggest mutual target-specific regulatory functions, e.g. hormone release and blood perfusion or maintenance of phenotype and plasticity responsiveness of adrenal medullary tissue.

Animals↗
PubMed1995

Magnesium deficiency induces joint cartilage lesions in juvenile rats which are identical to quinolone-induced arthropathy.

Quinolones accumulate in cartilage, and because they form chelate complexes with divalent cations, they possess the potential to induce a deficiency of functionally available magnesium. To test the hypothesis that quinolone-induced arthropathy is caused (or aggravated) by magnesium deficiency in cartilage, we induced magnesium deficiency by feeding juvenile rats a magnesium-deficient diet for 9 days and treated the rats with single oral doses of ofloxacin (0, 100, 300, 600, or 1,200 mg/kg of body weight) during this period. Additional groups of juvenile rats on a normal diet were treated with ofloxacin correspondingly. Typical cartilage lesions (e.g., swollen matrix, cleft formation) were found in knee joints of all magnesium-deficient rats, including those without ofloxacin treatment. Lesions in these groups were not distinguishable from lesions induced by a single dose of 600 mg of ofloxacin per kg of body weight or higher in rats on a normal diet. Ofloxacin levels in plasma after 600 mg/kg of body weight were approximately 10-fold higher than those in humans during therapy with this quinolone. Lesions in rats treated with ofloxacin plus magnesium deficiency were more pronounced than those in rats with normal magnesium concentrations. After intake of a magnesium-deficient diet for 9 days, the magnesium concentration in serum (mean +/- standard deviation) was 0.18 +/- 0.05 mmol/liter (control on normal diet, 0.82 +/- 0.10 mmol/liter). Magnesium concentrations in bone (femur) and cartilage (processus xiphoideus) samples were 64.7 +/- 10.5 and 14.3 +/- 3.9 mmol/kg of dry weight, respectively, which corresponded to approximately 50% of the concentrations measured in controls on a normal diet. It was concluded that quinolone-induced arthropathy is probably caused by a deficit of available magnesium in joint cartilage due to the formation of quinolone-magnesium chelate complexes. If juvenile patients must be treated with quinolones for serious infections, it seems prudent to ensure that these patients do not have a disturbed magnesium balance.

Animals↗
PubMed1995

Changes in integrin expression during chondrogenesis in vitro: an immunomorphological study.

Integrins are receptors composed of ligand-specific alpha-chains and cell type-specific beta-chains which are involved in cell-cell and cell-matrix interactions. The distribution of alpha 1- and alpha 3-integrins as well as collagen Types I and II, was investigated by immunofluorescence and immunoelectron microscopy during chondrogenesis in organ culture after various culture periods. Mesenchymal cells from limb buds of Day 12 mouse embryos were grown at high density. Within the first 2 days of the culture period, only alpha 1-integrin could be detected. Formation of cartilage-specific matrix on Day 3 was accompanied by the occurrence of alpha 3-integrin. On Day 7, alpha 3 was present only in cartilage nodules, whereas alpha 1 was strongly expressed in the perichondrium and was more or less homogeneously distributed in the surrounding mesenchyme. On Day 14, alpha 1-integrin was again detectable in cartilage. We suggest that the change in collagen formation from Type I to Type II during chondrogenesis is accompanied by a change in integrin expression from alpha 1 to alpha 3. Conversely, dedifferentiation of chondrocytes in aging cartilage is accompanied by the occurrence of collagen Type I and alpha 1-integrin. Therefore, a strict correlation between the collagen type synthesized by the cells and the appropriate receptor presented by the cells is suggested.

Animals↗
PubMed1995

Integrin expression on epiphyseal mouse chondrocytes in monolayer culture.

The expression of alpha 1-, alpha 3-, alpha v- and alpha 5 beta 1-integrins and their specific ligand binding were investigated in monolayer cultures of chondrocytes from 17-day-old mouse embryos using morphological and immunomorphological methods. After a 1-day culture period alpha v-, alpha 3- and alpha 5 beta 1-integrins were observed on these cells. Immunoelectronmicroscopic investigation revealed localization predominantly in the contact areas with extracellular structures on the cell surface. alpha 1-integrin could not be demonstrated on chondrocytes. After a 5-day culture period the number of fibroblast-like cells with alpha 1-, alpha v- and alpha 5 beta 1-integrins had increased. alpha 3-integrin was hardly recognizable on these cells. Collagen type I and fibronectin could be shown as ligands on the cell surface. The number of chondrocytes with collagen type II on their surface continuously decreased. alpha 3-integrin is obviously responsible for the binding of collagen type II, and alpha 1-integrin for the binding of collagen type I. Therefore, it can be concluded that the changes of chondrocytes to fibroblast-like cells in monolayer culture are accompanied by changes of integrin genes.

Animals↗
PubMed1995

Synovial and peritoneal macrophages in organoid culture.

Cultivation of macrophages and their progenitors has been very useful for elucidation of function, behaviour and morphology of these cells. The purpose of this contribution is to describe a new in vitro system (organoid, high density or micromass culture) which proved to be convenient for cultivation of macrophages derived from human synovial fluid and tissue and mouse peritoneal fluid. Using this method, highly differentiated and functionally active macrophages of marked purity and long maintenance (up to 2 weeks) could be obtained even after previous cultivation and subcultivation in monolayer culture. The macrophages were identified by electron microscopy and immunomorphology using HLA-DR-DP, CD-68 (markers for human macrophages), anti-human-polymorphonuclear leukocyte-gelatinase and F4/80 (a mouse macrophage surface marker). The significance of this method as a research tool in the study of cartilage degradation by macrophages in co-cultures is stressed.

Animals↗
PubMed1994

Influence of mouse peritoneal macrophages on cartilage matrix and integrins in vitro. A morphological study.

The problem of cartilage breakdown in rheumatoid arthritis can partially be studied using in vitro techniques. Among the numerous models, organoid (high density) cultures have proved to be quite suited for these purposes. Chondroblasts/chondrocytes, obtained by cultivating precartilage cells of limb buds from day-12 mouse embryos, were cocultivated with mouse peritoneal macrophages in high-density or monolayer cultures. The result was an extensive breakdown of the cartilage matrix. Numerous chondrocytes detached from the matrix and became fibroblast-like cells. Immunomorphological methods showed that collagen type II, fibronectin and several integrins (beta 1-, alpha 3-, and alpha 5 beta 1 types) disappeared from the surface of chondrocytes. These pathological findings in cartilage tissue could be due to degradative products of macrophages and also chondrocytes. The in vitro model introduced here should be useful for studying matrix components and the turnover of matrix receptors of cartilage tissue in vivo under pathological conditions.

Animals↗
PubMed1993

Integrins in ageing cartilage tissue in vitro.

Matrix-cell interactions are of great importance for numerous cell functions whereby integrins play an essential role as transmitters of extracellular signals. In cultures of ageing cartilage tissue (organoid or high density cultures) cartilage cells occur on the surface of which thick fibrils of collagen type I are deposited. Since integrins, in their role as receptors, cause an interaction between matrix components and cell membrane, we tried to demonstrate immunomorphologically (light and electron microscopically) the corresponding integrin receptors for collagen type I (beta 1 alpha 1 and beta 1 alpha 2) on the surface of these ageing cartilage cells. Cultures of normal, i.e. young cartilage tissue exhibit only beta 1 alpha 3- and beta 1 alpha 5-receptors; labelling against the integrins beta 1 alpha 1 and beta 1 alpha 2 is not possible in this case. Our results show that after the occurrence of thick fibrils cartilage cells express new receptors (beta 1 alpha 1 and beta 1 alpha 2) on the cell membrane. Thus, in ageing or dedifferentiating cartilage tissue it is not only the synthesis programme of matrix components (e.g. instead of collagen type II >> collagen type I) which changes but also the integrins (instead of alpha 3/beta 1, alpha 5/beta 1 >> alpha 1/beta 1, alpha 2/beta 1) so that new collagen types can be bound. These findings may also serve for a better understanding and interpretation of cartilage changes in vivo during ageing and under pathological conditions.

Animals↗