Production and characterisation of polyclonal antisera against feline IgE

To confirm the characteristics of the stromal cells of Wharton's jelly, we investigated the morphological changes in these cells during each trimester of pregnancy. We evaluated the cytoskeletal features of these cells by examining immunohistochemically the localization of one of the contractile proteins, alpha-smooth muscle actin (ASMA). After the second trimester, the stromal cells of Wharton's jelly were stained with ASMA antibody, exhibited the ultrastructural characteristics of the myofibroblasts, and began to express numerous microfilaments in the cytoplasm. Postembedding immunogold labeling detected immunoreactivity for ASMA on these microfilaments. The finding indicated that the stromal cells of Wharton's jelly undergo a time-dependent maturation involving the differentiation of myofibroblasts during the last 6 months of pregnancy. These cells possess a contractile function that may help to protect the umbilical vessels from compression, considering that ASMA was detected in the microfilamentous bundles.     

Calretinin in the cat retina: colocalizations with other calcium-binding proteins, GABA and glycine

Regeneration of skeletal muscle was studied in the sea bream Sparus aurata, in which extensive post-larval muscle hyperplasia contributes to its large adult size, and in the zebrafish Brachydanio rerio, which shows little post-larval hyperplasia and reaches only a small adult size. Small mechanical lesions of body wall muscle were made under general anaesthesia, and the progress of subsequent regeneration was assessed at various intervals by histology and electron microscopy (for general morphology), by immunostaining for desmin and myosin isoforms (to identify the phenotype of new fibres), and by 5'-bromo-2'-deoxyuridine (BrdU) incorporation (to identify proliferating cells). Despite the difference in normal growth-related hyperplasia in these fish, a vigorous regeneration occurred in both species, giving rise to new fibres with an initial myosin composition that differed from that in mature fast-white fibres. However, species differences in myosin expression in these fibres suggest that they may have derived from different myoblast populations. In sea bream, myosin expression in regenerating fibres resembled that seen in new fibres produced in post-larval white muscle, whereas in the zebrafish it resembled that of the primitive monolayer fibres formed during embryonic development. Subsequently, most regenerating fibres gradually transformed into the mature fast-white phenotype in both species.     

Expression of myosin heavy chain and of myogenic regulatory factor genes in fast or slow rabbit muscle satellite cell cultures

We investigated the myogenic properties of rabbit fast or slow muscle satellite cells during their differentiation in culture, with a particular attention to the expression of myosin heavy chain and myogenic regulatory factor genes. Satellite cells were isolated from Semimembranosus proprius (slow-twitch muscle; 100% type I fibres) and Semimembranosus accessorius (fast-twitch muscle; almost 100% type II fibres) muscles of 3-month-old rabbits. Satellite cells in culture possess different behaviours according to their origin. Cells isolated from slow muscle proliferate faster, fuse earlier into more numerous myotubes and mature more rapidly into striated contractile fibres than do cells isolated from fast muscle. This pattern of proliferation and differentiation is also seen in the expression of myogenic regulatory factor genes. Myf5 is detected in both fast or slow 6-day-old cell cultures, when satellite cells are in the exponential stage of proliferation. MyoD and myogenin are subsequently detected in slow satellite cell cultures, but their expression in fast cell cultures is delayed by 2 and 4 days respectively. MRF4 is detected in both types of cultures when they contain striated and contractile myofibres. Muscle-specific myosin heavy chains are expressed earlier in slow satellite cell cultures. No adult myosin heavy chain isoforms are detected in fast cell cultures for 13 days, whereas cultures from slow cells express neonatal, adult slow and adult fast myosin heavy chain isoforms at that time. In both fast and slow satellite cell cultures containing striated contractile fibres, neonatal and adult myosin heavy chain isoforms are coexpressed. However, cultures made from satellite cells derived from slow muscles express the slow myosin heavy chain isoform, in addition to the neonatal and the fast isoforms. These results are further supported by the expression of the mRNA encoding the adult myosin heavy chain isoforms. These data provide further evidence for the existence of satellite cell diversity between two rabbit muscles of different fibre-type composition, and also suggest the existence of differently preprogrammed satellite cells.     

Individual differences in behavior following amphetamine, GBR-12909, or apomorphine but not SKF-38393 or quinpirole

Cultures of dissociated Edinger Westphal nuclei, dissected from embryonic chick brainstems, were screened immunohistochemically for a variety of non-neuronal cell markers. In young cultures, small clusters of cells were stained by the oligodendrocyte-specific antibodies 04 and 01. In older cultures, larger groups of cells were 04 and 01 positive, sheets of myelin-like membrane were elaborated, and immunoreactivity for proteolipid protein appeared. This sequence resembles that observed in well-characterized rodent brain cultures and suggests that oligodendrocytes in chick Edinger Westphal cultures differentiate in a pattern similar to rodent oligodendrocytes in culture. Variable numbers of cells were immunoreactive for glial fibrillary acidic protein. Many vimentin positive cells were observed, some of which morphologically resembled flat astrocytes. Together with the widespread presence of vimentin, large patches of fibronectin-like immunoreactivity suggested the presence of fibroblasts and/or endothelial cells. An anti-thymocyte polyclonal antibody stained a subset of cobblestone-shaped cells, possibly endothelial cells, in both Edinger Westphal cultures and control cultures of skin fibroblasts. Staining for smooth muscle myosin was detected in several patches of cells, tentatively identifying them as pericytes or smooth muscle cells. In conclusion, Edinger Westphal cultures contain a diverse and varying population of non-neuronal cells loosely organized in large, overlapping islands of cell types and including oligodendrocytes, astrocytes, possibly fibroblasts, endothelial cells, pericytes and/or smooth muscle cells.     

Histochemical and immunohistochemical studies on the origin of the blue marlin heater cell phenotype

The superior rectus muscle fibers of marlins, swordfish, sailfish and spearfish are modified for heat production at the expense of contractile ability. Although 'heater cells' are a muscle derivative (Block, 1986, 1991), the myoblast origin and developmental pathway of these thermogenic cells is unknown. To gain insight into heater cell origins, we characterized blue marlin superior rectus muscle and its heater tissue derivative with histochemical and immunological techniques. We specifically employed myosin ATPase and succinate dehydrogenase histochemical assays, and myosin heavy chain immunohistochemistry. Results revealed that marlin superior rectus muscles contain at least six distinct fiber types, and suggested the presence of both twitch and tonic fibers. Immunological results indicate that myosin is present within the thermogenic cells but not in myofibrillar lattices. The antibodies that recognized myosin in heater cells also labeled myosin in the twitch fibers of swimming muscle. In contrast, antibodies that labeled histologically defined tonic fibers did not label heater cells. These results suggest that heater cells and twitch fibers express the same myosin isoform, and establish a phenotypic connection between heater cells and twitch fibers. This conclusion is discussed in the context of the muscle-to-heater trajectory and the muscle fiber-type origin of heater cells.     

Sarcomeric gene expression and contractility in myofibroblasts

Myofibroblasts are unusual cells that share morphological and functional features of muscle and nonmuscle cells. Such cells are thought to control liver blood flow and kidney glomerular filtration rate by having unique contractile properties. To determine how these cells achieve their contractile properties and their resemblance to muscle cells, we have characterized two myofibroblast cell lines. Here, we demonstrate that myofibroblast cell lines from kidney mesangial cells (BHK) and liver stellate cells activate extensive programs of muscle gene expression including a wide variety of muscle structural proteins. In BHK cells, six different striated myosin heavy chain isoforms and many thin filament proteins, including troponin T and tropomyosin are expressed. Liver stellate cells express a limited subset of the muscle thick filament proteins expressed in BHK cells. Although these cells are mitotically active and do not morphologically differentiate into myotubes, we show that MyoD and myogenin are expressed and functional in both cell types. Finally, these cells contract in response to endothelin-1 (ET-1); and we show that ET-1 treatment increases the expression of sarcomeric myosin.     

Myogenic cells express multiple myosin isoforms

In vivo and in vitro, proliferating motile myoblasts form aligned groups of cells, with a characteristic bipolar morphology, subsequently become post-mitotic, begin to express skeletal myosin and fuse. We were interested in whether members of the myosin superfamily were involved in myogenesis. We found that the myoblasts expressed multiple myosin isoforms, from at least five different classes of the myosin superfamily (classes I, II, V, VII and IX), using RT-PCR and degenerate primers to conserved regions of myosin. All of these myosin isoforms were expressed most highly in myoblasts and their expression decreased as they differentiated into mature myotubes, by RNAse protection assays, and Western analysis. However, only myosin I alpha, non-muscle myosin IIA and IIB together with actin relocalize in response to the differentiative state of the cell. In single cells, myosin I alpha was found at the leading edge, in rear microspikes and had a punctate cytoplasmic staining, and non-muscle myosin was associated with actin bundles as previously described for fibroblasts. In aligned groups of cells, all these proteins were found at the plasma membrane. Co-staining for skeletal myosin II, and myosin I alpha showed that myosin I alpha also appeared to be expressed at higher levels in post-mitotic myoblasts that had begun to express skeletal myosin prior to fusion. In early myotubes, actin and non-muscle myosin IIA and IIB remained localized at the membrane. All of the other myosin isoforms we looked at, myosin V, myosin IX and a second isoform of myosin I (mouse homologue to myr2) showed a punctate cytoplasmic staining which did not change as the myoblasts differentiated. In conclusion, although we found that myoblasts express many different isoforms of the myosin superfamily, only myosin I alpha, non-muscle myosin IIA and IIB appear to play any direct role in myogenesis.     

The association of personality characteristics with parenting problems among alcoholic couples

Phosphorylation of the regulatory light chain of myosin II (RMLC) at Serine 19 by a specific enzyme, MLC kinase, is believed to control the contractility of actomyosin in smooth muscle and vertebrate nonmuscle cells. To examine how such phosphorylation is regulated in space and time within cells during coordinated cell movements, including cell locomotion and cell division, we generated a phosphorylation-specific antibody. Motile fibroblasts with a polarized cell shape exhibit a bimodal distribution of phosphorylated myosin along the direction of cell movement. The level of myosin phosphorylation is high in an anterior region near membrane ruffles, as well as in a posterior region containing the nucleus, suggesting that the contractility of both ends is involved in cell locomotion. Phosphorylated myosin is also concentrated in cortical microfilament bundles, indicating that cortical filaments are under tension. The enrichment of phosphorylated myosin in the moving edge is shared with an epithelial cell sheet; peripheral microfilament bundles at the leading edge contain a higher level of phosphorylated myosin. On the other hand, the phosphorylation level of circumferential microfilament bundles in cell-cell contacts is low. These observations suggest that peripheral microfilaments at the edge are involved in force production to drive the cell margin forward while microfilaments in cell-cell contacts play a structural role. During cell division, both fibroblastic and epithelial cells exhibit an increased level of myosin phosphorylation upon cytokinesis, which is consistent with our previous biochemical study (Yamakita, Y., S. Yamashiro, and F. Matsumura. 1994. J. Cell Biol. 124:129-137). In the case of the NRK epithelial cells, phosphorylated myosin first appears in the midzones of the separating chromosomes during late anaphase, but apparently before the formation of cleavage furrows, suggesting that phosphorylation of RMLC is an initial signal for cytokinesis.     

Contractile events during inflammation

Contractile events during wound healing. During granulation tissue contraction, fibroblasts develop characteristics typical of smooth muscle; (1) they contain an extensive cytoplasmic fibrillar system, (2) they show immunofluorescent labeling of anti-actin antibodies, (3) there are cell and cell to stroma attachments, (4) strips of granulation tissue, when tested pharmacologically in vitro, behave similarly to smooth muscle. These data support the view that under certain conditions, fibroblasts can differentiate into a cell type structurally and functionally similar to smooth muscle and this cell, the 'myofibroblast', plays an important role in connective tissue contraction. During epithelialization, epidermal cells develop an extensive cytoplasmic contractile apparatus which has morphological and immunological characteristics similar to those of myofibroblasts. Such apparatus disappears as soon as epithelialization is completed. It is proposed that such a contractile apparatus plays a role in cell motility enabeling individual cells to rearrange themselves in an appropriate pattern. In conclusion, significant amounts of contractile proteins may be synthetized by fibroblasts and epithelial cells during wound healing and may play an important role in this process.     

Molecular cardiomyoplasty: potential cardiac gene therapy for chronic heart failure

In this study, we evaluated the feasibility of converting cardiac fibroblasts into skeletal muscle cells by forced expression of the MyoD gene, one of the basic helix-loop-helix myogenic factors. Primary cardiac fibroblasts, isolated from newborn rats, were infected with retrovirus-carrying sense or antisense MyoD gene. Ten days after infection, expression of MyoD protein was demonstrated in 95% of cells infected with sense MyoD virus by intense nuclear immunostaining with a MyoD polyclonal antibody. In contrast, none of the cells infected with antisense MyoD virus showed staining. On withdrawal of serum, 95% of MyoD positive cells became elongated and, in the presence of appropriate cell density, fused to form multinucleated myotubes, morphologically similar to striated muscle cell. Expression of downstream myogenic differentiation markers, myosin heavy chain and myocyte-specific enhancer factor 2, in 95% of these myotubes were detected by intense cytoplasmic and nuclear immunostaining, respectively, with specific antibodies. In contrast, no detectable staining was noted in MyoD negative cells. Spontaneous contractile movements were noted in a few clusters of myotubes. In summary, cardiac fibroblasts were able to be converted into bonafide potentially functional skeletal myocytes as shown by definitive morphologic and biochemical changes. Further studies with in vivo models are needed to explore this unique molecular strategy to treat patients with chronic heart failure.     

Constriction of the umbilical cord as a cause of fetal demise following midtrimester amniocentesis

Two cases of constriction of the umbilical cord resulting in fetal demise following midtrimester amniocentesis are presented. In both cases, real-time ultrasonography prior to amniocentesis revealed a viable fetus. Fetal demise was identified immediately following the procedure in the first case and one month later in the other. A localized constriction at the fetal end of the umbilical cord in both, with torsion of the constricted segment in the second case, was observed. Wharton's jelly was noted to be deficient in this segment of the cord in the first case. The mechanism of fetal demise is discussed. It is suggested that this abnormality should be considered when fetal demise follows midtrimester amniocentesis.     

Placental villous stroma as a model system for myofibroblast differentiation

Different subtypes of myofibroblasts have been described according to their cytoskeletal protein patterns. It is quite likely that these different subtypes represent distinct steps of differentiation. We propose the human placental stem villi as a particularly suitable model to study this differentiation process. During the course of pregnancy, different types of placental villi develop by differentiation of the mesenchymal stroma surrounding the fetal blood vessels. In order to characterise the differentiation of placental stromal cells in the human placenta, the expression patterns of the cytoskeletal proteins vimentin, desmin, alpha- and gamma-smooth muscle actin, pan-actin, smooth muscle myosin, and the monoclonal antibody GB 42, a marker of myofibroblasts, were investigated on placental tissue of different gestational age (7th-40th week of gestation). Proliferation patterns were assessed with the proliferation markers MIB 1 and PCNA. Additionally, dipeptidyl peptidase IV distribution was studied in term placenta and the ultrastructure of placental stromal cells was assessed by electron microscopy. Different subpopulations of extravascular stromal cells were distinguished according to typical co-expression patterns of cytoskeletal proteins. Around the fetal stem vessels in term placental villi they were arranged as concentric layers with increasing stage of differentiation. A variable layer of extravascular stromal cells lying beneath the trophoblast expressed vimentin (V) or vimentin and desmin (VD). They were mitotically active. The next layer co-expressed vimentin, desmin, and alpha-smooth muscle actin (VDA). More centrally towards the fetal vessels, extravascular stromal cells co-expressed vimentin, desmin, alpha- and gamma-smooth muscle actin, and GB 42 (VDAG). Cells close to the fetal vessels additionally co-expressed smooth muscle myosin (VDAGM). Ultrastructurally, V cells resembled typical mesenchymal cells. VD cells corresponded to fibroblasts, while VDA and VDAG cells developed features of myofibroblasts. Cells of the VDAGM-type revealed a smooth muscle cell-related ultrastructure. In earlier stages of pregnancy, stromal cell types with less complex expression patterns prevailed. The media smooth muscle cells of the fetal vessels showed a mixture of different co-expression patterns. These cells were separated from extravascular stromal cells by a layer of collagen fibres. The results obtained indicate a clearly defined spatial differentiation gradient with increasing cytoskeletal complexity in human placental stromal cells from the superficial trophoblast towards the blood vessels in the centre of the stem villi. The spatial distribution of the various stages of differentiation suggests that human placental villi could be a useful model for the study of the differentiation of myofibroblasts.     

Localization of 17-kDa myosin light chain isoforms in cultured aortic smooth muscle cells

Smooth muscle myosin II contains two 17-kDa essential light chain isoforms (LC17gi and LC17nm) of which the relative contents differ among myosins. To understand the roles of LC17 isoforms in the functions of myosin, we performed an immunofluorescence microscopic examination of their localization in primary cultured cells isolated from rat aortic smooth muscle. To identify the isoforms, rabbit polyclonal antibodies were prepared against C-terminal nonapeptides corresponding to either LC17gi or LC17nm from porcine aortic smooth muscle myosin. These isoforms differ in only 5 amino acid residues within the C-terminal 9 residues. These antibodies specifically recognize each LC17 isoform on urea-PAGE of total rat aortic cell lysates. Immediately after plating, the smooth muscle cells stained heterogeneously with each antibody, indicating differing contents of LC17 isoforms among cells. On double staining 1-2 d cultures with both antibodies, LC17nm was detected diffusely throughout the cytoplasm, whereas LC17gi was concentrated in specific regions such as the cell periphery and the base of cytoplasmic processes. These results support the suggestion that myosin containing LC17gi is essential for force-generation by aortic smooth muscle and that myosin containing LC17nm may play an important role in maintaining smooth muscle tension.     

Elastin of the umbilical cord arteries and its alterations in EPH gestosis (preeclampsia)

Edema, proteinuria, hypertension (EPH) gestosis is accompanied by an increase of collagen content and premature replacement of hyaluronic acid by sulfated glycosaminoglycans both in the umbilical cord arteries and in Wharton's jelly. The effect of EPH gestosis on elastin content and metabolism in the umbilical cord arterial wall was the aim of this work. Studies were performed on normal umbilical cord arteries and those taken from newborns of mothers with EPH gestosis. Elastin was isolated from the arterial wall and quantified by a dye-binding method. Biosynthesis and degradation of this protein was evaluated by a pulse-chase experiment with the use of 14C-proline. It was found that EPH gestosis is associated with a significant reduction of elastin content in the umbilical cord arteries as a result of decrease in elastin biosynthesis rate and accelerated degradation of this protein. The replacement of elastin by collagen, and hyaluronate by sulfated glycosaminoglycans, may decrease the hydration of arterial wall and reduce its elasticity. Such rearrangement of extracellular matrix of the umbilical cord arteries may affect mechanical properties of these vessels and disturb fetal blood circulation.     

Myosin synthesis increased by electrical stimulation of skeletal muscle cell cultures

When cultures of skeletal muscle cells of the chick embryo are subjected to repetitive, electrical stimulation, the contractions increase the amount of protein produced by these cells. The increase is greater for contractile proteins such as myosin heavy chain than for total cellular protein. This demonstrates that in a culture system of skeletal muscle cells that have differentiated in the absence of innervation, one can elicit the protein synthetic response associated with skeletal muscle hypertrophy in vivo.     

Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy

The absence of dystrophin at the muscle membrane leads to Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease that is inevitably fatal in early adulthood. In contrast, dystrophin-deficient mdx mice appear physically normal despite their underlying muscle pathology. We describe mice deficient for both dystrophin and the dystrophin-related protein utrophin. These mice show many signs typical of DMD in humans: they show severe progressive muscular dystrophy that results in premature death, they have ultrastructural neuromuscular and myotendinous junction abnormalities, and they aberrantly coexpress myosin heavy chain isoforms within a fiber. The data suggest that utrophin and dystrophin have complementing roles in normal functional or developmental pathways in muscle. Detailed study of these mice should provide novel insights into the pathogenesis of DMD and provide an improved model for rapid evaluation of gene therapy strategies.