Differential expression of receptor tyrosine kinases and Shc in fetal and adult rat fibroblasts: toward defining scarless versus scarring fibroblast phenotypes

The remarkable ability of the fetus to heal early gestation skin wounds without scarring remains poorly understood. Taking advantage of recent advances in signal transduction, the tyrosine phosphorylation patterns of fetal rat fibroblasts, representing the scarless cutaneous repair phenotype, and adult rat fibroblasts, representing scarforming phenotype, were examined whether there were inherent differences in cellular signaling. Specifically, correlation of the phosphorylation patterns with the expression levels of the signaling molecules that transmit information from the plasma membrane receptor to the nucleus was sought. By using three different cell lines of explanted fibroblasts from gestational day 13 fetal rat skin (n = 24) and 1-month-old postnatal adult rat skin (n = 3), immunoblotting was performed to compare tyrosine phosphorylation patterns. The results revealed five major protein bands of interest in fetal rat fibroblasts, but not in the adult rat fibroblasts. These phosphorylated protein bands are of interest because of their possible role in wound repair and may have the potential to regulate cellular responses to the extracellular matrix and their secondary signaling molecules. It was hypothesized that these bands represented receptor tyrosine kinases, epidermal growth factor receptor, and discoidin domain receptor 1, and their downstream adaptor protein Shc that binds receptor tyrosine kinases to transduce signals intracellularly. Furthermore, elevated expression of platelet-derived growth factor receptor-beta in adult compared with fetal fibroblasts was demonstrated, suggesting that decreased expression of certain growth factors may also be important for the scarless phenomenon to occur.     

Matrix metalloproteinases and the ontogeny of scarless repair: the other side of the wound healing balance

Early gestation mammalian fetuses possess the remarkable ability to heal cutaneous wounds in a scarless fashion. Over the past 20 years, scientists have been working to decipher the mechanisms underlying this phenomenon. Much of the research to date has focused on fetal correlates of adult wound healing that promote fibrosis and granulation tissue formation. It is important to remember, however, that wound repair consists of a balance between tissue synthesis, deposition, and degradation. Relatively little attention has been paid to this latter component of the fetal wound healing process.In this study, we examined the ontogeny of ten matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in nonwounded fetal rat skin and fibroblasts as a function of gestational age. We used a semiquantitative polymerase chain reaction protocol to analyze these important enzymes at time points that represent both the scarless and scar-forming periods of rat gestation. The enzymes evaluated were collagenase-1 (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), membrane-type matrix metalloproteinases (MT-MMPs) 1, 2, and 3, and TIMPs 1, 2, and 3.Results demonstrated marked increases in gene expression for MMP-1, MMP-3 and MMP-9 that correlated with the onset of scar formation in nonwounded fetal skin. Similar results were noted in terms of MMP-9 gene expression in fetal fibroblasts. These results suggest that differences in the expression of these matrix metalloproteinases may have a role in the scarless wound healing phenotype observed early in fetal rat gestation. Furthermore, our data suggest that the differential expression of gelatinase B (MMP-9) may be mediated by the fetal fibroblasts themselves.     

A Confocal microscopic study on colony morphology and sporulation of Bacillus sp.

Hexavalent chromium Cr(VI) is one of the dominant oxidation states of chromium that exist in the environment and is highly toxic to all forms of life. In the present study, we employ a confocal laser scanning microscope (CLSM) and investigate the effect of Cr(VI) on colony morphology of a Bacillus sp. isolated from soil exposed to tannery effluent. The colonies grown at chromium concentrations, control and 100 ppm are found to be opaque and beyond 200 ppm the colonies were translucent thus exhibiting phase variation. CLSM studies on colonies grown on control plates showed significant increase in height and in biovolume as a function of time whereas, the translucent colonies showed very little change in height and biovolume corresponding to the colony growth. Exopolymeric substance (EPS) content of translucent colonies was lesser than that of opaque colonies, indicating that EPS also plays a role in the observed phenomenon of phase variation. Studies on the effect of Cr(VI) on spore formation showed that Cr(VI) concentrations up to 100 ppm favored spore formation, while concentrations beyond 100 ppm showed a steady decline in spore formation.     

Confocal laser scanning microscopy of rat follicle development.

This study used confocal laser scanning microscopy (CLSM) to study follicular development in millimeter pieces of rat ovary. To use this technology, it is essential to stain the tissue before laser excitation with the confocal microscope. Various fluorescent stains (Yo-Pro, Bo-Pro, LysoTracker Red, hydroethidine, ethidium bromide, and 7-aminoactinomycin-d) were applied either to fresh tissue or to tissue that had been fixed with glutaraldehyde or paraformaldehyde. After fixation and staining, the tissue was dehydrated with MEOH and cleared with benzyl alcohol/benzyl aldehyde. CLSM was then used with the appropriate laser excitation, dichroics, and bandpass filters to acquire images of oocytes contained in follicles. Analysis of the data revealed three principal findings. First, a rapid increase in oocyte size occurred in the preantral stages of follicle development. In the antral stage of follicle development, there was a rapid increase in follicle size without any substantial increase in oocyte size. Second, accompanying these changes in oocyte and follicle growth was a differential staining pattern in which the nucleus stained more than the cytoplasm in a young follicle, but stained less than the cytoplasm as the follicle enlarged into the late antral stage. Lastly, using CLSM, atretic follicles showed increased LysoTracker Red staining in the granulosa region of the antral follicle, suggestive of cell death.     

Ontogenetic transition of wound healing pattern in rat skin occurring at the fetal stage

Full-thickness excisional wounds were made in the dorsal skin of rat fetuses at day 16 and day 18 of gestation. A small patch of skin surrounding the open wound was cut out, mounted on a plastic ring and incubated in an organ culture system. In the presence of serum, the open wound in the day-16 fetal skin closed within three days of culture. During the wound-closure process, no new structures were formed in the wound space, and no conspicuous changes were noted in the histological architecture of the surrounding skin during culture, indicating that the wound closure may result from a centripetal movement of the surrounding skin only. In contrast, the size of the open wound in the day-18 fetal skin remained almost unchanged for one week, but a thin acellular network spread over the wound space within one day of culture. The predominant component of the network was cross-linked fibrin, as disclosed by scanning electron microscopy and sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by immunoblotting. The network served as a scaffold for the ingrowth of fibroblast-like cells. These stage-dependent differences in fetal wound healing were consistent with an in vivo study showing that the day-16 wound was covered with the surrounding skin itself, whereas the day-18 wound was covered with newly formed epidermis and invaded by inflammatory cells. The present investigation strongly indicates the prenatal occurrence of a fetal-to-adult transition in the wound-healing pattern of rat skin.     

DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe.

We have tested mutants corresponding to 20 DNA repair genes of the fission yeast Schizosaccharomyces pombe for their ability to arrest in G2 after DNA damage. Of the mutants tested, four are profoundly defective in this damage dependent G2 arrest. In addition, these four mutants are highly sensitive to a transient inhibition of DNA synthesis by hydroxyurea. This suggests that the pathway responsible for the recognition of DNA damage and the subsequent mitotic arrest, shares many functions with the mechanism that controls the dependency of mitosis on the completion of S phase. The phenotype of these checkpoint rad mutants in wee mutant backgrounds indicate that the G2 arrest response is mediated either through, or in parallel with, the activity of the cdc2 gene product.     

Secretory phenomena at the ependyma of the sulcus hypothalamicus Monroi of the fetal rat. A histochemical and electron microscopic study

In a localized area above the sulcus hypothalamicus Monroi ventriculi tertii of the rat ependymal structures are observed, which only occur between the 18th and 20th day of gestation. They were investigated with the aid of lightmicroscopic, ultrastructural and histochemical technics. The surface of the ventricular wall is rendered irregular by protrusions and deposits. Neurosecretory stains and histochemical examination give no positive results. Apical parts of the ependymal cells are fingershaped, dome-like and of spheric appearance. The equipment of the protrusions with organels is variable. Some of them contain a fine granular ground plasma, polyribosomes and occasional profiles of the endoplasmatic reticulum. Others are impacted with optic empty or substantial vacuols. Vacuols merging in the cellular membrane evacuate their content into the ventricular space. Multivesicular bodys engorged with vesicles are together with those, which lie empty in the ventricular cavity. Empty, buddle like and frequently folded membrane protrusions suggest that cellular content has been delivered into the ventricle.     

Progenitors of oligodendrocytes: Limiting dilution analysis in fetal rat brain culture

In this paper, we describe the use of a combination of cell culture techniques and limiting dilution analysis to determine the number of oligodendrocyte progenitor cells and the oligodendrocyte clone size in primary dispersed cultures of 20- to 21-day-old fetal rat brain. Single-cell suspensions (1,2,3 × 10⁶ cells/ml) were plated in either microwell or 100 mm dishes. After 22 days in culture the number of differentiated oligodendrocytes was ascertained by determining the amount of myelin basic protein by radioimmunoassay. The total amount of myelin basic protein was the same in the two types of dish, indicating that proliferation and differentiation were unaffected when oligodendrocytes were grown in microwells. The fraction (F₀) of microwells containing no oligodendrocytes was determined at each cell dilution. F₀ decreased exponentially with increasing total cell concentration. The linearity of the plot of ln F₀ versus cell number indicates that the number of oligodendrocyte progenitor cells is limiting. From the equation describing the Poisson distribution of progenitor cells in microwells we calculated that, at the time of plating, primary cultures of fetal rat brain contain one oligodendrocyte progenitor cell per 1.3 × 10⁵ brain cells, or a total population of 300–500 progenitor cells per brain. The mean oligodendrocyte clone size was determined to be approximately 825 at 22 days and close to 2000 by 35 days in culture. Therefore, each progenitor cell must undergo approximately 11 divisions, on the average, during postnatal development.     

Confocal microscopic findings of cysteine protease calpain in Plasmodium falciparum

Pf-calpain, a cysteine protease of Plasmodium falciparum, is believed to be one of the central mediators for essential parasitic activity. However, the roles of calpain on parasitic activity have not been determined in P. falciparum. In the present study, the localization of Pf-calpain was investigated using polyclonal antibodies (anti-Pf-calpain antibody A and B) against peptides that distinguished it from human calpain-7 and rat calpain-10 protein. Recombinant Pf-calpain (rPf-calpain) was identified as a 46 kDa protein using an anti-Pf-calpain antibody A, which can recognize the Pf-calpain binding site. Confocal microscopy revealed calpain within cytoplasmic localized parasites in the erythrocytic cycle. The findings suggested that the expression of Pf-calpain would be proportional to all different parasites in the erythrocytic cycle. On the other hand, anti-human calpain-7 antibody detected Pf-calpain in schizonts, and the immunofluorescence was stronger than with anti-rat calpain-10 antibody. However, the antibodies reacted with calpains in human red blood cells. These results show that anti-Pf-calpain antibody A and B specifically recognize only Pf-calpain. Taken together, the results suggest that Pf-calpain is expressed in all erythrocytic stages. In particular, the expression of Pf-calpain is increased much more when the late ring matures into the early trophozoite. Moreover, anti-Pf-calpain antibody A and B against synthetic peptides of the catalytic domain of Pf-calpain are useful to specifically detect Pf-calpain in all erythrocytic stages, while human and rat calpain antibody are not useful.     

Confocal microscopic study of GABA(A) receptors in Xenopus oocytes after rat brain mRNA injection: modulation by tyrosine kinase activity

The expression of GABA(A) receptors in Xenopus oocytes injected with rat brain mRNA was studied by immunocytochemistry and evaluation of the distribution of fluorescent probes at the confocal microscope. The beta(2/3) subunit distributed exclusively on the membrane at the animal pole of the oocytes. Treatment of oocytes for 20 min with the protein tyrosine kinase inhibitor genistein, 200 microM, resulted in a lower presence of GABA(A) receptors on the membrane. The inactive genistein analogue daidzein, 200 microM, had no effect even with a 30 min treatment. Alkaline phosphatase but not a protein tyrosine phosphatase, when injected into oocytes, reduced GABA(A) receptor membrane expression. The data indicate that protein tyrosine phosphorylation modulates the expression on the plasma membrane of presynthesized GABA(A) receptors.     

Effects of space flight on the histological characteristics of the aortic depressor nerve in the adult rat: electron microscopic analysis.

The effects of microgravity on the histological characteristics of the aortic depressor nerve, which is the afferent of the aortic baroreflex arc, were determined in 10 female adult rats. The rats were assigned for nursing neonates in the Space Shuttle Columbia or in the animal facility on the ground (NASA Neurolab, STS-90), and were housed for 16 days under microgravity in space (microg, n=5) or under one force of gravity on Earth (one-g, n=5). In the Schwann cell unit in which the axons of unmyelinated fibers are surrounded by one Schwann cell, the average number of axons per unit in the microg group was 2.1 +/- 1.6 (mean +/- SD, n=312) and significantly less than that in the one-g group (3.0 +/- 2.9, n=397, p<0.05). The proportion of unmyelinated fibers in the aortic depressor nerve in the microg group was 64.5 +/- 4.4% and significantly less than that in the one-g group (74.0 +/- 7.3%, p<0.05). These results show that there is a decrease in the number of high-threshold unmyelinated fibers in the aortic depressor nerve in adult rats flown on the Shuttle Orbiter, suggesting that the aortic baroreflex is depressed under microgravity during space flight.     

Subcellular localization of xanthine oxidase in rat hepatocytes: high-resolution immunoelectron microscopic study combined with biochemical analysis.

Xanthine oxidase (XO), a molybdo-flavoprotein enzyme involved in purine degradation, was localized immunocytochemically in rat hepatocytes by high-resolution immunoelectron microscopy. XO was isolated from rat liver and a 150 KD polypeptide was purified. Antibodies were raised in rabbits. Small pieces of fresh liver were quickly frozen by contact with a copper block pre-cooled with liquid helium and were freeze-substituted with either 2.5% OsO4 or 0.2% glutaraldehyde in acetone. They were then warmed and embedded in Epon-Araldite or Araldite 6005. Resin sections were treated by indirect immunostaining using anti-rat liver XO antibody and protein A-gold. The labeling pattern was clearly over the cytosol and not on cell organelles. A few gold particles were found over the mitochondrial matrix, but not over the endoplasmic reticulum, Golgi apparatus, lysosomes, or peroxisomes, including their crystalloid core. These results are consistent with those of the biochemical assay of XO in this study. The significance of the occasional immunolabeling of the mitochondrial matrix remains obscure, since biochemical determinations in this study indicate no XO activity in the mitochondrial fraction.     

The fetal cleft palate: III. Ultrastructural and functional analysis of palatal development following in utero repair of the congenital model

The role of fetal surgery in the management of congenital anomalies and intrauterine abnormalities is appropriately restricted on the basis of feasibility and risk-to-benefit analyses of intrauterine intervention. Recently, the authors demonstrated that in utero cleft palate repair of the congenital caprine model is technically feasible and results in scarless healing of the mucoperiosteum and velum, with subsequent development of a potentially functional bilaminar palate with distinct oral and nasal mucosal layers, following single-layer repair of the fetal mucoperiosteal flaps. A slight indentation at the site of repair was the only remaining evidence of a cleft. At 6 months of age, normal palatal architecture, including that of mucosal, muscular, and glandular elements, was seen grossly and histologically. The present work investigated the ultrastructural and functional aspects of the palate following in utero cleft repair to determine what benefits might be derived from fetal intervention. Six goats pregnant with twins were gavaged twice daily for 10 days (gestational days 32 to 41; term, 145 days) with dry, ground Nicotiana glauca plant delivering between 2.4 and 14 mg/kg per day of anabasine, doses that were adjusted in response to mater-nal toxicity. At 85 days' gestation, six fetuses underwent in utero palatoplasty using a modified von Langenbeck technique with elevation of bilateral mucoperiosteal flaps and lateral relaxing incisions. A single-layer repair of the mucoperiosteal flaps was performed using interrupted 6-0 Vicryl sutures. Six fetuses remained as unrepaired clefted controls. Six months after in utero palatoplasty, each group of goats underwent nasoendoscopy to evaluate palatal function; two unclefted 6-month-old goats served as controls. Subsequently, soft palate muscle was harvested from each of the goats and was evaluated by light and electron microscopy. Velar muscle was also harvested from the unclefted control goats and was similarly studied. Nasoendoscopy demonstrated functional palates capable of dynamic velopharyngeal closure following in utero cleft repair; this motion was similar to that observed in unclefted animals. Unrepaired clefted goats did not demonstrate any evidence of velar motion or velopharyngeal closure. Soft palate muscle from this group demonstrated evidence of myofibril degeneration, atrophy, and loss compared with unclefted control velar muscle. Ultrastructural changes included sarcomere "scalloping, " partial Z-line degeneration and loss, and progressive I-band degeneration and loss. Repaired clefted soft palate muscle was remarkably similar to unclefted control muscle. Significantly less myofibril, Z-line, and I-band degeneration and loss were observed with minimal evidence of sarcomere scalloping. In utero cleft palate repair results in a functional soft palate with restoration of ultrastructural architecture of the velum. These findings were attributed to reconstitution of the velar muscular sling, which is disrupted during the clefting process and remains abnormally inserted into the posterior edge of the palatal bone and along the bony cleft. Although repaired velar muscle does demonstrate some evidence of ultrastructural change compared with control muscle, these findings are significantly less pronounced than those observed in the unrepaired clefted muscle.     

Ontogenetic transition of cardiac myosin heavy chain isoforms in rat ventricle: effects of fetal exposure to beta-adrenergic agonists or antagonists.

Cardiac myosin heavy chain (MHC) expression undergoes an ontogenetic transition from beta to alpha MHC isoforms. Although thyroid hormone plays a role in this change, the timing of the events suggests the participation of other factors. Using a new, denaturing SDS-PAGE procedure that cleanly resolves the beta and alpha heavy chains, we have assessed the role of beta-adrenergic stimulation on this transition in fetal and neonatal rat hearts. In control animals at embryonic day 20, less than 15% of the MHC was the alpha-form, and the proportion increased to approximately 35% by postnatal day 1 and to 80% by postnatal day 8. Although catecholamine levels rise abruptly at birth, and cyclic AMP levels increase the expression of alpha-MHC in vitro, neither premature beta-adrenergic stimulation (maternal treatment with terbutaline on embryonic days 17, 18 and 19) nor continuous prenatal blockade of beta-receptors (maternal propranolol infusions from embryonic day 7 onward) influenced the developmental profile. Because beta-receptors in fetal and neonatal heart are functionally linked to adenylate cyclase, and cyclic AMP has been shown to promote the expression of alpha-MHC, the lack of effect of terbutaline or propranolol suggests that activation of adenylate cyclase through fetal cardiac beta-receptors is not sufficient to mediate the switchover without participation of other factors, such as thyroid or steroid hormones, or hypoxia.     

Cysts in cultured fetal rat kidneys.

The formation of cysts has been studied in kidneys removed from day-15 and day-18 rat fetuses and cultured in a mixture of medium 199 (Morgan et al., '50) for periods of up to 15 days. Gas phases of 95% O2 and 5% CO2, and 95% air and 5% CO2, were employed, the latter being considered more satisfactory. In day-15 kidneys cysts formed from the ampullary portions of the collecting tubules after 2 days whereas cysts derived from nephrons were not seen until 9 days of culturing. The latter arose from developing juxtamedullary nephrons. In day-18 kidneys cysts from collecting tubules and nephrons were both present after 3 days of culturing. The latter, in this instance, originated mostly in immature midcortical nephrons, the juxtamedullary mephrons having undergone rapid degeneration. The tubular portion of the nephron seemed to be the primary site of dilatation. Under culture conditions cysts of nephrons thus formed from immature actively developing nephrons and not from those that were mature. Cysts associated with collecting tubules arose from the ampullary (terminal) portions of the latter in both day-15 and day-18 cultured kidneys. The study of cultured mammalian fetal kidneys can provide information on the nature and genesis of renal cysts. It is possible that the same technique also may be helpful for examining the effects of teratogens directly on the kidney.