Iatrogenic blood loss in critical care: A prospective observational study conducted at Universitas Academic Hospital in the Free State Province, South Africa.

Background: Prevention of iatrogenic blood loss is an essential component of patient blood management (PBM) in intensive care units (ICUs). The amount of iatrogenic blood loss from diagnostic phlebotomy in the ICUs at Universitas Academic Hospital, Free State Province, South Africa, is unknown. Objectives: To quantify diagnostic phlebotomy volumes, and volumes submitted in excess for diagnostic testing in the ICU. Methods: We conducted a prospective descriptive observational study on adults who were admitted to ICUs at a single centre over a period of 14 days. The weight of each filled phlebotomy tube was calculated using the specific gravity of blood and averages of empty phlebotomy tubes, establishing the total volume. Results: Data from 59 participants with a median length of stay at the ICU of 3 days were analysed. The median phlebotomy volume was 7.0 mL day and 13.6 mL/ICU admission. The volume of blood required for analysis daily and ICU admission was 0.7 mL and 2.2 mL, respectively. The median phlebotomy volume in excess of the amount required for analysis daily and ICU admission was 5.05 mL and 12.11 mL, respectively. Conclusion: While the median excess daily phlebotomy volume in this present study may seem insignificant and underestimating the true excess of phlebotomy volume, interventions to reduce phlebotomy volumes and development of a PBM guideline for appropriate phlebotomy volumes and preventing wastage of patients' blood in the ICU is required. Contributions of the study: We determined blood volume requirements for laboratory instrumentation, which allows phlebotomists to be cognisant of the true requirements for diagnostic tests to be undertaken accurately. We established diagnostic blood loss volumes in critical care units at a tertiary hospital in South Africa and we advocate for the introduction of patient blood management practice guidelines at local institutions.

Frequency and prediction of persistent urinary tract dilation in third trimester and postnatal urinary tract dilation in infants following diagnosis in second trimester.

OBJECTIVE: To determine the frequency, associated characteristics and prognostic value of the current risk stratification system for prenatal urinary tract dilation (UTD) for predicting persistent UTD in the third trimester and subsequent postnatal UTD in the infant, following diagnosis in the second trimester. METHODS: This was a single-institution retrospective cohort study of singleton pregnancies diagnosed with unilateral or bilateral UTD in the second trimester (before 28 weeks' gestation) with follow-up in the third trimester (at or after 28 weeks) between January 2017 and May 2019. In all cases, the prenatal diagnosis and stratification to low-risk (Grade A1) or increased-risk (Grade A2-3) UTD was made using the 2014 UTD consensus classification system. The primary outcomes included persistent prenatal UTD in the third trimester and postnatal UTD up to 6 months of age. We performed multivariable analysis to assess whether patient and second- and third-trimester sonographic characteristics (such as UTD laterality, other renal abnormality (calyceal dilation, abnormal parenchymal appearance, abnormal ureter or bladder) and anteroposterior renal pelvic diameter (AP-RPD)) were associated with the study outcomes. We assessed the predictive value of the current risk stratification system (Grade A1 vs Grade A2-3) in the second and third trimesters for persistent prenatal UTD and postnatal UTD using the area under the receiver-operating-characteristics curve (AUC). RESULTS: Of 26 620 second-trimester ultrasound assessments in the study period, 347 patients were diagnosed with UTD in the second trimester and had third-trimester follow-up, of whom 150/347 (43% (95% CI, 38-49%)) had persistent UTD in the third trimester. Among the 282/347 (81%) patients with postnatal follow-up available, the frequency of postnatal UTD was 49/282 (17% (95% CI, 13-22%)), and among the subset with persistent UTD in the third trimester, the frequency of postnatal UTD was 46/102 (45% (95% CI, 35-55%)). The most frequent postnatal diagnosis was transient UTD (76%), followed by duplicated collecting system (10%). Of infants originally diagnosed with UTD in the second trimester, 2% (7/347) required surgery; stated differently, of the 49 infants with postnatal UTD, 14% (7/49) required surgery. At second-trimester diagnosis, sonographic predictors of both persistent prenatal UTD and postnatal UTD included the presence of other renal abnormality and UTD Grade A2-3. At third-trimester follow-up, predictors of postnatal UTD were larger mean AP-RPD and UTD Grade A2-3, while all cases had other renal abnormality. Second-trimester diagnosis of UTD Grade A2-3 had satisfactory discrimination for predicting persistent prenatal UTD (AUC, 0.64 (95% CI, 0.58-0.70)) and postnatal UTD (AUC, 0.72 (95% CI, 0.63-0.81)), as did third-trimester UTD Grade A2-3 for predicting postnatal UTD (AUC, 0.66 (95% CI, 0.56-0.76)). CONCLUSIONS: The majority of cases of prenatal UTD did not result in postnatal UTD, and of those that did, very few required surgery. Follow-up third-trimester assessment after a second-trimester diagnosis of UTD is warranted. The current risk stratification system by UTD grade, based on the 2014 UTD consensus classification, can be used to predict postnatal UTD with fair accuracy. Further research is needed to determine whether the predictive performance of this system can be improved by incorporating additional risk factors. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Hypereosinophilic syndrome with characteristic left ventricular thrombus demonstrated by contrast echocardiography.

Hypereosinophilic syndrome (HES) is a rare condition characterised by idiopathic eosinophilia with organ system involvement. The condition is far more common in males, with a typical onset in the third to sixth decade. Cardiac damage may result in the formation of a characteristic apical thrombus readily visualised on two-dimensional echocardiography. Cardiac involvement portends a less favourable prognosis as it can be complicated by acute embolic events and progressive development of restrictive cardiomyopathy, valvular dysfunction, and heart failure. In this case report, we describe a middle-aged gentleman with HES and characteristic apical thrombus identified on contrast echocardiography. Although the use of contrast agents for assessment of left ventricular thrombus is documented in the literature,1 this case illustrates the application of contrast echocardiography in the evaluation of eosinophilia. (Neth Heart J 2009;17:169-70.).

Selective activation of nuclear factor kappa B in the cochlea by sensory and inflammatory stress.

Damage response pathways triggered by mechanical stress might reasonably be expected to be conserved throughout evolution. However, using a nuclear factor kappa B (NF-kappaB) reporter mouse we show here that this phylogenetically recent transcription factor plays a major role in the response to mechanosensory stress in the mammalian inner ear. The protective action of NF-kappaB is exerted in neither sensory nor non-sensory epithelial cells, but rather in connective tissue cells within the spiral ligament and spiral limbus. In the spiral ligament, predominantly type I fibrocytes are activated following noise exposure, whereas type II fibrocytes are activated following systemic inflammatory stress. Immune-mediated and acoustic trauma-mediated hearing loss syndromes in humans may in part result from the vulnerability of type II and type I fibrocytes to systemic inflammatory stress and acoustic trauma, respectively. Unexpected cell-specific and stress-specific NF-kappaB activation found in the inner ear in this in vivo study suggest that this approach may have wide applications in demonstrating similar specializations of stress responses in other tissues, including the brain.

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage.

BACKGROUND: Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes. RESULTS: A single tenascin gene was identified in the genome of C. intestinalis that encodes a polypeptide with domain features common to all vertebrate tenascins. Both pufferfish genomes encode five tenascin genes: two tenascin-C paralogs, a tenascin-R with domain organization identical to mammalian and avian tenascin-R, a small tenascin-X with previously undescribed GK repeats, and a tenascin-W. Four tenascin genes corresponding to tenascin-C, tenascin-R, tenascin-X and tenascin-W were also identified in the X. tropicalis genome. Multiple sequence alignment reveals that differences in the size of tenascin-W from various vertebrate classes can be explained by duplications of specific fibronectin type III domains. The duplicated domains are encoded on single exons and contain putative integrin-binding motifs. A phylogenetic tree based on the predicted amino acid sequences of the fibrinogen-related domains demonstrates that tenascin-C and tenascin-R are the most closely related vertebrate tenascins, with the most conserved repeat and domain organization. Taking all lines of evidence together, the data show that the tenascins referred to as tenascin-Y and tenascin-N are actually members of the tenascin-X and tenascin-W gene families, respectively. CONCLUSION: The presence of a tenascin gene in urochordates but not other invertebrate phyla suggests that tenascins may be specific to chordates. Later genomic duplication events led to the appearance of four family members in vertebrates: tenascin-C, tenascin-R, tenascin-W and tenascin-X.

A physiologically based toxicokinetic model of inhalation exposure to xylenes in Caucasian men.

Widespread exposure to the volatile aromatic hydrocarbons, ortho-, meta-, and para-xylene occurs in many industries including the manufacture of plastics, pharmaceuticals, and synthetic fibers. This paper describes the development of a physiologically based toxicokinetic model using biomonitoring data to quantify the kinetics of ortho-, meta-, and para-xylenes. Serial blood concentrations of deuterium-labeled xylene isomers were obtained over 4 days after 37 controlled, 2h inhalation exposures to different concentrations of the isomers. Peak toxicant concentrations in blood occurred in all subjects at the termination of exposure. Systemic clearance averaged 116 L/h+/-34 L/h, 117 L/h+/-23 L/h, and 129 L/h+/-33 L/h for ortho-, para-, and meta-xylene, respectively. The half-life of each toxicant in the terminal phase (>90 h post-exposure) was fit by the model, yielding values of 30.3+/-10.2 h for para-xylene, 33.0+/-11.7 h for meta-xylene and 38.5+/-18.2 h for ortho-xylene. Significant isomeric differences were found (p<0.05) for toxicant half-life, clearance and extrahepatic metabolism. Inter-individual variability seen in this study suggests that airborne concentration guidelines may not protect all workers. A Biological Exposure Index is preferred for this purpose since it is integrative and reflective of inter-individual kinetic variability.

Selective gentamicin uptake by cytochemical subpopulations of guinea-pig geniculate ganglion cells.

Cytochemical subpopulations of geniculate ganglion (GG) cells were identified in guinea-pigs using immunohistochemistry and selective gentamicin accumulation. Two subpopulations of GG cells were evident based upon their location and immunoreactivity for peptide 19 (PEP 19), for plasma membrane Ca2+-ATPase (PMCA-ATPase), and for neurofilament proteins. Cells within the posterior part of GG were positive for PEP 19 and PMCA-ATPase, but not for 68 kD or 160 kD neurofilament proteins. Cells within the anterior part showed complementary staining properties. Cells within these populations showed differences in accumulation of gentamicin, depending upon the administration route. Cells within the posterior part showed avid accumulation of gentamicin when animals received the drug systemically. When the drug was administered directly into the middle ear, cells within the anterior part showed avid gentamicin accumulation. Immunostaining for gentamicin in both cell populations was much more extreme and remained so for longer post-administration times when compared with spiral ganglion and vestibular ganglion cells. The results suggest that cells in the anterior part of GG have little exposure to gentamicin in the serum and that perhaps they innervate the middle ear mucosa or they absorb the drug through their axons within the middle ear. In contrast, cells in the posterior part of GG have greater access to systemically administered gentamicin either directly or via their axon terminals.

Thrombospondins: multifunctional regulators of cell interactions.

Thrombospondins are secreted, multidomain macromolecules that act as regulators of cell interactions in vertebrates. Gene knockout mice constructed for two members of this family demonstrate roles in the organization and homeostasis of multiple tissues, with particularly significant activities in the regulation of angiogenesis. This review discusses the functions of thrombospondins with regard to their cellular mechanisms of action and highlights recent advances in understanding how multifactorial molecular interactions, at the cell surface and within extracellular matrix, produce cell-type-specific effects on cell behavior and the organization of matrix and tissues.

Inner ear localization of mRNA and protein products of COCH, mutated in the sensorineural deafness and vestibular disorder, DFNA9.

Missense mutations in the COCH gene, which is expressed preferentially at high levels in the inner ear, cause the autosomal dominant sensorineural deafness and vestibular disorder, DFNA9 (OMIM 601369). By in situ hybridization of mouse and human inner ear sections, we find high-level expression of COCH mRNA in the fibrocytes of the spiral limbus and of the spiral ligament in the cochlea, and in the fibrocytes of the connective tissue stroma underlying the sensory epithelium of the crista ampullaris of the semicircular canals. A polyclonal antibody against the human COCH protein product, cochlin, was raised against the N-terminal 135 amino acid residues of cochlin, corresponding to the Limulus factor C-homology (cochFCH) domain; this domain harbors all five known point mutations in DFNA9. On western blots of human fetal cochlear extracts, anti-cochlin reacts with a cochlin band of the predicted full-length size as well as a smaller isoform. Immunohistochemistry performed with anti-cochlin shows staining predominantly in the regions of the fibrocytes of the spiral limbus and of the spiral ligament in mouse and in human fetal and adult tissue sections. These sites correspond to those areas that express COCH mRNA as determined by in situ hybridization, and to the regions of the inner ear which show histological abnormalities in DFNA9. The fibrocytes expressing mRNA and protein products of COCH are the very cell types which are either absent or markedly reduced and replaced by eosinophilic acellular material in temporal bone sections of individuals affected with DFNA9.

Cell-matrix contact structures.

Cell-extracellular matrix contacts are points on cell surfaces where adhesion receptors tether cells to matrix and are linked intracellularly to cytoskeletal components. These structures integrate cell organisation within tissues, support cell motility and specialised activities of differentiated cells, and transduce extracellular signals. Current characterisations of matrix contacts are based on morphological and biochemical criteria, yet the levels of definition of different contact types are very varied. Some contacts are surprisingly little-studied given their likely importance in vivo. Here, I describe the general features of matrix contacts, review the functions and molecular composition of major types of transient and stable matrix contacts, and discuss the information that is emerging on contact integration and dynamics in single cells.

A role for syndecan-1 in coupling fascin spike formation by thrombospondin-1.

An important role of cell matrix adhesion receptors is to mediate transmembrane coupling between extracellular matrix attachment, actin reorganization, and cell spreading. Thrombospondin (TSP)-1 is a modulatory component of matrix expressed during development, immune response, or wound repair. Cell adhesion to TSP-1 involves formation of biochemically distinct matrix contacts based on stable fascin spikes. The cell surface adhesion receptors required have not been identified. We report here that antibody clustering of syndecan-1 proteoglycan specifically transduces organization of cortical actin and fascin bundles in several cell types. Transfection of COS-7 cells with syndecan-1 is sufficient to stimulate cell spreading, fascin spike assembly, and extensive protrusive lateral ruffling on TSP-1 or on syndecan-1 antibody. The underlying molecular mechanism depends on glycosaminoglycan (GAG) modification of the syndecan-1 core protein at residues S45 or S47 for cell membrane spreading and on the VC2 region of the cytoplasmic domain for spreading and fascin spike formation. Expression of the VC2 deletion mutant or GAG-negative syndecan-1 showed that syndecan-1 is necessary in spreading and fascin spike formation by C2C12 cells on TSP-1. These results establish a novel role for syndecan-1 protein in coupling a physiological matrix ligand to formation of a specific matrix contact structure.

Stimulation of fascin spikes by thrombospondin-1 is mediated by the GTPases Rac and Cdc42.

Cell adhesion to extracellular matrix is an important physiological stimulus for organization of the actin-based cytoskeleton. Adhesion to the matrix glycoprotein thrombospondin-1 (TSP-1) triggers the sustained formation of F-actin microspikes that contain the actin-bundling protein fascin. These structures are also implicated in cell migration, which may be an important function of TSP-1 in tissue remodelling and wound repair. To further understand the function of fascin microspikes, we examined whether their assembly is regulated by Rho family GTPases. We report that expression of constitutively active mutants of Rac or Cdc42 triggered localization of fascin to lamellipodia, filopodia, and cell edges in fibroblasts or myoblasts. Biochemical assays demonstrated prolonged activation of Rac and Cdc42 in C2C12 cells adherent to TSP-1 and activation of the downstream kinase p21-activated kinase (PAK). Expression of dominant-negative Rac or Cdc42 in C2C12 myoblasts blocked spreading and formation of fascin spikes on TSP-1. Spreading and spike assembly were also blocked by pharmacological inhibition of F-actin turnover. Shear-loading of monospecific anti-fascin immunoglobulins, which block the binding of fascin to actin into cytoplasm, strongly inhibited spreading, actin cytoskeletal organization and migration on TSP-1 and also affected the motility of cells on fibronectin. We conclude that fascin is a critical component downstream of Rac and Cdc42 that is needed for actin cytoskeletal organization and cell migration responses to thrombospondin-1.

The thrombospondin type 1 repeat (TSR) superfamily: diverse proteins with related roles in neuronal development.

The thrombospondins are a family of proteins found widely in the embryonic extracellular matrix. Like most matrix proteins, thrombospondins are modular and contain a series of repeated domains arrayed between globular amino and carboxyl terminal domains. In recent years, other proteins that share thrombospondin type 1 repeats, or TSRs, have been identified. These include the F-spondin gene family, the members of the semaphorin 5 family, UNC-5, SCO-spondin, and others. Most of these are expressed in the developing nervous system, and many have expression patterns and in vitro properties that suggest potential roles in the guidance of cell and growth cone migration. Both cell- and matrix-binding motifs have been identified in the TSRs of thrombospondin-1, so it has been hypothesized that the properties of these diverse proteins may also depend on the presence of these repeats. Here, we review the cell biology of the TSR module, the extensive literature regarding the distribution and functions of thrombospondins and other TSR superfamily proteins, and evaluate their possible roles during the development of the nervous system.

Gap junction systems in the mammalian cochlea.

Recent findings that a high proportion of non-syndromic hereditary sensorineural hearing loss is due to mutations in the gene for connexin 26 indicate the crucial role that the gene product plays for normal functioning of the cochlea. Excluding sensory cells, most cells in the cochlea are connected via gap junctions and these gap junctions appear to play critical roles in cochlear ion homeostasis. Connexin 26 occurs in gap junctions connecting all cell classes in the cochlea. There are two independent systems of cells, which are defined by interconnecting gap junctions. The first system, the epithelial cell gap junction system, is mainly composed of all organ of Corti supporting cells, and also includes interdental cells in the spiral limbus and root cells within the spiral ligament. The second system, the connective tissue cell gap junction system, consists of strial intermediate cells, strial basal cells, fibrocytes in the spiral ligament, mesenchymal cells lining the bony otic capsule facing the scala vestibuli, mesenchymal dark cells in the supralimbal zone, and fibrocytes in the spiral limbus. One function of these gap junctional systems is the recirculation of K(+) ions from hair cells to the strial marginal cells. Interruption of this recirculation, which may be caused by the mutation in connexin 26 gene, would deprive the stria vascularis of K(+) and result in hearing loss.

Potassium ion recycling pathway via gap junction systems in the mammalian cochlea and its interruption in hereditary nonsyndromic deafness.

In the mammalian cochlea, there are two independent gap junction systems, the epithelial cell gap junction system and the connective tissue cell gap junction system. Thus far, four different connexin molecules, including connexin 26, 30, 31, and 43, have been reported in the cochlea. The two networks of gap junctions form the route by which K+ ions that pass through the sensory cells during mechanosensory transduction can be recycled back to the endolymphatic space, from which they reenter the sensory cells. Activation of hair cells by acoustic stimuli induces influx of K+ ions from the endolymph to sensory hair cells. These K+ ions are released basolaterally to the extracellular space of the organ of Corti, from which they enter the cochlear supporting cells. Once inside the supporting cells they move via the epithelial cell gap junction system laterally to the lower part of the spiral ligament. The K+ ions are released into the extracellular space of the spiral ligament by root cells and taken up by type II fibrocytes. This uptake incorporates K+ into the connective tissue gap junction system. Within this system, the K+ ions pass through the tight junctional barrier of the stria vascularis and are released within the intrastrial extracellular space. The marginal cells of the stria vascularis then take up K+ and return it to the endolymphatic space, where it can be used again in sensory transduction. It is highly probable that mutations of connexin genes that result in human nonsyndromic deafness cause dysfunction of cochlear gap junctions and thereby interrupt K+ ion recirculation pathways. In addition to connexin mutations, other conditions may disrupt gap junctions within the ear. For example, mice with a functionally significant mutation of Brain-4, which is expressed in the connective tissue cells within the cochlea, show marked depression of the endolymphatic potential and profound sensorineural hearing loss. It seems likely that disruption of connective tissue cells by this mutation disrupts K+ ion entry into the stria vascularis and thereby results in loss of endolymphatic potential. The association of sensorineural hearing loss with these genetic disorders provides strong evidence for the necessity of gap junction systems for the normal functioning of the cochlea.

Cell-matrix adhesions differentially regulate fascin phosphorylation.

Cell adhesion to individual macromolecules of the extracellular matrix has dramatic effects on the subcellular localization of the actin-bundling protein fascin and on the ability of cells to form stable fascin microspikes. The actin-binding activity of fascin is down-regulated by phosphorylation, and we used two differentiated cell types, C2C12 skeletal myoblasts and LLC-PK1 kidney epithelial cells, to examine the hypothesis that cell adhesion to the matrix components fibronectin, laminin-1, and thrombospondin-1 differentially regulates fascin phosphorylation. In both cell types, treatment with the PKC activator 12-tetradecanoyl phorbol 13-acetate (TPA) or adhesion to fibronectin led to a diffuse distribution of fascin after 1 h. C2C12 cells contain the PKC family members alpha, gamma, and lambda, and PKCalpha localization was altered upon cell adhesion to fibronectin. Two-dimensional isoelectric focusing/SDS-polyacrylamide gels were used to determine that fascin became phosphorylated in cells adherent to fibronectin and was inhibited by the PKC inhibitors calphostin C and chelerythrine chloride. Phosphorylation of fascin was not detected in cells adherent to thrombospondin-1 or to laminin-1. LLC-PK1 cells expressing green fluorescent protein (GFP)-fascin also displayed similar regulation of fascin phosphorylation. LLC-PK1 cells expressing GFP-fascin S39A, a nonphosphorylatable mutant, did not undergo spreading and focal contact organization on fibronectin, whereas cells expressing a GFP-fascin S39D mutant with constitutive negative charge spread more extensively than wild-type cells. In contrast, C2C12 cells coexpressing S39A fascin with endogenous fascin remained competent to form microspikes on thrombospondin-1, and cells that expressed fascin S39D attached to thrombospondin-1 but did not form microspikes. Blockade of PKCalpha activity by TPA-induced down-regulation led to actin association of wild-type fascin in fibronectin-adherent C2C12 and LLC-PK1 cells but did not alter the distribution of S39A or S39D fascins. The association of fascin with actin in fibronectin-adherent cells was also evident in the presence of an inhibitory antibody to integrin alpha5 subunit. These novel results establish matrix-initiated PKC-dependent regulation of fascin phosphorylation at serine 39 as a mechanism whereby matrix adhesion is coupled to the organization of cytoskeletal structure.

cDNA cloning of human muskelin and localisation of the muskelin (MKLN1) gene to human chromosome 7q32 and mouse chromosome 6 B1/B2 by physical mapping and FISH.

Muskelin is a novel intracellular protein, identified in mouse cells, that acts as a mediator of cell spreading and cytoskeletal responses to the extracellular matrix component thrombospondin-1. We report that human muskelin is 98% identical with mouse muskelin at the amino acid level. The human muskelin gene is located at 7q32. The mouse muskelin gene maps syntenically at chromosome 6 B1/B2.

Dense innervation of Deiters' and Hensen's cells persists after chronic deefferentation of guinea pig cochleas.

Innervation of Deiters' and Hensen's cells has been described in the organ of Corti of several mammalian species and has been suggested to arise from the olivocochlear (OC) efferent system (Wright and Preston [1976] Acta Otolaryngol. 82:41-47). In the present study, antineurofilament immunostaining was used to reveal these outer supporting cell fibers (OSCFs) in the normal guinea pig. In control ears, OSCFs were absent in the basal half of the cochlea but increased in number steadily toward the apex, peaking at values of over 1,200 fibers/mm. These values indicate a far more profuse innervation of supporting cells than has been described previously, suggesting that most OSCFs were not stained in previous immunohistochemical studies. Chronic cochlear deefferentation was used to test whether OSCFs are part of the OC system. The OC bundle was transected unilaterally, and the animals were allowed to survive for 4-8 weeks. Completeness of deefferentation was assessed by using acetylcholinesterase staining of the brainstem and measurement of the density of OC fascicles in the cochlea. By using these metrics, unilateral deefferentation was nearly complete in three animals. In successfully deefferented cases, the OSCF innervation density was not statistically different from control values. We conclude that the vast majority of OSCFs are not of OC origin. We speculate that they may be branches of type II afferent fibers to outer hair cells and that a smaller population of OSCFs with different morphology and immunoreactivity may arise from the OC system.

Muskelin, a novel intracellular mediator of cell adhesive and cytoskeletal responses to thrombospondin-1.

We have used an expression cloning strategy based on a cell-attachment assay screen to seek identification of molecules required in cellular responses to thrombospondin-1, a regulated macromolecular component of extracellular matrix. We report the identification and functional characterization of a novel, widely expressed, intracellular protein, named muskelin, which contains dispersed motifs with homology to the tandem repeats first identified in the Drosophila kelch ORF1 protein. In adherent C2C12 cells, muskelin localizes in the cytoplasm and at cell margins. Over-expression of muskelin in C2C12 cells promotes cell attachment to the thrombospondin-1 C-terminal domain, alters the mechanisms of attachment to intact thrombospondin-1 and correlates with decreased formation of fascin microspikes and increased assembly of focal contacts by cells adherent on thrombospondin-1. Reciprocally, cell attachment, spreading and cytoskeletal organization are specifically reduced in TSP-1-adherent cells after antisense depletion of muskelin. These results establish a requirement for muskelin in cell responses to thrombospondin-1 and demonstrate that such responses involve a novel process which is integrated into the regulation of cell-adhesive behaviour and cytoskeletal organization.

Cell-adhesive responses to tenascin-C splice variants involve formation of fascin microspikes.

Tenascin-C is an adhesion-modulating matrix glycoprotein that has multiple effects on cell behavior. Tenascin-C transcripts are expressed in motile cells and at sites of tissue modeling during development, and alternative splicing generates variants that encode different numbers of fibronectin type III repeats. We have examined the in vivo expression and cell adhesive properties of two full-length recombinant tenascin-C proteins: TN-190, which contains the eight constant fibronectin type III repeats, and TN-ADC, which contains the additional AD2, AD1, and C repeats. In situ hybridization with probes specific for the AD2, AD1, and C repeats shows that these splice variants are expressed at sites of active tissue modeling and fibronectin expression in the developing avian feather bud and sternum. Transcripts incorporating the AD2, AD1, and C repeats are present in embryonic day 10 wing bud but not in embryonic day 10 lung. By using a panel of nine cell lines in attachment assays, we have found that C2C12, G8, and S27 myoblastic cells undergo concentration-dependent adhesion to both variants, organize actin microspikes that contain the actin-bundling protein fascin, and do not assemble focal contacts. On a molar basis, TN-ADC is more active than TN-190 in promoting cell attachment and irregular cell spreading. The addition of either TN-190 or TN-ADC in solution to C2C12, COS-7, or MG-63 cells adherent on fibronectin decreases cell attachment and results in decreased organization of actin microfilament bundles, with formation of cortical membrane ruffles and retention of residual points of substratum contact that contain filamentous actin and fascin. These data establish a biochemical similarity in the processes of cell adhesion to tenascin-C and thrombospondin-1, also an "antiadhesive" matrix component, and also demonstrate that both the adhesive and adhesion-modulating properties of tenascin-C involve similar biochemical events in the cortical cytoskeleton. In addition to these generic properties, TN-ADC is less active in adhesion modulation than TN-190. The coordinated expression of different tenascin-C transcripts during development may, therefore, provide appropriate microenvironments for regulated changes in cell shape, adhesion, and movement.