p53-independent expression of p21Cip1 in muscle and other terminally differentiating cells.

Terminal differentiation is coupled to withdrawal from the cell cycle. The cyclin-dependent kinase inhibitor (CKI) p21Cip1 is transcriptionally regulated by p53 and can induce growth arrest. CKIs are therefore potential mediators of developmental control of cell proliferation. The expression pattern of mouse p21 correlated with terminal differentiation of multiple cell lineages including skeletal muscle, cartilage, skin, and nasal epithelium in a p53-independent manner. Although the muscle-specific transcription factor MyoD is sufficient to activate p21 expression in 10T1/2 cells, p21 was expressed in myogenic cells of mice lacking the genes encoding MyoD and myogenin, demonstrating that p21 expression does not require these transcription factors. The p21 protein may function during development as an inducible growth inhibitor that contributes to cell cycle exit and differentiation.

Nature of micellar calcium phosphate in cows' milk as studied by high-resolution electron microscopy.

The nature of the inorganic calcium phosphate in the casein micelle of cows' milk has been studied by high-resolution electron microscopy. No periodic lattice spacings could be imaged, and diffraction patterns were of the diffuse amorphous type. Short-range order of less than 15 A may be present, but the results indicate that there is no long-range order in micellar calcium phosphate.

Pressure mediates angiotensin II-induced arterial hypertrophy and PDGF-A expression.

Angiotensin II (Ang II) may induce arterial hypertrophy either directly or through an increase in arterial pressure. To separate these 2 mechanisms, rats were implanted with osmopumps delivering either Ang II (100 ng x kg-1 x min-1) or saline. 5-Bromo-2'-deoxyuridine (BrdU) was delivered to both groups by osmopump (2.5 microg x kg-1 x min-1). Half of the rats in each group were given minoxidil (9 mg x kg-1 . d-1) in their drinking water. After 14 days, systolic blood pressure was 117+/-2, 124+/-3, and 115+/-2 mm Hg in the control, Ang II-minoxidil, and minoxidil groups, respectively, and 181+/-6 mm Hg in the Ang II group (P<0.05). After perfusion-fixation, the thoracic aorta, carotid artery, small mesenteric artery, external spermatic artery, and kidneys were harvested, paraffin-embedded, and used for morphological measurements, immunohistochemistry for BrdU, and in situ hybridization with a 35S-labeled riboprobe for platelet-derived growth factor-A chain (PDGF-A) mRNA. The walls of the aorta and carotid arteries hypertrophied in the Ang II group only. There were no significant morphological differences in the small arteries. BrdU was negative in all arteries but positive in the renal tubules. Expression of PDGF-A was elevated 8-fold in the thoracic aorta of the Ang II group (P<0.05). These results show that (1) arterial hypertrophy from Ang II infusion occurs in response to elevated arterial pressure, (2) hypertrophy was not associated with hyperplasia or polyploidy of vascular smooth muscle cells, and (3) PDGF-A expression correlated with elevated pressure and arterial wall hypertrophy.

Failure of target heart rate to accurately monitor intensity during aerobic dance.

Fourteen untrained females (age 19 +/- 1, range 18-21) were studied to examine the heart rate-VO2 relationship during a single aerobic dance training session. These findings were used to help explain the changes in VO2max resulting from an aerobic dance training program. VO2max and body composition were determined before and after an 8 wk training period. In addition, the heart rate-VO2 responses to an aerobic dance training session were monitored and compared to the heart rate responses of treadmill jogging performed at the same VO2. The aerobic dance session elicited a significantly lower oxygen pulse than did treadmill exercise (7.2 +/- 0.3 vs 8.1 +/- 0.8 ml.beat-1; P less than 0.01). There were no significant changes in percent body fat, whereas VO2max increased by 11% (34.4 +/- 0.9 vs 38.1 +/- 0.8 ml.kg-1.min-1; P less than 0.05). No significant changes in any of the parameters tested were observed in 10 untrained controls. These findings indicate that the heart rate elicited from aerobic dance represents a lower relative exercise intensity (VO2) than that of running. Therefore, the assumption that aerobic dance training produces the same cardiovascular adaptations as running training when performed at the same target rate may be unwarranted.

Gap junction proteins exhibit early and specific expression during intramembranous bone formation in the developing chick mandible.

The spatial and temporal expression of three closely related members of the connexin family of gap junction proteins (connexin42, Cx42; connexin43, Cx43; and connexin45, Cx45) was evaluated during bone formation in the mandibular process of the chick embryo. Mandibles of chick embryos from Hamburger and Hamilton stage 25 (approximately 5 days) through 19 days of development were dissected, serially sectioned and processed for immunocytochemical localization, employing site-specific anti-connexin antibodies. Our data revealed that (1) Cx43 was present throughout mandibular bone formation; (2) although it appeared to be associated with all bone cell types, Cx43 was concentrated in mesenchymal cells during the earliest stages in the osteogenic lineage; (3) most importantly, the localization of Cx43 at sites of bone formation appeared to precede the overt expression of the osteogenic phenotype; (4) by contrast, Cx45 was more restricted, spatially and temporally, in its distribution; (5) Cx42 expression was not detected in osteogenic tissue during mandibular bone formation. From all of the data obtained, Cx45 appeared to be associated with stages of bone formation characterized by the elaboration of matrix and the progressive expression of the differentiated osteogenic phenotype. Cx43 appeared to be associated with condensation of mesenchyme and the earliest stages of osteogenesis. Because of these associations, we propose that connexin expression may be necessary for the initiation of bone formation and the full expression of the osteogenic phenotype.

Benton Controlled Oral Word Association Test: reliability and updated norms.

The aim of this paper is to update the over 20-year-old normative data for the Benton Controlled Word Association (COWA) Test. In a sample of 360 normal volunteers, the age ranged between 16-70 years, and the educational level ranged from 7-22 years. Care was taken to ensure that the population was heterogeneous, yet the two stratifications of gender, four age, and three educational groups led to 24 cells with 15 individuals in each. Test-retest reliability was established by testing 30% of the sample after a 6-month delay, which represents a typical follow up duration between testings in a clinical setting. The two forms of the COWA revealed significant test-retest reliability. Generally, our updated values fall above the original normative values, which were derived from a less well-educated and rural sample. No major gender or age trends were noted, but the COWA test performances were influenced by education, i.e., as the level of education increased, the performance on the COWA increased. The only gender differences that were found were for the women in the highest educational group ( > 16 years), who performed significantly better that men in the highest educational group. An error analysis of repetitions or perseverations is provided, with cut-off scores according to age levels. Finally, the updated COWA norms are compared to the original norms as well as to other measures of word fluency.

The psychological construct of word fluency.

Measures of word fluency have been convincingly linked in the literature to damage in the left prefrontal lobe region. Yet, a reduction in word fluency has also been reported with diffuse, multifocal and nonfrontal lobe damage. Despite the undisputed neuropsychological application of multiple word fluency measures, the psychological construct underlying this measure is not well understood. In a sample of 360 normal adults stratified by age, gender, and level of education, we found that auditory attention and word knowledge were among the most important determinants. With respect to memory, short-term memory was not significantly correlated, but long-term memory was. Despite these three determinants, a large share of the variance of the multiple regression was still not accounted for, which underscores the partial independence of word fluency per se. Thus, we propose a distinction between (1) poor word fluency secondary to deficient verbal attention, word knowledge, and/or verbal long-term memory and (2) impaired word fluency without these three areas concurrently affected. Based on a review of the literature, it seems likely that in the latter condition, the profile is more associated with prefrontal lobe impairment, versus in the former condition, diffuse multifocal or nonfrontal lobe factors can play a role.

An aviator with idiopathic dilated cardiomyopathy.

The clinical presentation, evaluation and diagnosis of an aviator with dilated cardiomyopathy of determined etiology are discussed. The aeromedical disposition and prognosis of this patient are also presented.

Gender- and age-specific changes in motor speed and eye-hand coordination in adults: normative values for the Finger Tapping and Grooved Pegboard Tests.

Normative values for the Finger Tapping and Grooved Pegboard Tests were developed on a sample of 360 normal volunteers stratified according to gender, three educational groups ranging from 7 to 22 years, and four age groups subdivided between the ages of 16 to 70 years. Retest reliability was estimated for both measures. The Finger Tapping Test showed significant gender differences, since women were substantially slower, particularly in the older age groups. On the Grooved Pegboard Test, a converse gender difference was noted, since women were substantially faster than men. A smaller effect with increasing age resulted, and better educated individuals performed faster. If these motor and visuomotor tests are to be applied, then stratified normative estimates need to be implemented to provide viable clinical judgements.

Modulation of gap junction-mediated intercellular communication in embryonic chick mesenchyme during tissue remodeling in vitro.

Gap junction-mediated intercellular communication was analyzed in a model system in which tissue necrosis and remodeling could be modulated. This in vitro system, previously used for analysis of epithelial-mesenchymal tissue interaction, was modified to permit analysis of the presence and extent of intercellular communication by monitoring intercellular transfer of the microinjected fluorescent dye, Lucifer Yellow. Light and transmission electronmicroscopy were employed to correlate the presence and degree of gap junctional communication (coupling) with tissue morphology. Digital image analysis was used to determine cell density and mitotic indices within the outgrowths of explants. Our results indicated that cell communication in outgrowths adjacent to necrotic foci within an explant was minimal or absent. Cell-coupling in outgrowths adjacent to a compartment of viable mesenchyme was significantly higher - equivalent to unseparated control cultures. A time-course study demonstrated correlation of increased levels of cell-coupling in outgrowths with the level of tissue remodeling within an explant. Our conclusions from these studies are that embryonic mesenchymal cell populations may be selectively uncoupled as a result of alterations in the microenvironment produced by a proximate impaired cell population. It is proposed that endogenous factors in the microenvironment ("wound signals"), emanating from impaired cell populations, regulate gap junction-mediated intercellular communication in adjacent viable tissue. Normal, unimpaired populations of cells surrounding an area of injury are thereby isolated from the effects of a potentially toxic environment. This could serve as a protective function in development and may represent, in a more general sense, part of the repertoire of events associated with tissue repair and remodeling.

Influence of epithelial-mesenchymal interaction on the viability of facial mesenchyme. II: Synthesis of basement-membrane components during tissue recombination.

The presence of basement-membrane components during tissue separation procedures was determined employing monoclonal antibodies to laminin and type IV collagen. In addition, the reconstitution of basement-membrane components and the formation of the basement-membrane were examined in isolated epithelium and mesenchyme and in tissue recombination. Epithelium and mesenchyme of maxillary processes of chick embryos were separated by a variety of protocols, including those employed in a prior study (Saber et al: Anat. Rec. 225:56-66, 1989). Results indicated that the protocol previously employed did not remove basement-membrane components after enzymatic tissue separation. A revised protocol in which the basement-membrane components (i.e., laminin and type IV collagen) were removed from isolated tissues prior to recombination revealed that a developmental compartment and a gradient of cell viability, comparable in size and dimensions to that observed in the study of Saber et al. (ibid.) was present in the mesenchyme of recombined explants. Type IV collagen and laminin, therefore, do not appear to be required initially during tissue recombination in order for subsequent growth-sustaining effects to be expressed. Additional studies revealed, however, that synthesis of basement-membrane components occurred not only in isolated tissues but was altered markedly by tissue recombination. Culture of isolated tissues demonstrated induction of laminin synthesis in separated epithelium by 24 hours and induction of collagen synthesis in isolated mesenchyme by 24 hours. Recombination of epithelium and mesenchyme, however, resulted in rapid induction of laminin synthesis within 1 hour. Recombination of epithelium and mesenchyme after 24 hours resulted in the presence of laminin not only in epithelium but in mesenchyme as well. Both tissues were required for basement-membrane formation which appeared to be fully reconstituted by 24 hours in culture. These observations indicate that recombination in culture alters the pattern of synthetic activity of these basement-membrane components. These can be characterized as "early" (temporal) and "late" spatial) responses by the recombined tissues.

Analysis of distribution patterns of gap junctions during development of embryonic chick facial primordia and brain.

Gap junction distribution in the facial primordia of chick embryos at the time of primary palate formation was studied employing indirect immunofluorescence localization with antibodies to gap junction proteins initially identified in rat liver (27 x 10(3) Mr, connexin 32) and heart (43 x 10(3) Mr, connexin 43). Immunolocalization with antibodies to the rat liver gap junction protein (27 x 10(3) Mr) demonstrated a ubiquitous and uniform distribution in all regions of the epithelium and mesenchyme except the nasal placode. In the placodal epithelium, a unique non-random distribution was found characterized by two zones: a very heavy concentration of signal in the superficial layer of cells adjacent to the exterior surface and a region devoid of detectable signal in the interior cell layer adjacent to the mesenchyme. This pattern was seen during all stages of placode invagination that were examined. The separation of gap junctions in distinct cell layers was unique to the nasal placode, and was not found in any other region of the developing primary palate. One other tissue was found that exhibited this pattern-the developing neural epithelium of the brain and retina. These observations suggest the presence of region-specific signaling mechanisms and, possibly, an impedance of cell communication among subpopulations of cells in these structures at critical stages of development. Immunolocalization with antibodies to the 'heart' 43 x 10(3) Mr gap junction protein also revealed the presence of gap junction protein in facial primordia and neural epithelium. A non-uniform distribution of immunoreactivity was also observed for connexin 43.

Distribution of type IV collagen, laminin, and fibronectin during maxillary process formation in the chick embryo.

The presence and distribution of laminin, type IV collagen, and fibronectin were analyzed in the facial primordia and developing primary palates of chick embryos from stages of development corresponding to maxillary process formation and primary palate closure. Frozen sections through the maxillary process and roof of the stomodeum were prepared for indirect immunofluorescence employing a biotin-avidin system using monoclonal antibodies against laminin, type IV collagen, and fibronectin. Light microscopic examination of sections stained with antibodies against type IV collagen revealed a much stronger fluorescent signal in the roof of the stomodeum than in the maxillary process at all stages examined. Regional differences in signal intensity and staining patterns were noted within the maxillary process; for example, the lateral surface of the maxillary process displayed a much less intense signal at most stages examined than the inferior and medial surfaces. The signal from sections of the maxillary process stained with laminin was much stronger than the signal from the same tissues stained with collagen. Regional differences in signal intensity within the maxillary process were minimal in sections stained with antibodies to laminin, in contrast to the differences seen in sections stained with antibodies to type IV collagen. Differences in signal intensity between the maxillary process and the roof of the stomodeum with laminin were slight. Sections stained with antibody to fibronectin displayed intense staining throughout the mesenchyme in both the maxillary process and the roof of the stomodeum. From comparison of the data of type IV collagen and laminin, the following hypothesis is proposed. In structures which undergo rapid change in form, such as the facial primordia, collagen distribution and/or organization is altered to a much greater extent than laminin, which is more uniformly distributed and which may be required for structural support of other developmentally regulated macromolecules. Where tissue morphology must be maintained, such as the roof of the stomodeum, the concentration and organization of type IV collagen is maintained in a manner that confers stability to these regions.

Influence of epithelial-mesenchymal interaction on the viability of facial mesenchyme in vitro.

Separation and recombination experiments, employing a variety of tissue configurations in organ culture, were performed to determine the extent to which the epithelium of the maxillary process influences the viability of the underlying mesenchyme during organogenesis. The results of these studies indicated that the viability of mesenchyme of the maxillary process of early stage embryos was severely impaired when separated from the overlying epithelium. The influence of epithelium on the viability of mesenchyme was stage dependent; that is, the requirement for the presence of epithelium for the maintenance of the viability of mesenchyme became progressively less pronounced at older developmental stages. The response of mesenchyme to the presence of recombined epithelium resulted in the appearance of a delimited zone of influence extending, within specific boundaries, from the epithelial-mesenchymal interface. Preliminary data from homotypic (maxillary epithelium-maxillary mesenchyme), heterotypic (limb apical ectodermal ridge-maxillary mesenchyme) and heterochronic (stage 28 epithelium-stage 22 mesenchyme) recombination experiments indicated that viability of mesenchyme could be achieved only through direct epithelial-mesenchymal contact which allowed restoration of normal morphological relationships at the interface of the two tissues.

Connexin expression in the developing avian cardiovascular system.

Recent observations have suggested that the patterns of expression of the gap junction protein connexin43 in the developing cardiovascular system of the avian embryo diverge significantly from the patterns previously seen in mammalian species. Therefore, a detailed analysis of connexin43 expression in the chicken embryo was performed by use of immunofluorescent localization with two different connexin43-specific antipeptide antibodies as well as Western and Northern blot analysis. Connexin43 protein was not detected in the avian myocardium, the venous system, or the smaller vessels of the arterial system. Rather, it was limited exclusively to the vessels of the arterial outflow tract in a concentric pattern that became evident by embryonic day 8. Double staining with anti-alpha-smooth muscle actin and connexin43 demonstrated colocalization in the media of outflow tract vessel walls. The developmental expression of connexin43 was found to mirror the spatial patterning of secondary actin; connexin43, however, preceded the expression of secondary actin by a period of 1-2 days. In contrast, antibodies to a related gap junction protein (connexin42) revealed an absence of immunostaining in the avian outflow tract. Double staining with anti-connexin42 and anti-A-cell adhesion molecule (specific for avian intercalated discs) demonstrated colocalization between cardiac myocytes, indicating that connexin42 is a constituent of avian myocardial gap junctions. In light of these findings, developmental expression of differing myocardial connexins may reconcile previous studies showing different physiological properties of avian and mammalian cardiac gap junctions.

Expression patterns of connexin43 protein during facial development in the chick embryo: associates with outgrowth, attachment, and closure of the midfacial primordia.

BACKGROUND: In a prior report, evidence was presented for the presence of gap junction proteins [connexin32 and connexin43 (Cx43)] in embryonic facial primordia. The purpose of the present study was, first, to examine in detail the patterns of distribution of Cx43 protein in embryonic chick facial primordia and, second, to consider the possible roles played by this protein during midfacial development. METHODS: Chick embryo heads were serially sectioned and processed for immunofluorescent localization of Cx43. The developmental stages examined encompassed the period of formation, enlargement, and union of the facial primordia. Western blot analysis of the facial primordia was also performed. RESULTS: Analysis of serial sections revealed the presence of signal in both epithelium and mesenchyme at sites of attachment in each of the midfacial primordia (i.e., the medial nasal, lateral nasal, and maxillary processes). Furthermore, although signal was concentrated in mesenchyme in the distal tips of the primordia at sites of attachment, immunoreactivity was absent, sparse, or less intense outside the areas of attachment. In some cases (i.e., the maxillary process), immunoreactive signal in mesenchyme did not appear in the distal tip until the primordia approximated each other or contact of the primordia was initiated. Most significantly, signal was also found between the facial primordia in nonprimordial epithelium and mesenchyme at sites where the primordia were joined. CONCLUSIONS: These data suggest that the expression of Cx43 protein is spatially and temporally regulated in the facial primordia and that the patterns of expression that were observed are significant to the cascade of events that ultimately lead to the attachment and union of the primordia that form the midface.

AT(1) receptor inhibition does not reduce arterial wall hypertrophy or PDGF-A expression in renal hypertension.

To separate the role of ANG II from pressure in hypertrophy of the vascular wall in one-kidney, one-clip (1K1C) hypertension, experimental and sham-operated rats were given the AT(1)-receptor antagonist losartan (20 mg x kg(-1) x day(-1)) or tap water for 14 days. Mean arterial pressure was elevated in both experimental groups compared with controls. Rats were anesthetized with pentobarbital sodium, and the thoracic aorta and carotid, small mesenteric, and external spermatic arteries were harvested and embedded in paraffin. Tissue sections were used for morphological analysis, immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) and platelet-derived growth factor (PDGF)-AA, stereological measurements, and in situ hybridization with a (35)S-labeled riboprobe for PDGF-A mRNA. Elevated cross-sectional areas of thoracic, carotid, and small mesenteric artery in 1K1C rats were not reduced by losartan. The internal diameter of the external spermatic artery and microvascular density of the cremaster muscle were reduced in 1K1C rats. The number of BrdU-positive nuclei per cross section did not differ between 1K1C and control arteries. PDGF-A mRNA was elevated in the arterial walls of 1K1C rats compared with controls and was hardly changed by losartan. PDGF-A protein stained strongly in the media of 1K1C arteries and was not inhibited by losartan; it appeared in the adventitia of all aortas and carotid arteries. These observations demonstrate that effects of ANG II mediated through the AT(1) receptor are not necessary for hypertrophy of the vascular wall during 1K1C hypertension or expression of PDGF-A.

The ultrastructure of the calcium carbonate balance organs of the inner ear: an ultra-high resolution electron microscopy study.

The balance organs of the inner ear of vertebrates, found as single, large growths of aragonite ('otoliths') in fish and small clumped masses ('otoconia') of either aragonite (amphibians) or calcite (mammals), have long been regarded as polycrystalline and single crystals respectively. The use of ultra-high resolution electron microscopy and electron diffraction to study comparatively crushed samples of these biominerals and samples of geological calcium carbonates, as examples of pure inorganic crystals, reveals that the biological structures are composed of microcrystals joined together by organic matrices to form composite crystals. Such structures either grow to a finite, controlled size (otoconia) or have daily growth patterns (otoliths). Mechanisms of growth are proposed to link these seemingly different patterns varying only in the number of nucleation sites and the degree of biological as against chemical control over the growth.

Mammalian p50Cdc37 is a protein kinase-targeting subunit of Hsp90 that binds and stabilizes Cdk4.

CDC37, an essential gene in Saccharomyces cerevisiae, interacts genetically with multiple protein kinases and is required for production of Cdc28p/cyclin complexes through an unknown mechanism. We have identified mammalian p50Cdc37 as a protein kinase-targeting subunit of the molecular chaperone Hsp90. Previously, p50 was observed in complexes with pp60v-src and Raf-1, but its identity and function have remained elusive. In mouse fibroblasts, a primary target of Cdc37 is Cdk4. This kinase is activated by D-type cyclins and functions in passage through G1. In insect cells, Cdc37 is sufficient to target Hsp90 to Cdk4 and both in vitro and in vivo, Cdc37/Hsp90 associates preferentially with the fraction of Cdk4 not bound to D-type cyclins. Cdc37 is coexpressed with cyclin Dl in cells undergoing programmed proliferation in vivo, consistent with a positive role in cell cycle progression. Pharmacological inactivation of Cdc37/Hsp90 function decreases the half-life of newly synthesized Cdk4, indicating a role for Cdc37/Hsp90 in Cdk4 stabilization. This study suggests a general role for p50Cdc37 in signaling pathways dependent on intrinsically unstable protein kinases and reveals a previously unrecognized chaperone-dependent step in the production of Cdk4/cyclin D complexes.

Association of cell and substrate adhesion molecules with connexin43 during intramembranous bone formation.

Prior studies in our laboratory have demonstrated an association of specific gap junction proteins with intramembranous bone formation in the avian mandible. The purpose of the present study was to extend these observations by determining if there was a relationship between the expression of one of the gap junction proteins examined previously (connexin43) and the expression of specific cell adhesion (CAM) and/or substrate adhesion (SAM) molecules [i.e. NCAM, A-CAM (N-cadherin) and tenascin (tenascin-C)] that have previously been shown to be associated with bone formation. Immunohistochemical localization of connexin43, tenascin, NCAM and N-cadherin was performed on serial sections of mandibles of chick embryos from 6 to 12 days of incubation. Analysis of adjacent serial sections revealed that the NCAM and tenascin immunostaining that appeared initially on the lateral aspect of Meckel's cartilage preceded the overt expression of trabecular bone. At subsequent stages, NCAM and tenascin staining gradually overlapped the region of connexin43 expression. In contrast, the expression of N-cadherin was found to colocalize with that of connexin43 from the first appearance of connexin43 expression. Most significantly, although the domains of NCAM and tenascin expression were initially separate from that of connexin43, bone formation originated only in the region where these domains intersected. These findings suggest that, of the CAMs and SAMs examined, N-cadherin appears to be associated with the establishment of cell contacts responsible for the presence and/or maintenance of connexin43-mediated gap junctional communication, while tenascin and NCAM appear to be associated, in a more specific manner, with processes that accompany the overt expression of the osteogenic phenotype.