Mechanical stretch increases L-type calcium channel stability in cardiomyocytes through a polycystin-1/AKT-dependent mechanism.

The L-type calcium channel (LTCC) is an important determinant of cardiac contractility. Therefore, changes in LTCC activity or protein levels could be expected to affect cardiac function. Several studies describing LTCC regulation are available, but only a few examine LTCC protein stability. Polycystin-1 (PC1) is a mechanosensor that regulates heart contractility and is involved in mechanical stretch-induced cardiac hypertrophy. PC1 was originally described as an unconventional Gi/o protein-coupled receptor in renal cells. We recently reported that PC1 regulates LTCC stability in cardiomyocytes under stress; however, the mechanism underlying this effect remains unknown. Here, we use cultured neonatal rat ventricular myocytes and hypo-osmotic stress (HS) to model mechanical stretch. The model shows that the Cavß2 subunit is necessary for LTCC stabilization in cardiomyocytes during mechanical stretch, acting through an AKT-dependent mechanism. Our data also shows that AKT activation depends on the G protein-coupled receptor activity of PC1, specifically its G protein-binding domain, and the associated Gßγ subunit of a heterotrimeric Gi/o protein. In fact, over-expression of the human PC1 C-terminal mutant lacking the G protein-binding domain blunted the AKT activation-induced increase in Cav1.2 protein in cardiomyocytes. These findings provide novel evidence that PC1 is involved in the regulation of cardiac LTCCs through a Gißγ-AKT-Cavß2 pathway, suggesting a new mechanism for regulation of cardiac function.

Presence of Langerhans cells in the central corneas of normal human infants.

Nine normal adult and seven normal infant human corneas were studied for the presence of dendritic epithelial Langerhans cells in a masked fashion. Epithelial flatmounts were separated from the underlying corneal stroma using EDTA. The epithelial Langerhans cell densities were determined in the limbus as well as the peripheral, pericentral, and central corneal regions following staining with ATPase. Segments of the flatmounts were also studied by immunofluorescence to confirm that the dendritic cells contained class II histocompatibility antigens. The limbus, peripheral, and pericentral zones of adult and infant flatmounts contained similar densities of Langerhans cells. However, the central corneal Langerhans cell densities in infants were significantly elevated as compared with those in adults. These results suggest that Langerhans cells are a constant constituent of the human central corneal epithelium during late gestation and early infancy. They further suggest that perturbations of the corneal epithelium are not required for the presence of Langerhans cells in the corneal epithelium.

HLA-DR+/T6- Langerhans cells of the human cornea.

We have investigated normal human corneas for the presence of T6-marker on Langerhans cells. With the exception of one pair of newborn corneas and two pairs of very young infant corneas, all HLA-DR-positive cells in central and peripheral corneal epithelium were T6-negative by double-labeled immunofluorescence. In contrast, epidermal sheets from normal human eyelid skin displayed positive staining for T6 on most of the HLA-DR-positive Langerhans cells. Since T6 antigen is considered to be a specific Langerhans cell differentiation marker, we interpret this finding to indicate a nonactivated or undifferentiated state of Langerhans cells in normal human corneas.

Phosphatic metabolites of the intact cornea by phosphorus-31 nuclear magnetic resonance.

The principal low molecular weight phosphatic metabolites of the intact cornea were identified and quantitated nondestructively by phosphorus-31 nuclear magnetic resonance (P-31 NMR) spectroscopy. As part of this analytical procedure, the intracorneal pH was approximated from the resonance shift position of inorganic orthophosphate. In addition the metabolic and pH stability of incubated corneas at 37 C in MK medium was evaluated during an 8-hr time course and compared to similar dynamic analyses performed on corneas with denuded endothelium. Perchloric acid extracts prepared from these same corneas were analyzed by P-31 NMR to verify the metabolite peak assignments and to quantitate the concentrations of minor corneal metabolites. The concentrations of phosphatic metabolites of the cornea, including three previously unidentified phosphorus-containing substances, were determined for freshly excised corneas. The initial corneal spectroscopic profile was not altered by removal of the endothelium. At 37 C the MK media-incubated intact whole corneas experienced a time-dependent decline in ATP levels with a concomitant rise in inorganic orthophosphate; however, the tissue levels of the other principal phosphatic metabolites were not altered by prolonged incubation. In contrast, removal of the endothelial layer of the cornea-induced progressive metabolic deterioration of intact corneas characterized, most prominantly, by time-dependent declines in ATP and glycerol 3-phosphorylcholine levels and concomitant increases in ADP and inorganic orthophosphate levels relative to intact whole corneas. This study has established the feasibility of monitoring the metabolic status of intact rabbit corneas nondestructively and noninvasively. As such, P-31 NMR spectroscopy offers a promising method that may enable analysis of the metabolic viability of intact human donor corneas to provide a basis for selecting donor corneas for transplantation.

Immunohistochemical localization of human tear lysozyme.

Possible local sources of human tear lysozyme were investigated using an indirect immunofluorescence technique. Lysozyme was identified in 20% to 50% of acinar and ductular epithelial cells of both main and accessory lacrimal glands. The staining was granular in character and confined to the apices of the cells. Cells that stained positive tended to be grouped. Interstitial tissues of main and accessory lacrimal tissues did not stain. Conjunctiva and all other ocular tissues examined were unstained by antilysozyme antisera. Our findings are compatible with lysozyme either being produced in lacrimal tissue or being concentrated from plasma. The absence of any other lysozyme-specific fluorescence in the interstitial elements of the lacrimal tissues supports the notion of local synthesis by acinar lacrimal tissue.

Secretory component in human ocular tissues.

Lacrimal tissue and accessory lacrimal tissue from seven autopsy cases, lacrimal tissue from three patients undergoing orbital exploration, and biopsy specimens from conjunctiva of 14 subjects were examined for the presence and distribution of secretory component by three anti-secretory-component antisera. Secretory component was present in all lacrimal and accessory lacrimal tissues but in no other ocular tissues. Over 60% of acinar cells stained for secretory component; about 5% of acinar cells stained brightly; about 30% of tubular cells stained brightly. Main and accessory lacrimal tissues appeared identical in their staining patterns. We concluded that the main sites of synthesis of secretory IgA in human ocular tissues are the lacrimal and accessory lacrimal tissues.

Lactoferrin in human ocular tissues.

The main lacrimal gland and accessory lacrimal tissue from seven autopsy cases, lacrimal biopsy specimens from three patients, and conjunctival biopsy specimens from ten patients were examined for lactoferrin by an immunohistologic technique. Lactoferrin was identified and localized to acinar epithelial cells of both main and accessory lacrimal tissue. Lactoferrin was not found in conjunctival tissue except within conjunctival neutrophils. Other possible sources of human tear lactoferrin were considered, but we concluded from our data that the primary source of lactoferrin in normal human tears is the acinar epithelium of the main and accessory lacrimal glands.

Histologic and immunohistologic comparison of main and accessory lacrimal tissue.

Main and accessory lacrimal tissues from autopsy and biopsy specimens were compared histologically and immunohistologically. Formaldehyde-fixed, paraffin-embedded specimens were studied by light microscopy with hematoxylinand-eosin and PAS staining. Glutaraldehyde-fixed, Epon-embedded specimens were sectioned at 1 micron, stained with alkaline Giemsa, and studied by light microscopy. Specimens fixed in a solution of alcohol and acetic acid were stained by immunofluorescence techniques for lactoferrin, lysozyme, secretory component, and the immunoglobulins IgG, IgA, IgM, IgD, and IgE. The main and the accessory lacrimal tissues were identical histologically and had identical distributions of secretory products and immunoglobulin-containing plasma cells. The finding of myoepithelial cells in 1-micron sections of accessory lacrimal tissue indicates autonomic innervation in that tissue. This finding, in conjunction with the identical immunohistology, indicates a common source for unstimulated and stimulated tears.

Recurrent keratoconus after keratoplasty.

A 35-year-old woman who had undergone bilateral penetrating corneal grafts for keratoconus had keratoconus in the eye that had received a graft 16 years earlier. A second penetrating keratoplasty was performed, and the excised button was examined with light and electron microscopy. Both the light and electron microscopic findings were consistent with the clinical diagnosis of keratoconus. Keratoconus in a graft for the same disease should be added to the list of late complications of successful keratoplasties.

Relationship of Body Core Temperature and Warm-up to Knee Range of Motion.

This study examined the effects of submaximal, treadmill exercise-induced body core temperature (BCT) increase on selected knee range of motion (ROM). Twenty males, 18-35 years old, were tested (randomized crossover) for ROM, BCT, and heart rate (HR), followed by either Treatment I (20 minutes of rest) or Treatment II (20 minutes of submaximal running). The two treatments were subsequently followed by a two-minute passive stretch. Range of motion was assessed before and after passive stretch treatment intervention. Treatment means differed for BCT and HR (p < 0.001) but not for ROM after exercise intervention. It was concluded that 20 minutes of exercise increased BCT (>1 degrees C) but had no effect on knee ROM. J Orthop Sports Phys Ther 1991;13(3):126-131.

Phlyctenular keratoconjunctivitis.

There is growing evidence that a variety of corneal disorders may be expressions of altered immune mechanisms. Phlyctenular keratoconjunctivitis is probably such a condition. Typically described as arising from hypersensitivity to tuberculin protein, other antigens clearly may participate, particularly staphylococcus products. When corneal involvement occurs, it need not be confined to the peripheral cornea. The symptoms of the process may be disproportionate to obvious findings and so exaggerated as to suggest a psychiatric disorder. Resultant visual deficits, if the disease is corneal, progressive, unrecognized, and untreated may be profound. Representative examples of this disease are cited. Immune mechanisms are reviewed. The importance of recognizing the characteristic sign and symptom complex is stressed. Appropriate diagnostic studies and treatment regimens are presented.

Cell death and survival through the endoplasmic reticulum-mitochondrial axis.

The endoplasmic reticulum has a central role in biosynthesis of a variety of proteins and lipids. Mitochondria generate ATP, synthesize and process numerous metabolites, and are key regulators of cell death. The architectures of endoplasmic reticulum and mitochondria change continually via the process of membrane fusion, fission, elongation, degradation, and renewal. These structural changes correlate with important changes in organellar function. Both organelles are capable of moving along the cytoskeleton, thus changing their cellular distribution. Numerous studies have demonstrated coordination and communication between mitochondria and endoplasmic reticulum. A focal point for these interactions is a zone of close contact between them known as the mitochondrial-associated endoplasmic reticulum membrane (MAM), which serves as a signaling juncture that facilitates calcium and lipid transfer between organelles. Here we review the emerging data on how communication between endoplasmic reticulum and mitochondria can modulate organelle function and determine cellular fate.

Immunologic defense mechanisms of the ocular surface.

The ocular surface represents an immunologic microcosm in which certain local immunologic and paraimmunologic defense systems analogous or identical to those of other mucosal surfaces are operative. These defenses include a resident normal flora; intrinsic anatomic barriers; secretion of mucous and certain chemical bacteriostatics and bacteriolytics; local humoral (slgA) antibody secretion; and local T-lymphocyte cellular responses. A series of sophisticated mechanisms has evolved to defend the ocular surface against diverse environmental pathogens. Often, the activation of one system leads to the subsequent activation of another, providing a highly integrated, series of mechanisms for host defense. And, it is clear that close integration of these various mechanisms provides a highly efficient amplification system for host defense, so that when one mechanism fails to deal with the invading pathogen the next mechanism is then efficiently initiated. Only in those situations where the pathogen overwhelms these defense systems, or is of such a persistent nature that it becomes resistant to removal by these various mechanisms, or alternatively is able to subvert its identity and, therefore, evade these mechanisms do organisms infect and cause disease. It is undoubtedly the complex interaction of all of these defense systems, both paraimmunologic and immunologic, which finally determines the integrity of the ocular surface.

Distinct roles of two binding sites for the bovine papillomavirus (BPV) E2 transactivator on BPV DNA replication.

The modulation of DNA replication by transcription factors was examined by using bovine papillomavirus type 1 (BPV). BPV replication in vivo requires two viral proteins: E1, an origin-binding protein, and E2, a transcriptional transactivator. In the origin, E1 interacts with a central region flanked by two binding sites for E2 (BS11 and BS12), of which only BS12 has been reported to be essential for replication in vivo. Using chemical interference and electrophoretic mobility shift assays, we found that the binding of E2 to each site stimulates the formation of distinct E1-origin complexes. A high-mobility C1 complex is formed by using critical E2 contacts to BS12 and E1 contacts to the dyad symmetry element. In contrast, interaction of E2 with the BS11 element on the other origin flank promotes the formation of the lower-mobility C3 complex. C3 is a novel species that resembles C2, a previously identified complex that is replication active and formed by E1 alone. The binding of E1 greatly differs in the C1 and C3 complexes, with E1 in the C1 complex limited to the origin dyad symmetry region and E1 in the C3 complex encompassing the region from the proximal edge of BS11 through the distal edge of BS12. We found that the presence of both E2-binding sites is necessary for wild-type replication activity in vivo, as well as for maximal production of the C3 complex. These results show that in the normal viral context, BS11 and BS12 play separate but synergetic roles in the initiation of viral DNA replication that are dependent on their location within the origin. Our data suggest a model in which the binding of E2 to each site sequentially stimulates the formation of distinct E1-origin complexes, leading to the replication-competent complex.

Induction of structural changes in the bovine papillomavirus type 1 origin of replication by the viral E1 and E2 proteins.

Chemical and enzymatic probing techniques were used to examine the interaction of the bovine papillomavirus type 1 E1 and E2 proteins with the viral origin of replication (ori). E1 was found to generate significant distortions to the structure of ori, as assayed by KMnO4 oxidation of DNA. The primary site of ori distortion was located within and adjacent to the AT-element of the core replicator sequence, although a number of minor structural transitions were also detected. The induction of these structural changes required ATP and appeared to require ATP hydrolysis. E2 was found to decrease the amount of E1 required for ori distortion but did not significantly alter the pattern of structural distortion. In contrast, the presence of E2 resulted in a biphasic mechanism for E1 binding to ori, as assayed by nuclease protection. Under these conditions, E1 bound preferentially to the dyad symmetry region containing the conserved Hpa I site. Higher levels of E1 were required for binding to the adjacent ori AT-rich region. Thus, these data suggest that E2 can order the stepwise binding of E1 to ori.

Langerhans cells of the ocular surface.

Recent evidence has been collected by several investigators defining a distinct population of dendritic cells (Langerhans cells) of mesenchymal origin residing in the epidermal surfaces of many mammalian species. These cells play a dominant role in the processing of antigens presented through cutaneous surfaces and carry a Class II histocompatability antigen felt to be of central importance in the afferent arm of allograft rejection. They also possess many of the characteristics of macrophages active in the efferent arm of immunologic responses. An equivalent subset of dendritic cells (Langerhans cells) in ocular surface epithelium of the human, mouse rat, and guinea pig has been identified by enzyme histochemistry, immunofluorescence, and electron microscopy. Ocular surface Langerhans cells proliferate in the setting of corneal inflammation (remote and recent) and are depleted by topical and systemic corticosteroids. Ocular surface Langerhans cells may play a central role in ocular contact hypersensitivity, corneal allograft rejection, and ocular surface immune surveillance.

Recognition of model DNA replication forks by the SV40 large tumor antigen.

The ability of the SV40 large tumor antigen (T antigen), a DNA helicase, to bind to model DNA replication forks was tested. DNA fork molecules were constructed either from two partially complementary oligonucleotides or from a single oligonucleotide able to form a 'panhandle' structure. T antigen specifically recognized the two-strand fork in a reaction dependent on the presence of ATP, dATP, or non-hydrolyzable analogs of ATP. T antigen asymmetrically bound the two-strand fork, protecting from nuclease cleavage a fork-proximal region on only one of the two strands. The asymmetric binding is consistent with the 3'-->5' directionality of the DNA helicase activity of T antigen. An analogous region on the one-strand fork was also bound by T antigen, suggesting that T antigen does not require a free single-stranded end to load onto the fork. Use of chemically modified DNA substrates indicated that T antigen binding to the fork utilized important contacts with the DNA sugar-phosphate backbone.