Scanning electron microscopy of the human cerebral ventricular system

Summary Scanning electron microscopy was used to assess the ultrastructural differences exhibited by the varigated ependymal lining of the near-term human fetal 4th ventricle. The central portion of the fourth ventricular floor, including the median sulcus is punctuated by numerous clumps of cilia. The density of cilia here is not as great as that described for other regions of the human cerebral ventricular system; accordingly, underlying substructure can be noted. There are distinct differences between ependymas that line the floor of the fourth ventricle with those of the adjacent area postrema. The latter region possesses not cilia, but instead exhibits a dense knap of microvilli. The ultra-architecture of the choroid plexus is relatively similar to that of other circumventricular organs with the exception that it possesses small isolated groups of cilia as well as microvilli. These findings are discussed with respect to the dynamics of local CSF movement and flow, ependymoabsorption and ependymosecretionSupported by U.S.P.H.S. Grant NS 08171.Career Development Awardee GM K04 70001.     

A sex-limited serum protein variant in the mouse: Hormonal control of phenotypic expression

The sex-limited protein (Slp) antigen of the mouse is first detected in the serum of strain DBA/2J males at 5–6 weeks of age and reaches full adult levels by 10 weeks. This antigen is normally absent in females. Immature DBA/2J males castrated at 3 1/2 weeks of age failed to develop Slp antigen, while DBA/2J females treated with testosterone propionate starting at 3 1/2 weeks developed normal adult male levels of Slp antigen. Similar hormone-influenced effects were demonstrated in adult males and females of the same strain. Experiments indicated that testosterone does not act directly in the serum to expose Slp antigenic sites. Testosterone treatment of both males and females of strain C57BL/10JSf, which does not carry the gene for the presence of the Slp antigen, failed to stimulate the appearance of the antigen. Thus, the presence of Slp antigen in the serum is dependent on both the proper genotype and the presence of male hormone.Supported by U.S.P.H.S. Research Grant GM-15419, U.S.P.H.S. Training Grant 2T01-GM-00071 (H.C.P.), and U.S.P.H.S. Career Development Award K3-HE-24,980 (D.C.S.).     

Sexual differences in the ependyma lining the third ventricle in the area of the anterior hypothalamus of adult rhesus monkeys

Summary Previous studies have shown that a circumscribed region of the anterior hypothalamus of the rhesus monkey is lined by tanycyte ependyma and it has been suggested that this ependyma which links the third ventricle with the pars tuberalis may have a functional role in the hypothalamic regulation of anterior pituitary function (Anand Kumar and Knowles, 1967a). In view of the known sexual differences in the hypothalamic regulation of pituitary gonadotropin secretion the present investigation was made to determine whether any structural differences were evident in the tanycyte ependyma in male and female rhesus monkeys.The results of this investigation are based on light and electron microscopic studies of the hypothalamus in 24 rhesus monkeys comprising 12 adult females, 11 sexually mature males and a two month old sexually immature male.The tanycyte ependyma in the rhesus monkey is double layered. There are bulbous projections on the ventricular surface of the cells in the ependymal layer nearest to the ventricle (the first layer of ependyma). These bulbous projections vary in size in relation to the menstrual cycle. They are well developed during mid-cycle and regressed during menstruation. In the males, where the secretion of pituitary gonadotropins does not occur cyclically as in the females, there was no marked variation in the bulbous projections between different individuals as in the female monkeys.In the sexually mature males, but not in the females, the two layers of ependyma are separated by a distinct space. The absence of such a space in the sexually immature male suggests that this difference may be related to sexual maturity.In the adult males the cells in the ependymal layer below the first layer of ependyma have microvilli which extend into the space between the ependymal layers. In the females where such a space is not present, microvilli were not evident.The precise functional significance of the tanycyte ependyma is not known. It is hoped that the results of the present investigation would draw attention to the need for a more detailed examination of the physiological role of the tanycyte ependyma in relation to reproduction.The expenses for this investigation were met from a grant made by the Ford Foundation to Professor Sir Solly Zuckerman and the electron microscope was provided by the Medical Research Council. I am indebted to Sir Solly for his interest in this work.     

Differential immunocytochemical staining for glial fibrillary acidic (GFA) protein,S-100 protein and glutamine synthetase in the rat subcommissural organ,nonspecialized ventricular ependyma and adjacent neuropil

Summary Antibodies raised against glial fibrillary acidic protein (GFA), S-100 protein (S100) and glutamine synthetase (GS) are currently used as glial markers. The distribution of GFA, S100 and GS in the ependyma of the rat subcommissural organ (SCO), as well as in the adjacent nonspecialized ventricular ependyma and neuropil of the periaqueductal grey matter, was studied by use of the immunocytochemical peroxidase-antiperoxidase technique. In the neuropil, GFA, S100 and GS were found in glial elements, i.e., in fibrous (GFA, S100) and protoplasmic astrocytes (S100, GS). The presence of S100 in the majority of the ventricular ependymal cells and tanycytes, and the presence of GFA in a limited number of ventricular ependymal cells and tanycytes confirm the glial nature of these cells. The absence of S100, GFA and GS from the ependymocytes of the SCO, which are considered to be modified ependymal cells, suggests either a non-astrocytic lineage of these cells or an extreme specialization of the SCO-cells as glycoprotein-synthesizing and secreting elements, a process that may have led to the disappearance of the glial markers.     

Morphological and neurochemical diversity of neuronal nitric oxide synthase-positive amacrine cells in the turtle retina

By gently scraping off the surface of the lateral ventricles of adult bovine brains, we obtained sheets containing the ependymal layer and some attached sub-ependymal cells. Explants were cultured in serum-free medium or in two media enriched with 20% fetal calf serum or 20% adult bovine cerebrospinal fluid, and processed for different time intervals from 4 h to 60 days. For characterization of the ependymal cells we used antisera against S-100 protein, vimentin and glial fibrillary acidic protein (GFAP). For comparison, the ependyma of adult bovines and of fetuses from days 60 to 120 post coitum was studied in situ. The adult ependyma consisted of a ciliated, cuboid cell monolayer with short basal processes; it displayed S-100 immunoreactivity but only scarce deposits of vimentin and no GFAP. The fetal ependyma had the appearance of a pseudostratified epithelium with elongated nuclei and basal processes containing S-100 and vimentin from day 80 post coitum and GFAP from day 100 post coitum. In explants, no differences were seen between the three culture media; the ependyma became pseudostratified, developed basal processes and showed increasing amounts of S-100 and vimentin first, and subsequently also GFAP. These changes were concomitant with the onset of mitotic activity in the subependymal layer leading to the production of numerous cells. The morphological and immunocytochemical features of ependymal cells in cultured explants resembled those of fetal ependyma. Our results indicate that the culture of ependymal explants from adult bovine lateral ventricles is an useful model system for morphological and functional studies of the ependyma and for the analysis of cell proliferation in the subependymal layer.     

Regional differences of proliferation activity in the spinal cord ependyma of adult rats

Increased proliferation activity in the central canal ependyma of adult rodent spinal cord was described after injury and is thought to participate in recovery processes. Proliferation activity is scarce under physiological conditions, but still could be of importance, as in vitro studies showed that the spinal cord ependyma is an internal source of neural stem cells. Data from these studies indicate that there are regional differences in the distribution of proliferation activity along the rostro-caudal axis. We analyzed the proliferation activities in the ependyma within the entire extent of intact adult rat spinal cord. To identify proliferating cells we performed immunohistochemistry either for cell cycle S-phase marker BrdU or for the nuclear protein Ki-67. BrdU and Ki-67 positive cells were counted on sections selected from four spinal cord regions — cervical, thoracic, lumbar and sacral/coccygeal. Analysis showed that the number of BrdU positive cells within the ependyma was very low in all subdivisions of the spinal cord. Both BrdU and Ki-67 labeling revealed a significantly higher number of proliferating cells in the ependyma of sacrococcygeal part in comparison to all other spinal cord regions, suggesting that the caudal spinal cord might have potentially higher regeneration capacity compared to more rostral parts.     

The crystalloid of Lubarsch in the human spermatogonium

Summary Spermatogonia of adult men contain frequently the so-called crystalloid of Lubarsch. This rod-shaped structure consists of a bundle of fine tubules running parallel to the long axis. The tubules (100 Å) are connected with one another. This structure is also observed in the spermatocyte occasionally and is similar to the crystalloid of Charcot-Böttcher in the cytoplasm of human Sertoli cells. In chick spermatogonia, a homologous crystalloid occurs infrequently. Morphological similarities of the crystalloids to those occurring in other cells are discussed. Mitochondria in the human spermatogonium show some aggregation; between them dense intermitochondrial material was noted.Study supported by grants (HD 00593-05) from U.S.P.H.S. and the Japanese Ministry of Education. The author wishes to thank members of Urology Department, Chiba University for supplying biopsy materials and Dr. G. Yasuzumi for assistance in the bibliography.     

Clusterin: a protective mediator for ischemic cardiomyocytes?

We examined the relationship between clusterin and activated complement in human heart infarction and evaluated the effect of this protein on ischemic rat neonatal cardiomyoblasts (H9c2) and isolated adult ventricular rat cardiomyocytes as in vitro models of acute myocardial infarction. Clusterin protects cells by inhibiting complement and colocalizes with complement on jeopardized human cardiomyocytes after infarction. The distribution of clusterin and complement factor C3d was evaluated in the infarcted human heart. We also analyzed the protein expression of clusterin in ischemic H9c2 cells. The binding of endogenous and purified human clusterin on H9c2 cells was analyzed by flow cytometry. Furthermore, the effect of clusterin on the viability of ischemically challenged H9c2 cells and isolated adult ventricular rat cardiomyocytes was analyzed. In human myocardial infarcts, clusterin was found on scattered, morphologically viable cardiomyocytes within the infarcted area that were negative for complement. In H9c2 cells, clusterin was rapidly expressed after ischemia. Its expression was reduced after reperfusion. Clusterin bound to single annexin V-positive or annexin V and propidium iodide-positive H9c2 cells. Clusterin inhibited ischemia-induced death in H9c2 cells as well as in isolated adult ventricular rat cardiomyocytes in the absence of complement. We conclude that ischemia induces the upregulation of clusterin in ischemically challenged, but viable, cardiomyocytes. Our data suggest that clusterin protects cardiomyocytes against ischemic cell death via a complement-independent pathway.     

Über eigentümliche neuronale Zellelemente im Ependym des Zentralkanals von Salamandra maculosa

Zusammenfassung Zwischen den eigentlichen Ependymzellen des Zentralkanals adulter Feuersalamander kommen amphorenartig gestaltete Elemente vor, die sich aufgrund ihrer synaptischen Kontakte mit Axonendigungen als Neurone identifizieren lassen. Diese intraependymalen Nervenzellen weisen einen apikalen Fortsatz auf, der sich mit einer warzen- oder knotenförmigen Protrusion in das Lumen des Zentralkanals erstreckt. Die Protrusion ist gewöhnlich mit stereozilienartigen Ausläufern besetzt. Die funktionelle Bedeutung der beschriebenen neuronalen Elemente konnte bisher nicht geklärt werden.

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On peculiar neuronal cells in the ependyma of the canalis centralis in the salamander

Summary The ependyma of the canalis centralis of adult salamanders was examined by electron microscopy. Between the ependymal cells occur amphora-like elements identifiable as neurons by their synaptic contacts with axon terminals. These intraependymal nerve cells exhibit an apical outgrowth extending into the lumen of the canalis centralis with a wart-like or knob-like protrusion. The latter usually bears extensions resembling stereocilia. The functional significance of the neuronal elements is still unknown.

Für ausgezeichnete technische Unterstützung danke ich Frl. S. Luh, Frl. R. Beck, Frl. D. Höhna und Fr. U. Qreini.     

Vesicle formation from the nuclear envelope in amphibian oocytes

Conclusions Electron microscopic evidence has been presented which suggests that the cytoplasmic vesicles observed in amphibian oocytes are derived by blebbing from the nuclear envelope and migrate to the periphery. The mechanism of bleb formation is discussed.This work was supported by a U.S.P.H.S. research grant RG5803(C2) and was carried out during the tenure of a U.S.P.H.S. Special Research Fellowship (5356).     

Generation of a new adenovirus type 12-inducible fragile site by insertion of an artificial U2 locus in the human genome.

Infection with adenovirus type 12 (Ad12) induces four fragile sites in the human genome (H.F. Stich, G.L. van Hoosier, and J.J. Trentin, Exp. Cell Res. 34:400-403, 1964; H. zur Hausen, J. Virol. 1:1174-1185, 1967). The major site, at 17q21-22, contains the U2 gene cluster, which is specifically disrupted by infection in at least a percentage of the cells (D.M. Durnam, J.C. Menninger, S.H. Chandler, P.P. Smith, and J.K. McDougall, Mol. Cell. Biol. 8:1863-1867, 1988). For direct assessment of whether the U2 locus is the target of the Ad12 effect, an artificial locus, constructed in vitro and consisting of tandem arrays of the U2 6-kbp monomer, was transfected into human cells. We report that integration of this artificial locus on the p arm of chromosome 13 creates a new Ad12-inducible fragile site.     

中国绒泡菌属的分类学研究 Ⅱ.产自新疆的新种和稀有种

关于新疆的粘菌,过去仅知８种,且无一为绒泡菌。本文第一作者于１９９４年在新疆进行了粘菌资源调查,在随后的研究中共明确了５０多个种,有１０种为绒泡菌,其中的几个种,如垂头绒泡菌Ｐｈｙｓａｒｕｍｎｕｔａｎｓ（Ｂｕｌｌ．）Ｐｅｒｓ．是常见和广布的,但也有几种是特殊的和稀有的。本文报告了５种绒泡菌：橙红绒泡菌Ｐ.ａｕｒａｎｔｉａｃｕｍ　Ｓ．Ｌ．Ｃｈｅｎ,Ｙ．ＬｉｅｔＨ．Ｚ．Ｌｉ和侧扁绒泡菌Ｐ．ｌｏｒａｔｕｍＳ．Ｌ．ＣｈｅｎＹ．Ｌｉ　ｅｔＨ．Ｚ．Ｌｉ是新种,黄白绒泡菌Ｐ．ａｌｂｅｓｃｅｎｓＥｌｌｉｓｅｘＴ．Ｍａｃｂｒ．和团聚绒泡菌Ｐ．ｃｏｎｇｌｏｍｅｒａｔｕｍ（Ｆｒ．）Ｒｏｓｔａｆ．是中国新记录种,已知种黄绿绒泡菌Ｐ．ｖｉｒｅｓｃｅｎｓＤｉｔｍａｒ显然少见,国内此前仅知分布于福建。橙红绒泡菌Ｐ．ａｕｒａｎｔｉａｃｕｍＳ．Ｌ．Ｃｈｅｎ,Ｙ．ＬｉｅｔH.Ｚ．Ｌｉ的孢丝近似钙丝菌状,由或大或小的石灰结将许多细短的孢丝线联成致密的白色网体,这使其被归入绒泡菌属,并与同属其它种相区分;侧扁绒泡菌Ｐ．ｌｏｒａｔｕｍＳ．Ｌ．Ｃｈｅｎ,Ｙ．ＬｉｅｔＨ．Ｚ．Ｌｉ的联囊体虽发达而侧扁,但顶部无预成开裂线,在相似种中也易识别。本文对这两个新种     

中国绒泡菌属的分类学研究II.产自新疆的新种和稀有种

关于新疆的粘菌,过去仅知８种,且无一为绒泡菌。本文第一作者于１９９４年在新疆进行了粘菌资源调查,在随后的研究中共明确了５０多个种,有１０种为绒泡菌,其中的几个种,如垂头绒泡菌Ｐｈｙｓａｒｕｍｎｕｔａｎｓ（Ｂｕｌｌ．）Ｐｅｒｓ．是常见和广布的,但也有几种是特殊的和稀有的。本文报告了５种绒泡菌：橙红绒泡菌Ｐ.ａｕｒａｎｔｉａｃｕｍ　Ｓ．Ｌ．Ｃｈｅｎ,Ｙ．ＬｉｅｔＨ．Ｚ．Ｌｉ和侧扁绒泡菌Ｐ．ｌｏｒａｔｕｍＳ．Ｌ．ＣｈｅｎＹ．Ｌｉ　ｅｔＨ．Ｚ．Ｌｉ是新种,黄白绒泡菌Ｐ．ａｌｂｅｓｃｅｎｓＥｌｌｉｓｅｘＴ．Ｍａｃｂｒ．和团聚绒泡菌Ｐ．ｃｏｎｇｌｏｍｅｒａｔｕｍ（Ｆｒ．）Ｒｏｓｔａｆ．是中国新记录种,已知种黄绿绒泡菌Ｐ．ｖｉｒｅｓｃｅｎｓＤｉｔｍａｒ显然少见,国内此前仅知分布于福建。橙红绒泡菌Ｐ．ａｕｒａｎｔｉａｃｕｍＳ．Ｌ．Ｃｈｅｎ,Ｙ．ＬｉｅｔH.Ｚ．Ｌｉ的孢丝近似钙丝菌状,由或大或小的石灰结将许多细短的孢丝线联成致密的白色网体,这使其被归入绒泡菌属,并与同属其它种相区分;侧扁绒泡菌Ｐ．ｌｏｒａｔｕｍＳ．Ｌ．Ｃｈｅｎ,Ｙ．ＬｉｅｔＨ．Ｚ．Ｌｉ的联囊体虽发达而侧扁,但顶部无预成开裂线,在相似种中也易识别。本文对这两个新种     

A sex-limited serum protein variant in the mouse: Inheritance and association with the H-2 region

An alloantiserum produced in the mouse has been used to detect an antigen which is present only in male serum from certain inbred strains of mice, e.g., DBA/2J, A/J, and BALB/c. Genetic tests reveal that the presence of this antigen is controlled by a dominant autosomal gene which is expressed only in males of the proper genotype. Test crosses and analysis of congenic resistant strains indicate close linkage between the sex-limited protein (Slp) and the histocompatibility-2 (H-2) region of linkage group IX. Analysis of seven intra-H-2 recombinant strains is consistent with the placement of the genetic determinant for Slp within the H-2 region in the same position as the Ss (serum substance) determinant. Immunological evidence suggests that the Slp antigenic sites reflect structural variation in the Ss component of mouse serum.Supported by U.S.P.H.S. Research Grant GM-15419, U.S.P.H.S. Career Development Award K3-HE-24, 980 (D.C.S.), and U.S.P.H.S. Training Grant 2T01-GM-00071 (H.C.P.).     

Modeling the cellular basis of altered excitation-contraction coupling in heart failure

Ca transients measured in failing human ventricular myocytes exhibit reduced amplitude and slowed relaxation [Beuckelmann, D.J., Nabauer, M., Erdmann, E., 1992. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation 85, 1046-1055; Gwathmey, J.K., Copelas, L., MacKinnon, R., Schoen, F.J., Feldman, M.D., Grossman, W., Morgan, J.P., 1987. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ. Res. 61, 70-76; Kaab, S., Nuss, H. B., Chiamvimonvat, N., O'Rourke, B., Pak, P.H., Kass, D.A., Marban, E., Tomaselli, G.F., 1996. Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure. Circ. Res. 78(2); Li, H.G., Jones, D.L., Yee, R., Klein, G.J., 1992. Electrophysiologic substrate associated with pacing-induced hert failure in dogs: potential value of programmed stimulation in predicting sudden death. J. Am. Coll. Cardiol. 19(2), 444-449; Vermeulen, J.T., McGuire, M.A., Opthof, T., Colonel, R., Bakker, J.M.T.d., Klopping, C., Janse, M.J., 1994. Triggered activity and automaticity in ventricular trabeculae of failing human and rabbit hearts. Cardiovasc. Res. 28, 1547-1554.] and blunted frequency dependence [Davies, C.H., Davia, K., Bennett, J.G., Pepper, J.R., Poole-Wilson, P.A., Harding, S.E., 1995. Reduced contraction and altered frequency response of isolated ventricular myocytes from patients with heart failure. Circulation, 92, 2540-2549; Hasenfuss, G., Reinecke, H., Studer, R., Meyer, M., Pieske, B., Holtz, J., Holubarsch, C., Posival, H., Just, H., Drexler, H., 1994. Relation between myocardial function and expression of sarcoplasmic reticulum Ca-ATPase in failing and nonfailing human myocardium. Circ. Res. 75, 434-442; Hasenfuss, G., Reinecke, H., Studer, R., Pieske, B., Meyer, M., Drexler, H., Just, H., 1996. Calcium cycling proteins and force-frequency relationships in heart failure. Basic Res. Cardiol. 91, 17-22; Monte, F.D., O'Gara, P., Poole-Wilson, P.A., Yacoub, M., Harding, S.E., 1995. Cell geometry and contractile abnormalities of myocytes from failing human left ventricle. Cardiovasc. Res. 30, 281-290; Philips, P.J., Gwathmey, J.K., Feldman, M.D., Schoen, F.J., Grossman, W., Morgan, J.P., 1990. Post-extrasystolic potentiation and the force-frequency relationships: differential augmentation of myocardial contractility in working myocardium from patients with end-stage heart failure. J. Mol. Cell. Cardiol. 22, 99-110; Pieske, B., Hasenfuss, G., Holubarsch, C., Schwinger, R., Bohm, M., Just, H., 1992. Alterations of the force-frequency relationship in the failing human heart depend on the underlying cardiac disease. Basic Res. Cardiol. 87, 213-221.]. Analyses of protein levels in these failing hearts reveal that the SR Ca-ATPase is down-regulated on average by 50% and that the Na/Ca exchanger is up-regulated on average by a factor of two. In this paper, we test the hypothesis that this altered pattern of expression of Ca handling proteins is sufficient to account for changes in excitation-contraction coupling properties measured experimentally at the cellular level. To do this, we present an integrated model of excitation-contraction coupling in the guinea pig ventricular cell. The model is used to determine the effects of SR Ca-ATPase down-regulation and Na/Ca exchanger up-regulation on action potential duration, Ca transient shape and amplitude, and isometric force. Model analyses demonstrate that changes in Ca handling proteins play a direct and critical role in prolongation of action potential duration, and in reduction of contractile force in heart failure.     

The nature of thymidine kinase in the human-mouse hybrid cell

In order to characterize the thymidine kinase in human-mouse somatic cell hybrids derived from mouse parental cells lacking thymidine kinase, we have examined the electrophoretic migration on starch gel and the heat sensitivity of this enzyme in human, mouse, and hybrid cells. The enzyme of hybrid cells migrates similarly to that of human fetal liver and human diploid fibroblasts and faster than that of either L or A₉ mouse cells. It is less sensitive to heat than that of the mouse cells. Therefore, the human group E chromosome provides human thymidine kinase for the hybrid cell. The electrophoresis of thymidine kinase makes possible the search for variants.Aided by U.S.P.H.S. Grant No. HD 00486.     

Aging alters the force-frequency relationship and toxicity of oxidative stress in rabbit heart

Adult (6 months) and senescent (greater than 5 years) rabbit atria were studied under conditions known to increase cytoplasmic calcium (increased frequency of contraction and oxidative stress). At a contraction frequency of 1/sec, cardiac relaxation (90% relaxation time) was similar in senescent and adult atria but at a frequency of 2 or 3/sec, relaxation was significantly slower in senescent preparations (P less than 0.05). Additional experiments indicated that H2O2 (500 microM), a powerful oxidant, increased resting force and decreased developed force (DF) much more rapidly in senescent than adult atria; the maximum decrease in DF, however, was less in senescent preparations (adult = 81 +/- 6% and senescent = 42 +/- 27% of pre-H2O2 values; P less than 0.05). Age-related differences in effects of H2O2 did not result simply from a decreased ability of senescent hearts to detoxify an oxidative stress by the glutathione pathway. Both basal glutathione (GSH) concentrations and the H2O2-mediated decreases in GSH were similar in adult and senescent ventricular preparations, as were activities of glutathione peroxidase and glutathione reductase. These observations suggest that interventions known to increase cytoplasmic calcium can amplify age-related impairments of cardiac relaxation through mechanisms that may be independent of the glutathione pathway.     

Modeling of the human intercellular adhesion molecule-1, the human rhinovirus major group receptor

A model has been built of the amino-terminal domain of the intercellular adhesion molecule-1 (ICAM-1), the receptor for most human rhinovirus serotypes. The model was based on sequence and presumed structural homology to immunoglobulin constant domains. It fits well into the putative receptor attachment site, the canyon, on the human rhinovirus-14 (HRV14) surface in a manner consistent with most of the mutational data for ICAM-1 (Staunton, D. E., Dustin, M. L., Erickson, H. P., Springer, T. A. Cell, in press, 1989) and HRV14 (Colonno, R. J., Condra, J. H., Mizutani, S., Callahan, P. L., Davies, M. E., Murcko, M. A. Proc. Natl. Acad. Sci. U.S.A. 85: 5449-5453, 1988).