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PubMed2003

Fer kinase/FerT and adherens junction dynamics in the testis: an in vitro and in vivo study.

Fer kinase is a 94-kDa cytoplasmic cell-cell actin-based adherens junction (AJ)-associated nonreceptor protein tyrosine kinase (PTK) found in multiple epithelia including the testis, whereas FerT kinase (51 kDa) is the truncated testis-specific form of Fer kinase, lacking the Fps/Fes/Fer/CIP4 (products of oncogenes identified in avian and feline sarcoma, encoding tyrosine protein kinases) and the three coiled-coil domains versus Fer kinase. Yet the role(s) of Fer kinase in AJ dynamics in the testis remains largely unexplored. We have used an in vitro model of AJ assembly with Sertoli-germ cell cocultures and an in vivo model of AJ disassembly in which adult rats were treated with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) to study changes in the expression and/or localization of Fer kinase during AJ restructuring. Fer kinase/FerT was expressed by Sertoli and germ cells when cultured in vitro. Using an antibody prepared against a synthetic peptide, NH2-SAPQNCPEEIFTIMMKCWDYK-COOH, corresponding to residues 779-799 of Fer kinase in the rat, which failed to cross-react with FerT kinase, for immunohistochemistry, Fer kinase was detected in the seminiferous epithelium in virtually all stages of the epithelial cycle. At stages XIII-VI, Fer kinase was associated largely with round and elongating spermatids. At stages VII-VIII, Fer kinase associated almost exclusively with round spermatids with very weak staining associated with elongated spermatids. This stage-specific localization of Fer kinase in the epithelium was confirmed by using staged tubules for semiquantitative reverse transcription-polymerase chain reaction. Studies by immunoprecipitation revealed that Fer kinase associated with N-cadherin, gamma-catenin, p120ctn, c-Src (a putative PTK and the product of the transforming, sarcoma-inducing gene of Rous sarcoma virus), Rab 8 (a GTPase), actin, vimentin, but not E-cadherin, afadin, nectin-3, and integrin beta1, suggesting Fer kinase associates not only with the actin-based cell-cell AJ structures, such as the N-cadherin/catenin complex (but not the alpha6beta1 integrin/laminin and the afadin/nectin complex), but also with intermediate filament-based cell-cell desmosomes. An induction in Fer kinase expression was detected during Sertoli-germ cell AJ assembly in vitro but not during AF-2364-induced AJ disruption in vivo. Yet this AF-2364-induced Fer kinase plummeting associated with an induction in N-cadherin, beta-catenin, and p120ctn, particularly at the base of the seminiferous epithelium. In summary, Fer kinase structurally associates with the N-cadherin/catenin protein complex in the testis and can possibly be used to mediate signaling function via the cadherin/catenin protein complex.

Adherens Junctions↗
PubMed2003

Interactions of proteases, protease inhibitors, and the beta1 integrin/laminin gamma3 protein complex in the regulation of ectoplasmic specialization dynamics in the rat testis.

During spermatogenesis, developing germ cells migrate progressively across the seminiferous epithelium. This event requires extensive restructuring of cell-cell actin-based adherens junctions (AJs), such as the ectoplasmic specialization (ES, a testis-specific AJ type), between Sertoli cells and elongating/elongate spermatids. It was postulated that proteases and protease inhibitors worked in a yin-yang relationship to regulate these events. If this is true, then it is anticipated that both proteases and protease inhibitors are found at the ES. Indeed, matrix metalloprotease (MMP)-2, membrane-type 1 (MT1)-MMP and their inhibitor, tissue-inhibitor of metalloproteases (TIMP)-2, were shown to localize at the apical ES. In order to identify the putative MMP substrate as well as the unknown binding ligand for alpha6beta1 integrin in the ES, immunofluorescent microscopy coupled with immunoprecipitation techniques were used to demonstrate that laminin gamma3, largely a germ cell product, was present at the apical ES and could form a bona fide complex with beta1-integrin. Furthermore, the structural interactions of MMP-2 and MT1-MMP with laminin gamma3 and beta1-integrin, but not with N-cadherin or nectin-3, have implicated the crucial role of MMP-2/MT1-MMP in the regulation of integrin/laminin-based ES dynamics. Using an in vivo model to study AJ dynamics where adult rats were treated with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) to disrupt Sertoli-germ cell adhesive function, an induction of active MMP-2, active MT1-MMP and TIMP-2 but not active MMP-9 was detected between 0.5 and 8 h after AF-2364 treatment. This time frame coincided with the depletion of elongating/elongate spermatids from the epithelium, illustrating the synergistic relationships between MMP-2, MT1-MMP, and TIMP-2 in AJ disassembly. Perhaps the most important of all, the use of a specific MMP-2 and MMP-9 inhibitor, (2R)-2-[(4-biphenylylsulfonyl)amino]-3-phenylpropionic acid, could effectively delay the AF-2364-induced elongating/elongate spermatid loss from the epithelium, demonstrating the pivotal role of MMP-2 activation in ES disassembly. Collectively, these studies illustrate that the beta1-integrin/laminin gamma3 complex is a putative ES-structural protein complex, which is regulated, at least in part, by the activation of MMP-2 involving MT1-MMP and TIMP-2 at the apical ES. The net result of this interaction likely regulates germ cell movement in the seminiferous epithelium.

Adherens Junctions↗
PubMed2005

Regulation of Sertoli-germ cell adherens junction dynamics in the testis via the nitric oxide synthase (NOS)/cGMP/protein kinase G (PRKG)/beta-catenin (CATNB) signaling pathway: an in vitro and in vivo study.

During spermatogenesis, extensive restructuring of cell junctions takes place in the seminiferous epithelium to facilitate germ cell movement. However, the mechanism that regulates this event remains largely unknown. Recent studies have shown that nitric oxide (NO) likely regulates tight junction (TJ) dynamics in the testis via the cGMP/protein kinase G (cGMP-dependent protein kinase, PRKG) signaling pathway. Due to the proximity of TJ and adherens junctions (AJ) in the testis, in particular at the blood-testis barrier, it is of interest to investigate if NO can affect AJ dynamics. Studies using Sertoli-germ cell cocultures in vitro have shown that the levels of NOS (nitric oxide synthase), cGMP, and PRKG were induced when anchoring junctions were being established. Using an in vivo model in which adult rats were treated with adjudin [a molecule that induces adherens junction disruption, formerly called AF-2364, 1-(2,4-dichlorobenzyl)-IH-indazole-3-carbohydrazide], the event of AJ disruption was also associated with a transient iNOS (inducible nitric oxide synthase, NOS2) induction. Immunohistochemistry has illustrated that NOS2 was intensely accumulated in Sertoli and germ cells in the epithelium during adjudin-induced germ cell loss, with a concomitant accumulation of intracellular cGMP and an induction of PRKG but not cAMP or protein kinase A (cAMP-dependent protein kinase, PRKA). To identify the NOS-mediated downstream signaling partners, coimmunoprecipitation was used to demonstrate that NOS2 and eNOS (endothelial nitric oxide synthase, NOS3) were structurally associated with the N-cadherin (CDH2)/beta-catenin (CATNB)/actin complex but not the nectin-3 (poliovirus receptor-related 3, PVRL 3)/afadin (myeloid/lymphoid or mixed lineage-leukemia tranlocation to 4 homolog, MLLT4) nor the integrin beta1 (ITB1)-mediated protein complexes, illustrating the spatial vicinity of NOS with selected AJ-protein complexes. Interestingly, CDH2 and CATNB were shown to dissociate from NOS during the adjudin-mediated AJ disruption, implicating the CDH2/CATNB protein complex is the likely downstream target of the NO signaling. Furthermore, PRKG, the downstream signaling protein of NOS, was shown to interact with CATNB in the rat testis. Perhaps the most important of all, pretreatment of testes with KT5823, a specific PRKG inhibitor, can indeed delay the adjudin-induced germ cell loss, further validating NOS/NO regulates Sertoli-germ cell AJ dynamics via the cGMP/PRKG pathway. These results illustrate that the CDH2/CATNB-mediated adhesion function in the testis is regulated, at least in part, via the NOS/cGMP/PRKG/CATNB pathway.

Adherens Junctions↗
PubMedDate not supplied

Effects of radiofrequency glow discharge and oligopeptides on the attachment of human endothelial cells to polyurethane.

Activation of the surface of small diameter polyurethane vascular grafts using radiofrequency glow discharge (RFGD) and covalent linkage of cell attachment oligopeptides may improve graft patency. The effects of RFGD treatment were investigated on a polyurethane-polydimethylsiloxane (PU-PDMS) copolymer (Cardiothane-51; Kontron Cardiovascular, Inc., Everett, MA) membrane fabricated using the spray, phase-inversion technique. RFGD using H2O vapor was used to functionalize the membrane surface, reacted with 1',1 carbonyldiimidazole and covalently bound with oligopeptides (RGDS, RGES). Membranes not subjected to RFGD, either unmodified or with adsorbed fibronectin (Fn), vitro-nectin (Vn), RGDS, RGDV, and RGES, were used as controls. RFGD treated membrane surfaces were evaluated using electron spectroscopy for chemical analysis, which demonstrated a qualitative increase in nitrogen and silicon compared to unmodified PU-PDMS. Indirect confirmation of surface hydroxylation was provided by metallization with palladium and nickel, demonstrating uniform metallization of RFGD treated PU-PDMS surfaces. Human umbilical vein endothelial cells (HUVEC) were seeded at a density of 10(4) cells/cm2 and cell attachment assessed at 3 hr, 24 hr, and 7 days. Untreated PU-PDMS and membrane with adsorbed oligopeptides demonstrated poor HUVEC attachment at all intervals. Adsorbed Fn and Vn had significantly better early cell attachment and growth (p < 0.01). RFGD improved initial attachment and growth over non-RFGD treated controls (p < 0.01) for RGDS bound membranes, which performed as well as Fn controls (N.S.). PU-PDMS membranes can be modified using RFGD to covalently link oligopeptides. RFGD treatment alone, or with covalent linkage of cell attachment oligopeptides, improves HUVEC attachment and growth in a static environment.

Amino Acid Sequence↗
PubMed2004

A role for 3-O-sulfated heparan sulfate in cell fusion induced by herpes simplex virus type 1.

Membrane fusion induced by herpes simplex virus (HSV) is required for both entry and cell-to-cell spread. It is mediated by the viral glycoprotein gB, gD, gH-gL and gD receptors. Although 3-O-sulfated heparan sulfate (3-OS HS) is a receptor for HSV-1 entry, the requirement for heparan sulfate in the fusion process has been ruled out. Here, it is demonstrated that cells expressing 3-OS HS, generated by D-glucosaminyl 3-O-sulfotransferase isoforms-3 and/or -5 (3-OST-3 and 3-OST-5), fused with cells expressing the four glycoproteins. The cell fusion observed exhibited similar requirements but was independent of protein receptors, HVEM or nectin-1. Additionally, removal of 3-OS HS from the cell surface by heparinase-I treatment and, in separate experiments, the presence of soluble 3-OST-3- and 3-OST-5-modified HS, significantly inhibited fusion. Taken together, these results indicate that 3-OS HS can play a crucial role in virus entry and cell fusion.

Animals↗
PubMed2005

Glycoprotein D receptor-dependent, low-pH-independent endocytic entry of herpes simplex virus type 1.

Two herpes simplex virus type 1 (HSV-1) entry pathways have been described: direct fusion between the virion envelope and the plasma membrane, as seen on Vero cells, and low-pH-dependent endocytosis, as seen on CHO nectin-1 and HeLa cells. In this paper, we studied HSV entry into C10 murine melanoma cells and identified a third entry pathway for this virus. During entry into C10 cells, virion envelope glycoproteins rapidly became protected from the membrane-impermeable chemical cross-linker BS3 and from proteinase K. Protection was gD receptor dependent, and the time taken to detect protected protein was proportional to the rate of virus entry. Ultrastructural examination revealed that virions attached to the surface of C10 cells were localized to membrane invaginations, whereas those on the surface of receptor-negative B78 cells were peripherally attached. Virus entry into C10 cells was energy dependent, and intracellular enveloped virions were seen within membrane-bound vesicles consistent with endocytic entry. Entry was not inhibited by bafilomycin A1 or ammonium chloride, showing that passage of the virion through a low-pH environment was not required for infection. Resistance to similar reagents should therefore not be taken as proof of HSV entry by a nonendosomal pathway. These data define a novel gD receptor-dependent acid-independent endocytic entry pathway for HSV.

Adenosine Triphosphate↗
PubMed2005

Herpes simplex virus glycoprotein B binds to cell surfaces independently of heparan sulfate and blocks virus entry.

Virion glycoproteins gB, gD, and gH/gL play essential roles for herpes simplex virus (HSV) entry. The function of gD is to interact with a cognate receptor, and soluble forms of gD block HSV entry by tying up cell surface receptors. Both gB and the nonessential gC interact with cell surface heparan sulfate proteoglycan (HSPG), promoting viral attachment. However, cells deficient in proteoglycan synthesis can still be infected by HSV. This suggests another function for gB. We found that a soluble truncated form of gB bound saturably to the surface of Vero, A431, HeLa, and BSC-1 cells, L-cells, and a mouse melanoma cell line expressing the gD receptor nectin-1. The HSPG analog heparin completely blocked attachment of the gC ectodomain to Vero cells. In contrast, heparin only partially blocked attachment of soluble gB, leaving 20% of the input gB still bound even at high concentrations of inhibitor. Moreover, heparin treatment removed soluble gC but not gB from the cell surface. These data suggest that a portion of gB binds to cells independently of HSPG. In addition, gB bound to two HSPG-deficient cell lines derived from L-cells. Gro2C cells are deficient in HSPG, and Sog9 cells are deficient in HSPG, as well as chondroitin sulfate proteoglycan (CSPG). To identify particular gB epitopes responsible for HSPG-independent binding, we used a panel of monoclonal antibodies (MAbs) to gB to block gB binding. Only those gB MAbs that neutralized virus blocked binding of soluble gB to the cells. HSV entry into Gro2C and Sog9 cells was reduced but still detectable relative to the parental L-cells, as previously reported. Importantly, entry into Gro2C cells was blocked by purified forms of either the gD or gB ectodomain. On a molar basis, the extent of inhibition by gB was similar to that seen with gD. Together, these results suggest that soluble gB binds specifically to the surface of different cell types independently of HSPG and CSPG and that by doing so, the protein inhibits entry. The results provide evidence for the existence of a cellular entry receptor for gB.

Animals↗
PubMed2005

Tumor rejection by the poliovirus receptor family ligands of the DNAM-1 (CD226) receptor.

The poliovirus receptor CD155 and its family member CD112 (nectin-2) are the ligands for the activating cell-surface receptor DNAM-1 on CD8+ T cells and natural killer (NK) cells. Here, we demonstrate that, whereas the RMA tumor grew in syngeneic mice, DNAM-1 ligand-transduced RMA was rejected, in which CD8+ T cells and NK cells played an essential role. Importantly, CD8+ memory cytotoxic T cells to parental RMA were generated in these mice. We found that DNAM-1 was also expressed on CD8alpha+, rather than CD8alpha-, dendritic cells (DCs). Cross-linking DNAM-1 induced maturation of CD8alpha+ DCs. Antigen presentation by these stimulated DCs drove Th1 cells. Moreover, the rejection of DNAM-1 ligand-transduced RMA was canceled in CD4+ T-cell-depleted and major histocompatibility complex class II-deficient mice. Taken together, these results suggest that DNAM-1 ligands stimulate innate immunity by CD8alpha+ DCs as well as NK cells, which efficiently prime cell-mediated tumor-specific immunity.

Animals↗
PubMed2005

Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation.

In recent years, mesenchymal stem cells (MSCs) have been shown to inhibit T-lymphocyte proliferation induced by alloantigens or mitogens. However, no substantial information is available regarding their effect on natural killer (NK) cells. Here we show that MSCs sharply inhibit IL-2-induced proliferation of resting NK cells, whereas they only partially affect the proliferation of activated NK cells. In addition, we show that IL-2-activated NK cells (but not freshly isolated NK cells) efficiently lyse autologous and allogeneic MSCs. The activating NK receptors NKp30, NKG2D, and DNAM-1 represented the major receptors responsible for the induction of NK-mediated cytotoxicity against MSCs. Accordingly, MSCs expressed the known ligands for these activating NK receptors-ULBPs, PVR, and Nectin-2. Moreover, NK-mediated lysis was inhibited when IFN-gamma-exposed MSCs were used as target cells as a consequence of the up-regulation of HLA class I molecules at the MSC surface. The interaction between NK cells and MSCs resulted not only in the lysis of MSCs but also in cytokine production by NK cells. These results should be taken into account when evaluating the possible use of MSCs in novel therapeutic strategies designed to improve engraftment or to suppress graft-versus-host disease (GVHD) in bone marrow transplantation.

Cell Culture Techniques↗
PubMed2004

Prevention of genital herpes in a guinea pig model using a glycoprotein D-specific single chain antibody as a microbicide.

BACKGROUND: Genital herpes (GH) is a recurrent sexually transmitted infection (STI) that causes significant morbidity and is also the major source of herpes simplex virus (HSV) in cases of neonatal herpes. Vaccination is a current goal which has had limited success so far in preventing GH and microbicides offer an attractive alternative. Treatment of primary disease cannot prevent establishment of latent infections and thus, cannot prevent subsequent recurrent disease. Recently, many of the molecular events leading to entry of HSV into cells have been elucidated, resulting in the description of a number of herpesvirus entry mediators (HVEMs) that interact with HSV glycoprotein D (gD) on the surface of virions. Described here is a strategy for interrupting the spread of HSV based on interfering with these interactions. The hypothesis addressed in the current report was that single chain antibody variable fragments (scFv) that interrupt associations between gD and HVEMs would not only prevent infection in vitro but could also be used as microbicides to interfere with acquisition GH. RESULTS AND CONCLUSIONS: Here we show that a scFv derived from a particular hybridoma, DL11, not only inhibits infection in vitro but also prevents development of GH in a guinea pig model when applied intravaginally in an inert vehicle. Comparison of different anti-gD single chain antibodies supported the hypothesis that the activity of DL11-scFv is based on its ability to disrupt the associations between gD and the two major receptors for HSV, nectin-1 and HveA. Further, the results predict that bacterial expression of active single chain antibodies can be optimized to manufacture inexpensively a useful microbicidal product active against HSV.

Animals↗
PubMed2004

Regulation of Sertoli-germ cell adherens junction dynamics via changes in protein-protein interactions of the N-cadherin-beta-catenin protein complex which are possibly mediated by c-Src and myotubularin-related protein 2: an in vivo study using an androgen suppression model.

Using a well characterized model of cell-cell actin-based adherens junction (AJ) disruption by suppressing the intratesticular testosterone level in adult rats with testosterone-estradiol implants, we have confirmed earlier findings that Sertoli-germ cell AJ dynamics are regulated by the activation of kinases via putative signaling pathways but with some unexpected findings as follows. First, the loss of germ cells from the seminiferous epithelium during androgen suppression was associated with a surge in myotubularin-related protein 2 (MTMR2, a lipid phosphatase, in which adult MTMR2-/- mice were recently shown to be azoospermic because of the loss of cell adhesion function between germ and Sertoli cells); kinases: phosphatidylinositol 3-kinase, c-Src, and C-terminal Src kinase; adaptors: alpha-actinin, vinculin, afadin, and p130 Crk-associated protein; and AJ-integral membrane proteins at the ectoplasmic specialization (ES, a testis-specific cell-cell actin-based AJ type) site: N-cadherin, beta-catenin, integrin beta1, and nectin 3. Second, MTMR2, instead of structurally interacting with phosphatidylinositol 3-kinase, a protein and lipid kinase, was shown to associate only with c-Src, a nonreceptor protein tyrosine kinase, as demonstrated by both coimmunoprecipitation and fluorescent microscopy at the site of apical ES, but none of the kinases, adaptors, and AJ-integral proteins that were examined. Collectively, these results suggest that the MTMR2/c-Src is an important phosphatase/kinase protein pair in AJ dynamics in the testis. Because c-Src is known to associate with the cadherin/catenin protein complex at the ES in the testis, we next sought to investigate any changes in the protein-protein interactions of this protein complex during androgen suppression-induced germ cell loss. Indeed, there was a loss of N-cadherin and beta-catenin association, accompanied by a surge in Tyr phosphorylation of beta-catenin, during germ cell loss from the epithelium. Third, and perhaps the most important of all, during natural recovery of the epithelium after removal of testosterone-estradiol implants when spermatids were reattaching to Sertoli cells, an increase in N-cadherin and beta-catenin association was detected with a concomitant loss in the increased Tyr phosphorylation in beta-catenin. In summary, these results illustrate that the cadherin/catenin is a crucial cell adhesion complex that regulates AJ dynamics in the testis, and its functionality is likely modulated by the MTMR2/c-Src protein complex.

Adherens Junctions↗
PubMed2004

Regulation of blood-testis barrier dynamics: an in vivo study.

An in vivo model was used to investigate the regulation of tight junction (TJ) dynamics in the testis when adult rats were treated with CdCl(2). It was shown that the CdCl(2)-induced disruption of the blood-testis barrier (BTB) associated with a transient induction in testicular TGF-beta2 and TGF-beta3 (but not TGF-beta1) and the phosphorylated p38 mitogen activated protein (MAP) kinase, concomitant with a loss of occludin and zonula occludens-1 (ZO-1) from the BTB site in the seminiferous epithelium. These results suggest that BTB dynamics in vivo are regulated by TGF-beta2/-beta3 via the p38 MAP kinase pathway. Indeed, SB202190, a specific p38 MAP kinase inhibitor, blocked the CdCl(2)-induced occludin and ZO-1 loss from the BTB. This result clearly illustrates that CdCl(2) mediates its BTB disruptive effects via the TGF-beta3/p38 MAP kinase signaling pathway. Besides, this CdCl(2)-induced occludin and ZO-1 loss from the BTB also associated with a significant loss of the cadherin/catenin and the nectin/afadin protein complexes at the site of cell-cell actin-based adherens junctions (AJs). An induction of alpha(2)-macroglobulin (a non-specific protease inhibitor) was also observed during BTB damage and when the seminiferous epithelium was being depleted of germ cells. These data illustrate that a primary disruption of the BTB can lead to a secondary loss of cell adhesion function at the site of AJs, concomitant with an induction in protease inhibitor, which apparently is used to protect the epithelium from unwanted proteolysis. alpha(2)-Macroglobulin was also shown to associate physically with TGF-beta3, afadin and nectin 3, but not occludin, E-cadherin or N-cadherin, indicating its possible role in junction restructuring in vivo. Additionally, the use of SB202190 to block the TGF-beta3/p-38 MAP kinase pathway also prevented the CdCl(2)-induced loss of cadherin/catenin and nectin/afadin protein complexes from the AJ sites, yet it had no apparent effect on alpha(2)-macroglobulin. These results demonstrate for the first time that the TGF-beta3/p38 MAP kinase signaling pathway is being used to regulate both TJ and AJ dynamics in the testis, mediated by the effects of TGF-beta3 on TJ- and AJ-integral membrane proteins and adaptors, but not protease inhibitors.

Adherens Junctions↗
PubMed2004

The acrosome-acroplaxome-manchette complex and the shaping of the spermatid head.

A combination of exogenous contractile forces generated by a stack of F-actin-containing hoops embracing the apical region of the elongating spermatid nucleus and an endogenous modulating mechanism dependent on the spermatid-containing acrosome-acroplaxome-manchette complex may play a cooperative role in the shaping of the spermatid head. In addition, the manchette is a key element in the transport of vesicles and macromolecules to the centrosome and developing spermatid tails as well as in nucleocytoplasmic transport. The proposed model of spermatid head shaping is based on: 1) currently known structural and molecular components of the F-actin hoops, the main cytoskeletal element of the Sertoli cell ectoplasmic specializations; 2) the molecular features of acrosome biogenesis; 3) the assembly of a subacrosomal cytoskeletal plate called the acroplaxome; and 4) the spatial relationship of the acrosome-acroplaxome complex with the manchette, a transient microtubular/actin-containing structure. During acrosome biogenesis, the acroplaxome becomes the nucleation site to which Golgi-derived proacrosomal vescicles tether and fuse. The acroplaxome has at least two functions: it anchors the developing acrosome to the elongating spermatid head. It may also provide a mechanical scaffolding plate during the shaping of the spermatid nucleus. The plate is stabilized by a marginal ring with junctional complex characteristics, adjusting to exogenous clutching forces generated by the stack of Sertoli cell F-actin-containing hoops applied to the elongating spermatid head. A tubulobulbar complex, formed by cytoplasmic processes protruding from the elongating spermatid head extending into the adjacent Sertoli cell, is located at the concave side of the spermatid head. The tubulobulbar complex might provide stabilizing conditions, together with the actin-afadin-nectin-2/nectin-3 adhesion unit, to enable sustained and balanced clutching exogenous forces applied during the elongation of the spermatid head.

Acrosome↗
PubMed2000

Changes in diabetic retinal matrix protein mRNA levels in a common transgenic mouse strain.

Recently, all the structural features of non-proliferative diabetic retinopathy have been demonstrated in mice fed 30% galactose for 21-26 months. To determine whether changes in retinal matrix protein mRNA levels occur early in the course of murine diabetes we used a competitive RT-PCR method to quantitate retinal mRNA levels in an inbred mouse strain (FVB) commonly used for transgenic studies. Retinal mRNA was prepared from STZ-diabetic and non-diabetic FVB mice at 4, 8, 12 and 16 weeks and cDNA encoding basement membrane components was quantitated using MIMIC constructs that compete for the same primer pairs. alpha1 (IV) collagen, the beta1 and gamma1 chains of laminin, fibronectin, and vitronectin mRNAs were quantitated. For alpha1 (IV) collagen, statistically significant diabetes-induced increases were apparent by 8 weeks (3.11 +/- 0.20 vs. 1.29 +/- 0.19 x 10(6) molecules/mg total RNA, p < 0.005). Similarly, diabetes-induced increases were observed by 8 weeks for the beta1 chain of laminin (4.54 +/- 0.22 vs. 1.85 +/- 0.43 x 10(5) molecules/mg total RNA, p < 0.005), the gamma1 chain of laminin (7. 33 +/- 0.29 vs. 4.84 +/- 0.76 x 10(4)/microg total RNA, p < 0.05), and for fibronectin (2.22 +/- 0.21 vs. 1.35 +/- 0.15 x 10(6) molecules/mg total RNA, p < 0.05). The magnitude of change was greatest for alpha1 (IV) collagen (2.4-fold) and beta1 laminin (2. 5-fold) at 8 weeks, and least for fibronectin (1.6-fold). A smaller diabetes-induced increase in vitro nectin mRNA was also observed, but it failed to reach statistical significance at 12 and 16 weeks. These data provide the basis for assessing the effects of genetic manipulation on diabetic retinopathy in transgenic mouse models.

Animals↗
PubMed2004

Germ-line mutation of Foxn5 gene in mouse lineage.

Amplified region (amplicon) around MLL gene is closely linked to the 11q23.3 commonly deleted region of neuroblastoma, which includes cancer-associated genes such as PHLDB1 (LL5A), BCL9L, FOXN5 (FOXR1), CBL, MFRP, and PVRL1 (Nectin) genes. FOXN6 (FOXR2) gene at human chromosome Xp11.21 is generated due to retrotransposition of ancestral Foxn5 gene during evolution. FOXN5 and FOXN6 orthologs share the common domain structure consisting of FN56 and Forhead-box (FOX) domains. Here, we identified and characterized mouse Foxn5 gene by using bioinformatics. Mouse Foxn5, consisting of six exons, was located within mouse genome sequences AC122428.4 and AC125129.5. Foxn5 locus at mouse chromosome 9B was synthenic to rat chromosome 8q22 and human chromosome 11q23.3. Mouse Foxn5 (180 aa) was C-terminally truncated compared with rat Foxn5 and human FOXN5. Mouse 'Foxn5' protein without FOX domain was generated due to a frame shift introduced by germ-line one-base deletion within exon 3. Mouse Foxn5 mRNA was expressed in embryonic germ cells and fertilized eggs. Germ-line mutation of Foxn5 gene in the mouse lineage might lead to divergent scenario of early embryogenesis between mouse and rat through the deregulation of Foxn5 target genes in mouse early embryos, and explain the difficulty in manipulation of rat embryonic stem (ES) cells based on the mouse equivalent system. This is the first report on identification and characterization of mouse Foxn5 gene as well as on species specific germ-line mutation of the Fox family gene.

Amino Acid Sequence↗
PubMed1989

Fibroblast matrix and surface components that mediate cell-to-cell interaction with lymphocytes.

The interaction between lymphocytes and fibroblasts in vitro has been examined using a quantitative ELISA assay to measure the binding of T and B cells to monolayer cultures of human dermal fibroblasts. This was carried out on microtiter culture plates, using an anti-Thy-1 monoclonal antibody, to determine the attachment of murine T lymphocytes and an affinity-purified polyclonal anti-IgM antibody to measure B cell binding. Both types of lymphocyte were found to adhere strongly to intact human fibroblasts, and also had high levels of attachment to purified fibroblast plasma membranes and extracts of the fibroblast extracellular matrix. Attachment, particularly of B lymphocytes, also took place onto plastic surfaces coated with fibronectin, but not to collagens or to intact fibroblasts that had been fixed with a low concentration of paraformaldehyde. Lymphocyte binding to fibroblasts was partially prevented by a monoclonal antibody against fibroblast MHC class II antigens, but not against the class I membrane complex, or by polyclonal antiserum to the cell surface mannose 6-phosphate receptor. In addition, although both lymphocyte types were able to adhere to fibro-nectin, the presence of antibody against fibronectin or the synthetic peptide Arg-Gly-Asp-Ser, had no effect on their attachment to fibroblasts. Thus, lymphocyte adhesion may occur by fibronectin, but other types of interactions with fibroblasts also appear to take place.

Animals↗
PubMed1999

Inhibition of dendritic cell maturation by herpes simplex virus.

Maturation of dendritic cells (DC), leading to migration and increased T cell stimulatory capacity, is essential for the initiation of immune responses. This process is triggered by a variety of stimuli, such as inflammatory cytokines, bacterial and viral products. Using a recombinant disabled infectious single cycle herpes simplex virus 1 (HSV-1) encoding green fluorescent protein, we show that the infected DC are defective in up-regulating co-stimulatory molecules, do not produce cytokines, and do not acquire responsiveness to chemokines required for migration to secondary lymphoid organs. These results reveal yet another strategy used by HSV-1 to evade the immune response, namely the inhibition of signaling pathways involved in DC maturation.

Cell Adhesion Molecules↗