An ABCA12 missense variant in a Shorthorn calf with ichthyosis fetalis.

Two clinical forms of ichthyosis in cattle have been reported, ichthyosis fetalis and congenital ichthyosis. Ichthyosis poses animal welfare and economic issues and the more severe form, ichthyosis fetalis, is lethal. A Shorthorn calf with ichthyosis fetalis was investigated and a likely causal missense variant on chromosome 2 in the ABCA12 gene (NM_001191294.2:c.6776T>C) was identified by whole genome sequencing. Mutations in the ABCA12 gene are known to cause ichthyosis fetalis in cattle and Harlequin ichthyosis in humans. Sanger sequencing of the affected calf and the dam confirmed the variant was homozygous in the affected calf and heterozygous in the dam. Further genotyping of 130 Shorthorn animals from the same property revealed an estimated allele frequency of 3.8%. The presented findings enable genetic testing for breeding and diagnostics.

Use of vascularized posterior rectus sheath allograft in pediatric multivisceral transplantation--report of two cases.

Restoring abdominal wall cover and contour in children undergoing bowel and multivisceral transplantation is often challenging due to discrepancy in size between donor and recipient, poor musculature related to birth defects and loss of abdominal wall integrity from multiple surgeries. A recent innovation is the use of vascularized posterior rectus sheath to enable closure of abdomen. We describe the application of this technique in two pediatric multivisceral transplant recipients--one to buttress a lax abdominal wall in a 22-month-old child with megacystis microcolon intestinal hypoperistalsis syndrome and another to accommodate transplanted viscera in a 10-month child with short bowel secondary to gastoschisis and loss of domain. This is the first successful report of this procedure with long-term survival. The procedure has potential application to facilitate difficult abdominal closure in both adults and pediatric liver and multivisceral transplantation.

Effects of cytoplasmic and luminal pH on Ca(2+) release channels from rabbit skeletal muscle.

Ryanodine receptor (RyR)-Ca(2+) release channels from rabbit skeletal muscle were incorporated into lipid bilayers. The effects of cytoplasmic and luminal pH were studied separately over the pH range 5-8, using half-unit intervals. RyR activity (at constant luminal pH of 7.5) was inhibited at acidic cytoplasmic pH, with a half-inhibitory pH (pH(I)) approximately 6.5, irrespective of bilayer potential and of whether the RyRs were activated by cytoplasmic Ca(2+) (50 microM), ATP (2 or 5 mM), or both. Inhibition occurred within approximately 1 s and could be fully reversed within approximately 1 s after brief inhibition or within approximately 30-60 s after longer exposure to acidic cytosolic pH. There was no evidence of any hysteresis in the cytoplasmic pH effect. Ryanodine-modified channels were less sensitive to pH inhibition, with pH(I) at approximately 5.5, but the inhibition was similarly reversible. Steady-state open and closed dwell times of RyRs during cytoplasmic pH inhibition suggest a mechanism where the binding of one proton inhibits the channel and the binding of two to three additional protons promotes further inhibited states. RyR activity was unaffected by luminal pH in the pH range 7.5 to 6.0. At lower luminal pH (5-5.5) most RyRs were completely inhibited, and raising the pH again produced partial to full recovery in only approximately 50% of cases, with the extent of recovery not detectably different between pH 7.5 and pH 9. The results indicate that isolated skeletal muscle RyRs are not inhibited as strongly by low cytoplasmic and luminal pH, as suggested by previous single-channel studies.

Cardiac ryanodine receptor activity is altered by oxidizing reagents in either the luminal or cytoplasmic solution.

The location of reactive cysteine residues on the ryanodine receptor (RyR) calcium release channel was assessed from the changes in channel activity when oxidizing or reducing reagents were added to the luminal or cytoplasmic solution. Single sheep cardiac RyRs were incorporated into lipid bilayers with 10(-7) m cytoplasmic Ca2+. The thiol specific-lipophilic-4,4'-dithiodipyridine (4,4'-DTDP, 1 mm), as well as the hydrophilic thimerosal (1 mm), activated and then inhibited RyRs from either the cis (cytoplasmic) or trans (luminal) solutions. Activation was associated with an increase in the (a) mean channel open time and (b) number of exponential components in the open time distribution from one ( approximately 2 msec) to three (approximately 1 msec; approximately 7 msec; approximately 15 msec) in channels activated by trans 4,4'-DTDP or cis or trans thimerosal. A longer component (approximately 75 msec) appeared with cis 4, 4'-DTDP. Activation by either oxidant was reversed by the thiol reducing agent, dithiothreitol. The results suggest that three classes of cysteines are available to 4,4'-DTDP or thimerosal, SHa or SHa* activating the channel and SHi closing the channel. SHa is either distributed over luminal and cytoplasmic RyR domains, or is located within the channel pore. SHi is also located within the transmembrane domain. SHa* is located on the cytoplasmic domain of the protein.

Activation of the cardiac ryanodine receptor by sulfhydryl oxidation is modified by Mg2+ and ATP.

The reactive disulfide 4,4'-dithiodipyridine (4,4' DTDP) was added to single cardiac ryanodine receptors (RyRs) in lipid bilayers. The activity of native RyRs, with cytoplasmic (cis) [Ca2+] of 10(-7) M (in the absence of Mg2+ and ATP), increased within approximately 1 min of addition of 1 mM 4,4'-DTDP, and then irreversibly ceased 5 to 6 min after the addition. Channels, inhibited by either 1 mM cis Mg2+ (10(-7) M cis Ca2+) or by 10 mM cis Mg2+ (10(-3) M cis Ca2+), or activated by 4 mM ATP (10(-7) M cis Ca2+), also responded to 1 mM cis 4,4'-DTDP with activation and then loss of activity. Po and mean open time (T(o)) of the maximally activated channels were lower in the presence of Mg2+ than in its absence, and the number of openings within the long time constant components of the open time distribution was reduced. In contrast to the reduced activation by 1 mM 4,4'-DTDP in channels inhibited by Mg2+, and the previously reported enhanced activation by 4,4'-DTDP in channels activated by Ca2+ or caffeine (Eager et al., 1997), the activation produced by 1 mM cis 4,4'-DTDP was the same in the presence and absence of ATP. These results suggest that there is a physical interaction between the ATP binding domain of the cardiac RyR and the SH groups whose oxidation leads to channel activation.

Actions of sulfhydryl reagents on single ryanodine receptor Ca(2+)-release channels from sheep myocardium.

Effects of the reactive disulfides, 2,2'- and 4,4'-dithiodipyridine, on single cardiac ryanodine receptor (RyR) ion channels incorporated into lipid bilayers are reported. RyRs are activated within minutes of addition of the reactive disulfides (10(-7) to 10(-3) M) with an irreversible loss of channel activity after the activation at concentrations > or = 10(-4) M. This activation, followed by loss of activity, is seen over a wide range of cytoplasmic (cis) Ca2+ concentration between 10(-9) and 2 x 10(-2) M and occurs more rapidly with higher reactive disulfide concentrations or when RyRs are initially active at 10(-5) or 10(-3) M Ca2+. The reactive disulfides increase the channel open probability by introducing long components into the open time distributions, increasing the mean channel open time by up to 50-fold. Closed time distributions are not altered by the sulfhydryl reagents. The effects of the reactive disulfides are prevented by the reducing agents dithiothreitol and glutathione (1-10 mM). The results suggest that cysteine residues on the RyR complex can regulate the ion channel gating mechanisms.

Ion channels in the sarcoplasmic reticulum of striated muscle.

This review provides a summary of current concepts about the structure and single-channel properties of ryanodine receptor calcium release channels and counter ion channels that facilitate Ca2+ release and reuptake by the sarcoplasmic reticulum. Some recent results, obtained with single ryanodine receptor ion channels incorporated into lipid bilayers from terminal cisternae vesicles of rabbit skeletal muscle and sheep ventricular myocardium, are described. The ryanodine receptor is the major Ca2+ release channel in skeletal and cardiac muscle and has been studied in far greater detail than other sarcoplasmic reticulum ion channel proteins. Several ryanodine receptor genes have been cloned and sequenced, and isoforms of the protein have been detected in muscle and in endoplasmic reticulum of brain and many other tissues from mammals, lower vertebrates, nematodes and drosophila. The proteins from all species are tetramers of a peptide with a molecular mass of approximately equal to 560 kDa, containing approximately equal to 5000 amino acids, with a similar maximum single-channel conductance of 500-800 row S for monovalent cations at 250mM. Results presented here include: Ca2+ activation and adaptation of activity in skeletal ryanodine receptors with rapid changes in [Ca2+] controlled by perfusion; activation by FK506 and regulation of cooperative gating of skeletal ryanodine receptor channel activity by FK506-binding proteins; activation and block of cardiac ryanodine receptors by addition of reactive disulphides and by bilayer voltage. Effects of phosphorylation, calmodulin, triadin, calsequestrin and interactions with the alpha 1 subunit of the dihydropyridine receptor on ryanodine receptor activity are summarized. Potassium and chloride channels in skeletal muscle sarcoplasmic reticulum, are described.

Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle.

Ca(2+)-dependent inhibition of native and isolated ryanodine receptor (RyR) calcium release channels from sheep heart and rabbit skeletal muscle was investigated using the lipid bilayer technique. We found that cytoplasmic Ca2+ inhibited cardiac RyRs with an average Km = 15 mM, skeletal RyRs with Km = 0.7 mM and with Hill coefficients of 2 in both isoforms. This is consistent with measurements of Ca2+ release from the sarcoplasmic reticulum (SR) in skinned fibers and with [3H]-ryanodine binding to SR vesicles, but is contrary to previous bilayer studies which were unable to demonstrate Ca(2+)-inhibition in cardiac RyRs (Chu, Fill, Stefani & Entman (1993) J. Membrane Biol. 135, 49-59). Ryanodine prevented Ca2+ from inhibiting either cardiac or skeletal RyRs. Ca(2+)-inhibition in cardiac RyRs appeared to be the most fragile characteristic of channel function, being irreversibly disrupted by 500 mM Cs+, but not by 500 mM K+, in the cis bath or by solublization with the detergent CHAPS. These treatments had no effect on channel regulation by AMP-PNP, caffeine, ryanodine, ruthenium red, or Ca(2+)-activation. Ca(2+)-inhibition in skeletal RyRs was retained in the presence of 500 mM Cs+. Our results provide an explanation for previous findings in which cardiac RyRs in bilayers with 250 mM Cs+ in the solutions fail to demonstrate Ca(2+)-inhibition, while Ca(2+)-inhibition of Ca2+ release is observed in vesicle studies where K+ is the major cation. A comparison of open and closed probability distributions from individual RyRs suggested that the same gating mechanism mediates Ca(2+)-inhibition in skeletal RyRs and cardiac RyRs, with different Ca2+ affinities for inhibition. We conclude that differences in the Ca(2+)-inhibition in cardiac and skeletal channels depends on their Ca2+ binding properties.

Endogenous opioid modulation of hypercapnic-stimulated respiration in the rat.

The role of endogenous opioids in respiratory control in the pentobarbital anaesthetised rat was investigated using a rebreathing technique to generate a progressively increasing hypercapnic stimulus to the respiratory centers following administration of an opioid antagonist or agonist. Respiratory output was measured by intraesophageal pressure (IEP) changes, and a ventilatory equivalent (VEq) was calculated by multiplying IEP by respiratory rate (mmHg.min-1). A non-selective opioid antagonist, naloxone (0.4 mg/kg i.v.), significantly enhanced the slope of the CO2 response curve for VEq (20 +/- 3 mmHg.min-1.%CO2-1) compared with the control (14 +/- 2 mmHg.min-1.%CO2(-1)) (P < 0.05; n = 14). A similar enhancement of the hypercapnic response by naloxone was found in rats anaesthetised with urethane (n = 5). The mu receptor agonist dermorphin (1 mg/kg i.v.) significantly depressed the slope of the CO2 response curve for IEP (-0.01 +/- 0.03) compared with the control (0.10 +/- 0.03) in pentobarbital anaesthetised rats (P < 0.05; n = 5) but had no significant effect on respiratory rate. These results suggest a role of endogenous opioids in the modulation of respiration during hypercapnia.

Immunological characterization of rat kininogens with monoclonal antibodies to T-kininogen. Distinction between the different domains of T-kininogen and the multiple rat kininogens.

A panel of 16 monoclonal antibodies (mAb) were produced against rat T-kininogen to characterize this family of proteins. These mAbs bound 125I-T-kininogen by radioimmunoassay as well as reacting strongly with immobilized T-kininogen in an enzyme-linked immunosorbent assay (ELISA). The reactivity of these antibodies with proteolytic fragments of T-kininogen demonstrated the recognition of several different epitopes. One antibody was specific for the domain 1 of the heavy chain and/or the light chain, twelve antibodies were specific for domain 2 and three antibodies were specific for domain 3. All monoclonal antibodies recognized the two forms of T-kininogen encoded by the two different T-kininogen genes, TI and TII kininogen, except antibody TK 16-3.1 which uniquely reacted with TII kininogen. Two antibodies recognizing domain 2 cross-reacted with the high-molecular-mass kininogen (H-kininogen), whereas all the other monoclonal antibodies were specific to T-kininogen and did not recognize the heavy chain of H-kininogen. None of the antibodies tested altered the thiol protease inhibitory activity of T-kininogen, its partial proteolysis by rat mast cell chymase or the hydrolysis of H-kininogen by rat urinary kallikrein. The use of these antibodies in the development of sensitive ELISA to measure T-kininogen levels in plasma, urine, liver microsomes and hepatocytes is described. Two different forms of T-kininogen were distinguished by these monoclonal antibodies in Western blotting using rat plasma. The localization of T-kininogen was defined using these monoclonal antibodies by immunohistochemistry in rat liver hepatocytes and rat kidney.

Molecular characterization of human trk proto-oncogene product monoclonal antibodies.

Monoclonal antibodies specific for the human trk protooncogene product, a tyrosine kinase receptor, have been produced from mice in which tumors were generated by injection of stably transfected NIH3T3 cells expressing the human trk proto-oncogene product. The panel of eleven antibodies are reactive in ELISA, immunostaining and immunoprecipitation. These antibodies bind to the extracellular domain of the human trk proto-oncogene product and demonstrate no cross-reactivity to the trk oncogene or murine trkB gene products. The antibodies are equally effective in recognizing human proto-trk when expressed by transfected human or mouse cell lines. These antibodies either bind to the carbohydrate moieties or are dependent upon the conformational structure created by the extensive glycosylations. The localization and nature of the epitopes recognized by these monoclonal antibodies were defined by immunoprecipitation analyses using several different trk oncoproteins. Potential applications of these antibodies are discussed.

Murine cell lines stably expressing the influenza virus hemagglutinin gene introduced by a recombinant retrovirus vector are constitutive targets for MHC class I- and class II-restricted T lymphocytes.

A retrovirus vector containing the hemagglutinin (HA) gene of influenza virus was constructed and used to infect murine cell lines of fibroblast, mastocytoma and B cell lineages which are able to present antigens to MHC-restricted T cells. Stable cell lines were selected in which the retrovirus vector integrated as a single copy in almost all of the individual cell clones examined. The HA mRNA was shown to be of the expected length by Northern blot analysis, but the levels varied among the cell clones. Although the HA transcript was difficult to detect in any of the retrovirus-infected cell clones derived from fibroblasts, HA Ag was easily detected on the cell surface by cytofluorographic analysis. Significantly, retrovirus-infected clones derived from each cell type were recognized by HA-specific class I and class II MHC-restricted T lymphocytes. HA produced in these cells was able to be acquired, processed, and presented to class II-restricted T cells by additional, non-HA-expressing APC. This indicates that HA endogenously synthesized within these cell lines is available for Ag processing by an exogenous route.

Modulation of major histocompatibility complex (MHC) class I genes in adenovirus 12 transformed cells: interferon-gamma increases class I expression by a mechanism that circumvents E1A induced-repression and tumor necrosis factor enhances the effect of interferon-gamma.

The protein products of the E1A gene of adenovirus type-12 (Ad12) block transcription of major histocompatibility (MHC) class I genes in both rodent and human transformed cells and interferon-gamma (IFN-gamma) is able to override this repression. Although IFN-gamma is known to stimulate class I transcription, we investigated whether its dominance over E1A repression could alternatively result from the ability of this cytokine to induce antiviral mechanisms. We show that this is not so, since the accumulation of Ad12 E1A mRNA and protein are unabated in the presence of IFN-gamma. Also, tumor necrosis factor (TNF) was shown to act synergistically with IFN-gamma to enhance class I antigen levels, although it had little effect alone. These results suggest that the normal pathway by which IFN-gamma acts to enhance the level of class I mRNAs, circumvents the block by which E1A represses class I transcription.

CTL recognition of adenovirus-transformed cells infected with influenza virus: lysis by anti-influenza CTL parallels adenovirus-12-induced suppression of class I MHC molecules.

We examined the ability of influenza-specific cytotoxic T lymphocytes to lyse adenovirus-transformed cells infected with influenza virus. Cytotoxic T lymphocyte lysis of Ad12-transformed cells was greatly reduced relative to that of Ad5-transformed cells. Lysability of adenovirus-12-transformed cells was restored in parallel with interferon-gamma induced increases in major histocompatibility complex class I gene products. These findings establish that recognition of foreign molecules by self-restricted cytotoxic T lymphocytes is reduced in adenovirus-12-transformed cells. This provides further evidence that Ad12 tumorigenicity is related to its ability to suppress class I major histocompatability complex molecule expression thereby avoiding recognition by the host immune system.

Adenovirus type 12 early region 1A proteins repress class I HLA expression in transformed human cells.

The adenovirus type 12 (Ad12) early region 1A (E1A) gene is thought to play a major role in repressing class I major histocompatibility complex expression in transformed rodent cells. However, since transformation by adenovirus requires both E1A and E1B genes, it has not been demonstrated whether the Ad12 E1A gene acts alone or synergistically with the E1B gene to accomplish this effect. Moreover, it is not known whether the repression of class I antigen synthesis by Ad12-transforming gene products occurs only in rodent cells. We show that the Ad12 E1A gene, in the absence of the E1B gene, is capable of greatly reducing the levels of class I HLA antigens and mRNAs in primary human cells transformed by the E1A gene of Ad12 and the large tumor antigen (T-antigen) gene of BK virus; control cells transformed by BK virus T-antigen gene alone or the highly related simian virus 40 T-antigen gene showed no apparent alteration in class I HLA expression. Human recombinant interferon gamma was able to restore synthesis of class I HLA antigens in transformed cells that produced Ad12 E1A proteins, indicating that these cells were not deficient for class I genes. These results strongly indicate that the Ad12 E1A proteins modulate class I gene expression by similar mechanisms in both transformed rodent and human cells.

Cellular protein differences between nontumorigenic Ad5 and tumorigenic Ad12 transformed mouse cells.

Murine fibroblasts transformed by adenovirus 12 (Ad12) show reduced class I major histocompatibility complex (MHC) antigen synthesis and form tumors in syngeneic mice whereas those transformed by adenovirus 5 (Ad5) show no alteration in class I antigen synthesis and do not form tumors (K. B. Eager, J. Williams, D. Breiding, S. Pan, B. Knowles, E. Appella, and R. P. Ricciardi (1985) Proc. Natl. Acad. Sci. USA 82, 5525-5529). Nearly 1500 metabolically labeled polypeptides from the Ad5 and Ad12 transformed cell lines as well as polypeptides from a nontransformed murine line of the same haplotype were compared by two-dimensional gel electrophoresis. In addition to the reduction of the class I H-2 transplantation antigens seen in the Ad12-transformed lines, we detect few but reproducible polypeptide differences between the tumorigenic and nontumorigenic cell lines.

The use of conventional antisera in the production of specific monoclonal antibodies.

We have shown here that conventionally produced antisera aids in the production of specific monoclonal antibodies. The complex of immunoglobulin plus antigen acted as an effective immunogen in both rats and mice. Stable cell lines producing anit-human alpha 2M monoclonal antibodies to numerous antigenic sites on the alpha 2M molecule have been generated by this method. This method has also been used to produce monoclonal antibodies to human complement components. This procedure of immunizing mice with an immunoprecipitated product derived from conventionally produced antisera is a unique approach in that the antigen is conveniently enriched without tedious and time-consuming biochemical purification. In addition, the use of the immunoprecipitated product in conjunction with the immunoprecipitating antisera allows for rapid screening of the hybridomas in the initial stages when cell growth and maintenance is critical. This method thus further simplifies the task of obtaining a specific monoclonal antibody from a complex mixture. A final consideration is the wide availability of conventionally produced antisera to numerous proteins and other biological substances that could be used in the production of monoclonal antibodies, and consequently these antibodies could be used in more detailed studies of these same molecules.

Expression of histocompatibility antigens H-2K, -D, and -L is reduced in adenovirus-12-transformed mouse cells and is restored by interferon gamma.

Primary mouse cells transformed by adenovirus type 12 (Ad12) expressed negligible amounts of class I antigens H-2K, -D, and -L on the cell surface and were capable of forming tumors in syngeneic animals, whereas cells transformed by Ad5 continued to express class I antigens and were nontumorigenic. Cells from a tumor, generated by injection of Ad12-transformed mouse cells into a syngeneic mouse, also expressed low levels of H-2 antigens, indicating that this phenotype is maintained in vivo. In all Ad12-transformed cells, synthesis of the H-2 heavy chain was not detected whereas the beta 2-microglobulin light chain was synthesized. Furthermore, the level of cytoplasmic H-2 mRNA in the Ad12 lines was greatly reduced. Reduction of H-2 expression is instructed solely by the transforming region of the viral genome, since this repression occurred in cells transformed by a DNA fragment containing only Ad12 E1A and E1B genes. Addition of recombinant murine interferon gamma strongly stimulated expression of class I antigens in the Ad12 transformants as well as in cells from the Ad12 tumor. This result indicates that Ad12 does not preferentially transform cells that are deficient for class I genes and that Ad12 does not mutate the class I genes in cells it transforms. The correlation between tumorigenicity and loss of H-2 expression in Ad12-transformed cells is discussed.