The TRP ion channel family.

Mammalian homologues of the Drosophila transient receptor potential (TRP) channel gene encode a family of at least 20 ion channel proteins. They are widely distributed in mammalian tissues, but their specific physiological functions are largely unknown. A common theme that links the TRP channels is their activation or modulation by phosphatidylinositol signal transduction pathways. The channel subunits have six transmembrane domains that most probably assemble into tetramers to form non-selective cationic channels, which allow for the influx of calcium ions into cells. Three subgroups comprise the TRP channel family; the best understood of these mediates responses to painful stimuli. Other proposed functions include repletion of intracellular calcium stores, receptor-mediated excitation and modulation of the cell cycle.

TRP-PLIK, a bifunctional protein with kinase and ion channel activities.

We cloned and characterized a protein kinase and ion channel, TRP-PLIK. As part of the long transient receptor potential channel subfamily implicated in control of cell division, it is a protein that is both an ion channel and a protein kinase. TRP-PLIK phosphorylated itself, displayed a wide tissue distribution, and, when expressed in CHO-K1 cells, constituted a nonselective, calcium-permeant, 105-picosiemen, steeply outwardly rectifying conductance. The zinc finger containing alpha-kinase domain was functional. Inactivation of the kinase activity by site-directed mutagenesis and the channel's dependence on intracellular adenosine triphosphate (ATP) demonstrated that the channel's kinase activity is essential for channel function.

Determination of the affinities between heterotrimeric G protein subunits and their phospholipase C-beta effectors.

Phosphatidylinositide-specific phospholipase C-betas play a key role in Ca2+ signaling and are specifically activated by the alphaq family of heterotrimeric G proteins and as well as betagamma subunits. We have determined the affinity between Gbetagamma subunits and GTPgammaS and GDP-liganded Galphaq subunits on membrane surfaces, and their respective affinities to PLC-beta1, -beta2 and -beta3 effectors by fluorescence spectroscopy. We find that activation of Galphaq by GTPgammaS decreases its affinity for Gbetagamma subunits at least 36-fold compared to the GDP-liganded form, but increases its affinity for PLC-betas at least 40-200-fold depending on the PLC-beta isoform. The affinity of Galphaq(GTPgammaS) is similar for PLC-beta1 and -beta3 and 10-fold stronger for PLC-beta2, which corresponds to the reported relationship between the concentration of Galphaq(GTPgammaS) and PLC-beta activation on lipid bilayers. We find that a large portion of the PLC-beta-Galphaq association energy lies within the 400 residue C-terminal region of PLC-beta1 since truncating this region reduces its Galphaq affinity. In contrast, the isolated N-terminal region does not interact with Galphaq. Gbetagamma subunits interact with all three PLC-beta isotypes, but only showed strong binding to PLC-beta2, and activation of the three PLC-betas by Gbetagamma subunits parallels this behavior. We also tested the possibility that both Galphaq and Gbetagamma can simultaneously bind PLC-beta2. Our data argue against simultaneous binding and show that Galphaq and Gbetagamma independently regulate this effector.

Regulation of the rate and extent of phospholipase C beta 2 effector activation by the beta gamma subunits of heterotrimeric G proteins.

The activity of mammalian phosphoinositide-specific phospholipase C beta 2 (PLC-beta 2) is regulated by the alpha q family of G proteins and by beta gamma subunits. We measured the affinity between the laterally associating PLC-beta 2 and G beta gamma on membrane surfaces by fluorescence resonance energy transfer. Using a simple model, we translated this apparent affinity to a bulk or three-dimensional equilibrium constant (Kd) and obtained a value of 3.2 microM. We confirmed this Kd by separately measuring the on and off (kf and kr) rate constants. The kf was slower than a diffusion-limited value, suggesting that conformational changes occur when the two proteins interact. The off rate shows that the PLC-beta 2.G beta gamma complexes are long-lived ( approximately 123 s) and that activation of PLC-beta 2 by G beta gamma would be sustained without a deactivating factor. The addition of alpha i1(GDP) subunits failed to physically dissociate the complex as determined by fluorescence. However, enzyme activity studies performed under similar conditions show that the addition of G alpha i1(GDP) results in reversal of PLC-beta 2 activation by G beta gamma during the time of the assay (30 s). From these results, we propose that G alpha(GDP) subunits can bind to the PLC-beta 2.G beta gamma complex to allow for rapid deactivation without complex dissociation. In support of this model, we show by fluorescence that G alpha i1(GDP).G beta gamma.PLC-beta 2 can form.

Phosphoinositide binding specificity among phospholipase C isozymes as determined by photo-cross-linking to novel substrate and product analogs.

We tested for the presence of high-affinity phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and PI(3,4,5)P3 binding sites in four phospholipase C (PLC) isozymes (delta1, beta1, beta2, and beta3), by probing these proteins with analogs of inositol phosphates, D-Ins(1,4,5)P3, D-Ins(1,3,4,5)P4, and InsP6, and polyphosphoinositides PI(4,5)P2 and PI(3,4,5)P3, which contain a photoactivatable benzoyldihydrocinnamide moiety. Only PLC-delta1 was specifically radiolabeled. More than 90% of the label was found in tryptic and chymotryptic fragments which reacted with antisera against the pleckstrin homology (PH) domain, whereas less than 5% was recovered in fragments that encompassed the catalytic core. In separate experiments, the isolated delta1-PH domain was also specifically labeled. Equilibrium binding of D-Ins(1,4,5)P3 to PLC-delta1 indicated the presence of a single, high-affinity binding site; binding of D-Ins(1,4,5)P3 to PLC-beta1, -beta2, or -beta3 was not detected. The catalytic activity of PLC-delta1 was inhibited by the product D-Ins(1,4,5)P3, whereas no inhibition of PLC-beta1, -beta2, or -beta3 activity was observed. These results demonstrate that the PH domain is the sole high-affinity PI(4,5)P2 binding site of PLC-delta1 and that a similar site is not present in PLC-beta1, -beta2, or -beta3. The data are consistent with the idea that the PH domain of PLC-delta1, but not the beta isozymes, directs the catalytic core to membranes enriched in PI(4,5)P2 and is subject to product inhibition.

Membrane binding of phospholipases C-beta 1 and C-beta 2 is independent of phosphatidylinositol 4,5-bisphosphate and the alpha and beta gamma subunits of G proteins.

We have measured the membrane binding affinities of purified phosphatidylinositol-specific phospholipases C-beta 1 and C-beta 2 to membranes of varying lipid composition using fluorescence methods. Our studies show that these proteins bind with affinities of 10(-5)-10(-4) M, with a small dependence on lipid type. Binding was relatively insensitive to the presence of phosphatidylinositol-specific phospholipases C-beta s' major physiological substrate, phosphatidylinositiol 4,5-bisphosphate, as well as the presence of Ca2+, which is required for activity. The presence of purified GTP gamma S-activated alpha 11 subunits of heterotrimeric guanine nucleotide binding proteins (G proteins) did not alter the membrane binding affinity of phosphatidylinositol-specific phospholipases C-beta 1, even though alpha 11 is a potent activator of this protein. Similarly, the presence of purified beta gamma subunits of G proteins did not alter the membrane association of phosphatidylinositol-specific phospholipases C-beta 2 even though these subunits strongly activate this isoform. These results argue against a recruitment model for PLC-beta activation by G proteins, negatively charged lipids, Ca2+, or substrate, and suggest that activation occurs through association of the membrane-bound species.

Myristoylated alanine-rich C kinase substrate (MARCKS) produces reversible inhibition of phospholipase C by sequestering phosphatidylinositol 4,5-bisphosphate in lateral domains.

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major protein kinase C (PKC) substrate in many different cell types. MARCKS is bound to the plasma membrane, and several recent studies suggest that this binding requires both hydrophobic insertion of its myristate chain into the bilayer and electrostatic interaction of its cluster of basic residues with acidic lipids. Phosphorylation of MARCKS by PKC introduces negative charges into the basic cluster, reducing its electrostatic interaction with acidic lipids and producing translocation of MARCKS from membrane to cytoplasm. The present study shows that physiological concentrations of MARCKS (<10 microM) inhibit phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in phospholipid vesicles. A peptide corresponding to the basic cluster, MARCKS(151-175), produces a similar inhibition, which was observed with both PLC-delta1 and -beta1. Direct fluorescence microscopy observations demonstrate that the MARCKS peptide forms lateral domains enriched in the acidic lipids phosphatidylserine and PIP2 but not PLC, which accounts for the observed inhibition of PIP2 hydrolysis. Phosphorylation of MARCKS(151-175) by PKC releases the inhibition and allows PLC to produce a burst of inositol 1,4, 5-trisphosphate and diacylglycerol.

Theory and application of fluorescence homotransfer to melittin oligomerization.

Fluorescence homotransfer (electronic energy transfer between identical fluorophores) has the potential to quantitate the number of subunits in membrane protein oligomers. Homotransfer strongly depolarizes fluorescence emission as a result of intermolecular excitation energy exchange between an initially excited, oriented molecule and a randomly oriented neighbor. We have theoretically treated fluorescein labeled subunits in an oligomer as a cluster of molecules that can exchange excitation energy back and forth among the subunits within that group. We find that the larger the number of subunits, the more depolarized is the emission. The general equations to calculate the expected anisotropy for complexes composed of varying numbers of labeled subunits are presented. Self-quenching of fluorophores, orientation, and changes in lifetime are also discussed and/or considered. To test this theory, we have specifically labeled melittin on its N-terminal with fluorescein and monitored its monomer to tetramer equilibrium both in solution and in lipid bilayers. The calculated anisotropies are close to the experimental values when non-fluorescent fluorescein dimers are taken into account. Our results show that homotransfer may be a promising method to study membrane-protein oligomerization.

Relationship to growth performance of pneumonia and atrophic rhinitis lesions detected in pigs at slaughter among four seasons.

A commercial swine herd was selected for study, because pigs at slaughter repeatedly had lung lesions consistent with enzootic pneumonia and had snout lesions typical of atrophic rhinitis. Pigs born during various seasons of the year were allotted to 4 investigations and were evaluated from birth to slaughter. Individual lungs and snouts were identified and collected at the slaughter plant and later examined for gross lesions of bronchopneumonia and atrophic rhinitis, respectively. Each lesion was scored, and the following comparisons were made within investigations: prevalence and mean scores for lung lesions; prevalence and mean grades for snout lesions; correlations between lung lesion scores and growth indicators; correlations between snout lesion grades and growth indicators; and correlations between lung lesion scores and snout grade scores. Included in the growth indicators were average daily gain during the growing phase, average daily gain during the finishing phase, average daily gain during growing and finishing phases, and days to attain 104.5 kg of body weight. Prevalence of lung or snout lesions, mean values for lung lesion scores, mean values for snout lesion grades, and mean values for the various growth indicators were tested for statistical differences among the 4 investigations. Prevalence of lung lesions was highest (96%) for winter-slaughtered and lowest (81%) for autumn-slaughtered pigs. Mean scores for lung lesions were 7% (summer), 5% (autumn), 9% (winter), and 16% (spring). Prevalence of snout lesions was highest (85%) for spring-slaughtered pigs and lowest (42%) for autumn-slaughtered pigs.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathologic response of the lung to irritant gases.

The pathologic response of the lung to irritant gases ranges from the acute exudative phase through the subacute proliferative phase to the chronic fibrosing phase. These responses are based on damage to the Type I cells, and possibly endothelial cells, and the subsequent proliferative and repair processes in the surviving animals. Responses to high dose exposures appear at the microscopic level as exudation of protein rich fluids into alveoli (alveolar edema) and subsequent death due to anoxia. Physiologically, this could be described as a mismatch of ventilation with perfusion, resulting in impaired gas exchange. Animals surviving this acute exudative phase resolve the alveolar edema to fibrin, and Type II cells become hypertrophic and hyperplastic in the process of replacing the damaged Type I cells. The acute and subacute responses also elicit inflammatory changes in the interstitium of the lung that may progress to fibrosis in the chronic stage of a survivable exposure. Diagnostic cases in livestock involving irritant gases reflect similar toxic injuries to the lung.

Relationship of growth performance to pneumonia and atrophic rhinitis detected in pigs at slaughter.

Three commercial swine herds were selected for study, because pigs at slaughter consistently had lung lesions typical of bronchopneumonia and snout lesions consistent with atrophic rhinitis. Pigs were reared in the conventional system for each herd except that they were identified at birth and weighted at various intervals. At slaughter, individual pig lungs and snout were examined for lesions of pneumonia and atrophic rhinitis, respectively. Lesions were scored and correlated with growth indicators for each pig. Included in the growth indicators were: average daily gain (growing phase), average daily gain (finishing phase), average daily gain (total), and days to reach 104.5-kg body weight. Additionally, for each pig, scores for lung lesions were correlated to grades for snout lesions. Three correlation coefficients for measurements of pigs within herd B were significant and included days to 104.5-kg body weight and grades for snout lesions, -0.15 (P less than 0.02); average daily gain (finishing) and grades for snout lesions, 0.17 (P less than 0.01); and average daily gain (total) and grades for snout lesions, 0.16 (P less than 0.01). Contrary to findings in other investigations, pigs that attained market weight at the youngest age did not have the lowest score for lung lesions, the lowest grade for snout lesions, or the least extensive or severe lesions. Combining data from all 3 herds, the mean scores for lung lesions and mean grades for snout lesions decreased significantly (P less than 0.05) as the age of pigs at slaughter increased. All other statistical correlations were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Monensin toxicosis in swine: potentiation by tiamulin administration and ameliorative effect of treatment with selenium and/or vitamin E.

Modulation of acute monensin toxicosis in swine was evaluated in 2 studies. In study 1, 56 weanling male pigs were allotted to 14 groups of 4 each. Pigs in 7 groups were given tiamulin in the drinking water (to supply 7.7 mg/kg of body weight/day) for 3 days before and for 2 days after monensin administration. Monensin was given as a single oral dose (at 0, 7.5, 15, 25, 50, 75, or 100 mg/kg) to pigs in groups with or without tiamulin exposure. Prominent acute clinical signs of monensin toxicosis (hypermetria, hind limb ataxia, paresis, knuckling of hind limbs, and recumbency) developed by 2 to 6 hours after dosing in pigs given 15 or 25 mg of monensin/kg with tiamulin exposure, but not in pigs given the 15 or 25 mg of monensin/kg without tiamulin exposure. Also, the extent of monensin-induced skeletal muscle damage at 4 days after monensin dosing was enhanced in pigs given 7.5, 15, or 25 mg of monensin/kg and exposed to tiamulin. In study 2, 48 weanling male pigs were allotted to 8 groups of 6 each. Four groups of pigs were given 20 mg of monensin/kg orally, and 4 groups were given 100 mg of monensin/kg orally. For each monensin dose, a group was treated 24 hours before monensin administration with (i) selenium (Se)-vitamin E preparation, 0.25 mg of Se and 68 IU of d-alpha-tocopheryl acetate (vitamin E)/kg, IM; (ii) vitamin E only, 68 IU of d-alpha-tocopheryl acetate/kg; (iii) Se only, 0.25 mg of Se/kg; or (iv) vehicle.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of criteria for the postmortem diagnosis of mycoplasmal pneumonia of swine.

Ten swine from each of five herds believed to be affected with mycoplasmal pneumonia of swine and ten swine from each of five herds believed to be mycoplasmal pneumonia-free were selected for postmortem study. Lungs from the 100 swine were examined; grossly and microscopically for lesions typical of mycoplasmal pneumonia of swine and culturally and by an indirect immunofluorescent procedure for the presence of Mycoplasma hyopneumoniae. Nineteen of the lungs had both gross and microscopic lesions typical of mycoplasmal pneumonia of swine and 13 (68%) of these were infected, i.e. were culturally and/or indirect immunofluorescent positive. Absence of gross lesions did not prove freedom from mycoplasmal pneumonia, 14 of 73 (19%) grossly normal lungs were found to be infected with M. hyopneumoniae. Comparison of the indirect immunofluorescent and cultural examination, as methods of diagnosing mycoplasma pneumonia, revealed that neither procedure alone was reliable in the case of negative results. Ten lungs were indirect immunofluorescent negative and culturally positive and seven were culturally negative and indirect immunofluorescent positive (11 lungs were positive by both procedures). It was concluded that a definitive diagnosis of mycoplasmal pneumonia of swine requires that M. hyopneumoniae be visualized in indirect immunofluorescent stained lung sections or that it be recovered culturally.

Comparison of the enzyme-linked immunosorbent assay and the indirect hemagglutination and complement fixation tests for detecting antibodies to Mycoplasma hyopneumoniae.

Caesarean-derived, colostrum-deprived swine were exposed to a broth culture of a low passage field isolate of Mycoplasma hyopneumoniae by intranasal inoculation. The intranasal-inoculated swine subsequently were commingled with their litter-mates to effect transmission via contact-exposure. Sera were collected from the swine at two to four week intervals for approximately one year postexposure and evaluated by the enzyme-linked immunosorbent assay (ELISA), indirect hemagglutination and complement fixation tests. The intranasal-exposed swine seroconverted earlier, developed higher titers and remained indirect hemagglutination and complement fixation positive longer than the contact-exposed swine. It was concluded that the antibody response of intranasal-exposed swine was artificially high and that sera from such swine were not suitable for evaluating the sensitivity of mycoplasmal pneumonia of swine serodiagnostic tests. The indirect hemagglutination test was relatively insensitive and technically cumbersome and the least promising as a practical field test. The complement fixation test appeared to be slightly more sensitive in detecting early antibody production (especially in contact-exposed swine) but it was the least sensitive in detecting late antibodies. The ELISA was generally the most sensitive procedure. Individual high ELISA titers were from ten to 32 times greater than maximum complement fixation and indirect hemagglutination titers. The most striking difference among the three tests was the persistence of high ELISA titers late in the study. All swine were ELISA positive at necropsy approximately one year postexposure despite the fact that lungs were devoid of lesions and culturally and immunofluorescent negative for M. hyopneumoniae.

Pseudorabies virus, porcine parvovirus, and porcine enterovirus interactions with the zona pellucida of the porcine embryo.

Porcine embryos (n = 93) were incubated on cell monolayers that had been previously inoculated with pseudorabies virus, porcine parvovirus (PPV), or each of 2 porcine enteroviruses. After 2, 24, or 48 hours of incubation, the embryos were fixed in glutaraldehyde and examined by electron microscopic procedures. It was found that pseudorabies virus adsorbed to the zona pellucida (ZP) and entered sperm tracks in the ZP. The PPV and both enteroviruses entered pores in the ZP and were associated with sperm that were at or near the outer surface of the ZP. In addition, PPV was seen enmeshed in cellular debris on the outer surface of the ZP. Evidence of a productive viral infection of the blastomeres of the embryos was not found.

A model for the packing of cells.

A model is presented for the statistical and geometrical aspects of the packing of cells. The model is closely related to lattice polymer models and the Ising model of ferromagnetism. Fundamental questions of existence and analyticity in the infinite volume limit are discussed, and the results of some initial numerical studies are given.

Equine granulosa cell tumors.

Unilateral ovariectomy was performed on 3 mares affected with granulosa cell tumors. Tumor fluid in each mare was found to contain estrogen, testosterone, and progesterone. In 2 mares, preoperative blood plasma concentrations of these hormones were comparable to those of a series of clinically normal mares. The other mare, which had a history of aggressive, masculine behavior, had higher testosterone content in the tumor fluid and in the preoperative blood sample. After surgical removal of the tumors, each mare developed follicles and ovulated with the remaining ovary. Each was eventually bred and 2 conceived. The probability of metastasis of these tumors in mares appears uncertain. Data from other species suggests a guarded long-term prognosis may be justified.