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PubMed1984

Common features of the yes and src gene products defined by peptide-specific antibodies.

Anti-peptide antibodies generated against a hydrophilic domain of pp60src comprising amino acid residues 498 through 512 were shown to be cross-reactive with the corresponding region in the yes transforming proteins encoded by Yamaguchi 73 and Esh sarcoma viruses. This cross-reactivity was demonstrated by immunoblot and immunoprecipitation analyses, and the identity of the proteins was verified by partial proteolytic mapping. By utilizing a combination of immunofluorescence and interference-reflection microscopy, these cross-reactive anti-peptide antibodies were shown to produce an immunofluorescence staining pattern in Yamaguchi 73 and Esh sarcoma virus-transformed chicken embryo fibroblasts remarkably similar to that pp60src in Rous sarcoma virus-infected chicken cells. Like the src gene products, the yes transformation-specific polyproteins were found to be concentrated within adhesion plaque structures and needle-like interdigitating cell-cell junctions. This analogous subcellular distribution suggests that these onc proteins are functionally related and may share common intracellular targets.

Amino Acid Sequence↗
PubMed2006

Mouse-adapted scrapie infection of SN56 cells: greater efficiency with microsome-associated versus purified PrP-res.

The process by which transmissible spongiform encephalopathy agents, or prions, infect cells is unknown. We employed a new differentiable cell line (SN56) susceptible to infection with three mouse-adapted scrapie strains to gain insight into the cellular infection process. The effect of disease-associated PrP (PrP-res) association with microsomal membranes on infection efficiency was examined by comparing sustained PrP-res production in cells treated with either scrapie brain microsomes or purified, detergent-extracted PrP-res. When normalized for quantity of input PrP-res, scrapie brain microsomes induced dramatically enhanced persistent PrP-res formation compared to purified PrP-res. Infected SN56 cells released low levels of PrP-res into the culture supernatant, which also efficiently initiated infection in recipient cells. Interestingly, microsomes labeled with a fluorescent marker were internalized by SN56 cells in small vesicles, which were subsequently found in neuritic processes. When bound to culture wells to reduce internalization during the infection process, scrapie microsomes induced less long-term PrP-res production than suspended microsomes. Long-term differentiation of infected SN56 cells was accompanied by a decrease in PrP-res formation. Our observations provide evidence that infection of cells is aided by the association of PrP-res with membranes and/or other microsomal constituents.

Animals↗
PubMed1995

A deficiency of the small GTPase rab8 inhibits membrane traffic in developing neurons.

One of the major activities of developing neurons is the transport of new membrane to the growing axon. Candidates for playing a key role in the regulation of this intense traffic are the small GTP-binding proteins of the rab family. We have used hippocampal neurons in culture and analyzed membrane traffic activity after suppressing the expression of the small GTP-binding protein rab8. Inhibition of protein expression was accomplished by using sequence-specific antisense oligonucleotides. While rab8 depletion resulted in the blockage of morphological maturation in 95% of the neurons, suppression of expression of another rab protein, rab3a, had no effect, and all neurons developed normal axons and dendrites. The impairment of neuronal maturation by rab8 antisense treatment was due to inhibition of membrane traffic. Thus, by using video-enhanced differential interference contrast microscopy, we observed in the rab8-depleted cells a dramatic reduction in the number of vesicles undergoing anterograde transport. Moreover, by incubating antisense-treated neurons with Bodipy-labeled ceramide, a fluorescent marker for newly formed exocytic vesicles, we observed fluorescence labeling restricted to the Golgi apparatus, whereas in control cells labeling was found also in the neurites. These results show the role of the small GTPase rab8 in membrane traffic during neuronal process outgrowth.

Animals↗
PubMed2005

Regulation of cell polarity by interactions of Msb3 and Msb4 with Cdc42 and polarisome components.

In Saccharomyces cerevisiae, polarized growth depends on interactions between the actin cytoskeleton and the secretory machinery. Here we show that the Rab GTPase-activating proteins (GAPs) Msb3 and Msb4 interact directly with Spa2, a scaffold protein of the "polarisome" that also interacts with the formin Bni1. Spa2 is required for the polarized localization of Msb3 and Msb4 at the bud tip. We also show that Msb3 and Msb4 bind specifically to Cdc42-GDP and Rho1-GDP in vitro and that Msb3 and Rho GDP dissociation inhibitor act independently but oppositely on Cdc42. Finally, we show that Msb3 and Msb4 are involved in Bni1-nucleated actin assembly in vivo. These results suggest that Msb3 and Msb4 regulate polarized growth by multiple mechanisms, directly regulating exocytosis through their GAP activity toward Sec4 and potentially coordinating the functions of Cdc42, Rho1, and Bni1 in the polarisome through their binding to these GTPases. A functional equivalent of the polarisome probably exists in other fungi and mammals.

Actins↗
PubMed1992

Upregulation of insulin-like growth factor I gene expression in the lesions of osteoarthritic human articular cartilage.

Expression of insulin-like growth factor I (IGF-I)mRNA and IGF-I protein was studied in human osteoarthritic and young articular cartilage by in situ hybridisation and immunohistochemistry. In situ hybridisation showed that relatively low amounts of IGF-I mRNA signal were present in anatomically normal regions of osteoarthritic and young cartilage. In fibrillated osteoarthritic cartilage, however, the signal intensity was significantly higher than in non-fibrillated cartilage. Particularly high levels of IGF-I mRNA were present in the surface cell clones of more advanced lesions, the amount of signal being about four to five times greater than in anatomically normal cartilage. The amount of message varied with cartilage depth. In young cartilage there was less IGF-I mRNA in the superficial zone than in the middle and deep zones. In fibrillated regions of osteoarthritic joints the amount of message in surface cells was greater than in deeper regions. A specific human IGF-I antibody was used to show the presence intracellularly of IGF-I protein in osteoarthritic and young cartilage. Raised levels of IGF-I message in osteoarthritic chondrocytes may represent an attempt at increased matrix repair, operating by an autocrine/paracrine mechanism.

Adolescent↗
PubMed2001

The Humphrey optical coherence tomography scanner: quantitative analysis and reproducibility study of the normal human retinal nerve fibre layer.

BACKGROUND/AIMS: To determine the reproducibility of the Humphrey optical coherence tomography scanner (OCT), software version 5.0, for measurement of retinal nerve fibre layer (RNFL) thickness in normal subjects and to compare OCT measurements with published histological thickness of the human RNFL. METHODS: Three independent measurements were obtained at each session for one eye from 15 normal subjects with a mean age of 30.8 (SD 10.9) years. Scans were taken in the peripapillary retina using the default setting (1.74 mm radius from centre of the optic disc) and were repeated 1 week later. Additional scans were obtained at the optic nerve head (ONH) margin overlying the scleral rim, for comparison with available histological data on the human RNFL. RESULTS: For the 1.74 mm circular scan, the mean coefficient of variation (COV) for the global RNFL thickness measurement was 5% (SD 3%). This increased to 8% (3%) for quadrant measurements and to 9% (3%) with further subdivision into 12 segments. Significant differences (p<0.05) between sessions were only found when the data were divided into segments. The mean RNFL thickness for the 1.74 mm scan was 127.87 (9.81) microm. The RNFL was maximal at the superior disc pole, 161.44 microm (14.8), and minimal at the temporal pole, 83.1 (12.8) microm. Peak thickness values occurred superior temporal and inferior temporal to the vertical axis. RNFL thickness for every sector of the disc was greatest at the margin of the optic disc (mean 185.79 microm; SD 32.61). Although the variation in RNFL thickness around the disc follows published histology data, the OCT underestimates RNFL thickness by an average of 37% (SD 11; range 21-48%). CONCLUSION: The OCT provides reproducible measurement of the retinal structures that are consistent with the properties of the RNFL. However, comparison with available studies of RNFL thickness in the human suggests that in its present form, the OCT underestimates RNFL thickness. Further refinement of this technology is required to improve the accuracy with which the OCT measures retinal nerve fibre layer thickness.

Adult↗
PubMed2002

Prediction of visual outcome after retinal detachment surgery using the Lotmar visometer.

AIM: To evaluate whether an achromatic interferometer, the Lotmar visometer, is useful in predicting postoperative visual outcome in patients with primary rhegmatogenous retinal detachment (RD) involving the macula. METHODS: This prospective study included 40 eyes of 40 non-consecutive patients with macula-off RD. The eyes were phakic or pseudophakic, had a clear optical media, and had a measurable potential vision on preoperative visometric examination. Preoperative variables included Snellen visual acuity, duration of macular detachment, extent of RD, and visometric potential acuity. Reattachment surgery consisted of radial scleral buckling in 33 patients, circumferential scleral buckling and encircling in seven patients, and subretinal fluid drainage in 10 patients. Retinal breaks were treated with cryotherapy or laser photocoagulation. Patients were followed up for at least 6 months after uncomplicated surgery. Best corrected visual acuity measured at any time during follow up was correlated with the preoperative variables. RESULTS: Preoperative visual acuity was less than 20/200 in 37 (93%) of 40 patients. Potential visual acuity of 20/200 or better was measured using the Lotmar visometer in 37 patients (93%). Postoperative visual acuity was correlated significantly with duration of macular detachment (r=0.55; p<0.001), and extent of RD approached statistical significance (r=0.31; p=0.05). There was a higher correlation between postoperative visual acuity and the visometric measurements (r=0.61; p<0.001). CONCLUSIONS: The Lotmar visometer may be a valuable method to estimate visual outcome after uncomplicated scleral buckling surgery in patients with RD involving the macula.

Adult↗
PubMed1995

Functional hyposplenism following allogeneic bone marrow transplantation.

AIMS: To investigate the incidence of functional hyposplenism in a group of patients who had undergone allogeneic bone marrow transplantation (BMT). METHODS: Splenic function was assessed by counting the number of gluteraldehyde fixed red blood cells containing pits or indentations as examined by interference phase microscopy. Normal values are < 2% whereas splenectomy patients have values of 25 to 40%. RESULTS: Twenty eight BMT recipients (17 men, 11 women) were studied at varying periods post-transplant and the results compared with 20 healthy volunteers and 10 patients who had undergone splenectomy or had splenic atrophy because of haematological conditions. Of the 28 BMT recipients, one had undergone a prior splenectomy; of the remaining 27 patients, four (15%) had evidence of functional hyposplenism with between 5.0 and 34.0% pitted cells. Of these four patients, one had active extensive chronic graft versus host disease (GvHD) which has been previously reported to be associated with functional hyposplenism following transplantation. Only one of the four patients had peripheral blood red cell changes typical of hyposplenism. CONCLUSION: These results confirm that extensive chronic GvHD is associated with hyposplenism. Intermediate degrees of functional hyposplenism may also occur following BMT in the absence of chronic GvHD and in the absence of haematological features of hyposplenism on routine blood films. This may be of significance in mediating the susceptibility to infection with encapsulating bacteria seen following allogeneic BMT.

Adolescent↗
PubMed1975

The development and structure of thick-walled, multicellular, aerial spores in Diheterospora chlamydosporia (=Verticillium chlamydosporium).

The aerial, thick-walled spores in Diheterospara chlamydosporia arose as terminal swellings on erect hyphae. Repeated septation of the continuously swelling spore resulted in a multicellular structure. Immediately after the onset of septation secondary wall material was laid down between the two-layered primary wall and the plasmalemma. The presence of secondary wall material indicates that the multicellular spore is a dictyochlamydospore and not an aleuriospore. The relationship between chlamydospores and aleuriospores in other fungi is discussed.

Cell Membrane↗
PubMed1986

Hypertonic cell volume regulation in mouse thick limbs. I. ADH dependency and nephron heterogeneity.

Differential interference contrast microscopy was used in combination with standard electrophysiological techniques in the in vitro perfused mouse medullary (mTALH) and cortical (cTALH) thick ascending limbs of Henle to evaluate the cell volume responses of these nephron segments to sudden increases in peritubular osmolality and to assess the role of antidiuretic hormone (ADH) and net NaCl absorption on hypertonic volume regulation. In the absence of CO2/HCO3- in external media, the cells of the mTALH behaved in a simple osmometric fashion, with an osmotic space equivalent to 70-80% of the total cell volume. However, in CO2/HCO3- -containing media, the cells of the mTALH, but not the cTALH, were able to increase their cell volume to the original volume after shrinkage in peritubular media made hypertonic with either NaCl or mannitol. This volume-regulatory increase response (VRI) in the mTALH was mediated by an increase in intracellular osmoles, and required peritubular ADH, at concentrations that stimulate maximally the rate of net NaCl absorption. This ADH effect on VRI could be mimicked by addition of dibutyryladenosine 3',5'-cyclic monophosphate to the bath in the absence of hormone. However, 10(-4) M luminal furosemide, a concentration that abolishes ADH-dependent NaCl absorption in the mTALH, had no effect on the VRI response. These results indicate that the cells of the mTALH, but not the cTALH, are capable of hypertonic volume regulation, that ADH (via adenosine 3',5'-cyclic monophosphate) is required for expression of the VRI response in the mTALH, and that the effects of ADH on net NaCl absorption and the VRI response in the mTALH are completely dissociable. Thus these results are consistent with a role for ADH in hypertonic VRI in the mammalian mTALH, which may operate to maintain constant cell volume in this nephron segment during antidiuresis.

Absorption↗
PubMed1986

Hypertonic cell volume regulation in mouse thick limbs. II. Na+-H+ and Cl(-)-HCO3- exchange in basolateral membranes.

Differential interference contrast microscopy and standard electrophysiological techniques were used to evaluate the transport processes involved in antidiuretic hormone (ADH)-dependent hypertonic cell volume regulation in the in vitro perfused mouse medullary thick ascending limb of Henle. Hypertonic cell volume regulation appeared to involve NaCl uptake into cells, since the cell volume increase after osmotic shrinkage in hypertonic media could be abolished either by symmetrical removal of Na+ from external solutions or by bath Cl- omission. The volume-regulatory process also required CO2/HCO3- in external media and could be abolished by the lipophilic carbonic anhydrase inhibitor, ethoxzolamide, in the presence of CO2/HCO3-. In addition, ADH-dependent hypertonic cell volume regulation was reduced or abolished by 10(-4) M amiloride, 10(-3) M ouabain, or 10(-4) M 4-acetamido-4'-isothiocyanostilbene-2,2-disulfonic acid in peritubular media or by cooling to 15 degrees C. In contrast, lumen Cl- omission or 10(-4) M amiloride addition to the perfusate had no effect on cell volume regulation in hypertonic peritubular media. These data suggest that ADH-dependent, hypertonic cell volume regulation in the mouse medullary thick limb depends on cell NaCl uptake via a secondary active transport process involving parallel Na+-H+ and Cl(-)-HCO3- exchangers in basolateral cell membranes. Finally, luminal furosemide (10(-4) M) abolished bath ouabain-mediated, rapid cell swelling in isotonic media containing ADH. Thus these exchangers do not appear to be active in the resting, isotonic state. The specific role of ADH in this NaCl transport process remains to be defined.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1986

Vasoactive agents affect mesangial cell adhesion.

The formation and maintenance of stress fibers in cultured mesangial cells is associated with myosin light chain phosphorylation [Kreisberg et al. Am. J. Physiol. 249 (Renal Fluid Electrolyte Physiol. 18): F227-F235, 1985], a biochemical indicator for activation of actin-myosin interactions. Agents that elevate intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) (e.g., isoproterenol) fragment stress fibers and cause myosin light chain dephosphorylation, whereas the addition of contractile agents such as arginine vasopressin (AVP) and prostaglandin E2 (PGE2) reverses these changes. Because stress fiber development in cultured cells is correlated with tight cell to substrate adhesion, we wanted to examine whether vasoactive agents have an effect on mesangial cell adhesion. Both isoproterenol and dibutyryl cAMP (DBcAMP) reduced mesangial cell adherence as measured by a trypsin assay (% detached cells: control 11 +/- 2.4%; isoproterenol plus isobutylmethylxanthine (IBMX) = 48.3 +/- 7.4%; DBcAMP = 29.3 +/- 3.7%; DBcAMP-IBMX = 73 +/- 4.4%). The areas of focal (adhesive) contacts between the cell and substratum as observed by interference-reflexion microscopy were also reduced, being replaced by areas of greater separation (% of the surface in contact with the substratum: control = 7.4 +/- 0.8%; isoproterenol-IBMX = 2.9 +/- 1.1%). Addition of PGE2 or AVP to the incubation medium containing the cAMP-elevating agents prevented the above changes. PGE2 or AVP alone increased mesangial cell adhesion (% detached cells: control 11 +/- 2.4%; PGE2 = 6.8 +/- 0.5%; AVP = 5.1 +/- 1.2%).(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1990

[Ca2+]i modulation of Cl- content controls cell volume in single salivary acinar cells during fluid secretion.

Differential interference contrast microscopy and low-light-level digital imaging of the fluorescent chloride indicator dye 6-methyl-1-(3-sulfonatopropyl)quinolinium (SPQ) were performed simultaneously in single mammalian salivary gland acinar cells to examine the relationship between cytoplasmic chloride concentration [( Cl-]i) and cell volume during stimulus-secretion coupling. Agonist stimulation of Cl(-)-driven fluid secretion is associated with rapid, Ca2(+)-dependent changes of cell volume, which are temporally coupled to changes of [Cl-]i. The agonist-induced changes in [Cl-]i, if accompanied by cations and water, quantitatively account for the cell volume changes, demonstrating in a single cell that cell volume is determined by cell solute content. Agonist-induced modulation of cell volume appears to be a consequence of the requirement to develop appropriate ion gradients necessary for vectorial salt (and fluid) transport.

Animals↗
PubMed1996

Protein phosphatases independently regulate vesicle movement and microtubule subpopulations in hepatocytes.

To investigate the regulation of microtubule (MT)-based vesicle transport and the interphase MT array in hepatocytes, we have used okadaic acid (OKA) and microcystin (MCYST), two toxins that inhibit serine-threonine protein phosphatases (PP) 1 and 2A, to alter cellular phosphorylation. Video-enhanced differential interference contrast microscopy analysis revealed that both toxins inhibited the frequency, velocity, and run length of MT-dependent vesicle movements dose dependently between 50 and 500 nM. At our maximum dose of 500 nM, both toxins significantly decreased PP2A activity (OKA to 45 +/- 12% and MCYST to 57 +/- 2%), whereas PP1 was inhibited only by MCYST. Because no additional effects on vesicle movements were caused by MCYST over the changes caused by OKA, these data implicate PP2A in the regulation of MT-dependent vesicle movement. To understand whether the changes in parameters of vesicle movements were due to changes in the MT array, the effects of these toxins on MT distribution were examined by immunofluorescence microscopy. Although lower doses of OKA produced no effects, treatment with 500 nM OKA altered MT organization and also caused fragmentation and loss of acetylated (stable) MTs. In contrast, MCYST concentrations up to 500 nM elicited no changes in MT organization in general or in the acetylated (stable) array. From these findings we conclude that inhibition of MT-dependent vesicle movement by the PP inhibitors, MCYST and OKA, in hepatocytes cannot result from changes or disruption in the MT array. Because OKA (an inhibitor of PP2A only in our system) at high doses caused loss of stable MTs, whereas MCYST (an inhibitor of both PP1 and PP2A) did not, we conclude that the control of the preservation of the stable MT array in hepatocytes is complex. Stable MTs require active PP2A for maintenance, but the disruption of the array through inhibition of PP2A can be prevented if PP1 is also inhibited, suggesting that the relative degree of phosphorylation of multiple cellular components is the determinant of MT stability.

Animals↗