A direct method for the simultaneous measurement of ceramide and phospholipase D activity.

Both ceramide and phospholipase D (PLD) have important roles in a variety of signal transduction pathways. Recent evidence suggests that ceramide is a novel second messenger with specific biological effects. Publications in this field have increased rapidly in the last few years. However, a method to directly and rapidly measure cermide production has been lacking. Herein, we report on a novel, inexpensive, direct and rapid assay for the measurement of ceramide and the simultaneous measurement of PLD activity. This method uses labeling of cells with [(14)C]myristic acid and a TLC solvent of ethyl acetate/acetic acid/trimethylpentane. This method avoids the loss of radioactivity and variability due to changes in DAG kinase activity that are associated with the commonly-used DAG kinase assay.

Arrays of multielement ultrasound applicators for interstitial hyperthermia.

Arrays of multielement ultrasound applicators for interstitial hyperthermia have been developed and tested both in vitro and in vivo. The system includes multielement applicators, a 64 channel RF driving unit, a power measuring unit, a 112 channel multisensor temperature measuring unit, and a water cooling unit. Ninety-five arrays of single-element and nine arrays of three-element ultrasound applicators were designed, built, and characterized by measuring transducer efficiency and ultrasound field distribution. Improved uniformity in the azimuthal direction was achieved by using multiple driving frequencies. In addition, production of ultrasound in a desired sector of the transducer was possible by selecting a suitable frequency. Both in vitro and in vivo experiments showed that 92% of monitored temperature points within the target volume of 30 mm x 30 mm x 35 mm achieved a therapeutic temperature rise (above 5 degrees C) when an array of five three-element applicators were used. These results indicated that the arrays of multielement ultrasound applicators have distinct advantages over present interstitial hyperthermia modalities in terms of the capability to control the temperature distribution with a large catheter spacing. As a conclusion, the feasibility of a practical arrays of multielement ultrasound applicators for interstitial hyperthermia was demonstrated.

Bradykinin inhibits ceramide production and activates phospholipase D in rabbit cortical collecting duct cells.

Recent reports suggest that inflammatory cytokines, growth factors, and vasoconstrictor peptides induce sphingomyelinase (SMase) activity. This results in the hydrolysis of sphingomyelin (SM) into ceramide, which is implicated in various cellular functions. Although ceramide regulates phospholipase D (PLD) activity, there is controversy about this relationship. Thus we investigated whether the effect of bradykinin (BK), a proinflammatory factor and vasodilator, was mediated by ceramide signal transduction and by PLD. In rabbit cortical collecting duct (RCCD) cells, BK increased SM levels and decreased ceramide levels in a time-dependent manner. Thus SMase activity was inhibited by BK. Also, the production of ceramide was regulated in a concentration-dependent manner. The BK-B1 antagonist [Lys-des-Arg9,Leu8]BK did not affect ceramide signal transduction but the BK-B2 antagonist (Hoe-140) blocked the effect of BK on SMase, suggesting that the BK-B2 receptor mediates BK-induced inhibition of ceramide generation. Our results show that exogenous SMase significantly hydrolyzed endogenous SM to form ceramide and weakly activated PLD. In contrast, BK induced a significant activation of PLD. However, additive effects of BK and ceramide on PLD activity were not observed. We concluded that in RCCD cells, the BK-induced second messengers ceramide and phosphatidic acid were generated by distinct signal transduction mechanisms, namely the SMase and PLD pathways.

Advances in the signal transduction of ceramide and related sphingolipids.

Recently, the sphingolipid metabolites ceramide, sphingosine, ceramide 1-P, and sphingosine 1-P have been implicated as second messengers involved in many different cellular functions. Publications on this topic are appearing at a rapidly increasing rate and new developments in this field are also appearing rapidly. It is thus important to summarize the results obtained from many different laboratories and from different fields of research to obtain a clearer picture of the importance of sphingolipid metabolites. This article reviews the studies from the last few years and includes the effects of a variety of extracellular agents on sphingolipid signal transduction pathways in different tissues and cells and on the mechanisms of regulation. Sphingomyelin exists in a number of functionally distinct pools and is composed of distinct molecular species. Sphingomyelin metabolites may be formed by many different pathways. For example, the generation of ceramide from sphingomyelin can be catalyzed by at least five different sphingomyelinases. A large variety of stimuli can induce the generation of ceramide, leading to activation or inhibition of various cellular events such as proliferation, differentiation, apoptosis, and inflammation. The effect of ceramide on these physiological processes is due to its many different downstream targets. It can activate ceramide-activated protein kinases and ceramide-activated protein phosphatases. It also activates or inhibits PKCs, PLD, PLA2, PC-PLC, nitric oxide synthase, and the ERK and SAPK/JNK signaling cascades. Ceramide activates or inhibits transcription factors, modulates calcium homeostasis and interacts with the retinoblastoma protein to regulate cell cycle progression. Most of the work in this field has involved the study of ceramide effects, but the roles of the other three sphingomyelin metabolites is now attracting much attention. The complex interactions between signaling components and ceramide and the controls regulating these interactions are now being identified and are presented in this review.

The presence of an unusual PKC isozyme profile in rat liver cells.

We have previously shown that protein kinase C (PKC) is involved in the mitogenic response of T51B cells to epidermal growth factor. In fact, epidermal growth factor was an excellent mitogen, even after prolonged pretreatment of cells with TPA, suggesting that the PKC isoform implicated in proliferation is not down-regulated by 12-O-tetradecanoyl phorbol-13-acetate (TPA). We have now determined that the PKC isozymes -alpha, -beta, -delta, -epsilon, and -zeta are present in T51B cells. All five isoforms are associated with the plasma membrane and the cytoplasm and are either in or around the nucleus. PKC-beta I has a slightly different subcellular profile from that of the other isoforms in that it is clearly and strongly associated with the nuclear membrane. Also, a unique and novel pattern is obtained from immunoblots with anti-PKC-beta I. PKC-beta I is detected as a single band of 70 kDa in the cytosolic fraction and as a doublet of 65 and 77 kDa in the membrane fraction. PKC-alpha, -delta, and -epsilon were down-regulated by pretreatment of cells with TPA, while PKC-zeta was unaffected. Of particular interest was the fact that TPA did not down-regulate PKC-beta I. In fact, the amount of this isoform associated with the plasma membrane increased. These findings indicate that it is probably PKC-beta I that is involved in the mitogenic response of T51B cells to epidermal growth factor. Since PKC-zeta is also not down-regulated by TPA, the possible involvement of this isoform needs to be resolved.

Computed tomography and magnetic resonance imaging of cavernous sinus enlargement in a dog with unilateral exophthalmos.

Computed tomography (CT), magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA) were performed on a dog with a two year history of unilateral exophthalmos occurring two years following head trauma. On CT images, an expansile enhancing mass was present along the right intracranial cavernous sinus and extended through the orbital fissure into the retrobulbar space. With MRI, the structure appeared as a signal void due to the presence of rapidly flowing blood. Gadolinium enhancement of the adjacent brain was not present. A vascular origin of the lesion was confirmed with MRA. Based on the CT and MRI findings, the enlarged cavernous sinus and associated ophthalmic plexus were believed to represent an arterialized aneurysm, most likely the result of traumatic arteriovenous fistulization. Treatment consisted of surgical enucleation. At the time of this report, 29 months later, the dog remains free of clinical signs.

Biological response of nonhuman primates to long-term repeated lung exposure to Ad2/CFTR-2.

We have assessed the safety and efficacy of repeated adenovirus vector administration by exposing the left caudal lung lobe of rhesus monkeys to as many as 17 exposures of Ad2/CFTR-2. After nine doses of either 3 x 10(9) or 3 x 10(10) infectious units, the monkeys were free of adverse effects as assessed by thoracic radiographs, CBCs, clinical chemistries, arterial blood gases, and physical and clinical signs. In some animals elevated protein levels and increased numbers of cells were recovered in bronchoalveolar lavage (BAL), and in all animals there were increased proportions of lymphocytes in the BAL. After 11 doses, two animals were killed. In the lower dose animal (3 x 10(9) IU), there was little histopathology evident. In the higher dose animal (3 x 10(10) IU), histopathology was largely confined to a focal fibrotic lesion that may have been associated with treatment. At the tenth exposure, the dose was increased to 6 x 10(10) or 3 x 10(11) IU. There was evidence of lung injury by thoracic radiographs after two additional exposures and an increase in protein and number of cells in the BAL. The animals were still free of evidence of adverse effects by other parameters, but histopathologic changes were noted upon death. After 15 or 17 doses, three animals were instilled with Ad2/beta gal-2 and killed 3 days later. These animals had greatly reduced levels of transgene expression when compared with controls.

In vivo characterization of cytotoxic intracellular edema by multicomponent analysis of transverse magnetization decay curves.

RATIONALE AND OBJECTIVES: We investigated the multicompartmental nature of T2 decay in a specific white matter edema model. METHODS: Triethyltin (TET) intoxication was produced in six male New Zealand White rabbits. Images were obtained over the 23-day study duration using a 64-echo Carr-Purcell-Meiboom-Gill (CPMG) sequence (repetition time = 3000 msec, echo time = 20 msec). T2 decay curves were extracted from 0.7 x 0.7 x 3.0 mm3 voxels in the corpus callosum and contiguous white matter tracts, cortex, thalamic nuclei, hypothalamic nuclei, and the masseter muscles. The curves were fit with biexponential functions. RESULTS: Increased signal intensity in the corpus callosum was evident 2-3 days after the first TET injection. At this time, a substantial slowly relaxing component appeared in the decay curves of the corpus callosum and, to a lesser extent, in the thalamus and hypothalamus. Changes in the rabbits' body weight, general physical condition, and neurologic state paralleled the growth and regression of the second, slowly relaxing component. CONCLUSION: The appearance and regression of a slowly decaying second component in the T2 decay curve is consistent with the formation and shrink-age of intracellular vesicles in the intramyelin sheaths of central white matter.

EGF causes hyperproliferation and apoptosis in T51B cells: involvement of high and low affinity EGFR binding sites.

We have recently described an in vitro model for liver homeostasis which involves growing T51B rat liver epithelial cells with 1 nM epidermal growth factor (EGF). There is an initial period of hyperplasia, lasting about 3 days, which is followed by an increase in apoptosis. The cell density returns to around the confluent control level 5 days after EGF addition. The dose response of T51B cells to EGF shows three distinct growth patterns. We have carried out EGF binding studies that suggest that the occupancy of the low affinity binding site of the EGF receptor, is responsible for the hyperproliferation seen when the cells are grown with high doses of EGF. These studies also suggest that the apoptosis could be triggered by down-regulation of the receptor, in a manner analogous to the removal of a trophic hormone in other systems.

Expression of clusterin (testosterone-repressed prostate message-2) mRNA during growth and regeneration of rat liver.

Clusterin has been used as a marker for apoptosis (often denoted "active" "or programmed" cell death) in the prostate, mammary gland and other solid organs. The protein is thought to be involved in membrane remodelling during separation of apoptotic cells from their vital neighbours and fragmentation into apoptotic bodies. In the present study, we have looked at the expression of clusterin during the growth and regression of rat liver induced by short term administration of the hepatomitogen, cyproterone acetate. The steady state level of clusterin mRNA, as measured by Northern and slot blot analysis, is low in control hepatocytes. Following administration of cyproterone acetate, the clusterin mRNA level is increased during both liver growth and regression. In situ hybridization reveals that clusterin is expressed in all hepatocytes, indicating that it is not confined to cell death by apoptosis. These results suggest that the gene product may be involved in maintaining membrane integrity, which is necessary during both mitosis and apoptosis. To determine whether clusterin mRNA is induced by membrane remodelling independent of either mitosis or apoptosis, we examined the expression of clusterin mRNA in the liver after a necrogenic dose of carbon tetrachloride. During the first 24-48 h of this time period, necrosis is the predominant form of cell death and liver regeneration starts after approximately 24 h. Elevated levels of clusterin mRNA are found as early as 12 h after carbon tetrachloride administration and persist for at least 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term exposure to epidermal growth factor results in apoptosis in T51B cells.

The major objective of this research was the development of an in vitro model for liver homeostasis that would allow the future study of early events in cell proliferation and cell death. The model that was set up involves growing T51B rat liver epithelial cells with a single dose of 1 nM epidermal growth factor (EGF). This results in a period of hyperplasia where the cells reach double the control cell numbers 2 days after EGF addition. This is then followed by a decrease in cell numbers and the cell density returns to around the confluent control level 5 days after EGF addition. The model was investigated to ascertain whether the decrease in cell numbers 3-5 days after EGF addition was due to an increase in apoptosis. The results from light and electron microscopy studies, electrophoresis of T51B cell DNA, and quantification of nuclear fragmentation indicated that the cells do die via an increase in apoptosis. The electron microscopy studies also show that healthy T51B cells can phagocytose apoptotic bodies. This suggests that the model is more physiological than other in vitro models of apoptosis. This work describes the development and characterization an in vitro model of liver homeostasis that closely parallels in vivo systems where animals are given mitogenic stimuli.

The epidermal growth factor mitogenic signal is modulated by protein kinase C in T51B rat liver cells.

The regulation of cell proliferation involves a complex interplay between several signal transduction pathways. The effect of EGF on DNA synthesis in serum starved quiescent, synchronized T51B cells was investigated by [3H]thymidine incorporation and flow cytometry. 1 nM EGF or readdition of serum initiated G1 progression and entry into S phase by 18 h and DNA synthesis reached a maximum by 28 h. Low concentrations of EGF markedly stimulated DNA synthesis, but EGF was not as potent as readdition of serum. The effect of EGF on DNA synthesis was only partially blocked by the tyrosine inhibitors genistein and tyrphostin, suggesting that other signalling pathways play a role in EGF-stimulated mitogenesis. 1 nM EGF caused a rapid, transient increase in the activity of membrane-associated protein kinase C (PKC) followed by a longer sustained increase that continued into S phase. TPA (12-O-tetradecanoyl-phorbol-13-acetate) did not mimic EGF, rather it caused a slight stimulation of membrane-associated PKC activity within 1 h followed by a dramatic downregulation of PKC within 4 h. TPA was without effect on DNA synthesis alone, but when added along with EGF or serum TPA caused a significant enhancement of DNA synthesis. Pretreatment of quiescent, serum-deprived T51B cells with TPA reduced the basal level of DNA synthesis; however, under these conditions EGF became as potent a mitogen as serum. We hypothesize that EGF via activation of PKC regulates the activity of its receptor by switching from high affinity to low affinity states. Downregulation of PKC by long term treatment with TPA removes this regulation thus rendering T15B cells more sensitive to exogenous EGF.

Bent subcutaneous peritoneal catheter shape for regular dialysis treatment in children.

A permanent bowing of the subcutaneous part of the Tenckhoff-type catheter (bent neck--Quinton, and swan neck--Accurate Surgical Instruments) enables the catheter to turn from an upward direction of the subcutaneous tunnel to a downward direction by a smooth 160 degrees-180 degrees bend creating a downward skin exit. We have used this catheter shape in combination with a coiled intra-abdominal edge. Two sizes are available for children. We use 2 cuffs and glue them ourselves according to the body size. In this study we compare the durability of the traditional subcutaneously straight catheter in 8 children aged 0.1-12.6 years (Group A) with the bent shaped catheter in 8 children aged 3.7-15.8 years (Group B). Median duration of function was 10.5 (2-34) and 8 (3-36) months, respectively. Frequency of complications was equal in both groups: peritonitis episodes 0.69/year in Group A and 0.53/year in Group B; tunnel infection 0.16 vs 0.11/year; skin exit infection 0.54 vs 0.53/year; noninfectious complications 0.16 vs 0.32/year; mean number of catheters used was 1.0 vs 1.1/treatment year. Treatment had to be terminated in some patients: kidney transplantation 5, kidney recovery 1, severe peritonitis 1. The bent subcutaneous catheter shape did not show any medical or technical disadvantage compared with the straight type, but the downward directed catheter skin exit can be covered invisibly under bikini or bermuda shorts which means aesthetic and social advantage. Whether the downward drainage of secretes and cell detritus influences the rate of tunnel infection positively cannot be answered to date.

Characterization of cerebral infarction by multicomponent analysis of transverse magnetization decay curves.

RATIONALE AND OBJECTIVES: Individual components of the transverse magnetization decay curve (TDC) were assessed for their ability to characterize ischemia in photochemically induced cerebral infarcts. METHODS: Fifty rats were randomly divided into equal-sized experimental and control groups, which were subdivided into groups studied at five different time points, ranging from 6 hours to 22 days. All the rats received transcalvarial irradiation with 560-nm light. Five rats in each time group also received a sensitizing dye before irradiation. In these latter animals, lesions of uniform size and location developed. Lesions were compared with tissue of similar volume and location from the contralateral cortex of the experimental animals and with tissue from both hemispheres of the control animals. TDCs of all the samples were measured and fit with mono- and bi-exponential functions. RESULTS: Unlike the control tissue, infarcted tissue displayed definitive two-component TDC behavior. The time course of the bi-exponential parameters yielded information unavailable from mono-exponential analyses. CONCLUSIONS: Bi-exponential analysis of TDCs may have diagnostic use as a more sensitive indicator of cerebral infarction than mono-exponential analyses.

Activation of protein kinase C sensitizes the cyclic AMP signalling system of T51B rat liver cells.

Activation of protein kinase C (PKC) by phorbol esters (TPA) results in a modification of the cyclic AMP system leading to either attenuation or amplification of the cyclic AMP signal. In the non-neoplastic T51B rat liver cell line, TPA, when added to intact cells, had no effect on the basal level of cyclic AMP synthesis but caused a 1.5 fold amplification of the stimulation induced by beta-adrenergic agents, cholera toxin and forskolin. The effect appeared to be mediated by PKC since diacylglycerols caused the same amplification as did TPA while inactive phorbol esters were without effect. Phosphorylation of Gs or the catalytic subunit of adenylate cyclase by PKC is likely to be responsible for the enhancement of cyclic AMP synthesis. TPA also caused translocation of PKC; however, the time course of the translocation was longer than the time course of the enhancement of adenylate cyclase activity. Thus, the ability of TPA to amplify cyclic AMP synthesis is probably mediated by activation of PKC that is already present in the membrane.

Nonsurgically managed caudal acetabular fractures in dogs: 15 cases (1979-1984).

Medical records of 15 dogs with nonsurgically managed caudal one-third acetabular fractures were reviewed. In-hospital evaluation of these dogs was possible 6 to 67 months after injury. Thirteen dogs had radiographic evidence of moderate to severe degenerative joint disease in affected hips. Twelve dogs had decreased hip joint range of motion and/or signs of pain on the affected side, and 7 dogs were lame. Because of limited pain-free motion and degenerative joint disease in most affected hips, results of nonsurgical management of caudal one-third acetabular fractures in dogs were considered unsatisfactory.

[Peritonitis and infection in children with idiopathic nephrotic syndrome]. / Peritonitis und Sepsis bei Kindern mit idiopathischem nephrotischem Syndrom.

We analyzed episodes of peritonitis and/or sepsis associated with the idiopathic nephrotic syndrome (ns) in 23 children treated between 1975 and 1985 at our clinic. 37.5% of the children with infantile ns, 16% of those with steroidsensible ns, and 13.6% of those with steroidresistant ns developed peritonitis. Children with infantile ns and one girl with gram-negative secondary peritonitis presented with sepsis. 3 septic children died. Four patients developed peritonitis secondary to intestinal perforation. The most common bacterial pathogen in primary peritonitis was S. pneumoniae (7 patients). 7 cases were culture-negative. All episodes of peritonitis coincided with an active nephrotic syndrome: in more than half of the patients therapy with corticosteroids had already been started. Eight patients underwent surgical exploration for presumed appendicitis, but none was confirmed by histological examination of the appendix. In 2 instances S. pneumoniae was cultured from ascitic fluid. Prophylactic polyvalent pneumococcal vaccination and early start of corticotherapy during the acute illness of ns is warranted.

Serum-activated T51B rat liver cells transiently accumulate cyclic AMP-dependent protein kinases on their surfaces during the G1 phase.

Confluent T51B rat liver epithelial cells promptly began accumulating cyclic AMP-binding sites on their surfaces when they were stimulated from quiescence by serum growth factors in medium containing 1.8 mM Ca2+, but they began losing the accumulated binding sites shortly before initiating DNA replication. When the medium contained only 0.02 mM Ca2+, the cells still accumulated surface cyclic AMP-binding sites, but they did not initiate DNA replication and tended to continue accumulating the binding sites. The cyclic AMP-binding sites were eliminated completely by treating intact cells for 5 minutes with 0.005% trypsin (which did not damage the cells), and cyclic AMP caused them to be released from intact, undamaged cells into the medium. The binding sites also comigrated electrophoretically with purified regulatory subunits of type I cyclic AMP-dependent protein kinase, and to a lesser extent the regulatory subunit of type II cyclic AMP-dependent protein kinase. Therefore, it is likely that a transient accumulation of cyclic AMP-dependent protein kinases on the outer surface of the plasma membrane is part of the T51B rat liver cell's prereplicate program.