Differential role of MAP kinases in stimulation of hepatocyte growth by EGF and G-protein-coupled receptor agonists

Several agonists acting on G-protein-coupled receptors (GPCR) enhance the mitogenic effect of EGF in rat hepatocytes. Previous studies have shown that mitogen-activated protein (MAP) kinases are involved in the mitogenic effect of EGF. In the present study on cultured rat hepatocytes we show that although the comitogenic GPCR agonists prostaglandin F(2alpha), vasopressin, angiotensin II, and norepinephrine all activated ERK, blocking of the ERK pathway with the MEK inhibitor PD 98059 did not abolish their comitogenic effects. These GPCR agonists also activated p38, but the p38 blocker SB 203580 did not reduce the comitogenic effects. The mitogenic effect of EGF was inhibited completely by PD 98059 and partially by SB 203580. These results suggest that, in contrast to the mitogenic effect of EGF, the comitogenic effect of a group of GPCR agonists is independent of ERK and p38 in these cells.     

Functional separation of the requirements for establishment and maintenance of centromeric heterochromatin

The establishment and maintenance of centromeric heterochromatin in fission yeast require the RITS complex. Comprised of centromeric siRNAs, the chromodomain protein Chp1, Argonaute (Ago1), and Tas3, RITS couples the cellular RNAi pathway with assembly of constitutive heterochromatin. However, the mechanisms governing RITS-dependent establishment versus maintenance of centromeric heterochromatin remain unresolved. Here, we report that a mutant Tas3 protein that cannot bind Ago1 supports the maintenance of centromeric heterochromatin but cannot mediate efficient de novo establishment from cells transiently depleted for the histone H3 lysine 9 methyltransferase Clr4. In contrast, centromeric heterochromatin efficiently assembles in mutant cells transiently depleted for dicer. This mutant therefore allows ordering of the events leading to establishment of centromeric heterochromatin and places lysine 9 methylation of histone H3 upstream of dicer function.     

Epac- and Rap- independent ERK1/2 phosphorylation induced by Gs-coupled receptor stimulation in HEK293 cells

Serotonin activates Ras and Ras-dependent ERK1/2 phosphorylation in HEK293 cells expressing G(s)-coupled 5-HT(4) or 5-HT(7) serotonin receptors through unknown mechanisms. Both Epac/Rap-dependent and -independent pathways for Ras-dependent ERK1/2 activation have been suggested. Epac overexpression or Epac-specific 8-CPT-2'-O-Me-cAMP did not cause ERK1/2 phosphorylation, despite Rap activation. The data did not support a role for PLCepsilon or DAG-dependent Ras GEFs of the Ras-GRP family in Ras-dependent ERK1/2 phosphorylation. However, serotonin stimulated phosphorylation of endogenous and recombinant Ras-GRF1, increased [Ca(2+)](i) and caused Ca(2+)- and calmodulin-dependent ERK1/2 phosphorylation. Different signalling pathways seem to be utilised by G(s)-coupled receptors in various isolates of HEK293 cells.     

Effects of pertussis toxin on extracellular signal-regulated kinase activation in hepatocytes by hormones and receptor-independent agents: evidence suggesting a stimulatory role of G(i) proteins at a level distal to receptor coupling

It was previously found that pertussis toxin (PTX) pretreatment inhibits the activation of extracellular signal-regulated kinases ERK1 (p44(mapk)) and ERK2 (p42(mapk)) in hepatocytes in response to either agonists that bind to heptahelical receptors or epidermal growth factor (EGF), suggesting a role of G(i) proteins in stimulatory mechanisms for ERK1/2. The present work shows that ERK1/2 is activated in a PTX-sensitive way not only by vasopressin, angiotensin II, prostaglandin (PG) F(2alpha), alpha(1)-adrenergic stimulation, and EGF but also by agents whose actions bypass receptors and stimulate protein kinase C (PKC) and/or elevate intracellular Ca(2+), such as 12-O-tetradecanoyl phorbol-13-acetate (TPA), exogenous phosphatidylcholine-specific phospholipase C (PC-PLC, from Bacillus cereus), thapsigargin, and the Ca(2+) ionophore A23187. Under the same conditions, PTX did not affect agonist stimulation of phosphoinositide-specific phospholipase C (PI-PLC) (IP(3) generation), and did not reduce the activation by these agents of phospholipase D (PLD). The results suggest that in hepatocytes a PTX-sensitive mechanism, presumably involving G(i) proteins, exerts a stimulatory effect on ERK at a level distal to receptor coupling, acting either as an integral part of the signaling pathway(s) or by a permissive, synergistic regulation.     

Normal ranges of some blood chemistry parameters in adult farmed Atlantic salmon, Salmo salar

Blood samples from healthy adult Atlantic salmon fed an optimal diet in net sea pens were collected at intervals from October to May. Haematological determinations and biochemical serum analyses were carried out on 20 fish in each of seven samples. The ranges of haemato-logical values for sample means were: haematocrit 44–49%, haemoglobin 8.9–10.4 g 100ml⁻¹, red blood cell count 0.85–1.10 × 10¹² l⁻¹, MCV 441–553 × 10⁻¹⁵ 1, MCH 94–106 × 10⁻⁶ g, MCHC 19.4–21.7 g 100ml⁻¹ and leucocrit 0.43–0.96%. The ranges of enzyme activities in serum, for sample means, were: alkaline phosphatase 647–988Ul⁻¹, aspartate aminotrans-ferase 202–351 Ul⁻¹ and alanine aminotransferase 4–8 Ul⁻¹. The ranges of the other parameters analyzed in serum were: total protein 41.6–56.6 gl⁻¹, albumin 18.3–24.3 gl⁻¹, albumin/total protein ratio 39.3–44.0%, creatinine 26–46 μmol, triglycerides 2.53–4.98 μmol and cholesterol 9.3–12.8 μmol. These values are considered to be the normal ranges in healthy fish. Variations due to seasonal changes, and the clinical significance of the selected parameters, are discussed. Data showing the reproducibility of the biochemical analyses in serum are presented.     

Role of diacylglycerol (DAG) in hormonal induction of S phase in hepatocytes: The DAG-dependent protein kinase C pathway is not activated by epidermal growth factor (EGF), but is involved in mediating the enhancement of responsiveness to EGF by vasopressin,angiotensin II,and norepinephrine

The role of diacylglycerol (DAG) in hormonal induction of S phase was investigated in primary cultures of rat hepatocytes. In this model, several agonists that bind to G protein-coupled receptors act as comitogens when added to the cells soon after plating (i.e., in G_o/early G_l phase), while the cells are most responsive to the mitogenic effect of epidermal growth factor (EGF) at 24–48 h of culturing (i.e., mid/late G_l). It was found that the cellular concentration of DAG rose markedly and progressively during the first 24 h of culturing. Exposure of the hepatocytes at 3 h to α_l-adrenergic stimulation (norepinephrine with timolol), vasopressin, or angiotensin II further increased this rise, producing a sustained increase in the DAG level. Norepinephrine, which was the most efficient comitogen, produced the most prolonged DAG elevation. In contrast, no significant increase of DAG was found in response to EGF, neither at 3 nor at 24 h, using concentrations that markedly stimulated the ERK subgroup of the mitogen-activated protein kinases (MAPK) and DNA synthesis. Addition of Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLC) strongly elevated DAG, while Streptomyces phospholipase D (PLD) increased phosphatidic acid (PA) but not DAG. B. cereus PC-PLC and the protein kinase C (PKC) activator tetradecanoyl phorbol-acetate (TPA), like norepinephrine, vasopressin, and angiotensin II, stimulated MAPK and enhanced the stimulatory effect of EGF on DNA synthesis. The PKC inhibitor GF109203X did not diminish the effect of EGF on MAPK or DNA synthesis, but strongly inhibited the effects of norepinephrine, vasopressin, angiotensin II, TPA and B. cereus PC-PLC on MAPK and almost abolished the enhancement by these agents of EGF-stimulated DNA synthesis. These results suggest that although generation of DAG is not a direct downstream response mediating the effects of the EGF receptor in hepatocytes, a sustained elevation of DAG with activation of PKC markedly increases the responsiveness to EGF. Mechanisms involving DAG and PKC seem to play a role in the comitogenic effects of various agents that bind to G protein-coupled receptors and activate the cells early in G_l, such as norepinephrine, angiotensin II, and vasopressin. J. Cell. Physiol. 180:203–214, 1999. © 1999 Wiley-Liss, Inc.     

Persistent Postural-Perceptual Dizziness: A Matter of Higher,Central Dysfunction?

Objective

Persistent postural-perceptual dizziness (PPPD) is the most common vestibular disorder in the age group between 30 and 50 years. It is considered to be based on a multisensory maladjustment involving alterations of sensory response pattern including vestibular, visual and motion stimuli. Previous data supported a link between vestibular and pain mechanism. The aim of the study was to investigate whether other sensory inputs such as pain stimuli might be altered in terms of a more widespread central perception dysfunction in this disorder.

Methods

Nociceptive blink reflex was measured in 27 patients with PPPD and compared with 27 healthy, age and gender matched controls. The habituation of the R2 component of the blink reflex was evaluated as the percentage area-under-the curve (AUC) decrease in ten consecutive blocks of five averaged rectified responses. Additionally, clinical characteristics were evaluated.

Results

In patients with PPPD a lack of habituation was observed compared to healthy controls. Relative AUC decreased between the first and the tenth block by 19.48% in PPPD patients and by 31.63% (p = 0.035) in healthy controls. There was no correlation between clinical data (course of disease, comorbid depression, medication, trigger factors) or electrophysiological data (perception threshold, pain threshold, stimulus intensity) and habituation pattern. No trigeminal sensitization in terms of facilitation of absolute values could be detected.

Conclusion

Our study results supports the hypothesis of the multisensory dimension of impaired sensory processing in patients with PPPD extends beyond vestibular/visual motion stimuli and reflexive postural/oculomotor control mechanisms to other sensory inputs such as pain perception in terms of a more generalized disturbed habituation pattern.     

The epidemiological investigation of Trichinella infection in brown rats (Rattus norvegicus) and domestic pigs in Croatia suggests that rats are not a reservoir at the farm level

Whether the brown rat (Rattus norvegicus) is a reservoir of Trichinella spp. infection or merely an accidental host, which may be vector of Trichinella spp., continues to be debated. We estimated the prevalence of Trichinella sp. infection in brown rat populations and in domestic pigs in 2 villages in Croatia, where Trichinella sp. infection in pigs has been endemic in the past 10 yr. Trichinella spiralis larvae, identified by a multiplex polymerase chain reaction analyses, were the only species detected in both rats and pigs. In 2001 and 2002, 2,287 rats were collected on 60 farms with different levels of sanitation and with, or without, T. spiralis-infected pigs. The prevalence of infection in rats ranged from 0.2 to 10.7%. Infected rats were detected only on farms with T. spiralis-positive pigs and low sanitation or formerly with low sanitation (P = 0.007, Fisher's exact test), yet no infected rat was detected on farms with T. spiralis-negative pigs. The finding that no infected rat was found on farms with T. spiralis-negative pigs suggests that, in the investigated area, the brown rat is not a reservoir but only a victim of improper pig slaughtering.