Trypanosoma musculi: tracking parasites and circulating lymphoid cells in host mice

Hyperglycemia can cause vascular dysfunctions by multiple factors including hyperosmolarity, oxidant formation, and protein kinase C (PKC) activation. We have characterized the effect of hyperglycemia on p38 mitogen-activated protein (p38) kinase activation, which can be induced by oxidants, hyperosmolarity, and proinflammatory cytokines, leading to apoptosis, cell growth, and gene regulation. Glucose at 16.5 mM increased p38 kinase activity in a time-dependent manner compared with 5.5 mM in rat aortic smooth muscle cells (SMC). Mannitol activated p38 kinase only at or greater than 22 mM. High glucose levels and a PKC agonist activated p38 kinase, and a PKC inhibitor, GF109203X, prevented its activation. However, p38 kinase activation by mannitol or tumor necrosis factor-alpha was not inhibited by GF109203X. Changes in PKC isoform distribution after exposure to 16.5 mM glucose in SMC suggested that both PKC-beta2 and PKC-delta isoforms were increased. Activities of p38 kinase in PKC-delta- but not PKC-beta1-overexpressed SMC were increased compared with control cells. Activation of p38 kinase was also observed and characterized in various vascular cells in culture and aorta from diabetic rats. Thus, moderate hyperglycemia can activate p38 kinase by a PKC-delta isoform-dependent pathway, but glucose at extremely elevated levels can also activate p38 kinase by hyperosmolarity via a PKC-independent pathway.     

Role of MEKK1 in cell survival and activation of JNK and ERK pathways defined by targeted gene disruption

Targeted disruption of the gene encoding MEK kinase 1 (MEKK1), a mitogen-activated protein kinase (MAPK) kinase kinase, defined its function in the regulation of MAPK pathways and cell survival. MEKK1(-/-) embryonic stem cells from mice had lost or altered responses of the c-Jun amino-terminal kinase (JNK) to microtubule disruption and cold stress but activated JNK normally in response to heat shock, anisomycin, and ultraviolet irradiation. Activation of JNK was lost and that of extracellular signal-regulated protein kinase (ERK) was diminished in response to hyperosmolarity and serum factors in MEKK1(-/-) cells. Loss of MEKK1 expression resulted in a greater apoptotic response of cells to hyperosmolarity and microtubule disruption. When activated by specific stresses that alter cell shape and the cytoskeleton, MEKK1 signals to protect cells from apoptosis.     

Hyperosmolar comatose states

A recently determined entity, hyperosmolar coma is a good example of a clinical candition attributable to a fundamental alteration, namely extracellular hyperosmolarity. The principal stages in its history are summarised. The laws governing osmosis are recalled and the various features of the sndrome are studied. Attention is drawn to the clinical peculiarities (skin dehydration), biological features (record glycaemia value), and EEG data noted in 17 cases treated in a resuscitation centre over a period of seven years, along with the treatment employed-this being still a matter of debate. Reference is also made to 52 cases of acute drunkenness in a study of the relation between blood alcohol values and plasma hyperosmolarity.     

Response of isolated rat liver mitochondria to variation of external osmolarity in KCl medium: regulation of matrix volume and oxidative phosphorylation

When isolated rat liver mitochondria are incubated in KCI medium, matrix volume, flux, and forces in both hypo- and hyperosmolarity are time-dependent. In hypoosmotic KCl medium, matrix volume is regulated via the K+/H+ exchanger. In hyperosmotic medium, the volume is regulated in such a manner that at steady state, which is reached within 4 min, it is maintained whatever the hyperosmolarity. This regulation is Pi- and deltamuH+-dependent, indicating Pi-K salt entry into the matrix. Under steady state, hyperosmolarity has no effect on isolated rat liver mitochondria energetic parameters such as respiratory rate, proton electrochemical potential difference, and oxidative phosphorylation yield. Hypoosmolarity decreases the NADH/NAD+ ratio, state 3 respiratory rate, and deltamuH+, while oxidative phosphorylation yield is not significantly modified. This indicates kinetic control upstream the respiratory chain. This study points out the key role of potassium on the regulation of matrix volume, flux, and forces. Indeed, while matrix volume is regulated in NaCl hyperosmotic medium, flux and force restoration in hyperosmotic medium occurs only in the presence of external potassium.     

Response to Pneumocystis infection in an immunocompetent host

Cell swelling is now admitted as being a new principle of metabolic control but little is known about the energetics of cell swelling. We have studied the influence of hypo- or hyperosmolarity on both isolated hepatocytes and isolated rat liver mitochondria. Cytosolic hypoosmolarity on isolated hepatocytes induces an increase in matricial volume and does not affect the myxothiazol sensitive respiratory rate while the absolute value of the overall thermodynamic driving force over the electron transport chain increases. This points to an increase in kinetic control upstream the respiratory chain when cytosolic osmolarity is decreased. On isolated rat liver mitochondria incubated in hypoosmotic potassium chloride media, energetic parameters vary as in cells and oxidative phosphorylation efficiency is not affected. Cytosolic hyperosmolarity induced by sodium co-transported amino acids, per se, does not affect either matrix volume or energetic parameters. This is not the case in isolated rat liver mitochondria incubated in sucrose hyperosmotic medium. Indeed, in this medium, adenine nucleotide carrier is inhibited as the external osmolarity increases, which lowers the state 3 respiration close to state 4 level and consequently leads to a decrease in oxidative phosphorylation efficiency. When isolated rat liver mitochondria are incubated in KCl hyperosmotic medium, state 3 respiratory rate, matrix volume and membrane electrical potential vary as a function of time. Indeed, matrix volume is recovered in hyperosmotic KCl medium and this recovery is dependent on Pi-Kentry. State 3 respiratory rate increases and membrane electrical potential difference decreases during the first minutes of mitochondrial incubation until the attainment of the same value as in isoosmotic medium. This shows that matrix volume, flux and force are regulated as a function of time in KCl hyperosmotic medium. Under steady state, neither matrix volume nor energetic parameters are affected. Moreover, NaCl hyperosmotic medium allows matrix volume recovery but induces a decrease in state 3 respiratory flux. This indicates that potassium is necessary for both matrix volume and flux recovery in isolated mitochondria. We conclude that hypoosmotic medium induces an increase in kinetic control both upstream and on the respiratory chain and changes the oxidative phosphorylation response to forces. At steady state, hyperosmolarity, per se, has no effect on oxidative phosphorylation in either isolated hepatocytes or isolated mitochondria incubated in KCl medium. Therefore, potassium plays a key role in matrix volume, flux and force regulation.     

Hyperosmolarity-induced interleukin-8 expression in human bronchial epithelial cells through p38 mitogen-activated protein kinase

The changes in airway osmolarity have been described to contribute to the production of exercise- induced bronchoconstriction (EIB) and the development of the late-phase response (LPR). The mechanism has been investigated; however, the responsiveness of bronchial epithelial cells (BEC) to hyperosmolarity and the intracellular signals leading to cell activation have not been determined. In this study, we examined the effect of hyperosmolar medium on interleukin-8 (IL-8) expression and the role of p38 mitogen-activated protein (MAP) kinase and c-Jun NH2 terminal kinase ( JNK) in human BEC in this response in order to clarify the intracellular signals regulating IL-8 expression in hyperosmolarity-stimulated BEC. The results showed that hyperosmolarity induced IL-8 expression in a concentration dependent manner, p38 MAP kinase phosphorylation and activation, and JNK activation whether NaCl or mannitol was used as the solute. SB 203580 as the specific p38 MAP kinase inhibitor inhibited hyperosmolarity-induced p38 MAP kinase activation and partially inhibited hyperosmolarity-induced IL-8 expression. These results indicate that p38 MAP kinase, at least in part, regulates hyperosmolarity-induced IL-8 expression in BEC. However, other signals such as JNK are possibly also involved. These results provide new evidence on the mechanism responsible for the development of the LPR induced by EIB, and a strategy for treatment with the specific p38 MAP kinase inhibitor.     

Changes in paO2 and of acid-base equilibrium of the arterial blood of hypoxemic polyglobulic patients subjected to acute blood depletion

Partial respiratory gas tension and acid-base equilibrium patterns were examined in polyglobulic patients subjected to acute blood depletion. The increase in oxyhaemoglobin concentration and fall in haematocrit value already described were confirmed. It was also noted that: 1) pH increased significantly in all subjects; 2) urinary osmolarity increased in all subjects; 3) blood sodium and potassium concentration increased significantly in 14 patients (1st group), but fell in the 2nd group (8 subjects). It is suggested, therefore, that acute blood depletion changes renal reabsorption of the main electrolytes, i.e. increased values in cases where a fall in glomerular arteriolar pressure leads to increased renal medullary osmolarity, and decreased values where a fall in circulation leads to increased arteriolar pressure, hyperosmolarity of the medulla, and retainment of liquids as opposed to electrolytes.     

Diabetic ketoacidosis and hyperosmolar nonketotic state: gaining control over extreme hyperglycemic complications

Decompensated hyperglycemia is a frequent, severe complication of diabetes mellitus. Ketoacidosis usually occurs in patients with insulin-dependent (type I) diabetes, and insulin therapy is required to correct their hyperglycemic derangement. Hyperosmolar nonketotic state is more common in patients with non-insulin-dependent (type II) diabetes, who usually present with severe dehydration and hyperosmolar plasma. They respond readily to aggressive volume expansion, and insulin has a lesser role in management. Some patients exhibit a mixture of ketoacidosis and hyperosmolarity, which suggests that the two conditions may represent variants of decompensated hyperglycemia that differ only by the magnitude of dehydration and the severity of acidosis. All diabetic patients with hyperglycemic decompensation should return to their usual hypoglycemic programs as soon as possible and receive close follow-up after hospitalization.     

Role of rpoS in stress survival and virulence of Vibrio cholerae

Vibrio cholerae is known to persist in aquatic environments under nutrient-limiting conditions. To analyze the possible involvement of the alternative sigma factor encoded by rpoS, which is shown to be important for survival during nutrient deprivation in several other bacterial species, a V. cholerae rpoS homolog was cloned by functional complementation of an Escherichia coli mutant by using a wild-type genomic library. Sequence analysis of the complementing clone revealed an 1.008-bp open reading frame which is predicted to encode a 336-amino-acid protein with 71 to 63% overall identity to other reported rpoS gene products. To determine the functional role of rpoS in V. cholerae, we inactivated rpoS by homologous recombination. V. cholerae strains lacking rpoS are impaired in the ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and carbon starvation. These results suggest that rpoS may be required for the persistence of V. cholerae in aquatic habitats. In addition, the rpoS mutation led to reduced production or secretion of hemagglutinin/protease. However, rpoS is not critical for in vivo survival, as determined by an infant mouse intestinal competition assay.     

Activation of p38 mitogen-activated protein kinase by c-Abl-dependent and -independent mechanisms

The p38 mitogen-activated protein (MAP) kinase defines a subgroup of the mammalian MAP kinases that are induced in response to lipopolysaccharide, hyperosmolarity, and interleukin 1. p38 MAP kinase appears to play a role in regulating inflammatory responses, including cytokine secretion and apoptosis. Here we show that diverse classes of DNA-damaging agents such as cisplatinum, 1-beta-D-arabinofuranosylcytosine, UV light, ionizing radiation, and methyl methanesulfonate activate p38 MAP kinase. We also demonstrate that cells deficient in c-Abl fail to activate p38 MAP kinase after treatment with cisplatinum and 1-beta-D-arabinofuranosylcytosine but not after exposure to UV and methyl methanesulfonate. Reconstitution of c-Abl in the Abl-/- cells restores that response. Similar results were obtained for induction of the Jun-NH2-kinase/stress-activated protein kinase. These findings indicate that p38 MAP and Jun-NH2-kinase/stress-activated protein kinases are differentially regulated in response to different classes of DNA-damaging agents.     

Extracellular acidification related to the stimulation of catecholamines release by strontium in the bovine adrenal medulla

The possible modifications of extracellular pH associated with the secretion of catecholamines evoked by the introduction of 2.2 mM Sr2+ to a Ca(2+)-free, buffer-free, Locke solution were investigated in decorticated perfused bovine adrenal glands. A progressive and reversible decrease of external pH accompanied the catecholamine release promoted by Sr(2+)-introduction into the perfusion fluid. This extracellular acid shift was practically undetected when the chromaffin tissue was stimulated by the addition of Sr2+ to a buffered medium. Both the secretory response as well as the extracellular pH drop mediated by Sr(2+)-introduction to a Ca(2+)-free, buffer-free, Locke solution were markedly inhibited by methoxyverapamil (0.3 mM), Mg2+ (20 mM) and hyperosmolarity (750 mOsm). The exposure of the adrenal medulla to a Ca(2+)-free, buffer-free, high-K+ solution containing 2.2 mM Sr2+ for 6 min promoted a significant enhancement of both the secretory response and the acidification of the perfusates compared with the responses evoked by Sr2+ in a 5.6 mM K+ medium. These results are consistent with the existence of a close relationship between extracellular acidification and the release of catecholamines triggered by the introduction of Sr2+ to the perfusion fluid.     

Effects of estrogen on tight junctional resistance in cultured human umbilical vein endothelial cells

OBJECTIVE: To study the effects of estrogen on transendothelial paracellular permeability in women. METHODS: Human umbilical vein endothelial cells (HUVEC) obtained from women were grown on filters. The paracellular permeability characteristics were determined in terms of changes in the permeability to the polar acid pyranine (Ppyr) and as changes in the transendothelial electrical resistance (RTE). Tight junctional resistance characteristics were assayed by lowering luminal NaCl and measuring the dilution potential, and were expressed as the ratio of monoion mobility uCl/uNa (cation selectivity). RESULTS: Low extracellular calcium and hyperosmolarity increased Ppyr and decreased RTE. The former but not the latter condition abolished the endothelium-specific cation selectivity. Treatment with 10 nM of estradiol-17 beta had no effect on RTE, but it increased the cation selectivity. The effect of estradiol required 1-6 hours' incubation with the hormone; it was dose dependent and saturable, with a median effective concentration of estradiol of 1 nM. Diethylstilbestrol, but not estriol, could mimic the effect of estradiol, and the estrogen receptor antagonist ICI-182, 780 blocked it. CONCLUSION: Cultured HUVEC cells form patent tight junctions. Estrogens increase the cation selectivity across HUVEC cultures. The effect of estrogen may be mediated by an estrogen receptor. These effects may be important for vasculoprotection in cases of sudden changes in ions levels across the capillary wall, such as ischemia or reperfusion.     

The RNA-binding protein HF-I, known as a host factor for phage Qbeta RNA replication, is essential for rpoS translation in Escherichia coli

The rpoS-encoded sigma(S) subunit of RNA polymerase in Escherichia coli is a global regulatory factor involved in several stress responses. Mainly because of increased rpoS translation and stabilization of sigma(S), which in nonstressed cells is a highly unstable protein, the cellular sigma(S) content increases during entry into stationary phase and in response to hyperosmolarity. Here, we identify the hfq-encoded RNA-binding protein HF-I, which has been known previously only as a host factor for the replication of phage Qbeta RNA, as an essential factor for rpoS translation. An hfq null mutant exhibits strongly reduced sigma(S) levels under all conditions tested and is deficient for growth phase-related and osmotic induction of sigma(S). Using a combination of gene fusion analysis and pulse-chase experiments, we demonstrate that the hfq mutant is specifically impaired in rpoS translation. We also present evidence that the H-NS protein, which has been shown to affect rpoS translation, acts in the same regulatory pathway as HF-I at a position upstream of HF-I or in conjunction with HF-I. In addition, we show that expression and heat induction of the heat shock sigma factor sigma(32) (encoded by rpoH) is not dependent on HF-I, although rpoH and rpoS are both subject to translational regulation probably mediated by changes in mRNA secondary structure. HF-I is the first factor known to be specifically involved in rpoS translation, and this role is the first cellular function to be identified for this abundant ribosome-associated RNA-binding protein in E. coli.     

Hypothyroidism and megacolon

A 71 years old woman, affected by ischemic heart disease from the age of 50 and by chronic constipation was admitted to the emergency department for drowsiness, intense dyspnea and acute abdominal distension. Laparotomy evidenced a megacolon. Because of the age and sex of the patient the congenital form of the megacolon was ruled out. No one of the more common causes of megacolon was recognized, but a severe hypothyroldism and Hashimoto's thyroiditis was discovered. Treatment with levothyroxine caused a progressive improvement of the general condition of the patient and of the megacolon so that the authors hypothesize that the intestinal pseudo-occlusion was caused by the hypothyroidism. In this paper the authors make a thorough analysis of the literature about the association between hypothyroidism and megacolon. Although many hypothesis have been put forward about the possible pathogenetic association between these two diseases, until now no definitive result has been reached. The authors, moreover, hypothesize that the pleural and pericardial effusion and the peculiar metabolic state characterized by plasma hyponatremia and hyposmolarity, with a constant urinary hyperosmolarity, were also caused by hypothyroidism; in fact the clinical and metabolic conditions improved after levothyroxine therapy. In the end the authors discuss if it is preferable to use tetraiodothyronine or triIodothyronine for the treatment of intense hypothyroidism in a patient in critical clinical state.     

Anisomycin selectively desensitizes signalling components involved in stress kinase activation and fos and jun induction

Anisomycin, a translational inhibitor secreted by Streptomyces spp., strongly activates the stress-activated mitogen-activated protein (MAP) kinases JNK/SAPK (c-Jun NH2-terminal kinase/stress-activated protein kinase) and p38/RK in mammalian cells, resulting in rapid induction of immediate-early (IE) genes in the nucleus. Here, we have characterized this response further with respect to homologous and heterologous desensitization of IE gene induction and stress kinase activation. We show that anisomycin acts exactly like a signalling agonist in eliciting highly specific and virtually complete homologous desensitization. Anisomycin desensitization of a panel of IE genes (c-fos, fosB, c-jun, junB, and junD), using epidermal growth factor (EGF), basic fibroblast growth factor, (bFGF), tumor necrosis factor alpha (TNF-alpha), anisomycin, tetradecanoyl phorbol acetate (TPA), and UV radiation as secondary stimuli, was found to be extremely specific both with respect to the secondary stimuli and at the level of individual genes. Further, we show that anisomycin-induced homologous desensitization is caused by the fact that anisomycin no longer activates the JNK/SAPK and p38/RK MAP kinase cascades in desensitized cells. In anisomycin-desensitized cells, activation of JNK/SAPKs by UV radiation and hyperosmolarity is almost completely lost, and that of the p38/RK cascade is reduced to about 50% of the normal response. However, all other stimuli produced normal or augmented activation of these two kinase cascades in anisomycin-desensitized cells. These data show that anisomycin behaves like a true signalling agonist and suggest that the anisomycin-desensitized signalling component(s) is not involved in JNK/SAPK or p38/RK activation by EGF, bFGF, TNF-alpha, or TPA but may play a significant role in UV- and hyperosmolarity-stimulated responses.     

Human mitogen-activated protein kinase kinase 7 (MKK7) is a highly conserved c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activated by environmental stresses and physiological stimuli

We report the cloning of a novel human activator of c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 7 (MKK7). The mRNA for MKK7 is widely expressed in humans and mice and encodes a 47-kDa protein (419 amino acids), as determined by immunoblotting endogenous MKK7 with an antibody raised against its N terminus. The kinase domain of MKK7 is closely related to a Drosophila JNK kinase dHep (69% identity) and to a newly identified ortholog from Caenorhabditis elegans (54% identity), and was more distantly related to MKK4, MKK3, and MKK6. MKK7 phosphorylated and activated JNK1 but failed to activate p38 MAPK in co-expression studies. In hematopoietic cells, endogenous MKK7 was activated by treatment with the growth factor interleukin-3 (but not interleukin-4), or by ligation of CD40, the B-cell antigen receptor, or the receptor for the Fc fragment of immunoglobulin. MKK7 was also activated when cells were exposed to heat, UV irradiation, anisomycin, hyperosmolarity or the pro-inflammatory cytokine tumor necrosis factor-alpha. Co-expression of constitutively active mutants of RAS, RAC, or CDC42 in HeLa epithelial cells or of RAC or CDC42 in Ba/F3 factor-dependent hematopoietic cells also activated MKK7, suggesting that MKK7 will be involved in many physiological pathways.     

Emergency stiuations connected with electrolyte disorders. Special aspects

Electrolyte-induced situations which are dangerous to life usually result from disturbances of the extracellular volume (ECV), osmolarity, the potassium level or the acid-base equilibrium. In recent years (thanks to the wide spread use of hormone therapy for mammary carcinoma) severe hypercalcemia has increased in importance as a life-threatening complication, while hypocalcemia, at least in adults, should only very seldom lead to unexpected emergencies. As long as serious clinical symptoms do not suggest an emergency, assessment of the threat to the patient as a result of the existing electrolyte disturbance often causes some difficulty. Besides the extent of the deviation from normal, the rate of development of the disturbance determines the resulting danger: chronic hypo-osmolarity, and especially hyperosmolarity are occasionally tolerated without symptoms while acute disturbances of the same or a less extent lead to severe central nervous symptoms. A similar state of affairs is also true of the emergency situations arising from disturbances of the acid-base equilibrium, among which the respiratory disorders are particularly important clinically. In the case of threatening disorders of the potassium metabolism, the accompanying circumstances (digitalis, simultaneous disorders and treatment of the acid-base equilibrium) often determine the clinical significance and danger to the patient. Clinical symptoms, anticipation ("expecting the unexpected"), prevention and treatment of emergency situations of fluid volume, osmolarity, potassium and acid-base equilibrium are the subjects of this paper.