Observation of few-cycle, strong-field phenomena in surface plasmon fields.

We present experimental evidence of the generation of few-cycle propagating surface plasmon polariton wavepackets. These ultrashort plasmonic pulses comprised of only 2-3 field oscillations were characterized by an autocorrelation measurement based on electron photoemission. By exploiting plasmonic field enhancement, we achieved plasmon-induced tunnelling emission from the metal surface at low laser intensity, opening perspectives for strong-field experiments with low pulse energies. All-optical electron acceleration up to keV kinetic energy is also demonstrated in these surface-confined, few-cycle fields with only 1.35×10(12) W/cm2 focused laser intensity. The experimental results are found to be in excellent agreement with the model.

Polymorphisms of beta defensins are associated with the risk of severe acute pancreatitis.

AIMS: Bacterial translocation from the intestinal tract plays an important role in severe acute pancreatitis (AP). Human ß-defensins are a family of antimicrobial peptides present at the mucosal surface. The aim of this study was to investigate the relevance of single nucleotide polymorphisms (SNPs) in the DEFB1 gene and copy number polymorphisms of the DEFB4 genes in AP. METHODS: 124 AP patients (30 with mild and 94 with severe disease) and 100 healthy controls were enrolled in the study. Three SNPs of the DEFB1 gene [G-20A (c.-20G→A), C-44G (c.-44C→G) and G-52A (c.-52G→A)] were genotyped by Custom TaqMan assay. The DEFB4 gene copy number was determined by means of a TaqMan real-time PCR assay. RESULTS: Significantly higher frequencies of the AA genotype of G-20A (c.-20G→A) and the AA genotype of G-52A (c.-52G→A) were observed among the patients with severe AP (SAP) compared with the healthy controls (38 vs. 20 and 41 vs. 18%, respectively). The GG protective genotype of C-44G (c.-44C→G) SNP was much less frequent (1%) among the patients than among the controls (9%). A higher frequency of a lower (<4) copy number of the DEFB4 gene was observed in the patients with SAP compared with the healthy controls (62 vs. 24%, respectively). CONCLUSIONS: The variations in the genes encoding human ß-defensin-1 and -2 may be associated with the risk of SAP. and IAP.

Plasma concentrations of high-mobility group box protein 1, soluble receptor for advanced glycation end-products and circulating DNA in patients with acute pancreatitis.

AIMS: High-mobility group box protein 1 (HMGB1), a late-acting proinflammatory cytokine, is secreted actively by inflammatory cells, and released passively from necrotic cells. From the aspect that both inflammation and necrosis are involved in the pathogenesis in acute pancreatitis, the aim of the study was a joint investigation of the plasma concentrations of HMGB1, its soluble receptor for advanced glycation end-products (sRAGE), and the circulating DNA as a marker of cell death. METHODS: 62 patients with acute pancreatitis (30 mild, 32 severe), 20 patients with sepsis, and 20 healthy controls were enrolled in the study. HMGB1 and sRAGE plasma levels were measured by means of ELISA. Plasma DNA concentrations were estimated by real-time quantitative PCR for the beta-globin gene. RESULTS: The circulating HMGB1 level was significantly higher in patients with severe acute pancreatitis (13.33 +/- 2.11 ng/ml) than in healthy controls (0.161 +/- 0.03 ng/ml) or than in patients with mild pancreatitis (2.64 +/- 0.185 ng/ml). The plasma concentration of sRAGE was highest in patients with sepsis (2,210 +/- 252 pg/ml), while the levels of sRAGE correlated inversely with that of HMGB1 in patients with acute pancreatitis. The plasma DNA level was significantly elevated in patients with severe acute pancreatitis (2,206 +/- 452 ng/ml). CONCLUSION: A complex study of the plasma levels of HMGB1, sRAGE and circulating DNA can be informative in evaluations of acute pancreatitis with different levels of severity.

Histochemical and biochemical correlates of ventilatory muscle fatigue in emphysematous hamsters.

Histochemical and biochemical characteristics of the ventilatory muscles were evaluated in control and elastase-induced emphysematous hamsters. The emphysematous group was divided into sedentary and endurance-trained groups. Endurance training consisted of treadmill running, 1 h a day, 7 d a week. The experimental period lasted 24 wk. Histochemically, the diaphragm from the sedentary emphysematous hamsters revealed a selective fast fiber atrophy which was prevented by endurance training. Training also led to a hypertrophy of the slow, high oxidative fibers. The external intercostals from both emphysematous groups revealed an increased proportion of fast oxidative fibers at the expense of a decreased number of fast glycolytic fibers. However, the fast fibers in both emphysematous groups were significantly atrophied as compared with controls. The internal intercostals revealed no adaptive changes in either size or proportion distribution of the various fiber types. Biochemically, the diaphragm of the emphysematous animals had a significantly improved oxidative potential as measured by citrate synthase, and a reduced glycolytic capacity as indicated by phosphofructokinase activity, compared with controls. The magnitudes of the biochemical changes were similar in both emphysematous groups and were consistent for diaphragmatic samples taken from the costal and crural segments. The combined internal and external intercostals also underwent significant biochemical increases in their oxidative capacity. In addition, training of the emphysematous group led to an increased glycolytic potential of the intercostals.

Diaphragm mechanics in dogs with unilateral emphysema.

We studied dogs with unilateral papain-induced emphysema to answer two questions: (1) Do emphysema lung-apposed hemidiaphragm (DiE) and normal lung-apposed hemidiaphragm (DiN) have equal capacities for lowering lung surface pressure? and (2) Are side-to-side differences in intrathoracic pressure the result of unequal force outputs by DiE and DiN or are they caused by differences in their mechanical efficiency as pressure generators? After the airways of the emphysematous and normal lungs were intubated with a dual lumen endotracheal tube, both phrenic nerves were maximally stimulated at rates between 1 and 50 Hz and the changes in airway occlusion pressure (delta PaoE,N) and diaphragm length (sonomicrometry) were recorded. In all animals, delta PaoN exceeded delta PaoE. Differences in pressure ranged from 1.2 +/- 0.6 cm H2O during a twitch to 6.0 +/- 2.9 cm H2O during a 50-Hz tetanus. Midcostal bundles of DiE shortened less than corresponding bundles of DiN, but both reached the same active length relative to their optimal lengths, which were measured in vitro. There was no significant difference in fiber type distribution, fiber cross-sectional area, or maximal isometric tetanic tensions among midcostal regions of DiE and DiN. We conclude that unilateral hyperinflation impairs the mechanical efficiency of the apposing hemidiaphragm as a pressure generator.

The 38 kDa Ca2+/membrane-binding protein of pig granulocytes needs a high Ca2+ concentration to be phosphorylated by protein kinase C.

The 38 kDa Ca2+/membrane-binding protein reported to be the dominant substrate of protein kinase C in the extracts of pig neutrophil granulocytes was purified partially and its phosphorylation was investigated. In pig granulocytes type II protein kinase C was the major isoform, while type III isoenzyme was present only as a minor activity. Phosphorylation of the 38 kDa protein was performed with rat brain protein kinase C. Each of the three isoenzymes purified from rat brain was able to phosphorylate this protein, though on the conditions used in our experiments it was phosphorylated most intensively by type II protein kinase C. A phospholipid-dependent, but Ca2(+)-independent, form of protein kinase C was demonstrated with the aid of a synthetic oligopeptide substrate. Phosphorylation of the 38 kDa protein by the Ca2(+)-independent enzyme proceeded exclusively in the presence of Ca2+. The Ca2+ concentration necessary for the phosphorylation of the 38 kDa by either form of protein kinase C was by orders of magnitude higher than that required for the activation of protein kinase C.

Lipocortin I is not accessible for protein kinase C bound to the cytoplasmic surface of the plasma membrane in streptolysin-O-permeabilized pig granulocytes.

We previously observed a 38 kDa protein that was a major protein component of the cytosolic extract of pig granulocytes and the dominant substrate of protein kinase C at supra-physiological Ca2+ concentrations. The purified 38 kDa protein itself required Ca2+ to be phosphorylated by protein kinase C. Now we demonstrate that this protein, which is also present in human granulocytes, is identical to lipocortin I. The identification is based on the chromatographic properties and immunoblot of the purified protein which is also a good substrate for tissue transglutaminase. Phosphorylation of lipocortin I by protein kinase C was investigated in granulocytes permeabilized with streptolysin-O. At physiological intracellular Ca2+ concentrations lipocortin I was not phosphorylated at all. At supra-physiological Ca2+ concentrations (0.5 mM), lipocortin I was also not phosphorylated when protein kinase C was translocated to the membrane by treatment of the cells with phorbol myristate acetate. Its phosphorylation was detectable only in control experiments when protein kinase C was activated in the cytosol by the addition of dioleoylglycerol and phosphatidylserine to the permeabilized cells. The data presented show that, in permeabilized granulocytes, Ca(2+)-lipocortin is not formed at physiological Ca2+ concentrations, and at supra-physiological Ca2+ concentrations the Ca(2+)-lipocortin I is not accessible to protein kinase C bound to the cytoplasmic surface of the plasma membrane.

Phosphorylation of poly(ADP-ribose)polymerase protein in human peripheral lymphocytes stimulated with phytohemagglutinin.

Intracellular phosphorylation of poly(ADP-ribose)polymerase was assayed in streptolysin-O-permeabilized human lymphocytes. Whereas 32P incorporation from [gamma-32P]ATP into immunoprecipitated enzyme protein was undetectable in resting cells, significant phosphorylation of this enzyme was observed in lymphocytes treated with phytohemagglutinin. The phosphorylation of poly(ADP-ribose)polymerase in permeabilized cells was not stimulated by phorbol ester, while phorbol-induced phosphorylation of other proteins and of a specific oligopeptide substrate of protein kinase C was observed. However, the specific inhibitory pseudosubstrate peptide of protein kinase C blocked the phosphorylation of poly(ADP-ribose)polymerase induced by phytohemagglutinin. Therefore, a potential role of a member of the protein kinase C family in the phytohemagglutinin stimulated intracellular phosphorylation of poly(ADP-ribose)polymerase is conceivable.

Simple and reliable conditions for routine, long-term culturing of fetal human pancreatic tissue fragments.

Fetal human pancreatic tissue fragments were isolated and cultured for 18 wk. Insulin production was almost continuous during this period. Multiplication of cells was observed at the second week, and these cells later aggregated as epithelioid cells and formed pseudoislets. The growth characteristics, insulin-like immunoreactivity, and endocrine properties of these cells were evidenced by light microscopy, immunocytochemistry, electron microscopic examination, and measurement of the total insulin content. These results indicate that long-term culture of fetal islets may be useful in clinical work and provides a possible method for increasing islet mass and reducing immunogenicity.

Phosphatidylinositol 3-kinase contributes to Erk1/Erk2 MAP kinase activation associated with hepatocyte growth factor-induced cell scattering.

MAP kinase cascade-dependent responses were investigated during scattering of HepG2 human hepatoma cells stimulated by HGF or phorbol ester. Inhibition of phosphatidylinositol 3-kinase with LY294002 prevented completely the dissociation of cells. Inhibition of MAP kinase kinase (MEK) with PD98059 prevented the development of characteristic morphological changes associated with cell migration. EGF, which failed to induce cell scattering, caused a short-term increase in the phosphorylation of Erk1/Erk2 MAP kinases. On the contrary, HGF or phorbol ester stimulated the phosphorylation of MAP kinases for a long time. Experiments performed with LY294002 indicated that phosphatidylinositol 3-kinase contributed to the HGF-stimulated phosphorylation of Erk1/Erk2. This finding was confirmed by the demonstration that the MAP kinase cascade-dependent expression of a high-Mr (>300 kDa) protein pair appearing in the course of cell scattering was inhibited by LY294002 in HGF-induced cells but was not inhibited in phorbol ester-treated cells.

Protein kinase C decreases the hepatocyte growth factor-induced activation of Erk1/Erk2 MAP kinases.

HGF and phorbol ester induce the scattering of HepG2 cells. Recently, we have reported that the motility and morphological responses that accompany this process require the activation of Erk1/Erk2 MAP kinases, and phosphatidylinositol 3-kinase contributes to the activation of Erk1/Erk2 in HGF-induced cells. The cell scattering-associated appearance of a high-M(r) (>300 kDa) protein pair has also been observed, and has been proven to be a sensitive marker of the intensity of Erk1/Erk2 activation. Our present study demonstrates that in HGF-induced cells protein kinase C and phosphatidylinositol 3-kinase regulate oppositely the expression of these cell scattering-associated proteins. While in phorbol ester-treated cells the sustained activation of protein kinase C is essential for this expression, in HGF-induced cells the inhibition of protein kinase C with bisindolylmaleimide I stimulates the expression. Protein kinase C reduces the HGF-induced phosphorylation of Erk1/Erk2, and in this way it can limit the intensity of Erk1/Erk2-dependent gene-expression

Effects of enhanced conventional therapy on metabolic control in children with insulin-dependent diabetes mellitus.

We implemented a three-phase, 32-wk program to improve both self-regulation of adherence behaviors and insulin delivery in children with diabetes. Twenty children, aged 8-12 yr (mean duration 3.6 yr), enrolled. Phase 1 (wk 1-12) used behavior modification to improve diet, exercise, urine testing, and insulin adjustment, targeting an increased percentage negative urines. Feedback training and parent checks were used to improve reliability; adherence was measured using Clinitest placebos. Phase 2 (wk 13-20) was a stabilization period. Phase 3 (wk 21-32) studied the effect of insulin dose adjustment, comparing once-versus twice-daily shots in 10 pairs of children matched for %GHb. GHb, fasting plasma glucose, and lipids were measured at baseline and at the end of each phase. Results revealed a significant and sustained increase in negative urine tests, but no change in % GHb or FBG. Reliability of and adherence to urine tests were 83% and 76%, respectively. During phase 3, no significant differences were noted between groups receiving once- or twice-daily insulin injections. Thus, behavior modification resulted in increased reliability and adherence to routines, associated with a reliable increase in negative urines. This did not, however, produce changes in other control measures. Furthermore, no differences between those receiving 1 or 2 daily shots were evident.

Stereoselective coordination: a six-membered P,N-chelate tailored for asymmetric allylic alkylation.

Six-membered chelate complexes [Pd(1a-b)Cl2], (2a-b) and [Pd(1a-b)(η(3)-PhCHCHCHPh)]BF4, (3a-b) of P,N-type ligands 1a, ((2S,4S)-2-diphenyl-phosphino-4-isopropylamino-pentane) and 1b, ((2S,4S)-2-diphenyl-phosphino-4-methylamino-pentane) have been prepared. The Pd-complexes have been characterized in solution by 1D and 2D NMR spectroscopy. The observed structures were confirmed by DFT calculations and in the case of 2a also by X-ray crystallography. Unexpectedly, the coordination of the all-carbon-backbone aminophosphine 1a resulted in not only a stereospecific locking of the donor nitrogen atom into one of the two possible configurations but also the conformation of the six-membered chelate rings containing three alkyl substituents was forced into the same single chair structure showing the axially placed isopropyl group on the coordinated N-atom. The stereodiscriminative complexation of 1a led to the formation of a palladium catalyst with a conformationally rigid chelate having a configurationally fixed nitrogen and electronically different coordination sites due to the presence of P and N donors. The stereochemically fixed catalyst provided excellent ee's (up to 96%) and activities in asymmetric allylic alkylation reactions. In contrast, the chelate rings formed by 1b exist in two different chair conformations, both containing axial methyl groups, but with the opposite configurations of the coordinated N-atom. Pd-complexes of 1b provided low enantioselectivities in similar alkylations, therefore emphasizing the importance of the stereoselective coordination of N-atoms in analogous P-N chelates. The factors determining the coordination of the ligands were also studied with respect to the chelate ring conformation and the nitrogen configuration.

Possible involvement of protein kinase C-epsilon in phorbol ester-induced growth inhibition of human lymphoblastic cells.

Sustained activation of members of the protein kinase C (PKC) family is known to influence the growth and differentiation of various cell types, however, the specific roles for individual isoforms mediating these cellular events have yet to be elucidated. Activation of PKC by phorbol esters leads to growth inhibition in certain cell lines. The HT58 human B lymphoblastic cell may serve as a cellular model system to investigate the participation of individual isoforms in the initial events of growth arrest induced by phorbol ester. Determination of cell cycle and investigation of apoptosis were performed by flow cytometric measurements. Phorbol ester-induced translocation and down-regulation of the conventional alpha, beta and the novel epsilon isoforms of PKC were demonstrated by Western blot analysis. At lower concentrations (o.5 ng/ml) phorbol myristate acetate (PMA) stimulated a G1 arrest with retention of viability in the human HT58 B lymphoblastic cell. The protein kinase inhibitor staurosporine at a concentration of 25 nM did not significantly alter HT58 cell viability. However, staurosporine (25 nM) induced apoptosis in cells preincubated for 4 hr with 0.5-1.0 ng/ml PMA. The translocation of PKC-epsilon was observed within 39 min exposure to 0.5 ng/ml PMA. After a 4 hr treatment, evidence for down-regulation and and altered phosphorylation state of PKC-epsilon was seen. In contrast, the conventional alpha and beta isoforms were practically uneffected by this PMA treatment. At higher PMA concentrations (50 ng/ml) the alpha and beta isoforms showed a significant down-regulation. The preferential alterations in PKC-epsilon observed under the conditions required for PMA to influence the growth and survival of HT58 cells suggest a role for the Ca(2+)-independent epsilon isoform in mediating the initial events of the phorbol ester stimulated cellular responses.

Chromatin organization and transcriptional control of gene expression in Drosophila.

It is increasingly clear that the packaging of DNA in nucleosome arrays serves not only to constrain the genome within the nucleus, but also to encode information concerning the activity state of the gene. Packaging limits the accessibility of many regulatory DNA sequence elements and is functionally significant in the control of transcription, replication, repair and recombination. Here, we review studies of the heat-shock genes, illustrating the formation of a specific nucleosome array at an activatable promoter, and describe present information on the roles of DNA-binding factors and energy-dependent chromatin remodeling machines in facilitating assembly of an appropriate structure. Epigenetic maintenance of the activity state within large domains appears to be a key mechanism in regulating homeotic genes during development; recent advances indicate that chromatin structural organization is a critical parameter. The ability to utilize genetic, biochemical and cytological approaches makes Drosophila an ideal organism for studies of the role of chromatin structure in the regulation of gene expression.

Interaction between hysteretic regulation and redox modulation of glucose-6-phosphate dehydrogenase from Anacystis nidulans.

The glucose-6-phosphate dehydrogenase (G6PDH) of cyanobacteria is a hysteretic enzyme which is also subject to redox modulation [FEBS Lett. 126 (1981) 85-88]. We have found that the hysteretic and redox properties of G6PDH exhibit specific interactions: (1) The hysteretic forms of G6PDH ('hypoactive' in equilibrium 'hyperactive'), obtained at pH 7.5 and 6.5, respectively, differ in their redox properties. The 'hypoactive' form is easily activated by oxidation whereas the 'hyperactive' form is easily deactivated by reduction. (2) At low G6P concentrations (greater than 1 mM) only the oxidized form of G6PDH has significant activity. An increase in G6P level diminishes the difference between the activity of oxidized and reduced G6PDH forms.

Isoenzyme patterns of protein kinase C and a phospholipid-dependent but Ca2+-inhibited enzyme fraction in the crude extracts of different tissues.

We compared the protein kinase C isoenzyme patterns of crude extracts of rabbit brain, cerebellum, spleen, thymus and human and pig granulocytes. The isoenzymes were fractionated by hydroxyapatite chromatography and the protein kinase C activity was determined with a synthetic oligopeptide substrate. In the extracts of several tissues we also observed an enzyme fraction which was activated by phosphatidylserine + diacylglycerol but inhibited by Ca2+.

Two components of type III protein kinase C with different substrate specificities and a phospholipid-dependent but Ca2+-inhibited protein kinase in rat brain.

The activities of rat brain protein kinase C isoenzymic fractions separated by hydroxyapatite chromatography were measured with histone H1 or the oligopeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide as substrates. The oligopeptide was a better substrate than histone H1 for nearly all of the protein kinase C fractions. Two subfractions of type III isoenzyme were resolved (IIIa and IIIb); type IIIb was characterized by a very low histone kinase activity compared to its peptide kinase activity. In some brain extracts a phospholipid-dependent but Ca2+-inhibited protein kinase was also observed which was eluted from the hydroxyapatite column between type II and III isoenzymes of protein kinase C.

Proteolytic activation of protein kinase C in the extracts of cells treated for a short time with phorbol ester.

A 10 min treatment of human neutrophils with phorbol 12-myristate 13-acetate (PMA) has been reported to induce accumulation of the proteolytically activated Ca2+/phospholipid-independent catalytic fragment of protein kinase C in the cytosol of intact cells [(1986) J. Biol. Chem. 261, 4101-4105]. We investigated the proteolytic conversion of protein kinase C to Ca2+/phospholipid-independent form in the cytosol and membrane fractions of pig neutrophils. The activity of protein kinase C was measured with its specific oligopeptide substrate Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide designed previously. In our experiments the short-term treatment of neutrophils with PMA did not induce the accumulation of the proteolytically activated form of protein kinase C in the cytosol of intact cells. However, treatment of cells with PMA enhanced the limited proteolysis of protein kinase C during the preparation of cell extracts.

Gadolinium chloride-induced macrophage blockade prevents rejection of human insulinoma cell xenograft in rats.

The effect of gadolinium chloride (GdCl3)-induced Kupffer cell blockade on the survival of discordant insulinoma cell xenografts was investigated. Insulinoma cells isolated by means of collagenase from human insulinoma and subsequently cultured were transplanted through the portal vein into the liver of streptozotocin-induced diabetic, male, CFY inbred rats. In the control, streptozotocin-treated rats, the decrease in blood glucose level was only transitory, in contrast with the GdCl3-pretreated diabetic rats, which remained normoglycemic during the 2-week observation period. Histologically, in the liver and lung of rats pretreated with GdCl3, large areas of extensively proliferating insulinoma cells were seen, whereas no insulinoma cells were seen in either the liver or the lung of diabetic control rats, not treated with GdCl3. These studies suggest that the Kupffer cells play significant roles in the recognition of xenoantigens and the induction of xenograft rejection.