Root deformation affects mineral nutrition but not leaf gas exchange and growth of Genipa americana seedlings during the recovery phase after soil flooding / A deformação de raízes afeta a nutrição mineral mas não as trocas gasosas foliares e o crescimento de mudas de Genipa americana durante a fase de recuperação após o alagamento do solo

Root deformation (RD) caused by errors in the pricking out process are irreversible and very difficult to detect in container-grown seedlings at the time of planting in the field. The objective of this study was to evaluate the effects of RD on leaf gas exchange, growth, biomass allocation and mineral nutrition of G. americana seedlings during the recovery phase after soil flooding. Four-months-old seedlings, with and without RD, were flooded for 42 days and their recovery was evaluated 28 days after soil drainage. There were no significant interactions between RD and soil flooding for all leaf gas exchange, growth and mineral nutrition after soil drainage, with the exception of leaf P concentrations. In plants with no RD, the P concentration in leaves of non-flooded plants was significantly higher than that of plants with RD. Soil flooding and RD did not influence leaf or root N concentrations or whole-plant N content. RD increased the K concentration in the roots, but not in the leaves. Changes in the nutrient concentrations in leaves and roots indicate that RD may affect physiological performance of seedlings after planting in the field.

A deformação da raiz (RD) causada por erros no processo de repicagem é irreversível e difícil de detectar em mudas produzidas em embalagens no momento do plantio no campo. O objetivo deste estudo foi avaliar os efeitos do RD nas trocas gasosas foliares, crescimento, alocação de biomassa e nutrição mineral de mudas de G. americana na fase de recuperação após o alagamento do solo. Mudas com quatro meses de idade, com e sem RD, foram alagadas por 42 dias e a sua recuperação foi avaliada 28 dias após a drenagem do solo. Não houve interação significativa entre RD e alagamento do solo nas trocas gasosas foliares, crescimento e nutrição mineral após a drenagem, com exceção das concentrações de P foliar. Em plantas sem RD, a concentração de P nas folhas de plantas não alagadas foi significativamente maior que a das plantas com RD. O alagamento do solo e a RD não influenciaram as concentrações de N nas folhas e raízes, e no conteúdo de N na planta inteira. A RD aumentou a concentração de K nas raízes, mas não nas folhas. Alterações nas concentrações de nutrientes nas folhas e raízes indicam que a RD pode afetar o desempenho fisiológico das mudas após o plantio no campo.

Root deformation affects mineral nutrition but not leaf gas exchange and growth of Genipa americana seedlings during the recovery phase after soil flooding

Abstract Root deformation (RD) caused by errors in the pricking out process are irreversible and very difficult to detect in container-grown seedlings at the time of planting in the field. The objective of this study was to evaluate the effects of RD on leaf gas exchange, growth, biomass allocation and mineral nutrition of G. americana seedlings during the recovery phase after soil flooding. Four-months-old seedlings, with and without RD, were flooded for 42 days and their recovery was evaluated 28 days after soil drainage. There were no significant interactions between RD and soil flooding for all leaf gas exchange, growth and mineral nutrition after soil drainage, with the exception of leaf P concentrations. In plants with no RD, the P concentration in leaves of non-flooded plants was significantly higher than that of plants with RD. Soil flooding and RD did not influence leaf or root N concentrations or whole-plant N content. RD increased the K concentration in the roots, but not in the leaves. Changes in the nutrient concentrations in leaves and roots indicate that RD may affect physiological performance of seedlings after planting in the field.

Resumo A deformação da raiz (RD) causada por erros no processo de repicagem é irreversível e difícil de detectar em mudas produzidas em embalagens no momento do plantio no campo. O objetivo deste estudo foi avaliar os efeitos do RD nas trocas gasosas foliares, crescimento, alocação de biomassa e nutrição mineral de mudas de G. americana na fase de recuperação após o alagamento do solo. Mudas com quatro meses de idade, com e sem RD, foram alagadas por 42 dias e a sua recuperação foi avaliada 28 dias após a drenagem do solo. Não houve interação significativa entre RD e alagamento do solo nas trocas gasosas foliares, crescimento e nutrição mineral após a drenagem, com exceção das concentrações de P foliar. Em plantas sem RD, a concentração de P nas folhas de plantas não alagadas foi significativamente maior que a das plantas com RD. O alagamento do solo e a RD não influenciaram as concentrações de N nas folhas e raízes, e no conteúdo de N na planta inteira. A RD aumentou a concentração de K nas raízes, mas não nas folhas. Alterações nas concentrações de nutrientes nas folhas e raízes indicam que a RD pode afetar o desempenho fisiológico das mudas após o plantio no campo.

Root deformation affects mineral nutrition but not leaf gas exchange and growth of Genipa americana seedlings during the recovery phase after soil flooding / A deformação de raízes afeta a nutrição mineral mas não as trocas gasosas foliares e o crescimento de mudas de Genipa americana durante a fase de recuperação após o alagamento do solo

Root deformation (RD) caused by errors in the pricking out process are irreversible and very difficult to detect in container-grown seedlings at the time of planting in the field. The objective of this study was to evaluate the effects of RD on leaf gas exchange, growth, biomass allocation and mineral nutrition of G. americana seedlings during the recovery phase after soil flooding. Four-months-old seedlings, with and without RD, were flooded for 42 days and their recovery was evaluated 28 days after soil drainage. There were no significant interactions between RD and soil flooding for all leaf gas exchange, growth and mineral nutrition after soil drainage, with the exception of leaf P concentrations. In plants with no RD, the P concentration in leaves of non-flooded plants was significantly higher than that of plants with RD. Soil flooding and RD did not influence leaf or root N concentrations or whole-plant N content. RD increased the K concentration in the roots, but not in the leaves. Changes in the nutrient concentrations in leaves and roots indicate that RD may affect physiological performance of seedlings after planting in the field.

A deformação da raiz (RD) causada por erros no processo de repicagem é irreversível e difícil de detectar em mudas produzidas em embalagens no momento do plantio no campo. O objetivo deste estudo foi avaliar os efeitos do RD nas trocas gasosas foliares, crescimento, alocação de biomassa e nutrição mineral de mudas de G. americana na fase de recuperação após o alagamento do solo. Mudas com quatro meses de idade, com e sem RD, foram alagadas por 42 dias e a sua recuperação foi avaliada 28 dias após a drenagem do solo. Não houve interação significativa entre RD e alagamento do solo nas trocas gasosas foliares, crescimento e nutrição mineral após a drenagem, com exceção das concentrações de P foliar. Em plantas sem RD, a concentração de P nas folhas de plantas não alagadas foi significativamente maior que a das plantas com RD. O alagamento do solo e a RD não influenciaram as concentrações de N nas folhas e raízes, e no conteúdo de N na planta inteira. A RD aumentou a concentração de K nas raízes, mas não nas folhas. Alterações nas concentrações de nutrientes nas folhas e raízes indicam que a RD pode afetar o desempenho fisiológico das mudas após o plantio no campo.

Root deformation affects mineral nutrition but not leaf gas exchange and growth of Genipa americana seedlings during the recovery phase after soil flooding.

Root deformation (RD) caused by errors in the pricking out process are irreversible and very difficult to detect in container-grown seedlings at the time of planting in the field. The objective of this study was to evaluate the effects of RD on leaf gas exchange, growth, biomass allocation and mineral nutrition of G. americana seedlings during the recovery phase after soil flooding. Four-months-old seedlings, with and without RD, were flooded for 42 days and their recovery was evaluated 28 days after soil drainage. There were no significant interactions between RD and soil flooding for all leaf gas exchange, growth and mineral nutrition after soil drainage, with the exception of leaf P concentrations. In plants with no RD, the P concentration in leaves of non-flooded plants was significantly higher than that of plants with RD. Soil flooding and RD did not influence leaf or root N concentrations or whole-plant N content. RD increased the K concentration in the roots, but not in the leaves. Changes in the nutrient concentrations in leaves and roots indicate that RD may affect physiological performance of seedlings after planting in the field.

Correction of EELS dispersion non-uniformities for improved chemical shift analysis.

We outline a simple routine to correct for non-uniformities in the energy dispersion of a post-column electron energy-loss spectrometer for use in scanning transmission electron microscopy. We directly measure the dispersion and its variations by sweeping a spectral feature across the full camera to produce a calibration that can be used to linearize datasets post-acquisition, without the need for reference materials. The improvements are illustrated using core excitation electron energy-loss spectroscopy (EELS) spectra collected from NiO and diamond samples. The calibration is rapid and will be of use in all EELS analysis, particularly in assessments of the chemical states of materials via the chemical shift of core-loss excitations.

Tree carbon allocation explains forest drought-kill and recovery patterns.

The mechanisms governing tree drought mortality and recovery remain a subject of inquiry and active debate given their role in the terrestrial carbon cycle and their concomitant impact on climate change. Counter-intuitively, many trees do not die during the drought itself. Indeed, observations globally have documented that trees often grow for several years after drought before mortality. A combination of meta-analysis and tree physiological models demonstrate that optimal carbon allocation after drought explains observed patterns of delayed tree mortality and provides a predictive recovery framework. Specifically, post-drought, trees attempt to repair water transport tissue and achieve positive carbon balance through regrowing drought-damaged xylem. Furthermore, the number of years of xylem regrowth required to recover function increases with tree size, explaining why drought mortality increases with size. These results indicate that tree resilience to drought-kill may increase in the future, provided that CO2 fertilisation facilitates more rapid xylem regrowth.

Electrical cardioversion of patients with implanted pacemaker or cardioverter-defibrillator: results of a survey of german centers and systematic review of the literature.

AIMS: A relevant number of patients presenting for electrical cardioversion carry a pacemaker (PM) or ICD. Case reports suggest a potential hazard of external cardioversion/defibrillation. The incidence of shock related device complications is unknown. No guidelines or recommendations by international medical societies for a cardioversion protocol of cardiovascular implantable electronic device (CIED) patients exist. We conducted a nationwide survey to gather real-world clinical data on the current clinical approach towards these patients during electrical cardioversion and to estimate the incidence of shock-related complications. METHODS AND RESULTS: Ninety hospitals with > 380 ECV in 2014 were identified from mandatory hospital quality reports and 60 were randomly selected. All centers were provided with a standardized questionnaire on the general proceedings and complications during electrical cardioversion of pacemaker, ICD and CRT patients (CIED patients). Thirty-two centers (53%) participated in the survey. In total, 16,554 ECV were reported (534 ± 314 per center). Biphasic cardioversion with a first shock energy of ≥ 150 J via adhesive patches in antero-posterior orientation was preferred by most centers (78%). Eleven percent (n = 1809) of pts were reported to carry a PM/ICD. The ECV protocol was heterogeneous among centers. Complications associated with electrical cardioversion were reported in 11/1809 patients (0.6%), all were transitory elevations of pacing thresholds. CONCLUSIONS: In this nationwide snapshot survey of cardioversion procedures in Germany, approximately 11% of patients presenting for elective electrical cardioversion were pacemaker or ICD carriers. Cardioversion protocols in these patients are heterogeneous throughout centers and mostly not in accordance with recommendation of the German Cardiac Society. Complications associated with external electrical cardioversion are rare. Controlled trials and large registries are necessary to provide evidence for future recommendations.

Influence of periprocedural anticoagulation strategies on complication rate and hospital stay in patients undergoing catheter ablation for persistent atrial fibrillation.

BACKGROUND: The use of non-vitamin K antagonists (NOACs), uninterrupted (uVKA) and interrupted vitamin K antagonists (iVKA) are common periprocedural oral anticoagulation (OAC) strategies for atrial fibrillation (AF) ablation. Comparative data on complication rates resulting from OAC strategies for solely persistent AF (persAF) undergoing ablation are sparse. Thus, we sought to determine the impact of these OAC strategies on complication rates among patients with persAF undergoing catheter ablation. METHODS: Consecutive patients undergoing persAF ablation were included. Depending on preprocedural OAC, three groups were defined: (1) NOACs (paused 48 h preablation), (2) uVKA, and (3) iVKA with heparin bridging. A combined complication endpoint (CCE) composed of bleeding and thromboembolic events was analyzed. RESULTS: Between 2011 and 2014, 1440 persAF ablation procedures were performed in 1092 patients. NOACs were given in 441 procedures (31 %; rivaroxaban 57 %, dabigatran 33 %, and apixaban 10 %), uVKA in 488 (34 %), and iVKA in 511 (35 %). Adjusted CCE rates were 5.5 % [95 % confidence interval (CI) (3.1-7.8)] in group 1 (NOACs), 7.5 % [95 % CI (5.0-10.1)] in group 2 (uVKA), and 9.9 % [95 % CI (6.6-13.2)] in group 3. Compared to group 1, the combined complication risk was almost twice as high in group 3 [odd's ratio (OR) 1.9, 95 % CI (1.0-3.7), p = 0.049)]. The major complication rate was low (0.9 %). Bleeding complications, driven by minor groin complications, are more frequent than thromboembolic events (n = 112 vs. 1, p < 0.0001). CONCLUSIONS: Patients undergoing persAF ablation with iVKA anticoagulation have an increased risk of complications compared to NOACs. Major complications, such as thromboembolic events, are generally rare and are exceeded by minor bleedings.

Cortical event-related potentials in preclinical familial Alzheimer disease.

OBJECTIVE: To define changes in cortical function in persons inheriting familial Alzheimer disease (FAD) mutations before the onset of cognitive decline. METHODS: Twenty-six subjects with a family history of FAD were divided into 2 subgroups according to genotype (FAD mutation carriers, n = 15; FAD noncarriers, n = 11). Subjects were given standardized tests of cognitive function and the Clinical Dementia Rating scale (CDR). Sensory (P50, N100, P200) and cognitive (N200, P300) event-related potentials were recorded during an auditory discrimination task. Amplitudes and latencies of cortical potentials were compared among FAD mutation carriers and noncarriers. RESULTS: FAD mutation carriers and noncarriers did not significantly differ in age or on measures of cognitive function, but FAD carriers had a greater incidence of 0.5 CDR scores (1/10 noncarriers, 5/15 carriers). Relative to noncarriers, FAD mutation carriers had significantly longer latencies of the N100, P200, N200, and P300 components, and smaller slow wave amplitudes. Subanalyses of subjects having CDR scores of 0.0 also showed latency increases in FAD mutation carriers. CONCLUSIONS: Auditory sensory and cognitive cortical potentials in persons with familial Alzheimer disease (FAD) mutations are abnormal approximately 10 years before dementia will be manifest. Longer event-related potential latencies suggest slowing of cortical information processing in FAD mutation carriers.

The retinoblastoma gene Rb and its family member p130 suppress lung adenocarcinoma induced by oncogenic K-Ras.

Mutations of the retinoblastoma tumor suppressor gene RB are frequently observed in human cancers, but rarely in non-small cell lung carcinomas (NSCLCs). Emerging evidence also suggests that the RB-related gene p130 is inactivated in a subset of human NSCLCs. To directly test the specific tumor suppressor roles of RB and p130 in NSCLC, we crossed Rb and p130 conditional mutant mice to mice carrying a conditional oncogenic K-Ras allele. In this model, controlled oncogenic K-Ras activation leads to the development of adenocarcinoma, a major subtype of NSCLC. We found that loss of p130 accelerated the death of mice, providing direct evidence in vivo that p130 is a tumor suppressor gene, albeit a weak one in this context. Loss of Rb increased the efficiency of lung cancer initiation and resulted in the development of high-grade adenocarcinomas and rapid death. Thus, despite the low frequency of RB mutations in human NSCLCs and reports that K-Ras activation and loss of RB function are rarely found in the same human tumors, loss of Rb clearly cooperates with activation of oncogenic K-Ras in lung adenocarcinoma development in mice.

Monochromated, spatially resolved electron energy-loss spectroscopic measurements of gold nanoparticles in the plasmon range.

Gold nanoparticles show optical properties different from bulk material due to resonance phenomena which depend on local structure and geometry. Electron energy-loss spectrometry (EELS) in scanning transmission electron microscopy (STEM) allows the spatially resolved measurement of these properties at a resolution of few nanometers. In this work, the first monochromated measurements of gold nanoparticles (spheres, rods and triangles) are presented. Due to the improved energy resolution of about 0.2 eV, surface plasmon excitations at energies below 1 eV could be accurately measured from raw experimental data.

Biochemical markers in persons with preclinical familial Alzheimer disease.

BACKGROUND: Persons at risk for familial Alzheimer disease (FAD) provide a model in which biomarkers can be studied in presymptomatic disease. METHODS: Twenty-one subjects at risk for presenilin-1 (n = 17) or amyloid precursor protein (n = 4) mutations underwent evaluation with the Clinical Dementia Rating (CDR) scale. We obtained plasma from all subjects and CSF from 11. Plasma (Abeta(40), Abeta(42), F(2)-isoprostanes) and CSF (F(2)-isoprostanes, t-tau, p-tau(181), Abeta(40), Abeta(42), and Abeta(42)/Abeta(40) ratio) levels were compared between FAD mutation carriers (MCs) and noncarriers (NCs). RESULTS: Plasma Abeta(42) levels (25.1 pM vs 15.5 pM, p = 0.031) and the ratio of Abeta(42)/Abeta(40) (0.16 vs 0.11, p = 0.045) were higher in presymptomatic MCs. Among MCs, those with CDR scores of 0.5 had lower plasma Abeta(42) levels than those with CDR scores of 0 (14.1 pM vs 25.1, p = 0.02). The ratio of Abeta(42) to Abeta(40) was also reduced in the CSF (0.08 vs 0.15, p = 0.046) of nondemented MCs compared to NCs. Total CSF tau and p-tau(181) levels were elevated in presymptomatic FAD MCs. CSF levels of F(2)-isoprostanes were also elevated in MCs (n = 7, 48.6 pg/mL) compared to NCs (n = 4, 21.6 pg/mL, p = 0.031). CONCLUSIONS: Our data indicate that Abeta(42) is elevated in plasma in familial Alzheimer disease (FAD) mutation carriers (MCs) and suggests that this level may decrease with disease progression prior to the development of overt dementia. We also demonstrated that the ratio of Abeta(42) to Abeta(40) was reduced in the CSF of nondemented MCs and that elevations of t-tau and p-tau(181) are sensitive indicators of presymptomatic disease. Our finding of elevated F(2)-isoprostane levels in the CSF of preclinical FAD MCs suggests that oxidative stress occurs downstream to mismetabolism of amyloid precursor protein.

Single crystalline nature of para-sexiphenyl crystallites grown on KCl(100).

This work focuses on studies of the single crystal nature of para-sexiphenyl structures grown on freshly cleaved KCl(100) surfaces. Two different kinds of morphologies, namely terrace like structures and needle like structures, are found by atomic force microscopy as well as by electron microscopy. Regardless of the morphology the individual crystallites show highly regular shapes. The crystalline alignment and the degree of order of the crystallites on the surface are determined by X-ray diffraction. Several epitaxial alignments of para-sexiphenyl on KCl(100) are observed and all of them are perfectly aligned on the surface. The rocking curve widths of the organic crystallites do not exceed 800" which is approximately only the four fold of the substrates' ones. The single crystalline nature of para-sexiphenyl crystallites is proven by transmission electron microscopy, diffraction patterns, dark field imaging and high resolution techniques. Single crystalline terraced mounds reach diameters of several microns and heights of 50 nm. Single crystal needles show heights and breadths of more than 100 nm and lengths of several microns.

Cerenkov losses: a limit for bandgap determination and Kramers-Kronig analysis.

Measuring low energy losses in semiconductors and insulators with high spatial resolution becomes attractive with the increasing availability of modern transmission electron microscopes (TEMs) equipped with monochromators, C(s) correctors and energy filters. In this paper, we demonstrate that Cerenkov losses pose a limit for the interpretation of low energy loss spectra (EELS) in terms of interband transistions and bandgap determination for many materials. If the velocity of a charged particle in a medium exceeds the velocity of light, photons are emitted leading to a corresponding energy loss of a few electronvolt. Since these losses are strong for energies below the onset of interband transitions, they change the apparent loss function of semiconductors and insulators, with the risk of erroneous interpretation of spectra. We measured low energy losses of Si and GaAs with a monochromated TEM demonstrating the effect of sample thickness on Cerenkov losses. Angle resolved EELS and energy filtered diffraction patterns (taken without a monochromator) show the extremely narrow angular distribution of Cerenkov losses. The latter experiment provides a method that allows to decide whether Cerenkov radiation masks the very low loss signal in EELS.

Scripting-customized microscopy tools for Digital Micrograph.

Software is an integral part of all electron microscopy systems, encompassing hardware control, data acquisition and processing. It is unlikely that any one software system will meet all the requirements of experienced users. However, if the software supports custom scripting, then users are well placed to address any shortcomings by writing their own software. In this paper, we highlight the scripting capability within Gatan Inc.'s Digital Micrograph (DM) software, a widely used program for TEM imaging and EELS spectroscopy. We show how scripting can greatly extend the capabilities of the DM software, in tasks ranging in complexity from simple image manipulation through to full-blown microscope/imaging filter control and data acquisition. Scripting enables customized software tools to be developed to meet individual experimental needs, something which no software manufacturer could ever hope to do on a commercial basis. In essence, scripting allows the microscopist to drive the software rather than the software drive the microscopist. To foster an increased awareness and interest in DM scripting we have developed a web-based archive for DM scripts, which is freely accessible via the internet.

Gene structure and alternative splicing of glycogen synthase kinase 3 beta (GSK-3beta) in neural and non-neural tissues.

Glycogen synthase kinase 3 beta (GSK-3beta) is a multifaceted serine-threonine kinase that is of interest both because of its role in the canonical Wnt signaling pathway, which is involved in mammalian brain regionalization, and its role in phosphorylating the microtubule-associated protein Tau. Because of the potential association of GSK-3beta with human developmental and neurodegenerative conditions, we determined its exon/intron boundaries by a combination of sequencing, polymerase chain reaction (PCR) and database mining. Study of GSK-3beta expression using reverse transcription-PCR, Western blotting and Northern blotting showed alternative splicing in nervous and non-nervous system tissues. Both at the protein and mRNA level we were able to identify two isoforms, one full length form containing exon 10 and one without exon 10. At the mRNA level we identified an additional exon that is sometimes seen between exons 8 and 9. Furthermore, rather than the reported 2-3 kb mRNA predominant in non-neural tissues, we identified the major brain isoforms of GSK-3beta as two high molecular weight RNAs (8.4 and 7.7 kb).

Elevated intrahepatic pressures and decreased hepatic tissue blood flow prevent gas embolus during limited laparoscopic liver resections.

BACKGROUND: As new techniques are emerging for laparoscopic liver resections, concerns have been raised about the development of gas embolus related to the CO(2) pneumoperitoneum. We hypothesized that elevated intrahepatic vascular pressures and decreased hepatic tissue blood flow (LQB) would prevent gas embolus during laparoscopic liver resections under conventional pneumoperitoneum. METHODS: Intrahepatic vascular pressures and LQB were measured in nine pigs with varying CO(2) pneumoperitoneum. Gas embolus was determined after hepatic incision by monitoring pulmonary arterial pressure (PAP), hepatic venous PCO(2), systemic blood pressure (SBP), and suprahepatic vena cava ultrasound. RESULTS: As the pneumoperitoneum was increased from 0 to 15 mmHg, intrahepatic vascular pressures increased significantly (p < 0.05), while LQB decreased significantly (p < 0.05). A 2.0-cm hepatic incision at 4, 8, 15, and 20mmHg produced no ultrasound evidence of gas embolus and no changes in PAP, SBP, or hepatic venous PCO(2) (p = NS). CONCLUSION: These data suggest that the risk of significant embolus under conventional pneumoperitoneum is minimal during laparoscopic liver resections.

Synthetic low-calcaemic vitamin D(3) analogues inhibit secretion of insulin-like growth factor II and stimulate production of insulin-like growth factor-binding protein-6 in conjunction with growth suppression of HT-29 colon cancer cells.

The aims of the present study were to compare the ability of various synthetic analogues of 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25-(OH)(2)D(3)] to inhibit proliferation of HT-29 cells, a human colon adenocarcinoma cell line. HT-29 cells were incubated for 144 h with various concentrations (0-100 nM) of 1 alpha,25-(OH)(2)D(3), or the analogues EB1089, CB1093 or 1 beta,25-(OH)(2)D(3). All these analogues except 1 beta,25-(OH)(2)D(3) inhibited cell proliferation, but relative potencies and efficacies of EB1089 and CB1093 were much greater than that of the native vitamin. Cells grew in serum-free medium, reaching a plateau density at day 10 of culture, and addition of 10 nM 1 alpha,25-(OH)(2)D(3) or 1 beta,25-(OH)(2)D(3) did not alter the long-term growth characteristics of HT-29 cells. However, cells treated with 10 nM EB1089 or CB1093 grew at a rate slower than control and reached final densities that were 53+/-1 and 36+/-2% lower than control, respectively. Immunoblot analysis of serum-free conditioned medium using a monoclonal anti-insulin-like growth factor-(IGF)-II antibody showed that both 10 nM EB1089 and CB1093 markedly inhibited secretion of both mature 7500 M(r) and higher M(r) forms of IGF-II. Ligand blot and immunoblot analyses of conditioned media revealed the presence of IGFBPs of M(r) 24,000 (IGFBP-4), 30,000 (glycosylated IGFBP-4), 35,000 (IGFBP-2) and 32,000-34,000 (IGFBP-6). The level of IGFBP-2 was decreased by 42+/-8 and 49+/-7% by 10 nM EB 1089 and CB1093, respectively, compared to controls. IGFBP-6 was increased approximately twofold by EB1089 and CB1093, and exogenously added IGFBP-6 inhibited HT-29 cell proliferation. These results suggest that inhibition of HT-29 cell proliferation by EB1089 and CB1093 may be attributed, at least in part, to the decreased secretion of IGF-II. The increase in IGFBP-6 concentration coupled with its high affinity for IGF-II may also contribute to decreased cellular proliferation by an indirect mechanism involving sequestration of endogenously produced IGF-II.

Protective effects of ischemic preconditioning on the cold-preserved liver are tyrosine kinase dependent.

BACKGROUND: Little data exist regarding the use of ischemic preconditioning before sustained hepatic cold storage. We hypothesized that ischemic preconditioning protects hepatic grafts via a tyrosine kinase-dependent pathway. METHODS: Six porcine livers underwent routine harvest (control). Five other livers underwent 15 min of in situ ischemia followed by 15 min of reflow before harvest (ischemic preconditioning). Another five livers were pretreated with a tyrosine kinase inhibitor (genistein) before preconditioning. Upon reperfusion and after 2 hours of cold storage, graft function, graft circulatory impairment, and markers of cellular damage were analyzed. Tissue cytoplasmic extracts were analyzed for tyrosine phosphorylation with Western blot. Significance was determined with t tests. RESULTS: Ischemic-preconditioned grafts demonstrated enhanced bile production, augmented responses to a bile acid challenge, and elevated O2 consumption (P<0.05) compared to controls. Also, preconditioned grafts demonstrated improved hepatic tissue blood flow and decreased hepatic vascular resistance (P<0.005) compared to controls. Endothelial cell preservation (factor VIII immunostain) was improved in preconditioned graft biopsies compared to controls. With genistein pretreatment, all observed improvements returned to control levels. Analysis of cytoplasmic extracts demonstrated an increase in tyrosine phosphorylation before cold ischemia in preconditioned grafts only, but not in control or genistein-pretreated grafts. CONCLUSIONS: The data indicate that ischemic preconditioning protects the liver from sustained cold ischemia and that tyrosine kinases are involved in preconditioning responses.

Protein kinase C inhibition abrogates hepatic ischemic preconditioning responses.

INTRODUCTION: A transient period of warm ischemia prior to a longer ischemic episode (ischemic preconditioning) protects the hepatic graft from cold ischemia. The mechanism for this protection is unknown, as is the role of protein kinase C in ischemic preconditioning responses. METHODS: Livers from 40 kg Yorkshire pigs were harvested and subjected to 2 h of cold ischemia (n = 6) (control). Another group of harvested livers was pretreated with a 15-min ischemic period followed by 15 min of in situ perfusion with (n = 5) or without (n = 5) a protein kinase C inhibitor, chelerythrine. Following cold ischemia, all grafts were reperfused on a perfusion circuit and the following variables evaluated: (1) hepatic graft function, (2) graft circulatory impairment, (3) hepatocellular damage, and (4) endothelial cell damage. Protein kinase C levels were also evaluated by Western blot in the cytoplasm of all grafts. RESULTS AND DISCUSSION: Ischemic preconditioned grafts demonstrate improved graft function, reduced graft circulatory impairment, and reduced endothelial cell damage as compared to cold ischemia controls. When preconditioned grafts were pretreated with chelerythrine, graft function, graft circulatory impairment, and endothelial cell damage were no different than cold ischemia controls. Ischemic preconditioned grafts demonstrated decreased levels of protein kinase C prior to cold ischemia. There was no change in protein kinase C levels in cold ischemia controls or chelerythrine-pretreated grafts prior to cold ischemia. These data indicate that modulation of protein kinase C is essential for ischemic preconditioning responses in the cold preserved hepatic graft.