[The Influence of Laparoscopic Fundoplication on Reflux-Associated Cough]. / Einfluss der laparoskopischen Fundoplikation auf den mit gastroösophagealem Reflux assoziierten Husten.

Introduction: The gastrooesophageal reflux disease (GERD) is a possible cause of chronic cough. The laparoscopic fundoplication is well established in the treatment of GERD. In a retrospective study, the effectivity of this operation on the GERD associated cough was examined and possible preoperative predictive factors concerning the post-surgical therapy effect were characterized. Patients and Methods: 85 patients after laparoscopic fundoplication due to GERD treated with proton pump inhibitors without (RS-H: n = 31) or with associated cough (RS+H: n = 54) were evaluated in a three-month follow-up by data analysis regarding an indication point score from typical symptoms as well as findings (gastroscopy, histology, 24-hour oesophagus pH-metry). Results: For the leading symptoms of heartburn and regurgitation a complete freedom from complaints was reached with 98.8 % of all patients postal-surgically. In the group RS+H 70.4 % of the patients were free of cough after 3 months, other 22.2 % with significant improvement and 7.4 % with unchanged irritant cough. Higher values of the typical reflux symptoms and a therapy resistance to proton pump inhibitors (PPI) were clearly seen in the RS-H patients. The RS+H patients showed less reflux complaints with lower PPI resistance, frequent allergies as well as significantly more often an acid or bitter taste and hoarseness. After further subdivision of the RS+H patients into the subgroups RS>H (mainly reflux, n = 31) and H>RS (mainly cough), the lowest values for heartburn, regurgitation and PPI resistance were found in subgroup H>RS. Diagnostics did not show any significiant differences between the groups although a trend could be seen towards fewer duodenogastric bile reflux, larger hiatus hernias and higher DeMeester scores in RS+H and H>RS. Also smokers, non-allergic asthmatics and polyallergic sufferers with cough profited from the intervention. Conclusion: Patients with reflux-associated respiratory symptoms present an own entity with good PPI therapy response to heartburn, but not to cough. They should be considered more often for surgery. Since the cough symptoms in more than two-thirds of appropriately selected patients disappear in a short time after surgery, laparoscopic antireflux surgery should also be considered from pneumological aspects. There are no individual predictors for the success of antireflux surgery, only the sum of all relevant individual case history and clinical criteria, as they are combined in the used score, can provide a reliable indication for surgery.

Human Islets Exhibit Electrical Activity on Microelectrode Arrays (MEA).

This study demonstrates for the first time that the microelectrode array (MEA) technique allows analysis of electrical activity of islets isolated from human biopsies. We have shown before that this method, i.e., measuring beta cell electrical activity with extracellular electrodes, is a powerful tool to assess glucose responsiveness of isolated murine islets. In the present study, human islets were shown to exhibit glucose-dependent oscillatory electrical activity. The glucose responsiveness could be furthermore demonstrated by an increase of insulin secretion in response to glucose. Electrical activity was increased by tolbutamide and inhibited by diazoxide. In human islets bursts of electrical activity were markedly blunted by the Na(+) channel inhibitor tetrodotoxin which does not affect electrical activity in mouse islets. Thus, the MEA technique emerges as a powerful tool to decipher online the unique features of human islets.Additionally, this technique will enable research with human islets even if only a few islets are available and it will allow a fast and easy test of metabolic integrity of islets destined for transplantation.

Role of K(ATP) channels in ß-cell resistance to oxidative stress.

The importance of K(ATP) channels in stimulus-secretion coupling of ß-cells is well established, although they are not indispensable for the maintenance of glycaemic control. This review article depicts a new role for K(ATP) channels by showing that genetic or pharmacological ablation of these channels protects ß-cells against oxidative stress. Increased production of oxidants is a crucial factor in the pathogenesis of type 2 diabetes mellitus (T2DM). T2DM develops when ß-cells can no longer compensate for the high demand of insulin resulting from excess fuel intake. Instead ß-cells start to secrete less insulin and ß-cell mass is diminished by apoptosis. Both, reduction of insulin secretion and ß-cell mass induced by oxidative stress, are prevented by deletion or inhibition of K(ATP) channels. These findings may open up new insights into the early treatment of T2DM.

BK channels affect glucose homeostasis and cell viability of murine pancreatic beta cells.

AIMS/HYPOTHESIS: Evidence is accumulating that Ca(2+)-regulated K(+) (K(Ca)) channels are important for beta cell function. We used BK channel knockout (BK-KO) mice to examine the role of these K(Ca) channels for glucose homeostasis, beta cell function and viability. METHODS: Glucose and insulin tolerance were tested with male wild-type and BK-KO mice. BK channels were detected by single-cell RT-PCR, cytosolic Ca(2+) concentration ([Ca(2+)](c)) by fura-2 fluorescence, and insulin secretion by radioimmunoassay. Electrophysiology was performed with the patch-clamp technique. Apoptosis was detected via caspase 3 or TUNEL assay. RESULTS: BK channels were expressed in murine pancreatic beta cells. BK-KO mice were normoglycaemic but displayed markedly impaired glucose tolerance. Genetic or pharmacological deletion of the BK channel reduced glucose-induced insulin secretion from isolated islets. BK-KO and BK channel inhibition (with iberiotoxin, 100 nmol/l) broadened action potentials and abolished the after-hyperpolarisation in glucose-stimulated beta cells. However, BK-KO did not affect action potential frequency, the plateau potential at which action potentials start or glucose-induced elevation of [Ca(2+)](c). BK-KO had no direct influence on exocytosis. Importantly, in BK-KO islet cells the fraction of apoptotic cells and the rate of cell death induced by oxidative stress (H(2)O(2), 10-100 µmol/l) were significantly increased compared with wild-type controls. Similar effects were obtained with iberiotoxin. Determination of H(2)O(2)-induced K(+) currents revealed that BK channels contribute to the hyperpolarising K(+) current activated under conditions of oxidative stress. CONCLUSIONS/INTERPRETATION: Ablation or inhibition of BK channels impairs glucose homeostasis and insulin secretion by interfering with beta cell stimulus-secretion coupling. In addition, BK channels are part of a defence mechanism against apoptosis and oxidative stress.

Activation of the Na+/K+-ATPase by insulin and glucose as a putative negative feedback mechanism in pancreatic beta-cells.

Pancreatic beta-cells of sulfonylurea receptor type 1 knock-out (SUR1(-/-)) mice exhibit an oscillating membrane potential (V (m)) demonstrating that hyper-polarisation occurs despite the lack of K(ATP) channels. We hypothesize that glucose activates the Na(+)/K(+)-ATPase thus increasing a hyper-polarising current. Elevating glucose in SUR1(-/-) beta-cells resulted in a transient fall in V (m) and [Ca(2+)](c) independent of sarcoplasmic and endoplasmic reticulum Ca(2+)-activated ATPase (SERCA) activation. This was not affected by K(+) channel blockade but inhibited by ATP depletion and by ouabain. Increasing glucose also reduced [Na(+)](c), an effect reversed by ouabain. Exogenously applied insulin decreased [Na(+)](c) and hyper-polarised V (m). Inhibiting insulin signalling in SUR1(-/-) beta-cells blunted the glucose-induced decrease of [Ca(2+)](c). Tolbutamide (1 mmol/l) disclosed the SERCA-independent effect of glucose on [Ca(2+)](c) in wild-type beta-cells. The data show that in SUR1(-/-) beta-cells, glucose activates the Na(+)/K(+)-ATPase presumably by increasing [ATP](c). Insulin can also stimulate the pump and potentiate the effect of glucose. Pathways involving the pump may thus serve as potential drug targets in certain metabolic disorders.

An adenylate kinase is involved in KATP channel regulation of mouse pancreatic beta cells.

AIMS/HYPOTHESIS: In a previous study, we demonstrated that a creatine kinase (CK) modulates K(ATP) channel activity in pancreatic beta cells. To explore phosphotransfer signalling pathways in more detail, we examined whether K(ATP) channel regulation in beta cells is determined by a metabolic interaction between adenylate kinase (AK) and CK. METHODS: Single channel activity was measured with the patch-clamp technique in the inside-out (i/o) and open-cell attached (oca) configuration. RESULTS: The ATP sensitivity of K(ATP) channels was higher in i/o patches than in permeabilised beta cells (oca). One reason for this observation could be that the local ATP:ADP ratio in the proximity of the channels is determined by factors not active in i/o patches. AMP (0.1 mmol/l) clearly increased open channel probability in the presence of ATP (0.125 mmol/l) in permeabilised cells but not in excised patches. This suggests that AK-catalysed ADP production in the vicinity of the channels is involved in K(ATP) channel regulation. The observation that the stimulatory effect of AMP on K(ATP) channels was prevented by the AK inhibitor P (1),P (5)-di(adenosine-5')pentaphosphate (Ap(5)A; 20 micromol/l) and abolished in the presence of the non-metabolisable ATP analogue adenosine 5'-(beta,gamma-imido)triphosphate tetralithium salt (AMP-PNP; 0.12 mmol/l) strengthens this idea. In beta cells from AK1 knockout mice, the effect of AMP was less pronounced, though not completely suppressed. The increase in K(ATP) channel activity induced by AMP in the presence of ATP was outweighed by phosphocreatine (1 mmol/l). We suggest that this is due to an elevation of the ATP concentration by CK. CONCLUSIONS/INTERPRETATION: We propose that phosphotransfer events mediated by AK and CK play an important role in determining the effective concentrations of ATP and ADP in the microenvironment of pancreatic beta cell K(ATP) channels. Thus, these enzymes determine the open probability of K(ATP) channels and eventually the actual rate of insulin secretion.

Crosstalk between membrane potential and cytosolic Ca2+ concentration in beta cells from Sur1-/- mice.

AIMS/HYPOTHESIS: Islets or beta cells from Sur1(-/-) mice were used to determine whether changes in plasma membrane potential (V(m)) remain coupled to changes in cytosolic Ca(2+) ([Ca(2+)](i)) in the absence of K(ATP) channels and thus provide a triggering signal for insulin secretion. The study also sought to elucidate whether [Ca(2+)](i) influences oscillations in V(m) in sur1(-/-) beta cells. METHODS: Plasma membrane potential and ion currents were measured with microelectrodes and the patch-clamp technique. [Ca(2+)](i) was monitored with the fluorescent dye fura-2. Insulin secretion from isolated islets was determined by static incubations. RESULTS: Membrane depolarisation of Sur1(-/-) islets by arginine or increased extracellular K(+), elevated [Ca(2+)](i) and augmented insulin secretion. Oligomycin completely abolished glucose-stimulated insulin release from Sur1(-/-) islets. Oscillations in V(m) were influenced by [Ca(2+)](i) as follows: (1) elevation of extracellular Ca(2+) lengthened phases of membrane hyperpolarisation; (2) simulating a burst of action potentials induced a Ca(2+)-dependent outward current that was augmented by increased Ca(2+) influx through L-type Ca(2+) channels; (3) Ca(2+) depletion of intracellular stores by cyclopiazonic acid increased the burst frequency in Sur1(-/-) islets, elevating [Ca(2+)](i) and insulin secretion; (4) store depletion activated a Ca(2+) influx that was not inhibitable by the L-type Ca(2+) channel blocker D600. CONCLUSIONS/INTERPRETATION: Although V(m) is largely uncoupled from glucose metabolism in the absence of K(ATP) channels, increased electrical activity leads to elevations of [Ca(2+)](i) that are sufficient to stimulate insulin secretion. In Sur1(-/-) beta cells, [Ca(2+)](i) exerts feedback mechanisms on V(m) by activating a hyperpolarising outward current and by depolarising V(m) via store-operated ion channels.

Direct interference of HIV protease inhibitors with pancreatic beta-cell function.

The aim of the present study was to evaluate whether HIV protease inhibitors directly interfere with stimulus-secretion coupling in pancreatic beta-cells. Insulin secretion was determined by a radioimmunoassay (RIA), cytosolic free Ca2+ concentration ([Ca2+]c) with the fluorescence dye fura-2 and whole-cell membrane currents with the patch-clamp technique. Glucose-induced insulin secretion was inhibited in a concentration-dependent manner by ritonavir and nelfinavir but not by indinavir. Ritonavir and nelfinavir lowered [Ca2+]c in the presence of a stimulatory glucose concentration whereas indinavir again had no effect. Ritonavir and nelfinavir completely inhibited the effect of tolbutamide, which normally increases [Ca2+]c by blocking KATP channels. This observation points to an action of both drugs on KATP channels or a step distal to these channels in stimulus-secretion coupling. Ritonavir was used to further evaluate the direct effects of HIV protease inhibitors on beta-cell ion channel currents. Unexpectedly, ritonavir inhibited neither the whole-cell KATP current nor the whole-cell L-type Ca2+ current. Tolbutamide almost completely suppressed the KATP current in the presence of ritonavir excluding that ritonavir alters the tolbutamide sensitivity of the KATP channel. Ritonavir increased the length and decreased the frequency of glucose-induced action potentials. This effect can be attributed to inhibition of voltage-dependent K+ currents. Intracellular stores seem not to be involved in the ritonavir-induced lowering of [Ca2+]c. In conclusion, different HIV protease inhibitors surprisingly reveal distinct effects on insulin secretion. Ritonavir inhibits insulin secretion by lowering [Ca2+]c but this effect is evidently independent of the opening of KATP channels or the closure of voltage-dependent Ca2+ channels, which are commonly considered to play a key role in stimulus-secretion coupling.

Oscillations of membrane potential and cytosolic Ca(2+) concentration in SUR1(-/-) beta cells.

AIMS/HYPOTHESIS: SUR1(ABCC8)(-/-) mice lacking functional K(ATP) channels are an appropriate model to test the significance of K(ATP) channels in beta-cell function. We examined how this gene deletion interferes with stimulus-secretion coupling. We tested the influence of metabolic inhibition and galanin, whose mode of action is controversial. METHODS: Plasma membrane potential (Vm) and currents were measured with microelectrodes or the patch-clamp technique; cytosolic Ca(2+) concentrations ([Ca(2+)](c)) and mitochondrial membrane potential (DeltaPsi) were measured using fluorescent dyes. RESULTS: In contrast to the controls, SUR1(-/-) beta cells showed electrical activity even at a low glucose concentration. Continuous spike activity was measured with the patch-clamp technique, but with microelectrodes slow oscillations in Vm consisting of bursts of Ca(2+)-dependent action potentials were detected. [Ca(2+)](c) showed various patterns of oscillations or a sustained increase. Sodium azide did not hyperpolarize SUR1(-/-) beta cells. The depolarization of DeltaPsi evoked by sodium azide was significantly lower in SUR1(-/-) than SUR1(+/+) cells. Galanin transiently decreased action potential frequency and [Ca(2+)](c) in cells from both SUR1(-/-) and SUR1(+/+) mice. CONCLUSION/INTERPRETATION: The strong dependence of Vm and [Ca(2+)](c) on glucose concentration observed in SUR1(+/+) beta cells is disrupted in the knock-out cells. This demonstrates that both parameters oscillate in the absence of functional K(ATP) channels. The lack of effect of metabolic inhibition by sodium azide shows that in SUR1(-/-) beta cells changes in ATP/ADP no longer link glucose metabolism and Vm. The results with galanin suggest that this peptide affects beta cells independently of K(ATP) currents and thus could contribute to the regulation of beta-cell function in SUR1(-/-) animals.

Auxiliary partial orthotopic liver transplantation: treatment of acute liver failure in a new rat model.

BACKGROUND AND AIMS: Liver regeneration and functional interaction between native liver and graft after auxiliary partial orthotopic liver transplantation (APOLT) are not entirely understood and, therefore, require an experimental model simulating the clinical features of acute liver failure (ALF) and the surgical technique of APOLT. MATERIALS AND METHODS: ALF was induced by subtotal hepatectomy in 50 Lewis rats (200-250 g). Sham operation (I), ALF without treatment (II), ALF with portocaval shunt for decreasing blood flow of the remnant liver (III), and ALF treated by APOLT (IV) were performed. The auxiliary graft represented a left donor liver lobe which was orthotopically implanted using a microsurgical technique including reconstruction of the graft artery and internal biliary drainage. Operative outcomes, serum chemistry and histopathological findings were examined up to the 14th day. RESULTS: ALF without treatment (groups II and III) led to a small droplet fatty degeneration in the hepatocytes and a significant increase of liver parameters until the death of the animals within the first two postoperative days ( P<0.05). After APOLT (group IV), 80% of the animals survived up to the 14th day, revealing significantly decreased liver parameters ( P<0.05), a well-perfused graft and an up to five times increased native liver size with normal architecture. CONCLUSION: This new rat model simulates the clinical features of an ALF treated by APOLT and is especially interesting for further basic research on the interaction between native liver and auxiliary graft after APOLT.

Diabetogenic effect of cyclosporin A is mediated by interference with mitochondrial function of pancreatic B-cells.

Treatment of patients after organ transplantation with the immunosuppressive drug cyclosporin A (CsA) is often accompanied by impaired glucose tolerance, thus promoting the development of diabetes mellitus. In the present article we show that 2 to 5 microM CsA diminishes glucose-induced insulin secretion of isolated mouse pancreatic islets in vitro by inhibiting glucose-stimulated oscillations of the cytoplasmic free-Ca(2+) concentration [Ca(2+)](c). This effect is not due to an inhibition of calcineurin, which mediates the immunosuppressive effect of CsA, because other calcineurin inhibitors, deltamethrin and tacrolimus, did not affect the oscillations in [Ca(2+)](c) of the B-cells. The CsA-induced decrease in [Ca(2+)](c) to basal values was not caused by a direct inhibition of L-type Ca(2+) channels. CsA is known to be a potent inhibitor of the mitochondrial permeability transition pore (PTP), which we recently suggested to be involved in the regulation of oscillations. Consequently, CsA also inhibited the oscillations of the cell membrane potential, and it is shown that these effects could not be ascribed to cellular ATP depletion. However, the mitochondrial membrane potential Delta Psi was affected by CsA by inhibiting the oscillations in Delta Psi. Interestingly, the observed reduction in [Ca(2+)](c) could be counteracted by the K(+)(ATP) channel blocker tolbutamide, indicating that the stimulus-secretion coupling was interrupted before the closure of K(+)(ATP) channels. It is concluded that CsA alters B-cell function by inhibiting the mitochondrial PTP. This terminates the oscillatory activity that is indispensable for adequate insulin secretion. Thus, CsA acts on different targets to induce the immunosuppressive and the diabetogenic effect.

Stimulation of leukotriene synthesis in intact polymorphonuclear cells by the 5-lipoxygenase inhibitor 3-oxo-tirucallic acid.

Commercially available extracts from Boswellia serrata resin used as anti-inflammatory drugs or phytonutrients show paradoxical concentration-dependent potentiating and inhibitory actions on 5-lipoxygenase (5-LO) product synthesis in stimulated PMNs. In our attempt to characterize the stimulating constituents, we identified the tetracyclic triterpene 3-oxo-tirucallic acid (3-oxo-TA), which, in the range from 2.5 to 15 microM, enhanced 5-LO product formation in ionophore-challenged polymorphonuclear cells (PMNs) (e.g., from 1981 +/- 177 to 3042 +/- 208 pmol at 10 microM 3-oxo-TA), and initiated Ca(2+) mobilization, MEK-1/2 phosphorylation, 5-LO translocation, and 5-LO product formation in resting cells (534 +/- 394 pmol/5 x 10(6) PMNs). In cell-free 5-LO assays, 3-oxo-TA acted only inhibitory (IC(50) value of about 3 microM), demonstrating the pivotal role of intact cell structure for its activating property. In 3-oxo-TA-challenged PMNs, the mitogen-activated protein kinase kinase (MEK)-1/2 inhibitor PD098059 abolished 5-LO product formation, along with inhibition of MEK-1/2 phosphorylation and 5-LO translocation. The 3-acetoxy derivative of 3-oxo-TA acted like 3-oxo-TA in intact PMNs, whereas 3-hydroxy-TA barely stimulated MEK phosphorylation in resting cells and showed only inhibition on ionophore-induced 5-LO product synthesis. Steroid-type tetracycles neither induced 5-LO activation nor had enhancing or inhibitory effects. In summary, defined natural tetracyclic triterpenes, which act as inhibitors of the 5-LO in the cell-free assay, initiate 5-LO activation by a MEK-inhibitor sensitive mechanism and potentiate stimulated product synthesis in intact cells. Because TAs contribute significantly to the overall biological effects of B. serrata resin extracts, special precaution for standardization is recommended when using B. serrata preparations as drugs or dietary supplements.

REVOLUTA regulates meristem initiation at lateral positions.

While the shoot apical meristem (SAM) is indirectly responsible for the initiation of all above-ground postembryonic organs, in most plants the vast majority of these organs are directly initiated by lateral meristems. In Arabidopsis thaliana, the lateral meristems include flower meristems (FMs), which form on the flanks of the SAM, and lateral shoot meristems (LSMs), which develop in leaf axils. While significant progress has been made on the molecular genetic basis of SAM initiation during embryo development, relatively little is known about the initiation of meristems at lateral positions. Here we have characterized the phenotypic consequences and genetic interactions of mutations in the REVOLUTA (REV) gene, with an emphasis on the role of REV in lateral meristem initiation. Our observations indicate that REV is required for initiation of both LSMs and FMs, and likely acts in the same pathway as, and upstream of, known meristem regulators. We identified the REV gene and found it encodes a predicted homeodomain/leucine zipper transcription factor that also contains a START sterol-lipid binding domain. REV is the same as the IFL gene. REV was expressed at the earliest stages of LSM and FM formation. Within the inflorescence shoot meristem, REV expression appeared to predict 3--5 incipient flower primordia on the flanks of the SAM, and REV expression at stage 1 and stage 2 matched that of WUS and STM, respectively. We propose that REV acts at lateral positions to activate the expression of known meristem regulators.

Contrasting effects of alloxan on islets and single mouse pancreatic beta-cells.

Alloxan is used to induce diabetes in animals; however, the underlying mechanisms are still a matter of debate. Alloxan evoked a rapid hyperpolarization of the plasma membrane potential and suppressed electrical activity elicited by 15 mM glucose, thus terminating voltage-dependent Ca(2+) influx. Accordingly, glucose-induced oscillations in intracellular free Ca(2+) concentration were abolished. The effect of alloxan on membrane potential could not be reversed by glucose but was reversed by tolbutamide. However, the sensitivity to tolbutamide was decreased after treatment of the cells with alloxan. These effects closely resemble those described earlier for H(2)O(2). H(2)O(2) and alloxan decreased the mitochondrial membrane potential, indicating a decrease in ATP production and thus interference with cell metabolism. A decrease in ATP synthesis would explain the plasma membrane hyperpolarization observed in intact islets, reflecting the activation of ATP-dependent K(+) channels. Surprisingly, alloxan inhibited the whole-cell K(+)(ATP) current measured in single cells and the single-channel K(+)(ATP) current registered in excised patches. This inhibitory effect of alloxan is not mediated by changes in cell metabolism but seems to be due to direct interactions with the K(+)(ATP) channels via thiol-group oxidation. We have monitored the appearance of reactive oxygen species in single cells and islets treated with alloxan and H(2)O(2) for comparison. In contrast to H(2)O(2), alloxan induced the appearance of measurable reactive oxygen species only in islets but not in single cells. The results show that alloxan evokes different effects in islets and single cells, giving a possible explanation for inconsistent results reported in the past. It is concluded that alloxan exerts its diabetogenic effect by the production of H(2)O(2) in intact islets.

Protective effects of ambroxol in hypothermic liver preservation: a transplant study.

The bronchosecretolytic agent ambroxol added to histidine-tryptophan-ketoglutarate (HTK) solution has recently been shown to protect cold stored rat hepatocytes. The aim of the present study was to confirm these observations in a rat liver transplantation model. Before orthotopic liver transplantation, donor livers from 30 syngeneic Wistar rats were assigned to three groups (n = 10): (A) in situ flush (ISF) and 1/2-h cold storage (CS) with HTK solution, (B) ISF and 3-h CS with HTK, and (C) ISF and 3-h CS with HTK + 10(-3) mol/L ambroxol. The efficacy of the drug was evaluated by postoperative survival (> 14 days) and liver enzyme release (ALT), bile flow, histomorphological injury, and malondialdehyde (MDA) level in the grafts 15 min after reperfusion. After 1/2-h CS with HTK solution (A), 90% of the transplanted rats survived. In comparison with donor conditions, bile flow in the reperfused grafts decreased to 87 +/- 5.3%, whereas postoperative ALT levels slightly increased. After 3-h HTK preservation (B), the survival rate decreased to 60%, while ALT values markedly increased and bile flow after reperfusion declined to 82 +/- 6.6%. Ambroxol added to HTK solution (C) enhanced bile flow to 106 +/- 3.4%(p < .05), and reduced ALT and MDA levels and histomorphological injury of the transplanted livers, so that its beneficial effect in organ preservation has been confirmed in the transplant model. However, survival rate was not improved by the agent, probably because of the low cold ischemia tolerance of the Wistar rat livers used.

The roots of microbiology and the influence of Ferdinand Cohn on microbiology of the 19th century.

The beginning of modern microbiology can be traced back to the 1870s, and it was based on the development of new concepts that originated during the two preceding centuries on the role of microorganisms, new experimental methods, and discoveries in chemistry, physics, and evolutionary cell biology. The crucial progress was the isolation and growth on solid media of clone cultures arising from single cells and the demonstration that these pure cultures have specific, inheritable characteristics and metabolic capacities. The doctrine of the spontaneous generation of microorganisms, which stimulated research for a century, lost its role as an important concept. Microorganisms were discovered to be causative agents of infectious diseases and of specific metabolic processes. Microscopy techniques advanced studies on microorganisms. The discovery of sexuality and development in microorganisms and Darwin's theory of evolution contributed to the founding of microbiology as a science. Ferdinand Cohn (1828-1898), a pioneer in the developmental biology of lower plants, considerably promoted the taxonomy and physiology of bacteria, discovered the heat-resistant endospores of bacilli, and was active in applied microbiology.