[Individualised correction of presbyopia by the use of phacoemulsification: monovision and multifocal lenses]. / Individualisierte Presbyopiekorrektur bei der Kataraktoperation: Monovision und Multifokallinsen.

The cataract operation has transformed from a procedure for correcting spherical and astigmatic errors to one for correcting even presbyopia. Higher demands by the patients and more and more complex and individual lifestyle options need customised concepts of presbyopic correction, taking also into account comorbidities and neuroadaption. One concept for achieving this goal is multifocal lenses, undergoing a renaissance these days. Monovision using monofocal lenses is a cost neutral alternative with very few side effects, if well performed. It is defined by the far focus of one eye and the near focus of the other. Binocularity of human vision enables multiple options by combining different means of presbyopia correction. But it also complicates making the right choice. This underlines the importance of an accurate patient selection and the precise definition of what to achieve for each individual patient.

Hepatocyte differentiation of mesenchymal stem cells from human adipose tissue in vitro promotes hepatic integration in vivo.

OBJECTIVE: The hepatic integration of human adipose tissue derived mesenchymal stem cells (hAT-MSCs) in vivo with or without prior differentiation to hepatocyte-like cells in vitro was investigated. METHODS AND RESULTS: Cells, isolated either from peritoneal or subcutaneous adipose tissue, expressed mesenchymal stem cell surface markers and featured multiple lineage differentiation. Under conditions favouring hepatocyte differentiation, hAT-MSCs gained hepatocytic functions in vitro including urea formation, glycogen synthesis, cytochrome P450 enzyme activity, and expression of hepatocyte-specific transcripts of carbamoylphosphate synthetase, albumin and cytochrome P450 type 3A4 (CYP3A4). Transgenic expression of green fluorescent protein emerged upon hepatocyte differentiation when driven by the hepatocyte-specific promoter of the cytosolic phosphoenolpyruvate carboxykinase gene but was constitutive from the ubiquitin gene promoter. Human AT-MSCs were transplanted into livers of immunodeficient Pfp/Rag2-/- mice with or without prior hepatocyte differentiation in vitro. Donor-derived human cells engrafted in the mouse host liver predominantly in the periportal region of the liver lobule. They expressed HepPar1 and albumin, typical features of differentiated human hepatocytes, in the otherwise negative mouse liver background. Engraftment was significantly more efficient using hAT-MSCs pre-differentiated to hepatocyte-like cells in vitro as compared with undifferentiated cells. CONCLUSIONS: Pre-differentiation of human MSCs from adipose tissue into hepatocyte-like cells in vitro facilitates long term functional hepatic integration in vivo.

Hepatic progenitor cells from adult human livers for cell transplantation.

OBJECTIVE: Liver regeneration is mainly based on cellular self-renewal including progenitor cells. Efforts have been made to harness this potential for cell transplantation, but shortage of hepatocytes and premature differentiated progenitor cells from extra-hepatic organs are limiting factors. Histological studies implied that resident cells in adult liver can proliferate, have bipotential character and may be a suitable source for cell transplantation. METHODS: Particular cell populations were isolated after adequate tissue dissociation. Single cell suspensions were purified by Thy-1 positivity selection, characterised in vitro and transplanted in immunodeficient Pfp/Rag2 mice. RESULTS: Thy-1(+) cells that are mainly found in the portal tract and the surrounding parenchyma, were isolated from surgical liver tissue with high yields from specimens with histological signs of regeneration. Thy-1(+) cell populations were positive for progenitor (CD34, c-kit, CK14, M2PK, OV6), biliary (CK19) and hepatic (HepPar1) markers revealing their progenitor as well as hepatic and biliary nature. The potential of Thy-1(+) cells for differentiation in vitro was demonstrated by increased mRNA and protein expression for hepatic (CK18, HepPar1) and biliary (CK7) markers during culture while progenitor markers CK14, chromogranin A and nestin were reduced. After transplantation of Thy-1(+) cells into livers of immunodeficient mice, engraftment was predominantly seen in the periportal portion of the liver lobule. Analysis of in situ material revealed that transplanted cells express human hepatic markers HepPar1 and albumin, indicating functional engraftment. CONCLUSION: Bipotential progenitor cells from human adult livers can be isolated using Thy-1 and might be a potential candidate for cell treatment in liver diseases.

Cultivation of hepatoma cell line HepG2 on nanoporous aluminum oxide membranes.

Nanoporous aluminum oxide membranes were prepared by anodic oxidation of aluminum for application as novel cell culture substrates. Self-supporting as well as mechanically stabilized nanoporous membranes were produced from aluminum plates and micro-imprinted aluminum foils, respectively. Membranes of two different pore sizes (70 and 260 nm) were selected to investigate cellular interactions with such nanoporous substrates using cells of hepatoma cell line HepG2. The membranes express excellent cell-growth conditions. As shown by scanning electron microscopy investigations, the cells could easily adhere to the membranes and proliferate during a 4 day cell culture period. The cells exhibit normal morphology and were able to penetrate into pores with a diameter of 260 nm by small extensions (filopodia). On mechanically stabilized aluminum oxide membranes it was observed that the cells even adhere to the walls of the small cavities. It was demonstrated experimentally that the nanoporous aluminum oxide membranes are well suited as substrates in cell culture model systems for metabolic, pharmacological/toxicological research, tissue engineering and studies on pathogens as well as bioartificial liver systems.

A microtiterplate-based screening assay to assess diverse effects on cytochrome P450 enzyme activities in primary rat hepatocytes by various compounds.

During the development of potential drugs it is useful to identify pharmacological and/or toxicological side effects of a compound as early as possible in order to exclude them from further development for reasons of time and cost. Activation or inactivation of members of the cytochrome P450-dependent monooxygenase system (CYP450) might indicate potential undesired effects of a given compound. However, results using CYP450 assay systems are often inconsistent because of different experimental settings. Therefore, it was the goal of the present study to optimize the CYP450 assay in primary rat hepatocytes with respect to the time point of addition of and duration of exposure to alpha-naphthoflavone (ANF) and beta-naphthoflavone (BNF) as well as trans-resveratrol (RES), which have well-described stimulatory and inhibitory effects on CYP450 enzymes of the 1A and 2B family, respectively. Hepatocytes were also treated with putative lipoxygenase (LOX)/cyclooxygenase (COX) inhibitors with unknown impact on CYP450 enzyme activity in order to detect potential side effects. Cells were cultured for up to 7 days on 96-well microtiter plates, and enzyme activity was determined by a conventional fluorescence spectroscopy assay. ANF and BNF, given to the cells after 4 days of culture, stimulated CYP1A and 2B activities significantly in a concentration-dependent fashion after long-term exposure for at least 1 day. However, during short-term exposure for 1-6 h, CYP1A activity was inhibited, while CYP2B was increased weakly by ANF but not BNF. RES inhibited CYP1A activity during short- and long-term exposure without affecting CYP2B activity. From the results it was concluded that primary rat hepatocytes should be cultured for at least 3-4 days but no longer prior to the assay. The assay should be performed at two different time points of exposure, i.e., 6 h for short-term and 24 h for long-term exposure. The compounds under investigation should be applied at two different concentrations, e.g., at one time and 10 times higher concentrations, which should be oriented to the ED50, provided it is known for the respective substance. Under these assay conditions the LOX/COX inhibitors tested activated CYP1A enzyme activity in long-term but instead inhibited it in short-term experiments. CYP2B activity was stimulated during short- and long-term exposure. These results indicated drug side effects recommending exclusion of the compounds from the drug developmental process. Hence, in order to assess the pharmacological potential of novel compounds it is adequate to perform both short- and long-term experiments to concisely describe the effect of a compound on the CYP450 system.

Immunoelectron microscopic localization of microsomal alanine aminopeptidase.

The localization of microsomal alanine aminopeptidase was investigated in the rat kidney. Resin embedding failed to demonstrate the localization of the enzyme by immunogold labelling. Using a cryo-ultramicrotomy method the enzyme could be detected on the luminal side of the brush border membrane of proximal tubular cells and to a lesser degree in their mitochondria. Furthermore, vesicular structures labelled with gold were found in the cytoplasma in the apical region of these cells.

[Piggyback lens implantation for anisometropia in pseudophakic eyes]. / Huckepacklinsenimplantation zur Korrektur einer Anisometropie bei Pseudophakie.

OBJECTIVE: Long standing pseudophakia or previous laser capsulotomy make lens exchange for anisometropia or incorrect target refraction difficult. Piggyback lens implantation might be a less traumatic surgical alternative. PATIENTS AND METHODS: Eight patients had a secondary posterior chamber lens implant (PMMA) into the ciliary sulcus. Lens power was estimated by the preoperative refraction according to the refractive vergence formula and was controlled intraoperatively by an automatic refractometer. Surgical access minimized preoperative astigmatism. RESULTS: All patients had disturbing anisometropia of 4.57+/-2.86 D and had undergone cataract surgery 1-13 years previously. Piggyback lens implantation was combined with surgical aspiration of the secondary cataract in three patients. Due to recurrent PVR retinal detachment, one patient received a silicon oil refilling procedure and piggyback lens implantation. The preoperative spherical equivalent was -3.34+/-4.9 D. Mean follow up time was 16.6 months. Postoperative anisometropia was reduced to 1.1+/-1.01 D. The mean absolute deviation from target refraction was 0+/-0.92 D. Elevated IOP or secondary cataract formation in the lens interface were not observed. One patient developed pigment dispersion. CONCLUSION: Secondary piggyback lens implantation into the ciliary sulcus in front of an existing posterior chamber lens is a safe and effective refractive surgical procedure for special cases with predictable refraction.

The behaviour of NAD+ and NADH in Acinetobacter calcoaceticus during n-alkane assimilation.

The behaviour of the nicotinamide adenine dinucleotides NAD+ and NADH in Acinetobacter calcoaceticus during n-alkane assimilation was studied, acetate and succinate being used as reference carbon sources. The intracellular concentration of the two nucleotides was found to increase during the exponential growth phase, reaching its maximum in the phase of decreasing growth rates. In the exponential phase, the NAD+/NADH quotients were less than 1 and showed only unimportant variations. In the phase of decreasing growth rates, the concentration of NADH showed a distinct decrease, reaching its minimum in the stationary phase. Parallel to this, the concentration of NAD+ showed a continuous increase until the stationary phase was reached. This resulted in an increase, during the phase of decreasing growth rates, of the NAD+/NADH quotients to values greater than 1, similarly as recorded in the stationary phase. There were no fundamental differences in this behaviour between the individual carbon sources.

[Iris sutures for refixation of decentered intraocular lenses]. / Irisnahtfixation bei dezentrierter Intraokularlinse.

BACKGROUND: Most revisions after intraocular lens (IOL) implantation are due to an insufficiency of the zonular apparatus. Frequently, an inferior decentration can be seen ("sunset syndrome"). In this clinical study, suture refixation of the haptics to the iris was assessed. Functional and morphological results were considered. METHOD: 21 eyes with subluxation of the IOL were treated with iris sutures for refixation. The operation was performed under topical and intracameral anaesthesia. Refixation was achieved by suturing one or both haptics to one or two fixation points in the outer periphery of the iris. Complete zonular dialysis made it necessary to suture at two fixation spots. All eyes were examined preoperatively, at one day and three months postoperatively considering functional results and postoperative IOL centration. RESULTS: In 15 eyes (71%), the IOL was optimally centred postoperatively. In 5 eyes (24%), a revision had to be performed due to instable centration. In one further case the IOL was minimally decentred, but its position was stable. Best corrected visual acuity was 0.3 +/- 0.2 preoperatively and 0.5 +/- 0.2 after three months. The mean refraction was stable in the postoperative course and astigmatism did not change significantly. There were no major complications intraoperatively or postoperatively, but a localised iris atrophy at the haptic fixation points was noted. CONCLUSIONS: In the presence of a partially intact zonular apparatus, iris sutures are a safe and minimally invasive method for fixing a decentred IOL. Postoperative centration and functional results were stable after 3 months.

Periplasmic aminopeptidases in Acinetobacter calcoaceticus and Pseudomonas aeruginosa.

The greater part of the intracellular aminopeptidases in Pseudomonas aeruginosa and Acinetobacter calcoaceticus is soluble. The localization of aminopeptidases in the cells was examined using the osmotic shock method with some modifications. When the cells of A. calcoaceticus and P. aeruginosa of the logarithmic phase were subjected to an osmotic shock, all aminopeptidases investigated were mainly localized in the sucrose supernatants and in the periplasm. Acid phosphatase as marker enzyme for periplasm showed a similar distribution between the fractions as the aminopeptidases. The periplasmic aminopeptidases of both microorganisms were separated by FPLC on Superose 12 and their molecular masses were determined. The results obtained show that at least four different aminopeptidases occur in the periplasm, a leucyl aminopeptidase (LAP, cleaving Leu-NH-NH2, 400 kDa), a glutamyl aminopeptidase (GAP, 200 kDa), an alanyl aminopeptidase (AAP, 80 kDa) and a prolyl aminopeptidase (PAP, 65 kDa). The results are in agreement for both species. Our results show clearly that aminopeptidases of these typical members of Gram-negative bacteria are mainly periplasmic like degrading enzymes (alkaline and acid phosphatases, 5'-nucleotidase, cyclic phosphodiesterase), detoxifying enzymes and binding proteins for amino acids and sugars.

Purification of a periplasmic insulin-cleaving proteinase from Acinetobacter calcoaceticus.

Cells of Acinetobacter calcoaceticus contain a constitutive periplasmic metalloproteinase showing similar properties as the periplasmic metalloproteinase of Escherichia coli. The periplasmic proteinase of A. calcoaceticus was purified, starting from periplasm, by ammonium sulfate precipitation, hydrophobic interaction chromatography and chromatofocusing up to the homogeneity of the enzyme in SDS-electrophoresis with a yield of 6.7% and a purification factor of 417. The enzyme has a molecular mass of 108,000 (gel filtration) or 112,000 (native electrophoresis), and consists of four identical subunits with a molecular mass of 27,000 (SDS-electrophoresis). The purified enzyme degrades preferentially polypeptides such as glucagon and insulin. Larger proteins are accepted as substrates to a considerably lower extent. All tested synthetic substrates with trypsin, chymotrypsin, elastase and thermolysin specificity were not cleaved. Therefore, the described enzyme was designated "insulin-cleaving proteinase" (ICP).

[Identification and localization of exopeptidase activities bound to membranes of Acinetobacter calcoaceticus]. / Nachweis und Lokalisation von Exopeptidase-Aktivitäten in Membranen von Acinetobacter calcoaceticus.

After gel filtration the supernatant of Triton X-100 treated membrane sediments of Acinetobacter calcoaceticus shows activities for alanyl aminopeptidase, leucyl aminopeptidase, glutamyl aminopeptidase, prolyl aminopeptidase, aminopeptidase My, gamma-glutamyl arylamidase, dipeptidylpeptidase IV and prolyl carboxypeptidase. The intracellular localization of the enzymes was analyzed by sucrose density gradient centrifugation of the DNase/RNase pretreated membrane sediments. Most of the exopeptidases are located in the inner membrane fractions (band L). Only gamma-glutamyl arylamidase is located with its main activity in the cytoplasmic membrane (band M). Only small exopeptidase activities could be found in the outer membrane (band H).

Characterization of a periplasmic insulin-cleaving metalloproteinase from Acinetobacter calcoaceticus.

In Acinetobacter calcoaceticus, a Gram-negative bacterial species, a soluble insulin-degrading proteinase, located in the periplasm as well as in the cytosol, could be established. The periplasmic and cytosolic enzymes agree in their inhibition pattern, pH-optimum and molecular weight. The insulin-degrading enzyme of Acinetobacter calcoaceticus resembles the corresponding proteinases of Escherichia coli. It is a metalloproteinase with a pH-optimum in the neutral range and can be reactivated by divalent ions after EDTA-inhibition, but it is not entirely identical with any of the described proteinases of Escherichia coli in its inhibitory behaviour.

[The effect of pH value and temperature on the stability of L-aminoacidoxidase from the venom of the sand viper]. / Einfluss von pH-Wert und Temperatur auf die Stabilität der L-Aminosäureoxydase aus dem Gift der Sandotter

The stability of highly purified L-amino acid oxidase from the sand viper venom remains practically unaffected by the pH-value at 4degreesC between pH 5 and 8, whereas a sharp activity fall was observed on both sides of this range. At temperatures above 30 degreesC the enzyme is stable only at pH 5.0--5.5. The inactivation pH values above 5.5 follows a first-order rate equation with characteristic changes in the absorption and emission spectra of the enzyme. The stability of the enzyme is dependent on the temperature of storage. At pH 7.5 there is a stability minimum at --10 degrees and -- 30 degreesC. At -- 72 degreesC the enzyme is stable practically for an unlimited period of time; temperatures exceeding 50 degrees C rapidly lead to complete inactivation. Also in the cold, the L-amino acid oxidase is most stable at pH 5.5. There are characteristic changes in absorption and emission spectra in the temperature-stability minimum (--15 degreesC) and at temperatures above 30degreesC. The inactivations follow a first-order rate equation. The cold inactivation is reversible. The stability of the enzyme is diminished by some anions and cations at 37 degreesC. The cold inactivation is promoted by several inorganic anions; organic anions and ammonium sulfate prevent cold inactivation.

Kinetics and reaction mechanism of yeast alcohol dehydrogenase with long-chain primary alcohols.

Kinetic studies of yeast alcohol dehydrogenase with NAD+ and ethanol, hexanol or decanol as substrates invariably result in non-linear Lineweaver-Burk plots if the alcohol is the variable substrate. The kinetic coefficients determined from secondary plots are consistent with an 'equilibrium random-order' mechanism for extremely low alcohol concentrations and for all alcohols, the transformation of the ternary complexes being the rate-limiting step of the reaction. This mechanism also applies to long-chain substrates at high concentrations, whereas the rate of the ethanol-NAD+ reaction at high ethanol concentrations is determined by the dissociation of the enzyme-NADH complex. The dissociation constants for the enzyme-NAD+ complex and for the enzyme-alcohol complexes obtained from the kinetic quotients satisfactorily correspond to the dissociation constants obtained by use of other techniques. It is suggested that the non-linear curves may be attributed to a structural change in the enzyme itself, caused by the alcohol.

[Membrane-bound alanine aminopeptidase from Acinetobacter calcoaceticus. 4. Amino acid chloromethyl ketones as pH-dependent, substrate-derived inhibitors of cysteine protease]. / Eine membrangebundene Alanylaminopeptidase aus Acinetobacter calcoaceticus: 4. Aminosäurechlormethylketone als pH-abhängige, substratanaloge Cysteinprotease-Inhibitoren.

The alanine aminopeptidase (AAP) from A. calcoaceticus is a metal-ion-dependent cysteine protease bound to the inner membranes of the cells. Amino acid chloromethyl ketones are highly specific inhibitors of the enzyme. The AAP is inhibited at pH 7.5 (pH-optimum) by mixed-type inhibition. A competitive inhibition type results if the pH value is decreased to 6.2. In contrast, the inhibition of the AAP by substrate-derived chloromethyl ketones is of the irreversible type at pH 8.25.