Mimicking phosphorylation at Ser-48 strongly reduces surface expression of human macrophage scavenger receptor class A: implications on cell motility.

The role of human macrophage scavenger receptor A1 (SRA1) in the development of atherosclerotic lesions is still scarcely defined. Substituting serine 48 in human SRA1 by an aspartate demonstrated that (1) surface expression of the mutated receptor was 13-fold decreased; (2) the amount of cell-associated Texas red-labeled acetylated low density lipoprotein (LDL) in mutant receptor-expressing cells was almost three-fold reduced; (3) the migration of mutant receptor-transfected cells towards surfaces coated with oxidized LDL decreased by almost 60% compared to cells that were transfected with the wild type receptor. Phosphorylation of the cytoplasmic part of SRA1 may help to modulate the residence time of macrophages in atherosclerotic lesions.

A chemiluminescence detection method of hantaviral antigens in neutralisation assays and inhibitor studies.

A protocol for chemiluminescence detection of hantaviral antigens in infected cell foci is described. This focus detection is based on the conversion of a substrate into a luminescent product by peroxidase-antibody conjugates; the emitted light of infected cell foci can easily be recorded by autoradiography or video imaging providing a hard copy for documentation. The main advantage of this method as compared to conventional immunochemical staining is a higher detection sensitivity due to the inherent magnification effect of luminescence causing an obvious boost in focal image and intensity. This enables reduction of (i) incubation time of virus-infected cells and (ii) amount of needed antibody for antigen detection in foci. This method is applied to a chemiluminescence focus reduction assay for the serotyping of hantavirus-specific neutralising antibodies in infected persons and for the determination of activity of antiviral agents against hantavirus.

Comparison of lipid-mediated and adenoviral gene transfer in human monocyte-derived macrophages and COS-7 cells.

Lipid-mediated transfection was compared to adenoviral-mediated gene transfer in COS-7 cells as well as human monocyte-derived macrophages (HMDM). For this purpose, we monitored enhanced green fluorescent protein (EGFP) expression by fluorescence microscopy and quantified gene transfer by competitive PCR. Transfection of COS-7 cells with a novel lipid formulation for DNA transfer was highly effective in COS-7 cells. On average, 30% of the cells were fluorescent 48 h after transfection. In HMDM, the same formulation resulted in the expression of EGFP in less than 0.5% of cells. We measured plasmid DNA by quantitative PCR in lipid-transfected macrophages and found that each macrophage contained on average 2 fg of plasmid DNA 24 h after transfection, that is, more than 400 molecules of plasmid DNA entered each cell. Despite the high level of reporter DNA in lipid transfected cells, expression of the fluorescent protein was suppressed in more than 99.5% of the macrophages. We also used adenoviral gene transfer to introduce the foreign DNA into both COS-7 cells and HMDM. Even though the multiplicity of infection was less than 30, expression of EGFP was observed in nearly all COS-7 cells and in more than 80% of HMDM 48 h after transfection. Despite major advances in the field of lipid-mediated transfection of HMDM, the lipid formulations that are available commercially cannot compete with the efficiency of adenoviral gene transfer.

Requirement of pyk2 for the activation of the MAP kinase cascade induced by Ca(2+) (but not by PKC or G protein) in PC12 cells.

The role of the Ca(2+)-activated tyrosine kinase, Pyk2, in the pleiotropic coupling of nerve cell stimulation to the MAP kinase cascade still remains undefined. Using a panel of PC12 clones, one of which was defective in Pyk2, we demonstrate (1) that the MAP kinase response induced by a [Ca(2+)](i) rise (following application of the Ca(2+) ionophore, ionomycin) is inappreciable in the defective clone and is re-established after Pyk2 transfection; and (2) that the responses to both protein kinase C and P(2y2) receptor activation occur normally even in the defective cells. We conclude that Pyk2 is the key mediator in the pathway activated by Ca(2+) but has minor roles with the other types of stimulation.

[Eye diseases in dwarf rabbits. 1. Anatomy, investigation procedures, diseases of the eyelid, the conjunctiva and the nasolacrimal duct]. / Augenkrankheiten beim Zwergkaninchen. Teil 1: Anatomie, Untersuchungsgang, Erkrankungen der Augenlider, der Konjunktiva und des Tränennasengangs.

The clinical ophthalmology of dwarf rabbits is reviewed in this continuing article. The first part contains the ophthalmological examination scheme and anatomical features of the rabbit's eye. Furthermore diseases of the eyelids, conjunctiva and of the nasolacrimal duct are described. These are illustrated by means of selected photographs.

The tomato Hsf system: HsfA2 needs interaction with HsfA1 for efficient nuclear import and may be localized in cytoplasmic heat stress granules.

In heat-stressed (HS) tomato (Lycopersicon peruvianum) cell cultures, the constitutively expressed HS transcription factor HsfA1 is complemented by two HS-inducible forms, HsfA2 and HsfB1. Because of its stability, HsfA2 accumulates to fairly high levels in the course of a prolonged HS and recovery regimen. Using immunofluorescence and cell fractionation experiments, we identified three states of HsfA2: (i) a soluble, cytoplasmic form in preinduced cultures maintained at 25 degrees C, (ii) a salt-resistant, nuclear form found in HS cells, and (iii) a stored form of HsfA2 in cytoplasmic HS granules. The efficient nuclear transport of HsfA2 evidently requires interaction with HsfA1. When expressed in tobacco protoplasts by use of a transient-expression system, HsfA2 is mainly retained in the cytoplasm unless it is coexpressed with HsfA1. The essential parts for the interaction and nuclear cotransport of the two Hsfs are the homologous oligomerization domain (HR-A/B region of the A-type Hsfs) and functional nuclear localization signal motifs of both partners. Direct physical interaction of the two Hsfs with formation of relatively stabile hetero-oligomers was shown by a two-hybrid test in Saccharomyces cerevisiae as well as by coimmunoprecipitation using tomato and tobacco whole-cell lysates.

[Eye diseases in dwarf rabbits. 2. Diseases of the cornea, intraocular and retrobulbar disorders, and neoplasms]. / Augenkrankheiten beim Zwergkaninchen. Teil 2: Erkrankungen der Kornea, intraokuläre und retrobulbäre Erkrankungen sowie Neoplasien.

In the second part of this review article the diseases affecting the cornea, intraocular and retrobulbar disorders, and eye-neoplasia of dwarf rabbits are described. These are illustrated by means of selected photographs.

Focus luminescence assay: macroscopically visualized foci of human cytomegalovirus and varicella zoster virus infection.

The plaques or foci of certain viruses due to their small size have to be counted microscopically, e.g., human cytomegalovirus (HCMV). The focus luminescence assay (FLA) described below generates macroscopic images as a result of the magnification due to scattered emitted light, and provides a hard copy using autoradiography or video imaging. Foci are detected according to an immunohistochemical protocol with horseradish peroxidase or alkaline phosphatase antibody conjugates which convert substrate into a luminescent product. Detection of varicella zoster virus (VZV) foci developed with a specific substrate-enhancer combination was so sensitive that 20-times lower primary antibody concentrations were effective than those required for conventional immunohistochemical staining. This method for HCMV and VZV may allow quantitative infectivity and focus reduction assays for viruses which produce little or no CPE.

[Intraocular metastasis of bronchial carcinoma in a cat]. / Intraokuläre Metastase eines Bronchialkarzinoms bei einer Katze.

A rare case of a ten-year-old neutered male Persian cat is described, in which metastasis of a pulmonary adenocarcinoma into the choroid of the right eye had led to visual impairment and major ophthalmoscopically detectable changes of the fundus. These were a generalized hyporeflectivity of the tapetal fundus due to retinal edema with multiple areas of retinal detachment and also severe edema of the papilla, furthermore an increased tortuosity and congestion of the retinal vessels. While the diagnosis of a primary lung tumor could be made intra vitam based on the result of an x-ray examination, the neoplastic nature of the fundic lesions could only be confirmed by histopathologic examination.

Different modes of inhibition by adamantane amine derivatives and natural polyamines of the functionally reconstituted influenza virus M2 proton channel protein.

The influenza virus M2 protein, target of the antiviral drugs amantadine and rimantadine, forms a proton channel which functions during virus uncoating and maturation by modifying the pH in virions as well as in trans-Golgi vesicles. We studied the influence of different ionic gradients on the inhibition of the proton translocation activity of isolated, baculovirus-expressed M2 protein reconstituted into liposomes. Two distinct patterns of inhibition were observed. A group of amphiphilic amines including amantadine, cyclooctylamine and rimantadine inhibited M2 effectively in the presence of physiological Na+ concentrations. The 10-fold greater activity of rimantadine over amantadine and the 100-fold stronger effect of cyclooctylamine compared to cyclopentylamine matched the relative activities in influenza virus-infected cells. A completely different inhibitory pattern emerged for the polyamines spermine, spermidine and putrescine. Polyamines have recently been identified as the 'intrinsic' rectifiers of a class of potassium channels and shown to interact with acidic amino acid residues lining and flanking the channel pore. In the presence of a physiological Na+/K+ gradient their minimal inhibitory concentrations for influenza virus M2 protein were 100, 400 and 500 microM, polyamine levels reported to exist in oocytes. In conditions depleted for Na+, polyamines inhibited M2 at concentrations two to three orders of magnitude lower. The data suggest that influenza virus M2 protein possesses a binding site for polyamines, distinct from the amantadine binding site, which is normally masked by Na+ and which could be targeted by selective antiviral inhibitors.

Immunohistochemical findings in eyes of cats serologically positive for feline immunodeficiency virus (FIV).

In this study the eyes of 15 cats in the terminal stage of FIV infection were examined. The findings were compared to those in cats, which were euthanized because of other infectious diseases for for non-infectious reasons. Thirteen FIV-infected cats showed an anterior uveitis by means of light microscopy. No accumulation of retinal lesions were found in FIV-infected cats compared to the other cats examined. Additionally, there were no indications of lesions caused by opportunistic infections. In the posterior segments of the eyes, immunohistochemical examinations proved the plasma proteins C3 and IgG to be predominantly intravascular. The eyes of 11 serologically FIV-positive cats were available for immunohistochemical examination. In all 11 cats at least one of the plasma proteins C3 or IgG could be detected in the extravascular tissue of the anterior uvea. The extravascular presence of plasma proteins within the tissue seemed to be caused by an increased permeability of the vessels due to inflammation. Furthermore, the similar extravascular distribution pattern of IgG and complement component C3 in four cases indicated that immune complexes may play a role in the anterior uveitis of FIV-infected cats.

[Findings in the eyes of serologically FIV (feline immunodeficiency virus) positive cats]. / Augenbefunde bei serologisch FIV (felines Immundefizienzvirus)-positiven Katzen.

The findings in the eyes of 19 serologically FIV-positive cats in the terminal stage of the disease were compared to those in 23 serologically FIV-negative cats. An inflammation of iris and ciliary body was more common in the FIV-group than in the other cats and could be seen in 16 out of 19 serologically FIV-positive cats. In most cases a diffuse infiltration of both lymphocytes and plasma cells could be observed. There were only few signs of acute inflammation in the posterior segment. Degenerative alterations of the retina were found in all groups. The most common degenerative finding was a cystoid atrophy of the retina. Due to these findings an association of FIV-infection and anterior uveitis seems to be very likely. It remained unclear whether the FIV was involved directly or whether indirect mechanisms like secondary infections or alterations in the immune system caused the inflammation.

Okadaic acid potentiates heat-induced activation of erk2.

Subjecting exponentially growing HeLa cells to heat shock at 45 degrees C for 30 min leads to retarded migration of erk1 and erk2, as revealed on immunoblots indicating their activation. Renaturation gels confirmed activation of erk2 but not erk1. Treatment of cells with okadaic acid (OA) alone marginally upregulated erk1 and erk2, whereas simultaneous exposure to heat shock and OA led to a considerably augmented response for erk2 which was approximately 3-fold higher than the sum of heat- and OA-induced activation. Chronic treatment of cells with 12-O-tetradecanoyl-phorbol 13-acetate marginally diminished the extent of erk2 stimulation, but had no influence on the OA-induced potentiation of heat-induced erk2 activity.

[Diagnosis of retinal diseases in dogs and cats]. / Retinadiagnostik bei Hund und Katze.

The article presents an overview on the different examination techniques that can be used to make up a diagnosis of retinal disease in the dog and cat. Patient history, examination for visual impairment, direct and indirect ophthalmoscopy, fluorescence angiography, electroretinography and ophthalmic ultrasonography are mentioned. The most important elements of the normal fundus of dogs and cats as well as the ophthalmoscopical features of the most common retinal diseases in these species are given, illustrated by coloured photographs.

A 40-kDa myelin basic protein kinase, distinct from erk1 and erk2, is activated in mitotic HeLa cells.

Mitotic HeLa cells showed an increased phosphorylation activity towards myelin basic protein compared to cells in G1 or S phases. Further investigation using renaturation gels revealed that, in mitotic cell lysates, a protein with an apparent molecular mass of around 40 kDa phosphorylates myelin basic protein. This kinase is active early in mitosis, but is then downregulated concomitantly with p34cdc2 kinase as mitosis proceeds, its activity decreasing to basal levels by early G1. The molecular mass of the kinase suggested that it might be one of the human homologues of rat erk1 or erk2. However, antibodies raised against C-terminal sequences of erk1 and erk2 failed to immunoprecipitate renaturable kinase activity from mitotic lysates. In addition, in immunoblots erk1 and erk2 failed to show the well established changes in electrophoretic migration that are consequences of their activation. These data indicate that these two mitogen-activated protein (MAP) kinases are not stimulated during HeLa cell mitosis and indicate that the 40-kDa kinase is either a new member of the MAP kinase family or it is a novel mitotic kinase that has not yet been described.

[Capsular sack suture PMMA intraocular lens. Comparative study of centering behavior after phacoemulsification and planned extracapsular cataract extraction]. / Kapselsackfixierte PMMA-Intraokularlinse. Vergleichende Untersuchung über das Zentrierverhalten nach Phakoemulsifikation und nach geplant extrakapsulärer Kataraktextraktion.

The position of bag-fixated intraocular lenses after phacoemulsification or extracapsular cataract extraction (ECCE) was compared in order to determine the relationship to the method of operation and the type of capsular opening. Photographs were taken from 71 eyes 6 months after in-the-bag implantation of a one-piece posterior chamber lens. Centering was determined with respect to the pupillary center. Forty-six eyes were operated on by phacoemulsification after continuous circular capsulorhexis with closed edges and 25 eyes by ECCE after capsulorhexis with a radial incision of the capsular rim at the 12 o'clock position. Ninety-four percent of the intraocular lenses implanted after phacoemulsification and 92% of the intraocular lenses implanted after ECCE were decentered less than 0.5 mm. The centering of bag-fixated intraocular lenses is related to the technique of opening the anterior capsule. Phacoemulsification with continuous capsulorhexis offers conditions similar to that in ECCE with capsulorhexis and radial capsular rim incision for good centering of one-piece posterior chamber lenses.

[Bilateral blindness after injury in a riding horse]. / Beidseitige Erblindung nach Trauma bei einem Reitpferd.

Bilateral blindness was diagnosed in a 5-year-old Hanoverian gelding presented for evaluation of a corneal opacity in one eye. About 12 months prior to the examination, the gelding had fallen head over, hitting his head and the cornea. Clinical and electroretinographic findings as well as pathohistologic and ultrastructural lesions of both eyes including the optic nerves are presented. Ophthalmoscopically visible pigment disruption of the non-tapetal fundus adjacent to the optic discs correlated morphologically with foci of degeneration and atrophy of the retina, whereas ophthalmoscopically visible accumulation of pigment was morphologically characterized by hypertrophy of the pigment epithelium, increased intracellular pigment accumulation and by migration of pigment cells into the inner lamina of the retina. Severe atrophy of the retinal neuronal layer was linked to traumatic optic nerve degeneration.

[Plasminogen activator and PAI. Detection in aqueous humor of the human eye]. / Gewebsplasminogenktivator und PAI. Nachweis im Kammerwasser des menschlichen Auges.

Tissue-type plasminogen activator (t-PA), a serine protease that catalyzes the conversion from plasminogen to plasmin, plays an important role in the fibrinolytic system and has also in recent years attracted attention in the field of ophthalmology. In order to examine the role of t-PA in the physiology of the anterior segment, we detected t-PA in aqueous humor by using immunoassays. The samples were taken by keratocentesis prior to cataract or glaucoma surgery. The sample volumes ranged from 50-200 microliters. The quantities of t-PA Ag and plasminogen-activator inhibitor (PAI) Ag were determined by using enzyme-linked immunosorbent assays. We determined t-PA and PAI in the aqueous humor of 54 patients between 32 and 87 years of age. The t-PA Ag levels ranged from 0.2 to 1.9 ng/ml (average 0.8 ng/ml), PAI-Ag from 0.2 to 1.7 ng/ml (average 0.9 ng/ml). The values measured in men were slightly higher than those measured in women. No association between t-PA and PAI levels and accompanying diseases or metabolic disorders was noted. Precise knowledge about the presence of t-PA in aqueous humor is a prerequisite for the recognition of pathological events following intraocular fibrin formation and may be an important basis for the therapeutic use of rt-PA in the intraocular inflammatory process.

Different glycosylation in acetylcholinesterases from mammalian brain and erythrocytes.

Acetylcholinesterases (EC 3.1.1.7, AChE) have varying amounts of carbohydrates attached to the core protein. Sequence analysis of the known primary structures gives evidence for several asparagine-linked carbohydrates. From the differences in molecular mass determined on sodium dodecyl sulfate-polyacrylamide gel before and after deglycosylation with N-glycosidase F (EC 3.2.2.18), it is seen that dimeric AChE from red cell membranes is more heavily glycosylated than the tetrameric brain enzyme. Furthermore, dimeric and tetrameric forms of bovine AChE are more heavily glycosylated than the corresponding human enzymes. Monoclonal antibodies 2E6, 1H11, and 2G8 raised against detergent-soluble AChE from electric organs of Torpedo nacline timilei as well as Elec-39 raised against AChE from Electrophorus electricus cross-reacted with AChE from bovine and human brain but not with AChE from erythrocytes. Treatment of the enzyme with N-glycosidase F abolished binding of monoclonal antibodies, suggesting that the epitope, or part of it, consists of N-linked carbohydrates. Analysis of N-acetylglucosamine sugars revealed the presence of N-acetylglucosamine in all forms of cholinesterases investigated, giving evidence for N-linked glycosylation. On the other hand, N-acetylgalactosamine was not found in AChE from human and bovine brain or in butyrylcholinesterase (EC 3.1.1.8) from human serum, indicating that these forms of cholinesterase did not contain O-linked carbohydrates. Despite the notion that within one species, the different forms of AChE arise from one gene by different splicing, our present results show that dimeric erythrocyte and tetrameric brain AChE must undergo different postsynthetic modifications leading to differences in their glycosylation patterns.

Relation between drug resistance and antigenicity among norakin-resistant mutants of influenza A (fowl plague) virus.

Norakin-resistant (NR) mutants of fowl plague virus (A/FPV/Weybridge, H7N7) have 1 to 2 (in one instance 3) amino acid substitutions in different positions of the heavy (HA 1) and/or light (HA 2) subunits of the haemagglutinin (HA) molecule. Investigation of NR mutants using the haemagglutination inhibition test with monoclonal antibodies (MAb) to the HA of A/seal/Massachusetts/80 (H7N7) virus revealed that one of the mutants (NR 1) differs antigenically from the wild-type fowl plague virus: its haemagglutination was not inhibited by MAb 55/2 and 58/6. By contrast, MAb-resistant (escape) mutants, selected from the wild-type fowl plague virus under pressure from MAb 55/2 or 58/6, showed reduced drug sensitivity. These findings suggest a possibility of correlation between alteration of influenza virus antigenicity and change of its sensitivity to drugs whose target is the haemagglutinin. This potential effect should be taken into account when antiviral substances directed to surface influenza virus antigens are being developed for use as antiviral drugs.