Imbalance of the nerve growth factor and its precursor as a potential biomarker for diabetic retinopathy.

Our previous studies have demonstrated that diabetes-induced oxidative stress alters homeostasis of retinal nerve growth factor (NGF) resulting in accumulation of its precursor, proNGF, at the expense of NGF which plays a critical role in preserving neuronal and retinal function. This imbalance coincided with retinal damage in experimental diabetes. Here we test the hypothesis that alteration of proNGF and NGF levels observed in retina and vitreous will be mirrored in serum of diabetic patients. Blood and vitreous samples were collected from patients (diabetic and nondiabetic) undergoing vitrectomy at Georgia Regents University under approved IRB. Levels of proNGF, NGF, and p75(NTR) shedding were detected using Western blot analysis. MMP-7 activity was also assayed. Diabetes-induced proNGF expression and impaired NGF expression were observed in vitreous and serum. Vitreous and sera from diabetic patients (n = 11) showed significant 40.8-fold and 3.6-fold increases, respectively, compared to nondiabetics (n = 9). In contrast, vitreous and sera from diabetic patients showed significant 44% and 64% reductions in NGF levels, respectively, compared to nondiabetics. ProNGF to NGF ratios showed significant correlation between vitreous and serum. Further characterization of diabetes-induced imbalance in the proNGF to NGF ratio will facilitate its utility as an early biomarker for diabetic complications.

The role of dynamic indocyanine green angiography in the diagnosis and treatment of retinal angiomatous proliferation.

AIMS: We compared the effectiveness and durability of indocyanine green angiography (ICG) directed focal thermal laser treatment of the afferent arteriole in the treatment of retinal angiomatous proliferation (RAP). METHODS: Sixteen consecutive patients presenting with stage I or II RAP lesions underwent optical coherence tomography, fluorescein angiography and dynamic ICG examination and were evaluated for response to treatment. Two groups were evaluated: focal laser as initial treatment; and focal laser as salvage treatment after recurrence with photodynamic therapy (PDT) and intravitreal triamcinolone. Five additional eyes with stage III RAP were evaluated separately. RESULTS: Seven eyes received focal laser as initial treatment, and nine eyes received focal laser as salvage treatment after failure of PDT with triamcinolone. Five of seven eyes in the initial focal laser group demonstrated resolution of oedema. All five of the responders recurred (mean 4.4 months). Salvage therapy with PDT and triamcinolone after focal laser failure transiently closed these recurrences. In contrast, eight of nine eyes receiving thermal ablation of the RAP lesion after recurrence from prior PDT/triamcinolone demonstrated initial improvement of the retinal oedema. Four eyes demonstrated no recurrence within a year. None of the five eyes with stage III RAP improved anatomically or visually. CONCLUSION: Focal thermal laser treatment of RAP arteriole can resolve retinal oedema. However, durability was longer in eyes with prior PDT and triamcinolone treatment than those receiving thermal laser as initial therapy. These results suggest that focal laser may be useful in treatment of RAP recurrences and that combination therapy with PDT/triamcinolone plus focal laser is better than focal laser alone.

Reduction of Cre recombinase toxicity in proliferating Drosophila cells by estrogen-dependent activity regulation.

The Cre/loxP site-specific recombination system has been used successfully for genome manipulation in a wide range of species. However, in Drosophila melanogaster, a major model organism for genetic analyses, the alternative FLP/FRT system, which is less efficient at least in mammalian cells, has been established, primarily for the generation of genetic mosaics for clonal analyses. To extend genetic methodology in D. melanogaster, we have created transgenic lines allowing tissue-specific expression of Cre recombinase with the UAS/GAL4 system. Surprisingly, chronic expression of Cre recombinase from these transgenes (UAST-cre) was found to be toxic for proliferating cells. Therefore, we also generated transgenic lines allowing the expression of Cre recombinase fused to the ligand-binding domain of the human estrogen receptor (UASP-cre-EBD). We demonstrate that recombination can be efficiently dissociated from toxicity by estrogen-dependent regulation of recombinase activity of the UASP-cre-EBD transgene products.

Cloning of the mouse 5-HT6 serotonin receptor and mutagenesis studies of the third cytoplasmic loop.

We have cloned the mouse 5-HT6 serotonin receptor and examined structure-function relationships in the C-terminal end of the third cytoplasmic (CIII) loop, introducing point mutations by site-directed mutagenesis at positions 264 to 268. We examined the ability of 5-HT6 wild type and receptor mutants to activate a cAMP responsive reporter gene when transiently expressed in JEG-3 or COS-7 cells. The wild type 5-HT6 receptor showed strong constitutive activity even when expressed at very low levels and which increased in proportion to the amount of receptor cDNA transfected. Three of the five mutants investigated (K264I, K267A and A268R) showed reduction in constitutive activity compared to wild type. These data suggest that constitutive activity may be important to 5-HT6 receptor activity in vivo and that, unlike some other G-protein coupled receptors, alteration in the BBXXB CIII-loop motif reduces rather than further activates basal activity of the murine 5-HT6 receptor.

Changes in 5-HT(7) serotonin receptor mRNA expression with aging in rat brain.

We examined 5-HT(7) receptor mRNA expression with in situ hybridization histochemistry in the brains of young (3 months), middle-aged (12 months) and old rats (24 months). In the ventral CA3 area of the hippocampus 5-HT(7) mRNA expression is reduced by approximately 30% between young and middle age without further decline between middle and old age. In other brain areas 5-HT(7) mRNA expression is unaffected by age.

A screen for modifiers of cyclin E function in Drosophila melanogaster identifies Cdk2 mutations, revealing the insignificance of putative phosphorylation sites in Cdk2.

In higher eukaryotes, cyclin E is thought to control the progression from G1 into S phase of the cell cycle by associating as a regulatory subunit with cdk2. To identify genes interacting with cyclin E, we have screened in Drosophila melanogaster for mutations that act as dominant modifiers of an eye phenotype caused by a Sevenless-CycE transgene that directs ectopic Cyclin E expression in postmitotic cells of eye imaginal disc and causes a rough eye phenotype in adult flies. The majority of the EMS-induced mutations that we have identified fall into four complementation groups corresponding to the genes split ends, dacapo, dE2F1, and Cdk2(Cdc2c). The Cdk2 mutations in combination with mutant Cdk2 transgenes have allowed us to address the regulatory significance of potential phosphorylation sites in Cdk2 (Thr 18 and Tyr 19). The corresponding sites in the closely related Cdk1 (Thr 14 and Tyr 15) are of crucial importance for regulation of the G2/M transition by myt1 and wee1 kinases and cdc25 phosphatases. In contrast, our results demonstrate that the equivalent sites in Cdk2 play no essential role.

Gs protein-coupled serotonin receptors: receptor isoforms and functional differences.

Three distinct mammalian Gs coupled serotonin receptor genes have been identified, 5-HT4, 5-ht6, and 5-HT7, which produce at least seven different functional receptors through alternative splicing. One of the chief questions facing workers in this area mirrors that confronting the serotonin receptor field as a whole: why so many subtypes? The answer to this question is made more elusive at present by two further considerations. First, there may well be additional Gs coupled receptor subtypes yet to be described. Secondly, although the various isoforms of 5-HT4 and 5-HT7 have been shown to be functional in in vitro assays, it remains to be shown that all isoforms have biological significance. This paper will summarize some of the differences at the molecular and cellular level that are becoming apparent among the 5-HT4, 5-ht6 and 5-HT7 receptor subtypes and their various isoforms. As an example, it will focus on the 5-HT7 system, and describe recent developments in ascribing particular functions to differences due to alternative splicing.

Function and distribution of three rat 5-hydroxytryptamine7 (5-HT7) receptor isoforms produced by alternative splicing.

Serotonin (5-HT7) receptor pre-mRNA is alternatively spliced in rat tissue to produce three isoforms, 5-HT(7a), 5-HT(7b) and 5-HT(7c), which differ in the amino acid sequences of their carboxyl terminal tails. Substantial species differences in structure and expression patterns exist for 5-HT7 isoforms. We have now compared some of the functional characteristics and level of expression for the three rat 5-HT7 receptor isoforms. Recombinant receptor isoforms were expressed in COS-7 cells for examination of [3H]5-HT binding characteristics and in JEG-3 cells to ascertain their ability to stimulate cAMP production. These studies showed that all three isoforms are functionally active and have similar agonist binding characteristics. Distribution of expression of the three rat receptor isoforms were examined in several brain regions and peripheral tissues using RT-PCR and in situ hybridization. The relative proportions of total 5-HT7 receptor message lent by each isoform varied little between these areas. In contrast to what has been observed in human tissue, the 5-HT(7a) isoform predominated in all regions examined, while the 5-HT(7c) isoform revealed a low level of expression (3% of total transcript). In situ hybridization was used to determine if the overall low level of expression of the 5-HT(7c) isoform by RT-PCR could be attributed to a small localized subpopulation of cells expressing high levels 5-HT(7c) message. In situ hybridization results indicate a generalized low level of expression of the 5-HT(7c) isoform throughout the CNS. These data suggest that while all three known 5-HT7 receptor isoforms in the rat are functionally competent, any functionally important differences between the three isoforms are not likely to involve differences in ligand binding or gross differences in adenylate cyclase coupling. However, differences in receptor phosphorylation, regulation or coupling to other effectors or cell trafficking could still exist.

Four 5-hydroxytryptamine7 (5-HT7) receptor isoforms in human and rat produced by alternative splicing: species differences due to altered intron-exon organization.

The serotonin (5-HT) 5-HT7 receptor subtype is thought to mediate a number of physiological effects in mammalian brain and periphery. Previous studies suggested that alternative splicing might contribute to 5-HT7 receptor diversity as well. We now report that alternative splicing in human and rat tissues produces four 5-HT7 receptor isoforms that differ in their predicted C-terminal intracellular tails. Human and rat partial 5-HT7 cDNAs and intronic sequences were identified and compared. In rat tissues, three 5-HT7 isoforms, here called 5-HT7(a), 5-HT7(b), and 5-HT7(c), are found. Rat 5-HT7(a) [448-amino acid (aa)] and 5-HT7(b) (435-aa) forms arise from alternative splice donor sites. A third new isoform found in rat, 5-HT7(c) (470-aa), results from a retained exon cassette. Three 5-HT7 mRNA isoforms were also identified in human tissues, where only one isoform was previously described. Two human isoforms represent 5-HT7(a) and 5-HT7(b) forms (445- and 432-aa), but the third form does not correspond to 5-HT7(c). Instead, it constitutes a distinct isoform, 5-HT7(d) (479-aa), resulting from retention of a separate exon cassette. 5-HT7(d) transcripts are not present in rat because the 5-HT7(d)-specifying exon is absent from the rat 5-HT7 gene. A frame-shifting homologue of the rat 5-HT7(c)-Specifying exon is present in the human gene but is not used in the human tissues examined. Tissue-specific splicing differences are present in human between brain and spleen. These studies suggest that alternative splicing may contribute to diversity of 5-HT7 receptor action and that the human and rat repertoires of 5-HT7 splice variants are substantially different.

[Flow rates of roentgen contrast media of different viscosity in 4.1 Charrière coronary catheters]. / Flussraten von Röntgenkontrastmitteln verschiedener Viskosität in 4.1-Charrière-Koronarkathetern.

The studies presented here investigated the obtainable flows of different contrast media (Iopromide 370 mg iodine/ml, ZK 119 095 370 mg iodine/ml, ZK 139 129 370 mg iodine/ml, Iopamidol 370 mg iodine/ml, Iopromide 300 mg iodine/ml, ZK 119 095 300 mg iodine/ml, ZK 139 129 300 mg iodine/ml, Iopamidol 300 mg iodine/ml, aqua dest.) in 4.1 Charrière coronary catheters. The measurements of the flow achieved by a standardised power of 100 N show that the highest values are reached with the substance ZK 119 095 (both for 300 mg iodine/ml and 370 mg iodine/ml). On comparison of the catheter types there are no differences in the delivery rate. The x-ray contrast-media, however, are significantly different: the lowest iodine delivery rate is found for iopromide 370 with 384.5 mg iodine/s; the highest rate for the test substance ZK 119 095 with 648.9 mg iodine/s. Although contrast media with low viscosity contain considerably less iodine/ml it is possible to achieve an iodine density in coronary vessels by about 86% higher than that achieved by contrast media with 370 mg iodine/ml. Therefore, the possibility to choose a viscosity-adapted x-ray contrast-medium allows the use of very thin cardiac catheter systems without leading to a worsening of picture quality.

Expression of T cell receptor gamma delta antigens in human brain tissue.

The human gamma delta T cell receptor (TCR) is normally expressed on lymphoid tissue. Since expression of different molecules of the T cell system has been described for human brain cells, we examined the expression of CD4 and TCR gamma delta antigens with a panel of various anti-gamma delta TCR monoclonal antibodies (mAbs) and an anti-CD4 mAb using immunohistochemistry on different regions of frozen human postmortem tissue from five different brains. We could confirm the expression of CD4 antigens. gamma delta TCR expression on brain tissue was found in different regions of the brain by immunohistochemistry. Double staining with anti-gamma delta TCR and antineuronal enolase (NSE) mAbs showed that gamma delta TCR+ cells were not stained by anti-NSE, although they were sometimes located near neurons and showed dendritic forms; they are possibly tissue-resident gamma delta T cells, as described in the skin. Polymerase chain reaction analysis using a highly sensitive primer sequence against the constant-region delta sequence supports, combined with immunohistochemistry findings, the notion that the gamma delta TCR is expressed in human brain.

[The flow of contrast media in coronary catheters: study of the structural quality in interventional cardiology]. / Kontrastmittelfluss in Koronarkathetern: Untersuchung zur Strukturqualität in der interventionellen Kardiologie.

In the present study we investigated the contrast medium flow in 8 different types of left heart catheter having two different diameters (each n = 5). Using a 10 ml syringe and the contrast medium Ultravist 370, we calculated a mean value of 94.24 N + 16.01 for the maximum manual injection force in 18 test subjects. For the sake of simplicity, the figure of 100 N was defined as standardized manual force (which is within the standard deviation). If the maximally admissible static pressure of 82.5 bar is not to be exceeded, flow rates of not more than 16-17 ml/s are possible with 5.2 Fr. catheters, and 21-23 ml/s and 1.46 ml/s for 5.2 Fr. catheters, and between 1.99 ml/s and 2.17 ml/s for 6 Fr. catheters. Thus, a 50% higher flow can be achieved with 6 Fr. catheters as compared with 5.2 Fr. catheters at the same injection force. The iodine delivery rates are between 506 mg iodine/s and 539 mg iodine/s for 5.2 Fr. catheters, and between 738 mg iodine/s and 804 mg iodine/s for 6 Fr. catheters. The figures for the jet stream.

[Torsion stability of coronary catheters]. / Torsionsstabilität von Koronarkathetern.

In the present study, the torsional moments of 8 different types of catheter having two different calibres (n = 5 in each case) were investigated. The catheters were made of linear polyurethane (Cordis, Netherlands). The transmitted torsional moments of the 5.2 Fr. catheters positioned in a straight line were 1.48 +/- 0.09 Ncm (VK = 6%), positioned in a 90 degree curve 1.39 +/- 0.11 Ncm (VK = 8%). The corresponding figures for the 6 Fr. catheters were 2.43 +/- 0.20 Ncm (VK = 8%) and 2.26 +/- 0.20 Ncm (VK = 9%). The highly stable transmission behaviour in particular of the 5 Fr. catheters--with a variation coefficient of the torsional moments of 6%--is due mainly to the accurate manufacture of the catheters, and the material chosen. The study is a first step towards achieving systematic quality control of the materials used for diagnosis and therapy.