Global flash multifocal electroretinogram: early detection of local functional changes and its correlations with optical coherence tomography and visual field tests in diabetic eyes.

PURPOSE: To investigate the correlations of the global flash multifocal electroretinogram (MOFO mfERG) with common clinical visual assessments--Humphrey perimetry and Stratus circumpapillary retinal nerve fiber layer (RNFL) thickness measurement in type II diabetic patients. METHODS: Forty-two diabetic patients participated in the study: Ten were free from diabetic retinopathy (DR), while the remainder suffered from mild to moderate nonproliferative diabetic retinopathy. Fourteen age-matched controls were recruited for comparison. MOFO mfERG measurements were made under high- and low-contrast conditions. Humphrey central 30-2 perimetry and Stratus OCT circumpapillary RNFL thickness measurements were also performed. Correlations between local values of implicit time and amplitude of the mfERG components [direct component (DC) and induced component (IC)], and perimetric sensitivity and RNFL thickness were evaluated by mapping the localized responses for the three subject groups. RESULTS: MOFO mfERG was superior to perimetry and RNFL assessments in showing differences between the diabetic groups (with and without DR) and the controls. All the MOFO mfERG amplitudes (except IC amplitude at high contrast) correlated better with perimetry findings (Pearson's r ranged from 0.23 to 0.36, p < 0.01) than did the mfERG implicit time at both high and low contrasts across all subject groups. No consistent correlation was found between the mfERG and RNFL assessments for any group or contrast conditions. The responses of the local MOFO mfERG correlated with local perimetric sensitivity but not with RNFL thickness. CONCLUSION: Early functional changes in the diabetic retina seem to occur before morphological changes in the RNFL.

Use of a quantitative product-enhanced reverse transcriptase assay to monitor retrovirus levels in mAb cell-culture and downstream processing.

Murine hybridoma cells used in the production of monoclonal antibodies (mAb's) produce endogenous type C retrovirus particles. Regulatory agencies require a demonstration that mAb's intended for human use are free of retrovirus with an adequate margin of safety. This is usually achieved by validation studies, performed at small scale, to demonstrate that the manufacturing process is capable of removing or inactivating several different model viruses, including a murine retrovirus. In this report, we assess the utility of the TaqMan fluorogenic 5'-nuclease Product-Enhanced Reverse Transcriptase (TM-PERT) assay for measuring reverse transcriptase (RT) activity in cell-culture samples and RT removal by models of processing steps. The levels of RT activity contained in laboratory-scale cell-culture harvests (10(8)-10(13) pU/mL) were substantially above the detection limit of the TM-PERT assay ( approximately 10(6) pU/mL). The nature of the RT activity from cell culture was complex, but the bulk of RT activity in clarified mAb harvests appears to be contained in large molecular weight viral particles. In laboratory-scale chromatographic runs, sufficient RT activity was present in mAb-containing eluates to accurately calculate its log(10) reduction value (LRV), typically between 2 and 4 log(10) per step. Monoclonal antibody purified using a model purification scheme consisting of three serial columns contained some residual RT activity near the limit of detection. The data indicate that the TM-PERT assay, because it is quantitative and highly sensitive and can be used to analyze a large number of samples in a short period, is ideally suited to investigate and optimize retrovirus clearance in purification processes.

Requirement for a negative charge at threonine 60 of the FcRgamma for complete activation of Syk.

Aggregation of FcepsilonRI on mast cells results in the phosphorylation of the FcepsilonRIgamma chain on tyrosine and threonine residues within the immunoreceptor tyrosine-based activation motif. In the present study we sought to identify the site of threonine phosphorylation in FcepsilonRIgamma and investigate its functional importance. We found that threonine 60 was phosphorylated in vitro and in vivo. Expression of a mutated FcepsilonRIgamma (T60A), in either FcepsilonRIgamma-deficient or gamma-null mast cells, resulted in a delay of FcepsilonRI endocytosis, inhibition of TNF-alpha mRNA production, and inhibition of degranulation but did not affect FcepsilonRI-induced cell adhesion. Tyrosine phosphorylation of the T60A mutant gamma chain was normal, but Syk phosphorylation was dramatically reduced in these transfectants. This correlated with reduced co-immunoprecipitation of FcepsilonRIgamma with Syk. Substitution of an aspartic residue for threonine 60 of the FcepsilonRIgamma reconstituted complete activation of Syk and co-immunoprecipitation of FcepsilonRIgamma with Syk. We conclude that the negative charge provided by phosphorylation of threonine 60 of the FcepsilonRIgamma is required for the appropriate interaction and activation of Syk. This is a likely requirement for immunoreceptor tyrosine-based activation motifs involved in Syk activation.

Tyrosine phosphorylation-dependent and -independent associations of protein kinase C-delta with Src family kinases in the RBL-2H3 mast cell line: regulation of Src family kinase activity by protein kinase C-delta.

Src kinases and protein kinase C (PKC) have been well studied for their role in oncogenic and normal cellular processes. Herein we report on a novel regulatory pathway mediated by the interaction of PKC-delta with p53/56Lsy (Lyn) and with p60Src (Src) that results in the phosphorylation and increased activity of Lyn and Src. In the RBL-2H3 mast cell line, the interaction of PKC-delta with Lyn required the activation of the high affinity receptor for IgE (FcsigmaRI) while the interaction with Src was constitutive. Increased complex formation of PKC-delta with Lyn or Src led to increased serine phosphorylation and activity of the Src family kinases. Conversely, Lyn was found to phosphorylate Lyn-associated and recombinant PKC-delta in vitro and the tyrosine 52 phosphorylated PKC-delta was recruited to associate with the Lyn SH2 domain. The constitutive association of PKC-delta with Src did not result in the tyrosine phosphorylation of PKC-delta prior to or after FsigmaRI engagement. However in cells over-expressing PKC-delta, FsigmaRI engagement resulted in the dramatic inhibition of Src activity and some inhibition of Lyn activity. Thus, the interaction and cross-talk of PKC-delta with Src family kinases suggests a novel and inter-dependent mechanism for regulation of enzymatic activity that may serve an important role in cellular responses.

A genetic basis for corneal sensitivity to ultraviolet light among recombinant SWXJ inbred strains of mice.

PURPOSE: To examine a possible genetic basis for corneal sensitivity to UV-B light exposure. METHODS: To this end, adult male mice from the 14 SWXJ recombinant inbred albino strains (originating from SJL/J and SWR/J parental strains) were subjected to ultraviolet (UV) radiation exposure of 0.078 J/cm2 and photographed four days post-exposure, to assess corneal opacity and the possible correlation with corneal aldehyde dehydrogenase (ALDH) activity, alcohol dehydrogenase (ADH) activity and soluble protein content. RESULTS: Those recombinant strains that exhibited the SWR/J strain phenotype of having low levels of ALDH and decreased soluble protein levels also exhibited greater levels of corneal clouding after UV-exposure than the other strains, which exhibited "normal" levels of both ALDH activity and soluble protein in the cornea. CONCLUSIONS: These data support an hypothesis for a major role for ALDH in assisting the cornea to protect the eye against UV-induced tissue damage.

Distinct tyrosine phosphorylation sites in JAK3 kinase domain positively and negatively regulate its enzymatic activity.

Cytokines are critically important for the growth and development of a variety of cells. Janus kinases (JAKs) associate with cytokine receptors and are essential for transmitting downstream cytokine signals. However, the regulation of the enzymatic activity of the JAKs is not well understood. Here, we investigated the role of tyrosine phosphorylation of JAK3 in regulating its kinase activity by analyzing mutations of tyrosine residues within the putative activation loop of the kinase domain. Specifically, tyrosine residues 980 and 981 of JAK3 were mutated to phenylalanine individually or doubly. We found that JAK3 is autophosphorylated on multiple sites including Y980 and Y981. Compared with the activity of wild-type (WT) JAK3, mutant Y980F demonstrated markedly decreased kinase activity, and optimal phosphorylation of JAK3 on other sites was dependent on Y980 phosphorylation. The mutant Y980F also exhibited reduced phosphorylation of its substrates, gammac and STAT5A. In contrast, mutant Y981F had greatly increased kinase activity, whereas the double mutant, YY980/981FF, had intermediate activity. These results indicate that Y980 positively regulates JAK3 kinase activity whereas Y981 negatively regulates JAK3 kinase activity. These observations in JAK3 are similar to the findings in the kinase that is closely related to the JAK family, ZAP-70; mutations of tyrosine residues within the putative activation loop of ZAP-70 also have opposing actions. Thus, it will be important to determine whether this feature of regulation is unique to JAK3 or if it is also a feature of other JAKs. Given the importance of JAKs and particularly JAK3, it will be critical to fully dissect the positive and negative regulatory function of these and other tyrosine residues in the control of kinase activity and hence cytokine signaling.

Nonspecific protease-catalyzed hydrolysis/synthesis of a mixture of peptides: product diversity and ligand amplification by a molecular trap.

We sought to develop a peptide library in solution and dynamically screen this library for peptides that would bind to macromolecules of interest. Peptide diversity was achieved in an initial stock solution of peptides by using proteases under conditions in which both hydrolysis and synthesis occurred. As an example, a simple reaction containing YGG, FL and thermolysin resulted in the synthesis of YGGFL as well as many other undefined products. When low molecular weight products of a reaction containing VA, AL, and thermolysin were subsequently exposed to dipeptidase, 7 out of 9 potential dipeptides were observed. Incubation of protease with an hydrolysate of albumin and a radiolabeled peptide resulted in the radiolabel participating in reactions other than simple hydrolysis and, after 24 h, the specific activity of radiolabel was shown by high performance liquid chromatography to disperse to a level that would be necessary in the event of maximum theoretical diversity. When a binding macromolecule was exposed to this system, ligand production was amplified relative to reactions run in the absence of binding macromolecule. This protease-based peptide scrambling and binding system was utilized for the discovery of novel peptides that bind to fibrinogen.

Differential corneal sensitivity to ultraviolet light among inbred strains of mice. Correlation of ultraviolet B sensitivity with aldehyde dehydrogenase deficiency.

Adult male mice from four inbred albino strains (SJL/J, NZW/BL, BALB/c HeA, and SWR/J) were subjected to ultraviolet radiation (UVR) exposure (302 nm peak wavelength, intensity 398 microW/cm2) for 3.25 min and photographed 4 days postexposure to assess corneal clouding. Corneal extracts from control (unexposed) mice from each strain, were also monitored for aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) activity and soluble protein content. The SWR/J strain exhibited more extensive corneal clouding after UV exposure than did the other strains, and control SWR/J mice exhibited a low activity variant phenotype for the major ocular ALDH AHD-4, and decreased levels of soluble protein in corneal extracts. These data support earlier proposals for a major role for ALDH in assisting the cornea in protecting the eye against UVR-induced tissue damage.

Investigation of parameters influencing intraocular pressure increases during sleep.

In previous studies, we have observed that young normal subjects show an increase in intraocular pressure (IOP) after sleep. Here we describe three experiments which investigated: (i) the effects of sleep in five groups of subjects: glaucoma, suspect glaucoma, young high-normal IOP, old high-normal IOP groups and an elderly control group, (ii) the effect of exposure to bright light (2500 lux) during sleep on associated IOP changes, and (iii) the relationship between changes in IOP and plasma melatonin during sleep. For all experiments IOP was measured before and after sleep. We found that IOP increased significantly after sleep. There was also a significant difference between the five groups with the old high-normal group showing the greatest increase, and the young high-normal group showing the lowest increase in IOP. The increase in IOP after sleep was reduced when the same subjects slept in bright light compared to that recorded when subjects slept in the dark. Plasma melatonin levels, as well as IOP, increased after sleep in the dark although there was no correlation between these changes for individual subjects.

Ultraviolet light-induced pathology in the eye: associated changes in ocular aldehyde dehydrogenase and alcohol dehydrogenase activities.

Adult male C57BL/6J inbred mice were subjected to ultraviolet radiation (UVR) exposure (302-nm peak wavelength; average intensity 282 microW/cm2) for 1 h and monitored for ocular aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) activity changes over a period of 25 days. Dramatic reductions in activities were observed by 4-6 days postexposure, resulting in enzyme levels of 15-16% of control animals. Major decreases in corneal enzyme levels were predominantly responsible for these changes. Ocular morphology was observed throughout using a photoslit-lamp biomicroscope, with maximum corneal clouding occurring at days 4-6. These data support earlier proposals for major roles for these corneal enzymes in assisting the cornea in protecting the eye against UVR-induced tissue damage.

Age-related maculopathy. II: The nature of the central visual field loss.

This is the second of three papers dealing with age-related maculopathy (ARM) and its effects on visual function (Swann, P.G. and Lovie-Kitchin, J.E. Age-related maculopathy. I: A review of its morphology and effects on visual function. Ophthal. Physiol. Opt. 10, 149-158 (1990)). We investigated the nature or character of the central visual field loss in ARM and pre-age related maculopathy (PARM) and compared them with age-matched normal subjects. Central visual fields were examined using the Friedmann Visual Field Analyser, Mark II, the Bausch and Lomb Autoplot Tangent Screen and Amsler charts. The central visual field defects in ARM were predominantly paracentral with a relative sparing of foveal sensitivity. PARM subjects did not show significant visual field defects. However, three PARM subjects did show slight distortions with the Amsler charts. The third paper in this series will compare the efficacy of the three methods of visual field investigation in the detection of these defects.

Enrichment of platelet phospholipids with eicosapentaenoic acid and docosahexaenoic acid inhibits thromboxane A2/prostaglandin H2 receptor binding and function.

Human platelet lipids were enriched in vitro with different amounts of either docosahexaenoic acid (22:6n-3), eicosapentaenoic acid (20:5n-3) or linoleic acid (18:2n-6). Of the total fatty acid incorporated, between 82 and 95% was associated with the phospholipid (PL) fraction, with the remainder as either neutral lipid or hydroxy fatty acid. Within the PL fraction, the majority (64% of total) of each fatty acid was incorporated into phosphatidylcholine. It was found that platelet aggregation induced by the thromboxane A2/prostaglandin H2 mimetic (15S)-hydroxy-11,9-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619) was inhibited after PL enrichment with 22:6n-3 or 20:5n-3, but not after 18:2n-6 enrichment. The specificity of 22:6n-3 and 20:5n-3 for U46619 activation was demonstrated by the finding that neither fatty acid significantly inhibited thromboxane A2/prostaglandin H2-independent aggregation induced by A23187 or thrombin. Furthermore, enrichment with 22:6n-3 or 20:5n-3 resulted in inhibition of [3H]U46619 specific binding, while enrichment with 18:2n-6 did not affect binding. Scatchard analysis revealed that thromboxane A2/prostaglandin H2 receptor affinity for [3H]U46619 decreased 4.8-fold following 22:6n-3 incorporation. These results demonstrate that platelet phospholipid enrichment with 22:6n-3 or 20:5n-3 results in a selective inhibition of thromboxane A2/prostaglandin H2 receptor function.

Age-related maculopathy. I: A review of its morphology and effects on visual function.

Age-related maculopathy (ARM) is a leading cause of permanent vision loss in elderly people. ARM therefore constitutes an important public health problem which will increase in magnitude as the number of aged people in the general population becomes greater. The consequences of this condition are exacerbated by the fact that treatment, especially of the atrophic form of the disease, is ineffective. While laser photocoagulation may be helpful in the exudative form of ARM, there is often an inexorable progression towards severe vision loss in these patients. Therefore considerable attention needs to be paid to the aetiology of ARM, the potential for its prevention or delayed onset and its recognition through functional disturbances. This is the first of three papers dealing with ARM and its effects on visual function. We review its morphology and the visual disturbances that may ensue. The second and third papers will discuss the nature and detection of the central visual field loss due to ARM.

Investigation of central visual fields in patients with age-related macular changes.

The effect of early macular pigmentary and drusen changes on the central visual field was investigated in elderly patients with normal visual acuities. Visual field measurements were taken with the Humphrey Field Analyser using its 24-2 and 10-2 full threshold programs. No significant differences were found between two patients groups, one with and one without the macular changes. We conclude that fine pigmentary changes and hard drusen do not cause changes in visual functioning and can be accepted as normal age-related changes.

Eicosapentaenoic acid and docosahexaenoic acid are antagonists at the thromboxane A2/prostaglandin H2 receptor in human platelets.

The present study investigated the mechanism by which eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) inhibit platelet activation induced by thromboxane A2. DHA was found to be more potent than EPA in blocking platelet aggregation induced by the stable thromboxane A2 mimetic, U46619. Furthermore, this inhibition by DHA or EPA was competitive. Binding studies using 3H-U46619 demonstrated that both EPA and DHA interact with the platelet thromboxane receptor. The potency of the inhibition of binding corresponded with that seen for the inhibition of aggregation. These results suggest that thromboxane receptor antagonism may be an important mechanism by which EPA and DHA modulate platelet reactivity in vivo.

Perinatal upregulation of benzodiazepine receptor ontogenesis: "fearless" and more efficient goal-directed behavior of adult rat progenies.

Pregnant and subsequently lactating rats had ad lib access to drinking water which contained either a benzodiazepine antagonist, Ro 15-1788 or diazepam (DZ). On the average, the rats consumed 2.9 mg/kg/day of Ro 15-1788 or 5.3 mg/kg/day of diazepam over the time period of 3 weeks, from gestation day 14 through postpartum day 14. The control group consumed equivalent volumes of the drug vehicle in water. While Ro 15-1788 had no apparent toxic effects, the numbers of viable pups in the DZ group were significantly reduced. The mean weight of the viable pups, and their gross behavior were not different in all three groups. However, the fully mature 4.5-month-old male progenies exposed to Ro 15-1788 were much more efficient in the radial arm maze test than the control or the diazepam-exposed animals; they rapidly habituated to the novel environment, their exploratory activity was uninhibited by distracting visual and auditory stimuli, they made fewer "working memory" errors in collecting baits, had a much better control over their emotional responses and the autonomic nervous system, as shown by very low defecation/urination scores, and, at the age of 5 months, they had a significant (66%) increase in the density of benzodiazepine receptors in the hippocampal formation, as compared to the control or the diazepam-exposed progenies. In conclusion, the upregulation of benzodiazepine receptor ontogenesis is retained in adult animals and resulted in improved "working memory" and better control over emotional responses that were particularly evident when the animals were challenged by novel and "intimidating" environmental stimuli.

Fluctuations in intra-ocular pressure with sleep: I. Time course of IOP increase after the onset of sleep.

Ten normal subjects slept over a series of four nights for time periods of 30 minutes, 1, 2 and 4 hours. Intra-ocular pressure (IOP) measurements were made using a non-contact tonometer before and after sleep. The subjects showed a significant increase in IOP of 3.45 mm Hg after 30 minutes of sleep and a further IOP increase thereafter to 6.41 mm Hg above baseline. Such increases in IOP after sleep in normal subjects suggest that glaucoma patients and those suspected of having glaucoma should be monitored overnight to assess their IOP control mechanisms.

Sample preparation procedure for determination of dopamine sulfate isomers in human urine by high-performance liquid chromatography with dual-electrode electrochemical detection.

We developed a procedure utilizing small columns of solid-phase extraction material for sample preparation for the determination of dopamine sulfate (DAS) isomers in human urine. Processed sample is then subjected to high-performance liquid chromatography (HPLC) with dual-series-electrode electrochemical detection. Dopamine 3-O-sulfate (DA-3-S) and dopamine 4-O-sulfate (DA-4-S) were determined using two different HPLC systems. The ratio of the urinary excretion rate of DA-3-S to DA-4-S was relatively constant, but the 24-h excretion rates of total DAS varied widely among individuals. This method should prove useful in future studies concerning the metabolic and physiologic roles of DAS isomers.

Keratoconus: the clinical spectrum.

This paper reports observations made on 57 keratoconus patients, 32 women and 25 men. Particular attention is paid to the retinoscopic reflex, intraocular pressure, and the patient's bodyweight. Diagnostic criteria are proposed.