A crop loss-related forecasting model for sclerotinia stem rot in winter oilseed rape.

Sclerotinia stem rot (SSR) is an increasing threat to winter oilseed rape (OSR) in Germany and other European countries due to the growing area of OSR cultivation. A forecasting model was developed to provide decision support for the fungicide spray against SSR at flowering. Four weather variables-air temperature, relative humidity, rainfall, and sunshine duration-were used to calculate the microclimate in the plant canopy. From data reinvestigated in a climate chamber study, 7 to 11 degrees C and 80 to 86% relative humidity (RH) were established as minimum conditions for stem infection with ascospores and expressed as an index to discriminate infection hours (Inh). Disease incidence (DI) significantly correlated with Inh occurring post-growth stage (GS) 58 (late bud stage) (r(2) = 0.42, P /= Inh(i). Historical field data (1994 to 2004) were used to assess the impact of agronomic factors on SSR incidence. A 2-year crop rotation enhanced disease risk and, therefore, lowered the infection threshold in the model by a factor of 0.8, whereas in 4-year rotations, the threshold was elevated by a factor 1.3. Number of plants per square meter, nitrogen fertilization, and soil management did not have significant effects on DI. In an evaluation of SkleroPro with 76 historical (1994 to 2004) and 32 actual field experiments conducted in 2005, the percentage of economically correct decisions was 70 and 81%, respectively. Compared with the common practice of routine sprays, this corresponded to savings in fungicides of 39 and 81% and to increases in net return for the grower of 23 and 45 euro/ha, respectively. This study demonstrates that, particularly in areas with abundant inoculum, the level of SSR in OSR can be predicted from conditions of stem infection during late bud or flowering with sufficient accuracy, and does not require simulation of apothecial development and ascospore dispersal. SkleroPro is the first crop-loss-related forecasting model for a Sclerotinia disease, with the potential of being widely used in agricultural practice, accessible through the Internet. Its concept, components, and implementation may be useful in developing forecasting systems for Sclerotinia diseases in other crops or climates.

Prion protein affects Ca2+-activated K+ currents in cerebellar purkinje cells.

The prion protein (PrPC) has a primary role in the pathogenesis of transmissible spongiform encephalopathies. Its physiological function is not known yet. Altered late afterhyperpolarization has been observed in hippocampal CA1 pyramidal cells of prion protein-deficient mice (Prnp(0/0) mice) presumably caused by a disruption of Ca2+-activated K+ currents. An alteration of these currents has been recently described in scrapie-infected animals, and loss of function of PrPC has been put forward as one possible pathophysiological mechanism in prion diseases. This work focuses on patch-clamp studies of Ca2+-activated K+ currents in cerebellar Purkinje cells in the slice preparation of Prnp(0/0) mice as well as of transgenic mice. A significant correlation between PrPC expression in Purkinje cells and the maximal amplitude of TEA-insensitive Ca2+-activated K+ currents was observed, with reduced current amplitudes in Prnp(0/0) mice and a rescue of the phenotype in transgenic mice where PrPC had been reintroduced. Further studies of the intracellular free calcium concentration revealed an alteration of the maximal increase of intracellular calcium concentration with depolarization in the Prnp(0/0) mouse Purkinje cells. These data provide strong evidence that Ca2+-activated K+ currents in Prnp(0/0) mice are reduced due to an alteration of intracellular calcium homeostasis.

Quantitative molecular characterization of bovine vitreous and lens with non-invasive dynamic light scattering.

The non-invasive technique of dynamic light scattering (DLS) was used to quantitatively characterize vitreous and lens structure on a molecular level by measuring the sizes of the predominant particles and mapping the three-dimensional topographic distribution of these structural macromolecules in three spatial dimensions. The results of DLS measurements in five fresh adult bovine eyes were compared to DLS measurements in model solutions of hyaluronan (HA) and collagen (Coll). In the bovine eyes DLS measurements were obtained from excised samples of gel and liquid vitreous and compared to the model solutions. Measurements in whole vitreous were obtained at multiple points posterior to the lens to generate a three-dimensional 'map' of molecular structure. The macromolecule distribution in bovine lens was similarly characterized.In each bovine vitreous (Bo Vit) specimen, DLS predominantly detected two distinct particles, which differed in diffusion properties and hence size. Comparisons with model vitreous solutions demonstrated that these most likely corresponded to the Coll and HA components of vitreous. Three-dimensional mapping of Bo Vit found heterogeneity throughout the vitreous body, with different particle size distributions for Coll and HA at different loci. In contrast, the three-dimensional distribution of lens macromolecules was more homogeneous. Thus, the non-invasive DLS technique can quantitate the average sizes of vitreous and lens macromolecules and map their three-dimensional distribution. This method to assess quantitatively the macromolecular structure of vitreous and lens should be useful for clinical as well as experimental applications in health and disease.

Neuron specific enolase in retinal detachment.

PURPOSE: Neuron Specific Enolase (NSE) is released following central nervous system (CNS) distress. As retina is part of the CNS, NSE levels were measured in the subretinal fluid (SRF), aqueous, and serum of patients with primary rhegmatogenous retinal detachment (RD). METHODS: Radioimmunoassay was used to determine NSE levels in the SRF, aqueous, and serum of 13 patients (28-92 years old, mean = 71 years) with RD. As controls, NSE was measured in the aqueous of 6 patients undergoing cataract surgery and in serum of 18 patients without ophthalmological or neurological diseases. RESULTS: SRF levels of NSE ranged from 50-200 microg/l (mean +/- s.d. = 150 +/- 57). NSE levels in aqueous from patients with RD were 2-140 microg/l (mean +/- s.d. = 39 +/- 42), significantly higher than in controls (0-6 microg/l; mean +/- s.d. = 1.58 +/- 2.24; p = 0.04). Serum NSE levels in RD patients ranged from 6.5-80 microg/l (mean +/- s.d. = 26 +/- 21) and was significantly higher than in controls (5.3 +/- 1.66 microg/l; p = 0.005). CONCLUSIONS: Retinal neuron injury in retinal detachment (RD) releases sufficient Neuron Specific Enolase (NSE) to be detected in subretinal fluid, aqueous, and even in serum. Thus, NSE could index disease severity in RD and provide a means by which to assess the response to neuroprotection in RD.

Altered intracellular calcium homeostasis in cerebellar granule cells of prion protein-deficient mice.

Previous studies have indicated that recombinant cellular prion protein (PrP(C)), as well as a synthetic peptide of PrP(C), affects intracellular calcium homeostasis. To analyze whether calcium homeostasis in neurons is also affected by a loss of PrP(C), we performed microfluorometric calcium measurements on cultured cerebellar granule cells derived from prion protein-deficient (Prnp(0/0)) mice. The resting concentration of intracellular free calcium [Ca(2+)](i) was found to be slightly, but significantly, reduced in Prnp(0/0) mouse granule cell neurites. Moreover, we observed a highly significant reduction in the [Ca(2+)](i) increase after high potassium depolarization. Pharmacological studies further revealed that the L-type specific blocker nifedipine, which reduces the depolarization-induced [Ca(2+)](i) increase by 66% in wild-type granule cell somas, has no effect on [Ca(2+)](i) in Prnp(0/0) mouse granule cells. Patch-clamp measurements, however, did not reveal a reduced calcium influx through voltage-gated calcium channels in Prnp(0/0) mice. These data clearly indicate that loss of PrP(C) alters the intracellular calcium homeostasis of cultured cerebellar granule cells. There is no evidence, though, that this change is due to a direct alteration of voltage-gated calcium channels.

Dynamic light scattering of diabetic vitreopathy.

BACKGROUND: Diabetes induces pathology throughout the body via nonenzymatic glycation of proteins. Vitreous, which is replete with type II collagen, undergoes significant changes in diabetes. The resultant diabetic vitreopathy plays an important role in diabetic retinopathy. Detecting these molecular changes could provide insight into diabetic eye disease as well as molecular effects elsewhere in the body. METHODS: Human eyes were obtained at autopsy and studied in the fresh, unfixed state. Sclera, choroid, and retina were dissected off the vitreous for dark-field slit microscopy and dynamic light scattering (DLS). For the former, the entire vitreous was exposed. For the latter, only a window at the equator was dissected in some specimens, and the anterior segment was removed leaving the posterior lens capsule intact in others. DLS was performed to determine particle sizes at multiple sites 0.5 mm apart, spanning the globe at the equator (window dissections) and along the antero-posterior axis. RESULTS: Dark-field slit microscopy in diabetic subjects detected findings typical of age-related vitreous degeneration, but at much younger ages than nondiabetic controls. Noninvasive DLS measurements found a greater heterogeneity and larger particle sizes in vitreous of subjects with diabetes as compared to age-matched controls. CONCLUSIONS: DLS can detect and quantify the early molecular effects that cause vitreous collagen fibrils to cross-link and aggregate. This could provide valuable insight into ocular and systemic effects of hyperglycemia, because the molecular changes in diabetic vitreopathy could serve as an index of such effects throughout the body. In addition to the diagnostic implications, this methodology could provide a rapid, reproducible way to monitor the response to therapy with novel agents intended to prevent the complications of diabetes on a molecular level.

Diabetic vitreopathy--findings using the celloidin embedding technique.

BACKGROUND: To evaluate the role of the vitreous in diabetic eyes, autopsy eyes from diabetic patients have been examined by means of light and electron microscopy. METHODS: Twenty-five eyes were obtained from 19 patients. The duration of diabetes was from 8 to 26 years (average 17 years). All patients had type II diabetes. The glycerol-dehydrated globes were embedded in celloidin and hardened with chloroform. For light microscopy, areas of interest were cut from 200-microm-thick celloidin sections and embedded in paraffin, and 10-microm sections were cut. Such 200-microm and 10-microm sections were also used for scanning electron microscopy. For transmission electron microscopy, areas of interest were cut from thick celloidin sections and embedded in Epon for thin sectioning. RESULTS: The vitreous in diabetic eyes exhibits delayed senile degeneration in case of early onset of diabetes. It shows intensified staining of the vitreous cortex. Posterior detachment is often incomplete without collapse. The vitreous collagen fibers are coarse and aggregated in the centrally located cortical region of the vitreous. Hyalocytes in diabetic eyes have a different shape than normally, and their number seems to be increased. All these structural changes were not demonstrated in nondiabetic eyes. CONCLUSION: The vitreous in diabetic patients is morphologically different from normal vitreous. Features of the diabetic vitreous are aggregated collagen fibers, increased density of the vitreous cortex and morphological changes of hyalocytes. It is discussed whether these findings arise from nonenzymatic glycosylation of collagen molecules resulting in diabetic vitreopathy.

[Immunohistologic studies of the vitreous humor]. / Immunhistologische Untersuchungen des Glaskörpers.

BACKGROUND: Immunohistological staining of the vitreous is difficult because of its high water content. We present a method for immunohistological staining of celloidin-embedded eyes. Results from diabetic and non-diabetic eyes are demonstrated. METHOD: Diabetic and non-diabetic eyes were firstly immersed in formol and then slowly dehydrated using rising concentrations of glycerine (sink method). Subsequently, the whole globe was embedded in celloidin and cut into 200-micron sections. Control areas of interest were dissected from the 200 microns sections under a lightmicroscope. These specimens were then embedded in paraffin and cut into 7-micron sections. The 7-micron sections were immunohistochemically stained for type I-collagen, type IV-collagen, fibronectin and laminin. RESULTS: This method makes immunohistochemical staining of the vitreous possible. Type IV collagen, laminin and fibronection were found at higher concentrations in diabetic eyes than in normal eyes. Type I collagen was detected in neither diabetic nor in normal eyes. CONCLUSIONS: Our method of examination allows immunohistological staining of the vitreous in its place of origin. Although our method is time consuming, it has some advantages over biochemical analysis: Even minimal changes and their exact distribution can be detected. Our first results show that the vitreous is built up inhomogeneously and that pathological influence can cause structural changes.

Morphologic studies of the peripheral vitreoretinal interface in humans reveal structures implicated in the pathogenesis of retinal tears.

PURPOSE: The ultrastructural nature of the preequatorial vitreoretinal interface was studied to elucidate the predisposing role for peripheral retinal tear formation. METHODS: Fourteen enucleated globes from seven decreased patients were examined. The patients ranged in age from 16-88 years, with an average age of 40 years. None of the patients had a history of ocular or systemic disease that could have affected the eyes. Globes were examined by stereomicroscopy and by light and electron microscopy using the celloidin embedding method. RESULTS: On examination we found several structures in addition to tufts and rosettes. In the vitreous cortex, fibrillar structures with no vitreoretinal attachment to the retina frequently were found. We propose that these structures be called "tubuli" because of their spiral appearance. Mushroom-like structures, which we propose be referred to as "spiculae," were found to arise from the intact internal limiting lamina of the retina and to insert into the vitreous cortex, constituting foci of vitreoretinal adhesion. Other structures, which we propose be called "verrucae," arose from the disrupted internal limiting lamina of the retina and inserted into disrupted areas of vitreous cortex. CONCLUSIONS: Tubuli appear to be remnants of the embryonic vasculature with no clinical or pathologic significance. Because of their pattern of inserting into the internal limiting lamina of the retina and the peripheral vitreous cortex, spiculae and verrucae may play an important role in the formation of retinal breaks.

The effect of retinal cryoapplication on the vitreous.

PURPOSE: The effect of retinal cryopexy on the vitreous was studied morphologically in an animal model. METHODS: The retina and vitreous were frozen with single cryolesions on one eye and 24 contiguous cryolesions on the contralateral eye in 16 rabbits. The cryoprobe was applied to the sclera from 3 mm to 6 mm posterior to the limbus at -60 degrees C until ophthalmoscopically visible whitening occurred. Two animals were killed on the first day; the third day; after 1, 2, and 4 weeks; and after 2, 3, and 6 months after surgery. The eyes were enucleated and prepared by the celloidin embedding method. Each 200-microgram section was examined by light microscopy. Areas of the specimens were dissected and studied by scanning and transmission electron microscopy. RESULTS: Single cryolesions did not have a significant generalized effect on the vitreous. Evidence of local collagen destruction and dispersion of cells was found near the area of cryoapplication. Contiguous cryoapplication led primarily to increased density in the vitreous and subretinal edema. The vitreoretinal border was invaded by mononuclear cells containing pigment granules. Thickened collagen fibers were attached to the coagulated retina in a perpendicular manner and traversed the whole vitreous body. After 4 weeks the increased vitreous density slowly diminished, and preretinal capillaries surrounded by vitreous collagen started to proliferate from the vitreoretinal interface. After 6 months central vitreous collagen fibers looked normal. In the area of cryoapplication, vitreoretinal membrane formation had occurred. CONCLUSION: Single cryolesions have no significant effect of the vitreous. Multiple cryolesions lead to neovascularization soon after the procedure (1 month) and membrane formation later (6 months after the procedure). This supports the concept that the extensive use of cryopexy in human retinal surgery could contribute to the development of proliferative vitreoretinopathy.

Prognostic value of magnetic resonance imaging in monosymptomatic optic neuritis.

PURPOSE: Magnetic resonance imaging is able to depict lesions in the optic nerve in the acute stage of monosymptomatic optic neuritis. Most patients have lesions located intraorbitally, intracanalicularly, and/or intracranially. The goal of this study is to determine whether these lesions resolve after visual recovery, change in length or localization, or could be correlated to the visual function. METHODS: Between 1987 and 1992, the authors examined 22 patients with acute optic neuritis using magnetic resonance imaging short-time inversion recovery sequences. Additionally, the authors determined visual acuity, visual field, color vision, contrast sensitivity, and visual-evoked responses. All patients were re-examined between 1993 and 1994 in the same manner. Visual recovery in the re-examination was divided into three groups: group 1 with complete visual recovery (visual acuity better than 20/25); group 2 with incomplete recovery (visual acuity better than 20/25 but defect in at least one of the other tests: visual field, color vision, and contrast sensitivity); and group 3 with partial recovery (visual acuity remained less than 20/25, defect in all the other tests). RESULTS: All group 1 patients initially had lesions less than 17.5 mm, group 2 patients had lesions greater than 17.5 mm (44%) and/or lesions located intracanalicularly (66%), and most of group 3 patients initially had lesions greater than 17.5 mm (79%). CONCLUSION: Eyes with lesions less than 17.5 mm in the optic nerve in acute optic neuritis have a good prognosis for visual recovery. Lesions greater than 17.5 mm or lesions involving the intracanalicular portion of the optic nerve lead to incomplete or partial visual recovery.

[Optic nerve involvement in chronic lymphatic leukemia of the B-cell series]. / Sehnervenbeteiligung bei chronisch-lymphatischer Leukämie der B-Zell-Reihe.

BACKGROUND: Malignant Non-Hodgkin lymphomas rarely manifest with ocular symptoms. We report on a patient with a low grade malignant lymphoma of the b-cell type with optic nerve involvement. PATIENT: In 1990 a 38-year old patient presented with typical symptoms of optic neuritis in the left eye. Magnetic resonance imaging (MRI) revealed a signal enhancement in the left optic nerve. Hematologically a well differentiated Non Hodgkin's lymphoma of the b-cell type was diagnosed. Neurological signs and symptoms in the following years were regarded as manifestations of multiple sclerosis (MS). In 1994 the patient presented with an acute visual loss in the right eye. MRI this time revealed a signal enhancement in the right optic nerve and a big CNS lesion. Gradually a bilateral partial optic nerve atrophy developed. The cerebrospinal fluid contained atypical lymphocytes, matching with the cells in the peripheral blood, morphologically and in the fluorescence-activated cell sorter (FACS). These findings suggest an intracerebral manifestation of a well differentiated Non-Hodgkin's lymphoma. CONCLUSION: Low grade malignant well-differentiated Non-Hodgkin lymphomas infiltrate the optic nerve. The clinical course in our case mimicked multiple sclerosis.

On describing microbial growth kinetics from continuous culture data: Some general considerations, observations, and concepts.

Analysis of continuous culture methodology suggests that this potentially powerful tool for kinetic analysis can be improved by minimizing several inherent shortcomings. Medium background substrates - organic carbon, phosphate, and manganese - were shown to dominate kinetic observations at concentrations below chemical detection methods. Reactor wall growth, culture size distribution changes, sample removal-induced steady state perturbations, and limiting substrate leakage from organisms are treated in terms of kinetic measurement errors. Large variations in maximal growth rates and substrate uptake rates found are attributed to experimental protocol-induced transient states. Relationships are presented for correcting limiting substrate concentrations for lability during sampling, contamination with unreacted medium, and background substrate effects. Analytical procedures are discussed for improved measurement of limiting substrate kinetics involving enzymes, isotopes, and material balance manipulation. Relaxation methods as applied to continuous culture are introduced as a means for isolating separate rate constants describing net substrate transport and for evaluating cellular metabolite leakage. Low velocity growth, multiple substrate metabolism, and endogenous metabolism are discussed along with measurements showing that 1-month generation times for aquatic microorganisms can be quite normal and that the kinetics are compatible withµg/liter limiting substrate concentrations. The concept of regarding growth kinetics as the sum of several net accumulation processes is suggested.

Continuous culture of Rhodotorula rubra: kinetics of phosphate-arsenate uptake, inhibition, and phosphate-limited growth.

The pink yeast Rhodotorula rubra of marine origin was found to be capable of extended growth at very low phosphate concentrations (K(0.5) = 10.8 nm). Average intracellular phosphate concentrations, based on isotope exchange techniques, were 15 to 200 nm, giving concentration gradients across the cell envelope of about 10(6). Sensitivity to metabolic inhibitors occurred at micromolar concentrations. Inability of the phosphate transport system, K(s) = 0.5 to 2.8 mum, V(max) = 55 mumoles per g of cells per min, to discriminate against arsenate transport led to arsenate toxicity at 1 to 10 nm, whereas environmental arsenate levels are reportedly much higher. Phosphate competitively prevented arsenate toxicity. The K(i) for phosphate inhibition of arsenate uptake was 0.7 to 1.2 mum. Phosphate uptake experiments showed that maximal growth rates could be achieved with approximately 4% of the total phosphate-arsenate transport system. Organisms adapted to a range both of concentration of NaCl and of pH. Maximal affinity for phosphate occurred at pH 4 and at low concentrations of NaCl; however, V(max) for phosphate transport was little affected. Maximal specific growth rates on minimal medium were consistent in batch culture but gradually increased to the much higher rates found with yeast extract media when the population was subjected to long-term continuous culture with gradually increasing dilution rates. Phosphate initial uptake rates that were in agreement with the steady-state flux in continuous culture were obtained by using organisms and medium directly from continuous culture. This procedure resulted in rates about 500 times greater than one in which harvested batch-grown cells were used. Discrepancies between values found and those reported in the literature for other organisms were even larger. Growth could not be sustained below a threshold phosphate concentration of 3.4 nm. Such thresholds are explained in terms of a system where growth rate is set by intracellular nutrient concentrations. Threshold concentrations occur in response to nutrient sinks not related to growth, such as efflux and endogenous metabolism. Equations are presented for evaluation of growth rate-limiting substrate concentrations in the presence of background substrate and for evaluating low inhibitor concentration inhibition mechanisms by substrate prevention of inhibitor flux.