In vivo uptake of chitosan microparticles by murine Peyer's patches: visualization studies using confocal laser scanning microscopy and immunohistochemistry.

Although oral vaccination has numerous advantages over parenteral injection, degradation of the vaccine and low uptake by the gut associated lymphoid tissue (GALT) still complicate the development of efficient oral vaccines. However, previous studies in our laboratory demonstrated that chitosan microparticles can have suitable size, charge, loading and release characteristics for oral vaccination using ovalbumin as model vaccine. In this study, two different approaches were used to investigate the in vivo uptake of chitosan microparticles by murine Peyer's patches. Firstly, a confocal laser scanning microscopy (CLSM) study was performed to visualize the uptake of fluorescent-labeled chitosan microparticles in the Peyer's patches after intragastrical feeding. Subsequently, the intestinal epithelial uptake of ovalbumin loaded chitosan microparticles was visualized using immunohistochemical staining of ovalbumin. Because the microparticles are biodegradable, this entrapped ovalbumin will be released after intracellular digestion in the Peyer's patches. CLSM visualization demonstrated that chitosan microparticles enhance the uptake of fluorescent-labeled ovalbumin by the epithelium of the Peyer's patches. No ovalbumin uptake by the intestinal epithelium was observed when the protein was administered without microparticles. Moreover, immunohistochemical visualization studies revealed that ovalbumin could only be transported into the Peyer's patches after association to chitosan microparticles. Since uptake by Peyer's patches is an essential step in oral vaccination, these in vivo experiments demonstrate that chitosan microparticles are very promising vaccine delivery systems.

APOE genotype: no influence on galantamine treatment efficacy nor on rate of decline in Alzheimer's disease.

Apolipoprotein E (APOE) has been extensively demonstrated to be a genetic risk factor for Alzheimer's disease (AD). Associations of APOE genotype have been reported with age at AD onset, rate of decline, and responsiveness to therapy. This study aimed to test these hypotheses in a large study population of AD patients. APOE genotype was determined from 1,528 Caucasian subjects, diagnosed by NINCDS/ADRDA criteria as probable AD patients, enrolled in four international placebo-controlled clinical trials of 3--12 months duration, designed to evaluate efficacy of treatment with galantamine or sabeluzole. In addition to patient demographics and baseline scores for Mini Mental State Examination, scores on the Disability Assessment for Dementia (DAD) and the cognitive subscale of the Alzheimer's Disease Assessment Scale (ADAS-cog) were recorded at the start, during, and at the end of the study. APOE epsilon 4 homozygotes had a significantly lower age at disease onset compared to patients with other APOE genotypes. The epsilon 4 allele was significantly over-represented in females compared to males, and in the group of subjects with an AD family history. Based on longitudinal data of 504 placebo-treated AD patients, the linear annual rate of change in score was 5 points on the ADAS-cog scale and 11 on the DAD scale. The epsilon 4 allele copy number did not influence these rates of decline. Sabeluzole treatment was not effective in the overall group compared to the placebo-treated group, nor in any subgroup stratified by epsilon 4 allele count. Galantamine produced cognitive and functional improvement that were not affected by epsilon 4 allele count. In conclusion, our data confirm a strong association between epsilon 4 homozygotes and age at onset of AD but do not support an effect of epsilon 4 allele copy number on rate of cognitive and functional decline nor on the efficacy of galantamine in patients with AD.

Two linked mutations in transcriptional regulatory elements of the CYP3A5 gene constitute the major genetic determinant of polymorphic activity in humans.

Cytochrome P450 3A subfamily members (CYP3A) are the most abundant liver cytochrome P450 forms, responsible for the biotransformation of over 50% of all drugs. The expression and activity of isoforms CYP3A4 and CYP3A5 show wide inter-individual variation, influencing both drug response and disease susceptibility. The molecular basis for this variation has never been defined. In this study, we used midazolam to characterize CYP3A5 phenotype in a panel of liver samples. A clear bimodality in metabolism was observed. Analysis of the 5' flanking region of the CYP3A5 gene identified two linked polymorphisms, T-369G and A-45G, located in transcriptional regulatory elements which are associated with increased expression and activity of the gene. A polymerase chain reaction based detection assay is described facilitating future studies into both the metabolic consequences of this variation and disease association studies relating to CYP3A5.

Analysis of the human KCNH2(HERG) gene: identification and characterization of a novel mutation Y667X associated with long QT syndrome and a non-pathological 9 bp insertion.

Long QT (LQT) syndrome is a potentially life-threatening disorder, characterized by a distinct cardiac arrhythmia known as torsades de pointes. Mutations within a number of genes linked to the familial form, including that coding for a cardiac potassium channel called KCNH2 (HERG), have been described based on the characterized genomic organization. A standardized method was developed to screen the entire gene for gene variants. We report a single base pair substitution, introducing a premature STOP codon at codon 667 of the gene in a healthy individual with an extended QTc interval (460 msec). In vitro expression of the codon Y667X variant in Xenopus oocyte suggests that the autosomal dominant variant does not function in a dominant/negative manner and cannot co-assemble to form a channel, resulting in a reduction of the KCNH2 current, and an extension of the QT interval. This indicates that pathogenic LQT gene variants exist in the apparently normal population, the prognosis and clinical consequences of which remain to be determined. The assays described should facilitate future studies into this area.

[An impacted bladder stone]. / Een afgezakte blaassteen.

A case of colic in a Haflinger gelding is reported. This was due to a calculus in the penis. The operation is described.

The use of monoclonal antibodies in the study of the interaction between adrenal medullary cell membranes and chromaffin granules.

Simultaneous incubation of bovine adrenal medullary plasma membranes (PM) with chromaffin granules (CG) resulted in the release of the soluble granular content. The molecular mechanism of this process was studied with several monoclonal antibodies (mAb) raised against different plasma membrane components. Specific inhibition of the catecholamine secretion was obtained upon incubation with the monoclonal antibody UIA/NEU/VI B17. The corresponding antigen had an apparent molecular weight of 54000 Dalton. These results suggest a specific recognition between proteins located on the plasma membrane and chromaffin granule membrane, the interaction of which mediates exocytosis.

The effect of colchicine treatment on the buoyant density of noradrenaline storage particles in the rabbit superior cervical ganglion.

The subcellular distribution of noradrenaline (NA) was studied in the rabbit superior cervical ganglion before and after colchicine treatment. One-third of the total NA could be sedimented with the microsomal material in control conditions. Upon colchicine treatment the percentage of the NA which was particle-bound increased only slightly. The absolute NA content of the superior cervical ganglion from colchicine-treated rabbits, however, showed a 3.5-fold increase compared to control animals. Density gradient centrifugation of the microsomal fraction revealed that the distribution of the NA-containing particles changed significantly. Before colchicine treatment the NA was found in a broad band covering the gradient fractions where 'light' and 'heavy' NA vesicles were expected to equilibrate. After colchicine treatment the NA was concentrated in the 'heavy' NA vesicles part of the gradient. The dopamine beta-hydroxylase activity and the NA content in the gradient are increased 3 times after colchicine treatment. These experimental results can be explained by the axoplasmic transport impairing action of colchicine. The neuronal cell body accumulates 'heavy' NA vesicles formed there, unable to transport them towards the periphery.

Metabolism and growth of chickens before and after hatch in relation to incubation temperatures.

Chick embryos were incubated at 35.8 C from days 1 to 10 and at 37.8 C from day 11 to hatching. Control embryos were incubated at 37.8 C. Other environmental conditions were standardized. Embryos incubated at 35.8 C showed compensatory growth when compared to embryos of the control group and showed a higher heat production (kJ/embryo/hr) expressed as a linear function of dry embryo weight (g) or incubation time (days). After hatching, the compensatory growth of the chickens incubated at 35.8 C (days 1 to 10) continued, as demonstrated by analyses of the weight of the body, liver, and the gastrointestinal tract. The same animals produced more heat per unit of time and body weight. Adult hens from this group showed a trend for higher heat production per unit body weight as well as a higher dry matter content. These results suggest a link between in ovo environmental conditions, growth processes, and metabolic level up to the reproduction period of domestic fowl.

Release of chromaffin granular content on interaction with plasma membranes.

Cell-free interaction between bovine adrenal medullary plasma membranes and chromaffin granules results in the release of the granular content, a process specifically controlled by micromolar concentrations of calcium and therefore regarded as a putative model for exocytosis. The density gradient distribution of interacted organelles (1 min at 37 degrees C) was compared with the distribution of noninteracted material (kept at 0 degree C). Self-generating Percoll gradients, in which intact chromaffin granules were separated from plasma membranes and empty granule "ghosts," were used. The plasma membrane-induced release of the chromaffin granular matrix upon concomitant incubation was demonstrated by the disappearance of the "intact granule" peak accompanied by a proportional rise of the "granule ghost" peak. The degree of granule disappearance depended on the amount of plasma membranes added. The soluble content of the fraction that had disappeared was dispersed throughout the gradient. No shift of the granule peak toward intermediate densities was observed; this suggests that the loss of each granule's content is an all-or-none phenomenon rather than a partial release.

Protein phosphorylation and the exocytosis-like interaction between isolated adrenal medullary plasma membranes and chromaffin granules.

The interaction between isolated adrenal medullary plasma membranes and chromaffin granules has been proposed as a cell-free model for exocytosis. Phosphorylation experiments showed that isolated chromaffin granules as well as isolated plasma membranes contain protein kinases and phosphate accepting membranous proteins. Upon joint incubation however, the chromaffin granule-located proteins are preferentially phosphorylated. beta-gamma-methylene-ATP, a non-hydrolysable analogue, was able to reduce both the plasma membrane-induced release of the soluble chromaffin granular content and the phosphate incorporation into the protein fraction. The results of these experiments on a cell-free model system fit in the hypothesis originating from work on several types of intact cells that the exocytotic event is linked with protein phosphorylation.

In vitro interaction between bovine adrenal medullary cell membranes and chromaffin granules: specific control by Ca2+.

The control of the in vitro interaction between bovine adrenal medullary plasma membranes and chromaffin granules by calcium has been studied. This interaction, which has previously been shown to result in the release of the soluble granular content, is a possible cell-free model for exocytosis. The plasma membrane-induced catecholamine release was stimulated when the [Ca2+] exceeded 2x10(-7) M. A maximal release was reached at 10(-5) M with a half maximal response around 10(-6)M. Mg2+ was not able to stimulate the system in the absence of Ca2+. These data suggest a high specificity of the calcium controlled exocytotic mechanism and the absence of an antagonism by high concentrations (10(-5)-10(-1) M) of magnesium at the exocytotic site itself.

The distribution of Mg2+ ATPase and its loss of specific activity upon gradient centrifugation of isolated chromaffin granules.

Preparations of purified chromaffin granules were subjected to isopycnic sucrose gradient centrifugation. Determination of Mg2+ ATPase activity and catecholamines showed that the distribution of ATPase almost parallelled the distribution of catecholamines. The distribution of ATPase was slightly shifted to lower densities and was suggested to be caused by the heterogeneity of the chromaffin granules. The results therefore provide evidence that ATPase is associated with chromaffin granules. Determination of the recovery of ATPase activity upon gradient centrifugation revealed losses of enzyme activity which were found to be proportional to the dilutions of the granule preparation subjected to gradient centrifugation.