Intraneuronal amyloid precursor protein (APP) and appearance of extracellular β‐amyloid peptide (aβ) in the brain of aging kokanee salmon

Antibodies to human amyloid precursor protein (APP₆₉₅) and beta‐amyloid peptide (Aβ_1‐42) were used to determine timing of amyloidosis in the brain of kokanee salmon (Oncorhynchus nerka kennerlyi) in one of four reproductive stages: immature (IM), maturing (MA), sexually mature (SM), and spawning (SP), representing a range of aging from somatically mature but sexually immature to spawning and somatic senescence. In IM fish, immunoreactive (ir) intracellular APP occurred in 18 of 23 brain regions. During sexual maturation and aging, the number of neurons expressing APP increased in 11 of these APP‐ir regions. Aβ‐ir was absent in IM fish, present in seven regions in MA fish, moderately abundant in 15 regions in SM fish, and was most abundant in all brain regions of SP fish exhibiting Aβ‐ir. Intracellular APP‐ir was observed in brain regions involved in sensory integration, olfaction, vision, stress responses, reproduction, and coordination. Intra‐ and extracellular Aβ_1‐42 immunoreactivity (Aβ‐ir) was present in all APP‐ir regions except the nucleus lateralis tuberis (hypothalamus) and Purkinje cells (cerebellum). APP‐ir and Aβ deposition increase during aging. APP‐ir is present in IM fish; Aβ‐ir usually appears first in MA or SM fish and increases in SM fish as does APP‐ir. Extracellular Aβ deposition dramatically increases between SM and SP stages (1–2 weeks) in all fish, indicating an extremely rapid and synchronized process. Rapid senescence observed in pacific salmon could make them a useful model to investigate timing of amyloidosis and neurodegeneration during brain aging. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 11–20, 2002     

Determination of the lactone and lactone plus carboxylate forms of 9-aminocamptothecin in human plasma by sensitive high-performance liquid chromatography with fluorescence detection

Two sensitive reversed-phase high-performance liquid chromatographic fluorescence methods, with simple sample handling at the site of the patient, are described for the determination of the lactone and lactone plus carboxylate forms of 9-aminocamptothecin (9AC). For 9AC lactone, the sample preparation was a liquid–liquid extraction with acetonitrile–n-butyl chloride (1:4, v/v), whereas the sample preparation for 9AC total (lactone plus carboxylate) was a simple deproteinization with 5% perchloric acid–methanol (1:1, v/v), which results in the conversion of the carboxylate into the lactone form. The lower limits of quantitation were 50 pg/ml and 100 pg/ml for 9AC lactone and 9AC total, respectively. The within-run precisions at four tested concentrations were ≤6.3% for 9AC lactone and ≤5.3% for 9AC total. The between-run precisions were ≤8.9% and ≤5.6%, respectively. The assays were developed to enable pharmacological analysis of 9AC in a bioavailability and oral phase I study in patients with solid tumors.     

Species specificity of iron delivery in hybridomas

Summary Studies with Human x Human (HxH), Human x Mouse (HxM), and Mouse x Mouse (MxM) hybridomas have enabled us to define specific factors that affect hybridoma growth in a species-specific manner. Three transferrins and three lipophilic iron chelates have been tested for their ability to support hybridoma proliferation and antibody production. The results of these studies demonstrate that HxH hybridomas do not respond to bovine transferrin a⁺ concentrations up to 100 μg/ml and are approximately 100-fold less responsive to mouse transferrin than to human transferrin. HxM and MxM hybridomas respond equally to human or mouse transferrin but are 100-fold less sensitive to bovine transferrin. An antibody to the human transferrin receptor inhibited the growth-promoting activity of human or mouse transferrin on HxH hybridomas but was ineffective on HxM hybridomas. This semonstrated the functionality of the human transferrin receptor in HxH hybridomas and that human, mouse, and bovine transferrin were interacting through the mouse transferrin receptor in HxM hybridomas. HxH and HxM hybridomas respond similarly to three different iron chelates exhibiting 80 to 110% of the growth response to human transferrin. MxM hybridomas fail to respond to the iron chelates at similar concentrations, suggesting that the human genome present in the other hybridoma species confers a unique ability for utilizing iron when delivered in this form.     

Hepatic autophagy and intracellular ATP. A morphometric study

In order to estimate the sensitivity of macroautophagy in liver toward changes in ATP we have analyzed the volume density of the autophagic/lysosomal system in isolated rat hepatocytes, incubated under conditions where intracellular ATP was partially depleted. (a) It appeared that reduction of the intracellular ATP concentration by 30-50% decreased the volume density of autophagic vacuoles by 70%. (b) Partial ATP depletion did not involve significant changes in the volume density of dense bodies. Together with studies showing that the rate of overall proteolysis via macroautophagy decreases with decreasing ATP concentration (P.J.A.M. Plomp, E.J. Wolvetang, A.K. Groen, A.J. Meijer, P.B. Gordon, and P.O. Seglen (1987) Eur. J. Biochem. 164, 197-203) our data indicate that changes in intracellular ATP primarily affect early steps in the autophagic/proteolytic pathway.     

Interstrain Variation in Amylase Gene Copy Number and mRNA Abundance in Three Mouse Tissues

Amylase expression in strain YBR differs in several respects from the standard mouse phenotype. The synthesis of salivary amylase is elevated twofold in YBR mice and the synthesis of pancreatic amylase is reduced to one-half the normal rate. We have compared the concentrations of amylase mRNA in the parotid, liver and pancreas of YBR mice with those in strains A/J and C3H. We observed differences in amylase mRNA abundance which can account for the levels of amylase protein synthesis in the parotid and pancreas of these strains. Unexpectedly, the concentration of amylase mRNA in the liver of YBR mice was also higher than in the other strains. Since liver amylase is transcribed from the same gene as parotid amylase, duplication of the Amy-1 locus could account for the elevated mRNA concentration in both tissues. Quantitative analysis of genomic DNA by Southern blotting provided direct evidence for duplication of Amy-1 in strain YBR.     

Quantitative changes in the lysosomal vacuolar system of rat hepatocytes during short-term starvation

Summary Ultrastructural morphometric analysis was used to study time-dependent variations in macro and microautophagy in rat hepatocytes. Except during periods of shortterm starvation for up to 24 h, animals were kept under standardized conditions of food intake.In hepatocytes of meal-fed rats the volume fraction of macroautophagic vacuoles is significantly higher at 23:00 h, i.e., immediately before food intake, compared to 11:00 h, i.e., 12 h following feeding. During fasting, macroautophagy drops to a low level.Microautophagic vacuoles in hepatocytes of meal-fed rats, sacrificed at 11:00 or 23:00 h respectively, do not show any significant quantitative differences. However, during 12 h of starvation, the volume fraction of microautophagic vacuoles rises significantly, whereas the numerical density remains constant. Subsequently, during the second 12-h period of fasting, the volume fraction of microautophagic vacuoles remains unchanged, but the numerical density increases. Over a period of 24 h of starvation the volume fraction of the total lysosomal system does not change significantly, whereas the numerical density rises.The time-dependent changes of the macroautophagic vacuolar system correlate with the circadian, food-related variations in the protein content of individual hepatocytes from meal-fed animals. The increase in volume fraction and thereafter in number of microautophagic vacuoles, as observed during starvation, coincides with a large decrease in protein content of individual hepatocytes.     

The use of unfixed cryostat sections for electron microscopic study of D-amino acid oxidase activity in rat liver.

Unfixed cryostat sections of rat liver were incubated to demonstrate D-amino acid oxidase activity at the ultrastructural level. Incubation was performed by mounting the sections on a semipermeable membrane which was stretched over a gelled incubation medium containing D-proline as substrate and cerium ions as capture reagent for hydrogen peroxide. After an incubation period of 30 min, ultrastructural morphology was retained to such an extent that the final reaction product could be localized in peroxisomes, whereas the crystalline core remained unstained. Control incubations were performed in the absence of substrate; the lack of final reaction product in peroxisomes indicated the specificity of the reaction. We conclude that the semipermeable membrane technique opens new perspectives for localization of enzyme activities at the ultrastructural level without prior tissue fixation, thus enabling localization of the activity of soluble and/or labile enzymes.