Abnormal subcellular distribution of β-glucuronidase in mice with a genetic alteration in enzyme structure

Liver -glucuronidase is structurally altered in inbred strain PAC so that a peptide subunit with a more basic isoelectric point, GUS-SN, is produced. This allele of -glucuronidase was transferred to strain C57BL/6J by 12 backcross matings to form the congenic line B6 · PAC-Gus ⁿ. Liver -glucuronidase activity was halved in males of the congenic strain compared to normal males. The lowered activity was specifically accounted for by a decrease in the lysosomal component. There was no alteration in the concentration of microsomal activity. This alteration in the subcellular distribution of -glucuronidase in Gus ⁿ/Gus ⁿ mice was confirmed by two independent gel electrophoretic systems which separate microsomal and lysosomal components. -Glucuronidase activity was likewise approximately halved in mutant spleen, lung, and brain, organs which contain exclusively or predominantly lysosomal -glucuronidase. The loss of liver lysosomal -glucuronidase activity was shown by immunotitration to be due to a decrease in the number of -glucuronidase molecules in lysosomes of the congenic strain. The Gus ⁿ structural alteration likely causes the lowered lysosomal -glucuronidase activity since the two traits remain in congenic animals. Heterozygous Gus ⁿ/Gus ^b animals had intermediate levels of liver -glucuronidase. Also, the effect was specific, in that three other lysosomal enzymes were not reproducibly lower in Gus ⁿ/Gus ⁿ mice. Gus ⁿ is, therefore, an unusual example of a mutation which causes a change in the subcellular distribution of a two-site enzyme.This work was supported by National Institutes of Health Grants GM-33559 and GM-33160 and National Science Foundation Grant PCM-8215808.     

Occurrence of bacterivory in Cryptomonas,a common freshwater phytoplankter

Summary Bacterivory was detected by incorporation of 0.57 m diameter, fluorescent polystyrene beads and fluorescently labeled bacteria (FLB) in two cultured species of Cryptomonas (C. ovata and C. erosa), and a population of Cryptomonas sp in a humic, mesotrophic lake. Rates of ingestion and clearance were very low, and similar for the cultures and the in situ population. The in situ population incorporated 0.7–1.7 bacteria cell^-1 h^-1, thereby ingesting 0.3%–2.0% of the total bacterial numbers present in the water per day, and receiving less than 2% of its carbon content per day through bacterivory. Thus, bacterivory by Cryptomonas was quantitatively important neither as a sink for bacterial biomass, nor as a carbon source for the algal cells. Possibly, it served in the uptake of essential nutrients.     

Evaluation of Diverse Antisera, Conjugates, and Support Media for Detecting Bradyrhizobium japonicum by Indirect Enzyme-Linked Immunosorbent Assay

We evaluated three antisera and four enzyme conjugates for the detection of Bradyrhizobium japonicum by an indirect enzyme-linked immunosorbent assay in microtiter plates. Nitrocellulose membrane sheets were then evaluated as an alternative support medium by using some combinations. Partially purified immunoglobulin G (IgG) or unpurified antisera to strain USDA 110 raised in rabbits, goats, or sheep was reacted in microtiter plates with alkaline phosphatase conjugated to protein A, goat anti-rabbit (GAR), sheep anti-rabbit (SAR), or rabbit anti-goat (RAG) IgG. Cultures or nodules containing homologous rhizobia were detected with equal sensitivity when protein A, GAR, or SAR was reacted with 5 μg of protein IgG per ml or a 1:800 titer of antisera from rabbits, but not goats or sheep. RAG reacted with IgG or antisera from goats or sheep. The detection limit was 2 × 10⁵ rhizobia per well. Rhizobia were spotted on nitrocellulose sheets as an alternative support medium, followed by soaking in 5 μg of protein per ml as IgG and 1:4,000 dilutions of protein A or GAR conjugate. Rhizobia in serogroup 110 were detected with the dye combination Nitro Blue Tetrazolium-5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP), and rhizobia in serogroup 122 were detected with fast red-naphthol phosphate (FR-NP). At the conclusion of the 5-h assay, purple (NBT-BCIP) or red (FR-NP) spots were visible in positive reactions. The sensitivity of detection was about 1,000 rhizobial cells or 3 μg of nodules tissue.     

Polyploid cells in blastocysts and early fetuses from Australian Merino sheep

Cytogenetic examination was made of 103 13-14-day-old blastocysts and 116 24-32-day-old fetuses from untreated and androstenedione-7-HSA-immunized Merino ewes. There were no differences in the chromosome composition of blastocysts or fetuses from treated or untreated ewes and so the data were combined. At Days 13-14 a 1N/2N mosaic and a 2N - 1/2N/4N mosaic embryo were observed. In addition, 52 of the blastocysts were 2N/4N mosaics, with 8 of these also containing 8N cells, and one blastocyst was a 2N/8N mosaic. No aneuploid fetuses were observed, but 80 of the 116 fetuses contained polyploid cells, including 4N, 6N and 8N cells. The polyploid cells observed in the blastocysts and fetuses should not be considered as abnormal cells as they appear to be a normal part of the developmental processes leading to trophoblast formation and fetal differentiation.     

Behavioral and biochemical assessment of time-related changes in globus pallidus and striatal dopamine induced by intranigrally administered neurotensin

Microinjection of neurotensin (NT; 2 and 5 μg) into the substantia nigra zona compacta caused an increase in dopamine (DA) and DA metabolites in the rodent globus pallidus and striatum which persisted for at least 20 hours after peptide administration. Similar NT treatments given unilaterally into the nigra caused circling away from the injected side in amphetamine-pretreated rats, but were without effect when microinjected into saline-pretreated animals. Circling also occurred when the animals were given amphetamine 20 hours after intranigral NT administration. Contralateral rotation was observed with unilateral intranigral injections of gamma-hydroxybutyric acid (GHB; 400 μg) or with lower intranigral GHB doses (250 μg) in amphetamine-pretreated animals. The effects of GHB and NT differed in the manner in which the animals rotated as well as in the profile of DA and DA metabolite changes induced by these drugs. These studies indicated that: (1) dopaminergic functions of the globus pallidus are influenced, like the striatum, by manipulations of the substantia nigra; (2) NT and GHB likely act via different mechanisms to effect nigral dopamine-containing cells; and (3) NT was capable of inducing changes in dopamine neurons which had long term consequences.     

Comparison of α1-Adrenergic Receptor-Stimulated Inositol Phosphate Formation in Primary Neuronal and Glial Cultures

alpha 1-Adrenergic receptor binding sites and norepinephrine-stimulated 3H-inositol phosphate (3H-InsP) accumulation were measured in primary cultures of neurons and glia from 1-day-old rat brains. The density of alpha 1-adrenergic receptor binding sites was approximately three times higher in membranes from neurons compared to glia. Although norepinephrine was slightly more potent in stimulating 3H-InsP formation in neurons than in glia, the maximal response was greater in glial cells. Norepinephrine-stimulated 3H-InsP formation remained constant for [3H]inositol prelabelling periods of 1-14 days in neurons, whereas the response increased with time in glia and was maximal after 7-10 days of prelabelling. Both the incorporation of [3H]inositol into lipid and basal levels of 3H-InsPs were lower in glial cells than in neurons, which accounted for the greater percent stimulation in glia. Pretreatment with phenoxybenzamine decreased norepinephrine-stimulated 3H-InsP formation in a dose-dependent manner in both neurons and glia by decreasing the maximal response without altering potency. HPLC separation showed that similar types of 3H-InsPs were accumulated in neurons and glial cells. These results demonstrate that alpha 1-adrenergic receptors exist on both neurons and glial cells and activate 3H-InsP accumulation in both cell types. Although receptor density is higher in neurons than in glia, the 3H-InsP response is higher in glia. This difference does not appear to be due to different receptor reserves, but may be due to differential coupling mechanisms in the two cell types.