An in situ study of beta-glucosidase activity in normal and gaucher fibroblasts with fluorogenic probes

Beta-glucosidase activity was evaluated in situ by means of fluorogenic probes in normal human fibroblasts and fibroblasts from homozygous carriers of the Gaucher trait. Probe internalization, targeting to lysosomes and post-cleavage probe retention were the primary concerns. Internalization and targeting were attempted by in situ photosensitized labilization of lysosomal membranes, lysosomotropic detergents and the use of low density lipid (LDL) or the receptor ligand apolipoprotein E (ApoE). Post-cleavage increase of fluorescence with fluoresceinyl (bis) betaglucopyranoside was appreciably above the rather large pre-cleavage emission. In cells incubated overnight with nonylumbelliferylbetaglucoside (UG9) in the presence of bovine serum albumin and in the absence of ApoE, the probe was dealt with as a cytotoxic agent, accumulating in a paranuclear cap, most likely comprising elements of the endoplasmic reticulum (ER) and Golgi apparatus. Targeting of UG9 to lysosomes occurred within 1 to 3 h of preincubation in the presence of ApoE. There was some evidence of specificity, as Gaucher fibroblasts exhibited weaker cleavage of UG9 (by 50 per cent or more) compared to normal fibroblasts, but in the Gaucher cells there was some residual beta-glucosidase activity. Cleavage of UG9 was nearly totally suppressed in Gaucher cells treated with the beta-glucosidase inhibitor, conduritol B epoxide, for 24 h to 7 days. Suppression in the control fibroblasts was evident but to a lesser degree. The in situ method of fluorogenic assay established for beta-glucosidase deficiency, is in principle applicable to enzyme deficiencies in other lysosomal storage diseases, or to evaluate enhanced enzyme activity following gene therapy.     

The molecular basis of potassium nutrition in plants

Over the last five years, the cloning and characterization of K⁺ transport genes corresponding to K⁺ channels (KAT1, AKT1, KST1, AKT2), associated subunits (KAB1) and a high-affinity transporter (HKT1) has opened up important new avenues for research on plant K⁺ nutrition. With the abundance of molecular data now available it seems timely to link this information with the wealth of data previously accumulated on the physiology of plant K⁺ acquisition. The ultimate goal of all this research is to gain a better understanding of K⁺ transport and nutrition in the intact plant. Thus it is important to begin to integrate the molecular research with results from biochemical and physiological research conducted at the cellular, root and whole plant levels. This article will focus on describing the features of the cloned K⁺ transporters and their possible roles in mediating high- and low-affinity K⁺ uptake from the soil, as well as how K⁺ acquisition may be regulated.Abbreviations NEM N-ethyl maleimide - PCMBS p-chloromercuribenzene sulphonic acid     

Bifurcation analysis of a two-compartment hippocampal pyramidal cell model

The Pinsky-Rinzel model is a non-smooth 2-compartmental CA3 pyramidal cell model that has been used widely within the field of neuroscience. Here we propose a modified (smooth) system that captures the qualitative behaviour of the original model, while allowing the use of available, numerical continuation methods to perform full-system bifurcation and fast-slow analysis. We study the bifurcation structure of the full system as a function of the applied current and the maximal calcium conductance. We identify the bifurcations that shape the transitions between resting, bursting and spiking behaviours, and which lead to the disappearance of bursting when the calcium conductance is reduced. Insights gained from this analysis, are then used to firstly illustrate how the irregular spiking activity found between bursting and stable spiking states, can be influenced by phase differences in the calcium and dendritic voltage, which lead to corresponding changes in the calcium-sensitive potassium current. Furthermore, we use fast-slow analysis to investigate the mechanisms of bursting and show that bursting in the model is dependent on the intermediately slow variable, calcium, while the other slow variable, the activation gate of the afterhyperpolarisation current, does not contribute to setting the intraburst dynamics but participates in setting the interburst interval. Finally, we discuss how some of the described bifurcations affect spiking behaviour, during sharp-wave ripples, in a larger network of Pinsky-Rinzel cells.     

X-ray absorption spectroscopy of selenium-containing amino acids

Received: 2 April 1999 / Accepted: 17 September 1999     

The development and ecology of the Japanese macaque gut microbiome from weaning to early adolescence in association with diet

Previously we have shown that the Japanese macaque gut microbiome differs not by obesity per se, but rather in association with high‐fat diet (HFD) feeding. This held true for both pregnant dams, as well as their 1‐year‐old offspring, even when weaned onto a control diet. Here we aimed to examine the stability of the gut microbiome over time and in response to maternal and postweaning HFD feeding from 6 months of age, and at 1 and 3 years of age. In both cross‐sectional and longitudinal specimens, we performed analysis of the V4 hypervariable region of the 16S rRNA gene on anus swabs collected from pregnant dams and their juveniles at age 6 months to 3 years (n = 55). Extracted microbial DNA was subjected to 16S‐amplicon‐based metagenomic sequencing on the Illumina MiSeq platform. We initially identified 272 unique bacterial genera, and multidimensional scaling revealed samples to cluster by age and diet exposures. Dirichlet multinomial mixture modeling of microbiota abundances enabled identification of two predominant enterotypes to which samples sorted, characterized primarily by Treponema abundance, or lack thereof. Approximating the time of initial weaning (6 months), the Japanese macaque offspring microbiome underwent a significant state type transition which stabilized from 1 to 3 years of age. However, we also found the low abundance Treponema enterotype to be strongly associated with HFD exposure, be it during gestation/lactation or in the postweaning interval. Examination of taxonomic co‐occurrences revealed samples within the low Treponema cluster were relatively permissive (allowing for increased interactions between microbiota) whereas samples within the high Treponema cluster were relatively exclusionary (suggesting decreased interactions amongst microbiota). Taken together, these findings suggest that Treponemes are keystone species in the developing gut microbiome of the gut, and susceptible to HFD feeding in their relative abundance.     

Fish prey of the Wandering Albatross Diomedea exulans at South Georgia

Summary The fish diet (45% of total diet by weight) of Wandering Albatrosses rearing chicks at South Georgia during the austral winters of 1983 and 1984 was investigated using otoliths retrieved from regurgitations. These provide the first quantitative data for this species and for any albatross. By number of identified otoliths (32% could be identified only as ?Macrouridae and ?Moridae), Pseudochaenichthys georgianus (35%), Muraenolepis microps (33%) and Chaenocephalus aceratus (20%) predominated, with Notothenia gibberifrons, Pagothenia hansoni and Champsocephalus gunnari (together 12%) also present. Composition by weight (estimated from otolith length) of the main species was Pseudochaenichthys 51%, Muraenolepis 14%, Chaenocephalus 27%; if digestion and wear had reduced otoliths by 10% the values would be Pseudochaenichthys 54%, Chaenocephalus 25%, Muraenolepis 13%. Composition by weight (actual or corrected values) was almost identical between years but epipelagic fish were significantly more abundant in 1983 than 1984. All identified fish eaten by Wandering Albatrosses are common on the South Georgia continental shelf and most of them are caught in the commercial fishery there. However, two of the three main target species of this fishery in 1983–1984, Notothenia rossii and Champsocephalus were not, or rarely, caught by Wandering Albatrosses. It seems unlikely, therefore, that the albatrosses depend greatly on the fishery for acquisition of fish prey but how they catch several species, including Muraenolepis, which are mainly benthic in habit is unknown.