Prion uptake in the gut: identification of the first uptake and replication sites

After oral exposure, prions are thought to enter Peyer's patches via M cells and accumulate first upon follicular dendritic cells (FDCs) before spreading to the nervous system. How prions are actually initially acquired from the gut lumen is not known. Using high-resolution immunofluorescence and cryo-immunogold electron microscopy, we report the trafficking of the prion protein (PrP) toward Peyer's patches of wild-type and PrP-deficient mice. PrP was transiently detectable at 1 day post feeding (dpf) within large multivesicular LAMP1-positive endosomes of enterocytes in the follicle-associated epithelium (FAE) and at much lower levels within M cells. Subsequently, PrP was detected on vesicles in the late endosomal compartments of macrophages in the subepithelial dome. At 7-21 dpf, increased PrP labelling was observed on the plasma membranes of FDCs in germinal centres of Peyer's patches from wild-type mice only, identifying FDCs as the first sites of PrP conversion and replication. Detection of PrP on extracellular vesicles displaying FAE enterocyte-derived A33 protein implied transport towards FDCs in association with FAE-derived vesicles. By 21 dpf, PrP was observed on the plasma membranes of neurons within neighbouring myenteric plexi. Together, these data identify a novel potential M cell-independent mechanism for prion transport, mediated by FAE enterocytes, which acts to initiate conversion and replication upon FDCs and subsequent infection of enteric nerves.     

Understanding the cellular uptake of phosphopeptides

Phosphopeptide-cellular uptake has been studied with a unique combination of tools designed to quantitate this phenomena and to understand properties that contribute to transmembrane penetration. High-affinity src-homology domain (SH2) hexapeptides for the phosphatidyl inositol 3-kinase system were used to judge cell penetration using red blood cells—a model system for the study of transmembrane cellular uptake. Hexapeptides without phosphate groups and devoid of charged residues poorly entered cells. N-terminal modification with bulky hydrophobic groups enhanced partitioning into octanol, an index of hydrophobicity, and allowed certain non-phosphorylated peptides to pass into red cells. However, tyrosine phosphorylation of hexapeptides markedly decreased octanol-water partitioning and completely eliminated cellular uptake. Inclusion of ion-pairing agents that masked the phosphate hydrophilic character enabled partitioning of phosphopeptides into octanol and achieved cellular uptake. This effect was demonstrated using fluorescent derivatives of phosphopeptides and CV1 cells in culture. The results validate the concept of facilitating cell entry by charge masking and open the way to future refinements of this principle. Various penetration techniques are compared and discussed in the context of maximizing cellular viability.     

Studying the uptake of cell-penetrating peptides

More than a decade ago, it was discovered that cationic peptides could traverse the cellular plasma membrane without specific transporter proteins or membrane damage. Subsequently, it was found that these peptides, known as cell-penetrating peptides (CPPs), were also capable of delivering cargos into cells, hence the great potential of these vectors was acknowledged. Today, many different research groups are working with CPPs, which necessitates efforts to develop unified assays enabling the comparison of data. Here we contribute three protocols for evaluation of CPPs which, if used in conjunction, provide complementary data about the amount and mechanism of uptake (fluorometric analysis and confocal microscopy, respectively), as well as the extent of degradation (HPLC analysis of cell lysates). All three protocols are based on the use of fluorescently labeled peptides and can be performed on the same workday.     

Inhibitors of the GABA uptake systems

Summary This review describes a novel class of heterocyclic GABA uptake inhibitor with no affinity for the GABA receptors. The parent compound nipecotic acid is a potent inhibitor of neuronal and glial GABA uptake, and nipecotic acid is a substrate for the transport carriers concerned. The structurally related cyclic amino acids guvacine and cis-4-hydroxynipecotic acid are also potent inhibitors of both GABA transport systems. Even minor structural alterations of these compounds result in considerable or complete loss of activity. Whereas homonipecotic acid is a weak but selective inhibitor of glial GABA uptake, homoguvacine is virtually inactive. Similarly the lower homologues of nipecotic acid and guvacine, -proline and 3-pyrroline-3-carboxylic acid, respectively, show some selectivity with respect to inhibition of glial GABA uptake, but these compounds are much weaker than the parent compounds. The bicyclic compounds THPO and THAO, in which the carboxyl groups of nipecotic acid and homonipecotic acid have been replaced by 3-isoxazolol units are moderately potent and practically specific inhibitors of glial GABA uptake. cis-4-Mercaptonipecotic acid is considerably weaker than the closely related analogue cis-4-hydroxynipecotic acid, but the former compound may interact irreversibly with the GABA transport carriers.The results demonstrate a pronounced substrate specificity of the glial and in particular the neuronal GABA transport system. It is evident that the GABA molecule is transported in a conformation different from that, in which it activates its receptors. These findings are of importance for the development of drugs for selective pharmacological regulation of the functions of central GABA-mediated synapses in certain neurological diseases.     

Topographic distribution of dopamine uptake,choline uptake,choline acetyltransferase,and GABA uptake in the striata of weaver mutant mice

The topographic distribution of dopamine (DA) uptake, choline uptake, choline acetyltransferase (ChAT) activity and GABA uptake within the striata of weaver mutant mice and control mice was determined. Uptake of [³H]dopamine, [³H]choline and [¹⁴C]GABA, as well as ChAT activity were determined in samples prepared from the dorsolateral, dorsomedial, ventrolateral and ventromedial portions of the striatum. In 45–60 day old control mice, dopamine uptake was homogeneously distributed throughout the striatum. On the other hand, striata from weaver mice exhibited an uneven distribution with the ventral aspects having greater uptake activity than the dorsal regions. Thus, although the ventral portion of the striatum is less severely affected than the dorsal portion, all areas of the striatum exhibited significantly reduced uptake rates. In 9 and 12 month old mice, choline uptake was higher in lateral than medial zones of the striatum of both genotypes and no differences were observed between genotypes. GABA uptake was higher in the ventral striatum than in the dorsal striatum but again no differences were found between weaver and control mice. The results of this study indicate that the entire weaver striatum is severely deficient in its ability to recapture dopamine and thus is functionally compromised. The results also indicate that the striatal cholinergic and GABAergic interneurons are not directly or indirectly affected by the weaver gene.Special ïssue dedicated to Dr. Morris H. Aprison     

Concerning the uptake of lipid by the nucleus

Summary Our previous radioautographic experiments suggesting absorption of lipid by the nucleus have been followed by direct examination of liver cell nuclei isolated from the animals at various times after administration of palmitic acid-9,10-³H. Twenty four hours post administration, the radioactivity in the isolated nuclei was still present in lipid. At this and all subsequent times examined, the total radioactivity in the nuclei did not exceed 1 percent of the total present in the liver. Treatment of the nuclei with Triton X100, to remove the nuclear membranes, led to the loss of most of the radioactivity; that remaining was associated primarily with the chromatin, with very little being found in the nucleolar fraction.Work supported by the U. S. Atomic Energy Commission.This work was started before the death of Dr. Wassermann on June 16, 1969; this paper is respectfully dedicated to his memory.     

On the mechanism of cellular cadmium uptake

Review of the available evidence on the mechanism of cellular Cd uptake in the rat jejunum supports the concept that this process consists of nonspecific binding to anionic sites on the membrane, followed by a temperature-dependent and rate-limiting internalization step. Because temperature-sensitive transmembrane movement of Cd can be demonstrated also in isolated brush-border vesicles and in erythrocyte ghosts, it is not likely to result from pinocytosis but may be related directly to membrane fluidity. There is no need to assume the existence of saturable Cd carriers, or competition of Cd with essential polyvalent cations for their specific transport systems. Uptake of Cd by tubular epithelium in the kidney of the intact rabbit appears to resemble that described for the jejunum, with the internalization step limiting the rate of uptake.     

Autoradiography of nucleoside uptake into the retina

A selective uptake mechanism for some nucleosides and related substances was found in retinae of light adapted rabbits and fish. After the intravitreal injection in vivo of [³H]adenosine, [³H]inosine, [³H]guanosine and certain related compounds, the distribution of radioactivity was studied by autoradiography. Retinae were also incubated in [³H]adenosine and [³H]inosine and then were similarly processed.In rabbits, the accumulation of radioactivity from [³H]adenosine and [³H]guanosine was predominantly into glial cells, but also into neurons. [³H]Inosine labelled glia almost exclusively. However, the adenosine analog, [³H]methylphenylethyl-adenosine, resulted in well-defined neuronal labelling in this species. In fish, a few photoreceptor cell bodies exhibited strong radioactivity with the nucleosides, presumably representing incorporation into nucleic acids of replicating cells. Labelling was also seen in horizontal cells, amacrine cells and ganglion cells after the injection of either [³H]adenosine, [³H]guanosine or [³H]inosine.To some extent, the selective accumulation of radioactivity is likely to be due to cell replication, but in most neurons, other factors must be responsible. Judging from what is known about the actions of adenosine in central nervous tissue, signal transmission in the retina could be such a factor.     

The water-vapour uptake system of the phthiraptera

The occurrence of active water-vapour uptake was examined in 16 Phthirapteran species representing the two groups of biting lice or Mallophaga (Amblycera and Ischnocera) and the sucking lice or Anoplura. Water-vapour uptake could not be demonstrated in the Anoplura and although it is frequently encountered in the biting lice, some members of both the Ischnocera and Amblycera lack the faculty. Uptake performance in the absorbing species of biting lice is exceptionally efficient showing higher rates and lower thresholds than other absorbing arthropods. The critical equilibrium humidities were found to range between 43 and 52% r.h., 43% being the lowest value so far reported. The uptake rates, expressed as a percentage of total body water mass, by far surpass those of all other arthropods investigated. The Phthirapteran uptake pattern closely resembles that of the Psocoptera, showing abrupt initiation and termination of the uptake process, limitation of continuous absorption to relatively short periods of time and constancy of the absorption rate over the entire period of continuous uptake. Studies on the uptake site and mechanism in Phthiraptera revealed an oral-uptake system which is congruous with that of the Psocoptera. The structural components of the uptake device, like a pair of oval lingual sclerites, representing the site of water-vapour condensation and a cibarial sucking pump which is connected by a paired or branching selerotized tubule to the lingual selerites and is responsible for the transfer of the condensed water to the gut, are unique acquisitions of the Psocodea.In those members of the biting lice which have lost the faculty of water-vapour uptake the structural components of the uptake system are modified or reduced.     

Pitfalls of the 45Ca uptake method

The interpretation of the data derived from ⁴⁵Ca uptake measurements by cells and tissues can be difficult. Several of these difficulties and possible misinterpretations are described: 1) ⁴⁵Ca uptake is not equivalent to calcium influx; 2) interpretations based on the sole visual examination of ⁴⁵Ca uptake curves can be misleading because an increased tracer uptake can coexist with a decreased calcium transport and vice-versa; 3) drugs and hormones can have diametrically opposite effects when they are tested at steady state on in nonsteady state conditions. It is concluded that ⁴⁵Ca uptake curves must be kinetically analyzed and that the steady or non-steady state of the system must be known for a valid interpretation of such data.     

Anniversary uptake

DNA at 50: Media editors have been thinking hard about the best ways to cover the DNA landmark and subsequent developments. Richard Harris looks at how the story came out.     

Nitrogen uptake

Summary Nitrogen uptake from applied nutrient solutions was evaluated in two old fields, in a pine plantation, and in a hardwood stand, to test the idea that plant communities become more efficient trappers and retainers of plant nutrients during succession. Uptake was estimated as the difference between nutrient concentrations in water collected from beneath soil profiles with and without roots by lysimeters within each successional stage. Results suggest that nitrate uptake decreased while ammonia uptake increased with succession. This apparent shift from a nitrate to an ammonia nitrogen economy during succession has been reported by other workers and is evolutionarily significant as an energy, nitrogen, and cation saving mechanism.     

Synaptosomal uptake

Using purified synaptosomal preparations from rat brain, the uptake ofl-tryptophan and norepinephrine was studied. We were unable to replicate some of the results of the experiments obtained with crude mitochondrial, fractions (P₂). Thus we examined the validity of the results of uptake studies obtained with the crude synaptosomes and established conditions which would simulate the biochemical milieu in which the nerve terminals functionin vivo, such as active substrate-dependent respiration, respiratory coupling on addition of ADP, low impurity of noncharacteristic markers, exogenous added proteins (e.g. bovine serum albumin), and verification by electron microscopy. All uptake studies withl-TRP and NE were completed in a system designed for simultaneous recording of respiration and the effect of added ADP. This system was also employed in comparative studies with mitochondria purified by multiple density gradients derived either from the perikaryon or from synaptosomes. Synaptosomal or mitochondrial preparations which did not conform to the above criteria invariably showed significantly lowered ability of uptake ofl-TRP or NE. This was found to be related to impairment in their respiratory and coupling ability. When the experimental conditions of others were employed, the time course of uptake of TRP for crude synaptosomes (P₂) was 100 nmol/g/min and was linear for 2.5 min, while for the purified synaptosomes it was 20 nmol/g/min with a l-min linearity. The mitochondria purified from P₂ displayed 30 nmol/g/min uptake withl-TRP with a linearity of 2.5 min. Reconstituted system of purified synaptosomes and mitochondria gave 60 nmol/g/min ofl-TRP transport with 2.5 min linearity. Also examined was the effect of eight different media. It was found that Krebs-Ringer solution containing glucose (40 mM), pyruvate and malate (10 mM), and ADP (250 nmol) gave optimal uptake of TRP both for synaptosomes and for mitochondria, increasing it to 60 and 86 nmol/g/min. The above conditions also enhanced the uptake of NE by synaptosomes and mitochondria. Uptake of NE was not proportional to protein concentration when the protein content exceeded 0.4 mg. Purified synaptosomal mitochondria accumulated NE more actively than the purified nonsynaptic free mitochondria, albeit at the same rate. Synaptic and free mitochondria had an impaired uptake of NE in presence of DNP, antimycin A, and rotenone, and unlike withl-TRP, pyruvate and malate also reduced uptake of NE. Significant differences were noted for the cytochrome oxidase activity between the synaptosomal and free michondria when compared to that of the homogenate.     

Kinetics of oxygen uptake at the onset of exercise near or above peak oxygen uptake.

We tested the hypothesis that kinetics of O(2) uptake (VO(2)) measured in the transition to exercise near or above peak VO(2) (VO(2 peak)) would be slower than those for subventilatory threshold exercise. Eight healthy young men exercised at approximately 57, approximately 96, and approximately 125% VO(2 peak). Data were fit by a two- or three-component exponential model and with a semilogarithmic transformation that tested the difference between required VO(2) and measured VO(2). With the exponential model, phase 2 kinetics appeared to be faster at 125% VO(2 peak) [time constant (tau(2)) = 16.3 +/- 8.8 (SE) s] than at 57% VO(2 peak) (tau(2) = 29. 4 +/- 4.0 s) but were not different from that at 96% VO(2 peak) exercise (tau(2) = 22.1 +/- 2.1 s). VO(2) at the completion of phase 2 was 77 and 80% VO(2 peak) in tests predicted to require 96 and 125% VO(2 peak). When VO(2) kinetics were calculated with the semilogarithmic model, the estimated tau(2) at 96% VO(2 peak) (49.7 +/- 5.1 s) and 125% VO(2 peak) (40.2 +/- 5.1 s) were slower than with the exponential model. These results are consistent with our hypothesis and with a model in which the cardiovascular system is compromised during very heavy exercise.