Differential opiate influences on food hoarding and intake in the deer mouse, Peromyscus maniculatus

The feeding behavior of the deer mouse, Peromyscus maniculatus, includes food hoarding as well as ingestion. In this animal the mu opiate agonist, morphine, and the kappa opiate agonist, U-50, 488H, selectively stimulate food hoarding and ingestion, respectively. This suggests that mu and kappa opiate systems may differentially mediate primary components of natural feeding behavior.     

The effect of alterations in haematocrit on tumour sensitivity to X-rays

Hypoxic cells in human tumours probably contribute to the failure of radiotherapy in some sites. Changes in the oxygen carrying capacity of the blood, such as in anaemia, have been shown to influence tumour response. The effect of acute and chronic changes in haematocrit on the radiosensitivity of three mouse tumours (EMT6, KHT and RIF-1) were studied. Alterations in haematocrit were achieved by bleeding followed by retransfusion. When radiation was preceded immediately by an acute reduction in haematocrit (anaemia), radiosensitivity was markedly reduced in each tumour. An acute rise in haematocrit (polycythaemia) increased or decreased X-ray sensitivity depending on its severity. The optimum haematocrit for maximum sensitivity was always found to be at a level 5-10 per cent above normal. When the time between induction of anaemia and irradiation was increased, simulating a progressively longer duration of anaemia, marked changes in radiosensitivity of all the tumours were observed. A short duration of anaemia resulted in a resistant tumour with each cell line, but the resistance was gradually lost as the anaemia was prolonged, even though no recovery in haematocrit occurred. The rate of recovery to normal radiosensitivity varied from 24 to 72 hours in the different tumours. Therefore, only haematocrit changes which occurred within 1-3 days of a dose of radiation affect the radiosensitivity of these tumours.     

Cellular location of heat-labile enterotoxin in Escherichia coli.

We demonstrated that both the A and B subunits of heat-labile enterotoxin from Escherichia coli are located in the periplasm. The toxin was shown to form aggregates in Tris-EDTA buffers which are routinely used for isolating membranes. The aggregates pellet upon centrifugation, and this may explain why several previous investigators have concluded that enterotoxin is associated with membranes.     

Triplet repeat expansion at the FRAXE locus and X-linked mild mental handicap.

We have recently shown that the expression of the FRAXE fragile site in Xq28 is associated with the expansion of a GCC trinucleotide repeat. In the families studied, FRAXE expression is also associated with mild mental handicap. Here we present data on families that previously had been diagnosed as having the fragile X syndrome but that later were found to be negative for trinucleotide repeat expansion at the FRAXA locus. In these families we demonstrate the presence of a GCC trinucleotide repeat expansion at the FRAXE locus. Studies of the FRAXE locus of normal individuals show that they have 6-25 copies of the repeat, whereas affected individuals have > 200 copies. As in the fragile X syndrome, the amplified CpG residues are methylated in affected males.     

L-Alanine absorption in human intestinal Caco-2 cells driven by the proton electrochemical gradient

In human Caco-2 intestinal epithelial layers, xxxl-alanine absorption can be energized by a proton gradient across the brush-border membrane. Acidification of the apical medium, even in Na⁺-free media, is associated with a saturable net transepithelial absorption of xxxl-alanine. xxxl-Alanine transport causes cytosolic acidification consistent with proton/amino acid symport. xxxl-Alanine transport in Na⁺-free media is rheogenic, stimulating an inward short-circuit current in voltageclamped epithelial monolayers. By measurement of rapid xxxl-alanine influx across the apical membrane, xxxl-alanine-stimulated inward short-circuit current and intracellular acidification in the same cell batch, we estimate xxxl-alanine/proton stoichiometry to be 10.62 ±0.25 (xxxsd) (short-circuit current) or 10.73 ±0.19 (intracellular acidification). From competition studies, it is likely that xxxl-proline, -aminoisobutyric acid, and -alanine, but not xxxl-valine and xxxl-serine, are substrates for protonlinked, substrate transport in the brush border of Caco-2 cells.This study was supported by the Wellcome Trust (to D.T.T. and N.L.S.) and the LINK Programme in Selective Drug Delivery and Targeting (funded by the SERC/MRC/DTI and Industry). Charlotte Ward gave excellent technical assistance.     

Epithelial M cells in the rabbit caecal lymphoid patch display distinctive surface characteristics

The follicle-associated epithelium (FAE) in the rabbit caecal lymphoid patch is characterised by the presence of membranous (M) cells, which are believed to be functionally equivalent to those present at other sites of gut-associated lymphoid tissue (GALT). Caecal patch M cells display distinctive features compared with those of other GALT sites, despite similar general morphology and expression of the M cell marker vimentin, suggesting marked heterogeneity in the apical surface of M cells at discrete GALT sites. Electron microscopy reveals that rabbit caecal patch M cells differ from those in the small intestinal Peyer's patch FAE: the former have a prominent aspect within the epithelium and possess microvilli which are longer than those of adjacent enterocytes. Many of the M cells in peripheral regions of the caecal patch FAE are not associated with leucocytes and may thus represent an immature M cell population. The M cells are also histochemically distinct from adjacent enterocytes and from Peyer's patch M cells, showing greater expression of brush-border alkaline phosphatase activity and affinity for certain lectins (peanut and wheat germ agglutinins, Bandeiraea simplicifolia agglutinin II). The differences in the brush-border morphology and glycocalyx structure between M cells at different GALT sites may affect their function at these sites by influencing the interaction of luminal antigens and microorganisms with the M cell surface. The present data also support the hypothesis that M cells arise directly from differentiation of crypt stem cells and not from the transformation of existing fully differentiated enterocytes.     

Structural studies of receptor binding by cholera toxin mutants.

The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33-->Arg complexed with the GM1 oligosaccharide, as well as the 2.2-A refinement of a Gly 33-->Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33-->Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GM1 oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33-->Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33-->Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33-->Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.     

Lectin binding defines and differentiates M-cells in mouse small intestine and caecum

M-cell surface glycoconjugate expression was investigated by applying a panel of lectins to whole fixed mouse Peyer's and caecal patches. While the majority of lectins failed to identify mouse M-cells, the lectinEuonymus europaeus differentially stained the surface of M-cells in both mouse Peyer's and caecal patches, and the lectinsUlex europaeus II andBandeiraea simplicifolia I isolectin B₄ identified M-cells in the Peyer's and caecal patch follicle associated epithelium, respectively. These three mouse M-cell markers failed to identify rat and rabbit Peyer's patch M-cells, although bothEuonymus europaeus andUlex europaeus II differentially stained M-cells in the periphery of rabbit caecal patch domes. These site and species related variations in M-cell surface glycoconjugate expression may reflect the local microorganism populations and will have important implications if orally delivered vaccines and drugs are to be targeted to M-cells via their surface glycoconjugates.     

A receptor-like binding macromolecule for 1 alpha, 25-dihydroxycholecalciferol in cultured mouse bone cells.

1alpha, 25-Dihydroxycholecalciferol (1,25-(OH)2D3), the active form of vitamin D, like other steroid hormones, initiates its action by binding to cytoplasmic receptors in target cells. Although the 1,25-(OH)2D3 receptor has been well studied in intestine, little information beyond sucrose gradient analyses is presently available from mammalian bone. We, therefore, employed primary cultures of mouse calvarial cells to characterize the mammalian receptor in bone. A hypertonic molybdate-containing buffer was found to protect receptor binding. On hypertonic sucrose gradients, the 1,25-(OH)2-[3H]D3 binder sedimented at 3.2 S. Scatchard analysis of specific 1,25-(OH)2[3H]D3 binding sites at 0 degrees C yielded an apparent Kd of 0.26 nM and an Nmax of 75 fmol/mg of cytosol protein. Competitive binding experiments revealed the receptor to prefer 1,25-(OH)2D3 greater than 25-(OH)-D3 = 1 alpha-(OH)-D3 greater than 24R,25-(OH)2D3; vitamin D3, dihydrotachysterol, sex steroids, and glucocorticoids exhibited negligible binding. As shown in other systems, the receptor could be distinguished from a 25-(OH)-[3H]D3 binder which sedimented at approximately 6 S. In summary, cultured mouse calvarial cells possess a macromolecule with receptor-like properties. This system appears to be an ideal model for the investigation of 1,25-(OH)2D3 receptor binding and action in mammalian bone.     

Minimal deletion of amino acids from the carboxyl terminus of the B subunit of heat-labile enterotoxin causes defects in its assembly and release from the cytoplasmic membrane of Escherichia coli

Minimal alterations at the carboxyl terminus of the B subunit (EtxB) of heat-labile enterotoxin from Escherichia coli were found to have a marked effect on the assembly and release of this polypeptide into the periplasm. Nine mutant EtxB polypeptides were obtained by genetic manipulation of the 3'-end of the etxB gene using Bal31 nuclease digestion and codon substitution. A correlation was observed between the magnitude of the changes introduced at the carboxyl terminus and the extent to which the mutant polypeptides were defective in assembly and release. Some of the mutant B subunits, exemplified by those in which the last 2 amino acids had been deleted or in which the last 4 residues had been replaced by three different ones, were found to be only partially defective, with a proportion being associated with the periplasmic face of the cytoplasmic membrane and the remainder being exported to the periplasm. The portion associated with membranes was detected as monomers on sodium dodecyl sulfate-polyacrylamide gels, whereas the portion exported to the periplasm were detected as assembled oligomers. We conclude that the last few amino acids at the carboxyl terminus of EtxB exert a profound influence on the assembly and release of the B subunit from the cytoplasmic membrane during export in E. coli.