Characterization of porcine intestinal receptors for the K88ac fimbrial adhesin of Escherichia coli as mucin-type sialoglycoproteins.

We have previously identified two K88ac adhesion receptors (210 and 240 kDa) which are present in membrane preparations from adhesive but not nonadhesive porcine intestinal brush border cells; these adhesin receptors are postulated to be important determinants of the susceptibility of pigs to K88ac+ enterotoxigenic Escherichia coli infections (A.K. Erickson, J.A. Willgohs, S.Y. McFarland, D.A. Benfield, and D.F. Francis, Infect. Immun. 60:983-988, 1992). We now describe a procedure for the purification of these two receptors. Receptors were solubilized from adhesive intestinal brush border vesicles using deoxycholate and were purified by gel filtration chromatography on Sepharose CL-4B and then by hydroxyapatite chromatography. Amino acid compositional analyses indicated that the two receptors have similar amino acid compositions. The most distinguishing characteristic of both receptors is a high percentage of threonine and proline residues. Neuraminidase treatment caused the K88ac adhesin receptors to migrate with a slower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, indicating that these receptors are sialoglycoproteins. Results from lectin-binding studies indicated that the receptors contain O-linked oligosaccharides composed of galactosyl (beta-1,3)N-acetylgalactosamine, alpha-linked fucose, galactosyl(beta-1,4)N-acetylglucosamine, sialic acid, galactose, and N-acetylgalactosamine. Collectively, these characteristics indicate that the K88ac adhesin receptors are mucin-type sialoglycoproteins.     

Sodium-coupled neutral amino acid (System N/A) transporters of the SLC38 gene family

The sodium-coupled neutral amino acid transporters (SNAT) of the SLC38 gene family resemble the classically-described System A and System N transport activities in terms of their functional properties and patterns of regulation. Transport of small, aliphatic amino acids by System A subtypes (SNAT1, SNAT2, and SNAT4) is rheogenic and pH sensitive. The System N subtypes SNAT3 and SNAT5 also countertransport H(+), which may be key to their operation in reverse, and have narrower substrate profiles than do the System A subtypes. Glutamine emerges as a favored substrate throughout the family, except for SNAT4. The SLC38 transporters undoubtedly play many physiological roles including the transfer of glutamine from astrocyte to neuron in the CNS, ammonia detoxification and gluconeogenesis in the liver, and the renal response to acidosis. Probing their regulation has revealed additional roles, and recent work has considered SLC38 transporters as therapeutic targets in neoplasia.     

Concept of neuron types in gustation in the rat.

1. In taste neurophysiology, from Pfaffmann's (49, 50) pioneering work until the present, the possibility of types of neurons corresponding in some sense with the "primary" taste qualities of Henning (33) has been entertained: recently types of gustatory neurons in peripheral nerves have been established according to which of the four classical stimuli is the "best stimulus." However, considerable variation occurs in the response profiles within neurons classified as belonging to the same type. The purpose of this research is to determine, using mathematical techniques where appropriate, if the within-type variation is spurious or, instead, indicates the absence of a typology of taste neurons. The data used were counts of the spike discharges of 50 individual taste neurons in the nucleus of the solitary tract of the rat, evoked by 32 diverse chemical stimuli. 2. Using as input the matrix of Pearson r correlation coefficients calculated for the responses of all pairings of neurons to all stimuli, multidimensional scaling analysis revealed a two-dimensional space in which no clear groupings of neurons occurred. 3. In a hierarchical cluster analysis of the neuron response profile similarities, no evidence of grouping was found, suggesting a more-or-less continuous variation among neurons. 4. When the organization of the 32 stimuli utilized was studied by the same techniques, no clear evidence for stimulus types was found, although the possibility of two stimulus types--"sweet" and "nonsweet"--was raised. 5. Construction of a joint neuron-stimulus space supported a spatial model of taste neuron-stimulus interaction, while analysis of the number and pattern of high correlations among neurons--even after allowance for attenuation due to measurement error--failed to support the notion of types of taste neurons with identical response profiles. 6. Aspects of the logical role of types of neurons in gustatory coding were discussed, and the results and methods of the present investigation were related to classification schemes for neurons in general. Suggestions for a formal taxonomy of neurons were given. 7. It should be emphasized that the present study and conclusions are of second-order, CNS neurons, whereas the studies advocating the presence of neurons types were of peripheral neurons. Taken together, the implication to be drawn from these studies is that if neural types do exist in peripheral taste nerves, the typology is lost at the first synapse and is thus unavailable to the CNS for coding purposes, at least in the rat.     

Female pseudohermaphroditism with multiple caudal anomalies: Absence of Y-specific DNA sequences as pathogenetic factors

46,XX female pseudohermaphrodites have been previously described with nearly complete masculinization of the external genitalia and no apparent source of testosterone. Multiple malformations of internal genital, urinary, and gastrointestinal tracts are associated. We have evaluated four such infants with female pseudohermaphroditism and multiple caudal anomalies. Three cases had apparently normal chromosomes (46,XX); one had a 46,XX,del(10)(q25.3→qter) chromosome constitution. The chromosome breakpoint is in the region of PAX2, a developmentally important paired box gene which is expressed in urogenital tissue. Using the polymerase chain reaction, we screened for the presence of multiple Y specific sequences, including SRY (sex determining region, Y chromosome), that could explain masculinization of the external genitalia. All were negative for Y centromeric sequences, ZFY(Zinc finger Y), and SRY. Furthermore, there was no evidence for adrenal or other sources of testosterone. We suggest that the masculinization in these cases is the result of abnormal expression of genes which would normally be regulated by testosterone. © 1994 Wiley-Liss, Inc.     

The self-assembly of papaya mosaic virus.

The conditions for the in vitro reconstitution of papaya mosaic virus (PMV) from its isolated constituents are described and are related to the formation of coat protein subassembly products. PMV assembles best in 0.01 M, pH 8.0, Tris buffer at 25° at a protein to RNA ratio of 20:1 (w/w). At lower pH levels, faulty, segmented particles are formed which are sensitive to ribonuclease. The assembly process at pH 8.0 is composed of two energetically distinct phases corresponding to helix initiation and elongation. Both reactions may be stopped by low levels of NaCl, whereas only elongation requires elevated temperatures. If the growth of elongating particles is stopped by lowering the temperature, long and thin “extended particles” are detected; if NaCl is used, the arrested particles terminate with a “brush” at one end only. Under virus assembly conditions, the coat protein exists in an equilibrium among several polymeric species, most notably 14 S and 25 S polymers. The latter is not required for reconstitution. The equilibrium mixture is very sensitive to changes in pH, ionic strength, temperature, and protein concentration.     

Cell interactions in the initial contact between cultured melanoma cells and syngeneic macrophages.

Thioglycolate-induces peritoneal macrophages from melanoma-bearing mice (immune macrophages) or from control mice (control macrophages) were cultured with syngeneic melanoma cells (P51) to determine the surface characteristics of the effector cells during interaction and destruction of the target cells. After a short culture period (3 hours), immune macrophages had extensive connections via filopodia and ruffled membranes to the surfaces of the melanoma cells; control macrophages did not exhibit the same behavior. A dense region in the cytoplasm immediately beneath the macrophage plasmalemma was observed at the point of contact with the target tumor cell. With longer periods of culture (24 hours), effector cells began phagocytosis of the target cells; immune macrophages, however, had more fine filopodial connections and were more cytostatic than were controls. These observations indicate that one of the initial mechanisms of tumor cell destruction was contact-induced lysis, with phagocytosis playing a minor part.     

Morphogenesis of the fetal membranes of the white-tailed deer

Morphogenesis of the fetal membranes of the white-tailed deer was studied throughout pregnancy. In placentomes, the long, branched, fetal villi occupied corresponding maternal crypts. The bases of the villi and the arching areas connecting them (arcades) were covered with high columnar cytotrophoblast, which apparently had phagocytosed material from the adjacent degenerating rims of the crypts. This arcade cytotrophoblast contained much glycogen and occasional mitochondria, free ribosomes, and pigment granules. Elsewhere, the columnar cytotrophoblast cells usually contained three to five rows of rod-shaped mitochondria and other cytoplasmic organelles. Interspersed among them were numerous binucleate giant cells which usually contained peripheral lace-like granular endoplasmic reticulum and many complex lipoprotein droplets. Cryptal epithelium, deep to the degenerate rims, was low columnar, had infranuclear osmiophilic lipid droplets, and sparsely distributed cytoplasmic organelles. The microvilli of cryptal and cytotrophoblastic epithelia interdigitated and appeared as a brush border under light microscopy. The microvilli and their PAS-positive mucopolysaccharide material appeared capable of holding uterine and chorionic epithelia together during pregnancy. The placentomes were epitheliochorial, but showed “intra-epithelial” capillaries. Interplacenomal uterine and cytotrophoblasitc epithelia resembled those of the placentomes. Their microvill were inundated by endometrial gland secretion (uterine milk). The cytotrophoblast contained pigment granules and much absorbed uterine milk. The amniotic epithelium showed short microvilli, complexly folded lateral plasma membranes, many desmosomes, abundant glycogen granules, foot processes and other cytoplasmic organelles.