Studies on the chemical nature of the lipidic J blood-group substance of cattle

Total lipids extracted from J-positive cattle serum, erythrocytes or spleen exhibit J blood-group activity. The J subsance is concentrated in a lipid fraction obtained by column chromatography. Following mild alkaline hydrolysis or reduction with complex hydrides (LiAlH₄, LiBH₄), the J activity remains detectable in this lipid fraction even though all acyl ester groups have been destroyed as revealed by ester group determination. This disagrees with the suggestion that fatty acyl esters are essential for J activity. This was confirmed by experiments with a water-soluble J-active product prepared by ozone treatment of glycosphingolipids from bovine spleen. The results of these experiments are in favour of a glycosphingolipid containing an unusually lang oligosaccharide chain. Furthermore, it appears that the terminal moiety of the J determinant is not necessarily an N-acetyl galactos-amine unit as suggested previously.     

Studies on the chemical nature of the lipidic J blood-group substance of cattle

Total lipids extracted from J-positive cattle serum, erythrocytes or spleen exhibit J blood-group activity. The J subsance is concentrated in a lipid fraction obtained by column chromatography. Following mild alkaline hydrolysis or reduction with complex hydrides (LiAlH4, LiBH4), the J activity remains detectable in this lipid fraction even though all acyl ester groups have been destroyed as revealed by ester group determination. This disagrees with the suggestion that fatty acyl esters are essential for J activity. This was confirmed by experiments with a water-soluble J-active product prepared by ozone treatment of glycosphingolipids from bovine spleen. The results of these experiments are in favour of a glycosphingolipid containing anunusually lang oligosaccharide chain. Furthermore, it appears that the terminal moiety of the J determinant is not necessarily an N-acetyl galactosamine unit as suggested previously.     

The inactivation of the bovine J blood group substance by the periodate ion

1. Treatment of J-positive (Jcs) bovine erythrocytes with periodate (0.25 mmol/l final concentration, 1 hour, room temperature) has no effect on the J activity. Higher periodate concentrations cause spontaneous haemolyses. 2. Treatment of the lipids extracted from (and containing all J activity of) Jcs erythrocytes with periodate leads to a decrease of J activity even with lower periodate concentrations. 3. Treatment of the stroma prepared from Jcs erythrocytes with periodate demonstrated the relative stability of the J antigen up to 0.25 mmol/l periodate. At the same time the sialic acid concentration of stroma is reduced to about 13% of the initial concentration. 4. Desialylation of Jcs erythrocytes or Jcs stroma with sialidase does not affect the J activity thus confirming previous findings. On the other hand, the J activity of desialylated Jcs stroma is much more susceptible to periodate. 5. It is concluded that membrane-bound sialic acid shields the membrane-bound J antigen from being attacked by periodate.     

The inactivation of the bovine J blood group substance by the periodate ion

• 1 Treatment of J-positive (JR) bovine erythrocytes with periodate (0.25 mmol/1 final concentration, 1 hour, room temperature) has no effect on the J activity. Higher periodate concentrations cause spontaneous haemolyses.
• 2 Treatment of the lipids extracted from (and containing all J activity of) J^cs erythrocytes with periodate leads to a decrease of J activity even with lower periodate concentrations.
• 3 Treatment of the stroma prepared from J^cs erythrocytes with periodate demonstrated the relative stability of the J antigen up to 0.25 mmol/l periodate. At the same time the sialic acid concentration of stroma is reduced to about 13 % of the initial concentration.
• 4 Desialylation of J^cs erythrocytes or J^cs stroma with sialidase does not affect the J activity thus confirming previous findings. On the other hand, the J activity of desialylated J^cs stroma is much more susceptible to periodate.
• 5 It is concluded that membrane-bound sialic acid shields the membrane-bound J antigen from being attacked by periodate.

     

Molecular cloning of the murine substance K and substance P receptor genes.

The peptides substance K and substance P evoke a variety of biological responses via distinct, guanosine-nucleotide-binding-regulatory-protein-coupled receptors. We have screened a murine genomic cosmid library using oligonucleotide probes and have isolated, cloned and characterized the substance K receptor and the substance P receptor genes. The coding portion of the substance K receptor gene consists of five exons distributed over 13 kbp. The substance P receptor gene is considerably larger than that of substance K (more than 30 kbp), however, the boundaries of the four exons that have been characterized in the substance P receptor gene correspond exactly to the homologous exons in the substance K receptor gene. To verify the identity of the isolated genes, we have cloned the corresponding cDNA by means of the polymerase chain reaction and we have expressed these cDNA species in Xenopus laevis oocytes. The ligand binding characteristics determined in this system pharmacologically confirm the identity of the two receptors. The deduced amino acid sequence of the mouse substance K receptor is 94% identical to the rat sequence and 85% identical to the bovine and human sequences. The mouse substance P receptor amino acid sequence is 99% identical to the rat sequence. The cloning of the murine substance K and substance P receptor genes should contribute substantially to the generation of in vivo models for the detailed analysis of the functional significance of these receptors.     

Properties of substance P aggregates. Application to the synthesis and purification of substance P

By 1H-NMR spectroscopy it has been shown that Substance P is largely aggregated at basic and acid pH and in saline solutions. These SP polymers dissociate rapidly by addition of pyridine and acetonitrile and slowly by addition of methanol. The difficulties previously encountered in the purification of SP and SP analogs may be attributed to this aggregation and can be overcome under disaggregating conditions. As a first application of our study we propose a reliable method for obtaining SP with good yield.     

The gynostemium of Bulbophyllum ecornutum (J. J. Smith) J. J. Smith (Orchidaceae)

RASMUSSEN, F. N., 1985. The gynostemium of Bulbophyllum ecornutum (J. J. Smith) J.J. Smith (Orchidaceae) . Stages in the development of the gynostemium of Bulbophyllum ecornutum demonstrate that the pollinium stalk is a hamulus in this and in a closely related species, B. gibbolabium . A hamulus arises by apical growth and reflexion of the median carpel. Hamuli have recently been discovered in several orchid genera, and a transverse fold of the rostellar apex is already known from a large group of orchids. The closely related B. cornutum has a quite different gynostemium structure.     

Photoaffinity labeling the substance P receptor using a derivative of substance P containing p-benzoylphenylalanine.

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)] in place of the Phe8 residue of SP. [Phe8(pBz)]SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the 125I-labeled Bolton-Hunter conjugate of [Phe8(pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites (Bmax = 0.2 pmol/mg of membrane protein) with an affinity KD = 0.4 nM. The binding of 125I-[Phe8(pBz)]SP was inhibited competitively by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70% of the specifically bound 125I-[Phe8(pBz)]SP underwent covalent linkage to two polypeptides of Mr = 53,000 and 46,000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on 125I-[Phe8(pBz)]SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. In addition, the labeling of the two polypeptides was equally sensitive to inhibition by guanyl-5'-yl imidodiphosphate, a nonhydrolyzable analogue of GTP. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies: the Mr = 46,000 polypeptide contains N-linked carbohydrates and is derived most likely from the higher molecular weight species by proteolytic nicking.(ABSTRACT TRUNCATED AT 250 WORDS)