Secondary structure and orientation of the surfactant protein SP-B in a lipid environment. A Fourier transform infrared spectroscopy study.

Attenuated total reflection Fourier transform infrared spectroscopy was used to investigate the secondary structure of the surfactant protein SP-B. Nearly half of the polypeptide chain is folded in an alpha-helical conformation. No significant change of the secondary structure content was observed when the protein is associated to a lipid bilayer of dipalmitoylphosphatidylcholine (DPPC)/phosphatidylglycerol (PG) or of dipalmitoylphosphatidylglycerol (DPPG). The parameters related to the gamma w(CH2) vibration of the saturated acyl chains reveal no modification of the conformation or orientation of the lipids in the presence of SP-B. A model of orientation of the protein at the lipid/water interface is proposed. In this model, electrostatic interactions between charged residues of SP-B and polar headgroups of PG, and the presence of small hydrophobic alpha-helical peptide stretches slightly inside the bilayers, would maintain SP-B at the membrane surface.     

Humoral anti-idiotypic and anti-anti-idiotypic immune response in cancer patients treated with monoclonal antibody 17-1A

 A group of 96 patients with advanced colorectal carcinoma were treated with the mouse (m) or chimeric (c) (mouse variable regions × human IgG1 constant regions) monoclonal antibody (mAb) 17-1A recognizing the tumour-associated antigen GA733-2. Eighty-two of the 83 patients treated with mmAb17-1A and 69% of the patients given cmAb17-1A (n = 13) developed anti-idiotypic antibodies (ab₂). Auto-antibodies binding to tumour cells expressing GA733-2 were found in 7% of the patients. In a further 38 patients (40%) antitumour-cell antibodies, i.e. anti-anti-idiotypic antibodies (ab₃), were induced by the mAb17-1A therapy. Patients with detectable ab₃ after treatment had significantly higher ab₂ levels than those not developing ab₃. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of ab₂ as well as induction of anti-anti-idiotypic antibodies (ab₃), compared to mmAb17-1A alone. Patients with a high increase in antitumour-cell antibodies (ab₃) induced by the therapy lived significantly longer than patients with no or a low level of induction of ab₃ (P = 0.016). The results indicate that induction of an idiotypic network response might be an important effector mechanism in mAb therapy. Received: 20 October 1995 / Accepted: 18 December 1995     

A mannose-specific adherence mechanism in Lactobacillus plantarum conferring binding to the human colonic cell line HT-29.

Two Lactobacillus plantarum strains of human intestinal origin, strains 299 (= DSM 6595) and 299v (= DSM 9843), have proved to be efficient colonizers of the human intestine under experimental conditions. These strains and 17 other L. plantarum strains were tested for the ability to adhere to cells of the human colonic cell line HT-29.L.plantarum 299 and 299v and nine other L. plantarum strains, including all six strains that belong to the same genetic subgroup as L. plantarum 299 and 299v, adhered to HT-29 cells in a manner that could be inhibited by methyl-alpha-D-mannoside. The ability to adhere to HT-29 cells correlated with an ability to agglutinate cells of Saccharomyces cerevisiae and erythrocytes in a mannose-sensitive manner and with adherence to D-mannose-coated agarose beads. L. plantarum 299 and 299v adhered to freshly isolated human colonic and ileal enterocytes, but the binding was not significantly inhibited by methyl-alpha-D-mannoside. Periodate treatment of HT-29 cells abolished mannose-sensitive adherence, confirming that the cell-bound receptor was of carbohydrate nature. Proteinase K treatment of the bacteria also abolished adherence, indicating that the binding involved protein structures on the bacterial cell surface. Thus, a mannose-specific adhesin has been identified in L. plantarum; this adhesin could be involved in the ability to colonize the intestine.     

Target-specific arrest of mRNA translation by antisense 2'-O-alkyloligoribonucleotides.

We describe a novel experimental approach to investigate mRNA translation. Antisense 2'-O-allyl oligoribonucleotides (oligos) efficiently arrest translation of targeted mRNAs in rabbit reticulocyte lysate and wheat germ extract while displaying minimal non-specific effects on translation. Oligo/mRNA-hybrids positioned anywhere within the 5' UTR or the first approximately 20 nucleotides of the open reading frame block cap-dependent translation initiation with high specificity. The thermodynamic stability of hybrids between 2'-O-alkyl oligos and RNA permits translational inhibition with oligos as short as 10 nucleotides. This inhibition is independent of RNase H cleavage or modifications which render the mRNA untranslatable. We show that 2'-O-alkyl oligos can also be employed to interfere with cap-independent internal initiation of translation and to arrest translation elongation. The latter is accomplished by UV-crosslinking of psoralen-tagged 2'-O-methyloligoribonucleotides to the mRNA within the open reading frame. The utility of 2'-O-alkyloligoribonucleotides to arrest translation from defined positions within an mRNA provides new approaches to investigate mRNA translation.     

Tetrasomy 15q: two marker chromosomes with no detectable alpha-satellite DNA.

Two patients with specific and similar phenotypes were both found to have an unusual marker chromosome present in 70%-80% of their lymphocytes at routine cytogenetic examination. The marker chromosomes were isolated by flow sorting and were amplified by degenerate oligonucleotide-primed PCR. These libraries and a cosmid probe located at 15q26 were used to characterize the marker chromosomes by FISH. Both marker chromosomes were found to consist of duplicated chromosome material from the distal part of chromosome 15q and were identified as inv dup(15) (qter-->q23::q23-->qter) and inv dup(15) (qter-->q24::q24-->qter), respectively. Hence, the markers did not include any known centromere region, and no alpha-satellite DNA could be detected at the site of the primary constriction. Tetrasomy 15q may be a new syndrome, associated with a specific type of marker chromosome. In addition, further analyses of this type of marker chromosome might give new insight into the structure and function of the mammalian centromere.     

A Primary Linkage Map of the Porcine Genome Reveals a Low Rate of Genetic Recombination

A comprehensive genetic linkage map of the porcine genome has been developed by typing 128 genetic markers in a cross between the European Wild Boar and a domestic breed (Large White). The marker set includes 68 polymerase chain reaction-formatted microsatellites, 60 anchored reference markers informative for comparative mapping and 47 markers which have been physically assigned by in situ hybridization. Novel multipoint assignments are provided for 54 of the markers. The map covers about 1800 cM, and the average spacing between markers is 11 cM. We used the map data to estimate the genome size in pigs, thereby addressing the total recombination distance in a third mammalian species. A sex-average genome length of 1873 +/- 139 cM was obtained by comparing the recombinational and physical distances in defined regions of the genome. This is strikingly different from the length of the human genome (3800-4000 cM) and is more similar to the mouse estimate (1600 cM). The recombination rate in females was significantly higher than in males.     

Concentrations of Amino Acids in Extracellular Fluid After Opening of the Blood-Brain Barrier by Intracarotid Infusion of Protamine Sulfate

Abstract: This article evaluates the influence of an opening of the blood-brain barrier (BBB) on compounds in brain extracellular fluid. The concentrations of amino acids and some other primary amines were determined in dialysates sampled from the right parietal cortex of rats before and after an intracarotid infusion of protamine sulfate. Extravasated plasma proteins were visualized by Evans blue/albumin and immunohistochemistry. CSF albumin— an indicator of blood-CSF barrier opening—was quantified with immunoelectrophoresis. The brains were macroscopically edematous after 10 mg but not after 5 mg of protamine sulfate. The higher dose led to a 50% death rate. The concentrations of amino acids did not change 10 min after the BBB opening. No significant alterations in the amino acid concentrations were observed after the lower dose. The concentrations of glutamate, aspartate, GABA, glycine, taurine, and phosphoethanolamine increased significantly within 50–80 min after the infusion of 10 mg of protamine sulfate. CSF albumin levels were significantly increased 1 h after infusion. We conclude that a dysfunction of the BBB, of a degree known to induce brain edema (10 mg of protamine sulfate), significantly increases the extracellular concentration of excitatory amino acids, GABA, taurine, and phosphoethanolamine in the extracellular space.     

Four cytosolic-type CuZn-superoxide dismutases in germinating seeds of Pinus sylvestris

A differential analysis of CuZn-superoxide dismutase (SOD. EC 1.15.1.1) isozymes after native-polyacry lamide gel elecrrophoresis (PAGE) and isoelectric focusing (IEF) indicated that germinating seeds of Scots pine (Pinus sylvestris L.) 3 days after the start of imbibition (3 DAI) contain five CuZn-SOD isozymes. Two isozymes co-migrated on native–PAGE but were separated after IEF. CuZn-SODs of Scots pine were purified from germinating seeds (3 DAI) by anion-exchange chromatography, hydrophobic interaction chromatography and chromatofocusing. The final separation of CuZn-SOD isozymes was accomplished by native-PAGE. CuZn-SOD isozymes were electroblotted and their NH₂-terminal amino acid sequence was determined. Comparisons of the amino acid sequences with sequences of CuZn-SOD isozymes from other plant sources indicated that one CuZn-SOD isozyme was of the chloroplastic type whereas the other four isozymes belonged to the cytosolic-type CuZn-SODs, The NH₂-terminal amino acid sequence of the chloroplastic CuZn-SOD and of one cytosolic-type CuZn-SOD were identical to those of two previously isolated, sequenced and localized CuZn-SOD isozymes from Scots pine needles. Two cytosolic-type CuZn-SOD isozymes showed a homology at 20 out of 21 NH₂-terminal amino acids. Mitochondria and glyoxysomes were isolated by differential and Percoll density-gradient centrifugation from germinating seeds (3 DAI). The cell fractionation experiments did not suggest that a major part of the CuZn-SOD activity in germinating seeds was derived from glyoxysomes or mitochondria.     

Linkage maps of porcine Chromosomes 3, 6, and 9 based on 31 polymorphic markers

Linkage maps of porcine Chromosomes (Chrs) 3, 6, and 9, based on 31 polymorphic markers, are reported. The markers include 14 microsatellites, 12 RFLPs, three protein polymorphisms, and two blood group loci. The genetic interpretations of 11 RFLPs are documented. The markers were scored in a three-generation Wild Boar/Large White pedigree, and genetic maps were constructed on the basis of two-point and multi-point linkage analysis. Altogether the maps span a genetic distance of 216 cM, and previous physical assignments indicate that the linkage groups cover major parts of the three chromosomes. Significant differences in recombination rates between the sexes were observed for all three chromosomes. The recombination rate on the q arm of Chr 6 was markedly low. Sixteen loci are informative with regard to comparative mapping, that is, they have previously been mapped in the human and/or mouse genomes.     

Immunohistochemical studies of neurochemical markers in normal human buccal mucosa

The content of various substances, such as regulatory peptides, hormones and structural proteins, was investigated in normal buccal mucosa using indirect immunofluorescence. Thin nerve fibres, which from a morphological point of view were most probably sensory, showed immunoreactivity for substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide K (NPK) and neurokinin A (NKA). Also galanin (GAL), -melanocyte stimulating hormone (-MSH) and somatostatin (SOM) stained thin fibres were found in the propria, which were, however, few in number and the -MSH staining was weak. CGRP, vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine amide (PHI) and neuropeptide Y (NPY) immunoreactive nerve fibres were observed in close connection to blood vessels. SOM positive cells with processes were found, mostly scattered, in the connective tissue. A population of cells within the epithelium also showed somatostatin immunoreactivity. Protein S-100 (S-100) stained distinct populations of cells at two separate locations. In the propria, cells with one or two slender processes were seen, being mostly single but sometimes forming groups. In the epithelium, dendritic cells with many processes with or without spines were observed, mainly located to the basal layer of the lamina epithelialis. Single nerve fibres and nerve bundles were also stained. Neurofilament (NF) positive fibres, singly and in bundles, as well as endorgan-like structures were seen. Neuron-specific enolase (NSE) and protein gene product 9.5 (PGP 9.5) both stained the same structures, namely single fibres, nerve bundles, nerves surrounding vessels and innervating muscles and glands (if present in the section), as well as Merkel cells. Also with these two markers endorgan-like structures were seen. No clear innervation of the epithelium could be observed with the markers used. No methionine-enkephalin (ENK) or synaptophysin (SYN) immunoreactive material was found.