Purification of the neurotensin receptor from bovine brain

The neurotensin receptor protein, solubilized with digitonin/asolectin from bovine cerebral cortex membranes, was purified to apparent homogeneity by affinity chromatography using immobilized neurotensin. The product exhibits saturable and specific binding of [3,11-tyrosyl-3,5-3H]neurotensin with an apparent affinity (Kd = 5.5 nM) comparable to that measured in intact membranes and crude soluble extracts. The affinity-purified material, after reduction with 100 mM dithiothreitol, in denaturing gel electrophoresis showed a single polypeptide of Mr 72,000. Under nonreducing conditions the apparent Mr, however, was 50,000, suggesting the presence of intramolecular disulfide bonds. The purified neurotensin receptor was judged to be homogeneous, in that (i) only a single polypeptide was detectable; and (ii) the overall purification was 30,000-50,000-fold, giving a specific neurotensin-binding activity close to the theoretical maximum.     

Joint Report of the First International Comparison Test on Swine Lymphocyte Alloantigens (SLA)

The first international comparison test on swine lymphocyte alloantigens (SLA) was held in Helsinki, Finland in July 1986. The results reported were based on a comparison of 157 alloantisera originating from six laboratories. The antisera were tested against a selected panel of 264 lymphocyte samples belonging to four laboratories. The most common breeds in Europe were chosen for this first comparison test (Landrace and Large White). Eighteen of the 31 previously known specificities were confirmed and a new nomenclature was established.     

Conductance measurements of the lag phase of injured Salmonella typhimurium

The duration of the lag phase of Salmonella typhimurium injured by heating, freezing, acidification or drying was measured using the 'Malthus' conductance meter. Results confirmed those previously obtained by viable counting and additionally, revealed the very wide variation in lag between and within populations and the extreme length of lag that can occur in some severely injured cells. In an extreme example, the measured lag times of low numbers of bacteria taken from the same heat-injured population ranged from 16 h to 70 h. The implications for the detection of injured micro-organisms in food are discussed.     

ELISA absorbance cut-off method affects malaria sporozoite rate determination in wild Afrotropical Anopheles

ABSTRACT. Malaria sporozoite infection rates in a mixed species group of 244 Anopheles gambiae Giles sensu lato and 115 An. funestus Giles wild female mosquitoes were compared using three methods to determine cutoff absorbance values for positivity of a Plasmodium falciparum Welch enzyme-linked immunosorbent assay (ELISA). Positive controls were based on P. falciparum circumsporozoite protein. As negative controls, four wild male Anopheles were included on each microtitre plate; tests were repeated on four consecutive days for each plate.
Infection rates were estimated at 13.1–22.8% using the mean absorbance value of negative controls plus three standard deviations, 11.7–12.8% using double the mean and 12.5–13.6% using the fixed cut-off value of 0.20 (allowing for 20% variation in negative control absorbance values).
Observed agreement for positivity or negativity among samples tested four times was 98.6% for the 2× mean method, 97.2% for the fixed cut-off 0.20 value, but only 82.7% for the mean +3 SD method. It was concluded that the 2× mean cut-off method is most reliable for field studies. P. falciparum sporozoite rates of 12.2% in An. funestus and 11.9% in An. gambiae s. l . were thus determined on the basis of the 2× mean cut-off method.
This comparative evaluation demonstrates that vector infectivity rates can be seriously over-estimated from sporozoite ELISA tests, by as much as 87% in one case considered here, depending on the absorbance cut-off method applied for negative controls.     

Kinetics of D-glucose and 2-deoxy-D-glucose transport by Rhodotorula glutinis

The yeast Rhodotorula glutinis (Rhodosporidium toruloides) is capable of accumulative transport of a wide variety of monosaccharides. Initial velocity studies of the uptake of 2-deoxy-D-glucose were consistent with the presence of at least two carriers for this sugar in the Rhodotorula plasma membrane. Non-linear regression analysis of the data returned maximum velocities of 0.8 +/- 0.2 and 2.0 +/- 0.2 nmol/min per mg (wet weight) and Km values of 18 +/- 4 and 120 +/- 20 microM, respectively, for the two carriers. Kinetic studies of D-glucose transport also revealed two carriers with maximum velocities of 1.1 +/- 0.4 and 2.4 +/- 0.4 nmol/min per mg (wet weight) and Km values of 12 +/- 3 and 55 +/- 12 microM. As expected, 2-deoxy-D-glucose was a competitive inhibitor of D-glucose transport. Ki values for the inhibition were 16 +/- 8 and 110 +/- 40 microM. These Ki values were in good agreement with the Km values for 2-deoxy-D-glucose transport. D-Xylose, the 5-deoxymethyl analog of D-glucose, appears to utilize the D-glucose/2-deoxy-D-glucose carriers. This pentose was observed to be a competitive inhibitor of D-glucose (Ki values = 0.14 +/- 0.06 and 5.6 +/- 1.6 mM) and 2-deoxy-D-glucose (Ki values = 0.15 +/- 0.07 and 4.6 +/- 1.2 mM) transport.     

Distribution of newly synthesized DT-diaphorase in rat liver

The distribution, synthesis transport, and glycosylation of rat-liver DT-diaphorase has been investigated. The enzyme could be isolated using specific antibodies, mainly from the soluble supernatant but also from microsomal vesicles, Golgi membrane, and mitochondria. 40% of the microsomal enzyme was located in the lumen or on the interior side of the membrane, the rest remaining as an integral non-extractable part of the membrane. Synthesis of DT-diaphorase takes place on both free and bound ribosomes, although it was found to be transported in a sequential manner from the rough to the smooth endoplasmic reticulum and also subsequently to the mitochondria. The rough and smooth microsomal DT-diaphorase contains covalently bound carbohydrate, but no sugar moiety could be detected bound to the cytoplasmic form of the enzyme.     

Permeability of the foetal capillary endothelium of the guinea-pig placenta to haem proteins of various molecular sizes

Summary Haem proteins of different molecular sizes were perfused into the foetal circulation of the guinea-pig placenta to study the permeability of the foetal endothelium.The smallest molecules tested, microperoxidase (a_e 1.0 nm) and cytochrome C (a_e 1.5 nm), readily penetrated the endothelium; tracer-reaction product was found in the subendothelial space of the capillaries. However, there was no uptake of these two tracers into the syncytiotrophoblast layer of the placenta. An intermediate-sized molecule, myoglobin (a_e 1.7 nm), produced only a weak reaction product in the subendothelial space even when perfused at high concentration. The largest molecule tested, haemoglobin (a_e 2.8 nm), did not penetrate the foetal endothelium at any of the concentrations employed.The foetal capillary endothelium thus provided a barrier to protein penetration from the foetal circulation, dependent on molecular size. There was evidence that the site of this barrier was located in the lateral intercellular spaces between the endothelial cells.The syncytiotrophoblast of this haemomonochorial placenta provided an almost absolute barrier to protein penetration from the foetal circulation. As other workers have described maternal-to-foetal transmission of proteins across this layer in the guinea-pig, a working hypothesis of the role of endothelium and syncytiotrophoblast in maternal/foetal protein exchange is discussed.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.