Resolution of the extracellular α-glucosidase system ofBacillus sp. NCIB 11203

Summary Two extracellular -glucosidases (EC 3.2.1.20, -D-glucoside glucohydrolase) of the alkalophilic bacterium,Bacillus sp. NCIB 11203, were separated, purified and partially characterised. Resolution of the system into two separate enzymes was achieved by fractionation with (NH₄)₂SO₄ and chromatography on DEAE-Biogel A. The first of these activities, an -glucosidase, hydrolysed p-nitrophenyl--D-glucopyranoside preferentially and had minor activity on isomaltose and isomaltotriose. The second enzyme was a maltase and displayed highest activity on maltose and maltotriose and some activity on p-nitrophenyl--D-glucopyranoside.     

Pi Mheerlen,a Pi M allele resulting in very low a 1-antitrypsin serum levels

Summary A patient with pulmonary emphysema is described, who had a very low ₁-antitrypsin serum concentration (2% of normal). After isoelectric focusing and staining, the patient's serum revealed no visible ₁-antitrypsin bands. Immunofixation, following isoelectric focusing, gave a banding pattern identical to that of a normal M type. The existence of this deficient M-allele was confirmed by family studies. Low ₁-antitrypsin concentrations, due to the presence of the deficient allele, were coupled with low serum antitrypsin activities.     

Formation of a raw starch-hydrolyzing alpha-amylase by Clostridium 2021: Effect of carbon sources

Summary Clostridium 2021 was found to produce -amylase effective at hydrolyzing raw starch. Of the carbohydrates examined, starch at 3 % concentration was found to be the best carbon source for enzyme production. The products of -amylase action on starch were: maltose. glucose and higher dextrins.     

Chromosomes in old age: A six year longitudinal study

Summary A new variant of ₁-antitrypsin has been detected with the aid of isoelectric fucosing on polyacrylamide slab gels. In contrast with many other variants this new ₁-antitrypsin allele is found in 10–15% of the phenotypes examined. The electrophoretic properties of the new ₁-antitrypsin variant in isofocusing polyacrylamide gels differ only silightly from the most common ₁-antitrypsin allele M. On the basis of its electroforetic behaviour we propose the term M_N to indicate this new protease inhibitor variant. The isofocusing technique employed, provides an easy to handle, very reproducible method for determining the ₁-antitrypsin phenotype and can be employed for large scale screening.     

High frequency of triplicated α-globin loci and absence or low frequency of α thalassemia in Polynesian Samoans

Summary Most of the population in certain areas of Melanesia have one -globin gene deletion ( thal₂). It is thought that the high frequencies of thal₂ in this population is due to a selective advantage given by malaria infection to carriers of thal₂. We are interested in neighboring Polynesia which, although adjacent to Melanesia, has always been free of malaria due to the absence of the vector anopheles. We studied 60 Polynesian Samoans and 150 Malaysians by restriction endonuclease gene mapping using Eco RI, Bam HI, and Bgl II and hybridization to ³²P-labeled -globin gene probe. Seven among the 60 (11.7%) Samoans had triplicated -globin loci type 1, while none had thal₂. On digestion with Bgl II the third -globin gene was found in an additional 3.7kb fragment in all seven Samoans with triplicated -globin loci, while digestion with Bam HI produced an abnormal elongated 18.2 kb fragment carrying -globin genes in addition to the normal 14.5 kb fragment. None of the Polynesian Samoans had thal₂ or thal₁. Only two of the Malaysians had triplicated -globin loci.     

In-vitro processing of yeast α-factor leader fusion proteins using a soluble yscF (Kex2) variant

Summary The Saccharomyces cerevisiae KEX2 gene encodes the membrane-bound endoprotease yscF, which is responsible for the site-specific endoproteolytic cleavages at pairs of basic amino acid residues in the -factor precursor. In order to obtain soluble yscF activity, a mutant KEX2 gene lacking 600 bp coding for the C-terminal 200 amino acids was constructed. Expression of the truncated KEX2 gene in yeast led to the secretion of an active soluble yscF protein (yscF_s). The soluble yscF protein is able to efficiently cleave heterologous protein precursors in-vitro, as demonstrated for -factor leader-hIGF1 and -factor leader-hirudin fusion proteins. Offprint requests to: P. G. Seeboth     

Enhanced alpha - amylase production by chromosomal integration of pTVA1 in industrial strain in Bacillus subtilis

Summary Strain Bacillus subtilis MS was constructed with 12–22 fold increase of -amylase production, caused by presence of multiple -amylase gene copies in the chromosome of industrial strain Bacillus subtilis CCM2722, as demonstrated by DNA hybridization. The enhanced production is a result of multiple integration of plasmid pTVA1, carrying a temperature sensitive origin of replication from pE194, and containing the -amylase gene and a modified transposon Tn917.     

Soluble Expression and Affinity Purification of Functional Domain of Human Acetylcholine Receptor <Emphasis Type="Italic">α</Emphasis>-subunit by the Modulation of Maltose Binding Protein

An open reading frame of the -subunit 1-205 residues (₂₀₅) of human acetylcholine receptor (AchR) was amplified by PCR with pUC-AChR₂₀₅ as the template and inserted into vector pMAL-c2X. The constructed pMAR₂₀₅ was transferred into E. coli BL21 which were then grown in LB medium. The amount of soluble MBP-AChR₂₀₅ protein reached about 25% of total soluble proteins from the cell lysate. Using amylose-affinity chromatography, about 35 mg MBP-AChR₂₀₅ could be obtained from 1 l culture. Western blot analysis and ELISA showed that immunoreactivities of both MBP-AChR₂₀₅ and AChR₂₀₅ were similar to that of AChR -subunit from Torpedo.Revisions received 23 September 2004     

MHC class II sequences of an HLA-DR2 narcoleptic

Narcolepsy has a 98% association with the DR2-Dw2/DQw1 haplotype. To establish if a disease-specific allele is present in narcolepsy, a cDNA library was made from a B-cell line from a DR2,4/DQw1,3 narcoleptic. Clones encoding the two expressed DR2 chains, along with DQw1 and chains, were isolated and completely sequenced. The coding regions of these four genes were similar to published nucleotide and protein sequences from corresponding healthy controls, with some minor exceptions. The 3 untranslated region of one of the DR2 genes in the narcoleptic was extended by 42 bp. Complete sequences were not available for DQw1.2 or from healthy individuals, but first domain nucleotide sequences showed only a single nonproductive difference in DQ. Partial protein sequences of both DQ and from published data were identical. Although the effects of minor differences cannot be ruled out completely, it is concluded that there are probably no narcolepsy-specific DR or DQ / sequences, and that the alleles found in narcolepsy are representative of those found in the healthy population.     

The transport of class I major histocompatibility complex antigens is determined by sequences in the α1 and α2 protein domains

The transport of human-mouse hybrid class I histocompatibility antigens has been studied in a mutant human cell line, 174 × CEM.T2 (T2). T2, a somatic cell hybrid of human B- and T-lymphoblastoid cell lines (B-LCL and T-LCL, respectively), synthesizes HLA-A2 and HLA-B5 glycoproteins, but expresses only low levels of A2 and undetectable levels of B5 at the cell surface. We have previously shown that the products of human class I genes introduced into T2 by transfection behave like the endogenous HLA-B5 glycoproteins, while the products of mouse class I alleles similarly introduced are transported normally to the cell surface. We have now determined that the surface expression of class I glycoproteins in T2 depends on the origin of the ₁ and ₂ domains. Human (HLA-B7) and mouse (H-2D ^p ) hybrid class I genes, encoding the leader, ₁, and ₂ sequences of one species fused to the ₃, transmembrane, and cytoplasmic domains of the other, were transfected into T2. Normal surface expression of the hybrid class I molecule was observed in T2 only when the leader, ₁, and ₂-encoding exons were derived from the mouse gene. The reciprocal construct, encoding human leader, ₁, and ₂ domains fused to the mouse ₃, transmembrane, and cytoplasmic regions, resulted in biosynthesis of a hybrid glycoprotein which was not transported to the cell surface. The products of both constructs were expressed normally in control cells. The effects of glycosylation on class I antigen transport were also studied using mutant class I constructs with altered glycosylation sites. Two mutant B7 genes encoding either an extra glycosylation site at position 176 or no glycosylation sites were transfected into T2. These mutant products were expressed at the cell surface in control cells, but were synthesized and not surface-expressed in T2. These data demonstrate that the HLA/H-2 transport dichotomy in T2 is a function of the origin of the ₁ and/or ₂ domains of the class I glycoprotein, and is not a reflection of glycosylation differences between the human and mouse molecules. Offprint requests to: P. Cresswell.     

Degradation pathways from n-tridecane to α, ω-tridecanedioic acid in a mutant of Candida tropicalis

Summary The metabolic formation of ,-tridecanedioic acid via n-tridecanoic acid and via ,-tridecanediol from n-tridecane in the mutant S 76 of Candida tropicalis was studied. It was found that resting cells of S 76 produced ,-tridecanediol from n-tridecane.With n-tridecanol as substrate, the ,-diol could also be detected. The mutant S 76 was able to produce ,-tridecanedioic acid using either n-tridecanol or n-tridecanoic acid as the sole carbon source. Quantitative changes in the concentration of -hydroxy tridecanoic acid and other intermediates were recorded during the formation of ,-dioic acid.The results confirm the existence of two metabolic pathways mentioned above in the course of ,-dioic acid formation from odd n-alkane in the mutant S 76 of C. tropicalis.     

α-Amylase structural genes in rye

Summary Rye -Amy1, -Amy2, and -Amy3 genes were studied in the cross between inbred lines using wheat -amylase cDNA probes. The -Amy1 and -Amy2 probes uncovered considerable restriction fragment length polymorphism, whereas the -Amy3 region was much more conserved. The numbers of restriction fragments found and the F₂ segregation data suggest that there are three -Amy1 genes, two or three -Amy2 genes, and three -Amy3 genes in rye. These conclusions were supported by a simultaneous study of -amylase isozyme polymorphism. The F₂ data showed the three individual -Amy1 genes to span a distance of 3cM at the locus on chromosome 6RL. The genes were mapped relative to other RFLP markers on 6RL. On chromosome 7RL two -Amy2 genes were shown to be separated by 5 cM. Linkage data within -Amy3 on 5RL were not obtained since RFLP could be detected at only one of the genes.     

Integrins: cell adhesives and modulators of cell function

Summary Integrins encompass a family of cell-surface molecules which play a crucial role in cell-cell and cell-extracellular matrix interaction. Of these heterodimeric transmembrane glycoproteins (consisting of an and chain) as yet at least 20 different types have been described, all with a different pattern of reactivity with extracellular matrix components. In this review the cell and tissue distribution of the integrins is discussed, with special emphasis on immunohistochemical localization of the ₁ integrins and the ₆₄ integrin. The ₁ integrins comprise a subfamily in which eight chains combine with one (the ₁) chain. The ₂₁, ₃₁ and ₆₁ and the ₆₄ integrins are expressed on a wide variety of epithelia on the basolateral surface or exclusively on the basal surface facing the basement membrane (e.g. ₆₁ and ₆₄). Leucocyte integrins, which share a common ₂ chain, occur almost exclusively on white blood cells and their precursors. The vitronectin receptors, which share a common _v chain, occur in a wide variety of cell types. Integrins play a major role in the interaction of the cell with the extracellular matrix in order to create and maintain tissue architecture. It has become clear, however, that through integrin-ligand interaction cell function is also modulated. Furthermore, in pathological conditions integrins play a role of some significance. Integrins mediate leucocyte traffic in developing inflammatory processes and function in neoplastic growth when it comes to invasion and metastasis.     

Expression of pAMα1 tetracycline resistance gene inCorynebacterium glutamicum: Segregation of antibiotic resistance due to intramolecular recombination

Summary AnEscherichia coli/Corynebacterium glutamicum chimeric plasmid has been constructed containing the tetracycline resistance (Tc^R) determinant from pAM1 and the kanamycin resistant (Km^R) determinant from pBD10. The paAM1 tetracycline resistance determinant is expressed inC. glutamicum although the transformed population segregates readily into tetracycline and kanamycin resistant populations. The segregation event is the result of an intramolecular recombination between the pAM1 and pUB110 regions of the chimera.     

Protease inhibitor localization in control and streptozotocin-diabetic skeletal muscles

Summary ₁-Antitrypsin and ₁-inhibitor-3 were localized for the first time inside skeletal muscle cells. Their content, especially that of ₁-inhibitor-3, was greatly reduced following streptozotocin-induced diabetes. ₁-Antitrypsin and ₁-inhibitor-3 were also observed in the vascular components and interstitial space surrounding both control and diabetic soleus muscles as revealed by immunofluorescence. In diabetic muscles, the non-myofibre locale of ₁-inhibitor-3 was reduced, and to a lesser extent, ₁-antitrypsin. Both myofibre and extracellular patterns were reversed to control levels by insulin replacement.     

Desensitization of prostaglandin F2α receptor-mediated phosphoinositide hydrolysis in cultured rat astrocytes

Desensitization of prostaglandin (PG) F₂ receptor-mediated phosphoinositide (PI) hydrolysis was investigated in cultured rat astrocytes. Prolonged exposure of astrocytes differentiated by dibutyryl cyclic AMP-treatment to PGF₂ caused the desensitization of subsequent PGF₂-induced PI hydrolysis. The desensitization was time- and PGF₂ dose-dependent; maximal decrease in the PI hydrolysis was observed after exposure to 10 M PGF₂ for 4 h and the degree of the desensitization was 31.7±2.7% of control. Pretreatment with either PGD₂ or PGE₂ also induced the desensitization of subsequent PGF₂-stimulated PI hydrolysis and conversely pretreatment of PGF₂ decreased the PI responses to PGD₂ and PGE₂. The desensitization prevented by phloretin and was reversible upon removal of the agonist. Protein synthesis inhibitors blocked the recovery of the desensitization. Treatment of the cells with phorbol 12-myristate 13-acetate had no effect on the desensitization. These results suggest that prolonged exposure of the astrocytes to PGF₂ caused the desensitization of the receptors.     

α-Subunit Composition of Nicotinic Cholinoreceptors of Neurons of the Guinea Pig Inferior Mesenteric Ganglion: an Immunohistochemical Study

Using immunoperoxidase labeling, we studied the subunit composition of nicotinic acetylcholine receptors, nAChR, in preparations of the inferior mesenteric ganglion, IMG, of the guinea pig. Antibodies against synthetic peptides corresponding to agonist-binding membrane components of the ₃, ₄, ₅, and ₇ nAChR subunits were used. The presence of ₃-specific antibodies was revealed on the membranes of about 58% of large neurons and of all small ganglionic cells (means of the greater and smaller diameters of the somata 53.8 ± 1.8 vs 33.6 ± 1.4 m, n = 20, and 14.1 ± 0.5 vs 7.5 ± 0.4 m, n = 50, respectively). Labeled cells of the rostral node were distributed evenly, while those of the caudal node were localized mostly within the regions of branching of the lumbar, colonic, and both hypogastric tracts. Immune labels to the ₄ subunit were observed only on the membranes of small ganglionic cells distributed mostly in the region of the internodal commissural tracts. ₅-Specific labeling was found on the membranes of about 63% large neurons, whose distribution was similar to that of the ₃-labeled units, and on all small cells. Immunoreactivity to the ₇ subunit was observed only on the membranes of small cells concentrated around unlabeled large neurons in the region of branching of the intermesenteric, colonic, and both hypogastric tracts. Thus, nAChR in the guinea pig IMG include ₃, ₄, ₅, and ₇ subunits. The nAChR with ₃ and ₅ subunits are localized on the membranes of large ganglionic neurons, whose number and topographical distribution are very close to each other. Our data agree with our results of earlier electrophysiological experiments and are indicative of the crucial role of the ₃- and ₅-containing nAChR in synaptic transmission via the ganglion under study. The presence of the ₄- and ₇-containing nAChR was found only on small ganglionic cells (which are, probably, not the relay units) and their processes.     

Studies of α- and βL-Crystallin Complex Formation in Solution at 60°C

Studies of molecular mechanisms of chaperone-like activity of -crystallin became an active field of research over last years. However, fine interactions between -crystallin and the damaged protein and their complex organization remain largely uncovered. Complexation between - and _L-crystallins was studied during thermal denaturation of _L-crystallin at 60°C using small-angle X-ray scattering (SAXS), light scattering, gel-permeation chromatography, and electrophoresis. A mixed solution of - and _L-crystallins at concentrations about 10 mg/ml incubated at 60°C was found to contain their soluble complexes with a mean radius of gyration 14 nm, mean molecular mass 4 MDa and maximal size over 40 nm. In pure _L-crystallin solution, no complexes were observed at 60°C. In SAXS studies, transitions in the -crystallin quaternary structure at 60°C were shown to occur and result in doubling of the molecular weight. This suggests that during the temperature-induced denaturation of _L-crystallin it binds with modified -crystallin or, alternatively, _L-crystallin complexation and -crystallin modifications are concurrent. Estimates of the -_L-crystallin complex size and relative contents of - and -_L-crystallins in the complex suggest that several -crystallin molecules are involved in complex formation.     

α-Globin gene deletions associated with αAand αG Philadelphiagenes in an Algerian family that includes two Hb G homozygotes

Summary An Algerian family with a high degree of consanguinity and including two homozygotes for Hb-G Philadelphia is presented. Whether homozygotes or heterozygotes, all subjects displayed microcytosis (with various degrees of poikilocytosis) and a moderately depressed -globin chain synthesis. Hb H and Heinz bodies were absent. DNA mapping revealed the presence of 3.7 kb deletion resulting from the rightward type of recombination event between ₂ and ₁ genes on both the ^A/and the ^G chromosomes. Such data indicate that the –^A/ and –^G/ haplotypes are involved and suggest that the –^G/ haplotype, which is very rare in Algeria, has an African Black origin. In subjects with genotype (–^A/–^G/) or (–^G/–^G/), the output of the remaining genes is sufficiently high to avoid the appearance of Hb H. This situation contrasts with that reported in an Algerian patient, who had a (–^A/–^A/) genotype but who was producing Hb H (Whitelaw et al. 1980). The data collected from this family suggest that the –^A/ haplotypes are heterogeneous in Algerians.     

Measurement of residual dipolar couplings from 1Hα to 13Cα and 15N using a simple HNCA-based experiment

Novel NMR pulse schemes for simultaneous measurement of ¹ D _CHand ² D _NHresidual dipolar couplings in proteins is presented. We show that ² D _NHcoupling can be very useful for protein structure determination. The ² D _NHcoupling can be measured from ¹⁵N dimension with good accuracy on a slowly relaxing TROSY resonance, utilizing HNCA-TROSY-based experiments, which concomitantly supply large ¹ D _CHcoupling. The dynamic range of ² D _NHcoupling is comparable to ¹ D _NC coupling, but instead, it also serves non-redundant information on the course of protein backbone, thanks to rotational degree of freedom with respect to peptide bond. The HNCA-TROSY-based experiments are optimal for measuring residual dipolar couplings at high magnetic fields owing to absence of rapid transverse relaxation of carbonyl carbon. The reliability of the proposed approach was tested on ¹⁵N/¹³C human ubiquitin. A very good correlation with ubiquitin solution as well as crystal structure, for both ¹ D _CHand ² D _NHcouplings, was obtained.