Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

An elongated segment of DNA observed between two human α globin genes

Summary Detailed restriction enzyme analysis of the DNA from a Chinese female showed that one of her chromosomes had a >17.5 kb deletion of DNA, including the , 2, and 1 globin genes, which is present in many Southeast Asians with an -thalassemia-1 chromosome. Her normal chromosome had the expected cluster of -like globin genes (5----2-1-3), but the segment of DNA between the two globin genes was elongated by some 0.5–0.7 kb. Analyses of various restriction sites suggested that this normal variant of the human globin gene complex is due to a crossover between a normal chromosome with () and a chromosome with an -thalassemia-2 (–^3.7) and an -21-hybrid gene.     

Average phase difference theory and 1∶1 phase entrainment in interlimb coordination

The dynamics of coupled biological oscillators can be modeled by averaging the effects of coupling over each oscillatory cycle so that the coupling depends on the phase difference between the two oscillators and not on their specific states. Average phase difference theory claims that mode locking phenomena can be predicted by the average effects of the coupling influences. As a starting point for both empirical and theoretical investigations, Rand et al. (1988) have proposed d/dt= — K sin ), with phase-locked solutions =arcsin( /K), where is the difference between the uncoupled frequencies and K is the coupling strength. Phase-locking was evaluated in three experiments using an interlimb coordination paradigm in which a person oscillates hand-held pendulums. was controlled through length differences in the left and right pendulums. The coupled frequency _c was varied by a metronome, and scaled to the eigenfrequency _v of the coupled system K was assumed to vary inversely with _c. The results indicate that: (1) and K contribute multiplicatively to (2) =0 or = regardless of K when =0; (3) 0 or regardless of when K is large (relative to ); (4) results (1) to (3) hold identically for both in phase and antiphase coordination. The results also indicate that the relevant frequency is _c/_v rather than _c. Discussion high-lighted the significance of confirming =arcsin(/K) for more general treatments of phase-locking, such as circle map dynamics, and for the 11 phase-entrainment which characterizes biological movement systems.     

Thermal stability of soluble mitochondrial H+-ATPase

ATPase melting has been studied by circular dichroism and differential scanning microcalorimetry. Decomposition of the -helix of H⁺-ATPase (in which about 80% of the peptide groups of the enzyme are involved) following thermal treatment is shown to proceed gradually, beginning with room temperature. Effect of nucleotides upon melting is detected in the range of 20–40 C. Above 40 C, the pattern of thermal decomposition of the three-dimensional structure of H⁺-ATPase is independent of the nature of nucleotides present. Highly stable -helical sites have been found in the enzyme molecule. Possible mechanism of formation of such sites is discussed, and the results obtained are compared with data on thermal stability of ATPase from thermophilic bacteria. Structural changes in the molecule following thermal treatment are compared with ATPase activity changes under similar experimental conditions.     

Physiological relationship between the temperate phages lambda and phi80

Summary This work deals with the ability of phage 80 to provide defective mutants of with their missing functions. Functions Involved in Recombination. As shown by others, the Int mechanism of 80 cannot excise prophage . However, 80 efficiently excises recombinants from tandem dilysogens, using its Ter mechanism. Likewise, the nonspecific mechanism Red is interchangeable between 80 and . Maturation of DNA by 80. The Ter recombinants excised by 80 from tandem dilysogens are packaged into a 80 protein coat. This contrasts with the fact, already mentionned by Dove, that 80 is extremely inefficient for packaging phage superinfecting a -lysogen. The latter result is also found when the helper phage is a hybrid with the left arm of (80hy4 or 80hy41 — see Fig. 1). However, the maturation of the superinfecting is much more efficient if the 80hy used as a helper has the att-N region of (like 80hy1). Conversely a with the att-N region of 80 (hy6 — see Fig. 1) is packaged more efficiently by 80 or 80hy4 than by 80hy1. It is suggested that the maturation of chromosome superinfecting an immune cell requires a recombination with the helper phage. Vegetative Functions. Among the replicative functoons O and P, the latter only can be supplied by 80. That N mutants are efficiently helped by 80 does not tell that 80 provides the defective with an active N product; the chromosomes are simply packaged into a 80 coat. This shows that 80 is unable to switch on the late genes of . That neither 80 nor any of the 80hy tested can provide an active N product is shown in a more direct way by their complete failure to help N ^-r₁₄; this phage carries a polar mutation which makes the expression of genes O and P entirely N-dependant. The maturation of a N ^- by 80 contrasts with the fact that mutants affected in late genes (A, F or H) are not efficiently helped by 80. This suggests that the products coded by these genes are not interchangeable between 80 and , and that packaging of DNA into 80 coats is possible but inhibited when late proteins are present in the cell. Activation of the Late Genes. Among the im ₈₀ h ⁺ hybrids tested, only 80hy41 is able to switch on the late genes of a N defective mutant. This hybrid differs from the other hybrids studied here, by the fact that it has the Q-S-R region of (see Fig. 1). The results are consistant with the view that the product of Q gene is sufficient for activating the late genes of a DNA. N would thus control the expression of late genes only indirectly by controlling the expression of gene Q (Couturier & Dambly have independantly reached the same conclusion, 1970). Furthermore the failure of 80 and of the 80hy1 and 80hy4 to activate the late genes of would imply that these phages are unable to provide an Q product active on the chromosome Reciprocally, switches on the late genes of prophage 80hy41, but not of prophages 80hy1 and 80hy4. This suggests that the initiation of late genes expression takes place at a main specific site located in the Q-S-R region of the chromosome. The expression of the late genes would thus be sequential, and proceed through the left arm only when steaky ends cohere. Similar conclusions were reached independantly by Toussaint (1969) and by Herskowitz and Signer (1970).

---

---

Ce travail a été réalisé dans le cadre du contrat d'association Euratom-U. L. B. 007-61-10 ABIB et avec l'aide du Fonds de la Recherche Fondamentale Collective.     

Carotenoid pigments of Stigmatella aurantiaca (Myxobacterales)

Summary In this first article on the carotenoids of Myxobacterales we report on the minor carotenoids of Stigmatella aurantiaca: phytoene, phytofluene, lycopene, -carotene, 4-keto--carotene, 1,2-dihydro-1-hydroxy--carotene, 4-keto-1,2-dihydro-1-hydroxy--carotene, 4-keto-1,2-dihydro-1-hydroxy-torulene, and 1,2,1,2-tetrahydro-1,1-dihydroxy-lycopene. These pigments account for about 10% of total carotenoids.     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

Stimulation by hexose esters of lactate production by rat erythrocytes,insensitivity to 3-O-methyl-D-glucose and inhibition by 2-deoxy-D-glucose and its tetraacetic ester

Selected esters of D-glucose were recently proposed as tools to provide the sugar to cells, whilst bypassing the carrier system for hexose transport across the plasma membrane. In the present study, -D-glucose pentaacetate, -D-glucose pentaacetate, -D-mannose pentaacetate and, to a lesser extent, 6-O-acetyl-D-glucose, all tested at a 1.7 mM concentration, were found to increase lactate production above basal value in rat erythrocytes. Over 90 min incubation, the increment in lactate production ranged from about 1.2 (-D-glucose pentaacetate) to 0.6 (6-O-acetyl-D-glucose) mol/l of erythrocytes. Little or no change in lactate production was observed in cells exposed to -L-glucose pentaacetate, -D-glucose pentaethylsuccinate, -D-galactose pentaacetate or -D-galactose pentaacetate. The metabolic response to -D-glucose pentaacetate was resistant to 3-O-methyl-D-glucose (10-80 mM) which suppressed, however, that evoked by D-glucose. D-mannoheptulose (10 mM) virtually failed to affect the response to D-glucose and its pentaacetate ester. On the contrary, 2-deoxy-D-glucose (10.6 mM) inhibited to the same relative extent (55% decrease) lactate production in erythrocytes exposed to either unesterified D-glucose or -D-glucose pentaacetate. The tetraacetic ester of 2-deoxy-D-glucose was more efficient than unesterified 2-deoxy-D-glucose in inhibiting lactate production from -D-glucose pentaacetate. It is proposed that selected esters of saccharides represent useful tools to bypass defects in hexose transport, and to increase their nutritional or therapeutic efficiency.     

Adoptions expérimentales de larves entre des fourmis de genres différents (II):Myrmica laevinodis Nylander etAnergates atratulus Schenck

Résumé Nous avons fait élever des larves d'Anergates atratulus par des ouvrières deMyrmica laevinodis à 22°C. Pour y parvenir, il n'est pas utile de faire hivernerensemble les larves d'Anergates et les ouvrières deMyrmica. La présence de larves autochtones n'empêche pas lesMyrmica d'élever des larves d'Anergates. Dans toutes les expériences lesMyrmica ont été soumises au fridavant de recevoir des larves d'Anergates. Aucune reine deMyrmica n'a été utilisée dans ces expériences.Sur les 64 larves d'Anergates que nous avons utilisées, 38 se sont transformées en imagos. C'est au début de l'adoption et au moment des métamorphoses que périrent la plupart des 26Anergates perdus. Les femelles vécurent en général 2 ou 3 jours et cherchèrent très tôt à quitter le nid natal. Les mâles vécurent 2 à 3 semaines.

---

Summary Larvae ofAnergates atratulus were experimentally reared by workers ofMyrmica laevinodis, at 22°C. An overwintering of both larvae ofAnergates and workers ofMyrmica is not necessary for the success of that experiment. The presence of larvae ofMyrmica does not keep theMyrmica from rearing larvae ofAnergates. The workers ofMyrmica have been cooled, in all the experiments, before receiving larvae ofAnergates. No queen ofMyrmica have been used in that experiments.38 of the 64 larvae ofAnergates used became imagos. Most of the 26 lostAnergates died at the beginning of the adoption and during the metamorphosis. The females lived generally 2 or 3 days and tried, very early, to leave their native nest. The males lived 2 or 3 weeks.

---

Anergates atratulus Myrmica laevinodis, 22 . bmecme Anergates Myrmica. Myrmica Anergates. Myrmica Anergates. Myrmica . 64 Anergates , 38 . 26 Anergates 2 3 . 2 3 .

     

C(alpha) chemical shift tensors in helical peptides by dipolar-modulated chemical shift recoupling NMR

The C chemical shift tensors of proteins contain information on the backbone conformation. We have determined the magnitude and orientation of the C chemical shift tensors of two peptides with -helical torsion angles: the Ala residue in G*AL (=–65.7°, =–40°), and the Val residue in GG*V (=–81.5°, =–50.7°). The magnitude of the tensors was determined from quasi-static powder patterns recoupled under magic-angle spinning, while the orientation of the tensors was extracted from C–H and C–N dipolar modulated powder patterns. The helical Ala C chemical shift tensor has a span of 36 ppm and an asymmetry parameter of 0.89. Its ₁₁ axis is 116° ± 5° from the C–H bond while the ₂₂ axis is 40° ± 5° from the C–N bond. The Val tensor has an anisotropic span of 25 ppm and an asymmetry parameter of 0.33, both much smaller than the values for -sheet Val found recently (Yao and Hong, 2002). The Val ₃₃ axis is tilted by 115° ± 5° from the C–H bond and 98° ± 5° from the C–N bond. These represent the first completely experimentally determined C chemical shift tensors of helical peptides. Using an icosahedral representation, we compared the experimental chemical shift tensors with quantum chemical calculations and found overall good agreement. These solid-state chemical shift tensors confirm the observation from cross-correlated relaxation experiments that the projection of the C chemical shift tensor onto the C–H bond is much smaller in -helices than in -sheets.     

Purification and characterization of (1→3, 1→4)-β-glucan endohydrolases from germinated wheat (Triticum aestivum)

A (13, 14)--glucan 4-glucanohydrolase [(13, 14)--glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (13, 14)--glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (13, 14)--glucanase isoenzyme EI from barley. The complete primary structure of the wheat (13, 14)--glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)⁺ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH₂-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32085 and a predicted pI of 8.1. The other cDNA, designated LW1, carries a 109 nucleotide pair sequence at its 5 end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.     

Inheritance and linkage of isozyme loci in almond

The segregation of seven isozyme marker genes was investigated using eight controlled crosses in almond. The cultivar Nonpareil was the maternal parent in all crosses. Pollination was achieved using eight different cultivars, and a total of 3200 individual kernels were assessed. For each isozyme the goodness-of-fit test was used to test for departure from the expected frequencies assuming Mendelian inheritance. Given a higher than expected number of significant results for individual isozymes, independent segregation between pairs of isozymes was tested using the chi-square statistic on the resulting two-way contingency tables. In all crosses a highly significant association (P value< 0.001) was observed between (1) the AAT- 1 and IDH isozymes loci and (2) the LAP-1 and PGM-2 isozymes loci, which leads to the conclusion that the respective isozyme pairs are linked.In addition, a significant association (P value < 0.001) was observed between LAP-1 and GPI-2 when the pollen sources were Fritz, Mission, or Price, but this could not be tested for the remaining five pollen sources, Carmel, Grant, Keane, Ne plus Ultra, Peerless, because they are homozygous at these loci. If LAP-1 is linked with GPI-2 and PGM-2, it might be expected that we should find evidence of linkage between GPI-2 and PGM-2. The lack of a significant association between these two isozymes suggests that LAP-1 is located centrally on the chromosome. These three pairs of linked loci are the first to be reported in almond.     

The origin of Q-independent derivatives of phage lambda

Summary qsr (Q-independent) phages are characterised by the replacement of the region of the genome that contains Q, S, R, and the late gene promoter, P_R, with host-derived DNA that codes for functions analogous to those deleted. Restriction endonuclease analysis and DNA/DNA hybridisation methods have been used to show that p4 and qin A 3, two such Q-independent phages, are the product of recombination between and a defective lambdoid prophage (the qsr prophage) located at an as yet unidentified site in the E. coli K 12 chromosome. The qsr prophage is distinct from the defective lambdoid prophage Rac (Kaiser and Murray 1979). In the E. coli K 12 strain AB1157 from which qsr phages cannot be generated, the qsr prophage has suffered an internal deletion. That the qsr prophage appears not to carry a full complement of essential late genes suggests one explanation for its apparently defective nature.     

Heritable variation in the phaseolin protein of nondomesticated common bean,Phaseolus vulgaris L.

Summary Crude protein extracts from single seeds of nondomesticated Mexican bean accessions were analysed by SDS polyacrylamide gel electrophoresis for variability in phaseolin protein. Six new phaseolin types; M1, M2, M3, M4, M5, M6, which contained polypeptides within the same range of molecular weights (51,000 to 45,000 daltons) as occur in the S, T and C phaseolin types of cultivated beans were identified. No T and C types were found among the non-domesticated Mexican accessions, and the S type occurred in less than 7% of the seeds screened. Genetic analyses of F₂ progenies from crosses between Sanilac (S), and five of the M types showed that each M phaseolin phenotype was allelic to the S type and expressed codominantly.     

Aislamiento de antigenos cationicos de Paracoccidioides brasiliensis mediante intercambio ionico

Resumen El procedimiento de intercambio iónico empleado en este trabajo ha mostrado ser un método eficiente, sencillo y rápido, para eliminar las fracciones antigénicas aniónicas de un extracto crudo deP. brasiliensis.Su empleo permitió la obtención de un antígeno purificado que contiene algunas de las fracciones catiónicas del extracto crudo. El antígeno responsable de la formación del arco E fue identificado en el extracto purificado, mediante análisis inmunoelectroforético.     

Factors affecting transient gene expression in protoplasts isolated from very slowly growing embryogenic callus cultures of wheat (Triticum aestivum L.)

Protoplasts isolated from embryogenic (Mustang and Chinese Spring) and non-embryogenic (Mit) calli of wheat (Triticum aestivum L.) genotypes transiently expressed -glucuronidase (GUS) activity when electroporated with a plasmid containing the GUS gene and driven by an enhanced 35S promoter and a TMV leader sequence. Conditions for the maximum expression of GUS activity were: electroporation of the freshly isolated protoplasts at 250 Vcm^-1 and 250 F for 2 s using 50 g/ml of plasmid DNA; incubation of the protoplasts with the plasmid before the pulse for 2 h; and a 15-min recovery period on ice after the pulse. In general, a higher GUS activity was obtained in protoplasts of non-embryogenic (NE) callus origin than in those of embryogenic (E) callus origin. Only GUS constructs containing a duplicate 35S promoter derivative resulted in a significant level of GUS expression. The presence of the TMV viral leader sequence in the pAGUS1-TN2 plasmid construct resulted in a significant increase of GUS activity in the electroporated protoplasts of both callus types. On the other hand, protoplasts electroporated with the Adh1 promoter and intron showed a threefold less GUS activity than those electroporated with pAGUS1-TN2. Optimized conditions for DNA uptake and expression were very similar for protoplasts of both callus types. The importance of these findings for the successful regeneration of transgenic and fertile wheat plants is discussed.     

Synthesis and secretion of caseins by the mouse mammary gland: Production and characterization of new polyclonal antibodies

Polyclonal antibodies to mouse - and /-caseins were raised in rabbits. These antibodies display tissue- and species specificity as shown by immunoblotting. Immunohistochemical analyses demonstrate that both - and /-caseins were synthesized and secreted from virtually all lactating mammary epithelial cells, in a pattern very similar to that of the mouse -lactalbumin. Residual amounts of caseins were located also in the apical surface of epithelial cells surrounding the ducal lumen of virgin mammary gland sections. In contrast to the significant level of -casein in the milk, the amount of this protein compared to - or -caseins was extremely low in medium conditioned for 24 h by mammary explants of mid-pregnant mice immediately after explantation or after 4 days.     

The influence of interferon alpha and gamma,singly or in combination on human natural cell mediated cytotoxicity

The influence of interferon alpha and gamma alone or in combination on the augmentation of human natural cytotoxicity was studied. Treatment of peripheral blood lymphocytes with IFN- led to a rapid augmentation of NK activity, in contrast to IFN- where target cell killing was observed only following 18 hrs exposure of lymphocytes to IFN-. The results of the single cell assay paralleled those obtained using the Chromium release test, but neither interferon type caused an increase in the number of target binding lymphocytes. The combined effect of IFN- and IFN- in stimulating human natural cytotoxicity demonstrated individual lymphocyte responses to be variable. Exposure of lymphocytes to IFN- and IFN- for 18 hrs prior to assay for cytotoxicity usually decreased the level of cytotoxicity compared with control values, whereas other treatment regimes gave an additive and sometimes synergistic effect. Only treatment with IFN- for 18 hrs and IFN- for one hr produced a synergistic response in the majority of individuals tested. We conclude from this study that individual responses to IFN- and IFN- alone or in combination are variable and dependent upon timing of exposure of lymphocytes to individual interferon types, and possibly reflects the donor status at the time of sampling.     

Sex identification by male-specific growth hormone pseudogene (GH-Ψ) in Oncorhynchus masou complex and a related hybrid

It is often difficult to identify sexes of many fish species by conventional cytological method because of the lack of heteromorphic sex chromosomes. Isolation of sex-specific molecular markers is thus important for sexing and for understanding sex chromosome evolution in these species. We have identified genetic sexes by PCR-based male-specificity of a growth hormone pseudogene (GH-) in masu and Biwa salmon, two subspecies of the Oncorhynchus masou complex, and their hybrid Honmasu. PCRs with primers designed from sequences of chinook salmon GH genes amplified GH-I and GH-II fragments in both sexes, but a third GH- fragment was detected in predominant proportion of males and very few phenotypic females. The consistency of phenotypic sex with genetic sex identified by GH- for masu salmon, Biwa salmon and Honmasu was 93.1, 96.7 and 94%, respectively. The remaining individuals showed inconsistency or deviation from sex-specificity: a few phenotypic males lacked the GH-, whereas a few phenotypic females possessed the GH-. Sequence of the putative GH- fragment from such females was identical to that from genetic males, and shared about 95% homology with the corresponding GH- fragment from chinook salmon. This result confirmed that these females were really GH--bearing individuals. PCR analyses with primers designed from masu salmon GH- gave identical results, indicating that the absence of GH- in a few males was not resulted from primer mismatching. These GH--bearing females and GH--absent males were more likely to originate from spontaneous sex reversion than from crossing-over between GH- and the sex determination gene/region.     

Production of pineapple plants in vitro

In vitro culture of pineapple (Ananas comosus) was studied to determine the efficiency of axillary bud culture for rapid propagation of several cultivars. The technique used maximizes the success rate of various steps in the production of pineapple plants. Rapid mass multiplication of plantlets started 9 months after explanting with a significant log phase. The number of plantlets obtained from the culture of a single bud by the thirteenth month ranged from 210 to 380 for Perolera; 300 to 350 for PR-1-67; and 40 to 85 for Smooth Cayenne. The method permits culture of a range of pineapple cultivars. Little morphological variation was observed in young regenerated plants.