The frequency of the γ chain variant AγT in different populations,and its use in evaluating γ gene expression in association with thalassemia

Summary The occurrence of the ^AT chain (i.e. ^A75 Ile Thr) in different populations was evaluated through a study of 4250 cord blood samples and blood samples from more than 350 SS¹ patients. High frequencies were observed in Italy, Yugoslavia, Turkey, Holland, but also in Japan, Vietnam, and India. The chain is (nearly) absent in the Black population of Ghana and Kenya, and low frequencies were observed in China and Australian aborigines. Only a few adult SS patients (18 out of 357) were ^AT heterozygotes. The chromosomes with the ^AT globin gene were mapped through an evaluation of the presence of 10 different restriction sites. The ^AT chromosomes from different populations were closely related and had the same subhaplotypes of [--++-+] (Hinc II 5 to ; Xmn I 5 to ^G; Hind III in ^G and ^A; Hinc II in and 3 to ), quite different from the subhaplotypes seen for ^AT negative chromosomes.² This suggests a common ancestor which may have originated in Southern Europe. An evaluation of the chain production by both chromosomes in SS patients and -thalassemia heterozygotes was possible for subjects with an ^AT heterozygosity. It was concluded that in -thalassemia trait, the chain synthesis is directed for about two-thirds by the thalassemic chromosome and for about onethird by the normal chromosome; the contribution by the normal chromosome decreases with a decrease in total chain production.This is contribution #0890 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA     

A model of associative memory in the brain

A model of associative memory for time varying spatial patterns is proposed and simulated on a digital computer. This is a network composed of many neuron-like elements, and shows an ability for associative memory similar to that of the brain.Suppose a number of sequences of spatial patterns are presented to this network, for example, 12345, ABC, and so on. Then, these patterns are memorized in the network. After that, if any part of one of these sequences, say 23, is presented to the circuit, the rest of the sequence, 45, is recalled following to it. It resembles to such a situation — if we hear a part of a melody which we have memorized in the past, the rest of the melody is recalled even after it is stopped half-way. Although the recalled patterns are not always 100% correct, they are not completely destroyed even if the presented patterns are imperfect.     

The energy transmission in ATP synthase: From the γ-c rotor to the α3β3 oligomer fixed by OSCP-b stator via the βDELSEED sequence

ATP synthase (F₀F₁) is driven by an electrochemical potential of H⁺ (H⁺). F₀F₁ is composed of an ion-conducting portion (F₀) and a catalytic portion (F₁). The subunit composition of F₁ is ₃₃. The active ₃₃ oligomer, characterized by X-ray crystallography, has been obtained only from thermnophilic F₁ (TF₁). We proposed in 1984 that ATP is released from the catalytic site (C site) by a conformational change induced by the DELSEED sequence via -F₀. In fact, cross-linking of DELSEED to stopped the ATP-driven rotation of in the center of ₃₃. The torque of the rotation is estimated to be 420 pN·å from the H⁺ and H⁺-current through F₀F₁. The angular velocity () of is the rate-limiting step, because H⁺ increased theV _max of H⁺ current through F₀, but not theK _m (ATP). The rotational unit of F₀ (=ab₂c₁₀) is /5, while that in ₃₃ is 2/3. This difference is overcome by an analog-digital conversion via elasticity around DELSEED with a threshold to release ATP. The distance at the C site is about 9.6 å (2,8-diN₃-ATP), and tight Mg-ATP binding in ₃₃ was shown by ESR. The rotational relaxation of TF₁ is too rapid (=100 nsec), but the rate of AT(D)P-induced conformational change of ₃₃ measured with a synchrotron is close to . The ATP bound between the P-loop and E188 is released by the shift of DELSEED from RGL. Considering the viscosity resistance and inertia of the free rotor (-c), there may be a stator containing OSCP (= of TF₁) and F₀-d to hold free rotation of ₃₃.     

Novel prebiotic systems: Nucleotide oligomerization in surfactant entrapped water pools

Summary Oligomerization of 5-TMP in water pools entrapped by dodecyl-ammonium chloride surfactant aggregates in benzene: hexane in the presence of dicyanodiimide at temperatures ranging from 21°–72° resulted in the formation of linear and cyclic oligonucleotides containing up to pentamers. Effects of temperature, time and surfactants have been examined. Rate constants for the formation of oligomers have been determined at five different temperatures. These data afforded values of H = 11.8 ± 1.9 Kcal mole^–1, S=–53 6 e.u. and G = 27.4 4.0 Kcal mole^–1. Prebiotic significance of these results are discussed.     

Die Hypokotylfarbe als Markierungsfaktor von Genomstufen der Zuckerrübe

Zusammenfassung In zunehmendem Maße werden anisoploideBeta-Rübensorten angebaut, deren zytologische Kontrolle zwecks Feststellung der Genomstufenprozentanteile recht arbeitszeitaufwendig ist. Übereinstimmend mit polnischen Autoren wurde festgestellt, daß die Hypokotylfarbe ein geeigneter Markierungsfaktor für die einzelnen Genomstufen darstellt. Kreuzt man tetraploide Pflanzen, die ein grünes Hypokotyl besitzen, mit diploiden Pflanzen, die ein rosa Hypokotyl aufweisen, so erhält man von dem tetraploiden Partner tetraploide grüne und triploide hellbraune, von dem diploiden Partner diploide rosa und triploide hellbraune Nachkommenschaften. Die in bezug auf die Hypokotylfarbe heterozygoten Pflanzen kann man demnach von den homozygot grünen und homozygot rosa Individuen unterscheiden. Die Kreuzung diploid grünxtetraploid rosa ist für diese Zwecke nicht brauchbar, da sich die triploiden Heterozygoten mit einem grünen und zwei rosa Allelen in der Hypokotylfarbe nicht deutlich von den homozygoten rosa Pflanzen abheben. Auf die Bedeutung dieser Markierungsmöglichkeit für bestimmte Forschungsprobleme, die Züchtung und die Saatgutkontrolle wird hingewiesen.     

Heteronuclear NMR studies of 13C-labeled yeast cell wall β-glucan oligosaccharides

Summary The structures of uniformly ¹³C-labeled -glucan octa- and undeca-oligosaccharides enzymatically prepared by the yeast cell wall glucanosyl transferase of Candida albicans were characterized by using a combination of HCCH-COSY, HCCH-TOCSY, and HMBC experiments. The oligosaccharide structures indicate that the cell wall glucanosyl transferase cleaves two glucosyl units from the reducing end of the initial linear (13) penta-oligosaccharide and subsequently transfers the remainder to another oligosaccharide at the nonreducing end via a (16) linkage. These results indicate that the combined action of cell wall glucanase and glucanosyl transferase activities could not only introduce intrachain (16) linkages within a single glucan strand, but also result in cross-linking of two initially separate glucan strands with concurrent introduction of intrachain (16) linkages. Since isolated fungal membranes only synthesize linear (13) glucan strands, wall-associated enzymes probably participate in the assembly of the final wall glucan structure during cell growth and division.     

Filipino β-thalassemia due to a large deletion: identification of the deletion end points and polymerase chain reaction (PCR)-based diagnosis

The gene frequency of -thalassemia among Filipinos is estimated to be 0.02, although little is known about the mutations involved. Recently, an extensive -thalassemia deletion was reported in several unrelated individuals of Filipino descent. The deletion begins approximately 4 kb upstream of the -globin gene, and extends 3 beyond the -globin gene. In this report, we identify the 5 and 3 deletion endpoints and present a polymerase chain reaction (PCR) strategy for rapid DNA diagnosis of the Filipino -thalassemia deletion.     

Allospecific T cell recognition of HLA-A2 antigens: Evidence for group-specific and subgroup-specific epitopes

Interleukin 2-dependent alloreactive cytotoxic T cell lines, with activity predominantly directed against the HLA-A2 antigen, have been generated in vitro by stimulating blood mononuclear cells from donors nonimmune to the Epstein-Barr (EB) virus with appropriate numbers of EB virus-transformed B cells from A2-homozygous individuals. Such effector cells were tested against a panel of EB virus-transformed target cell lines all expressing the serologically defined A2 antigen but typed into common A2 and variant A2 subgroups on the basis of their recognition by A2-restricted EB virus-specific cytotoxic T cells. Variant A2 responder cells cocultivated with common A2-bearing stimulators gave rise to effector T cell lines which recognized only the common A2-bearing subgroup of targets. By contrast, responder cells from A2-negative donors stimulated with common A2-bearing cells produced effector T cell lines in which the strong lysis of common A2-bearing targets was accompanied by a lower, but still significant, lysis directed against all targets within the variant A2 subgroup. In both cases, lysis of the target cells was blocked equally well by the anti-A2-specific monoclonal antibody MA2.1 as by the monoclonal antibody W6/32 specific for HLA-A, -B, and -C determinants. This suggests that HLA-A2 molecules possess at least two distinct sets of epitopes capable of inducing alloreactive T cell cytotoxicity: first, epitopes probably associated with T cell-restricting sites, which generate subgroup-specific responses, and second, epitopes shared by all A2 molecules, and perhaps associated with serologically defined sites, which generate pan A2 group-specific responses.Abbreviations used in this paper EB Epstein-Barr - IL-2 Interleukin 2 - UM unfractionated mononuclear - AET aminoethylisothiouroniumbromide hydrobromide     

Fromβ-lactams toα- andβ-amino acid derived peptides

Summary The potential of-lactams as intermediates for the access to- and-amino acid-derived peptides is shortly reviewed, with major focus on the technologies developed in our group. The two general strategies lie, on one side, in the oxidative ring expansion of 3-hydroxy-lactams toN-carboxy-amino acid anhydrides or Leuch's anhydrides and subsequent coupling with-amino acid esters and, on the other side, in the nucleophilic ring opening ofN-Boc--lactams. Both approaches have been successfully applied to the synthesis of,-diamino acid,-amino--hydroxy acid, polyhydroxylated-amino acid,,-disubstituted-amino acid,-amino acid,-amino--hydroxy acid and,-disubstituted-amino acid derived peptides. Because of the mild reaction conditions needed for the above transformations and the highly stereoselective procedures employed for the construction of the starting-lactam ring, the whole process allows the production of optically pure final products.     

Hydrogen isotope discrimination in higher plants: Correlations with photosynthetic pathway and environment

Summary The ratio of deuterium to hydrogen (expressed as D) in hydrogen released as water during the combustion of dried plant material was examined. The D value (metabolic hydrogen) determined on plant materials grown under controlled conditions is correlated with pathways of photosynthetic carbon metabolism. C₃ plants show mean D values of-132 for shoots and -117 for roots; C₄ plants show mean D values of -91 for shoots and-77 for roots and CAM plants a D value of-75 for roots and shoots. The difference between the D value of shoot material from C₃ and C₄ plants was confirmed in species growing under a range of glasshouse conditions. This difference in D value between C₃ and C₄ species does not appear to be due to differences in the D value (tissue water) in the plants as a result of physical fractionation of hydrogen isotopes during transpiration. In C₃ and C₄ plants the hydrogen isotope discrimination is in the same direction as the carbon isotope discrimination and factors contributing to the difference in D values are discussed. In CAM plants grown in the laboratory or collected from the field D values range from-75 to +50 and are correlated with ¹³C values. When deprived of water, the D value (metabolic hydrogen) in both soluble and insoluble material in leaves of Kalanchoe daigremontiana Hamet et Perr., becomes less negative. These changes may reflect the deuterium enrichment of tissue water during transpiration, or in field conditions, may reflect the different D value of available water in areas of increasing aridity. Whatever the origin of the variable D value in CAM plants, this parameter may be a useful index of the water relations of these plants under natural conditions.     

Conformational changes of α-lactalbumin and its fragment, Phe31-Ile59, induced by sodium dodecyl sulfate

Conformational changes of bovine -lactalbumin in sodium dodecyl sulfate (SDS) solution were studied with the circular dichroism (CD) method using a dilute phosphate buffer ofpH 7.0 and ionic strength 0.014. The proportions of -helix and -structure in -lactalbumin were 34% and 12%, respectively, in the absence of SDS. In the SDS solution, the helicity increased to 44%, while the -structure disappeared. In order to verify the structural change from -structure to -helix, the moiety, assuming the -structure in the -lactalbumin, was isolated by a chymotryptic digestion. The structure of this -lactalbumin fragment, Phe³¹-Ile⁵⁹, was almost disordered. However, the fragment adopted a considerable amount of -helical structure in the SDS solution. On the other hand, the tertiary structure of -lactalbumin, detected by changes of CD in the near-ultraviolet region, began to be disrupted before the secondary structural change in the surfactant solution. Dodecyl sulfate ions of 80 mol were cooperatively bound to -lactalbumin. Although the removal of the bound dodecyl sulfate ions was tried by the dialysis against the phosphate buffer for 5 days, 4 mol dodecyl sulfates remained per mole of the protein. The remaining amount agreed with the number of stoichiometric binding site, determined by the Scatchard plot, indicating that the stoichiometric binding was so tight.     

On age morphological changes of males of Chydoridae (Cladocera)

Summary Young and adult males of 11 species of Chydoridae are studied, their figures being published here (fig. 1–15). The necessity is stressed to distinguish young forms of males and gynandromorphic individuals.Pleuroxus balatonicus is considered to be described from the population ofPleuroxus unicatus having under Balaton Lake conditions retarded transformation of young males into adult form, and accordingly having unusually numerous young males. \qO\qs\qn\qo\qv\qn\qy\ye \qr\ye\qz\qu\ql\Qj\qt\qa\qt\qy 11 (. 1–15). . , Pleuroxus uncinatus , Pleuroxus balatonicus.     

Dependence of transfection efficiency of calcium treated Escherichia coli cells on bacterial genotype and form of lambda DNA

Summary The transfecting activity of linear DNA is 100 times higher in calcium treated E. coli K 12 (ⁱ⁴³⁴) than in non-lysogenic strains: the levels of transfection are 1–2.10⁷ and 1–2.10⁵ infective centers per 1 g of DNA, respectively. The high efficiency of lysogenic cells transfection is not due to the spontaneously liberated helper phage. Evidently, it is called forth by transfecting DNA-prophage recombination or/and by inhibition of nuclease activity in lysogenic cells. Both ring forms DNA (supercoiled and open circles) show very low infectivity, if any, in calcinated cells.     

Circadian changes in protein-synthesis rate and protein phosphorylation in cell-free extracts of Gonyaulax polyedra

The polysomal pattern of the dinoflagellate Gonyaulax polyedra, cultured under constant conditions, demonstrates a circadian rhythm. The relative amount of polysomes increases during the phase corresponding to the previous night period (=subjective night phase) when the rate of protein synthesis reaches its maximum (Cornelius et al., 1985, Planta 160, 365–370). Cell-free extracts were isolated at different circadian phase. The rate of protein synthesis in the extracts changed rhythmically in the same manner as the rate of protein synthesis in vivo. Substances in the postribosomal supernatants influenced the protein-synthesis rate of the cell-free system, depending on the phase when they were isolated: night factors stimulated protein synthesis in day extracts whereas day factors inhibited protein synthesis in night extracts. These effects were abolished by heating the postribosomal supernatant. In-vitro phosphorylation in parallel probes showed changes in the pattern of phosphorylated proteins. Phosphorylation of one of the proteins (95 kDa) was decreased after addition of night factor(s) and increased after addition of day factor(s). Cyclic-AMP enhanced the rates of protein synthesis and phosphorylation in the day extracts.Abbreviations cAMP cyclic-AMP - CT circadian time - D (N) subjective day (night)phase     

A cyto-biochemical study of the ‘canal’ formation in the yeast cell wall

Summary A distinction between the chemical composition of ultrastructurally modified regions and the rest of the cell wall (canals) of the yeast Schwanniomyces occidentalis was shown by cytochemical staining of cell wall polysaccharides. The formation of canals was induced by cultivation of yeasts on hydrocarbons and was parallelled by the enhancement of -glucosidase, -glucanase and -mannosidase activities which were all capable of degrading cell wall polysaccharides. The presence of cycloheximide prevented canal formation. We assume that these hydrolases modified definite cell wall regions transforming them into canals.     

Localization of Transforming Growth Factor β in Association with Neuromuscular Junctions in Adult Human Muscle

Transforming growth factors- 1, 2, and 3 are known for their regulatory function in embryogenesis, fibrogenesis, and tissue repair of different cell types. A trophic function of TGF- subclasses for motoneurons has been shown in vitro. TGF- 1 is a potent survival factor for cultured embryonic rat motoneurons. In addition, TGF- 1 stimulates proliferation of rat Schwann cells. Recently, TGF- 2 has been reported to be associated with the subsynaptic nuclei of mature rat neuromuscular junctions. In this study, we investigated the expression of TGF- 1, 2, and 3 at neuromuscular junctions in skeletal muscle of 11 adults without neuromuscular disease. On muscle biopsies, neuromuscular junctions were depicted by acetylcholine esterase reaction and acetylcholine receptor antibodies. TGF- 1, 2, and 3 were stained immunohistochemically with monoclonal antibodies. Some muscle fibers showed low levels of inhomogeneous immunoreactivity for both TGF- 1 and TGF- 3. Intense immunoreactivity of TGF- 1 and 3 was shown at the postsynaptic area of neuromuscular junctions. TGF- 2 was expressed in the same subcellular distribution, but less strongly. In conclusion, the colocalization of TGF- with neuromuscular junctions may suggest a significant function in neuromuscular communication.     

Partial mispairing and crossing-over between β0 and δ genes as the origin of the δ β0 thalassemia gene

Summary Two double heterozygous ⁰/⁰ thalassemic sibs of Mexican descent were studied. The father had a ⁰/⁰ genotype, while the mother, one sib and several maternal relatives were ⁰/⁰ heterozygotes. Parental consanguinity and an apparently low frequency of thalassemia among Mexicans suggested a possible common origin of both ⁰ and ⁰ genes. A hypothesis to explain such a possibility is proposed on the basis of a partial mispairing between ⁰ and genes followed by a crossing-over which would results in a ⁰ recombinant gene. This hypothesis could also be extended to explain the 22 gluala, 22 alaglu and 116 arghis Hb variants as recombinants from double crossing-over between and mispaired genes for which the name interstitial-Lepore is proposed.     

Control of glycoprotein synthesis: substrate specificity of rat liver UDP-GlcNAc:Manα3R β2-N-acetylglucosaminyl-transferase I using synthetic substrate analogues

UDP-GlcNAc: Man3R 2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) is the key enzyme in the synthesis of complex and hybrid N-glycans. Rat liver GlcNAc-T I has been purified more than 25,000-fold (M _r 42,000). TheV _max for the pure enzyme with [Man6(Man3)Man6](Man3)Man4GlcNAc4GlcNAc-Asn as substrate was 4.6 µmol min^–1 mg^–1. Structural analysis of the enzyme product by proton nuclear magnetic resonance spectroscopy proved that the enzyme adds anN-acetylglucosamine (GlcNAc) residue in 1–2 linkage to the Man3Man-terminus of the substrate. Several derivatives of Man6(Man3)Man-R, a substrate for the enzyme, were synthesized and tested as substrates and inhibitors. An unsubstituted equatorial 4-hydroxyl and an axial 2-hydroxyl on the -linked mannose of Man6(Man3)Man-R are essential for GlcNAc-T I activity. Elimination of the 4-hydroxyl of the 3-linked mannose (Man) of the substrate increases theK _M 20-fold. Modifications on the 6-linked mannose or on the core structure affect mainly theK _M and to a lesser degree theV _max, e.g., substitutions of the Man6 residue at the 2-position by GlcNAc or at the 3- and 6-positions by mannose lower theK _M, whereas various other substitutions at the 3-position increase theK _M slightly. Man6(Man3)4-O-methyl-Man4GlcNAc was found to be a weak inhibitor of GlcNAc-T I.Abbreviations BSA Bovine serum albumin - Bn benzyl - Fuc, F l-fucose - Gal, G d-galactose - GalNAc, GA N-acetyl-d-galactosamine - Glc d-glucose - GlcNAc, Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man, M d-mannose - mco 8-methoxycarbonyl-octyl, (CH₂)₈ COOOCH₃ - Me methyl - MES 2-(N-morpholino)ethanesulfonate - NMR nuclear magnetic resonance - PMSF phenylmethylsulfonylfluoride - pnp p-nitrophenyl - SDS sodium dodecyl sulfate - T transferase - Tal d-talose - Xyl d-xylose; - {0, 2 + F} Man6 (GlcNAc2Man3) Man4GlcNAc4 (Fuc6) GlcNAc - {2, 2} GlcNAc2Man6 (GlcNAc2Man3) Man4GlcNAc4GlcNAc; M₅-glycopeptide, Man6 (Man3) Man6 (Man3) Man4 GlcNAc4GlcNAc-Asn Enzymes: GlcNAc-transferase I, EC 2.4.1.101; GlcNAc-transferase II, EC 2.4.1.143; GlcNAc-transferase III, EC 2.4.1.144; GlcNAc-transferase IV, EC 2.4.1.145; GlcNAc-transferase V, UDP-GlcNAc: GlcNAc2 Man6-R (GlcNAc to Man) 6-GlcNAc-transferase; GlcNAc-transferase VI, UDP-GlcNAc: GlcNAc6(GlcNAc2) Man6-R (GlcNAc to Man) 4-GlcNAc-transferase; Core 1 3-Gal-transferase, EC 2.4.1.122; 4-Gal-transferase, EC 2.4.1.38; 3-Gal-transferase, UDP-Gal: GlcNAc-R 3-Gal-transferase; blood group i 3-GlcNAc-transferase, EC 2.4.1.149; blood group I 6-GlcNAc-transferase, UDP-GlcNAc: GlcNAc3Gal-R (GlcNAc to Gal) 6-GlcNAc-transferase.