Sequences of the VH and VL regions of murine monoclonal antibodies against 3-fucosyllactosamine

Many mAb that bind the carbohydrate antigenic determinant 3-fucosyl-lactosamine (3-FL), Gal beta 1-4[Fuc alpha-3]GlcNAc-R have been raised in BALB/c mice, and we are studying the structure and regulation of these antibodies. In this report, we present the first information about their amino acid sequences and the Ig gene segments used to encode them. V regions of the H and L chains of three anti-3-FL antibodies, PMN6, PMN29, and PM81, were sequenced by a combination of mRNA and amino acid sequencing. The L chain sequences of PMN6 and PM81 antibodies indicate that their VK and JK regions are encoded by VK24B and JK1 germ-line genes, respectively. The nucleotide and amino acid sequences of the H chains suggest that the three anti-3-FL antibodies are encoded by the VH441 gene segment of the X24 VH family, and this conclusion was supported by Southern filter hybridization with VH441 and JH3-JH4 probes. PMN29 has at least 11 amino acid substitutions, which is an unusually large amount of somatic mutation for an IgM antibody. Previous analyses of BALB/c genomic libraries with VHX24 and VH441 probes make it unlikely that this VH family contains additional germ-line genes, but this possibility cannot be excluded. All three antibodies use the DQ52 and JH4 gene segments. The single VH and VL gene segments used to encode the anti-3-FL antibodies is in contrast to the multiple VH and VL segments used by antibodies against other carbohydrate Ag such as alpha 1-6 dextran and group A streptococcal carbohydrate. VH441 also encodes the VH regions of antibodies against galactan and levan (beta 2-6 fructosan). The similarities among VH segments of antibodies against 3-FL, levan, and galactan, and the striking differences in their CDR3 sequences, suggest that CDR3 plays an important role in the formation of the Ag binding site. The use of a single VH segment from the smallest VH gene family by antibodies against at least three different carbohydrate determinants is noteworthy. It raises the possibility that the amino acid sequence encoded by VH441 has some general structural features that make it particularly well adapted for binding to carbohydrate sequences.     

Membrane glycophorins in Sta blood group erythrocytes

Structural and immunochemical studies of glycophorins isolated from erythrocytes of an individual homozygous for the M Sta blood group phenotype are described. Reactivities with specific monoclonal antibodies indicated that two major M and N glycophorins were present. The M and N Sta glycophorins were resolved by Lens culinaris lectin affinity chromatography. The N species was not held on the lectin but the M species, like control alpha glycophorins, was retained and could be eluted with alpha-methylmannoside. The two proteins were present in almost equimolar amounts. Studies of the CNBr fragments provided evidence that the structure of M Sta glycophorin is the same as that of the usual M alpha glycophorin but that the N Sta glycophorin is a variant. The amino-terminal octapeptides of the M and N species were similar in amino acid and carbohydrate composition to those isolated, respectively, from M and N alpha glycophorins. The studies focused on CNBr glycopeptide B that, in control alpha glycophorins, extends from amino acid residues 9 to 81. The fragment from the M species exhibited properties identical to those of the corresponding fragment of control alpha glycophorins in terms of size, chromatographic behavior, amino acid and carbohydrate contents and compositions, the presence of O-glycosidically linked saccharides and a single Asn-linked carbohydrate unit. The structures of the O-linked units were inferred experimentally to be NeuAc(alpha 2,3)Gal-(beta 1,3)GalNAc and NeuAc(alpha 2,3)Gal(beta 1,3) [NeuAc(alpha 2,6)]GalNAc, present in a ratio similar to that found in controls; and the Asn-linked unit also appeared to be as in the control. The tryptic glycopeptide pattern of the M Sta glycophorin CNBr fragment B was identical to the pattern of the corresponding control fragment, and the composition of the tryptic peptides suggested sequence identity with the control fragment. In contrast, the N Sta glycophorin yielded two CNBr glycopeptides B; both contained fewer amino acid residues and virtually lacked Man and GlcNAc, indicating the absence of the Asn-linked carbohydrate. The much decreased levels of these carbohydrates in the intact N protein, corroborated the latter finding. The O-glycosidic saccharides appeared similar to those found in control alpha glycophorins. However, the tryptic glycopeptide pattern of the variant differed from control M or N alpha glycophorins, suggesting a deletion of a large segment of the molecule near residues 40-61 and/or a substitution of methionine for a residue upstream from residue 40.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and characterization of antithrombin III from human, porcine and rabbit plasma, and rat serum.

1. Human, porcine, rabbit, and rat antithrombin III have been purified by affinity chromatography using heparin-agarose. The amino acid and carbohydrate compositions, amino-terminal sequences, immunological cross-reactivities, and inhibitions of human thrombin were studied. 2. Human, porcine, rabbit, and rat antithrombin III are single-chain glycoproteins containing hexose, glucosamine, and neuraminic acid. 3. The total carbohydrate contents were 17, 16, 14, and 15% for human, porcine, rabbit, and rat antithrombin III, respectively. 4. Molecular weights estimated from the migration in sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis were 59,000, 58,000, 63,000, and 63,000 for human, porcine rabbit, and rat antithrombin III, respectively. 5. These four proteins have similar amino acid compositions, although some minor differences were noted. 6. Human, porcine, and rabbit antithrombin III have a histidine residue at the amino-terminus, while rat antithrombin III contains an amino-terminal asparagine residue. 7. The amino-terminal sequences up to the first 17 residues showed high homology among the four proteins. 8. Some immunological cross-reactivity was observed only between human and porcine antithrombin III. 9. The apparent dissociation constants (KI) for the complexes between human thrombin and human, porcine, rabbit, and rat antithrombin III were about 1.2 x 10(-10) M, 9.5 X 10 (-9) M, 1.4 X 10(-7) M, and 2.8 X 10(-9) M, respectively.     

Conformation of the oligosaccharide chain of G(M1) ganglioside in a carbohydrate-enriched surface.

The solution structure of ganglioside G(M1) carbohydrate moiety at the surface of a 102-kDa lipid-modified-G(M1) micelle is investigated by high-resolution 1H-NMR in H2O. The micellar surface can be considered a cluster-like lateral distribution of the gangliosides, each single monomer being anchored in a carbohydrate-enriched model membrane matrix. 1H NOESY measurements at short mixing times reveal a rigid trisaccharide core -beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)]-beta-Gal- and a more flexible beta-Gal-(1-3)-beta-GalNAc- terminal glycosidic bond. In the lipid-modified G(M1) ganglioside micellar system, there is no evidence that intermolecular side-by-side carbohydrate interactions modulate, or alter in any way, the head-group spatial arrangement. Possible intermonomer interactions at the level of the branched trisaccharide portion were further investigated on mixed micelles of natural N-glycolyl- and N-acetylneuraminic acid containing G(M1) in D2O, taking advantage of the different NMR features of N-glycolyl- and N-acetylneuraminic acids, which allow discrimination between sialic acid ring proton signals. Measurements of the water/ganglioside-OH proton chemical exchange rates suggest hydroxyl group involvement at position 8 of sialic acid in strong intramolecular interaction processes.     

Preparation and chemical characterisation of calf Tamm-Horsfall glycoprotein.

Tamm-Horsfall glycoprotein preparations were obtained from calf urine by 1.0 M NaCl precipitation followed by 4 M urea/Sepharose 4B chromatography. By using 0.1% sodium dodecyl sulfate polyacrylamide gel electrophoresis a molecular weight of 86 500 +/- 4500 (n = 12) was calculated for the glycoprotein. Amino acid and carbohydrate analyses were performed, the carbohydrate composition being (in residues per 100 amino acid residues in the glycoprotein): fucose, 0.90; galactose, 4.82; mannose, 4.63;N-acetylglucosamine, 7.36; N-acetylgalactosamine, 1.38; sialic acid, 2.93. Under conditions of mild acid hydrolysis (0.05 M H2SO4, 80 degrees C, 1 h) the calf Tamm-Horsfall glycoprotein preparations were degraded partially into two lower molecular weight fragments (approximate Mr 66 000 and 51 000), as shown by polyacrylamide gel electrophoresis, both fragments being periodic acid-Schiff reagent positive.     

Polylactosamine glycosylation on human fetal placental fibronectin weakens the binding affinity of fibronectin to gelatin

Gelatin-binding chymotryptic fragments from placental fibronectin contain polylactosamine carbohydrates (Zhu, B.C.R., Fisher, S.R., Pande, H., Calaycay, J. Shively, J.E., and Laine, R.A. (1984) J. Biol. Chem. 259, 3962-3970). We have separated polylactosamine-containing gelatin-binding fragments of placental fibronectin from their counterparts containing smaller "complex" N-linked saccharides using Sephadex G-200 gel permeation chromatography. The peptide portions of both fragments have similar amino acid composition and N-terminal sequence (see reference above). The strength of binding of these two glycosylation types of chymotryptic fragments to gelatin differs as shown by the following experiments. 1) Upon urea gradient elution of affinity-bound fibronectin fragments from gelatin-Sepharose chromatography, the apex of the elution peak for polylactosamine-containing fragments occurs at 2.0 M urea while the peak for complex N-linked carbohydrate-containing fragments maximized at 2.5 M urea indicating a tighter binding. Removal of polylactosamine sequences from the former glycopeptide by endo-beta-galactosidase digestion caused the elution peak for this fraction to change from 2.0 to 2.5 M, the same as for the complex N-linked carbohydrate-containing glycopeptide. 2) Competitive displacement experiments give an apparent dissociation constant of polylactosamine-containing fragments at 3 X 10(-9) M whereas this constant for complex carbohydrate-containing fragments is 1 X 10(-9) M. These results indicate that the binding of placental fibronectin to gelatin is weakened by the presence of high molecular weight polylactosamine carbohydrate. To our knowledge this is the first report that the type and extent of glycosylation of a glycoprotein can affect its binding affinity to a proteinacious ligand. Thus, fetal placental fibronectin may have different biological properties than fibronectins containing only the smaller N-linked complex carbohydrate.     

Study of the glycosylation of apolipoprotein H

Apolipoprotein H is a single chain polypeptide composed of 326 amino acids highly glycosylated. Its carbohydrate content is approximately 19% of the molecular weight. We show that it is rich in sialic acid linked alpha (2-6) to galactose or N-acetylgalactosamine. Sialic acid is not alpha (2-3) linked to galactose. Galactose is beta (1-4) linked to N-acetylglucosamine and beta (1-3) linked to N-acetylgalactosamine. Carbohydrate O-linked chains (mainly sialic acid) are alpha (2-6) linked to galactose or N-acetylgalactosamine. Galactose is also organised in O-linked chains and beta (1-4) linked to N-acetylglucosamine and beta (1-3) linked to acetylgalactosamine. Concanavalin A lectin was used to isolate two groups of apolipoprotein H molecules bearing biantennary and truncated hybrids and high mannose and hybrid oligosaccharides. Apolipoprotein H fails to bind lysine-Sepharose. Our results thus show that it presents truncated hybrid or hybrid-type carbohydrate chains which bear few unmasked mannose residues as a terminal sugar. Biochemical analysis of carbohydrate structures conducted on single isoforms separated through IEF revealed that no specific carbohydrate complex is bound to a single isoform.     

Plasma amino acid and ammonia responses to altered dietary intakes prior to prolonged exercise in humans.

This study examined the effects of altered dietary intakes on amino acid and ammonia (NH3) responses prior to and during prolonged exercise in humans. Six male recreational cyclists rode to exhaustion at 75% of VO2max following 3 days on a low carbohydrate (LC), mixed (M), or high carbohydrate (HC) diet in a latin square design. There were differences (p less than 0.05) in exercise times among all treatments (58.8 +/- 3.7, 112.1 +/- 7.3, and 152.9 +/- 10.3 min for the LC, M, and HC treatments, respectively). The rate of increase in plasma NH3 during exercise was greater (p less than 0.05) during the LC trial. The LC trial was also characterized by higher (p less than 0.05) resting plasma concentrations of branched chain amino acids (BCAA) and a greater decrease in these amino acids during exercise (p less than 0.05), as compared with the other two treatments. Both plasma BCAA and NH3 were susceptible to dietary manipulations. These findings suggest that limited carbohydrate availability in association with increased BCAA availability results in enhanced BCAA metabolism during exercise. This is reflected in a greater rate of increase in plasma NH3 and is consistent with the hypothesis that a significant fraction of the NH3 released during a prolonged, submaximal exercise bout is from amino acid catabolism.     

Interaction of leucine with the plasma membranes of catfish taste buds

The binding of L-[3H]leucine by the plasma membranes from the catfish Ictalurus nebulosus taste organ was studied. The two types of specific binding centers for amino acid with high (KD = 2,5 X 10(-10) M) and low (KD = 1,04 X 10(-9) M) affinities were found. Concentrations of high and low affinity centers were 11,85 nmol/mg and 26 nmol/mg respectively. The structural rearrangement of the surface layer of the chemoreceptor membrane was revealed by spin label technique using 5-doxylstearic acid at leucine concentrations 10(-4)-10(-9) M.     

Coordination abilities of the 1-16 and 1-28 fragments of beta-amyloid peptide towards copper(II) ions: a combined potentiometric and spectroscopic study

Stoichiometry, stability constants and solution structures of the copper(II) complexes of the (1-16H), (1-28H), (1-16M), (1-28M), (Ac-1-16H) and (Ac-1-16M) fragments of human (H) and mouse (M) beta-amyloid peptide were determined in aqueous solution in the pH range 2.5-10.5. The potentiometric and spectroscopic data (UV-Vis, CD, EPR) show that acetylation of the amino terminal group induces significant changes in the coordination properties of the (Ac-1-16H) and (Ac-1-16M) peptides compared to the (1-16H) and (1-16M) fragments, respectively. The (Ac-1-16H) peptide forms the 3N [N(Im)(6), N(Im)(13), N(Im)(14)] complex in a wide pH range (5-8), while for the (Ac-1-16M) fragment the 2N [N(Im)(6), N(Im)(14)] complex in the pH range 5-7 is suggested. At higher pH values sequential amide nitrogens are deprotonated and coordinated to copper(II) ions. The N-terminal amino group of the (1-16) and (1-28) fragments of human and mouse beta-amyloid peptide takes part in the coordination of the metal ion, although, at pH above 9 the complexes with the 4N [N(Im), 3N(-)] coordination mode are formed. The phenolate -OH group of the Tyr(10) residue of the human fragments does not coordinate to the metal ion.     

Gustatory responses of eel palatine receptors to amino acids and carboxylic acids

The gustatory receptors of the eel palate were found to be extremely sensitive to amino acids and carboxylic acids. The results obtained are as follows: (a) 11 amino acids which are among naturally occurring amino acids elicited responses in the palatine nerve, but 9 amino acids did not elicit a response even at a high concentration. The effect of D-amino acids was always much less than that of their corresponding L-isomers. There was no appreciable difference in the effectiveness of an alpha-amino acid (alpha-alanine) and beta-amino acid (beta-alanine). (b) The threshold concentrations of the most potent amino acids (arginine, glycine) were between 10(-8) and 10(-9) M. A linear relation between the magnitude of the response and log stimulus concentration held for a wide concentration range for all the amino acids examined. (c) The palatine receptors responded sensitively to various carboxylic acid solutions whose pH was adjusted to neutral. The threshold concentrations varied between 10(-4) and 10(-7) M. The magnitude of the response at 10(-2) M increased with an increase of carbon chain length. (d) The extent of cross-adaptation was examined with various combinations of amino acids. A variety of the response patterns showing complete cross-adaptation, no cross-adaptation, or synergetic interaction was observed. The synergetic interaction was also observed when one amino acid below its threshold concentration was added to the other amino acid below its threshold concentration was added to the other amino acid. No cross-adaptation was observed between amino acids and fatty acids. (e) The treatment of the palate with papain led to loss of the responses to arginine, glycine, and histidine without affecting those to proline and acetic acid. The treatment with pronase E eliminated selectively the response to proline. The possibility that the eel gustatory receptors are responsible for sensing food at a distance was discussed.     

H9N2亚型禽流感病毒基因组的遗传进化分析

2009～2011年从江苏省、湖北省和安徽省等地来源于鸡、鸭、鹌鹑和鸽子的样品中分离鉴定出16株H9N2亚型禽流感病毒。通过反转录聚合酶链式反应(RT-PCR)扩增出分离株的全基因片段,并对其进行测序及遗传进化分析。序列分析显示,16株病毒HA基因裂解位点氨基酸序列为P-S-R/K-S-S-R,符合低致病性禽流感的分子特征;226位均为L,具有与哺乳动物唾液酸α,2-6受体结合的特性。M2基因均出现了对金刚烷胺产生耐药性的N31S突变。不同宿主来源的H9亚型AIV的主要分子特征一致。全基因遗传进化分析表明16株H9N2亚型禽流感病毒全基因发生了3配体重组,即以F98亚系AIV为骨架,HA来源于Y280亚系,PB2和M基因来源于G1亚系,形成了2种新的基因型。因此,要加强对H9N2亚型禽流感病毒的监测,密切关注它的重组趋势。     

Cloning, expression and some properties of alpha-carbonic anhydrase from Helicobacter pylori

The alpha-carbonic anhydrase gene from Helicobacter pylori strain 26695 has been cloned and sequenced. The full-length protein appears to be toxic to Escherichia coli, so we prepared a modified form of the gene lacking a part that presumably encodes a cleavable signal peptide. This truncated gene could be expressed in E. coli yielding an active enzyme comprising 229 amino acid residues. The amino acid sequence shows 36% identity with that of the enzyme from Neisseria gonorrhoeae and 28% with that of human carbonic anhydrase II. The H. pylori enzyme was purified by sulfonamide affinity chromatography and its circular dichroism spectrum and denaturation profile in guanidine hydrochloride have been measured. Kinetic parameters for CO2 hydration catalyzed by the H. pylori enzyme at pH 8.9 and 25 degrees C are kcat=2.4x10(5) s(-1), KM=17 mM and kcat/KM=1.4x10(7) M(-1) x s(-1). The pH dependence of kcat/KM fits with a simple titration curve with pK(a)=7.5. Thiocyanate yields an uncompetitive inhibition pattern at pH 9 indicating that the maximal rate of CO2 hydration is limited by proton transfer between a zinc-bound water molecule and the reaction medium in analogy to other forms of the enzyme. The 4-nitrophenyl acetate hydrolase activity of the H. pylori enzyme is quite low with an apparent catalytic second-order rate constant, k(enz), of 24 M(-1) x s(-1) at pH 8.8 and 25 degrees C. However, with 2-nitrophenyl acetate as substrate a k(enz) value of 665 M(-1) x s(-1) was obtained under similar conditions.     

Purification and characterization of a prokaryotic glucoprotein from the cell envelope of Halobacterium salinarium.

The glycoprotein which accounts for approximately 50% of the protein and all of the nonlipid carbohydrate of the cell envelope of Halobacterium salinarium (Mescher, M. F., Strominger, J. L., and Watson S. W. (1974) J. Bacteriol. 120, 945-954) has been purified and partially characterized. The glycoprotein has an apparent molecular weight of 200,000, is extremely acidic, and has a carbohydrate content of approximately 10 to 12%. The carbohydrate included neutral hexoses, amino sugar, and uronic acid. Information regarding the number, composition, and mode of attachment of the carbohydrate chains was obtained by isolation and examination of the glycopeptides derived from degradation of cell envelope protein with trypsin and pronase. Trypsin digestion resulted in two glycopeptides. One of these was large (approximately 55,000 daltons) and had most of the neutral hexose linked to it. The carbohydrate moieties consisted of di- and trisaccharides of glucosylgalactose and (uronic acid, glucose)-galactose attached via O-glycosidic linkages between galactose and threonine. The other tryptic glycopeptide had a relatively large heterosaccharide attached to it via an alkaline-stable linkage. The heterosaccharide contained 1 glucose, 8 to 9 galactose, 1 mannose, and 10 to 11 glucosamine residues, and approximately 6 residues of an unidentified amino augar. The alkaline stability of the linkage and the amino acid composition of glycopeptides resulting from Pronase digestion of the tryptic glycopeptide showed that the heterosaccharide was attached to an asparagine residue, presumably via an N-glycosylamine bond to the amide group. The intact glycoprotein has a single N-linked heterosaccharide, 22 to 24 O-linked disaccharides, and 12 to 14 O-linked trisaccharides per molecule. N- and O-glycosidic linkages are the most common carbohydrate-protein linkages in mammalian glycoproteins but, to our knowledge, this is the first report of either type of linkage in a prokaryotic cell envelope protein.     

Characterization of the combining site of mouse myeloma protein M315

The interaction of M315 with 2,4-dinitrophenyl haptens was studied. 2,4-Dinitroaniline (DNP-NH2) showed maximum affinity to M315 at about pH 4. The pH dependence of the association constant of DNP-NH2 to M315 showed three transitions at pH 4.7, at pH 7.2, and below pH 9, respectively. Since the DNP-NH2 molecule has no charged group in this pH range, the transitions were explained in terms of amino acid residues with ionizable side chains in M315. Judging from the pK values and the effect of succinylation, these transitions were concluded to be related to ionizations of carboxyl, imidazole, and phenol groups, respectively. Measurement of the fluorescence of affinity-labeled M315 suggested that the transition at pH 4.7 reflected an equilibrium between two forms of M315 with different conformations of the combining site. The contribution of the amino acid sequence on the light (L) chain to the interaction with haptens was studied by use of antibodies (Abs) reconstituted from the heavy chain of M315 (H315) and either a homologous or a heterologous L chain. The reconstituted heterologous Ab (H315L952) showed similar pH dependence of binding to DNP-NH2 to that of the homologous Ab (H315L315). Moreover, the two Abs showed no appreciable difference in binding to DNP-haptens of different sizes. These results suggested that the difference in the amino acid sequences of L315 and L952, which originated by a somatic hypermutation, has little effect on the ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

广东人禽流感H5N1毒株M基因特性、进化和变异

通过对人禽流感H5N1毒株M基因序列的变异分析,揭示毒株M基因特征与进化。检测广东地区人禽流感H5N1毒株M基因核苷酸序列,同时检索全球人禽流感H5N1毒株M基因序列,采用DNAStar5.0软件对检索的人禽流感H5N1毒株M基因核苷酸序列进行比对和分析;并结合流行病学资料对变异毒株进行进化速度分析。结果发现,1997~2006年53株毒株M1基因和51株毒株M2核苷酸序列同源性均分成两组,1997年毒株为第一组(GⅠ),2003~2006年香港、越南、泰国、印尼、中国大陆、土耳其、伊拉克、阿塞拜疆、埃及毒株为第二组(GⅡ)。M1基因20个氨基酸位点置换,占7.94%(20/252),其中2003~2006年毒株M1基因有9个氨基酸位点不同于1997年毒株;M2基因22个氨基酸置换,占22.7%,其中2003~2006年毒株M2基因有4个氨基酸不同于1997年毒株。M2基因Ks值为26.8×10-6~42.6×10-6Nt/d,Ka值为4.39×10-6~6.98×10-6Nt/d;而M1基因的同义突变速度均远高于错义突变速度,显示M1基因受到机体免疫压力较小;检验发现M1基因进化存在负选择性压力。2003~2006年毒株M1基因通过氨基酸S224N置换,增加一个糖基化位点NSS224-226;而来自印尼的8株毒株M2基因发生C50F置换,引起蛋白二级结构改变。1997年中国香港人禽流感毒株自当时出现后,便未在以后人禽流感疫情中出现。2003~2006年毒株M1基因增加糖基化位点NSS224-226,可能与毒株致病性有关。人禽流感H5N1毒株M基因在自然界变异频繁,可能影响H5N1毒株的人-人传播能力。     

Variation in resistance of Mycobacterium paratuberculosis to acid environments as a function of culture medium

Acid resistance of Mycobacterium paratuberculosis was examined as a function of growth conditions (i.e., in vitro growth medium and pH). M. paratuberculosis was cultured in either fatty acid-containing medium (7H9-OADC) or glycerol-containing medium (WR-GD or 7H9-GD) at two culture pHs (pHs 6.0 and 6.8). Organisms produced in these six medium and pH conditions were then tested for resistance to acetate buffer at pHs 3, 4, 5, and 6 at 20 degrees C. A radiometric culture method (BACTEC) was used to quantify viable M. paratuberculosis cell data at various acid exposure times, and D values (decimal reduction times, or the times required to kill a 1-log(10) concentration of bacteria) were determined. Soluble proteins of M. paratuberculosis grown under all six conditions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to identify proteins that may be associated with acid resistance or susceptibility. The culture medium affected growth rate and morphology: thin floating sheets of cells were observed in 7H9-OADC versus confluent, thick, waxy, and wrinkled pellicles in WR-GD. Culture medium pH affected growth rate (which was highest at pH 6.0), but it had little or no effect on D values for M. paratuberculosis at any test pH. When grown in 7H9-OADC, M. paratuberculosis was more acid resistant at all test pHs (higher D values) than when grown in WR-GD. Glycerol appeared to be the culture medium component most responsible for lower levels of M. paratuberculosis acid resistance. When glycerol was substituted for OADC in the 7H9 medium, D values were significantly lower than those of 7H9-OADC-grown M. paratuberculosis and were approximately the same as those for M. paratuberculosis grown in WR-GD medium. Comparison of the SDS-PAGE protein profiles for M. paratuberculosis cultures grown in 7H9-OADC, WR-GD, or 7H9-GD medium revealed that increased expression of 34.2- and 14.0-kDa proteins was associated with higher levels of acid resistance of M. paratuberculosis grown in 7H9-OADC medium and that 56.6- and 41.3-kDa proteins were associated with lower levels of acid resistance. This is the first report showing that in vitro culture conditions significantly affect growth characteristics, acid resistance, and protein expression of M. paratuberculosis, and the results emphasize the importance of culture conditions for in vitro susceptibility studies.     

A single point mutation reverses the donor specificity of human blood group B-synthesizing galactosyltransferase

Blood group A and B antigens are carbohydrate structures that are synthesized by glycosyltransferase enzymes. The final step in B antigen synthesis is carried out by an alpha1-3 galactosyltransferase (GTB) that transfers galactose from UDP-Gal to type 1 or type 2, alphaFuc1-->2betaGal-R (H)-terminating acceptors. Similarly the A antigen is produced by an alpha1-3 N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine from UDP-GalNAc to H-acceptors. Human alpha1-3 N-acetylgalactosaminyltransferase and GTB are highly homologous enzymes differing in only four of 354 amino acids (R176G, G235S, L266M, and G268A). Single crystal x-ray diffraction studies have shown that the latter two of these amino acids are responsible for the difference in donor specificity, while the other residues have roles in acceptor binding and turnover. Recently a novel cis-AB allele was discovered that produced A and B cell surface structures. It had codons corresponding to GTB with a single point mutation that replaced the conserved amino acid proline 234 with serine. Active enzyme expressed from a synthetic gene corresponding to GTB with a P234S mutation shows a dramatic and complete reversal of donor specificity. Although this enzyme contains all four "critical" amino acids associated with the production of blood group B antigen, it preferentially utilizes the blood group A donor UDP-GalNAc and shows only marginal transfer of UDP-Gal. The crystal structure of the mutant reveals the basis for the shift in donor specificity.     

Purification, composition, and structure of macrophage adhesion molecule

Macrophage adhesion molecule (MAM) is a surface heterodimer consisting of the trypsin- and plasmin-sensitive glycopeptide gp160 (MAM-alpha) and the glycopeptide gp93 (MAM-beta). MAM, which is the guinea pig analogue of Mo1 and Mac-1, was purified from detergent lysates of peritoneal neutrophils by lentil lectin chromatography and M2-antibody chromatography. The pure heterodimer molecule was dissociated by acidic conditions (pH 3.5), and MAM-alpha and MAM-beta were separated by M7-antibody chromatography. MAM-beta is an approximately 640 amino acid residue polypeptide with exceptionally high cysteine content. At 7.2 residues per 100 amino acids, Cys/2 of MAM-beta is more than 3 times the mean for 200 purified proteins. Reactivity with six beta-subunit-specific monoclonal antibodies recognizing at least four epitopes demonstrated that intrapeptide disulfide bonds are required to maintain the structure of MAM-beta. All six antibodies failed to react when MAM-beta was treated with reducing agents. MAM-beta is 18% carbohydrate; the major monosaccharides are mannose, N-acetylglucosamine, galactose, and sialic acid. MAM-beta is estimated to contain five to six N-linked carbohydrate units. MAM-alpha is an approximately 1100-residue polypeptide with lower Cys/2 content (2.0 residues per 100 amino acid residues). MAM-alpha is 21% carbohydrate. The major monosaccharides are mannose, N-acetylglucosamine, galactose, and sialic acid; the mannose content is higher in MAM-alpha than MAM-beta. MAM-alpha is estimated to contain 12 N-linked carbohydrate units.     

Spectroscopic and redox properties of sym1 and (M)F195H: Rhodobacter capsulatus reaction center symmetry mutants which affect the initial electron donor.

The redox properties, absorption, electroabsorption, CD, EPR, and P+QA- recombination kinetics have been measured for the special pairs of two mutants of Rhodobacter capsulatus reaction centers involving amino acid changes in the vicinity of the special pair, P. Both mutants symmetrize amino acid residues so that portions of the M-sequence are replaced with L-sequence: sym1 symmetrizes all residues between M187 and M203, whereas (M)F195H is a single amino acid subset of the sym1 mutation. (M)F195H introduces a His residue in a position where it is likely to form a hydrogen bond to the acetyl group of the M-side bacteriochlorophyll of P. For both mutants compared with wild-type, (i) the redox potential is at least 100 meV greater, (ii) the P+QA- recombination rate is about twice as fast at room temperature, and (iii) the large electroabsorption feature for the QY band of P is shifted relative to the absorption spectrum. The comparison of the properties observed for the sym1 and (M)F195H reaction center mutants and the differences between these mutants and wild-type suggest that residue M195 is an important determinant of the properties of the special pair.