Recombination activating gene 1 (Rag1) in zebrafish and shark.
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Amino Acid Sequence↗
Biomedical subjects
L A Steiner
Publications and source records attributed to L A Steiner.
Comparative effects of esmolol and labetalol to attenuate hyperdynamic states after electroconvulsive therapy.
We studied 18 patients (age range, 53-90 yr) with at least one cardiovascular risk factor who were treated with electroconvulsive therapy (ECT) and compared effects of five pretreatments: no drug; esmolol, 1.3 or 4.4 mg/kg; or labetalol, 0.13 or 0.44 mg/kg. Each patient received all five treatments, during a series of five ECT sessions. Pretreatment was administered as a bolus within 10 s of induction or anesthesia. Doses of methohexital and succinylcholine were constant for the series of treatments and the assignment to no drug or to drug and dose was determined by randomized block design. Measurements of systolic and diastolic blood pressure (SBP, DBP) and heart rate (HR) were recorded during the awake state and 1, 3, 5, and 10 min after the seizure. The deviation of ST segments from baseline was measured by an electrocardiogram (ECG) monitor equipped with ST-segment analysis software. The results (mean +/- SEM) show that without pretreatment, there were significant (P < 0.05) peak increases in SBP and HR (55 +/- 5 mm Hg and 37 +/- 6 bpm, respectively), recorded 1 min after the seizure. Comparable reductions (by approximately 50%) in these peak values were achieved after esmolol (1.3 mg/kg) or labetalol (0.13 mg/kg), and cardiovascular responses were nearly eliminated after the same drugs in doses of 4.4 and 0.44 mg/kg, respectively. The deviation of ST-segment values from baseline in any lead was not measurably influenced by either antihypertensive drug. SBP values were lower after labetalol 10 min after the seizure, but not after esmolol. Asystolic time after the seizure was not significantly longer with either drug.(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenergic beta-Antagonists↗
Primary structure of Xenopus laevis S10, a ribosomal protein that cross-reacts with antibodies to immunoglobulin light chains.
Screening of a cDNA expression library from Xenopus laevis splenocytes with purified antibodies to Xenopus immunoglobulin light chains unexpectedly led to the isolation of a clone with an insert whose deduced amino acid sequence is similar to that of a segment of a protein, S10, from the small (40S) subunit of rat ribosomes. A clone containing an insert encoding the corresponding complete protein was isolated from another cDNA library by nucleic acid hybridization. The deduced amino acid sequence of this insert is 94% identical to that of rat S10; no similarity to immunoglobulin sequences could be discerned. The reactivity of the anti-light chain antibodies with the putative Xenopus S10 facilitated the purification of the protein, by high-pressure liquid chromatography, from the 40S subunit of Xenopus ribosomes. Amino-terminal sequence analysis established the identity of the ribosomal protein with the protein encoded by the cDNA insert. To explore the basis for this unexpected cross-reaction, an "antibody transfer" experiment was carried out. Antibodies to Xenopus light chains were adsorbed to Xenopus S10 on a nitrocellulose strip, which was incubated with another strip containing separated heavy and light chains from Xenopus IgM. Antibodies migrated from the strip carrying S10 to the light chains, but not the heavy chains, on the second strip. These results suggest that this unexpected cross-reaction is due to the sharing of one or more epitopes by Xenopus immunoglobulin light chains and the ribosomal protein, S10.
Amino Acid Sequence↗
Genes encoding Xenopus laevis Ig L chains. Implications for the evolution of kappa and lambda chains.
Xenopus laevis Ig contain two distinct types of L chains, designated rho or L1 and sigma or L2. We have analyzed Xenopus genomic DNA by Southern blotting with cDNA probes specific for L1 V and C regions. Many fragments hybridized to the V probe, but only one or two fragments hybridized to the C probe. Corresponding C, J, and V gene segments were identified on clones isolated from a genomic library prepared from the same DNA. One clone contains a C gene segment separated from a J gene segment by an intron of 3.4 kb. The J and C gene segments are nearly identical in sequence to cDNA clones analyzed previously. The C segment is somewhat more similar and the J segment considerably more similar in sequence to the corresponding segments of mammalian kappa chains than to those of mammalian lambda chains. Upstream of the J segment is a typical recombination signal sequence with a spacer of 23 bp, as in J kappa. A second clone from the library contains four V gene segments, separated by 2.1 to 3.6 kb. Two of these, V1 and V3, have the expected structural and regulatory features of V genes, and are very similar in sequence to each other and to mammalian V kappa. A third gene segment, V2, resembles V1 and V3 in its coding region and nearby 5'-flanking region, but diverges in sequence 5' to position -95 with loss of the octamer promoter element. The fourth V-like segment is similar to the others at the 3'-end, but upstream of codon 64 bears no resemblance in sequence to any Ig V region. All four V segments have typical recombination signal sequences with 12-bp spacers at their 3'-ends, as in V kappa. Taken together, the data suggest that Xenopus L1 L chain genes are members of the kappa gene family.
Amino Acid Sequence↗
Wild mice express an Ig V lambda gene that differs from any V lambda in BALB/c but resembles a human V lambda subgroup.
The lambda L chain locus in the inbred mouse strains commonly used in the laboratory contains a limited number of germ-line genes; only three V lambda and three functional J lambda-C lambda genes have been identified in BALB/c mice. Previous studies indicated that wild mice may have a considerably expanded number of C lambda genes, as judged by the number of DNA restriction fragments that hybridize to C lambda probes derived from BALB/c. In order to evaluate the expression of these putative lambda genes, we have determined sequences of cDNA encoding lambda-chains in hybridomas from wild mice of the subspecies Mus musculus musculus from two different geographic regions, Denmark and Czechoslovakia. Two of these hybridomas produce L chains with J and C regions that are very similar to those of BALB/c lambda 1 chains, but the V regions of these L chains are only approximately 40% identical in amino acid sequence to the known murine V lambda. Indeed, these wild mouse V lambda are closer in sequence to human V lambda than they are to BALB/c V lambda, especially to human V lambda of subgroup VI, with which they share an unusual two-residue insertion in framework 3; L chains bearing V regions of this rare human type have a marked tendency to enter into amyloid deposits. These findings suggest that similar V lambda may be widespread in mammalian populations, although analysis by Southern blotting indicates that they are not found in BALB/c mice. A third hybridoma produces a L chain whose V lambda resembles BALB/c V lambda 1. The J lambda and C lambda segments of the cDNA encoding all three hybridoma L chains are identical; evidently, of the several putative genes that hybridize to C lambda 1 probes, one is expressed preferentially.
Amino Acid Sequence↗
Sequence of C region of L chains from Xenopus laevis Ig.
A cDNA expression library, prepared from Xenopus laevis splenocytes, was screened with antibodies to Xenopus Ig. One clone, lambda XIg23, reacted with antibodies to IgY and to IgM; the insert hybridized to approximately 1.3-kb RNA from spleen, the approximate size expected for L chain mRNA. An additional clone, lambda XIg31, was identified by cross-hybridization. The inserts of lambda XIg23 and lambda XIg31 begin in the third framework region of the V region and extend through the C region to the poly(A) tail. Except for a single nucleotide difference, the two C region sequences are identical. The amino acid sequence of the C region was compared with the sequences of a variety of C kappa and C lambda, as well as to C region sequences of L chains from Rana catesbeiana and from two species of shark. The Xenopus C region resembles mouse and human C kappa slightly more than C lambda. The similarity of the Xenopus and Rana C regions to each other is approximately the same as that of either amphibian sequence to mammalian CL. The data are discussed in terms of the evolution of kappa and lambda C regions.
Amino Acid Sequence↗
Adult lumbar scoliosis. Treatment with combined anterior-posterior spinal fusion.
Innovative surgical approaches to current adult lumbar scoliosis result in faster recovery time and greater satisfaction regarding patient abilities after surgery. Combined ASF/PSF requires expert surgical team coordination throughout the perioperative period to achieve positive patient results.
Evoked Potentials, Somatosensory↗
J chain in Rana catesbeiana high molecular weight Ig.
A polypeptide homologous to human and mouse J chain has been identified in the high molecular weight (HMW) Ig of the bullfrog, Rana catesbeiana. In previous studies, we had detected a component that was similar in size to mammalian J chains and that, relative to L chains, migrated rapidly to the anode in alkaline-urea PAGE; however, its mobility was less than that of mammalian J chains. We now demonstrate that this component is covalently linked to the H chain of R. catesbeiana HMW Ig. All of the disulfide bridges of this polypeptide, like those of human and mouse J chain, can be cleaved by reducing agents even in the absence of denaturing solvents. The putative frog J chain was isolated by a procedure that did not require preliminary purification of the HMW Ig. The chain differed in amino acid composition from L chains but resembled J chains from several other species. Tryptic peptides were isolated and sequenced. Except for a single heptapeptide, the peptides could be aligned by virtue of their similarity to segments of human and mouse J chain. Of the 116 residues that were placed, 55 were identical with residues in human J chain and 60 with residues in mouse J chain. The six cysteine residues identified in the frog J chain are at the same positions as six of the eight cysteines in the human and mouse J chains. The results indicate significant conservation in structure between amphibian and mammalian Ig J chains.
Amino Acid Sequence↗
Amino acid sequence of the constant region of immunoglobulin light chains from Rana catesbeiana.
Little is known about the detailed structure of immunoglobulins in non-mammalian vertebrates. We have determined the amino acid sequence of the constant region of immunoglobulin light chains from the bullfrog, Rana catesbeiana. There appears to be one major type of light chain in this species. However, at position 153, about half of the chains have lysine, the remainder having arginine. Variation at this same or at an adjacent position is responsible for allotypic or isotypic variation in human kappa and lambda chains. At three positions (118, 119, and 181), residues that occur in all known kappa and lambda chains in other species are replaced in the Rana catesbeiana sequence. One of these unusual substitutions--the replacement of proline by cysteine at position 119--allows the formation of an extra intrachain disulfide bond within the constant domain, between positions 119 and 214. This bond appears to replace the usual disulfide bridge between heavy and light chains so that, in the immunoglobulins of this species, the light chains are not covalently bonded to heavy chains. The sequence of the Rana catesbeiana constant region is compared to sequences of a variety of mammalian light chain constant regions. We consider the implications of these comparisons for the timing of the divergence of kappa and lambda chains relative to the divergence of the lineages leading to amphibians and to mammals and birds.
Amino Acid Sequence↗
Amino acid sequence of heavy chain from Xenopus laevis IgM deduced from cDNA sequence: implications for evolution of immunoglobulin domains.
Present understanding of the evolution of immunoglobulins is derived almost entirely from studies of a few mammalian species. To obtain information about immunoglobulin genes in Xenopus laevis, a cDNA library was prepared in the expression vector lambda gt11 from mitogen-stimulated splenocytes of this species. Of approximately equal to 50,000 clones screened, 18 were found to express IgM epitopes. One of these, lambda XIg14, hybridized with RNA of RNA of approximately equal to 2 kilobases from splenocytes. The insert of this clone appears to encode a variable region and part of a mu constant region; that of another clone, lambda XIg8, appears to encode a variable region and a complete mu constant region. Both inserts contain sequence corresponding to the three gene segments (VH, DH, and JH) that encode heavy-chain variable regions. The heavy-chain constant region (CH) encoded by lambda XIg8 has the characteristic features of C mu, including a four-domain structure and a carboxyl-terminal tail. The amino acid sequences of two mu-chain peptides agree with the cDNA sequence. The identity in amino acid sequence between the corresponding Xenopus and mouse C mu domains ranges from 31 to 47%. The C mu domains vary in the extent to which their sequences resemble the sequences of other immunoglobulins, consistent with previous suggestions that the immunoglobulin domains have an independent evolutionary history.
Amino Acid Sequence↗
Primary structure of the reaction center from Rhodopseudomonas sphaeroides.
The reaction center is a pigment-protein complex that mediates the initial photochemical steps of photosynthesis. The amino-terminal sequences of the L, M, and H subunits and the nucleotide and derived amino acid sequences of the L and M structural genes from Rhodopseudomonas sphaeroides have previously been determined. We report here the sequence of the H subunit, completing the primary structure determination of the reaction center from R. sphaeroides. The nucleotide sequence of the gene encoding the H subunit was determined by the dideoxy method after subcloning fragments into single-stranded M13 phage vectors. This information was used to derive the amino acid sequence of the corresponding polypeptide. The termini of the primary structure of the H subunit were established by means of the amino and carboxy terminal sequences of the polypeptide. The data showed that the H subunit is composed of 260 residues, corresponding to a molecular weight of 28,003. A molecular weight of 100,858 for the reaction center was calculated from the primary structures of the subunits and the cofactors. Examination of the genes encoding the reaction center shows that the codon usage is strongly biased towards codons ending in G and C. Hydropathy analysis of the H subunit sequence reveals one stretch of hydrophobic residues near the amino terminus; the L and M subunits contain five such stretches. From a comparison of the sequences of homologous proteins found in bacterial reaction centers and photosystem II of plants, an evolutionary tree was constructed. The analysis of evolutionary relationships showed that the L and M subunits of reaction centers and the D1 and D2 proteins of photosystem II are descended from a common ancestor, and that the rate of change in these proteins was much higher in the first billion years after the divergence of the reaction center and photosystem II than in the subsequent billion years represented by the divergence of the species containing these proteins.
Amino Acid Sequence↗
Evidence that wild mice (Mus musculus musculus) express lambda genes that differ from those in BALB/c.
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Animals↗
An extra disulfide bridge in the constant domain of Rana catesbeiana immunoglobulin light chains.
The immunoglobulins of the bullfrog Rana catesbeiana are unusual in that, in all classes, the light chains are not disulfide bonded to heavy chains or to other light chains. Moreover, the light chains contain six, rather than the usual five, residues of half-cystine. As none of these half-cystines is in the sulfhydryl form or is alkylated after mild reduction, we suggested that the light chains probably contain three intrachain disulfide bridges. We have now carried out experiments to confirm the existence of an extra intrachain disulfide bridge in Rana catesbeiana light chains and to determine its location. Disulfide bridge assignments were based on 1) isolation and sequence analysis of S-(carboxymethyl)cysteine-containing peptides and 2) isolation, from unreduced light chains, of peptides containing a disulfide bridge. Half-cystine residues were found at positions 134 and 194, and these were shown to be joined in the conserved intradomain disulfide bridge. In addition, we found that a residue of half-cystine, located at the third position from the carboxy-terminus, forms a disulfide bridge with a half-cystine at position 119, near the amino-terminus of the domain, the latter residue replacing a proline that has been found at this position in all other light chains. An intrachain disulfide bridge has not been found at this location in any other light chain.
Amino Acid Sequence↗
Primary structure of the L subunit of the reaction center from Rhodopseudomonas sphaeroides.
The reaction center is an integral membrane protein that, together with several cofactors, mediates the primary photochemical events in bacterial photosynthesis. The amino-terminal sequences of the three subunits, L, M, and H, of the reaction center protein and the sequence of the structural gene encoding the M subunit have been reported previously. In the present study, we found that the 3' end of the structural gene encoding the L subunit overlaps by eight bases the 5' end of the gene encoding the M subunit. The primary structure of the L subunit has been determined from the nucleotide sequence of the gene and from analyses of the amino and carboxyl termini of the protein. The sequences of a number of tryptic and chymotryptic peptides were used to corroborate the nucleotide sequence. The L subunit was found to be composed of 281 amino acids (Mr 31,319) and to contain five hydrophobic segments. It is homologous to the M subunit and to a plant thylakoid protein referred to as the QB or Mr 32,000 protein.
Amino Acid Sequence↗
Amino- and carboxy-terminal sequence of mouse J chain and analysis of tryptic peptides.
Mouse J chain was isolated from an IgM-producing hybridoma by gel filtration and ion-exchange chromatography. The sequence of the amino-terminal 25 residues was determined. At these positions, the results agree with the amino acid sequence deduced from the cDNA sequence determined previously by Koshland and co-workers and indicate that a leader sequence terminating in glycine is removed to form the mature J chain. Tryptic peptides of J chain were isolated by high pressure liquid chromatography and their amino acid compositions were compared with those expected from the cDNA sequence. The amino acid sequence of the carboxy-terminal peptide and a mixture of two other peptides was determined. The results were consistent with the cDNA sequence except that we found valine, not leucine, at position 67, and arginine, not glycine, at position 117. The presence of aspartic acid at the carboxy-terminus, as predicted from the cDNA, indicates that processing does not occur at this end of the polypeptide chain. Upon amino acid analysis, glucosamine was found in tryptic peptides 47-57 and 47-58. J chain was also cleaved at aspartylproline bonds with formic acid and the unfractionated digest was subjected to automated Edman degradation. The mixed sequence was consistent with the sequence deduced from the cDNA at positions 1 to 13, 28 to 40, 52 to 64, and 73 to 85. In conjunction with the results obtained previously by analysis of cDNA, these data show that mouse J chain is a polypeptide containing 137 amino acid residues, 93 of which are identical to residues in human J chain.
Amino Acid Sequence↗
Noncovalent association of heavy and light chains in Rana catesbeiana immunoglobulins.
The unreduced immunoglobulins (Ig) in the bullfrog, Rana catesbeiana, dissociate into two components when subjected to electrophoresis or molecular sieving in dissociating solvents. One of these components is monomeric light chain and the other is a disulfide-bonded complex of heavy chains. This unusual behavior has been observed with all classes of bullfrog Ig that have been isolated and characterized previously: a high m.w. Ig that resembles mammalian IgM and two antigenically distinct varieties of low m.w. Ig. Light chains, isolated from the high m.w. Ig by gel filtration in 8 M urea, 1 M acidic acid, were found to contain, on average, 5.7 residues of half-cystine. None of these residues were in the free sulfhydryl form nor were they blocked by half-cystine. Moreover, none was alkylated after mild reduction of the high m.w. Ig. These findings indicate that none of the light chain half-cystine residues participate in an interchain disulfide bridge, and that most of the light chains contain three intrachain bridges. This unusual pattern of disulfide bonding appears to be responsible for the noncovalent association of heavy and light chains in this species.
Amino Acids↗
Amino acid sequence diversity in mouse lambda 2 variable regions.
The lambda-chains of immunoglobulins from BALB/c mice constitute the simplest system presently available for studying patterns of variable-region diversity. The limited number of V lambda and J lambda germ-line gene segments facilitates comparison of expressed and germ-line sequences. We report here the complete amino acid sequence of the variable regions of three lambda 2 chains and of one chain representing a V lambda 2----J lambda 3 rearrangement. Together with the previously determined sequence of the lambda 2 chain from myeloma MOPC-315, the results illustrate the following types of variable-region diversification: expression of a single V gene segment with more than one J segment, variability at the V-J junction, and presumably, somatic mutation in V and in J. The extent of somatic diversification in these lambda 2 chains is limited, consistent with results obtained previously with lambda 1 chains.
Amino Acid Sequence↗
Electron microscopy of the complement protein C1q from the bullfrog, Rana catesbeiana.
The complement protein C1q, isolated from bullfrog (Rana catesbeiana) serum, was found by electron microscopy to resemble human C1q; peripheral globular units, probably six in number, are connected by thin strands to a hollow stem-like central structure. The dimensions of frog and human C1q were also found to be very similar. These results are consistent with earlier observations that frog and human C1q are similar, although not identical, in overall size, subunit structure, amino acid composition, and functional properties. Evidently this protein, which binds to antigen-antibody complexes and to C1r and C1s, thereby forming a physical link between the immune and complement systems, has been highly conserved in evolution.
Animals↗