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L A Steiner

Publications and source records attributed to L A Steiner.

At least 19 recordsLinked to original sources
PubMed1997

Expression of zebrafish rag genes during early development identifies the thymus.

Recent experiments have demonstrated that zebrafish is a vertebrate in which it is possible to carry out large-scale mutagenic screens to identify genes involved in specific developmental pathways. To follow development of the immune system in zebrafish, we have analyzed the expression of the recombination activating genes, rag1 and rag2, which we have previously isolated and characterized. These genes catalyze the rearrangement of immunoglobulin genes in immature B lymphocytes and of T cell receptor genes in immature T lymphocytes and are therefore appropriate markers to follow the development of organs containing these cells. By whole-mount in situ hybridization, we detected expression of both rag genes in a paired organ in the head, beginning on the fourth day after fertilization. Histological examination of this organ indicated that it corresponds to the thymus, as described for other fish, an organ that has not previously been identified in zebrafish. By histological analysis, the thymus primordium appears at 54 hr but does not enlarge significantly until 30 hr later. The thymus continues to enlarge and reaches its mature histological organization at 1 month. The pronephros, the major hematopoietic organ in the adult fish, begins to develop hematopoietic tissue about 2 weeks after fertilization. By 1 month, mature lymphocytes are distinguishable in the tissue surrounding renal tubules. Lymphocytes appear in the kidney too late for screening by whole-mount in situ hybridization; however, the pattern of rag1 expression in the thymus forms the basis of an assay for mutations affecting development of the thymus or its constituent lymphocytes.

Animals↗
PubMed1997

Characterization and expression of the recombination activating genes (rag1 and rag2) of zebrafish.

The closely linked recombination activating genes, rag1 and rag2, encode components of the recombinase involved in V(D)J recombination of the immunoglobulin and T-cell receptor genes. These genes are expressed together exclusively in immature lymphocytes and are useful markers for following the development of lymphoid tissues. We cloned the rag locus of the zebrafish Danio rerio and sequenced the open reading frames of the rag1 and rag2 genes. Although the gene organization is similar to that in other species, the rag1 gene is unusual in possessing two introns within the coding region. In another teleost, the rainbow trout, the rag1 gene is interrupted by a single intron. Introns are not present in the rag1 gene of any other species examined to date. Expression of both rag1 and rag2 begins late in embryonic development, on day 4, by northern RNA blot analysis. Expression of rag1 was detected in the adult zebrafish thymus, pronephros, mesonephros, and ovary. This pattern of expression is consistent with previous histological studies of adult teleosts, which implicate the kidney as the major site of hematopoiesis and the thymus as the major lymphocyte-containing organ.

Amino Acid Sequence↗
PubMed1997

Sequence and expression pattern of J chain in the amphibian, Xenopus laevis.

We have determined the cDNA sequence encoding J chain, a polypeptide accessory molecule associated with polymeric Ig, from the anuran amphibian, Xenopus laevis (South African clawed frog). The translated polypeptide consists of 164 amino acid residues, including the signal sequence, and is somewhat longer than the corresponding sequence in mouse and cow, the two mammalian species in which the signal sequence of J chain has been determined. J chain in several mammalian species (human, mouse, cow and rabbit) has eight Cys residues. In the human chain, two of these Cys residues, the second and third in the sequence, have been shown to form disulfide bridges to heavy chains in IgM or IgA; the remaining Cys residues form intrachain disulfide bonds. The Xenopus J chain contains only seven of these Cys residues. Ser is found at the position corresponding to the third Cys in mammalian J chains. Northern blot analysis, performed on RNA isolated from various organs of 3-month old frogs, indicated that the highest level of expression was in the intestine. Transcripts corresponding to J chain were also detected in the spleen and at very low levels in the thymus.

Amino Acid Sequence↗
PubMed1996

Renovascular responses to high and low perfusate calcium steady-state experiments in the isolated perfused rat kidney with baseline vascular tone.

Acute hypercalcemia is commonly observed in surgical patients after calcium infusion while acute hypocalcemia is common during rapid citrated blood transfusion. Although high and low ionized calcium ([Ca2+]) within the clinical range produce an increase or decrease in cardiac performance and systemic vessel resistance, respectively, their effects on renal vessels have not been quantified. A possible renal vasoconstriction that might occur with high [Ca2+] is of clinical interest because it is a factor which may contribute to impaired renal circulation and decreased function. In this study we examined the renovascular responses to [Ca2+], which was varied within the clinical range under hemodynamically controlled conditions. We instituted high and low [Ca2+] in the per fusate, which consisted of Krebs-Henseleit buffer containing albumin, 60-65 g/liter. Stable high (n = 10) or low (n = 7) [Ca2+] (1.93 +/- 0.02 and 0.59 +/- 0.01 mM, respectively) was instituted for 10 min and preceded and followed by normal [Ca2+] of the same duration. In a separate protocol (n = 8) verapamil (10(-5) M) was added to the perfusate 10 min before high [Ca2+] was tested. We measured changes in renal flow at a constant perfusion pressure of 110 mm Hg and also characterized the renal vessels over a range of pressures by pressure vs flow plots. High [Ca2+] was associated with a small decrease in flow (from 28.8 +/- 2.4 to 26.9 +/- 2.6 ml/min/g, P < 0.02), indicating a small vasopressor effect. This effect was also shown by a leftward shift in the pressure vs flow plots. These changes were prevented by verapamil. GFR decreased (from 0.35 +/- 0.04 to 0.28 +/- 0.06 ml/min/ g, P < 0.01) without a significant change in sodium excretion or fractional sodium excretion. Low [Ca2+] was associated with increased renal flow (from 30.8 +/- 2.1 to 35.2 +/- 2.7 ml/min/g, P < 0.02), indicating a vasodilator effect. This effect was also shown by a rightward displacement of the pressure vs flow plots. GFR increased from 0.51 +/- 0.03 to 0.56 +/- 0.04 ml/min/ g, P < 0.01, as did sodium excretion (from 2.32 +/- 0.22 to 3.87 +/- 0.49 microEq/min, P < 0.01) and fractional sodium excretion (from 2.33 +/- 0.26 to 3.61 +/- 0.49%, P < 0.01). We conclude, first, that in the isolated perfused rat kidney, high [Ca2+] is a weak vasopressor while low [Ca2+] has vasodilator action. Second, high [Ca2+] effects are abolished by verapamil pretreatment. These findings illuminate mechanisms of high [Ca2+] effects on renovascular tone.

Animals↗
PubMed1996

Renal vascular responses to high and low ionized calcium: influence of norepinephrine in the isolated perfused rat kidney.

OBJECTIVE AND DESIGN: The aim of this study was to examine the influence of norepinephrine (NE) on renal vascular responses to high (1.88 mmol/L) and low (0.56 mmol/L) perfusate-ionized calcium ([Ca2+]) in the isolated perfused kidney of the rat. High and low [Ca2+] encompassed the clinical concentration range in this multiexperiment, randomized trial. MATERIALS AND METHODS: Rats (n = 25), ranging in age from 3 to 4 months, were anesthetized and the ureter and renal artery were cannulated. The right kidney was perfused with oxygenated, warmed albumin (67 g/L) containing Krebs-Henseleit buffer and placed in a thermostated chamber without interruption of flow. In protocol A (n = 7), steady-state high [Ca2+] (1.88 mmol/L) and low [Ca2+] (0.56 mmol/L) were instituted in randomized order in each experiment under basal conditions. In protocol B (n = 9), the same interventions were instituted during constant rate NE infusion. Changes in renal flow were measured at constant perfusion pressure (110 mm Hg), and renal vascular resistance (RVR) was calculated. Renal function was assessed by clearance of [14C]inulin and by fractional excretion of sodium. With NE-induced preconstriction, the increase in RVR observed during high [Ca2+] was +17.8 +/- 1.8% of control, and the decrease in RVR observed during low [Ca2+] was -35.9 +/- 8.2% of control. Both values were greater by a factor of 2 than corresponding results obtained under basal conditions (7 +/- 2.1% vs. -13.5 +/- 4.1% of control, respectively, p < 0.05). Whereas the decrease in glomerular filtration rate with high [Ca2+] was not significantly influenced by NE pretreatment (-9 +/- 1.8% of control with high [Ca2+] in combination with NE vs. 4.1 +/- 0.7% of control under basal conditions), the increase in glomerular filtration rate with low [Ca2+] was significantly greater in the presence of NE (12 +/- 0.7 vs. 102 +/- 8.5% of control, p < 0.01). CONCLUSIONS: Whereas under basal conditions renal vascular effects of high and low [Ca2+] (varied within the clinical concentration range) are small, the changes recorded with the same interventions after NE pretreatment are increased by a factor of > 2. Hypercalcemia-induced renovascular constriction and decreased function are unfavorable, especially in patients who are at risk for renal dysfunction from other causes.

Animals↗
PubMed1995

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↗
PubMed1993

Characterization and expression of recombination activating genes (RAG-1 and RAG-2) in Xenopus laevis.

The primary repertoire of B and T cells is established by V(D)J recombination. Two closely linked genes, RAG-1 and RAG-2, are essential for this process, and have been identified in mice, humans, and chickens. To study lymphocyte development in Xenopus laevis, we have characterized RAG-1 and RAG-2 in this species and examined their patterns of expression. Degenerate oligonucleotides, based on the known highly conserved RAG-1 sequences, were used to amplify, by the polymerase chain reaction, a segment of Xenopus RAG-1 from genomic DNA. A product of expected size was obtained and used to identify a genomic clone that contained the complete coding region of RAG-1 (1045 codons), and approximately the 3'-half of the coding region of RAG-2. The coding regions of RAG-1 and RAG-2 each lie on a single exon, are in opposite transcriptional orientation, and are separated by approximately 6 kb. The sequence of the remainder of RAG-2 was determined by PCR amplification of genomic DNA, with primers based on sequence analysis of RAG-2 cDNA clones. The predicted Xenopus RAG-1 protein is 71% identical in amino acid sequence to the sequences of each of the mouse, human, and chicken proteins; from position 392 to 1012 the identity is 88%. The coding region of Xenopus RAG-2 (520 codons) is somewhat less conserved among the different species. Tissue-specific expression of Xenopus RAG-1 and RAG-2 was examined both by Northern blotting and by a reverse transcription-polymerase chain reaction assay. In juvenile frogs, the highest levels of RAG-1 and RAG-2 expression were observed in the thymus, with lower levels in liver and spleen, and even lower levels in the kidneys. In adults, the thymus and bone marrow were found to be the principal sites of expression of both genes. RAG-2, but not RAG-1, was expressed in oocytes.

Age Factors↗
PubMed1993

Diversity of expressed V and J regions of immunoglobulin light chains in Xenopus laevis.

In Xenopus laevis, two immunoglobulin light chain isotypes, designated L1 or rho and L2 or sigma, have been identified. The genomic organization of the L1 locus has been described previously: a constant (C) gene segment is preceded by a joining (J) gene segment; in addition, there are many cross-hybridizing variable (V) gene segments. To evaluate the extent of sequence diversity of L1 V regions, we screened three cDNA libraries, constructed from mitogen-stimulated Xenopus splenocytes, with probes for the C or the J gene segment. Eighteen cDNA clones that contain complete or truncated V regions were chosen for sequence analysis. The C regions of all clones are identical or nearly identical to the genomic C gene segment; the V regions are greater than 80% identical in nucleotide sequence and are presumably derived from a single family of V gene segments. Although framework regions are nearly identical, complementarity-determining regions are quite diverse. The expressed J segments fall into distinct groups, suggesting the presence of more than one germ-line J segment. Therefore, a genomic library was screened with a J region probe. A clone overlapping with the previously identified J-C clone, and containing four additional J gene segments, was isolated. All five J gene segments are very similar and three are identical in nucleotide sequence. Each of the three distinct germ-line J sequences is represented in the set of cDNA clones, suggesting that combinatorial diversification occurs; imprecision of V-J joining also appears to contribute to variability. Overall, these results suggest that the immunoglobulin repertoire in this species is not significantly restricted by a limitation in the diversity of light chain V regions.

Amino Acid Sequence↗
PubMed1993

Reliability of eccentric isokinetic knee flexion and extension measurements.

This study assessed the test-retest reliability of knee isokinetic eccentric muscle performance in subjects with and without a history of tibio-femoral pathology. Nineteen adults were tested at 60 degrees/sec and 180 degrees/sec on three occasions using a standardized protocol that incorporates a same-session learning phase. Results revealed moderate to excellent reliability for average peak torque test-retest ICC (2,1) = .58 to .96, total work ICC = .63 to .93, and power ICC = .67 to .93. Joint angle at peak torque was unreliable (ICC = .01 to .69) for both muscle groups at both angular velocities. Knee flexion reliability was higher than extension reliability at both 60 degrees/sec and 180 degrees/sec. Subjects with tibio-femoral pathologies had ICC values lower than the healthy subjects. Reliable eccentric isokinetic measurements can be obtained for average peak torque, total work, and power. Clinicians should not assume the same degree of reliability in testing patients as in testing healthy subjects.

Adult↗
PubMed1993

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1988

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↗