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Biomedical subjects

A Light

Publications and source records attributed to A Light.

At least 19 recordsLinked to original sources
PubMed2001

Evidence for a common binding cavity for three general anesthetics within the GABAA receptor.

The GABA(A) receptor is an important target for a variety of general anesthetics (Franks and Lieb, 1994) and for benzodiazepines such as diazepam. Specific point mutations in the GABA(A) receptor selectively abolish regulation by benzodiazepines (Rudolph et al., 1999; McKernan et al., 2000) and by anesthetic ethers (Mihic et al., 1997; Krasowski et al., 1998; Koltchine et al., 1999), suggesting the existence of discrete binding sites on the GABA(A) receptor for these drugs. Using anesthetics of different molecular size (isoflurane > halothane > chloroform) together with complementary mutagenesis of specific amino acid side chains, we estimate the volume of a proposed anesthetic binding site as between 250 and 370 A(3). The results of the "cutoff" analysis suggest a common site of action for the anesthetics isoflurane, halothane, and chloroform on the GABA(A) receptor. Moreover, the data support a crucial role for Leu232, Ser270, and Ala291 in the alpha subunit in defining the boundaries of an amphipathic cavity, which can accommodate a variety of small general anesthetic molecules.

Anesthetics↗
PubMed2001

MGA2 is involved in the low-oxygen response element-dependent hypoxic induction of genes in Saccharomyces cerevisiae.

Eukaryotes have the ability to respond to changes in oxygen tension by alterations in gene expression. For example, OLE1 expression in Saccharomyces cerevisiae is upregulated under hypoxic conditions. Previous studies have suggested that the pathway regulating OLE1 expression by unsaturated fatty acids may involve Mga2p and Spt23p, two structurally and functionally related proteins. To define the possible roles of each of these genes on hypoxia-induced OLE1 expression, we examined OLE1 expression under normoxia, hypoxia, and cobalt treatment conditions in Deltamga2 or Deltaspt23 deletion strains. The results of OLE1 promoter-lacZ reporter gene and Northern blot analyses showed that hypoxia- and cobalt-induced OLE1 expression was dramatically decreased in a Deltamga2 strain but not in a Deltaspt23 strain. Further analyses using low-oxygen response element (LORE)-CYC1-lacZ fusion reporter assays and electrophoretic mobility shift assays (EMSAs) demonstrated that MGA2 significantly affects the LORE-dependent hypoxic induction pathway of gene expression. When MGA2 was supplied by a plasmid, the LORE-dependent hypoxia-inducible reporter expression was recovered, as was the hypoxia-inducible complex in EMSAs in the S. cerevisiae Deltamga2 strain. Supershift analysis of EMSAs using crude extracts containing mycMga2p indicated that Mga2p is a component of the LORE-binding complex. Another LORE-dependent, hypoxia-inducible gene, ATF1, was similarly affected in the Deltamga2 strain. These results indicate that MGA2 is required for the LORE-dependent hypoxic gene induction in S. cerevisiae.

Cell Hypoxia↗
PubMed2000

Single cell sorting and cloning.

Cell sorters now allow the selection of cells and other bodies according to a range of quite diverse criteria. The additional refinement that allows the sorting of individual cells based on these criteria has seen application in many fields of research. Single cells may be sorted for microscopy, for culture and for genetic analysis by way of single cell PCR (polymerase chain reaction). In practical terms, in the setting up of an instrument for single cell sorting, there are additional requirements to ensure that each detected event is indeed a single cell or body, that this cell can be reliably sorted via saline droplet, separate from its fellow travelers, that the aiming of the droplet deflection is sufficiently precise to find the target vessel and that the cell will be undamaged on arrival. Among the diverse reported applications of the technique, two fields which have benefited greatly are lymphocyte development and haemopoiesis. In the former case, the analysis of gene rearrangements in lymphocytes, both in the pre- and post-antigenic phases of development, has been enabled by the combined technologies of single cell sorting and PCR. It is argued that such experiments could not have been done without that partnership. In a similar way, the single cell sorting technique has been found to be the perfect way to demonstrate precursor/progeny relationships between haemopoietic cells and, further, to demonstrate rigorously the effects of particular cytokines on the haemopoietic system.

Cell Separation↗
PubMed2000

bcl-2 transgene expression inhibits apoptosis in the germinal center and reveals differences in the selection of memory B cells and bone marrow antibody-forming cells.

Immunization with T cell-dependent antigens generates long-lived memory B cells and antibody-forming cells (AFCs). Both populations originate in germinal centers and, predominantly, produce antibodies with high affinity for antigen. The means by which germinal center B cells are recruited into these populations remains unclear. We have examined affinity maturation of antigen-specific B cells in mice expressing the cell death inhibitor bcl-2 as a transgene. Such mice had reduced apoptosis in germinal centers and an excessive number of memory B cells with a low frequency of V gene somatic mutation, including those mutations encoding amino acid exchanges known to enhance affinity. Despite the frequency of AFCs being increased in bcl-2-transgenic mice, the fraction secreting high-affinity antibody in the bone marrow at day 42 remained unchanged compared with controls. The inability of BCL-2 to alter selection of bone marrow AFCs is consistent with these cells being selected within the germinal center on the basis of their affinity being above some threshold rather than their survival being due to a selective competition for an antigen-based signal. Continuous competition for antigen does, however, explain formation of the memory compartment.

ADP-ribosyl Cyclase↗
PubMed1997

The extent of affinity maturation differs between the memory and antibody-forming cell compartments in the primary immune response.

Immunization with protein-containing antigens results in two types of antigen-specific B cell: antibody forming cells (AFCs) producing antibody of progressively higher affinity and memory lymphocytes capable of producing high affinity antibody upon re-exposure to antigen. The issue of the inter-relationship between affinity maturation of memory B cells and AFCs was addressed through analysis of single, antigen-specific B cells from the memory and AFC compartments during the primary response to a model antigen. Only 65% of splenic memory B cells were found capable of producing high affinity antibody, meaning that low affinity cells persist into this compartment. In contrast, by 28 days after immunization all AFCs produced high affinity antibody. We identified a unique, persistent sub-population of bone marrow AFCs containing few somatic mutations, suggesting they arose early in the response, yet highly enriched for an identical affinity-enhancing amino acid exchange, suggesting strong selection. Our results imply that affinity maturation of a primary immune response occurs by the early selective differentiation of high affinity variants into AFCs which subsequently persist in the bone marrow. In contrast, the memory B-cell population contains few, if any, cells from the early response and is less stringently selected.

Amino Acid Sequence↗
PubMed1997

Immunohistochemical processing of tissue which allows for embedding in Araldite, and orientation and correlation between light and electron microscopic staining.

This paper describes a simple and efficient method for the immunohistochemical processing of tissue sections on glass slides and the subsequent transfer of the entire section to an Araldite medium. These sections present in one plane can then be orientated and when cut provide the user with whole tissue, free from gaps due to folding. This procedure therefore enables the changes in protein concentration and distribution to be assessed and correlated within and between different cells at the light and electron microscopic level. This procedure may be of use in other microscopic techniques studies.

Animals↗
PubMed1996

Urinary findings and renal function in adult Navajo Indians and associations with type 2 diabetes.

We screened a sample of adult Navajo Indians for signs of renal disease that might underlie their increasing rates of renal failure. Nondiabetics had modest rates of hypertension, which was more common in males and increased with age. Microscopic hematuria was very common, and only a fraction was associated with progressive nephropathy. Microalbuminuria, mostly undetected by routine dipstick, was present in 14.6% of subjects; overt albuminuria was present in 2%. Increasing albuminuria was related to renal insufficiency, which was more common in males. Hypertension was associated with greater then threefold increases in both albuminuria and renal insufficiency. Cardiovascular disease was uncommon and had no discernible relationship to albuminuria. Most diabetic patients (58.4%) had hypertension, with equal rates for males and females. Fully half of all diabetic patients had unsatisfactory blood pressure levels at screening. Rates and patterns of hematuria were like those of nondiabetics. Microalbuminuria was present in 36.1% and overt albuminuria in 17.9%, four and eight times the rates in matched nondiabetics, respectively; these differences persisted after controlling for blood pressure. Renal insufficiency was associated with progressive albuminuria and was present in 10.6%, with equal rates in males and females. Hypertension, albuminuria, and renal insufficiency, but not hematuria, increased with increasing diabetes duration. Hypertension was associated with a twofold increase in albuminuria, a threefold increase in overt albuminuria, and an eightfold increase in renal insufficiency. Cardiovascular disease had no detectable association with microalbuminuria, but had a strong relationship to overt albuminuria. The high rates of hematuria are not well explained. It probably has nonrenal as well as renal origins, the latter including mesangial proliferative glomerulonephritis. The impressive rates of albuminuria among diabetic patients mark a large reservoir of renal disease and fore-shadow even larger burdens of end-stage renal disease and cardiovascular disease in the near future. Improved detection and treatment of hypertension is needed to slow the progression of renal disease in nondiabetics and diabetics, together with screening and treatment protocols for albuminuric diabetic patients. Prevention of albuminuria probably involves population-based modification of blood pressure and metabolic profiles.

Adolescent↗
PubMed1994

Blood pressure in Navajo Indians and its association with type 2 diabetes and renal and cardiovascular disease.

In mid-1990 we evaluated blood pressure and its associations in 366 nondiabetic adult Navajos and 400 Navajos with type 2 diabetes attending Indian Health Service outpatient clinics in Tuba City, Arizona. In nondiabetics, systolic blood pressure (SBP) rose with increasing age while diastolic blood pressure (DBP) fell; 13.4% had hypertension by diagnosis or treatment. Female nondiabetics had lower blood pressures than males. SBP and DBP correlated with age, body mass index (BMI), and urinary albumin excretion (UAE). Hypertension was associated with a sixfold increase in nephropathy, a threefold increase in renal insufficiency, and an almost sixfold increase in cardiovascular disease. Diabetics had higher blood pressures than age- and sex-matched nondiabetics; 58.4% had hypertension by diagnosis or treatment, and, in spite of widespread antihypertensive treatment, blood pressures in almost 50% were suboptimal from the perspectives of cardiovascular and renal protection. Blood pressures of female diabetics were similar to those of males. Blood pressures correlated with age, BMI, and increasing UAE. Rates of nephropathy and cardiovascular disease were much higher in diabetics than nondiabetics, and within the diabetic population hypertension was associated with a greater than threefold increase in nephropathy, an eightfold increase in renal insufficiency, a five-fold increase in peripheral and cerebrovascular disease, and more than doubling of the rate of heart disease. The relationship of blood pressure to renal and cardiovascular disease suggest similar mechanisms in nondiabetics and diabetics, with diabetes contributing an accentuated susceptibility. Albuminuria and cardiac disease are generated at "subhypertensive" blood pressures, while established hypertension appears to drive overt renal, cerebrovascular, and peripheral vascular disease, and to further increase heart disease risk.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase.

Enterokinase (enteropeptidase) is a heterodimeric serine protease that is responsible for the physiological activation of trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)4-Lys. In this paper, we report the cloning and functional expression of a cDNA encoding the catalytic domain (light chain) of bovine enterokinase. The nucleotide sequence of this cloned cDNA predicts a 235-amino acid polypeptide that shares a high degree of homology with a variety of mammalian serine proteases involved in digestion, coagulation, and fibrinolysis. We have developed a novel expression method for the enzyme which utilizes the secretory leader and propeptide of the mammalian serine protease PACE fused to the enterokinase light chain amino terminus. Efficient cleavage of the paired dibasic amino acid cleaving enzyme (PACE) propeptide was achieved by coexpression with human PACE or yeast KEX2. The mature product migrates at 43,000 Da on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, comparable to light chain derived from bovine duodena, and exhibited high levels of activity in cleaving the enterokinase-specific fluorogenic substrate Gly-(Asp)4-Lys-beta-naphthylamide. The recombinant single-chain form of enterokinase was also capable of activating trypsinogen, indicating that the specificity of the enzyme for its natural substrate is retained even in the absence of the noncatalytic enterokinase heavy chain.

Amino Acid Sequence↗
PubMed1991

Further evidence for independent folding of domains in serine proteases.

The folding pathway of pancreatic serine proteases was clarified from kinetic studies on the refolding of the glutathione-mixed disulfide derivative of bovine neochymotrypsinogen. Neochymotrypsinogen is prepared from a limited proteolysis of native chymotrypsinogen A by cleavage at Tyr146-Thr147 (Duda and Light (1982) J. Biol. Chem. 257, 9866-9871). The mixed disulfide methodology (Odorzynski and Light (1979) J. Biol. Chem. 254, 4291-4295) was necessary to successfully refold chymotrypsinogen and neochymotrypsinogen. Mixtures of the chromatographically purified amino- and carboxyl-terminal polypeptides of neochymotrypsinogen, as the mixed disulfide derivatives, were refolded at varying molar ratios of the polypeptides. The regeneration of native structure was followed as a function of time from activity measurements and from the regain of the molecular weight of the zymogen. The rate data fit first-order kinetics. The kinetic analysis is compatible with a folding mechanism that supports (a) independent folding of the amino- and carboxyl-terminal domains; (b) identical rates of folding of each domain; and (c) the rate-limiting step is the formation of the interdomain disulfide. The formation of a stable complex of the folded domains was favored by complementary hydrophobic and hydrogen bonding interactions and the formation of the last disulfide bond. The geometric arrangement of the active site residues was regained and the zymogen could be converted to the active enzyme, namely, alpha-chymotrypsin.

Amino Acids↗
PubMed1991

The amino-terminal sequence of the catalytic subunit of bovine enterokinase.

Bovine enterokinase (enteropeptidase) is a serine protease and functions as the physiological activator of trypsinogen. The enzyme has a heavy chain (115 kD) covalently linked to a light or catalytic subunit (35 kD). The amino acid composition showed that the light chain has nine half-cystine residues (four as intramolecular disulfides) and that one half-cystine was in a disulfide link between the light and heavy subunits. The amino-terminal 27 residues of the S-vinylpyridyl derivative of the light chain were determined by gas-phase Edman degradation. The sequence has homologies with other serine proteases containing one or two chains. The homologies suggest that the catalytic subunit has the same three-dimensional structure and, therefore, the same mechanism of enzymatic action as pancreatic chymotrypsin, trypsin, and elastase. The presence of the conserved amino-terminal activation peptide sequence (IVGG) shows that enterokinase must have a zymogen precursor and that the two-chain enzyme arises from limited proteolysis during posttranslational processing.

Amino Acid Sequence↗
PubMed1989

A direct high-performance liquid chromatography assay of the enzymatic activity of enterokinase (enteropeptidase).

Bovine enterokinase (enteropeptidase) activates trypsinogen to trypsin at pH 8.0. In the presence of chicken ovomucoid, a stable complex of ovomucoid-trypsin is produced, inactivating trypsin and eliminating autoactivation of trypsinogen. The molecular size of trypsin (24,000 Da) is increased twofold on forming the ovomucoid-trypsin complex (52,000 Da). Size-exclusion chromatography on a Toya Soda TSK G2000SW column in an HPLC system and with computer-assisted analyses gives a direct quantitative determination of the amount of substrate (trypsinogen) and product (ovomucoid-trypsin). The rate of disappearance of substrate is equal to the rate of formation of product in agreement with kinetic theory. The simultaneous determination of both rates increases the reliability of the assay. The HPLC assay has an extended linear range for the velocity of the activation process as a function of enzyme concentration. The assay is reliable and accurate for highly purified preparations, samples at different steps in the purification scheme, and for a direct assay of the intestinal contents. The assay should be useful in clinical analyses.

Animals↗
PubMed1989

Enterokinase (enteropeptidase): comparative aspects.

The serine protease enterokinase is the physiological activator of trypsinogen and has a specificity for the sequence (Asp)4-Lys-Ile. The enzyme consists of two subunits linked by a disulfide bond. The heavy chain achors enterokinase in the intestinal brush border membrane and the light chain is the catalytic subunit, which has the same mechanism of action as trypsin and chymotrypsin. Many properties of enterokinase resemble blood-clotting enzymes, suggesting that enterokinase lies on the same phylogenetic branch as the blood-clotting proteins.

Animals↗
PubMed1988

Size-exclusion high performance liquid chromatography of native trypsinogen, the denatured protein, and partially refolded molecules. Further evidence that non-native disulfide bonds are dominant in refolding the completely reduced protein.

Size-exclusion high performance liquid chromatography was used to compare the Stokes radius of the mixed disulfide of trypsinogen refolded for 10 min with the Stokes radius of denatured trypsinogen in high concentrations of urea. After folding for 10 min, rechromatography of a collection of sequential fractions of an initial separation showed that the fractions display microheterogeneity as seen in the value of the Stokes radius of each fraction. These intermediate species differed in their Stokes radius, and each had a globular structure cross-linked by disulfide bonds. In contrast, when trypsinogen with the native disulfides intact was equilibrated at different concentrations of urea (0-8 M), a progressive increase in Stokes radius was observed with extent of unfolding. Rechromatography of a series of fractions collected at a specific urea concentration showed that each had the same Stokes radius as the fraction in the initial separation. Urea-denatured trypsinogen and partially refolded trypsinogen must therefore differ in the disulfide pairing that links regions of the polypeptide chain. These observations support the suggestion that non-native disulfide bonds are responsible for the many stable conformations that form early in the folding of the mixed disulfide of trypsinogen (Light, A., and Higaki, J.N. (1987) Biochemistry 26, 5556-5564). These intermediates initially are loose structures (large Stokes radius) that become more compact with time (decreasing Stokes radius). The intermediates must therefore undergo a continuing disulfide interchange until native disulfides form late in the process when the stable conformation of the native molecule is reached.

Chromatography, High Pressure Liquid↗
PubMed1987

Detection of intermediate species in the refolding of bovine trypsinogen.

The mixed disulfide of bovine trypsinogen and glutathione was refolded at pH 8.6 and 4 degrees C with a mixture of 3 mM cysteine and 1 mM cystine catalyzing disulfide interchange. The folding process was monitored by analysis of quenched samples with isoelectric focusing and size-exclusion chromatography. Isoelectric focusing showed a progressive change from a pI of 5.2 for the mixed disulfide derivative to a pI of 9.3 for native trypsinogen. A number of principal intermediates were detected as a function of the refolding time. These intermediates were also separated and further characterized by size-exclusion chromatography on columns of TSK G2000 SW operated in the high-performance liquid chromatographic mode. Rechromatography of a series of sequential fractions taken from the parental peak was necessary to resolve and characterize the principal intermediates. The loss of glutathione moieties produced a partly folded structure with an apparent hydrodynamic volume (Stokes radius, Rs) of 33.9 A. These structures became compact with time, and more intermediates were detected between 33.9 and 29.2 A. Finally, a change in conformation, resembling a two-state transition, changed the molecules of Rs 29.2 to the compact structure of native trypsinogen (22.4 A). The rate of formation of the native structure was determined from the progress curves derived from isoelectric focusing and size-exclusion chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Independent refolding of domains in the pancreatic serine proteinases.

Ser-neotrypsinogen and Val-neotrypsinogen are two-chain modifications of bovine trypsinogen produced on limited proteolysis with trypsin. Ser-neotrypsinogen has Lys131-Ser132 cleaved in the connecting peptide (the autolysis loop) linking the amino- and carboxyl-terminal domains. Val-neotrypsinogen has Arg105-Val106 cleaved which is located within the amino-terminal domain. The mixed disulfide derivative of Ser-neotrypsinogen was successfully refolded. A functional molecule was regenerated from the polypeptide fragments with the correct molecular weight of neotrypsinogen in an overall yield of 7%. Val-Neotrypsinogen could not be refolded. The first-order rate constants for the regeneration of Ser-neotrypsinogen were determined from the formation of active enzyme molecules as a function of time and from the regain of the correct molecular weight. Both kinetic values were the same indicating that refolding of the polypeptide chains first forms globular domain structures. The two domains then associate and the disulfide bonds between the domains and the correct geometry of the active site residues are formed last. The same kinetic results were also found in refolding Thr-neochymotrypsinogen (Duda, C. T., and Light, A. (1982) J. Biol. Chem. 257, 9866-9871) where peptide bond cleavage also occurred in the connecting peptide. These observations support the hypothesis that the pathway of folding of serine proteinases proceeds with the independent refolding of domains.

Animals↗