California justices limit families' right to end life support.
Explore the source record for details and available documents.
California↗
Biomedical subjects
E Nieves
Publications and source records attributed to E Nieves.
A transition-state analogue reduces protein dynamics in hypoxanthine-guanine phosphoribosyltransferase.
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is the key enzyme in purine base salvage in humans and in purine auxotrophs, including Plasmodium falciparum, the leading cause of malaria. Hydrogen/deuterium (H/D) exchange into amide bonds, quantitated by on-line HPLC and mass spectrometry, has been used to compare the dynamic and conformational properties of human HGPRT alone, the HGPRT-GMP-Mg(2+) complex, the HGPRT-IMP-MgPPi <==> HGPRT-Hx-MgPRPP equilibrating mixture, and the transition-state analogue complex HGPRT-ImmGP-MgPPi. The rate and extent of H/D exchange of 26 peptic peptides, spanning 91% of the primary structure, have been monitored. Human HGPRT has 207 amide H/D exchange sites. After 1 h in D2O, HGPRT alone exchanges 160, HGPRT-GMP-Mg(2+) exchanges 154, the equilibrium complex exchanges 139, and the transition-state analogue complex exchanges 126 of these amide protons. H/D exchange rates are correlated with structure for peptides in (1) catalytic site loops, (2) a connected peptide of the subunit interface of the tetramer, and (3) a loop buried in the catalytic site. Structural properties related to H/D exchange are defined from crystallographic studies of the HGPRT-GMP-Mg(2+) and HGPRT-ImmGP-MgPPi complexes. Transition-state analogue binding strengthens the interaction between subunits and tightens the catalytic site loops. The solvent exchange dynamics in specific peptides correlates with hydrogen bond patterns, solvent access, crystallographic B-factors, and ligand exchange rates. Solvent exchange reveals loop dynamics in the free enzyme, Michaelis complexes, and the complex with the bound transition-state analogue. Proton transfer paths, rather than dynamic motion, are required to explain exchange into a buried catalytic site peptide in the complex with the bound transition-state analogue.
Amino Acid Sequence↗
Identification by mass spectrometry of a new alpha-tubulin isotype expressed in human breast and lung carcinoma cell lines.
The extensive C-terminal molecular heterogeneity of alpha- and beta-tubulin is a consequence of multiple isotypes, the products of distinct genes, that undergo several posttranslational modifications. These include polyglutamylation and polyglycylation of both subunits, reversible tyrosination and removal of the penultimate glutamate from alpha-tubulin, and phosphorylation of the beta III isotype. A mass spectrometry-based method has been developed for the analysis of the C-terminal diversity of tubulin from human cell lines. Total cell extracts are resolved by SDS--PAGE and transferred to nitrocellulose, and the region of the blot corresponding to tubulin (approximately 50 kDa) was excised and digested with CNBr to release the highly divergent C-terminal tubulin fragments. The masses of the human alpha- and beta-tubulin CNBr-derived C-terminal peptides are all in the 1500--4000 Da mass range and can be analyzed directly by MALDI-TOF mass spectrometry in the negative ion mode without significant interference from other released peptides. In this study, the tubulin isotype diversity in MDA-MB-231, a human breast carcinoma cell line, and A549, a human non-small lung cancer cell line, is reported. The major tubulin isotypes present in both cell lines are k-alpha 1 and beta 1. Importantly, we report a previously unknown alpha isotype present at significant levels in both cell lines. Moreover, the degree of posttranslational modifications to all isotypes was limited. Glu-tubulin, in which the C-terminal tyrosine of alpha-tubulin is removed, was not detected. In contrast to mammalian neuronal tubulin which exhibits extensive polyglutamylation, only low-level monoglutamylation of the k-alpha 1 and beta 1 isotypes was observed in these two human cell lines.
Amino Acid Sequence↗
Primary structure of Noetia ponderosa hemoglobins: functional correlates.
Homo- and heterodimeric hemoglobins have been isolated from the red cells of the arcid clam Noetia ponderosa (Np). These hemoglobins bind oxygen cooperatively. An extensively studied dimeric hemoglobin from another arcid clam, Scapaharca inaequivalvis, exhibits a molecular mechanism for cooperative ligand binding that is radically different from tetrameric vertebrate hemoglobins. In this study, the two chains found in both Noetia hemoglobins are sequenced and compared to the hemoglobins of the related clam S. inaequivalvis to determine whether Noetia hemoglobins have the structural basis for the same unusual mechanism for cooperative ligand binding and to inquire about the structural basis of absence of tetramers. Although the Noetia sequences are homologous to the Scapharca sequences, critical differences exist. The lack of tetramerization of Np subunits is most likely related to the absence of critical residues in the A and G helices that stabilize the interdimer contact seen in the Scapharca Hb tetramer. The lower affinity of the homodimer (Np-I), but particularly the heterodimer (Np-II) with respect to the homodimer and heterotetramer of Scapharca, can be due to (i) changes in the proximal heme environment and (ii) changes in the dimer interface. Interactions between Asn 100 and the heme of the other subunit are altered in Np-II due to the substitution of this residue by methionine, possibly causing the reduced O(2) affinity of the heterodimer of Noetia. (iii) Sequence changes in the E and F helices present in Np-I and Np-II could also contribute to the effect through interfacial changes. In particular, the substitution of Val for Thr in position 72 is expected to have a substantial influence on the interface. We conclude that Np dimers have the structural basis for a direct heme-heme interaction mechanism for cooperativity, as in Scapharca, but there are enough sequence changes to suggest that the pathway of interaction might be somewhat different.
Amino Acid Sequence↗
Immucillin-H binding to purine nucleoside phosphorylase reduces dynamic solvent exchange.
The rate and extent of hydrogen/deuterium (H/D) exchange into purine nucleoside phosphorylase (PNP) was monitored by electrospray ionization mass spectrometry (ESI-MS) to probe protein conformational and dynamic changes induced by a substrate analogue, products, and a transition state analogue. The genetic deficiency of PNP in humans is associated with severe T-cell immunodeficiency, while B-cell immunity remains functional. Inhibitors of PNP have been proposed for treatment of T-cell leukemia, to suppress the graft-vs.-host response, or to counter type IV autoimmune diseases without destroying humoral immunity. Calf spleen PNP is a homotrimer of polypeptide chains with 284 amino residues, molecular weight 31,541. Immucillin-H inhibits PNP with a Kd of 23 pM when only one of the three catalytic sites is occupied. Deuterium exchange occurs at 167 slow-exchange sites in 2 h when no catalytic site ligands are present. The substrate analogue and product prevented H/D exchange at 10 of the sites. Immucillin-H protected 32 protons from exchange at full saturation. When one of the three subunits of the homotrimer is filled with immucillin-H, and 27 protons are protected from exchange in all three subunits. Deuterium incorporation in peptides from residues 132-152 decreased in all complexes of PNP. The rate and/or extent of deuterium incorporation in peptides from residues 29-49, 50-70, 81-98, and 112-124 decreased only in the complex with the transition state analogue. The peptide-specific H/D exchange demonstrates that (1) the enzyme is most compact in the complex with immucillin-H, and (2) filling a single catalytic site of the trimer reduces H/D exchange in the same peptides in adjacent subunits. The peptides most highly influenced by the inhibitor surround the catalytic site, providing evidence for reduced protein dynamic motion caused by the transition state analogue.
Amino Acid Sequence↗
Development of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis in the sand fly Lutzomyia migonei (Diptera: Psychodidae).
Development of Leishmania braziliensis (Vianna) and Leishmania amazonensis (Lainson and Shaw) in the sand fly Lutzomyia migonei (França) was compared by studying the parasite microhabitats in the alimentary tract, the sequence of parasite morphological changes leading to the metacyclogenesis process, and the parasite transmission to the vertebrate susceptible host. Although the infections by the 2 Leishmania species were initiated with the same number of amastigotes, Le. amazonensis developed a higher population. Infections with Le. braziliensis were typically peripylarian and those with Le. amazonensis suprapylarian but with an unusual invasion of an organ other than the gut, the Malpighian tubules. The life cycle of the 2 parasites within the sand fly vector included the development of all promastigote forms: procyclics, haptomonads, nectomonads, paramastigotes and infective metacyclics, the last of which are uniquely adapted for transmission to the vertebrate hosts. Appearance of metacyclics coincided with the presence of large number of procyclics and haptomonads, low numbers of nectomonads and the appearance of paramastigotes. In both type of infections, there was a high mortality of the promastigotes inside the bloodmeal during digestion but once infection became established metacyclic forms appeared. Although the numbers of metacyclics that developed in sand flies were low for both parasites they were able to transmit the infection to vertebrates, a key event in the vector competence. We suggest that L. migonei is a true biological host and a possible vector of the 2 Leishmania species, which coexist in extensive geographic areas.
Animal Feed↗
Collagenase degrades collagen in vivo in the ischemic heart.
Previously, we showed that ischemic rat heart contains an activated procollagenase capable of degrading collagen in vitro. We now demonstrate that the collagen resident in such hearts (in vivo) also becomes degraded, producing characteristic fragments implicating the action of an activated collagenase. The evidence is the appearance of amino-terminal dansyl-Ile (+dansyl-Leu) residues in pepsin digests of re-oxygenated rat hearts and immunoblots showing 3/4 length (alphaA) fragments from type I collagen. Also, in ischemic rat myocardium, alphaA(I) and alphaA(III) fragments were detected in pepsin digests. The time periods required for the cleavage and degradation of collagen suggest the participation of a procollagenase that becomes activated. Results demonstrate for the first time that an interstitial collagenase in such hearts initiates in vivo degradation of types I and III collagens.
Animals↗
Human testicular glutathione S-transferases: insights into tissue-specific expression of the diverse subunit classes.
Cytosolic glutathione S-transferase (GST) subunits from human testis were resolved by HPLC and unambiguously identified by combined use of peptide sequence-specific antisera and electrospray ionization mass spectrometry (ESI MS). Allelic variants of hGSTP1, hGSTM1 and hGSTA2 were distinguished on the basis of observed differences in their molecular masses. Relative amounts of the multiple different subunit types in various human tissues were determined from HPLC profiles. From this type of analysis, tissues from hGSTM1 null allele individuals were readily discerned at the protein level; liver was the only tissue in which the hGSTM1 subunit was the major mu-class GST. hGSTM4 and hGSTM5 subunits were found at very low levels in all tissues examined. By far the tissue richest in the unique hGSTM3 subunit was testis, although brain also has significant levels.
Aged↗
Identification of the covalent flavin attachment site in sarcosine oxidase.
Sarcosine oxidase from Corynebacterium sp. P-1 is a heterotetrameric enzyme (alphabetagammadelta) that contains two noncovalently bound coenzymes (FAD, NAD+) and covalently bound FMN [8alpha-(N3-histidyl)FMN] which is attached to the beta subunit. Chlumsky et al. [(1995) J. Biol. Chem. 270, 18252-18259] tentatively identified His175 as the covalent FMN attachment site in the beta subunit, based on an alignment of the sequence of C. sp. P-1 beta subunit with a highly homologous flavin-containing peptide from another corynebacterial sarcosine oxidase (C. sp. U-96). To test this hypothesis, His175 in the C. sp. P-1 beta subunit was mutated to an alanine. Unexpectedly, the mutant enzyme was found to contain 1 mol of covalently bound flavin and to exhibit catalytic activity similar to wild-type enzyme. Covalent flavin-containing peptides were isolated from wild-type and mutant enzymes and analyzed by electrospray mass spectrometry. The mass observed for the mutant peptide (1152.4 Da) matched that predicted for an FMN-containing hexapeptide, corresponding to residues 173-178 (1152.1 Da). In the mutant, this region (HDAVAW) contains a single histidine (His173) which must be the covalent flavin attachment site. The mass observed for the wild-type peptide (1218.6 Da) matched that predicted for an FMN-containing hexapeptide, also corresponding to residues 173-178 in the beta subunit (1218.2 Da). This region in the wild-type enzyme includes two histidine residues (HDHVAW). Attempts to sequence the wild-type or mutant peptides by automated Edman degradation were unsuccessful. Instead, the peptide sequences were investigated by collisional-activated dissociation (CAD) and tandem mass spectrometry. The CAD mass spectral data with the mutant peptide confirmed the sequence deduced based on the mass of the intact peptide. The CAD mass spectral results with the wild-type peptide showed that FMN was covalently attached to the N-terminal histidine in the hexapeptide, which corresponds to His173 in the beta subunit.
Amino Acid Sequence↗
Identification of in vivo phosphorylation sites for basic-directed kinases in murine mdr1b P-glycoprotein by combination of mass spectrometry and site-directed mutagenesis.
Explore the source record for details and available documents.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Subunit diversity and tissue distribution of human glutathione S-transferases: interpretations based on electrospray ionization-MS and peptide sequence-specific antisera.
Uncertainties about the composition and identities of glutathione S-transferases (GSTs) in human tissue have impeded studies on their biological functions. A rigorous protocol has therefore been developed to characterize the human proteins. Cytosolic GST subunits were resolved by reverse-phase HPLC methods, individual components were assigned to Alpha, Mu and Pi classes on the basis of their immunoreactivities, and peptide-sequence-specific antisera were used to distinguish among five different Mu-class subunits (GSTM1-GSTM5). Each subunit type was characterized and identified unambiguously by electrospray ionization-MS. Acetylation of N-terminal residues in the GSTA1, GSTA2, GSTM3 and GSTM4 subunits were the only natural post-translational modifications detected. The unique structure of GSTM3, with N- and C-terminal peptide extensions predicted from cDNA sequences, was confirmed. Only testis and brain were rich sources of GSTM3 subunits. Subunit profiles were distinct and characteristic of the particular tissue type, and this tissue specificity in GST expression was evident even in organs from different individuals. For instance, livers had relatively simple GST compositions, consisting of a preponderance of Alpha-class subunits and GSTM1 (when present). By contrast, representation of most subunit types was a characteristic feature of testis, which had the highest levels of GSTs. GSTM4 and GSTM5 subunits, here identified for the first time in human tissue extracts, were minor components, with GSTM5 found only in brain, lung and testis. Specimens devoid of GSTM1 subunits, particularly those from null-genotype individuals, were readily discerned at the protein level. Liver was the only rich source of the GSTM1 subunit (although it also constituted a major fraction of adrenal GSTs), and so the functional consequences of the GSTM1 gene deletion are likely to vary in extrahepatic tissues.
Amino Acid Sequence↗
[Dental treatment under general anesthesia offered at the Hospital Pediátrico Universitario de Puerto Rico during the years 1989-1994].
One hundred and fourteen mentally retarded (MR) and non-mentally retarded (NMR) patients were divided into two groups and categorized according to the condition presented. Age, sex, and type of procedure performed were recorded for each patient. On the MR group 32% were over 17 years of age. On the NMR group 51% were under 6 years of age. The sex distribution was similar in both groups. Exodontia was the most frequently performed dental procedure. The MR group was composed of those who presented only mental retardation (42%), cerebral palsy (17%), epilepsy (15%), syndromes (7%), endocrinopathies (7%), hydrocephalus (5%) and other conditions (7%). The NMR group was composed of those who presented cardiopathy (7%), bottle syndrome (42%), hemotopathy (11%), maxillofacial disorders (24%) and other conditions (16%).
Adolescent↗
Natural and synthetic non-peptide antigens recognized by human gamma delta T cells.
T lymphocytes express either alpha beta or gamma delta T-cell receptor heterodimers. Most alpha beta T cells recognize antigenic peptides bound to major histocompatibility complex molecules but the antigen recognition and biological function of gamma delta T cells is unknown. A major human gamma delta T-cell subset expressing V gamma 2 and V delta 2 germline genes, but having diverse junctional sequences, is found in human mycobacterial lesions and responds in vitro to antigens of bacteria and parasites. In addition, certain haematopoietic tumour cells are specifically recognized and lysed by these T cells. V gamma 2V delta 2-bearing T cells were shown to recognize mycobacterial antigens that are protease resistant and phosphatase sensitive. Because of the difficulty in isolating natural antigens from mycobacterial culture filtrates or extracts, we synthesized a series of monoalkyl phosphates, and found that some, particularly monoethyl phosphate, could mimic the activity of mycobacterial antigens in stimulating these gamma delta T cells. Here we report the identification of natural antigens produced by mycobacteria recognized by human V gamma 2V delta 2-bearing T cells as isopentenyl pyrophosphate and related prenyl pyrophosphate derivatives, compounds involved in the synthesis of complex polyisoprenoid compounds in microbial and mammalian cells. Substitution of phosphate for the pyrophosphate moiety, or elimination of the double bond, greatly reduced antigenic activity of these compounds. These results provide formal evidence that, in contrast to recognition of major histocompatibility complex-bound peptide antigens by alpha beta T cells, human gamma delta T cells can recognize naturally occurring small non-peptidic antigens.
Antigens, Bacterial↗
Nonpeptide ligands for human gamma delta T cells.
gamma delta T cells respond to a variety of microbial pathogens and transformed cells. Their limited receptor repertoire and activation by mycobacterial antigens resistant to proteases suggest that they may recognize nonpeptide antigens. We have tested a variety of nonpeptide molecules for stimulation of human gamma delta T cells. Synthetic alkyl phosphates, particularly monoethyl phosphate (MEP), selectively activated gamma delta T cells and stimulated their proliferation in vitro. All gamma delta T cells stimulated by MEP expressed V gamma 2/V delta 2 receptors. The purified natural ligand of mycobacteria is chemically similar to, though distinct from, MEP and contains a phosphate residue that is critical for biological activity. Recognition and expansion of a specific T-cell receptor-bearing population to non-peptide ligands is unprecedented among T cells. We suggest that MEP mimics small natural ligands capable of expanding one subset of gamma delta T cells and that this recognition of nonpeptide antigens may play an important role in human immunity to pathogens.
Antibodies↗
Purification and characterization of three isolectins of soybean agglutinin. Evidence for C-terminal truncation by electrospray ionization mass spectrometry.
Soybean agglutinin (SBA) is a tetrameric D-Gal/D-GalNAc-specific lectin possessing one Man9 oligomannose-type chain/monomer. SBA exists as multiple isolectins having similar binding and immunochemical properties. The present study shows that native SBA consists of at least five isolectins. Three of these isoforms have been purified by chromatofocusing and designated as SBA-I, SBA-II and SBA-III in order of their elution from a chromatofocusing column. The pI of the isolectins are 7.0, 6.85 and 6.7, respectively, as determined by isoelectric focusing. Each isolectin was denatured in 6 M guanidine hydrochloride into their individual subunits which were separated by reverse-phase high performance liquid chromatography (RP-HPLC). The HPLC profiles were similar for all three isoforms which showed two major peaks (peak 1 and peak 3) along with a minor peak (peak 2). The first peak of SBA-II existed as a double labeled as 1 a and 1 b. Each peak was analyzed by electrospray ionization mass spectrometry to characterize each isoform and determine their structural differences. The calculated mass of an intact lectin monomer from the amino acid sequence (253 residues) derived from cDNA of the lectin including a Man9 oligomannose chain is 29438 Da. The present results show that peak 3 of each isoform corresponds to an intact subunit (alpha) while peak 1 of each isoform shows lower masses which are assigned to C-terminal fragmentation of the protein. Peak 1 of SBA-I has a molecular mass of 28000Da corresponding to a fragmented subunit (beta) consisting of 240 residues (calculated molecular mass 28001Da). Peak 1a of SBA-II shows a molecular mass of 28000Da corresponding to a fragmented beta subunit, while peak 1b showed two major species: a 28000-Da (beta subunit) and a 28327-Da subunit which corresponds to 243 residues (calculated mass 28326Da) designated as a gamma subunit. In addition, peak 1b showed the presence of a molecular species of 28627Da corresponding to a 246-residue subunit (gamma'). Peak 1 of SBA-III showed a major molecular species corresponding to a fragmented gamma subunit. The minor peak in the HPLC profile (peak 2) represented a subunit of 252 residues for all three isoforms. The results suggest that the subunit compositions of SBA-I, SBA-II and SBA-III are approximately alpha 2 beta 2, alpha 2 beta gamma and alpha 2 gamma 2, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Sequence↗
Epidemiology of cutaneous leishmaniasis in Merida, Venezuela. III. Altitudinal distribution, age structure, natural infection and feeding behaviour of sandflies and their relation to the risk of transmission.
An entomological survey revealed that the distribution of the Lutzomyia species in Merida, Venezuela, varies with altitude. Of the 21 species encountered, 12 (57%) are known to be anthropophilic. The significance of the predominant man-biting species at any altitude is discussed in terms of the risk of transmission of cutaneous leishmaniasis. The general parous rate (and perhaps the ability of a female sandfly to survive oviposition and transmit Leishmania parasites during a second or subsequent bloodmeal) was found to be related to rainfall and altitude. The finding of Leishmania infections in wild-caught parous females of anthropophilic species indicates the high risk of acquiring leishmaniasis in the study area. Observations on the feeding habits of the sandflies indicate that some species are opportunistic feeders, attracted to a variety of hosts, including man.
Altitude↗
Sugars in the alimentary canal of Lutzomyia youngi (Diptera: phlebotominae).
Gas chromatography (GC) analysis of 159 specimens (144 females and 15 males) of Lutzomyia youngi collected in Shannon traps in a coffee plantation in the Andean region of western Venezuela, where leishmaniasis is endemic, revealed the presence of fructose, sucrose, glucose and maltose in the gut and crop of the wild sandflies. The identification of the sugars was confirmed by comparing retention times with those observed for standard sugars and those obtained from sandflies experimentally fed on known sugar solutions. Although the sandflies in nature may ingest each of the four sugars, the results suggest that it is more probable there is an invertase enzyme (glycosidase?) in the gut or crop of the sandfly which hydrolyses ingested disaccharides (e.g. sucrose) to the constituent monosaccharides (i.e. fructose and glucose). Ecological and altitudinal distributions of sandfly species may be related to the availability of specific sugar sources, with epidemiological implications. Identification of the preferred sugar could make breeding easier and would facilitate further research on Leishmania-vector relationships.
Animals↗
Identification of the major phosphorylation domain of murine mdr1b P-glycoprotein. Analysis of the protein kinase A and protein kinase C phosphorylation sites.
P-glycoprotein is phosphorylated in cells, and it has been suggested that phosphorylation may regulate the drug transport activity of P-glycoprotein. Domain mapping, utilizing a combination of cyanogen bromide digestion and immunoblot analysis, was used to reveal the major phosphorylation sites in murine mdr1b P-glycoprotein. After labeling of J7.V1-1 cells with [32P]Pi, or labeling membranes with [gamma-32P]ATP and either protein kinase A or protein kinase C, it was found that the majority of the label was contained within a single cyanogen bromide fragment (amino acid 627-682) that encompassed the majority of the linker region. The in vitro protein kinase C phosphorylation sites within this fragment were analyzed by a combination of fast atom bombardment mass spectrometry (FABMS) and two-dimensional phosphopeptide mapping. FABMS analysis of a protein kinase C-phosphorylated synthetic peptide, corresponding to a segment of the linker region of P-glycoprotein, identified serine 669 as the single site of phosphorylation. Comparison of two-dimensional tryptic phosphopeptide maps prepared from synthetic peptide and P-glycoprotein, both of which were phosphorylated in vitro with protein kinase C, revealed that serine 669 was also the major phosphorylation site in the intact glycoprotein. The in vitro protein kinase A phosphorylation site was identified as serine 681 by site-directed mutagenesis. Inspection of the gene organization and the deduced amino acid sequence of mdr1b P-glycoprotein revealed that the linker region, although shorter than the R domain (55 versus 241 amino acids), fits the operational definition of the R domain of cystic fibrosis conductance regulator. Like the R domain, the linker region is encoded by a single exon, is highly charged with alternating acidic and basic side chains, and contains several protein kinase A/protein kinase C consensus phosphorylation sites. Since the R domain is believed to be involved in the regulation of cystic fibrosis conductance regulator function by phosphorylation, it is possible that the linker region plays a similar regulatory role in P-glycoprotein function.
ATP Binding Cassette Transporter, Subfamily B, Mem↗