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Biomedical subjects

B Santos

Publications and source records attributed to B Santos.

15 recordsLinked to original sources
PubMed2006

Alternatives for coupling sequential injection systems to commercial capillary electrophoresis-mass spectrometry equipment.

On-line coupling of an automated flow system with a commercially available capillary electrophoresis (CE) system with an electrospray interface (ESI) for mass spectroscopic (MS) detection is described. The peculiarities of CE-ESI-MS interfaces, in which a high electrical field must be applied to the capillary end where the sample is provided by the flow system, introduce significant difficulties for the appropriate work of the entire arrangement. Experimental strategies are proposed for achieving stable conditions for on-line sample pre-treatment, conditioning of the separation capillary, sample injection, as the proper separation. The versatility and robustness of the proposed arrangement is discussed, taken as example the separation of a variety of amines. Connection of the CE system's pressure to the automated flow system enables hydrodynamic introduction of sample with high precision. The developed hyphenated system is of practical relevance as it opens an avenue for the simplification and automation of the whole analytical process required when using powerful CE-ESI-MS equipments.

Butylamines↗
PubMed2000

Sbe2p and sbe22p, two homologous Golgi proteins involved in yeast cell wall formation.

The cell wall of fungal cells is important for cell integrity and cell morphogenesis and protects against harmful environmental conditions. The yeast cell wall is a complex structure consisting mainly of mannoproteins, glucan, and chitin. The molecular mechanisms by which the cell wall components are synthesized and transported to the cell surface are poorly understood. We have identified and characterized two homologous yeast proteins, Sbe2p and Sbe22p, through their suppression of a chs5 spa2 mutant strain defective in chitin synthesis and cell morphogenesis. Although sbe2 and sbe22 null mutants are viable, sbe2 sbe22 cells display several phenotypes indicative of defects in cell integrity and cell wall structure. First, sbe2 sbe22 cells display a sorbitol-remediable lysis defect at 37 degrees C and are hypersensitive to SDS and calcofluor. Second, electron microscopic analysis reveals that sbe2 sbe22 cells have an aberrant cell wall structure with a reduced mannoprotein layer. Finally, immunofluorescence experiments reveal that in small-budded cells, sbe2 sbe22 mutants mislocalize Chs3p, a protein involved in chitin synthesis. In addition, sbe2 sbe22 diploids have a bud-site selection defect, displaying a random budding pattern. A Sbe2p-GFP fusion protein localizes to cytoplasmic patches, and Sbe2p cofractionates with Golgi proteins. Deletion of CHS5, which encodes a Golgi protein involved in the transport of Chs3p to the cell periphery, is lethal in combination with disruption of SBE2 and SBE22. Thus, we suggest a model in which Sbe2p and Sbe22p are involved in the transport of cell wall components from the Golgi apparatus to the cell surface periphery in a pathway independent of Chs5p.

Amino Acid Sequence↗
PubMed1998

Spa2p interacts with cell polarity proteins and signaling components involved in yeast cell morphogenesis.

The yeast protein Spa2p localizes to growth sites and is important for polarized morphogenesis during budding, mating, and pseudohyphal growth. To better understand the role of Spa2p in polarized growth, we analyzed regions of the protein important for its function and proteins that interact with Spa2p. Spa2p interacts with Pea2p and Bud6p (Aip3p) as determined by the two-hybrid system; all of these proteins exhibit similar localization patterns, and spa2Delta, pea2Delta, and bud6Delta mutants display similar phenotypes, suggesting that these three proteins are involved in the same biological processes. Coimmunoprecipitation experiments demonstrate that Spa2p and Pea2p are tightly associated with each other in vivo. Velocity sedimentation experiments suggest that a significant portion of Spa2p, Pea2p, and Bud6p cosediment, raising the possibility that these proteins form a large, 12S multiprotein complex. Bud6p has been shown previously to interact with actin, suggesting that the 12S complex functions to regulate the actin cytoskeleton. Deletion analysis revealed that multiple regions of Spa2p are involved in its localization to growth sites. One of the regions involved in Spa2p stability and localization interacts with Pea2p; this region contains a conserved domain, SHD-II. Although a portion of Spa2p is sufficient for localization of itself and Pea2p to growth sites, only the full-length protein is capable of complementing spa2 mutant defects, suggesting that other regions are required for Spa2p function. By using the two-hybrid system, Spa2p and Bud6p were also found to interact with components of two mitogen-activated protein kinase (MAPK) pathways important for polarized cell growth. Spa2p interacts with Ste11p (MAPK kinase [MEK] kinase) and Ste7p (MEK) of the mating signaling pathway as well as with the MEKs Mkk1p and Mkk2p of the Slt2p (Mpk1p) MAPK pathway; for both Mkk1p and Ste7p, the Spa2p-interacting region was mapped to the N-terminal putative regulatory domain. Bud6p interacts with Ste11p. The MEK-interacting region of Spa2p corresponds to the highly conserved SHD-I domain, which is shown to be important for mating and MAPK signaling. spa2 mutants exhibit reduced levels of pheromone signaling and an elevated level of Slt2p kinase activity. We thus propose that Spa2p, Pea2p, and Bud6p function together, perhaps as a complex, to promote polarized morphogenesis through regulation of the actin cytoskeleton and signaling pathways.

Binding Sites↗
PubMed1997

Targeting of chitin synthase 3 to polarized growth sites in yeast requires Chs5p and Myo2p.

Chitin is an essential structural component of the yeast cell wall whose deposition is regulated throughout the yeast life cycle. The temporal and spatial regulation of chitin synthesis was investigated during vegetative growth and mating of Saccharomyces cerevisiae by localization of the putative catalytic subunit of chitin synthase III, Chs3p, and its regulator, Chs5p. Immunolocalization of epitope-tagged Chs3p revealed a novel localization pattern that is cell cycle-dependent. Chs3p is polarized as a diffuse ring at the incipient bud site and at the neck between the mother and bud in small-budded cells; it is not found at the neck in large-budded cells containing a single nucleus. In large-budded cells undergoing cytokinesis, it reappears as a ring at the neck. In cells responding to mating pheromone, Chs3p is found throughout the projection. The appearance of Chs3p at cortical sites correlates with times that chitin synthesis is expected to occur. In addition to its localization at the incipient bud site and neck, Chs3p is also found in cytoplasmic patches in cells at different stages of the cell cycle. Epitope-tagged Chs5p also localizes to cytoplasmic patches; these patches contain Kex2p, a late Golgi-associated enzyme. Unlike Chs3p, Chs5p does not accumulate at the incipient bud site or neck. Nearly all Chs3p patches contain Chs5p, whereas some Chs5p patches lack detectable Chs3p. In the absence of Chs5p, Chs3p localizes in cytoplasmic patches, but it is no longer found at the neck or the incipient bud site, indicating that Chs5p is required for the polarization of Chs3p. Furthermore, Chs5p localization is not affected either by temperature shift or by the myo2-66 mutation, however, Chs3p polarization is affected by temperature shift and myo2-66. We suggest a model in which Chs3p polarization to cortical sites in yeast is dependent on both Chs5p and the actin cytoskeleton/Myo2p.

Carrier Proteins↗
PubMed1997

CHS5, a gene involved in chitin synthesis and mating in Saccharomyces cerevisiae.

The CHS5 locus of Saccharomyces cerevisiae is important for wild-type levels of chitin synthase III activity. chs5 cells have reduced levels of this activity. To further understand the role of CHS5 in yeast, the CHS5 gene was cloned by complementation of the Calcofluor resistance phenotype of a chs5 mutant. Transformation of the mutant with a plasmid carrying CHS5 restored Calcofluor sensitivity, wild-type cell wall chitin levels, and chitin synthase III activity levels. DNA sequence analysis reveals that CHS5 encodes a unique polypeptide of 671 amino acids with a molecular mass of 73,642 Da. The predicted sequence shows a heptapeptide repeated 10 times, a carboxy-terminal lysine-rich tail, and some similarity to neurofilament proteins. The effects of deletion of CHS5 indicate that it is not essential for yeast cell growth; however, it is important for mating. Deletion of CHS3, the presumptive structural gene for chitin synthase III activity, results in a modest decrease in mating efficiency, whereas chs5delta cells exhibit a much stronger mating defect. However, chs5 cells produce more chitin than chs3 mutants, indicating that CHS5 plays a role in other processes besides chitin synthesis. Analysis of mating mixtures of chs5 cells reveals that cells agglutinate and make contact but fail to undergo cell fusion. The chs5 mating defect can be partially rescued by FUS1 and/or FUS2, two genes which have been implicated previously in cell fusion, but not by FUS3. In addition, mating efficiency is much lower in fus1 fus2 x chs5 than in fus1 fus2 x wild type crosses. Our results indicate that Chs5p plays an important role in the cell fusion step of mating.

Amino Acid Sequence↗
PubMed1996

Transcriptional responses to tubule challenges.

Adaptation to physiological stimuli often involves changes in gene transcription. Studies of hyperosmolar stress in renal epithelial cells have provided an ideal paradigm for understanding regulation of gene expression. Renal epithelial cells respond very differently to hyperosmolar NaCl and urea and several strategies including cloning based on known biological function, candidate gene analysis, and differential display analysis have successfully identified many genes induced by these hyperosmolar challenges. Hyperosmolar NaCl produces adverse effects on cellular biosynthetic processes and compensatory increases are observed in transcription of transporters, stress proteins, and metabolic enzymes. In contrast, hyperosmolar urea fails to inhibit biosynthetic processes but, nonetheless, initiates a very specific program of gene expression in renal epithelial cells. This program appears to involve a urea sensor/receptor system which activates transcription and translation of the zinc-finger transcription factor Egr-1. This work highlights the concept that rapid analysis of differential gene expression will enable one to define cellular programs of gene expression involving up- and down-regulation of functionally-related gene families.

Animals↗
PubMed1994

Are yeast chitin synthases regulated at the transcriptional or the posttranslational level?

The three chitin synthases of Saccharomyces cerevisiae, Chs1, Chs2, and Chs3, participate in septum and cell wall formation of vegetative cells and in wall morphogenesis of conjugating cells and spores. Because of the differences in the nature and in the time of execution of their functions, the synthases must be specifically and individually regulated. The nature of that regulation has been investigated by measuring changes in the levels of the three synthases and of the messages of the three corresponding genes, CHS1, CHS2, and CAL1/CSD2/DIT101/KTI2 (referred to below as CAL1/CSD2), during the budding and sexual cycles. By transferring cells carrying CHS2 under the control of a GAL1 promoter from galactose-containing medium to glucose-containing medium, transcription of CHS2 was shut off. This resulted in a rapid disappearance of Chs2, whereas the mRNA decayed much more slowly. Furthermore, Chs2 levels experienced pronounced oscillations during the budding cycle and were decreased in the sexual cycle, indicating that this enzyme is largely regulated by a process of synthesis and degradation. For CHS1 and CAL1/CSD2, however, a stop in transcription was followed by a slow decrease in the level of zymogen (Chs1) or an increase in the level of activity (Chs3), despite a rapid drop in message level in both cases. In synchronized cultures, Chs1 levels were constant during the cell cycle. Thus, for Chs1 and Chs3, posttranslational regulation, probably by activation of latent forms, appears to be predominant. Since Chs2, like Chs1, is found in the cell in the zymogenic form, a posttranslational activation step appears to be necessary for this synthase also.

Aminoglycosides↗
PubMed1985

Posterior capsular-zonular disruption in planned extracapsular surgery.

The incidence of inadvertent posterior capsule-zonular disruption at each stage of planned extracapsular cataract extraction was studied in a consecutive series of 250 cases. Disruption occurred during nucleus expression in 0.8%, during cortical cleanup in 3.2%, during removal of anterior capsular flaps in 0.4%, during polishing of the posterior capsule in 0.4%, and during posterior chamber lens (Sinskey-style) implantation in 0%. No cases of subluxation of the posterior chamber lens were observed (mean follow-up, 22 months). The overall incidence of posterior capsule-zonular disruption was 4.8%. Vitreous loss occurred in one half of these cases (2.4% of eyes). No cases of dislocation of the nucleus into the vitreous occurred. In 50% of the cases of posterior capsule-zonular disruption (2.4% of eyes), prompt recognition and proper management avoided disruption of the vitreous face. In 80% of these cases with posterior capsule-zonular disruption but intact vitreous face, it was possible to remove sufficient cortex and to proceed with uncomplicated posterior chamber lens implantation.

Cataract Extraction↗
PubMed1982

Nutrition and public health in the Dominican Republic.

A nutrition survey was carried out in a mountainous region southwest of Santiago, Dominican Republic, to determine the extent of malnutrition in the area and to obtain information regarding food beliefs and practices needed to design a viable nutrition program. A stratified cluster sampling technique was used to interview 295 mothers of preschool children and examine 448 of their children under five years of age. The incidence of moderate and severe malnutrition was less than expected. Only 12.3% of the children were less than or equal to 75% of the National Center for Health Statistics (NCHS) reference standard for weight/age; however, 6.5% were greater than 110% of the standard. Children from large, poor families were most vulnerable to growth retardation. Definite meal patterns were identified that included varied sources of cereal products and animal and mixed vegetable proteins. Milk and sausage consumption had a significant positive association with all three growth parameters. Suggestions were made for further research and program development.

Child, Preschool↗
PubMedDate not supplied

Survival and medical intervention in southern Brazilian AIDS patients.

The aim of the study was to describe survival patterns of Southern Brazilian AIDS patients: 224 predominantly working class AIDS patients were treated in an AIDS referral centre in Porto Alegre between October 1986 and September 1991. The caseload increased progressively, as did the number of female AIDS cases treated at the Hospital during the study period. Self-referred patients were more likely to present with an AIDS defining condition (P < 0.03) and they (n = 106) had significantly worse survival patterns compared with patients referred by other health care professionals (n = 112; P < 0.04). Median survival from the time of AIDS diagnosis was 5 months which did not change significantly during the study period (P = 0.38). Patients (n = 42) presenting with opportunistic infections other than mycobacterial disease (n = 42), Pneumocystis carinii pneumonia (n = 37) or candidiasis (n = 18), had significantly worse survival patterns (P = 0.001). Patients treated with zidovudine (n = 33) survived significantly longer from time of AIDS diagnosis than those not on zidovudine (n = 185; P = 0.0002). No significant survival differences were observed from time of AIDS diagnosis between those who commenced on zidovudine before developing AIDS (n = 17) and those who were treated with zidovudine since diagnosed with AIDS (n = 16; P = 0.80). During the study period zidovudine was only available through private prescriptions. Survival of Southern Brazilian AIDS patients has not improved: earlier access to HIV-related services and the provision of effective and affordable therapeutic interventions are two measures which could improve future survival patterns.

AIDS-Related Opportunistic Infections↗
PubMedDate not supplied

[Coma associated with migraine].

INTRODUCTION: Basilar migraine is a particular form of migraine with an aura in which crises of headache are accompanied by symptoms of dysfunction in the vertebro-basilar territory, including alteration of consciousness in the form of stupor or coma. CLINICAL CASES: We report four patients, three men of 14, 17 and 83 years of age and one woman of 21. All had previous histories of migraine and presented with transitory episodes of coma. During the coma, the woman woke up spontaneously with intense bulimia. In the three men, it was seen, on injecting flumazenil, that the state of consciousness and the EEG returned to normal transiently. Neuroimaging studies (CT and MR) were normal in all patients. CONCLUSIONS: Migraine-coma is an exceptional, emergency condition in which structural, infectious, toxic and metabolic pathology of the Central Nervous System should be ruled out. As well as in patients with basilar migraine, the association of migraine and coma may also be seen in patients with familial hemiplegic migraine and CADASIL and MELAS syndromes. It may be that gabaergic mechanisms are involved in the theoretical dysfunction of the ascending reticular activating system causing alteration of consciousness, since in the three patients in whom flumazenil was injected, there was a response.

Adolescent↗