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Biomedical subjects

M Losada

Publications and source records attributed to M Losada.

At least 19 recordsLinked to original sources
PubMed2001

Simultaneous occurrence of two different glyceraldehyde-3-phosphate dehydrogenases in heterocystous N(2)-fixing cyanobacteria.

Enzyme activity determinations and Western and Northern blot analyses have shown the presence of two catalytically different glyceraldehyde-3-phosphate dehydrogenases (GAPDH) in both vegetative cells and heterocysts of several N(2)-fixing Anabaena strains: (a) the gap2-encoded NAD(P)-dependent GAPDH2 (EC 1.2.1.59), the enzyme involved in the photosynthetic carbon assimilation pathway, which is present at higher levels in vegetative cells, and (b) the gap3-encoded NAD-dependent GAPDH3 (EC 1.2.1.12), presumably involved in carbohydrate anabolism and catabolism, which is the predominant GAPDH in heterocysts. In contrast, the gap1-encoded GAPDH1, which is the other NAD-dependent cyanobacterial GAPDH, is virtually absent in both cell types. These findings are discussed in the context of carbon metabolism of heterocystous N(2)-fixing cyanobacteria.

Anabaena↗
PubMed2001

A thermostable K(+)-stimulated vacuolar-type pyrophosphatase from the hyperthermophilic bacterium Thermotoga maritima.

Current evidence suggests the occurrence of two classes of vacuolar-type H(+)-translocating inorganic pyrophosphatases (V-PPases): K(+)-insensitive proteins, identified in eukaryotes, bacteria and archaea, and K(+)-stimulated V-PPases, identified to date only in eukaryotes. Here, we describe the functional characterization of a thermostable V-PPase from the anaerobic hyperthermophilic bacterium Thermotoga maritima by heterologous expression in Saccharomyces cerevisiae. The activity of this 71-kDa membrane-embedded polypeptide has a near obligate requirement for K(+), like the plant V-PPase, and its thermostability depends on the binding of Mg(2+). Phylogenetic analysis of protein sequences consistently assigned the T. maritima V-PPase to the K(+)-sensitive class of V-PPases so far only known for eukaryotes. The finding of a K(+)-stimulated V-PPase also in a member of a primitive eubacterial lineage strongly supports an ancient evolutionary origin of this group of pyrophosphate-energized proton pumps.

Bacterial Proteins↗
PubMed2001

Enzymatic systems of inorganic pyrophosphate bioenergetics in photosynthetic and heterotrophic protists: remnants or metabolic cornerstones?

An increasing body of biochemical and genetic evidence suggests that inorganic pyrophosphate (PPi) plays an important role in protist bioenergetics. In these organisms, two types of inorganic pyrophosphatases [EC 3.6.1.1, namely soluble PPases (sPPases) and proton-translocating PPases (H+-PPases)] that hydrolyse the PPi generated by cell anabolism, thereby replenishing the orthophosphate pool needed for phosphorylation reactions, are present in different cellular compartments. Photosynthetic and heterotrophic protists possess sPPases located in cellular organelles (plastids and mitochondria), where many anabolic and biosynthetic reactions take place, in addition to H+-PPases, which are integral membrane proteins of the vacuolysosomal membranes and use the chemical energy of PPi to generate an electrochemical proton gradient useful in cell bioenergetics. This last category of proton pumps was considered to be restricted to higher plants and some primitive photosynthetic bacteria, but it has been found recently in many protists (microalgae and protozoa) and bacteria, thus indicating that H+-PPases are much more widespread than previously thought. No cytosolic sPPase (in bacteria, fungi and animal cells) has been shown to occur in these lower eukaryotes. The widespread occurrence of these key enzymes of PPi metabolism among evolutionarily divergent protists strongly supports the ancestral character of the bioenergetics based on this simple energy-rich compound, which may play an important role in survival under different biotic and abiotic stress conditions.

Animals↗
PubMed2001

Natural evolution of Elschnig pearl posterior capsule opacification after posterior capsulotomy.

PURPOSE: To study the natural evolution of Elschnig pearl posterior capsule opacification (PCO) after neodymium:YAG posterior capsulotomy. SETTING: Instituto Oftalmológico Murciano, Murcia, Spain. METHODS: This prospective study comprised 173 eyes that had a posterior capsulotomy to treat Elschnig pearl PCO. Patients were included in the study at different stages after posterior capsulotomy and followed using serial anterior segment photographs for 18 to 48 months. RESULTS: During the first year after the capsulotomy, pearls proliferated and migrated toward the capsulotomy edge in 83% of the cases, leading to the formation of a collar-like ring of pearls around the capsulotomy. During the second year, an increase in cells was observed in 45% of cases; in 45% of eyes, there were fewer pearls. The amount of pearls decreased in 48%, 52%, and 26% of cases at 3, 4, and 5 years, respectively, and were completely gone in 6%, 17%, and 45%. After 5 years, 17% of eyes had a reduction in pearls and 67% had no pearls. CONCLUSIONS: Elschnig pearl PCO is a self-limiting process. After posterior capsulotomy, cells proliferated and attempted to occlude the capsular opening. Later, a reduction in the amount of cells was observed, leading to a completely clear posterior capsule in most eyes.

Adolescent↗
PubMed2001

Factors determining the special redox properties of photosynthetic cytochrome b559.

Factors controlling the redox properties of the two conventional forms of cytochrome b559, i.e. the unstable high-potential form and the stable low-potential form, have been further investigated using PSII-enriched membranes from pea and spinach chloroplasts. The redox potential of the stable form of cytochrome b559 is pH independent both above pH 7.5 (E'm approximately +110 mV) and below pH 6.0 (E'm approximately +203 mV), but it changes with a slope of 58 mV per pH unit between these two pH values. Thus, cytochrome b559 seems to have a single ionizing group influencing its redox potential, with a higher affinity for protons in the reduced form (pK(red) = 7.5) and a lower affinity in the oxidized form (pK(ox) = 6.0); consequently, one unprotonated low-potential form (LP) and one protonated intermediate-potential form (IP). The redox potential of the high-potential form (HP) is pH-independent between pH 5.0 and 8.0, but its relative content (compared to the total amount of protein) decreases progressively above pH 7.0. This conversion to the stable LP form is interpreted as corresponding to the loss of a proton by one ionizing group, the protonation of which is essential for maintaining the unstable HP state. According to chemical modification experiments with diethylpyrocarbonate, one of the two histidine ligands of the heme seems to be the ionizing group responsible for the existence of both the protonated IP and HP forms. It is proposed that the difference between the IP and HP forms is due to the formation of an additional hydrogen bond between the protonated histidine and the protein in the HP state that stabilizes a special hydrophobic heme environment responsible for its high redox potential.

Cytochrome b Group↗
PubMed2000

Baryogenesis at low reheating temperatures

We note that the maximum temperature during reheating can be much greater than the reheating temperature T(r) at which the universe becomes radiation dominated. We show that the standard model anomalous (B+L)-violating processes can therefore be in thermal equilibrium for 1 GeV less, similarT(r)<<100 GeV. Electroweak baryogenesis could work and the traditional upper bound on the Higgs mass coming from the requirement of the preservation of the baryon asymmetry may be relaxed. Alternatively, the baryon asymmetry may be reprocessed by sphaleron transitions either from a (B-L) asymmetry generated by the Affleck-Dine mechanism or from a chiral asymmetry between e(R) and e(L) in a B-L = 0 universe.

Journal Article↗
PubMed1999

Light-induced degradation of cytochrome b559 during photoinhibition of the photosystem II reaction center.

The behaviour of cytochrome (cyt) b559 during acceptor- and donor-side photoinhibition has been investigated in oxygen-evolving and non-evolving photosystem II (PSII) membranes. Strong illumination at 20 degrees C under aerobiosis induced a strong decrease in the absorbance of the cyt b559 alpha-band in the two preparations. This absorbance decline was observed only in non-oxygen-evolving PSII samples when illumination was performed under aerobiosis but at 4 degrees C, or under anaerobiosis at 20 degrees C. These results suggest that acceptor-side photoinhibition induces the degradation of cyt b559 by a mechanism related to an enzymatic reaction mediated by singlet oxygen. Donor-side photoinhibition may induce, however, a non-enzymatic photocleavage of the protein.

Cytochrome b Group↗
PubMed1999

Engineering a central metabolic pathway: glycolysis with no net phosphorylation in an Escherichia coli gap mutant complemented with a plant GapN gene.

A cDNA fragment containing the Pisum sativum GapN gene, which encodes the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase, was cloned in a prokaryote expression vector. This construct enabled Escherichia coli strain W3CG, a mutant which lacks the glycolytic phosphorylating G3P dehydrogenase, to grow aerobically on sugars. The functionally complemented mutant exhibited high levels of the catalytically active plant enzyme, which renders 3-phosphoglycerate and NADPH, thus bypassing the first substrate level phosphorylation step of the glycolysis. As expected if such a glycolytic bypass would be operative in vivo, this clone failed to grow anaerobically on sugars in contrast to W3CG clones complemented with phosphorylating glyceraldehyde-3-phosphate dehydrogenases. According to the irreversible catabolic character of the non-phosphorylating reaction, the GapN-complemented clone was unable to grow on gluconeogenic substrates. This metabolic engineering approach demonstrates that a pure catabolic Embden-Meyerhof pathway with no net energy yield is feasible.

Escherichia coli↗
PubMed1998

Parathyroid function in long-term renal transplant patients: importance of pre-transplant PTH concentrations.

Lack of resolution of hyperparathyroidism after long-term renal transplantation is common. The relative roles of the graft function attained and the degree of pre-transplant hyperparathyroidism have not been established. Intact parathyroid hormone (iPTH) and several clinical parameters were studied before and 68.6+/-26.8 months (range: 30-124) after renal transplantation in 62 patients (20 females/42 males) with good renal function (creatinine <2 mg/dl). iPTH decreased from 214+/-229 pre-transplantation to 116+/-70 pg/ml post-transplantation (P<0.01). However, only 22.6% of patients had PTH concentrations in the normal range, and values greater than twice the upper normal limit were not uncommon (27.4%). Of the many variables analysed, creatinine (r=0.43; P=0.001) and pre-transplant PTH (r=0.31; P=0.02) significantly correlated with post-transplant PTH. After selecting patients with serum creatinine <1.5 mg/dl (n=46), pre-transplant PTH emerged as the more important predictor of post-transplant PTH (r=0.58; P<0.0001). After controlling for creatinine, the partial correlation was r=0.53, P<0.0001. We concluded that spontaneous resolution of hyperparathyroidism after renal transplantation is uncommon. In addition, the magnitude of pre-transplant hyperparathyroidism and the renal function determine the long-term post-transplant parathyroid function.

Adult↗
PubMed1998

Subclavian vascular access stenosis in dialysis patients: natural history and risk factors.

Stenosis of the subclavian vein (SVS) after cannulation occurs in 15 to 50% of chronic hemodialysis patients, and impedes the placement of an arteriovenous fistula in the ipsilateral arm. Its natural history and pathogenic mechanisms are not well established. This study examined 42 consecutive chronic renal failure patients (28 men and 14 women; 46+/-19 yr) in whom subclavian catheters had been placed as the initial vascular access for hemodialysis. All patients underwent sequential venography studies: at baseline (24 to 48 h after removal of the catheter) and 1, 3, and 6 mo thereafter. Venograms were considered abnormal when there was evidence of unequivocal strictures (more than 30% narrowing), with or without collateral circulation. At baseline, 52.4% (n=22) of patients showed stenotic vein lesions (n=19) or total thrombosis (n=3), and identical lesions were also observed after 1 mo. Surprisingly, 10 of 22 patients with initial SVS (45.4%) showed spontaneous recanalization of venous lesions in the venographies performed 3 mo after removal. The patients with normal baseline venograms (n=20) showed no change during follow-up. Patients with definitive stenosis at 6 mo (n=12) had a higher number of inserted catheters (1.58+/-0.6 versus 1.2+/-0.48; P < 0.05), longer time in place (49.08+/-32.2 versus 29.03+/-26.6 d; P < 0.05), and higher number of dialysis sessions (21+/-13.8 versus 12.4+/-11.4; P < 0.05) than those without SVS or with spontaneous recanalization of venous lesions during follow-up. Furthermore, a higher number of catheter-related infections were observed in patients with definitive SVS (66.6% versus 33.3%; P < 0.05). In summary, SVS is observed in more than half of patients 24 to 48 h after catheter removal and 1 mo later. Even when recanalization occurs in many cases, a definitive stenosis is seen in 28% of patients by the third month. Thus, the creation of an ipsilateral vascular access is possible provided that venography is normal at this time. Finally, mechanical factors and catheter-related infections are the major risk factors for the development of late SVS.

Adult↗
PubMed1997

Functional complementation of an Escherichia coli gap mutant supports an amphibolic role for NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase of Synechocystis sp. strain PCC 6803.

The gap-2 gene, encoding the NAD(P)-dependent D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH2) of the cyanobacterium Synechocystis sp. strain PCC 6803, was cloned by functional complementation of an Escherichia coli gap mutant with a genomic DNA library; this is the first time that this cloning strategy has been used for a GAPDH involved in photosynthetic carbon assimilation. The Synechocystis DNA region able to complement the E. coli gap mutant was narrowed down to 3 kb and fully sequenced. A single complete open reading frame of 1,011 bp encoding a protein of 337 amino acids was found and identified as the putative gap-2 gene identified in the complete genome sequence of this organism. Determination of the transcriptional start point, identification of putative promoter and terminator sites, and orientation of the truncated flanking genes suggested the gap-2 transcript should be monocystronic, a possibility further confirmed by Northern blot studies. Both natural and recombinant homotetrameric GAPDH2s were purified and found to exhibit virtually identical physicochemical and kinetic properties. The recombinant GAPDH2 showed the dual pyridine nucleotide specificity characteristic of the native cyanobacterial enzyme, and similar ratios of NAD- to NADP-dependent activities were found in cell extracts from Synechocystis as well as in those from the complemented E. coli clones. The deduced amino acid sequence of Synechocystis GAPDH2 presented a high degree of identity with sequences of the chloroplastic NADP-dependent enzymes. In agreement with this result, immunoblot analysis using monospecific antibodies raised against GAPDH2 showed the presence of the 38-kDa GAPDH subunit not only in crude extracts from the gap-2-expressing E. coli clones and all cyanobacteria that were tested but also in those from eukaryotic microalgae and plants. Western and Northern blot experiments showed that gap-2 is conspicuously expressed, although at different levels, in Synechocystis cells grown in different metabolic regimens, even under chemoheterotrophic conditions. A possible amphibolic role of the cyanobacterial GAPDH2, namely, anabolic for photosynthetic carbon assimilation and catabolic for carbohydrate degradative pathways, is discussed.

Amino Acid Sequence↗
PubMed1996

HLA-DR class II and ICAM-1 expression on tubular cells taken by fine-needle aspiration biopsy in renal allograft dysfunction.

BACKGROUND: Percutaneous biopsy is the method of choice for differential diagnosis of renal allograft dysfunction, although it is not risk-free. The use of less aggressive methods for diagnosis should limit the need for percutaneous biopsy to some specific situations. METHODS: We analysed 42 fine-needle aspiration biopsies from 36 kidney allograft recipients immunosuppressed with quadruple sequential therapy who suffered renal allograft dysfunction. Seven cases with stable renal function were used as controls and included as non-rejection cases in the analysis. In all aspirates the Corrected Increment was calculated and an immunocytochemical analysis of renal tubular cells with the monoclonal antibodies HLA-DR and ICAM-1 was performed. RESULTS: The Corrected Increment was increased in 13 out of 18 acute rejection cases and in one out of 31 non-rejection cases. HLA-DR expression was found in more than 30% of tubular cells from the aspirates in 16 out of 18 acute rejection cases and in eight out of 31 cases without rejection (P < 0.001). ICAM-1 expression was detected in more than 30% of tubular cells in 14 out of 18 cases with acute rejection, and in four out of 31 cases without acute rejection (P < 0.001). Interestingly, all acute vascular rejection cases (n = 6), and six out of 12 acute cellular rejection cases expressed both, HLA-DR and ICAM-1, in more than 30% of tubular cells. On the other hand, none of the non-rejection allograft dysfunctions or control samples showed more than 30% of tubular cells immunostained with both HLA-DR and ICAM-1 antibodies. CONCLUSIONS: The immunocytochemical analysis of HLA-DR and ICAM-1 on renal tubular cells taken by fine-needle aspiration biopsy, allows the diagnosis of acute cellular rejection and acute vascular rejection even when the Corrected Increment is not increased. Moreover, the risk of a core renal biopsy can be avoided when both tests are negative since an acute rejection is a remote possibility.

Acute Disease↗
PubMed1995

Resting metabolic rate in subjects with paraplegia: the effect of pressure sores.

OBJECTIVE: To determine the overall effect of paraplegia and pressure sores on resting metabolic rate. DESIGN: Unblinded, case-control study using a convenience sample. SETTING: Hospital primary care setting. PATIENTS: Fourteen individuals with paraplegia and pressure sores (PS-Para), 24 with paraplegia in good health (NPS-Para), and 23 non-spinal cord injury (SCI) controls. MAIN OUTCOME MEASURES: The planned outcome measures consisted of resting metabolic rate, percent of predicted resting metabolic rate, resting metabolic rate per kilogram body weight, and resting metabolic rate per meter squared body surface area. Post hoc analyses were used to identify the effect of completeness of lesion, smoking, and pressure sores on percent of predicted resting metabolic rate and resting metabolic rate per kilogram body weight. RESULTS: Percent of predicted resting metabolic rate and resting metabolic rate per kilogram body weight were significantly higher in the PS-Para group than in the NPS-Para or control groups (115% +/- 4% vs 100% +/- 2% or 107% +/- 2%, p < .05) and (25.9 +/- 1.2 vs 21.4 +/- 0.6 or 22.5 +/- 0.4 kcal/kg, p < .05, respectively). The resting metabolic rate per meter squared body surface area was significantly higher in the PS-Para group than in NPS-Para group (973 +/- 39 vs 874 +/- 20kcal/m2, p < .05). In the PS-Para group, current smokers had significantly higher resting metabolic rate per kilogram body weight than nonsmokers (27.3 +/- 1.7 vs 24.0 +/- 1.4kcal/kg, p < .01). Controlling for the effects of smoking in a multiple regression model, those in the PS-Para group had significantly (p < .001) greater percent of predicted resting metabolic rate and resting metabolic rate per kilogram body weight than those in the NPS-Para group. CONCLUSIONS: These findings indicate that individuals with SCI may have a decreased percent of predicted resting metabolic rate and those with pressure sores may have a hypermetabolic state. This hypermetabolic state is significantly higher than that resulting from smoking. Because ordinary prediction equations for energy expenditure may not be accurate when applied to subjects with paraplegia and pressure sores, quantification of energy needs by indirect calorimetry is recommended.

Basal Metabolism↗
PubMed1995

Long-term decentration of intraocular lenses implanted with envelope capsulotomy and continuous curvilinear capsulotomy: a comparative study.

Intraocular lens (IOL) decentration was studied in a series of 569 consecutive eyes that had extracapsular cataract extraction (ECCE) and posterior chamber IOL implantation. In 383 of the eyes, an envelope capsulotomy (EC) was performed; in 186, a continuous curvilinear capsulotomy (CCC). In 33 eyes, the anterior capsule had one radial tear that reached the lens equator. In all eyes, IOL decentration was determined more than six months after surgery. Mean IOL decentration was 0.42 +/- 0.02 mm in the EC eyes and 0.27 +/- 0.01 mm in the CCC eyes (P < .001). In the CCC eyes, mean IOL decentration was 0.23 +/- 0.02 mm when the anterior capsule had no tears and 0.42 +/- 0.06 mm when it had one radial tear (P < .01). In the EC eyes, mean decentration was less with lenses with a total diameter of 11.0 mm or less and 360 degree circular loops (compressible disc and circular open-loop lenses) than with C- and J-loop lenses with a total length of 13.5 mm to 14.0 mm. With the C- and J-loop lenses in all three capsulotomy groups, mean decentration was related to the presence and number of tears in the anterior capsule: 0.58 mm in the EC eyes, 0.41 mm in the CCC with tear eyes, and 0.23 mm in the CCC without tear eyes. A similar relationship was seen with the bag-implanted lenses: 0.48 mm in the EC eyes, 0.28 mm in the CCC with tear eyes; 0.22 mm in the CCC without tear eyes.

Cataract Extraction↗
PubMed1995

Effect of ovarian hormones on the hypothalamic excitatory amino acids system during sexual maturation in female rats.

The present experiments were designed to study in female rats during sexual maturation: (1) the hypothalamic release of aspartate (Asp), glutamate (Glu) and glycine (Gly) which are the excitatory amino acids (EAAs) involved in NMDA neurotransmission and of taurine (Tau), a putative inhibitory amino acid of GnRH secretion; (2) the relationships between the effect of estrogen-progesterone (EP) on the release of these EAAs and the secretion of gonadotropins, and (3) the effect of hypothalamic NMDA receptor stimulation on EAAs release by the hypothalamus as well as the effect of EP on this release. The release of EAAs by the anterior preoptic and medial-basal hypothalamic areas (APOA-MBH) is significantly higher in peripubertal than in prepubertal rats (p < 0.01). EP treatment in prepubertal rats (16 days of age) decreased LH and FSH plasmatic levels and also the in vitro release of Asp, Glu, Gly and Tau. Contrary to the observations in prepubertal rats, in 30-day-old peripubertal rats the ovarian hormones significantly (p < 0.01) increased the levels of LH and FSH as well as the release to the medium of these amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Presentation, clinical course, and outcome of the congenital form of myotonic dystrophy.

We report the clinical experience of 18 patients with the congenital form of myotonic dystrophy, the majority of whom were diagnosed during the neonatal period and monitored from 5 to 14 years. Prematurity associated with congenital myotonic dystrophy gives rise to the severest clinical manifestations. Among them, respiratory involvement is common and is the leading cause of death in the neonatal period. Weakness and foot deformities secondary to muscle involvement are the predominant clinical features of this group of patients from birth to age 3 or 4 years. Once muscle strength improves, learning disabilities and behavioral disturbances become the main clinical problems. All our patients, when tested after 5 years of age, had intelligence quotients under 65, clearly below the average intelligence quotient of their mothers (IQ = 80). There is no relationship between the degree of mothers' and patients' disease. No patient has presented problems with routine immunizations, and no complications were observed in the 7 patients who underwent surgery under general anesthesia. Among the surviving patients, no correlation can be established between severity of disease in the neonatal period and the magnitude of sequelae as teenagers. Mental and behavioral disturbances are the factors which mainly influence the long-term management and prognosis of this cohort of individuals.

Adolescent↗
PubMed1994

[Pneumonia in patients undergoing heart surgery].

OBJECTIVE: We performed a follow-up study of 104 consecutive patients who underwent cardiac surgery to ascertain the following: 1) the incidence of nosocomial pneumonia (NP) in this patient population; 2) the differences in the incidence of NP between patients who underwent coronary artery bypass grafting (CABG) vs. valvular replacement (VR), and 3) the identification of risk factors which predispose patients to the development of NP. RESULTS: The study included 104 patients of which 49 underwent VR, 43 CABG and 12 who had both procedures performed simultaneously. Six of the 104 patients developed NP (5.7%). Five of these patients had undergone VR where as opposed to only one in the CABG group. Pulmonary hypertension preoperatively was a risk factor for the development of NP. Of the 49 patients in the VR group, 46 had pulmonary artery pressures (PAP) recorded, and from their 23 (50%) had pulmonary hypertension. However, 4 of the 5 (80%) patients who developed NP had elevated PAP. The mortality among patients with NP was high. Sixty six percent of patients (4/6) with NP died in comparison to the 10 deaths (10.2%) among 92 patients without NP (p = 0.002). CONCLUSIONS: A trend in the development of NP was observed in patients who underwent valve replacement as opposed to CABG. Because of the small number of patients who developed pneumonia in the study population statistical significance cannot be reached. Pulmonary hypertension in the post operative period is a risk factor for the development of NP. Mortality among patients who develop NP is significantly high (p = 0.002).

Adult↗