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Biomedical subjects

Javier Garcia

Publications and source records attributed to Javier Garcia.

16 recordsLinked to original sources
PubMed2007

Characterisation of Plasmodium falciparum RESA-like protein peptides that bind specifically to erythrocytes and inhibit invasion.

The Plasmodium falciparum ring-erythrocyte surface antigen (RESA)-like putative protein was identified and characterised. PCR and RT-PCR assays revealed that the gene encoding this protein was both present and being transcribed in P. falciparum strain FCB-2 16 h after erythrocyte invasion. Indirect immunofluorescence studies detected this protein in infected erythrocyte (IE) cytosol in dense fluorescent granules similar to Maurer's clefts at 16-20 h (parasites in ring and trophozoite stages) and very strongly on IE membranes at 22 h, suggesting that it is synthesised during early ring stages (16 h) and transported to the infected red blood cell (RBC) membrane surface during the trophozoite stage (22 h). Western blotting showed that antisera produced against polymerised synthetic peptides of this protein recognised a 72-kDa band in P. falciparum schizont lysate. P. falciparum RESA-like peptides used in normal RBC binding assays revealed that peptides 30326 ((101)NAEKI LGFDD KNILE ALDLFY(120)), 30334 ((281)RVTWK KLRTK MIKAL KKSLTY(300)) and 30342 ((431)SSPQR LKFTA GGGFC GKLRNY(450)) bind with high activity and saturability, presenting nM affinity constants. These peptides contain alpha-helical structural elements, as determined by circular dichroism, and inhibit P. falciparum in vitro invasion of normal RBCs by up to 91%, suggesting that some RESA-like protein regions are involved in intra-erythrocyte stage P. falciparum invasion.

Amino Acid Sequence↗
PubMed2006

Nanoliter high throughput quantitative PCR.

Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols.

Cells, Cultured↗
PubMed2006

Pattern projection for subpixel resolved imaging in microscopy.

In this paper, we present a new approach providing super resolved images exceeding the geometrical limitation given by the detector pixel size of the imaging camera. The concept involves the projection of periodic patterns on top of the sample, which are then investigated under a microscope. Combining spatial scanning together with proper digital post-processing algorithm yields the improved geometrical resolution enhancement. This new method is especially interesting for microscopic imaging when the resolution of the detector is lower than the resolution due to diffraction.

Journal Article↗
PubMed2006

Speckle random coding for 2D super resolving fluorescent microscopic imaging.

In this manuscript we present a novel super resolving approach based upon projection of a random speckle pattern onto samples observed through a microscope. The projection of the speckle pattern is created by coherent illumination of the inspected pattern through a diffuser. Due to local interference of the coherent wave front with itself, a random speckle pattern is superimposed on the sample. This speckle pattern can be scanned over the object. A super-resolved image can be extracted from a temporal sequence of images by appropriate digital processing of the image stream. The resulting resolution is significantly higher than the diffraction limitation of the microscope objective. The new super-resolution method is demonstrated by application to fluorescence of biological samples.

Journal Article↗
PubMed2006

Septic thrombophlebitis: diagnosis and management.

Septic thrombophlebitis, as a result of invasion from adjacent nonvascular infections, includes conditions such as Lemierre syndrome (internal jugular vein septic thrombophlebitis), pylephlebitis (portal vein septic thrombophlebitis), and septic thrombophlebitis of the dural sinuses and the pelvic veins. All of these conditions are associated with a very high mortality if untreated. Appropriate antibacterial therapy dramatically improves the outcome of these infections and results in a low mortality rate, with the notable exception of septic thrombophlebitis of the dural sinuses. The endovascular nature of these infections results in secondary metastatic disease, including pneumonia, endocarditis, and arthritis due to septic embolization and/or hematogenous bacterial spread. The appropriate diagnosis and management of these infections depends on a high degree of clinical suspicion, the use of imaging studies, and early initiation of empiric antibacterial therapy. In this article, we review the diagnosis and management of septic thrombophlebitis, focusing on Lemierre syndrome, pylephlebitis, and septic thrombophlebitis of the pelvic veins.

Anti-Bacterial Agents↗
PubMed2005

Analysis of PM10, PM2.5, and PM2 5-10 concentrations in Santiago, Chile, from 1989 to 2001.

Daily particle samples were collected in Santiago, Chile, at four urban locations from January 1, 1989, through December 31, 2001. Both fine PM with da < 2.5 microm (PM2.5) and coarse PM with 2.5 < da < 10 microm (PM2.5-10) were collected using dichotomous samplers. The inhalable particle fraction, PM10, was determined as the sum of fine and coarse concentrations. Wind speed, temperature and relative humidity (RH) were also measured continuously. Average concentrations of PM2.5 for the 1989-2001 period ranged from 38.5 microg/m3 to 53 microg/m3. For PM2.5-10 levels ranged from 35.8-48.2 microg/m3 and for PM10 results were 74.4-101.2 microg/m3 across the four sites. Both annual and daily PM2.5 and PM10 concentration levels exceeded the U.S. National Ambient Air Quality Standards and the European Union concentration limits. Mean PM2.5 levels during the cold season (April through September) were more than twice as high as those observed in the warm season (October through March); whereas coarse particle levels were similar in both seasons. PM concentration trends were investigated using regression models, controlling for site, weekday, month, wind speed, temperature, and RH. Results showed that PM2.5 concentrations decreased substantially, 52% over the 12-year period (1989-2000), whereas PM2.5-10 concentrations increased by approximately 50% in the first 5 years and then decreased by a similar percentage over the following 7 years. These decreases were evident even after controlling for significant climatic effects. These results suggest that the pollution reduction programs developed and implemented by the Comisión Nacional del Medio Ambiente (CONAMA) have been effective in reducing particle levels in the Santiago Metropolitan region. However, particle levels remain high and it is thus imperative that efforts to improve air quality continue.

Air Pollutants↗
PubMed2005

Identifying Plasmodium falciparum merozoite surface antigen 3 (MSP3) protein peptides that bind specifically to erythrocytes and inhibit merozoite invasion.

Receptor-ligand interactions between synthetic peptides and normal human erythrocytes were studied to determine Plasmodium falciparum merozoite surface protein-3 (MSP-3) FC27 strain regions that specifically bind to membrane surface receptors on human erythrocytes. Three MSP-3 protein high activity binding peptides (HABPs) were identified; their binding to erythrocytes became saturable, had nanomolar affinity constants, and became sensitive on being treated with neuraminidase and trypsin but were resistant to chymotrypsin treatment. All of them specifically recognized 45-, 55-, and 72-kDa erythrocyte membrane proteins. They all presented alpha-helix structural elements. All HABPs inhibited in vitro P. falciparum merozoite invasion of erythrocytes by ~55%-85%, suggesting that MSP-3 protein's role in the invasion process probably functions by using mechanisms similar to those described for other MSP family antigens.

Animals↗
PubMed2005

P. falciparum pro-histoaspartic protease (proHAP) protein peptides bind specifically to erythrocytes and inhibit the invasion process in vitro.

Plasmodium falciparum histoaspartic protease (HAP) is an active enzyme involved in haemoglobin degradation. HAP is expressed as an inactive 51-kDa zymogen and is cleaved into an active 37-kDa enzyme. It has been proposed that this kind of protease might be implicated in the parasite's invasion of erythrocytes; however, this protein's role during invasion has still to be determined. Synthetic peptides derived from the HAP precursor (proHAP) were tested in erythrocyte binding assays to identify their possible function in the invasion process. Two proHAP high-activity binding peptides (HABPs) specifically bound to erythrocytes; these peptides were numbered 30609 (101LKNYIKESVKLFNKGLTKKS120) and 30610 (121YLGSEFDNVELKDLANVLSF140 ). The binding of these two peptides was saturable, presenting nanomolar affinity constants. These peptides interacted with 26- and 45-kDa proteins on the erythrocyte surface; the nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. The HABPs showed greater than 90% merozoite invasion inhibition in in vitro assays. Goat serum containing proHAP polymeric peptide antibodies inhibited parasite invasion in vitro .

Amino Acid Sequence↗
PubMed2004

Identifying gp85-regions involved in Epstein-Barr virus binding to B-lymphocytes.

Epstein-Barr virus lacking glycoprotein gp85 cannot infect B-cells and epithelial cells. The gp85 belongs to the molecular complex required for virus invasion of B-lymphocyte or epithelial cells. Moreover, there is evidence that gp85 is necessary for virus attachment to epithelial cells. Thirty-six peptides from the entire gp85-sequence were tested in epithelial and lymphoblastoid cell line binding assays to identify gp85-regions involved in virus-cell interaction. Five of these peptides presented high binding activity to Raji, Ramos, P3HR-1, and HeLa cells, but not to erythrocytes; Raji-cell affinity constants were between 80 and 140nM. Of these five peptides, 11435 ((181)TYKRVTEKGDEHVLSLVFGK(200)), 11436 ((201)TKDLPDLRGPFSYPSLTSAQ(220)), and 11438 ((241)YFVPNLKDMFSRAVTMTAAS(260)) bound to a 65kDa protein on Raji-cell surface. These peptides and antibodies induced by them (recognising live EBV-infected cells) inhibited Epstein-Barr virus interaction with cord blood lymphocytes. It is thus probable that gp85-regions defined by peptides 11435, 11436, and 11438 are involved in EBV invasion of B-lymphocytes.

Animals↗
PubMed2003

Identification of specific Hep G2 cell binding regions in Plasmodium falciparum sporozoite-threonine-asparagine-rich protein (STARP).

It has been demonstrated that Plasmodium falciparum sporozoite threonine-asparagine-rich protein (PfSTARP) is located on the sporozoite surface. This protein's non-overlapping consecutive peptides were synthesised and tested in Hep G2 cell binding assays. Twelve high activity binding peptides (HABPs) were identified in the resulting 31 peptides. Three were found in 5' non-repeat region (amino acids 41-80). Peptides 20546 (41VIKHNRFLSEYQSNFLGGGY(60)), 20547 (61SAALKLVNSKKSGTNVNVTKY(80)) and 20548 (81NSENTNTNNNIPESSSTYTN(100)) were located in the conserved amino terminal region, as well as peptide 20548 which shared the sequence with the M region (amino acids 85-134). Six HABPs were located in region 10 (Rp10) (STDNNNTKTI). HABPs 20569 (501TSDDELNKDSCDYSEEKENI(520)) and 20570 (521KSMINAYLDKLDLETVRKIH(40)) were found in 3' non-repeat region. All these HABPs showed saturable binding and presented dissociation constants between 18 and 219 nM. The number of binding sites per Hep G2 cell ranged from 45000 to 370000. High binding peptides' critical amino acids involved in Hep G2 cell binding were determined by competition binding assays. SDS-PAGE results showed that both peptides 20570 and 20547 had at least two different sets of 44 and 38 kDa HABP receptors on Hep G2 cells. Specific modification of peptide 20546 and 20570 critical binding residues rendered these peptides immunogenic in Aotus monkeys, inducing high antibody titres against sporozoites, as assessed by IFA.

Amino Acid Sequence↗
PubMed2003

Synthesis and SAR of cis-1-benzoyl-1,2,3,4-tetrahydroquinoline ligands for control of gene expression in ecdysone responsive systems.

A library of 35 cis-1-benzoyl-2-methyl-4-(phenylamino)-1,2,3,4-tetrahydroquinolines was prepared. The compounds bore various substitutuents on the benzoyl ring, at the 4-position of the phenylamino ring and at the 6-position of the tetrahydroquinoline ring. The compounds were assayed for their ability to cause expression of a reporter gene downstream of an ecdysone response element in a mammalian cell line engineered to express the ecdysone receptor from Aedes aegypti. In general, compounds with small lipophilic substituents at the meta and para-positions of the benzoyl ring and hydrogen or fluorine at the 4-position of the phenylamino ring and the 6-position of the tetrahydroquinoline ring were the most potent.

Aedes↗
PubMed2003

Synthesis and SAR of alpha-acylaminoketone ligands for control of gene expression.

A lead discovery library and a follow-up focused library of alpha-acylaminoketones were designed based on known dibenzoylhydrazine ecdysone agonists, including GS(TM)-E. The compounds were assayed in mammalian cells expressing the ecdysone receptor from Bombyx mori for their ability to cause expression of a reporter gene downstream of an ecdysone response element. The most potent alpha-acylaminoketones were comparable to GS(TM)-E in this assay.

Amines↗
PubMed2002

Plasmodium vivax Duffy binding protein peptides specifically bind to reticulocytes.

Plasmodium vivax Duffy Binding Protein (Pv-DBP) is essential during merozoite invasion of reticulocytes. Reticulocyte binding region identification is important for understanding Pv-DBP reticulocyte recognition. Fifty 20 mer non-overlapping peptides, spanning Pv-DBP sequences, were tested in erythrocyte and reticulocyte binding assays. Ten HARBPs, mainly located in region II (Kd 50-130 nM), were High Activity Reticulocyte Binding Peptides (HARBPs); one bound to erythrocytes. Reticulocyte trypsin-, chymotrypsin- or neuraminidase- treatment affects HARBP binding differently, suggesting that these peptides have different reticulocyte-binding-sites. Some peptides bound to a Coomasie non-stainable 40 Kda band. Some HARBPs were able to block recombinant PvRII binding (Pv-DBP region II) to Duffy positive reticulocytes.

Amino Acid Sequence↗
PubMed2002

The evolution of Lemierre syndrome: report of 2 cases and review of the literature.

Lemierre syndrome (postanginal septicemia) is caused by an acute oropharyngeal infection with secondary septic thrombophlebitis of the internal jugular vein and frequent metastatic infections. A high degree of clinical suspicion is necessary for diagnosis. Fusobacterium necrophorum is the usual etiologic agent. The disease progresses in several steps. The first stage is the primary infection, which is usually a pharyngitis (87.1% of cases). This is followed by local invasion of the lateral pharyngeal space and IJV septic thrombophlebitis (documented in 71.5% of cases), and finally, the occurrence of metastatic complications (present in 90% of cases at the time of diagnosis). A sore throat is the most common symptom during the primary infection (82.5% of cases). During invasion of the lateral pharyngeal space and IJV septic thrombophlebitis, a swollen and/or tender neck is the most common finding (52.2% of patients) and should be considered a red flag in patients with current or recent pharyngitis. The most common site of metastatic infection is the lungs (79.8% of cases). In contrast to the preantibiotic era, cavitating pneumonia and septic arthritis are now uncommon. Most patients (82.5%) had fever at some stage during the course of the disease. Gastrointestinal complaints such as abdominal pain, nausea, and vomiting were common (49.5% of cases). An elevated white blood cell count occurred in 75.2% of cases. Hyperbilirubinemia with slight elevation of liver enzyme levels occurred in one-third of patients, but frank jaundice was uncommon, in contrast to its high frequency reported in the preantibiotic era. We conclude that, most likely as a consequence of widespread antibiotic use for pharyngeal infections, the typical course of the disease has changed since Lemierre's original description. The typical triad in our series was: pharyngitis, a tender/swollen neck, and noncavitating pulmonary infiltrates. The previous classical description of severe sepsis with cavitating pneumonia and septic arthritis was not commonly seen in our review. Mortality was low in our series (6.4%), but significant morbidity occurred, which was likely preventable by early diagnosis and treatment. The pathophysiology, natural history, diagnostic methods for internal jugular vein thrombosis, and management are discussed.

Abdominal Pain↗
PubMed2002

Complementary roles of multiple nuclear targeting signals in the capsid proteins of the parvovirus minute virus of mice during assembly and onset of infection.

This report describes the distribution of conventional nuclear localization sequences (NLS) and of a beta-stranded so-called nuclear localization motif (NLM) in the two proteins (VP1, 82 kDa; VP2, 63 kDa) forming the T=1 icosahedral capsid of the parvovirus minute virus of mice (MVM) and their functions in viral biogenesis and the onset of infection. The approximately 10 VP1 molecules assembled in the MVM particle harbor in its 142-amino-acid (aa) N-terminal-specific region four clusters of basic amino acids, here called BC1 (aa 6 to 10), BC2 (aa 87 to 90), BC3 (aa 109 to 115), and BC4 (aa 126 to 130), that fit consensus NLS and an NLM placed toward the opposite end of the polypeptide (aa 670 to 680) found to be necessary for VP2 nuclear uptake. Deletions and site-directed mutations constructed in an infectious MVM plasmid showed that BC1, BC2, and NLM are cooperative nuclear transport sequences in singly expressed VP1 subunits and that they conferred nuclear targeting competence on the VP1/VP2 oligomers arising in normal infection, while BC3 and BC4 did not display nuclear transport activity. Notably, VP1 proteins mutated at BC1 and -2, and particularly with BC1 to -4 sequences deleted, induced nuclear and cytoplasmic foci of colocalizing conjugated ubiquitin that could be rescued from the ubiquitin-proteasome degradation pathway by the coexpression of VP2 and NS2 isoforms. These results suggest a role for VP2 in viral morphogenesis by assisting cytoplasmic folding of VP1/VP2 subviral complexes, which is further supported by the capacity of NLM-bearing transport-competent VP2 subunits to recruit VP1 into the nuclear capsid assembly pathway regardless of the BC composition. Instead, all four BC sequences, which are located in the interior of the capsid, were absolutely required by the incoming infectious MVM particle for the onset of infection, suggesting either an important conformational change or a disassembly of the coat for nuclear entry of a VP1-associated viral genome. Therefore, the evolutionarily conserved BC sequences and NLM domains provide complementary nuclear transport functions to distinct supramolecular complexes of capsid proteins during the autonomous parvovirus life cycle.

Animals↗