Search PubMed⌕ Search

Biomedical subjects

M Espinosa

Publications and source records attributed to M Espinosa.

At least 37 records · Page 2Linked to original sources
PubMed2000

Expression of green fluorescent protein in Lactococcus lactis.

The gfp gene from Aequorea victoria, encoding the green fluorescent protein (GFP) has been expressed in Lactococcus lactis subsp. lactis biovar cremoris MG1363, upon construction and introduction of plasmid pLS1GFP into this host. GFP was monitored in living cells during growth to evaluate its use in molecular and physiological studies. Quantification of the levels of GFP expressed by cultures was feasible by fluorescence spectroscopy. Phase-contrast and fluorescence microscopy allowed us to distinguish, in mixed cultures, lactococcal cells expressing GFP. Our results indicate that GFP can be used as a reporter in L. lactis.

Genes, Reporter↗
PubMed2000

A functional lagging strand origin does not stabilize plasmid pMV158 inheritance in Escherichia coli.

Plasmid rolling circle replication generates single-stranded DNA intermediates. The intracellular amount of these molecules depends upon the efficiency of the conversion of single-stranded into double-stranded plasmid forms, that is, the functionality of the lagging strand origin (sso). The broad-host-range streptococcal plasmid pMV158 harbors two different ssos, both of which function efficiently in Streptococcus pneumoniae but poorly in Escherichia coli. Plasmid pMV158 is stably inherited in the pneumococcal host, but it is unstable in E. coli. A pMV158 derivative lacking its two ssos is unstable in both strains. We have cloned into this derivative the coliphage f1 lagging strand origin. Whereas the f1 sso was fully functional in E. coli, it did not show any activity in S. pneumoniae, a bacteria closely related to the pMV158 natural host. The presence of the f1 sso did not stabilize pMV158 inheritance in either the gram-positive or the gram-negative host.

Cloning, Molecular↗
PubMed2000

Construction of a tightly regulated plasmid vector for Streptococcus pneumoniae: controlled expression of the green fluorescent protein.

We have constructed a regulated plasmid vector for Streptococcus pneumoniae, based on the streptococcal broad-host-range replicon pLS1. As a reporter gene, we subcloned the gfp gene from Aequorea victoria, encoding the green fluorescent protein. This gene was placed under the control of the inducible P(M) promoter of the S. pneumoniae malMP operon which, in turn, is regulated by the product of the pneumococcal malR gene. Binding of MalR protein to the P(M) promoter is inactivated by growing the cells in maltose-containing media. Highly regulated gene expression was achieved by cloning in the same plasmid the P(M)-gfp cassette and the malR gene, thus providing the MalR regulator in cis. Pneumococcal cells harboring this vector gave a linear response of GFP synthesis in a maltose-dependent mode without detectable background levels in the absence of the inducer.

Bacterial Proteins↗
PubMed2000

Plasmid copy number control: an ever-growing story.

Bacterial plasmids maintain their number of copies by negative regulatory systems that adjust the rate of replication per plasmid copy in response to fluctuations in the copy number. Three general classes of regulatory mechanisms have been studied in depth, namely those that involve directly repeated sequences (iterons), those that use only antisense RNAs and those that use a mechanism involving an antisense RNA in combination with a protein. The first class of control mechanism will not be discussed here. Within the second class (the most 'classical' one), exciting insights have been obtained on the molecular basis of the inhibition mechanism that prevents the formation of a long-range RNA structure (pseudoknot), which is an example of an elegant solution reached by some replicons to control their copy number. Among the third class, it is possible to distinguish between (i) cases in which proteins play an auxiliary role; and (ii) cases in which transcriptional repressor proteins play a real regulatory role. This latter type of regulation is relatively new and seems to be widespread among plasmids from Gram-positive bacteria, at least for the rolling circle-replicating plasmids of the pMV158 family and the theta-replicating plasmids of the Inc18 streptococcal family.

DNA Replication↗
PubMed2000

IDO induction in IFN-gamma activated astroglia: a role in improving cell viability during oxidative stress.

In the central nervous system (CNS), astrocytes play an integral role in the maintenance of neuronal viability and function. Inflammation within the CNS increases the concentration of oxidative metabolites and, therefore, the potential for NAD depletion through increased poly-(ADP-ribose) polymerase (PARP) activity. However, the activity of indoleamine 2,3-dioxygenase (IDO), the rate limiting enzyme for de novo NAD synthesis, is also markedly increased in astrocytes during inflammation. This study investigated the role of IDO induction in the maintenance of intracellular NAD and its relationship to improved cell viability under conditions of increased oxidative stress in the human astroglioma cell line, HTB-138. Treatment with the pro-inflammatory cytokine IFN-gamma increased IDO activity in these cells. Intracellular NAD levels also increased significantly after treatment with IFN-gamma in the presence of a PARP inhibitor. Pretreatment of astroglial cells with IFN-gamma significantly moderated both the drop in intracellular NAD concentration and cell death following exposure to hydrogen peroxide. These results suggest that induction of IDO and subsequent de novo NAD synthesis may contribute to the maintenance of intracellular NAD levels and cell viability under conditions of increased oxidative stress.

Astrocytes↗
PubMed2000

High ALT levels predict viremia in anti-HCV-positive HD patients if a modified normal range of ALT is applied.

AIMS: Some studies have reported that ALT determination is of little value in the study of chronic hepatitis C in hemodialysis (HD) patients. This could be due to the fact that ALT values are lower in HD patients than in healthy individuals; ALT in HCV infection follows a fluctuating pattern and the HCV viremia may be intermittent. The aim of this study was to establish the reference ALT values in a large group of hepatitis-free HD patients, and to determine their role in predicting viremia in anti-HCV-positive HD patients. METHODS: Four subject groups were studied: group I, patients with normal renal function (n = 88); group II, hepatitis-free HD patients (n = 218); group III, non-viremic anti-HCV+ HD patients (n = 9); and group IV, viremic anti-HCV+ HD patients (n = 24). The ALT used for calculation purposes was the mean value of the twelve previous months for each individual patient. PCR screening for HCV-RNA was performed at least twice for anti-HCV+ patients; these were deemed viremic (HCV-RNA+) if at least one screening was positive, and non-viremic (HCV-RNA) if all PCR results were negative. RESULTS: Mean ALT in group II was lower than in subjects with normal renal function (15.6 +/- 12 vs. 22.7 +/- 18 IU/l, p < 0.05). No significant differences were observed between group III and group II (17.7 +/- 6 vs. 15.6 +/- 6 IU/l, ns). ALT levels in group IV patients were higher than those of groups II and III (38.5 +/- 39 IU/l, p < 0.05). The upper limit (mean + 2 SD and 95th percentile) for ALT in hepatitis-free HD patients was 27 IU/l. Sensitivity of a mean ALT value > or = 27 IU/l in the diagnosis of HCV viremia was 50%, and specificity was 100%. The positive predictive value of this test in the diagnosis of hepatitis C viremia was 100%. CONCLUSIONS: ALT values are lower in HD patients and a high ALT level can constitute an excellent tool in predicting viremia in anti-HCV-positive HD patients once other causes of liver disease have been excluded.

Alanine Transaminase↗
PubMed1999

Expression of the mobM gene of the streptococcal plasmid pMV158 in Lactococcus lactis subsp. lactis.

The streptococcal plasmid pMV158 is not auto-transferable, but it can be mobilised between bacteria by the use of functions supplied by plasmids of the pIP501/pAM beta 1 family. Plasmid pMV158 encodes a protein, MobM, which is involved in its mobilisation. This process initiates when MobM specifically cleaves supercoiled pMV158 plasmid DNA at the origin of transfer, oriT. Plasmid pMV158 has been transferred to Lactococcus lactis by conjugation aided by plasmid pAM beta 1. In the lactococcal host, MobM-mediated specific pMV158-relaxed molecules were detected. The intracellular amount of MobM has been quantified by immunoblot analyses and shown to be about 3500 molecules per cell. In the same host, we have mapped the initiation point of transcription of mobM. Transcription of this gene is directed from a promoter with an extended--10 region which overlaps with the pMV158-oriT.

Bacterial Proteins↗
PubMed1999

Mobilisation of the streptococcal plasmid pMV158: interactions of MobM protein with its cognate oriT DNA region.

The streptococcal plasmid pMV158 encodes the relaxase protein, MobM, involved in its mobilisation. Purified MobM protein specifically cleaved supercoiled or single-stranded DNA containing the plasmid origin of transfer, oriT. Gel retardation and DNase I footprinting assays performed with DNA fragments containing the plasmid oriT provided evidence for specific binding of MobM by oriT DNA. Dissection of the MobM-binding sequence revealed that the oriT region protected by MobM spanned 28 nucleotides, and includes an inversely repeated sequence, termed IR2. MobM exhibits a high degree of similarity with the mob gene product of the Streptococcus ferus plasmid pVA380-1. Although the origins of transfer of pMV158 and pVA380-1 show 20% sequence divergence in a 24-bp sequence included in their oriT regions, the pMV158 MobM was able to cleave a supercoiled derivative of pVA380-1 in vitro.

Amino Acid Sequence↗
PubMed1999

Psychopathology, mother-child interaction, and infant development: substance-abusing mothers and their offspring.

The course of severe depressive symptoms from pregnancy to 6 months postpartum, as well as the occurrence of severe paranoid symptoms prenatally, were examined by the Millon Clinical Multiaxial Inventory I and the Beck Depression Inventory, in 78 women who were heavy, chronic cocaine users and who retained custody of their children after birth. Six months postpartum, the quality of caregiving was observed and assessed in the home, and the children were assessed on the Bayley MDI Index in the laboratory. Mothers who were depressed and paranoid prenatally, regardless of whether the depression continued to 6 months postpartum, were less sensitive in caregiving than women without severe symptoms of paranoia or depression during pregnancy or those who reported only depression that lifted by 6 months postpartum. Mothers who were depressed prenatally and continued to be depressed by 6 months postpartum, regardless of the presence or absence of paranoia, had infants who earned lower Bayley MDI scores than the offspring of women without severe psychological symptoms or women whose depression had lifted. Severe depressive symptoms during pregnancy, if they did not continue to 6 months postpartum, did not appear to adversely influence either caregiving or infant functioning.

Adult↗
PubMed1999

Characterization of a single-strand origin, ssoU, required for broad host range replication of rolling-circle plasmids.

Single-stranded DNA (ssDNA) promoters are the key components of the single-strand origins (ssos) of replication of rolling-circle (RC) replicating plasmids. The recognition of this origin by the host RNA polymerase and the synthesis of a short primer RNA are critical for initiation of lagging-strand synthesis. This step is thought to be a limiting factor for the establishment of RC plasmids in a broad range of bacteria, because most of the ssos described are fully active only in their natural hosts. A special type of sso, the ssoU, is unique in the sense that it can be efficiently recognized in a number of different Gram-positive hosts. We have experimentally deduced the folded structure and characterized the ssDNA promoter present within the ssoU using P1 nuclease digestion and DNase I protection assays with the Bacillus subtilis and Staphylococcus aureus RNA polymerases. We have also identified the RNA products synthesized from this ssDNA promoter and mapped the initiation points of lagging-strand synthesis in vivo from ssoU-containing plasmids. Through gel mobility shift experiments, we have found that ssDNA containing the ssoU sequence can efficiently interact with the RNA polymerase from two different Gram-positive bacteria, S. aureus and B. subtilis. We have also realigned the narrow and broad host range sso sequences of RC plasmids, and found that they contain significant homology. Our data support the notion that the strength of the RNA polymerase-ssoU interaction may be the critical factor that confers the ability on the ssoU to be fully functional in a broad range of bacteria.

Bacillus subtilis↗
PubMed1999

Evaluation of algorithms for the registration of 99Tcm-HMPAO brain SPET studies.

Five functions of merit were used for the registration of 99Tcm-HMPAO brain SPET studies. The correlation coefficient (COR), the stochastic sign change (SSC), the standard deviation of ratios (SDR), the sum of the absolute differences (SAD) and a new function based on a local correlation coefficient (LOC) were tested in the registration of photic neuroactivation (ACT), epilepsy (EPL) and Wada (WAD) SPET studies. The comparison included simulated and real studies. The translation error in registration was 0.1 +/- 0.1 pixels (mean +/- S.D.) for all functions of merit for the complete set of simulated studies (10 runs for each ACT, EPL and WAD). For rotation, LOC yielded the best results with a mean error of 0.3 +/- 0.2 degree and a maximum error of 0.6 degree. Slightly higher errors were found with SAD (0.4 +/- 0.2 degree, maximum 1.0 degree) and COR (0.5 +/- 1.0 degree, maximum 1.0 degree). The highest errors were found with SDR (0.8 +/- 1.0 degree, maximum 4.8 degrees) and SSC (0.8 +/- 1.1 degrees, maximum 4.7 degrees). The results obtained from five real studies of ACT, of EPL and of WAD were in agreement with the findings from the simulated studies, thus confirming the robustness of LOC, SAD and COR for the registration of 99Tcm-HMPAO brain SPET studies.

Algorithms↗
PubMed1999

Continuity and change in the social competence of children with autism, Down syndrome, and developmental delays.

The aims of this longitudinal study were: (1) to assess the continuity and change in diagnosis, intelligence, and language skills in children with autism, Down syndrome, and other developmental delays, (2) to specify the deficits in social competence and language skills in these children, and (3) to identify precursors in the preschool period of gains in language skills and of peer engagement in the mid-school years. The initial sample consisted of 70 children with autism, 93 children with Down syndrome, 59 children with developmental delays, and 108 typically developing children, with the first three groups of children studied when they were between 2 and 6 years of age. At follow-up, 51 children with autism, 71 children with Down syndrome, and 33 children with developmental delays were assessed at mean ages around 10-13 years. The long-term follow-up showed little change in the diagnosis of autism but sizeable improvements in intellectual and language abilities within the autistic group, a pattern that was not seen in the children with Down syndrome. Unique deficits in joint attention, some forms of representational play, responsiveness to the emotions of others, and initiation of peer engagement were identified in the autistic children, whereas the children with Down syndrome seemed to have a specific deficit only in language. Joint attention skills were concurrently associated with language abilities in all groups and predicted long-term gains in expressive language for the children with autism. Children with autism, regardless of their level of functioning, were less socially engaged with classmates than the other developmentally disabled children because they infrequently initiated and accepted play bids, not because they were rebuffed by peers. Early nonverbal communication and play skills were predictors of the frequency of initiations of peer play for the children with Down syndrome as well as the extent of peer engagement of the children with autism. These results suggest that improvements in early communication and play skills may have long-term consequences for later language and social competence in these groups of children.

Adolescent↗
PubMed1998

The structure of plasmid-encoded transcriptional repressor CopG unliganded and bound to its operator.

The structure of the 45 amino acid transcriptional repressor, CopG, has been solved unliganded and bound to its target operator DNA. The protein, encoded by the promiscuous streptococcal plasmid pMV158, is involved in the control of plasmid copy number. The structure of this protein repressor, which is the shortest reported to date and the first isolated from a plasmid, has a homodimeric ribbon-helix-helix arrangement. It is the prototype for a family of homologous plasmid repressors. CopG cooperatively associates, completely protecting several turns on one face of the double helix in both directions from a 13-bp pseudosymmetric primary DNA recognition element. In the complex structure, one protein tetramer binds at one face of a 19-bp oligonucleotide, containing the pseudosymmetric element, with two beta-ribbons inserted into the major groove. The DNA is bent 60 degrees by compression of both major and minor grooves. The protein dimer displays topological similarity to Arc and MetJ repressors. Nevertheless, the functional tetramer has a unique structure with the two vicinal recognition ribbon elements at a short distance, thus inducing strong DNA bend. Further structural resemblance is found with helix-turn-helix regions of unrelated DNA-binding proteins. In contrast to these, however, the bihelical region of CopG has a role in oligomerization instead of DNA recognition. This observation unveils an evolutionary link between ribbon-helix-helix and helix-turn-helix proteins.

Amino Acid Sequence↗
PubMed1998

Lagging strand replication of rolling-circle plasmids: specific recognition of the ssoA-type origins in different gram-positive bacteria.

Many bacterial plasmids replicate by a rolling-circle mechanism that involves the generation of single-stranded DNA (ssDNA) intermediates. Replication of the lagging strand of such plasmids initiates from their single strand origin (sso). Many different types of ssos have been identified. One group of ssos, termed ssoA, which have conserved sequence and structural features, function efficiently only in their natural hosts in vivo. To study the host specificity of sso sequences, we have analyzed the functions of two closely related ssoAs belonging to the staphylococcal plasmid pE194 and the streptococcal plasmid pLS1 in Staphylococcus aureus. The pLS1 ssoA functioned poorly in vivo in S. aureus as evidenced by accumulation of high levels of ssDNA but supported efficient replication in vitro in staphylococcal extracts. These results suggest that one or more host factors that are present in sufficient quantities in S. aureus cell-free extracts may be limiting in vivo. Mapping of the initiation points of lagging strand synthesis in vivo and in vitro showed that DNA synthesis initiates from specific sites within the pLS1 ssoA. These results demonstrate that specific initiation of replication can occur from the pLS1 ssoA in S. aureus although it plays a minimal role in lagging strand synthesis in vivo. Therefore, the poor functionality of the pLS1 in vivo in a nonnative host is caused by the low efficiency rather than a lack of specificity of the initiation process. We also have identified ssDNA promoters and mapped the primer RNAs synthesized by the S. aureus and Bacillus subtilis RNA polymerases from the pE194 and pLS1 ssoAs. The S. aureus RNA polymerase bound more efficiently to the native pE194 ssoA as compared with the pLS1 ssoA, suggesting that the strength of RNA polymerase-ssoA interaction may play a major role in the functionality of the ssoA sequences in Gram-positive bacteria.

Bacillus subtilis↗
PubMed1998

Structural features of the plasmid pMV158-encoded transcriptional repressor CopG, a protein sharing similarities with both helix-turn-helix and beta-sheet DNA binding proteins.

The small transcriptional repressor CopG protein (45 amino acids) encoded by the streptococcal plasmid pMV158 was purified to near homogeneity. Gel filtration chromatography and analytical ultracentrifugation showed that the native protein is a spherical dimer of identical subunits. Circular dichroism measurements of CopG indicated a consensus average content of more than 50% alpha-helix and 10-35% beta-strand and turns, which is compatible with the predicted secondary structure of the protein. CopG exhibited a prolonged intracellular half-life, but deletions in regions other than the C-terminal affected the global structure of the protein, severely reducing the half-lives of the CopG variants. This indicates that CopG has a compact structure, perhaps constituted by a single domain. Molecular modeling of CopG showed a good fitting between the helix-turn-helix motifs of well-known repressor proteins and a bihelical unit of CopG. However, modeling of CopG with ribbon-helix-helix class of DNA binding proteins also exhibited an excellent fit. Eleven out of the 12 replicons belonging to the pMV158 plasmid family could also encode Cop proteins, which share features with both helix-turn-helix and beta-sheet DNA binding proteins.

Amino Acid Sequence↗
PubMed1998

Overexpression, purification, crystallization and preliminary X-ray diffraction analysis of the pMV158-encoded plasmid transcriptional repressor protein CopG.

Plasmid pMV158 encodes a 45 amino acid transcriptional repressor, CopG, which is involved in copy number control. A new procedure for overproduction and purification of the protein has been developed. The CopG protein thus obtained retained its ability to specifically bind to DNA and to repress its own promoter. Purified CopG protein has been crystallized using the sitting-drop vapor diffusion method. The crystals, belonging to orthorhombic space group C222(1) (cell constants a = 67.2 A, b = 102.5 A, c = 40.2 A), were obtained from a solution containing methylpentanediol, benzamidine and sodium chloride, buffered to pH 6.7. Complete diffraction data up to 1.6 A resolution have been collected. Considerations about the Matthews parameter account for the most likely presence of three molecules in the asymmetric unit (2.27 A3/Da).

Amino Acid Sequence↗