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Biomedical subjects

J Jacob

Publications and source records attributed to J Jacob.

At least 55 records · Page 3Linked to original sources
PubMed2000

Anti-HIV and anti-tumor protein MAP30, a 30 kDa single-strand type-I RIP, shares similar secondary structure and beta-sheet topology with the A chain of ricin, a type-II RIP.

MAP30 is a 30 kDa single-stranded, type-I ribosome inactivating protein (RIP) possessing anti-tumor and anti-HIV activities. It binds both ribosomal RNA and the HIV-1 long-terminal repeat DNA. To understand the structural basis for MAP30 activities, we undertook the study of MAP30 by solution NMR spectroscopy. We report nearly complete 1H, 13C, and 15N chemical shift assignments of its 263 amino acids. Based upon an analysis of secondary 13C chemical shifts, 3J(HNHA) coupling constants, hydrogen exchange data, and nuclear Overhauser effect patterns, we find that the secondary structure and beta-sheet topology of MAP30 are very similar to those of the ricin A chain, a subunit of the well-known type-II RIP, even though two proteins display distinct activities. We therefore suggest that MAP30 and ricin A chain share a similar three-dimensional fold, and that the reported functional differences between two proteins arise primarily from differences in local three-dimensional structure and other structural properties such as surface electrostatic potentials.

Amino Acid Sequence↗
PubMed2000

Delivery room management of the apparently vigorous meconium-stained neonate: results of the multicenter, international collaborative trial.

OBJECTIVE: Disagreement exists concerning the appropriate delivery room management of the airway of vigorous meconium-stained infants. Some suggest a universal approach to intubation and suctioning of the airway in all such neonates, whereas others advocate a selective approach. We performed this investigation: 1) to assess whether intubation and suctioning of apparently vigorous, meconium-stained neonates would reduce the incidence of meconium aspiration syndrome (MAS); and 2) to determine the frequency of complications from delivery room intubation and suctioning of such infants. METHODS: Inclusion criteria included: 1) gestational age >/=37 weeks; 2) birth through meconium-stained amniotic fluid of any consistency; and 3) apparent vigor immediately after birth. Subjects were randomized to be intubated and suctioned (INT) or to expectant management (EXP). Primary outcome measures included: 1) the incidence of respiratory distress, including MAS, and 2) the incidence of complications from intubation. RESULTS: A total of 2094 neonates were enrolled from 12 participating centers (1051 INT and 1043 EXP). Meconium-stained amniotic fluid consistency was similar in both groups. Of the 149 (7.1%) infants that subsequently demonstrated respiratory distress, 62 (3.0%) had MAS and 87 (4.2%) had findings attributed to other disorders. There were no significant differences between groups in the occurrence of MAS (INT = 3.2%; EXP = 2.7%) or in the development of other respiratory disorders (INT = 3.8%; EXP = 4.5%). Of 1098 successfully intubated infants, 42 (3.8%) had a total of 51 complications of the procedure. In all cases, the complications were mild and transient in nature. CONCLUSIONS: Compared with expectant management, intubation and suctioning of the apparently vigorous meconium-stained infant does not result in a decreased incidence of MAS or other respiratory disorders. Complications of intubation are infrequent and short-lived.

Adult↗
PubMed1999

Solution structure of anti-HIV-1 and anti-tumor protein MAP30: structural insights into its multiple functions.

We present the solution structure of MAP30, a plant protein with anti-HIV and anti-tumor activities. Structural analysis and subsequent biochemical assays lead to several novel discoveries. First, MAP30 acts like a DNA glycosylase/apurinic (ap) lyase, an additional activity distinct from its known RNA N-glycosidase activity toward the 28S rRNA. Glycosylase/ap lyase activity explains MAP30's apparent inhibition of the HIV-1 integrase, MAP30's ability to irreversibly relax supercoiled DNA, and may be an alternative cytotoxic pathway that contributes to MAP30's anti-HIV/anti-tumor activities. Second, two distinct, but contiguous, subsites are responsible for MAP30's glycosylase/ap lyase activity. Third, Mn2+ and Zn2+ interact with negatively charged surfaces next to the catalytic sites, facilitating DNA substrate binding instead of directly participating in catalysis.

Anti-HIV Agents↗
PubMed1999

Interactions controlling the membrane binding of basic protein domains: phenylalanine and the attachment of the myristoylated alanine-rich C-kinase substrate protein to interfaces.

Basic residues are known to play a critical role in the attachment of protein domains to membrane interfaces. Many of these domains also contain hydrophobic residues that may alter the binding and the position of the domain on the interface. In the present study, the role of phenylanine in determining the membrane position, dynamics and free energy of a peptide derived from the effector domain of the myristoylated alanine-rich C-kinase substrate (MARCKS) protein was examined. Deuterium NMR in membranes containing phosphatidylcholine (PC) and phosphatidylserine (PS) indicates that this peptide, MARCKS(151-175), partially penetrates the membrane interface when bound and alters the effective charge density on the membrane interface by approximately 2 charges per bound peptide. However, a derivative of this peptide in which the five phenylalanines are replaced by alanine, MARCKS-Ala, does not penetrate the interface when membrane-bound. This result was confirmed by depth measurements by electron paramagnetic resonance spectroscopy on several spin-labeled derivatives of the Phe-less derivative. In contrast to nitroxides on MARCKS(151-175), nitroxides on the derivative lacking Phe do not reside within the bilayer but are in the aqueous phase when the peptide is bound to the membrane. The Phe to Ala substitutions shift the position of the labeled side chains by approximately 10-15 A. The side-chain dynamics of MARCKS-Ala are strongly influenced by membrane charge density and indicate that this peptide is drawn closer to the membrane interface at higher charge densities. As expected, MARCKS-Ala binds more weakly to membranes composed of PS/PC (1:9) than does the native MARCKS peptide; however, each phenylalanine contributes only 0.2 kcal/mol to the binding energy difference, far less than the 1.3 kcal/mol expected for the binding of phenylalanine to the membrane interface. This energetic discrepancy and the differences in membrane position of these peptides can be accounted for by a dehydration energy that is encountered as the peptide approaches the membrane interface. This energy likely includes a Born repulsion acting between the charged peptide and the low dielectric membrane interior. The interplay between the long-range attractive Coulombic force, the short-range repulsive force and the hydrophobic effect controls the position and energetics of protein domains on acidic membrane interfaces.

Amino Acid Sequence↗
PubMed1999

Modelling T-cell memory by genetic marking of memory T cells in vivo.

Immunological memory is the ability of the immune system to respond with enhanced vigour to pathogens that have been encountered in the past. Following infection or immunization, most effector T cells undergo apoptotic cell death, but a small fraction of these cells, proportional to the early antigen load and initial clonal burst size, persist in the host as a stable pool of memory T cells. The existence of immunological memory has been recognized for over 2,000 years, but our understanding of this phenomenon is limited, primarily because memory lymphocytes cannot be unequivocally identified as they lack specific, permanent markers. Here we have developed a transgenic mouse model system whereby memory T cells and their precursors can be irreversibly marked with a reporter gene and thus can be unambiguously identified. Adoptive transfer of marked CD8+ T cells specific for lymphocytic choriomeningitis virus protected naive recipients following viral challenge, demonstrating that we have marked memory T cells. We also show that cytotoxic effector lymphocytes that develop into memory T cells can be identified in the primary response.

Adoptive Transfer↗
PubMed1999

Mosaicism with a normal cell line and an unbalanced structural rearrangement.

Mosaicism with a normal cell line (N) and an unbalanced autosomal structural rearrangement (UASR) is rare. This report describes a case of a newborn female with a karyotype of 46,XX,der(4)t(4;15)(q35;q22)/46,XX. Molecular cytogenetic analysis confirmed the origin of the derivative chromosome 4. Here we discuss this case as well as other cases of mosaic karyotypes involving N/UASR.

Chromosome Aberrations↗
PubMed1999

A case of sporadic Pick disease with onset at 27 years.

BACKGROUND: Pick disease is an uncommon cause of dementia in middle age, and young-onset cases have rarely been reported. SETTING: A specialist hospital. PATIENT: Patient with onset of cognitive impairment at the age of 27 years whose cerebral biopsy specimen demonstrated Pick cells and tau-positive Pick bodies. CONCLUSION: Pick disease should be considered in the differential diagnosis of dementia in young adults.

Adult↗
PubMed1999

Coenzyme Q10, a cutaneous antioxidant and energizer.

The processes of aging and photoaging are associated with an increase in cellular oxidation. This may be in part due to a decline in the levels of the endogenous cellular antioxidant coenzyme Q10 (ubiquinone, CoQ10). Therefore, we have investigated whether topical application of CoQ10 has the beneficial effect of preventing photoaging. We were able to demonstrate that CoQ10 penetrated into the viable layers of the epidermis and reduce the level of oxidation measured by weak photon emission. Furthermore, a reduction in wrinkle depth following CoQ10 application was also shown. CoQ10 was determined to be effective against UVA mediated oxidative stress in human keratinocytes in terms of thiol depletion, activation of specific phosphotyrosine kinases and prevention of oxidative DNA damage. CoQ10 was also able to significantly suppress the expression of collagenase in human dermal fibroblasts following UVA irradiation. These results indicate that CoQ10 has the efficacy to prevent many of the detrimental effects of photoaging.

Antioxidants↗
PubMed1999

Detection of polycyclic aromatic hydrocarbon metabolites by high-pressure liquid chromatography after purification on immunoaffinity columns in urine from occupationally exposed workers.

OBJECTIVE: The objective in our study was to quantitate benzo[a]pyrene (B[a]P) metabolites by a combination of immunoaffinity chromatography and high-pressure liquid chromatography (HPLC) with fluorescence detection in urine from workers exposed to high levels of polycyclic aromatic hydrocarbons (PAH). Furthermore, by the simultaneous quantitation of 1-hydroxypyrene, the correlation between the B[a]P-tetrol and 1-hydroxypyrene would provide a means of evaluating the validity of 1-hydroxypyrene as a surrogate biomarker for occupational exposure to the potent carcinogen B[a]P in an electrode paste plant. METHODS: The study was carried out at an electrode paste plant that produces electrode paste for Söderberg electrodes. A total of 34 pre- and post-shift urine samples and 17 personal air samples were collected from 17 workers during a normal work week. The concentration of 1-hydroxypyrene was measured in all urine samples. A recent method of quantitating B[a]P-r-7, t-8, t-9, c-10-tetrol in urine of humans exposed to low levels of PAH has been described. A modified version of this method involving purification of urine samples on immunoaffinity columns and HPLC analysis with fluorescence detection was used on urine samples from workers exposed to high levels of PAH. A monoclonal antibody (8E11) with binding affinity to B[a]P-tetrols was used. This antibody also binds several PAH-DNA adducts and metabolites, including 1-hydroxypyrene. Gas chromatography/mass spectroscopy (GC/MS) was also used for identification of metabolites isolated by HPLC fractionation. RESULTS: From personal air sampling the mean exposure to particulate PAHs was 38 microg/m3. The mean concentration of urinary 1-hydroxypyrene was 3.9 micromol/mol creatinine in preshift samples and 10.2 micromol/mol creatinine in postshift samples. We could not identify detectable amounts of urinary B[a]P-tetrol by HPLC or fluorescence spectroscopy after purification on immunoaffinity columns. However, in the HPLC analysis we identified several hydroxyphenantrene metabolites that were detected at relatively high concentrations in all of the workers' urine samples. We could not separate 2- and 3-hydroxyphenanthrene (2 + 3-OH-Phe) in peak 1, and peak 2 contained both 1- and 9-hydroxyphenanthrene (1 + 9-OH-Phe). The phenanthrene metabolites were mainly conjugated to glucuronic acid and sulfate. There was a significant correlation between the 1-hydroxypyrene concentration and 2 + 3-OH-Phe (r = 0.73) and 1 + 9-OH-Phe (r = 0.64) in the urine samples. 1-Hydroxypyrene was measured in all post-shift urine samples but was not significantly correlated with workplace pyrene exposure, indicating that skin exposure is an important route of pyrene exposure in this factory. As with 1-hydroxypyrene, dermal PAH uptake may also account for the poor correlation between 2 + 3- and 1 + 9-OH-Phe and ambient phenanthrene. DISCUSSION: Since dermal uptake is likely to be important in occupational PAH exposure in addition to inhalation, estimation of total PAH exposure is best achieved by quantitation of PAHs excreted into body fluids. However, it remains unclear whether there might be a difference in uptake and urinary excretion of 3-ring, 4-ring, or 5-ring PAHs and in the correlation between these metabolites and ambient-air PAH measurements. In summary, using immunaffinity chromatography, we did not find detectable amounts of B[a]P-tetrol in urine from workers occupationally exposed to PAH. However, by an HPLC/immunoaffinity method, relatively high amounts of 1-hydroxypyrene as well as 2 + 3- and 1 + 9-OH-Phe were quantitated in the urine samples, both of which are relevant as biomarkers of PAH exposure.

Air Pollutants, Occupational↗
PubMed1999

Distance estimates from paramagnetic enhancements of nuclear relaxation in linear and flexible model peptides.

The distance dependence of electron-nuclear dipole-dipole coupling was tested using a series of poly-L-proline based peptides of different length. The poly-proline based peptides were synthesized with a nitroxide spin label on the N-terminus and a tryptophan on the C-terminus, and paramagnetic enhancements of nuclear spin-lattice relaxation rates were measured for the aromatic protons on the tryptophan as a function of the number of proline spacers in the sequence. As expected, paramagnetic enhancements decrease with distance, but the distances deduced from the NMR relaxation rates were shorter than expected for every peptide studied compared to a rigid linear poly-L-proline type II helix structure. Calculations of cross-relaxation rates indicate that this difference is not the result of spin-diffusion or the creation of a spin-temperature gradient in the proton spins caused by the nitroxide. Molecular dynamics simulations were used to estimate dynamically averaged value of (2). These weighted average distances were close to the experimentally determined distances, and suggest that molecular motion may account for differences between the rigid linear models and the distances implied by the NMR relaxation data. A poly-L-prolone peptide synthesized with a central glycine hinge showed dramatic relaxation rate enhancements compared to the peptide of the same length lacking the hinge. Molecular dynamics simulations for the hinged peptide support the notion that the NMR data is a representation of the weighted average distance, which in this case is much shorter than that expected for an extended conformation. These results demonstrate that intermoment distances based on NMR relaxation rates provide a sensitive indicator of intramolecular motions.

Amino Acid Sequence↗
PubMed1999

The role of proline and glycine in determining the backbone flexibility of a channel-forming peptide.

Alamethicin is a helical 20-amino acid voltage-gated channel-forming peptide, which is known to exhibit segmental flexibility in solution along its backbone near alpha-methylalanine (MeA)-10 and Gly-11. In an alpha-helical configuration, MeA at position 10 would normally hydrogen-bond with position 14, but the presence of proline at this position prevents the formation of this interhelical hydrogen bond. To determine whether the presence of proline at position 14 contributes to the flexibility of this helix, two analogs of alamethicin were synthesized, one with proline 14 replaced by alanine and another with both proline 14 and glycine 11 replaced by alanine. The C-termini of these peptides were derivatized with a proxyl nitroxide, and paramagnetic enhancements produced by the nitroxide on the Calpha protons were used to estimate r-6 weighted distances between the nitroxide and the backbone protons. When compared to native alamethicin, the analog lacking proline 14 exhibited similar C-terminal to Calpha proton distances, indicating that substitution of proline alone does not alter the flexibility of this helix; however, the subsequent removal of glycine 11 resulted in a significant increase in the averaged distances between the C- and N-termini. Thus, the G-X-X-P motif found in alamethicin appears to be largely responsible for mediating high-amplitude bending motions that have been observed in the central helical domain of alamethicin in methanol. To determine whether these substitutions alter the channel behavior of alamethicin, the macroscopic and single-channel currents produced by these analogs were compared. Although the substitution of the G-X-X-P motif produces channels with altered characteristics, this motif is not essential to achieve voltage-dependent gating or alamethicin-like behavior.

Alamethicin↗
PubMed1999

Metabolic activation of dibenzo[a,l]pyrene by human cytochrome P450 1A1 and P450 1B1 expressed in V79 Chinese hamster cells.

Metabolic activation of the strongly carcinogenic polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P) and its trans-8,9-dihydrodiol (trans-8,9-diol) catalyzed by human cytochromes P450 (P450) 1A1 and 1B1 was investigated. DNA binding of DB[a,l]P in mammalian cell lines has previously been shown to be preferentially mediated by fjord region DB[a,l]P-11,12-dihydrodiol 13,14-epoxides (DB[a,l]PDE). In order to elucidate different capabilities of both P450 enzymes for metabolic activation of DB[a, l]P V79 Chinese hamster cells, stably expressing human P450s 1A1 or 1B1 have been exposed to the parent PAH or its racemic trans-8, 9-diol. For this purpose, synthesis and spectroscopic characterization of the trans-DB[a,l]P-8,9-diol and its individual enantiomers have been achieved. Both human P450-expressing cell lines were capable of transforming DB[a,l]P to its fjord region DB[a, l]PDE, but the extent of metabolism to DB[a,l]PDE catalyzed by human P450 1B1 was higher compared to human P450 1A1 at all times measured. On the other hand, cytotoxicity studies performed with the same incubation systems emerged stronger effects by DB[a,l]P and its enantiomeric trans-11,12-diols in human P450 1A1-expressing cells. Both human P450 enzymes stereospecifically catalyzed the formation of the (-)-DB[a,l]P-11,12-diol with R,R-configuration, whereas only the human P450 1A1-expressing cells form small amounts of the K-region trans-8,9-diol with high excess of the (+)-(8R, 9R)-enantiomer. Application of trans-DB[a,l]P-8,9-diol in metabolism studies revealed that this compound is converted by human P450s 1A1 and 1B1 to several diol phenols and bis-diols. However, and even at concentrations as high as 10 microM, in both cell lines the trans-DB[a,l]P-8,9-diol showed no cytotoxicity at all, suggesting that an activation of DB[a,l]P via further oxidation of the K-region trans-8,9-diol plays a minor role.

Animals↗
PubMed1999

Measurement of 3hJNC' connectivities across hydrogen bonds in a 30 kDa protein.

A method is described which permits detection of 3hJNC' scalar couplings across hydrogen bonds in larger, perdeuterated proteins. The experiment is demonstrated for the uniformly 2H/13C/15N-enriched 30 kDa ribosome inactivating protein MAP30. The 3hJNC' interactions are smaller than 1 Hz, but their detection in an HNCO experiment is made possible through the use of constructive interference between the 15N chemical shift anisotropy and 1H-15N dipole-dipole relaxation mechanisms in a manner similar to that of recently proposed TROSY schemes. Sensitivity of the HNCO experiment depends strongly on the 15N transverse relaxation rate of the downfield 15N multiplet component and on the amide proton T1. In perdeuterated MAP30 at 40 degrees C, the average TROSY T2 was 169 ms at 750 MHz 1H frequency, and a wide range of longitudinal relaxation rates was observed for the amide protons.

Carbon Isotopes↗
PubMed1999

The use of digital cameras in a mobile retinal screening environment.

AIMS: To assess how successfully digital camera technology might be incorporated into a mobile screening environment. METHODS: One hundred and ninety-seven people had their fundi photographed using a Topcon/Imagenet digital system and 534 using a Canon CR5/Ris-Lite system in addition to concurrent 45 degrees CR4NM Polaroid photography. One hundred and eighteen randomly selected patients were also sent for 7 field stereo photography as a gold standard. An acceptability questionnaire was answered by a random sample of those photographed. RESULTS: For the detection of any retinopathy, digital pictures had a sensitivity of 0.74 (95% confidence interval (CI) 0.68-0.80) whilst Polaroid was 0.72 (95% CI 0.66-0.78) and for referable retinopathy digital pictures had a sensitivity of 0.85 (95% CI 0.80-0.90) and Polaroid was 0.90 (95% CI 0.86-0.94). A concurrent ophthalmoscopic evaluation improved the sensitivity to 0.92 (95% CI 0.86-0.98) for detection of any retinopathy whilst the sensitivity at the referral level was improved to 0.95 (95% CI 0.91-0.99). Twenty-nine of 176 respondents experienced flash discomfort with the Polaroid system with only four of 154 describing 'some' discomfort from the digital systems which have a lower flash power (10 W vs. 300 W) and a faster recovery time. CONCLUSIONS: This study indicates that digital systems are a feasible and acceptable alternative to Polaroid-based cameras for use in a mobile environment.

Adult↗
PubMed1999

Gene organisation determines evolution of function in the chicken MHC.

Some years ago, we used our data for class I genes, proteins and peptide-binding specificities to develop the hypothesis that the chicken B-F/B-L region represents a "minimal essential MHC". In this view, the B locus contains the classical (highly expressed and polymorphic) class I alpha and class II beta multigene families, which are reduced to one or two members, with many other genes moved away or deleted from the chicken genome altogether. We found that a single dominantly expressed class I gene determines the immune response to certain infectious pathogens, due to peptide-binding specificity and cell-surface expression level. This stands in stark contrast to well-studied mammals like humans and mice, in which every haplotype is more-or-less responsive to every pathogen and vaccine, presumably due to the multigene family of MHC molecules present. In order to approach the basis for a single dominantly expressed class I molecule, we have sequenced a portion of the B complex and examined the location and polymorphism of the class I (B-F) alpha, TAP and class II (B-L) beta genes. The region is remarkably compact and simple, with many of the genes expected from the MHC of mammals absent, including LMP, class II alpha and DO genes as well as most class III region genes. However, unexpected genes were present, including tapasin and putative natural killer receptor genes. The region is also organised differently from mammals, with the TAPs in between the class I genes, the tapasin gene in between the class II (B-L) beta genes, and the C4 gene outside of the class I alpha and class II beta genes. The close proximity of TAP and class I alpha genes leads to the possibility of co-evolution, which can drive the use of a single dominantly expressed class I molecule with peptide-binding specificity like the TAP molecule. There is also a single dominantly expressed class II beta gene, but the reason for this is not yet clear. Finally, the presence of the C4 gene outside of the classical class I alpha and class II beta genes suggests the possibility that this organisation was ancestral, although a number of models of organisation and evolution are still possible, given the presence of the Rfp-Y region with non-classical class I alpha and class II beta genes as well as the presence of multigene families of B-G and rRNA genes.

Animals↗
PubMed1999

[Data protection as a personal right. Government Agency].

The sector-specific secrets are traditionally regarded as the historical predecessors of general data protection law. The lecture demonstrates that they are not only aimed at protecting the individual's right to self-determination of the handling of his personal data. It also indicates that those particular secrets are, at the same time, serving for further purposes which are of importance in their respective sectors. The general (omnibus) data protection statues are given attention in the light of the beginning of automated information processing and in their role of guaranteeing the right to personal privacy, a role which the Federal Constitutional Court underlined in its census decision of 1983 and which became of general relevance in the course of the further developments. In conclusion, the report particularizes that, inter alia, statutory adaptations and amendments are necessary in order to preserve the effects of the doctor-patient confidentiality under the conditions of modern data processing. The examples given are comprising: health data on chip cards, health data in networks and the electronic record of the patient.

Confidentiality↗
PubMed1998

Specific signals generated by the cytoplasmic domain of the granulocyte colony-stimulating factor (G-CSF) receptor are not required for G-CSF-dependent granulocytic differentiation.

Granulocyte colony-stimulating factor (G-CSF) is the principal growth factor regulating the production of neutrophils, yet its role in lineage commitment and terminal differentiation of hematopoietic progenitor cells is controversial. In this study, we describe a system to study the role of G-CSF receptor (G-CSFR) signals in granulocytic differentiation using retroviral transduction of G-CSFR-deficient, primary hematopoietic progenitor cells. We show that ectopic expression of wild-type G-CSFR in hematopoietic progenitor cells supports G-CSF-dependent differentiation of these cells into mature granulocytes, macrophages, megakaryocytes, and erythroid cells. Furthermore, we show that two mutant G-CSFR proteins, a truncation mutant that deletes the carboxy-terminal 96 amino acids and a chimeric receptor containing the extracellular and transmembrane domains of the G-CSFR fused to the cytoplasmic domain of the erythropoietin receptor, are able to support the production of morphologically mature, chloroacetate esterase-positive, Gr-1/Mac-1-positive neutrophils in response to G-CSF. These results demonstrate that ectopic expression of the G-CSFR in hematopoietic progenitor cells allows for multilineage differentiation and suggest that unique signals generated by the cytoplasmic domain of the G-CSFR are not required for G-CSF-dependent granulocytic differentiation.

Animals↗