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Biomedical subjects

L Lerner

Publications and source records attributed to L Lerner.

At least 19 recordsLinked to original sources
PubMed2001

Creation of genome-wide protein expression libraries using random activation of gene expression.

Here we report the use of random activation of gene expression (RAGE) to create genome-wide protein expression libraries. RAGE libraries containing only 5 x 10(6) individual clones were found to express every gene tested, including genes that are normally silent in the parent cell line. Furthermore, endogenous genes were activated at similar frequencies and expressed at similar levels within RAGE libraries created from multiple human cell lines, demonstrating that RAGE libraries are inherently normalized. Pools of RAGE clones were used to isolate 19,547 human gene clusters, approximately 53% of which were novel when tested against public databases of expressed sequence tag (EST) and complementary DNA (cDNA). Isolation of individual clones confirmed that the activated endogenous genes can be expressed at high levels to produce biologically active proteins. The properties of RAGE libraries and RAGE expression clones are well suited for a number of biotechnological applications including gene discovery, protein characterization, drug development, and protein manufacturing.

Cell Line↗
PubMed1999

Clinical, epidemiological, and microbiological features of Vibrio vulnificus biogroup 3 causing outbreaks of wound infection and bacteraemia in Israel. Israel Vibrio Study Group.

BACKGROUND: Vibrio vulnificus is a gram-negative bacterium that causes septicaemia and wound infection. Cases occur sporadically, and no previous outbreaks due to a common source or a clonal strain have been reported. In the summer and autumn of 1996 and 1997, an outbreak of invasive V. vulnificus infection occurred in Israel in people who had recently handled fresh, whole fish purchased from artificial fish-ponds. METHODS: We reviewed clinical and epidemiological information, and undertook an environmental investigation to assess disease characteristics, modes of transmission, phenotypic characteristics of the bacterium, and fish-marketing policy. The clonal nature of 19 isolates was studied by biotyping, pulsed-field gel electrophoresis, and restriction-fragment length polymorphism (RFLP) analysis of a PCR fragment. FINDINGS: During 1996-97, 62 cases of wound infection and bacteraemia occurred. 57 patients developed cellulitis, four had necrotising fasciitis, and one developed osteomyelitis. In all cases, the fish were cultivated in inland fish-ponds. In the summer of 1996, fish-pond managers initiated a new marketing policy, in which fish were sold alive instead of being packed in ice. Phenotypically, the isolates had five atypical biochemical test results. The isolates were non-typeable by pulsed-field gel electrophoresis, and all had the same PCR-RFLP pattern which had not been seen previously. INTERPRETATION: The cause of the outbreak was a new strain of V. vulnificus, classified as biogroup 3. A new fish-marketing policy that began in 1996 may have exposed susceptible people to the organism.

Adolescent↗
PubMed1999

A convenient microscale colorimetric method for terminal galactose on immunoglobulins.

A new approach for quantitative determination of terminal galactose (Gal) residues of immunoglobulins was developed by combining exoglycosidase digestion with the classical colorimetric estimation of reducing sugars. The ferricyanide colorimetric method was modified to increase the stability of the chromophore (Prussian blue) and adapted to determine the amount of terminal Gal residues present in immunoglobulins. The method involves the release of covalently bound Gal from immunoglobulins by Diplococcus pneumoniae beta-D-galactosidase (specific for beta(1,4) linked galactose), removal of the glycoprotein and enzyme from the reaction mixture by heat denaturation or ethanol precipitation, followed by colorimetric measurement of the released sugar using the ferricyanide assay. The ferricyanide method was modified to enhance the solubility and stability of the chromophore by increasing the concentration of aqueous sulfuric acid and sodium dodecyl sulfate (SDS). The linear range of the modified method was from approximately 11 to 111 microM Gal. Typical variation in assay results was on the order of 5%. Using the modified method, the terminal Gal content of a recombinant chimeric monoclonal antibody (anti-CD20, rIgG) expressed in Chinese hamster ovary (CHO) cells was determined and evaluated for batch-to-batch consistency. The method was used to optimize pH, time, temperature, and enzyme concentration for beta-galactosidase digestion for maximal release of terminal Gal residues from rIgG.

Animals↗
PubMed1999

The effect of O-fucosylation on the first EGF-like domain from human blood coagulation factor VII.

The first epidermal growth factor-like domain (EGF-1) from blood coagulation factor VII (FVII) contains two unusual O-linked glycosylation sites at Ser-52 and Ser-60. We report here a detailed study of the effect of O-fucosylation at Ser-60 on the structure of FVII EGF-1, its Ca2+-binding affinity, and its interaction with tissue factor (TF). The in vitro fucosylation of the nonglycosylated FVII EGF-1 was achieved by using O-fucosyltransferase purified from Chinese hamster ovary cells. Distance and dihedral constraints derived from NMR data were used to determine the solution structures of both nonglycosylated and fucosylated FVII EGF-1 in the presence of CaCl2. The overall structure of fucosylated FVII EGF-1 is very similar to the nonfucosylated form even for the residues near the fucosylation site. The Ca2+ dissociation constants (Kd) for the nonfucosylated and fucosylated FVII EGF-1 were found to be 16.4 +/- 1.8 and 8.6 +/- 1.4 mM, respectively. The FVII EGF-1 domain binds to the extracellular part of TF with a low affinity (Kd approximately 0. 6 mM), and the addition of fucose appears to have no effect on this affinity. These results indicate that the FVII EGF-1 alone cannot form a tight complex with TF and suggest that the high binding affinity of FVIIa for TF requires cooperative interaction among the four domains in FVII with TF. Although the fucose has no significant effect on the interaction between TF and the individual FVII EGF-1 domain, it may affect the interaction of full-length FVIIa with TF by influencing its Ca2+-binding affinity.

Amino Acid Sequence↗
PubMed1997

Incidentally detected renal tumors.

Asymptomatic renal masses are increasingly being found during radiography ordered for other reasons. Unfortunately, radiography cannot always provide precise identification of the masses, some of which are renal cell carcinomas. Whether to choose surveillance, surgery, or other therapy depends on the tumor and on the patient.

Adenoma, Oxyphilic↗
PubMed1997

Stereoselectivity of the Chinese hamster ovary cell sialidase: sialoside hydrolysis with overall retention of configuration.

The stereochemical course of enzymatic hydrolysis by the soluble sialidase from Chinese hamster ovary cells, expressed as a recombinant protein in insect Sf9 cells, was determined using proton nuclear magnetic resonance spectroscopy. 4-Methyl umbelliferyl-N-acetyl neuraminic acid was employed as substrate, and the stereoselectivity of the enzyme catalysis was ascertained by monitoring the H3 axial and equatorial protons of the sialic acid product over the reaction course. At both high (3 U) and low concentrations (1 U) of the enzyme, the alpha anomer of the sialic acid was clearly observed as the initial reaction product. The corresponding beta anomer of sialic acid appeared much later in the reaction, arising from mutarotation of the alpha anomer. Similar studies were also carried out using the Salmonella typhimurium LT 2 sialidase, a protein of similar size and substrate specificity. Both enzymes apparently cleave the alpha linked sialoside substrate with retention of configuration. Based on the observations of a wide variety of other glycohydrolytic enzymes that have shown a strong correlation of the stereoselectivity of catalysis with active site topology (Gebler et al., J. Biol. Chem. 267, 12559-12561, 1992), the results obtained here suggest that the microbial and mammalian sialidases have a homologous active site architecture even though the molecules do not share significant primary sequence similarities.

Animals↗
PubMed1995

The effect of diagnosis and treatment delay on prognostic factors and survival in endometrial carcinoma.

OBJECTIVE: The aim of this study was to assess the association of diagnosis and treatment delay with established prognostic factors and survival. STUDY DESIGN: The study group comprised 181 consecutive patients with endometrial carcinoma diagnosed between 1970 and 1986, whose records contained details with regard to diagnosis delay; 174 of them also contained details with regard to treatment delay. RESULTS: The significant prognostic factors that we found, namely, age, clinical stage, grade, depth of myometrial invasion, and histologic type, are in line with those of other studies. However, no significant correlation was found between the duration of delay and these prognostic factors or with survival. CONCLUSION: We conclude that delay of diagnosis (< 1 year) and of treatment of < 4 months do not compromise survival of patients with endometrial cancer.

Adenocarcinoma↗
PubMed1995

Effect of diclofenac sodium (Voltaren) on hypoxia-induced corneal edema in humans.

We evaluated the effect of diclofenac sodium (Voltaren) drops on patients with hypoxia-induced corneal edema. Thirty age- and sex-matched subjects were randomly assigned to one of three groups. Members of each group received masked solutions of either Voltaren, Voltaren vehicle, or a non-preserved lubricant (Cellufresh) every 6 hours for 24 hours and then hourly for 2 hours immediately prior to inducing corneal edema in the experimental eye. Bilateral ultrasonic pachymetry was performed prior to applying a thick contact lens and light patch on the experimental eye of all subjects for 3 hours. The fellow eye served as the control. Following lens removal, bilateral corneal thickness was measured every 30 minutes. The percentage change in corneal swelling for each subject and group was calculated. The findings were also normalized to the control eye to minimize diurnal and individual variability. The results were plotted both as percentage change from hour 0 and percentage change normalized to the control eye. Corneal swelling ranged from 9-11% in all 3 groups, with recovery at 2-3 hours. No significant difference was found among the three groups (P > 0.05, ANOVA). There was a slight trend toward reduced thickness in the Cellufresh group, but this was not statistically significant. Voltaren does not appear to have an effect on the hypoxia-induced corneal edema associated with the production of arachidonic acid pathway metabolites.

Adult↗
PubMed1993

A detailed 1H and 13C NMR study of a repeating disaccharide of hyaluronan: the effects of temperature and counterion type.

For the first time, a detailed NMR study of the conformation of methyl 2-acetamido-2-deoxy-3-O-(beta-D-glucopyranosyluronic acid)-beta-D-glucopyranoside (disaccharide 1) in aqueous solution is reported. This disaccharide is a repeating unit of hyaluronan, a polysaccharide with widespread biological and pharmaceutical applications. Relatively small changes in temperature, over typical experimental conditions (0-37 degrees C), completely change the appearance of its one-dimensional 1H NMR spectrum at 500 MHz. To determine the underlying cause for this temperature sensitivity, we analyzed 1H and 13C chemical shifts, temperature coefficients (delta gamma/delta T), 1H-1H coupling constants, and interglycosidic 1H-13C coupling constants for 1 as a function of temperature. For comparison, we measured the temperature dependence of 1H chemical shifts and coupling constants for related monosaccharides: glucuronate (GlcUA or U) and N-acetylglucosamine (GlcNAc or N), and glucose (Glc). The temperature sensitivity of the 1H spectrum of 1 is caused by relatively larger values of delta delta/delta T for some ring protons, rather than a conformational change. The effect is mediated by strong coupling. To detect the presence of long-lived intramolecular hydrogen bonds in the disaccharide, we measured chemical shifts, delta delta/delta T, and coupling constants for hydroxyl protons of 1, GlcUA, and GlcNAc in 1:1 H2O-acetone-d6 at low temperature. We compared 1H NMR parameters for 1, GlcUA, and GlcNAc in water with published values measured in Me2SO-d6 and concluded that interactions with water predominated. We found no evidence for long-lived intramolecular hydrogen bonds occurring in 1 in aqueous solution.

Acetylglucosamine↗
PubMed1993

Teaching high-resolution nuclear magnetic resonance to graduate students in biophysics.

Teaching modern methods of high resolution, multi-dimensional nuclear magnetic resonance (NMR) spectroscopy in the limited time usually available is a challenge because of the breadth and complexity of the subject. Here we present two outlines for lectures on basic principles and biophysical applications of NMR, either in one lecture or in six to twelve lectures. We advocate emphasizing the versatility of NMR, and its numerous applications to biophysical questions. An annotated list of references is provided.

Biophysics↗
PubMed1991

Antibodies to seven rotavirus serotypes in cord sera, maternal sera, and colostrum of German women.

Forty percent of colostrum samples from German women showed neutralizing antibody titers of greater than or equal to 50 to rotavirus (RV) serotypes 1, 3, 4, and 6. Antibody to serotypes 2, 8, and 9 was less prevalent. Titers are, however, too low to indicate an important effect of colostrum on the RV vaccine take rate. On the other hand, about 50% of the cord serum samples showed high neutralizing-antibody titers to serotypes 1, 3, and 4, which could interfere with the take rate of RV vaccines based on these serotypes in very young infants.

Adult↗
PubMed1990

Significant conformational changes in an antigenic carbohydrate epitope upon binding to a monoclonal antibody.

Transferred nuclear Overhauser enhancement spectroscopy (TRNOE) was used to observe changes in a ligand's conformation upon binding to its specific antibody. The ligands studied were methyl O-beta-D-galactopyranosyl(1----6)-4-deoxy-4-fluoro-beta-D-galactopyra nos ide (me4FGal2) and its selectively deuteriated analogue, methyl O-beta-D-galactopyranosyl(1----6)-4-deoxy-2-deuterio-4-fluoro-beta -D- galactopyranoside (me4F2dGal2). The monoclonal antibody was mouse IgA X24. The solution conformation of the free ligand me4F2dGal2 was inferred from measurements of vicinal 1H-1H coupling constants, long-range 1H-13C coupling constants, and NOE cross-peak intensities. For free ligand, both galactosyl residues adopt a regular chair conformation, but the NMR spectra are incompatible with a single unique conformation of the glycosidic linkage. Analysis of 1H-1H and 1H-13C constants indicates that the major conformer has an extended conformation: phi = -120 degrees; psi = 180 degrees; and omega = 75 degrees. TRNOE measurements on me4FGal2 and me4F2dGal2 in the presence of the specific antibody indicate that the pyranose ring pucker of each galactose ring remains unchanged, but rotations about the glycosidic linkage occur upon binding to X24. Computer calculations indicate that there are two sets of torsion angles that satisfy the observed NMR constraints, namely, phi = -152 +/- 9 degrees; psi = -128 +/- 7 degrees; and omega = -158 +/- 6 degrees; and a conformer with phi = -53 +/- 6 degrees; psi = 154 +/- 10 degrees; and omega = -173 +/- 6 degrees. Neither conformation is similar to any of the observed conformations of the free disaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Systematic chemical synthesis and n.m.r. spectra of methyl alpha-glycosides of isomalto-oligosaccharides and related compounds.

Acid-catalyzed thiophenolysis of 1,6-anhydro-2,3,4-tri-O-benzyl-beta-D-glucopyranose and acetylation of the resulting phenyl 2,3,4-tri-O-benzyl-1-thio-alpha-D-glucopyranoside (4) gave phenyl 6-O-acetyl-2,3,4-tri-O-benzyl-1-thio-alpha-D-glucopyranoside (5). Reaction of 5 with chlorine gave, stereospecifically, the corresponding beta-glycosyl chloride, which was treated with 4 in the presence of silver perchlorate and 2,4,6-trimethylpyridine to afford phenyl O-(6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl)-(1----6)-2,3,4- tri-O- benzyl-1-thio-alpha-D-glucopyranoside (17). Crystalline O-(6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl)-(1----6)-2,3,4- tri- O-benzyl-beta-D-glucopyranosyl chloride, readily obtainable in a stereo-specific manner from 17 by treatment with chlorine, was used as the key glycosyl (isomaltosyl) donor in the blockwise synthesis of methyl glycosides of isomalto-oligosaccharides, up to and including the octasaccharide. The methyl alpha-glycoside of isomaltotetraose fluorinated at C-6 of the terminal D-glucopyranosyl group was prepared by using SnCl2-activated 2,3,4-tri-O-benzyl-6-deoxy-6-fluoro-alpha,beta-D-glucopyranosyl fluoride as the glycosyl donor, a suitably protected methyl alpha-isomaltotrioside as the nucleophile, and silver perchlorate as the promoter. The n.m.r. spectra (1H- and 13C-) of numerous synthetic intermediates were analyzed and completely assigned by a variety of two-dimensional homo- and hetero-nuclear n.m.r.-spectroscopic techniques, and the final deprotected title oligosaccharides were characterized by 13C-n.m.r. data. Silver perchlorate-mediated glycosylation reactions involving beta-glycosyl chlorides were high-yielding and showed high stereo-selectivity for the formation of an alpha-(cis)-glycosidic linkage. The practical limitation of obtaining high isomalto-oligosaccharides in this way appears to lie solely in the separation technique applied for the resolution of the crude products formed.

Carbohydrate Conformation↗