Predicting XAFS scattering path cumulants and XAFS spectra for metals (Cu, Ni, Fe, Ti, Au) using molecular dynamics simulations.

The ability of molecular dynamics (MD) simulations to support the analysis of X-ray absorption fine-structure (XAFS) data for metals is evaluated. The low-order cumulants (ΔR, σ(2), C3) for XAFS scattering paths are calculated for the metals Cu, Ni, Fe, Ti and Au at 300 K using 28 interatomic potentials of the embedded-atom method type. The MD cumulant predictions were evaluated within a cumulant expansion XAFS fitting model, using global (path-independent) scaling factors. Direct simulations of the corresponding XAFS spectra, χ(R), are also performed using MD configurational data in combination with the FEFF ab initio code. The cumulant scaling parameters compensate for differences between the real and effective scattering path distributions, and for any errors that might exist in the MD predictions and in the experimental data. The fitted value of ΔR is susceptible to experimental errors and inadvertent lattice thermal expansion in the simulation crystallites. The unadjusted predictions of σ(2) vary in accuracy, but do not show a consistent bias for any metal except Au, for which all potentials overestimate σ(2). The unadjusted C3 predictions produced by different potentials display only order-of-magnitude consistency. The accuracy of direct simulations of χ(R) for a given metal varies among the different potentials. For each of the metals Cu, Ni, Fe and Ti, one or more of the tested potentials was found to provide a reasonable simulation of χ(R). However, none of the potentials tested for Au was sufficiently accurate for this purpose.

SARS-CoV-2 seroprevalence and antibody trends in vaccinated, multi-ethnic healthcare employees.

Understanding of antibody kinetics against SARS-CoV-2 and its vaccines is rapidly evolving. This study aims to (1) determine post-vaccination seroprevalence; (2) compare antibody levels between vaccine types and various clinical/demographic determinants; and (3) determine post-vaccination antibody concentrations against time. This is a retrospective cross-sectional study involving 148 healthcare employees all over Malaysia. IgG Spike (RBD), IgM Spike and IgG Nucleocapsid concentration medians were compared using Mann-Whitney U or Kruskal-Wallis tests. Chi Square and Spearman correlation coefficient tests were performed to identify variables associated with antibody titers. A scatter plot of IgG Spike (RBD) against time from last vaccine dose was also plotted. At 1-month post-vaccination, all employees successfully seroconverted regardless of vaccine type, health status and COVID- 19 history. Comirnaty, convalescent, female or Malay vaccinees had significantly higher IgG Spike (RBD) titers compared to their respective counterparts. No correlation was found between age and IgG Spike (RBD) levels. Concentration of all three antibodies waned with time post-vaccination, with IgM Spike and IgG Nucleocapsid waning faster than IgG Spike (RBD).

Primary structure of the human fgr proto-oncogene product p55c-fgr.

Normal human c-fgr cDNA clones were constructed by using normal peripheral blood mononuclear cell mRNA as a template. Nucleotide sequence analysis of two such clones revealed a 1,587-base-pair-long open reading frame which predicted the primary amino acid sequence of the c-fgr translational product. Homology of this protein with the v-fgr translational product stretched from codons 128 to 516, where 32 differences among 388 codons were observed. Sequence similarity with human c-src, c-yes, and fyn translational products began at amino acid position 76 of the predicted c-fgr protein and extended nearly to its C-terminus. In contrast, the stretch of 75 amino acids at the N-terminus demonstrated a greatly reduced degree of relatedness to these same proteins. To verify the deduced amino acid sequence, antibodies were prepared against peptides representing amino- and carboxy-terminal regions of the predicted c-fgr translational product. Both antibodies specifically recognized a 55-kilodalton protein expressed in COS-1 cells transfected with a c-fgr cDNA expression plasmid. Moreover, the same protein was immunoprecipitated from an Epstein-Barr virus-infected Burkitt's lymphoma cell line which expressed c-fgr mRNA but not in its uninfected fgr mRNA-negative counterpart. These findings identified the 55-kilodalton protein as the product of the human fgr protooncogene.

Tissue-specific expression and developmental regulation of the human fgr proto-oncogene.

In this study, we show that c-fgr proto-oncogene expression is limited to normal peripheral blood granulocytes, monocytes, and alveolar macrophages, all of which contain 50 to 100 copies of c-fgr mRNA per cell. The c-fgr RNA molecules in these cells consisted of partially spliced transcripts containing intron 7 and completely spliced molecules capable of encoding the predicted p55 c-fgr protein. The splicing of intron 7 appeared to occur after the splicing of most of the other introns; partially spliced molecules containing intron 7 did not appear to be transported into the cytoplasm. Very low levels of fgr transcripts were also present in U937 promonocytic cells and increased in abundance with 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation. The level of fgr transcripts began to increase 2 to 4 h after TPA addition, peaked at 8 h, and subsequently declined. Since we found that the half-life of fgr mRNA was longer than 8 h, these changes are best explained by transient transcriptional activation of fgr during TPA-induced differentiation, although nuclear runoff experiments were not sensitive enough to detect this event. Cycloheximide also caused accumulation of c-fgr transcripts in U937 cells; no superinduction was observed when TPA and cycloheximide were added at the same time. Induction by either agent was blocked with actinomycin D. These results demonstrate that the c-fgr gene is expressed in a tissue- and development-specific fashion and suggest that constitutive expression of c-fgr in U937 cells is regulated by a labile transcriptional repressor.

Hypomethylation of DNA in human cancer cells: a site-specific change in the c-myc oncogene.

Site-specific methylation of the human c-myc oncogene was investigated in a set of cultured human tumor cell lines and normal human fibroblast strains. As previously reported, all of the tumor cell lines in contrast to normal cells were hypomethylated to various degrees in their total genomic DNA. The presence of methylation in specific regions of the c-myc gene was analyzed by use of the restriction endonuclease isoschizomers MspI and HpaII, which recognize the sequence 5'-CCGG-3' (CCGG = DNA sequence of 2 cytosine bases followed by 2 guanine bases) but differ in their abilities to cleave at the internal cytosine residue when it is methylated. The first exon, first intervening sequence, and the second exon were hypomethylated in all cell types, regardless of whether the cells were normal or oncogenically transformed and regardless of the degree of total genomic methylation of the cell. However, the solitary CCGG site in the third exon was fully methylated in normal cell strains. In contrast, in 3 of 5 tumor cell lines measured, this site was hypomethylated. This is the first demonstration of a site-specific DNA methylation defect in a cellular oncogene. Some possible implications relating disruption of DNA methylation to oncogene control and oncogenesis are discussed.

Immunoglobulin delta expression in Burkitt's lymphoma cell lines.

Eighteen Burkitt's lymphoma (BL) cell lines were analyzed for rearrangement and expression of the delta gene. None had rearrangement of the delta gene locus within the 9.0-kb BamHI restriction fragment. Cell lines that expressed the delta gene contained both mu alleles, with at least one productively rearranged. Thirteen of 18 cell lines had detectable transcripts hybridizing with the delta probe. In 10 of the 13 cell lines with delta transcripts, cytoplasmic delta chains were detected, but only two of these expressed delta chains strongly on the surface. All 13 lines made cytoplasmic mu chains, and all except one made cytoplasmic light chains. Surface IgM was detected in all except two of the 13 cell lines. Although BL has generally been considered not to express IgD, except in occasional cases, previous studies have been confined to examination of the cell surface. Many of the cell lines that we examined express delta mRNA transcripts as well as produce cytoplasmic delta chains but no detectable surface IgD. This suggests that delta chains are detectable in the cytoplasm prior to being apparent on the surface. Our findings argue against an origin of BL from germinal center cells since IgD is almost totally lacking in normal B cells present in germinal centers.

Extent of DNA methylation in human tumor cells.

The total genomic DNA methylation, i.e., the percentage of methylated cytosines, was measured in 20 cell lines derived from different types of human tumors. The measurements were obtained by cation-exchange liquid chromatography of bases released by formic acid hydrolysis. These experiments were done to determine if altered methylation is a prevalent and large defect in oncogenic transformation. A majority of the tumor cells measured had decreased levels of methylated DNA in comparison to our laboratory's and other laboratories' published measurements of normal cells and tissues. In fact, tumor cell DNA ranged as low as 1.2% of cytosines methylated compared to a value of 3% or more for normal cells and tissues. HpaII and MspI DNA restriction enzyme analysis confirmed for all tumor cell lines tested that their DNA was hypomethylated in comparison to the DNA from normal diploid fibroblasts tested. The results obtained by liquid chromatography and restriction enzyme analysis were strikingly similar. The reduced methylation of the tumor and DNA correlated with the recent observation of other laboratories that individual genes are undermethylated in human cancer cells and that a number of different carcinogens can lower DNA methylation directly.

Estrogen receptor gene methylation in human breast tumors.

DNA methylation is known to be involved in eukaryotic gene control; it may thus exert effects during development and tumorigenesis. We have examined the methylation status of the estrogen receptor (ER) gene in different human tissues. The ER gene was found to be methylated in placental tissues, but normal breast tissues exhibited a different methylation pattern. In addition, specific sites in the hormone-binding domain of the ER gene were observed to be differently methylated in different human breast tumor specimens. We did not detect, however, any association between the ER status of a tumor and ER gene methylation at these sites. Interestingly, a difference in the methylation status between normal and adjacent breast tumor tissues was observed. Thus, DNA methylation may be considered an additional molecular measure of the genetic heterogeneity in breast cancer.

Childhood undifferentiated leukemia with early erythroid markers and c-myb duplication.

Ultrastructural, flow cytometric, and molecular studies were performed on leukemia cells from bone marrow and pleural effusion of a 6-year-old boy diagnosed with undifferentiated (MO) leukemia, using routine histology and immunostains at diagnosis and relapse. Ultrastructurally, surface and/or intracellular ferritin particles were present on or in some blasts and the majority of blasts contained identifiable acid ferrocyanide reactive inorganic iron comparable to that seen in normal early erythroblasts. The cells lacked other evidence of differentiation, including diaminobenzidine-reactive or immunoreactive hemoglobin. Flow cytometric analysis of malignant cells showed a lack of lymphoid or myeloid markers. Anti-transferrin receptor antibody was positive on 93% of cells and antibody to glycophorin A reacted with 23% of cells. RNA blot analysis of leukemia cells with myeloperoxidase (MPO) showed an absence of appreciable levels of MPO mRNA. Chromosome analysis showed 51,XY, t(1;16)(p31;q24), +6, +10, +15, +19, +21. The oncogene c-myb, which is specifically expressed and regulated in hematopoietic cells and produces a DNA-binding protein responsible for myeloid differentiation, was found to be duplicated in the patient's tumor cells. Expression of c-jun, N-ras, c-myc, and p53 was normal. The data indicate that the malignant cells in this patient are of early erythroid lineage at diagnosis and relapse and that classification of cell lineage can be enhanced by ultrastructural Prussian blue staining. The failure of this otherwise undifferentiated leukemia to express or evolve into a myeloid phenotype is biologically and clinically distinct from previously described cases of erythroid and myeloid leukemia and may represent a previously unidentified phenotype which should be included in the spectrum of 'undifferentiated' childhood leukemia.

Effects of nonsteroid antiinflammatory drugs on the specific binding of platelet activating factor to membrane preparations of rabbit platelets.

The inhibitory effects of several antiinflammatory agents on the specific binding of tritiated 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphorylcholine, (platelet activating factor, PAF), with its receptor on isolated rabbit platelet plasma membranes were investigated. Several potent cyclooxygenase inhibitors do not inhibit 3H-PAF binding to its receptor sites. Yet, three others, indomethacin, phenylbutazone and sulfinpyrazone, as well as three non-cyclooxygenase inhibitors, the 3',4'-dimethoxy analog of indomethacin, the prodrug sulindac and its sulfone metabolite, are moderately active at relatively high concentrations (50 - 100 microM). Parallel inhibitions of 3H-PAF binding and PAF-induced platelet aggregation by derivatives of these antiinflammatory agents suggest that these inhibitors are probably interacting with the functional binding sites of PAF. The results clearly indicate that the configuration of PAF binding site is very different from the inhibitory site of cyclooxygenase. A preference for oxygenated substituents in these hydrophobic molecules to inhibit the PAF-receptor binding is noted. Some binding characteristics of the receptor are briefly discussed.

Obesity: basic science and medical aspects relevant to anaesthetists.

Obesity is becoming a major public health problem throughout the world. It is now the second leading cause of death in the United States and is associated with significant, potentially life-threatening co-morbidities. Significant advances in the understanding of the physiology of body weight regulation and the pathogenesis of obesity have been achieved. A better understanding of the physiology of appetite control has enabled advances in the medical and surgical treatment of obesity. Visceral or abdominal obesity is associated with an increased risk of cardiovascular disease and type 2 diabetes. Various drugs are used in the treatment of mild obesity but they are associated with adverse effects. Surgery has become an essential part of the treatment of morbid obesity, notwithstanding the potential adverse events that accompany it. An appreciation of these problems is essential to the anaesthetist and intensivist involved in the management of this group of patients.

fgr proto-oncogene mRNA induced in B lymphocytes by Epstein-Barr virus infection.

Several acute transforming retroviruses encode tyrosine-specific protein kinases which possess structural and functional relationships to cell-surface receptors for certain growth factors. One such tyrosine kinase is encoded by the onc gene, v-fgr, of Gardner-Rasheed feline sarcoma virus (GR-FeSV). Recently, we have isolated and characterized the human gene, c-fgr, corresponding to the viral onc sequence and have shown that c-fgr is a unique gene located on the short arm of chromosome 1 (ref. 7). Here we report that certain lymphomas (but not sarcomas or carcinomas) express fgr-related messenger RNA. This transcript is detected in Burkitt's lymphoma cell lines naturally infected with Epstein-Barr virus (EBV), but not in EBV-negative Burkitt's lymphoma cells. Normal umbilical cord or peripheral blood lymphocyte lines established in vitro by EBV infection also contain detectable c-fgr mRNA. Moreover, a 50-fold increase of the steady-state c-fgr mRNA concentration is observed when uninfected Burkitt's lymphoma cell lines are deliberately infected with EBV. These findings demonstrate for the first time the induction of a proto-oncogene in response to infection by a DNA tumour virus.

Specific receptor sites for 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) on rabbit platelet and guinea pig smooth muscle membranes.

By using tritiated 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (3H-PAF), we have directly identified its specific binding sites on rabbit platelet plasma membranes. The equilibrium dissociation constant for 3H-PAF is 1.36 (+/- 0.05) X 10(-9) M at 0 degrees C. The number of binding sites is 1.61 (+/- 0.34) X 10(12)/mg of membrane, which corresponds to approximately 150-300 receptors/platelet (depending on membrane vesicle orientation). Binding of 3H-PAF to rabbit platelet plasma membrane is rapid (t1/2 less than 5 min at 0 degrees C) and reversible. For a series of PAF analogues, their affinity for the receptor sites parallels with their relative potency to induce platelet aggregation. PAF can cause contraction of smooth muscle of heart, parenchymal strip, trachea, and ileum. Specific PAF receptor binding was demonstrated with purified plasma membrane from several smooth muscles and from polymorphonuclear leukocytes but not from presumably PAF nonresponsive cells such as erythrocytes and alveolar macrophages. It is likely that the interaction of PAF with these binding sites initiates the specific responses of platelets, polymorphonuclear leukocytes, and smooth muscles.

Acute undifferentiated leukemia (AUL): a case report and a proposed system of classification.

This report describes a case of HLADR+, CD34- acute undifferentiated leukemia (AUL) diagnosed in an 18-year-old male. A definition of AUL and a system for its classification are proposed on the basis of the current state of knowledge about phenotypic features of AUL cells and their clonal counterparts that exist during early stages of normal hematopoiesis.

Cytogenetic and molecular evaluation of clinically aggressive esthesioneuroblastoma.

We report a 16-year-old boy with esthesioneuroblastoma that presented with a unilateral tumor extending to the maxillary sinus and periorbital region. Despite initial therapy with gross resection, 5,682 cGy to the tumor bed and chemotherapy, the patient subsequently had a rapid local recurrence with distant metastases. Immunocytochemical, ultrastructural, cytogenetic, and molecular techniques were performed to determine if this tumor was biologically similar to childhood neuroblastoma. Urinary excretion of vanillylmandelic acid (VMA) and homovanillic acid (HVA) were markedly elevated. Chromogranin and neuron specific enolase immunostaining of tumor cells was positive, as seen in neuroblastoma. Electron microscopic studies showed cells that were closely packed and connected by occasional cell junctions. The cell cytoplasm contained moderate amounts of filaments and microtubules. Numerous electron dense granules were observed; however, these granules lacked distinct nucleoids and generally reacted strongly for acid phosphatase, indicating a lysosomal rather than a secretory function. Tumor cells contained near-pseudotetraploid chromosomes, with all chromosomes represented at least three times, and chromosome 5 was present in multiples of eight. Clonal structural abnormalities included 2q+ and 5q+ and multiple double minutes. Northern blot analysis revealed both c-myc and N-myc expression; however, N-myc amplification was not demonstrated, and c-myc expression appeared increased, unlike cases of rapidly progressive neuroblastoma. These results suggest that despite biologic similarities to neuroblastoma in catecholamine excretion and some ultrastructural features, molecular genetic abnormalities differ in this comparatively aggressive case of estesioneuroblastoma.

Disseminated primitive neuroectodermal tumor: diagnosis using immunocytochemistry, electron microscopy, and molecular probes.

A patient with a disseminated small cell tumor presented with hyperuricemia, gingival hypertrophy, lymphadenopathy, and bone marrow replacement with tumor cells. Initial histologic examination and clinical presentation were consistent with presumed marker silent lymphoma/leukemia. Despite initial treatment with and response to lymphoma/leukemia therapy the patient relapsed in the testis, bone marrow, pancreas, and skin whereupon subsequent and retrospective immunocytochemical, ultrastructural, cytogenetic, and molecular analysis led to the diagnosis of primitive neuroectodermal tumor (PNET). Despite extensive investigation and autopsy no primary site of tumor could be found demonstrating that PNET should be considered in the differential diagnosis of disseminated small cell tumors without an apparent primary.

Isolation and chromosomal localization of the human fgr protooncogene, a distinct member of the tyrosine kinase gene family.

The cell-derived domain of Gardner-Rasheed feline sarcoma virus (GR-FeSV) consists of a gamma-actin- and a tyrosine-specific protein kinase-encoding sequence designated v-fgr. By utilizing a v-fgr probe, it was possible to detect related sequences present at low copy number in DNAs of a variety of mammalian species and to isolate a human fgr homologue. Comparative studies revealed that this human DNA clone represented all but 200 base pairs of v-fgr. Analysis of human genomic DNA demonstrated that the fgr protooncogene was distinct from the cellular homologues of other retrovirus onc genes. In addition, the fgr protooncogene was localized to the distal portion of the short arm of human chromosome 1 at p36.1-36.2 by in situ hybridization. Taken together, our findings establish that the fgr protooncogene is a unique member of the tyrosine kinase gene family.