Prospective identification of erythroid elements in cultured peripheral blood.

We have developed a prospective approach to identify the generation of erythroid cells derived from cultured peripheral blood mononuclear cells (PBMC) by monitoring the expression of the cell surface protein CD48. Unpurified populations of PBMC obtained from the buffy coats of normal volunteers were grown in suspension culture in the absence or presence of erythropoietin. A profile of surface CD48 expression permitted a flow cytometric identification of erythropoietin responsive populations at various stages of their maturation. In the absence of erythropoietin (EPO) supplemented media, the CD48- cells represented <5% of the total population of PBMC remaining in culture. In cultures supplemented with 1 U/mL EPO, the mean percentage of CD48- cells increased to 34.7 + 14.9% (p < 0.01) after 14 days in culture. Coordinated CD34 and CD71 (transferrin receptor) expression, morphology, gamma-globin transcription, and colony formation in methylcellulose were observed during the 14-day culture period. Flow cytometric monitoring of bulk cultured PBMC provides a simple and reliable means for the prospective or real-time study of human erythropoiesis.

Synapsin I phosphorylated by Ca2+, calmodulin-dependent protein kinase II is able to self-degrade.

Phosphorylation of the neurospecific protein synapsin I (SI) by various cellular protein kinases was studied. The analysis of functional properties of phosphosynapsins showed that in the case of PK II-mediated modification of the protein, it becomes capable of self-degradation. The latter process was found to be specific and did not appear to be characteristic of the phosphoforms emerging after the protein modification by PK C or PK A. A possible involvement of the process in the regulation of neurotransmitter secretion is discussed.

Stable expression of green fluorescent protein after liposomal transfection of K562 cells without selective growth conditions.

Little is known about the durability of plasmid DNA transgene expression in mammalian cells in the absence of growth selection. For this purpose, we have begun the study of liposomal transfer and expression of plasmid DNA encoding green fluorescent protein (GFP) in human erythroleukemia K562 cells. Detection and selection of GFP expression were accomplished visually and by flow cytometry. GFP expression was noticeable in cells within 4 h of transfection. In nine separate transfections, approximately 20% of the transfected cells expressed GFP with a mean fluorescence 40-50x that of control cells (15 fluorescent units [FU] vs. 0.3 FU) during the first five days after transfection. The percentage of GFP positive cells dropped rapidly to 0.1% by day 14 post-transfection, but fluorescence activated cell sorting on this day resulted in the identification of stable transfectants expressing GFP for an additional 6-12 months in culture. GFP expression is adequate for the identification, isolation and monitoring of stable transfection events after lipid-mediated transfection of eukaryotic cells.

Ca(2+)-dependent phosphorylation of synapsin I as a possible regulatory mechanism of neurosecretion.

Phosphorylation of homogeneous synapsin I isolated from human brain by Ca2+, phospholipid-dependent protein kinase (protein kinase C) from the same source was studied. The inhibitory effect of calmodulin on this process was demonstrated. The kinetics of activation of synapsin I phosphorylation by acidic phospholipids, phosphatidylserine and phosphatidylinositol, in the absence and presence of phosphatidylinositol-4,5-bisphosphate and diacylglycerol was compared. The proteolytic effect of degradation of the synapsin I molecule phosphorylated by Ca2+, calmodulin-dependent protein kinase II was revealed. No proteolysis of synapsin phosphorylated under similar conditions either by protein kinase C or cAMP-dependent protein kinase was detected. In view of the process specificity, the physiological significance of the observed effect is suggested. The inter-relationship between two ways of neurosecretion regulation is discussed: an earlier known, conventional way, mediated by synapsin I phosphorylation by Ca2+, calmodulin-dependent protein kinase II, and another one, mediated by synapsin I phosphorylation by protein kinase C. The modulating role of polyphosphoinositides in the PK C-dependent way of regulation is considered.

[Endoscopic studies in lymphosarcoma in children]. / Endoskopicheskie issledovaniia pri limfosarkome u detei.

The value of various endoscopic methods in diagnosing lymphosarcoma was assessed in 149 suspects aged 3-14 years. Fiberscopy of the upper digestive tract was performed in 130 children of whom 43 revealed lymphosarcoma in Waldeyer's ring whereas in 87 lymphoid hyperplasia was detected. Laparoscopy was carried out in 17 children, esophagogastroduodenoscopy--2 and colonoscopy--in one case Endoscopic methods proved to be of high diagnostic value, easily available and safe for examination of children suspected for lymphosarcoma.

[The role of endoscopic study in the diagnosis of precancerous gastric pathology and identification of a risk group]. / Rol' éndoskopicheskogo issledovaniia v diagnostike predopukholevoi patologii zheludka i formirovanii gruppy riska.

The results of endoscopic diagnosis and case follow-up of 686 patients with precancerous gastric conditions usually identified as a risk group (chronic gastritis, polyps, peptic ulcer, conditions following distal stomach resections) have shown that in patients with symptoms of epithelial dysplasia of gastric mucosa indices of cancer detectability significantly exceeded those in similar pathological conditions without symptoms of epithelial dysplasia. They were 22.0 +/- 2.5% and 2.5 +/- 0.8%, respectively; indices of stage I stomach cancer detectability were 9.9 +/- 1.8% and 0.2 +/- 0.2%, respectively. Patients with precancerous stomach diseases with moderate and severe epithelial dysplasia found in their gastric biopsies, should be attributed to a risk group, and these changes of gastric mucosa must be regarded as the main criterion for the identification of such a group.

[Laparoscopic diagnosis of neoplastic diseases in ambulatory patients]. / Laparoskopicheskaia diagnostika opukholevykh zabolevanii u ambulatornykh bol'nykh.

The article deals with the results of the first experience in using laparoscopy for establishing the diagnosis of neoplastic diseases of the abdominal organs in 32 patients in an out-patient clinic. The contraindications were determined and some organizational measures in conducting laparoscopy in this group of patients were mapped out. A method is suggested for closing the operative wound after laparoscopy with sutures applied through all layer under laparoscopic control. The authors consider strict selection of patients for laparoscopy in out-patient clinics to be expedient.

Human erythroid porphobilinogen deaminase exists in 2 splice variants.

Human porphobilinogen deaminase (PBGD) is, reportedly, encoded by 2 distinct messenger RNAs (mRNAs) transcribing from a single gene. The ubiquitous form of the PBGD gene product is often used as an endogenous reference in gene expression studies because it is pseudogene free and has minimal transcriptional variability among tissues. A distinct erythroid-specific gene product has also been described because of the alternate splicing of the gene. Here is reported the existence of an additional erythroid-specific isoform of PBGD mRNA in primary cells.

[Cytologic diagnosis of metastases of stomach cancer during laparoscopy]. / Tsitologicheskaia diagnostika metastazov raka zheludka pri laparoskopii.

The detection of gastric carcinoma metastases to the peritoneum necessitates the development of a specific treatment strategy. The present paper reviews the results of analyses of ascitic fluid and impressions obtained in laparoscopy from 42 patients. Eighty-one investigations have been carried out. Malignant cells have been detected in 93.1% of impressions and in 50% of ascites. Three types of mesothelial cells are described, all of them present both in the impressions and in the fluid. Type I cells are more incident in proliferation of the mesothelial cells. They are often similar to adenocarcinoma cells as regards their chromatin structure, hyperchromatism, eccentric position of the nucleus, and cytoplasmic basophilia. In distinction from the adenocarcinoma cells, the mesothelial cells are monomorphous and are much smaller. Type I mesothelial cells differ from signet ring cell carcinoma in the following: nuclear hyperchromatism in mesothelial cells is combined with cytoplasmic basophilia, whereas in signet ring cells a colorless vacuole occupies the whole of the cytoplasm in case of a hyperchromatic eccentric nucleus.

Gene expression in proliferating human erythroid cells.

A complete understanding of human erythropoiesis will require a robust description of transcriptional activity in hematopoietic cells that proliferate and differentiate in response to erythropoietin (EPO). For this purpose, we cultured peripheral blood mononuclear cells in the presence or in the absence of EPO and examined the transcriptional profile of those cells arising only in response to EPO. A distinct population of CD71( +) cells that demonstrated an average of six additional doublings in suspension culture and erythroid colony formation in methylcellulose was isolated. Suppression subtractive hybridization of mRNA isolated from those cells permitted the identification of transcribed genes. A summary of 719 expressed sequence tags (ESTs) describing 505 independent transcripts is provided here with a full analysis of each EST available at http://hembase.niddk.nih.gov. Several transcripts that matched genes previously reported in the context of erythroid differentiation including 4 cell surface proteins were expressed at this developmental stage. Active chromatin remodeling was suggested by the identification of 4 histone proteins, 4 high-mobility group proteins, 13 transcription factors, and 6 genes involved in DNA recombination and repair. Numerous genes associated with leukemic translocations were also recognized including topoisomerases I and II, nucleophosmin, Translin, EGR1, dek, pim-1, TFG, and MLL. In addition to known transcripts, 44 novel EST were discovered. This transcriptional profile provides the first genomic-scale description of gene activity in erythroid progenitor cells.

Long-term, stable expression of green fluorescent protein in mammalian cells.

Despite the proven utility of green fluorescent protein (GFP) as a reporter molecule for transient gene expression, the adequacy of this marker for models requiring durable, high-level gene expression has not been fully tested. To address this issue, we performed the transfection of Chinese Hamster Ovary (CHO) cells with plasmid DNA encoding both GFP and neomycin phosphotransferase (neo) cassettes. The expression of GFP was measured after the cells were cultured in the presence or absence of G418-mediated selective pressure. After removal of G418 from the growth medium, the percentage of pooled G418 resistant transfectants which co-expressed the GFP transgene increased or remained unchanged. Flow cytometric and visual isolation of GFP-expressing cells was possible without continued selection in G418. One cloned cell line, C463, maintained high-level green fluorescence for 18 weeks in G418 and an additional 12 weeks in nonselective medium. Our data suggest expression of GFP does not confer a growth disadvantage in mammalian cells.

Micelles of poly(oxyethylene)-poly(oxypropylene) block copolymer (pluronic) as a tool for low-molecular compound delivery into a cell: phosphorylation of intracellular proteins with micelle incorporated [gamma-32P]ATP.

Pluronic P85 (poly(oxyethylene)-poly(oxypropylene) block copolymer) was used for in vitro delivery of [gamma-32P]ATP into intact Jurkat cells. Negatively charged ATP molecules are not able to penetrate cell plasma membrane. Hence, exogenous [gamma-32P]ATP added to a cell culture does not participate in phosphorylation of intracellular proteins. The addition to cells of [gamma-32P]ATP solubilized in positively charged (containing dodecylamine) pluronic micelles results in considerable increase of protein phosphorylation. In this case the treatment of intact cells with alkaline phosphatase (resulting in dephosphorylation of external proteins) causes no essential decrease of [32P]-incorporation in cell proteins. That gives an evidence of delivery of solubilized ATP into a cell. Under the experimental conditions used, pluronic micelles neither influence the viability of cells nor permeabilize cell plasma membrane.

[Lymphosarcoma of the oropharynx and nasopharynx in children]. / Limfosarkoma roto- i nosoglotki u detei.

From 1983 to 1989, 22 patients aged 3 to 15 years with oro- and rhinopharyngeal lymphosarcomas were under observation. This number accounted for 10% of the total children's population with the same disease, with the observation period being the same. As a result of the examination, stage II was diagnosed in 13, stage III in 5, and stage IV in 4 patients. All the patients received chemo- and radiotherapy for 6-18 months. The relapse-free survival amounted to 50 months on the average, the total one to 52 months. It is stressed that early diagnosis and adequate therapy allow the cure of the majority of children with oro- and rhinopharyngeal lymphosarcomas.

Transcription patterning of uncoupled proliferation and differentiation in myelodysplastic bone marrow with erythroid-focused arrays.

Because abnormal erythroid differentiation is the most common manifestation of the myelodysplastic syndromes (MDS), it was hypothesized that erythroid gene expression may be used to illustrate myelodysplastic transcription patterns. Ten normal bone marrow aspirates (NBM) were first analyzed using an erythroid-focused cDNA array to define steady-state transcription levels. Proliferation and differentiation gene subsets were identified by statistically significant differences between NBM and erythroleukemia gene expression. Next, cDNAs from 5 separate MDS aspirates were studied: refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation (RAEB-T), and RAEB-T/secondary MDS. A distinct pattern of significantly increased proliferation-associated and reduced differentiation-associated gene activity was established for MDS.

Identification of the dombrock blood group glycoprotein as a polymorphic member of the ADP-ribosyltransferase gene family.

Identification of the 25 known human blood group molecules is of fundamental importance for the fields of erythroid cell biology and transfusion medicine. Here we provide the first molecular description of the "Dombrock" blood group system. A candidate gene was identified by in silico analyses of approximately 5000 expressed sequence tags (ESTs) from terminally differentiating human erythroid cells. Transfection experiments demonstrated specific binding of anti-Dombrock and confirmed glycosylphosphatidylinositol membrane attachment. Dombrock expression is developmentally regulated during erythroid differentiation and occurs at highest levels in the fetal liver. Homology studies suggest that the Dombrock molecule is a member of the adenosine 5'-diphosphate (ADP)-ribosyltransferase ectoenzyme gene family. Genotypic comparisons suggest Do(a) versus Do(b) antigenicity results from a single amino acid substitution within an encoded arginine-glycine-aspartic acid (RGD) motif of the molecule.