Influence of epidermal growth factor/transforming growth factor alpha and polyamines on Caco-2 cell proliferation.
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Caco-2 Cells↗
Biomedical subjects
J Stein
Publications and source records attributed to J Stein.
A piece of my mind. The holy society.
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Attitude to Death↗
Post-translational processing of the insulin-like growth factor-2 precursor. Analysis of O-glycosylation and endoproteolysis.
Insulin-like growth factor-2 (IGF-2) is expressed in most embryonic tissues and is required for normal development during gestation. After birth IGF-2 expression is extinguished in most tissues, but the gene is often reactivated during tumorigenesis. Tumors secrete high molecular weight forms of IGF-2 that result from aberrant post-translational processing of pro-IGF-2. As a first step toward understanding how high molecular weight IGF-2 peptides might contribute to tumor progression, we have characterized the biosynthesis of IGF-2 in a human embryonic cell line. We have found that pro-IGF-2 can initially form two disulfide isomers that undergo rearrangement to a single conformation in vivo. The addition of N-acetylgalactosamine to Ser71, Thr72, Thr75, and Thr139 likely occurs in the cis- Golgi apparatus. Sialic acid addition begins in the trans- Golgi apparatus, but IGF-2 peptides must reach the trans-Golgi network for oligosaccharide maturation to be completed. Endoproteolysis occurs concomitant to or slightly after oligosaccharide maturation. Cleavage was observed only at Arg104, resulting in the secretion of IGF-2-(1-104) and free E-peptide. Proteolysis required basic residues in the P1 (Arg104) and P4 (Arg101) positions, was completely blocked by a furin inhibitor, and was enhanced by coexpression with furin, PACE4, PC6A, PC6B, and LPC. These data suggest that members of the subtilisin-related proprotein convertase family mediate processing of pro-IGF-2 at Arg104. We did not detect the IGF-2 peptides that are most abundant in normal serum, mature IGF-2, and IGF-2-(1-87), in this expression system, which indicates that novel endoproteases are responsible for generating these products.
Arginine↗
Home intravenous antibiotic treatment for febrile episodes in immune-compromised pediatric patients.
The purpose of this work was to assess the feasibility of home intravenous antibiotic treatment (HIAT) for febrile episodes in immune-compromised (neutropenic, splenectomized), low-risk pediatric patients. Thirty hematology-oncology patients who presented to our emergency room from January 1993 to January 1995 and who suffered from a febrile episode and were considered at low risk for septic complications were immediately discharged on HIAT. Patients were followed for at least 3 weeks after recovery. Patients and parents were retrospectively questioned about adverse effects and about their degree of satisfaction with home treatment. Patients who required hospitalization during this period were considered unresponsive to HIAT and were analyzed for causes and adverse effects. Thirteen out of 60 (22%) febrile episodes, or eight out of 42 (19%) episodes of fever and neutropenia eventually led to hospitalization. Pseudomonas species infections were associated with the highest rate of unresponsiveness (88%). A central venous catheter infection developed in two cases following HIAT (two cases out of 640 days of therapy). No other complications were identified. No infection-related morbidity was observed. Patients and parents were highly satisfied with HIAT and wanted to use it again, if necessary. Immediate discharge on HIAT for low-risk pediatric immune-compromised patients suffering from a febrile episode is feasible, safe, and well accepted by patients and families. Patients who are found to have Pseudomonas infections should probably be hospitalized. Our results are preliminary and must be confirmed by a prospective, randomized trial before definite recommendations can be made.
Adolescent↗
[Inverse radiotherapy planning for intensity modulated photon fields].
Substantial improvement in conformal radiotherapy is possible using modulated irradiation fields. Such modulated fields may be generated even with conventional accelerators by means of individual metal compensators or with the recently available dynamic multileaf collimators (MLC). For treatment planning a new kind of planning program is required that can calculate the 2 D-intensity matrices for each photon field. At the German Cancer Research Center (Deutsches Krebsforschungszentrum) such a program has been developed under the name of "KonRad" (Conformal Radiotherapy). Although it is an independent application, it is proposed for clinical usage to supplement a planning system that is already present. So-called inverse planning differs from conventional 3 D planning, as the trial-and-error approach for finding good field parameters is nearly completely avoided. Instead, the radio-oncologist is given the chance directly to specify medically oriented criteria like the prescription dose in the target volume, maximal tolerance dose values for each organ at risk and their weighting factors. In addition, the so-called DVH optimization allows aimed, partial overdosage, especially in parallelly structured organs in order to obtain better overall planning results. Because of very fast dose calculation in connection with rapidly converging gradient optimization and an intuitive use interface, the planning is done in a comfortable and interactive manner. Using a workstation or a PC, a typical plan can be created within a few minutes.
Equipment Design↗
Risk of infection with Borrelia burgdorferi sensu lato for a host in relation to the duration of nymphal Ixodes ricinus feeding and the method of tick removal.
The objectives of the present study were to investigate the risk of B. burgdorferi s.1. (Bb)-transmission by I. ricinus-nymphs to a host (i) after different periods of feeding, and (ii) with regard to the particular method of tick removal. On each of 72 Mongolian gerbils 3 tick nymphs taken from a highly infected batch were allowed to feed in a small capsule. Feeding ticks were removed 16.7, 28.9, 47.0, and 65.2 hrs post-attachment. In each of these 4 groups 3 sub-groups with 6 gerbils each were deticked by (a) pulling ticks out with forceps without any pretreatment, (b) pulling ticks out after 3 min of intensive squeezing, and (c) applying nail polish to ticks 1.1 hrs before removal. The infection status in each gerbil was subsequently determined by larval xenodiagnosis. All gerbils with ticks removed > or = 47 hrs post-attachment were found to be infected. After 16.7 hrs as well as after 28.9 hrs of tick feeding, approximately 50% of the gerbils had acquired a transmissible infection, thus Bb-transmission to a host may even occur in the early phases of I. ricinus feeding. There is no evidence from this study that the tick removal method used has any significant influence on a host's Bb-infection risk.
Animals↗
The role of descending basal ganglia connections to the brain stem in parkinsonian akinesia.
Akinesia is the most disabling symptom of Parkinson's disease. The neural mechanisms underlying it probably involve the descending projections of the basal ganglia to the brain stem as it improves after a pallidotomy or subthalamic nucleotomy but not after a thalamotomy. We describe the effects of lesioning the pedunculopontine nucleus in the normal primate in generating an akinetic syndrome. The possible clinical implications of this study are discussed.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
The microdissection cautery needle versus the cold scalpel in bicoronal incisions.
Craniofacial procedures commonly use scalp incisions to optimize surgical access and aesthetic results. Although the use of traditional electrocautery instruments on hair-bearing tissue has been limited secondary to the width of resulting alopecia, needle-tipped electrocautery devices with decreased energy transmission have been developed. This study investigates the cosmetic effect of such instruments on scalp incisions. Twelve children undergoing craniosynostotic correction via bicoronal incisions were included. One side of the incision was completed with the cold scalpel whereas the contralateral portion was effected using the Colorado MicroDissection Needle (Colorado Biomedical Inc., Evergreen, CO, U.S.A.) according to optimal usage instructions. At the conclusion of the study period, precise measurements of the resultant width of alopecia were obtained from the parietal and temporal regions bilaterally, and were analyzed statistically. Also, parents completed a questionnaire concerning subjective observations of the surgical incision and its healing. The portions of the incision completed with the Colorado MicroDissection Needle demonstrated a wider area of peri-incisional alopecia (5.8 +/- 1.8 mm) than that produced by the cold scalpel (3.5 +/- 0.87 mm). Not only was this increased width significant statistically (P < 0.05), in addition the disparity was noted by the majority of parents (10 of 12) either on the patient questionnaire or with nonsuggestive verbal questioning. The benefit of the hemostatic incisional technique of electrocautery with even needle-tipped instruments must be weighed carefully against its cosmetic consequences.
Alopecia↗
Disruption of intestinal barrier function associated with experimental colitis: possible role of mast cells.
The objective was to characterize changes in barrier and transport function in an experimental model of colitis, and to determine whether mast cells contribute to these changes. Colitis was induced in rats with intracolonic 2,4,6-trinitrobenzenesulfonic acid (TNBS, 30 mg) in 50% ethanol. Controls received 0.9% saline or the ethanol vehicle alone. In vivo loop perfusion was used to assess colonic water flux (in microliter.cm-1.h-1) and lumen-to-blood 51Cr-labeled EDTA clearance (% administered dose) after TNBS. Myeloperoxidase (MPO) was used as an index of granulocyte influx. TNBS or its vehicle caused a marked decrease in water absorption and an increase in permeability at 4 h after administration compared with saline. Neither dexamethasone (anti-inflammatory control) nor doxantrazole (mast cell stabilizer) was able to attenuate these early changes likely caused by the vehicle. In contrast, at later times, TNBS (but not its vehicle) also increased 51Cr-EDTA permeability and decreased water absorption; both effects were significantly attenuated by dexamethasone or doxantrazole. These drugs also significantly reduced TNBS-induced MPO accumulation and release of rat mast cell protease II. We conclude that experimental colitis is associated with severe defects in intestinal transport and barrier functions and that mast cells may contribute to the pathogenesis of these changes.
Animals↗
Polyamine uptake across the basolateral membrane of the enterocyte is mediated by a high-affinity carrier: a study using isolated basolateral membrane vesicles.
BACKGROUND/AIMS: Polyamine uptake from the circulation plays an important role in the maintenance of the intracellular polyamine content during extensive proliferation in intestinal mucosal cells. METHODS: Isolated basolateral membrane vesicles of the rabbit enterocyte were used to characterize polyamine transport across the basolateral side of the intestinal epithelium. Incorporation of spermidine and spermine into the basolateral membrane was rapid, although 30-60% of polyamines were initially bound to the basolateral membrane. In order to avoid the influence of binding on the actual uptake into the vesicles, polyamine incorporation was measured at 37 and 4 degrees C, and kinetic parameters were calculated from the difference in polyamine incorporation rates at these temperatures. RESULTS: Uptake kinetics was saturable, with Km values of 13.34 and 12.35 micromol/l and Vmax of 159 and 105 pmol/mg protein/ min for spermidine and spermine, respectively. It was also temperature dependent, with Q10 values (calculated between uptake velocities at 37 and 25 degrees C) of 2.56 for spermidine and 1.90 for spermine. At physiological pH, polyamine uptake was at its highest. Since at this pH polyamines are fully charged, charge might be essential for polyamines to be taken up across the basolateral membrane. Polyamine uptake was inhibited by di-, tri- and tetracations, and there was no evidence for sodium cotransport. Transport of putrescine was not inhibited by spermine and spermidine, although spermidine inhibited spermine uptake in a competitive manner, with Ki of 127 micromol/l. CONCLUSION: These results imply that a saturable high-affinity transport system for polyamine does exist at the basolateral side of the enterocyte. Such a transport system may be responsible for the active transport of polyamine into rapidly proliferating enterocytes.
Animals↗
Surveillance of drug overdose deaths using medical examiner data.
This paper describes the epidemiology of drug overdose deaths investigated by the medical examiner in one of the cities participating in the Canadian Community Epidemiology Network on Drug Use and assesses the quality of the information obtained from medical examiner charts with respect to drug overdose deaths, for surveillance purposes. Information was abstracted from medical examiner charts of all deaths involving drugs from 1993 to 1995 in Halifax, Nova Scotia. During these three years, 636 deaths from all causes were investigated by the medical examiner. Of the 42 overdose deaths, 47.6% were suicides. Ethanol was detected in 47.8% of overdose deaths, and 61.9% of all overdose deaths involved psychotropic medications. Two deaths were attributed to an illicit drug (cocaine). An independent review performed by a toxicologist and a medical examiner revealed poor overall agreement concerning overdose as a cause of death (Kappa coefficient: 0.27). In conclusion, the average crude mortality rate due to drug overdose in Halifax from 1993 to 1995 was 4.1 deaths per 100,000 population. Potential threats to the quality of data were the lack of standardization concerning toxicological testing and the definition of drug overdose.
Adult↗
Doppler color-flow imaging: screening of a patent foramen ovale in children scheduled for neurosurgery in the sitting position.
A patent foramen ovale (PFO) is the most common cause of paradoxical air embolism during neurosurgical procedures in the posterior fossa in the sitting position in both adults and children. To detect right-to-left shunting, we performed Doppler color-flow imaging preoperatively in 30 children scheduled for neurosurgical procedures in the sitting position. A PFO was diagnosed on the basis of color alterations indicating an immediate blood shunt through a PFO. Six of 30 children (20%) had a PFO; in 4 of these children the sitting position then was avoided, and 2 children were operated on in a special supine position with minimal elevation of the head. Venous air embolism occurred in 9 of 24 (37%) children operated on in the sitting position and in none of the 6 children operated on in a nonsitting position. We conclude that Doppler color-flow mapping could be a useful noninvasive technique to screen children scheduled for neurosurgery in the sitting position for the presence of a PFO.
Adolescent↗
[Stress proteins and molecular chaperones--protectors of intestinal epithelial damage?].
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Animals↗
S-adenosylmethionine decarboxylase activity and utilization of exogenous putrescine are enhanced in colon cancer cells stimulated to grow by EGF.
UNLABELLED: Polyamines spermidine and spermine and their precursor putrescine are necessary for cell growth. Polyamine content is high in rapidly growing malignant cells, due to enhanced putrescine synthesis by ornithine decarboxylase (ODC), and increased uptake. In contrast to other cells of the body, colon cancer cells are exposed to high putrescine concentrations from the lumen. AIMS: To investigate the utilization of luminal putrescine in colon cancer, we studied the effect of a potent mitogen, epidermal growth factor (EGF), on the activity of the enzyme responsible for putrescine conversion, S-adenosylmethionine decarboxylase (SAMDC), in Caco-2 cells. METHODS: Cell counts, ODC and SAMDC activities and intracellular polyamines were evaluated in the presence and absence of exogenous putrescine in concentrations resembling those normally present in the colonic lumen. RESULTS: ODC and SAMDC activity and putrescine uptake were strongly stimulated by EGF. Both synthesized and absorbed putrescine was rapidly converted to spermidine and spermine after EGF. Conversion pattern was identical in the cells stimulated with EGF only and EGF plus exogenous putrescine, indicating that, if stimulated to proliferate, colon cancer cells utilize the entire available putrescine pool. SAMDC inhibitor, methylglyoxal-bis-guanylhydrazone, induced growth arrest which was not reversed by exogenous putrescine, but only by high concentrations of spermidine. CONCLUSION: Enhanced proliferation in colon cancer cells is associated with increased SAMDC activity and rapid conversion of putrescine to spermidine and spermine. SAMDC might be a preferable target for therapeutic attempts to impair growth by reducing intracellular polyamine pools in colon cancer.
Adenosylmethionine Decarboxylase↗
Planning, delivery, and quality assurance of intensity-modulated radiotherapy using dynamic multileaf collimator: a strategy for large-scale implementation for the treatment of carcinoma of the prostate.
PURPOSE: To improve the local control of patients with adenocarcinoma of the prostate we have implemented intensity modulated radiation therapy (IMRT) to deliver a prescribed dose of 81 Gy. This method is based on inverse planning and the use of dynamic multileaf collimators (DMLC). Because IMRT is a new modality, a major emphasis was on the quality assurance of each component of the process and on patient safety. In this article we describe in detail our procedures and quality assurance program. METHODS AND MATERIALS: Using an inverse algorithm, we have developed a treatment plan consisting five intensity-modulated (IM) photon fields that are delivered with DMLC. In the planning stage, the planner specifies the number of beams and their directions, and the desired doses for the target, the normal organs and the "overlap" regions. Then, the inverse algorithm designs intensity profiles that best meet the specified criteria. A second algorithm determines the leaf motion that would produce the designed intensity pattern and produces a DMLC file as input to the MLC control computer. Our quality assurance program for the planning and treatment delivery process includes the following components: 1) verification of the DMLC field boundary on localization port film, 2) verification that the leaf motion of the DMLC file produces the planned dose distribution (with an independent calculation), 3) comparison of dose distribution produced by DMLC in a flat phantom with that calculated by the treatment planning computer for the same experimental condition, 4) comparison of the planned leaf motions with that implemented for the treatment (as recorded on the MLC log files), 5) confirmation of the initial and final positions of the MLC for each field by a record-and-verify system, and 6) in vivo dose measurements. RESULTS: Using a five-field IMRT plan we have customized dose distribution to conform to and deliver 81 Gy to the PTV. In addition, in the overlap regions between the PTV and the rectum, and between the PTV and the bladder, the dose is kept within the tolerance of the respective organs. Our QA checks show acceptable agreement between the planned and the implemented leaf motions. Correspondingly, film and TLD dosimetry indicates that doses delivered agrees with the planned dose to within 2%. As of September 15, 1996, we have treated eight patients to 81 Gy with IMRT. CONCLUSION: For complex planning problems where the surrounding normal tissues place severe constraints on the prescription dose, IMRT provides a powerful and efficient solution. Given a comprehensive and rigorous quality-assurance program, the intensity-modulated fields can be efficaciously and accurately delivered using DMLC. IMRT treatment is now ready for routine implementation on a large scale in our clinic.
Adenocarcinoma↗
Pentoxifylline promotes replication of human cytomegalovirus in vivo and in vitro.
OKT3 monoclonal antibody (MoAb) therapy is well established in the prevention and therapy of acute rejection in transplant patients. Unfortunately, this therapy is associated with several short-term (cytokine release syndrome) and long-term (infections, EBV-related lymphoma) side effects. Recently, we were able to demonstrate an association between the TNF alpha release following the first OKT3 MoAb infusions and the appearance of human cytomegalovirus (HCMV) reactivation several days later. In order to prevent this TNF alpha associated HCMV reactivation patients were additionally treated with pentoxifylline (PTX), a methylxanthine derivative that has been shown to suppress TNF alpha induction. Although the TNF alpha peak plasma level following OKT3 MoAb treatment was markedly reduced, the incidence of HCMV reactivation and HCMV disease was not influenced. In transient transfection experiments using HCMV immediate early enhancer/promoter CAT reporter gene constructs PTX enhanced the promoter activity independently from TNF alpha in premonocytic cells. Furthermore, PTX acted synergistically with TNF alpha. In virus-infected human embryonal lung fibroblasts HCMV replication was triggered in the presence of both PTX and TNF alpha, while either substance alone had only marginal effects. The stimulatory effect of PTX on the immediate early (IE) enhancer/promoter was mediated via CREB/ ATF, a eukaryotic transcription factor that binds to the 19 bp sequence motif in the enhancer region, while TNF alpha stimulation was mediated by activation of the transcription factor NF-kappaB and its binding to the 18 bp sequence motif in the enhancer. These data suggest a potential side effect of cAMP-elevating drugs such as PTX.
Acute Disease↗
A novel germ line p53 mutation in intron 6 in diverse childhood malignancies.
Screening for p53 mutations in exons 5 to 8 in 124 pediatric malignancies identified 18 abnormal shifts using single strand conformation polymorphism: 12 were missense mutations and in 6, no mutation was detected in the exon or in the splice donor acceptor sequences. Sequencing was then performed in the adjacent introns, revealing a G to A base substitution at 39 base pairs upstream to exon 7. This mutation was identified in the germ line of five of the patients, and also in the father of one, whose parents were available. For comparison, of the 184 normal controls similarly screened, only one had this mutation (P=0.036). Positive staining of p53 protein was observed in three of the paraffin embedded tissues that were available: brain tumor, rhabdomyosarcoma, and lymphocytes from a normal lymph node from the rhabdomyosarcoma patient. All tumors with the identified intron mutation were Li-Fraumeni syndrome tumors. Sequencing of all exons including splice sites was performed and revealed no mutation. We suggest that this mutation in intron 6 of the p53 gene stabilizes the wild type p53 protein, resulting in its abnormal accumulation. Mutations in the noncoding region of p53 should be further studied.
Brain Neoplasms↗