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Biomedical subjects

A Klein

Publications and source records attributed to A Klein.

At least 145 records · Page 8Linked to original sources

Induction of apoptosis by chlorodeoxyadenosine and dexamethasone in B-chronic lymphocytic leukemia.

Apoptosis, programmed cell death, occurs in a variety of cellular systems and in response to many different stimuli. In the present study we examined the ability of dexamethasone (Dex) and chlorodeoxyadenosine (2-CdA) to induce apoptosis in lymphocytes of patients with B-chronic lymphocytic leukemia (B-CLL). Lymphocytes of 29 untreated patients and 9 healthy controls were isolated and incubated for 24 hours in the presence or absence of either Dex (2 microM) (n = 15) or 2-CdA (3 microM) (n = 14). Following incubation the cells were harvested and their DNA extracted and analysed for internucleosomal DNA cleavage by UV illumination after electrophoresis on agarose slab gel containing ethidium bromide. In the Dex group, 10 patients showed dexamethasone independent spontaneous apoptosis appearing 24 hours after the start of incubation. These were the only instances of dexamethasone-enhanced apoptosis. Five patients showed no spontaneous or dexamethasone induced apopto sis. Of the 2-CdA group, 5 showed spontaneous apoptosis enhanced by 2-CdA. No spontaneous apoptosis was observed in the cells from 9 other patients, however, 2-CdA induced apoptosis in 8 cases in this group. This study shows that monitoring of apoptosis in CLL may provide important information regarding susceptibility of the cells to drug induced apoptosis.

Antineoplastic Agents, Hormonal↗

Accumulation of sphingolipids in SAP-precursor (prosaposin)-deficient fibroblasts occurs as intralysosomal membrane structures and can be completely reversed by treatment with human SAP-precursor.

The degradation of glycosphingolipids takes place in lysosomes by action of specific exohydrolases, with the assistance of sphingolipid activator proteins (SAPs). Four of the SAPs, SAP-A to -D (also called saposins A to D), are synthesized from a single protein, the SAP-precursor (prosaposin). Deficiency in this precursor protein, a rare inherited disease in humans, results in the storage of sphingolipids with short oligosaccharide head groups within the patients' tissues, and electron microscopy revealed the accumulation of large multivesicular storage organelles. In this study we analyze the multivesicular storage organelles in cultivated fibroblasts from these patients. The results support our hypothesis that endocytosis of plasma membrane-derived lipids occurs via small intraendosomal and intralysosomal vesicles and membrane structures that are then digested within the lysosomes (Sandhoff, K., T. Kolter, Trends in Cell Biol. 6, 98-103 (1996). First, we show that the storage compartment consists of late endosomes and lysosomes by immunogold labeling for marker proteins of these organelles. The transport of endocytosed bovine serum albumin-colloidal gold or cationized ferritin into the compartment occurs with the timing expected for transport to late endocytic organelles. Second, complementation of the medium of the SAP-precursor-deficient fibroblasts with only nanomolar concentrations of purified SAP-precursor nearly completely reversed the aberrant accumulation of multivesicular structures, thereby abolishing most of the intralysosomal membrane structures. Analysis of the sphingolipid pattern of the cells after metabolic labeling with [14C]serine reveals that the cells' ability to degrade glycosphingolipids is completely restored by feeding of SAP-precursor at the same concentrations. This is the first demonstration in vivo that endocytosed SAP-precursor is processed into functional active SAPs A,- B,- C, and D and that the degradation of the vesicular structures within the lysosomes depends on the presence of the SAPs. Moreover, these studies suggest that a therapy program based on feeding purified SAP-precursor may be valuable in treating the entire family of diseases which result from the loss of one or more of the SAPs.

Endosomes↗

Hepatitis C virus genotypes in patients with persistent infection--a preliminary report.

Hepatitis C virus (HCV) has nucleotide sequence diversity distributed throughout the viral genome, with variants showing even less than 70% homology. There is some evidence that sequence variation of HCV genotypes partly determines the course of infection and response to treatment with interferon. We studied the sera of 29 Israeli HCV patients, all suffering from chronic liver disease, and 34 patients with renal failure necessitating hemodialysis. HCV genotypes were detected using a reverse hybridization assay (LiPA), after reverse transcription polymerase chain reaction, using primers spanning the 5' UTR of the HCV genome. In this preliminary report the predominant HCV type detected was type 1, found in 65% of the chronic hepatitis patients and in 88% of the hemodialysis patients. Subtype 1b was the most prevalent and was detected in >40% of the chronic hepatitis patients and in >70%of the dialysis patients. Other types detected were 2a and 3, and in only two patients was type 4 found. More than 50% of patients with type 1 (1a or 1b) among patients with chronic hepatitis had received blood transfusion in the past, but only 16.6% of patients bearing subtype 2a HCV had such a history. Our preliminary evaluation revealed that patients bearing subtype 1b seemed to have a better response to interferon treatment, as compared with patients infected with subtypes 1a, 2a, who displayed a low response rate.

Adult↗

[Ultrasound fracture diagnosis--an experimental study].

Thanks to the increasing use of ultrasonography in the diagnostic evaluation of the musculoskeletal system, and the development of high-resolution ultrasonic transducers with ever better image quality, as well as the greater general trend towards non-invasive techniques, the use of ultrasonography in the diagnosis and follow-up of fractures would appear to make good sense. The aim of the present experimental study was, therefore, to identify the typical acoustic pattern of fractures and areas of bone impression and defects, while taking account of possible sources of error due to artefacts. Experiments were carried out on cadaver bones immersed in a water bath, using 7.5 and 10 MHz linear transducers. Depending on its size, the bony defect is represented by an interruption of the cortical echo, or a dorsal band of echoes limited to the fracture zone. Of interest is the fact that fractures and bony defects are not represented as such when the transducer is directed parallel to the line of the fracture or the impression area. Particular attention needs to be paid to the numerous artefacts occurring at the margins of the fracture. In summary, it may be noted that using resolution transducers under standardised experimental examination conditions, cortical interruptions of not less than 1 mm can be identified.

Artifacts↗

The effects of predetermined payment rates for Medicare home healthcare.

OBJECTIVE: To assess the effects of an alternative method of paying home health agencies for services to Medicare beneficiaries, based on a demonstration program. DATA SOURCES/STUDY SETTING: Primary and secondary data collected on participating home health agencies in five states and their patients during the three-year demonstration period. Primary data included patient surveys at discharge and six months later, and two rounds of interviews with executive staff of the agencies. Secondary data included agencies' Medicare cost reports, quality assurance reviews, Medicare claims data, demonstration claims data, demonstration patient intake forms, and plan of treatment forms. STUDY DESIGN: The 47 agencies volunteering to participate in the demonstration were each randomly assigned to the treatment or control group. Treatment group agencies were paid a predetermined rate based on their inflation-adjusted cost per visit during the year preceding the demonstration; control group agencies were paid under Medicare's conventional cost reimbursement method. Demonstration impacts were estimated by comparing outcomes for the two groups of agencies and their respective patients, using regression models to control for any remaining differences. PRINCIPAL FINDINGS: Agencies paid under prospective rate setting were slightly better at holding per-visit cost increases below inflation than were control group agencies. The change in payment method had no effect on agencies' volume of Medicare visits or quality of care, nor on patients' use of Medicare services or other formal or informal care services. CONCLUSION: Changing from cost-based reimbursement to predetermined payment rates for Medicare home healthcare visits would not lead to large savings for the Medicare program, but would not increase costs to Medicare or adversely affect patients or their caregivers.

Centers for Medicare and Medicaid Services, U.S.↗

The Second Canadian Consensus Conference on the Management of Patients with Gastroesophageal Reflux Disease.

The Second Canadian Consensus Conference on the Management of Patients with Gastroesophageal Reflux Disease (GERD) was organized by the Canadian Association of Gastroenterology to address major advances in the understanding of the pathophysiology of GERD, to review the new methods of investigation and therapy introduced since the first conference in 1992 and to examine the issue of relevant health economics. The changes that have taken place over the past four years have been sufficiently dramatic to necessitate reassessment of the recommendations made following the first conference. The second conference dealt with the investigation and treatment of uncomplicated GERD and the complex issues of esophageal and extraesophageal complications such as chest pain, Barrett's esophagus, and reflux-related pulmonary and laryngeal disorders. The role of laparoscopic surgery was also discussed. A decision tree for investigation and treatment of patients with GERD was developed. The 38 participants represented a broad spectrum of experience, location of practice and special interests. The distribution of participants conformed to the recommendations of the Canadian Medical Association guidelines for consensus documents in that there should be input from all possible interested parties. A list of the state-of-the-art lectures presented during the conference, the small group sessions, the session chairpersons and participants are appended to this document. CONCLUSIONS. UNCOMPLICATED GERD: GERD with alarm symptoms must be investigated immediately. There was no consensus about when to investigate uncomplicated GERD, ie, whether to perform endoscopy immediately or after initial therapy fails. There was controversy regarding 'step up' (H2 receptor antagonist [H2RA] or prokinetic [PK] first therapy) versus 'step down' therapy (proton pump inhibitor [PPI] first therapy). The majority decision was for short term 'step up' therapy and investigation if symptoms do not improve or recur. Maintenance therapy should be carried out with the initial therapy that was effective. H2RAs and PKs may suffice for maintenance therapy in milder GERD; however, for severe esophagitis, PPIs should be used. SURGERY: Indications for laparoscopic surgery should be the same as for conventional antireflux operations. NONCARDIAC ANGINA-LIKE CHEST PAIN: After exclusion of nonesophageal causes, the majority decided that eight weeks of therapy with a PPI should be performed, while some suggested work-up before a therapeutic test. In the absence of response or recurrence, esophagogastroduodenoscopy (EGD) and, depending on the circumstances, 24 h ambulatory pH/motility may be indicated. BARRETT'S ESOPHAGUS: Only patients who, in case of future discovery of cancer or dysplasia, are able or willing to undergo therapy should have surveillance. In the absence of dysplasia EGD should be performed every two years, and in the presence of mild dysplasia every three to six months. All agreed that for severe dysplasia, esophagectomy or poor risk patients, esophageal mucosal ablation is indicated. ESTRAESOPHAGEAL COMPLICATONS (EECs): Asthma, chronic cough and posterior laryngitis were considered EECs. Although PPIs may decrease symptoms, improvement alone is not diagnostic of the presence of EEC. Ambulatory pH studies with two pH probes or ambulatory pH/motility may be useful in establishing causation. HEALTH ECONOMICS: There are limited data for an economic comparison among the different drugs or between medical and surgical therapy.

Canada↗

[Technical aspects of extraction of platelet concentrates from dogs with an automated cell separator].

The results of this work point out that the cell separator AS 104 (Fresenius AG, Bad Homburg), which is developed to obtain platelet concentrates from human beings, can be used for dogs. The base adjustment of the cell separator for human beings (adjustment I) is useful, because of the high yield [platelet count (median): 1.75 x 10(11)] and quality [low leucocyte- (40/microliter) and erythro count (10 x 10(3)/microliter)]. In addition, there was no clear difference between the base adjustment (adjustment I) and an empirical modification (adjustment II). The platelet count of the donor dog decreased during the separation by approximately 140,000/microliter and reached the initial value after four days. Supplementary, the time course of the haematocrit and the concentrations of albumin, total protein and total calcium of the donor dog were measured. It should be mentioned that the concentrations of ionized calcium distinctly decreased during the separation. Synchronously, the concentrations of ionized calcium and of citrat reached the starting-counts 3-4 hours after the ending of the separation.

Animals↗

Turnover of endogenous ceramide in cultured normal and Farber fibroblasts.

De novo synthesis and turnover of endogenous ceramide in cultured skin fibroblasts from patients affected with Farber lipogranulomatosis were studied by biosynthetical labeling of cellular sphingolipids with [14C]serine. The cellular uptake of [14C]serine and incorporation into de novo synthesized ceramide was similar in normal and Farber fibroblasts, with a half life of newly synthesized ceramide of 2.7 h in normal and diseased cells. Newly synthesized ceramide was found to be channeled directly into biosynthesis of complex sphingolipids rather than contributing to the pool of accumulated ceramide in Farber fibroblasts. The degradation of ceramide generated by the catabolism of complex sphingolipids in Farber cells was greatly delayed compared with control fibroblasts, with differences in the amount of radiolabeled cellular ceramide becoming evident after 6 h chase time. Individual Farber cell lines differed from each other in the amount of accumulated ceramide; however, no correlation was found between ceramide accumulation and residual acid ceramidase activity as determined in vitro. In addition, the amount of radiolabeled sphingomyelin was significantly increased in Farber fibroblasts suggesting a delayed degradation of this compound in this ceramide storage disorder. We propose biosynthetical labeling of endogenous ceramide with [14C]serine, in addition to other established methods, as a highly sensitive and reliable method for the diagnosis of Farber disease, allowing semiquantitative measurement of ceramide accumulation in cultured skin fibroblasts of patients affected with Farber lipogranulomatosis.

Acid Ceramidase↗

Paracrine activation of the HIV-1 LTR promoter by the viral Tat protein is mechanistically similar to trans-activation within a cell.

The HIV-1 Tat protein activates transcription of the viral LTR promoter through interaction with the nuclear transcription machinery of the host cell. Tat can also activate the LTR promoter in a paracrine or inter-cellular manner by a yet unknown mechanism. One possibility is that Tat protein itself is secreted by cells and taken up by other cells. According to this mechanism, inter-cellular transcriptional activation by Tat should be very similar to intra-cellular trans-activation in Tat-producing cells. A large number of cytokine genes was recently reported to be Tat-responsive, raising the possibility that such cytokines and the corresponding cellular transduction pathways are involved in inter-cellular Tat action. The transcriptional events in such an indirect route are likely to differ from intra-cellular Tat action. To discriminate between a direct or indirect mechanism of inter-cellular Tat action, we compared the activity of a set of Tat mutants and different promoter constructs in inter-cellular and intra-cellular transcriptional activation. Identical results were obtained in both assays, suggesting that Tat protein itself is exported by one and transported into the nucleus of another cell. The demonstration that Tat antibodies specifically inhibit the inter-cellular route is also consistent with cell-to-cell transport of the Tat protein. Furthermore, we found that the second Tat coding exon, including the RGD motif that has been proposed to interact with an integrin receptor, is not required for cellular uptake of the Tat protein.

Cell Line↗

Interactions of 77Se and 13CO with nickel in the active site of active F420-nonreducing hydrogenase from Methanococcus voltae.

The selenium-containing F420-nonreducing hydrogenase from Methanococcus voltae was prepared in the Nia(I) middle dotCO state. The effect of illumination on this light-sensitive species was studied. EPR studies were carried out with enzyme containing natural selenium or with enzyme enriched in 77Se. Samples were prepared with either CO or 13CO. In the Nia(I) middle dotCO state, the nuclear spins of both 77Se (I = 1/2) and 13C (I = 1/2) interacted with the nickel-based unpaired electron, suggesting that they are positioned on opposite sites of the nickel ion. In the light-induced signal, the interaction with 13CO was lost. The 77Se nuclear spin introduced an anisotropic hyperfine splitting in both the dark and light-induced EPR signals. The data on the active enzyme of M. voltae are difficult to reconcile with the crystal structure of the inactive hydrogenase of Desulfovibrio gigas (Volbeda, A., Charon, M. H., Piras, C., Hatchikian, E. C., Frey, M., and Fontecilla Camps, J. C. (1995) Nature 373, 580-587) and suggest a structural change in the active site upon activation of the enzyme.

Binding Sites↗

Only sphingolipid activator protein B (SAP-B or saposin B) stimulates the degradation of globotriaosylceramide by recombinant human lysosomal alpha-galactosidase in a detergent-free liposomal system.

The degradation of globotriaosylceramide (GbO-se3Cer) by insect-cell derived recombinant human alpha-galactosidase (EC 3.2.1.22) was carried out in a detergent-free liposomal system in order to mimic intralysosomal conditions. GbOse3Cer incorporated into unilamellar liposomes was used as the substrate, and naturally occurring sphingolipid activator proteins, rather than detergents, were used to stimulate the enzyme reaction. The degradation of GbOse3Cer was dependent on the presence of both alpha-galactosidase and sphingolipid activator protein B (SAP-B or saposin B). It proceeded optimally at pH 4.6, and was enhanced by increasing amounts of both alpha-galactosidase (0.24-24 mU/50 microliters assay) and SAP-B (0-5 micrograms/50 microliters assay). The enzyme reaction was not affected by SAP-A, SAP-C, or SAP-D. Therefore, our results indicate that only SAP-B is essential for the degradation of GbOse3Cer by alpha-galactosidase.

Animals↗

A nascent micronuclear pseudogene in the ciliate Euplotes crassus.

The macronuclear genome of Euplotes crassus contains two different genes, EFA1 and EFA2, encoding EF-1alpha proteins. They are derived from micronuclear precursors in the course of a sexual process termed conjugation. We have found that two apparent micronuclear precursors exist for EFA1. They differ in their potential coding sequences and in the internal sequences interrupting the genes, which are normally removed during the processing from micronuclear into macronuclear genes. One of these genes is not processed into a macronuclear gene and has accumulated C-->T transitions in a limited section of the coding region. The gene obviously constitutes a recent duplication which has lost its ability to be processed into a macronuclear gene and has therefore become a micronuclear pseudogene. The true EFA1 precursor harbours a novel type of internal sequence in addition to a classical AT-rich IES. As usual, only one micronuclear EFA2 precursor gene was found. Its coding sequence is interrupted by a 79 bp TeIIES.

Amino Acid Sequence↗

Expression of the three alternative forms of the sphingolipid activator protein precursor in baby hamster kidney cells and functional assays in a cell culture system.

Sphingolipid activator proteins (SAPs) are non-enzymatic glycoproteins required for lysosomal degradation of various sphingolipids with short oligosaccharide chains by their respective exohydrolases. Four of these (SAP-A to SAP-D or saposins A to D) are derived from a common precursor by proteolytic processing. Alternative splicing of the SAP-precursor gene results in insertion of additional 6 or 9 bases of exon 8' or 8, respectively, into the SAP-B coding region of the transcribed mRNAs. To examine the features of the three different SAP-precursor proteins (prosaposins), the respective cDNAs were stably expressed in baby hamster kidney cells. Pulse-chase experiments with transfected cells and endocytosis studies on human fibroblasts showed that synthesis, transport, and maturation of all SAP-precursor led to formation of the four mature SAPs (SAP-A to SAP-D). In order to determine the biological function of the three different SAP-B isoforms, SAP-precursor-deficient human fibroblasts were loaded with recombinant SAP-precursor proteins with or without 2- and 3-amino acid insertions, respectively, purified from the medium of the baby hamster kidney cells. They were found to stimulate at nanomolar concentrations the turnover of biosynthetically labeled ceramide, glucosylceramide, and lactosylceramide. Since the physiological function of SAP-B is to stimulate the degradation of sulfatide by arylsulfatase A (EC 3.1.6.1) and globotriaosylceramide by beta-galactosidase (EC 3.2.1.23) loading studies with the respective exogenously labeled lipids on SAP-precursor-deficient fibroblasts were performed. Addition of different purified SAP-precursors to the medium of the lipid-loaded fibroblasts showed positive stimulation of the lipid degradation by all three SAP-B isoforms derived from the SAP-precursors. These findings establish that all three forms of the SAP-B can function as sulfatide/globotriaosylceramide activator.

Alternative Splicing↗

Two different macronuclear EF-1 alpha-encoding genes of the ciliate Euplotes crassus are very dissimilar in their sequences, copy numbers and transcriptional activities.

Genes (EFA) encoding the translation elongation factor EF-1 alpha (EFA) or the prokaryotic homolog EFTu frequently occur in multiple copies in the same organism. This has been interpreted either in terms of a potential of differential gene expression during different phases of development, or as gene dosage adaptation to the need of high-level production of the gene products. Since ciliates can differentially amplify their genes, the latter argument would lead to the expectation of only one EFA gene in the macronucleus. However, we have found two such genes which strongly differ in both copy number and codon usage. Both transcripts are detectable at very different levels. The expression of the genes takes place both in the vegetative and sexual phases, i.e.,during conjugation.

Amino Acid Sequence↗

Response of Bacillus subtilis spores to dehydration and UV irradiation at extremely low temperatures.

Spores of Bacillus subtilis have been exposed to the conditions of extreme dehydration (argon/silica gel; simulated space vacuum) for up to 12 weeks at 298 K and 80 K in the dark. The inactivation has been correlated with the production of DNA-double strand-breaks. The temperature-dependence of the rate constants for inactivation or production of DNA-double strand-breaks is surprisingly low. Controls kept in the frozen state at 250 K for the same period of time showed no sign of deterioration. In another series of experiments the spores have been UV irradiated (253.7 nm) at 298 K, 200 K and 80 K after exposure to dehydrating conditions for 3 days. Fluence-effect relationships for inactivation, production of DNA-double strand-breaks and DNA-protein cross-links are presented. The corresponding F37-values for inactivation and production of DNA lesions are significantly increased only at 80 K (factor of 4 to 5). The data indicate that the low temperatures that prevail in the outer parts of the Solar System or at the nightside of Mars or the Moon are not sufficiently low to crucially inhibit inactivation by dehydration. Our data place further constraints on the panspermia hypothesis.

Bacillus subtilis↗

Timing of differential amplification of macronucleus-destined sequences during macronuclear development in the hypotrichous ciliate Euplotes crassus.

The change in copy numbers of macronucleus-destined gene sequences was followed in anlagen DNA during postconjugational development in Euplotes crassus. As noted earlier, copy numbers increase during the polytene stage. During this replication process major differential amplification of different genes is not observed. Instead it is only achieved during or shortly after the fragmentation of the polytene chromosomes. This process is not totally synchronous with respect to different genes. Highly amplified genes are excised earlier than genes with a low final macronuclear copy number. Unexpectedly, the pattern of processing of the newly added oversized telomeres also appears to correlate with the degree of gene amplification. These observations are discussed in terms of a limited replication period after polytene chromosome fragmentation leading to preferential amplification of early excised genes.

Animals↗