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Biomedical subjects

Robert Zimmermann

Publications and source records attributed to Robert Zimmermann.

At least 37 records · Page 2Linked to original sources
PubMed2005

New insights into the interaction of ribosomal protein L1 with RNA.

The RNA-binding ability of ribosomal protein L1 is of profound interest, since L1 has a dual function as a ribosomal structural protein that binds rRNA and as a translational repressor that binds its own mRNA. Here, we report the crystal structure at 2.6 A resolution of ribosomal protein L1 from the bacterium Thermus thermophilus in complex with a 38 nt fragment of L1 mRNA from Methanoccocus vannielii. The conformation of RNA-bound T.thermophilus L1 differs dramatically from that of the isolated protein. Analysis of four copies of the L1-mRNA complex in the crystal has shown that domain II of the protein does not contribute to mRNA-specific binding. A detailed comparison of the protein-RNA interactions in the L1-mRNA and L1-rRNA complexes identified amino acid residues of L1 crucial for recognition of its specific targets on the both RNAs. Incorporation of the structure of bacterial L1 into a model of the Escherichia coli ribosome revealed two additional contact regions for L1 on the 23S rRNA that were not identified in previous ribosome models.

Amino Acid Sequence↗
PubMed2005

Obese yeast: triglyceride lipolysis is functionally conserved from mammals to yeast.

Storage and degradation of triglycerides are essential processes to ensure energy homeostasis and availability of precursors for membrane lipid synthesis. Recent evidence suggests that an emerging class of enzymes containing a conserved patatin domain are centrally important players in lipid degradation. Here we describe the identification and characterization of a major triglyceride lipase of the adipose triglyceride lipase/Brummer family, Tgl4, in the yeast Saccharomyces cerevisiae. Elimination of Tgl4 in a tgl3 background led to fat yeast, rendering growing cells unable to degrade triglycerides. Tgl4 and Tgl3 lipases localized to lipid droplets, independent of each other. Serine 315 in the GXSXG lipase active site consensus sequence of the patatin domain of Tgl4 is essential for catalytic activity. Mouse adipose triglyceride lipase (which also contains a patatin domain but is otherwise highly divergent in primary structure from any yeast protein) localized to lipid droplets when expressed in yeast, and significantly restored triglyceride breakdown in tgl4 mutants in vivo. Our data identify yeast Tgl4 as a functional ortholog of mammalian adipose triglyceride lipase.

Amino Acid Sequence↗
PubMed2005

The lipolytic proteome of mouse adipose tissue.

Hydrolysis of triacylglycerols and cholesteryl esters is a key event in energy homeostasis of animals. However, many lipolytic activities still await their molecular identification. Here we report on a novel tool for concomitant analysis of lipases in complex proteomes. Fluorescent activity tags mimicking lipid substrates were used to label the proteome of mouse adipose tissue. Analysis by two-dimensional gel electrophoresis and LC-MS/MS led to the identification of all known intracellular lipases as well as a number of novel candidates. One of them was recently shown to be involved in triacylglycerol mobilization in adipocytes and therefore named adipose triglyceride lipase. Functional characterization of expressed enzymes demonstrated that lipolytic and esterolytic activities could be well discriminated. Thus our results show the first map of the lipolytic proteome of mouse adipose tissue and demonstrate the general applicability of our method for rapid profiling and identification of lipolytic activities in complex biological samples.

4-Chloro-7-nitrobenzofurazan↗
PubMed2005

The lipid droplet enzyme Tgl1p hydrolyzes both steryl esters and triglycerides in the yeast, Saccharomyces cerevisiae.

Based on sequence homology to mammalian acid lipases, yeast reading frame YKL140w was predicted to encode a triacylglycerol (TAG) lipase in yeast and was hence named as TGL1, triglyceride lipase 1. A deletion of TGL1, however, resulted in an increase of the cellular steryl ester content. Fluorescently labeled lipid analogs that become covalently linked to the enzyme active site upon catalysis were used to discriminate between the lipase and esterase activities of Tgl1p. Tgl1p preferred single-chain esterase inhibitors over lipase inhibitors in vitro. Under assay conditions optimal for acid lipases, Tgl1p exhibited steryl esterase activity only and lacked any triglyceride lipase activity. In contrast, at pH 7.4, Tgl1p also exhibited TAG lipase activity; however, steryl ester hydrolase activity was still predominant. Tgl1p localized exclusively to lipid droplets which are the intracellular storage compartment of steryl esters and triacylglycerols in the yeast S. cerevisiae. In a tgl1 deletion mutant, the mobilization of steryl esters in vivo was delayed, but not abolished, suggesting the existence of additional enzymes involved in steryl ester mobilization.

Amino Acid Sequence↗
PubMed2005

Lipolysis: pathway under construction.

PURPOSE OF REVIEW: The lipolytic catabolism of stored fat in adipose tissue supplies tissues with fatty acids as metabolites and energy substrates during times of food deprivation. This review focuses on the function of recently discovered enzymes in adipose tissue lipolysis and fatty acid mobilization. RECENT FINDINGS: The characterization of hormone-sensitive lipase-deficient mice provided compelling evidence that hormone-sensitive lipase is not uniquely responsible for the hydrolysis of triacylglycerols and diacylglycerols of stored fat. Recently, three different laboratories independently discovered a novel enzyme that also acts in this capacity. We named the enzyme 'adipose triglyceride lipase' in accordance with its predominant expression in adipose tissue, its high substrate specificity for triacylglycerols, and its function in the lipolytic mobilization of fatty acids. Two other research groups showed that adipose triglyceride lipase (named desnutrin and Ca-independent phospholipase A2zeta, respectively) is regulated by the nutritional status and that it might exert acyl-transacylase activity in addition to its activity as triacylglycerol hydrolase. Adipose triglyceride lipase represents a novel type of 'patatin domain-containing' triacylglycerol hydrolase that is more closely related to plant lipases than to other known mammalian metabolic triacylglycerol hydrolases. SUMMARY: Although the regulation of adipose triglyceride lipase and its physiological function remain to be determined in mouse lines that lack or overexpress the enzyme, present data permit the conclusion that adipose triglyceride lipase is involved in the cellular mobilization of fatty acids, and they require a revision of the concept that hormone-sensitive lipase is the only enzyme involved in the lipolysis of adipose tissue triglycerides.

Adipose Tissue↗
PubMed2005

Mononuclear cell variability and recruitment in non-cytokine-stimulated donors after serial 10-liter leukapheresis procedures.

BACKGROUND: We introduced monitoring of mononuclear cell (MNC) counts to obtain enhanced donor control and a stable quality of MNC products, because there are limited data available about blood donors after serial leukapheresis (LP) procedures. STUDY DESIGN AND METHODS: In a prospective paired study, 13 male healthy blood donors underwent 10-L LP procedures performed on two apheresis devices by use of two MNC program settings (COBE Spectra, Gambro BCT, SF 250 vs. SF 500; and AS.TEC 204, Fresenius Hemocare, CP 129 vs. CP 194). Donors' pre- and postdonation MNC counts were analyzed by fluorescence-activated cell sorting. RESULTS: After each 10-L LP procedure, a transient decline (p < 0.05) of CD14+ monocyte and platelet counts appeared in donors. Loss of donors' CD3+ T cells, CD19+ B cells, and CD16+56+ natural killer (NK) cells during MNC collection was partly compensated by cell recruitment. The MNC recruitment factor (RF) seems to be higher with high-yield MNC program settings. Negative correlations (p < 0.01) were noticed between predonation counts and RFs of CD3+ T cells and CD16+56+ NK cells. Four serial 10-L LP procedures did not result in long lasting MNC depletion for donors. CONCLUSION: MNC recruitment seems to depend on MNC program settings and collected cell yields. Low MNC counts could result in high cell recruitment that may contribute to stable collection results to some degree. Nevertheless, there seems to be a considerable individual variation of MNC recruitment in donors that should be investigated in more detail.

Adult↗
PubMed2005

Plateletpheresis does not cause long-standing platelet-derived growth factor release into the donor blood.

BACKGROUND: Recently, long-standing elevations of soluble growth factors released from platelets (PLTs) after contact with artificial surfaces during dialysis were described. They could be jointly responsible for the high frequency of death from cardiovascular diseases in dialysis patients. There are no comparable data on the extent and the duration of a growth factor release by plateletpheresis procedures. STUDY DESIGN AND METHODS: A total of 37 plateletpheresis procedures were performed with two different devices. PLT-derived growth factor (PDGF) isoform AB, transforming growth factor (TGF)-beta1, and beta-thromboglobulin (beta-TG) were measured in the donors' plasma samples, and PLT activation and function were measured by cytometry and aggregometry before and after plateletpheresis and 1 and 24 hours later. RESULTS: Before apheresis, the following mean plasma levels were found: beta-TG, 98.6 +/- 37.3 IU per mL; PDGF-AB, 71.5 +/- 38.5 pg per mL; and TGF-beta1, 2.24 +/- 0.80 ng per mL. At the end of the apheresis procedures, the mean PDGF-AB level had increased by a factor of 1.8 (p < 0.05). One hour later, the mean PDGF-AB level had normalized again. No significant change in the levels of beta-TG and TGF-beta1 was found by the apheresis procedures. There was no influence of the blood cell separator type on the results. CONCLUSION: Only a slight and rapidly reversible increase in soluble PDGF-AB was found during plateletpheresis and no increase in soluble TGF-beta1 and beta-TG was found. This change should not be harmful to the donor.

Blood Donors↗
PubMed2005

Frequently used plateletpheresis techniques result in variable target yields and platelet recruitment of donors.

BACKGROUND: Standard plateletpheresis techniques and effects on platelet (PLT) donors were investigated to provide an informative basis for advancement of apheresis software. STUDY DESIGN AND METHODS: Three paired groups with 33 male and 22 female blood donors were prospectively investigated by analyzing blood counts of donors and products. Four apheresis platforms, the COBE Spectra LRS and the Trima v4 (Gambro BCT) and the AS.TEC204 and the COM.TEC (Fresenius Hemocare), were compared. Deviations of the collected from programmed PLT targets and donor PLT recruitment were calculated for single-unit PLT concentrates (SU-PCs; 3 x 10(11) PLTs) and double-unit PLT concentrates (DU-PCs; 6 x 10(11) PLTs). RESULTS: Regarding SU-PCs, the productivity of the COM.TEC machine was superior to the AS.TEC204 machine, because of shorter processing time (54 min vs. 67 min) and higher yields (2.90 x 10(11) PLTs vs. 2.75 x 10(11) PLTs). Compared to the Spectra machine, the Trima v4 machine showed higher collection efficiencies (CEs) and shorter processing time and complied better with the programmed target (SU-PCs, 3.24 x 10(11) PLTs vs. 3.70 x 10(11) PLTs; DU-PCs, 6.87 x 10(11) PLTs vs. 7.56 x 10(11) PLTs). Harvests of the Spectra machine (DU-PCs) exceeded the target by 40 percent, which resulted in high PLT loss for donors. A longer processing time resulted in some higher CEs (SU-PCs, 53%; DU-PCs, 58%), which could contribute to this result. PLT recruitment compensated PLT loss to some extent. CONCLUSION: The major finding was that the newer devices (COM.TEC and Trima) gave more predictable yields than the older devices (AS.TEC204 and Spectra) and resulted in lower PLT deficit. PLT software should be improved to minimize relevant variations of collected yields regarding the programmed target.

Adult↗
PubMed2005

Endothelial lipase provides an alternative pathway for FFA uptake in lipoprotein lipase-deficient mouse adipose tissue.

Lipoprotein lipase (LPL) is thought to be the only enzyme responsible for the catabolism of triglycerides (TGs) associated with TG-rich lipoproteins in adipose tissue (AT). However, LPL deficiency in humans and induced mutant mice is not associated with decreased fat mass. We investigated whether endothelial lipase (EL), a recently discovered phospholipase, might represent an alternative mechanism for the uptake of phospholipid-derived fatty acids in murine lipoprotein-deficient AT. When LPL was expressed in AT and isolated murine adipocytes, EL mRNA was not detectable. In contrast, mouse AT and isolated adipocytes that lacked LPL expressed large amounts of EL mRNA. The cellular phospholipase activity in LPL-deficient fat pads was increased 4-fold compared with control fat pads and could be inhibited to control levels by a specific EL antibody. Fatty acids produced by EL activity were absorbed by adipocytes and incorporated into the TG moiety of AT. Our results suggest that EL activity in AT and other peripheral tissues might contribute to the tissue uptake of free fatty acids, which could have important implications for the metabolism of plasma lipoproteins.

3T3-L1 Cells↗
PubMed2004

Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase.

Mobilization of fatty acids from triglyceride stores in adipose tissue requires lipolytic enzymes. Dysfunctional lipolysis affects energy homeostasis and may contribute to the pathogenesis of obesity and insulin resistance. Until now, hormone-sensitive lipase (HSL) was the only enzyme known to hydrolyze triglycerides in mammalian adipose tissue. Here, we report that a second enzyme, adipose triglyceride lipase (ATGL), catalyzes the initial step in triglyceride hydrolysis. It is interesting that ATGL contains a "patatin domain" common to plant acyl-hydrolases. ATGL is highly expressed in adipose tissue of mice and humans. It exhibits high substrate specificity for triacylglycerol and is associated with lipid droplets. Inhibition of ATGL markedly decreases total adipose acyl-hydrolase activity. Thus, ATGL and HSL coordinately catabolize stored triglycerides in adipose tissue of mammals.

3T3-L1 Cells↗
PubMed2004

Long-term results following pediatric distal forearm fractures.

INTRODUCTION: The purpose of this retrospective study was to investigate the frequency and extent of clinical and radiological late sequelae and to identify predicting factors. MATERIALS AND METHODS: A total of 220 patients of growing age with 232 closed, conservatively treated fractures were re-examined clinically and radiologically at a median follow-up time of 10 years (range 5-16 years). Clinical and radiological findings were summarised as an overall result. RESULTS: Of the total of patients, 19% reported pain in the injured wrist, and wrist mobility was limited in 5% of patients. Forearm rotation was decreased in 16%, primarily in epiphyseal separation of the ulna ( p=0.0033). Radial inclination was different in 6% of patients, palmar tilt in 2%, and ulnar variance in 37%, compared with the contralateral side. Ulnocarpal impaction syndrome was present in 75% of the patients with positive ulnar variance. Overall outcome was excellent in 72%, good in 19%, moderate in 6%, and poor in 3% of patients. The younger the children were at the time of injury, the more favourable the results were ( p=0.009). Children who were older than 10 years when they suffered a severe fracture dislocation had the poorest results ( p=0.008). Further factors having a negative influence on outcome were repeated reduction maneuvers and an additional fracture of the ulna. CONCLUSION: Our follow-up examinations showed that the majority of patients achieved good results, especially in children under 10 years old. Large dislocations at the time of fracture healing do not influence long-term results in this age group and thus can be tolerated. Patients over 10 years old, whose fractures healed with an angular deformity of more than 20 degrees and/or fragment dislocation over half the breadth of the shaft showed the poorest results. Thus, such dislocations should not be tolerated, and reduction should be attempted in this age group by only one reduction maneuver.

Adolescent↗
PubMed2004

Comparison of two mononuclear cell program settings on two apheresis devices intended to collect high yields of CD14+ and CD3+ cells.

BACKGROUND: In cancer and transplantation therapy apheresis devices and software of optimum standards are required for the collection of high cell yields with high purity of the desired cell fraction. STUDY DESIGN AND METHODS: In a paired study, 15 healthy blood donors underwent four 10-L leukapheresis procedures (197 +/- 33 min) with an inlet blood flow rate of 60 mL per minute by use of two different MNC program settings of the COBE Spectra (Gambro BCT) and the AS.TEC 204 (Fresenius Hemocare) cell separators. RESULTS: Use of the standard MNC program of both apheresis devices resulted in significantly higher (p < 0.01) collection efficiencies of CD14+ monocytes, CD3+ cells, CD4+ cells, CD8+ T cells, CD16+ CD56+ natural killer (NK) cells, and residual PLTs (p < 0.001), owing to higher centrifuge speed. The mean MNC purity of all components was more than 90 percent. By use of standard programs of either device, significant correlations (p < 0.01) between donor monocytes and preleukapheresis NK cell counts and the corresponding component cell yields were found. CONCLUSION: Compared to the program modifications with lower centrifuge velocities the standard MNC programs were significantly more efficient regarding CD14+, CD3+, and CD16+ CD56+ cells. Enhanced centrifuge speed and inlet blood flow rate in MNC programs resulted in higher, similar composed MNC concentrations of the products.

Adult↗
PubMed2004

Effects of platelet-rich plasma on bone healing in combination with autogenous bone and bone substitutes in critical-size defects. An animal experiment.

OBJECTIVES: An animal study was carried out to investigate the influence of platelet-rich plasma (PRP) on the regeneration of bony defects. MATERIAL AND METHODS: Critical-size defects in the forehead region of a mini-pig were filled with randomly distributed combinations of autogenous bone, tricalcium-phosphate granules (CeraSorb), bovine spongious blocks (BioOss) and a bovine bone inducing collagenous sponge (Colloss) with and without PRP in two preparations (Cusasan, 3i). The animals were killed after 2, 4 and 12 weeks. The specimens were evaluated microradiographically and immunohistologically. RESULTS: Autologous bone (38 +/- 9.9%) and Colloss (52.6 +/- 4.0%) showed the highest remineralization rates at 2 weeks. The initial high expression of BMP-2 in the Colloss-group gives evidence of an early initiation of bony regeneration. At 2 weeks PRP ad modum 3i was able to enhance bone healing significantly (P=0.028) only when applied in combination with autogenous bone (62.8 +/- 1.6%). Four weeks after surgery, both PRP preparations did no longer increase bony regeneration in the autogenous groups. The osteoconductive effect of Bio-Oss (38.7 +/- 5.5%) and CeraSorb (41+/-4.9%) was remarkable as well 4 weeks after surgery. Nevertheless, the addition of PRP hardly influenced bony regeneration, ceramic degradation or cytokine expression when bone substitutes were applied. At 12 weeks, the level of reossification had adjusted similarly in all groups. CONCLUSION: PRP did not add additional benefit when xenogenic bone substitutes were used. However, a significant effect on bone regeneration was found in the autogenous group initially when PRP is added.

Animals↗
PubMed2003

Remodeling capacity and functional outcome of palmarly versus dorsally displaced pediatric radius fractures in the distal one-third.

INTRODUCTION: The purpose of this retrospective study was to compare the remodeling capacity and functional outcome of palmarly and dorsally displaced pediatric radius fractures in the distal one-third. MATERIALS AND METHODS: Fifty-three children with a residual dorsal angulation of 15 degrees (range 10 degrees -28 degrees, +/-SD 5.32) and 31 children with a residual palmar angulation of 15 degrees (range 10 degrees -30 degrees, +/-SD 4.88) at fracture healing were re-examined clinically and radiologically with a median follow-up time of 10 years (range 5-15 years). RESULTS: There was no difference in the remodeling capacity, palmar tilt, radial inclination, and ulnar variance between both groups at follow-up. Pain as well as flexion/extension of the wrist and pronation showed no difference in both groups. Palmarly displaced fractures showed a significantly higher restriction of supination ( p=0.01). CONCLUSION: We conclude that remodeling of residual palmar angulation occurs to the same extent as it does in dorsal angulation. Functional outcome differs in forearm supination.

Adolescent↗
PubMed2003

Class B scavenger receptors CD36 and SR-BI are receptors for hypochlorite-modified low density lipoprotein.

The presence of HOCl-modified epitopes inside and outside monocytes/macrophages and the presence of HOCl-modified apolipoprotein B in atherosclerotic lesions has initiated the present study to identify scavenger receptors that bind and internalize HOCl-low density lipoprotein (LDL). The uptake of HOCl-LDL by THP-1 macrophages was not saturable and led to cholesterol/cholesteryl ester accumulation. HOCl-LDL is not aggregated in culture medium, as measured by dynamic light scattering experiments, but internalization of HOCl-LDL could be inhibited in part by cytochalasin D, a microfilament disrupting agent. This indicates that HOCl-LDL is partially internalized by a pathway resembling phagocytosis-like internalization (in part by fluid-phase endocytosis) as measured with [14C]sucrose uptake. In contrast to uptake studies, binding of HOCl-LDL to THP-1 cells at 4 degrees C was specific and saturable, indicating that binding proteins and/or receptors are involved. Competition studies on THP-1 macrophages showed that HOCl-LDL does not compete for the uptake of acetylated LDL (a ligand to scavenger receptor class A) but strongly inhibits the uptake of copper-oxidized LDL (a ligand to CD36 and SR-BI). The binding specificity of HOCl-LDL to class B scavenger receptors could be demonstrated by Chinese hamster ovary cells overexpressing CD36 and SR-BI and specific blocking antibodies. The lipid moiety isolated from the HOCl-LDL particle did not compete for cell association of labeled HOCl-LDL to CD36 or SR-BI, suggesting that the protein moiety of HOCl-LDL is responsible for receptor recognition. Experiments with Chinese hamster ovary cells overexpressing scavenger receptor class A, type I, confirmed that LDL modified at physiologically relevant HOCl concentrations is not recognized by this receptor.

Animals↗
PubMed2003

Decreased fatty acid esterification compensates for the reduced lipolytic activity in hormone-sensitive lipase-deficient white adipose tissue.

It has been observed previously that hormone-sensitive lipase-deficient (HSL-ko) mice have reduced white adipose tissue (WAT) stores compared to control mice. These findings contradict the expectation that the decreased lipolytic activity in WAT of HSL-ko mice would cause accumulation of triglycerides (TGs) in that tissue. Here we demonstrate that the cellular TG synthesis in HSL-deficient WAT is markedly reduced due to downregulation of the enzymatic activities of glycerophosphate acyltransferase, dihydroxyacetonphosphate acyltransferase, lysophosphatidate acyltransferase, and diacylglycerol acyltransferase. Fatty acid de novo synthesis is also decreased due to reduced cellular glucose uptake, reduced glucose incorporation into adipose tissue lipids, and reduced activities of acetyl:CoA carboxylase and fatty acid synthase. Finally, the activities of phosphoenolpyruvate carboxykinase (PEPCK), acyl:CoA synthetase (ACS), and glucose 6-phosphate dehydrogenase, the enzymes that provide glycerol-3-phosphate, acyl-CoA, and NADPH for TG synthesis, respectively, are decreased in HSL-ko mice. The reduced expression of the peroxisome proliferator-activated receptor gamma (PPAR gamma) target genes PEPCK, ACS, and aP2, as well as reduced mRNA levels of PPAR gamma itself, suggest the involvement of this transcription factor in the downregulation of lipogenesis. Taken together, these results establish that in the absence of HSL, the reduced NEFA production is counteracted by a drastic reduction of NEFA reesterification that provides sufficient quantities of NEFA for release into the circulation. These metabolic adaptations result in decreased fat mass in HSL-ko mice.

Acyltransferases↗