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In the nervous system, NMDA receptors (NMDARs) participate in neurotransmission and modulate the viability of neurons. In contrast, little is known about the role of NMDARs in pancreatic islets and the insulin-secreting beta cells whose functional impairment contributes to diabetes mellitus. Here we found that inhibition of NMDARs in mouse and human islets enhanced their glucose-stimulated insulin secretion (GSIS) and survival of islet cells. Further, NMDAR inhibition prolonged the amount of time that glucose-stimulated beta cells spent in a depolarized state with high cytosolic Ca(2+) concentrations. We also noticed that, in vivo, the NMDAR antagonist dextromethorphan (DXM) enhanced glucose tolerance in mice, and that in vitro dextrorphan, the main metabolite of DXM, amplified the stimulatory effect of exendin-4 on GSIS. In a mouse model of type 2 diabetes mellitus (T2DM), long-term treatment with DXM improved islet insulin content, islet cell mass and blood glucose control. Further, in a small clinical trial we found that individuals with T2DM treated with DXM showed enhanced serum insulin concentrations and glucose tolerance. Our data highlight the possibility that antagonists of NMDARs may provide a useful adjunct treatment for diabetes. more...

Aim: We hypothesized that 4 days of normal daily activity after 21 days of experimental bed rest (BR) will not reverse BR induced impaired glucose tolerance., Design: Glucose tolerance of seven male, healthy, untrained test subjects (age: 27.6 (3.3) years (mean (SD)); body mass: 78.6 (6.4) kg; height: 1.81 (0.04)  m; VO2 max: 39.5 (5.4) ml/kg body mass/min) was studied. They stayed twice in the metabolic ward (crossover design), 21 days in bed and 7 days before and after BR each. Oral glucose tolerance tests were applied before, on day 21 of BR, and 5 and 14 days after BR., Results: On day 21 of BR, AUC(120 min) of glucose concentration was increased by 28.8 (5.2)% and AUC(120 min) of insulin by 35.9 (10.2)% (glucose: P < 0.001; insulin: P = 0.02). Fourteen days after BR, AUC(120 min) of serum insulin concentrations returned to pre-bed-rest concentrations (P = 0.352) and AUC(120 min) of glucose was still higher (P = 0.038). Insulin resistance did not change, but sensitivity index was reduced during BR (P = 0.005)., Conclusion: Four days of light physical workload does not compensate inactivity induced impaired glucose tolerance. An individually tailored and intensified training regime is mandatory in patients being in bed rest to get back to normal glucose metabolism in a reasonable time frame. more...

Objective: Unrestricted somatic stem cells (USSCs) were successfully identified from human cord blood. However, the efficacy of USSC transplantation for improving left ventricular (LV) function post myocardial infarction (MI) is still controversial., Methods and Results: PBS, 1x10(6) human fibroblasts (Fbr), 1x10(5) USSCs (LD), or 1x10(6) USSCs (HD) were transplanted intramyocardially 20 minutes after ligating the LAD of nude rats. Echocardiography and a microtip conductance catheter at day 28 revealed a dose-dependent improvement of LV function after USSC transplantation. Necropsy examination revealed dose-dependent augmentation of capillary density and inhibition of LV fibrosis. Dual-label immunohistochemistry for cardiac troponin-I and human nuclear antigen (HNA) demonstrated that human cardiomyocytes (CMCs) were dose-dependently generated in ischemic myocardium 28 days after USSC transplantation. Similarly, dual-label immunostaining for smooth muscle actin and class I human leukocyte antigen or that for von Willebrand factor and HNA also revealed a dose-dependent vasculogenesis after USSC transplantation. RT-PCR indicated that expression of human-specific genes of CMCs, smooth muscle cells, and endothelial cell markers in infarcted myocardium were significantly augmented in USSC-treated animals compared with control groups., Conclusions: USSC transplantation leads to functional improvement and recovery from MI and exhibits a significant and dose-dependent potential for concurrent cardiomyogenesis and vasculogenesis. more...

Valproic acid (VPA) is a histone deacetylase inhibitor previously shown to promote the proliferation and self-renewal of CD34(+) hematopoietic cells. We tested the effect of VPA in conjunction with the selective amplification technology developed by Viacell Inc. Stem cells enriched from frozen cord blood were cultured for 7 d, subjected to reselection and grown in fresh medium for a further 7 d. Treatment with VPA resulted in an average two-fold higher expansion of CD45(+)34(+) cells compared with control. Furthermore, VPA-treatment induced higher numbers of CD45(+)34(+) cells to reside in the S phase than control cultured cells and resulted in a 2.5-fold upregulation in HOXB4 expression. Importantly, VPA-treated cells reconstituted hematopoiesis in non-obese diabetic/severe combined immunodeficient mice with a six-fold higher efficiency than control cells. Collectively, our results indicate that VPA, already used clinically for neurologic disorder treatment, is a useful additive for the ex vivo culture of hematopoietic stem/progenitor cells to enhance engraftment efficiency. more...

Regeneration of infarcted myocardium by injecting stem cells has been proposed to prevent heart failure. We studied the i.c. administration of human umbilical cord blood stem cells (USSC) in a porcine model of myocardial infarction (MI) and reperfusion. In 15 swine, MI was induced by balloon-occlusion of the left circumflex coronary artery (LCX) for 2 h followed by reperfusion. Five swine served as healthy controls. One week later, magnetic resonance imaging (MRI) was performed to assess left ventricular (LV) function and infarct size. Then, under immune suppression, 6 of the 12 surviving MI swine received intracoronary injection of approximately 10(8) human USSC in the LCX while the other MI-swine received medium. Four weeks later all swine underwent follow-up MRI, and were sacrificed for histology. One week after MI, end-diastolic volume (92+/-3 mL) and LV mass (75+/-2 g) were larger, while ejection fraction (42+/-2%) was smaller than in healthy control (68+/-3 mL, 66+/-3 g and 55+/-3%, all P<0.05). Regional wall thickening (-7+/-2%) in the LCX area became akinetic. No difference in global and regional LV function at 5 weeks was observed between MI animals receiving USSC or medium. Infarct size after USSC treatment was significantly larger (20+/-3 g vs. 8+/-2 g, P<0.05). USSC survived only in the infarct border zone at 5 weeks and did not express cardiomyocyte or endothelial markers. Histology showed that intracoronary injection of USSC caused micro infarctions by obstructing blood vessels. In swine with a 1 week old MI, injection of USSC via the intracoronary route does not improve LV function 4 weeks later. more...

Mesenchymal stem cells have been implicated as playing an important role in stem cell engraftment. Recently, a new pluripotent population of umbilical cord blood (UCB) cells, unrestricted somatic stem cells (USSCs), with intrinsic and directable potential to develop into mesodermal, endodermal, and ectodermal fates, has been identified. In this study, we evaluated the capacity of ex vivo expanded USSCs to influence the homing of UCB-derived CD34(+) cells into the marrow and spleen of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. USSCs induced a significant enhancement of CD34(+) cell homing to both bone marrow and spleen (2.2 +/- 0.3- and 2.4 +/- 0.6-fold, respectively; p < .05), with a magnitude similar to that induced by USSCs that had been thawed prior to transplantation. The effect of USSCs was dose-dependent and detectable at USSC:CD34(+) ratios of 1:1 and above. Enhanced marrow homing by USSCs was unaltered by extensive culture passaging of the cells, as similar enhancement was observed for both early-passage (passage 5 [p5]) and late-passage (p10) USSCs. The homing effect of USSCs was also reflected in an increased proportion of NOD/SCID mice exhibiting significant human cell engraftment 6 weeks after transplantation, with a similar distribution of myeloid and lymphoid components. USSCs enhanced the homing of cellular products of ex vivo expanded UCB lineage-negative (lin(-)) cells, generated in 14-day cultures by Selective Amplification. The relative proportion of homing CD34(+) cells within the culture-expanded cell population was unaltered by USSC cotransplantation. Production of stromal-derived factor-1 (SDF-1) by USSCs was detected by both gene expression and protein released into culture media of these cells. Knockdown of SDF-1 production by USSCs using lentiviral-SiRNA led to a significant (p < .05) reduction in USSC-mediated enhancement of CD34(+) homing. Our findings thus suggest a clinical potential for using USSCs in facilitating homing and engraftment for cord blood transplant recipients. more...

Local strategies directed against vascular smooth muscle cell (VSMC) proliferation such as drug-eluting stents reduce the occurrence of restenosis. However, these approaches may also inhibit endothelial cell (EC) proliferation and, thus, impair reendothelialization. We compared the effects of tacrolimus on human VSMC and EC proliferation and migration to sirolimus, a compound with similar molecular structure. Thymidine incorporation was determined in growth factor-stimulated VSMC and EC. The drug concentration at which maximal VSMC proliferation was inhibited by 50% (IC50) was about 10-fold higher for tacrolimus (3.8 x 10 M) than for sirolimus (4.1 x 10 M; P = 0.055). It is interesting that the molar IC50 value in EC was around 10-fold higher for tacrolimus (2.3 x 10 M) than for sirolimus (7.1 x 10 M; P < 0.01). The profile of these drugs on VSMC and EC migration was similar to the one found in the proliferation assays. Inhibition of VSMC proliferation by both tacrolimus and sirolimus was associated with upregulation of the cell-cycle inhibitor p27. Thus, tacrolimus is less potent than sirolimus for inhibiting VSMC proliferation or migration. However, tacrolimus exerts markedly less antiproliferative effects on EC compared with sirolimus. In combination with its potent antiinflammatory effects, tacrolimus may represent a promising compound for the use in drug-eluting stents. more...

In-stent restenosis remains an unresolved problem which occurs in 5-20% of patients undergoing coronary stenting within the first 3-6 months. Neointimal formation is the main contributor to in-stent restenosis. Stent-induced arterial injury and peri-strut inflammation are involved in the process of neointimal formation by activating cytokines and growth factors which induce smooth muscle cell dedifferentiation, migration, and proliferation. Histopathological studies found that neointimal hyperplasia is principally composed of smooth muscle cells, inflammatory cells, and extracellular matrix. Stent-based delivery of anti-proliferative and/or anti-inflammatory agents have shown beneficial effects on neointimal hyperplasia in experimental studies and clinical trials. Tacrolimus (FK506) is a water-insoluble macrolide immunosuppressant discovered in 1984. It has been widely used in reducing the incidence and severity of allograft rejection after organ transplantation. It has also been used to treat other inflammatory conditions such as atopic dermatitis. In this study, we evaluated the efficacy of stent-based delivery of tacrolimus on inflammation and neointimal formation in an overstretched coronary stent model. more...

Polymer stent coatings may not be suitable for drug elution because of inherent proinflammatory effects. A previous study suggested a beneficial effect of a stent eluting tacrolimus from a nanoporous ceramic aluminum oxide coating in a rabbit restenosis model. We investigated whether this stent is effective in preventing in-stent restenosis in a porcine restenosis model. Thirty-four juvenile swine underwent balloon overstretch injury and were subjected to implantation of either stainless steel (bare) stents, bare stents coated with nanoporous aluminum oxide alone, and coated stents eluting 50 and 180 mug of tacrolimus (FK506). In-stent restenosis was quantified at 1 and 3 months after stent placement by histomorphometry. A significant increase of neointimal hyperplasia was noted with the stents coated with aluminum oxide alone compared with bare stents (2.92 +/- 1.02 and 1.38 +/- 0.51 mm(2), respectively; P < 0.02). In all arteries containing coated stents, particle debris was found in the media and neointima, resulting in augmented vascular inflammation. In the group of stents coated with aluminum oxide, FK506 elution at a dose 180 mug reduced neointimal hyperplasia vs. no drug elution (1.66 +/- 0.49 vs. 2.92 +/- 1.02 mm(2); 180 mug vs. ceramic alone; P < 0.03). At a dose of 50 mug stent-based delivery of FK506, no reduction of neointimal hyperplasia was found (2.88 +/- 1.31 and 2.92 +/- 1.02 mm(2), respectively; P = NS; FK506 vs. ceramic alone). In summary, particle debris shed from a drug-eluting aluminum oxide coating of a stainless steel stent counteracts potential antiproliferative effects of stent-based tacrolimus delivery in a porcine model of restenosis. We propose that stent coatings eluting drugs need to be routinely tested for being tightly anchored into the stent surface. Alternatively, omission of any coating used as a drug reservoir may eliminate inflammatory particle debris after placement of drug-eluting stents. more...

To overcome the problem of in-stent restenosis, the concept of local delivery of antiproliferative or immunosuppressive drugs has been introduced into interventional cardiology. Local drug delivery can be achieved by drug-eluting stents coated with polymer surfaces used for controlled drug release. However, several polymer coatings have shown an induction of inflammatory response and increased neointima formation. In the present study, the effect of a new inorganic ceramic nanoporous aluminum oxide (Al(2)O(3)) coating on neointima proliferation and its suitability as a carrier for the immunosuppressive drug tacrolimus have been investigated. 316 L stainless steel coronary stents were coated with a 500 nm thin nanoporous aluminum oxide layer. This ceramic nanolayer was used as a carrier for tacrolimus. Bare stents (n = 6), ceramic coated stents (n = 6), and ceramic coated stents loaded with 60 (n = 7) and 120 mug (n = 6) tacrolimus were implanted in the common carotid artery of New Zealand rabbits. The ceramic coating caused no significant reduction of neointimal thickness after 28 days. Loading the ceramic stents with tacrolimus led to a significant reduction of neointima thickness by 52% for 60 mug (P = 0.047) and 56% for 120 mug (P = 0.036) as compared to the bare stents. The ceramic coating alone as well as in combination with tacrolimus led to a reduced infiltration of lymphocytes and macrophages in the intima in response to stent implantation. Ceramic coating of coronary stents with a nanoporous layer of aluminum oxide in combination with tacrolimus resulted in a significant reduction in neointima formation and inflammatory response. The synergistic effects of the ceramic coating and tacrolimus suggest that this new approach may have a high potential to translate into clinical benefit., (Copyright 2003 Wiley-Liss, Inc.) more...

Background: Since its introduction in clinical cardiology, several studies have shown the superiority of coronary stent implantation as compared to conventional angioplasty. However, restenosis still remains a major drawback of this new technique. Basic research in animal models could identify stent-related factors like stent-material and stent-design as major determinants of intima proliferation. Since materials with good biocompatibility often have unsuitable mechanical properties and vice versa, the concept of stent coating has been developed to allow the combination of favorable characteristics from different materials., Passive Coating: In general, passive coatings, which only serve as a barrier between the stainless steel and the tissue, and active coatings, which directly interfere with the process of intima proliferation have been identified. Currently there are several passive coatings commercially available with good results in animal models and preliminary reports from clinical studies., Active Coating: As any surface induces some kind of tissue reaction promoting restenosis, an active stent coating with antiproliferative drugs has been proposed. However, while animal studies revealed convincing results, preliminary clinical studies not only showed active stent coating effective in preventing restenosis, but also demonstrated the potential risks of this new approach. Although this technique may harbor some specific risks, with the introduction of stent coating a new chapter of interventional cardiology has been flipped open. more...

Three new polypeptides were isolated from the venom of the Thailand cobra Naja kaouthia and their amino-acid sequences determined. They consist of 65-amino-acid residues and have four disulfide bridges. A comparison of the amino-acid sequences of the new polypeptides with those of snake toxins shows that two of them (MTLP-1 and MTLP-2) share a high degree of similarity (55-74% sequence identity) with muscarinic toxins from the mamba. The third polypeptide (MTLP-3) is similar to muscarinic toxins with respect to the position of cysteine residues and the size of the disulfide-confined loops, but shows less similarity to these toxins (30-34% sequence identity). It is almost identical with a neurotoxin-like protein from Bungarus multicinctus (TrEMBL accession number Q9W727), the sequence of which has been deduced from cloned cDNA only. The binding affinities of the isolated muscarinic toxin-like proteins towards the different muscarinic acetylcholine receptor (mAChR) subtypes (m1-m5) was determined in competition experiments with N-[3H]methylscopolamine using membrane preparations from CHO-K1 cells, which express these receptors. We found that MTLP-1 competed weakly with radioactive ligand for binding to all mAChR subtypes. The most pronounced effect was observed for the m3 subtype; here an IC50 value of about 3 microM was determined. MTLP-2 had no effect on ligand binding to any of the mAChR subtypes at concentrations up to 1 microM. MTLP-1 showed no inhibitory effect on alpha-cobratoxin binding to the nicotinic acetylcholine receptor from Torpedo californica at concentrations up to 20 microM. more...

The present study was conducted to characterise the centrally active analgesic drug tramadol hydrochloride [(1RS,2RS)-2-[(dimethyl-amino)-methyl]-1-(3-methoxyphenyl)-cyclohe xanol hydrochloride] and its metabolites M1, M2, M3, M4 and M5 at the cloned human mu-opioid receptor. Membranes from stably transfected Chinese hamster ovary (CHO) cells were used to determine the four parameters of the ligand-receptor interaction: the affinity of (+/-)-tramadol and its metabolites was determined by competitive inhibition of [3H]naloxone binding under high and low salt conditions. The agonist-induced stimulation of [35S]GTPgammaS binding permits the measurement of potency (EC50), efficacy (Emax = maximal stimulation) and relative intrinsic efficacy (effect as a function of receptor occupation). The metabolite (+)-M1 showed the highest affinity (Ki=3.4 nM) to the human mu-opioid receptor, followed by (+/-)-M5 (Ki=100 nM), (-)-M1 (Ki=240 nM) and (+/-)-tramadol (Ki=2.4 microM). The [35S]GTPgammaS binding assay revealed an agonistic activity for the metabolites (+)-M1, (-)-M1 and (+/-)-M5 with the following rank order of intrinsic efficacy: (+)-M1>(+/-)-M5>(-)-M1. The metabolites (+/-)-M2, (+/-)-M3 and (+/-)-M4 displayed only weak affinity (Ki> 10 microM) and had no stimulatory effect on GTPgammaS binding. These data indicate that the metabolite (+)-M1 is responsible for the mu-opioid-derived analgesic effect. more...

The role of the opioid-like receptor 1 (ORL1) and its endogenous ligand, nociceptin/orphanin FQ (N/OFQ), in nociception, anxiety, and learning remains to be defined. To allow the rapid identification of agonists and antagonists, a reporter gene assay has been established in which the ORL1 receptor is functionally linked to the cyclic AMP-dependent expression of luciferase. N/OFQ and N/OFQ(1-13)NH(2) inhibited the forskolin-induced luciferase gene expression with IC(50) values of 0.81 +/- 0.5 and 0.87 +/- 0.16 nM, respectively. Buprenorphine was identified as a full agonist at the ORL1 receptor with an IC(50) value of 8.4 +/- 2.8 nM. Fentanyl and 7-benzylidenenaltrexone displayed a weak agonistic activity. The ORL1 antagonist [Phe(1)Psi(CH(2)-NH)Gly(2)]N/OFQ((1-13))NH(2) clearly behaved as an agonist in this assay with an IC(50) value of 85 +/- 47 nM. Thus, there is still a need for antagonistic tool compounds that might help to elucidate the neurophysiological role of N/OFQ. more...

The synthesis of 5'-substituted thalidomide analogs is described. The amino acids 2 necessary to synthesize the target compounds were prepared by Michael reaction. Condensation of 2 with phthalic anhydrides followed by reaction with urea yielded 4 as diastereomeric mixtures. Furthermore glutethimide (5) was brominated by an improved method and the resulting compound 6 was reacted in several steps with sodium azide, hydrogen, and phthalic anhydride to give 8. In a similar manner, 6 was reacted with sodium azide and various phthalic anhydrides to give 9, 10, and 11. All final compounds were tested in vitro for their inhibitory activity on the release of TNF-alpha, using stimulated peripheral mononuclear blood cells (PBMCs). Compounds with an additional aromatic substituent in position 5' of the thalidomide molecule were more active than thalidomide. Compound 11 was able to reduce increased levels of IL-2 in vitro. more...

The chimaeric molecule rscu-PA-40 kDA/Hir (M23) comprises the kringle and protease domain of saruplase (rscu-PA) and a thrombin inhibitory domain fused to the C-terminus of the protease domain. The 27 amino cid long thrombin inhibitory domain contains a sequence directed to the active site of thrombin and a fragment from the C-terminal region of hirudin. 125I-radiolabelled M23 (0.03 microM) bound to thrombin that was immobilised onto CNBr-activated sepharose beads. Unlabelled M23 (0.01-10 microM) and hirudin (0.001-10 microM) concentration-dependently displaced 125I-M23 from its binding to thrombin. Saruplase (up to 10 microM) did not influence the thrombin binding of M23. The fibrinolytic properties of M23 and saruplase were compared in anaesthetized dogs with femoral artery and saphenous vein thrombosis. Under concomitant heparinization, the intravenous bolus injections of 1 mg/kg M23 or saruplase induced reperfusion of thrombotically occluded femoral arteries in 4 out of 5 treated animals in each case. There was one reocclusion in the M23-treated group. Time to reperfusion (23 +/- 4 vs 25 +/- 11 min) and maximal height of reperfusion blood flow (98 +/- 21 vs 108 +/- 15% of baseline flow) did not differ significantly between the treatment groups. The time course of the lysis of incorporated 125I-fibrin radioactivity in thrombosed saphenous-veins was similar after bolus injections of M23 and saruplase. The maximal dissolution of 125I-fibrin in the venous thrombosis model was 91 +/- 1% in M23- and 88 +/- 5% in saruplase-treated animals. Plasma levels of fibrinogen were not influenced and alpha 2-antiplasmin levels were slightly reduced (-27 +/- 3%) after bolus injection of M23. In contrast, bolus injection of saruplase was accompanied by a significant decrease of fibrinogen (-55 +/- 19%) and alpha 2-antiplasmin (-75 +/- 11%) plasma levels. Template bleeding times virtually did not differ before (2.8 +/- 0.3 min) and 60 min after bolus injection of M23 (3.1 +/- 0.3 min), whereas treatment with saruplase resulted in a significant prolongation of template bleeding time from 2.6 +/- 0.2 min to 28 +/- 13 min. It is concluded that the saruplase derivative M23, while inducing equieffective thrombolysis after intravenous bolus injection in dogs, causes much fewer haemostatic side effects than its parent molecule. The high thrombus-specific activity of M23 is tentatively attributed to its affinity to clot-bound thrombin. more...

Tissue-type plasminogen activator (rt-PA) and prourokinase (rscu-PA) have been tested with respect to the influence of amyloid beta peptides on plasminogen activation which was monitored by cleavage of the chromogenic plasmin substrate S-2251. It was shown that rt-PA is stimulated by amyloid beta peptides at concentrations of 10 micrograms/ml in contrast to prourokinase, which does not alter its catalytic properties in presence of amyloid beta peptides. The stimulation of rt-PA can be inhibited by tranexamic acid indicating a molecular mode of stimulation similar to the fibrin mediated stimulation of rt-PA. more...

The blood clotting enzyme thrombin plays a central role in the aetiology of occlusive disorders such as stroke and acute myocardial infarction. During fibrinolytic therapy with plasminogen activators, thrombin is neutralized by anticoagulative drugs. In order to combine plasminogen-activating and thrombin-inhibitory activities we constructed chimeric derivatives of recombinant single-chain, urokinase-type plasminogen activator (rscu-PA) which comprise the kringle and protease domain of rscu-PA fused via a linker sequence to a thrombin-inhibitory domain. The inhibitory domain contains a sequence element directed to the active site of thrombin and a sequence taken from either hirudin or the human thrombin receptor both binding to the fibrinogen recognition site of thrombin. Analysing different sets of point mutants showed that the linker between the protease domain and the active site-directed sequence is contributing significantly to the thrombin-inhibitory potential. Kinetic analysis of thrombin inhibition revealed that most of the chimeras tested competitively inhibit the thrombin-mediated cleavage of a peptide substrate in a concentration-dependent manner; however, in two examples the insertion of one glycine residue into the active site directed-sequence abolished the blockade of the active site. This supports the conclusion that the chimeras with high thrombin-inhibitory potential interact with the active site and the fibrinogen recognition site of thrombin. more...

The question whether the immunomodulating activity of rac-thalidomide resides in either the (-)-(S)- or the (+)-(R)-enantiomer was addressed by synthesis and separation of pure enantiomers of thalidomide analogues which carry a methyl-group at the asymmetric carbon atom and are thus prevented from racemization. The effect of the pure enantiomers of the thalidomide-analogues and also of the enantiomers of thalidomide on release of TNF-alpha was tested in vitro by using stimulated peripheral mononuclear blood cells. Both enantiomers of thalidomide inhibited the release of TNF-alpha equally well at low concentrations (5 and 12.5 micrograms/ml) but at higher concentrations (25 and 50 micrograms 50 micrograms/ml) there was a weak but statistically significant selectivity towards the (-)-(S)-enantiomer. In the case of the configuration-stable thalidomide-analogues there was a very pronounced and statistically significant enantioselectivity towards the (S)-form even at lower concentrations (> or = 5 micrograms/ml). The (S)-enantiomers of the thalidomide-analogues differed in their inhibitory potency from (-)-(S)-thalidomide suggesting that the introduction of the methyl-group increases the TNF-alpha-inhibitory activity while the reduction of one of the carbonyl-functions in the glutarimide-moiety to a methylene-group decreases activity. The effect of these small molecular alterations on activity and the enantioselectivity towards the (S)-enantiomers may indicate that thalidomide and its analogues directly interact with one or several cellular target-proteins. more...

A chimeric protein (rscu-PA-40-kDa/Hir), consisting of the C-terminal amino acids 53-65 of hirudin (Hir), fused via a 14-amino-acid linker sequence to the C-terminal of a 40-kDa fragment (Ser47-Leu411) of recombinant (r) single-chain (sc) urokinase-type plasminogen activator (rscu-PA), was produced by expression of the corresponding chimeric cDNA in Escherichia coli cells. The thrombin inhibitory potential of purified rscu-PA-40-kDa/Hir was confirmed by complete inhibition of the coagulant activity of thrombin at 20-30-fold molar excess of the chimera, and by the resistance of rscu-PA-40-kDa/Hir to proteolytic cleavage by thrombin, rscu-PA-40-kDa/Hir prolonged the thrombin time of normal human plasma in a dose-dependent way (reduction of the apparent thrombin concentration to 50% with 95 nM chimeric protein as compared to 4.7 nM hirudin), and inhibited thrombin-mediated platelet aggregation (reduction of the apparent thrombin concentration to 50% with 40 nM chimeric protein). The chimera had a specific activity on fibrin films of 57,000 IU/mg as compared to 95,000 IU/mg for rscu-PA. The urokinase-like amidolytic activity of the single-chain protein was only 220 IU/mg but increased to 169,000 IU/mg after treatment with plasmin, which resulted in quantitative conversion to a two-chain (tc) derivative (rtcu-PA-40-kDa/Hir). Corresponding values for rscu-PA were 270 and 226,000 IU/mg. The catalytic efficiencies for plasmin-mediated conversion to two-chain molecules were comparable for rscu-PA-40-kDa/Hir and rscu-PA (0.63 and 0.65 microM-1.s-1, respectively). The plasminogen-activating potential of the single-chain chimera was comparable to that of rscu-PA; the catalytic efficiencies for plasminogen activation by their two-chain counterparts were also similar (0.55 and 0.73 microM-1.s-1, respectively). In 2 h, 50% lysis of 125I-fibrin-labeled clots prepared from platelet-poor human plasma and immersed in normal plasma was obtained with 1.3 micrograms/ml rscu-PA-40-kDa/Hir and with 0.67 micrograms/ml rscu-PA, with corresponding residual fibrinogen levels of 74% and 87%, respectively. In the absence of fibrin, 50% fibrinogenolysis in 2 h in normal human plasma required 2.1 micrograms/ml rscu-PA, but 7.9 micrograms/ml rscu-PA-40-kDa/Hir. Thus, the chimera rscu-PA-40-kDa/Hir has maintained the specific fibrinolytic and plasminogen activating activity of rscu-PA as well as its fibrinolytic potency in plasma, whereas it displayed a similar or somewhat better fibrin specificity.(ABSTRACT TRUNCATED AT 250 WORDS) more...

Three decades of immunological investigations using thalidomide are reviewed. Both in vitro and in vivo investigations are in accordance with the clinical finding that thalidomide does not impede T-cell competence in the control of infection by mycobacteriae. The term immunosuppressant does not apply. The immunomodulatory effects of thalidomide are evident in a myriad of phenomenological changes, and a molecularly defined common denominator of these activities is not known at present. Critical assessment with the objective to account for the clinical activity of thalidomide in specific human diseases leads to a focus on effects of thalidomide on phagocytic leukocytes and endothelia. The former are responsive to thalidomide by modulation of cytokine synthesis in vitro and in vivo; this activity can be shown using monocyte-specific stimuli in peripheral blood mononuclear cells but also in other phagocytic cells like microglia. For technical reasons, endothelial cells have until now been tested primarily in vitro. However, there is solid evidence now from intravital microscopy that the induction of adhesivity in postcapillary venules by LPS is modulated by thalidomide. Altered surface antigen expression has been described on leukocytes obtained from humans and experimental animals treated with thalidomide, but convincing evidence is lacking for in vitro modulation of surface antigen expression on leukocytes (as opposed to the modulation of adhesion antigens on endothelial cells stimulated by LPS or exogenous TNF alpha in the presence of thalidomide). Therefore, in vivo redistribution is likely to account for some, if not all, changes in circulating leukocyte phenotypes. The immunopathological conditions most clearly responsive to thalidomide are vasculitic alterations of post-capillary venules either in the context of mycobacterial infection (in the case of erythema nodosum leprosum) or mucocutaneous aphths. In both instances (as in the majority of focal inflammatory lesions), leukocyte infiltration and cytokine responses, in particular TNF alpha, are present. Thalidomide acts clinically not only by palliation of existing lesions but also by prevention of recurrence. The mechanism operates in skin, mucosa and parts of the nervous system and is most readily explained by synergism of TNF alpha modulation and a separate point of action on leukocyte migration patterns. more...

The DNA-directed RNA polymerase from the extremely thermophilic eubacterium Thermus thermophilus HB8 was purified employing a new and rapid method. The subunit pattern of the enzyme, analyzed by SDS gel electrophoresis, was interpreted as: 140 kDa and 170 kDa for beta and beta', 40 kDa for alpha and 92 kDa for sigma. The RNA polymerase is active at elevated temperatures (65 degrees C). Kinetic data provide evidence for the existence of two NTP binding sites with very strong cooperativity. The promoter site specificity of the isolated enzyme has been proven by in vitro transcription employing two T. thermophilus templates whose in vivo starts of transcription were characterized by nuclease S1 mapping. more...

A wild strain of A. giganteus was transformed to hygromycin B resistance using a bacterial resistance gene under the control of A. nidulans sequences. Stable transformants arose by heterogenous integration, mainly of tandem repeats of vector DNA at various sites in the host genome. Between 6 and 30 resistant colonies were obtained per microgram DNA per 3 x 10(3) viable protoplasts. Vector DNA could be recovered by transformation of Escherichia coli with undigested genomic DNA from Aspergillus giganteus transformants. more...