Three alternatively spliced variants of the gene coding for the human bone morphogenetic protein-1.

The human bone morphogenetic protein-1 was originally identified as a protein with the capacity to stimulate bone and cartilage growth in vitro. Its gene sequence identified it as an alternatively spliced human homolog of the Drosophila dorsal-ventral patterning tolloid gene and suggested that it activates transforming growth factor-beta-like molecules by proteolytic cleavage. Its expression pattern and its recently identified activity as a procollagen C proteinase, however, suggest that it has a more general function in the early stages of embryogenesis. This view is strengthened by the previous observation of a third alternatively spliced isoform of the gene, called bone morphogenetic protein 1/His. We now show that the gene is expressed in three additional variants, leading to shorter and slightly modified C-termini. The three variants are preferentially expressed in placenta but show individual differences in their expression profiles in other soft tissues.

The effect of anoxia on cardiomyocyte glucose transport does not involve an adenosine release or a change in energy state.

The action of anoxia on glucose transport was investigated in isolated resting rat cardiomyocytes. Incubation of these cells in the absence of oxygen for 30 min resulted in a 4- to 5-fold increase in glucose transport (with a lag period of 5-10 min). Up to 40 min of anoxia failed to alter the cellular concentrations of ATP, phosphocreatine, and creatine. Adenosine deaminase (1.5 U/ml), the A1-adenosine receptor antagonist 1,3-diethyl-8-phenylxanthine (1 microM), or the A2-selective antagonist 3,7-dimethyl-1-propargylxanthine (20 microM) had no effect on anoxia-dependent glucose transport. Moreover, adenosine (10-300 microM, added under normoxia) did not stimulate glucose transport. Wortmannin (1 microM) did not influence the effect of anoxia, but completely suppressed that of insulin. On the other hand, the effects of anoxia and insulin were not additive. These results indicate (i) that the effect of anoxia on cardiomyocyte glucose transport is not mediated by a change in energy metabolism, nor by an adenosine release; (ii) that it probably does not involve a phosphatidylinositol 3-kinase, in contrast to the effect of insulin, and (iii) that the signal chains triggered by anoxia or insulin may converge downstream of this enzyme, or, alternatively, that anoxic conditions may impair the action of the hormone.

Electrocardiograph-triggered two-dimensional time-of-flight versus optimized contrast-enhanced three-dimensional MR angiography of the peripheral arteries.

We determined whether the accuracy of magnetic resonance angiography (MRA) in the peripheral run-off vessels can be improved by using contrast-enhanced (CE) three-dimensional (3D) technique in comparison to electrocardiograph (ECG)-triggered two-dimensional (2D) time-of-flight (TOF) technique. In a prospective study 20 patients with occlusions of the pelvic and/or femoral arteries underwent a CE 3D MRA (repetition time (TR): 5 ms, (TE) echo time: 2 ms, flip angle (FA): 30 degrees ) and an ECG-triggered 2D time-of-flight (TOF) technique (TR: 408 resp. 608 ms, TE: 7 ms, FA: 70 degrees) of the run-off vessels on a 1.5 T MR system. Each patient received a contrast material volume of 0.15 mmol/kg of body weight of gadolinium (Gd)/DTPA using an automatic injector. The tube system to the patient was flushed by 50 mL of a saline solution applied with the same injection rate as the contrast material administration. The start of the 3D MR sequence was tailored individually to the applied contrast material after determination of circulation times by a prior bolus. All patients underwent each conventional or digital arteriography for comparison, as well. The visualization of the run-off vessels was ranked on a scale of 0-3 (0 = poor, 1 = fair, 2 = good, 3 = excellent) by three blinded reviewers. They also graded the vascular segments as either occluded or significantly altered (>50% reduction in diameter) or free of significant stenosis. CE 3D MRA was significantly faster in imaging the run-off vessels in comparison to the ECG-triggered 2D TOF technique. All 160 vascular segments were visualized with the 3D method, whereas only 142/160 segments were seen with 2D technique. The resulting image quality ranking of all vascular segments was significantly higher (p < 0.05) using CE 3D MRA (2.8) than with the 2D TOF technique (2.4). The detection of the stenoses was possible with both techniques. The grading of seven of seven stenoses was correct with 3D method and in five of seven cases with the 2D TOF technique. All vessel occlusions were detected by using both techniques. Small collaterals were visualized in more detail with the CE 3D MR angiography. These data demonstrate an improvement in image quality and accuracy of MRA of the peripheral arteries using a CE 3D technique in comparison to an ECG-triggered 2D TOF sequence.

A simple method of generating variable T1 contrast images using temporally reordered phase encoding.

A rapid method of generating T1 images by means of an inversion pulse followed by a fast low-angle imaging experiment is presented. The T1 contrast is manipulated by a temporal reordering of the phase-encoding gradient, resulting in an almost completely free choice of T1 contrast, without any extra restriction on the size of the data acquisition matrix. The intrinsic rapidity of the sequence renders it insensitive to motion artifacts and lends itself to multipoint T1 calculations. The method is particularly well suited to high-field imaging.

Experimental non-A, non-B hepatitis: four types of cytoplasmic alteration in hepatocytes of infected chimpanzees.

On the occasion of an outbreak of non-A, non-B hepatitis in a plasmapheresis centre (81 cases, incubation period: 3--6 weeks) a pool of 12 plasma samples was obtained in the early phase of increasing transaminases. Two chimpanzees were inoculated, each receiving 12 ml of the pooled plasma. After an incubation period of 10--12 weeks a mild non-A, non-B hepatitis developed. Serum transaminases were slightly elevated. Needle biopsies, taken fortnightly, showed a slight activation of Kupffer cells (6--8 weeks), single cell necroses, and infiltration of the portal tracts (10--13 weeks). Electron microscopically four types of cytoplasmic change, were found in hepatocytes and assumed to be specific for the infection, Type I: Sponge-like inclusion (6 weeks after inoculation) composed of a dense matrix and irregularly arranged membranes. Type II: Attaching curved membranes (8 weeks), developing by close apposition of two cisternae of smooth endoplasmic reticulum. Type III: Cylindrical complexes (10 weeks), already described in literature. Type IV: Microtubular aggregates, usually neighbouring type III structures. The findings suggest 1) that the agent of the present infection is, at least in part, identical with that of the long incubation type of experimental non-A, non-B hepatitis, and 2) that ultrastructural alterations may precede manifest hepatitis.

Glucose transport and glucose transporter GLUT4 are regulated by product(s) of intermediary metabolism in cardiomyocytes.

Alternative substrates of energy metabolism are thought to contribute to the impairment of heart and muscle glucose utilization in insulin-resistant states. We have investigated the acute effects of substrates in isolated rat cardiomyocytes. Exposure to lactate, pyruvate, propionate, acetate, palmitate, beta-hydroxybutyrate or alpha-oxoglutarate led to the depression of glucose transport by up to 50%, with lactate, pyruvate and propionate being the most potent agents. The percentage inhibition was greater in cardiomyocytes in which glucose transport was stimulated with the alpha-adrenergic agonist phenylephrine or with a submaximal insulin concentration than in basal or fully insulin-stimulated cells. Cardiomyocytes from fasted or diabetic rats displayed a similar sensitivity to substrates as did cells from control animals. On the other hand, the amination product of pyruvate (alanine), as well as valine and the aminotransferase inhibitors cycloserine and amino-oxyacetate, stimulated glucose transport about 2-fold. In addition, the effect of pyruvate was counteracted by cycloserine. Since reversible transamination reactions are known to affect the pool size of the citrate cycle, the influence of substrates, amino acids and aminotransferase inhibitors on citrate, malate and glutamate content was examined. A significant negative correlation was found between alterations in glucose transport and the levels of citrate (P < 0.01) or malate (P < 0.01), and there was a positive correlation between glucose transport and glutamate levels (P < 0.05). In contrast, there was no correlation with changes in [1-(14)C]pyruvate oxidation or in glucose-6-phosphate levels. Finally, pyruvate decreased the abundance of GLUT4 glucose transporters at the surface of phenylephrine- or insulin-stimulated cells by 34% and 27 % respectively, as determined by using the selective photoaffinity label [3H]ATB-BMPA [[3H]2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-man nos-4-yloxy)propyl-2-amine]. In conclusion, cardiomyocyte glucose transport is subject to counter-regulation by alternative substrates. The glucose transport system appears to be controlled by (a) compound(s) of intermediary metabolism (other than glucose 6-phosphate), but in a different way than pyruvate dehydrogenase. Transport inhibition eventually occurs via a decrease in the amount of glucose transporters in the plasma membrane.

Recurrent inhibition of individual Ia inhibitory interneurones and disinhibition of their target alpha-motoneurones during muscle stretches.

The effects of ramp stretches applied to triceps surae muscle on the discharge patterns of single Ia inhibitory interneurones, monosynaptically invaded from various nerves, were studied in either decerebrate or anesthetized cats. Interneurones which received direct excitatory Ia input from the stretched muscle exhibited augmented activity both during the dynamic and static phase of stretch, which was, however, interrupted by a transient inhibitory influence during the dynamic phase of stretch. The influences on Ia inhibitory interneurones, monosynaptically invaded from hamstring or tibial nerve, were exclusively inhibitory. These stretch-induced inhibitions were better demonstrable in decerebrate than in anesthetized preparations. The timing of the discharge patterns of additionally recorded Renshaw cells during stretch, and the disappearance or reduction of the above described inhibitory effects after administration of DHE, strongly support the idea that these inhibitory actions are caused by Renshaw inhibition. In Ia inhibitory interneurones, monosynaptically activated from the antagonistic peroneal nerve, stretch induced also pronounced inhibitory effects, which were most probably caused by mutual inhibition between Ia inhibitory interneurones. The suppression of agonistic Ia inhibitory interneurone activity below the tonic resting activity corresponded to an enhancement of the monosynaptic reflex amplitude of the antagonistic motoneurone pool. The findings suggest that normal orthodromic activation of Renshaw cells, and consequently the recurrent inhibition of the Ia inhibitory interneurones, is predominantly linked with rapid phasic, rather than slow tonic, motoneuronal firing. The functional role of this mechanism for the performance of rapidly alternating movements and the damping of ballistic agonist contractions is discussed.

[Modification of cardiovascular dynamics in orthostatic dysregulation in childhood by oxilofrin]. / Die Beeinflussung der Herzkreislaufdynamik bei orthostatischer Dysregulation im Kindesalter durch Oxilofrin.

In 10 children with clinically and hemodynamically proven orthostatic dysregulation of the sympathetic type, non invasive hemodynamics using sphygmographic techniques as well as systolic time intervals were evaluated. Measurements were taken during 10 minutes in the supine and a 65 degrees position on a tilting table before and after acute administration of Oxilofrin, 20 mg as well as after 2 and 4 weeks of chronic treatment with 20 mg t.i.d. During orthostatic stress, Oxilofrin caused a significant increase in pulse pressure, cardiac output, stroke volume, and systolic ejection rate with a reduction in peripheral vascular resistance compared to control. Pre-ejection period and isovolumic contraction time, while prolonged on baseline orthostatic stress, were only slightly shortened by Oxilofrin. These hemodynamic actions of the drug are explained by a stimulation of adrenergic beta and alpha receptions with the consequence of decreasing venous capacitance and increased venous return, thus providing a useful hemodynamic profile for the treatment of orthostatic dysfunction.

[Preparation and testing of a hepatitis B vaccine (author's transl)]. / Herstellung und Erprobung eines Hepatitis-B-Impfstoffes.

Starting with 41.5 l of plasma from anti-HBe positive carriers of HBs antigen, 11,400 doses of a hepatitis B vaccine with 42 micrograms HBsAg-protein and 11,300 national units HBsAg activity per dose were obtained. After purification, HBsAg is obtained in 99% purity with a yield of more than 90% protein. A possible residual infectivity was inactivated by a diluted formalin solution. The infectivity test in chimpanzees confirmed the absence of infectious hepatitis viruses (HBV and nonA-nonB). In guinea pigs the immunogenicity of the vaccine was comparable to that of the reference preparation from the U.S. National Institute of Health. The presence of Al(OH)3 in the vaccine increased the anti-HBs titre by factors of 30-50. After vaccination with two doses 41 of 45 persons became anti-HBs positive, with three doses 42 of 45 persons developed anti-HBs. Median anti-HBs titre after the third doses: schedule I (three doses in intervals of 6 weeks) 427 mWHO-U/ml; schedule II (two doses at an interval of 4 weeks, third doses 4 months after the first doses) 1535 mWHO-U/ml. The vaccine was well tolerated. There were minor local reactions only.

Recognition of chlamydial antigen by HLA-B27-restricted cytotoxic T cells in HLA-B*2705 transgenic CBA (H-2k) mice.

OBJECTIVE: The association of reactive arthritis (ReA) with HLA-B27 and the presence of bacterial antigen in joints with ReA suggest that bacterial peptides might be presented by the HLA-B27 molecule and thus stimulate CD8 T cells. This study was performed to investigate the B27-restricted cytotoxic T lymphocyte (CTL) response to Chlamydia trachomatis, using the model of HLA-B27 transgenic mice. METHODS: CBA (H-2k) mice homozygous for HLA-B*2705 and human beta2-microglobulin expression were immunized with C trachomatis or with the chlamydial 57-kd heat-shock protein (hsp57) coupled to latex beads. Cytotoxicity of lymphocytes from in vivo-primed transgenic mice was tested against C trachomatis-infected targets. Blocking experiments were performed with monoclonal antibodies (MAb) against class I major histocompatibility complex molecules. RESULTS: A Chlamydia-specific lysis of both B27-transfected and nontransfected target cells was observed. This response could be inhibited by anti-B27 and anti-H2 MAb. CTL from mice immunized with hsp57 were not able to lyse Chlamydia-infected target cells, and Chlamydia-specific CTL could not destroy targets loaded with hsp57. CONCLUSION: These results suggest the existence of at least 2 CTL populations in this mouse model: one recognizing peptide of bacteria-infected cells restricted by HLA-B*2705 and the other recognizing peptide of bacteria-infected cells restricted by the murine H-2Kk molecule. It does not appear that hsp57 is a major target for the CD8 T cell response directed against Chlamydia. This animal model opens the way for identifying bacterial epitopes presented by HLA-B27, and might thus help to clarify the pathogenesis of B27-associated diseases.

Safety and potency aspects in the preparation of an experimental HBsAg vaccine.

No experimental setting is available to exclude residual infectivity in HBsAg vaccines derived from human plasma. Thus, safety can be achieved only by means of their preparation. To reduce infectivity of the starting material, only plasma from healthy anti-HBe positive donors was used. In the FRG, 50% of all healthy HBsAg carriers with anti-HBe have a suitable serum level of 5 to 20 micrograms/ml. The purification procedure removed hepatitis B virus by a factor greater than 10(4). The purified product contained only the HBsAg proteins and no serum protein, as shown by SDS gel electrophoresis. The pure HBsAg was treated with formalin 1:500 at 37 degrees C for 4 days. A loss of 30 to 50% antigenicity was tolerated to achieve the highest possible destruction of known and unknown infectious agents. After inactivation, the HBsAg was bound to aluminium hydroxide gel. The gel was washed repeatedly to remove the formalin. Doses of 40 micrograms or 20 micrograms absorbed HBsAg protein were given to greater than 2500 persons without serious side effects. In greater than 97% anti-HBs was formed with a median titer of 1900 I.U./ml.

Characterization of the synovial T cell response to various recombinant Yersinia antigens in Yersinia enterocolitica-triggered reactive arthritis. Heat-shock protein 60 drives a major immune response.

OBJECTIVE: In Yersinia enterocolitica-triggered reactive arthritis (Yersinia ReA), the synovial T cell response is primarily directed against bacterial components, which are mostly unknown. This study was performed to investigate the synovial proliferative T cell response to a panel of recombinant Yersinia antigens in patients with Yersinia ReA and in controls. METHODS: Synovial fluid mononuclear cells (SFMC) were obtained from 4 patients with Yersinia ReA and from 14 patients with arthritides of different etiology. SFMC were stimulated with 5 recombinant Yersinia antigens (the 19-kd urease beta subunit, 13-kd ribosomal L23 protein, 32-kd ribosomal L2 protein, 18-kd outer membrane protein H, and Y. enterocolitica heat-shock protein 60 [hsp60]), and with human, Chlamydia trachomatis, and Borrelia burgdorferi hsp60. Three T cell clones specific for Y. enterocolitica hsp60 were generated from 1 patient with Yersinia ReA. Antigen-induced cytokine release was measured by enzyme-linked immunosorbent assay. RESULTS: SFMC from all 4 patients with Yersinia ReA responded to each of the Yersinia antigens except the 13-kd protein. These antigens were also recognized by SFMC from a subgroup of patients with undifferentiated arthritis (n = 4), but not by SFMC from other patients with arthritis of different etiology (n = 10). Y. enterocolitica hsp60 induced the strongest proliferative response in all cases. Two types of hsp60-reactive T cell clones could be obtained. One clone responded to all hsp60 variants, including the human variant, and showed a type 2 T helper (Th2)-like cytokine-secretion pattern. In contrast, another clone with specificity for the bacterial hsp60 proteins, but not the human equivalent, reacted with a more Th1-like pattern. CONCLUSION: In Y. enterocolitica-triggered ReA, at least 4 immunodominant T cell antigens exist, which might be used in lymphocyte proliferation assays to identify patients with Yersinia ReA. The hsp60 is a strong antigen, inducing both bacteria-specific and potentially autoreactive CD4+ T cells of both the Th1 and Th2 type.