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Biomedical subjects

J Heim

Publications and source records attributed to J Heim.

At least 19 recordsLinked to original sources
PubMed2001

SH3GLB, a new endophilin-related protein family featuring an SH3 domain.

A new cDNA encoding a protein of 362 amino acids designated SH3GLB1, for SH3 domain GRB2-like endophilin B1, was identified in a yeast two-hybrid screen devoted to the identification of new partners interacting with the apoptosis inducer Bax. SH3GLB1 shows strong similarities to the SH3 domain-containing proteins of the endophilin family and presumably represents the human homologue of the potential Caenorhabditis elegans SH3 containing-protein identified by systematic translation of the C. elegans genome (GenBank Accession No. U46675). Reversing prey to bait in the yeast screen, a second protein, SH3GLB2, of 395 amino acids showing 65% identity to SH3GLB1 was identified as an interacting partner of SH3GLB1. The discovery of SH3GLB1 itself in the screening with SH3GLB1 as a bait and further mapping experiments demonstrated that a core coiled-coil-type region is required for the formation of SH3GLB homo- and/or heterodimers, whereas the SH3 domain is not involved in these interactions. Interestingly, the similarities with the endophilin proteins cover the entire sequence of the SH3GLB family, suggesting a common fold and presumably a common mode of action. Furthermore, SH3GLB members colocalize to the cytoplasmic compartment of the cell together with Bax and are excluded from the nucleus. SH3GLB1 and SH3GLB2 do not significantly influence the onset and time course of Bax-mediated apoptosis in HeLa or 293T cells.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Psychological model for judicial decision making in emergency or temporary child placement.

In emergencies, family court judges must often make rapid decisions, without benefit of thorough information, that have significant impact on people's lives. Action-oriented research was used to develop a model that would bring psychosocial factors to the legal system for the purpose of enhancing the judicial decision-making process in emergency and temporary child placement cases.

Child↗
PubMed2000

[Reversible pulmonary hypertension in Basedow's disease].

BACKGROUND: An association between pulmonary hypertension and hyperthyroidism has been described. An autoimmune mechanism is usually advocated but a direct effect thyroid hormones on the pulmonary vascular system is also suggested. CASE REPORT: A 49-year-old woman with a history of Graves' disease was admitted for sudden dyspnea. There was no evidence of ischemic stroke and the electrocardiogram and cardiac enzymes were normal. The lung scintiscan did not show any sign of pulmonary embolism. The chest computed tomography was normal. Doppler echocardiography found pulmonary hypertension with systolic pulmonary artery pressure of 68.7 mmHg. Thyroid function tests showed hyperthyroidism with recurrent Graves' disease. Pulmonary hypertension markedly decreased after the euthyroid state was achieved with antithyroid therapy. DISCUSSION: This patient had pulmonary hypertension associated with hyperthyroidism due to Graves' disease. The rapid reversibility of pulmonary hypertension in this case after the achievement of the euthyroid state suggest a pathogenic link between these two disorders and a possible direct effect of thyroid hormones on the pulmonary vascular system.

Antithyroid Agents↗
PubMed2000

Comparative microarray analysis of gene expression during apoptosis-induction by growth factor deprivation or protein kinase C inhibition.

The transcriptional response of mouse pro-B cells to two different apoptotic stimuli was investigated. First, interleukin-3 (IL-3) deprivation was used to trigger programmed cell death in IL-3 dependent FL5.12 cells. Alternatively, cells were treated with the protein kinase C (PKC) inhibitor staurosporine. The temporal pattern of gene expression was followed with cDNA microarrays, covering over 8700 different mouse cDNA sequences corresponding to approximately 7900 unique genes. Messenger RNA levels of 315 genes were found to be regulated by more than twofold upon IL-3 removal, while 125 genes reacted to staurosporine treatment. Cross-comparison revealed an intersection of 34 genes similarly regulated in both pathways and thus representing candidates for common apoptosis regulators. For many expressed sequence tags (ESTs) our data suggest for the first time functions in the control of apoptosis, stress response or the cell cycle. IL-3 removal led to the repression of genes required for proliferation and to the induction of genes, linked to apoptotic and signaling pathways. Staurosporine caused predominantly activation of genes, some of which had previously been described to be involved in inflammation. Our findings indicate that cellular responses to both apoptotic stimuli influence various physiological pathways which had not previously been known to be linked.

Animals↗
PubMed2000

Structural basis of BFL-1 for its interaction with BAX and its anti-apoptotic action in mammalian and yeast cells.

BFL-1 is the smallest member of the BCL-2 family and has been shown to retard apoptosis in various cell lines. However, the structural basis for its function remains unclear. Molecular modeling showed that BFL-1 could have a similar core structure as BCL-xL, consisting of seven alpha helices, although both proteins share only the conserved BCL-2 homology domains (BH1 and BH2 domains), but otherwise have very limited sequence homology, particularly in the N-terminal region. We demonstrated in the yeast two-hybrid system that BFL-1 interacts strongly with human BAX but is not able to form homodimers nor to interact with human BCL-2 or BCL-xL. Overexpression experiments in REF52 rat fibroblasts showed that BFL-1 conferred increased resistance to apoptosis induced by serum deprivation. BFL-1 had also the ability to neutralize BAX lethality in yeast. BAX requires the BH3 domain for interaction with BFL-1. However, the minimal region of BFL-1 for the interaction with BAX in coimmunoprecipitation experiments was not sufficient to protect cells from apoptosis. Further examination of BFL-1 and several other anti-apoptotic proteins suggests a more general type of structure based on structural motifs, i.e. a hydrophobic pocket for the binding of proapoptotic proteins, rather than extended sequence homologies.

Amino Acid Sequence↗
PubMed2000

Factor X fusion proteins: improved production and use in the release in vitro of biologically active hirudin from an inactive alpha-factor-hirudin fusion protein.

Many recombinant proteins are synthesized as fusion proteins containing affinity tags to aid in the downstream processing. After purification, the affinity tag is often removed by using a site-specific protease such as factor Xa (FXa). However, the use of FXa is limited by its expense and availability from plasma. To develop a recombinant source of FXa, we have expressed two novel forms of FXa using baby hamster kidney (BHK) cells as host and the expression vector pNUT. The chimeric protein FIIFX consisted of the prepropeptide and the Gla domain of prothrombin linked to the activation peptide and protease region of FXa, together with a cellulose-binding domain (CBD(Cex)) as an affinity tag. A second variant consisted of the transferrin signal peptide linked to the second epidermal growth factor-like domain and the catalytic domain of FX and a polyhistidine tag. Both FX variants were secreted into the medium, their affinity tags were functional, and following activation, both retained FXa-specific proteolytic activity. However, the yield of the FIIFX-CBD(Cex) fusion protein was 10-fold higher than that of FX-CBD(Cex) and other forms of recombinant FX reported to date. The FXa derivatives were used to cleave two different fusion proteins, including a biologically inactive alpha-factor-hirudin fusion protein secreted by Saccharomyces cerevisiae. After cleavage, the released hirudin demonstrated biological activity in a thrombin inhibition assay, suggesting that this method may be applicable to the production of toxic or unstable proteins. The availability of novel FX derivatives linked to different affinity tags allows the development of a versatile system for processing fusion proteins in vitro.

Amino Acid Sequence↗
PubMed2000

Uncoupling proteins 2 and 3 interact with members of the 14.3.3 family.

Uncoupling proteins (UCPs) are members of the superfamily of the mitochondrial anion carrier proteins (MATP). Localized in the inner membrane of the organelle, they are postulated to be regulators of mitochondrial uncoupling. UCP2 and 3 may play an important role in the regulation of thermogenesis and, thus, on the resting metabolic rate in humans. To identify interacting proteins that may be involved in the regulation of the activity of UCPs, the yeast two-hybrid system was applied. Segments of hUCP2 containing the hydrophilic loops facing the intermembrane space, or combinations of these, were used to screen an adipocyte activation domain (AD) fusion library. The 14.3.3 protein isoforms theta, beta, zeta were identified as possible interacting partners of hUCP2. Screening of a human skeletal muscle AD fusion library, on the other hand, yielded several clones all of them encoding the gamma isoform of the 14.3.3 family. Mapping experiments further revealed that all these 14.3.3 proteins interact specifically with the C-terminal intermembrane space domain of both hUCP2 and hUCP3 whereas no interactions could be detected with the C-terminal part of hUCP1. Direct interaction between UCP3 and 14.3.3 theta could be demonstrated after in vitro translation by coimmunoprecipitation. When coexpressed in a heterologous yeast system, 14.3.3 proteins potentiated the inhibitory effect of UCP3 overexpression on cell growth. These findings suggest that 14.3.3 proteins could be involved in the targeting of UCPs to the mitochondria.

14-3-3 Proteins↗
PubMed2000

Survival and distribution of Echinostoma caproni in the small intestine of ICR mice up to 36 hours after the death of the host.

This study examined survival and distribution of Echinostoma caproni in the small intestine of ICR mice at various times up to 36 hr following the death of the host. Adult worms were obtained at 2-wk postinfection of 21 ICR mice each infected with 50 metacercarial cysts. Mice were killed with light ether anesthetization and cervical dislocation and maintained at room temperature (22 +/- 1C) until examination at 0 (controls), 2, 4, 6, 12, 24, and 36 hr postmortem. Survival was based on worm activity and distribution was assessed on the basis of worm location in 1 of 5 equal intestinal segments numbered from the pylorus to the ileocecal valve. Worms were alive up to 36 hr post-mortem and were distributed mainly in segments 3 and 4 at all times postmortem. Histochemical Oil Red O studies on whole control and experimental worms showed neutral lipids localized in the protone-phridial tubules and the excretory bladder. Eggs from experimental worms at all times produced miracidia that infected Biomphlaria glabrata snails.

Animals↗
PubMed1999

Demonstration of frequent occurrence of clonal T cells in the peripheral blood but not in the skin of patients with small plaque parapsoriasis.

Clinical, immunohistological, and molecular biological data suggest the chronic dermatosis small plaque parapsoriasis (SPP) to be a precursor of mycosis fungoides (MF). However, most data are contradictory and confusing due to inexact definition of SPP. Recently, clonal T cells were detected in skin and blood samples of early MF. Because demonstration of identical T-cell clones in skin and blood of SPP patients would indicate a close relationship of SPP to MF, we investigated the clonality of skin and blood specimens from 14 well-defined SPP patients. By a polymerase chain reaction (PCR) amplifying T-cell receptor gamma rearrangements and subsequent high-resolution electrophoresis, clonal T cells were detected in 9 of 14 initial and 32 of 49 follow-up blood samples, but in 0 of 14 initial skin specimens. Even a clone-specific PCR showing the persistence of the initial blood T-cell clone in 20 of 20 follow-up samples, failed to detect the T-cell clone in the skin. In 2 patients, the clonal T cells were shown to be CD4(+). For the first time, the majority of SPP patients was shown to carry a T-cell clone in the peripheral blood. Although a relation between circulating clonal T cells and SPP cannot directly be proven by the applied techniques, our results indicate blood T-cell clonality to be a characteristic feature of SPP and CTCL because analysis of multiple controls and clinical workup of our SPP patients excluded other factors simulating or causing a clonal T-cell proliferation. A sufficient cutaneous antitumor response but also an extracutaneous origin of the T-cell clones might explain the failure to detect skin infiltrating clonal T cells.

Aged↗
PubMed1999

Novel BNIP1 variants and their interaction with BCL2 family members.

By PCR and EST database searches we have identified three novel BNIP1 splice variants, and found that one of them, BNIP1-b, contains a highly conserved BH3 domain. The BNIP1 gene has been assigned to chromosome 5q33-34. Using in vitro protein-protein interaction assays, all BNIP1 variants were shown to interact with BCL2 and also with BCL2L1 (previously Bcl-xL). These interactions are BH3-independent. Furthermore, the BNIP1 variants cannot interact with BAX. The results suggest that the BNIP1 variants are novel members of the BCL2 family but function through a mechanism different from other BH3-only members.

Alternative Splicing↗
PubMed1999

Breath ethane as a marker of reactive oxygen species during manipulation of diet and oxygen tension in rats.

Breath ethane, O2 consumption, and CO2 production were analyzed in 24-mo-old female Fischer 344 rats that had been fed continuously ad libitum (AL) or restricted 30% of AL level (DR) diets since 6 wk of age. Rats were placed in a glass chamber that was first flushed with air, then with a gas mixture containing 12% O2. After equilibration, a sample of the outflow was collected in gas sampling bags for subsequent analyses of ethane and CO2. The O2 and CO2 levels were also directly monitored in the outflow of the chamber. O2 consumption and CO2 production increased for DR rats. Hypoxia decreased O2 consumption and CO2 production for the AL-fed and DR rats. These changes reflect changes in metabolic rate due to diet and PO2. A significant decrease in ethane generation was found in DR rats compared with AL-fed rats. Under normoxic conditions, breath ethane decreased from 2.20 to 1.61 pmol ethane/ml CO2. During hypoxia the levels of ethane generation increased, resulting in a DR-associated decrease in ethane from 2.60 to 1.90 pmol ethane/ml CO2. These results support the hypothesis that DR reduces the level of oxidative stress.

Aging↗
PubMed1999

Mutations in FL5.12 cells conferring resistance to apoptosis induced by interleukin-3 deprivation.

In an attempt to dissect the signal pathway in which Bax increases cellular responses to apoptotic stimuli and leads to the activation of the caspase cascade, we mutated FL5.12 Bax CL16 cells with a chemical mutagen. In this report we characterize two mutant clones, FL5.12 ms1 and m3. Both clones are resistant to IL-3 deprivation exhibiting no changes in mitochondrial membrane potential, annexin V and propidium iodide binding. FL5.12 ms1 is also resistant to staurosporine and anti-Fas antibody. In cell fusion experiments m3 behaves genetically dominant and ms1 is recessive. The results suggest that m3 has a mutation in a specific function upstream of Bax, while ms1 has a mutational block in the general pathway downstream of the 'Bcl-2 checkpoint'.

Animals↗
PubMed1998

Redistribution of Bax from cytosol to membranes is induced by apoptotic stimuli and is an early step in the apoptotic pathway.

It is known that overexpression of Bax accelerates apoptosis, but the biochemical mechanism of the signal transduction from Bax to downstream targets has still not been fully determined. In the present study, we demonstrate that upon apoptotic stimuli, Bax moves from the cytosolic to the membrane fraction. The redistribution of Bax is not inhibited by a caspase inhibitor, zVAD-fmk, which blocks caspase-3 activity and prevents apoptosis in vivo. A FL5.12 Bax CL16 mutant cell, ms3, which is resistant to apoptosis induced by staurosporine, retains the activity of Bax redistribution but shows no caspase-3 activity. Our results revealed that Bax accumulation on membranes precedes caspase-3 activation, indicating that redistribution of Bax is an early event in apoptosis. These results suggest that Bax may be functionally significant in the regulation of caspase-3 activation.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Functional expression of a mammalian acetylcholinesterase in Pichia pastoris: comparison to acetylcholinesterase, expressed and reconstituted from Escherichia coli.

The mature rat brain acetylcholinesterase gene (T subunit, AChE) was subcloned downstream of the temperature-inducible lambda promoter PL and fused to the signal peptide of the OmpA protein. Three different expression vectors were constructed: (i) pCompmA containing the mature AChE, (ii) pComp delta TA containing a truncated AChE and (iii) pComp delta TAH containing the truncated AChE C-terminal fused to a 6xHis-tag. With all expression vectors the overexpression of AChE in Escherichia coli resulted mainly in cytoplasmic inclusion bodies (IB). However, some activity was found in the periplasmic space. The inclusion bodies were refolded in vitro, yielding up to 1.42 U/mg IB of active AChE. The refolded AChE was partially purified (approx. 300-fold) by affinity chromatography with a specific activity of approx. 250 U/mg. Removing the cysteine residue near the C-terminus (truncated AChE, delta TAChE) assuming to affect the refolding, did not increase the amount of active enzyme obtained after refolding. Purification of denatured delta TAChE-6xHis prior to refolding by Ni-NTA-chromatography increased the refolding efficiency by a factor of 1.5. Functional expression and secretion of rat brain acetylcholinesterase into the medium was achieved in Pichia pastoris. By optimizing the culture conditions, 100 mU/ml AChE in the medium was produced. In this work we are describing the functional expression of a mammalian AChE in a microbial host in good yields for the first time. The physico-chemical properties of both, the bacterial and yeast expressed AChE were compared with those of the native AChE. The properties of the yeast expressed AChE and the native AChE were similar, whereas the E. coli expressed enzyme was found to be less stable and had different inhibition properties.

Acetylcholinesterase↗
PubMed1997

The BH3 domain of Bax is sufficient for interaction of Bax with itself and with other family members and it is required for induction of apoptosis.

bax is an apoptosis-inducing member of the bcl-2 multigene family. We have studied interactions of human Bax with itself, and with the apoptosis-preventing members Bcl-2 and Bcl-xL using a yeast two-hybrid system. Exhaustive Bax truncations were constructed and their interactions with full-length family members studied. Bax interacted similarly with itself as with the apoptosis-suppressing family members Bcl-2 and Bcl-xL in quantitative two-hybrid studies. A region of 41 amino acids covering the recently discovered BH3 domain of Bax was found to be necessary and sufficient for all interactions of Bax. Bax truncations containing BH3, but lacking BH1 and BH2 homology domains, interacted with the other family members markedly more strongly than full-length Bax, which may reflect conformational changes required for the interactions of full-length Bax. The minimum requirements for Bax homodimerization were found to be the BH3 domain from one Bax molecule and a region covering BH3 plus BH1 from another. We also studied the apoptosis-inducing activity of the Bax truncations upon microinjection of expression plasmids into rat fibroblasts. The BH3 region was required for the apoptosis-inducing activity of Bax, whereas BH1, BH2 and the N-terminus of Bax were dispensable.

Animals↗
PubMed1997

A new structural class of serine protease inhibitors revealed by the structure of the hirustasin-kallikrein complex.

BACKGROUND: Hirustasin belongs to a class of serine protease inhibitors characterized by a well conserved pattern of cysteine residues. Unlike the closely related inhibitors, antistasin/ghilanten and guamerin, which are selective for coagulation factor Xa or neutrophil elastase, hirustasin binds specifically to tissue kallikrein. The conservation of the pattern of cysteine residues and the significant sequence homology suggest that these related inhibitors possess a similar three-dimensional structure to hirustasin. RESULTS: The crystal structure of the complex between tissue kallikrein and hirustasin was analyzed at 2.4 resolution. Hirustasin folds into a brick-like structure that is dominated by five disulfide bridges and is sparse in secondary structural elements. The cysteine residues are connected in an abab cdecde pattern that causes the polypeptide chain to fold into two similar motifs. As a hydrophobic core is absent from hirustasin the disulfide bridges maintain the tertiary structure and present the primary binding loop to the active site of the protease. The general structural topography and disulfide connectivity of hirustasin has not previously been described. CONCLUSIONS: The crystal structure of the kallikrein-hirustasin complex reveals that hirustasin differs from other serine protease inhibitors in its conformation and its disulfide bond connectivity, making it the prototype for a new class of inhibitor. The disulfide pattern shows that the structure consists of two domains, but only the C-terminal domain interacts with the protease. The disulfide pattern of the N-terminal domain is related to the pattern found in other proteins. Kallikrein recognizes hirustasin by the formation of an antiparallel beta sheet between the protease and the inhibitor. The P1 arginine binds in a deep negatively charged pocket of the enzyme. An additional pocket at the periphery of the active site accommodates the sidechain of the P4 valine.

Amino Acid Sequence↗
PubMed1997

Recombinant hirustasin: production in yeast, crystallization, and interaction with serine proteases.

A synthetic gene coding for the 55-amino acid protein hirustasin, a novel tissue kallikrein inhibitor from the leech Hirudo medicinalis, was generated by polymerase chain reaction using overlapping oligonucleotides, fused to the yeast alpha-factor leader sequence and expressed in Saccharomyces cerevisiae. Recombinant hirustasin was secreted mainly as incompletely processed fusion protein, but could be processed in vitro using a soluble variant of the yeast yscF protease. The processed hirustasin was purified to better than 97% purity. N-terminal sequence analysis and electrospray ionization mass spectrometry confirmed a correctly processed N-terminus and the expected amino acid sequence and molecular mass. The biological activity of recombinant hirustasin was identical to that of the authentic leech protein. Crystallized hirustasin alone and in complex with tissue kallikrein diffracted beyond 1.4 A and 2.4 A, respectively. In order to define the reactive site of the inhibitor, the interaction of hirustasin with kallikrein, chymotrypsin, and trypsin was investigated by monitoring complex formation in solution as well as proteolytic cleavage of the inhibitor. During incubation with high, nearly equimolar concentration of tissue kallikrein, hirustasin was cleaved mainly at the peptide bond between Arg 30 and Ile 31, the putative reactive site, to yield a modified inhibitor. In the corresponding complex with chymotrypsin, mainly uncleaved hirustasin was found and cleaved hirustasin species accumulated only slowly. Incubation with trypsin led to several proteolytic cleavages in hirustasin with the primary scissile peptide bond located between Arg 30 and Ile 31. Hirustasin appears to fall into the class of protease inhibitors displaying temporary inhibition.

Amino Acid Sequence↗