Biomedical subjects

D Rapaport

Publications and source records attributed to D Rapaport.

At least 19 recordsLinked to original sources

PubMed2002

Antiviral prophylaxis may prevent human herpesvirus-6 reactivation in bone marrow transplant recipients.

Human herpesvirus-6 (HHV-6) infects the majority of children under the age of 2 years causing roseola infantum. Following short self-limited disease, the virus enters into a latency phase in peripheral blood lymphocytes (PBL). It has been previously reported that HHV-6 reactivation from latency, in immunocompromised patients undergoing bone marrow transplantation (BMT), could result in febrile illness, pneumonitis, meningitis, and/or encephalitis. In our study, 14 BMT patients received two different antiviral prophylactic therapies: 8 patients received acyclovir, whereas 6 patients received ganciclovir. Clinical manifestations and virus recovery were monitored pre- and post-BMT by polymerase chain reaction tests of cord blood cells cultured with the patients' PBL. No HHV-6 recovery was shown in the 6 patients treated with ganciclovir, whereas 3 of the 8 acyclovir-treated patients experienced virus reactivation 20-21 days post-BMT. One of the 3 patients was asymptomatic but had late engraftment; the second patient had prolonged fever, skin rash, and hemorrhage; the third patient experienced prolonged fever, pneumonitis, marrow rejection, and fatal encephalitis. It is concluded that viral reactivation may be prevented by prophylactic treatment with ganciclovir. Our observation awaits further documentation in prospective randomized trials in high-risk BMT recipients.

Acyclovir↗

PubMed2001

Assembly of Tom6 and Tom7 into the TOM core complex of Neurospora crassa.

Translocation of preproteins across the mitochondrial outer membrane is mediated by the translocase of the outer mitochondrial membrane (TOM) complex. We report the molecular identification of Tom6 and Tom7, two small subunits of the TOM core complex in the fungus Neurospora crassa. Cross-linking experiments showed that both proteins were found to be in direct contact with the major component of the pore, Tom40. In addition, Tom6 was observed to interact with Tom22 in a manner that depends on the presence of preproteins in transit. Precursors of both proteins are able to insert into the outer membrane in vitro and are assembled into authentic TOM complexes. The insertion pathway of these proteins shares a common binding site with the general import pathway as the assembly of both Tom6 and Tom7 was competed by a matrix-destined precursor protein. This assembly was dependent on the integrity of receptor components of the TOM machinery and is highly specific as in vitro-synthesized yeast Tom6 was not assembled into N. crassa TOM complex. The targeting and assembly information within the Tom6 sequence was found to be located in the transmembrane segment and a flanking segment toward the N-terminal, cytosolic side. A hybrid protein composed of the C-terminal domain of yeast Tom6 and the cytosolic domain of N. crassa Tom6 was targeted to the mitochondria but was not taken up into TOM complexes. Thus, both segments are required for assembly into the TOM complex. A model for the topogenesis of the small Tom subunits is discussed.

Amino Acid Sequence↗

PubMed2001

Connection of the mitochondrial outer and inner membranes by Fzo1 is critical for organellar fusion.

Mitochondrial membrane fusion is a process essential for the maintenance of the structural integrity of the organelle. Since mitochondria are bounded by a double membrane, they face the challenge of fusing four membranes in a coordinated manner. We provide evidence that this is achieved by coupling of the mitochondrial outer and inner membranes by the mitochondrial fusion machinery. Fzo1, the first known mediator of mitochondrial fusion, spans the outer membrane twice, exposing a short loop to the intermembrane space. The presence of the intermembrane space segment is required for the localization of Fzo1 in sites of tight contact between the mitochondrial outer and inner membranes. Mutations in the intermembrane space domain of yeast Fzo1 relieve the association with the inner membrane. This results in a loss of function of the protein in vivo. We propose that the mitochondrial fusion machinery forms membrane contact sites that mediate mitochondrial fusion. A fusion machinery that is in contact with both mitochondrial membranes appears to be functionally important for coordinated fusion of four mitochondrial membranes.

Cell Polarity↗

PubMed2001

Biogenesis of porin of the outer mitochondrial membrane involves an import pathway via receptors and the general import pore of the TOM complex.

Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro-imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.

Amino Acid Sequence↗

PubMed2001

Structural requirements of Tom40 for assembly into preexisting TOM complexes of mitochondria.

Tom40 is the major subunit of the translocase of the outer mitochondrial membrane (the TOM complex). To study the assembly pathway of Tom40, we have followed the integration of the protein into the TOM complex in vitro and in vivo using wild-type and altered versions of the Neurospora crassa Tom40 protein. Upon import into isolated mitochondria, Tom40 precursor proteins lacking the first 20 or the first 40 amino acid residues were assembled as the wild-type protein. In contrast, a Tom40 precursor lacking residues 41 to 60, which contains a highly conserved region of the protein, was arrested at an intermediate stage of assembly. We constructed mutant versions of Tom40 affecting this region and transformed the genes into a sheltered heterokaryon containing a tom40 null nucleus. Homokaryotic strains expressing the mutant Tom40 proteins had growth rate defects and were deficient in their ability to form conidia. Analysis of the TOM complex in these strains by blue native gel electrophoresis revealed alterations in electrophoretic mobility and a tendency to lose Tom40 subunits from the complex. Thus, both in vitro and in vivo studies implicate residues 41 to 60 as containing a sequence required for proper assembly/stability of Tom40 into the TOM complex. Finally, we found that TOM complexes in the mitochondrial outer membrane were capable of exchanging subunits in vitro. A model is proposed for the integration of Tom40 subunits into the TOM complex.

Amino Acid Sequence↗

PubMed2000

Recognition of preproteins by the isolated TOM complex of mitochondria.

A multisubunit complex in the mitochondrial outer membrane, the TOM complex, mediates targeting and membrane translocation of nuclear-encoded preproteins. We have isolated the TOM holo complex, containing the preprotein receptor components Tom70 and Tom20, and the TOM core complex, which lacks these receptors. The interaction of recombinant mitochondrial preproteins with both types of soluble TOM complex was analyzed. Preproteins bound efficiently in a specific manner to the isolated complexes in the absence of chaperones and lipids in a bilayer structure. Using fluorescence correlation spectroscopy, a dissociation constant in the nanomolar range was determined. The affinity was lower when the preprotein was stabilized in its folded conformation. Following the initial binding, the presequence was transferred into the translocation pore in a step that required unfolding of the mature part of the preprotein. This translocation step was also mediated by protease-treated TOM holo complex, which contains almost exclusively Tom40. Thus, the TOM core complex, consisting of Tom40, Tom22, Tom6 and Tom7, is a molecular machine that can recognize and partially translocate mitochondrial precursor proteins.

Binding Sites↗

PubMed1999

Biogenesis of Tom40, core component of the TOM complex of mitochondria.

Tom40 is an essential component of the preprotein translocase of the mitochondrial outer membrane (TOM complex) in which it constitutes the core element of the protein conducting pore. We have investigated the biogenesis of Tom40. Tom40 is inserted into the outer membrane by the TOM complex. Initially, Tom40 is bound as a monomer at the mitochondrial surface. The import receptor Tom20 is involved in this initial step; it stimulates both binding and efficient insertion of the Tom40 precursor. This step is followed by the formation of a further intermediate at which the Tom40 precursor is partially inserted into the outer membrane. Finally, Tom40 is integrated into preexisting TOM complexes. Efficient import appears to require the Tom40 precursor to be in a partially folded conformation. Neither the NH(2) nor the COOH termini are necessary to target Tom40 to the outer membrane. However, the NH(2)-terminal segment is required for Tom40 to become assembled into the TOM complex. A model for the biogenesis of Tom40 is presented.

Biological Transport↗

PubMed1999

Tamplicon-7, a novel T-lymphotropic vector derived from human herpesvirus 7.

We describe the derivation of a novel T-cell-defective virus vector employing the human herpesvirus 7 (HHV-7). The new vector, designated Tamplicon-7, replicates in CD4(+) T cells. The system is composed of a helper virus and defective virus genomes derived by the replication of the input Tamplicon vector. There are two cis-acting functions required for the replication and packaging of the defective virus genomes in the presence of the helper virus: the viral DNA replication origin and the composite cleavage and packaging signal, which directs the cleavage and packaging of defective virus genomes. Viral DNA replication is compatible with the rolling circle mechanism, producing large head-to-tail concatemers of the Tamplicon vector. Thus, in the presence of the helper virus, the replicated vectors are packaged and secreted into the medium. Furthermore, we have shown that the vector can be employed to express a foreign gene, encoding the green fluorescent protein, in the T cells infected with the HHV-7 helper virus. We predict that the Tamplicon-7 vector might be potentially useful for gene therapy of diseases affecting the human CD4(+) T cells, including autoimmune diseases, T-cell lymphomas, and AIDS.

Base Sequence↗

PubMed1998

Fzo1p is a mitochondrial outer membrane protein essential for the biogenesis of functional mitochondria in Saccharomyces cerevisiae.

Fzo1p is a novel component required for the biogenesis of functional mitochondria in the yeast Saccharomyces cerevisiae. The protein is homologous to Drosophila Fzo, the first known protein mediator of mitochondrial fusion. Deletion of the FZO1 gene results in a petite phenotype, loss of mitochondrial DNA, and a fragmented mitochondrial morphology. Fzo1p is an integral protein of the mitochondrial outer membrane exposing its major part to the cytosol. It is imported into the outer membrane in a receptor-dependent manner. Fzo1p is part of a larger protein complex of 800 kDa, and presumably is the first identified component of the yeast mitochondrial fusion machinery.

Fungal Proteins↗

PubMed1998

The DNA sequence of the RK strain of human herpesvirus 7.

The complete DNA sequence of human herpesvirus-7 (HHV-7) strain RK was determined following direct cloning of virion DNA fragments into a sequencing vector. The sequence was compared with the previously published complete sequences of HHV-7 strain JI and human herpesvirus-6 (HHV-6) strain U1102. Despite a very close relationship between the two HHV-7 strains, differences are apparent in regions containing tandem reiterations, particularly in the "telomeric" reiterations located near the termini of the large direct repeat at the genome ends, and in a total of 179 additional positions distributed throughout the genome (i.e., about one nucleotide difference per kbp). This extent of divergence implies that the two strains arose from an ancestral virus several thousands of years ago. Differences that affect coding potential do not cluster in particular protein-coding regions, indicating that specific HHV-7 genes have not been measurably subject to unusual evolutionary pressures since divergence. Reassessments of genetic content indicated that the HHV-7 genome contains 84 different genes, whereas the HHV-6 genome contains 85. All HHV-7 genes but 1 have direct HHV-6 counterparts, and all but 2 HHV-6 genes have HHV-7 homologues. Sequence comparisons between HHV-7 and HHV-6 provided evidence that the protein-coding regions of 11 genes are expressed by splicing.

Amino Acid Sequence↗

PubMed1998

cis and trans sites of the TOM complex of mitochondria in unfolding and initial translocation of preproteins.

Translocation of preproteins across the mitochondrial outer membrane is mediated by the TOM complex. Our previous studies led to the concept of two preprotein binding sites acting in series, the surface-exposed cis site and the trans site exposed to the intermembrane space. We report here that preproteins are bound to the cis site in a labile fashion even at low ionic strength, whereas intermediates arrested at the trans site remained firmly bound at higher salt concentration. The stability of the trans site intermediate results from interactions of both the presequence and unfolded parts of the mature part of the preprotein with the TOM complex. Binding to the trans site proceeded at rates comparable with those of unfolding of the mature domain and appeared to be kinetically limited by the unfolding reaction. Efficient binding to the trans site and unfolding were observed with both outer membrane vesicles and intact mitochondria whose membrane potential, DeltaPsi, was dissipated. Upon re-establishing DeltaPsi, trans site-bound preprotein resumed translocation into the matrix. The rates of unfolding and binding to the trans site were the same as those for translocation into intact energized mitochondria. We conclude that preprotein unfolding in intact mitochondria can take place without the involvement of the translocation machinery of the inner membrane and, in particular, the matrix Hsp70 chaperone. Further, preprotein unfolding at the outer membrane can be a rate-limiting step for formation of the trans site intermediate and for the entire translocation reaction.

Animals↗

PubMed1998

Efficacy of hyperthermic isolated limb perfusion for extremity-confined recurrent melanoma.

BACKGROUND: Recurrent melanoma of the extremity has been treated by local excision, systemic chemotherapy, amputation, or a combination of these approaches. Hyperthermic isolated limb perfusion (HILP) provides a method of limb preservation through isolation, allowing the administration of chemotherapy in higher doses than is possible through systemic treatment. METHODS: An experimental group of 59 HILP patients with melanoma recurrences of the extremity was studied prospectively. A control group of 248 melanoma patients with similar recurrences was excluded from HILP because their recurrences were in non-extremity locations. The experimental group underwent HILP and excision; the control group had excision only. The experimental procedure consisted of vascular isolation of the affected extremity and a 1-hour perfusion with melphalan. Temperatures were maintained at 40 degrees C in the perfusion circuit. RESULTS: The HILP patients had a lower rate of locoregional recurrence (P=.028) and demonstrated increased survival (P=.026) compared to the control group. In multivariate regression analysis, which included age, ulceration and thickness of the primary, and the treatment variable of perfusion, age (P=.02) and perfusion for the treatment of recurrence (P=.006) were significant predictors of survival. CONCLUSIONS: HILP improves prognosis by sterilizing the treated extremity, controlling locoregional disease, and perhaps preventing metastasis, thus having a positive impact on overall survival.

Antineoplastic Agents↗

PubMed1998

The dorsal cutaneous branch of the ulnar nerve. An anatomic clarification with six case reports.

We studied the anatomy and pathology of the dorsal cutaneous branch of the ulnar nerve by dissecting 10 fresh cadaver upper limbs and reviewing 6 cases of injury or entrapment of the dorsal cutaneous branch of the ulnar nerve. In all of the cadavers and in our series of cases, several anatomical features were apparent: 1) the dorsal cutaneous branch of the ulnar nerve arises from the main ulnar nerve an average of 5.5 centimeters proximal to the head of the ulna; 2) the dorsal cutaneous branch of the ulnar nerve reaches the dorsum of the hand after coursing volar to the ulnar head; 3) there was no communication between the dorsal cutaneous branch of the ulnar nerve and the superficial sensory branch of the radial nerve; and 4) no volar branches were noted. Based on our experience, disorders of this nerve are more prevalent than previously reported. This clarification of the anatomy will help prevent unnecessary injury during surgery and will be valuable in the diagnosis of disorders of the dorsal cutaneous branch of the ulnar nerve.

Adult↗

PubMed1998

Role of the negative charges in the cytosolic domain of TOM22 in the import of precursor proteins into mitochondria.

TOM22 is an essential mitochondrial outer membrane protein required for the import of precursor proteins into the organelles. The amino-terminal 84 amino acids of TOM22 extend into the cytosol and include 19 negatively and 6 positively charged residues. This region of the protein is thought to interact with positively charged presequences on mitochondrial preproteins, presumably via electrostatic interactions. We constructed a series of mutant derivatives of TOM22 in which 2 to 15 of the negatively charged residues in the cytosolic domain were changed to their corresponding amido forms. The mutant constructs were transformed into a sheltered Neurospora crassa heterokaryon bearing a tom22::hygromycin R disruption in one nucleus. All constructs restored viability to the disruption-carrying nucleus and gave rise to homokaryotic strains containing mutant tom22 alleles. Isolated mitochondria from three representative mutant strains, including the mutant carrying 15 neutralized residues (strain 861), imported precursor proteins at efficiencies comparable to those for wild-type organelles. Precursor binding studies with mitochondrial outer membrane vesicles from several of the mutant strains, including strain 861, revealed only slight differences from binding to wild-type vesicles. Deletion mutants lacking portions of the negatively charged region of TOM22 can also restore viability to the disruption-containing nucleus, but mutants lacking the entire region cannot. Taken together, these data suggest that an abundance of negative charges in the cytosolic domain of TOM22 is not essential for the binding or import of mitochondrial precursor proteins; however, other features in the domain are required.

Alleles↗

PubMed1998

Dynamics of the TOM complex of mitochondria during binding and translocation of preproteins.

Translocation of preproteins across the mitochondrial outer membrane is mediated by the TOM complex. This complex consists of receptor components for the initial contact with preproteins at the mitochondrial surface and membrane-embedded proteins which promote transport and form the translocation pore. In order to understand the interplay between the translocating preprotein and the constituents of the TOM complex, we analyzed the dynamics of the TOM complex of Neurospora crassa and Saccharomyces cerevisiae mitochondria by following the structural alterations of the essential pore component Tom40 during the translocation of preproteins. Tom40 exists in a homo-oligomeric assembly and dynamically interacts with Tom6. The Tom40 assembly is influenced by a block of negatively charged amino acid residues in the cytosolic domain of Tom22, indicating a cross-talk between preprotein receptors and the translocation pore. Preprotein binding to specific sites on either side of the outer membrane (cis and trans sites) induces distinct structural alterations of Tom40. To a large extent, these changes are mediated by interaction with the mitochondrial targeting sequence. We propose that such targeting sequence-induced adaptations are a critical feature of translocases in order to facilitate the movement of preproteins across cellular membranes.

Antibodies↗

PubMed1997

Mitochondrial protein import. Tom40 plays a major role in targeting and translocation of preproteins by forming a specific binding site for the presequence.

During preprotein transport across the mitochondrial outer membrane, the N-terminal presequence initially binds to a surface-exposed site, termed cis site, of the protein translocation complex of this membrane (the TOM complex). The presequence then moves into the translocation pore and becomes exposed at the intermembrane space side. Membrane passage is driven by specific interaction of the presequence with the trans site. We have used chemical cross-linking to identify components in the vicinity of the translocating presequence. Preproteins bound to the surface-exposed cis site can be cross-linked via their N-terminal presequence to Tom20 and Tom22, demonstrating their direct association with this part of the preprotein. In addition, the presequence establishes an early contact to Tom40, a membrane-embedded protein of the TOM complex. Upon further entry of the preprotein into the translocation pore, the presequence loses its contact with Tom20/Tom22, but remains in firm association with Tom40. Our study suggests that Tom40 plays an important function in guiding the presequence of a preprotein across the mitochondrial outer membrane. We propose that Tom40 forms a major part of the trans presequence binding site.

Animals↗

PubMed1997

Fusion peptides derived from the HIV type 1 glycoprotein 41 associate within phospholipid membranes and inhibit cell-cell Fusion. Structure-function study.

The fusion domain of human immunodeficiency virus (HIV-1) envelope glycoprotein (gp120-gp41) is a conserved hydrophobic region located at the N terminus of the transmembrane glycoprotein (gp41). A V2E mutant has been shown to dominantly interfere with wild-type envelope-mediated syncytium formation and virus infectivity. To understand this phenomenon, a 33-residue peptide (wild type, WT) identical to the N-terminal segment of gp41 and its V2E mutant were synthesized, fluorescently labeled, and characterized. Both peptides inhibited HIV-1 envelope-mediated cell-cell fusion and had similar alpha-helical content in membrane mimetic environments. Studies with fluorescently labeled peptide analogues revealed that both peptides have high affinity for phospholipid membranes, are susceptible to digestion by proteinase-K in their membrane-bound state, and tend to self- and coassemble in the membranes. In SDS-polyacrylamide gel electrophoresis the WT peptide formed dimers as well as higher order oligomers, whereas the V2E mutant only formed dimers. The WT, but not the V2E mutant, induced liposome aggregation, destabilization, and fusion. Moreover, the V2E mutant inhibited vesicle fusion induced by the WT peptide, probably by forming inactive heteroaggregates. These data form the basis for an explanation of the mechanism by which the gp41 V2E mutant inhibits HIV-1 infectivity in cells when co-expressed with WT gp41.

Amino Acid Sequence↗

PubMed1997

Multiple primary melanomas: implications for screening and follow-up programs for melanoma.

BACKGROUND: Once individuals are diagnosed with malignant melanoma, they are at an increased risk of developing another melanoma when compared with the normal population. METHODS: To determine the impact of an intensive follow-up protocol on the stage of disease at diagnosis of subsequent primary melanomas, a retrospective query was performed of an electronic medical record database of 2,600 consecutively registered melanoma patients. RESULTS: Sixty-seven patients (2.6%) had another melanoma diagnosed at the time of presentation to the clinic or within 2 months (synchronous) and another 44 patients (1.7%) developed a second primary melanoma during the follow-up period (metachronous). For the 44 patients diagnosed with metachronous lesions, the Breslow mean tumor thickness for the first invasive melanoma was 2.27 mm compared with 0.90 mm for the second melanoma. The first melanomas diagnosed are thicker by an average of 3.8 mm (p = 0.008). The mean Clark level for the initial melanoma was greater than the mean level for subsequently diagnosed melanomas (p = 0.002). Twenty-three percent of the initial melanomas were ulcerated, whereas only one of the second primary lesions showed this adverse prognostic factor (p = 0.002). CONCLUSIONS: Once individuals are diagnosed with melanoma, they are in a high-risk population for having other primary site melanomas diagnosed and should be placed in an intensive follow-up protocol consisting of a complete skin examination.

Adult↗