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PubMed2006

Efficient enantioselective synthesis of condensed and aromatic-ring-substituted tyrosine derivatives.

An efficient access to both condensed and conjugated tyrosine analogues of high enantiomeric purity is described. Novel ring-substituted tyrosines were synthesized by Suzuki cross couplings of appropriately protected l-3-iodotyrosine with a series of activated and deactivated boronic acid derivatives to achieve the target compounds in high yields. d- and l-4-hydroxy-1-naphthylalanines were readily prepared from the corresponding alpha-enamide in two different approaches, by asymmetric hydrogenation as well as by unselective hydrogenation and enzymatic resolution of the racemic mixture.

Catalysis↗

PubMed2006

S-adenosylhomocysteine analogues with the carbon-5' and sulfur atoms replaced by a vinyl unit.

Cross-metathesis of suitably protected 5'-deoxy-5'-methyleneadenosines with racemic and chiral N-Boc-protected six-carbon amino acids bearing a terminal double bond in the presence of the Hoveyda-Grubbs catalyst gave adenosylhomocysteine analogues with the C5'-C6' double bond. Bromination with pyridinium tribromide and dehydrobromination with DBU followed by standard deprotections yielded the 5'-(bromo)vinyl analogue. [structure: see text]

Adenosine↗

PubMed2005

The exon junction core complex is locked onto RNA by inhibition of eIF4AIII ATPase activity.

The multiprotein exon junction complex (EJC) is assembled on mRNAs as a consequence of splicing. EJC core components maintain a stable grip on mRNAs even as the overall EJC protein composition evolves while mRNAs travel to the cytoplasm. Here we show that recombinant EJC subunits MLN51, MAGOH and Y14, together with the DEAD-box protein eIF4AIII bound to ATP, are necessary and sufficient to form a highly stable complex on single-stranded RNA. Cross-linking and RNase protection studies indicate that this recombinant complex recapitulates the EJC core. The stable association of the recombinant EJC core with RNA is maintained by inhibition of eIF4AIII ATPase activity by MAGOH-Y14. We elucidate the modalities of EJC binding to RNA and provide the first example of how cellular machineries may use RNA helicases to clamp several proteins onto RNA in stable and sequence-independent manners.

Adenosine Triphosphatases↗

PubMed1994

Large-scale production of Ba(2+)-alginate-coated islets of Langerhans for immunoisolation.

Islet xenografts immunisolated in alginate capsules have been proposed by many groups for clinical islet transplantation. However, diffusion limitations and the total volume of microcapsules required for transplantation are, among other things, factors which have so far prevented successful clinical application. In this study, these problems have been overcome by immobilisation of rat and porcine islets in a Ba(2+)-cross-linked alignate matrix using an air jet droplet generator technique in combination with subsequent density-gradient purification. This procedure leads to high yields of islets coated with a tailored, thin layer of cross-linked alginate which protects the islets against cytotoxic compounds present in human plasma. The recovery of encapsulated rat and porcine islets was about 70%. Empty capsules were nearly completely removed. Using this technique, the total volume of the resulting microcapsules increased only by a factor of about 1.5 compared to the volume of free islets. The technique can be used for large-scale production of coated islets.

Alginates↗

PubMed1980

Comparison of three methods of myocardial protection.

Ischemic arrest (45 minutes), injection cardioplegia (2-3 ml/kg body weight within 1 minute) using Cardioplegin (magnesium-aspartate-procaine-sorbitol) according to Kirsch (90 minutes), and infusion cardioplegia (30 ml/kg body weight within 5 to 7 minutes) employing solution LK 352 (sodium-potassium-magnesium-aspartate-sorbitol) according to Bretschneider (90 minutes) were investigated in dogs in order to evaluate their efficacy of protection during myocardial ischemia and their effects on post-ischemic cardiac function. Parameters studied were myocardial tissue levels of adenine nucleotides, creatine phosphate, total creatine and glycogen as well as heart rate, systemic pressure, cardiac output, LVEDP, left ventricular pressure-volume-work, dp/dtmax, t-dp/dtmax, (dp/dtmax)/IP, and Vpm. According to biochemical data corrected for differences in myocardial temperature during arrest, myocardial protection during aortic cross-clamping at a core temperature of 30 degrees C was increased 1.8-fold by Cardioplegin and 3.4-fold by LK 352 administration respectively when compared to ischemic arrest. Functional recovery was poor after ischemic arrest and better after Cardiplegin arrest. Infusion cardioplegia with LK 352 did not result in a post-arrest depression of functional parameters.

Animals↗

PubMed1988

Anomalous carotid-basilar anastomoses in cerebrovascular surgery.

Primitive embryonic anastomotic vessels between the internal carotid and basilar arterial systems occasionally persist into adult life. We encountered six such patients in a series of 765 carotid endarterectomies (0.8%). These arteries are named for the cranial nerves with which they run. The most commonly found vessel is the trigeminal artery, followed by the hypoglossal and proatlantal intersegmental arteries. The primitive otic artery is the rarest of the four anomalous vessels. These vessels are usually large and are associated with hypoplasia or aplasia of the normal intracranial anastomotic channels. Cerebral ischemia is common during temporary intraoperative carotid occlusion and was encountered in two of three patients who had intraoperative electroencephalographic monitoring. Thus cerebral ischemia should be anticipated during carotid cross-clamping and cerebral protective measures should be instituted when persistent primitive cranial vessels are encountered.

Basilar Artery↗

PubMed1999

Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA.

The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from cross-linking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism.

Aerobiosis↗

PubMed2003

Regulation of tension-induced mechanotranscriptional signals by the microtubule network in fibroblasts.

Mechanical loading of connective tissues induces the expression of extracellular matrix and cytoskeletal genes that are involved in matrix remodeling. These processes depend in part on force transmission through beta1 integrins and actin filaments, but the role of microtubules in regulating mechanotranscriptional responses is not well defined. We assessed the involvement of microtubules in the mechanotranscriptional regulation of filamin A, an actin-cross-linking protein that protects cells against force-induced apoptosis by stabilizing cell membranes. Collagen-coated magnetite beads and magnetic fields were used to apply tensile forces to cultured fibroblasts at focal adhesions. Force enhanced recruitment of alpha-tubulin and the plus end microtubule-binding protein cytoplasmic linker protein-170 (CLIP-170) at focal adhesions. Immunoprecipitation studies demonstrated no direct binding of tubulin to actin or filamin A, but CLIP-170 interacted with tubulin, filamin A, and beta-actin. The association of CLIP-170 with beta-actin was enhanced by force. Force activated the p38 mitogen-activated protein kinase, increased filamin A expression, and induced the relocation of p38 and filamin A to focal adhesions. Disruption of microtubules with nocodazole, independent of force application, enhanced filamin A expression and Sp1-mediated filamin A promoter activity, while stabilization of microtubules with Taxol inhibited force induction of both filamin A mRNA and protein. We conclude that in response to tensile forces applied through beta1 integrins and actin the microtubule network modulates mechanotranscriptional coupling of filamin A.

Actins↗

PubMed2004

Telosome, a mammalian telomere-associated complex formed by multiple telomeric proteins.

In mammalian cells, telomere-binding proteins TRF1 and TRF2 play crucial roles in telomere biology. They interact with several other telomere regulators including TIN2, PTOP, POT1, and RAP1 to ensure proper maintenance of telomeres. TRF1 and TRF2 are believed to exert distinct functions. TRF1 forms a complex with TIN2, PTOP, and POT1 and regulates telomere length, whereas TRF2 mediates t-loop formation and end protection. However, whether cross-talk occurs between the TRF1 and TRF2 complexes and how the signals from these complexes are integrated for telomere maintenance remain to be elucidated. Through gel filtration and co-immunoprecipitation experiments, we found that TRF1 and TRF2 are in fact subunits of a telomere-associated high molecular weight complex (telosome) that also contains POT1, PTOP, RAP1, and TIN2. We demonstrated that the TRF1-interacting protein TIN2 binds TRF2 directly and in vivo, thereby bridging TRF2 to TRF1. Consistent with this multi-protein telosome model, stripping TRF1 off the telomeres by expressing tankyrase reduced telomere recruitment of not only TIN2 but also TRF2. These results help to unify previous observations and suggest that telomere maintenance depends on the multi-subunit telosome.

Blotting, Western↗

PubMed2006

Induction of the heat shock pathway during hypoxia requires regulation of heat shock factor by hypoxia-inducible factor-1.

Activation of heat shock proteins (Hsps) is critical to adaptation to low oxygen levels (hypoxia) and for enduring the oxidative stress of reoxygenation. Hsps are known to be regulated by heat shock factor (Hsf), but our results demonstrate an unexpected regulatory link between the oxygen-sensing and heat shock pathways. Hsf transcription is up-regulated during hypoxia due to direct binding by hypoxia-inducible factor-1 (HIF-1) to HIF-1 response elements in an Hsf intron. This increase in Hsf transcripts is necessary for full Hsp induction during hypoxia and reoxygenation. The HIF-1-dependent increase in Hsps has a functional impact, as reduced production of Hsps decreases viability of adult flies exposed to hypoxia and reoxygenation. Thus, HIF-1 control of Hsf transcriptional levels is a regulatory mechanism for sensitizing heat shock pathway activity in order to maximize production of protective Hsps. This cross-regulation represents a mechanism by which the low oxygen response pathway has assimilated complex new functions by regulating the key transcriptional activator of the heat shock pathway.

Animals↗

PubMed2005

Private specificities of CD8 T cell responses control patterns of heterologous immunity.

CD8 T cell cross-reactivity between viruses can play roles in protective heterologous immunity and damaging immunopathology. This cross-reactivity is sometimes predictable, such as between lymphocytic choriomeningitis virus (LCMV) and Pichinde virus, where cross-reactive epitopes share six out of eight amino acids. Here, however, we demonstrate more subtle and less predictable cross-reactivity between LCMV and the unrelated vaccinia virus (VV). Epitope-specific T cell receptor usage differed between individual LCMV-infected C57BL/6 mice, even though the mice had similar epitope-specific T cell hierarchies. LCMV-immune mice challenged with VV showed variations, albeit in a distinct hierarchy, in proliferative expansions of and down-regulation of IL-7Ralpha by T cells specific to different LCMV epitopes. T cell responses to a VV-encoded epitope that is cross-reactive with LCMV fluctuated greatly in VV-infected LCMV-immune mice. Adoptive transfers of splenocytes from individual LCMV-immune donors resulted in nearly identical VV-induced responses in each of several recipients, but responses differed depending on the donor. This indicates that the specificities of T cell responses that are not shared between individuals may influence cross-reactivity with other antigens and play roles in heterologous immunity upon encounter with another pathogen. This variability in cross-reactive T cell expansion that is unique to the individual may underlie variation in the pathogenesis of infectious diseases.

Adoptive Transfer↗

PubMed2006

Vaccines for seasonal and pandemic influenza.

Seasonal influenza continues to have a huge annual impact in the United States, accounting for tens of millions of illnesses, hundreds of thousands of excess hospitalizations, and tens of thousands of excess deaths. Vaccination remains the mainstay for the prevention of influenza. In the United States, 2 types of influenza vaccine are currently licensed: trivalent inactivated influenza vaccine and live attenuated influenza vaccine. Both are safe and effective in the populations for which they are approved for use. Children, adults <65 years of age, and the elderly all receive substantial health benefits from vaccination. In addition, vaccination appears to be cost-effective, if not cost saving, across the age spectrum. Despite long-standing recommendations for the routine vaccination of persons in high-priority groups, US vaccination rates remain too low across all age groups. Important issues to be addressed include improving vaccine delivery to current and expanded target groups, ensuring timely availability of adequate vaccine supply, and development of even more effective vaccines. Development of a vaccine against potentially pandemic strains is an essential part of the strategy to control and prevent a pandemic outbreak. The use of existing technologies for influenza vaccine production would be the most straightforward approach, because these technologies are commercially available and licensing would be relatively simple. Approaches currently being tested include subvirion inactivated vaccines and cold-adapted, live attenuated vaccines. Preliminary results have suggested that, for some pandemic antigens, particularly H5, subvirion inactivated vaccines are poorly immunogenic, for reasons that are not clear. Data from evaluation of live pandemic vaccines are pending. Second-generation approaches designed to provide improved immune responses at lower doses have focused on adjuvants such as alum and MF59, which are currently licensed for influenza or other vaccines. Additional experimental approaches are required to achieve the ultimate goal for seasonal and pandemic influenza prevention--namely, the ability to generate broadly cross-reactive and durable protection in humans.

Adjuvants, Immunologic↗

PubMed1982

Characterization of two new plasmid DNAs found in mitochondria of wild-type Neurospora intermedia strains.

Mitochondria from two Neurospora intermedia strains (P4O5-Labelle and Fiji N6-6) were found to contain plasmid DNAs in addition to the standard mitochondrial DNA species. The plasmid DNAs consist of monomeric circles (4.1-4.3 kbp and 5.2-5.3 kbp for Labelle and Fiji, respectively) and oligomers in which monomers are organized as head-to-tail repeats. DNA-DNA hybridization experiments showed that the plasmids have no substantial sequence homology to mtDNA, to each other, or to a previously characterized mitochondrial plasmid from N. crassa strain Mauriceville-lc (Collins et al. Cell 24, 443-452, 1981). The intramitochondrial location of the plasmids was established by cell fractionation and nuclease protection experiments. In sexual crosses, the plasmids showed strict maternal inheritance, the same as Neurospora mitochondrial DNA. The plasmids may represent a novel class of mitochondrial genetic elements.

Base Composition↗

PubMed2005

Novel modified purine bases and nucleosides: new methodologies of synthesis and biological activity.

Novel modified purine derivatives (bases or nucleosides) bearing C-substituents in position(s) 6, 2 and/or 8 were prepared by Pd- or Fe-catalyzed cross-coupling reactions of halopurines with organometallics. Reactions of di- and trihalopurines are regioselective and applicable to the synthesis of di- or trisubstituted purines bearing different substituents. Cross-coupling reactions of protected functionalized organometallics were used for the preparation of purines bearing functionalized C-substituents. Some of the title modified purine bases and nucleosides display cytostatic activity.

Antineoplastic Agents↗

PubMed2004

New devices for percutaneous coronary intervention are rapidly making bypass surgery obsolete.

PURPOSE OF REVIEW: This review summarizes recent and cumulative progress in the success, safety, applicability, and durability of percutaneous coronary intervention. RECENT FINDINGS: Improvements in basic percutaneous coronary intervention (PCI) equipment and the availability of bare metal stents in the mid-1990s improved acute procedural success to 98%, reduced the emergency surgery rate to 0.2%, and reduced the incidence of recurrent symptoms due to restenosis at the treated site to 15 to 20%. The recent availability of drug-eluting stents has reduced the in-stent neointimal proliferation that causes restenosis and reduced the incidence of symptomatic recurrence to less than 5%, rivaling that of bypass surgery. The work on better antithrombotic pharmacology, distal embolic protection, and devices for crossing chronic total occlusions will further add to the armamentarium for catheter-based revascularization. SUMMARY: Based on progress over the past decade, PCI has grown to represent about two-thirds of all coronary revascularization (800,000 PCI vs 350,000 bypass surgeries). Recent and ongoing progress will make bypass surgery largely obsolete within the next several years.

Angioplasty, Balloon, Coronary↗

PubMed2005

Identification of karyopherin beta as an immunogenic antigen of the malaria parasite using immune mice and human sera.

A differential immunoscreening of the lambdagt11 Plasmodium falciparum genomic expression library was carried out using anti-P. yoelii sera (convalescent-phase mouse sera) and immune sera collected from healthy adults, to identify novel cross-reactive and possibly protective antigens of the parasite. One clone, with an insert size of 1132 bp that reacted strongly with both the sera was selected. The insert was found to be a part of the P. falciparum karyopherin beta (PfKbeta) homologue. RT-PCR and Northern blot analysis confirmed the expression of PfKbeta in the blood stages of the parasite. The approximately 110 kDa protein was localized in the cytoplasm at the ring and trophozoite, and in the parasitophorous vacuole at the schizont stage. Two large fragments of PfKbeta representing the N- and C-terminal halves were expressed in E. coli. The recombinant proteins were highly immunogenic in mice, and also found to be the target for immune response in natural infections of Plasmodium spp. Anti-sera against the protein showed a low level of anti-parasitic activity. Immunization with recombinant PfKbeta fragments was only partially protective against a heterologous challenge infection in mice. Our results show that the parasite releases a highly immunogenic, cytoplasmic protein into the host which may not contribute to the development of protective immunity.

Animals↗

PubMed2005

Induction of systemic and mucosal cross-clade neutralizing antibodies in BALB/c mice immunized with human immunodeficiency virus type 1 clade A virus-like particles administered by different routes of inoculation.

We have recently developed a candidate human immunodeficiency virus type 1 (HIV-1) vaccine model, based on virus-like particles (VLPs) expressing gp120 from a Ugandan HIV-1 isolate of clade A (HIV-VLP(A)s), which shows the induction of neutralizing antibodies as well as cytotoxic T lymphocytes (CTL) in BALB/c mice by intraperitoneal (i.p.) administration. In the present study, immunization experiments based on a multiple-dose regimen have been performed with BALB/c mice to compare different routes of administration. i.p. and intranasal (i.n.), but not oral, administration induce systemic as well as mucosal (vaginal and intestinal) immunoglobulin G (IgG) and IgA responses. These immune sera exhibit >50% ex vivo neutralizing activity against both autologous and heterologous primary isolates. Furthermore, the administration of HIV-VLP(A)s by the i.n. immunization route induces a specific CTL activity, although at lower efficiency than the i.p. route. The HIV-VLP(A)s represent an efficient strategy to stimulate both arms of immunity; furthermore, the induction of specific humoral immunity at mucosal sites, which nowadays represent the main port of entry for HIV-1 infection, is of great interest. All these properties, and the possible cross-clade in vivo protection, could make these HIV-VLP(A)s a good candidate for a mono- and multicomponent worldwide preventive vaccine approach not restricted to high-priority regions, such as sub-Saharan countries.

AIDS Vaccines↗

PubMed2005

Relation of sexual risks and prevention practices with individuals' stigmatising beliefs towards HIV infected individuals: an exploratory study.

OBJECTIVE: To investigate how an individual's stigmatising beliefs towards people living with HIV are related to his or her own sexual risk and protective behaviours. METHODS: A cross sectional survey was conducted to assess HIV related stigmatising beliefs, risk sexual behaviours, and preventive practices among sexually experienced rural to urban migrants aged 18-30 years in 2002 in Beijing and Nanjing, two large Chinese cities. RESULTS: Among 2153 migrants, 7.2% reported having had more than one sexual partner in the previous month, 9.9% had commercial sex partners, and 12.5% had an episode of a sexually transmitted disease (STD). Only 18% reported frequently or always using condoms, with 20% sometimes or occasionally using them. 57% of the Chinese migrants were willing to take a voluntary HIV test, and 65% had HIV related stigmatising beliefs towards people living with HIV. Multiple logistic regression analysis depicts that individual's stigmatising beliefs towards people with HIV were positively associated with having had an episode of an STD, having multiple sex partners, or having had commercial sex partners, and were negatively associated with condom use and the willingness to accept an HIV test. CONCLUSION: The finding that one's own stigmatising belief is a potential barrier to HIV related preventive practices highlights the difficulties and challenges in implementing behavioural interventions.

Adolescent↗