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PubMed1996

Toward cloning genes by complementation in Paramecium.

Conventional methods of gene cloning by complementing mutant defects is made difficult by the 800 ploidy of the Paramecium macronucleus. However, this nucleus is some 30 microns in diameter and readily propagates exogenous DNA fragments as cells divide. These attributes allow for massive injection of engineered DNA fragments and their maintenance in the transformed descendant. If a genomic DNA fraction injected into a mutant macronucleus effects complementation, it should be possible to sort a fractional library to isolate the complementing gene. Here, we investigated four aspects of establishing this method for general use. First, using the cloned CAM gene as a test case, we further investigated transformation by macronuclear injection and showed that phenotypic reversion is directly correlated with the copy number of the transgene, even when it is of a recessive allele, cam2, which has a missense mutation but produces a partially functional protein. Second, we examined the copy number of the transgene established in cells of older clonal age and discussed the likely dilution of the transgene in younger descendants of the injected cell. Third, we showed that the degree of phenotypic reversion is correlated with the transgene product, the cam2 calmodulin protein in the cell. Fourth, we extended the investigation to very recessive mutants whose genes are to be cloned. We showed that size fractions of wild-type genomic DNA digests effect strong phenotypic reversions in several pawn mutants, setting the stage for cloning these Ca(2+)-channel related genes. The general usefulness of this method in cloning genes that complement recessive alleles and current limitations of this method in dealing with dominant alleles are assessed and discussed.

Animals↗
PubMed1975

[Cellular responses to vibration during dental drilling with belt drive motor engine (in vitro) (author's transl)].

There have been many reports on the pulpal reaction caused by vibration during high speed dental drilling with air turbine hand-piece. It is, however, not clear pulpal responses to vibration of low speed drilling with 7 000 rpm of belt drive motor engine which produces a fundamental vibration of about 170 cps having most unpleasant sensation at vestibular apparatus. Here, cellular responses of L strain cells to vibration derived from 7 000 rpm and 15 000 rpm of belt drive motor engine were observed in vitro system. There is no difference between the both cellular responses to the vibration of 7 000 rpm and 15 000 rpm. Suspended free cells were easily influenced and degenerated by the vibration, and remarkably checked in their cell-multiplication. On the contrary, in the cells adhered to the substratum of glassware, no degeneration and no inhibitory effect were observed. And the cells showed reversible response of cytoplasmic shrinkage only.

Cell Adhesion↗
PubMed2006

Cre recombinase-mediated restoration of nigrostriatal dopamine in dopamine-deficient mice reverses hypophagia and bradykinesia.

A line of dopamine-deficient (DD) mice was generated to allow selective restoration of normal dopamine signaling to specific brain regions. These DD floxed stop (DDfs) mice have a nonfunctional Tyrosine hydroxylase (Th) gene because of insertion of a NeoR gene flanked by lox P sites targeted to the first intron of the Th gene. DDfs mice have trace brain dopamine content, severe hypoactivity, and aphagia, and they die without intervention. However, they can be maintained by daily treatment with l-3,4-dihydroxyphenylalanine (L-dopa). Injection of a canine adenovirus (CAV-2) engineered to express Cre recombinase into the central caudate putamen restores normal Th gene expression to the midbrain dopamine neurons that project there because CAV-2 efficiently transduces axon terminals and is retrogradely transported to neuronal cell bodies. Bilateral injection of Cre recombinase into the central caudate putamen restores feeding and normalizes locomotion in DDfs mice. Analysis of feeding behavior by using lickometer cages revealed that virally rescued DDfs mice are hyperphagic and have modified meal structures compared with control mice. The virally rescued DDfs mice are also hyperactive at night, have reduced motor coordination, and are thigmotactic compared with controls. These results highlight the critical role for dopamine signaling in the dorsal striatum for most dopamine-dependent behaviors but suggest that dopamine signaling in other brain regions is important to fine-tune these behaviors. This approach offers numerous advantages compared with previous models aimed at examining dopamine signaling in discrete dopaminergic circuits.

Adenoviridae↗
PubMed2001

Lymphotactin expression by engineered myeloma cells drives tumor regression: mediation by CD4+ and CD8+ T cells and neutrophils expressing XCR1 receptor.

The C chemokine lymphotactin has been characterized as a T cell chemoattractant both in vitro and in vivo. To determine whether lymphotactin expression within tumors could influence tumor growth, we transfected an expression vector for lymphotactin into SP2/0 myeloma cells and tested their ability to form tumors in BALB/c and nude mice. Transfection did not alter cell growth in vitro. Whereas SP2/0 cells gave rise to a 100% tumor incidence, lymphotactin-expressing SP2/0-Lptn tumors invariably regressed in BALB/c mice and became infiltrated with CD4(+) and CD8(+) T cells and neutrophils. Regression of the SP2/0-Lptn tumors was associated with a type 1 cytokine response and dependent on both CD4(+) and CD8(+) T cells, but not NK cells. Both SP2/0 and SP2/0-Lptn tumors grew in nude mice, but growth of the latter tumors was retarded and associated with heavy neutrophil responses; this retardation of SP2/0-Lptn tumor growth was reversed by neutrophil depletion of the mice. Our data also indicate that mouse neutrophils express the lymphotactin receptor XCR1 and that lymphotactin specifically chemoattracts these cells in vitro. Thus, lymphotactin has natural adjuvant activities that may augment antitumor responses via effects on both T cells and neutrophils and thereby could be important in gene transfer immunotherapies for some cancers.

Animals↗
PubMedDate not supplied

Dynamic deformational loading results in selective application of mechanical stimulation in a layered, tissue-engineered cartilage construct.

The application of dynamic physiologic loading to a bilayered chondrocyte-seeded agarose construct with a 2% (wt/vol) top layer and 3% (wt/vol) bottom layer was hypothesized to (1) improve overall construct properties and (2) result in a tissue that mimics the mechanical inhomogeneity of native cartilage. Dynamic loading over the 28 day culture period was found to significantly increase bulk mechanical and biochemical properties versus free-swelling culture. The initial depth-distribution of the compressive Young's modulus (EY) reflected the intrinsic properties of the gel in each layer and a similar trend to the native tissue, with a softer 2% gel layer and a much stiffer 3% gel layer. After 28 days in culture, free-swelling conditions maintained this general trend while loaded constructs possessed a reverse profile, with significant increases in EY observed only in the 2% gel. Histological analysis revealed preferential matrix formation in the 2% agarose layer, with matrix localized more pericellularly in the 3% agarose layer. Finite element modeling revealed that, prior to significant matrix elaboration, the 2% layer experiences increased mechanical stimuli (fluid flow and compressive strain) during loading that may enhance chondrocyte stimulation and nutrient transport in that layer, consistent with experimental observations. From these results, we conclude that due to the limitations in 3% agarose, the use of this type of bilayered construct to construct depth-dependent inhomogeneity similar to the native tissue is not likely to be successful under long-term culture conditions. Our study underscores the importance of other physical properties of the scaffold that may have a greater influence on interconnected tissue formation than intrinsic scaffold stiffness.

Animals↗
PubMed2000

Characterization of novel safe lentiviral vectors derived from simian immunodeficiency virus (SIVmac251) that efficiently transduce mature human dendritic cells.

We describe the generation and the characterization of new lentiviral vectors derived from SIVmac251, a simian immunodeficiency virus (SIV). A methodical approach was used to engineer both efficient and safe packaging constructs allowing the production of SIV viral core proteins. SIV-vectors encoding GFP (green fluorescent protein) were generated as VSV-G-pseudotyped particles upon transient expression of the vector construct and helper functions in 293 cells. The SIV vectors were able to transduce efficiently various target cell types at low multiplicity of infection, including monocyte-differentiated human dendritic cells (DCs) which retained their capacity to differentiate into mature DCs after gene transfer. Transduction of the DCs by the SIV vectors was prevented when infections were performed in the presence of AZT, a reverse-transcriptase inhibitor. After gene transfer, expression of the GFP in the target cells remained constant after several weeks, indicating that the vectors had been stably integrated into the genome of the host cells. Preparations of SIV vectors were systematically checked for the absence of replication-competent and recombinant retroviruses but remained negative, suggesting the innocuousness of these novel gene delivery vectors. Side-to-side comparisons with vectors derived from HIV-1 (human immunodeficiency virus) indicated that the SIV vectors were equally potent in transducing proliferating target cells. Finally, we have determined the infectivity of SIV vectors pseudotyped with surface glycoproteins of several membrane-enveloped viruses.

Animals↗
PubMed2000

Expression of hTERT and hTR in cis reconstitutes and active human telomerase ribonucleoprotein.

Telomeres in eukaryotic cells are generally synthesized and maintained by the ribonucleoprotein (RNP) telomerase. This enzyme is composed of at least two subunits, the telomerase reverse transcriptase (TERT) and the telomerase RNA. Human telomerase activity can be reconstituted in vitro by the expression of the telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. The hTERT and hTR subunits are independently expressed in vivo, and little is known about the mechanism of their assembly. To facilitate recombinant telomerase RNP formation and reconstitution, we engineered a construct, termed hTERT-hTR cis, in which the 3' end of the hTERT coding sequence was extended by the addition of the sequence encoding hTR. Expression of the hTERT-hTR cis construct in vitro (in RRL) and in vivo (in the yeast Saccharomyces cerevisiae) produced hTERT-hTR transcripts of the predicted size. Active human telomerase was reconstituted by hTERT-hTR cis expression in both RRL and S. cerevisiae. Assembly of functional human telomerase by the bicistronic expression of the protein and RNA components may facilitate the overexpression and reconstitution of this enzyme in heterologous systems.

Animals↗
PubMedDate not supplied

Mechanistic investigation of smart polymer-protein conjugates.

Many affinity separation and diagnostic applications rely upon both capture and release steps. There is thus a need for methods to enhance the reversibility of biomolecular interactions. We have previously demonstrated that stimuli-responsive polymers can be used to gate biomolecular reactions when conjugated near the active site of proteins. Here we have used a new smart polymer, N,N-dimethyl acrylamide-co-4-phenylazophenylacrylate that has allowed a mechanistic investigation of the smart polymer switches. This polymer was conjugated via a vinyl sulfone terminus to cysteine residues of genetically engineered streptavidin mutant E116C, where the polymer is conjugated close to the biotin-binding site, and streptavidin mutant S139C, where the conjugation site is distant. The biotin binding switching activity was strongly dependent on conjugation position, as the E116C conjugate displayed a large thermal response while the S139C conjugate displayed only small effects. Kinetic measurements of biotin release demonstrated that the off-rate of biotin was unperturbed and that the thermally triggered release of biotin with the E116C conjugate was due to the blocking the reassociation of biotin. The addition of free polymer to purified E116C conjugates was also shown to increase the blocking and release properties of the switch. This effect was site dependent, suggesting that the conjugated polymers were directing a physical aggregation near the binding site that effectively enhanced the switching activity. These investigations provide mechanistic insight that can be utilized to design better molecular switches for a variety of stimuli-responsive polymer-protein conjugates.

Acrylates↗
PubMed1970

Mechanochemical turbine: a new power cycle.

A basic thermodynamic cycle for the production of mechanical power from materials that may be made to contract forcibly and reversibly is described. The cycle differs from existing mechanical power cycles which generally employ expanding fluid as working substances. A "contraction turbine" operating on this cycle has been devised, which has collagen fiber as its working substance and salt solution as fuel and produces mechanical work directly from chemical free energy. Direct conversion of chemical to mechanical energy is routinely effected in muscle but not in usual man-made engines.

Bromides↗
PubMed2006

Trastuzumab in combination with heregulin-activated Her-2 (erbB-2) triggers a receptor-enhanced chemosensitivity effect in the absence of Her-2 overexpression.

PURPOSE: The decision for treating breast cancer patients with trastuzumab is based on HER-2 amplification and/or overexpression. METHODS: Using MCF-7 cells (Her-2 +/-) engineered to overexpress heregulin (MCF-7/HRG), a ligand for the Her-2/3/4 network, we investigated whether HRG-induced transactivation of Her-2 affected breast cancer cell sensitivity to chemotherapy and whether trastuzumab trigger receptor-enhanced chemosensitivity (REC) when combined with chemotherapy without Her-2 overexpression. RESULTS: MCF-7/HRG cells were more than 10-fold resistant to the alkylating agent cisplatin (CDDP), while trastuzumab coexposure completely reversed HRG-promoted CDDP resistance. A synergistic interaction between trastuzumab in combination with CDDP (paclitaxel or vincristine) was obtained in MCF-7/HRG cells. Trastuzumab prevented activation of the antiapoptotic and proliferative cascades and inhibited HRG-induced Her-2/3 phosphorylation. CDDP efficacy was enhanced by trastuzumab in cells expressing endogenously high levels of HRG. Conversely, trastuzumab coexposure was ineffective in enhancing chemotherapy efficacy in cells that did not secrete HRG, such as MCF-7 cells overexpressing a structural mutated HRG isoform. Therefore, trastuzumab-induced REC, in the absence of Her-2 overexpression, occurs through the kinase activity of Her-2/3. Interestingly, HRG expression in tumor biopsies from invasive breast carcinomas (n = 189) revealed that, whereas the minority (12%) of Her-2 positive tumors (n = 60; 32%) demonstrated Her-2 phosphorylation, the majority (67%) of HRG-overexpressing and Her-2 tumors (n = 57; 30%) were in active Her-2 status. CONCLUSION: We demonstrate that assessment of HRG expression and Her-2 activation define a particular breast cancer patient population for which trastuzumab plus CDDP or taxol are extremely efficient without Her-2 overexpression.

Antibodies, Monoclonal↗
PubMed2004

A suspected case of delayed onset malignant hyperthermia with desflurane anesthesia.

UNLABELLED: Desflurane has been identified as a weak triggering anesthetic of malignant hyperthermia that, in the absence of succinylcholine, may produce a delayed onset of symptoms. The prolonged interval after exposure may occur more than 6 h after the induction of anesthesia. The unintended underdosing of this patient with dantrolene and the prompt reversal of symptoms may be an attribute of the genetic expression of a weak triggering volatile anesthetic such as desflurane. IMPLICATIONS: There are multiple genetic variations for malignant hyperthermia (MH) at the ryanodine receptor. Desflurane, as a sole trigger of MH, is weak, and on two occasions in the literature (including this case), less than optimal doses of dantrolene were given with a good result. There may be possible to engineer the risk of MH out of an anesthetic once the genetics of the ryanodine receptor are better understood.

Anesthesia, Inhalation↗
PubMed2004

Hsp70 mutant proteins modulate additional apoptotic pathways and improve cell survival.

Although wild-type Hsp70 (Hsp70WT) inhibits procaspase-3 processing by preventing apoptosome formation, Hsp70WT does not block active caspase-3. Because all caspase-3 inhibitors bear canonical DXXD caspase-3 recognition motifs, we determined whether mutated Hsp70s with caspase-binding motifs act as direct caspase-3 inhibitors. Based on Hsp70 molecular modeling, the DNQP, DEVQ, and EEVD regions localized on the surface of Hsp70WT were chosen, allowing us to design mutants while trying to avoid disrupting the global fold of the molecule and losing the possibility of protein-protein interactions. We replaced DNQP with DQMD, and DEVQ and EEVD with DEVD residues that should be optimal substrates for caspase-3. The resultant Hsp70 mutants directly interacted with active caspase-3 and blocked its proteolytic activity while retaining the ability to reverse protein denaturation and disrupt the interaction between Apaf-1 and procaspase-9. The Hsp70C-terminal mutants interacted with Apaf-1 and active caspase-3 significantly longer than Hsp70WT. The Hsp70 DXXD mutants protected neuron and teratocarcinoma (NT) cells against cell death much better than Hsp70WT whether given before or after serum withdrawal. Hsp70 mutants represent a possible approach to antiapoptotic biotherapeutics. Similar rational designs could be used to engineer inhibitors of additional caspase family members.

Amino Acid Motifs↗
PubMed2003

CYP83A1 and CYP83B1, two nonredundant cytochrome P450 enzymes metabolizing oximes in the biosynthesis of glucosinolates in Arabidopsis.

In the glucosinolate pathway, the postoxime enzymes have been proposed to have low specificity for the side chain and high specificity for the functional group. Here, we provide biochemical evidence for the functional role of the two cytochromes P450, CYP83A1 and CYP83B1, from Arabidopsis in oxime metabolism in the biosynthesis of glucosinolates. In a detailed analysis of the substrate specificities of the recombinant enzymes heterologously expressed in yeast (Saccharomyces cerevisiae), we show that aliphatic oximes derived from chain-elongated homologs of methionine are efficiently metabolized by CYP83A1, whereas CYP83B1 metabolizes these substrates with very low efficiency. Aromatic oximes derived from phenylalanine, tryptophan, and tyrosine are metabolized by both enzymes, although CYP83B1 has higher affinity for these substrates than CYP83A1, particularly in the case of indole-3-acetaldoxime, where there is a 50-fold difference in K(m) value. The data show that CYP83A1 and CYP83B1 are nonredundant enzymes under physiologically normal conditions in the plant. The ability of CYP83A1 to metabolize aromatic oximes, albeit at small levels, explains the presence of indole glucosinolates at various levels in different developmental stages of the CYP83B1 knockout mutant, rnt1-1. Plants overexpressing CYP83B1 contain elevated levels of aliphatic glucosinolates derived from methionine homologs, whereas the level of indole glucosinolates is almost constant in the overexpressing lines. Together with the previous characterization of the members of the CYP79 family involved in oxime production, this work provides a framework for metabolic engineering of glucosinolates and for further dissection of the glucosinolate pathway.

Arabidopsis↗
PubMed1993

Genetic manipulation of negative-strand RNA virus genomes.

Negative-strand RNA viruses have been refractory to genetic manipulation using recombinant DNA techniques. Recently, new techniques were developed that allowed the rescue of synthetic RNA molecules into influenza A viruses and, subsequently, into other negative-strand RNA viruses. These techniques are presently being used to study the molecular biology of these viruses. Questions concerning cis- and trans-acting elements that are involved in transcription and replication of negative-sense RNA viral genomes can now be addressed with reverse genetic approaches. Further development of this methodology has enabled the construction--by recombinant DNA techniques--of influenza A viruses that contain altered genomes. The phenotypic characteristics and possible applications of these novel transfectant viruses are also discussed.

Base Sequence↗
PubMed1999

Reevaluation of nucleotide sequences of wild-type and attenuated polioviruses of type 3.

Published sequences of wild-type and attenuated Sabin strains of type 3 poliovirus (Leon/37 and Leon 12a(1)b) were derived from cDNA clones. Recent direct sequencing of Sabin 3 RNA showed that it differed from the published sequence in at least two sites. Here results of direct sequencing of genomes of three independently re-derived sub-strains of attenuated Sabin 3 poliovirus used for oral poliovirus vaccine (OPV) production in addition to the most widely used Pfizer sub-strain are reported. The results showed that all four sub-strains of attenuated type 3 poliovirus contain unique patterns of mutations. Two stocks of the wild-type progenitor Leon/37 strain were also sequenced. Analysis of the two samples of Leon/37 virus showed that one of them is much closer to the Sabin 3 strain, and is an intermediate product of the attenuation process. In addition, we created genetically engineered constructs which contained some of the mutations suspected for their possible role in neurovirulence, and tested them in monkeys and in transgenic mice sensitive to poliovirus. The results suggested that none of them increased neurovirulence of the virus, but some may improve virus replication. Therefore the only mutation occurring in Sabin 3 under vaccine production conditions that appears to affect neurovirulence of the virus is the well known U-->C reversion at nucleotide 472.

Animals↗
PubMed2005

Self-assembled polymer films for controlled agent-driven motion.

Reliable stimuli-responsive materials make up a vital part of molecular medicine and on-chip diagnostics. Here, we describe such a material which exhibits rapid, large-amplitude, reversible deformations and which is formed in a simple, one-material, one-step, self-assembly process. The material is a polymer network comprised of discrete molecular actuators which anisotropically expand in response to their driving stimuli. Tuning the relative orientation of the actuators with respect to one another creates expansion variations throughout a sample, and this is exploited to induce macroscopic motion. The deformation directions are pre-engineered by the molecular positioning, and extremely fast response times and high sensitivity are observed. We describe water- and pH-controlled motion, and we anticipate that the techniques are extendable to other biologically or industrially relevant agents.

Hydrogen-Ion Concentration↗
PubMed1992

Signalling through the MHC class II cytoplasmic domain is required for antigen presentation and induces B7 expression.

Class II major histocompatibility complex (MHC) molecules function as antigen-presenting elements as well as signal transducers on B lymphocytes. We previously reported that a B lymphoma cell transfectant, 5C2, expressing genetically engineered I-Ak molecules with truncated cytoplasmic domains was severely impaired in both antigen presentation and in anti-Ia-induced intracytoplasmic signalling. These two functions could be restored by preculturing 5C2 cells with cyclic AMP analogues. Here we demonstrate that impaired signal transduction by truncated class II molecules results in a deficiency in induction of the newly defined B-cell accessory molecule B7 (ref. 8), which can be reversed by restoration of B7 expression. These data imply that contact of the T-cell antigen receptor with MHC/antigen ligand results in signal transmission through the class II cytoplasmic domain. This signal, which can be mimicked by dibutyryl cAMP, induces expression of B7, resulting in effective antigen presentation. The fact that crosslinking of surface class II MHC also induces B7 expression on normal resting human B cells supports this contention.

Animals↗
PubMed2005

[Culturing and cryopreservation of pre-cartilaginous stem cells from neonate rat].

OBJECTIVE: To establish an effective way to cryopreserve pre-cartilaginous stem cells (PSCs) of neonate rat. METHODS: PSCs [fibroblast growth factor-3 (FGFR-3) positive cells] were isolated and purified by magnetic cell sorting method. PSCs were cultured and amplified to the third generation. PSCs were preserved in liquid nitrogen. The biological properties of cryopreserved PSCs were investigated by reverse transcriptase polymerase chain reaction (RT-PCR), immunohistochemistry, and immunofluorescence. RESULTS: Immunohistochemical and immunofluorescent analysis showed widespread expression of FGFR-3 in cryopreserved PSCs. FGFR-3 could be detected by RT-PCR in cryopreserved PSCs. Cryopreserved PSCs kept high cell viability, and phenotypic and proliferation characteristics of PSCs in vivo. CONCLUSION: Cryopreservation of PSCs can supply adequate qualified cells for repairing the defects of epiphyseal growth plate by tissue engineering technique.

Animals↗