Speaking up for the profession.
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Public Relations↗
Biomedical subjects
J M Shaw
Publications and source records attributed to J M Shaw.
Longer-term regulation of branched-chain-2-oxoacid dehydrogenase complex studied in rat hepatocytes in culture.
The effect of protein-free diet to decrease liver activity of branched-chain (-2-oxoacid) dehydrogenase (BCD) complex (active form) and increase BCD kinase activity was unaffected by preparation of hepatocytes, but partially reversed by 25 h of culture of hepatocytes in medium 199. Activation of BCD complex preceded loss of BCD kinase. The effect of culture on BCD complex was completely prevented by omission of branched-chain amino acids and partially prevented by 1 mM-alpha-cyano-4-hydroxycinnamate or 0.2 mM-pyruvate/2 mM-lactate. Protein-free diet decreased plasma branched-chain amino and oxo ('keto') acids and increased plasma pyruvate and lactate. It is concluded: (1) that branched-chain amino acids are involved directly in regulation of activities of BCD complex and BCD kinase; (2) that mitochondrial uptake of branched-chain oxo acids is necessary for regulation of BCD complex activity; and (3) that the stable increase in BCD kinase may function as a hysteresis mechanism.
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
Parthenogenetic activation of unfertilized mouse oocytes by exposure to 1,2-propanediol is influenced by temperature, oocyte age, and cumulus removal.
Cumulus-intact and -denuded unfertilized oocytes from two mouse strains were exposed to 1.5 M ethanol (EtOH) or two cryoprotectant solutions, 1.5 M propanediol (PROH) or 1.5 M dimethylsulfoxide (DMSO), for 4.5 min at 27 degrees C, and the proportion of activating or degenerating oocytes studied. Exposure to DMSO did not significantly increase activation above that of oocytes not exposed to DMSO. Treatment of oocytes in PROH resulted in the activation of up to 87% of viable oocytes. This was significantly higher (P less than .01) than in control oocytes and comparable to the rate of activation after treatment with EtOH (59-96% activation). In solutions at 1 degree C, 47% of control oocytes were activated, which was not significantly different from the rate of activation in EtOH (36%) or PROH (50%) at 1 degree C. Following treatment with PROH, up to 87% of oocytes degenerated within a period of 6 h in vitro. The age of the oocytes (h post hCG) and the time of cumulus removal with the enzyme hyaluronidase, relative to the time of exposure to the chemicals, influenced the level of degeneration in most groups. Significantly fewer oocytes degenerated when cumulus cells were removed before treatment (0-31%) than when the cumulus was left intact throughout the treatment and 6 h culture period (10-87%). Exposure to PROH at 1 degree C reduced oocyte degeneration to 5%. We conclude that PROH causes significantly greater losses of oocytes as a result of parthenogenetic activation and degeneration than of exposure to DMSO.
Animals↗
Effect of dimethyl sulfoxide and protein concentration on the viability of two-cell mouse embryos frozen with a rapid freezing technique.
Two-cell mouse embryos were frozen by direct plunging into liquid nitrogen after a 3-min exposure to solutions containing 0.25 M sucrose with 1.5, 3, or 4.5 M dimethyl sulfoxide (Me2SO), and 0, 4, 8, 16, or 32 mg/ml bovine serum albumin (BSA). In the absence of BSA, significantly more embryos were lost or damaged during freezing and thawing. Increasing the BSA concentration from 4 to 32 mg/ml had no significant effect on subsequent embryo viability in vivo or in vitro. Blastocyst formation in vitro was greater than 90% in embryos exposed to the cryoprotective solutions only. Although development to blastocysts was not significantly different to nonfrozen controls in most groups frozen in 3 and 4.5 M Me2SO (up to 92% blastocysts), it was significantly reduced when embryos were frozen in 1.5 M Me2SO (up to 65% blastocysts). The development to fetuses of embryos frozen in 3 M Me2SO (64 to 74% fetuses) was not significantly different from nonfrozen controls (68 to 79% fetuses) or embryos frozen by a conventional slow cooling method (70%). Frozen thawed two-cell embryos developed into normal adults which were able to reproduce normally. We conclude that this freezing method can efficiently cryopreserve early cleavage stage mouse embryos.
Animals↗
Characterization of a protein fraction containing cytochromes b and c1 from mitochondria of Leishmania tarentolae.
A soluble red band fraction was obtained from Leishmania tarentolae cells by sucrose gradient sedimentation of a Triton X-100 lysate. Spectral analysis indicated that cytochrome b was present in the red band: the reduced minus oxidized difference spectra revealed absorption maxima at 562,527, and 431 nm at room temperature and 562, 530, and 422 nm at 77K. In addition, a 28-kDa protein was identified in this fraction which retained heme-associated peroxidase activity even after denaturation on SDS-polyacrylamide gels. The amino acid composition of this protein showed a strong similarity to cytochrome c1 of both bovine and yeast.
Amino Acids↗
Successful single-cell biopsy and cryopreservation of preimplantation mouse embryos.
We have previously observed that preimplantation embryo biopsy in the mouse causes a reduction in implantation rate in utero. After minor modifications to the technique, we now find that sampling a single blastomere from the 4-cell mouse embryo does not compromise continued development in vitro or in vivo. When transferred to pseudopregnant foster mice, 60.3% and 64.3% of biopsied and control embryos, respectively, implanted into the uterine wall, and 52.6% and 52.4% of biopsied and control embryos, respectively, developed into fetuses. In a separate series of experiments, we have demonstrated that biopsied mouse embryos can be successfully cryopreserved by ultrarapid freezing even though they have a punctured zona pellucida. Biopsied (frozen-thawed), control (frozen-thawed), and nonfrozen embryos had an implantation rate of 81.1%, 74.3%, and 74.1%, respectively, and a fetal formation rate of 62.2%, 62.9%, and 66.7%, respectively.
Animals↗
Internal frameshifts within the mitochondrial genes for cytochrome oxidase subunit II and maxicircle unidentified reading frame 3 of Leishmania tarentolae are corrected by RNA editing: evidence for translation of the edited cytochrome oxidase subunit II mRNA.
The Leishmania tarentolae cytochrome oxidase (EC 1.9.3.1) subunit II (COII) and maxicircle unidentified reading frame 3 (MURF3) mRNAs are edited internally by the addition of four and five uridine residues, respectively, which eliminate -1 and +1 reading frameshifts in the gene sequences. The editing events in COII are conserved in three kinetoplastid species, and those in MURF3 are conserved in two species. A primer extension assay showed that the ratio of edited to unedited RNA differed for each gene: 89% of the COII and 36% of the MURF3 transcripts are edited. Preliminary evidence was obtained for translation of the edited COII transcript into protein: antibodies generated against a synthetic peptide with the predicted carboxyl-terminal amino acid sequence reacted with a polypeptide of the correct molecular weight in immunoblots of a mitochondrial lysate.
Amino Acid Sequence↗
Short-term buspirone treatment in disinhibition with dementia.
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Aged↗
Successful outcome of combined organochlorine and organomercury poisoning in a dog.
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Animals↗
Editing of kinetoplastid mitochondrial mRNAs by uridine addition and deletion generates conserved amino acid sequences and AUG initiation codons.
RNA editing is a novel type of mRNA processing in the kinetoplastid mitochondrion that involves the co- or posttranscriptional addition of uridine residues within transcripts. The result is an mRNA nucleotide sequence which differs from that of the corresponding gene. We have found that RNA editing may also occur through the deletion of genome-encoded uridines from the RNA sequence. Uridine additions and deletions in the 5' ends of the COIII, MURF2, and MURF3 transcripts create new N-terminal amino acid sequences that are conserved between species, and new AUG initiation codons in several cases. These results suggest that the creation of new amino acid sequences and methionine initiation codons may be important functions of RNA editing in the kinetoplastid mitochondrion.
Amino Acid Sequence↗
Induction of macrophage antitumor activity by acetylated low density lipoprotein containing lipophilic muramyl tripeptide.
A method has been developed for the selective delivery of lipophilic immunomodulators to macrophages, which results in the induction of antitumor activity. This method utilizes exhaustively acetylated low density lipoprotein (acetyl-LDL) to deliver the lipophilic immunomodulator, muramyl tripeptide phosphatidylethanolamine (MTP-PtdEtn; amide composed of N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine and dipalmitoyl phosphatidylethanolamine) to macrophages (M phi) by means of a scavenger lipoprotein receptor pathway. The binding of acetyl-LDL:MTP-PtdEtn to M phi showed specificity since minimal competition was observed in the presence of excess native LDL or phosphatidylcholine/cholesterol liposomes. Additional binding studies showed that acetyl-LDL may serve as a suitable delivery vehicle to a wide variety of M phi in different stages of activation. Cytostatic and tumoricidal activities by thioglycolate-elicited M phi against two tumor cell lines were examined in vitro following incubation with the acetyl-LDL:MTP-PtdEtn complex. Cytostatic activity against B16F10 melanoma cells was induced after the incubation of thioglycolate-elicited M phi with a minimum of 25 micrograms of acetyl-LDL protein containing 2.5 micrograms of bound MTP-PtdEtn (approximately equal to 40 molecules per particle of acetyl-LDL). The induction of cytostasis was not affected by liposome bilayers, which were also endocytosed by the M phi. In addition, tumoricidal activity against P815 mastocytoma cells was demonstrated at a 40:1 effector-to-target ratio using 18 micrograms of the acetyl-LDL:MTP-PtdEtn complex containing 3.6 micrograms of MTP-PtdEtn (approximately equal to 80 molecules per particle). These studies describe a method for the induction of antitumor activity by use of a chemically modified serum component, acetyl-LDL, to direct lipophilic immunomodulators to M phi.
Acetylmuramyl-Alanyl-Isoglutamine↗
Creation of AUG initiation codons by addition of uridines within cytochrome b transcripts of kinetoplastids.
The cytochrome b gene of Trypanosoma brucei has an ATG codon near its 5' end but the cytochrome b genes of the related kinetoplastids Leishmania tarentolae and Crithidia fasciculata lack an ATG. Recent results have shown that 34 uridines that are not encoded in the genome are added within the 5' end of T. brucei cytochrome b transcripts during or after transcription. These additions create an AUG in the transcript that is 20 amino acids upstream of the AUG predicted from the genomic sequence. We report here that the cytochrome b transcripts of L. tarentolae and C. fasciculata also contain added uridines within their 5' ends. The additions occur in similar numbers and positions and an in frame AUG is created at a similar site in all three species. These data strongly suggest that the created AUG functions as the initiation codon for cytochrome b in these species. Since some other kinetoplastid mitochondrial genes also lack conventional initiation codons, creation of initiation codons may be an important function of uridine addition.
Amino Acid Sequence↗
Ultrarapid embryo freezing: effect of dissolved gas and pH of the freezing solutions and straw irradiation.
While optimizing the ultrarapid embryo freezing procedure we noted that embryo survival was lowest when gas bubbles formed in the straws. Here we report the influences of gassing the freezing solutions with 5% CO2 in air, degassing the solution, the pH of the medium and straw irradiation on the survival and development in vitro of 2-cell mouse embryos. Embryos were ultrarapidly frozen in medium M2 containing 3 M dimethyl sulphoxide and 0.25 M sucrose. Significantly fewer embryos survived freezing and thawing in gassed solutions. The subsequent development of intact embryos to blastocysts was similar in gassed and non-gassed solutions. Survival and development of embryos frozen in solutions of pH 7.0, 7.6 or 8.0 was similar, but fewer embryos developed to blastocysts after freezing in media at pH 6.0. In straws sterilized by gamma irradiation before use, embryo survival after freezing and thawing was dramatically reduced compared with that seen when non-irradiated straws were used. With non-gassed solutions significantly more gas bubbles formed in irradiated straws than in non-irradiated straws. Although bubble formation was significantly reduced in all straws by using degassed freezing solutions, this did not improve embryo survival. We conclude that under conditions normally used for ultrarapid freezing, dissolved gas and pH have less effect on embryo viability than the use of irradiated straws.
Animals↗
Results using medium supplemented with either fresh or frozen stored serum in human in vitro fertilization.
This study retrospectively compared the success of in vitro fertilization (IVF) among patients whose gametes had been incubated either in medium supplemented with freshly prepared pooled serum (331 cases) or in pooled serum which had been stored at -20 degrees C prior to use (728 cases). Frozen stored serum was as effective as fresh serum with regard to the proportion of oocytes which fertilized and embryos which implanted and was not associated with any increased incidence of fetal loss during postimplantation development.
Blood Physiological Phenomena↗
Delivery of lipophilic drugs using lipoproteins.
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Aging↗
Suspected cyanide poisoning in two goats caused by ingestion of crab apple leaves and fruits.
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Animals↗
Rat tissue concentrations of branched-chain 2-oxo acid dehydrogenase complex. Re-evaluation by immunoassay and bioassay.
A rabbit polyclonal antibody to purified ox kidney branched-chain oxo acid dehydrogenase complex was shown by a variety of techniques to be an antibody to the E2 (acyltransferase) component. Rocket immunoelectrophoresis showed that the antibody does not discriminate between phosphorylated (inactive) or dephosphorylated (active) complex, and the same technique is used to assay total branched-chain complex (sum of active and inactive forms) in rat liver and heart mitochondrial extracts. The values obtained in normal rats fed on normal diet were comparable with those obtained by spectrophotometric assay of the holocomplex reaction after conversion of inactive complex into active complex. The values obtained in liver mitochondria from rats fed on 0%-casein diet or starved for 48 h were comparable with those in rats fed on normal diet, whereas earlier studies using spectrophotometric assay had shown substantial decreases in rats fed on 0%-casein diet or starved for 48 h. It has been shown that conversion of inactive complex into active complex requires prolonged incubation (120 min) in the presence of ketoleucine (4-methyl-2-oxopentanoate; to inhibit branched-chain oxo acid dehydrogenase kinase) to effect complete conversion in mitochondria from rats fed on 0%-casein diet, or starved for 48 h, or made diabetic with alloxan. By this technique, total activity of the complex in rat liver mitochondria was unaffected by diet or diabetes. The effects of diet and diabetes to decrease the activity of branched-chain complex in rat liver are therefore apparently mediated wholly through inactivation of the complex by phosphorylation.
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗