Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving
cerebellar degeneration, immunodeficiency, chromosomal instability,
radiosensitivity and cancer predisposition. The responsible gene, ATM, was
recently identified by positional cloning and found to encode a putative
350 kDa protein with a Pl 3-kinase-like domain, presumably involved in
mediating cell cycle arrest in response to radiation-induced DNA damage.
The nature and location of A-T mutations should provide insight into the
function of the ATM protein and the molecular basis of this pleiotropic
disease. Of 44 A-T mutations identified by us to date, 39 (89%) are
expected to inactivate the ATM protein by truncating it, by abolishing
correct initiation or termination of translation, or by deleting large
segments. Additional mutations are four smaller in-frame deletions and
insertions, and one substitution of a highly conserved amino acid at the Pl
3-kinase domain. The emerging profile of mutations causing A-T is thus
dominated by those expected to completely inactivate the ATM protein. ATM
mutations with milder effects may result in phenotypes related, but not
identical, to A-T.
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As more mutations are identified in genes of known sequence, there is a
crucial need in the areas of medical genetics and genome analysis for
rapid, accurate and cost-effective methods of mutation detection. We have
developed a multiplex allele-specific diagnostic assay (MASDA) for analysis
of large numbers of samples (> 500) simultaneously for a large number of
known mutations (> 100) in a single assay. MASDA utilizes
oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA
samples are immobilized on a solid support and a single hybridization is
performed with a pool of allele-specific oligonucleotide (ASO) probes. Any
probes complementary to specific mutations present in a given sample are in
effect affinity purified from the pool by the target DNA. Sequence-specific
band patterns (fingerprints), generated by chemical or enzymatic sequencing
of the bound ASO(s), easily identify the specific mutation(s). Using this
design, in a single diagnostic assay, we tested samples for 66 cystic
fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell
anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations,
four mutations in Canavan disease, four mutations in Fanconi anemia, and
five mutations in BRCA1. Each mutation was correctly identified. Finally,
in a blinded study of 106 of these mutations in > 500 patients, all
mutations were properly identified. There were no false positives or false
negatives. The MASDA assay is capable of detecting point mutations as well
as small insertion or deletion mutations. This technology is amenable to
automation and is suitable for immediate utilization for high-throughput
genetic diagnostics in clinical and research laboratories.
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In this study, we have used time-lapse video cinematography to study
fertilization in 50 human oocytes that had undergone intracytoplasmic sperm
injection (ICSI). Time-lapse recording commenced shortly after ICSI and
proceeded for 17-20 h. Oocytes were cultured in an environmental chamber
which was maintained under standard culture conditions. Overall, 38 oocytes
(76%) were fertilized normally, and the fertilization rate and embryo
quality were not significantly different from 487 sibling oocytes cultured
in a conventional incubator. Normal fertilization followed a defined course
of events, although the timing of these events varied markedly between
oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular
waves of granulation within the ooplasm which had a periodicity of 20-53
min. The sperm head decondensed during this granulation phase. The second
polar body was then extruded, and this was followed by the central
formation of the male pronucleus. The female pronucleus formed in the
cytoplasm adjacent to the second polar body at the same time as, or
slightly after, the male pronucleus, and was subsequently drawn towards the
male pronucleus until the two abutted. Both pronuclei then increased in
size, the nucleoli moved around within the pronuclei and some nucleoli
coalesced. During pronuclear growth, the organelles contracted from the
cortex towards the centre of the oocyte, leaving a clear cortical zone. The
oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during
the course of the observation period. The female pronucleus was
significantly smaller in diameter than the male pronucleus (24.1 and 22.4
microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0
respectively, P < 0.0001). After time-lapse recording, oocytes were
cultured for 48 h prior to embryo transfer or cryopreservation. Embryo
quality was related to fertilization events and periodicity of the
cytoplasmic wave, and it was found that good quality embryos arose from
oocytes that had more uniform timing from injection to pronuclear abuttal
and tended to have a longer cytoplasmic wave. In conclusion, we have shown
that time-lapse video cinematography is an excellent tool for studying
fertilization and early embryo development, and have demonstrated that
human fertilization comprises numerous complex dynamic events.
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Background: Atracurium has four chiral centers and the marketed product is a mixture of ten optical and geometric isomers. Six of the isomers were prepared and evaluated for neuromuscular blocking activity and autonomic effects in anesthetized cats. This study reports the comparative pharmacology of the six isomers and atracurium that led to the selection of one isomer, cisatracurium (Nimbex, 51W89) for clinical development.
Methods: Purpose bred cats, anesthetized with alpha-chloralose (80 mg/kg) and pentobarbital sodium (7 mg/kg) administered intraperitoneally, were used in this study. Neuromuscular blocking effects were assessed from the effects on the tibialis anterior twitch evoked at 0.15 Hz. Inhibition of the autonomic nervous system was assessed from the effects on the nictitating membrane contraction, in response to preganglionic sympathetic nerve stimulation and the bradycardia/vasodepressor responses to vagal nerve stimulation. Cardiovascular effects and plasma histamine concentrations were determined after a bolus injection of cisatracurium or atracurium.
Results: Like atracurium, all six isomers produced dose-dependent neuromuscular block (NMB). The calculated ED95 NMB values varied approximately tenfold (43+/-2 micro gram/kg-488+/-56 micro gram/kg). The "R-series" isomers were more potent than the corresponding "S series" isomers. With the exception of the S,Trans-S', Trans isomer, the NMB effects, i.e., onset times (range 2.6+/-0.2 min to 4.7+/-0.3 min) and total durations (range 9.9+/- 1.4 min to 14+/-0.9 min), of the other five isomers were very similar to that of atracurium. The former isomer had a relatively short duration of action. The 25-75% recovery times after cisatracurium at 1x ED sub 95 (4.4+/-0.4 min), 4x ED95 (4.5+/-0.4 min), and continuous infusions lasting at least 60 min that maintained 95-99% NMB (4.8+/-0.4 min) indicated a noncumulative effect. The vagal ID50: NMB ED95 ratios for atracurium and the six isomers ranged from 2 to 27. The sympathetic ID25: NMB ED95 ranged from 2.7 to 60. Atracurium and all of the isomers, except cisatracurium, produced cardiovascular effects after intravenous bolus administration at large doses (700-4,800 micro gram/kg). In contrast to atracurium, there were no changes in plasma histamine concentrations associated with the administration of doses of cisatracurium equivalent to 60x the NMB ED95 (62+/-8 micro gram/kg). 相似文献
The palpation and enucleation of occult insulinomas (less than 15 mm) can be a difficult surgical problem even with good arteriographic localization. In the authors' limited experience, confirmation of arteriographic findings by pancreatic venous sampling provided little additional localizing information. However, if arteriography is negative or equivocal, venous sampling can indicate the segment of pancreas to be "blindly" resected if the adenoma is not palpable. Venous sampling may be misleading in polyendocrine syndromes because of the frequency of multiple adenomas and variable hormone production. 相似文献