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31.
Optimization of a method for deactivation of platelet-activating factor:acetylhydrolase in serum for use in in-vitro fertilization culture media 总被引:1,自引:0,他引:1
Embryos produced by in-vitro fertilization (IVF) may produce less
platelet-activating factor (PAF) than is optimal for development. It was
previously shown that supplementation of culture media with PAF results in
a significant increase in pregnancy rate. Human embryos are often cultured
in media supplemented with serum containing the enzyme PAF:acetylhydrolase
(PAF:AH; EC 3.1.1.47), which hydrolyses PAF to its inactive form, lyso-PAF.
Thus, effective supplementation of media with PAF requires inactivation of
this enzyme. In this study we examine the efficacy of the methods of PAF:AH
deactivation used for PAF supplementation of IVF culture medium. When the
effectiveness of a commonly used acid treatment protocol (pH 3.0 at room
temperature for 5 min) was examined, it was found that it was not
completely effective for the majority of sera. When synthetic PAF was added
to 18 serum samples which had been acid treated, five had 90-100% of the
original PAF remaining after 24 h (showing that the acid treatment was
effective), eight had from 10-90% of the original PAF remaining after 24 h,
and five samples had 0-10%. The extent to which PAF:AH was susceptible to
deactivation was not associated with the activity in the serum prior to
treatment, the serum oestradiol concentration, or the cause of infertility.
The period of acidification and the incubation temperature were assessed to
develop a new acid-treatment protocol (20 min acid treatment at 37 degrees
C) which was able to deactivate PAF:AH effectively in all sera (53/53)
examined. A trial was performed to assess the effect of acid treatment of
serum for 5 min at room temperature compared with the new protocol (20 min
at 37 degrees C) on IVF outcome, following PAF supplementation of IVF
culture medium. Oocyte recovery, fertilization and embryo development rates
were equivalent for both groups and approximately equal numbers of embryos
were transferred or cryopreserved. Pregnancy rates were not significantly
different (14.6 versus 20.0%) for the two treatments, with a trend towards
a higher pregnancy rate with the new acid- treatment protocol. The results
show that this new procedure for acid treatment of serum in combination
with PAF supplementation does not have detrimental effects on embryos and
their pregnancy outcome and is therefore suitable for use in IVF.
相似文献
32.
Positional cloning of the gene for X-linked retinitis pigmentosa 3: homology with the guanine-nucleotide-exchange factor RCC1 总被引:6,自引:7,他引:6
Roepman R; van Duijnhoven G; Rosenberg T; Pinckers AJ; Bleeker-Wagemakers LM; Bergen AA; Post J; Beck A; Reinhardt R; Ropers HH; Cremers FP; Berger W 《Human molecular genetics》1996,5(7):1035-1041
The gene for retinitis pigmentosa 3 (RP3), the most frequent form of X-
linked RP (XLRP), has been mapped previously to a chromosome interval of
less than 1000 kbp between the DXS1110 marker and the OTC locus at
Xp21.1-p11.4. Employing a novel technique, YAC Representation Hybridization
(YRH)', we have recently identified a small XLRP associated microdeletion
in this interval, as well as several putative exons including the 3' end of
a gene that was truncated by the deletion. cDNA library screening and
sequencing of a cosmid centromeric to the deletion has now enabled us to
identify numerous additional exons and to detect several point mutations in
patients with XLRP. The predicted gene product shows homology to RCC1, the
guanine-nucleotide- exchange factor (GEF) of the Ras-like GTPase Ran. Our
findings suggest that we have cloned the long-sought RP3 gene, and that it
may encode the GEF of a retina-specific GTP-binding protein.
相似文献
33.
A. Soriano J. Gómez L. Gómez J.R. Azanza R. Pérez F. Romero M. Pons F. Bella M. Velasco J. Mensa 《European journal of clinical microbiology & infectious diseases》2007,26(5):353-356
The aim of the study presented here was to assess the efficacy and tolerability of linezolid in the treatment of orthopedic
implant infections (OII). Eighty-five patients with an OII treated with linezolid were prospectively followed up for a minimum
of 12 months from the end of antibiotic therapy. Outcome was evaluated in relation to the duration and type of symptoms (acute
or chronic) and the retention or removal of the implant. For acute and chronic infections, the respective success rates were
100 and 92.3% when the implant was removed and 72.2 and 42.8% when it was not. The median length of linezolid treatment in
acute and chronic infections was 47 and 60 days, respectively. Thrombocytopenia was observed in four (4.7%) patients and anemia
in five (5.8%). The results suggest oral linezolid is an effective and well-tolerated alternative for treating OII. 相似文献
34.
The t(X;1)(p11.2;q21.2) translocation in papillary renal cell carcinoma fuses a novel gene PRCC to the TFE3 transcription factor gene 总被引:4,自引:2,他引:4
35.
Somatic mutation processes at a human minisatellite 总被引:6,自引:3,他引:6
Germline instability at human minisatellites frequently involves complex
inter-allelic transfers of repeat units usually restricted to one end of
the repeat array and apparently regulated by flanking DNA. In contrast,
nothing is known about the structural basis of somatic instability at
minisatellites. An electrophoretic size-enrichment strategy was therefore
developed at minisatellite MS32 (D1S8) to enable rare abnormal-length
mutants to be detected, validated and quantitated in blood DNA by single
molecule PCR. Structural analysis of rare mutant alleles in blood revealed
simple deletions/duplications of repeat unit blocks located at random along
the tandem repeat array, a mode of mutation completely different from that
seen in sperm. Furthermore, allele-specific suppression of sperm
instability at MS32 did not affect somatic instability. These data suggest
that conversion-based minisatellite mutation in sperm is completely
germline-specific and most likely meiotic in origin. Somatic instability
appears to occur by a separate pathway involving replication slippage or,
more likely, intra-allelic unequal crossing over.
相似文献
36.
Mutations in the TSC2 gene: analysis of the complete coding sequence using the protein truncation test (PTT) 总被引:4,自引:0,他引:4
Mutations in the TSC2 gene on chromosome 16p13.3 are responsible for
approximately 50% of familial tuberous sclerosis (TSC). The gene has 41
small exons spanning 45 kb of genomic DNA and encoding a 5.5 kb mRNA. Large
germline deletions of TSC2 occur in <5% of cases, and a number of small
intragenic mutations have been described. We analysed mRNA from 18
unrelated cases of TSC for TSC2 mutations using the protein truncation test
(PTT). Three cases were predicted to be TSC2 mutations on the basis of
linkage analysis or because a hamartoma from the patient showed loss of
heterozygosity for 16p13.3 markers. Three overlapping PCR products,
covering the complete coding sequence of mRNA, were generated from
lymphoblastoid cell lines, translated into 35S-methionine labelled protein,
and analysed by SDS-PAGE. PCR products showing PTT shifts were directly
sequenced, and mutations confirmed by restriction enzyme digestion where
possible. Six PTT shifts were identified. Five of these were caused by
mutations predicted to produce a truncated protein: (i) a sporadic case
showed a 32 bp deletion in exon 11, and a mutant mRNA without exon 11 was
produced; the normal exon 10 was also spliced out; (ii) a sporadic case had
a 1 bp deletion in exon 12 (1634delT); (iii) a TSC2-linked mother and
daughter pair had a G-->T transversion in exon 23 (G2715T) introducing a
cryptic splice site causing a 29 bp truncation of mRNA from exon 23; (iv) a
sporadic case showed a 2 bp deletion in exon 36; (v) a sporadic case showed
a 1 bp insertion disrupting the donor splice site of exon 37 (5007+2insA),
resulting in the use of an upstream exonic cryptic splice site to cause a
29 bp truncation of mRNA from exon 37. In one case, the PTT shift was
explained by in-frame splicing out of exon 10, in the presence of a normal
exon 10 genomic sequence. Alternative splicing of exon 10 of the TSC2 gene
may be a normal variant. Three 3rd base substitution polymorphisms were
also detected during direct sequencing of PCR products. Confirmed mutations
were identified in 28% of the families studied and on the assumption that
half of the sporadic cases should have TSC2 mutations, a crude estimate of
the detection rate would be 60%. This compares favourably with other
screening methods used for TSC2, notably SSCP, and since PTT involves much
less work it may be the method of choice.
相似文献
37.
Detecting pre-ovulatory luteinizing hormone surges in urine 总被引:2,自引:1,他引:2
Kesner JS; Knecht EA; Krieg EF Jr; Wilcox AJ; O'Connor JF 《Human reproduction (Oxford, England)》1998,13(1):15-21
The study objectives were to determine (i) if pre-ovulatory luteinizing
hormone (LH) surges, undetected in urine by two immunoradiometric assays
(IRMA), were detectable by an ultrasensitive immunofluorometric assay
(IFMA) and (ii) the influence of creatinine adjustment on the detection and
timing of the urinary LH surges. Daily urine specimens were contributed by
healthy 25-36 year old volunteers during 14 ovulatory menstrual cycles for
an epidemiological study conducted in 1983-1985. Specimens were selected as
having been previously assayed by two IRMA without consistently detecting
LH surges. These urine specimens were remeasured using an IFMA and adjusted
for creatinine concentration. IFMA measurements revealed unambiguous LH
surges in all cycles. Adjusting IRMA urinary LH values for creatinine
concentrations revealed previously undetected LH surges in four of eight
cycles. Creatinine adjustment also altered the timing of IRMA and IFMA LH
surges by 1-5 days. These results demonstrate an IFMA that detects pre-
ovulatory LH surges in unpreserved, frozen urine from cycles where such
surges were previously undetectable. Further, creatinine adjustment can
markedly affect detection and timing of the onset and peak of the urinary
LH surge. While our analysis suggests that this adjustment improves the
validity of the LH measure, this requires further investigation.
相似文献
38.
39.
40.
J. Fortún A. López -San Román J. J. Velasco A. Sánchez-Sousa E. de Vicente J. Nuño C. Quereda R. Bárcena G. Monge A. Candela A. Honrubia A. Guerrero 《European journal of clinical microbiology & infectious diseases》1997,16(4):314-318
The cases of four liver transplant recipients who developed invasive candidiasis (2 cholangitis, 1 perihepatic abscess, 1 candidemia) due to azole-resistantCandida glabrata are reported. Three patients were receiving azolic compounds (2 itraconazole, 1 fluconazole) when the infection was diagnosed. All four patients received fluconazole as intestinal decontamination during the first three weeks post transplantation. The infections occurred two months after transplantation in all patients, and in one patientCandida infection was the direct cause of death. Infection of the biliary tree was the origin of candidiasis in three patients; the fourth patient developed neutropenic-related candidemia. Fluconazole MICs exceeded 16 g/ml in all cases; itraconazole MICs were 16, 2, 1, and 2 g/ml, respectively. The potential role ofCandida species other thanalbicans in these patients after administration of azole agents is discussed. 相似文献