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21.
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
22.
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
23.
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
24.
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
25.
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
26.
T A Spiegel A J Stunkard E E Shrager C P O'Brien M F Morrison E Stellar 《Physiology & behavior》1987,40(2):135-141
Increasing doses of naltrexone (25 to 200 mg) given over 4 consecutive days reduced intake of laboratory luncheon meals by 30% in 17 obese men. Meal size remained suppressed in the laboratory during the week following naltrexone administration. Water intake in the laboratory and body weight were not affected. Rates of ingestion and subjective ratings suggested that naltrexone reduced appetite rather than promoted early satiation. Nausea and other side effects occurred on 1 or 2 days during the naltrexone week in seven subjects whose food intake was reduced. Food intake was also reduced in seven of the remaining 10 subjects who reported no adverse reactions. These results suggest that a conditioned taste aversion or a conditioned anorexia may have developed in some subjects. 相似文献
27.
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
28.
29.
30.
Stunkard AJ Berkowitz RI Stallings VA Schoeller DA 《The American journal of clinical nutrition》1999,69(3):524-530
BACKGROUND: It has been proposed that the primary determinants of body weight at 1 y of age are genetic background, as represented by parental obesity, and low total energy expenditure. OBJECTIVE: The objective was to determine the relative contributions of genetic background and energy intake and expenditure as determinants of body weight at 1 y of age. DESIGN: Forty infants of obese and 38 infants of lean mothers, half boys and half girls, were assessed at 3 mo of age for 10 risk factors for obesity: sex, risk group (obese or nonobese mothers), maternal and paternal body mass index, body weight, feeding mode (breast, bottle, or both), 3-d energy intake, nutritive sucking behavior during a test meal, total energy expenditure, sleeping energy expenditure, and interactions among them. RESULTS: The only difference between risk groups at baseline was that the high-risk group sucked more vigorously during the test meal. Four measures accounted for 62% of the variability in weight at 12 mo: 3-mo weight (41%, P = 0.0001), nutritive sucking behavior (9%, P = 0.0002), 3-d food intake (8%, P = 0.0002), and male sex (3%, P = 0.05). Food intake and sucking behavior at 3 mo accounted for similar amounts of variability in weight-for-length, body fat, fat-free mass, and skinfold thickness at 12 mo. Contrary to expectations, neither total nor sleeping energy expenditure at 3 mo nor maternal obesity contributed to measures of body size at 12 mo. CONCLUSIONS: Energy intake contributes significantly to measures of body weight and composition at 1 y of age; parental obesity and energy expenditure do not. 相似文献