Expression and role of the ether-à-go-go-related (MERG1A) potassium-channel protein during preimplantation mouse development.

Potassium channels play important roles in many cellular processes, including cell-cycle progression and cell differentiation. In the present study, we investigated the pattern of expression of the mouse ether-à-go-go-related (KCNH2; MERG1A) potassium channel during mouse embryogenic development. Analysis by reverse transcription-polymerase chain reaction revealed maternal MERG1A transcripts until the late 2-cell stage of development, after which MERG1A expression from the zygotic genome was low until the 8-cell stage, then rose in the morula, but was low in trophoblast compared to inner cell mass cells. A trophoblast stem cell line also was shown to express MERG1A mRNA. Immunoblotting of oocytes, blastocysts, and the trophoblast stem cell line revealed different posttranslationally processed forms of MERG1A. Immunofluorescence analysis showed that the subcellular localization of MERG1A varied at different stages of the embryogenic cell cycle. In addition, MERG1A protein levels increased following compaction at the 8-cell stage, and its distribution became polarized. This relocalization of MERG1A was affected by treatment with specific inhibitors of ether-à-go-go-related gene (ERG)-channel function and of actin polymerization. Puromycin treatment of morulae indicated that membrane-associated MERG1A had a half-life of greater than 24 h. The ERG-specific inhibitor E-4031 reduced the incidence of blastocyst formation and the number of cells per blastocyst. These results show that MERG1A is developmentally regulated and suggest that it might play a role in early mouse embryogenic development.

Stewardship or clinical freedom? variations in dialysis decision making.

BACKGROUND: It is generally agreed that acceptance criteria for dialysis have varied and changed over time and that implicit rationing, to some extent forced on clinicians by limited capacity, has been widely practised. Our objective was to study the basis and extent of variation in dialysis decision making among nephrologists in one NHS region. DESIGN AND METHODS: In a clinical judgement analysis, linear regression models were employed to reflect the impact of clinical and non-clinical cues on nephrologists' decisions to offer dialysis to 60 'paper patients' under current capacity constraints and under an assumption of no capacity limit. A short questionnaire was also completed by eight nephrologists to elicit their expressed decision drivers, which were subsequently compared with those tacitly derived from the appraisal of the 60 clinical vignettes. RESULTS: Doctors showed substantial variation in their propensity to offer dialysis and in their perceptions of the benefits of dialysis. Even for the five patients where the discordance in propensity to offer dialysis was least, the range in perceived gain in life expectancy was from 24 to 264 months (mean 91 months). The decision models had relatively good explanatory power with an average r(2) of 0.67 (0.39-0.90) and 0.70 (0.47-0.95) for decisions made under current capacity constraints and under an assumption of no limit capacity respectively. Surprisingly, for most doctors, the patient's age had very little impact on dialysis decisions but the magnitude of the beta-coefficients for the patient's mental state (mean -30.7) was of a similar order of magnitude to the coefficient for the principal 'renal' drivers (e.g. the mean coefficient for uraemic symptomatology under current capacity constraints was 47.7). The influence of other non-renal factors on the doctor's likelihood to offer dialysis was largely independent of the capacity assumption. A comparison of the doctor's stated decision drivers with those tacitly derived from their decision models showed only modest correlation. CONCLUSIONS: The extent to which doctors vary in their propensity to offer dialysis is substantial. Very few non-clinical cues appear to influence the decision to offer dialysis. The most important non-renal factor in determining dialysis decisions was the patient's mental state.

Experimental manipulations of compaction and their effects on the phosphorylation of uvomorulin.

Compaction of the eight-cell stage mouse embryo is a critical event in the generation of different cell types within the preimplantation embryo. Uvomorulin, a member of the cadherin family of cell adhesion molecules, is important during compaction and its phosphorylation increases early in the eight-cell stage, suggesting that this posttranslational modification may be important for compaction to proceed. We have assessed the importance of the phosphorylation of uvomorulin during compaction by preventing, reversing, or inducing adhesion prematurely. The only condition that affected the overall level of uvomorulin phosphorylation was the prevention of compaction through prolonged exposure of four-cell embryos to low Ca2+. This treatment reduced the level of uvomorulin phosphorylation in eight-cell embryos, and perturbed its localization to regions of cell-cell contact. Thus, whilst the phosphorylation of uvomorulin does not appear to regulate directly uvomorulin's adhesive function, it may be associated with the redistribution of uvomorulin during compaction.

Hypoplasia of the cerebellar vermis in neurogenetic syndromes.

There are conflicting reports on the relationship between cerebellar vermal lobule hypoplasia and autism. Using quantitative magnetic resonance image analysis, we measured the cerebellar vermis in 125 normal individuals with a broad age range and 102 patients with a variety of neurogenetic abnormalities. We conclude that hypoplasia of cerebellar vermal lobules VI and VII is a nonspecific finding that even occurs in several conditions with-out autistic behavior. This suggests that it is not a specific neuroanantomical marker for autism, nor is cerebellar dysgenesis likely to be solely responsible for clinical autistic behaviors.

Control of the surface expression of uvomorulin after activation of mouse oocytes.

Uvomorulin (E-cadherin) is the major cell adhesion molecule responsible for intercellular adhesion in early mouse embryos. In contrast to other cell adhesion molecules, it is not detectable on the cell surface until around 6 h after fertilisation or parthenogenetic activation, at the time when pronuclear formation occurs (Clayton, L., Stinchcombe, S.V. and Johnson, M.H., Zygote 1, 333-44, 1993). In order to investigate this developmental control of surface expression of uvomorulin, we examined the effects of inhibitors of various cellular processes on the appearance of uvomorulin at the oocyte surface, as assessed immunocytochemically. Inhibitors of cytoskeletal assembly (cytochalasin D and nocodazole), protein synthesis (puromycin and anisomycin), and DNA synthesis (aphidicolin) had no effect on surface expression. Brefeldin A, which inhibits intracellular transport and secretion, did prevent surface expression, but monensin did not. The effects of brefeldin were reversible; following 8 h of treatment, recovery of surface expression after removal of brefeldin began within 2 h. The time-course of surface expression post-activation suggested a link with pronuclear formation. However, when pronuclear formation was advanced experimentally using 6-dimethylaminopurine (DMAP), concomitant advancement of surface uvomorulin was not observed. Similarly, surface expression of uvomorulin did not accompany puromycin-induced pronuclear formation in maturing meiotic metaphase 1 (MI) oocytes in vitro. Thus, surface uvomorulin expression does not appear to be linked simply to pronuclear formation. Proteolytic processing of both newly synthesised and total uvomorulin to generate mature molecule from precursor increased within 30 min to 1 h after activation, and also occurred in the continued presence of brefeldin, suggesting that uvomorulin processing appears to be controlled independently of its surface expression.

Capacity of mouse oocytes to become activated depends on completion of cytoplasmic but not nuclear meiotic maturation.

The ability of mouse oocytes to become activated after exposure to the calcium ionophore A23187 has been investigated at different stages of meiotic maturation. The potential to respond to ionophore has been studied in relation to the time since resumption of meiotic maturation, the chromosomal conformation of the DNA within each cell and the protein synthetic profile of the maturing oocyte. Our studies demonstrate that when maturing oocytes from an MF1 strain of mice were treated with A23187 activation occurred only in oocytes which had reached second meiotic metaphase (MII). However, development of the ability to respond to ionophore was not dependent on an orderly progression through normal chromosomal rearrangements such as separation at metaphase I (MI) and subsequent polar body extrusion, since these processes could be prevented and the capacity to be activated became apparent in such oocytes at a time when control cells had reached MII. These data suggest that the ability to respond to ionophore depends on the development of a cytoplasmic component or complex capable of monitoring the time since initiation of germinal vesicle breakdown. Metabolic radiolabelling of oocytes which were able to respond to calcium ionophore, even though they had been prevented from undergoing normal chromosomal rearrangements, showed them to be synthesising a group of proteins known as the 35 kDa complex.

Use of a polymorphic dinucleotide repeat sequence to detect non-blastomeric contamination of the polymerase chain reaction in biopsy samples for preimplantation diagnosis.

Using the polymerase chain reaction (PCR), amplification of two different target DNA sequences has been achieved with high frequency using single human blastomeres as template for the duplex reaction. One sequence is located within the beta-globin gene and contains the sickle cell locus, the other is a polymorphic dinucleotide repeat, which, as well as acting as a positive control for amplification, was used to check the origin of the amplified DNA. A comparison of the sequences amplified from the blastomere with sequences amplified from parental samples confirmed that amplification of blastomeric sequences, but not extraneous contaminating DNA, had taken place in most cases. The efficacy of this system for detecting extraneous DNA was checked by deliberately contaminating single blastomeres with foreign cells. The presence of contamination was detected by the amplification of sequences not present in blastomeric DNA and which therefore must have been amplified from extraneous contaminating DNA.

Staurosporine advances interblastomeric flattening of the mouse embryo.

Staurosporine, an inhibitor of protein kinase activity, causes premature intercellular flattening of blastomeres but does not induce their premature polarisation. The flattening induced is calcium dependent, is reversed transiently at mitosis and requires the continuing presence of the drug. Staurosporine also blocks the decompacting effect of phorbol ester on 8-cell embryos.

Reliability of detection by polymerase chain reaction of the sickle cell-containing region of the beta-globin gene in single human blastomeres.

Human preimplantation embryos at various stages of development have been analysed using the polymerase chain reaction to amplify a 680 base pair fragment of the beta-globin gene. Successful amplification was achieved more frequently with DNA from intact embryos containing between one and 11 cells, single cumulus cells, oocytes which had failed to fertilize and polar bodies than from single blastomeres disaggregated from intact embryos and treated in an identical manner. The distribution of nuclei demonstrated using the nuclear chromophore diamino-phenyl-indole showed considerable inter-blastomere variation; however, no clear correlation between staining pattern and successful amplification was observed. The reason for the unreliable amplification of DNA from single blastomeres is unclear but this finding has important implications for preimplantation diagnosis of genetic disease.

Effect of sodium hyaluronate on trabeculectomy filtration blebs.

A prospective, randomized study was carried out to investigate the effect of 1% sodium hyaluronate on the formation and function of trabeculectomy filtration blebs. Using a standardized operation, a group of patients had sodium hyaluronate introduced below the scleral and conjunctival flaps. In a control group the operation was done without sodium hyaluronate. Analysis of anterior chamber depth, postoperative intraocular pressure and the size and appearance of the bleb was carried out. The results showed that the use of sodium hyaluronate gives a significantly deeper postoperative anterior chamber but also suggested that it results in a filtration bleb which tends to be thinner walled and loculated.

Nd:YAG laser augmented pneumatic retinopexy.

We present the first ten consecutive cases of rhegmatogenous retinal detachment treated by one surgeon using pneumatic retinopexy with Nd:YAG laser disruption of vitreoretinal adhesions. In nine cases the retina has remained reattached, after a follow-up period of 11 to 18 months. The use of the Nd:YAG laser to disrupt vitreoretinal adhesions at the sites of retinal tears is discussed.

Unplugging of prolene IOL loops.

From end 1981 until early 1984, 415 Severin-type lenses were implanted in our two centres. In 10 instances i.e. 2.4%, we experienced unplugging of the anterior 12 o'clock positioned loop, apparently a result of the loop being held with the insertions forceps during insertion. The unplugging of the loop possibly affected the visual outcome in one instance.

Pseudophakic cystoid macular edema with the Severin lens.

Clinically significant cystoid macular edema confirmed by fluorescein angiography has been studied in a consecutive series of 285 eyes having intracapsular cataract extraction and Severin intraocular lens implantation. The incidence and natural course of the condition is described and associated factors are discussed.

Posterior chamber intraocular lens implantation: further review of the Severin lens.

A consecutive series of 116 Severin lens implantations with a mean follow-up time of 9 months was reported 2 years ago. In view of the excellent early outcome the series was continued and the results of the initial series is further reviewed at a mean follow-up time of 28 months. The entire series of 305 Severin lens implantations with a mean follow-up time of 21 months is reported. The early results are compared with the longer term results. Intraocular lens implantation in the United Kingdom is equally divided between extracapsular extractions with a posterior chamber lens and intracapsular extractions with an iris fixated lens, anterior chamber lenses taking third place. While the advantages of the extracapsular technique are undisputed, until YAG Lasers are more widely available for dealing with thickened posterior capsules, an intracapsular technique with an iris fixated lens may still have a place.

Posterior chamber intraocular lens implantation. Report on 116 consecutive intracapsular operations using Severin lens.

Experience with 116 consecutive posterior chamber Severin intraocular lenses following intracapsular extraction is described. The low incidence of complications in this series of selected patients and the good visual results are emphasized.