Differential distribution of N-acetylaspartylglutamate and N-acetylaspartate immunoreactivities in rat forebrain

Summary Contradictory immunohistochemical data have been reported on the localization of N-acetylaspartylglutamate in the rat forebrain, using different carbodiimide fixation protocols and antibody purification methods. In one case, N-acetylaspartylglutamate immunoreactivity was observed in apparent interneurons throughout all allocortical and isocortical regions, suggesting possible colocalization with GABA. In another case, strong immunoreactivity was observed in numerous pyramidal cells in neocortex and hippocampus, suggesting colocalization with glutamate or aspartate. Reconciling these disparate findings is crucial to understanding the role of N-acetylaspartylglutamate in nervous system function. Antibodies to N-acetylaspartylglutamate and a structurally related molecule, N-acetylaspartate, were purified in stages, and their cross-reactivities with protein conjugates of N-acetylaspartylglutamate and N-acetylaspartate were monitored at each stage by solidphase immunoassay. Reduction of the cross-reactivity of the anti-N-acetylaspartylglutamate antibodies to N-acetylaspartateprotein conjugates to about 1% eliminated significant staining of most pyramidal neurons in the rat forebrain. Utilizing highly purified antibodies, the distributions of N-acetylaspartylglutamate and N-acetylaspartate were examined in several major telencephalic and diencephalic regions of the rat, and were found to be distinct. N-acetylaspartylglutamate-immunoreactivity was observed in specific neuronal populations, including many groups thought to use GABA as a neurotransmitter. Among these were the globus pallidus, ventral pallidum, entopeducular nucleus, thalamic reticular nucleus, and scattered non-pyramidal neurons in all layers of isocortex and allocortex. N-acetylaspartate-immunoreactivity was more broadly distributed than N-acetylaspartylglutamate-immunoreactivity in the rat forebrain, appearing strongest in many pyramidal neurons. Although N-acetylaspartate-immunoreactivity was found in most neurons, it exhibited a great range of intensities between different neuronal types.     

Ciprofloxacin-induced renal insufficiency in cystic fibrosis.

Acute renal insufficiency is known to occur in patients who are taking ciprofloxacin, particularly the elderly. We report two young patients with cystic fibrosis who presented with acute renal insufficiency after 2-3 weeks of oral ciprofloxacin therapy. The incidence of this adverse effect in children and young adults who have cystic fibrosis is unknown. Multiple mechanisms for ciprofloxacin-induced nephrotoxicity have been proposed.     

Relative carriage rates of nuclear dehydrogenating clostridia in two populations of different colorectal cancer risk

Carriage of nuclear dehydrogenating clostridia has been associated with colon cancer and implicated in its aetiology. This study has compared the carriage of these organisms in a British population at high risk for the development of colon cancer with a low risk Nigerian population. Clostridia were found in all of the stools from both populations. Nuclear dehydrogenating clostridia were only found in the stools of the British subjects (32%). These results support the suggestion that the carriage rate of nuclear dehydrogenating clostridia in a population is related to the risk of colon cancer.     

Development of hatching blastocysts from immature human oocytes following in-vitro maturation and fertilization using a co-culture system

Recently, in-vitro maturation (IVM) of immature human oocytes recovered from non-stimulated follicles has been applied in the treatment of infertility. However, in previous reports, very few embryos cultured in conventional medium have reached the expanded blastocyst stage following in-vitro maturation and fertilization (IVM/IVF). The objective of this study was to investigate whether the developmental competence of human embryos following IVM/IVF could be enhanced by the use of a human ampullary cell co-culture system. Immature human oocytes were aspirated from small follicles at Caesarean section and then cultured in medium containing human menopausal gonadotrophin for 36 to 48 h, followed by insemination. Zygotes were randomly cultured either in conventional culture medium alone or in the co-culture system. Of 48 embryos cultured in conventional medium alone, all arrested at the 2-16- cell stage on day 3 after insemination. Of 46 embryos cultured in the co-culture system, 26 embryos (56.5%) arrested at the 2-16-cell stage. Six embryos (13%) developed to the morula stage. Fourteen embryos (30.4%) developed to expanded blastocysts and two blastocysts were hatching on day 7 after insemination. We conclude that co-culture significantly enhances the development of blastocysts in embryos resulting from IVM/IVF.      

Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.      

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

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.      

Circovirus-infected geese studied by in situ hybridization.

It has now been established that circovirus infection is common in farmed geese, but little is known about the clinicopathological significance of such infections. Ten clinically diseased geese suspected of being infected by circovirus were studied by in situ hybridization using a goose circovirus DNA probe. Circovirus DNA was demonstrated in the bursa of Fabricius (BF), spleen, thymus, bone marrow, liver, kidney, lung and heart, indicating that infection can be multisystemic. In some birds, virus DNA was present in very large quantities, most notably in the BF, liver and small intestine. With the exception of BF and thymus, there were no histological findings that would have suggested the presence of such quantities of circovirus DNA. In view of the very large quantities of virus DNA labelling present in some tissues, and by analogy to porcine circovirus type 2 infection and psittacine beak and feather virus infections, which are known to cause severe disease, and which have similar virus distribution to that found in our geese, it seems probable that the circovirus was important in the disease manifestations shown by the infected geese.     

Individuals with low back pain: how do they view physical activity?

BACKGROUND: Recent guidelines for those with acute low back pain have advocated early resumption of normal activity and increased physical activity. Little is known about the relationship between low back pain and physical activity, and on the impact of that relationship on the promotion of increased levels of physical activity within a general practice population. OBJECTIVES: We aimed to explore associations between factors that influence changes in physical activity and the way individuals perceive and behave with their low back pain, and the impact of those perceptions and behaviour on physical activity. METHODS: Twenty-seven informants were chosen using a purposive sample from a larger group of individuals who, because of their low back trouble, had been referred by their GPs to a community-based, single-blind, randomized controlled trial (RCT) at the University of York, which is evaluating the effectiveness and cost-effectiveness of a progressive exercise programme. Fifty-four interviews were conducted with this subgroup of the RCT; four informants were interviewed once, 19 twice and four of them three times. Interviews were transcribed and analysed using manual and computer-aided approaches. RESULTS: Physical activity was perceived as (i) activities of daily living, (ii) activities causing breathlessness that they went out of the way to do and (iii) more competitive-type activity. The avoidance of physical activity and fear of pain returning were the two main factors directly associated with informants' backs and changes in physical activity. These two factors hindered increases in physical activity, even though the majority of informants believed strongly that being physically active helped ease their low back pain. CONCLUSIONS: When advocating that individuals with acute low back pain return to or increase physical activity, it is important that clinicians identify avoidance of physical activity and/or fear of pain at the earliest stage in order to tailor advice and reassurance appropriately. If avoidance of activity and fear of pain is identified and clinicians want to encourage patients to take up and sustain increased physical activity, they should explore issues of fear of pain, and avoidance of and confidence to do physical activities, in addition to other factors influencing physical activity.