Prospective randomised comparison of photocoagulation and rubber band ligation in treatment of haemorrhoids.

Two hundred and sixty eight patients with haemorrhoids were allocated at random to treatment by either photocoagulation (group 1, n=141) or rubber band ligation (group 2, n=127) and followed up for one year. There was no significant difference in the symptomatic outcome of treatment between the two groups at one, four, or 12 months, irrespective of whether first or second degree haemorrhoids were treated. Side effects of treatment (bleeding or severe pain) were significantly more common after rubber band ligation (n=11) than after photocoagulation (n=2; p less than 0.01). Further outpatient treatment, however, was required significantly more often after photocoagulation (n=23) than rubber band ligation (n=6) (p greater than 0.02), and 19 patients (14 in group 1 and five in group 2; NS) subsequently had a haemorrhoidectomy. At one year 26 of 103 patients were dissatisfied after photocoagulation compared with 20 of 88 after rubber band ligation. Photocoagulation is a safe and comfortable treatment which gives long term results that are as good as those of rubber band ligation. Complications are more common after rubber band ligation, but further treatment is required more commonly after photocoagulation.     

Effects of Erythropoietin in Murine-Induced Pluripotent Cell-Derived Panneural Progenitor Cells

Induced cell fate changes by reprogramming of somatic cells offers an efficient strategy to generate autologous pluripotent stem (iPS) cells from any adult cell type. The potential of iPS cells to differentiate into various cell types is well established, however the efficiency to produce functional neurons from iPS cells remains modest. Here, we generated panneural progenitor cells (pNPCs) from mouse iPS cells and investigated the effect of the neurotrophic growth factor erythropoietin (EPO) on their survival, proliferation and neurodifferentiation. Under neural differentiation conditions, iPS-derived pNPCs gave rise to microtubule-associated protein-2 positive neuronlike cells (34% to 43%) and platelet-derived growth factor receptor positive oligodendrocytelike cells (21% to 25%) while less than 1% of the cells expressed the astrocytic marker glial fibrillary acidic protein. Neuronlike cells generated action potentials and developed active presynaptic terminals. The pNPCs expressed EPO receptor (EPOR) mRNA and displayed functional EPOR signaling. In proliferating cultures, EPO (0.1–3 U/mL) slightly improved pNPC survival but reduced cell proliferation and neurosphere formation in a concentration-dependent manner. In differentiating cultures EPO facilitated neurodifferentiation as assessed by the increased number of β-III-tubulin positive neurons. Our results show that EPO inhibits iPS pNPC self-renewal and promotes neurogenesis.     

The Mcf1 toxin induces apoptosis via the mitochondrial pathway and apoptosis is attenuated by mutation of the BH3-like domain

Photorhabdus are Gram-negative, nematode-vectored bacteria that produce toxins to kill their insect hosts. The expression of one of these, Makes caterpillars floppy 1 (Mcf1), is sufficient to allow Escherichia coli to survive within, and kill, caterpillars which are otherwise able to clear E. coli infection. Mcf1 treated caterpillars show rapid loss of body turgor (the 'floppy' phenotype) and death is associated with massive apoptosis of both the midgut epithelium and insect phagocytes. Mammalian tissue culture cells treated with Mcf1 also display key features of apoptosis including zeiosis, apoptotic nuclear morphology, DNA laddering, activation of the effector caspase-3 and PARP cleavage. As Mcf1 carries a single BH3-like domain, here we investigate the hypothesis that this toxin promotes apoptosis via the mitochondrial pathway by mimicking a BH3 domain-only protein. Consistent with this hypothesis, a double mutant within the BH3-like domain causes a dramatic decline in apoptosis. Mcf1 also alters mitochondrial membrane potential and triggers the release of cytochrome c. Cells overexpressing Bcl-x(L), an anti-apoptotic Bcl-2 family member, are resistant to Mcf1-mediated apoptosis, as are cells deficient in Bax. In addition, translocation of Bax to the mitochondrion is observed in response to Mcf1 treatment. Together, these results show that Mcf1 mediates apoptosis via the mitochondrial pathway, and are consistent with the hypothesis that the BH3-like domain in Mcf1 is a functional requirement for the pro-apoptotic activity of Mcf1.     

Mesenchymal stem cells restore frataxin expression and increase hydrogen peroxide scavenging enzymes in Friedreich ataxia fibroblasts

Dramatic advances in recent decades in understanding the genetics of Friedreich ataxia (FRDA)--a GAA triplet expansion causing greatly reduced expression of the mitochondrial protein frataxin--have thus far yielded no therapeutic dividend, since there remain no effective treatments that prevent or even slow the inevitable progressive disability in affected individuals. Clinical interventions that restore frataxin expression are attractive therapeutic approaches, as, in theory, it may be possible to re-establish normal function in frataxin deficient cells if frataxin levels are increased above a specific threshold. With this in mind several drugs and cytokines have been tested for their ability to increase frataxin levels. Cell transplantation strategies may provide an alternative approach to this therapeutic aim, and may also offer more widespread cellular protective roles in FRDA. Here we show a direct link between frataxin expression in fibroblasts derived from FRDA patients with both decreased expression of hydrogen peroxide scavenging enzymes and increased sensitivity to hydrogen peroxide-mediated toxicity. We demonstrate that normal human mesenchymal stem cells (MSCs) induce both an increase in frataxin gene and protein expression in FRDA fibroblasts via secretion of soluble factors. Finally, we show that exposure to factors produced by human MSCs increases resistance to hydrogen peroxide-mediated toxicity in FRDA fibroblasts through, at least in part, restoring the expression of the hydrogen peroxide scavenging enzymes catalase and glutathione peroxidase 1. These findings suggest, for the first time, that stem cells may increase frataxin levels in FRDA and transplantation of MSCs may offer an effective treatment for these patients.     

Potentiation and cellular phenotypes of the insecticidal Toxin complexes of Photorhabdus bacteria

The toxin complex (tc) genes of bacteria comprise a large and growing family whose mode of action remains obscure. In the insect pathogen Photorhabdus, tc genes encode high molecular weight insecticidal toxins with oral activity against caterpillar pests. One protein, TcdA, has recently been expressed in transgenic plants and shown to confer insect resistance. These toxins therefore represent alternatives to toxins from Bacillus thuringiensis (Bt) for deployment in transgenic crops. Levels of TcdA expression in transgenic plants were, however, low and the full toxicity associated with the native toxin was not reconstituted. Here we show that increased activity of the toxin TcdA1 requires potentiation by either of two pairs of gene products, TcdB1 and TccC1 or TcdB2 and TccC3. Moreover, these same pairs of proteins can also cross-potentiate a second toxin, TcaA1B1. To elucidate the likely functional domains present in these large proteins, we expressed fragments of each 'toxin' or 'potentiator' gene within mammalian cells. Several domains produced abnormal cellular morphologies leading to cell death, while others showed specific phenotypes such as nuclear translocation. Our results prove that the Tc toxins are complex proteins with multiple functional domains. They also show that both toxin genes and their potentiator pairs will need to be expressed to reconstitute full activity in insect-resistant transgenic plants. Moreover, they suggest that the same potentiator pair will be able to cross-potentiate more than one toxin in a single plant.     

Phosphoinositide 3-kinase mediated signalling contributes to development of diabetes-induced abnormal vascular reactivity of rat carotid artery

Diabetes mellitus is associated with vascular complications, including an impairment of vascular function and alterations in the reactivity of blood vessels to vasoactive agents. Phosphatidylinositol 3-kinase (PI3K) is a signalling enzyme that plays key roles in vascular growth, proliferation and cellular apoptosis and is implicated in modulating vascular smooth muscle contractility. The aim of this study was to determine whether PI3K plays a role in development of diabetes-induced altered vascular reactivity to selected vasoconstrictors and vasodilators. The effect of 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a selective PI3K inhibitor, on isolated segments of carotid arteries from streptozotocin (STZ)-diabetic rats was investigated. Ring segments of the isolated carotid arteries were mounted in organ baths to measure changes in isometric tension. Our results showed that STZ treatment produced an increase in the vasoconstrictor response to norepinephrine (NE), angiotensin II (Ang II) and endothelin-1 (ET-1) and an attenuated vasodilator response to carbachol and histamine in the isolated carotid arteries from STZ-diabetic animals. Diabetes-induced impaired vascular responsiveness to the vasoactive agonists was prevented by chronic inhibition of PI3K by LY294002 even though blood glucose levels remained high. This is the first study to show that selective inhibition of PI3K can attenuate the development of diabetes-induced abnormal vascular reactivity in the isolated carotid arteries of diabetic rats.     

Human bone marrow-derived mesenchymal stem cells secrete brain-derived neurotrophic factor which promotes neuronal survival in vitro

Bone marrow-derived mesenchymal stem cells (MSCs) are of therapeutic interest in a variety of neurological diseases. In this study, we wished to determine whether human MSCs secrete factors which protect cultured rodent cortical neurons from death by trophic factor withdrawal or nitric oxide (NO) exposure. Medium conditioned by MSCs attenuated neuronal death under these conditions, a process which was dependent on intact PI₃kinase/Akt pathway signaling. Trophic withdrawal and NO exposure in cultured cortical neurons led to reduction in Akt signaling pathways, whereas NO administration activated p38 MAPkinase in neuronal cultures. Addition of MSC-conditioned medium significantly activated the PI3kinase/Akt pathway and in neurons exposed to NO, MSC-conditioned medium reduced p38 signaling. We show that MSCs secrete brain-derived neurotrophic factor (BDNF) and addition of anti-BDNF neutralising antibodies to MSC-conditioned medium attenuated its neuroprotective effect. Exposure of neurons to BDNF increased activation of Akt pathways and protected neurons from trophic factor withdrawal. These observations determine the mechanisms of neuroprotection offered by MSC-derived factors and suggest an important role for BDNF in neuronal protection.     

Genetic Analysis of Circadian Responses to Low Frequency Electromagnetic Fields in Drosophila melanogaster

The blue-light sensitive photoreceptor cryptochrome (CRY) may act as a magneto-receptor through formation of radical pairs involving a triad of tryptophans. Previous genetic analyses of behavioral responses of Drosophila to electromagnetic fields using conditioning, circadian and geotaxis assays have lent some support to the radical pair model (RPM). Here, we describe a new method that generates consistent and reliable circadian responses to electromagnetic fields that differ substantially from those already reported. We used the Schuderer apparatus to isolate Drosophila from local environmental variables, and observe extremely low frequency (3 to 50 Hz) field-induced changes in two locomotor phenotypes, circadian period and activity levels. These field-induced phenotypes are CRY- and blue-light dependent, and are correlated with enhanced CRY stability. Mutational analysis of the terminal tryptophan of the triad hypothesised to be indispensable to the electron transfer required by the RPM reveals that this residue is not necessary for field responses. We observe that deletion of the CRY C-terminus dramatically attenuates the EMF-induced period changes, whereas the N-terminus underlies the hyperactivity. Most strikingly, an isolated CRY C-terminus that does not encode the Tryptophan triad nor the FAD binding domain is nevertheless able to mediate a modest EMF-induced period change. Finally, we observe that hCRY2, but not hCRY1, transformants can detect EMFs, suggesting that hCRY2 is blue light-responsive. In contrast, when we examined circadian molecular cycles in wild-type mouse suprachiasmatic nuclei slices under blue light, there was no field effect. Our results are therefore not consistent with the classical Trp triad-mediated RPM and suggest that CRYs act as blue-light/EMF sensors depending on trans-acting factors that are present in particular cellular environments.     

A single amino acid can switch the oligomerization state of the alpha-helical coiled-coil domain of cartilage matrix protein.

We have studied the oligomerization of an alpha-helical coiled-coil using as an example a peptide corresponding to the C-terminal domain of cartilage matrix protein. By replacing one arginine residue, which forms an interchain ionic interaction with a glutamic acid residue, with glutamine, we found that this peptide assembles into a homotetramer at neutral pH in contrast to the native molecule which forms homotrimers. At acidic and basic pH, however, we again observed the trimer conformation. Another arginine, which is probably involved in an intrachain salt bridge, has no effect on the assembly. Our data demonstrate that besides the specific distribution of hydrophobic residues, interchain ionic interactions can be crucial in modulating the association behavior of alpha-helical coiled-coil domains.     

Surface morphology, leaf and cuticle thickness of four dwarf shrubs from a sub-Arctic heath following long-term exposure to enhanced levels of UV-B

In 1991 a field experiment was established in subarctic heathland at Abisko (68°35'N, 18°82'E), northern Sweden, to investigate the effects of enhanced UV-B (280–315 nm) radiation, simulating 15% ozone depletion, on plants in their natural environment. Leaves of the four dominant dwarf shrubs, the deciduous Vaccinium myrtillus L. and V. uliginosum L. and the evergreen V. vitis-idaea L. and Empetrum hermaphroditum Hagerup were examined after 7 years of UV-B treatment. SEM and ESEM were used to visualize surface features and to determine trichome density. Multiphoton laser scanning microscopy showed that UV-B absorbing compounds were localized in the trichomes of all species. Trichomes varied in size, number and distribution between the species. Enhanced UV-B reduced adaxial trichome density significantly (by approximately 25%) in only one species, V. uliginosum . This effect could be of importance for the UV-B absorbing potential of the adaxial epidermis of V. uliginosum . Epicuticular wax structures were found only on the abaxial surface of V. uliginosum and were unaffected by enhanced UV-B. The cuticular surfaces of all other species were smooth and featureless. Leaf thickness, adaxial and abaxial cuticle thickness varied between the species although there was no apparent effect of enhanced UV-B. It is concluded that long-term enhancement of UV-B has an effect on adaxial trichome density in V. uliginosum , but that there is no general effect on leaf morphology of the other species.