MSTO1 mutations cause mtDNA depletion, manifesting as muscular dystrophy with cerebellar involvement.

MSTO1 encodes a cytosolic mitochondrial fusion protein, misato homolog 1 or MSTO1. While the full genotype-phenotype spectrum remains to be explored, pathogenic variants in MSTO1 have recently been reported in a small number of patients presenting with a phenotype of cerebellar ataxia, congenital muscle involvement with histologic findings ranging from myopathic to dystrophic and pigmentary retinopathy. The proposed underlying pathogenic mechanism of MSTO1-related disease is suggestive of impaired mitochondrial fusion secondary to a loss of function of MSTO1. Disorders of mitochondrial fusion and fission have been shown to also lead to mitochondrial DNA (mtDNA) depletion, linking them to the mtDNA depletion syndromes, a clinically and genetically diverse class of mitochondrial diseases characterized by a reduction of cellular mtDNA content. However, the consequences of pathogenic variants in MSTO1 on mtDNA maintenance remain poorly understood. We present extensive phenotypic and genetic data from 12 independent families, including 15 new patients harbouring a broad array of bi-allelic MSTO1 pathogenic variants, and we provide functional characterization from seven MSTO1-related disease patient fibroblasts. Bi-allelic loss-of-function variants in MSTO1 manifest clinically with a remarkably consistent phenotype of childhood-onset muscular dystrophy, corticospinal tract dysfunction and early-onset non-progressive cerebellar atrophy. MSTO1 protein was not detectable in the cultured fibroblasts of all seven patients evaluated, suggesting that pathogenic variants result in a loss of protein expression and/or affect protein stability. Consistent with impaired mitochondrial fusion, mitochondrial networks in fibroblasts were found to be fragmented. Furthermore, all fibroblasts were found to have depletion of mtDNA ranging from 30 to 70% along with alterations to mtDNA nucleoids. Our data corroborate the role of MSTO1 as a mitochondrial fusion protein and highlight a previously unrecognized link to mtDNA regulation. As impaired mitochondrial fusion is a recognized cause of mtDNA depletion syndromes, this novel link to mtDNA depletion in patient fibroblasts suggests that MSTO1-deficiency should also be considered a mtDNA depletion syndrome. Thus, we provide mechanistic insight into the disease pathogenesis associated with MSTO1 mutations and further define the clinical spectrum and the natural history of MSTO1-related disease.

Selective oxidative dehydrogenation of propane to propene using boron nitride catalysts.

The exothermic oxidative dehydrogenation of propane reaction to generate propene has the potential to be a game-changing technology in the chemical industry. However, even after decades of research, selectivity to propene remains too low to be commercially attractive because of overoxidation of propene to thermodynamically favored CO2 Here, we report that hexagonal boron nitride and boron nitride nanotubes exhibit unique and hitherto unanticipated catalytic properties, resulting in great selectivity to olefins. As an example, at 14% propane conversion, we obtain selectivity of 79% propene and 12% ethene, another desired alkene. Based on catalytic experiments, spectroscopic insights, and ab initio modeling, we put forward a mechanistic hypothesis in which oxygen-terminated armchair boron nitride edges are proposed to be the catalytic active sites.

Antibiotic cement-coated rods: an effective treatment for infected long bones and prosthetic joint nonunions.

The treatment of infected nonunions is difficult. Antibiotic cement-coated (ACC) rods provide stability as well as delivering antibiotics. We conducted a review of 110 infected nonunions treated with ACC rods. Patients were divided into two groups: group A (67 patients) with an infected arthrodesis, and group B (43 patients) with an infected nonunion in a long bone. In group A, infected arthrodesis, the success rate after the first procedure was 38/67 (57%), 29/67 (43%) required further surgery for either control of infection or non-union. At last follow-up, five patients required amputation, representing a limb salvage rate of 62/67 (93%) overall. In all, 29/67 (43%) presented with a bone defect with a mean size of 6.78 cm (2 to 25). Of those with a bone defect, 13/29 (45%) required further surgery and had a mean size of defect of 7.2 cm (3.5 to 25). The cultures were negative in 17/67 (26%) and the most common organism cultured was methicillin-resistant staphylococcus aureus (MRSA) (23/67, (35%)). In group B, long bones nonunion, the success rate after the first procedure was 26/43 (60%), 17/43 (40%) required further surgery for either control of infection or nonunion. The limb salvage rate at last follow-up was 43/43 (100%). A total of 22/43 (51%) had bone defect with a mean size of 4.7 cm (1.5 to 11.5). Of those patients with a bone defect, 93% required further surgery with a mean size of defect of 5.4 cm (3 to 8.5). The cultures were negative in 10/43 (24%) and the most common organism cultured was MRSA, 15/43 (35%). ACC rods are an effective form of treatment for an infected nonunion, with an acceptable rate of complications.

Eotaxin-1 is involved in parasite clearance during chronic filarial infection.

Eosinophil migration as key feature of helminth infection is increased during infection with filarial nematodes. In a mouse model of filariasis, we investigated the role of the eosinophil-attracting chemokine Eotaxin-1 on disease outcome. BALB/c and Eotaxin-1(-/-) mice were infected with the rodent filaria Litomosoides sigmodontis, and parasitic parameters, cellular migration to the site of infection, and cellular responsiveness were investigated. We found increased parasite survival but unaffected eosinophil migration to the site of infection in Eotaxin-1(-/-) mice. Expression of CD80 and CD86 was reduced on eosinophils from Eotaxin-1(-/-) mice after in vitro TLR2 stimulation and exposure to filarial antigen, respectively, suggesting a potential reduced activation state of eosinophils in Eotaxin-1 deficient mice. We further demonstrated that macrophages from Eotaxin-1(-/-) mice produce decreased amounts of IL-6 in vitro, a cytokine found to be associated with parasite containment, suggesting possible mechanisms by which Eotaxin-1 regulates activation of inflammatory cells and thus parasite survival.

The effect on mechanical axis deviation of femoral lengthening with an intramedullary telescopic nail.

Internal lengthening devices in the femur lengthen along the anatomical axis, potentially creating lateral shift of the mechanical axis. We aimed to determine whether femoral lengthening along the anatomical axis has an inadvertent effect on lower limb alignment. Isolated femoral lengthening using the Intramedullary Skeletal Kinetic Distractor was performed in 27 femora in 24 patients (mean age 32 years (16 to 57)). Patients who underwent simultaneous realignment procedures or concurrent tibial lengthening, or who developed mal- or nonunion, were excluded. Pre-operative and six-month post-operative radiographs were used to measure lower limb alignment. The mean lengthening achieved was 4.4 cm (1.5 to 8.0). In 26 of 27 limbs, the mechanical axis shifted laterally by a mean of 1.0 mm/cm of lengthening (0 to 3.5). In one femur that was initially in varus, a 3 mm medial shift occurred during a lengthening of 2.2 cm. In a normally aligned limb, intramedullary lengthening along the anatomical axis of the femur results in a lateral shift of the mechanical axis by approximately 1 mm for each 1 cm of lengthening.

Differential expression of toll-like receptor 2 on dendritic cells from asymptomatic and symptomatic atopic donors.

BACKGROUND: Early microbial exposure may reduce the risk for developing allergies on an atopic genetic background. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns of microbes and modulate innate and adaptive immunity. Different expression of TLRs in symptomatic and asymptomatic atopic donors may contribute to the development of allergic disease. METHODS: Monocytes and monocyte-derived dendritic cells (DCs) from symptomatic (n = 12) and asymptomatic atopic donors (n = 11), healthy nonatopics (n = 14) and from patients with psoriasis (n = 13) were analyzed for their expression of TLR2, TLR4 and TLR9 by real-time PCR. RESULTS: Monocytes did not show any differences in TLR2, TLR4 and TLR9 expression between the 4 groups. In contrast, DCs from asymptomatic donors showed an enhanced expression of TLR2 over DCs from nonatopics (p = 0.038) and just failed to reach significance when compared to symptomatic atopic patients (p = 0.060). TLR2 expression kinetics from monocytes to monocyte-derived DCs showed sustained expression of TLR2 in DCs only from asymptomatic donors but downregulation in the other groups. In DCs from symptomatic atopic donors, the expression of TLR2 correlated significantly with total IgE values in the serum (p = 0.01994). CONCLUSION: Differential expression and functional regulation of TLR2 expression by DCs from symptomatic and asymptomatic atopic donors may be important for the manifestation of allergic disease. Increased and sustained TLR2 expression on DCs, possibly as a result of an increased exposure to microorganisms or as a mechanism enhancing the sensitivity of microbe detection, may be of functional importance for the maintenance of clinical unresponsiveness toward allergens.

Granulocytes in helminth infection -- who is calling the shots?

Helminths are parasitic organisms that can be broadly described as "worms" due to their elongated body plan, but which otherwise differ in shape, development, migratory routes and the predilection site of the adults and larvae. They are divided into three major groups: trematodes (flukes), which are leaf-shaped, hermaphroditic (except for blood flukes) flatworms with oral and ventral suckers; cestodes (tapeworms), which are segmented, hermaphroditic flatworms that inhabit the intestinal lumen; and nematodes (roundworms), which are dioecious, cylindrical parasites that inhabit intestinal and peripheral tissue sites. Helminths exhibit a sublime co-evolution with the host's immune system that has enabled them to successfully colonize almost all multicellular species present in every geographical environment, including over two billion humans. In the face of this challenge, the host immune system has evolved to strike a delicate balance between attempts to neutralize the infectious assault versus limitation of damage to host tissues. Among the most important cell types during helminthic invasion are granulocytes: eosinophils, neutrophils and basophils. Depending on the specific context, these leukocytes may have pivotal roles in host protection, immunopathology, or facilitation of helminth establishment. This review provides an overview of the function of granulocytes in helminthic infections.

Filariasis in Africa--treatment challenges and prospects.

Lymphatic filariasis (LF) and onchocerciasis are parasitic nematode infections that are responsible for a major disease burden in the African continent. Disease symptoms are induced by the immune reactions of the host, with lymphoedema and hydrocoele in LF, and dermatitis and ocular inflammation in onchocerciasis. Wuchereria bancrofti and Onchocerca volvulus, the species causing LF and onchocerciasis in Africa, live in mutual symbiosis with Wolbachia endobacteria, which cause a major part of the inflammation leading to symptoms and are antibiotic targets for treatment. The standard microfilaricidal drugs ivermectin and albendazole are used in mass drug administration programmes, with the aim of interrupting transmission, with a consequent reduction in the burden of infection and, in some situations, leading to regional elimination of LF and onchocerciasis. Co-endemicity of Loa loa with W. bancrofti or O. volvulus is an impediment to mass drug administration with ivermectin and albendazole, owing to the risk of encephalopathy being encountered upon administration of ivermectin. Research into new treatment options is exploring several improved delivery strategies for the classic drugs or new antibiotic treatment regimens for anti-wolbachial chemotherapy.

Mechanical failure of the Intramedullary Skeletal Kinetic Distractor in limb lengthening.

Between October 2001 and September 2009 we lengthened 242 lower-limb segments in 180 patients using the Intramedullary Skeletal Kinetic Distractor (ISKD). Mechanical failure was defined either as breakage of the ISKD or failure of the internal mechanism to activate. Retrieved nails which failed mechanically were examined by the manufacturer for defects. In all, 15 ISKDs in 12 patients (13 limbs) failed mechanically representing an overall failure rate of 6.2%, with fracture of the device occurring in ten of the 15 failures. Two nails in one patient failed to lengthen and had to be replaced. The manufacturer detected an error in the assembly of the nail, which prompted a wide recall. One nail jammed after being forcefully inserted, and two nails failed to lengthen fully. Lengthening was achieved in all 12 patients, although three required a second operation to exchange a defective nail for a new, functioning device. The ISKD is a complex mechanical device which lengthens by the oscillation of two telescopic sections connected by a threaded rod. The junction between these sections is surrounded by a keyring collar. This keyring collar is the weakest part of the device.

Filariasis and lymphoedema.

Among the causes of lymphoedema (LE), secondary LE due to filariasis is the most prevalent. It affects only a minority of the 120 million people infected with the causative organisms of lymphatic filariasis (LF), Wuchereria bancrofti and Brugia malayi/timori, but is clustered in families, indicating a genetic basis for development of this pathology. The majority of infected individuals develop filarial-specific immunosuppression that starts even before birth in cases where mothers are infected and is characterized by regulatory T-cell responses and high levels of IgG4, thus tolerating high parasite loads and microfilaraemia. In contrast, individuals with this pathology show stronger immune reactions biased towards Th1, Th2 and probably also Th17. Importantly, as for the aberrant lymph vessel development, innate immune responses that are triggered by the filarial antigen ultimately result in the activation of vascular endothelial growth factors (VEGF), thus promoting lymph vessel hyperplasia as a first step to lymphoedema development. Wolbachia endosymbionts are major inducers of these responses in vitro, and their depletion by doxycycline in LF patients reduces plasma VEGF and soluble VEGF-receptor-3 levels to those seen in endemic normals preceding pathology improvement. The search for the immunogenetic basis for LE could lead to the identification of risk factors and thus, to prevention; and has so far led to the identification of single-nucleotide polymorphisms (SNP) with potential functional relevance to VEGF, cytokine and toll-like receptor (TLR) genes. Hydrocele, a pathology with some similarity to LE in which both lymph vessel dilation and lymph extravasation are shared sequelae, has been found to be strongly associated with a VEGF-A SNP known for upregulation of this (lymph-)angiogenesis factor.

New insights into the biology of filarial infections.

Recent successes in the control of lymphatic filariasis and onchocerciasis need continuing research in order to sustain the achievements and to develop further tools to tackle the new questions that arise when only reduced infection prevalences prevail. In this regard, in a symposium held at the Xth European Multicolloquium of Parasitology (August 2008, Paris) questions such as the impact of filarial immunosuppression, and its lack following filarial control, on the outcome of co-infections were addressed, as were new approaches to treatment with promising drugs such as moxidectin or the antibiotic chemotherapy against Wolbachia endosymbionts in filariae. In particular, longer treatment courses of doxycycline could be carried out by community-directed treatment at high coverage, thus potentially allowing its use in restricted areas with suboptimal responses to ivermectin against onchocerciasis, or in areas with co-infection by loiasis where onchocerciasis or lymphatic filariasis need to be controlled. New, more potent drugs, or eventually vaccines, will be of importance because in many vector-filarial parasite relationships worldwide, transmission efficacy increases with low numbers of ingested microfilariae, and since ivermectin may render treated hosts more susceptible to new infection.

Ultrasonography in filaria-infected rodents: detection of adult Litomosoides sigmodontis and Brugia malayi filariae.

OBJECTIVE: To evaluate the usefulness of ultrasonography (USG) in the detection of adult filariae in rodents. Wuchereria bancrofti are frequently detected using USG in humans, whereas adult Brugia malayi have not been so far. METHODS: A Meriones unguiculatus with Litomosoides sigmodontis infection was examined to visualize adult filariae of a similar length as W. bancrofti. Similarly, three Mastomys coucha, infected with B. malayi, were examined using USG to verify whether the adult worms, which are far smaller than W. bancrofti and L. sigmodontis, can be located using USG in the animals. RESULTS: Adult L. sigmodontis were detected using USG in the pleural cavity of M. unguiculatus, and in M. coucha adult B. malayi were visualized in the hearts, lungs, axillary lymph nodes and scrotum. Ultrasound findings were verified by dissection of the rodents. CONCLUSIONS: Although adult B. malayi are far smaller than L. sigmodontis and W. bancrofti, they can be detected using USG in rodents. USG may serve as an adjunctive tool to support parasitological examinations and can add information on filarial infections at any time point of an observation period, particularly in cryptic infections and without the need for invasive measures or killing of the rodent. Thus, USG can support the early detection of macrofilaricidal activities of new compounds and can be used to determine the location of adult worms in the animals. It is possible to give a rough estimate of the number of adult worms, but determination of the exact numbers of adult filariae in various locations is impossible with USG.

Immunomodulation by filarial nematodes.

In order to chronically infect their hosts, filarial nematodes have generated a range of strategies to evade and down-modulate the host's immune system. The recent concept of suppression of immune responses by regulatory T cells has in part benefited from examinations in human and murine filariasis. Its further development in basic immunology animal models has in turn helped to better understand down-regulatory immune mechanisms in filariasis. Thus, filarial nematodes orchestrate down-regulation by inducing regulatory T cells and alternatively activated macrophages, which are able to suppress both Th1 and Th2 responses. Regulatory T cells can also induce the secretion of IgG4 from B cells as another arm of modulation. Dendritic cells are down-regulated upon first encounter with infective L3 larvae. Failure to respond to down-regulatory induction is based on genetic traits in hosts and leads to reduced parasite loads, albeit at the expense of pathology and disease. Since down-regulation in chronically and heavily infected hosts extends to third-party antigens, it is essential to analyse the impact of filarial infection for vaccination, allergy and important coinfections such as malaria, in order to foresee and avert potentially disastrous consequences of filariasis control programmes.

Continuing damage to rat retinal DNA during darkness following light exposure.

The damaging effects of visible light on the mammalian retina can be detected as functional, morphological or biochemical changes in the photoreceptor cells. Although previous studies have implicated short-lived reactive oxygen species in these processes, the termination of light exposure does not prevent continuing damage. To investigate the degenerative processes persisting during darkness following light treatment, rats were exposed to 24 h of intense visible light and the accumulation of DNA damage to restriction fragments containing opsin, insulin 1 or interleukin-6 genes was measured as single-strand breaks (ssb) on alkaline agarose gels. With longer dark treatments all three DNA fragments showed increasing DNA damage. Treatment of rats with the synthetic antioxidant dimethylthiourea prior to light exposure reduced the initial development of alkali-sensitive strand breaks and allowed significant repair of all three DNA fragments. The time course of double-strand DNA breaks was also examined in specific genes and repetitive DNA. Nucleosomal DNA laddering was evident immediately following the 24 h light treatment and increased during the subsequent dark period. The increase in the intensity of the DNA ladder pattern suggests a continuation of enzymatically mediated apoptotic processes triggered during light exposure. The protective effects of antioxidant suggests that the light-induced DNA degradative process includes both early oxidative reactions and enzymatic processes that continue after cessation of light exposure.

Inhibition of the allograft response by donor specific blood transfusion: association with reduced local TH1 cytokines and nitric oxide but enhanced prostaglandin E2 production.

BACKGROUND: Donor-specific blood transfusion (DST) may improve allograft survival in human and animal models, but the mechanisms for this graft protective effect are incompletely understood. The sponge matrix allograft model was used to determine if DST induces regulatory factors within the allograft. METHODS: C57BL/6 (H-2b) recipients received donor-specific (DBA/2J, H-2d) or syngeneic (C57BL/6) blood 7 days before sponge matrix allograft (DBA/2J) implantation. Fourteen days postgrafting, the sponge infiltrating cells (SIC) were examined for cytotoxic T cell (CTL) and natural killer (NK) activity, and sponge exudate fluid (SEF) was assessed for nitric oxide (.N=O) and prostaglandin E2 (PGE2) content. Interleukin- (IL) 2, IL-4, IL-10, and interferon-gamma (IFN-gamma) production by SIC was also determined. Recipient splenocytes were simultaneously assessed for anti-donor cytotoxic and proliferative responses and .N=O production. RESULTS: SIC from mice receiving syngeneic transfusions (ST) acquired both CTL and NK activity postgrafting, with maximal activity by day 14. DST suppressed both CTL and NK activity throughout the postgrafting period. Limiting dilution analysis (LDA) of SIC to determine precursor and native CTL frequency showed significantly lower responder cell frequency after DST compared with ST. SEF .N=O levels and SIC production of IL-2 and IFN-gamma in grafted DST mice were significantly lower than in grafted mice receiving ST. No significant amounts of IL-4 and very low levels of IL-10 were produced by SIC from grafted mice after either ST or DST. Conversely, PGE2 content of sponge fluid and serum from DST mice was higher than in mice receiving ST. Antigen stimulated splenocyte proliferation and CTL development assessed by LDA were also inhibited by DST. CONCLUSIONS: Reduction in local TH1 cytokines, absence of detectable TH2 cytokines, with enhanced PGE2 and depressed .N=O were observed in the local graft environment after DST. These data support the hypothesis that DST induces donor-specific intragraft suppressor factors, accompanied by reduced local and systemic immune activation.

Graft hyporeactivity induced by immature donor-derived dendritic cells.

Immature dendritic cells (DCs) are deficient in surface co-stimulatory molecules and have been shown to exhibit a 'tolerogenic' potential. We investigated the allostimulatory activity of immature DCs in one-way mixed leukocyte reactions and their capacity to inhibit anti-donor cytolytic activity in the sponge matrix allograft model. Immature DCs (CD80 and CD86 deficient) were derived from bone marrow cells propagated in GM-CSF and TGF-beta1. Mature DCs (CD80+ and CD86+) were derived from bone marrow cells propagated in GM-CSF and IL-4. Either 2 x 10(6) DBA/2J (DBA, H-2d) immature DCs or 2 x 10(6) mature DCs were injected intravenously into C57BL/6J (B6, H-2b) mice 7 days prior to sponge matrix allograft implantation. On day 12, the sponge was harvested and the graft-infiltrating cells were tested in vitro for cytotoxic T lymphocyte (CTL) activity. Immature dendritic cell (DC) infused significantly and markedly inhibited intra-graft CTL activity compared to mature DCs and syngeneic bone marrow control cells. The administration of immature DCs directly into the sponge allograft failed to induce hyporeactivity. Thus, the only systemic infusion of immature donor DCs was able to recapitulate the donor-specific transfusion effect, and the capacity of donor bone marrow cells to induce donor-specific hyporeactivity in the sponge allograft model.

Human taste contrast and self-reported measures of anxiety.

Successive negative taste contrast in humans was demonstrated with a common taste stimulus, i.e., cherry-flavored Kool-Aid. A total of 31 male and female college-aged participants rated a 7% sucrose solution which was cherry-flavored as less sweet when it was preceded by a 28% rather than a 7% sucrose solution which was cherry-flavored. Because drugs such as the benzodiazepines affect taste contrast in rats and act as anxiolytics in humans, the present experiment also examined whether several self-reported measures of anxiety were related to taste contrast in humans. Neither scores on Taylor's Manifest Anxiety Survey nor those on the State-Trait Anxiety Inventory were related to "sweetness" ratings or contrast effects.

Damage to rat retinal DNA induced in vivo by visible light.

Intense visible light can damage retinal photoreceptor cells by photochemical or thermal processes, leading to cell death. The precise mechanism of light-induced damage is unknown; however, oxidative stress is thought to be involved, based on the protective effect of antioxidants on the light-exposed retina. To explore the in vivo effects of light on retinal DNA, rats were exposed to intense visible light for up to 24 h and the time courses of single-strand breaks in restriction fragments containing the opsin, insulin 1 and interleukin-6 genes were measured. All three gene fragments displayed increasing single-strand modifications with increasing light exposure. Treatment with the antioxidant dimethylthiourea prior to light exposure delayed the development of net damage. The time course of double-strand DNA damage was also examined in specific genes and in repetitive DNA. The appearance of discrete 140-200 base-pair DNA fragments after 20 h of light exposure implicated a nonrandom, possibly enzymatic damaging mechanism. The generation of nucleosome core-sized DNA fragments, in conjunction with single-strand breaks, suggests two phases of light-induced retinal damage, with random attack on DNA by activated oxygen species preceding enzymatic degradation.

Effect of high ionic strength and inhibitors of H,K-ATPase on the ouabain-sensitive K-p-nitrophenylphosphatase activity in the sea anemone Stichodactyla helianthus.

The ouabain-sensitive, K-stimulated p-nitrophenyl phosphatase (K-pNPPase) activity, an associated activity of the Na,K-ATPase, was assayed in tentacles of the sea anemone Stichodactyla helianthus to investigate the possibility that the sea anemone Na,K-ATPase activity is an associated activity of an H,K-ATPase. Activity was maximal at pH 6.5-7.0, decreasing only slightly in acidic medium but falling abruptly in alkaline medium to 60% of maximum at pH 7.4. The pH of maximum activity was not remarkably altered in high ionic strength medium (560 mM choline chloride), but ouabain-sensitive K-pNPPase activity of both rat and sea anemone was strongly inhibited. Inhibitors of the gastric H,K-ATPase, 100 microM omeprazole and 10 microM SCH 28080, did not inhibit the ouabain-sensitive K-pNPPase activity. Activity of the sea anemone enzyme was inhibited by 10 microM ammonium vanadate, an inhibitor of P-type ATPase, and not by 2.5 mM sodium azide, an inhibitor of both F-type and V-type ATPase. Because the sea anemone K-pNPPase activity was previously found to be more sensitive to ouabain than the Na,K-ATPase activity, K(+)-ouabain antagonism was investigated and found to be relatively muted, whereas K(+)-Na+ competition was stronger than in the rat kidney.