Palaeoclimatic changes resulted in range expansion and subsequent divergence in brown fur seals, Arctocephalus pusillus.

Past climatic change as a driving force of marine diversification is still largely unclear, particularly for Southern Hemisphere species. Here, we present a case using the brown fur seal, Arctocephalus pusillus, assessing the geographical structure and demographic history using mitochondrial and nuclear data. Results show the two previously defined subspecies (one from Australia and the other from southern Africa) are phylogeographically distinct. Migration analyses based on nuclear data suggest the absence of migrants among the two genetically close assemblages. The demographic history of A. pusillus is characterized by a glacial population expansion (approx. 18 kya) in the southern African lineage, which coincides with time estimates of population expansion of prey species of seals. Approximate Bayesian calculations support an eastward dispersal event during the Last Glacial Maximum when sea levels were lower, followed by a postglacial divergence event, approximately 13 kya. The demographic history of the brown fur seal in the Southern Oceans provides support that recent palaeoclimatic changes could have facilitated expansions in some marine species and that postglacial sea-level rise may have acted as a dispersal barrier for species mostly confined to continental shelves.

Functional optimality of the sulcus pattern of the human brain.

We develop a mathematical model of information transmission across the biological neural network of the human brain. The overall function of the brain consists of the emergent processes resulting from the spread of information through the neural network. The capacity of the brain is therefore related to the rate at which it can transmit information through the neural network. The particular transmission model under consideration allows for information to be transmitted along multiple paths between points of the cortex. The resulting transmission rates are governed by potential theory. According to this theory, the brain has preferred and quantized transmission modes that correspond to eigenfunctions of the classical Steklov eigenvalue problem, with the reciprocal eigenvalues quantifying the corresponding transmission rates. We take the model as a basis for testing the hypothesis that the sulcus pattern of the human brain has evolved to maximize the rate of transmission of information between points in the cerebral cortex. We show that the introduction of sulci, or cuts, in an otherwise smooth domain indeed increases the overall transmission rate. We demonstrate this result by means of numerical experiments concerned with a spherical domain with a varying number of slits on its surface.

How many daddies: microsatellite genotyping reveals polyandry in a live-bearing clinid fish Muraenoclinus dorsalis.

Fishes belonging to the family Clinidae in South Africa display super-embryonation, a rare reproductive mode were females gestate broods at different gestational stages, but little is known regarding the mating systems of this family. Here we tested the hypothesis that multiple males would contribute not only to the offspring of each female, but that several males would contribute to each brood, by sampling Muraenoclinus dorsalis from three sampling locations along the west and south-west coast of South Africa. Larval (n = 97) and maternal (n = 14) genotpyes, generated with newly developed microsatellites, were used to estimate the number of potential mates per female. Our results show that up to 78% of females displayed multiple mating with an average of 2·1-2·2 males. In addition, 39-42% of females displayed polyandry with an average of 1·5-1·6 sires per brood. This study provides the evidence for multiple mating and polyandry within a clinid fish characterized by super-embryonation that offers important baseline information regarding rare reproductive strategies, highlighting several gaps in our knowledge concerning clinid reproduction and mating systems.

Phylogeny of Parablennius Miranda Ribeiro, 1915 reveals a paraphyletic genus and recent Indo-Pacific diversification from an Atlantic ancestor.

A molecular phylogeny of 15 (out of 26 recognized) species of Parablennius Miranda Ribeiro, 1915 was constructed based on two mitochondrial and two nuclear gene fragments, and using maximum parsimony, maximum likelihood and Bayesian approaches. The closely related genera Hypleurochilus, Salaria and Scartella were also studied to ascertain their relationship with Parablennius. Phylogenetic analyses were compared with morphology-based taxonomical studies. Hypleurochilus, Salaria and Scartella appear well supported within a clade including all Parablennius, indicating that this genus is paraphyletic. The species pairs P. parvicornis-P. sanguinolentus and P. gattorugine-P. ruber are well-supported and relatively distant from remaining Parablennius. Remaining Parablennius form two distinct well-supported groups: (1) a clade of Atlantic-Mediterranean Parablennius (P. pilicornis, P. marmoreus, P. rouxi, P. salensis and P. tentacularis); (2) a clade including Hypleurochilus, the Indo-Western Pacific Parablennius (P. cornutus, P. intermedius, P. tasmanianus and P. yatabei) and the Atlantic-Mediterranean P. incognitus and P. zvonimiri. Use of a relaxed molecular clock suggests that Indo-Pacific Parablennius originated recently from an Atlantic Parablennius that may have dispersed via southern Africa, rather than via the Tethys seaway.

Phylogeographic patterns and cryptic speciation across oceanographic barriers in South African intertidal fishes.

Biogeographic boundaries are the meeting zone of broadly distributed faunas, or the actual cause of a faunal break. In the latter case, closely related sister species should be found across such a boundary. To achieve such a situation, preliminary stages are expected, where phylogeographic breaks followed by genetic cryptic speciation would be observed. Biogeographic boundaries, in the Cape Point/Cape Agulhas region of southern Africa, offer an ideal system to test such predictions. Here, we studied two intertidal clinid fish species that are endemic to southern Africa, Clinus superciliosus (n = 127) and Muraenoclinus dorsalis (n = 114). Using mitochondrial control region, 16S rRNA, 12S rRNA and NADH2 genes and the nuclear rhodopsin and the first intron of the S7 ribosomal protein gene, we show both phylogeographic breaks and likely cryptic speciation in each species. Pairwise Φ(st) results suggest population genetic structuring for both species, with higher levels for M. dorsalis (Φ(st) = 0.34-0.93) than for C. superciliosus (Φ(st) = 0.1-0.74). Further, we recover two and three distinct lineages within M. dorsalis and C. superciliosus, respectively. Phylogenetic topologies, concordance between nuclear and mitochondrial markers and levels of sequence divergence, which are consistent with closely related sister species pairs, suggest the presence of cryptic species. Our results therefore meet the expectation for reduced gene flow at a biogeographic barrier, which translates into significant genetic breaks and cryptic sister species.

Multilocus phylogenetic analysis of the genus Atherina (Pisces: Atherinidae).

Sand-smelts are small fishes inhabiting inshore, brackish and freshwater environments and with a distribution in the eastern Atlantic and Mediterranean Sea, extending south into the Indian Ocean. Here, we present a broad phylogenetic analysis of the genus Atherina using three mitochondrial (control region, 12S and 16S) and two nuclear markers (rhodopsin and 2nd intron of S7). Phylogenetic analyses fully support the monophyly of the genus. Two anti-tropical clades were identified, separating the South African Atherina breviceps from the north-eastern Atlantic and Mediterranean Atherina' species. In European waters, two groups were found. The first clade formed by a well supported species-pair: Atherina presbyter (eastern Atlantic) and Atherina hepsetus (Mediterranean), both living in marine waters; a second clade included Atherina boyeri (brackish and freshwater environments) and two independent lineages of marine punctated and non-punctated fishes, recently proposed as separate species. Sequence divergence values strongly suggest multiple species within the A. boyeri complex.

Expression of P-glycoprotein in the intestinal epithelium of dogs with lymphoplasmacytic enteritis.

Inflammatory bowel disease (IBD) is an idiopathic chronic inflammatory disease of the stomach, the small intestine and/or the large intestine. Loss of integrity of the intestinal barrier may be an important factor in the pathogenesis of IBD. In dogs, lymphoplasmacytic enteritis (LPE) is one of the recognized forms of IBD. P-glycoprotein (P-gp) is a membrane-bound efflux pump constituting an important component of the intestinal barrier. Changes in P-gp expression at the level of the intestinal barrier may be important in the pathogenesis of canine LPE, as this may lead to variable protection against xenobiotics and bacterial products in the intestine. The aim of the present study was to evaluate the expression of epithelial P-gp in the intestine in dogs with LPE compared with disease-free animals. Formalin-fixed intestinal biopsy samples from 57 dogs with histopathological evidence of LPE were immunolabelled with anti-P-gp antibodies (C494 and C219). Endoscopic biopsy samples of the duodenum and colon from 16 healthy beagles were used as controls. None of the control dogs had P-gp expression in the apical membrane of duodenal enterocytes, but all had P-gp labelling at the colonic epithelial surface. Twenty out of 57 dogs with LPE had P-gp expression at the apical surface membrane of villus epithelial cells in the duodenum, jejunum and/or ileum. Six out of 16 colonic samples from dogs with LPE had decreased P-gp expression at the epithelial surface compared with controls. It is unclear whether these changes in P-gp expression in dogs with LPE are a cause or a consequence of the inflammation. The observed changes could affect bioavailability of therapeutic drugs used in LPE.

The influence of modulation of P-glycoprotein and /or cytochrome P450 3A on the pharmacokinetics and pharmacodynamics of orally administered morphine in dogs.

The influence of pretreatment with ketoconazole [cytochrome P450 3A (CYP3A) + P-glycoprotein (P-gp) inhibitor], elacridar (selective P-gp inhibitor) and rifampicin (CYP3A + P-gp inducer) on oral morphine pharmacokinetics and pharmacodynamics was investigated in experimental dogs. Seven beagles were used in a four-way crossover design. Morphine hydrochloride was administered orally (2.5 mg/kg) alone (control group CON) or after pretreatment with ketoconazole (group KETO), elacridar (group ELA) or rifampicin (group RIF). Morphine plasma concentrations were analysed by liquid chromatography-tandem mass spectrometry. Sedation scores (none, mild, moderate or severe) were evaluated subjectively. Dogs were significantly (P < 0.05) more sedated after ketoconazole pretreatment. There were no significant differences between group CON and the other pretreatment groups in pharmacokinetic parameters taking both sexes into account. Sex differences were apparent in some pharmacokinetic parameters of morphine. The area under the plasma concentration time curve (AUC(0-∞) ) was significantly higher, and the total body clearance was significantly lower in male compared to female dogs in all treatment groups. Ketoconazole, rifampicin and elacridar pretreatment had no significant effects on morphine pharmacokinetics, although dogs in the ketoconazole group showed higher sedation scores.

Expression of multidrug resistance-associated P-glycoprotein in feline tumours.

Permeability glycoprotein (P-gp) is a membrane-bound efflux pump that mediates the active transmembrane transport of a variety of substrates. Several studies in human and canine normal and neoplastic tissues indicate that P-gp is involved in resistance to chemotherapy and in the development of multidrug resistance (MDR). The purpose of this study was to evaluate P-gp expression with the monoclonal antibody C494 in common feline tumours from 88 patients not pretreated with chemotherapy. Tumours arising from tissues with intrinsic P-gp expression consistently showed positive labelling for P-gp in a cellular pattern identical to that described for the normal feline tissues. Both P-gp positive and negative tumour cells, however, were found in mammary gland tumours, lymphomas, mastocytomas and squamous cell carcinomas. Feline mammary gland tumours in particular showed strong membranous P-gp labelling, especially in areas with infiltrative growth and atypical cells, although this was not correlated with the grade of malignancy. These findings might have implications for future response to chemotherapy.

Tissue distribution of p-glycoprotein in cats.

Permeability glycoprotein (P-gp) is a membrane-bound efflux pump that exports various substances out of the cell. Variations in P-gp expression play an important role in susceptibility to toxic substances, drug efficacy and disease risk. In the present study, the distribution of the MDR1-gene product P-gp was determined in normal tissues of domestic shorthair cats using immunohistochemistry. Two monoclonal antibodies C494 and C219 were used, recognizing a different epitope on the human P-gp molecule. A consistent positive immunolabelling was obtained. The tissue distribution and cellular locations with antibody C494 were similar to those in man and dogs; with liver, colon, adrenal cortex and brain capillaries being consistently and intensely labelled. However, the immunolabelling in the kidney was in contradiction to man and dogs. The C219 antibody seems to react with a specific form of P-gp, only expressed in feline tissues with a barrier function, i.e. endothelia of the brain, testes and ovaries, and intestinal epithelial cells in contact with the lumen.

Bluetongue virus serotype 8-associated congenital hydranencephaly in calves.

Hydranencephaly, the almost complete absence of the cerebral parenchyma, induced by infection with modified live bluetongue virus (BTV) crossing the placenta has previously been reported in sheep and rarely in cattle in the USA and in South Africa. The current study describes 29 cases of hydranencephaly in bovine foetuses and 'dummy' calves up to 3 months of age in Belgium associated with natural BTV serotype 8 infection very early in gestation. Histological examination of the remaining cerebral parenchyma showed moderate to severe atrophy of the neural tissue. The lesions observed support the hypothesis of BTV-induced destruction of precursor cells. However, in several calves a slight infiltration of the walls of venules and arterioles with T lymphocytes (vasculitis) was observed as well, which seems to be responsible for at least some of the lesions. Bluetongue viral RNA was detected in 15 animals using a BTV-specific real-time RT-PCR with a much higher success rate in brain tissues compared with blood and spleen samples. Virus isolation in embryonated eggs was unsuccessful. In conclusion, hydranencephaly in calves can be associated with natural wild-type BTV-8 intra-uterine infection.

Concurrent atypical myopathy and equine dysautonomia in two horses.

This report concerns 2 horses that suffered typical clinical signs of atypical myopathy (AM) and equine grass sickness (EGS) concurrently. Clinical details and pathological lesions of the cases are described. EGS and AM are relatively rare diseases and the concurrency of the diseases in the same animals is therefore considered unlikely to be a coincidence. However, it is not suggested that the evidence shows a common aetiology but rather the existence of common predisposing causes.

Listeria monocytogenes and recurrent mycobacterial infections in a child with complete interferon-gamma-receptor (IFNgammaR1) deficiency: mutational analysis and evaluation of therapeutic options.

We describe the history of a girl with interferon-gamma-receptor (IFNgammaR1) deficiency and studies performed to identify the molecular and clinical characteristics of this recently discovered disorder. This is the first report of a child from Northern Europe with IFNgammaR1 deficiency. The patient, now 7 years old, first presented with swelling and reddening at the Bacille Calmette-Guerin (BCG) vaccination site, swelling of lymph nodes, hepatomegaly, and an unusually severe varicella rash at the age of 4 months. At that time, she was diagnosed with BCG histiocytosis without typical granuloma formation and was treated with antituberculous agents. During the clinical course of her illness, several different types of atypical mycobacteria and (for the first time in an IFNgammaR1-deficient patient) Listeria monocytogenes were detected. Flow cytometric analysis showed that the patient's monocytes could not bind a monoclonal antibody specific for the IFNgamma-receptor. Our analysis of mRNA derived from the alpha-chain (IFNgammaR1) gene of this receptor revealed deletions of 173 bp and 4 bp in cDNA sequences originating from individual alleles. The 173 bp deletion was located between nucleotide positions 200 and 372, exactly matching those of exon 3, and the 4 bp deletion was located between nucleotide positions 561 and 564 of the coding region of the cDNA. Analysis of genomic DNA revealed the presence of a G to T transition at the 5'end of the splice consensus sequence of intron 3, which explains the absence of exon 3. The other allele carried the 4-base-pair deletion (ACTC) at nucleotide positions 15-18 of exon 5. Twelve months after an allo\geneic bone marrow transplantation, the patient had clinically improved.

Pharmacokinetics and bioavailability of oral 5'-deoxy-5-fluorouridine in cancer patients.

AIMS: Oral administration of 5-fluorouracil (FUra), an important cytotoxic agent, is limited by a wide variation in bioavailability. 5'-deoxy-5-fluorouridine (dFUrd), a masked form of FUra, has shown promise clinically when given intravenously or orally as a solution or tablet. This study investigates the efficacy of an oral capsule formulation of dFUrd in generating continuous systemic levels of this compound in cancer patients. METHODS: Six patients with advanced intestinal or ovarian malignancies were given three cycles of dFUrd, days 1-5, at intervals of 4 weeks. The doses of dFUrd were 600 mg m-2 three times daily, 800 mg m-2 three times daily, and 1000 mg m-2 three times daily, on cycles one, two and three, respectively (total dose 36 g m-2 ). The initial dose in each cycle was given as a slow intravenous injection over 10 min, and the remainder orally. Plasma and urine levels of dFUrd and two of its metabolites, FUra and 5,6-dihydro-5-fluorouracil (FUraH2 ), were monitored in six patients at each dose level. RESULTS: All six patients completed the study, receiving three different doses over a 3 month period, following which one had achieved a partial response, one had stable disease, and four had developed progressive disease. Side-effects were negligible, and only two instances of transient diarrhoea WHO grade 1 were seen. Total body clearance (CLtot) of intravenous dFUrd decreased with increasing dose; 2.7, 2.0 and 1.3 l min-1 m-2, following doses of 600, 800 and 1000 mg m-2, respectively. The mean elimination half-life of intravenous dFUrd increased with the dose from 15 to 22 min. Oral dFUrd was rapidly absorbed with a lag time of less than 20 min. The mean elimination half-life (t1/2, z ) of oral dFUrd was 32-45 min in the dose range 600-1000 mg m-2. The AUC of FUra and FUraH2 increased overproportionally with increasing intravenous doses of dFUrd. The mean systemic bioavailability of oral dFUrd was 34-47%. CONCLUSIONS: dFUrd, which selectively releases the antimetabolite FUra in tumour cells, can be given orally at doses of 600-1000 mg m-2 three times daily for 5 days. The systemic levels achieved are equivalent to those seen following continuous infusions of dFUrd or FUra. Toxicity is tolerable, and further clinical investigation of oral dFUrd is warranted.

Uncommon missense and splice mutations and resulting biochemical phenotypes in German patients with X-linked chronic granulomatous disease.

Chronic granulomatous disease is an inherited disease characterized by the inability of phagocytes to generate normal amounts of superoxide, leaving patients susceptible to opportunistic, life-threatening infections. In the majority of cases, cytochrome b558 is absent in the X-chromosomal form of CGD. However, the neutrophils from six of nine X-linked CGD patients, reported here, expressed normal or decreased amounts of this cytochrome and are referred to as "variant" forms. In three of these six variant patients, a roughly proportional decrease in cytochrome b558 expression and production of H2O2 were found. In two cases this phenotype could be well explained by special splice mutations, whereas in the third case it was caused by a missense mutation, predicting Ser 193-->Phe. In the other three variant patients, cytochrome b558 expression and H2O2 production were clearly disproportionate as the generation of H2O2 was much more decreased than cytochrome expression. Missense mutations also were found in these cases. One of these mutations, predicting Leu 546-->Pro and affecting the putative nicotinamide adenine dinucleotide phosphate binding site, led to normal levels of cytochrome b558 expression and reduced H2O2 production. In the other two mutations, predicting Pro 339-->His and His 338-->Tyr, the putative flavin adenine dinucleotide binding site was affected. This could explain the corresponding uncommon phenotypes, characterized by zero or trace amounts of H2O2 production and the expression of relatively high amounts of nonfunctional or low functional cytochrome b558, respectively. The only missense mutation found that prevented the expression of any cytochrome b558 was caused by a predicted His 222-->Arg exchange in one of the three classic cases. The two other classic phenotypes were caused by splice mutations.

An in-frame triplet deletion within the gp91-phox gene in an adult X-linked chronic granulomatous disease patient with residual NADPH-oxidase activity.

In an adult patient suffering from X-linked chronic granulomatous disease (X-CGD) with residual activity of the NADPH-oxidase we found an unusual biochemical constellation with a defective gp91-phox gene. As shown by Western blot using a specific antibody the gp91-phox protein was normal in PMN. However, NADPH-oxidase activity was reduced and no heme spectrum was detectable. By Southern blot and RFLP analysis of genomic DNA a larger defect within the gp91-phox gene was excluded. Sequencing of the gp91-phox cDNA revealed an in-frame deletion of a TTC triplet in exon VI of the gp91-phox gene. This mutation indicates the loss of one amino acid (phenylalanine 215 or 216) in the gp91-phox protein. Sequencing of genomic DNA from the heterozygous daughter of the propositus confirmed this mutation. The absence of a functional cytochrome b558-spectrum in granulocytes of the patient suggests an involvement of the phenylalanine 216 area in heme binding by gp91 phox. This is the first mutation described in a X-CGD patient with absence of a functional cytochrome b558-spectrum but with detectable gp91-phox protein and residual NADPH-oxidase activity.

Comparison of daunorubicin and Fluo-3 for detection of multidrug resistance in human tumor cells.

The detection of multidrug resistance (MDR) in clinical samples is still a topic for discussion. One method, proven extremely useful for detection of membrane proteins in patients with hematological malignancies is the flow cytometrical analysis of individual tumor cells. Recently an assay was described based on the labeling of the P-glycoprotein (P-gp) with the monoclonal antibody MRK16, combined with detection of active daunorubicin (DNR) extrusion. In order to improve the specificity of the assay, on line with the results obtained by Wall et al., we exploited staining with Fluo-3. Both assays prove to be able to discriminate between drug-resistant and drug-sensitive cells. A major drawback of labeling with Fluo-3 in combination with the monoclonal antibody MRK16 is the important overlap of emission spectra of both fluorochromes. Moreover, using Fluo-3 for the detection of MDR might be complicated by the fact that differences in fluorescence intensities are not solely dependent on the presence of P-gp, but also on the activity of cytosolic esterases and the intracellular calcium concentration. Combination of the detection of structural and functional aspects of the MDR-associated protein may lead to a more precise detection of the MDR-positive patient.

[In vitro effects of piracetam on the radiosensitivity of hypoxic cells (adaptation of MTT assay to hypoxic conditions)]. / Effets in vitro du piracetam sur la radiosensibilité des cellules hypoxiques (adaptation du test au MTT aux conditions d'hypoxie).

This paper describes the adaptation of the MTT assay to hypoxic conditions in order to test the in vitro effect of piracetam on hypoxic cells and particularly on the radiosensitivity of hypoxic cells since this drug has shown clinical effect on acute and chronic hypoxia. The V79 cell line was selected by reference to preliminary hypoxic experiments using clonogenic assay and euoxic experiments using clonogenic and MTT assays. Cell growth and survival in our hypoxic conditions were assessed using MTT assay with an enclosure and special 48-well plates both made of glass. Growth curves on glass versus reference polystyrene plates were comparable and confirm the validity of using special glass plates. Growth curves on glass plates after 1-hour exposure to nitrogen versus air were comparable, so there is no bias effect due to gas composition. Survival curves using MTT versus reference clonogenic assay were comparable after radiation exposure in eu- and hypoxic conditions, and confirm the validity of our original technique for creating hypoxia. The Oxygen Enhancement Ratio was of about 3 for 1-hour hypoxic exposure. Piracetam gave no cytotoxic effect up to 10 mM of piracetam. Growth curves after continuous drug exposure and 1-hour euoxic versus hypoxic exposure gave no cytotoxic effect up to 10 mM of piracetam. Survival curves after continuous drug exposure to 10 mM of piracetam gave no significant effect on the radiosensitivity of hypoxic V79 cells using MTT or clonogenic assay. However, this does not preclude a potential in vivo effect of piracetam on the radiosensitivity owing to its action on microcirculation and its rheologic properties. The adaptation of the MTT assay to hypoxic irradiation conditions yields the easy screening of radiosensitizing drugs: shorter incubation, semi-automatic method and simultaneous analysis with different serial concentrations thanks to the special 48-well glass plates.

Lack of correlation between mdr-1 expression and volume-activation of cloride-currents in rat colon cancer cells.

Correlation between expression of the mdr-1 genes (a and b) at the mRNA and protein level and volume-activation of chloride-channels was studied in rat colon cancer CC531 cells by means of RT-PCR, Western blotting and patch clamp, respectively. Three different kinds of cell lines were used: CC531-PAR, CC531-COL and CC531-REV. At the mRNA level, the parental cell line CC531-PAR showed significantly less mdr-1a expression in comparison with CC531-COL, a drug-resistant cell line induced from the parental CC531 cells by growth in the presence of colchicin. The third cell line, CC531-REV, was a spontaneous revertant of the drug-resistant cell line to a drug-sensitive one, but with a maintained level of mdr-1a mRNA. In none of the three cell lines, mdr-1b mRNA could be detected. At the protein level, a clear difference in mdr1 expression between CC531-PAR/REV and CC531-COL was observed. Although the amount of mdr-1a mRNA detected in CC531-REV was comparable to that found in CC531-COL, the amount of mdr-1 encoded protein in CC531-REV was remarkably reduced. In all three cell types, cell swelling activated chloride-currents which could be blocked by NPPB.(ABSTRACT TRUNCATED AT 250 WORDS)