Tyrosinemia type II: Mutation update, 11 novel mutations and description of 5 independent subjects with a novel founder mutation.

BACKGROUND: Tyrosinemia type II, also known as Richner-Hanhart Syndrome, is an extremely rare autosomal recessive disorder, caused by mutations in the gene encoding hepatic cytosolic tyrosine aminotransferase, leading to the accumulation of tyrosine and its metabolites which cause ocular and skin lesions, that may be accompanied by neurological manifestations, mostly intellectual disability. AIMS: To update disease-causing mutations and current clinical knowledge of the disease. MATERIALS AND METHODS: Genetic and clinical information were obtained from a collection of both unreported and previously reported cases. RESULTS: We report 106 families, represented by 143 individuals, carrying a total of 36 genetic variants, 11 of them not previously known to be associated with the disease. Variants include 3 large deletions, 21 non-synonymous and 5 nonsense amino-acid changes, 5 frameshifts and 2 splice variants. We also report 5 patients from Gran Canaria, representing the largest known group of unrelated families sharing the same P406L mutation. CONCLUSIONS: Data analysis did not reveal a genotype-phenotype correlation, but stressed the need of early diagnosis: All patients improved the oculocutaneous lesions after dietary treatment but neurological symptoms prevailed. The discovery of founder mutations in isolated populations, and the benefits of early intervention, should increase diagnostic awareness in newborns.

Inhibition of auxin transport and auxin signaling and treatment with far red light induces root coiling in the phospholipase-A mutant ppla-I-1. Significance for surface penetration?

When grown on a non-penetretable at a surface angle of 45°, Arabidopsis roots form wave-like structures and, in wild type rarely, but in certain mutants the tip root even may form circles. These circles are called coils. The formation of coils depends on the complex interaction of circumnutation, gravitropism and negative thigmotropism where - at least - gravitropism is intimately linked to auxin transport and signaling. The knockout mutant of patatin-related phospholipase-AI-1 (pplaI-1) is an auxin-signaling mutant which forms moderately increased numbers of coils on tilted agar plates. We tested the effects of the auxin efflux transport inhibitor NPA (1-naphthylphtalamic acid) and of the influx transport inhibitor 1-NOA (1-naphthoxyacetic acid) which both further increased root coil formation. The pPLAI-1 inhibitors HELSS (haloenol lactone suicide substrate=E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one) and ETYA (eicosatetraynoic acid) which are auxin signaling inhibitors also increased coil formation. In addition, far red light treatment increased coil formation. The results point out that a disturbance of auxin transport and signaling is one potential cause for root coils. As we show that the mutant pplaI-1 penetrates horizontal agar plates better than wild type plants root movements may help penetrating the soil.

[Prehospital analgesia by paramedics in Rhineland-Palatinate : Feasability, analgesic effectiveness and safety of intravenous paracetamol]. / Prähospitale Analgesie durch Rettungsassistenten in Rheinland-Pfalz : Praktikabilität, analgetische Wirkung und Sicherheit bei i.v.-verabreichtem Paracetamol.

BACKGROUND: In contrast to the widespread practice in life-threatening emergencies, delegation of medical pain therapy to paramedics by the medical director  of Emergency Medical Services, EMS, are still the exception in Germany. This is due to the fact that in non-life-threatening situations, the expected benefit and potential side effects of drug therapy have to be carefully weighed. In addition, in Germany federal law generally restricts the administration of opiates to physicians. METHODS: In 2011 the medical directors of EMS in the German state of Rhineland- Palatinate (4 million inhabitants) developed and implemented a standard operating procedure (SOP) for paramedics related to the prehospital parenteral administration of paracetamol for patients with isolated limb trauma. After a 2 h training session and examination, paramedics were authorized to administer 1 g of paracetamol to patients with a pain score > 5 points on an 11-point numerical rating scale (NRS). For purposes of quality management, every administration of paracetamol had to be prospectively documented on a specific electronic mission form. RESULTS: A total of 416 mission forms could be analyzed. After administration of paracetamol the median NRS score decreased from 8 points (interquartile range: 6; 8) to 4 points (interquartile range: 3; 7). In 51.2 % of the patients the pain intensity was reduced by at least 3 NRS points and in 50.5 % of the patients the NRS was less than 5 points after treatment. The extent of pain reduction was positively correlated with the initial NRS value (r = 0.31, p < 0.0001). No serious side effects were noted. The percentage of patients with an initial heart rate > 100/min declined from 14.6 % to 5.2 % after the administration of paracetamol (p < 0.0001), 18.7 % of the patients received paracetamol for trauma not related to the extremities and 7 % of the patients for nontraumatic pain. An emergency physician was involved in 50 % of the EMS missions and 98.6 % of the patients were transported to a hospital for further diagnostics and treatment. CONCLUSION: The prehospital intravenous administration of paracetamol by paramedics to patients with limb trauma is simple, safe and in 50 % of the patients effective in achieving a NRS value < 5; however, further improvements in prehospital pain therapy initiated by paramedics are desirable, especially in patients with an initial NRS value > 7.

Gravity-dependent differentiation and root coils in Arabidopsis thaliana wild type and phospholipase-A-I knockdown mutant grown on the International Space Station.

Arabidopsis roots on 45° tilted agar in 1-g grow in wave-like figures. In addition to waves, formation of root coils is observed in several mutants compromised in gravitropism and/or auxin transport. The knockdown mutant ppla-I-1 of patatin-related phospholipase-A-I is delayed in root gravitropism and forms increased numbers of root coils. Three known factors contribute to waving: circumnutation, gravisensing and negative thigmotropism. In microgravity, deprivation of wild type (WT) and mutant roots of gravisensing and thigmotropism and circumnutation (known to slow down in microgravity, and could potentially lead to fewer waves or increased coiling in both WT and mutant). To resolve this, mutant ppla-I-1 and WT were grown in the BIOLAB facility in the International Space Station. In 1-g, roots of both types only showed waving. In the first experiment in microgravity, the mutant after 9 days formed far more coils than in 1-g but the WT also formed several coils. After 24 days in microgravity, in both types the coils were numerous with slightly more in the mutant. In the second experiment, after 9 days in microgravity only the mutant formed coils and the WT grew arcuated roots. Cell file rotation (CFR) on the mutant root surface in microgravity decreased in comparison to WT, and thus was not important for coiling. Several additional developmental responses (hypocotyl elongation, lateral root formation, cotyledon expansion) were found to be gravity-influenced. We tentatively discuss these in the context of disturbances in auxin transport, which are known to decrease through lack of gravity.

Increased endocytosis of fluorescent phospholipid in tobacco pollen in microgravity and inhibition by verapamil.

Gravity sensing in plants occurs in specialised tissues, like in the columella in root tips or the endodermis in shoots. Generally, dense organelles, acting as statoliths, are thought to interact with the cytosekeleton and ion channels in gravitropism. We examined the possibility that tobacco pollen tubes (Nicotiana sylvestris) having an elaborate cytoskeleton could perceive gravity through interaction of the cytoskeleton and the endomembrane system and organelles. Using lipid endocytosis as a quantitative parameter, we show that endocytosis is increased transiently in microgravity within 3 min. This increase is inhibited by the calcium blocker verapamil, suggesting that calcium is lowered in the tip, which is known to increase endocytosis in the pollen tube.

Genome-wide identification of Sox8-, and Sox9-dependent genes during early post-natal testis development in the mouse.

The SOX8 and SOX9 transcription factors are involved in, among others, sex differentiation, male gonad development and adult maintenance of spermatogenesis. Sox8(-/-) mice lacking Sox9 in Sertoli cells fail to form testis cords and cannot establish spermatogenesis. Although genetic and histological data show an important role for these transcription factors in regulating spermatogenesis, it is not clear which genes depend upon them at a genome-wide level. To identify transcripts that respond to the absence of Sox8 in all cells and Sox9 in Sertoli cells we measured mRNA concentrations in testicular samples from mice at 0, 6 and 18 days post-partum. In total, 621 and 629 transcripts were found at decreased or increased levels, respectively, at different time points in the mutant as compared to the control samples. These mRNAs were categorized as preferentially expressed in Sertoli cells or germ cells using data obtained with male and female gonad samples and enriched testicular cell populations. Five candidate genes were validated at the protein level. Furthermore, we identified putative direct SOX8 and SOX9 target genes by integrating predicted SOX-binding sites present in potential regulatory regions upstream of the transcription start site. Finally, we used protein network data to gain insight into the effects on regulatory interactions that occur when Sox8 and Sox9 are absent in developing Sertoli cells. The integration of testicular samples with enriched Sertoli cells, germ cells and female gonads enabled us to broadly distinguish transcripts directly affected in Sertoli cells from others that respond to secondary events in testicular cell types. Thus, combined RNA profiling signals, motif predictions and network data identified putative SOX8/SOX9 target genes in Sertoli cells and yielded insight into regulatory interactions that depend upon these transcription factors. In addition, our results will facilitate the interpretation of genome-wide in vivo SOX8 and SOX9 DNA binding data.

[The White Paper on treating medical emergencies preclinically and at hospital : how can it be implemented?]. / Eckpunktepapier zur notfallmedizinischen Versorgung der Bevölkerung in der Prä- und Hospitalphase : Wie ist es praktisch umsetzbar?

Up-to-date management of medical emergencies implies primarily that definitive diagnoses and treatment are performed in a timely manner. These claims have been reconfirmed in 2007 by the leading German language medical associations in their "White Paper on Emergency Treatment". To actually realize the demands described in this paper a timely, transsectoral and close collaboration of all involved organizations is mandatory. To illustrate this race against cell death the phrase relay of survival is proposed and launched to replace the hitherto used but rigid concept of chain of survival. The tasks of each member of this relay of survival are critically scrutinized one after the other from a patient perspective. The paper presents tangible recommendations for improving the respective individual performance as well as, in particular, the cooperation and coordination between the team members which is comparable to handing over the baton in a relay race.

Using 2H labeling with neutron radiography for the study of solid polymer electrolyte water transport properties.

A method combining (2)H labeling of different sources of H atoms (hydrogen, water vapor) with neutron imaging for the analysis of transport parameters in the bulk and at the interfaces of Nafion polymer electrolyte membranes is proposed. The use of different isotope compositions in the steady state allows evaluation of the relation between bulk and interface transport parameters, but relies on literature data for evaluating absolute values. By using transients of isotope composition, absolute values of these parameters including the self-diffusion coefficient of H can be extracted, making this method an attractive alternative to self-diffusion measurements using nuclear magnetic resonance (NMR), allowing measurements in precisely controlled conditions in real fuel cell structures. First measurements were realized on samples with and without electrodes and we report values of the self-diffusion coefficient of the same order of magnitude as values measured using NMR, although with slightly higher numbers. In our particular case, lower interfacial exchange rates for water transport were observed for samples with an electrode.

A novel SOX9 nonsense mutation, q401x, in a case of campomelic dysplasia with XY sex reversal.

Campomelic dysplasia (CD, MIM 114290) is a rare, often lethal, dominantly inherited, congenital skeletal dysplasia, associated with male-to-female autosomal sex reversal and due to de novo mutations of the SOX9 gene, a tissue-specific transcription factor gene involved both in skeletogenesis and male sexual differentiation. Here we report on a 4 months-old 46,XY sex reversed infant with typical clinical features for CD due to a novel mutation of the SOX9 gene, Q401X, leading to synthesis of a truncated SOX9 protein that completely lacks the C-terminal transactivation domain.

Human NK cells require caspases for activation-induced proliferation and cytokine release but not for cytotoxicity.

Natural killer (NK) cells are innate immune cells involved in antiviral defence and tumour surveillance. To fulfil these tasks, NK cells make use of two major effector functions, cytokine and chemokine release and cytotoxicity. In addition, NK cells proliferate in response to cytokines such as IL-2. NK cells possess a large array of activating and inhibitory receptors and their activation demands a complex crosstalk between those receptors. The signalling pathways leading to NK-cell activation are a field of intensive research. The first clue for signal specificity was provided by studies showing that a pathway leading to NF-κB activation selectively induces cytokine release, but is dispensable for cytotoxicity. Here, we demonstrate that in human NK cells caspase activity is required for the upregulation of select activation markers and IFN-γ and TNF production, but not for cytotoxicity. Interestingly, caspases have previously been linked in T cells to the same mechanism of NF-κB induction that is active in NK cells. Moreover, we provide evidence that caspases are involved in IL-2-induced proliferation. Thus, our data provide the basis for a novel approach using caspase inhibitors to generate cytotoxic NK cells, while simultaneously suppressing cytokine release.

Array-CGH analysis in patients with syndromic and non-syndromic XY gonadal dysgenesis: evaluation of array CGH as diagnostic tool and search for new candidate loci.

BACKGROUND: XY gonadal dysgenesis (XY-GD) is a heterogeneous disorder characterized by failure of testicular development despite a normal male karyotype. Non-syndromic and syndromic forms can be delineated. Currently, only a minority of cases can be explained by gene mutations. METHODS: The aim of this study was to detect microdeletions and duplications by using high-resolution Agilent oligonucleotide arrays in a cohort of 87 patients with syndromic or non-syndromic 46,XY-GD. RESULTS: In 26 patients, we identified gains or losses in regions including genes involved in XY-GD (DMRT1, SOX9, DAX1) or in regions, which have not been described as polymorphic copy number variants (CNVs). CONCLUSIONS: This study shows that array comparative genomic hybridization (CGH) analysis is a useful tool for the molecular diagnosis of XY-GD as well as for the identification of potential candidate genes involved in male sexual development.

Use of a dissociative potential to simulate hydration of Na+ and Cl- ions.

We have developed interatomic interaction parameters for Na+ and Cl- hydration using the dissociative water potential of Mahadevan and Garofalini [J. Phys. Chem. B 2007, 111, 8919] suitable for molecular dynamic simulations. Simulations were performed for small ion-water clusters Na(H2O)n+ (n=1-6) and Cl(H2O)m- (m=1-5), as well as dilute aqueous solutions of the ions in water, reproducing the structure and energies found in the literature. A simulation of an HCl molecule in water demonstrated the dissociation of the molecule. The Na+ and Cl- ion-ion interaction parameters also reproduce the energy and density of crystalline NaCl. A series of simulations of NaCl at progressively increasing temperatures from 300 to 1400 K produced solid densities varying by less than 1% from experiment.

Localized endocytosis in tobacco pollen tubes: visualisation and dynamics of membrane retrieval by a fluorescent phospholipid.

Two modes of endocytosis are known to occur in eucaryotic cells: fluid phase and receptor-mediated endocytosis. Fluid-phase endocytosis in plant cells resembles the retrieval of excess plasma membrane material previously incorporated by exocytosis. Pollen tubes need to carry out strong membrane retrieval due to their fast polar tip growth. Plasma membrane labelling of pollen tubes, grown in suspension, was achieved by the incorporation of a fluorescently modified phospholipid, 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine (20 microM) and measured with a confocal laser-scanning microscope. Time course experiments revealed a highly localised and relatively fast plasma membrane retrieval below the tip within the first 5 min after phospholipid application. The retrieved fluorescent plasma membrane was quickly re-integrated into parts of the endomembrane pool and then redistributed to the pollen tube base and very tip of the apex, with the exception of the cortical endoplasmic reticulum (ER) and the mitochondria even after 1-h incubation period. Low temperature (10 degrees C) and the actin filament depolymerizing cytochalasin D (2 microM) completely abolished plasma membrane retrieval, whereas the microtubule destabilizing herbicide oryzalin (1 microM) had no effect. Our results provide strong support for a highly localised endocytotic pathway in tobacco pollen tubes. Passive uptake of bis-Bodipy FL C(11)-phosphocholine by mere penetration can be excluded. It is a valuable alternative to the styryl dyes often used in endocytotic studies, and may also be used to follow lipid turnover because membrane flow of labelled membranes occurs apparently not in a default manner as ascertained by its fast distribution.

Determination of two mercapturic acids related to crotonaldehyde in human urine: influence of smoking.

Crotonaldehyde, an alpha,beta-unsaturated aldehyde, and a potent alkylating agent, is present in many foods and beverages, ambient air and tobacco smoke. A previous study indicated that two metabolites, 3-hydroxy-1-methylpropylmercapturic acid (HMPMA) and 2-carboxyl-l-methylethylmercapturic acid (CMEMA), were excreted in rat urine after subcutaneous injection of crotonaldehyde. Herein, we report the development of a method based on liquid chromatography with tandem mass spectrometry (LC-MS/MS) and deuterated analytes as internal standards, for the determination of HMPMA and CMEMA in human urine. The limits of quantification of the method were 92 and 104 ng/mL for HMPMA and CMEMA, respectively. The calibration curves for both compounds were linear up to 7500 ng/mL with R2 >0.99. It was found that cigarette smokers excreted about three to five-fold more HMPMA, and only slightly elevated amounts of CMEMA, in their urine compared to nonsmokers. In smokers, we also found significant correlations between the urinary excretion levels of HMPMA (but not CMEMA) and several markers of exposure for smoking, including the daily cigarette consumption, carbon monoxide in exhaled breath, salivary cotinine, and nicotine plus five of its major metabolites in urine. Smoking cessation or switching from smoking conventional cigarettes to experimental cigarettes with lower crotonaldehyde delivery led to significant reductions of urinary HMPMA excretion, but not CMEMA excretion. Alcohol consumption did not influence either urinary HMPMA or CMEMA excretion. We conclude that HMPMA is a potentially useful biomarker for smoking-related exposure to crotonaldehyde.

Two novel translocation breakpoints upstream of SOX9 define borders of the proximal and distal breakpoint cluster region in campomelic dysplasia.

The semilethal skeletal malformation syndrome campomelic dysplasia (CD) with or without XY sex reversal is caused by mutations within the SOX9 gene on 17q24.3 or by chromosomal aberrations (translocations, inversions or deletions) with breakpoints outside the SOX9 coding region. The previously published CD translocation breakpoints upstream of SOX9 fall into two clusters: a proximal cluster with breakpoints between 50-300 kb and a distal cluster with breakpoints between 899-932 kb. Here, we present clinical, cytogenetic and molecular data from two novel CD translocation cases. Case 1 with karyotype 46,XY,t(1;17)(q42.1;q24.3) has characteristic symptoms of CD, including mild tibial bowing, cryptorchidism and hypospadias. By standard fluorescence in situ hybridization (FISH) and by high-resolution fiber FISH, the 17q breakpoint was mapped 375 kb from SOX9, defining the centromeric border of the proximal breakpoint cluster region. Case 2 with karyotype 46,X,t(Y;17)(q11.2;q24.3) has the acampomelic form of CD and complete XY sex reversal. By FISH and somatic cell hybrid analysis, the 17q breakpoint was mapped 789 kb from SOX9, defining the telomeric border of the distal breakpoint cluster region. We discuss the structure of the 1 Mb cis-control region upstream of SOX9 and the correlation between the position of the 14 mapped translocation breakpoints with respect to disease severity and XY sex reversal.

Extended pedigree with multiple cases of XX sex reversal in the absence of SRY and of a mutation at the SOX9 locus.

It is well established that testicular differentiation of the human embryonic gonad depends on the action of the Y-chromosomal gene SRY. However, exceptional cases such as SRY-negative cases of 46,XX testicular disorder of sexual development (DSD), and of 46,XX ovotesticular DSD document that testicular tissue can develop in the absence of the SRY gene. These SRY-negative XX sex reversal cases are very rare and usually sporadic, but a few familial cases have been reported. We present a large, consanguineous family with nine affected individuals with phenotypes ranging from 46,XX testicular DSD to 46,XX ovotesticular DSD, with predominance of male characteristics. Absence of SRY in peripheral blood was documented by fluorescence in situ hybridization (FISH) and PCR analysis in all nine affected individuals, and by FISH analysis on gonadal sections with testicular tissue in four affected individuals. By quantitative PCR, a duplication of the SOX9 gene was excluded. In addition, as linkage analysis showed that the nine affected members of the family do not share a common SOX9 haplotype, any mutation at the SOX9 locus could be ruled out. Together, these findings implicate a mutation at a sex-determining locus other than SRY and SOX9 as the cause for the XX sex reversal trait in this family.

Halotolerance is enhanced in carrot callus by sensing hypergravity: influence of calcium modulators and cytochalasin D.

Carrot callus was centrifuged at 10 g and compared to callus growing at 1 g on agar in the presence of increasing sodium chloride concentrations. Growth after 14 days was enhanced in the centrifuged samples versus samples kept at 1 g. This effect was not found when the samples were grown on potassium chloride. At 50 mM NaCl, the calcium ionophore ionomycin was applied to centrifuged and noncentrifuged callus samples. In both experiments, the growth of callus increased with increasing ionomycin concentrations but under 10 g this increase was more enhanced. As inhibitors of calcium influx, lanthanum and gadolinium chloride were chosen in the presence of 50 mM NaCl. Both inhibitors inhibited growth at 1 g at low concentrations of around 2 microM, whereas the centrifuged samples were not or much less so inhibited. We tested an involvement of actin by application of cytochalasin D to callus grown in the presence of 50 mM NaCl. In both types of samples, growth at 1 g and growth at 10 g, cytochalasin D enhanced growth but the effect was clearly stronger at 10 g than at 1 g. As increased halotolerance was only observed in the presence of increased sodium ions, not potassium ions, and as halotolerance is known to be induced by an influx of calcium, the data suggest that a calcium influx induced by hypergravity and possibly modulated by actin caused the observed increase in halotolerance at 10 g.

Evaluation of urinary 1-hydroxypyrene, S-phenylmercapturic acid, trans,trans-muconic acid, 3-methyladenine, 3-ethyladenine, 8-hydroxy-2'-deoxyguanosine and thioethers as biomarkers of exposure to cigarette smoke.

The objective was to evaluate the utility of urinary 1-hydroxypyrene (1-OHP), S-phenylmercapturic acid (S-PMA), trans,trans-muconic acid (t,t-MA), 3-methyladenine (3-MeAd), 3-ethyladenine (3-EtAd), 8-hydroxy-2'-deoxyguanosine (8-OHdG) and thioethers as biomarkers for assessing the exposure in adult smokers who switched from smoking conventional cigarettes to candidate potential reduced exposure products (PREP) or who stopped smoking. Two electrically heated smoking systems (EHCSS) were used as prototype cigarettes that have significant reductions in a number of mainstream smoke constituents as measured by smoking machines relative to those from conventional cigarettes. Urine samples were collected from a randomized, controlled, forced-switching study in which 110 adult smokers of a conventional cigarette brand (CC1) were randomly assigned to five study groups. The groups included the CC1 smoking group, a lower-tar conventional cigarette (CC2) smoking group, EHCSS1 group, EHCSS2 group and a no smoking group that were monitored for 8 days. Biomarkers were measured at baseline and day 8. The daily excretion levels of these biomarkers were compared among the groups before and after switching, and the relationships between the daily excretion levels of these biomarkers and cigarette smoking-related exposure were investigated using Pearson product-moment correlation and multiple regression analyses. It was concluded that under controlled study conditions: (1) 1-OHP, S-PMA and t,t-MA are useful biomarkers that could differentiate exposure between smoking conventional and EHCSS cigarettes or between smoking conventional cigarettes and no smoking; between S-PMA and t,t-MA, the former appeared to be more sensitive; (2) 3-MeAd could only differentiate between smoking conventional cigarettes and no smoking; the results for 3-EtAd were not conclusive because contradictory results were observed; (3) 8-OHdG had a questionable association with smoking and therefore the utility of this biomarker for smoking-related exposure could not be established; and (4) urinary excretion of thioethers as a biomarker lacked sensitivity to demonstrate a clear dose-response relationship in conventional cigarette smokers, although it could differentiate the excretion levels between those subjects who smoked a conventional cigarette and those who stopped smoking.

Phosphoinositides and phosphatidic acid regulate pollen tube growth and reorientation through modulation of [Ca2+]c and membrane secretion.

The maintenance of a calcium gradient and vesicle secretion in the apex of pollen tubes is essential for growth. It is shown here that phosphatidylinositol-4,5-bisphosphate (PIP2) and D-myo-inositol-1,4,5-trisphosphate (IP3), together with phosphatidic acid (PA), play a vital role in the regulation of these processes. Changes in the intracellular concentration of both PIP2 and IP3 (induced by photolysis of caged-probes), modified growth and caused reorientation of the growth axis. However, measurements of cytosolic free calcium ([Ca2+]c) and apical secretion revealed significant differences between the photo-release of PIP2 or IP3. When released in the first 50 mum of the pollen tube, PIP2 led to transient growth perturbation, [Ca2+]c increases, and inhibition of apical secretion. By contrast, a concentration of IP3 which caused a [Ca2+]c transient of similar magnitude, stimulated apical secretion and caused severe growth perturbation. Furthermore, the [Ca2+]c transient induced by IP3 was spatially different causing a pronounced elevation in the sub-apical region. These observations suggest different targets for the two phosphoinositides. One of the targets is suggested to be PA, a product of PIP2 hydrolysis via phospholipase C (PLC) or phospholipase D (PLD) activity. It was found that antagonists of PA accumulation (e.g. butan-1-ol) and inhibitors of PLC and PLD reversibly halted polarity. Reduction of PA levels caused the dissipation of the [Ca2+]c gradient and inhibited apical plasma membrane recycling. It was also found to cause abolition of the apical zonation. These data suggest that phosphoinositides and phospholipids regulate tip growth through a multiple pathway system involving regulation of [Ca2+]c levels, endo/exocytosis, and vesicular trafficking.

Biological activity and physicochemical parameters of marine halogenated natural products 2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole and 2,4,6-tribromoanisole.

Physicochemical parameters (vapor pressure, water solubility, Henry's law constant) and biological activities of two halogenated natural products frequently detected in marine samples and food were determined. Synthetic 2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole (Q1) and 2,4,6-tribromoanisole (TBA) were available in pure form. The physicochemical parameters were in the range of anthropogenic chlorinated compounds of concern. The aqueous solubilities at 25 degrees C (S(w,25)) of Q1 and TBA were 4.6 microg/L and 12,200 microg/L, respectively, whereas subcooled liquid vapor pressures were 0.00168 Pa (Q1) and 0.06562 Pa (TBA) as measured by the gas chromatographic-retention time technique. Q1 was negative by established test systems for the determination of ethoxyresorufin-O-deethylase (EROD) induction and by sulforhodamine B assay. EROD induction potency was at least 10(-7) times lower than that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). At a relatively high concentration (20 microM), Q1 inhibited specific binding of 2 nM [(3)H]TCDD to the in vitro-expressed human aryl hydrocarbon receptor (AHR) by 18%; lower concentrations showed no effect. Molecular modeling showed that Q1 is nonplanar, consistent with its relatively modest affinity as an AHR ligand. When tested for cell-growth inhibitory/cytocidal activity in human tumor cells, Q1 was only marginally, if at all, active with an IC(50) value >50 microM compared with five to ten times lower IC(50) values for potent cytotoxins tested in the test system used. Furthermore, standard pesticide tests on insecticidal, herbicidal, and fungicidal activity did not provide any significant activity at highest concentrations. For TBA, the results in all tests were comparable with Q1. The SRB assay was also applied to the halogenated natural product 4,6-dibromo-2-(2',4'-dibromo)phenoxyanisole, but no toxic response was found. Although it was apparent that Q1 and TBA had been proven to have relatively low biological activity in all tests performed, further research is necessary to clarify whether metabolites of the compounds eventually may possess a risk to humans or other living organisms. Nevertheless, the role of Q1 in nature remains uncertain.