Identification of suitable ionic liquids for application in the enzymatic hydrolysis of rutin by an automated screening.

An automated method in milliliter scale was developed for the screening of process parameters concerning the hydrolysis of the flavonoid rutin catalyzed by the rhamnosidase activity of naringinase from Penicillium decumbens. Besides the effect of additives such as ionic liquids and low molecular salts, the productivity in a multiple phase system as well as the recyclability of the enzyme in repetitive batches were studied. The hydrophobic ionic liquid (IL) trihexyl(tetradecyl)phosphonium bis(trifluormethylsulfonyl)imide [P(h(3))t][Tf(2)N] was identified to combine the most favorable characteristics out of 23 investigated ILs with regard to enzyme compatibility, substrate solubility and enzyme partition coefficient. Also, for the corresponding cations 1-ethyl-3-methylimidazolium [EMIM], 1-butyl-3-methylimidazolium [BMIM], 1-butyl-1-methylpyrrolidinium [BMPL] and 1-octyl-3-methylimidazolium [OMIM], the entity with the [Tf(2)N] anion was best tolerated by the naringinase. With increasing IL content, higher space time yields with up to 1.5 g/(L h) for 80% (v/v) [P(h(3))t][Tf(2)N] were achieved. Enhanced specific enzyme activity was observed in the presence of Ca(2+) ions. By addition of [P(h(3))t][Tf(2)N] and calcium chloride, the reactive aqueous phase was successfully used in three repetitive batches with full conversion.

A novel oncogenic mechanism in Ewing sarcoma involving IGF pathway targeting by EWS/Fli1-regulated microRNAs.

MicroRNAs (miRs) are a novel class of cellular bioactive molecules with critical functions in the regulation of gene expression in normal biology and disease. MiRs are frequently misexpressed in cancer, with potent biological consequences. However, relatively little is known about miRs in pediatric cancers, including sarcomas. Moreover, the mechanisms behind aberrant miR expression in cancer are poorly understood. Ewing sarcoma is an aggressive pediatric malignancy driven by EWS/Ets fusion oncoproteins, which are gain-of-function transcriptional regulators. We employed stable silencing of EWS/Fli1, the most common of the oncogenic fusions, and global miR profiling to identify EWS/Fli1-regulated miRs with oncogenesis-modifying roles in Ewing sarcoma. In this report, we characterize a group of miRs (100, 125b, 22, 221/222, 27a and 29a) strongly repressed by EWS/Fli1. Strikingly, all of these miRs have predicted targets in the insulin-like growth factor (IGF) signaling pathway, a pivotal driver of Ewing sarcoma oncogenesis. We demonstrate that miRs in this group negatively regulate the expression of multiple pro-oncogenic components of the IGF pathway, namely IGF-1, IGF-1 receptor, mammalian/mechanistic target of rapamycin and ribosomal protein S6 kinase A1. Consistent with tumor-suppressive functions, these miRs manifest growth inhibitory properties in Ewing sarcoma cells. Our studies thus uncover a novel oncogenic mechanism in Ewing sarcoma, involving post-transcriptional derepression of IGF signaling by the EWS/Fli1 fusion oncoprotein via miRs. This novel pathway may be amenable to innovative therapeutic targeting in Ewing sarcoma and other malignancies with activated IGF signaling.

TRPV6.

The ion channel TRPV6 is likely to function as an epithelial calcium channel in organs with high calcium transport requirements such as the intestine, kidney, and placenta. Transcriptional regulation of TRPV6 messenger RNA (mRNA) is controlled by 1,25-dihydroxyvitamin D, which is the active hormonal form of vitamin D3, and by additional calcium-dependent and vitamin D3-independent mechanisms. Under physiological conditions, the conductance of the channel itself is highly calcium-selective and underlies complex inactivation mechanisms triggered by intracellular calcium and magnesium ions. There is growing evidence that transcriptional regulation of TRPV6 in certain tissues undergoing malignant transformation, such as prostate cancer, is linked to cancer progression.

Glycolipids from a colloid chemical point of view.

Glycolipids are a group of compounds with a broad range of applications. Two types of glycolipids (alkylpolyglycosides and gangliosides) were examined with regard to their physicochemical properties. Despite their structural differences, they have in common that they are amphiphilic molecules and able to aggregate to form monolayers, bilayers, micelles, lyothropic mesophases or vesicles. The structures of glycolipid micelles were investigated by different experimental techniques in addition to molecular dynamic simulations. The knowledge of the physicochemical properties of gangliosides enables a better understanding of their biological functions. Structural features were obtained for the monosialogangliosides GM1, GM2 and GT1b from bovine brain by means of mass spectrometry. Further the aggregation behaviour was determined by small-angle neutron and dynamic light scattering experiments. Interaction studies of these compounds were carried out by means of surface plasmon resonance using gangliosides incorporated liposomes. They were used as model membranes that interact with the lectins WGA, RCA and HPA. The interaction of lectins immobilized to a modified silicon surface was investigated by in-situ ellipsometry.

Development of a basic procedure to design sorption processes.

The intention of this work is to offer, within the shortest time, an appropriate sorption separation process for almost any odour problem. The development is based on the preparation and characterisation of new adsorbents, the strategy for the selection of the best adsorbent, the process engineering and the choice of a suitable regeneration procedure. In this context a new method for the characterisation of the adsorbents - the adsorption profile analysis - was developed. The classification of the adsorbents was carried out by means of a cluster analysis, which simplifies the selection of the most suitable adsorbent for a particular problem. The physical and chemical behaviour of silica-adsorbents could be tailored by silanisation of the surfaces. Methods for the determination of process engineering parameters were developed, established and used. Adsorption kinetics and isotherms were determined with a magnetic adsorption balance. In a laboratory-scale fixed bed adsorber, breakthrough curves of different support materials were investigated and compared. For the investigations of different regeneration procedures, four innovative methods were employed: microwave desorption, ultrasonic desorption, ultrasonic-water desorption and extraction with water. Of the four desorption methods examined, microwave desorption and ultrasonic-water desorption demonstrated the best results.

Preparation and evaluation of Ricinus communis agglutinin affinity adsorbents using polymeric supports.

A practicable and efficient procedure for preparation of Ricinus communis agglutinin (RCA) affinity adsorbents has been developed. For immobilization of RCA two different polymer-based supports, Toyopearl and TSKgel (TosoHaas), were used. RCA has been successfully immobilized onto these supports with amounts of coupled ligand between 15 and 23 mg/g dry support and corresponding coupling yields of 69-93% (w/w). The prepared affinity adsorbents were characterized concerning their binding capacity for the glycoprotein asialofetuin (ASF) and accessibility of the ligand binding sites. The high accessibility of 80% showed that steric hindrance was negligible at the present ligand density. RCA-Toyopearl was successfully applied in affinity chromatography of glycoproteins indicating its high specificity. A long-term stability test proved no change in capacity for a period of at least 12 months. High-performance affinity chromatography (HPLAC) was carried out using RCA-TSKgel. Experimental results showed that the prepared adsorbents are suitable for selective separation of glycoproteins and oligosaccharides and therefore can be used for investigations of adsorption characteristics of glycoconjugates and for laboratory-scale preparations.

Expression of CaT-like, a novel calcium-selective channel, correlates with the malignancy of prostate cancer.

The regulation of intracellular Ca(2+) plays a key role in the development and growth of cells. Here we report the cloning and functional expression of a highly calcium-selective channel localized on the human chromosome 7. The sequence of the new channel is structurally related to the gene product of the CaT1 protein cloned from rat duodenum and is therefore called CaT-like (CaT-L). CaT-L is expressed in locally advanced prostate cancer, metastatic and androgen-insensitive prostatic lesions but is undetectable in healthy prostate tissue and benign prostatic hyperplasia. Additionally, CaT-L is expressed in normal placenta, exocrine pancreas, and salivary glands. New markers with well defined biological function that correlate with aberrant cell growth are needed for the molecular staging of cancer and to predict the clinical outcome. The human CaT-L channel represents a marker for prostate cancer progression and may serve as a target for therapeutic strategies.

Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin.

A finely tuned Ca(2+) signaling system is essential for cells to transduce extracellular stimuli, to regulate growth, and to differentiate. We have recently cloned CaT-like (CaT-L), a highly selective Ca(2+) channel closely related to the epithelial calcium channels (ECaC) and the calcium transport protein CaT1. CaT-L is expressed in selected exocrine tissues, and its expression also strikingly correlates with the malignancy of prostate cancer. The expression pattern and selective Ca(2+) permeation properties suggest an important function in Ca(2+) uptake and a role in tumor progression, but not much is known about the regulation of this subfamily of ion channels. We now demonstrate a biochemical and functional mechanism by which cells can control CaT-L activity. CaT-L is regulated by means of a unique calmodulin binding site, which, at the same time, is a target for protein kinase C-dependent phosphorylation. We show that Ca(2+)-dependent calmodulin binding to CaT-L, which facilitates channel inactivation, can be counteracted by protein kinase C-mediated phosphorylation of the calmodulin binding site.

SNAP-24, a Drosophila SNAP-25 homologue on granule membranes, is a putative mediator of secretion and granule-granule fusion in salivary glands.

Fusion of vesicles with target membranes is dependent on the interaction of target (t) and vesicle (v) SNARE (soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor) proteins located on opposing membranes. For fusion at the plasma membrane, the t-SNARE SNAP-25 is essential. In Drosophila, the only known SNAP-25 isoform is specific to neuronal axons and synapses and additional t-SNAREs must exist that mediate both non-synaptic fusion in neurons and constitutive and regulated fusion in other cells. Here we report the identification and characterization of SNAP-24, a closely related Drosophila SNAP-25 homologue, that is expressed throughout development. The spatial distribution of SNAP-24 in the nervous system is punctate and, unlike SNAP-25, is not concentrated in synaptic regions. In vitro studies, however, show that SNAP-24 can form core complexes with syntaxin and both synaptic and non-synaptic v-SNAREs. High levels of SNAP-24 are found in larval salivary glands, where SNAP-24 localizes mainly to granule membranes rather than the plasma membrane. During glue secretion, the massive exocytotic event of these glands, SNAP-24 containing granules fuse with one another and the apical membrane, suggesting that glue secretion utilizes compound exocytosis and that SNAP-24 mediates secretion.

The Drosophila light-activated conductance is composed of the two channels TRP and TRPL.

SUMMARY: Drosophila phototransduction is a G protein-coupled, calcium-regulated signaling cascade that serves as a model system for the dissection of phospholipase C (PLC) signaling in vivo. The Drosophila light-activated conductance is constituted in part by the transient receptor potential (trp) ion channel, yet trp mutants still display a robust response demonstrating the presence of additional channels. The transient receptor potential-like (trpl) gene encodes a protein displaying 40% amino acid identity with TRP. Mammalian homologs of TRP and TRPL recently have been isolated and postulated to encode components of the elusive I(crac) conductance. We now show that TRP and TRPL localize to the membrane of the transducing organelle, together with rhodopsin and PLC, consistent with a role in PLC signaling during phototransduction. To determine the function of TRPL in vivo, we isolated trpl mutants and characterized them physiologically and genetically. We demonstrate that the light-activated conductance is composed of TRP and TRPL ion channels and that each can be activated on its own. We also use genetic and electrophysiological tools to study the contribution of each channel type to the light response and show that TRP and TRPL can serve partially overlapping functions.

A novel protein encoded by the InaD gene regulates recovery of visual transduction in Drosophila.

InaDp215 is a point mutation that affects photoreceptor function in Drosophila. To understand the molecular basis of the defect, we isolated the InaD gene and found it encodes a photoreceptor-specific polypeptide of 674 residues. Within its sequence are two repeats that share remarkable homology with a family of cytoskeleton-associated proteins that are involved in signal transduction. Patch-clamp recordings from isolated photoreceptor cells of InaDp215 show a slow deactivation of the light-induced current. This defective deactivation of InaD appears dependent on calcium influx; removal of extracellular calcium masks its abnormal phenotype. Moreover, InaD photoreceptors show increases sensitivity to dim light. We propose that InaD is involved in the negative feedback regulation of the light-activated signaling cascade in Drosophila photoreceptors.

Regulation of PLC-mediated signalling in vivo by CDP-diacylglycerol synthase.

CDP-diacylglycerol synthase (CDS) is an enzyme required for the regeneration of the signalling molecule phosphatidylinositol-4,5-bisphosphate (PtdlnsP2) from phosphatidic acid. A photo-receptor cell-specific isoform of CDS from Drosophila is a key regulator of phototransduction, a G-protein-coupled signalling cascade mediated by phospholipase C. cds mutants cannot sustain a light-activated current as a result of depletion of PtdlnsP2. Overexpression of CDS increases the amplitude of the light response, demonstrating that availability of PtdlnsP2 is a determinant in the gain of this pathway. cds mutants undergo light-dependent retinal degeneration which can be suppressed by a mutation in phospholipase C. Thus, enzymes involved in PtdlnsP2 metabolism regulate phosphoinositide-mediated signalling cascades in vivo.

Disturbing GTP-binding protein function through microinjection into the visual cell of Limulus.

We have tested the action of three agents microinjected into the ventral nerve photoreceptor of Limulus on the electrical response to dim light. 1. A monoclonal antibody (mAb 4A) against the G alpha subunit of frog transducin reduces the size of the receptor current to 60%, suggesting an interaction with G alpha in the Limulus photoreceptor. 2. Injection of Clostridium botulinum ADP-ribosyltransferase C3 reduces the size to 46%; latency is not affected. The results imply that small GTP-binding proteins play a functional role in photoreception of invertebrates. 3. Injection of GDP-beta-S reduces dose-dependently the size of the receptor current to 15% and prolongs the latency to 200%, presumably by reducing number and rate of G-protein activations.

High-dose intravenous immune globulin therapy for hyperbilirubinemia caused by Rh hemolytic disease.

We conducted a multicenter controlled trial to test the hypothesis that high-dose intravenous immune globulin (HDivIG) therapy can modulate bilirubin production and reduce the frequency of exchange transfusions in newborn infants with Rh hemolytic disease. Thirty-four patients with Rh incompatibility proved by positive direct antiglobulin test (Coombs test) results were randomly assigned to receive conventional treatment including phototherapy, with or without additional HDivIG therapy at 500 mg/kg given for a 2-hour period as soon as the diagnosis was established. Exchange transfusions were performed if serum bilirubin concentrations exceeded the modified curves of Polácek by more than 2 mg/dl. Two patients were excluded because of protocol violations. The results in 32 infants were analyzed. In the HDivIG group, 2 (12.5%) of 16 children required exchange transfusions, whereas it became necessary in 11 (69%) of 16 children in the control group (p less than 0.005). Bilirubin levels in the HDivIG group were lower despite reduced frequency of exchange transfusions. No side effects of HDivIG treatment were observed. We conclude that HDivIG therapy by a yet unknown mechanism reduces serum bilirubin levels and the need for blood exchange transfusions in children with Rh hemolytic disease.

Recent trends in neonatal mortality in South Carolina.

This study examines some of the primary factors responsible for the decline in South Carolina's neonatal mortality rate during the 1980s. Essentially all of the observed decline between 1980-82 and 1984-86 could be attributed to improved birthweight-specific survival rather than improvements in the infant birthweight distribution. Improved survival of 500-1,499 g infants accounted for 64% of the decline in white neonatal mortality and 70% of the decline among blacks. Also, during this period, the percentage of 500-1,499 g infants delivered at Level III hospitals increased significantly for both race groups. Comparisons with other southeastern states suggest that further reductions in South Carolina's neonatal mortality rate are possible through continued efforts aimed at improving birthweight-specific survival. Existing state-supported programs such as regional perinatal referral networks and the High Risk Channeling Project will continue to play an important role in maintaining the decline in the state's neonatal mortality rate.

Smoking in a public health agency: its relationship to sick leave and other life-style behavior.

A survey questionnaire was administered to employees of a public health agency regarding their involvement with smoking and other life-style behavior. Responses were analyzed and combined with sick leave data to determine the use of sick leave among employees who currently smoke, those who never smoked, and those who formerly smoked. Current smokers took significantly more sick leave than those who have never smoked ("nonsmokers") or former smokers. Using an analysis of variance model, only smoking status, education level, and sex, among selected demographic variables, were significantly related to the amount of sick leave taken. Current smokers took excess sick leave (amounting to nearly $40,000) as compared with nonsmokers and ex-smokers combined over a 21-month period. This study also characterizes the relationship between smoking status and selected life-style behavior. Smokers were less active, less likely to use seat belts, less likely to believe that smoking is related to health, and more likely to be heavier and to eat a poor diet than their nonsmoking or former smoking counterparts. We make suggestions regarding the reduction of smoking and other deleterious behavior as a means of controlling costs and reducing employee morbidity.

Underreporting of AIDS cases in South Carolina, 1986 and 1987.

An evaluation of the completeness and accuracy of case reporting of the acquired immunodeficiency syndrome (AIDS) for the 18-month period from January 1, 1986, through June 30, 1987, was conducted for South Carolina. A total of 596,585 hospital discharge billing records were searched by computer for conditions defining AIDS. The resulting 1513 records were manually reviewed. Of these, 349 discharges for 163 individuals were classified as being definitely AIDS related, the clinical features of these cases meeting Centers for Disease Control criteria for AIDS diagnosis. Of these cases, 153 were reportable to the South Carolina AIDS registry at the time of their diagnosis. Comparison of this case list with registry records revealed that only 91 (59.5%) had been reported. Reporting was significantly poorer for cases among blacks (53.1%) than for cases among whites (71.6%). These findings may have important implications for the interpretation of AIDS surveillance data and for planning activities in which awareness of complete case counts may be critical.