Chlamydomonas Inner-Arm Dynein Mutant, ida5, Has a Mutation in an Actin-encoding Gene

Chlamydomonas flagellar inner-arm dynein consists of seven subspecies (a–g), of which all but f contain actin as subunits. The mutant ida5 and a new strain, ida5-t, lack four subspecies (a, c, d, and e). These mutants were found to have mutations in the conventional actin gene, such that its product is totally lost; ida5 has a single-base deletion that results in a stop codon at a position about two-thirds from the 5′ end of the coding region, and ida5-t lacks a large portion of the entire actin gene. Two-dimensional gel electrophoresis patterns of the axonemes and inner-arm subspecies b and g of ida5 lacked the spot of actin (isoelectric point [pI] = ~5.3) but had two novel spots with pIs of ~5.6 and ~5.7 instead. Western blot with different kinds of anti-actin antibodies suggested that the proteins responsible for the two novel spots and conventional actin are different but share some antigenicity. Since Chlamydomonas has been shown to have only a single copy of the conventional actin gene, it is likely that the novel spots in ida5 and ida5-t originated from another gene(s) that codes for a novel actin-like protein(s) (NAP), which has hitherto been undetected in wildtype cells. These mutants retain the two inner-arm subspecies b and g, in addition to f, possibly because NAP can functionally substitute for the actin in these subspecies while they cannot in other subspecies. The net growth rate of ida5 and ida5-t cells did not differ from that of wild type, but the mating efficiency was greatly reduced. This defect was apparently caused by deficient growth of the fertilization tubule. These results suggest that NAP can carry out some, but not all, functions performed by conventional actin in the cytoplasm and raise the possibility that Chlamydomonas can live without ordinary actin.     

Interaction ofN-acetyl-4-, 7-, 8- or 9-deoxyneuraminic acids andN-acetyl-4-, 7- or 8-mono-epi- and-7,8-di-epineuraminic acids withN-acetylneuraminate lyase

Various deoxy- and epi-derivatives ofN-acetylneuraminic acid were synthesized and tested for their substrate properties withN-acetylneuraminate lyase fromClostridium perfringens.N-Acetyl-9-deoxyneuraminic acid is a good substrate,N-acetylneuraminic acid derivatives with epimeric configuration at C-7, C-8 or both are cleaved slowly, whileN-acetyl-4-epi-,N-acetyl-4-deoxy-,N-acetyl-7-deoxy-andN-acetyl-8-deoxyneuraminic acid are resistant to enzyme action.N-Acetyl-4-deoxyneuraminic acid andN-acetyl-4-epineuraminic acid competitively inhibit the enzyme. These studies give further insight into a mechanism proposed for the reversible cleavage of sialic acids byN-acetylneuraminate lyase.     

Divergent expression of claudin -1, -3, -4, -5 and -7 in developing human lung

Background

Claudins are the main components of tight junctions, structures which are associated with cell polarity and permeability. The aim of this study was to analyze the expression of claudins 1, 3, 4, 5, and 7 in developing human lung tissues from 12 to 40 weeks of gestation.

Methods

47 cases were analyzed by immunohistochemisty for claudins 1, 3, 4, 5 and 7. 23 cases were also investigated by quantitative RT-PCR for claudin-1, -3 and -4.

Results

Claudin-1 was expressed in epithelium of bronchi and large bronchioles from week 12 onwards but it was not detected in epithelium of developing alveoli. Claudin-3, -4 and -7 were strongly expressed in bronchial epithelium from week 12 to week 40, and they were also expressed in alveoli from week 16 to week 40. Claudin-5 was expressed strongly during all periods in endothelial cells. It was expressed also in epithelium of bronchi from week 12 to week 40, and in alveoli during the canalicular period. RT-PCR analyses revealed detectable amounts of RNAs for claudins 1, 3 and 4 in all cases studied.

Conclusion

Claudin-1, -3, -4, -5, and -7 are expressed in developing human lung from week 12 to week 40 with distinct locations and in divergent quantities. The expression of claudin-1 was restricted to the bronchial epithelium, whereas claudin-3, -4 and -7 were positive also in alveolar epithelium as well as in the bronchial epithelium. All claudins studied are linked to the development of airways, whereas claudin-3, -4, -5 and -7, but not claudin-1, are involved in the development of acinus and the differentiation of alveolar epithelial cells.     

Enantioseparation, absolute configuration determination, and anticonvulsant activity of (+/-)-1-(4-aminophenyl)-7,8-methylenedioxy-1,2,3,5-tetrahydro-4H-2,3-benzodiazepin-4-one

The resolution of 1-(4-aminophenyl)-7,8-methylenedioxy-1,2,3,5-tetrahydro-4H-benzodiazepin-4-one (+/-)-(R,S)-2 was accomplished by chiral HPLC. The absolute configuration of (+)-2, determined by X-ray crystallographic analysis, was R. The in vivo anticonvulsant activity of the enantiomers (+)-(R)-2 and (-)-(S)-2 is reported. It has been also demonstrated that compound (+/-)-(R,S)-2 in vivo undergoes oxidative metabolism to derivative 1.     

Determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, by gas chromatography with electron-capture detection

An electron-capture gas chromatographic procedure was developed for the analysis of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), a metabolite of haloperidol. The assay involved basic extraction of this metabolite from the biological samples, followed by back-extraction with HCl. After basification of the acid phase, extractive derivatization with pentafluorobenzoyl chloride in toluene was conducted. The pentafluorobenzoyl derivative was quantified on a gas chromatograph equipped with a fused-silica capillary column, an electron-capture detector and a printer-integrator. N-(3-Trifluoromethylphenyl)piperazine was carried through the procedure as an internal standard and calibration curves were determined for each assay run. The procedure was demonstrated to be linear and reproducible and was utilized to detect and quantify CPHP in urine, plasma, brain and liver samples from rats treated with haloperidol. The structure of the derivatized metabolite was confirmed by gas chromatography-mass spectrometry.     

Expression, purification and osteogenic bioactivity of recombinant human BMP-4, -9, -10, -11 and -14

Bone morphogenetic proteins (BMPs) are cytokines from the TGF-β superfamily, with important roles during embryonic development and in the induction of bone and cartilage tissue differentiation in the adult body. In this contribution, we report the expression of recombinant human BMP-4, BMP-9, BMP-10, BMP-11 (or growth differentiation factor-11, GDF-11) and BMP-14 (GDF-5), using Escherichia coli pET-25b vector. BMPs were overexpressed, purified by affinity his-tag chromatography and shown to induce the expression of early markers of bone differentiation (e.g. smad-1, smad-5, runx2/cbfa1, dlx5, osterix, osteopontin, bone sialoprotein and alkaline phosphatase) in C2C12 cells and in human adipose stem cells. The described approach is a promising method for producing large amounts of different recombinant BMPs that show potential for novel biomedical applications.     

Simultaneous determination of polyamines, N-acetylated polyamines and the polyamine analogues BE-3-3-3 and BE-4-4-4-4 by capillary gas chromatography with nitrogen-phosphorus detection

We describe a method for the profiling of polyamines, N-acetylated polyamines and the polyamine analogues N¹,N¹¹-bis(ethyl)norspermine (BE-3-3-3) and 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) in L1210 murine leukaemia cells by capillary gas chromatography with nitrogen-phosphorus detection. The method makes use of four internal standards. Prepurification comprises deproteinization, isolation with Sep-Pak silica at pH 9.0, conversion to heptafluorobutyryl derivatives and postderivatization organic fluid extraction. Within- and between-series precisions (given as C.V.s) for analysis of 1–2×10⁶ cells were: putrescine 5.5 and 29.4%; spermidine 1.6 and 7.1%; and spermine 3.2 and 7.6%, respectively. Recoveries relative to the respective internal standards, were in the 70.6–104.7% range. Accuracy and precision of measurements of BE-4-4-4-4 can probably be improved by the introduction of a separate pentamine internal standard. We conclude that the method can be used for studying the effect of BE-3-3-3 and BE-4-4-4-4, and possibly their metabolites, on polyamine homeostasis (biosynthesis, retroconversion, transport, terminal catabolism) and polyamine function.     

Centrally administered N-terminal fragments of ACTH (1-10, 4-10, 4-9) display convulsant properties in rabbits

Electroencephalographic and behavioral effects of the following ACTH fragments: 1-4, 4-9, 4-11, 1-10, 4-10, 1-13, 1-17 and 1-24 were studied in rabbits. Sequences 4-9, 1-10 and 4-10 displayed some epileptic properties, i.e., they induced epileptic seizures (only electrographic or also behavioral) or increased hippocampal spiking. The 4-9 sequence seemed to be the common sequence responsible for these proconvulsant effects. The possible involvement of the enkephalinergic system is discussed.     

Differential distribution of synaptotagmin-1, -4, -7, and -9 in rat adrenal chromaffin cells

Neurons and certain kinds of endocrine cells, such as adrenal chromaffin cells, have large dense-core vesicles (LDCVs) and synaptic vesicles or synaptic-like microvesicles (SLMVs). These secretory vesicles exhibit differences in Ca(2+) sensitivity and contain diverse signaling substances. The present work was undertaken to identify the synaptotagmin (Syt) isoforms present in secretory vesicles. Fractionation analysis of lysates of the bovine adrenal medulla and immunocytochemistry in rat chromaffin cells indicated that Syt 1 was localized in LDCVs and SLMVs, whereas Syt 7 was the predominant isoform present in LDCVs. In contrast to PC12 cells and the pancreatic β cell line INS-1, Syt 9 was not immunodetected in LDCVs in rat chromaffin cells. Double-staining revealed that Syt 9-like immunoreactivity was nearly identical with fluorescent thapsigargin binding, suggesting the presence of Syt 9 in the endoplasmic reticulum (ER).The exogenous expression of Syt 1-GFP in INS-1 cells, which had a negligible level of endogenous Syt 1, resulted in an increase in the amount of Syt 9 in the ER, suggesting that Syt 9 competes with Syt 1 for trafficking from the ER to the Golgi complex. We conclude that LDCVs mainly contain Syt 7, whereas SLMVs contain Syt 1, but not Syt 7, in rat and bovine chromaffin cells.     

Metabolic stability of 6,7-dialkoxy-4-(2-, 3- and 4-[18F]fluoroanilino)quinazolines, potential EGFR imaging probes

Epidermal growth factor receptors (EGFR), upregulated in many tumor types, have been a target for therapeutic development and molecular imaging. The objective of this study was to evaluate the distribution and metabolic characteristics of fluorine-18 labeled anilinoquinazolines as potential imaging agents for EGFR tyrosine kinase expression. Fluorine-18 labeled fluoronitrobenzenes were prepared by reaction of potassium cryptand [(18)F]fluoride with 1,2- and 1,4-dinitrobenzenes, and 3-nitro-N,N,N-trimethylanilinium triflate in 5min. Decay-corrected radiochemical yields of [(18)F]fluoride incorporation into the nitro-aromatic compounds were 81±2%, 44±4% and 77±5% (n=3-5) for the 2-, 3- and 4-fluoro isomers, respectively. Sodium borohydride reduction to the corresponding [(18)F]fluoroanilines was achieved with greater than 80% conversion in 5min. Coupling of [(18)F]fluoroaniline-hydrochlorides to 6,7-dimethoxy-4-chloro-quinazoline gave the corresponding 6,7-dimethoxy-4-(2-, 3- and 4-[(18)F]fluoroanilino)quinazolines in 31±5%, 17±2% and 55±2% radiochemical yield, respectively, while coupling to the 6,7-diethoxy-4-chloro-quinazoline produced 6,7-diethoxy-4-(2-, 3- and 4-[(18)F]fluoroanilino)quinazolines in 19±6%, 9±3% and 36±6% radiochemical yield, respectively, in 90min to end of synthesis from [(18)F]fluoride. Biodistribution of 2- and 4-[(18)F]fluoroanilinoquinazolines was conducted in tumor-bearing mice (MDA-MB-435 and MDA-MB-468 xenografts). Low tumor uptake (<1% injected dose per gram (ID/g) of tissue up to 3h postinjection of the radiotracers) was observed. High bone uptake (5-15% ID/g) was noted with the 4-[(18)F]fluoroanilinoquinazolines. The metabolic stabilities of radiolabeled quinazolines were further evaluated by incubation with human female cryopreserved isolated hepatocytes. Rapid degeneration of the 4-fluoro-substituted compounds to baseline polar metabolites was observed by radio-TLC, whereas, the 2- and 3-[(18)F]fluoroaniline derivatives were significantly more stable, up to 2h, corroborating the in vivo biodistribution studies. para-Substituted [(18)F]fluoroanilines, a common structural motif in radiopharmaceuticals, are highly susceptible to metabolic degradation.     

Substituted 3-((Z)-2-(4-nitrophenyl)-2-(1H-tetrazol-5-yl) vinyl)-4H-chromen-4-ones as novel anti-MRSA agents: synthesis, SAR, and in-vitro assessment

In search for a new antibacterial agent with improved antimicrobial spectrum and potency, we designed and synthesized a series of novel 3-((Z)-2-(4-nitrophenyl)-2-(1H-tetrazol-5-yl) vinyl)-4H-chromen-4-ones 7a-h by convergent synthesis approach. All the synthesized compounds were assayed for their in-vitro antibacterial activities against gram-negative and gram-positive bacteria. The preliminary structure-activity relationship, to elucidate the essential structure requirements for the antimicrobial activity that results into anti-MRSA (methicillin-resistant S. aureus) potential, has been described. Amongst the synthesized compounds 7d, 7e, 7f and 7h were found to possess activity against methicillin-resistant S. aureus in addition to the activity against other bacterial strains such as E. faecalis, S. pneumoniae, and E. coli.     

The stage-specific expression of TEC-1, -2, -3, and -4 antigens on bovine preimplantation embryos

The preimplantation developmental period is associated with constant changes within the embryo, and some of these changes are apparent on the embryo cell surface. For example, during transition from maternal to embryonic genome control and the compaction and differentiation of embryonic cells, the cell surface undergoes morphologic alterations that reflect changes in gene control. In order to gain insight into the events occurring during embryonic development and cellular differentiation, monoclonal antibodies specific for cell surface antigens (TEC antigens) of embryonic cells have been generated previously and shown to recognise either the carbohydrate moiety of embryoglycan or a developmentally regulated protein epitope. The TEC antigens have been identified on mouse preimplantation embryos, and their expression is specific to particular developmental stages. To determine whether these antigens are conserved in higher mammals, we examined the expression of four TEC antigens (TEC-1 to TEC-4) on in vitro–derived bovine and murine embryos during the preimplantation stage of development. It was found that bovine oocytes and embryos derived from in vitro maturation (IVM) and in vitro fertilisation (IVF) showed stage-specific expression of each of the TEC antigens investigated, with the pattern of expression overlapping but not identical to that seen in the mouse. Immunoprecipitation together with Western blot analysis showed that the TEC monoclonal antibodies recognised a single glycoprotein band with an apparent molecular weight of 70 kDa. Confocal microscopy of immunofluorescence staining of the bovine cells showed this protein to be located on the cell surface. The apparent negative expression of these TEC antigens by immunohistochemistry and immunoprecipitation at particular stages of development appears to be due to the epitopes being inaccessible to the TEC antibodies, since Western blotting revealed the TEC antigens to be present at all stages of development examined. Antibodies identifying stage-specific antigens will provide useful markers to characterise early embryonic cells, monitor normal embryonic development in vitro, and identify cell surface structures having a function in cell-cell interactions during embryogenesis and differentiation. Mol. Reprod. Dev. 49:19–28, 1998. © 1998 Wiley-Liss, Inc.     

Synthesis, cytotoxicity, and anti-inflammatory evaluation of 2-(furan-2-yl)-4-(phenoxy)quinoline derivatives. Part 4

A number of 2-(furan-2-yl)-4-phenoxyquinoline derivatives have been synthesized and evaluated for anti-inflammatory evaluation. 4-[(2-Furan-2-yl)quinolin-4-yloxy]benzaldehyde (8), with an IC(50) value of 5.0 microM against beta-glucuronidase release, was more potent than its tricyclic furo[2,3-b]quinoline isomer 3a (>30 microM), its 4'-COMe counterpart 7 (7.5 microM), and its oxime derivative 13a (11.4 microM) and methyloxime derivative 13b (>30 microM). For the inhibition of lysozyme release, however, oxime derivative 12a (8.9 microM) and methyloxime derivative 12b (10.4 microM) are more potent than their ketone precursor 7 and their respective tricyclic furo[2,3-b]quinoline counterparts 4a and 4b. Among them, 4-[4-[(2-furan-2-yl)-quinolin-4-yloxy]phenyl]but-3-en-2-one (10) is the most active against lysozyme release with an IC(50) value of 4.6 microM, while 8 is the most active against beta-glucuronidase release with an IC(50) value of 5.0 microM. (E)-1-[3-[(2-Furan-2-yl)quinolin-4-yloxy]phenyl] ethanone oxime (11a) is capable of inhibiting both lysozyme and beta-glucuronidase release with IC(50) values of 7.1 and 9.5 microM, respectively. For the inhibition of TNF-alpha formation, 1-[3-[(2-furan-2-yl)quinolin-4-yloxy]phenyl]ethanone (6) is the most potent with an IC(50) value of 2.3 microM which is more potent than genistein (9.1 microM). For the inhibitory activity of fMLP-induced superoxide anion generation, 11a (2.7 microM), 11b (2.8 microM), and 13b (2.2 microM) are three of the most active. None of above compounds exhibited significant cytotoxicity.