Round-cell mutants of Salmonella typhimurium produced by transposition mutagenesis: Lethality of rodA and mre mutations

Summary Thirty-three insertions of transposon Tn10l6l7 into genes involved in the control of rod cell shape were isolated in Salmonella typhimurium by the characteristic glossy appearance of colonies composed of spherical cells. Genetic tests demonstrated that 25 (76%) were insertions in the rodA gene, 7 (21 %) were mre mutants, and 1 (3%) was a divD mutant. No insertion in the pbpA gene were found. Insertions in cell shape genes only appeared when strains displaying resistance to mecillinam (not caused by -lactamase production) were employed. Neither rodA nor mre insertions could be transduced to wild-type strains but they were normally accepted by mecillinam-resistant derivatives and by cya and crp mutants, which, unlike the corresponding Escherichia coli strains, did not display resistance to mecillinam. On the other hand, the divD insertion could be efficiently transduced to any strain. It is concluded that the rodA, mre, and divD genes are involved in the control of rod cell shape but, in addition, the RodA and Mre products perform some function(s) that is essential for wild-type cells but dispensable for some mecillinam-resistant strains, and for cya and crp mutants.     

Opa-like repeats in the genome of the Medfly Ceratitis capitata

The sequence determination of several genomic clones isolated from the Mediterranean fruitfly Ceratitis capitata identified the existence of opa-like repeats, often more than one being clustered in small chromosomal segments. These repeats have previously been shown to consist of stretches of tandemly reiterated glutamine-encoding residues, and they are found in multiple genes of several organisms. Most of the repeats described here are flanked or interrupted by stop codons in all reading frames and, thus, could not possibly be part of protein-coding sequences. Furthermore, these repeats, of which there are several hundred in the genome of the Medfly, can be used effectively for the determination of sequence polymorphisms, providing a convenient approach to obtain additional landmarks for the construction of genomic maps of this economically important insect.This paper is dedicated to the memory of our colleague and friend Dr. Jim Flach who took part in the initial phase of this work and died during the course of the investigation.     

Spatial and temporal coding in single neurons

Convergence between cells which differ in both spatial and temporal properties create higher order neurons with response properties that are distinctly different from those of the input neurons. The spatial properties of target neurons are not necessarily cosinetuned. In addition, unlike the independence between spatial and temporal properties in cosine-tuned afferent neurons, higher-order target cells generally exhibit a dependence of temporal dynamics on spatial properties. The response properties of target neurons receiving spatio-temporal convergence (STC) from tonic and phasic-tonic or phasic afferents is investigated here by considering a general case where the dynamic input is represented by a fractional, leaky, derivative transfer function. It is shown that, at frequencies below the corner frequency of the dynamic input, the temporal properties of target neurons can be described by leaky differentiators having time constants that are a function of spatial direction. Thus, STC target neurons exhibit tonic temporal response properties during stimulation along some spatial directions (having small time constants) and phasic properties along other directions (having large time constants). Specifically, target neurons encode the complete derivative of the stimulus along certain spatial directions. Thus, STC acts as a directionally specific high-pass filter and produces complete derivatives from fractional, leaky derivative afferent signals. In addition, spatio-temporal transformations can generate novel temporal dynamics in the central nervous system. These observations suggest that spatio-temporal computations might constitute an alternative to parallel, independent spatial and temporal channels.     

Phytochemical Characterization of Cannabis sativa L. Roots from Northeastern Brazil

This article describes the phytochemical study of Cannabis sativa roots from northeastern Brazil. The dried plant material was pulverized and subjected to exhaustive maceration with ethanol at room temperature, obtaining the crude ethanolic extract (Cs-EEBR). The volatile compounds were analyzed by gas chromatography coupled with mass spectrometry (GC/MS), which allowed to identify 22 compounds by comparing the linear retention index (LRI), the similarity index (SI) and the fragmentation pattern of the constituents with the literature. By this technique the major compounds identified were: friedelan-3-one and β-sitosterol. In addition, two fractions were obtained from Cs-EEBR by classical column chromatography and preparative thin layer chromatography. These fractions were analyzed by NMR and IR and together with the mass spectrometry data allowed to identify the compounds: epifriedelanol, friedelan-3-one, β-sitosterol and stigmasterol. The study contributed to the phytochemical knowledge of Cannabis sativa, specifically the roots, as there are few reports on the chemical constituents of this part of the plant.     

Natural lignin modulators improve bagasse saccharification of sugarcane and energy cane in field trials

The burgeoning cellulosic ethanol industry necessitates advancements in enzymatic saccharification, effective pretreatments for lignin removal, and the cultivation of crops more amenable to saccharification. Studies have demonstrated that natural inhibitors of lignin biosynthesis can enhance the saccharification of lignocellulose, even in tissues generated several months post-treatment. In this study, we applied daidzin (a competitive inhibitor of coniferaldehyde dehydrogenase), piperonylic acid (a quasi-irreversible inhibitor of cinnamate 4-hydroxylase), and methylenedioxy cinnamic acid (a competitive inhibitor of 4-coenzyme A ligase) to 60-day-old crops of two conventional Brazilian sugarcane cultivars and two energy cane clones, bred specifically for enhanced biomass production. The resultant biomasses were evaluated for lignin content and enzymatic saccharification efficiency without additional lignin-removal pretreatments. The treatments amplified the production of fermentable sugars in both the sugarcane cultivars and energy cane clones. The most successful results softened the most recalcitrant lignocellulose to the level of the least recalcitrant of the biomasses tested. Interestingly, the softest material became even more susceptible to saccharification.     

Plant regeneration from stem and petiole-derived callus ofHumulus lupulus L. (hop) clone Bragança and var. Brewer’s Gold

Summary Plant regeneration was achieved from both a spontaneous clone (Bragan?a) and Brewer's Gold variety ofHumulus lupulus. The results obtained for these two different genotypes were compared. The organogenic ability of petiole and stem segments was tested on three different basal media supplemented with 0.025 mg (0.14 μM) indole-3-acetic acid/L and 2 mg (8.87 μM) 6-benzylaminopurine (N⁶-benzyladenine)/L. These conditions induced rather heterogeneous responses, which depended mainly on the explant source and the genotype. Because of the high organogenic competence revealed by the spontaneous clone on modified Murashige and Skoog medium, several hormones in different combinations were tested to optimize conditions for adventitious shoot regeneration in this clone. The best relation between the average shoot number/callus and the regeneration rate was achieved with 0.025 mg (0.14 μM) indole-3-acetic acid/L and 2 mg (8.87 μM) 6-benzylaminopurine/L or with 0.02 mg (0.11 μM) indole-3-acetic acid/L and 1.5 mg (6.97 μM) kinetin/L, which enabled 72 and 59% of regeneration, respectively. The regenerated plantlets could be acclimatized with 90% success.     

On the Modeling of Some Anti-inflammatory and Anti-thrombotic Drugs by AM1

Two families of autacoids from cell membrane phospholipids have been identified. The first, the icosanoids, which are formed from arachidonic acid, include prostaglandins and leukotrienes. The other includes modified phospholipids, as the platelet aggregating factor (PAF). These compounds are related to inflammatory and cardiovascular diseases. We review in this paper some of the work that has been done in our laboratories in the last few years relating to the modeling of new potential thromboxane synthase (TXS) and 5-lipoxygenase (5-LO) and cyclooxygenase (COX) inhibitors, and TXA₂ receptor antagonists derived from nitrogenated heterocycles. We include the results of the modeling of a group of proposed PAF antagonists, and compare their structures with PAF itself and with a recently proposed PAF antagonist model. This revised version was published online in June 2006 with corrections to the Cover Date.     

Product of aromatase activity in intact LNCaP and MCF-7 human cancer cells

We investigated conversion rates of androgens to estrogens in cultured, hormone-responsive prostate (LNCaP) and breast (MCF-7) human cancer cells. For this purpose, we adopted an intact cell analysis, whereby cells were incubated for different incubation times in the presence of close-to-physiological (1 nM) or supraphysiological (1 μM) concentrations of labelled androgen precursors, i.e. testosterone (T) and androstenedione (Δ⁴Ad). The aromatase activity, as measured by estrogen formation, was detected in LNCaP cells (0.5 pmol/ml), even though to a significantly lower extent than in MCF-7 cells (5.4 pmol/ml), using 1 μM T after 72 h incubation. Surprisingly, LNCaP cells displayed a much higher aromatase activity when T was used as a substrate with respect to Δ⁴Ad. In either cell line, T transformation to Δ⁴Ad was relatively low, attaining only 2.8% in LNCaP and 7.5% MCF-7 cells. However, T was mostly converted to conjugates (over 95%), glucuronides and some sulphates, in LNCaP cells, whereas it was only partly converted to sulphates (<10%) in MCF-7 cells. Aromatase activity seems to be inconsistent in LNCaP cells, being strongly affected by culture conditions, especially by fetal calf serum (FCS). Further studies should assess the regulation of aromatase expression by serum or growth factors in different human cancer cells, also using anti-aromatase and/or anti-estrogen compounds, in different culture conditions.     

The Apical Submembrane Cytoskeleton Participates in the Organization of the Apical Pole in Epithelial Cells

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145–3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40–70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical–basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37°C or in n-octyl-β-d-glycoside at 4°C (representative of GPIanchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na⁺– K⁺ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.Cell polarity (asymmetry) is a broadly distributed and highly conserved feature of many different cell types, from prokaryotes to higher eukaryotes (Nelson, 1992). In multicellular organisms it is more conspicuous in, but not restricted to, neurons and epithelial cells. In the latter, the plasma membrane is organized in two different domains, apical and basolateral. This characteristic enables epithelia to accomplish their most specialized roles including absorption and secretion and, in general, to perform the functions of organs with an epithelial parenchyma such as the kidney, liver, intestine, stomach, exocrine glands, etc. (Simons and Fuller, 1985; Rodriguez-Boulan and Nelson, 1989).The acquisition and maintenance of epithelial polarity is based on multiple interrelated mechanisms that may work in parallel. Although the origin of polarization depends on the sorting of apical and basolateral membrane proteins at the trans-Golgi network (Simons and Wandinger-Ness, 1990), the mechanisms involved in the transport of apical or basolateral carrier vesicles, the specific fusion of such vesicles to the appropriate domain, and the retention of membrane proteins in their correct positions are also important (Wollner and Nelson, 1992). Various components of the cytoskeleton seem to be especially involved in these mechanisms (Mays et al., 1994). Among them, the microtubules, characteristically oriented in the apical–basal axis with their minus ends facing toward the apical domain, appear in a strategic position to transport carrier vesicles (Bacallao et al., 1989). This orientation is largely expected because of the apical distribution of centrioles and microtubule organizing centers in epithelial cells (Buendia et al., 1990). The molecular interactions responsible for that localization, however, are unknown.Actin is a widespread component of the membrane skeleton found under apical, lateral, and basal membranes in a nonpolarized fashion (Drenckhahn and Dermietzel, 1988; Vega-Salas et al., 1988). Actin bundling into microvillus cores in the presence of villin/fimbrin, on the other hand, is highly polarized to the apical domain (Ezzell et al., 1989; Louvard et al., 1992). In fact, different isoforms of plastins determine microvillus shape in a tissue-specific manner (Arpin et al., 1994b ). Why this arrangement is not found in other actin-rich regions of the cell is unclear (Louvard et al., 1992; Fath and Burgess, 1995).Fodrin, the nonerythroid form of spectrin, underlies the basolateral domain (Nelson and Veshnock, 1987a ,b) and is known to participate in the anchoring/retention of basolateral proteins (Drenckhahn et al., 1985; Nelson and Hammerton, 1989). Although different groups have found specific cytoskeletal anchoring of apical membrane proteins at the “correct” domain (Ojakian and Schwimmer, 1988; Salas et al., 1988; Parry et al., 1990), no specific apical counterpart of the basolateral fodrin cytoskeleton is known. This is especially puzzling since we showed that MDCK cells can maintain apical polarity in the absence of tight junctions, an indication that intradomain retention mechanisms are operational for apical membrane proteins (Vega-Salas et al., 1987a ).It is known that a network of intermediate filament (IF)¹, the major component of the terminal web, bridges the desmosomes under the apical membrane in brush border cells (Franke et al., 1979; Hull and Staehelin, 1979; Mooseker, 1985), although no specific protein has been identified with this structure. The observation of a remarkable resistance to extractions of apical proteins anchored to cytoskeletal preparations (Salas et al., 1988) comparable to that of intermediate filaments, led us to the study of cytokeratins in polarized cells. We developed an antibody against a 53-kD intermediate filament protein in MDCK cells. This protein was found to be distributed exclusively to the apical domain and to form large (2,900 S) multi-protein complexes with apical plasma membrane proteins. Internal microsequencing of the 53-kD protein showed very high (95– 100%) homology with two polypeptides in the rod domain of cytokeratin 19 (CK19; Moll et al., 1982) a highly conserved and peculiar intermediate filament protein (Bader et al., 1986). A complete identification however, could not be achieved (Rodriguez et al., 1994). The present study was undertaken to establish that identity and to determine the possible functions of this apical membrane skeleton. Because cytokeratins have been poorly characterized in canine cells, and no cytokeratin sequences are available in this species, we decided to switch from MDCK cells to two human epithelial cell lines, CACO-2, an extensively studied model of epithelial polarization that differentiates in culture to form brush border containing cells (Pinto et al., 1983), and MCF-10A (Tait et al., 1990), a nontumorigenic cell line derived from normal mammary epithelia, as a model of nonbrush border cells.To assess possible functions of cytokeratin 19, we chose to selectively reduce its synthesis using anti-sense phosphorothioate oligodeoxy nucleotides, an extensively used approach in recent years (e.g., Ferreira et al., 1992 ; Hubber et al., 1993; Takeuchi et al., 1994). Although we could not achieve a complete knock out, the steady-state levels of cytokeratin 19 were decreased to an extent that enabled us to detect significant changes in the phenotype of CACO-2 and MCF-10A cells.