The specific transport system for lysine is fully inhibited by ammonium in Penicillium chrysogenum: An ammonium-insensitive system allows uptake in carbon-starved cells

The regulation exerted by ammonium and other nitrogen sources on amino acid utilization was studied in swollen spores of Penicillium chrysogenum. Ammonium prevented the L-lysine, L-arginine and L-ornithine utilization by P. chrysogenum swollen spores seeded in complete media, but not in carbon-deficient media. Transport of L-[¹⁴C]lysine into spores incubated in presence of carbon and nitrogen sources was fully inhibited by ammonium ions (35 mM). However, in carbon-derepressed conditions (growth in absence of sugars, with amino acids as the sole carbon source) L-[¹⁴C]lysine transport was only partially inhibited. Competition experiments showed that L-lysine (1 mM) inhibits the utilization of L-arginine, and vice versa, L-arginine inhibits the L-lysine uptake. High concentrations of L-ornithine (100 mM) prevented the L-lysine and L-arginine utilization in P. chrysogenum swollen spores. In summary, ammonium seems to prevent the utilization of basic amino acids in P. chrysogenum spores by inhibiting the transport of these amino acids through their specific transport system(s), but not through the general amino acid transport system that is operative under carbon-derepression conditions.     

Benthic macrofauna associated to Thalassia testudinum in Bahía de Mochima, Sucre, Venezuela

Diversity and abundance of benthic macrofauna associated to Thalassia testudinum were studied at Ensenada de Reyes, Mochima Bay, in the northeastern coast of Venezuela. Samples were taken monthly in six stations, three at 1 m in depth and three at 6 m, between December 1992 and February 1994, using a quadrat of 0.25 m2 for collecting plants and sediment; each sample was washed with seawater through a 1 mm sieve. The specimens were fixed in 6% formaldehyde. A total of 1722 organisms (6 888 ind x m2) and 127 species of macroinvertebrates were collected. Mollusks dominated with 53 species, followed by polychaetes (40), crustaceans (18) and echinoderms (8). Remaining groups were represented by 1-2 species. The highest abundance was in October (214 specimens), and the lowest in December 1993 (79 specimens). Specific richness was between 47 species in October and 18 in May 1993. Mean species diversity was 2.79-1.36 bits/ind. There were differences (ANOVA p<0.01) in number of specimens at the two depths but not throughout the 15 month study period (p>0.05). There were more specimens and species at the lowest depth and in stations with higher Thalassia testudinum biomass.     

Antimicrobial activity of organic extracts isolated from Aplysina fistularis (Demospongiae: Aplysinidae)

Organic extracts of the sponge Aplysina fistularis (Pallas 1766) were tested for antimicrobial activity against Gram positive bacteria (Staphylococcus aureus) and Gram negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa). The minimal inhibitory concentration (MIC) and toxic activity of extract were determined. Susceptibility trials of organic fractions obtained by VLC: Hexane, EtOAc and CHCl3 showed that EtOAc fraction has antibacterial activity against E. coli, while CHCl3 fraction inhibited E. coli and S. aureus growth. The later refractioning of EtOAc fraction and the biodirected assays showed that fractions F12 and F13 of EtOAc/Hex and EtOAc F14 were bioactive against Gram positive and Gram negative bacteria. Only EtOAc/MeOH Sf2 from subfractionig of EtOAc F14 produced inhibition for E. coli and S. aureus. In Sf2 EtOAc/MeOH, MIC was moderate for S. aureus (MIC > 256 g/ml). F4 CHCl3/MeOH produced a high inhibition in S. aureus (MIC = 0.125 g/ml) and for E. coli (MIC > 16 g/ml). F10 CHCl3/MeOH showed a moderate activity against S. aureus (MIC > 128 g/ml) and low activity against E. coli (MIC = 512 g/ml). F10 CHCL3/MeOH did no present toxic activity against Artemia salina. The fractiorts F4 CHCL3/MeOH and Sf2 EtOAc/MeOH were toxic for this organism when the concentration was higher than 100 microg/ml. LC50 in both cases was 548.4 and 243.4 microg/ml respectively. Secondary metabolites of medium polarity obtained from A. fistularis have a wide spectrum of anti bacterial activity. Toxicity analysis suggests that only F10 CHCL3/MeOH has potential as an antimicrobial agent for clinical use.     

Estrogen and progesterone modulation of eosinophilic infiltration of the rat uterine cervix

Ripening of the rat cervix involves widespread collagenolysis that follows an eosinophilic leukocyte infiltration. The hormonal control of these events is not well understood. The aims of this study were to investigate the mechanism through which progesterone (P) and 17beta-estradiol (E(2)) modulate eosinophilic invasion and to determine if this event is protein synthesis mediated. Cervical eosinophilic invasion was measured in intact rats during the second half of pregnancy and compared with values from ovariectomized (O) pseudopregnant (PSP) rats treated with P and E(2) in doses that mimicked the levels of pregnancy. Other O-PSP rats were treated with an E(2) antagonist (tamoxifen) and the antiprogestin RU-486. To study the role of protein synthesis in eosinophilic invasion of the cervix, rats were treated with actinomycin-D (an inhibitor of mRNA synthesis), and animals were sacrificed on D21 or D22 to evaluate eosinophilic invasion. Rats treated with E(2) showed high levels of infiltration and tamoxifen blocked this E(2) effect. On the other hand, P antagonized the stimulatory effects of E(2) on eosinophilic invasion, however when the P and E(2) treated rats were injected with RU-486 the inhibitory effect of P was reversed. In intact pregnant rats a sharp rise in eosinophilic infiltration was detected on D23, 20 h after the fall of serum P. Finally, E(2) treated rats injected with actinomycin-D had no invasion of eosinophils. In conclusion, the estrogen-triggered eosinophil invasion is affected by the classic estrogen receptor antagonist tamoxifen and by the mRNA synthesis blocker actinomycin-D suggesting a genomic action of E(2). Furthermore, the estrogen effect is blocked by P and this inhibition is reversed by RU-486.     

Expression of intercellular adhesion molecule 1 (ICAM-1) on the human oviductal epithelium and mediation of lymphoid cell adherence

The epithelium of the human oviduct expresses the major histocompatibility complex (MHC) class II and shows endocytic properties towards luminal antigens. Therefore, the epithelial cells might behave as antigen-presenting cells, inducing a local immune response. The activation of antigen-specific T cells not only requires presentation of the peptide antigen by MHC class II, but also the presence of co-stimulatory molecules in the antigen-presenting cells. Therefore, the expression of the intercellular adhesion molecule 1 (ICAM-1) was examined in the epithelium of the human oviduct. Most oviducts showed epithelial ICAM-1 expression, as assessed by immunocytochemistry, western blot analysis and RT-PCR assay, and the expression was restricted to the luminal border of ciliated and secretory cells. Interferon gamma, interleukin 1 and lipopolysaccharide treatments increased the percentage of ICAM-1-positive cells in primary cultures, indicating that the expression of ICAM-1 in the oviduct might be upregulated in vivo by inflammatory cytokines or bacterial infections. Binding assays between allogenic phytohaemagglutinin-activated lymphocytes and epithelial monolayers expressing ICAM-1 demonstrated that this molecule stimulated lymphocyte adherence. The presence of ICAM-1, in addition to MHC class II, supports the putative role of the oviductal epithelium in antigen presentation. The exclusive apical distribution of ICAM-1 indicates that T-cell activation would occur in a polarized manner. Binding of lymphoid cells to the surface of the oviductal epithelium may help to retain these immune cells that are required for the clearance of pathogens.     

Autoxidation and neurotoxicity of 6-hydroxydopamine in the presence of some antioxidants: potential implication in relation to the pathogenesis of Parkinson's disease

6-Hydroxydopamine (6-OHDA) is a dopaminergic neurotoxin putatively involved in the pathogenesis of Parkinson's disease (PD). Its neurotoxicity has been related to the production of reactive oxygen species. In this study we examine the effects of the antioxidants ascorbic acid (AA), glutathione (GSH), cysteine (CySH), and N-acetyl-CySH (NAC) on the autoxidation and neurotoxicity of 6-OHDA. In vitro, the autoxidation of 6-OHDA proceeds rapidly with the formation of H2O2 and with the participation of the H2O2 produced in the reaction. The presence of AA induced a reduction in the consumption of O2 during the autoxidation of 6-OHDA and a negligible presence of the p-quinone, which demonstrates the efficiency of AA to act as a redox cycling agent. The presence of GSH, CySH, and NAC produced a significant reduction in the autoxidation of 6-OHDA. In vivo, the presence of sulfhydryl antioxidants protected against neuronal degeneration in the striatum, which was particularly remarkable in the case of CySH and was attributed to its capacity to remove the H2O2 produced in the autoxidation of 6-OHDA. These results corroborate the involvement of oxidative stress as the major mechanism in the neurotoxicity of 6-OHDA and the putative role of CySH as a scavenger in relation to PD.     

Alcohol exposure alters the expression pattern of neural cell adhesion molecules during brain development

Neural cell adhesion molecules (NCAMs) play critical roles during development of the nervous system. The aim of this study is to investigate the possible effect of ethanol exposure on the pattern of expression and sialylation of NCAM isoforms during postnatal rat brain development because alterations in NCAM content and distribution have been associated with defects in cell migration, synapse formation, and memory consolidation, and deficits in these processes have been observed after in utero alcohol exposure. The expression of NCAM isoforms in the developing cerebral cortex of pups from control and alcohol-fed mothers was assessed by western blotting, ribonuclease protection assay, and immunocytochemistry. The highly sialylated form of NCAM [polysialic acid (PSA)-NCAM] is mainly expressed during the neonatal period and then is down-regulated in parallel with the appearance of NCAM 180 and NCAM 140. Ethanol exposure increases PSA-NCAM levels during the neonatal period, delays the loss of PSA-NCAM, decreases the amount of NCAM 180 and NCAM 140 isoforms, and reduces sialyltransferase activity during postnatal brain development. Neuraminidase treatment of ethanol-exposed neonatal brains leads to more intense band degradation products, suggesting a higher content of NCAM polypeptides carrying PSA in these samples. However, NCAM mRNA levels are not changed by ethanol. Immunocytochemical analysis demonstrates that ethanol triggers an increase in PSA-NCAM immunolabeling in the cytoplasm of astroglial cells, accompanied by a decrease in immunogold particles over the plasma membrane. These findings indicate that ethanol exposure during brain development alters the pattern of NCAM expression and suggest that modification of NCAM could affect neuronal-glial interactions that might contribute to the brain defects observed after in utero alcohol exposure.     

Immunoelectrotransfer blot assay in acute and chronic human trichinellosis

An immunoelectrotransfer blot assay (IETB) using excretory secretory products of muscle larvae of Trichinella spiralis (ML-ESP) and the avidin biotin system was developed in order to characterize reactivity against ML-ESP in sera from patients with acute and chronic trichinellosis. A complete pattern of up to 13 bands was developed by sera from individuals with trichinellosis where doublets, triplets, or single bands were shown to have molecular weights of roughly 66, 55, 45, 36, 29, 24, and 14 kDa. The bands at approximately 55, 36, 29, and 14 kDa proved specific for T. spiralis. The band at approximately 55 kDa was present in all trichinellosis sera, whereas the approximately 14-kDa band was present in only a small percentage of sera. The development of approximately 36- and 29-kDa bands suggests a modulation of the reactivity against ML-ESP over time. IETB proved more sensitive for the population of chronic trichinellosis under study than a conventional diagnostic enzyme-linked immunosorbent assay, allowing negative or borderline serum samples to be determined. Thus, this technique, when applied for human trichinellosis surveillance, should provide a useful tool in endemic areas.     

Small-scale isolation of high molecular weight DNA from Leishmania braziliensis

In this paper, we report a method for isolation of high molecular weight DNA from Leishmania promastigotes. This technique is especially indicated for small-scale purification of DNA suitable for the construction of highly representative genomic libraries. In our protocol, lysis buffer is compatible with RNase treatment, avoiding an additional precipitation step and consequent shearing of DNA. In order to prove the quality of the DNA isolated by this method, a Leishmania braziliensis genomic library was constructed, and an L. braziliensis KMP-11 gene was cloned after screening the library with a heterologous probe.