The identification of a caffeine-induced Ca2+ influx pathway in rat primary sensory neurons

Caffeine-induced Ca²⁺ transients (CICTs) in rabbit nodose ganglion neurons (NGNs) are produced by two distinct mechanisms: release from intracellular stores via ryanodine receptors and Ca²⁺ influx across the plasma membrane, due to activation of an unknown receptor. In isolated rat NGNs, we used single-cell microfluorimetry to measure changes in intracellular Ca²⁺ and to test whether TRPV1 receptors underlie the Ca²⁺ influx pathway. Caffeine (10 mM) evoked CICTs in all NGNs tested (n = 47) averaging 365 ± 32 nM. CICTs were partially dependent upon a Ca²⁺ influx pathway that ranged between 33% and 98% of the total Ca²⁺ transient. Application of two selective TRPV1 antagonists significantly attenuated CICTs. The peak average amplitudes of CICTs in Ca²⁺-free Locke solution and Ca²⁺-free Locke solution with IRTX or with BCTC were not significantly different from one another (n = 5 and 7, respectively). These observations suggest that caffeine can induce Ca²⁺ influx by activating TRPV1 channels.     

Expression of homeobox genes in oral squamous cell carcinoma cell lines treated with all‐trans retinoic acid

Oral squamous cell carcinoma (OSCC) may arise from potentially malignant oral lesions. All‐trans retinoic acid (atRA), which plays a role in cell growth and differentiation, has been studied as a possible chemotherapeutic agent in the prevention of this progression. While the mechanism by which atRA suppresses cell growth has not been completely elucidated, it is known that homeobox genes are atRA targets. To determine if these genes are involved in the atRA‐mediated OSCC growth inhibition, PCR array was performed to evaluate the expression of 84 homeobox genes in atRA‐sensitive SCC‐25 cells compared to atRA‐resistant SCC‐9 cells following 7 days with atRA treatment. Results showed that the expression of 8 homeobox genes was downregulated and expression of 4 was upregulated in SCC‐25 cells but not in SCC‐9 cells. Gene expression levels were confirmed for seven of these genes by RT‐qPCR. Expression of three genes that showed threefold downregulation was evaluated in SCC‐25 cells treated with atRA for 3, 5, and 7 days. Three different patterns of atRA‐dependent gene expression were observed. ALX1 showed downregulation only on day 7. DLX3 showed reduced expression on day 3 and further reduced on day 7. TLX1 showed downregulation only on days 5 and 7. Clearly the expression of homeobox genes is modulated by atRA in OSCC cell lines. However, the time course of this modulation suggests that these genes are not direct targets of atRA mediating OSCC growth suppression. Instead they appear to act as downstream effectors of atRA signaling. J. Cell. Biochem. 111: 1437–1444, 2010. © 2010 Wiley‐Liss, Inc.     

TRANSPARENT EXOPOLYMER PARTICLES FORMATION FROM CAPSULES OF ANABAENA SPIROIDES (CYANOBACTERIA) IN CULTURE1

We report the production of large numbers of transparent exopolymer particles (TEP) from polysaccharidic capsules of Anabaena spiroides Kleb. in cultures. Two biotic pathways of TEP formation were observed: (1) fragmentation of small portions of the capsules, which occurred throughout the growth phases; and (2) transformation of the whole polysaccharidic capsules into TEP, following cellular lyses in the aging culture. Photographic documentation of these processes was performed after staining small aliquots of the samples with Alcian Blue and negative staining with India ink. Concentrations of TEP were determined in distinct culture growth phases using semiquantitative Alcian Blue staining. Concentrations of TEP increased throughout the experimental time, while Alcian Blue remaining in solution decreased. Decreasing concentrations of chl a indicated cellular death, and by the end of the experiment, TEP formed by both pathways accumulate in the culture medium. These results show that virtually all dead chains of A. spiroides are transformed into TEP in the aged culture.     

Arachidonic acid cytotoxicity: can arachidonic acid be a physiological mediator of cell death?

Arachidonic acid is a polyunsaturated fatty acid that mediates inflammation and the functioning of several organs and systems either directly or upon its conversion into eicosanoids. However, arachidonic acid is found to be cytotoxic in vitro at concentrations that overlap physiological ones. It is tempting therefore to speculate that arachidonic acid may be a physiological inducer of apoptosis and that such cytotoxic action may be another of its roles in vivo. Nevertheless its pro-inflammatory and oxidative stress-inducing features are characteristic of necrosis and pathological conditions. We hereby review the cytotoxic action of arachidonic acid, indicate the possible pathways that lead to cell death and contemplate the cytotoxic role of arachidonic acid in vivo.     

Fine structure and cytochemistry of peroxisomes (microbodies) in Leptomonas samueli

Summary Leptomonas samueli possesses in its cytoplasm a membrane-bounded organelle which can reach a length of 2.8 m and a diameter of 0.2 m. Catalase activity, which is inhibited by 3-amino-1, 2, 4-triazole, was detected at the ultrastructural level in the matrix of the organelle by using an alkaline diaminobenzidine medium. Freeze-fracture studies showed the presence of a large number of intramembranous particles on both the P and the E faces of the membrane of the organelle. Based on these data as well as on previous observations, it is suggested that the trypanosomatids possess an organelle that can be considered to be a peroxisome.     

Expression of Metalloproteinase 2 in the Cell Response to Porous Demineralized Bovine Bone Matrix

Summary The purpose of the study was to analyze the involvement of metalloproteinase 2 (MMP-2) and macrophages in the tissue and cell response to the organic graft material produced from bovine cancellous bone. Thirty adult male white Wistar rats (Rattus norvegicus) received implants of blocks of demineralized bovine bone matrix between the fasciae of the quadriceps muscle. The specimens collected at 3, 7, 14, 21 and 28 days after implantation (n = 6/period). Sections of 6 μm thick were stained with hematoxylin and eosin and immunolabeled with anti-MMP-2 and anti-CD68 using standard avidin–biotin–peroxidase method. The tissue response to the material was initially mediated by polymorphonuclear neutrophils, evolving to a mononuclear inflammatory infiltrate with macrophages and few lymphocytes and plasma cells and presence of inflammatory multinucleated giant cells (GC) in contact with the material that exhibited signs of resorption. The number of cells immunolabeled to MMP-2 was highest at day 7 (103.2 ± 39.1), but significantly decreased (F = 3.67; p = 0.044) until day 28 (45.9 ± 13.1). CD68 immunostaining also significantly decreased (F = 6.75; p = 0.007) from day 7 (49.5 ± 10.4) to day 28 (19.5 ± 8.9). A positive and statistically significant correlation was observed between the evolutions of these two variables. The material had been almost completely resorbed at day 28. Among cells present at the granuloma, anti-MMP-2 immunostaining was predominant and more intense in macrophages, yet lightly immunolabeled multinucleated giant cells were found in close contact with the material. Thus, considering the experimental limitations of this study, we concluded that MMP-2 produced by macrophages participates in the resorption of demineralized bovine bone.     

CYP21 mutations in Brazilian patients with 21-hydroxylase deficiency

Congenital adrenal hyperplasia caused by 21-hydroxylase deficiency is a common autosomal recessive disorder resulting from mutations in the 21-hydroxylase (CYP21) gene. To develop a strategy to screen for the most commonly occurring CYP21 mutations in Brazil, we performed molecular genotype analysis on 73 children with CAH representing 71 unrelated families. The techniques used for CYP21 molecular genotype analysis were: restriction fragment length polymorphism, single-strand conformational polymorphism, allele-specific oligonucleotide hybridization, allele-specific polymerase chain reaction amplification, and heteroduplex analyses. Mutations were identified on all but eight affected alleles. The intron 2 splicing mutation was the most frequently identified mutation. Screening for the most common mutations detected at least one mutation on 132/142 (93%) alleles. Multiple CYP21 mutations were detected on 16.2% of alleles. The high frequency of multiple mutations on a single allele emphasizes the importance of thorough and accurate molecular genotype analysis of the complex CYP21 locus.     

Thermo-search: lifestyle and thermostability analysis

Thermo-search is an online web tool for the analysis of proteomes and individual proteins according to the ratio of two couplets of preferred and avoided amino acids in hyperthermophiles, thermophiles and mesophiles. It displays the ratio between glutamic acid plus lysine (E+K) and glutamine plus histidine (Q+H), which is higher in thermophilic proteomes and thermostable proteins than in mesophilic proteomes and thermo labile proteins. Thermo-search allows a rapid screen of the CRM database for thermostable proteins in their functional categories and a visualization of the (E+K)/(Q+H) average ratio between organisms, allowing a comparison of their lifestyles.