ROCK inhibitor (Y27632) increases apoptosis and disrupts the actin cortical mat in embryonic avian corneal epithelium.

The embryonic chicken corneal epithelium is a unique tissue that has been used as an in vitro epithelial sheet organ culture model for over 30 years (Hay and Revel [1969] Fine structure of the developing Avian cornea. Basel, Switzerland: S. Karger A.G.). This tissue was used to establish that epithelial cells could produce extracellular matrix (ECM) proteins such as collagen and proteoglycans (Dodson and Hay [1971] Exp Cell Res 65:215-220; Meier and Hay [1973] Dev Biol 35:318-331; Linsenmayer et al. [1977] Proc Natl Acad Sci U S A 74:39-43; Hendrix et al. [1982] Invest Ophthalmol Vis Sci 22:359-375). This historic model was also used to establish that ECM proteins could stimulate actin reorganization and increase collagen synthesis (Sugrue and Hay [1981] J Cell Biol 91:45-54; Sugrue and Hay [1982] Dev Biol 92:97-106; Sugrue and Hay [1986] J Cell Biol 102:1907-1916). Our laboratory has used the model to establish the signal transduction pathways involved in ECM-stimulated actin reorganization (Svoboda et al. [1999] Anat Rec 254:348-359; Chu et al. [2000] Invest Ophthalmol Vis Sci 41:3374-3382; Reenstra et al. [2002] Invest Ophthalmol Vis Sci 43:3181-3189). The goal of the current study was to investigate the role of ECM in epithelial cell survival and the role of Rho-associated kinase (p160 ROCK, ROCK-1, ROCK-2, referred to as ROCK), in ECM and lysophosphatidic acid (LPA) -mediated actin reorganization. Whole sheets of avian embryonic corneal epithelium were cultured in the presence of the ROCK inhibitor, Y27632 at 0, 0.03, 0.3, 3, or 10 microM before stimulating the cells with either collagen (COL) or LPA. Apoptosis was assessed by Caspase-3 activity assays and visualized with annexin V binding. The ROCK inhibitor blocked actin cortical mat reformation and disrupted the basal cell lateral membranes in a dose-dependent manner and increased the apoptosis marker annexin V. In addition, an in vitro caspase-3 activity assay was used to determine that caspase-3 activity was higher in epithelia treated with 10 microM Y-27632 than in those isolated without the basal lamina or epithelia stimulated with fibronectin, COL, or LPA. In conclusion, ECM molecules decreased apoptosis markers and inhibiting the ROCK pathway blocked ECM stimulated actin cortical mat reformation and increased apoptosis in embryonic corneal epithelial cells.     

Two independent mutations in a family with neurofibromatosis type 1 (NF1)

We report on two independent alterations of the NF1 gene in a three-generation kindred with neurofibromatosis type 1 (NF1). Using temperature gradient gel electrophoresis (TGGE) in a mutation analysis of exon 31 of the NF1 gene we detected the previously reported nonsense mutation R1947X. This C-to-T transition at codon 1947 in exon 31 is considered to represent a mutation "hot spot" of the NF1 gene due to 5mCpG deamination. All living family members together with their genomic DNA were included in this investigation. However, the mutation R1947X was absent from two undoubtedly affected siblings of the propositus. Another NF1 mutation (889-2A-->G) was identified in the two sibs by the protein truncation test (PTT). The novel splice site mutation 889-2A-->G results in a skip of NF1 exon 7 during splicing and protein truncation due to frameshift. The two NF1 alterations are linked to different paternal haplotypes. In our study of a three-generation kindred, R1947X represents a de novo mutation whereas 889-2A-->G is an inherited splice mutation. Implications for phenotype variation are discussed.     

Methodical problems for evidence of the hepatitis-B-surface and hepatitis-B-core antigens in tissue (author's transl)]

Liver biopsy material of 22 in the serum HBsAg positive patients was tested with the fluorescent antibody technique for the localization of HBcAg and HBsAg in the liver tissue. Comparative studies were done with the following tissue preparation techniques: Cryostat technique, freeze drying, freeze substitution, cold ethanol paraffin embedding technique (SAINTE MARIE) and isolated liver cells. The investigations revealed the following results: 1. No HB-components could be detected with the cold ethanol paraffin embedding technique and freeze substitution. 2. Using the cryostat technique HBsAg could be demonstrated in 16/22 (cytoplasmatic localization) and HBcAg in 8/22 (nuclear localization). 3. With freeze drying HBsAg and HBcAg could be found in the same cases. The excellent tissue preparation allowed a correct localization of the HB-components to the cell structure. 4. In comparison to cryostat sections in isolated liver cells HBcAg could be demonstrated in 11/16 and HBcAg in 8/8 cases.     

Information processing in long delay memory-guided saccades: further insights from TMS

The performance of memory-guided saccades with two different delays (3 s and 30 s of memorisation) was studied in eight subjects. Single pulse transcranial magnetic stimulation (TMS) was applied simultaneously over the left and right dorsolateral prefrontal cortex (DLPFC) 1 s after target presentation. In both delays, stimulation significantly increased the percentage of error in amplitude of memory-guided saccades. Furthermore, the interfering effect of TMS was significantly higher in the short delay compared to that of the long delay paradigm. The results are discussed in the context of a mixed model of spatial working memory control including two components: First, serial information processing with a predominant role of the DLPFC during the early period of memorisation and, second, parallel information processing, which is independent from the DLPFC, operating during longer delays.     

Molecular evolutionary relationships of enteroinvasive Escherichia coli and Shigella spp

Enteroinvasive Escherichia coli (EIEC), a distinctive pathogenic form of E. coli causing dysentery, is similar in many properties to bacteria placed in the four species of Shigella. Shigella has been separated as a genus but in fact comprises several clones of E. coli. The evolutionary relationships of 32 EIEC strains of 12 serotypes have been determined by sequencing of four housekeeping genes and two plasmid genes which were used previously to determine the relationships of Shigella strains. The EIEC strains were grouped in four clusters with one outlier strain, indicating independent derivation of EIEC several times. Three of the four clusters contain more than one O antigen type. One EIEC strain (an O112ac:H- strain) was found in Shigella cluster 3 but is not identical to the Shigella cluster 3 D2 and B15 strains with the same O antigen. Two forms of the virulence plasmid pINV have been identified in Shigella strains by using the sequences of ipgD and mxiA genes, and all but two of our EIEC strains have pINV A. The EIEC strains were grouped in two subclusters with a very low level of variation, generally not intermingled with Shigella pINV A strains. The EIEC clusters based on housekeeping genes were reflected in the plasmid gene sequences, with some exceptions. Two strains were found in the pINV B form by using the ipgD sequence, with one strain having an mxiA sequence similar to the divergent sequence of D1. Clearly, EIEC and Shigella spp. form a pathovar of E. coli.     

Females experiencing sexual and drug vulnerabilities are at elevated risk for HIV infection among youth who use injection drugs

OBJECTIVES: To compare sociodemographic, drug-related, and sexual risk variables between young (13-24 years of age) and older (> or =25 years of age) injection drug users (IDUs); and to determine HIV prevalence and associated risk factors for HIV infection among young IDUs. METHODS: Data were collected through the Vancouver Injection Drug Users Study (VIDUS). To date, over 1400 Vancouver area IDUs have been enrolled and observed during follow-up. Sociodemographic, drug-related, and sexual risk variables were compared between younger and older IDUs using nonparametric methods. Mantel-Haenszel and logistic regression methods were used to compare HIV-positive and HIV-negative female youth. RESULTS: Younger injectors (N = 232) were more likely to be female; work in the sex trade; report condom use; inject heroin daily; smoke crack cocaine daily; and need help injecting. HIV prevalence at baseline among the youth was 10%. HIV prevalence was associated with female gender; history of sexual abuse; engaging in survival sex; injecting heroin daily; injecting speedballs (a mixture of heroin and cocaine) daily; and having numerous lifetime sexual partners. CONCLUSION: Our data show that HIV positivity among young IDUs is concentrated among females engaged in dual sexual and drug-related risk exposure categories. Over half the HIV-positive youth were Aboriginal (a classification used by the federal government in Canada to include native peoples of all ethnic groups). Targeted interventions that take into account sexual and drug risk for young female and Aboriginal peoples are urgently needed.     

A novel mutation L1425P in the GAP-region of the NF1 gene detected by temperature gradient gel electrophoresis (TGGE). Mutation in brief no. 230. Online

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder with an incidence of between 1: 3000 and 1: 4000. Common clinical signs include more than six café-au-lait spots, multiple cutaneous neurofibromas and iris Lisch nodules. Rarer are skeletal anomalies, learning disabilities and an increased risk of malignancy. The NF1 gene contains at least 60 exons with intron sizes ranging from 60 bp to more than 40 kb. Despite using different techniques including PTT, SSCP heteroduplex analyses and direct sequencing, only a relatively small number of mutations have been reported world-wide. Using the more sensitive technique of temperature gradient gel electrophoresis (TGGE), we analysed a part of the NF1-GAP-region, namely exon 25, in DNA samples from 131 unrelated patients. We have identified a novel mutation L1425P in exon 25 of the NF1 gene in a 12-year-old boy (clinically diagnosed with NF1 at the age of 7). In contrast to those cases diagnosed with having both GAP-region mutations and malignant tumours, neither the proband nor four clinically affected family members with this mutation showed any evidence of malignancies.     

Secretion of salivary immunoglobulin a in relation to age,saliva flow,mood states,secretion of albumin,cortisol, and catecholamines in saliva

Salivary immunoglobulin A (IgA) is one characteristic humoral factor of the local immune system in the upper respiratory tract. Epidemiological studies emphasize the importance of secretory IgA in the protection from infections of the upper respiratory tract. However, due to high interindividual variability of secretion of salivary IgA, it remains difficult to define normal ranges. This series of studies focused on identification of factors influencing basal secretion of salivary IgA. The results indicate a significant relationship between age and salivary IgA concentration. Children below 7 years have lower salivary IgA concentration than children above 7 years or adults. Furthermore, a significant inverse relationship between saliva flow and salivary IgA concentration was found. Gender, mood states, salivary albumin, salivary catecholamines, and salivary cortisol were not associated with salivary IgA. It can be concluded that for defining normal ranges of salivary IgA, age and saliva flow have to be considered.     

TLR8 and TLR7 are involved in the host's immune response to human parechovirus 1

Toll-like receptors (TLR) have a key role in regulating immunity against microbial agents. Engagement of TLR by bacterial, viral or fungal components leads to the production and release of inflammatory cytokines. In this study we show that mainly TLR8 and also TLR7 act as the host sensors for human parechovirus 1, a single-stranded RNA (ssRNA) virus. Furthermore, we see that the viral ssRNA genome is detected in endosomal compartments by these TLR, which activate signalling that lead to the synthesis of pro-inflammatory molecules by the host.