Endothelial cell junctions

In the course of a freeze-cleave study on intercellular junctions in the regenerating rat liver, we observed an unusual array of intramembranous particles located in regions of contact between endothelial cells lining the hepatic sinusoids. These arrays were characterized by an accumulation of particles which resembled a zonula occludens in their linear deployment but differed in that the contact regions were composed of individual particles which remained separated from each other by regular particle-free intervals.     

Proliferative activity in the cerebellar external granular layer evaluated by bromodeoxyuridine labeling

We have optimised an indirect immunoperoxidase technique demonstrating bromodeoxyuridine (BrdU) incorporation into dividing cells for cerebellar tissue sections of four-day-old rats injected with this marker. This permits confident identification of granule-cell precursors engaged in DNA synthesis in the external granular layer of the developing cerebellum. Preservation of BrdU immunoreactivity is attained using methanol/acetic acid fixation and different pretreatments before immunostaining, while unlabeled nuclei can be recognized clearly after Feulgen or hematoxylin counterstaining. We established conditions to ensure satisfactory BrdU uptake without affecting cell-cycle progression during the postlabeling time period. The dose of BrdU employed provides saturation S-phase labeling from at least 1 h after BrdU delivery. Various kinetic parameters and phase durations have been determined in experiments involving a single injection or cumulative labeling sequences, and the cycle time was calculated based on two models of generative behavior: steady-state and exponential growth. The working hypothesis of steadystate kinetics can be adopted successfully if the existence of neuroblasts with different proliferation rates is taken into account.     

Phylogenetic diversity of stress signalling pathways in fungi

Background  

Microbes must sense environmental stresses, transduce these signals and mount protective responses to survive in hostile environments. In this study we have tested the hypothesis that fungal stress signalling pathways have evolved rapidly in a niche-specific fashion that is independent of phylogeny. To test this hypothesis we have compared the conservation of stress signalling molecules in diverse fungal species with their stress resistance. These fungi, which include ascomycetes, basidiomycetes and microsporidia, occupy highly divergent niches from saline environments to plant or mammalian hosts.     

Vital role for Plasmodium berghei Kinesin8B in axoneme assembly during male gamete formation and mosquito transmission

Sexual development is an essential phase in the Plasmodium life cycle, where male gametogenesis is an unusual and extraordinarily rapid process. It produces 8 haploid motile microgametes, from a microgametocyte within 15 minutes. Its unique achievement lies in linking the assembly of 8 axonemes in the cytoplasm to the three rounds of intranuclear genome replication, forming motile microgametes, which are expelled in a process called exflagellation. Surprisingly little is known about the actors involved in these processes. We are interested in kinesins, molecular motors that could play potential roles in male gametogenesis. We have undertaken a functional characterization in Plasmodium berghei of kinesin‐8B (PbKIN8B) expressed specifically in male gametocytes and gametes. By generating Pbkin8B‐gfp parasites, we show that PbKIN8B is specifically expressed during male gametogenesis and is associated with the axoneme. We created a ΔPbkin8B knockout cell line and analysed the consequences of the absence of PbKIN8B on male gametogenesis. We show that the ability to produce sexually differentiated gametocytes is not affected in ΔPbkin8B parasites and that the 3 rounds of genome replication occur normally. Nevertheless, the development to free motile microgametes is halted and the life cycle is interrupted in vivo. Ultrastructural analysis revealed that intranuclear mitoses are unaffected whereas cytoplasmic microtubules, although assembled in doublets and elongated, fail to assemble in the normal axonemal ‘9+2' structure and become motile. Absence of a functional axoneme prevented microgamete assembly and release from the microgametocyte, severely reducing infection of the mosquito vector. This is the first functional study of a kinesin involved in male gametogenesis. These results reveal a previously unknown role for PbKIN8B in male gametogenesis, providing new insights into Plasmodium flagellar formation.     

GMDD: a database of GMO detection methods

Background  

Since more than one hundred events of genetically modified organisms (GMOs) have been developed and approved for commercialization in global area, the GMO analysis methods are essential for the enforcement of GMO labelling regulations. Protein and nucleic acid-based detection techniques have been developed and utilized for GMOs identification and quantification. However, the information for harmonization and standardization of GMO analysis methods at global level is needed.     

Expression of matrix metalloproteinases and their tissue inhibitors in the serum and cerebrospinal fluid of patients with HIV-1 infection and syphilis or neurosyphilis

The potential mechanisms for altered matrix metalloproteinase (MMP) or tissue inhibitors of matrix metalloproteinase (TIMP) function in patients with syphilis and HIV-1 co-infection (HIV-S) was unclear. To determine the expression of MMP-2, 9 and TIMP-1, 2, 4 in the serum and cerebrospinal fluid (CSF) of HIV-S patients, a total of 20 HIV-S patients and 8 controls were enrolled in a HIV-1 clinical cohort for diagnosis of neurosyphilis in Taiwan. Serum and CSF concentrations of MMP-2, 9, and TIMP-1, 2, 4 were determined by ELISA. Gelatin zymography was used to detect the expression of MMP-2 and MMP-9 in the CSF. Neurosyphilis was defined as a CSF white blood cell count ≥ 20 cells/μL or a reactive CSF Venereal Disease Research Laboratory (VDRL). All the patients with HIV-S were males. Most (85%) had sex with men (MSM) and serum rapid plasma reagin (RPR) titers of ≥ 1:32. The median age was 35 years (IQR 30-43). The median CD4 T cell counts at the time of the diagnosis of syphilis were 270 cells/μL (IQR 96-484). Ten patients (50%) had neurosyphilis based on a reactive CSF VDRL test (n=8) or increased CSF white cell counts ≥ 20/μL (n=2). The concentrations of CSF MMP-9, TIMP-1, and TIMP-2 were significantly higher in patients with HIV-S than the controls (P<0.05). The CSF TIMP-4 concentrations were significantly lower in those with HIV-S (452 pg/ml) than controls (3101 pg/ml), P<0001. There were no significant differences in serum concentrations between the groups. The only finding that distinguished HIV-1 patients with from those without neurosyphilis is a significant higher expression of CSF MMP-9. In conclusion, the MMP/TIMP system was found to be dysregulated in patients with HIV-S regardless of whether they met the laboratory definition of neurosyphilis. The CSF level of MMP-9 was the only measure that distinguished those with or without neurosyphilis.