Recognition of HLA-B27 by mouse cytotoxic T-cell clones: a transgenic mouse

As a basis for the characterization of mouse T cells involved in the recognition of xenogeneic HLA molecules, a panel of HLA-B27-reactive cytotoxic T-cell clones was generated upon stimulation by cells from HLA-B27-transgenic mice. The HLA-B27-induced T-cell response was found to comprise two categories of clones: some recognizing HLA-B27 independent of H-2 molecules expressed by the target cells (unrestricted clones), others recognizing HLA-B27 in an H-2 restricted manner. The unrestricted clones exhibited diverse specificities, as judged from their various cross-reactivities with other xenogeneic (HLA) or allogeneic (H-2) molecules. In addition, although most of the unrestricted clones were able to react with both mouse and human HLA-B27-transgenic mice. The HLA-B27 induced T-cell which reacted only with HLA-B27-positive mouse, and not human cells. These findings illustrate that both H-2-restricted and unrestricted T cells with diverse species contribute to HLA-B27-xenorecognition.     

The human 5-HT1A receptor expressed in HeLa cells stimulates sodium-dependent phosphate uptake via protein kinase C

Regulation of phosphate uptake was studied in HeLa cell lines after transfection with DNA encoding the human 5-HT1A receptor. Phosphate uptake was saturable and greater than 90% sodium-dependent, with Vmax approximately 30-35% without changing Km. Treatment with 5-HT or the 5-HT1A-specific agonist 8-OH-2-(di-n-propylamino)1,2,3,4-tetrahydronaphthalene increased Vmax approximately 40% without affecting Km. This effect was blocked by pretreatment with the 5-HT1 antagonists, methiothepine and spiperone, or pertussis toxin. Surprisingly, the stimulation was not secondary to an inhibition of adenylyl cyclase because 5-HT stimulated phosphate uptake approximately 20% in the presence of 1 mM 8-Br-cAMP. Rather, the primary pathway linked to the stimulation of phosphate uptake involved activation of protein kinase C because (i) 5-HT measurably activated protein kinase C in these cells, (ii) activators of protein kinase C (phorbol esters and diacylglycerol analogues) stimulated phosphate uptake in these cells (iii) the half-maximal doses for 5-HT-induced phosphatidylinositol hydrolysis and stimulation of phosphate uptake were virtually equivalent, and both effects were equally sensitive to pertussis toxin, and (iv) the stimulation was markedly attenuated in cells made deficient in protein kinase C. These results demonstrate that the stimulation of phosphatidylinositol hydrolysis by the 5-HT1A receptor can generate physiologically measurable effects on cellular transport and suggest that such accessory pathways may play a prominent role in signal transduction.     

Identifying historical and future global change drivers that place species recovery at risk

Ecosystem management in the face of global change requires understanding how co-occurring threats affect species and communities. Such an understanding allows for effective management strategies to be identified and implemented. An important component of this is differentiating between factors that are within (e.g. invasive predators) or outside (e.g. drought, large wildfires) of a local manager's control. In the global biodiversity hotspot of south-western Australia, small- and medium-sized mammal species are severely affected by anthropogenic threats and environmental disturbances, including invasive predators, fire, and declining rainfall. However, the relative importance of different drivers has not been quantified. We used data from a long-term monitoring program to fit Bayesian state-space models that estimated spatial and temporal changes in the relative abundance of four threatened mammal species: the woylie (Bettongia penicillata), chuditch (Dasyurus geoffroii), koomal (Trichosurus vulpecula) and quenda (Isoodon fusciventor). We then use Bayesian structural equation modelling to identify the direct and indirect drivers of population changes, and scenario analysis to forecast population responses to future environmental change. We found that habitat loss or conversion and reduced primary productivity (caused by rainfall declines) had greater effects on species' spatial and temporal population change than the range of fire and invasive predator (the red fox Vulpes vulpes) management actions observed in the study area. Scenario analysis revealed that a greater extent of severe fire and further rainfall declines predicted under climate change, operating in concert are likely to further reduce the abundance of these species, but may be mitigated partially by invasive predator control. Considering both historical and future drivers of population change is necessary to identify the factors that risk species recovery. Given that both anthropogenic pressures and environmental disturbances can undermine conservation efforts, managers must consider how the relative benefit of conservation actions will be shaped by ongoing global change.     

Radioimmunoassay of 2-hydroxyestrone

Under the protection of ascorbic acid a 2-hydroxyestrone bovine serum albumin conjugate was prepared containing intact 2-hydroxyestrone as determined by gas chromatographymass spectrometry. Using this antigen highly specific antibodies were raised in rabbits. Cross-reactivity for 2-hydroxyestradiol and 2-hydroxyestriol was 26 and 4.5%, respectively. An assay procedure of 2-hydroxyestrone in human plasma is described. Using special precautions the assay allows the determination of 2-hydroxyestrone in plasma samples of women (50–95 pg/ml), pregnant women (105–220 pg/ml), men (45–65 pg/ml) and children (20–40 pg/ml).     

Characteristics of the F52 protein, a MARCKS homologue.

A recently cloned mouse cDNA designated F52 encodes a putative protein with striking sequence similarity to the MARCKS protein, a major cellular substrate for protein kinase C (PKC). Major regions of sequence similarity include the amino-terminal myristoylation consensus sequence and the central calmodulin-binding/PKC phosphorylation site domain. The F52 protein was expressed in Escherichia coli with apparent M(r) 50,000; it was a substrate for PKC and comigrated on two-dimensional electrophoresis with a myristoylated protein whose phosphorylation was stimulated by phorbol 12-myristate 13-acetate in mouse neuroblastoma cells. The F52 protein also was myristoylated in E. coli by co-expression with N-myristoyltransferase. A 24-amino acid peptide derived from the protein's phosphorylation site domain was a good substrate for PKC; like the cognate MARCKS peptide, it was phosphorylated with high affinity (S0.5 = 173 nM) and positive cooperativity (KH = 5.4). The F52 peptide also bound calmodulin with high affinity (Kd = less than 3 nM); this binding could be disrupted by phosphorylation of the peptide with PKC, with a half-time of 8 min. The F52 protein is clearly a member of the MARCKS family as defined by primary sequence; in addition, the two proteins share several key attributes that may be functionally important.     

Inter-helical hydrogen bond formation during membrane protein integration into the ER membrane

Recent work has shown that efficient di- or trimerization of hydrophobic transmembrane helices in detergent micelles or lipid bilayers can be driven by inter-helix hydrogen bonding involving polar residues such as Asn or Asp. Using in vitro translation in the presence of rough microsomes of a model integral membrane protein, we now show that the formation of so-called helical hairpins, two tightly spaced transmembrane helices connected by a short loop, can likewise be promoted by the introduction of Asn-Asn or Asp-Asp pairs in a long transmembrane hydrophobic segment. These observations suggest that inter-helix hydrogen bonds can form within the context of the Sec61 translocon in the endoplasmic reticulum, implying that hydrophobic segments in a nascent polypeptide chain in transit through the Sec61 channel have immediate access to a non-aqueous subcompartment within the translocon.     

Die Chloroplastendrehung beiMougeotia

Summary Mougeotia cells with chloroplasts oriented in profile have been irradiated with small spots of monochromatic red polarized light in order to induce chloroplast movement.In these experiments, four factors have been varied: 1. the orientation of the vibration plane of the light in relation to the cell axis, 2. the localization of the spot, i. e. irradiation of the chloroplast or the cytoplasm, 3. the spot size, and 4. the duration of the irradiation.As a result of our experiments, we conclude that the photoreceptor molecules responsible for the light-induced chloroplast movement are localized in the cytoplasm.As the photoreceptor of this reaction is the well known phytochromesystem, we may assume that also in other plants the phytochrome is localized in the cytoplasm rather than in the chloroplast.

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