Disruption of pubertal onset by exogenous testosterone and estrogen in two species of rodents

The administration of adult physiological levels of testosterone (T) and 17beta-estradiol (E(2)) to male Siberian hamsters was previously shown to delay the onset of puberty. To examine whether this is a response common to other rodents, we investigated whether exogenous steroids also alter the onset of puberty in Syrian hamsters and mice. Juvenile male Syrian hamsters and mice were implanted with Silastic capsules containing T, E(2), or cholesterol control. After 15 days, plasma, pituitaries, and testes were processed for histological analysis or measurements of gonadotropins and circulating steroid hormones. T and E(2) implants reduced testis mass and gonadotropin stores in both species and arrested spermatogenesis in Syrian hamsters. In contrast, spermatogenesis in mice was unaffected by T and only modestly affected by E(2). Although E(2) inhibited circulating follicle-stimulating hormone (FSH) in both species, T inhibited circulating FSH in mice only. Overall, our results demonstrate that the hypothalamic-pituitary-gonadal axis of each rodent species responds uniquely to T and E(2) during the pubertal transition. Despite the highly varied effects of T and E(2) in these two species, the ability of steroid hormones to disrupt the onset of puberty appears to be a feature common to many rodents.     

Metalloporphyrin probes for antimalarial drug action

Metal-substituted protoporphyrin IXs (Co(III)PPIX (1), Cr(III)PPIX (2), Mn(III)PPIX (3), Cu(II)PPIX (4), Mg(II)PPIX (5), Zn(II)PPIX (6) and Sn(IV)PPIX (7)), phthalocyanine tetrasulfonates (PcS (8) and Ni(II)PcS (9)), and anionic and cationic porphyrins (meso-tetra(4-sulfonatophenyl)porphine (TPPS4, 10), meso-tetra(4-carboxyphenyl)porphine (TPPC4, 11), tetrakis(4-N-trimethylaminophenyl)porphine (TMAP, 12) and meso-tetra(N-methyl-4-pyridyl)porphine (TMPyP4, 13)) have been used as probes to compare two different assays for the inhibition of beta-hematin formation. The results demonstrate that the efficacy of these probes in either the beta-hematin inhibition assay (9, 7, 6, 5>4>11, 3>10, 8>2, 1; 12 and 13 did not inhibit.) or the bionucleating template assay (8>1>11>9, 2>4>3>7>10>5>6; 12 and 13 did not inhibit.) differ significantly. These differences are examined in light of possible interactions between the inhibitor probes, heme, beta-hematin and the bionucleating template. This detailed analysis highlights the fact that while dominant modes of interactions may be occasionally identified, the precise mechanism of inhibition undoubtedly consists of the interplay between multiple interactions.     

Human CD30: structural implications from epitope mapping and modeling studies

The human CD30 molecule is expressed transiently at very low levels on intrafollicular and perifollicular T and B cell blasts in lymphoid tissues, but is specifically upregulated on certain tumor cells, e.g. Hodgkin and Reed-Sternberg (H-RS) cells. With its specific expression pattern and easy accessibility on the surface of H-RS cells CD30 is a valuable diagnostic marker and holds considerable promise as a target for in vivo immunotherapy. Knowledge of epitopes on the CD30 molecule is expected to facilitate the design of novel non-immunogenic anti-CD30 reagents. Therefore, we have mapped the epitopes of several monoclonal antibodies (mAb) applying a peptide array of overlapping CD30-derived peptides. For the mAb Ber-H2, two linear epitopes with identical sequence were found, while the mAb Ki-2 and the single chain Fv fragment R4-4 each recognized a single linear antigenic determinant, respectively. On the other hand, the mAb Ki-1 bound to a discontinuous epitope composed of two regions, one located near the N-terminus and the other near the membrane-spanning region of CD30. Using molecular modeling, it was possible to visualize the location of the epitopes on exposed loop regions of the molecule within the N-terminal domain. Finally, the results obtained with the mAb Ki-1 imply that the ends of the N- and C-terminal parts of the extracellular portion of CD30 are in close vicinity of each other, suggesting a flower-like structure for the membrane-bound homotrimeric CD30 molecule.     

Silencing of death receptor and caspase-8 expression in small cell lung carcinoma cell lines and tumors by DNA methylation

Small cell lung cancer cell lines were resistant to FasL and TRAIL-induced apoptosis, which could be explained by an absence of Fas and TRAIL-R1 mRNA expression and a deficiency of surface TRAIL-R2 protein. In addition, caspase-8 expression was absent, whereas FADD, FLIP and caspases-3, -7, -9 and -10 could be detected. Analysis of SCLC tumors revealed reduced levels of Fas, TRAIL-R1 and caspase-8 mRNA compared to non-small cell lung cancer (NSCLC) tumors. Methylation-specific PCR demonstrated methylation of CpG islands of the Fas, TRAIL-R1 and caspase-8 genes in SCLC cell lines and tumor samples, whereas NSCLC samples were not methylated. Cotreatment of SCLC cells with the demethylating agent 5'-aza-2-deoxycytidine and IFNgamma partially restored Fas, TRAIL-R1 and caspase-8 expression and increased sensitivity to FasL and TRAIL-induced death. These results suggest that SCLC cells are highly resistant to apoptosis mediated by death receptors and that this resistance can be reduced by a combination of demethylation and treatment with IFNgamma.     

Two plant-viral movement proteins traffic in the endocytic recycling pathway

Many plant viruses exploit a conserved group of proteins known as the triple gene block (TGB) for cell-to-cell movement. Here, we investigated the interaction of two TGB proteins (TGB2 and TGB3) of Potato mop-top virus (PMTV), with components of the secretory and endocytic pathways when expressed as N-terminal fusions to green fluorescent protein or monomeric red fluorescent protein (mRFP). Our studies revealed that fluorophore-labeled TGB2 and TGB3 showed an early association with the endoplasmic reticulum (ER) and colocalized in motile granules that used the ER-actin network for intracellular movement. Both proteins increased the size exclusion limit of plasmodesmata, and TGB3 accumulated at plasmodesmata in the absence of TGB2. TGB3 contains a putative Tyr-based sorting motif, mutations in which abolished ER localization and plasmodesmatal targeting. Later in the expression cycle, both fusion proteins were incorporated into vesicular structures. TGB2 associated with these structures on its own, but TGB3 could not be incorporated into the vesicles in the absence of TGB2. Moreover, in addition to localization to the ER and motile granules, mRFP-TGB3 was incorporated into vesicles when expressed in PMTV-infected epidermal cells, indicating recruitment by virus-expressed TGB2. The TGB fusion protein-containing vesicles were labeled with FM4-64, a marker for plasma membrane internalization and components of the endocytic pathway. TGB2 also colocalized in vesicles with Ara7, a Rab5 ortholog that marks the early endosome. Protein interaction analysis revealed that recombinant TGB2 interacted with a tobacco protein belonging to the highly conserved RME-8 family of J-domain chaperones, shown to be essential for endocytic trafficking in Caenorhabditis elegans and Drosophila melanogaster. Collectively, the data indicate the involvement of the endocytic pathway in viral intracellular movement, the implications of which are discussed.     

HLA-B27 subtypes differentially associated with disease exhibit subtle structural alterations

The reasons for the association of the human major histocompatibility complex protein HLA-B27 with spondyloarthropathies are unknown. To uncover the underlying molecular causes, we determined the crystal structures of the disease-associated B*2705 and the nonassociated B*2709 subtypes complexed with the same nonapeptide (GRFAAAIAK). Both differ in only one residue (Asp(116) and His(116), respectively) in the F-pocket that accommodates the peptide C terminus. Several different effects of the Asp(116) --> His replacement are observed. The bulkier His(116) induces a movement of peptide C-terminal pLys(9), allowing the formation of a novel salt bridge to Asp(77), whereas the salt bridge between pLys(9) and Asp(116) is converted into a hydrogen bond with His(116). His(116) but not Asp(116) adopts two alternative conformations, one of which leads to breakage of hydrogen bonds. Water molecules near residue 116 differ with regard to number, position, and contacts made. Furthermore, F-pocket atoms exhibit higher B-factors in B*2709 than in B*2705, indicating an increased flexibility of the entire region in the former subtype. These changes induce subtle peptide conformational alterations that may be responsible for the immunobiological differences between these HLA-B27 subtypes.     

Caspase-3-dependent and caspase-3-independent pathways leading to chromatin DNA fragmentation in HL-60 cells

Apoptosis induced by etoposide (VP-16) in HL-60 cells was confirmed to be caspase-dependent. It was fully inhibited by the broad-spectrum caspase inhibitor Z-VAD-fmk. However, the caspase-3-specific inhibitor Z-DEVD-fmk only partially inhibited apoptosis. This indicated that a second caspase is required in vivo for full activation of the apoptotic nucease CAD. Aurin tricarboxylic acid (ATA) did not inhibit VP-16-induced apoptosis. In contrast, apoptosis induced by hydroxychloroquine (HCQ) in HL-60 cells was caspase-3 independent and was fully inhibited by ATA. Thus, CAD does not appear to be involved in chromatin DNA degradation in this case. A second apoptotic nuclease is postulated to degrade the DNA, likely endo-exonuclease, an abundant nuclear enzyme that acts on both DNA and RNA and is present in latent form. HCQ, but not VP-16, stimulated DNA degradation (laddering) in isolated nuclei. This indicates that the drug can act directly in the nuclei to trigger activation of the second latent apoptotic nuclease.     

Facile assessment of cDNA constructs for expression of functional antibodies in plants using the potato virus X vector

Antibodies have been expressed in plants to confer novel traits such as virus resistance or altered phenotype. However, not every antibody is suitable for plant expression, and successful intracellular expression of antibody fragments depends primarily on their amino acid sequence in a way that is as yet unpredictable. Therefore it is desirable to assess different constructs before embarking on the production of transgenic plants. We have used a transient expression system based on potato virus X to compare different cDNA constructs for expression and stability of antibody variable gene fragments in plants. Constructs contained an anti-plant enzyme (granule-bound starch synthase I) scFv sequence derived from a naive phage display library together with different combinations of sequences encoding the human IgG constant domain, a murine IgG secretory signal sequence, or the endoplasmic reticulum retention signal peptide KDEL. The results obtained with the potato virus X vector correlated with those from Agrobacterium-mediated stable transformation of potato. The best expression levels were obtained by incorporating sequences that target scFv to the lumen of the endoplasmic reticulum and the secretory pathway. The anti-enzyme scFv retained activity during storage of potato tubers for more than five months. The results demonstrate the utility of the potato virus X vector for the analysis and comparison of many scFv with different epitope specificities or sequence modifications. Evaluation of scFv by transient expression from the PVX vector should aid progress in selection of functional scFv for applications in plant biotechnology.     

Accumulation of a thermostable endo-1,4-β-D-glucanase in the apoplast of Arabidopsis thaliana leaves

Plant biomass, the most abundant renewable resource on earth, is a potential source of fermentable sugars for production of alternative transportation fuels and other chemicals. Bioconversion of plant biomass to fermentable glucose involves enzymatic hydrolysis of cellulose, a major polysaccharide constituent. Because commercially available microbial cellulases are prohibitively expensive for bioethanol processes, we have investigated the feasibility of producing these enzymes in plants as a low-cost, potentially high-volume alternative to traditional production methods. We have successfully expressed the catalytic domain of a thermostable (T _opt=81 °C) endo-1,4--D-glucanase from the eubacterium, Acidothermus cellulolyticus, in the apoplast of tobacco BY-2 suspension cells and leaves of Arabidopsis thaliana plants. The apoplast-targeting cassette designed for this work consists of the cauliflower mosaic virus 35S promoter, the tobacco mosaic virus translational enhancer, the sequence encoding the tobacco Pr1a signal peptide, and the polyadenylation signal of nopaline synthase. Recombinant E1 catalytic domain was targeted to the ER by the signal peptide and secreted into the apoplast via the default pathway. Secretion of the enzyme did not detectably affect the growth rate of transgenic BY-2 cells, although the protein was enzymatically active at elevated temperatures. Similarly, transgenic plants exhibited no abnormal phenotypes correlating with expression of the enzyme. Close agreement between independent immunochemical and activity-based assays indicates that the enzyme accumulated to concentrations up to 26% of the total soluble protein in leaves of primary A. thaliana transformants. The amount of functional endoglucanase produced illustrates that plants can accumulate very large quantities of enzyme for commercial biomass conversion.     

Larval release rhythm of the mole crab Emerita talpoida (Say)

Ovigerous mole crabs Emerita talpoida (Say) were monitored in the laboratory to determine if the time of larval release is synchronous and under endogenous control. To determine the time of larval release, ovigerous females were placed under a 14:10 light/dark cycle simulating the ambient photoperiod. Hatching was rhythmic, occurring as a quick burst lasting about 5-15 min shortly after the onset of darkness. An individual mole crab will release batches of larvae for up to three successive nights, suggesting that the rhythm is under endogenous control. Mole crabs monitored under constant low-level red light displayed the same release pattern with hatching occurring near the time of expected sunset, indicating the presence of a circadian rhythm in larval release. To investigate whether the female or the embryos control hatching, a portion of the egg mass (50-100 embryos) was separated from the female. The time of hatching of the detached embryos subjected to either a still or shaken treatment was compared with the hatching time of embryos still attached to the female. Detached eggs in both treatments hatched within 1.5-2 h of the time of the female-attached eggs, which suggests that embryos control the timing of hatching.