Structural characteristics and refolding of in vivo aggregated hyperthermophilic archaeon proteins

Several recombinant proteins in inclusion bodies expressed in Escherichia coli have been measured by Fourier transform infrared and solid-state nuclear magnetic resonance spectra to provide the secondary structural characteristics of the proteins from hyperthermophilic archaeon Pyrococcus horikoshii OT3 (hyperthermophilic proteins) in inclusion bodies. The beta-strand-rich single chain Fv fragment (scFv) and alpha-helix-rich interleukin (IL)-4 lost part of the native-like secondary structure in inclusion bodies, while the inclusion bodies composed of the hyperthermophilic proteins of which the native form is alpha-helix rich, are predominated by alpha-helix structure. Further, the secondary structure of the recombinant proteins solubilized from inclusion bodies by detergent or denaturant was observed by circular dichroism (CD) spectra. The solubilization induced the denaturation of the secondary structure for scFv and IL-4, whereas the solubilized hyperthermophilic proteins have retained the alpha-helix structure with the CD properties resembling those of their native forms. This indicates that the hyperthermophilic proteins form native-like secondary structure in inclusion bodies. Refolding of several hyperthermophilic proteins from in vivo aggregated form without complete denaturation could be accomplished by solubilization with lower concentration (e.g. 2 M) of guanidine hydrochloride and removal of the denaturant via stepwise dialysis. This supports the existence of proteins with native-like structure in inclusion bodies and suggests that non-native association between the secondary structure elements leads to in vivo aggregation. We propose a refolding procedure on the basis of the structural properties of the aggregated archaeon proteins.     

Statistical characteristics of climbing fiber spikes necessary for efficient cerebellar learning

 Mean firing rates (MFRs), with analogue values, have thus far been used as information carriers of neurons in most brain theories of learning. However, the neurons transmit the signal by spikes, which are discrete events. The climbing fibers (CFs), which are known to be essential for cerebellar motor learning, fire at the ultra-low firing rates (around 1 Hz), and it is not yet understood theoretically how high-frequency information can be conveyed and how learning of smooth and fast movements can be achieved. Here we address whether cerebellar learning can be achieved by CF spikes instead of conventional MFR in an eye movement task, such as the ocular following response (OFR), and an arm movement task. There are two major afferents into cerebellar Purkinje cells: parallel fiber (PF) and CF, and the synaptic weights between PFs and Purkinje cells have been shown to be modulated by the stimulation of both types of fiber. The modulation of the synaptic weights is regulated by the cerebellar synaptic plasticity. In this study we simulated cerebellar learning using CF signals as spikes instead of conventional MFR. To generate the spikes we used the following four spike generation models: (1) a Poisson model in which the spike interval probability follows a Poisson distribution, (2) a gamma model in which the spike interval probability follows the gamma distribution, (3) a max model in which a spike is generated when a synaptic input reaches maximum, and (4) a threshold model in which a spike is generated when the input crosses a certain small threshold. We found that, in an OFR task with a constant visual velocity, learning was successful with stochastic models, such as Poisson and gamma models, but not in the deterministic models, such as max and threshold models. In an OFR with a stepwise velocity change and an arm movement task, learning could be achieved only in the Poisson model. In addition, for efficient cerebellar learning, the distribution of CF spike-occurrence time after stimulus onset must capture at least the first, second and third moments of the temporal distribution of error signals. Received: 28 January 2000 / Accepted in revised form: 2 August 2000     

EGFP-Tagged Vasopressin Precursor Protein Sorting Into Large Dense Core Vesicles and Secretion From PC12 Cells

1. Hypothalamic magnocellular neurons synthesize, store, and secrete large quantities of the neuropeptides, vasopressin (VP) and oxytocin (OT), which are synthesized as protein precursors also containing proteins called neurophysins. These protein precursors are sorted through the regulated secretory pathway (RSP), packaged into large dense core vesicles LDCVs, and their peptide products are secreted from nerve terminals in the posterior pituitary.2. It has been hypothesized that this efficient packaging is dependent on the interaction of the peptide with neurophysin in a complex that forms the granule core. To test this, PC12 cells were transfected with vasopressin precursor DNA constructs that either contained or deleted the neurophysin moiety and tagged with enhanced green fluorescent protein (EGFP) as reporters. The intracellular routing and secretion of the EGFP-tagged VP precursor proteins were studied by in differentiated PC12 cells by fluorescence microscopy, electron microscopic immunocytochemistry, and fluorescent imaging techniques.3. The data showed that only when the neurophysin was present in the VP precursor construct did the fluorescent fusion protein become routed to the RSP and get efficiently packaged into LDCVs and secreted. These data are consistent with the view that routing of the precursor to LDCVs requires the amino acids that encode the intravesicular chaperone, neurophysin.     

Human XPC-hHR23B interacts with XPA-RPA in the recognition of triplex-directed psoralen DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) represent a severe form of damage that blocks DNA metabolic processes and can lead to cell death or carcinogenesis. The repair of DNA ICLs in mammals is not well characterized. We have reported previously that a key protein complex of nucleotide excision repair (NER), XPA-RPA, recognizes DNA ICLs. We now report the use of triplex technology to direct a site-specific psoralen ICL to a target DNA substrate to determine whether the human global genome NER damage recognition complex, XPC-hHR23B, recognizes this lesion. Our results demonstrate that XPC-hHR23B recognizes psoralen ICLs, which have a structure fundamentally different from other lesions that XPC-hHR23B is known to bind, with high affinity and specificity. XPC-hHR23B and XPA-RPA protein complexes were also observed to bind psoralen ICLs simultaneously, demonstrating not only that psoralen ICLs are recognized by XPC-hHR23B alone, but also that XPA-RPA may interact cooperatively with XPC-hHR23B on damaged DNA, forming a multimeric complex. Since XPC-hHR23B and XPA-RPA participate in the recognition and verification of DNA damage, these results support the hypothesis that interplay between components of the global genome repair sub-pathway of NER is critical for the recognition of psoralen DNA ICLs in the mammalian genome.     

Movement and fixation of intestinal microbiota after administration of human feces to germfree mice

Human flora-associated (HFA) mice have been considered a tool for studying the ecology and metabolism of intestinal bacteria in humans, although they have some limitations as a model. Shifts in dominant species of microbiota in HFA mice after the administration of human intestinal microbiota was revealed by 16S rRNA gene sequence and terminal restriction fragment length polymorphism (T-RFLP) analyses. Characteristic terminal restriction fragments (T-RFs) were quantified as the proportion of total peak area of all T-RFs. Only the proportion of the T-RF peak at bp 366, identified as the Gammmaproteobacteria group and the family Coriobacteriaceae, was reduced in this study. Increased T-RFs over time at bp 56, 184, and 196 were affiliated with the Clostridium group. However, most of the isolated bacteria with unique population shifts were phylotypes. The vertical transmission of the intestinal microbiota of the mouse offspring was also investigated by dendrogram analysis derived from the similarity of T-RFLP patterns among samples. As a result, the intestinal microbiota of HFA mice and their offspring reflected the composition of individual human intestinal bacteria with some modifications. Moreover, we revealed that human-derived lactobacilli (HDL), which have been considered difficult to colonize in the HFA mouse intestine in previous studies based on culture methods, could be detected in the HFA mouse intestine by using a lactic acid bacterium-specific primer and HDL-specific primers. Our results indicate that the intestinal microbiota of HFA mice represents a limited sample of bacteria from the human source and are selected by unknown interactions between the host and bacteria.     

Two functional heads are required for full activation of smooth muscle myosin

The motor activity of smooth muscle myosin II is regulated by the regulatory light chain phosphorylation, but it is not understood how phosphorylation activates motor activity. To address this question, we produced asymmetric heavy meromyosin (HMM), which is composed of a wild-type (WT) heavy chain and a mutant heavy chain having no motor activity (i.e. S236T or G457A). The actin-activated ATPase activities (Vmax) of asymmetric HMMs were only 21.8 and 8.4% of the wild-type HMM for S236A/WT HMM and G456A/WT HMM, respectively. If the two heads of HMM are independent for their ATPase activities, asymmetric HMM should show 50% of the activity of wild-type HMM; however, the activity of asymmetric HMM was much lower than the expected value. The results suggest that the activity of the wild-type head is attenuated by the presence of inactive head. Consistently, the actin-gliding velocity of the asymmetric HMM (i.e. S236T/WT or G457A/WT) was less than one-fifth of the wild-type HMM. The present study supports an idea that the two heads of smooth muscle myosin II interact with each other and the presence of two active heads is required for full activation.     

The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity

An anti-human interleukin 5 receptor (hIL-5R) humanized immunoglobulin G1 (IgG1) and an anti-CD20 chimeric IgG1 produced by rat hybridoma YB2/0 cell lines showed more than 50-fold higher antibody-dependent cellular cytotoxicity (ADCC) using purified human peripheral blood mononuclear cells as effector than those produced by Chinese hamster ovary (CHO) cell lines. Monosaccharide composition and oligosaccharide profiling analysis showed that low fucose (Fuc) content of complex-type oligosaccharides was characteristic in YB2/0-produced IgG1s compared with high Fuc content of CHO-produced IgG1s. YB2/0-produced anti-hIL-5R IgG1 was subjected to Lens culinaris aggulutin affinity column and fractionated based on the contents of Fuc. The lower Fuc IgG1 had higher ADCC than the IgG1 before separation. In contrast, the content of bisecting GlcNAc of the IgG1 affected ADCC much less than that of Fuc. In addition, the correlation between Gal and ADCC was not observed. When the combined effect of Fuc and bisecting GlcNAc was examined in anti-CD20 IgG1, only a severalfold increase of ADCC was observed by the addition of GlcNAc to highly fucosylated IgG1. Quantitative PCR analysis indicated that YB2/0 cells had lower expression level of FUT8 mRNA, which codes alpha1,6-fucosyltransferase, than CHO cells. Overexpression of FUT8 mRNA in YB2/0 cells led to an increase of fucosylated oligosaccharides and decrease of ADCC of the IgG1. These results indicate that the lack of fucosylation of IgG1 has the most critical role in enhancement of ADCC, although several reports have suggested the importance of Gal or bisecting GlcNAc and provide important information to produce the effective therapeutic antibody.     

Role of the thrombopoietin (TPO)/Mpl system: c-Mpl-like molecule/TPO signaling enhances early hematopoiesis in Xenopus laevis

Multiple organs are induced in the primitive embryonic ectoderm excised from blastula stage Xenopus laevis embryos, under the strict control of mesoderm inducing factors. This in vitro system is useful for exploring the mechanisms of development. In this study, the function of thrombopoietin (TPO)/c-Mpl signaling in the development of hematopoietic cells was investigated. An optimal hematopoietic cell induction system was established to evaluate the influence of growth factors on hematopoiesis. It was found that exogenous TPO enhanced hematopoiesis in explants induced by activin and bone morphogenetic protein (BMP)-4 and increased the number of both erythrocytes and leukocytes in a dose-dependent manner. Addition of anti-c-Mpl antibody completely inhibited the expansion of hematopoietic cells stimulated by TPO, and the antibody specifically recognized blood-like cells. These results demonstrate that TPO acts on hematopoietic progenitors induced in explants and the c-Mpl-like molecule in Xenopus mediates the cellular function of TPO. We also found that forced expression of TPO in embryos promoted hematopoiesis in the ventral blood island and the dorsal-- lateral plate mesoderm. These results suggest that hematopoietic stem and progenitor cells are regulated by TPO/c-Mpl signaling from when they appear in their ontogeny. They also suggest that TPO/c-Mpl signaling play a crucial role in the formation of hematopoietic cells in Xenopus.