Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions

Bacteriophage P22 is a prototypical biological machine used for studying protein complex assembly and capsid maturation. Using cryo-EM, we solved the structures of P22 before and after the capsid maturation at 8.5 A and 9.5 A resolutions, respectively. These structures allowed visualization of alpha-helices and beta-sheets from which the capsid protein fold is derived. The capsid fold is similar to that of the coat protein of HK97 bacteriophage. The cryo-EM shows that a large conformational change of the P22 capsid during maturation transition involves not only the domain movement of individual subunits, but also refolding of the capsid protein.     

Silymarin inhibits TNF-alpha-induced expression of adhesion molecules in human umbilical vein endothelial cells

Silymarin is known to have an anti-atherosclerotic activity, but the mechanism responsible for it remains unclear. Here, we demonstrate a possible mechanism involved in the anti-atherosclerotic activity of silymarin. Silymarin inhibited THP-1 cell adhesion to human umbilical vein endothelial cells (HUVECs). Silymarin also suppressed the TNF-alpha-induced protein and mRNA expression of adhesion molecules, such as VCAM-1, ICAM-1 and E-selectin, in HUVECs. Moreover, silymarin suppressed the TNF-alpha-induced DNA binding of NF-kappaB/Rel in HUVECs. Taken together, these results demonstrate that silymarin exerts an anti-atherosclerotic activity, at least in part, by inhibiting the expression of adhesion molecules.     

Inhibitory effect of carbon dioxide on the fed-batch culture of Ralstonia eutropha: evaluation by CO2 pulse injection and autogenous CO2 methods

In order to see the effect of CO(2) inhibition resulting from the use of pure oxygen, we carried out a comparative fed-batch culture study of polyhydroxybutyric acid (PHB) production by Ralstonia eutropha using air and pure oxygen in 5-L, 30-L, and 300-L fermentors. The final PHB concentrations obtained with pure O(2) were 138.7 g/L in the 5-L fermentor and 131.3 g/L in the 30-L fermentor, which increased 2.9 and 6.2 times, respectively, as compared to those obtained with air. In the 300-L fermentor, the fed-batch culture with air yielded only 8.4 g/L PHB. However, the maximal CO(2) concentrations in the 5-L fermentor increased significantly from 4.1% (air) to 15.0% (pure O(2)), while it was only 1.6% in the 30-L fermentor with air, but reached 14.2% in the case of pure O(2). We used two different experimental methods for evaluating CO(2) inhibition: CO(2) pulse injection and autogenous CO(2) methods. A 10 or 22% (v/v) CO(2) pulse with a duration of 3 or 6 h was introduced in a pure-oxygen culture of R. eutropha to investigate how CO(2) affects the synthesis of biomass and PHB. CO(2) inhibited the cell growth and PHB synthesis significantly. The inhibitory effect became stronger with the increase of the CO(2) concentration and pulse duration. The new proposed autogenous CO(2) method makes it possible to place microbial cells under different CO(2) level environments by varying the gas flow rate. Introduction of O(2) gas at a low flow rate of 0.42 vvm resulted in an increase of CO(2) concentration to 30.2% in the exit gas. The final PHB of 97.2 g/L was obtained, which corresponded to 70% of the PHB production at 1.0 vvm O(2) flow rate. This new method measures the inhibitory effect of CO(2) produced autogenously by cells through the entire fermentation process and can avoid the overestimation of CO(2) inhibition without introducing artificial CO(2) into the fermentor.     

Nicotinamide Attenuates Focal Ischemic Brain Injury in Rats: With Special Reference to Changes in Nicotinamide and NAD+ Levels in Ischemic Core and Penumbra

We investigated the neuroprotective action of nicotinamide in focal ischemia. Male spontaneously hypertensive rats (5–7 months old) were subjected to photothrombotic occlusion of the right distal middle cerebral artery (MCA). Either nicotinamide (125 or 250 mg/kg) or vehicle was injected IV before MCA occlusion. Changes in the cerebral blood flow (CBF) were monitored using laser-Doppler flowmetry, and infarct volumes were determined with TTC staining 3 days after MCA occlusion. In another set of experiments, the brain nicotinamide and nicotinamide adenine dinucleotide (NAD⁺) levels were analyzed by HPLC using the frozen samples dissected from the regions corresponding to the ischemic core and penumbra. In the 250-mg/kg nicotinamide group, the ischemic CBF was significantly increased compared to that the untreated group, and the infarct volumes were substantially attenuated (–36%). On the other hand, the ischemic CBF in the 125 mg/kg nicotinamide group was not significantly different from the untreated CBF, however, the infarct volumes were substantially attenuated (–38%). Cerebral ischemia per se did not affect the concentrations of nicotinamide and NAD⁺ both in the penumbra and ischemic core. In the nicotinamide groups, the brain nicotinamide levels increased significantly in all areas examined, and brain NAD⁺ levels increased in the penumbra but not in the ischemic core. Increased brain levels of nicotinamide are considered to be primarily important for neuroprotection against ischemia, and the protective action may be partly mediated through the increased NAD⁺ in the penumbra.     

Sex differences on the progressive matrices: some data from Hong Kong

There has been a debate between N. J. Mackintosh and the first author of this paper on whether the sex difference on the Progressive Matrices is zero or, at most, 1-2 IQ points in favour of either sex, as maintained by Mackintosh, or whether from the age of 15 years onwards males obtain higher average scores than females by more than 2 IQ points, as maintained by Lynn. New data relevant to this controversy are presented from Hong Kong consisting of sex differences on the standardization sample of the Advanced Progressive Matrices on 15- to 18-year-olds. The results are that males obtained a higher mean score than females of 1.6 raw score points, equivalent to an advantage of 3.2 or 4.1 IQ points, according to two alternative methods of calculation. The results provide further confirmation that in later adolescence and among adults, males obtain significantly higher mean IQs on the Progressive Matrices than females.     

The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state

Molecular chaperones assist protein folding by facilitating their "forward" folding and preventing aggregation. However, once aggregates have formed, these chaperones cannot facilitate protein disaggregation. Bacterial ClpB and its eukaryotic homolog Hsp104 are essential proteins of the heat-shock response, which have the remarkable capacity to rescue stress-damaged proteins from an aggregated state. We have determined the structure of Thermus thermophilus ClpB (TClpB) using a combination of X-ray crystallography and cryo-electron microscopy (cryo-EM). Our single-particle reconstruction shows that TClpB forms a two-tiered hexameric ring. The ClpB/Hsp104-linker consists of an 85 A long and mobile coiled coil that is located on the outside of the hexamer. Our mutagenesis and biochemical data show that both the relative position and motion of this coiled coil are critical for chaperone function. Taken together, we propose a mechanism by which an ATP-driven conformational change is coupled to a large coiled-coil motion, which is indispensable for protein disaggregation.     

A thaumatin-like gene in nonclimacteric pepper fruits used as molecular marker in probing disease resistance,ripening, and sugar accumulation

During pepper (Capsicum annuum) fruit ripening, the ripe fruit interaction with the anthracnose fungus, Colletotrichum gloeosporioides, is generally incompatible. However, the unripe fruit can interact compatibly with the fungus. A gene, designated PepTLP (for pepper thaumatin-like protein), was isolated and characterized by using mRNA differential display. The PepTLP gene encodes a protein homologous to other thaumatin-like proteins and contains 16 conserved cysteine residues and the consensus pattern of thaumatin. PepTLP gene expression is developmentally regulated during ripening. The accumulation of PepTLP mRNA and PepTLP protein in the incompatible interaction was higher than that in the compatible one. Furthermore, PepTLP gene expression was stimulated by both jasmonic acid treatment and wounding during ripening, but by wounding only in the unripe fruit. Immunolocalization studies showed that it is localized to the intercellular spaces among cortical cells. The expression of the PepTLP gene upon fungal infection was a rise from the early-breaker fruit. The development of anthracnose became significantly prevented with beginning of fruit ripening, and the sum total of sugar accumulation increased. The results suggest that the PepTLP gene can be used as a molecular marker in probing for disease resistance, ripening, and sugar accumulation in nonclimacteric pepper fruits.     

cDNA microarray analysis of the differential gene expression in the neuropathic pain and electroacupuncture treatment models

Partial nerve injury is the main cause of neuropathic pain disorders in humans. Acupuncture has long been used to relieve pain. It is known to relieve pain by controlling the activities of the autonomic nervous system. Although the mechanism of neuropathic pain and analgesic effects of electroacupuncture (EA) have been studied in a rat model system, its detailed mechanism at the molecular level remains unclear. To identify genes that might serve as either markers or explain these distinct biological functions, a cDNA microarray analysis was used to compare the expression of 8,400 genes among three sample groups. Messenger RNAs that were pooled from the spinal nerves of 7 normal, 7 neuropathic pain, and 7 EA treatment rat models were compared. Sixty-eight genes were differentially expressed more than 2-fold in the neuropathic rat model when compared to the normal, and restored to the normal expression level after the EA treatment. These genes are involved in a number of biological processes, including the signal transduction, gene expression, and nociceptive pathways. Confirmation of the differential gene expression was performed by a dotblot analysis. Dot-blotting results showed that the opioid receptor sigma was among those genes. This indicates that opioid-signaling events are involved in neuropathic pain and the analgesic effects of EA. The potential application of these data include the identification and characterization of signaling pathways that are involved in the EA treatment, studies on the role of the opioid receptor in neuropathic pain, and further exploration on the role of selected identified genes in animal models.     

Dose-dependent biphasic activity of tRNA synthetase-associating factor,p43, in angiogenesis

Mammalian aminoacyl tRNA synthetases form a macromolecular protein complex with three non-enzymatic cofactors. Among these factors, p43 is also secreted to work as a cytokine on endothelial as well as immune cells. Here we investigated the activity of p43 in angiogenesis and determined the related mediators. It promoted the migration of endothelial cells at low dose but induced their apoptosis at high dose. p43 at low concentration activated extracellular signal-regulating kinase, which resulted in the induction and activation of matrix metalloproteinase 9. In contrast, p43 at high concentration activated Jun N-terminal kinase, which mediated apoptosis of endothelial cells. These results suggest that p43 is a novel cytokine playing a dose-dependent biphasic role in angiogenesis.     

Establishment of two immortalized nasopharyngeal epithelial cell lines using SV40 large T and HPV16E6/E7 viral oncogenes

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, especially in southern China. One of the most striking features of this disease is its close relationship with Epstein-Barr Virus (EBV). However, to date there is no direct study on the mechanisms involved in the role of EBV in the tumorigenesis of NPC, largely due to lack of an experimental model. Available hypotheses on the association between EBV and NPC are generated from non-nasopharyngeal epithelial cell systems such as human keratinocytes or mouse epithelial cells, which may not truly represent the biological properties of nasopharyngeal epithelial (NP) cells. In this study, we report the establishment of two immortalized NP cell lines, NP69SV40T and NP39E6/E7, using SV40T and HPV16E6/E7 oncogenes. We found that NP60SV40T and NP39E6/E7 cell lines not only maintained many characteristics of normal NP cells (i.e. keratin profile and responsive to TGFbeta inhibition) but also highly responsive to one of the EBV encoded genes, LMP1. Comparative genome hybridization (CGH) analysis showed that these two cell lines contained multiple genetic alterations, some of which have been described in NPC. The immortalized NP cell lines are non-tumorigenic and exhibit anchorage-dependent growth. These cell lines may provide a possible cell model system for studying the mechanisms involved in the tumorigenesis of NPC.