Purification and Characterization of Two Dihydroxyacetone Kinases from Schizosaccharomyces pombe IFO 0354

Two dihydroxyacetone kinases (DHAKs), DHAK I and DHAK II, were purified to homogeneity from Schizosaccharomyces pombe IFO 0354. They were immunologically different from each other. Although both of the enzymes had some affinity for glycerol and dl-glyceraldehyde in addition to dihydroxyacetone and glyceraldehyde, V(infmax) values for dihydroxyacetone were much higher than those for glycerol and dl-glyceraldehyde. On the basis of the K(infm) values of both enzymes for dihydroxyacetone, DHAK II plays a more important role than DHAK I in dissimilation of glycerol via dihydroxyacetone.     

Fecal microbiota of a dugong (Dugong dugong) in captivity at Toba Aquarium

A captive female dugong at Toba Aquarium (Japan) was examined to describe the microbiota of its lower digestive tracts using the molecular-biological technique, a culture-independent method. The phylogenetic analysis of bacterial 16S rRNA genes was conducted for fecal samples, which were taken at 3 different periods. Based on phylogenetic analysis of these sequences, the representatives of six bacterial phyla could be identified: Actinobacteria (0.7%), Bacteroidetes (15%), Firmicutes (83.1%), Lentisphaerae (0.1%), Proteobacteria (0.1%), and Verrucomicrobia (1.0%), suggesting the existence of bacterial species newly found not only in the digestive tract but also in natural field.     

Human Immunodeficiency Virus Type 1 Elite Neutralizers: Individuals with Broad and Potent Neutralizing Activity Identified by Using a High-Throughput Neutralization Assay together with an Analytical Selection Algorithm

The development of a rapid and efficient system to identify human immunodeficiency virus type 1 (HIV-1)-infected individuals with broad and potent HIV-1-specific neutralizing antibody responses is an important step toward the discovery of critical neutralization targets for rational AIDS vaccine design. In this study, samples from HIV-1-infected volunteers from diverse epidemiological regions were screened for neutralization responses using pseudovirus panels composed of clades A, B, C, and D and circulating recombinant forms (CRFs). Initially, 463 serum and plasma samples from Australia, Rwanda, Uganda, the United Kingdom, and Zambia were screened to explore neutralization patterns and selection ranking algorithms. Samples were identified that neutralized representative isolates from at least four clade/CRF groups with titers above prespecified thresholds and ranked based on a weighted average of their log-transformed neutralization titers. Linear regression methods selected a five-pseudovirus subset, representing clades A, B, and C and one CRF01_AE, that could identify top-ranking samples with 50% inhibitory concentration (IC₅₀) neutralization titers of ≥100 to multiple isolates within at least four clade groups. This reduced panel was then used to screen 1,234 new samples from the Ivory Coast, Kenya, South Africa, Thailand, and the United States, and 1% were identified as elite neutralizers. Elite activity is defined as the ability to neutralize, on average, more than one pseudovirus at an IC₅₀ titer of 300 within a clade group and across at least four clade groups. These elite neutralizers provide promising starting material for the isolation of broadly neutralizing monoclonal antibodies to assist in HIV-1 vaccine design.Since the identification of human immunodeficiency virus type 1 (HIV-1) as the cause of AIDS, one of the greatest challenges has been the development of a vaccine that will prevent infection and/or ameliorate disease progression (38, 43). Although over 100 phase I, II, and III vaccine clinical trials of different candidates have been conducted all over the world, only a few candidates have advanced to efficacy testing and none has yet to show any benefit in prevention or control of HIV-1 (HIV Vaccine Database; www.iavi.org). In other viral diseases (such as polio, influenza, and measles), neutralizing antibodies are generated as part of either the natural immune response to infection or the response to immunization, and their role in protective immunity is well established (10, 12, 15, 22, 37, 42, 45, 47, 49, 52). For HIV-1, studies in animal models indicate that both broadly neutralizing antibodies and cell-mediated responses may be required to provide vaccine protection (7, 14, 16, 20, 29, 31, 33, 34, 39, 53). Unlike many other viruses, HIV-1 is highly variable, with multiple subtypes and recombinant forms circulating in different regions of the world. This high level of HIV-1 genetic variability, particularly in the envelope glycoproteins (gp120 and gp41), has been one of the greatest obstacles in development of a safe and effective HIV-1 vaccine and in particular in the elicitation of broadly neutralizing antibodies. In addition, HIV-1 has other mechanisms of immune escape preventing elicitation of broadly neutralizing antibodies, including the heavy glycosylation of the envelope glycoproteins, instability of such glycoproteins, and conformational masking of receptor-binding sites (6, 25, 32).Despite the enormous diversity of HIV-1, a relatively small number of broadly neutralizing monoclonal antibodies (bnMAbs) have been isolated, providing evidence that broad neutralization by single antibody specificities can be achieved (3-5, 8, 9, 17, 21, 23, 24, 29, 35, 36, 40, 41, 44, 50, 51, 55). Structures for such bnMAbs have been determined in complex with HIV-1 Env (26, 54) and provide starting points for the design of immunogens capable of eliciting broadly neutralizing antibodies. However, since there are only a few such bnMAbs, we established a global program as part of International AIDS Vaccine Initiative''s (IAVI''s) Neutralizing Antibody Consortium (6), aimed at screening HIV-1⁺ subjects with the goal of identifying individuals with broad and potent neutralizing activities as a potential source of novel bnMAbs, with an emphasis placed on individuals infected with non-clade B viruses. This paper describes the screening algorithm implemented to successfully identify HIV-1⁺ subjects with broadly neutralizing antibodies, including a subset of individuals termed “elite neutralizers.” These volunteers will be studied further to characterize the specificities of serum antibodies and will provide source materials for isolation of bnMAbs.     

Redox state of tumor suppressor p53 regulates its sequence-specific DNA binding in DNA-damaged cells by cysteine 277

Using a bio-oligo pull-down DNA-binding assay we investigated the binding capacity of endogenous, DNA damage-induced p53 in human diploid fibroblasts to several p53-responsive elements (REs) present in p53-regulated genes. During the course of p53 accumulation, we observed a decrease in p53 binding to the GADD45 but not to the p21^WAF1/CIP1 RE. Using mutated GADD45 sequences we show that this change is dependent on the presence of cytosines at position 3 in RE pentamers and on the p53 redox state. Site-directed mutagenesis experiments demonstrated that Cys277 (a residue directly contacting base 3 in a RE pentamer) is critical for differential regulation of GADD45 in DNA-damaged cells. These data represent a novel mechanism for differential affinity of p53 to distinct REs.     

Recognition of receptor lipopolysaccharides by spike G protein of bacteriophage phiX174

The spike G protein of bacteriophage phiX174 was prepared as a hexa histidine-tagged G protein (HisG). In the enzyme-linked plate assay, HisG bound specifically to lipopolysaccharides (LPSs) of the phiX174-sensitive strains, and did not bind to LPSs of the phiX174-insensitive strains. The truncated G protein obtained after trypsin digestion of HisG had the similar affinity to the LPSs to HisG, indicating that eight amino acid residues from the N-terminus are not essential to the binding with the LPSs.     

Human leukocyte antigen variants B*44 and B*57 are consistently favorable during two distinct phases of primary HIV-1 infection in sub-Saharan Africans with several viral subtypes

As part of an ongoing study of early human immunodeficiency virus type 1 (HIV-1) infection in sub-Saharan African countries, we have identified 134 seroconverters (SCs) with distinct acute-phase (peak) and early chronic-phase (set-point) viremias. SCs with class I human leukocyte antigen (HLA) variants B*44 and B*57 had much lower peak viral loads (VLs) than SCs without these variants (adjusted linear regression beta values of -1.08 ± 0.26 log(10) [mean ± standard error] and -0.83 ± 0.27 log(10), respectively; P < 0.005 for both), after accounting for several nongenetic factors, including gender, age at estimated date of infection, duration of infection, and country of origin. These findings were confirmed by alternative models in which major viral subtypes (A1, C, and others) in the same SCs replaced country of origin as a covariate (P ≤ 0.03). Both B*44 and B*57 were also highly favorable (P ≤ 0.03) in analyses of set-point VLs. Moreover, B*44 was associated with relatively high CD4(+) T-cell counts during early chronic infection (P = 0.02). Thus, at least two common HLA-B variants showed strong influences on acute-phase as well as early chronic-phase VL, regardless of the infecting viral subtype. If confirmed, the identification of B*44 as another favorable marker in primary HIV-1 infection should help dissect mechanisms of early immune protection against HIV-1 infection.     

Characterization of lignocellulose particles during lignocellulose solubilization by Clostridium thermocellum

Clostridium thermocellum is a candidate bacterium for lignocellulose utilization due to its efficient lignocellulose solubilization ability. It has been reported that C. thermocellum efficiently degrades purified cellulose substrates, but cannot completely degrade milled lignocellulose powders. Evaluation of cellulose and hemicellulose contents in a lignocellulose residue after the cultivation of C. thermocellum indicated that C. thermocellum degraded cellulose and hemicellulose equally. Microscopic observations demonstrated that C. thermocellum significantly degraded small-sized lignocellulose particles, but it only partially degraded the larger sized particles. The lignin content of the large-sized particles was higher than that of the small particles. The remained large-sized particles included vascular tissues. These results suggest that the lignified structures such as vascular tissues in milled lignocellulose were less susceptible to bacterial lignocellulose solubilization.     

Kinetically trapped metastable intermediate of a disulfide‐deficient mutant of the starch‐binding domain of glucoamylase

Refolding of a thermally unfolded disulfide‐deficient mutant of the starch‐binding domain of glucoamylase was investigated using differential scanning calorimetry, isothermal titration calorimetry, CD, and ¹H NMR. When the protein solution was rapidly cooled from a higher temperature, a kinetic intermediate was formed during refolding. The intermediate was unexpectedly stable compared with typical folding intermediates that have short half‐lives. It was shown that this intermediate contained substantial secondary structure and tertiary packing and had the same binding ability with β‐cyclodextrin as the native state, suggesting that the intermediate is highly‐ordered and native‐like on the whole. These characteristics differ from those of partially folded intermediates such as molten globule states. Far‐UV CD spectra showed that the secondary structure was once disrupted during the transition from the intermediate to the native state. These results suggest that the intermediate could be an off‐pathway type, possibly a misfolded state, that has to undergo unfolding on its way to the native state.