A single amino acid of human immunodeficiency virus type 2 capsid protein affects conformation of two external loops and viral sensitivity to TRIM5α

We previously reported that human immunodeficiency virus type 2 (HIV-2) carrying alanine or glutamine but not proline at position 120 of the capsid protein (CA) could grow in the presence of anti-viral factor TRIM5α of cynomolgus monkey (CM). To elucidate details of the interaction between the CA and TRIM5α, we generated mutant HIV-2 viruses, each carrying one of the remaining 17 possible amino acid residues, and examined their sensitivity to CM TRIM5α-mediated restriction. Results showed that hydrophobic residues or those with ring structures were associated with sensitivity, while those with small side chains or amide groups conferred resistance. Molecular dynamics simulation study revealed a structural basis for the differential TRIM5α sensitivities. The mutations at position 120 in the loop between helices 6 and 7 (L6/7) affected conformation of the neighboring loop between helices 4 and 5 (L4/5), and sensitive viruses had a common L4/5 conformation. In addition, the common L4/5 structures of the sensitive viruses were associated with a decreased probability of hydrogen bond formation between the 97th aspartic acid in L4/5 and the 119th arginine in L6/7. When we introduced aspartic acid-to-alanine substitution at position 97 (D97A) of the resistant virus carrying glutamine at position 120 to disrupt hydrogen bond formation, the resultant virus became moderately sensitive. Interestingly, the virus carrying glutamic acid at position 120 showed resistance, while its predicted L4/5 conformation was similar to those of sensitive viruses. The D97A substitution failed to alter the resistance of this particular virus, indicating that the 120th amino acid residue itself is also involved in sensitivity regardless of the L4/5 conformation. These results suggested that a hydrogen bond between the L4/5 and L6/7 modulates the overall structure of the exposed surface of the CA, but the amino acid residue at position 120 is also directly involved in CM TRIM5α recognition.     

Microbial Production of l-Glutamic Acid by Glycerol Auxotrophs

To establish a novel process for the production of l-glutamic acid from n-paraffins, a glycerol auxotroph GL-21, a new type mutant, was successfully obtained from Corynebacterium alkanolyticum No. 314 by treatment with N-methyl-N′-nitro-N-nitrosoguanidine. This auxotroph required glycerol for its growth regardless of the carbon source used.

At 72 hr, this mutant GL-21 produced about 40 mg/ml of l-glutamic acid from n-paraffins in the culture broth at 0.01 per cent addition of glycerol in the absence of penicillin.

A thiamine auxotroph, a biotin auxotroph and an oleic acid auxotroph were also obtained by a similar technique, but these auxotrophs were found to be inapplicable for the production of l-glutamic acid from n-paraffins.     

Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase

Background

Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C₂-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK _a values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis.

Methodology/Principal Findings

Using His-HDX-MS, the pK _a values and the half-lives (t _1/2) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP⁺ (DHFR-folate-NADP⁺) were determined. The results showed that the two parameters (pK _a and t _1/2) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK _a, t _1/2 or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK _a and t _1/2 changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP⁺. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings.

Conclusions/Significance

Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.     

Effects of grandinol and related phloroglucinol derivatives on transpiration and stomatal closure

Grandinol, an inhibitor of seed germination and photosynthesis in Eucalyptus sp., inhibits transpiration and stomatal opening. The acylphloroglucinol structure in grandinol seemed to be essential for these activities. Enhancement of activity was achieved by the introduction of a formyl group into the molecule. Therefore, structural requirements for these activities were very similar to that for the inhibition of seed germination and photosynthesis. Other grandinol-related compounds having two electron-withdrawing groups on the phloroglucinol nuclei were also active in these assays.     

Characterization of a Human Glycoprotein with a Potential Role in Sperm–Egg Fusion: cDNA Cloning, Immunohistochemical Localization, and Chromosomal Assignment of the Gene (AEGL1)

Acidic epididymal glycoprotein (AEG), thus far identified only in rodents, is one of the sperm surface proteins involved in the fusion of the sperm and egg plasma membranes. In the present study, we describe the isolation and characterization of cDNA encoding a human glycoprotein related to AEG. Although this protein, designated ARP (AEG-related protein), is not the ortholog of rodent AEG, it resembles AEG in that it is an epididymal secretory glycoprotein that binds to the postacrosomal region of the sperm head. The fact that noAEGmRNA can be detected in the human epididymis suggests that ARP might be the functional counterpart of rodent AEG. The gene encoding ARP (AEGL1) was mapped by fluorescencein situhybridization to 6p21.1–p21.2. This result indicates thatAEGL1and the mouse gene for AEG are located in the chromosomal segments with conserved syntenies.     

Inbreeding and true seed in tetrasomic potato. I. Selfing and open pollination in Andean landraces (Solanum tuberosum Gp. Andigena)

 True potato seed (TPS) may be an alternative method of potato production in developing countries. A breeding method for the sexual propagation of this vegetatively propagated crop should consider the development of parental lines and the type of cultivar to be released. Open-pollinated (OP) cultivars seem to be an inexpensive procedure to produce potato from true seed. However, OP progenies are the result of selfing and outcrossing in male-fertile tetraploid potatoes. The aim of the present research was to establish the effect of inbreeding and open pollination in TPS. Ten Andigena clones were used as parental material to derive hybrid (S₀), inbred (S₁ and S₂), and open-pollinated (OP₁ and OP₂) generations. Significant differences among generations were found for pollen production, pollen viability (as determined by its stainability with aceto-carmine glycerol), number of flowers and berries plant^-1, number of seeds berry^-1, weight of 1000 seeds, and tuber yield plant^-1. The parental populations were significantly different for most of the traits, but not for flower production and berry weight. The interaction of population ×generation was significant for pollen and seed production as well as for weight for 1000 seeds. All the traits evaluated except seed weight showed a strong inbreeding depression, while the OP progenies had intermediate values between the S₀ and the S₁. This demonstrates that open pollination in potatoes is not exclusively the product of selfing; it also results from outcrossing. Received: 10 November 1997 / Accepted: 3 March 1998     

Molecular characterization of the Ran-binding zinc finger domain of Nup153

The nuclear pore complex is the gateway for selective traffic between the nucleus and cytoplasm. To learn how building blocks of the pore can create specific docking sites for transport receptors and regulatory factors, we have studied a zinc finger module present in multiple copies within the nuclear pores of higher eukaryotes. All four zinc fingers of human Nup153 were found to bind the small GTPase Ran with dissociation constants ranging between 5 and 40 mum. In addition a fragment of Nup153 encompassing the four tandem zinc fingers was found to bind Ran with similar affinity. NMR structural studies revealed that a representative Nup153 zinc finger adopts the same zinc ribbon structure as the previously characterized Npl4 NZF module. Ran binding was mediated by a three-amino acid motif (Leu(13)/Val(14)/Asn(25)) located within the two zinc coordination loops. Nup153 ZnFs bound GDP and GTP forms of Ran with similar affinities, indicating that this interaction is not influenced by a nucleotide-dependent conformational switch. Taken together, these studies elucidate the Ran-binding interface on Nup153 and, more broadly, provide insight into the versatility of this zinc finger binding module.     

Clonal sublines of rat neurotumor RT4 and cell differentiation: I. Isolation and characterization of cell lines and cell type conversion

A neurotumor of a peripheral nerve origin, RT4, was induced by subcutaneous injection of a newborn BDIX strain rat with ethylnitrosourea. The tumor cells were adapted to cell culture and, after about 2 months, were found to consist of cells displaying four distinct morphologies (AC, B, D, and E). Clonal lines of the four cell types were established. Their morphological stability suggested that there was a stem cell type (AC) which differentiated into other types of cells (B, D, and E). Presumptive stem cell lines were established from single cells. In 10 of the 12 clonal stem cell lines, colonies of differentiated cells appeared in approximately 40 days. One stem cell culture was maintained for 55 days, and all three differentiated cell types resulted in the same culture. The differentiated cells are morphologically indistinguishable from the cells isolated from the original tumor. We are thus able to demonstrate a stem cell in the RT4 neurotumor apparently capable of multipotential differentiation in vitro. We call this phenomenon cell type conversion. The stem cells (AC) and one type (D) of the differentiated cells produce a nervous system specific protein, S100 protein, while its production is arrested when the stem cells differentiate into two other cell types (B and E). No appreciable levels of neurotransmitter synthesizing enzymes (choline acetyltransferase, tyrosine hydroxylase, and glutamic acid decarboxylase) are detected in any cells. The chromosome number of each line is predominantly that of normal diploid rat cells, which is 42.     

rAAV-mediated shRNA ameliorated neuropathology in Huntington disease model mouse

Huntington disease (HD) is a fatal progressive neurodegenerative disorder associated with expansion of a CAG repeat in the first exon of the gene coding the protein huntingtin (htt). Although the feasibility of RNA interference (RNAi)-mediated reduction of htt expression to attenuate HD-associated symptoms is suggested, the effects of post-symptomatic RNAi treatment in the HD model mice have not yet been certified. Here we show the effects of recombinant adeno-associated virus (rAAV)-mediated delivery of RNAi into the HD model mouse striatum after the onset of disease. Neuropathological abnormalities associated with HD, such as insoluble protein accumulation and down-regulation of DARPP-32 expression, were successfully ameliorated by the RNAi transduction. Importantly, neuronal aggregates in the striatum were reduced after RNAi transduction in the animals comparing to those at the time point of RNAi transduction. These results suggest that the direct inhibition of mutant gene expression by rAVV would be promising for post-symptomatic HD therapy.