A modular domain of NifU,a nitrogen fixation cluster protein,is highly conserved in evolution

HnifU, a gene exhibiting similarity tonifU genes of nitrogen fixation gene clusters, was identified in the course of expressed sequence tag (EST) generation from a human fetal heart cDNA library. Northern blot of human tissues and polymerase chain reaction (PCR) using human genomic DNA verified that the hnifU gene represented a human gene rather than a microbial contaminant of the cDNA library. Conceptual translation of the hnifU cDNA yielded a protein product bearing 77% and 70% amino acid identity to NifU-like hypothetical proteins fromHaemophilus influenzae andSaccharomyces cerevisiae, respectively, and 40–44% identity to the N-terminal regions of NifU proteins from several diazatrophs (i.e., nitrogen-fixing organisms). Pairwise determination of amino acid identities between the NifU-like proteins of nondiazatrophs showed that these NifU-like proteins exhibited higher sequence identity to each other (63–77%) than to the diazatrophic NifU proteins (40–48%). Further, the NifU-like proteins of non-nitrogenfixing organisms were similar only to the N-terminal region of diazatrophic NifU proteins and therefore identified a novel modular domain in these NifU proteins. These findings support the hypothesis that NifU is indeed a modular protein. The high degree of sequence similarity between NifU-like proteins from species as divergent as humans andH. influenzae suggests that these proteins perform some basic cellular function and may be among the most highly conserved proteins. Correspondence to: C.-C. Liew     

NMR Studies of Pi-Containing Extracellular and Cytoplasmic Compartments in Brain

Abstract: The inorganic phosphate (P_i) NMR peak in brain has an irregular shape, which suggests that it represents more than a single homogeneous pool of P_i. To test the ability of the Marquardt-Levenberg (M-L) nonlinear curve fit algorithm software (Peak-Fit) to separate multiple peaks, locate peak centers, and estimate peak heights, we studied simulated P_i spectra with defined peak centers, areas, and signal-to-noise (S/N) ratios ranging from ∞ to 5.8. As the S/N ratio decreased below 15, the M-L algorithm located peak centers accurately when they were detected; however, small peaks tended to grow smaller and disappear, whereas the amplitudes of larger peaks increased. We developed an in vitro three-compartment model containing a mixture of P_i buffer, phosphocreatine, phosphate diester, and phosphate monoester (PME), portions of which were adjusted to three different pHs before addition of agar. Weighed samples of each buffered gel together with phospholipid extract and bone chips were placed in an NMR tube and covered with mineral oil. Following baseline correction, it was possible to separate the P_i peaks arising from the three compartments with different pH values if each peak made up 10–35% of total P_i area. In vivo, we identified the plasma compartment by intraarterial infusion of P_i. It was assumed that intracellular compartments contained high-energy phosphates and took up glucose. Based on these assumptions we subjected the brains to complete ischemia and observed that P_i compartments at pH 6.82, 6.92, 7.03, and 7.13 increased markedly in amplitude. If the brain cells took up and phosphorylated 2-deoxyglucose (2-DG), 2-DG-6-phosphate (2-DG-6-P) would appear in the PME portion of the spectrum ionized according to pH_i. Four 2-DG-6-P peaks with calculated pH values of 6.86, 6.94, 7.04, and 7.15 did appear in the spectrum, thereby confirming that the four larger P_i peaks represented intracellular spaces.     

vif-negative human immunodeficiency virus type 1 persistently replicates in primary macrophages, producing attenuated progeny virus.

The vif gene of human immunodeficiency virus type 1 (HIV-1) is required for efficient infection of primary T lymphocytes. In this study, we investigated in detail the role of vif in productive infection of primary monocyte-derived macrophages (MDM). Viruses carrying missense or deletion mutations in vif were constructed on the background of the monocytotropic recombinant NLHXADA-GP. Using MDM from multiple donors, we found that vif mutants produced in complementing or partially complementing cell lines were approximately 10% as infectious as wild-type virus when assayed for incomplete, complete, and circularized viral DNA molecules by quantitative PCR amplification or for viral core antigen p24 production by enzyme-linked immunosorbent assay. We then determined the structure and infectivity of vif mutant HIV-1 by using MDM exclusively both for virus production and as targets for infection. Biosynthetic labeling and immunoprecipitation analysis of sucrose cushion-purified vif-negative HIV-1 made in MDM revealed that the virus had reduced p24 content compared with wild-type HIV-1. Cell-free MDM-derived vif mutant HIV-1 was infectious in macrophages as determined by the synthesis and maintenance of full-length viral DNA and by the produc- tion of particle-associated viral RNA, but its infectivity was approximately 2,500-fold lower than that of wild-type virus whose titer was determined in parallel by measurement of the viral DNA burden. MDM infected with MDM-derived vif-negative HIV-1 were able to transmit the virus to uninfected MDM by cocultivation, confirming the infectiousness of this virus. We conclude that mutations in vif significantly reduce but do not eliminate the capacity of HIV-1 to replicate and produce infectious progeny virus in primary human macrophages.     

ATPase, GTPase, and RNA binding activities associated with the 206-kilodalton protein of turnip yellow mosaic virus.

The 206-kDa protein of turnip yellow mosaic virus belongs to an expanding group of proteins containing a domain which includes the consensus nucleotide binding site GxxxxGKS/T. A portion of this protein (amino acids [aa] 916 to 1259) was expressed in Escherichia coli and purified by affinity chromatography to near homogeneity. In the absence of any other viral factors, it exhibited ATPase and GTPase activities in vitro. A mutant protein with a single amino acid substitution in the consensus nucleotide binding site (Lys-982 to Ser) exhibited only low levels of both activities, implying that Lys-982 is important for nucleoside triphosphatase activity. The protein also possessed nonspecific RNA binding capacity. Deletion mutants revealed that an N-terminal domain (aa 916 to 1061) and a C-terminal domain (aa 1182 to 1259) participate in RNA binding. The results presented here provide the first experimental evidence that turnip yellow mosaic virus encodes nucleoside triphosphatase and RNA binding activities.     

The fate of immunoglobulin G fed to larvae of Ostrinia nubilalis

Since plants can be transformed genetically to produce functional antibodies, an immunological approach may be developed for controlling their arthropod pests. Specific antibodies would protect plants from arthropods if they could gain access to the pest antigen in sufficient amounts such that the normal function of the antigen is disrupted. In order to study the fate of ingested antibodies in the body of the European corn borer (ECB), Ostrinia nubilalis (Hübner), (Lepidoptera: Pyralidae), we fed the larvae on serum-containing diet. When larvae were fed on the serum-containing diet for various lengths of time between 12 and 96 h, no significant differences were noted in the immunoglobulin G (IgG) concentration in their body. Immediately after the larvae stopped feeding, the concentrations of the IgG in their midgut was about one half that of the diet itself, but it decreased significantly after 6 h and again after 18 h (about 3 and 10 fold, respectively). Immediately after the larvae stopped feeding, the concentration of the IgG in their hemolymph was about 1/500 that in the diet. The concentration of IgG in the hemolymph of ECB larvae was influenced directly by the titer of antibodies in their diet. During the first 6 h after the larvae stop feeding the concentration of IgG in their hemolymph did not decrease significantly; however, it did so after 18 h (about 6 fold). The possibility that specific antibodies will gain access to antigens in the ECB body is discussed.     

Effect of larval fish and nutrient enrichment on plankton dynamics in experimental ponds

The hypotheses that larval fish density may potentially affect phytoplankton abundance through regulating zooplankton community structure, and that fish effect may also depend on nutrient levels were tested experimentally in ponds with three densities of larval walleye, Stizostedion vitreum (0, 25, and 50 fish m^–3), and two fertilizer types (inorganic vs organic fertilizer). A significant negative relationship between larval fish density and large zooplankton abundance was observed despite fertilizer types. Larval walleye significantly reduced the abundances of Daphnia, Bosmina, and Diaptomus but enhanced the abundance of various rotifer species (Brachionus, Polyarthra, and Keratella). When fish predation was excluded, Daphnia became dominant, but Daphnia grazing did not significantly suppress blue-green algae. Clearly, larval fish can be an important regulator for zooplankton community. Algal composition and abundance were affected more by fertilizer type than by fish density. Inorganic fertilizer with a high N:P ratio (20:1) enhanced blue-green algal blooms, while organic fertilizer with a lower N:P ratio (10:1) suppressed the abundance of blue-green algae. This result may be attributed to the high density of blue-green algae at the beginning of the experiment and the fertilizer type. Our data suggest that continuous release of nutrients from suspended organic fertilizer at a low rate may discourage the development of blue-green algae. Nutrient inputs at a low N:P ratio do not necessarily result in the dominance of blue-green algae.     

Significant extension in northern Australia of the known geographic range of the Shield Shrimp Triops australiensis (Crustacea: Notostraca)

The program BIOCLIM predicts the total geographic distribution of species, based upon the biogeoclimatic characteristics common to the localities at which they are known to occur. Field studies in the Northern Territory have located the Shield Shrimp Triops australiensis at localities substantially north of its known and predicted geographic distribution.