Emergence of viruses resistant to neutralization by V3-specific antibodies in experimental human immunodeficiency virus type 1 IIIB infection of chimpanzees.

Emergence in two chimpanzees of human immunodeficiency virus type 1 (HIV-1) IIIB variants resistant to neutralization by the preexisting antibody is described. Viruses isolated from the HIV-1 IIIB gp120-vaccinated and -challenged animal were more resistant to neutralization by the chimpanzee's own serum than viruses isolated from the naive infected animal, indicating immune pressure as the selective mechanism. However, all reisolated viruses were 16- to 256-fold more neutralization resistant than the inoculum virus to antibodies binding to the third variable domain (V3) of the HIV-1 external envelope. Early chimpanzee serum samples that neutralized the inoculum strain but not the reisolated viruses were found to bind an HIV-1 IIIB common nonapeptide (IQRGPGRAF) derived from the gp120 isolate-specific V3 domain shown to induce isolate-specific neutralization in other animals. Amplification of the V3 coding sequence by polymerase chain reaction and subsequent sequence analysis of the neutralization-resistant variants obtained from in vivo-infected animals indicated that early resistance to neutralization by an HIV-1 IIIB monoclonal antibody (0.5 beta) was conferred by changes outside the direct binding site for the selective neutralizing antibody. The reisolated neutralization-resistant isolates consisted of the lower-replication-competent virus subpopulations of the HIV-1 IIIB stock, as confirmed by biological and sequence analyses. In vitro passage of the HIV-1 IIIB stock through chimpanzee and human peripheral blood mononuclear cell cultures void of HIV-specific antibody resulted in homogenic amplification of the more-replication-competent subpopulation preexisting in the original viral stock, suggesting a role for the immune system in suppressing the more-replication-competent viruses.     

Carbonic anhydrase activity in leaves and its role in the first step of c(4) photosynthesis

In C₄ plants carbonic anhydrase catalyzes the critical first step of C₄ photosynthesis, the hydration of CO₂ to bicarbonate. The maximum activity of this enzyme in C₄ leaf extracts, measured by H⁺ production with saturating CO₂ and extrapolated to 25°C, was found to be 3,000 to 10,000 times the maximum photosynthesis rate for these leaves. Similar activities were found in C₃ leaf extracts. However, the calculated effective activity of this enzyme at in vivo CO₂ concentrations was apparently just sufficient to prevent the rate of conversion of CO₂ to HCO₃⁻ from limiting C₄ photosynthesis. This conclusion was supported by the mass spectrometric determination of leaf carbonic anhydrase activities.     

Photosynthesis in phosphoenolpyruvate carboxykinase-type C4 plants: photosynthetic activities of isolated bundle sheath cells from Urochloa panicoides

A method has been developed for rapidly preparing bundle sheath cell strands from Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase-type C4 plant. These cells catalyzed both HCO3(-)- and oxaloacetate-dependent oxygen evolution; oxaloacetate-dependent oxygen evolution was stimulated by ATP. For this activity oxaloacetate could be replaced by aspartate plus 2-oxoglutarate. Both oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution were accompanied by PEP production and both were inhibited by 3-mercaptopicolinic acid, an inhibitor of PEP carboxykinase. The ATP requirement for oxaloacetate- and aspartate plus 2-oxoglutarate-dependent oxygen evolution could be replaced by ADP plus malate. The increased oxygen evolution observed when malate plus ADP was added with oxaloacetate was accompanied by pyruvate production. These results are consistent with oxaloacetate being decarboxylated via PEP carboxykinase. We suggest that the ATP required for oxaloacetate decarboxylation via PEP carboxykinase may be derived by phosphorylation coupled to malate oxidation in mitochondria. These bundle sheath cells apparently contain diffusion paths for the rapid transfer of compounds as large as adenine nucleotides.     

Purified envelope glycoproteins from human immunodeficiency virus type 1 variants induce individual, type-specific neutralizing antibodies.

Repeated immunizations of goats, horses, or chimpanzees with envelope glycoprotein gp120 isolated from human immunodeficiency virus type 1 (HIV-1) resulted in type-specific neutralizing-antibody responses, which began to decay approximately 20 days following the administration of antigen. This was true repeatedly for serum samples from animals hyperimmunized with gp120s from either the HTLV-IIIB (IIIB) or the envelope-divergent HTLV-IIIRF (RF) HIV-1 isolates. Animals previously immunized with the IIIB gp120 were then inoculated with purified RF gp120. The first response in these animals was an anamnestic resurgence of neutralizing antibody to IIIB without detectable neutralizing antibody for RF. However, with later RF gp120 boosts, the IIIB neutralizing-antibody titers fell and an RF type-specific neutralizing-antibody response developed. When assessed with other HIV-1 variants, no group-specific neutralizing antibody was seen in any of the vaccination protocols evaluated. These results will pose real obstacles in the development of an effective vaccine for HIV.     

Aberrant cardiolipin metabolism is associated with cognitive deficiency and hippocampal alteration in tafazzin knockdown mice

Cardiolipin (CL) is a key mitochondrial phospholipid essential for mitochondrial energy production. CL is remodeled from monolysocardiolipin (MLCL) by the enzyme tafazzin (TAZ). Loss-of-function mutations in the gene which encodes TAZ results in a rare X-linked disorder called Barth Syndrome (BTHS). The mutated TAZ is unable to maintain the physiological CL:MLCL ratio, thus reducing CL levels and affecting mitochondrial function. BTHS is best known as a cardiac disease, but has been acknowledged as a multi-syndrome disorder, including cognitive deficits. Since reduced CL levels has also been reported in numerous neurodegenerative disorders, we examined how TAZ-deficiency impacts cognitive abilities, brain mitochondrial respiration and the function of hippocampal neurons and glia in TAZ knockdown (TAZ kd) mice. We have identified for the first time the profile of changes that occur in brain phospholipid content and composition of TAZ kd mice. The brain of TAZ kd mice exhibited reduced TAZ protein expression, reduced total CL levels and a 19-fold accumulation of MLCL compared to wild-type littermate controls. TAZ kd brain exhibited a markedly distinct profile of CL and MLCL molecular species. In mitochondria, the activity of complex I was significantly elevated in the monomeric and supercomplex forms with TAZ-deficiency. This corresponded with elevated mitochondrial state I respiration and attenuated spare capacity. Furthermore, the production of reactive oxygen species was significantly elevated in TAZ kd brain mitochondria. While motor function remained normal in TAZ kd mice, they showed significant memory deficiency based on novel object recognition test. These results correlated with reduced synaptophysin protein levels and derangement of the neuronal CA1 layer in hippocampus. Finally, TAZ kd mice had elevated activation of brain immune cells, microglia compared to littermate controls. Collectively, our findings demonstrate that TAZ-mediated remodeling of CL contributes significantly to the expansive distribution of CL molecular species in the brain, plays a key role in mitochondria respiratory activity, maintains normal cognitive function, and identifies the hippocampus as a potential therapeutic target for BTHS.     

The coupling of the relative movement of thea andc subunits of the F0 to the conformational changes in the F1-ATPase

F₀F₁-ATPase structural information gained from X-ray crystallography and electron microscopy has activated interest in a rotational mechanism for the F₀F₁-ATPase. Because of the subunit stoichiometry and the involvement of both thea- andc-subunits in the mechanism of proton movement, it is argued that relative movement must occur between the subunits. Various options for the arrangement and structure of the subunits involved are discussed and a mechanism proposed.     

Mineralization of nitrogen in long-term pasture soils: effects of management

A field incubation technique with acetylene to inhibit nitrification was used to estimate net N mineralization rates in some grassland soils through an annual cycle. Measurements were made on previously long-term grazed pastures on a silty clay loam soil in S.W. England which had background managements of +/– drainage and +/– fertilizer (200 kg N ha^–1 yr^–1). The effect of fertilizer addition on mineralization during the year of measurement was also determined. Small plots with animals excluded, and with herbage clipped and removed were used as treatment areas and measurements were made using an incubation period of 7 days at intervals of 7 or 14 days through the year. Soil temperature, moisture and mineral N contents were also determined. Mineralization rates fluctuated considerably in each treatment. Maximum daily rates ranged from 1.01 to 3.19 kg N ha^–1, and there was substantial net release of N through the winter period (representing, on average, 27% of the annual release). Changes in temperature accounted for 35% of the variability but there was little significant effect of soil moisture. Annual net release of N ranged from 135 kg ha^–1 (undrained soil, no previous or current fertilizer) to 376 (drained soil, +200 kg N ha^–1 yr^–1 previous and current fertilizer addition). Addition of fertilizer N to a previously unfertilized sward significantly increased the net release of N but there was no immediate effect of withholding fertilizer on mineralization during the year in which measurements were made.     

Use of spectral analysis to test hypotheses on the origin of pinnipeds

The evolutionary origin of the pinnipeds (seals, sea lions, and walruses) is still uncertain. Most authors support a hypothesis of a monophyletic origin of the pinnipeds from a caniform carnivore. A minority view suggests a diphyletic origin with true seals being related to the mustelids (otters and ferrets). The phylogenetic relationships of the walrus to other pinniped and carnivore families are also still particularly problematic. Here we examined the relative support for mono- and diphyletic hypotheses using DNA sequence data from the mitochondrial small subunit (12S) rRNA and cytochrome b genes. We first analyzed a small group of taxa representing the three pinniped families (Phocidae, Otariidae, and Odobenidae) and caniform carnivore families thought to be related to them. We inferred phylogenetic reconstructions from DNA sequence data using standard parsimony and neighbor-joining algorithms for phylogenetic inference as well as a new method called spectral analysis (Hendy and Penny) in which phylogenetic information is displayed independently of any selected tree. We identified and compensated for potential sources of error known to lead to selection of incorrect phylogenetic trees. These include sampling error, unequal evolutionary rates on lineages, unequal nucleotide composition among lineages, unequal rates of change at different sites, and inappropriate tree selection criteria. To correct for these errors, we performed additional transformations of the observed substitution patterns in the sequence data, applied more stringent structural constraints to the analyses, and included several additional taxa to help resolve long, unbranched lineages in the tree. We find that there is strong support for a monophyletic origin of the pinnipeds from within the caniform carnivores, close to the bear/raccoon/panda radiation. Evidence for a diphyletic origin was very weak and can be partially attributed to unequal nucleotide compositions among the taxa analyzed. Subsequently, there is slightly more evidence for grouping the walrus with the eared seals versus the true seals. A more conservative interpretation, however, is that the walrus is an early, but not the first, independent divergence from the common pinniped ancestor.      

Primary partitioning and storage of photosynthate in sucrose and starch in leaves of C4 plants

A procedure involving pulse labelling of leaves with ¹⁴CO₂ was developed to measure the primary (initial) partitioning of photosynthate between sucrose and starch. Partitioning of photosynthate into sucrose and starch was determined in leaves of C₄ plants and compared with the patterns of storage of carbon in these products during the light period. The ratio of primary partitioning into sucrose and starch varied from about 0.5 in those species that accumulated mostly starch in the leaves (Amaranthus edulis L., Atriplex spongiosa F. Muell. and Flaveria trinervia (Spreng.) C. Mohr) to about 8 in Eleusine indica (L.) Gaertn., which accumulated mostly sucrose. No label was detected in free glucose or fructose. Generally there was a reasonable link between the primary partitioning of photosynthate and the type of carbohydrate stored in the leaf during the day. However, the ratio of carbon initially partitioned into sucrose versus starch was about 3 to 4 times higher in leaves of NADP-malic enzyme-type monocotyledonous species compared with phosphoenolpyruvate carboxykinase-type species, although the ratio of sucrose to starch accumulated in leaves during the day was very similar in the two groups. Sucrose and starch were the principal carbohydrates accumulated in leaves during the day. None of the species examined contained significant amounts of fructan and only one species, Atriplex spongiosa, contained substantial amounts of hexose sugars. In most of the species studied, the proportion of photosynthate partitioned into starch was greater at the end of the day than at the beginning. With the exception of Flaveria trinervia, the rate of CO₂ assimilation did not decline during the day, showing that, under our conditions, accumulation of carbohydrate in the leaves did not lead to feedback inhibition of photosynthesis in these C₄ species.Abbreviations Chl chlorophyll - NAD-ME NAD-malic enzyme - NADP-ME NADP-malic enzyme - PCK phosphoenolpyruvate carboxykinase We thank Prof. H.W. Heldt (Pflanzenphysiologisches Institut, Universität Göttingen) for discussions and advice during the course of this work.     

Relationship between the Genomic Organization and the Overlapping Embryonic Expression Patterns of the ZebrafishdlxGenes

To understand the relationship between the expression and the genomic organization of the zebrafishdlxgenes, we have determined the genomic structure of thedlx2anddlx4loci. This led to the identification of the zebrafishdlx1anddlx6genes, which are closely linked todlx2anddlx4,respectively. Therefore, the inverted convergent configuration ofDlxgenes is conserved among vertebrates. Analysis of the expression patterns ofdlx1anddlx6showed striking similarities to those ofdlx2anddlx4,respectively, the genes to which they are linked. Furthermore, the expression patterns ofdlx3anddlx7,which likely constitute a third pair of convergently transcribed genes, are indistinguishable. Thus, the overlapping expression patterns of linkedDlxgenes during embryonic development suggest that they sharecis-acting sequences that control their spatiotemporal expression. The evolutionary conservation of the genomic organization and combinatorial expression ofDlxgenes in distantly related vertebrates suggest tight control mechanisms that are essential for their function during development.