Estrogen induces thymic atrophy by eliminating early thymic progenitors and inhibiting proliferation of beta-selected thymocytes

Although it has been established that high levels of estrogen can induce thymic involution, the mechanism by which this happens is not known. We have found that daily i.p. injections of the synthetic estrogen 17-beta-estradiol reduce thymus cellularity by 80% over a period of 4-6 days. Although the atrophy is most strikingly observed in the CD4/CD8 double-positive (DP) thymic subset, the loss of thymocytes is not accompanied by a significant increase in thymocyte apoptosis, suggesting that direct killing of cells may not be the dominant means by which estrogens induce thymic atrophy. Instead, we find that estradiol drastically reduces the lineage-negative, Flt3(+)Sca-1(+)c-Kit(+) population in the bone marrow, a population that contains thymic homing progenitors. Within the thymus, we observe that estradiol treatment results in a preferential depletion of early thymic progenitors. In addition, we find that estradiol leads to a significant reduction in the proliferation of thymocytes responding to pre-TCR signals. Reduced proliferation of DN3 and DN4 cell subsets is likely the major contributor to the reduction in DP thymocytes that is observed. The reduction in early thymic progenitors is also likely to contribute to thymic atrophy, as we show that estradiol treatment can reduce the size of Rag1-deficient thymuses, which lack pre-TCR signals and DP thymocytes.     

Regulation of the xylan-degrading apparatus of Cellvibrio japonicus by a novel two-component system

The microbial degradation of lignocellulose biomass is not only an important biological process but is of increasing industrial significance in the bioenergy sector. The mechanism by which the plant cell wall, an insoluble composite structure, activates the extensive repertoire of microbial hydrolytic enzymes required to catalyze its degradation is poorly understood. Here we have used a transposon mutagenesis strategy to identify a genetic locus, consisting of two genes that modulate the expression of xylan side chain-degrading enzymes in the saprophytic bacterium Cellvibrio japonicus. Significantly, the locus encodes a two-component signaling system, designated AbfS (sensor histidine kinase) and AbfR (response regulator). The AbfR/S two-component system is required to activate the expression of the suite of enzymes that remove the numerous side chains from xylan, but not the xylanases that hydrolyze the beta1,4-linked xylose polymeric backbone of this polysaccharide. Studies on the recombinant sensor domain of AbfS (AbfS(SD)) showed that it bound to decorated xylans and arabinoxylo-oligosaccharides, but not to undecorated xylo-oligosaccharides or other plant structural polysaccharides/oligosaccharides. The crystal structure of AbfS(SD) was determined to a resolution of 2.6A(.) The overall fold of AbfS(SD) is that of a classical Per Arndt Sim domain with a central antiparallel four-stranded beta-sheet flanked by alpha-helices. Our data expand the number of molecules known to bind to the sensor domain of two-component histidine kinases to include complex carbohydrates. The biological rationale for a regulatory system that induces enzymes that remove the side chains of xylan, but not the hydrolases that cleave the backbone of the polysaccharide, is discussed.     

Bovine papilloma virus plasmids replicate randomly in mouse fibroblasts throughout S phase of the cell cycle

Bovine papilloma virus (BPV) replicates as a multicopy nuclear plasmid in mouse fibroblasts. Using fluorescence activated cell sorting and mitotic selection procedures, we show that the replication of BPV occurs throughout S phase of the cell cycle and that replication is confined to S phase. After one round of chromosomal DNA replication, almost one quarter of BPV plasmids have replicated more than once, while a similar number of plasmids have not replicated at all. While multiple forms of BPV exist in the cell, all forms show the same pattern of replication. These results are consistent with a model in which BPV plasmids are chosen at random for replication throughout, and only during, S phase and support the view that the completion of S phase is a specifically activated event in the cell cycle rather than simply the end of one round of chromosomal DNA replication.     

Risks of Avian Influenza Transmission in Areas of Intensive Free-Ranging Duck Production with Wild Waterfowl

For decades, southern China has been considered to be an important source for emerging influenza viruses since key hosts live together in high densities in areas with intensive agriculture. However, the underlying conditions of emergence and spread of avian influenza viruses (AIV) have not been studied in detail, particularly the complex spatiotemporal interplay of viral transmission between wild and domestic ducks, two major actors of AIV epidemiology. In this synthesis, we examine the risks of avian influenza spread in Poyang Lake, an area of intensive free-ranging duck production and large numbers of wild waterfowl. Our synthesis shows that farming of free-grazing domestic ducks is intensive in this area and synchronized with wild duck migration. The presence of juvenile domestic ducks in harvested paddy fields prior to the arrival and departure of migrant ducks in the same fields may amplify the risk of AIV circulation and facilitate the transmission between wild and domestic populations. We provide evidence associating wild ducks migration with the spread of H5N1 in the spring of 2008 from southern China to South Korea, Russia, and Japan, supported by documented wild duck movements and phylogenetic analyses of highly pathogenic avian influenza H5N1 sequences. We suggest that prevention measures based on a modification of agricultural practices may be implemented in these areas to reduce the intensity of AIV transmission between wild and domestic ducks. This would require involving all local stakeholders to discuss feasible and acceptable solutions.     

Synthesis and degradation of phenylalanine ammonia-lyase of Rhodosporidium toruloides.

The regulation of the enzyme phenylalanine ammonia-lyase (PAL), which is of potential use in oral treatment of phenylketonuria, was investigated. Antiserum against PAL was prepared and was shown to be monospecific for the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme and two inactive mutant forms of the enzyme were purified to homogeneity by immunoaffinity chromatography, using anti-PAL immunoglobulin G-Sepharose 4B. Both mutant enzymes contained intact prosthetic groups. The formation of PAL catalytic activity after phenylalanine was added to yeast cultures was paralleled by the appearance of enzyme antigen. During induction, uptake of [3H]leucine into the enzyme was higher than uptake into total protein. Our results are consistent with de novo synthesis of an enzyme induced by phenylalanine, rather than activation of a proenzyme. The half-lives of PAL and total protein were similar in both exponential and stationary phase cultures. No metabolite tested affected the rate of enzyme degradation. Glucose repressed enzyme synthesis, whereas ammonia reduced phenylalanine uptake and pool size and so may repress enzyme synthesis through inducer exclusion. The synthesis of enzyme antigen by a mutant unable to metabolize phenylalanine indicated that this amino acid is the physiological inducer of the enzyme.     

Optimizing clinical dosing of combination broadly neutralizing antibodies for HIV prevention

Broadly neutralizing antibodies (bNAbs) are promising agents to prevent HIV infection and achieve HIV remission without antiretroviral therapy (ART). As with ART, bNAb combinations are likely needed to cover HIV’s extensive diversity. Not all bNAbs are identical in terms of their breadth, potency, and in vivo longevity (half-life). Given these differences, it is important to optimally select the composition, or dose ratio, of combination bNAb therapies for future clinical studies. We developed a model that synthesizes 1) pharmacokinetics, 2) potency against a wide HIV diversity, 3) interaction models for how drugs work together, and 4) correlates that translate in vitro potency to clinical protection. We found optimization requires drug-specific balances between potency, longevity, and interaction type. As an example, tradeoffs between longevity and potency are shown by comparing a combination therapy to a bi-specific antibody (a single protein merging both bNAbs) that takes the better potency but the worse longevity of the two components. Then, we illustrate a realistic dose ratio optimization of a triple combination of VRC07, 3BNC117, and 10–1074 bNAbs. We apply protection estimates derived from both a non-human primate (NHP) challenge study meta-analysis and the human antibody mediated prevention (AMP) trials. In both cases, we find a 2:1:1 dose emphasizing VRC07 is nearly optimal. Our approach can be immediately applied to optimize the next generation of combination antibody prevention and cure studies.     

Binocular interactions of motion detection fibers in the optic lobes of flies

Zusammenfassung Nervenfasern, die die optischen Ganglien beider Kopfseiten von Musca und Phaenicia verbinden und die selektiv auf Musterbewegungen antworten, zeigen binoculare Wechselwirkungen. Details dieser Wechselwirkungen und der Ort an dem sie in den optischen Ganglien stattfinden wurden ermittelt. Die Bedeutung dieser Einheiten für die direkte Steuerung von Wendereaktionen beim Flug wurde ebenfalls untersucht.

---

---

This research was supported by the National Institutes Health, United States Public Health Service Grant NB 03627.     

Specificity of phosphatidylserine-containing membrane binding sites for factor VIII. Studies with model membranes supported by glass microspheres (lipospheres).

Factor VIII functions in an enzyme complex upon the activated platelet membrane where phosphatidylserine exposure correlates with expression of receptors for factor VIII. To evaluate the specificity of phosphatidylserine-containing membrane binding sites for factor VIII, we have developed a novel membrane model in which phospholipid bilayers are supported by glass microspheres (lipospheres). The binding of fluorescein-labeled factor VIII to lipospheres with membranes of 15% phosphatidylserine was equivalent to binding to phospholipid vesicles (KD = 4.8 nM). Purified von Willebrand factor (vWf), a carrier protein for factor VIII, decreased membrane binding of factor VIII with a Ki of 10 micrograms/ml. Likewise, normal plasma decreased bound factor VIII by more than 90% whereas plasma lacking vWf decreased the binding of factor VIII by only 20%. Proteolytic activation of factor VIII by thrombin, which releases factor VIII from vWf, increased liposphere binding in the presence of vWf and in the presence of normal plasma. Although factor V is homologous to factor VIII and binds to lipospheres with the same affinity, purified factor V was not an efficient competitor for the membrane binding sites of factor VIII. These results indicate that phosphatidylserine-containing membrane sites have sufficient specificity to select thrombin-activated factor VIII from the range of phospholipid-binding proteins in plasma.     

Lactate dehydrogenase in preimplantation rabbit embryos

Unfertilized eggs and early embryos up to the 2-day (16-cell) cleavage stage of development in the rabbit contain predominantly the most cathodal lactate dehydrogenase isoenzyme made up of A-type subunits. Following early cleavage there is a progressing increase in total LDH activity in the embryo as development proceeds through 4- and 6-day blastocyst stages. This is accompanied by an increase in the amount of B-type subunits and a concomitant shift in the lactate dehydrogenase isoenzyme electrophoretic pattern toward the anodal isoenzyme types.