William F. Loomis

The genome of the social amoeba Dictyostelium discoideum

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal–fungal lineage after the plant–animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

Skin-Pigment Regulation of Vitamin-D Biosynthesis in Man: Variation in solar ultraviolet at different latitudes may have caused racial differentiation in man

The known correlation between the color of human skin and latitude (Fig. 2) is explainable in terms of two opposing positive adaptations to solar ultraviolet radiation, weak in northern latitudes in winter yet powerful the year around near the equator. In northern latitudes there is selection for white skins that allow maximum photoactivation of 7-dehydrocholesterol into vitamin D at low intensities of ultraviolet radiation. In southern latitudes, on the other hand, there is selection for black skins able to prevent up to 95 percent of the incident ultraviolet from reaching the deeper layers of the skin where vitamin D is synthesized. Selection against the twin dangers of rickets on the one hand and toxic doses of vitamin D on the other would thus explain the world-wide correlation observed between skin pigmentation and nearness to the equator.

Unconventional secretion of Acb1 is mediated by autophagosomes

Evidence is presented for an unconventional protein secretion pathway that is conserved from yeast to Dictyostelium discoideum in which Acb1 may be sequestered into autophagosomal vesicles, which then fuse (either directly or indirectly) with the plasma membrane (see also the companion paper from Manjithaya et al. in this issue).

Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein, peroxisomal functions, and autophagosome formation

Evidence is presented for an unconventional protein secretion pathway that is conserved from yeast to Dictyostelium discoideum in which Acb1 may be sequestered into autophagosomal vesicles, which then fuse (either directly or indirectly) with the plasma membrane (see also the companion paper from Duran et al. in this issue).

Hypertonic Saline Resuscitation Decreases Susceptibility to Sepsis after Hemorrhagic Shock

BACKGROUND We hypothesized that improvements in cellular immune function after hypertonic saline (HTS) resuscitation will alter the outcome of sepsis after hemorrhage. METHODS To test this hypothesis, a two-hit model was used. Hemorrhage was induced in BALB/c mice by catheterizing the femoral artery and bleeding until a mean arterial pressure = 35 mm Hg was reached and maintained for 1 hour. Resuscitation was performed with HTS (NaCl 7.5%, 4 mL/kg) or lactated Ringers (LR, twice the shed blood volume), plus the shed blood. Cecal ligation and puncture (CLP) was performed 24 hours after hemorrhage. Mortality was assessed for 72 hours, comparing HTS (n = 14) and LR (n = 13) resuscitation. Another set of animals (n = 10 in each group at each time point) were killed at 2 and 24 hours after blood collection. Liver and blood were cultured for the presence of bacteria, and lung and liver samples were scored on a scale from 0 (normal) to 4 (most severe) in a blind fashion by a pathologist. RESULTS Mortality 72 hours after CLP was 14.3% in HTS and 76.9% in LR treated animals (p < 0.002). At 24 hours after CLP, 44% of HTS, but 77% of LR treated animals had > 1,000 colony forming units/mL of blood. Positive liver cultures (> 100,000 colony forming units/g) also showed the same trend (HTS = 30%, LR = 60%). Autopsies revealed a better containment of the infection (abscess formation) in the HTS group. At 2 hours, lung scores were 1.2 +/- 0.25 and 2.6 +/- 0.31 for HTS and LR, respectively (p < 0.002). At 24 hours, HTS treated animals showed marked improvement of lung injury, while the scores in the LR group remained high. A significant difference was also observed regarding liver injury. At 2 hours, scores were 0.4 +/- 0.22 and 2.3 +/- 0.16 for HTS and LR, respectively (p < 0.002). At 24 hours, HTS treated animals showed normal hepatic architecture, although mild injury was still observed in the LR group. CONCLUSION HTS resuscitation leads to increased survival after hemorrhage and CLP. Marked improvements were observed in lung and liver injury compared with isotonic resuscitation. The better containment of the infection observed with HTS resuscitation corresponds to a marked decreased in bacteremia. HTS resuscitation stands as an alternative resuscitation regimen with immunomodulatory potential.

Hypertonic saline resuscitation diminishes lung injury by suppressing neutrophil activation after hemorrhagic shock.

Hypertonic saline (HS) resuscitation after hemorrhage and sepsis has been shown to markedly reduce the development of lung injury in animals, compared with traditional resuscitation with lactated Ringers (LR). These experiments examined the effect of HS on lung injury after hemorrhage without sepsis. The effects of HS and LR resuscitation on neutrophil trafficking, neutrophil adhesion, and neutrophil oxidative burst were studied. Methods: BALB/c mice were hemorrhaged to a mean arterial pressure of 40 torr for 1 h. Animals were resuscitated with shed blood and either 4 mL/kg of 7.5% HS or LR in twice the volume of the shed blood. Lung histology was examined 24 h after hemorrhage. Lung myeloperoxidase content and bronchoalveolar lavage fluid neutrophil counts were obtained. Peripheral blood smears were obtained to determine the neutrophil percentage. Peripheral blood neutrophil CD11b expression and neutrophil H2O2 production were assayed by flow cytometry. Results: HS animals had less lung injury than LR animals. The mean myeloperoxidase activity in HS versus LR animals was 1.79 ± 1.33 U/100 mg versus 3.0 ± 1.33 U/100 mg, respectively. The percentage of neutrophils in the bronchoalveolar lavage fluid of HS animals (3.8% ± .8) was significantly less than that of LR animals (10.8% ± 2.1). This corresponded to a significantly higher peripheral blood neutrophil count in HS animals compared with LR animals, 41% vs. 20%, respectively. There was no difference in neutrophil expression of the CD11b integrin between the HS and LR groups. The neutrophils of LR animals had basal H2O2 production that was 107% greater than that of controls; HS suppressed this hemorrhage-induced activation by > 60%. HS resuscitation after hemorrhagic shock protects against the development of lung injury. This protection is due, in part, to suppression of the hemorrhage-induced neutrophil oxidative burst. HS resuscitation offers immunomodulatory potential after hemorrhagic shock.

Hypertonic saline resuscitation: a tool to modulate immune function in trauma patients?

Hypertonic saline (HS) resuscitation has recently gained attention from trauma physicians because it may benefit the immune system of trauma patients. We have found that HS augments in vitro and in vivo immune function of healthy T-cells. In addition, HS restored the function of suppressed T-cells in vitro and in vivo and reduced immunosuppression after hemorrhage, protecting mice from subsequent sepsis. These effects of HS are based on its direct influence on cellular signaling events through specific signaling pathway(s) that include protein tyrosine kinase and mitogen-activated protein kinase p38 activation. HS provides a costimulatory signal that enhances the proliferation of activated T-cells. HS may be able to substitute signals lost through blockage as a result of trauma induced suppressive factors, thereby restoring the function of suppressed T-cells. Although further work is needed to determine the optimal conditions and possible risks of HS resuscitation, the data presented in this short review of our recent work shed a favorable light on HS as a simple but effective tool to modulate cellular immune function after trauma.

Sensitivity of Dictyostelium discoideum to nucleic acid analogues

Abstract Growth of axenic cultures of Dictyostelium discoideum A3 was shown to be inhibited by 5-fluorouracil, 5-fluorodeoxyuridine and 5-bromodeoxyuridine. Drug-resistant mutants were isolated and described.

The Golgi-Associated Protein GRASP Is Required for Unconventional Protein Secretion during Development

During Dictyostelium development, prespore cells secrete acyl-CoA binding protein (AcbA). Upon release, AcbA is processed to generate a peptide called spore differentiation factor-2 (SDF-2), which triggers terminal differentiation of spore cells. We have found that cells lacking Golgi reassembly stacking protein (GRASP), a protein attached peripherally to the cytoplasmic surface of Golgi membranes, fail to secrete AcbA and, thus, produce inviable spores. Surprisingly, AcbA lacks a signal sequence and is not secreted via the conventional secretory pathway (endoplasmic reticulum-Golgi-cell surface). GRASP is not required for conventional protein secretion, growth, and the viability of vegetative cells. Our findings reveal a physiological role of GRASP and provide a means to understand unconventional secretion and its role in development.

Periodic Signaling Controlled by an Oscillatory Circuit That Includes Protein Kinases ERK2 and PKA

Self-regulating systems often use robust oscillatory circuits. One such system controls the chemotactic signaling mechanism of Dictyostelium, where pulses of adenosine 3′,5′-monophosphate (cAMP) are generated with a periodicity of 7 minutes. We have observed spontaneous oscillations in activation of the mitogen-activated protein (MAP) kinase ERK2 that occur in phase with peaks of cAMP, and we show that ERK2 modulates cAMP levels through the phosphodiesterase RegA. Computer modeling and simulations of the underlying circuit faithfully account for the ability of the cells to spontaneously generate periodic pulses during specific stages of development. Similar oscillatory processes may occur in cells of many different species.