Developmentally induced changes of the proteome in the protozoan parasite Leishmania donovani

In order to proceed through their life cycle, protozoan parasites of the genus Leishmania cycle between sandflies and mammals. This change of environment correlates with the differentiation from the promastigote stage (insect form) to the amastigote stage (intracellular mammalian form). The molecular basis underlying this major transformation is poorly understood so far; however, heat shock protein 90 (HSP90) appears to play a pivotal role. To further elucidate this process we identified proteins expressed preferentially in either of the two life cycle stages. By using two-dimensional (2-D) gel electrophoresis we observed defined changes in the protein pattern. A total of approximately 2000 protein spots were visualized. Of these, 31 proteins were present only in promastigotes. The abundance of 65 proteins increased during heat-induced in vitro amastigote differentiation, while a decreased abundance is observed for four proteins late in amastigote differentiation. Further analyses using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and peptide mass fingerprinting 67 protein spots were identified representing 41 different proteins known from databases and eight hypothetical proteins. Further studies showed that most of the stage-specific proteins fall into five groups of functionally related proteins. These functional categories are: (i) stress response (e.g. heat, oxidative stress); (ii) cytoskeleton and cell membrane; (iii) energy metabolism and phosphorylation; (iv) cell cycle and proliferation; and (v) amino acid metabolism. Very similar changes in the 2-D protein pattern were obtained when in vitro amastigote differentiation was induced either by pharmacological inhibition of HSP90 or by a combination of heat stress and acidic pH supporting the critical role for HSP90 in life cycle control.     

Stimulation of T7 DNA polymerase by a new phage-coded protein

Summary A bacteriophage-induced DNA-binding protein was purified from T7 infected E. coli. The protein has a molecular weight of about 25000, as judged by SDS-polyacrylamide gel electrophoresis. The purified protein binds to single-stranded but not to native T7 DNA. Like the T4 gene-32 protein and the 22000-dalton unwinding protein of E. coli, the T7 25000 protein lowers the melting temperature of poly d(A-T). Using partially single-stranded T7 DNA as template-primer, the protein stimulates in vitro DNA synthesis by T7 DNA polymerase about five-fold. It was also found that the DNA-unwinding protein of E. coli stimulates T7 DNA polymerase to approximately the same extent. However, neither of the unwinding proteins stimulate DNA polymerase I of E. coli.     

Temporal sampling,resetting, and adaptation orchestrate gradient sensing in sperm

Sperm, navigating in a chemical gradient, are exposed to a periodic stream of chemoattractant molecules. The periodic stimulation entrains Ca²⁺ oscillations that control looping steering responses. It is not known how sperm sample chemoattractant molecules during periodic stimulation and adjust their sensitivity. We report that sea urchin sperm sampled molecules for 0.2–0.6 s before a Ca²⁺ response was produced. Additional molecules delivered during a Ca²⁺ response reset the cell by causing a pronounced Ca²⁺ drop that terminated the response; this reset was followed by a new Ca²⁺ rise. After stimulation, sperm adapted their sensitivity following the Weber–Fechner law. Taking into account the single-molecule sensitivity, we estimate that sperm can register a minimal gradient of 0.8 fM/µm and be attracted from as far away as 4.7 mm. Many microorganisms sense stimulus gradients along periodic paths to translate a spatial distribution of the stimulus into a temporal pattern of the cell response. Orchestration of temporal sampling, resetting, and adaptation might control gradient sensing in such organisms as well.     

Loss of CCR7 Expression on CD56bright NK Cells Is Associated with a CD56dimCD16+ NK Cell-Like Phenotype and Correlates with HIV Viral Load

NK cells are pivotal sentinels of the innate immune system and distinct subpopulations in peripheral blood have been described. A number of studies addressed HIV-induced alterations of NK cell phenotype and functionality mainly focusing on CD56^dimCD16⁺ and CD56⁻CD16⁺ NK cells. However, the impact of HIV-infection on CD56^bright NK cells is less well understood. Here we report a rise of CD56^bright NK cells in HIV-infected individuals, which lack CCR7-expression and strongly correlate with HIV viral load. CCR7⁻CD56^bright NK cells were characterized by increased cytolytic potential, higher activation states and a more differentiated phenotype. These cells thus acquired a number of features of CD56^dimCD16⁺ NK cells. Furthermore, CD56^bright NK cells from HIV patients exhibited higher degranulation levels compared to uninfected individuals. Thus, chronic HIV-infection is associated with a phenotypic and functional shift of CD56^bright NK cells, which provides a novel aspect of HIV-associated pathogenesis within the NK cell compartment.     

Responsiveness of mesolimbic, mesocortical, septal and hippocampal cholecystokinin and substance P neuronal systems to stress, in the male rat

The effects of acute and subchronic stress upon discrete cholecystokinin (CCK) and Substance P (SP) neuronal systems have been studied. Adult male rats were exposed to foot-shock stress for periods of 2, 4, 10, 30 or 60 min, immediately following which they were decapitated; brains were rapidly removed and frozen, and subsequently microdissected and extracted. CCK and SP were determined by RIA. In the olfactory tubercule, stress had no effect upon CCK content, but induced a rapid depletion of SP. In the prefrontal cortex, increased CCK concentrations were found following 30 min of stress exposure. In the medial septum, foot-shock led to a rapid increase in CCK content, and to a similar but delayed change in SP levels. A rapid rise in CCK concentrations was also seen in the lateral septum, but no stress effect whatsoever upon SP occurred in this structure. In the dentate gyrus, CCK exhibited a biphasic responsiveness to stress, while SP levels were increased only at the later time intervals. These data demonstrate that discrete CCK and SP neuronal systems are responsive to stress, and thereby support a functional role for these peptides in the processing of neural and hormonal signals by the CNS.     

Psb27, a cyanobacterial lipoprotein, is involved in the repair cycle of photosystem II

Photosystem II (PSII) performs one of the key reactions on our planet: the light-driven oxidation of water. This fundamental but very complex process requires PSII to act in a highly coordinated fashion. Despite detailed structural information on the fully assembled PSII complex, the dynamic aspects of formation, processing, turnover, and degradation of PSII with at least 19 subunits and various cofactors are still not fully understood. Transient complexes are especially difficult to characterize due to low abundance, potential heterogeneity, and instability. Here, we show that Psb27 is involved in the assembly of the water-splitting site of PSII and in the turnover of the complex. Psb27 is a bacterial lipoprotein with a specific lipid modification as shown by matrix-assisted laser-desorption ionization time of flight mass spectrometry. The combination of HPLC purification of four different PSII subcomplexes and (15)N pulse label experiments revealed that lipoprotein Psb27 is part of a preassembled PSII subcomplex that represents a distinct intermediate in the repair cycle of PSII.     

The influence of externally supplied sucrose on phloem transport in the maize leaf strip

Sucrose (2,5–1000 mmol l^–1), labeled with [¹⁴C]sucrose, was taken up by the xylem when supplied to one end of a 30-cm-long leaf strip of Zea mays L. cv. Prior. The sugar was loaded into the phloem and transported to the opposite end, which was immersed in diluted Hoagland's nutrient solution. When the Hoagland's solution at the opposite end was replaced by unlabeled sucrose solution of the same molarity as the labeled one, the two solutions met near the middle of the leaf strip, as indicated by radioautographs. In the dark, translocation of ¹⁴C-labeled assimilates was always directed away from the site of sucrose application, its distance depending on sugar concentration and translocation time. When sucrose was applied to both ends of the leaf strip, translocation of ¹⁴C-labeled assimilates was directed toward the lower sugar concentration. In the light, transport of ¹⁴-C-labeled assimilates can be directed (1) toward the morphological base of the leaf strip only (light effect), (2) toward the base and away from the site of sucrose application (light and sucrose effect), or (3) away from the site of sucrose application independent of the (basipetal or acropetal) direction (sucrose effect). The strength of a sink, represented by the darkened half of a leaf strip, can be reduced by applying sucrose (at least 25 mmol l^–1) to the darkened end of the leaf strip. However, equimolar sucrose solutions applied to both ends do not affect the strength of the dark sink. Only above 75 mmol l^–1 sucrose was the sink effect of the darnened part of the leaf strip reduced. Presumably, increasing the sucrose concentration replenishes the leaf tissue more rapidly, and photosynthates from the illuminated part of the leaf strip are imported to a lesser extent by the dark sink.Supported by Deutsche Forschungsgemeinschaft