Phagocytosis of bacteria by polymorphonuclear leukocytes: a freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure

The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25μm(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane. During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.     

Leishmania donovani Argininosuccinate Synthase Is an Active Enzyme Associated with Parasite Pathogenesis

Background

Gene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo.

Methodology/Principal Findings

Two parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.

Conclusion/Significance

Our study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target.     

Deletion of mitochondrial associated ubiquitin fold modifier protein Ufm1 in Leishmania donovani results in loss of β‐oxidation of fatty acids and blocks cell division in the amastigote stage

Recently, we described the existence of the ubiquitin fold modifier 1 (Ufm1) and its conjugation pathway in Leishmania donovani. We demonstrated the conjugation of Ufm1 to proteins such as mitochondrial trifunctional protein (MTP) that catalyses β‐oxidation of fatty acids in L. donovani. To elucidate the biological roles of the Ufm1‐mediated modifications, we made an L. donovani Ufm1 null mutant (Ufm1^?/?). Loss of Ufm1 and consequently absence of Ufm1 conjugation with MTP resulted in diminished acetyl‐CoA, the end‐product of the β‐oxidation in the Ufm1^?/? amastigote stage. The Ufm1^?/? mutants showed reduced survival in the amastigote stage in vitro and ex vivo in human macrophages. This survival was restored by re‐expression of wild‐type Ufm1 with concomitant induction of acetyl‐CoA but not by re‐expressing the non‐conjugatable Ufm1, indicating the essential nature of Ufm1 conjugation and β‐oxidation. Both cell cycle analysis and ultrastructural studies of Ufm1^?/? parasites confirmed the role of Ufm1 in amastigote growth. The defect in vitro growth of amastigotes in human macrophages was further substantiated by reduced survival. Therefore, these studies suggest the importance of Ufm1 in Leishmania pathogenesis with larger impact on other organisms and further provide an opportunity to test Ufm1^?/? parasites as drug and vaccine targets.     

Redescription and palaeobiology of Palaeoscorpius devonicus Lehmann, 1944 from the Lower Devonian Hunsrück Slate of Germany

Abstract: Palaeoscorpius devonicus Lehmann, 1944 is known from only a single specimen, found in the Eschenbach Pit near Bundenbach in the Lower Devonian Hunsrück Slate of Germany. It is a key fossil, having been interpreted both as the most basal member of the Scorpiones and as one of the order’s most likely candidates for an aquatic mode of life. Prepared both ventrally and dorsally, some aspects of its morphology remain problematic. Here, with the aid of new techniques, including computed tomography, we present a re‐investigation of this scorpion’s anatomy and a new reconstruction, with a particular focus on the species’ original habitat. On the basis of the environmental interpretation of the Hunsrück Slate and the completeness of the specimen, previous authors concluded that P. devonicus was marine, but none offered convincing morphological evidence. Recent studies of the deposit’s environment suggest that the Hunsrück Sea was part of an intrashelf basin, relatively close to the coastline, and fossils of land plants show that terrestrial wash‐in occasionally occurred. Our revised interpretation of the fossil’s morphology demonstrates that the scorpion was most probably terrestrial. Internal mesosomal organs are interpreted as book lungs, but other terrestrial adaptations are lacking. The absence of both coxapophyses and gnathobases makes determining the scorpion’s feeding mechanism difficult. Interpreting the scorpion’s character states within a phylogenetic framework, especially the possible presence of book lungs, implies either that the plesiomorphic position of P. devonicus is no longer supported or that the development of book lungs had already taken place early in the scorpion lineage.     

17beta-Estradiol responsiveness of MCF-7 laboratory strains is dependent on an autocrine signal activating the IGF type I receptor

BACKGROUND: Human MCF-7 cells have been studied extensively as a model for breast cancer cell growth. Many reports have established that serum-starved MCF-7 cells can be induced to proliferate upon the sole addition of 17beta-estradiol (E2). However, the extent of the mitogenic response to E2 varies in different MCF-7 strains and may even be absent. In this study we compared the E2-sensitivity of three MCF-7 laboratory strains. RESULTS: The MCF-7S line is non-responsive to E2, the MCF-7 ATCC has an intermediate response to E2, while the MCF-7 NKI is highly E2-sensitive, although the levels and activities of the estrogen receptor (ER) are not significantly different. Both suramin and IGF type I receptor blocking antibodies are able to inhibit the mitogenic response to E2-treatment in MCF-7 ATCC and MCF-7 NKI cells. From this we conclude that E2-induced proliferation is dependent on IGF type I receptor activation in all three MCF-7 strains. CONCLUSIONS: The results presented in this article suggest that E2-responsiveness of MCF-7 cells is dependent on the secretion of an autocrine factor activating the IGF-IR. All three strains of MCF-7 breast cancer cells investigated do not respond to E2 if the IGF-RI-pathway is blocked. Generally, breast cancer therapy is targeted at inhibiting estrogen action. This study suggests that inhibition of IGF-action in combination with anti-estrogen-treatment may provide a more effective way in treatment or even prevention of breast cancer.     

Autoantigens interact with cis-acting elements of rubella virus RNA.

Rubella virus (RV) infections in adult women can be associated with acute and chronic arthritic symptoms. In many autoimmune individuals, antibodies are found targeting endogenous proteins, called autoantigens, contained in ribonucleoprotein complexes (RNPs). In order to understand the molecular mechanisms involved in the RV-associated pathology, we investigated the nature of cellular factors binding RV RNA and whether such RNPs were recognized by antibodies in infected individuals. Previously, we noted that cellular proteins associated with the RV 5'(+) stem-loop (SL) RNA are recognized by serum with Ro reactivity. To better understand the nature of the autoantigens binding RV cis-acting elements, serum samples from individuals with various autoimmune diseases were tested for their ability to immunoprecipitate RNPs containing labeled RV RNAs. A subset of serum samples recognizing autoantigen La, or Ro and La, immunoprecipitated both the RV 5'(+)SL and 3'(+)SL RNA-protein complexes. Autoantigens binding the RV 5'(+)SL and 3'(+)SL RNAs differed in molecular mass, specificities for respective RNA binding substrates, and sensitivity to alkaline phosphatase treatment. The La autoantigen was found to interact with the RV 5'(+)SL RNA as determined by immunological techniques and binding reactions with mixtures containing recombinant La protein. To test whether there is a correlation between La binding to an RV RNA element and the appearance of an anti-La response, we measured anti-La titers in RV-infected individuals. Significant anti-La activity was detected in approximately one-third of RV-infected individuals 2 years postinfection.     

Specific high-affinity binding of host cell proteins to the 3' region of rubella virus RNA

Replication of rubella virus is initiated at the 3' end of the genomic RNA. An inverted repeat sequence of 12 nucleotides that is capable of forming a stem-loop structure is located at the 3' end of the RNA, 59 nucleotides upstream from the poly (A) tail. We screened the 158-bp region of the 3' end of the virus, including the stem-loop structure, for its ability to bind to host-cell proteins. Specific high-affinity binding of three cytosolic proteins with relative molecular masses (Mr) of 61, 63 and 68 kD to the stem-loop structure was observed by UV-induced covalent crosslinking. Altering the stem structure by removal of specific bases abolished the binding interactions. The binding of the host proteins is greatly increased after infection and coincides with the appearance of negative strand RNA synthesis. The increase in binding is dependent on new protein synthesis. The amount of the 61-kD protein that binds varies in uninfected cells and is maximal in cells that are in the stationary phase of growth. All binding activity could be abrogated by alkaline phosphatase treatment of cell lysates. A possible role of these host proteins in the replication of rubella virus is discussed.