The unseen invaders: introduced earthworms as drivers of change in plant communities in North American forests (a meta‐analysis)

Globally, biological invasions can have strong impacts on biodiversity as well as ecosystem functioning. While less conspicuous than introduced aboveground organisms, introduced belowground organisms may have similarly strong effects. Here, we synthesize for the first time the impacts of introduced earthworms on plant diversity and community composition in North American forests. We conducted a meta‐analysis using a total of 645 observations to quantify mean effect sizes of associations between introduced earthworm communities and plant diversity, cover of plant functional groups, and cover of native and non‐native plants. We found that plant diversity significantly declined with increasing richness of introduced earthworm ecological groups. While plant species richness or evenness did not change with earthworm invasion, our results indicate clear changes in plant community composition: cover of graminoids and non‐native plant species significantly increased, and cover of native plant species (of all functional groups) tended to decrease, with increasing earthworm biomass. Overall, these findings support the hypothesis that introduced earthworms facilitate particular plant species adapted to the abiotic conditions of earthworm‐invaded forests. Further, our study provides evidence that introduced earthworms are associated with declines in plant diversity in North American forests. Changing plant functional composition in these forests may have long‐lasting effects on ecosystem functioning.     

Isolated trout liver cells: Establishing short-term primary cultures exhibiting cell-to-cell interactions

Summary Composition and interactions of cell types in rainbow trout (Oncorhynchus mykiss) liver digested with collagenase and cultured in serum-free media were investigated. Suspensions obtained after digesting trout liver with collagenase contained all the cell types present in the liver, including liver parenchymal cells (hepatocytes), biliary epithelial cells, sinusoidal endothelium, fat-storing cells of Ito, and macrophages. A major cell pellet, mainly hepatocytes but containing significant numbers of biliary epithelial cells, was obtained by centrifuging the cell suspension at 120×g for 1 min. Cells present in this pellet quantitatively attached to culture plates coated with a trout skin extract and remain attached for 4 to 6 d with good retention of intracellular enzymes and DNA. When in culture, significant changes in and among the cells were observed. Initial preparations were rounded, single cells. Within several hours, however, cellular interactions leading to aggregation became evident and aggregates increased in size for 2 to 3 d. Scanning electron microscopy (EM) showed frequent shaftlike projections from margins of the aggregates. Transmission EM indicated that these projections represent biliary ductules forming in vitro. Adjacent hepatocytes also showed plasma membrane specializations forming junctional complexes and canaliculi characteristics of normal trout liver. After 5 to 6 d in culture, significant numbers of the cell aggregates dislodged from the plate. Analysis showed the dislodged cells were viable but vacuolated. The reestablishment in vitro of morphologic relationships resembling in situ tissue components suggest these culture preparations may have significant utility in cooperative metabolic studies of cell interactions in trout liver. Supported by grant CA45131 from the National Cancer Institute, Bethesda, MD.     

Biotechnological advances in the diagnosis,species differentiation and phylogenetic analysis of Schistosoma spp.

Schistosomiasis is a serious parasitic disease caused by blood-dwelling flukes of the genus Schistosoma. Throughout the world, schistosomiasis is associated with high rates of morbidity and mortality, with close to 800 million people at risk of infection. Precise methods for identification of Schistosoma species and diagnosis of schistosomiasis are crucial for an enhanced understanding of parasite epidemiology that informs effective antiparasitic treatment and preventive measures. Traditional approaches for the diagnosis of schistosomiasis include etiological, immunological and imaging techniques. Diagnosis of schistosomiasis has been revolutionized by the advent of new molecular technologies to amplify parasite nucleic acids. Among these, polymerase chain reaction-based methods have been useful in the analysis of genetic variation among Schistosoma spp. Mass spectrometry is now extending the range of biological molecules that can be detected. In this review, we summarize traditional, non-DNA-based diagnostic methods and then describe and discuss the current and developing molecular techniques for the diagnosis, species differentiation and phylogenetic analysis of Schistosoma spp. These exciting techniques provide foundations for further development of more effective and precise approaches to differentiate schistosomes and diagnose schistosomiasis in the clinic, and also have important implication for exploring novel measures to control schistosomiasis in the near future.     

Function of Protonatable Residues in the Flagellar Motor of Escherichia coli: a Critical Role for Asp 32 of MotB

Rotation of the bacterial flagellar motor is powered by a transmembrane gradient of protons or, in some species, sodium ions. The molecular mechanism of coupling between ion flow and motor rotation is not understood. The proteins most closely involved in motor rotation are MotA, MotB, and FliG. MotA and MotB are transmembrane proteins that function in transmembrane proton conduction and that are believed to form the stator. FliG is a soluble protein located on the cytoplasmic face of the rotor. Two other proteins, FliM and FliN, are known to bind to FliG and have also been suggested to be involved to some extent in torque generation. Proton (or sodium)-binding sites in the motor are likely to be important to its function and might be formed from the side chains of acidic residues. To investigate the role of acidic residues in the function of the flagellar motor, we mutated each of the conserved acidic residues in the five proteins that have been suggested to be involved in torque generation and measured the effects on motility. None of the conserved acidic residues of MotA, FliG, FliM, or FliN proved essential for torque generation. An acidic residue at position 32 of MotB did prove essential. Of 15 different substitutions studied at this position, only the conservative-replacement D32E mutant retained any function. Previous studies, together with additional data presented here, indicate that the proteins involved in motor rotation do not contain any conserved basic residues that are critical for motor rotation per se. We propose that Asp 32 of MotB functions as a proton-binding site in the bacterial flagellar motor and that no other conserved, protonatable residues function in this capacity.     

Effect of parenterally injected benzimidazole compounds on Echinococcus multilocularis and Taenia crassiceps metacestodes in laboratory animals.

In mice infected with metacestodes of Taenia crassiceps, the following compounds were at least partially effective when injected intraperitoneally at the dosage indicated: cambendazole (500 mg/kg), mebendazole (6.25 mg/kg), oxibendazole (500 mg/kg), 5-benzamido-2(4-thiazolyl)benzimidazole (500 mg/kg), 2-carboethoxyamino benzimidazole (125 mg/kg), and 2-carbomethoxyamino benzimidazole (500 mg/kg). The following were inactive at the dosage indicated: parbendazole (500 mg/kg), thiabendazole (1,000 mg/kg), and fenbendazole (1,000 mg/kg). Mebendazole, which showed some activity at 6.25 mg/kg, was highly active as a single intraperitoneal dose at 25 mg/kg. When injected subcutaneously, mebendazole was much less active than when given intraperitoneally. In mice infected with metacestodes of Echinococcus multilocularis, intraperitoneal injection of mebendazole at 75 to 150 mg/kg, daily for 3 days, was highly effective (95 to 100% reduction in cyst mass). In contrast, oral administration at 1,000 mg/kg, daily for 3 days, was only partially effective. The drug was also effective when given intraperitoneally to infected cotton rats. A water-soluble benzimidazole, carboxymethyleneamino cambendazole, was approximately 50% effective in mice when injected daily for 3 days at a dosage of 75 or 150 mg/kg. The results suggest that, in metacestode infections of medical importance, it may be possible to kill the parasite by delivering a drug to its immediate vicinity, and so to reduce the required dosage with respect to the host.     

Chronosequence predictions are robust in a Neotropical secondary forest,but plots miss the mark

Tropical secondary forests (TSF) are a global carbon sink of 1.6 Pg C/year. However, TSF carbon uptake is estimated using chronosequence studies that assume differently aged forests can be used to predict change in aboveground biomass density (AGBD) over time. We tested this assumption using two airborne lidar datasets separated by 11.5 years over a Neotropical landscape. Using data from 1998, we predicted canopy height and AGBD within 1.1 and 10.3% of observations in 2009, with higher accuracy for forest height than AGBD and for older TSFs in comparison to younger ones. This result indicates that the space‐for‐time assumption is robust at the landscape‐scale. However, since lidar measurements of secondary tropical forest are rare, we used the 1998 lidar dataset to test how well plot‐based studies quantify the mean TSF height and biomass in a landscape. We found that the sample area required to produce estimates of height or AGBD close to the landscape mean is larger than the typical area sampled in secondary forest chronosequence studies. For example, estimating AGBD within 10% of the landscape mean requires more than thirty 0.1 ha plots per age class, and more total area for larger plots. We conclude that under‐sampling in ground‐based studies may introduce error into estimations of the TSF carbon sink, and that this error can be reduced by more extensive use of lidar measurements.     

A cellular trafficking signal in the SIV envelope protein cytoplasmic domain is strongly selected for in pathogenic infection

The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-based trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting. Despite extensive analysis, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which this signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239ΔGY), replicates acutely to high levels in pigtail macaques (PTM) but is rapidly controlled. However, we previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239ΔGY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the ΔGY deletion and a nine nucleotide deletion encoding amino acids 734–736 (ΔQTH) that overlaps the rev and tat open reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected that generated a new signal for polarized sorting but not endocytosis. This genotype, together with the ΔGY mutation, was conserved in association with high viral loads for several months when introduced into naïve PTMs. For the first time, our findings reveal strong selection pressure for Env endocytosis and particularly for polarized sorting during pathogenic SIV infection in vivo.     

Tumorigenicity of the met proto-oncogene and the gene for hepatocyte growth factor.

The met proto-oncogene is the tyrosine kinase growth factor receptor for hepatocyte growth factor/scatter factor (HGF/SF). It was previously shown that, like the oncogenic tpr-met, the mouse met proto-oncogene transforms NIH 3T3 cells. We have established NIH 3T3 cells stably expressing both human (Methu) and mouse (Metmu) met proto-oncogene products. The protein products are properly processed and appear on the cell surface. NIH 3T3 cells express endogenous mouse HGF/SF mRNA, suggesting an autocrine activation mechanism for transformation by Metmu. However, the tumor-forming activity of Methu in NIH 3T3 cells is very low compared with that of Metmu, but efficient tumorigenesis occurs when Methu and HGF/SFhu are coexpressed. These results are consistent with an autocrine transformation mechanism and suggest further that the endogenous murine factor inefficiently activates the tumorigenic potential of Methu. The tumorigenicity observed with reciprocal chimeric human and mouse receptors that exchange external ligand-binding domains supports this conclusion. We also show that HGF/SFhu expressed in NIH 3T3 cells produces tumors in nude mice.     

Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection.

Human immunodeficiency virus (HIV) has been associated with acquired immunodeficiency syndrome and related disorders. Assays to detect antibodies to HIV proteins have been developed and used to screen sera for the identification of individuals who have been exposed to the virus. Although these serological tests have significant sensitivity and specificity for detecting exposure to the virus, they do not provide direct identification of HIV. We report here the application of recently developed nucleic acid amplification and oligonucleotide-based detection procedures for the identification of HIV sequences in established infected cell lines and in cells cultured from infected individuals.