Mutations Altering a Structurally Conserved Loop-Helix-Loop Region of a Viral Packaging Motor Change DNA Translocation Velocity and Processivity

Many double-stranded DNA viruses employ ATP-driven motors to translocate their genomes into small, preformed viral capsids against large forces resisting confinement. Here, we show via direct single-molecule measurements that a mutation T194M downstream of the Walker B motif in the phage λ gpA packaging motor causes an 8-fold reduction in translocation velocity without substantially changing processivity or force dependence, whereas the mutation G212S in the putative C (coupling) motif causes a 3-fold reduction in velocity and a 6-fold reduction in processivity. Meanwhile a T194M pseudorevertant (T194V) showed a near restoration of the wild-type dynamics. Structural comparisons and modeling show that these mutations are in a loop-helix-loop region that positions the key residues of the catalytic motifs, Walker B and C, in the ATPase center and is structurally homologous with analogous regions in chromosome transporters and SF2 RNA helicases. Together with recently published studies of SpoIIIE chromosome transporter and Ded1 RNA helicase mutants, these findings suggest the presence of a structurally conserved region that may be a part of the mechanism that determines motor velocity and processivity in several different types of nucleic acid translocases.     

NK cell intrinsic regulation of MIP-1α by granzyme M

Granzymes are generally recognized for their capacity to induce various pathways of perforin-dependent target cell death. Within this serine protease family, Granzyme M (GrzM) is unique owing to its preferential expression in innate effectors such as natural killer (NK) cells. During Listeria monocytogenes infection, we observed markedly reduced secretion of macrophage inflammatory protein-1 alpha (MIP-1α) in livers of GrzM-deficient mice, which resulted in significantly impaired NK cell recruitment. Direct stimulation with IL-12 and IL-15 demonstrated that GrzM was required for maximal secretion of active MIP-1α. This effect was not due to reduced protein induction but resulted from heightened intracellular accumulation of MIP-1α, with reduced release. These results demonstrate that GrzM is a critical mediator of innate immunity that can regulate chemotactic networks and has an important role in the initiation of immune responses and pathogen control.     

Enumeration of Mycobacterium leprae using real-time PCR

Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae-infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories.     

Cytoskeleton of human embryonal carcinoma cells

Monoclonal antibodies to cytoskeletal proteins were used to study the intermediate filament proteins of human embryonal carcinoma (EC) cell lines, tumors produced in nude mice from these cell lines, and surgically removed testicular germ cell tumors. It was found that all cells of tumor lines 2102Ep, 1156 and Tera 1 react with antibodies to low molecular weight keratin proteins. By immunoblotting of SDS gels it was found that these lines expressed three keratin polypeptides (40K, 45K and 52K). Clonal line NTera-2 derived from Tera-2 differed from the above listed cell lines in that only 10% of the cells expressed the 40K keratin polypeptide. Upon treatment with retinoic acid 70% of NTera-2 cells became reactive with the antibody to the 40K keratin polypeptide. All cell lines contained a small population of vimentin-positive cells. The number of vimentin-positive cells could be increased by retinoic acid treatment of NTera-2 cells or by seeding the 2102Ep cells at low cell density. Neurofilament-positive cells could be induced in the cell line NTera-2 by retinoic acid treatment. Tumors produced from NTera-2 cells injected into nude mice contained cells reacting with antibodies to keratin, vimentin, neurofilament proteins and desmin. Keratin polypeptides were immunohistochemically demonstrated in embryonal carcinoma, yolk sac carcinoma and trophoblastic components of solid human germ cell tumors. Atypical intratubular cells ('carcinoma in situ') also reacted with antibodies to keratin.     

Nanotechnology Nexus—Intersection of Research,Science, Technology,and Regulation

Not a day passes where nanotechnology does not make headlines in the popular press, scientific journals, as well as in the regulatory arena. Environmental and public health activists are voicing a growing concern and focus on the risks potentially posed by nanotechnology and the ability of the government to regulate these new and exciting technologies. Whereas such concerns state the need for stringent, precautionary, and almost exclusionary approaches to the regulation of nanotechnology, many entities believe that a voluntary approach to these often novel materials and technologies is the appropriate and sensible path. In this editorial, we discuss the importance of nanotechnology, who cares, and the available options for approaching the regulation of this often novel technology. We focus on the U.S. Environmental Protection Agency (USEPA) and its voluntary regulatory data submission program as the preferred alternative. ^{2 2}Comments are those of the authors and do not necessarily represent those of their employers. View all notes     

Comparative effects of cytokines and cytokine combinations on complement component C3 secretion by HepG2 cells

The mechanisms that control complement protein synthesis are incompletely understood. Recent evidence suggests that cytokines are involved in the regulation of hepatic synthesis of circulating complement components. Therefore, we compared the effects of human recombinant IL-1alpha, IL-1beta, IL-6, IFN-gamma, and TNF-alpha individually or in combination, on HepG2 secretion of complement component C3, the major opsonic protein of the complement system. HepG2 cells were incubated with each cytokine alone and with various combinations of the cytokines. At 24, 48, 72, and 96 h of incubation, the C3 and albumin secreted by the HepG2 cells were quantified by a sandwich ELISA. IL-1alpha and IFN-gamma significantly enhanced C3 secretion by the cells (P<0.02 vs. control cells). IL-1beta when combined with either IL-6 or IFN-gamma also increased C3 secretion (P<0.03 vs. control cells). The stimulatory effect on HepG2 cells by the IL-1beta/IL-6 combination was synergistic. With the exception of IL-1alpha, which increased albumin secretion, HepG2 secretion of albumin was not affected by incubation with individual cytokines or the cytokine combinations. Therefore, IL-1alpha, IFN-gamma, and the combination of IL-1beta with IL-6 or IFN-gamma specifically enhanced C3 secretion by HepG2 cells. The greatest magnitude of C3 secretion was induced by the combination of IL-1beta and IL-6.     

Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca^2+-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D.