The use of additive and subtractive approaches to examine the nuclear localization sequence of the polyomavirus major capsid protein VP1.

A nuclear localization signal (NLS) has been identified in the N-terminal (Ala1-Pro-Lys-Arg-Lys-Ser-Gly-Val-Ser-Lys-Cys11) amino acid sequence of the polyomavirus major capsid protein VP1. The importance of this amino acid sequence for nuclear transport of VP1 protein was demonstrated by a genetic "subtractive" study using the constructs pSG5VP1 (full-length VP1) and pSG5 delta 5'VP1 (truncated VP1, lacking amino acids Ala1-Cys11). These constructs were used to transfect COS-7 cells, and expression and intracellular localization of the VP1 protein was visualized by indirect immunofluorescence. These studies revealed that the full-length VP1 was expressed and localized in the nucleus, while the truncated VP1 protein was localized in the cytoplasm and not transported to the nucleus. These findings were substantiated by an "additive" approach using FITC-labeled conjugates of synthetic peptides homologous to the NLS of VP1 cross-linked to bovine serum albumin or immunoglobulin G. Both conjugates localized in the nucleus after microinjection into the cytoplasm of 3T6 cells. The importance of individual amino acids found in the basic sequence (Lys3-Arg-Lys5) of the NLS was also investigated. This was accomplished by synthesizing three additional peptides in which lysine-3 was substituted with threonine, arginine-4 was substituted with threonine, or lysine-5 was substituted with threonine. It was found that lysine-3 was crucial for nuclear transport, since substitution of this amino acid with threonine prevented nuclear localization of the microinjected, FITC-labeled conjugate.     

Preprocessor Adapts Cardiovascular Signals to a Minicomputer

A laboratory-developed analog signal processor, driven by a conventional polygraph recorder and associated signal conditioning devices, provides automatic heart beat-by-heart beat preprocessing of various cardiovascular functions for input to a laboratory-type minicomputer. The technique of preprocessing individual functions, integrated with the minicomputer system which includes an A/D converter and teletype as input-output peripherals, provides a low-cost data acquisition and reduction system for the on-line computation and analysis of cardiovascular functions in experimental research applications. Such preprocessing more efficiently uses the minicomputer's memory to handle large amounts of information since the digitized data is in the form of one data sample, per function, per heart beat. Preprocessing analog data provides a low density data format and simplified software programs that are ideally suited for the utilization of a minicomputer in this on-line application.     

Nucleic acid vaccines

There are no adequate vaccines against some of the new or reemerged infectious scourges such as HIV and TB. They may require strong and enduring cell-mediated immunity to be elicited. This is quite a task, as the only known basis of protection by current commercial vaccines is antibody. As DNA or RNA vaccines may induce both cell-mediated and humoral immunity, great interest has been shown in them. However, doubt remains whether their efficacy will suffice for their clinical realization. We look at the various tactics to increase the potency of nucleic acid vaccines and divided them broadly under those affecting delivery and those affecting immune induction. For delivery, we have considered ways of improving uptake and the use of bacterial, replicon or viral vectors. For immune induction, we considered aspects of immunostimulatory CpG motifs, coinjection of cytokines or costimulators and alterations of the antigen, its cellular localization and its anatomical localization including the use of ligand-targeting to lymphoid tissue. We also thought that mucosal application of DNA deserved a separate section. In this review, we have taken the liberty to discuss these enhancement methods, whenever possible, in the context of the underlying mechanisms that might argue for or against these strategies.     

Genome-wide examination of myoblast cell cycle withdrawal during differentiation.

Skeletal and cardiac myocytes cease division within weeks of birth. Although skeletal muscle retains limited capacity for regeneration through recruitment of satellite cells, resident populations of adult myocardial stem cells have not been identified. Because cell cycle withdrawal accompanies myocyte differentiation, we hypothesized that C2C12 cells, a mouse myoblast cell line previously used to characterize myocyte differentiation, also would provide a model for studying cell cycle withdrawal during differentiation. C2C12 cells were differentiated in culture medium containing horse serum and harvested at various time points to characterize the expression profiles of known cell cycle and myogenic regulatory factors by immunoblot analysis. BrdU incorporation decreased dramatically in confluent cultures 48 hr after addition of horse serum, as cells started to form myotubes. This finding was preceded by up-regulation of MyoD, followed by myogenin, and activation of Bcl-2. Cyclin D1 was expressed in proliferating cultures and became undetectable in cultures containing 40% fused myotubes, as levels of p21(WAF1/Cip1) increased and alpha-actin became detectable. Because C2C12 myoblasts withdraw from the cell cycle during myocyte differentiation following a course that recapitulates this process in vivo, we performed a genome-wide screen to identify other gene products involved in this process. Using microarrays containing approximately 10,000 minimally redundant mouse sequences that map to the UniGene database of the National Center for Biotechnology Information, we compared gene expression profiles between proliferating, differentiating, and differentiated C2C12 cells and verified candidate genes demonstrating differential expression by RT-PCR. Cluster analysis of differentially expressed genes revealed groups of gene products involved in cell cycle withdrawal, muscle differentiation, and apoptosis. In addition, we identified several genes, including DDAH2 and Ly-6A, whose expression specifically was up-regulated during cell cycle withdrawal coincident with early myoblast differentiation.     

The class A macrophage scavenger receptor is a major pattern recognition receptor for Neisseria meningitidis which is independent of lipopolysaccharide and not required for secretory responses

Macrophages (Mphi) play a key role in the pathogenesis of invasive meningococcal infections. The roles of two pattern recognition molecules, the Mphi scavenger receptor (SR-A) and Toll-like receptor 4 (TLR-4), have been investigated using bone marrow culture-derived Mphi (BMMphi). Surprisingly, a comparison of BMMphi from wild-type and SR-A knockout (SR-A(-/-)) mice showed that nonopsonic phagocytosis of meningococci was mediated almost exclusively via SR-A. Previous studies have demonstrated only a partial involvement of the receptor in the uptake of other bacteria, such as Escherichia coli. Interestingly, we also show that lipopolysaccharide (LPS) was not the ligand for the receptor on these organisms. Further study of the downstream events of SR-A-mediated ingestion of Neisseria meningitidis demonstrated that SR-A was not required for cytokine production. To determine the bacterial and host factors required to stimulate Mphi activation, we examined TLR-4-deficient Mphi from C3H/HeJ mice and LPS-deficient meningococci. TLR-4-deficient cells elaborated reduced amounts of tumor necrosis factor alpha, interleukin-12 (IL-12), and IL-10, even though ingestion via SR-A was unaffected in these cells. Similarly, although there was no change in SR-A-mediated ingestion of LPS-deficient meningococci, the mutant failed to stimulate a Mphi-dependent cytokine response. Thus, we show that Mphi SR-A mediates opsonin-independent uptake of N. meningitidis independently of lipid A and that this activity is uncoupled from the Mphi secretion of proinflammatory cytokines, which provides a basis for further investigation of the role of this receptor in meningococcal disease in humans.     

Osteogenesis imperfecta lethal in infancy: Case report and scanning electron microscopic studies of the deciduous teeth

Radiologic evaluation of the skeleton and scanning electron microscopic studies of the teeth were performed on an infant boy with a lethal osteogenesis imperfecta (OI) syndrome who died at 10 mo of pneumonia. The skeletal findings included ribs that were focally expanded by fracture calluses, flat vertebral bodies, and wide limb bones. On fractured tooth surfaces, the enamel and dentin were normal as was the dentin calcification front. Although microscopic abnormalities have been noted in teeth from previously reported infants with lethal OI, a few studies also report infants with normal teeth. These differences in dental findings may indicate heterogeneity in OI lethal in infancy. Results of our study indicate that, until the primary biochemical defects in the OI syndromes are elucidated, examination of teeth from other infants with lethal OI and detailed evaluation of other clinical and skeletal features will aid in delineating heterogeneity and variation in expression in lethal OI.     

A Prophylactic Tibial Stem Reduces Rates of Early Aseptic Loosening in Patients with Severe Preoperative Varus Deformity in Primary Total Knee Arthroplasty

BackgroundPatients with a preoperative varus deformity >8 degrees are at increased risk of aseptic loosening after total knee arthroplasty. This study analyzes the effect of a tibial stem on the rate of aseptic loosening in patients with a severe preoperative varus deformity.MethodsPatients with a preoperative varus deformity of >8 degrees and 2-year minimum follow-up with a stemmed tibial component (n = 67) were matched 1:2 to patients with a similar preoperative varus deformity with a standard tibial component (n = 134). Radiolucent lines were measured on the tibia at 6 weeks, 1 year, and 2 years postoperatively using the Knee Society Radiographic Evaluation System. Failure was defined as revision due to aseptic loosening of the tibial component. Outcomes were evaluated using Student’s t-tests and log-rank tests.ResultsPatients with tibial stems had greater preoperative deformity (12.9 vs 11.3 degrees, P = .004). There was no difference in postoperative alignment (1.7 vs 2.1 degrees varus, P = .25) or tibial component angle (1.8 vs 2.1 degrees varus, P = .33). Patients with stems were more likely to have more constraint (44.8% vs 1.5%, P < .001). Progression of radiolucent lines >2 mm was observed in 17.6% (23/134) vs 5.97% (4/67) of patients in the stem group (P = .03). Rates of aseptic loosening were lower in the stem group (0% vs 5.15%, P = .05).ConclusionDespite worse preoperative deformity and higher utilization of constraint, tibial stem use in patients with severe preoperative varus deformity resulted in lower rates of aseptic loosening. Prophylactic use of stems in these patients may help increase implant survival.