Orthopedic management of osteopetrosis: results of a survey and review of the literature

Osteopetrosis or Albers-Schonberg disease is a rare hereditary disorder of osteoclast function in which resorption of bone is diminished, resulting in abnormally dense bones. The condition is known to occur in at least four recognizable clinical patterns, each of which is variable. The optimal treatment of fractures and of bone deformity in these patients has not previously been made clear. To determine appropriate orthopedic management of the condition, we conducted a survey of the membership of the Pediatric Orthopedic Society of North America. The combined experience of 57 surgeons who treated 79 patients with osteopetrosis was compiled. Four femoral neck fractures treated by closed reduction and internal fixation had a satisfactory result, but three treated nonoperatively developed varus and required osteotomy. A total of 20 hips was treated for coxa vara by various means, none of which was free of complications. Valgus osteotomy, when used as the primary treatment for coxa vara, was the most consistently satisfactory procedure, whereas in situ pinning failed in two of three hips. Fourteen subtrochanteric fractures and 31 other fractures of the femur were treated. Good results were reported with traction or casting or both in the majority of those fractures. Twenty-nine tibia fractures were treated successfully, the majority by nonoperative means. Upper extremity fractures healed well with closed reduction and casting. Vertebral fractures, spondylolysis, and back pain were most frequently treated without surgery.     

Retrospective analysis of the Beijing family of Mycobacterium tuberculosis in preserved lung tissues

Direct repeat spoligotyping of 85 paraffin-embedded lung biopsies was used to investigated the occurrence around Beijing of the Beijing family of Mycobacterium tuberculosis. Samples ranged in time from 1956 to 1990. Hybridization patterns were found with 49 (58%) samples, and 45 (92%) produced typical Beijing family patterns extending over the 34-year period.     

Assembly and analysis of conical models for the HIV-1 core

The genome of the human immunodeficiency virus (HIV) is packaged within an unusual conical core particle located at the center of the infectious virion. The core is composed of a complex of the NC (nucleocapsid) protein and genomic RNA, surrounded by a shell of the CA (capsid) protein. A method was developed for assembling cones in vitro using pure recombinant HIV-1 CA-NC fusion proteins and RNA templates. These synthetic cores are capped at both ends and appear similar in size and morphology to authentic viral cores. It is proposed that both viral and synthetic cores are organized on conical hexagonal lattices, which by Euler's theorem requires quantization of their cone angles. Electron microscopic analyses revealed that the cone angles of synthetic cores were indeed quantized into the five allowed angles. The viral core and most synthetic cones exhibited cone angles of approximately 19 degrees (the narrowest of the allowed angles). These observations suggest that the core of HIV is organized on the principles of a fullerene cone, in analogy to structures recently observed for elemental carbon.     

Developing a culture of lifelong learning in a library environment

Between 1995 and 1996, the Annette and Irwin Eskind Biomedical Library (EBL) at Vanderbilt University Medical Center (VUMC) radically revised the model of service it provides to the VUMC community. An in-depth training program was developed for librarians, who began to migrate to clinical settings and establish clinical librarianship and information brokerage services beyond the library's walls. To ensure that excellent service would continue within the library, EBL's training program was adapted for library assistants, providing them with access to information about a wide variety of work roles and processes over a four to eight-month training period. Concurrently, customer service areas were reorganized so that any question--whether reference or circulation--could be answered at any of four service points, eliminating the practice of passing customers from person to person between the reference and circulation desks. To provide an incentive for highly trained library assistants to remain at EBL, management and library assistants worked together to redesign the career pathway based on defined stages of achievement, self-directed participation in library-wide projects, and demonstrated commitment to lifelong learning. Education and training were the fundamental principles at the center of all this activity.     

Stable association of PYK2 and p130(Cas) in osteoclasts and their co-localization in the sealing zone

Bone resorption is initiated by osteoclast attachment to the mineralized matrix, cytoskeletal reorganization, cellular polarization, and the formation of the sealing zone. The present study examines the interaction between PYK2 and p130(Cas) (Crk-associated substrate), suggested to be part of the signaling pathway initiated by osteoclast adhesion. Using murine osteoclast-like cells (OCLs) and their mononuclear precursors (pOCs), generated in a co-culture of bone marrow and osteoblastic MB1.8 cells, we show that: 1) p130(Cas) is tyrosine-phosphorylated upon adhesion of pOCs to vitronectin or ligation of beta3 integrins; 2) p130(Cas) colocalizes with PYK2 and the cytoskeletal proteins F-actin, vinculin, and paxillin in the podosomal-rich ring-like structures of OCLs plated on glass and in the sealing zone in actively resorbing OCLs on bone; 3) p130(Cas) and PYK2 form a stable complex in pOCs, independent of tyrosine phosphorylation of either molecule, and this complex is present in Src (-/-) OCLs, in which neither protein is phosphorylated or associated with the osteoclast adhesion structure; 4) the association of p130(Cas) and PYK2 is mediated by the SH3 domain of p130(Cas) and the C-terminal domain of PYK2. These findings suggest that p130(Cas) and its association with PYK2 may play an important role in the adhesion-dependent signaling that leads to cytoskeletal reorganization and formation of the sealing zone during osteoclast activation.     

Enhanced oxygen delivery reverses anaerobic metabolic states in prolonged sandwich rat hepatocyte culture

It must be assumed that current petri dish primary hepatocyte culture models do not supply sufficient amounts of oxygen and thus cause anaerobic metabolism of the cells. This is contrary to the physiologic state of the cells. In vivo the liver is a highly vascularized organ with a rather high blood flow rate of a mixture of arterial and venous blood. The aim of the present study was to show the oxygen dependence of primary rat hepatocytes in long-term culture and to define appropriate conditions that could allow hepatocytes to maintain tissue specific functions in an aerobic environment. To this purpose matrix overlaid hepatocytes were either cultured on gas-permeable (fluorinated hydrocarbon films) or gas-impermeable (polystyrene) supports at 10% and 20% ambient oxygen concentration (v/v), respectively. Tissue-specific functions were assessed by studying albumin and urea secretion as well as xenobiotic metabolism. The mRNA expression and catalytic activities of the cytoprotective antioxidant enzymes mitochondrial manganese superoxide dismutase (MnSOD), cytosolic copper and zinc superoxide dismutase, peroxisomal catalase, and cytosolic glutathione peroxidase were investigated to assess intracellular responses to the defined variations in oxygen supply. Hepatocytes could successfully be maintained at aerobic conditions in long-term culture on gas-permeable PTFE films. At 50% (10%, v/v) of currently used oxygen levels lactate accumulation was prevented, a plateau-like albumin secretion reestablished, urea secretion improved, and xenobiotic metabolism proceeded at physiological rates. mRNA expression of cytoprotective enzymes responded to the pericellular availability of oxygen and was most pronounced in the case of MnSOD. However, the biggest stress factor for the hepatocytes still appeared to be the isolation procedure, as mRNA expression and catalytic activities were most elevated shortly thereafter. In conclusion, this study clearly shows the oxygen dependence of primary rat hepatocytes in long-term culture and indicates means to establish appropriate conditions for the aerobic culture of primary rat sandwich hepatocytes with full maintenance of function. The long-term culture of hepatocytes on oxygenating supports at in vivo-like oxygen tensions therefore appears to be more physiologic and beneficial for the cells.     

Dynamic compatibility testing of DMP 840, an experimental antitumor agent

The purpose of this study is to evaluate the potential for DMP 840, a novel experimental antitumor agent, to precipitate during injection or dilution with infusion solutions. The influence of predilution of the drug solution before injection and addition of buffers to the drug vehicle were also investigated. The compatibility of normal saline solution, pH 7.4 phosphate buffers, and human plasma with DMP 840 was examined in vitro under both static conditions and dynamic flow. The combination of DMP 840 solutions with normal saline solution resulted in conversion of the drug to an insoluble dihydrochloride salt. Under conditions of dynamic flow, precipitation, accompanied by large changes in turbidity, occurred at relatively high concentrations of the drug in the injection solution. Dilution of the injection solution below 2 mg/mL or slow injection avoided precipitation. As was the case with the normal saline system, turbidity changes after injection into protein-phosphate buffer (PPB) were dependent on the initial concentration of DMP 840 solution as well as the rate of administration. In addition, the maximum injection rate at which complete miscibility occurred increased exponentially as the drug injection solution was made more dilute. Buffering the DMP 840 injection solution with acetate buffer improved the miscibility of DMP 840 with PPB, which indicated that the turbidity increases were most likely due to conversion of the drug to its insoluble free base form. The observed effects of the buffer on the turbidity response agreed qualitatively with predictions from a graphical approach that considers the effects of dilution and pH changes on drug solubility. Despite these observations, no evidence for the formation of a solid precipitate could be found after injection of the unbuffered drug solution into PPB. Further investigation indicated that the presence of albumin in the PPB prevented the formation of a solid phase during injection. Likewise, fresh human plasma, spiked with 1 and 2 mg/mL solutions of DMP 840, showed no evidence for the formation of a solid precipitate.     

Cellular levels of factor 390 and methanogenic enzymes during growth of Methanobacterium thermoautotrophicum deltaH

Methanobacterium thermoautotrophicum deltaH was grown in a fed-batch fermentor and in a chemostat under a variety of 80% hydrogen-20% CO2 gassing regimes. During growth or after the establishment of steady-state conditions, the cells were analyzed for the content of adenylylated coenzyme F420 (factor F390-A) and other methanogenic cofactors. In addition, cells collected from the chemostat were measured for methyl coenzyme M reductase isoenzyme (MCR I and MCR II) content as well as for specific activities of coenzyme F420-dependent and H2-dependent methylenetetrahydromethanopterin dehydrogenase (F420-MDH and H2-MDH, respectively), total (viologen-reducing) and coenzyme F420-reducing hydrogenase (FRH), factor F390 synthetase, and factor F390 hydrolase. The experiments were performed to investigate how the intracellular F390 concentrations changed with the growth conditions used and how the variations were related to changes in levels of enzymes that are known to be differentially expressed. The levels of factor F390 varied in a way that is consistently understood from the biochemical mechanisms underlying its synthesis and degradation. Moreover, a remarkable correlation was observed between expression levels of MCR I and II, F420-MDH, and H2-MDH and the cellular contents of the factor. These results suggest that factor F390 is a reporter compound for hydrogen limitation and may act as a response regulator of methanogenic metabolism.     

Isolation of MutSbeta from human cells and comparison of the mismatch repair specificities of MutSbeta and MutSalpha

A human MSH2-human MSH3 (hMSH2.hMSH3) complex of approximately 1:1 stoichiometry (human MutSbeta (hMutSbeta)) has been demonstrated in several human tumor cell lines and purified to near homogeneity. In vitro, hMutSbeta supports the efficient repair of insertion/deletion (I/D) heterologies of 2-8 nucleotides, is weakly active on a single-nucleotide I/D mispair, and is not detectably active on the eight base-base mismatches. Human MutSalpha (hMutSalpha), a heterodimer of hMSH2 and hMSH6, efficiently supports the repair of single-nucleotide I/D mismatches, base-base mispairs, and all substrates tested that were repaired by hMutSbeta. Thus, the repair specificities of hMutSalpha and hMutSbeta are redundant with respect to the repair of I/D heterologies of 2-8 nucleotides. The hMutSalpha level in repair-proficient HeLa cells (1.5 microg/mg nuclear extract) is approximately 10 times that of hMutSbeta. In HCT-15 colorectal tumor cells, which do not contain hMSH6 and consequently lack hMutSalpha, the hMutSbeta level is elevated severalfold relative to that in HeLa cells and is responsible for the repair of I/D mismatches that has been observed in this cell line. LoVo tumor cells, which are genetically deficient in hMSH2, lack both hMutSalpha and hMutSbeta, and hMSH3 and hMSH6 levels are less than 4% of those found in repair-proficient cells. Coupled with previous findings (J. T. Drummond, J. Genschel, E. Wolf, and P. Modrich (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10144-10149), these results suggest that hMSH2 partitions between available pools of hMSH3 and hMSH6 and indicate that hMSH2 positively modulates hMSH6 and hMSH3 levels, perhaps by stabilization of the polypeptides upon heterodimer formation.