Acoustic stiffness and change in plug cartilage over time after autologous osteochondral grafting: correlation between ultrasound signal intensity and histological score in a rabbit model

We investigated quantitative changes over time in ultrasound signal intensity (an index of stiffness), signal duration (an index of surface irregularity), and interval between signals (an index of thickness) of plug cartilage in an animal model of autologous osteochondral grafting. A full-thickness osteochondral plug was surgically removed and replaced in male Japanese white rabbits (n = 22). Specimens obtained at day 0 and weeks 2, 4, 8, 12 and 24 postoperatively were assessed using an ultrasound system and by macroscopic and histological evaluation (modified Mankin's score). Histology revealed that the plug sank until 2 weeks postoperatively, and that newly formed cartilage-like tissue covered the plug, but at 24 weeks the tissue detached. The plug itself survived well throughout the period of observation. Although the signal intensity at the plug site was same as that in the sham operated contralateral knee at day 0, from 2 to 24 weeks postoperatively it was less than that in the sham knee. At 8 weeks, this difference was significant (P < 0.05). Modified Mankin's score revealed early degenerative changes at the site, but macroscopic examination did not. Signal intensity correlated significantly with score (both at day 0 and at the five postoperative time points [P < 0.05, r = -0.91] and as a whole [P < 0.05, r = -0.36]). Signal intensity also significantly correlated with the individual subscores for 'cartilage structure' (P < 0.05, r = -0.32) and 'cartilage cells' (P < 0.05, r = -0.30) from the modified Mankin's score, but not significantly with subscores for 'staining' and 'tidemark'. Signal duration correlated significantly with total score (as a whole [P < 0.05, r = 0.34]), but not significantly with the score for cartilage structure (P = 0.0557, r = 0.29). The interval between signals reflected well the actual thickness of the plug site. The significant relationships between ultrasound signal intensity and scores suggest that early degenerative changes in plug cartilage and cartilage-like tissue, especially in the superficial layer, are detectable by high-frequency ultrasound assessment.     

Dorsal telencephalon-specific expression of Cre recombinase in PAC transgenic mice

The ability to restrict gene expression or disruption to specific regions of the brain would enhance understanding of the molecular basis for brain development and function. For this purpose, brain region-restricted promoters are essential. Here we report the isolation of a DNA fragment containing the Emx1 gene promoter, which is responsible for dorsal telencephalon-specific expression. The Cre recombinase gene was inserted into a mouse PAC (P1-derived artificial chromosome) Emx1-locus clone (PAC-Emx1#1 clone) and utilized to generate three transgenic mouse lines. In all three lines, especially Tg3, Cre-mediated recombination was highly restricted to Emx1-expressing cell lineages, from embryonic stages to adulthood. Immunohistochemical analyses showed that Cre protein is expressed in the dorsal telencephalon in all three lines in adulthood. Thus, the PAC-Emx1#1 clone contains essentially all regulatory elements necessary for Emx1 gene expression. Our results suggest that Emx1-Cre Tg3 mice and the PAC-Emx1#1 clone constitute powerful tools for dorsal telencephalon-specific gene manipulation.     

Caspase-1 and -3 mRNAs are differentially upregulated in motor neurons and glial cells in mutant SOD1 transgenic mouse spinal cord: a study using laser microdissection and real-time RT-PCR

Amyotrophic lateral sclerosis is characterized by selective motor neuron degeneration. An apoptotic pathway is thought to be involved. It is difficult, however, to analyze the molecular pathogenic mechanism in single motor neurons because of complexity in the neural tissue, which consists of multiple lineages of cells neighboring motor neurons. We quantified the caspase-1 and -3 mRNA in single motor neurons and neighboring glial cells isolated from the spinal ventral horn of mutant SOD1 transgenic (Tg) mice and littermates. Motor neurons and neighboring glial cells were isolated from spinal sections by laser microdissection, and the mRNAs were quantified by RT-PCR. In the Tg mice, caspase-1 mRNA was first upregulated in motor neurons and second in glial cells. The caspase-3 mRNA was increased in motor neurons following the caspase-1 mRNA. These results indicated that caspase-1 and -3 mRNAs are differentially upregulated in motor neurons and glial cells of the Tg mice, and that mRNAs in isolated cells can be accurately assessed using our procedures.     

Isolation and molecular characterization of a nelfinavir (NFV)-resistant human immunodeficiency virus type 1 that exhibits NFV-dependent enhancement of replication

During the use of a phenotypic anti-human immunodeficiency virus type 1 (HIV-1) drug resistance assay in a large set of clinical virus isolates, we found a unique variant (CL-4) that exhibited a high level of nelfinavir (NFV) resistance and rather enhanced replication under subinhibitory concentrations of NFV (0.001 to 0.1 micro M). Comparison of gag-pol sequences of the CL-4 variant and its predecessor virus isolates showed a stepwise accumulation of a total of 19 amino acid substitutions in protease (PR) and Gag p17 during 32-month NFV-containing antiretroviral therapy, while other Gag regions including the cleavage sites of the p55 precursor remained highly conserved. To understand the relationship between the genetic and phenotypic changes in CL-4, we constructed chimeric viruses using pNL4-3, replacing the PR, p24PR, or p17PR gene segment of CL-4 or its predecessor. A series of tissue culture infections with the chimeras in the absence or presence of increasing concentrations of NFV demonstrated that only the p17PR segment of CL-4 could confer the NFV-dependent replication enhancement phenotype on NL4-3. Our data suggest a novel adaptation mechanism of HIV-1 to NFV, in which coevolution of Gag and PR genes generates a variant that replicates more efficiently in the cellular environment in the presence of NFV than without the drug.     

Protein region important for regulation of lipid metabolism in angiopoietin-like 3 (ANGPTL3): ANGPTL3 is cleaved and activated in vivo

Angiopoietin-like 3 (ANGPTL3) is a secreted protein that is mainly expressed in the liver and regulates lipid metabolism by inhibiting the lipolysis of triglyceriderich lipoproteins. Using deletion mutants of human ANGPTL3, we demonstrated that the N-terminal coiled-coil domain-containing fragment-(17-207) and not the C-terminal fibrinogen-like domain-containing fragment-(207-460) increased the plasma triglyceride levels in mice. We also found that the N-terminal region 17-165 was required to increase plasma triglyceride levels in mice and that a substitution of basic amino acid residues in the region 61-66 of the fragment showed no increase in the plasma triglyceride levels and no inhibition of lipolysis by lipoprotein lipase. In addition, when we analyzed ANGPTL3 in human plasma, we detected cleaved fragments of ANGPTL3. By analyzing recombinant ANGPTL3 in mouse plasma, we found that it was cleaved at two sites, Arg221 downward arrow Ala222 and Arg224 downward arrow Thr225, which are located in the linker region between the coiled-coil domain and the fibrinogen-like domain. Furthermore, a cleavage-resistant mutant of ANGPTL3 was determined to be less active than wild-type ANGPTL3 in increasing mouse plasma triglyceride levels but not in inhibiting lipoprotein lipase activity. These findings suggest that the cleavage of ANGPTL3 is important for the activation of ANGPTL3 in vivo.     

Synthesis of a high-density lipoprotein in the developing blue crab (Callinectes sapidus)

An important lipoprotein in the hemolymph of crustaceans is LpI. It transports lipid to peripheral tissues and also has a role in crustacean immune recognition. We employed a monoclonal antibody specific for the LpI peptide to demonstrate by ELISA, western blot and immunohistochemistry the appearance of LpI during development of Callinectes sapidus, the blue crab. LpI was first found in stage 5 embryos and appeared to be synthesized by lateral basophilic cuboidal cells that demonstrated cytoplasmic immunoreactivity for LpI at their interface with the yolk mass. The embryonic cuboidal cells bore a strong cytologic resemblance to the hepatopancreas cells of later stages (zoea, megalopae, adults), which were also immunoreactive for LpI.     

Increase in IP3 and intracellular Ca2+ induced by phosphate depletion in LLC-PK 1 cells

The mechanisms by which Pi depletion rapidly regulates gene expression and cellular function have not been clarified. Here, we found a rapid increase in intracellular ionized calcium [Ca(2+)](i) by phosphate depletion in LLC-PK(1) cells using confocal microscopy with the green-fluorescence protein based calcium indicator "yellow cameleon 2.1." The increase of [Ca(2+)](i) was observed in the presence or absence of extracellular Ca(2+). At the same time, an approximately twofold increase in intracellular inositol 1,4,5-triphosphate (IP(3)) occurred in response to the acute Pi depletion in the medium. Furthermore, 2-aminoethoxydiphenyl borate completely blocked the [Ca(2+)](i) increase induced by Pi depletion. These results suggest that Pi depletion causes IP(3)-mediated release of Ca(2+) from intracellular Ca(2+) pools and rapidly increases [Ca(2+)](i) in LLC-PK(1) cells.     

Structure and physicochemical properties of starches from kidney bean seeds at immature,premature and mature stages of development

Starches from kidney bean (Phaseolus vulgaris L. cv. Toramame) seeds at the immature, premature, mature stages of development were examined. The starch content increased from 94, 219 to 265 mg per seed. Starches showed the C(a)-crystalline type composed of small (<5 micrometer) and large (10-35 micrometer) granules, with the large granules largely increasing with maturity. The amylose content increased from 21, 26 to 27%, and rapid viscograms and DSC thermograms suggested that the mature-stage starch was gelatinized with ease. The amylose increased in size from DPn 820, 1000 to 1080 and a number of chains per molecule (NC) from 3.3, 4.2 to 4.5. The branched amylose was a minor component (11-18% by mole) with NC 20-22. The amylopectin was similar in CL (23), beta-amylolysis limit (59%), and chain-length distribution, but reduced in size (DPn 17,100-5270) and increased in content of phosphorus (114-174 ppm) with an increase in the amount of phosphorus linked to C-6 of the glucose residue (8-66%).     

Proteasome inhibition induces inclusion bodies associated with intermediate filaments and fragmentation of the Golgi apparatus

The ubiquitin-proteasome system is involved in a variety of biological processes. Inclusion bodies associated with intermediate filaments (IFs) and ubiquitin are observed in various diseases; however, the precise mechanisms of formation and the pathological significance of inclusion bodies have not been fully understood. We examined the effect of proteasome inhibitors on the structure of IF using anti-cytokeratin antibodies or transfection of green fluorescent protein-fused cytokeratin 18 in a hepatoma cell line, Huh7. Intracellular organelles were visualized by immunofluorescent and electron microscopies. Proteasome inhibitors induced IF inclusions associated with ubiquitin. Electron microscopic examination revealed inclusion bodies surrounded by filamentous structures. Autophagic vacuoles and lysosomes were frequently observed, and the organization of the Golgi apparatus was disrupted in these cells. After the removal of the proteasome inhibitors, the IF network and organization of the Golgi apparatus were restored. The IF inclusions could be induced by inhibition of the proteasome function. IF inclusions induced fragmentation of the Golgi apparatus and might inhibit the function of this important station of membrane traffic. The IF inclusions disappeared by restoring proteasome function, and autophagy and lysosomal degradation might be, at least in part, associated with the elimination of inclusion bodies.