Collagen in tissue-engineered cartilage: Types,structure, and crosslinks

The function of articular cartilage as a weight-bearing tissue depends on the specific arrangement of collagen types II and IX into a three-dimensional organized collagen network that can balance the swelling pressure of the proteoglycan/ water gel. To determine whether cartilage engineered in vitro contains a functional collagen network, chondrocyte-polymer constructs were cultured for up to 6 weeks and analyzed with respect to the composition and ultrastructure of collagen by using biochemical and immunochemical methods and scanning electron microscopy. Total collagen content and the concentration of pyridinium crosslinks were significantly (57% and 70%, respectively) lower in tissue-engineered cartilage that in bovine calf articular cartilage. However, the fractions of collagen types II, IX, and X and the collagen network organization, density, and fibril diameter in engineered cartilage were not significantly different from those in natural articular cartilage. The implications of these findings for the field of tissue engineering are that differentiated chondrocytes are capable of forming a complex structure of collagen matrix in vitro, producing a tissue similar to natural articular cartilage on an ultrastructural scale. J. Cell. Biochem. 71:313–327, 1998. © 1998 Wiley-Liss, Inc.     

Postmortem diagnosis and toxicological validation of illicit substance use.

The present study examines the diagnostic challenges of identifying ante-mortem illicit substance use in human postmortem cases. Substance use, assessed by clinical case history reviews, structured next-of-kin interviews, by general toxicology of blood, urine and/or brain, and by scalp hair testing, identified 33 cocaine, 29 cannabis, 10 phencyclidine and nine opioid cases. Case history identified 42% cocaine, 76% cannabis, 10% phencyclidine and 33% opioid cases. Next-of-kin interviews identified almost twice as many cocaine and cannabis cases as Medical Examiner (ME) case histories, and were crucial in establishing a detailed lifetime substance use history. Toxicology identified 91% cocaine, 68% cannabis, 80% phencyclidine and 100% opioid cases, with hair testing increasing detection for all drug classes. A cocaine or cannabis use history was corroborated by general toxicology with 50% and 32% sensitivity, respectively, and with 82% and 64% sensitivity by hair testing. Hair testing corroborated a positive general toxicology for cocaine and cannabis with 91% and 100% sensitivity, respectively. Case history corroborated hair toxicology with 38% sensitivity for cocaine and 79% sensitivity for cannabis, suggesting that both case history and general toxicology underestimated cocaine use. Identifying ante-mortem substance use in human postmortem cases are key considerations in case diagnosis and for characterization of disorder-specific changes in neurobiology. The sensitivity and specificity of substance use assessments increased when ME case history was supplemented with structured next-of-kin interviews to establish a detailed lifetime substance use history, while comprehensive toxicology, and hair testing in particular, increased detection of recent illicit substance use.     

Functional replacement of a retroviral late domain by ubiquitin fusion

Retroviral Gag polyprotein precursors are both necessary and sufficient for the assembly and release of virus-like particles (VLPs) from infected cells. It is well established that small Gag-encoded motifs, known as late domains, promote particle release by interacting with components of the cellular endosomal sorting and ubiquitination machinery. The Gag proteins of a number of different retroviruses are ubiquitinated; however, the role of Gag ubiquitination in particle egress remains undefined. In this study, we investigated this question by using a panel of equine infectious anemia virus (EIAV) Gag derivatives bearing the wild-type EIAV late domain, heterologous retroviral late domains or no late domain. Ubiquitin was fused in cis to the C-termini of these Gag polyproteins, and the effects on VLP budding were measured. Remarkably, fusion of ubiquitin to EIAV Gag lacking a late domain (EIAV/DeltaYPDL-Ub) largely rescued VLP release. We also determined the effects of ubiquitin fusion on the sensitivity of particle release to budding inhibitors and to depletion of key endosomal sorting factors. Ubiquitin fusion rendered EIAV/DeltaYPDL-Ub sensitive to depletion of cellular endosomal sorting factors Tsg101 and Alix and to overexpression of dominant-negative fragments of Tsg101 and Alix. These findings demonstrate that ubiquitin can functionally compensate for the absence of a retroviral late domain and provide insights into the host-cell machinery engaged by ubiquitin during particle egress.     

How much the eye tells the brain

In the classic "What the frog's eye tells the frog's brain," Lettvin and colleagues showed that different types of retinal ganglion cell send specific kinds of information. For example, one type responds best to a dark, convex form moving centripetally (a fly). Here we consider a complementary question: how much information does the retina send and how is it apportioned among different cell types? Recording from guinea pig retina on a multi-electrode array and presenting various types of motion in natural scenes, we measured information rates for seven types of ganglion cell. Mean rates varied across cell types (6-13 bits . s(-1)) more than across stimuli. Sluggish cells transmitted information at lower rates than brisk cells, but because of trade-offs between noise and temporal correlation, all types had the same coding efficiency. Calculating the proportions of each cell type from receptive field size and coverage factor, we conclude (assuming independence) that the approximately 10(5) ganglion cells transmit on the order of 875,000 bits . s(-1). Because sluggish cells are equally efficient but more numerous, they account for most of the information. With approximately 10(6) ganglion cells, the human retina would transmit data at roughly the rate of an Ethernet connection.     

Molecular origin of electron paramagnetic resonance line shapes on β-barrel membrane proteins: the local solvation environment modulates spin-label configuration

In this work, electron paramagnetic resonance (EPR) spectroscopy and X-ray crystallography were used to examine the origins of EPR line shapes from spin-labels at the protein-lipid interface on the β-barrel membrane protein BtuB. Two atomic-resolution structures were obtained for the methanethiosulfonate spin-label derivatized to cysteines on the membrane-facing surface of BtuB. At one of these sites, position 156, the label side chain resides in a pocket formed by neighboring residues; however, it extends from the protein surface and yields a single-component EPR spectrum in the crystal that results primarily from fast rotation about the fourth and fifth bonds linking the spin-label to the protein backbone. In lipid bilayers, site 156 yields a multicomponent spectrum resulting from different rotameric states of the labeled side chain. Moreover, changes in the lipid environment, such as variations in bilayer thickness, modulate the EPR spectrum by modulating label rotamer populations. At a second site, position 371, the labeled side chain interacts with a pocket on the protein surface, leading to a highly immobilized single-component EPR spectrum that is not sensitive to hydrocarbon thickness. This spectrum is similar to that seen at other sites that are deep in the hydrocarbon, such as position 170. This work indicates that the rotameric states of spin-labels on exposed hydrocarbon sites are sensitive to the environment at the protein-hydrocarbon interface, and that this environment may modulate weak interactions between the labeled side chain and the protein surface. In the case of BtuB, lipid acyl chain packing is not symmetric around the β-barrel, and EPR spectra from labeled hydrocarbon-facing sites in BtuB may reflect this asymmetry. In addition to facilitating the interpretation of EPR spectra of membrane proteins, these results have important implications for the use of long-range distance restraints in protein structure refinement that are obtained from spin-labels.     

Pathological Mechanism for Delayed Hyperenhancement of Chronic Scarred Myocardium in Contrast Agent Enhanced Magnetic Resonance Imaging

Objectives

To evaluate possible mechanism for delayed hyperenhancement of scarred myocardium by investigating the relationship of contrast agent (CA) first pass and delayed enhancement patterns with histopathological changes.

Materials and Methods

Eighteen pigs underwent 4 weeks ligation of 1 or 2 diagonal coronary arteries to induce chronic infarction. The hearts were then removed and perfused in a Langendorff apparatus. The hearts firstly experienced phosphorus 31 MR spectroscopy. The hearts in group I (n = 9) and II (n = 9) then received the bolus injection of Gadolinium diethylenetriamine pentaacetic acid (0.05 mmol/kg) and gadolinium-based macromolecular agent (P792, 15 µmol/kg), respectively. First pass T₂ ^* MRI was acquired using a gradient echo sequence. Delayed enhanced T₁ MRI was acquired with an inversion recovery sequence. Masson''s trichrome and anti- von Willebrand Factor (vWF) staining were performed for infarct characterization.

Results

Wash-in of both kinds of CA caused the sharp and dramatic T₂ ^* signal decrease of scarred myocardium similar to that of normal myocardium. Myocardial blood flow and microvessel density were significantly recovered in 4-week-old scar tissue. Steady state distribution volume (ΔR₁ relaxation rate) of Gd-DTPA was markedly higher in scarred myocardium than in normal myocardium, whereas ΔR₁ relaxation rate of P792 did not differ significantly between scarred and normal myocardium. The ratio of extracellular volume to the total water volume was significantly greater in scarred myocardium than in normal myocardium. Scarred myocardium contained massive residual capillaries and dilated vessels. Histological stains indicated the extensively discrete matrix deposition and lack of cellular structure in scarred myocardium.

Conclusions

Collateral circulation formation and residual vessel effectively delivered CA into scarred myocardium. However, residual vessel without abnormal hyperpermeability allowed Gd-DTPA rather than P792 to penetrate into extravascular compartment. Discrete collagen fiber meshwork and loss of cellularity enlarged extracellular space accessible to Gd-DTPA, resulting in the delayed hyper-enhanced scar.     

Differential glycosylation, virion incorporation, and sensitivity to neutralizing antibodies of human immunodeficiency virus type 1 envelope produced from infected primary T-lymphocyte and macrophage cultures.

Two primary cell targets for human immunodeficiency virus type 1 (HIV-1) infection in vivo are CD4+ T lymphocytes and monocyte-derived macrophages (MDM). HIV-1 encodes envelope glycoproteins which mediate virus entry into these cells. We have utilized infected and radiolabelled primary peripheral blood mononuclear cell (PBMC) and MDM cultures to examine the biochemical and antigenic properties of the HIV-1 envelope produced in these two cell types. The gp120 produced in MDM migrates as a broad, diffuse band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels compared with that of the more homogeneous gp120 released from PBMCs. Glycosidase analyses indicated that the diffuse appearance of the MDM gp120 is due to the presence of asparagine-linked carbohydrates containing lactosaminoglycans, a modification not observed with the gp120 produced in PBMCs. Neutralization experiments, using isogeneic PBMC and MDM-derived macrophage-tropic HIV-1 isolates, indicate that 8- to 10-fold more neutralizing antibody, directed against the viral envelope, is required to block virus produced from MDM. These results demonstrate that HIV-1 released from infected PBMC and MDM cultures differs in its biochemical and antigenic properties.