Electron microscopic investigations on aging and osteoarthrotic human articular cartilage

Summary The submicroscopic morphology of freeze-etched aging human articular cartilage is reported. In contrast to chemically fixed and thin sectioned cartilage tissue, chondrocytes and matrix of freeze-etched specimens exhibit new morphological aspects.The surface of superficial chondrooytes shows invaginations and bulges, producing a cauliflower-like appearance of the cell body. Finger-like protrusions as found in thin sections are absent. The matrix adjacent to the pericellular halo, the latter consisting of a fine granular material, is characterized by the presence of globular, membrane-bounded vesicles of variable size. This vesicle containing zone is called corona. The corona vesicles as well as the cellular membranes are occupied by two types of particles (85 and 135 Å in diameter). Based on the presence of these particles, the nature and possible origin of the corona vesicles are discussed.     

Induction and prevention of chondrocyte hypertrophy in culture

Primary chondrocytes from whole chick embryo sterna can be maintained in suspension culture stabilized with agarose for extended periods of time. In the absence of FBS, the cells remain viable only when seeded at high densities. They do not proliferate at a high rate but they deposit extracellular matrix with fibrils resembling those of authentic embryonic cartilage in their appearance and collagen composition. The cells exhibit many morphological and biochemical characteristics of resting chondrocytes and they do not produce collagen X, a marker for hypertrophic cartilage undergoing endochondral ossification. At low density, cells survive in culture without FBS when the media are conditioned by chondrocytes grown at high density. Thus, resting cartilage cells in agarose cultures can produce factors required for their own viability. Addition of FBS to the culture media leads to profound changes in the phenotype of chondrocytes seeded at low density. Cells form colonies at a high rate and assume properties of hypertrophic cells, including the synthesis of collagen X. They extensively deposit extracellular matrix resembling more closely that of adult rather than embryonic cartilage.     

Isolation of a subfraction of rough endoplasmic reticulum closely associated with mitochondria : Evidence for its role in cytochrome P450 synthesis

A subfraction of rough endoplasmic reticulum (RER) structurally associated with mitochondria (mito-RER complexes) was isolated from crude nuclear fractions of rat liver homogenate. When apocytochrome P450 synthesis (which presumably occurs in RER) and mitochondrial heme synthesis was dissociated by concomitant treatment of rats with phenobarbital and cobaltous chloride, apocytochrome P450 accumulated predominantly in mito-RER complexes. These data suggest that cytochrome P450 synthesis requires structural interaction of mitochondria and RER.     

MAHRP-1, a novel Plasmodium falciparum histidine-rich protein,binds ferriprotoporphyrin IX and localizes to the Maurer's clefts

Using a stage-specific cDNA library from Plasmodium falciparum we have identified a gene coding for a novel histidine-rich protein (MAHRP-1). The gene is exclusively transcribed during early erythrocyte stages and codes for a small transmembrane protein. The C-terminal region contains a polymorphic stretch of histidine-rich repeats. Fluorescence microscopy studies using polyclonal mouse sera revealed that MAHRP-1 is located at the Maurer's clefts, which represent parasite-induced structures within the cytosol of infected erythrocytes. Biochemical studies showed that recombinant MAHRP-1 binds the toxic hemoglobin degradation product, ferriprotoporphyrin (FP) with a submicromolar dissociation constant and a stoichiometry determined by the number of DHGH motifs. The bound FP has increased peroxidase-like activity and is 10-fold more susceptible to H2O2-induced degradation compared with unbound FP. These properties of MAHRP-1 suggest it may play a protective role against oxidative stress, and its location at the Maurer's clefts suggests a function in promoting the correct trafficking of exported proteins, such as P. falciparum erythrocyte membrane protein-1.     

Selective Condensation Drives Partitioning and Sequential Secretion of Cyst Wall Proteins in Differentiating Giardia lamblia

Controlled secretion of a protective extracellular matrix is required for transmission of the infective stage of a large number of protozoan and metazoan parasites. Differentiating trophozoites of the highly minimized protozoan parasite Giardia lamblia secrete the proteinaceous portion of the cyst wall material (CWM) consisting of three paralogous cyst wall proteins (CWP1–3) via organelles termed encystation-specific vesicles (ESVs). Phylogenetic and molecular data indicate that Diplomonads have lost a classical Golgi during reductive evolution. However, neogenesis of ESVs in encysting Giardia trophozoites transiently provides basic Golgi functions by accumulating presorted CWM exported from the ER for maturation. Based on this “minimal Golgi” hypothesis we predicted maturation of ESVs to a trans Golgi-like stage, which would manifest as a sorting event before regulated secretion of the CWM. Here we show that proteolytic processing of pro-CWP2 in maturing ESVs coincides with partitioning of CWM into two fractions, which are sorted and secreted sequentially with different kinetics. This novel sorting function leads to rapid assembly of a structurally defined outer cyst wall, followed by slow secretion of the remaining components. Using live cell microscopy we find direct evidence for condensed core formation in maturing ESVs. Core formation suggests that a mechanism controlled by phase transitions of the CWM from fluid to condensed and back likely drives CWM partitioning and makes sorting and sequential secretion possible. Blocking of CWP2 processing by a protease inhibitor leads to mis-sorting of a CWP2 reporter. Nevertheless, partitioning and sequential secretion of two portions of the CWM are unaffected in these cells. Although these cysts have a normal appearance they are not water resistant and therefore not infective. Our findings suggest that sequential assembly is a basic architectural principle of protective wall formation and requires minimal Golgi sorting functions.     

Superior wood for violins--wood decay fungi as a substitute for cold climate

Violins produced by Antonio Stradivari during the late 17th and early 18th centuries are reputed to have superior tonal qualities. Dendrochronological studies show that Stradivari used Norway spruce that had grown mostly during the Maunder Minimum, a period of reduced solar activity when relatively low temperatures caused trees to lay down wood with narrow annual rings, resulting in a high modulus of elasticity and low density. The main objective was to determine whether wood can be processed using selected decay fungi so that it becomes acoustically similar to the wood of trees that have grown in a cold climate (i.e. reduced density and unchanged modulus of elasticity). This was investigated by incubating resonance wood specimens of Norway spruce (Picea abies) and sycamore (Acer pseudoplatanus) with fungal species that can reduce wood density, but lack the ability to degrade the compound middle lamellae, at least in the earlier stages of decay. Microscopic assessment of the incubated specimens and measurement of five physical properties (density, modulus of elasticity, speed of sound, radiation ratio, and the damping factor) using resonance frequency revealed that in the wood of both species there was a reduction in density, accompanied by relatively little change in the speed of sound. Thus, radiation ratio was increased from 'poor' to 'good', on a par with 'superior' resonance wood grown in a cold climate.