Importance of collagen orientation and depth-dependent fixed charge densities of cartilage on mechanical behavior of chondrocytes

The collagen network and proteoglycan matrix of articular cartilage are thought to play an important role in controlling the stresses and strains in and around chondrocytes, in regulating the biosynthesis of the solid matrix, and consequently in maintaining the health of diarthrodial joints. Understanding the detailed effects of the mechanical environment of chondrocytes on cell behavior is therefore essential for the study of the development, adaptation, and degeneration of articular cartilage. Recent progress in macroscopic models has improved our understanding of depth-dependent properties of cartilage. However, none of the previous works considered the effect of realistic collagen orientation or depth-dependent negative charges in microscopic models of chondrocyte mechanics. The aim of this study was to investigate the effects of the collagen network and fixed charge densities of cartilage on the mechanical environment of the chondrocytes in a depth-dependent manner. We developed an anisotropic, inhomogeneous, microstructural fibril-reinforced finite element model of articular cartilage for application in unconfined compression. The model consisted of the extracellular matrix and chondrocytes located in the superficial, middle, and deep zones. Chondrocytes were surrounded by a pericellular matrix and were assumed spherical prior to tissue swelling and load application. Material properties of the chondrocytes, pericellular matrix, and extracellular matrix were obtained from the literature. The loading protocol included a free swelling step followed by a stress-relaxation step. Results from traditional isotropic and transversely isotropic biphasic models were used for comparison with predictions from the current model. In the superficial zone, cell shapes changed from rounded to elliptic after free swelling. The stresses and strains as well as fluid flow in cells were greatly affected by the modulus of the collagen network. The fixed charge density of the chondrocytes, pericellular matrix, and extracellular matrix primarily affected the aspect ratios (height/width) and the solid matrix stresses of cells. The mechanical responses of the cells were strongly location and time dependent. The current model highlights that the collagen orientation and the depth-dependent negative fixed charge densities of articular cartilage have a great effect in modulating the mechanical environment in the vicinity of chondrocytes, and it provides an important improvement over earlier models in describing the possible pathways from loading of articular cartilage to the mechanical and biological responses of chondrocytes.     

<Emphasis Type="Italic">MEFV</Emphasis> gene mutations spectrum among Lebanese patients referred for Familial Mediterranean Fever work-up: Experience of a major tertiary care center

Familial Mediterranean Fever (FMF) is an autosomal recessive inflammatory disorder predominantly affecting people living in or originating from areas around the Mediterranean Sea, mainly Jews, Armenians, Turks, and Arabs. It is characterized by recurrent attacks of inflammation of serosal membranes and fever resulting in acute abdominal, chest, or joint pain. Over 50 MEditerranean FeVer (MEFV) mutations and polymorphisms have been identified in FMF patients. The objective of this study was to analyze the distribution and frequencies of 12 MEFV mutations in 266 referred Lebanese patients using a reverse-hybridization assay. Of the 266 patients, 129 (48.5%) were positive for at least one mutation and 137 (51.5%) had no mutations detected. Of the 129 patients with mutations, 35 were homozygous, 41 were compound heterozygous and 53 were heterozygous. The five most common mutations M694V, E148Q, V726A, M694I and M680I (G/C) accounted for 26.1, 22.2, 21.3, 9.6 and 7.7%, respectively. The A744S, F479L, R761H and I692del were encountered in 2.9% of patients; P369S and M680I (G/A) were found in 1.2% of patients while K695R was absent. The spectrum of the MEFV mutations among our sampled Lebanese FMF patients shows the high heterogeneity at the allelic level when compared to Arab and non-Arab populations. The most important feature was the relatively high frequency of the E148Q in our study group that allows us to question it as a mutation rather than a polymorphism. Further studies should be conducted to evaluate the role of the E148Q allele.     

Frequency distribution of the <Emphasis Type="Italic">G/A</Emphasis> alleles of the β-fibrinogen gene in the Lebanese population

Fibrinogen is a plasma protein that has been reported to be associated with an increased risk of atherothrombotic diseases and venous thrombosis. The most common polymorphism that has been studied so far in different populations is the G-455-->A polymorphism in the promoter region of the beta-fibrinogen gene. We studied 160 healthy unrelated Lebanese individuals for the prevalence of -455G/G, -455G/A and -455A/A genotypes of the beta-fibrinogen gene and the frequency of G and A alleles using a reverse hybridization PCR assay. The prevalence of the G/G, G/A, and A/A genotypes were found to be 60.6, 31.9 and 7.5%, respectively. The frequency of the G and A alleles were found to be 0.77 and 0.23, respectively. As compared to other ethnic groups, the Lebanese individuals were found to have a relatively high prevalence of the A allele which may predispose them to develop cardiovascular diseases as well as thrombotic events. This study provides additional unique genetic information pertaining to the Lebanese population.     

Cardiac magnetic resonance imaging of myocardial contrast uptake and blood flow in patients affected with idiopathic or familial dilated cardiomyopathy

Idiopathic dilated cardiomyopathy (IDC) is characterized by left ventricular (LV) enlargement with systolic dysfunction, other causes excluded. When inherited, it represents familial dilated cardiomyopathy (FDC). We hypothesized that IDC or FDC would show with cardiac magnetic resonance (CMR) increased myocardial accumulation of gadolinium contrast at steady state and decreased baseline myocardial blood flow (MBF) due to structural alterations of the extracellular matrix compared with normal myocardium. CMR was performed in nine persons affected with IDC/FDC. Healthy controls came from the general population (n = 6) or were unaffected family members of FDC patients (n = 3) without signs or symptoms of IDC/FDC or any structural cardiac abnormalities. The myocardial partition coefficient for gadolinium contrast (lambda(Gd)) was determined by T1 measurements. LV shape and function and MBF were assessed by standard CMR methods. lambda(Gd) was elevated in IDC/FDC patients vs. healthy controls (lambda(Gd) = 0.56 +/- 0.15 vs. 0.41 +/- 0.06; P = 0.002), and correlated with LV enlargement (r = 0.61 for lambda(Gd) vs. end-diastolic volume indexed by height; P < 0.01) and with ejection fraction (r = -0.80; P < 0.001). The extracellular volume fraction was higher in IDC patients than in healthy controls (0.31 +/- 0.05 vs. 0.24 +/- 0.03; P = 0.002). Resting MBF was lower in IDC patients (0.64 +/- 0.13 vs. 0.91 +/- 0.22; P = 0.01) than unaffected controls and correlated with both the partition coefficient (r = -0.57; P = 0.012) and the extracellular volume fraction (r = -0.56; P = 0.019). The expansion of the extracellular space correlated with reduced MBF and ventricular dilation. Expansion of the extracellular matrix may be a key contributor to contractile dysfunction in IDC patients.     

A direct comparison between the genetic maps of sorghum and rice

A direct comparison of the genetic linkage maps of sorghum and rice is proposed. It is based on the mapping of a common set of 123 RFLP probes scattered on the genomes of both species. For each species a composite map was established by merging two individual maps comprising many common loci. This enabled us to confirm the global correspondence scheme that had previously been established between the chromosomes of sorghum and rice. It also provided a more detailed insight into the conservation of synteny and colinearity: 69% of the loci mapped on a given rice chromosome mapped to the corresponding homoeologous chromosome in sorghum; among them, 84% formed a colinear arrangement between the two species. Local inversions and translocations were detected. Received: 27 April 2000 / Accepted: 26 May 2000     

pH-jump-induced folding and unfolding studies of barstar: evidence for multiple folding and unfolding pathways.

Equilibrium and kinetic characterization of the high pH-induced unfolding transition of the small protein barstar have been carried out in the pH range 7-12. A mutant form of barstar, containing a single tryptophan, Trp 53, completely buried in the core of the native protein, has been used. It is shown that the protein undergoes reversible unfolding above pH 10. The pH 12 form (the D form) appears to be as unfolded as the form unfolded by 6 M guanidine hydrochloride (GdnHCl) at pH 7 (the U form): both forms have similar fluorescence and far-UV circular dichroism (CD) signals and have similar sizes, as determined by dynamic light scattering and size-exclusion chromatography. No residual structure is detected in the D form: addition of GdnHCl does not alter its fluorescence and far-UV CD properties. The fluorescence signal of Trp 53 has been used to monitor folding and unfolding kinetics. The kinetics of folding of the D form in the pH range 7-11 are complex and are described by four exponential processes, as are the kinetics of unfolding of the native state (N state) in the pH range 10.5-12. Each kinetic phase of folding decreases in rate with increase in pH from 7 to 10.85, and each kinetic phase of unfolding decreases in rate with decrease in pH from 12 to 10.85. At pH 10.85, the folding and unfolding rates for any particular kinetic phase are identical and minimal. The two slowest phases of folding and unfolding have identical kinetics whether measured by Trp 53 fluorescence or by mean residue ellipticity at 222 nm. Direct determination of the increase in the N state with time of folding at pH 7 and of the D form with time of unfolding at pH 12, by means of double-jump assays, show that between 85 and 95% of protein molecules fold or unfold via fast pathways between the two forms. The remaining 5-15% of protein molecules appear to fold or unfold via slower pathways, on which at least two intermediates accumulate. The mechanism of folding from the high pH-denatured D form is remarkably similar to the mechanism of folding from the urea or GdnHCl-denatured U form.     

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2α phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2α phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2α to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2α phosphorylation-dependent and -independent pathways that target translation initiation.     

Osmotic loading of articular cartilage modulates cell deformations along primary collagen fibril directions

Osmotic loading is known to modulate chondrocyte (cell) height, width and volume in articular cartilage. It is not known how cartilage architecture, especially the collagen fibril orientation, affects cell shape changes as a result of an osmotic challenge.Intact patellae of New Zealand white rabbits (n=6) were prepared for fluorescence imaging. Patellae were exposed to a hypotonic osmotic shock and cells were imaged before loading and 5–60 min after the osmotic challenge. Cell volumes and aspect ratios (height/width) were analyzed. A fibril-reinforced poroelastic swelling model with realistic primary collagen fibril orientations, i.e. horizontal, random and vertical orientation in the superficial, middle and deep zones, respectively and cells in different zones was used to estimate cell aspect ratios theoretically.As the medium osmolarity was reduced, cell aspect ratios decreased and volumes increased in the superficial zone of cartilage both experimentally (p<0.05) and theoretically. Theoretically determined aspect ratios of middle zone cells remained virtually constant, while they increased for deep zone cells as osmolarity was reduced.Findings of this study suggest that osmotic loading modulates chondrocyte shapes in accordance with the primary collagen fibril directions in articular cartilage.     

Bilateral transverse sinus thrombosis secondary to a homozygous C677T MTHFR gene mutation

We describe the case of a previously healthy young man who presented with headache, diplopia, nausea, vomiting, and bilateral papilledema. Magnetic resonance venography of the brain revealed thrombosis of the right transverse sinus. Blood tests showed elevated homocysteine levels, and coagulation studies revealed a homozygous C677T mutation and a heterozygous A1298C mutation of the methylenetetrahydrofolate reductase (MTHFR) gene. The patient had no other etiology for venous thrombosis. We recommend screening patients who present with sinus thrombosis for MTHFR gene mutations.