Effects of diurnal loading on the transport of charged antibiotics into intervertebral discs

The objective of this study was to quantitatively analyze the effect of diurnal loading on the transport of various charged antibiotics into negatively charged human intervertebral disc (IVD). Transport of charged antibiotics into a human lumbar disc was analyzed using a 3D finite element model. The valence (z) of the electrical charge of antibiotics varied from z = +2 (positively charged) to z = −2 (negatively charged). An uncharged antibiotic (z = 0) was used as a control. Cases with transient antibiotic concentration at disc boundaries [to mimic intravenous (IV) infusion] were simulated. Our results showed that diurnal compression increased the concentrations in the nucleus pulposus (NP) region, but degreased the concentrations in the annulus fibrosus (AF) region for all charged or non-charged drugs. The overall concentration (averaged over disc) increased with diurnal compression. The diurnal compression had more effects on negatively charged antibiotics than positively charged ones. For example, at day 5 with diurnal compression, the diurnal compression increased the concentration of negatively charged drug (z = −1) in NP by 18.3%, but only by 6.6% for positively charged one (z = +1). In AF, diurnal compression decreased the concentration by 13.2% for negatively charged drug (z = −1) versus 1.2% for positively charged one (z = +1). Note these percentages are the averaged values over day 5. This study provides quantitative information on understanding the mechanisms of charged drug transport in human IVDs.     

Self-diffusion of water-soluble fullerene derivatives in mouse erythrocytes

Self-diffusion of water-soluble fullerene derivative (WSFD) C₆₀[S(CH₂)₃SO₃Na]₅H in mouse red blood cells (RBC) was characterized by ¹H pulsed field gradient NMR technique. It was found that a fraction of fullerene molecules (~13% of the fullerene derivative added in aqueous RBC suspension) shows a self-diffusion coefficient of (5.5 ± 0.8)·10⁻¹² m²/s, which is matching the coefficient of the lateral diffusion of lipids in the erythrocyte membrane (D_L = (5.4 ± 0.8)·10⁻¹² m²/s). This experimental finding evidences the absorption of the fullerene derivative by RBC. Fullerene derivative molecules are also absorbed by RBC ghosts and phosphatidylcholine liposomes as manifested in self-diffusion coefficients of (7.9 ± 1.2)·10⁻¹² m²/s and (7.7 ± 1.2)·10⁻¹² m²/s, which are also close to the lateral diffusion coefficients of (6.5 ± 1.0)·10⁻¹² m²/s and (8.5 ± 1.3)·10⁻¹² m²/s, respectively. The obtained results suggest that fullerene derivative molecules are, probably, fixed on the RBC surface. The average residence time of the fullerene derivative molecule on RBC was estimated as 440 ± 70 ms. Thus, the pulsed field gradient NMR was shown to be a versatile technique for investigation of the interactions of the fullerene derivatives with blood cells providing essential information, which can be projected on their behavior in-vivo after intravenous administration while screening as potential drug candidates.     

Genetic and Functional Studies of the Intervertebral Disc: A Novel Murine Intervertebral Disc Model

Intervertebral disc (IVD) homeostasis is mediated through a combination of micro-environmental and biomechanical factors, all of which are subject to genetic influences. The aim of this study is to develop and characterize a genetically tractable, ex vivo organ culture model that can be used to further elucidate mechanisms of intervertebral disc disease. Specifically, we demonstrate that IVD disc explants (1) maintain their native phenotype in prolonged culture, (2) are responsive to exogenous stimuli, and (3) that relevant homeostatic regulatory mechanisms can be modulated through ex-vivo genetic recombination. We present a novel technique for isolation of murine IVD explants with demonstration of explant viability (CMFDA/propidium iodide staining), disc anatomy (H&E), maintenance of extracellular matrix (ECM) (Alcian Blue staining), and native expression profile (qRT-PCR) as well as ex vivo genetic recombination (mT/mG reporter mice; AdCre) following 14 days of culture in DMEM media containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. IVD explants maintained their micro-anatomic integrity, ECM proteoglycan content, viability, and gene expression profile consistent with a homeostatic drive in culture. Treatment of genetically engineered explants with cre-expressing adenovirus efficaciously induced ex vivo genetic recombination in a variety of genetically engineered mouse models. Exogenous administration of IL-1ß and TGF-ß3 resulted in predicted catabolic and anabolic responses, respectively. Genetic recombination of TGFBR1^fl/fl explants resulted in constitutively active TGF-ß signaling that matched that of exogenously administered TGF-ß3. Our results illustrate the utility of the murine intervertebral disc explant to investigate mechanisms of intervertebral disc degeneration.     

Variation in protein lateral diffusion coefficients is related to variation in protein concentration found in mitochondrial inner membranes

The electrophoretic freeze-fracture electron microscopy method (Sowers, A.E. and Hackenbrock, C.R. (1984) Proc. Natl. Acad. Sci. USA 78, 6246–6250) for measuring the lateral diffusion coefficient of integral proteins was applied to a large population of spherical-shaped mitochondrial inner membranes. Membrane integral protein concentration was estimated by determining the intramembrane particle concentration. Analysis of the data reveals that: (a) the radii of the spherical inner membranes in the selected population ranged from 0.22 to 1.2 μm, (b) the intramembrane particle concentrations ranged from 2300 to 6400 per μm², and (c) the calculated lateral diffusion coefficients of the intramembrane particles ranged from 1.3·10⁻¹⁰ to 3.35·10⁻⁹ cm²/s. The data clearly show a naturally occurring large range in protein concentration in the mitochondrial inner membrane and an inverse correlation of lateral diffusion coefficient with the membrane protein concentration. This study is the first to show that the lateral diffusion coefficient of integral proteins in a native membrane varies as the membrane protein concentration.     

Permeability of lipid bilayer to anthracycline derivatives. Role of the bilayer composition and of the temperature

The uptake of three anthracycline derivatives: doxorubicin, daunorubicin and pirarubicin, into large unilamellar vesicles (LUV) in response to a driving force provided by DNA encapsulated inside the LUV has been investigated as a function of the temperature and of the bilayers lipid composition. The kinetics of the decay of the anthracycline fluorescence in the presence of DNA-containing liposome was used to follow the diffusion of the drug through the membrane. For the three drugs, the permeability coefficient of the neutral form of the drug (P⁰) decreases as the amount of negatively charged phospholipid in the bilayers increases. This can be explained by the fact that the kinetics of passive diffusion of the drugs depends on the amount of neutral form embedded in the polar head group region, which decreases as the quantity of negatively charged phospholipids increases. P⁰ also decreases as the amount of cholesterol, that makes the bilayer more rigid, increases. The activation energies, E_a, for the passage of the neutral form of these anthracyclines through the bilayers lie within 100±15 kJ·mol⁻¹, except for pirarubicin and doxorubicin through anionic phospholipid-rich membranes (E_a=57 kJ·mol⁻¹) and cholesterol-rich membranes (E_a=167 kJ·mol⁻¹).     

Measurement of the apparent diffusion coefficient of N‐butane in soil

Measurements of in‐soil diffusion coefficients and the application of an appropriate diffusional model can allow for a more accurate prediction of soil gas concentrations and movement to locate subterranean contamination of volatile materials. The present study was undertaken to measure and evaluate the “apparent in‐soil diffusion coefficient”; for n‐butane through soil columns under non‐steady‐state conditions. The term “apparent in‐soil diffusion coefficient”; refers to a numerical coefficient that primarily describes the movement of the material by diffusion but also contains effects due to other mechanisms (e.g., adsorption and solubility).

Six test columns were evaluated at three soil porosity levels ranging from 0.30 to 0.43 and at two column temperature conditions, nominally 18°C and 7°C. Soil columns measured 25.4 cm in diameter by 84 cm in height and contained a moist sand/silt/clay mixture. The numerical range for the apparent in‐soil diffusion coefficients for n‐butane was 0.447 × 10^‐3cm²/s to 0.561 × 10^‐3cm²/s. The lower coefficient values were associated with lower soil porosity levels and cooler column conditions.     

Obtaining the concentration-dependent diffusion coefficient from ultrafiltration experiments: Application to hyaluronate

A method for determining the concentration-dependent mutual diffusion coefficient D(C) of a macromolecule-solvent combination over a wide macromolecular concentration range is presented. All necessary data are gathered from a single experiment, in which polymer concentration profiles are measured during one-dimensional dead-end ultrafiltration. Based on these profiles, the convection-diffusion equation is used to deduce the dependence of D on C. To demonstrate the utility of the approach, studies using sodium hyaluronate dissolved in either 10 mM NaCl or a phosphate buffer were carried out. For hyaluronate (HA) in 10 mM NaCl, the mutual diffusion coefficient varies approximately linearly with concentration according to D(C) = 4.1 × 10⁻⁶ C^0.96 cm²/s in the range 0 ≤ C ≤ 0.6 mass %, for C expressed in mass %. However, transition points (slope changes) in the D(C) curve are present at C ≅ 0.7 mass % and C ≅ 1.4 mass %. For HA in the phosphate buffer, the mutual diffusion coefficient is well described by D(C) = 1.9 × 10⁻⁶ C^0.825, for 0 ≤ C ≤ 1.8 mass %. These values agree well with previously published data. The technique is robust, and permits reasonably high polymer concentrations to be easily studied. © 1996 John Wiley & Sons, Inc.     

Hydrodynamic properties of nitrogenase — the MoFe protein from Azotobacter vinelandii studied by dynamic light scattering and hydrodynamic modelling

Experimental results for the nitrogenase MoFe protein from Azotobacter vinelandii obtained by dynamic light scattering (DLS) are presented. The translational diffusion coefficient was determined to D=(4.0±0.2)×10⁻⁷ cm²/s. Complementary, we have performed hydrodynamic model calculations based on the X-ray crystallographic data of the MoFe protein. The calculated transport coefficient suggests that the size and shape of the protein in solution is consistent with that in the crystal structure.     

Detection of Methicillin-Resistant Staphylococcus aureus Using mecA/nuc Genes and Antibiotic Susceptibility Profile of Malaysian Clinical Isolates

The aim of this study is to compare methicillin-resistant Staphylococcus aureus (MRSA) detection methods and to generate antibiogram profile of S. aureus clinical isolates from two teaching hospitals in Malaysia including three reference isolates from American Type Culture Collection (ATCC). The mecA/nuc gene PCR amplification, spot inoculation test and oxacillin disc diffusion test were applied to compare its MRSA detection abilities. No disagreement between the three methods was observed. From 29 bacterial isolates (including the ATCC strains) tested, 19 isolates were confirmed as S. aureus with 14 isolates exhibiting multidrug-resistance. All isolates are still susceptible to vancomycin as indicated by the E-test result. Current biochemical tests are comparable with the molecular detection method for MRSA used in this study while multidrug-resistance traits are present in both MRSA and MSSA clinical isolates. Presently, mupirocin seems to be the best alternative for vancomycin against multidrug-resistant S. aureus infections in Malaysia. Susceptibility profile of 19 S. aureus isolates acquired from two teaching hospitals and ATCC towards 16 selected antibiotics was analyzed and an antibiogram was generated. Findings also indicated resistance against many of the available antibiotics and thus an urgent need to search for alternative antibiotics.     

Translational diffusion of lipid monomers determined by spin label electron spin resonance

The collision rates between spin-labelled valeric acid in water, and between the corresponding mixed-chain, spin-labelled phosphatidylcholine in water-methanol mixtures, and also between spin-labelled phosphatidylcholine monomers and micelles in water have been determined from the spin-spin broadening of the electron spin resonance spectrum. In each case the second order rate constants are consistent with a diffusion-controlled process. For spin-labelled valeric acid in water the translational diffusion coefficient at 20°C is 3.4 · 10⁻⁶ cm² · s⁻¹, and for spin-labelled phosphatidylcholine varies between 2.3 · 10⁻⁶ and 3.8 · 10⁻⁶ cm² · s⁻¹ within the range 44 to 88 wt% methanol. The spin-labelled phosphatidylcholine monomer diffusion coefficient in water at 20°C is 2.4 · 10⁻⁶ cm² · s⁻¹, deduced from the monomer-micelle association rate, with an activation energy of 4.0 kcal · mol⁻¹. The much slower on-rates for association of lipid monomers with phospholipid bilayer vesicles reported in the literature, therefore indicate that incorporation into bilayers is not a diffusion-controlled process.     

The direction of human mesenchymal stem cells into the chondrogenic lineage is influenced by the features of hydrogel carriers

Low back pain is a major public health issue in the Western world, one main cause is believed to be intervertebral disc (IVD) degeneration. To halt/diminish IVD degeneration, cell therapy using different biomaterials e.g. hydrogels as cell carriers has been suggested. In this study, two different hydrogels were examined (in vitro) as potential cell carriers for human mesenchymal stem cells (hMSCs) intended for IVD transplantation. The aim was to investigate cell-survival and chondrogenic differentiation of hMSCs when cultured in hydrogels Puramatrix^® or Hydromatrix^® and potential effects of stimulation with growth hormone (GH). hMSCs/hydrogel cultures were investigated for cell-viability, attachment, gene expression of chondrogenic markers SOX9, COL2A1, ACAN and accumulation of extracellular matrix (ECM). In both hydrogel types, hMSCs were viable for 28 days, expressed integrin β1 which indicates adhesion of hMSCs. Differentiation was observed into chondrocyte-like cells, in a higher extent in hMSCs/Hydromatrix^® cultures when compared to hMSCs/Puramatrix^® hydrogel cultures. Gene expression analyses of chondrogenic markers verified results. hMSCs/hydrogel cultures stimulated with GH displayed no significant effects on chondrogenesis.In conclusion, both hydrogels, especially Hydromatrix^® was demonstrated as a promising cell carrier in vitro for hMSCs, when directed into chondrogenesis. This knowledge could be useful in biological approaches for regeneration of degenerated human IVDs.     

Interaction between Alzheimer's disease βA4 precursor protein (APP) and the extracellular matrix: Evidence for the participation of heparan sulfate proteoglycans

The interaction between the Alzheimer amyloid precursor protein (APP) and an intact extracellular matrix (ECM), matrigel, obtained from Engelbreth-Holm-Swarm tumors was evaluated. Based on quantitative analyses of the binding data obtained from solid phase binding assays, two binding sites on the ECM were identified for [¹²⁵I]-APP (with apparent Kd₁ of 1.0 × 10 ⁻¹¹ M and Kd₂ of 1.6 × 10 ⁻⁹ M respectively). Over 70% of [¹²⁵I]-APP was displaced by heparin and N-desulfated heparin but not by chondroitin sulfate. Pretreatment of matrigel with heparitinase decreased the binding of [¹²⁵I]-APP by 80%. β-amyloid peptides (residues 1–40, 1–28, and 1–16) containing a heparin binding domain also displaced 80% of bound [¹²⁵I]-APP, which was totally displaced by intact APP. The binding of [¹²⁵I]-APP to matrigel increased by 210% with a decrease in the pH. These observations suggest that [¹²⁵I]-APP interacts mainly with heparan sulfate proteoglycan present in the ECM. The binding of [¹²⁵I]-APP to individual ECM components was also analyzed. [¹²⁵I]-APP was found to bind laminin and collagen type IV but not fibronectin. However, when these ECM constituents were combined, the extent of APP-binding decreased significantly, to levels comparable to those obtained with intact matrigel, suggesting that multiple interactions may occur between ECM constituents and [¹²⁵I]-APP. The results are discussed in terms of APP function and amyloidogenesis. J. Cell. Biochem. 65:145–158. © 1997 Wiley-Liss, Inc.     

Determination of internal diffusion limitation and its macroscopic kinetics of the transesterification of CPB alcohol catalyzed by immobilized lipase in organic media

Optically active cyanohydrin esters such as (S)-α-cyano-3-phenoxybenzyl (CPB) acetate can be obtained with enzymatic transesterification of racemic cyanohydrins in organic media. The influence of internal diffusion limitation on transesterification of CPB alcohol with vinyl acetate catalyzed by immobilized lipase was studied. Internal diffusion limitation could not be ignored since the immobilized lipase granule was not small enough. In order to express the effect of internal diffusion limitation quantitatively, a method was proposed which calculates the effective diffusion coefficient (D_e) first and then calculates apparent Thiele modulus (Φ) and finally obtains internal diffusion effectiveness factor, η value (0.55). D_e was calculated to be 3.1 × 10⁻¹⁰ m² s⁻¹ by supposing that the immobilized lipase was a sphere granule and the diffusion only existed in one dimension. Φ was calculated to be 2.2 by presuming that transport of substrate through the catalyst could be described by Fick's law. Using King–Altman method, a kinetic model of the transesterification of CPB alcohol with vinyl acetate in organic media in the presence of internal diffusion limitation was proposed based on ping-pong bi–bi mechanism. The relative error of the model was 11.18%. A practicable method to evaluate η value and relatively simple model for such kind of reaction system was proposed in this paper.     

Vancomycin heteroresistance in coagulase negative <Emphasis Type="Italic">Staphylococcus</Emphasis> blood stream infections from patients of intensive care units in Mansoura University Hospitals,Egypt

Background

Vancomycin heteroresistance in coagulase negative Staphylococci (CoNS) is a recent health concern especially in serious infections like bloodstream infections as it may lead to failure of therapy. Little information is available about the prevalence vancomycin heteroresistance in CoNS causing bloodstream infections in intensive care units (ICUs) patients of Mansoura University Hospitals (MUHs).

Methods

This prospective study enrolled 743 blood samples collected from ICUs patients presented with clinical manifestations of bloodstream infections over the period extending from January 2014 to March 2016. Samples were processed, coagulase negative Staphylococci were identified by routine microbiological methods and the absence of coagulase activity. Species were identified by API Staph 32. Oxacillin resistant CoNS were identified by cefoxitin disc diffusion method. Susceptibility testing of isolated CoNS to vancomycin was carried out using vancomycin agar dilution method. Mec A gene detection by PCR was done for oxacillin resistant isolates. Screening for vancomycin heteroresistance was done on brain heart infusion (BHI) agar containing 4 μg/mL vancomycin. Confirmation of vancomycin heteroresistance was carried out by population analysis profile (PAP).

Results

A total of 58 isolates were identified as CoNS from patients of clinically suspected bloodstream infections. The identified species were 33 (56.9%) Staphylococcus epidermidis, 12 (20.7%) Staphylococcus capitis, 7 (12.1%) Staphylococcus haemolyticus, and 3 isolates (5.2%) Staphylococcus lugdunesis. Three isolates were unidentified by API Staph 32. Forty-four (75.9%) isolates were oxacillin resistant. Mec A gene was detected in all oxacillin resistant isolates. All isolates had susceptible vancomycin MICs by agar dilution. Nine isolates (15.5%) could grow on BHI agar containing 4 μg/mL vancomycin. These isolates showed heterogeneous profile of resistance to vancomycin by population analysis profile.

Conclusions

Vancomycin heteroresistant CoNS causing bloodstream infections is growing unrecognized health hazard in ICUs patients. These isolates have susceptible vancomycin MICs. Screening methods are recommended and should be considered to improve clinical outcome in these high risk patients.

     

Purification and some properties of γ-conglycinin in soybean seeds

A 7S globulin (γ-conglycinin) which was one of four major antigenic components in soybean globulins was purified and found to be homogeneous on ultracentrifugation, disc electrophoresis, immunoelectrophoresis and disc electrofocusing by gel filtration, preparative-scale disc electrophoresis and two kinds of affinity chromatography. Subsequently, some physico-chemical properties of the protein were determined. The sedimentation coefficient, isoelectric point, MW and diffusion constant were 6·55S, pH 5·80, 104000 and 5·80 × 10^?7 cm²/sec, respectively. The protein was a glycoprotein which contained 5·49% total carbohydrate per protein. The protein did not aggregate and dissociate with a change of ionic strength from 0·1 to 0·5.     

Targeting the extracellular matrix: Matricellular proteins regulate cell–extracellular matrix communication within distinct niches of the intervertebral disc

The so-called “matricellular” proteins have recently emerged as important regulators of cell–extracellular matrix (ECM) interactions. These proteins modulate a variety of cell functions through a range of interactions with cell-surface receptors, hormones, proteases and structural components of the ECM. As such, matricellular proteins are crucial regulators of cell phenotype, and consequently tissue function. The distinct cell types and microenvironments that together form the IVD provide an excellent paradigm to study how matricellular proteins mediate communication within and between adjacent tissue types. In recent years, the role of several matricellular proteins in the intervertebral disc has been explored in vivo using mutant mouse models in which the expression of target matricellular proteins was deleted from either one or all compartments of the intervertebral disc. The current review outlines what is presently known about the roles of the matricellular proteins belonging to the CCN family, SPARC (Secreted Protein, Acidic, and Rich in Cysteine), and thrombospondin (TSP) 2 in regulating intervertebral disc cell–ECM interactions, ECM synthesis and disc tissue homeostasis using genetically modified mouse models. Furthermore, we provide a brief overview of recent preliminary studies of other matricellular proteins including, periostin (POSTN) and tenascin (TN). Each specific tissue type of the IVD contains a different matricellular protein signature, which varies based on the specific stage of development, maturity or disease. A growing body of direct genetic evidence links IVD development, maintenance and repair to the coordinate interaction of matricellular proteins within their respective niches and suggests that several of these signaling modulators hold promise in the development of diagnostics and/or therapeutics targeting intervertebral disc aging and/or degeneration.     

Effect of Cartilage Endplate on Cell Based Disc Regeneration: A Finite Element Analysis

This study examines the effects of cartilage endplate (CEP) calcification and the injection of intervertebral disc (IVD) cells on the nutrition distributions inside the human IVD under physiological loading conditions using multiphasic finite element modeling. The human disc was modeled as an inhomogeneous mixture consisting of a charged elastic solid, water, ions (Na⁺ and Cl⁻), and nutrient solute(oxygen,glucose and lactate) phases. The effect of the endplate calcification was simulated by a reduction of the tissue porosity (i.e., water volume faction) from 0.60 to 0.48. The effect of cell injection was simulated by increasing the cell density in the nucleus pulposus (NP) region by 50%, 100%, and 150%. Strain-dependent transport properties(e.g., hydraulic permeability and solute diffusivities) were considered to couple the solute transport and the mechanical loading. The simulation results showed that nutrient solute distribution inside the discis maintained at a stable state during the day and night. The physiological diurnal cyclic loading does not change the nutrient environment in the human IVD. The cartilage endplate plays a significant role in the nutrient supply to human IVD. Calcification of the cartilage endplate significantly reduces the nutrient levels in human IVD. Therefore, in cell based therapy for IVD regeneration, theincreased nutrient demand as a result of cell injection needs to be addressed. Excessive numbers of injected cells may cause further deterioration of the nutrient environment in the degenerated disc. This study is important for understanding the pathology of IVD degeneration and providing new insights into cell based therapies for low back pain.     

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. V—Rabbit (Oryctolagus Cuniculus)

• 1.1. The diffusional water permeability (P_d) of rabbit red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition with p-chloromercuribenzene sulfonate (PCMBS).
• 2.2. The values of P_d were around 6.3 × 10⁻³ cm/sec at 15°C, 7.0 × 10⁻³cm/sec at 20°C, 8.0 × 10⁻³ cm/sec at 25°C, 9.1 × 10⁻³ cm/sec at 30°C and10.7 × 10⁻³ cm/sec at 37°C.
• 3.3. Systematic studies on the effects of PCMBS on water diffusion indicated that the maximal inhibition was reached in 15 min at 37°C with 0.5 mM PCMBS.
• 4.4. The values of maximal inhibition were around 71–74% at all temperatures.
• 5.5. The basal permeability to water was estimated as 1.6 × 10⁻³cm/sec at 15°C, 2.0 × 10⁻³cm/sec at 20°C, 2.4 × 10⁻³cm/sec at 25°C, 2.6 × 10⁻³cm/sec at 30°C, and 3.1× 10^{−3 cm/secat 37°C}.
• 6.6. The activation energy of water diffusion was around 18 kJ/mol and increased to 27 kcal/mol after incubation with PCMBS in conditions of maximal inhibition of water diffusion.
• 7.7. The membrane polypeptide electrophoretic pattern of rabbit RBCs has been compared with its human counterpart.
• 8.8. The rabbit membrane contained a higher amount of spectrin (bands 1 and 2), while the band 6 (glyceraldehyde-3-phosphate dehydrogenase) was markedly less intense.
• 9.9. Considerable differences in the electrophoretic patterns of the two sources of RBC membranes appeared in the bands migrating in the band 4.5 region and in front of band 7, where some polypeptides were apparent in higher amounts in the rabbit RBC membrane.

     

Comparative immunolocalisation of fibrillin-1 and perlecan in the human foetal, and HS-deficient hspg2 exon 3 null mutant mouse intervertebral disc

The aim of this study was to examine the comparative localisations of fibrillin-1 and perlecan in the foetal human, wild-type C57BL/6 and HS-deficient hspg2Δ^3?/Δ^3? exon 3 null mouse intervertebral disc (IVD) using fluorescent laser scanning confocal microscopy. Fibrillin-1 fibrils were prominent components of the outer posterior and anterior annulus fibrosus (AF) of the foetal human IVD. Finer fibrillin-1 fibrils were evident in the inner AF where they displayed an arcade-type arrangement in the developing lamellae. Relatively short but distinct fibrillin-1 fibrils were evident in the central region of the IVD and presumptive cartilaginous endplate and defined the margins of the nuclear sheath in the developing nucleus pulposus (NP). Fibrillin-1 was also demonstrated in the AF of C57BL/6 wild-type mice but to a far lesser extent in the HS-deficient hspg2Δ^3?/Δ^3? exon 3 null mouse. This suggested that the HS chains of perlecan may have contributed to fibrillin-1 assembly or its deposition in the IVD. The cell–matrix interconnections provided by the fibrillin fibrils visualised in this study may facilitate communication between disc cells and their local biomechanical microenvironment in mechanosensory processes which regulate tissue homeostasis. The ability of fibrillin-1 to sequester TGF-β a well-known anabolic growth factor in the IVD also suggests potential roles in disc development and/or remodelling.     

Uptake of GABA by neuronal and nonneuronal cells in dispersed cell cultures of postnatal rat cerebellum

A study was made of the time course and kinetics of [³H]GABA uptake by dispersed cell cultures of postnatal rat cerebellum with and without neuronal cells. The properties of GABA neurons were calculated from the biochemical difference between the two types of cultures. It was found that for any given concentration of [³H]GABA, or any time up to 20 min, GABA neurons in cultures 21 days in vitro had an average velocity of uptake several orders of magnitude greater than that of nonneuronal cells. In addition, the apparent K_m values for GABA neurons for high and low affinity uptake were 0.33 × 10⁻⁶ M and 41.8 × 10⁻⁴ M, respectively. For nonneuronal cells, the apparent K_m for high affinity uptake was 0.29 × 10⁻⁶ M. The apparent V_max values for GABA neurons for high and low affinity uptake were 28.7 × 10⁻⁶ mol/g DNA/min and 151.5 mmol/g DNA/min, respectively. For nonneuronal cells, the apparent V_max for high affinity uptake was 0.06 × 10⁻⁶ mol/g DNA/min. No low affinity uptake system for nonneuronal cells could be detected after correcting the data for binding and diffusion. By substituting the apparent kinetic constants in the Michaelis-Menten equation, it was determined that for GABA concentrations of 5 × 10⁻⁹ M to 1 mM or higher over 99% of the GABA should be accumulated by GABA neurons, given equal access of all cells to the label. In addition, high affinity uptake of [³H]GABA by GABA neurons was completely blocked by treatment with 0.2 mM ouabain, whereas that by nonneuronal cells was only slightly decreased. Most (75–85%) of the [³H]GABA (4.4 × 10⁻⁶ M) uptake by both GABA neurons and nonneuronal cells was sodium and temperature dependent.