3D Differentiation of Neural Stem Cells in Macroporous Photopolymerizable Hydrogel Scaffolds

Neural stem/progenitor cells (NSPCs) are the stem cell of the adult central nervous system (CNS). These cells are able to differentiate into the major cell types found in the CNS (neurons, oligodendrocytes, astrocytes), thus NSPCs are the mechanism by which the adult CNS could potentially regenerate after injury or disorder. Microenviromental factors are critical for guiding NSPC differentiation and are thus important for neural tissue engineering. In this study, D-mannitol crystals were mixed with photocrosslinkable methacrylamide chitosan (MAC) as a porogen to enhance pore size during hydrogel formation. D-mannitol was admixed to MAC at 5, 10 and 20 wt% D-mannitol per total initial hydrogel weight. D-mannitol crystals were observed to dissolve and leave the scaffold within 1 hr. Quantification of resulting average pore sizes showed that D-mannitol addition resulted in larger average pore size (5 wt%, 4060±160 µm², 10 wt%, 6330±1160 µm², 20 wt%, 7600±1550 µm²) compared with controls (0 wt%, 3150±220 µm²). Oxygen diffusion studies demonstrated that larger average pore area resulted in enhanced oxygen diffusion through scaffolds. Finally, the differentiation responses of NSPCs to phenotypic differentiation conditions were studied for neurons, astrocytes and oligodendrocytes in hydrogels of varied porosity over 14 d. Quantification of total cell numbers at day 7 and 14, showed that cell numbers decreased with increased porosity and over the length of the culture. At day 14 immunohistochemistry quantification for primary cell types demonstrated significant differentiation to the desired cells types, and that total percentages of each cell type was greatest when scaffolds were more porous. These results suggest that larger pore sizes in MAC hydrogels effectively promote NSPC 3D differentiation.     

Design and green synthesis of novel quinolinone derivatives of potential anti-breast cancer activity against MCF-7 cell line targeting multi-receptor tyrosine kinases

A new set of 4,6,7,8-tetrahydroquinolin-5(1H)-ones were designed as cytotoxic agents against breast cancer cell line (MCF-7) and synthesised under ultrasonic irradiation using chitosan decorated copper nanoparticles (CS/CuNPs) catalyst. The new compounds 4b, 4j, 4k, and 4e exhibited the most potent cytotoxic activity of IC₅₀ values (0.002 − 0.004 µM) comparing to Staurosporine of IC₅₀; 0.005 μM. The latter derivatives exhibited a promising safety profile against the normal human WI38 cells of IC₅₀ range 0.0149 − 0.048 µM. Furthermore, the most promising cytotoxic compounds 4b, 4j were evaluated as multi-targeting agents against the RTK protein kinases; EGFR, HER-2, PDGFR-β, and VEGFR-2. Compound 4j showed promising inhibitory activity against HER-2 and PDGFR-β of IC₅₀ values 0.17 × 10⁻³, 0.07 × 10⁻³ µM in comparison with the reference drug sorafenib of IC₅₀; 0.28 × 10⁻³, 0.13 × 10⁻³ µM, respectively. In addition, 4j induced apoptotic effect and cell cycle arrest at G2/M phase preventing the mitotic cycle in MCF-7 cells.     

Apoptosis through Bcl-2/Bax and Cleaved Caspase Up-Regulation in Melanoma Treated by Boron Neutron Capture Therapy

Boron neutron capture therapy (BNCT) is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET) particles, alpha and ⁷Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM) modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT.     

Transport of glucose and galactose in kidney-cortex cells

1. The aerobic transport of d-glucose and d-galactose in rabbit kidney tissue at 25° was studied. 2. In slices forming glucose from added substrates an accumulation of glucose against its concentration gradient was found. The apparent ratio of intracellular ([S]_i) and extracellular ([S]_o) glucose concentrations was increased by 0·4mm-phlorrhizin and 0·3mm-ouabain. 3. Slices and isolated renal tubules actively accumulated glucose from the saline; the apparent [S]_i/[S]_o fell below 1·0 only at [S]_o higher than 0·5mm. 4. The rate of glucose oxidation by slices was characterized by the following parameters: K_m 1·16mm; V_max. 4·5μmoles/g. wet wt./hr. 5. The active accumulation of glucose from the saline was decreased by 0·1mm-2,4-dinitrophenol, 0·4mm-phlorrhizin and by the absence of external Na⁺. 6. The kinetic parameters of galactose entry into the cells were: K_m 1·5mm; V_max 10μmoles/g. wet wt./hr. 7. The efflux kinetics from slices indicated two intracellular compartments for d-galactose. The galactose efflux was greatly diminished at 0°, was inhibited by 0·4mm-phlorrhizin, but was insensitive to ouabain. 8. The following mechanism of glucose and galactose transport in renal tubular cells is suggested: (a) at the tubular membrane, these sugars are actively transported into the cells by a metabolically- and Na⁺-dependent phlorrhizin-sensitive mechanism; (b) at the basal cell membrane, these sugars are transported in accordance with their concentration gradient by a phlorrhizin-sensitive Na⁺-independent facilitated diffusion. The steady-state intracellular sugar concentration is determined by the kinetic parameters of active entry, passive outflow and intracellular utilization.     

Understanding the effect of mean pore size on cell activity in collagen-glycosaminoglycan scaffolds

Mean pore size is an essential aspect of scaffolds for tissue-engineering. If pores are too small cells cannot migrate in towards the center of the construct limiting the diffusion of nutrients and removal of waste products. Conversely, if pores are too large there is a decrease in specific surface area available limiting cell attachment. However the relationship between scaffold pore size and cell activity is poorly understood and as a result there are conflicting reports within the literature on the optimal pore size required for successful tissue-engineering. Previous studies in bone tissue-engineering have indicated a range of mean pore sizes (96–150 µm) to facilitate optimal attachment. Other studies have shown a need for large pores (300–800 µm) for successful bone growth in scaffolds. These conflicting results indicate that a balance must be established between obtaining optimal cell attachment and facilitating bone growth. In this commentary we discuss our recent investigations into the effect of mean pore size in collagen-glycosaminoglycan (CG) scaffolds with pore sizes ranging from 85–325 µm and how it has provided an insight into the divergence within the literature.Key words: bone tissue engineering, cell adhesion, collagen, extracellular matrix, pore size, scaffoldThe goal of tissue engineering is to develop cell, construct and living system technologies to restore the structure and functional mechanical properties of damaged or degenerated tissue. While the field of tissue engineering may be relatively new, the idea of replacing tissue with another goes as far back as the 16^th century when an Italian, Gasparo Tagliacozzi (1546–99), Professor of Surgery and Anatomy at the Bologna University, described a nose replacement that he had constructed from a forearm flap in his work “De Custorum Chirurigia per Insitionem” (The Surgery of Defects by Implantation) which was published in 1597. In modern times, the techniques of transplanting tissue from one site to another in the same patient (an autograft) or from one individual to another (transplant or allograft) have been revolutionary and lifesaving. However major problems exist with both techniques. Harvesting autografts is expensive, painful, constrained by anatomical limitations and associated with donor-site morbidity due to infection and hemorrhage. Transplants have serious constraints. The major problem is accessing enough tissue and organs for all of the patients who require them. Transplants are strongly associated with rejection by the patient''s immune system and they are also limited by the potential risks of introducing infection or disease.Tissue engineering was born from the belief that primary cells could be isolated from a patient, expanded in vitro and seeded onto a substrate that could be grafted back into the patient.¹ It provides a biological alternative to transplantations and prosthesis. One of the first scaffolds pioneered for tissue regeneration was synthesized as a graft co-polymer of type I collagen and chondroitin 6-sulphate, a glycosaminoglycan. The development of these scaffolds, which are capable of supporting tissue synthesis when seeded with cells, marks the beginning of the field of tissue engineering.^2,3 Since this early work, there have been rapid advances in bone tissue engineering with the development of porous, biocompatible, three-dimensional scaffolds. Regardless of the application, the scaffold should be biocompatible and imitate both the physical and biological function of the native extracellular matrix (ECM), as the ECM provides a substrate with specific ligands for cell adhesion as well as physical support for cells.⁴ When designing scaffolds for any tissue engineering application, a major consideration is the mean pore size. Scaffolds must be permeable with interconnecting pores to facilitate cell growth, migration and nutrient flow. A previous study demonstrated that permeability increases with increasing pore size due to a reduction in specific surface area.⁵ If pores are too small, cell migration is limited, resulting in the formation of a cellular capsule around the edges of the scaffold. This in turn can limit the distribution of nutrients and removal of waste products resulting in necrotic regions within the construct. Conversely if pores are too large there is a decrease in specific surface area.³ It has been proposed that a reduction in specific surface area reduces the ligand density available for cells to bind to.⁶ Cellular activity is influenced by specific integrin-ligand interactions between cells and surrounding ECM. Initial cell adhesion mediates all subsequent events such as proliferation, migration and differentiation within the scaffold. As a result the mean pore size within a scaffold affects cell adhesion and ensuing proliferation, migration and infiltration. Therefore maintaining a balance between the optimal pore size for cell migration and specific surface area for cell attachment is essential.^4,7In our laboratory we use a composite scaffold fabricated from collagen and a glycosaminoglycan (GAG) for bone tissue engineering applications produced by a lyophilisation (freeze-drying) fabrication process. The first generation of this collagen-GAG (CG) scaffold was originally developed for skin regeneration but has since been applied to a number of other tissue engineering applications, due to its high biological activity and resultant ability to promote cell growth and tissue development.^2,8–12 Originally CG scaffolds were fabricated using a rapid uncontrolled quench process during lyophilisation which resulted in heterogeneous porous scaffolds with a large variation of pore size within certain areas of the scaffold.² When these scaffolds were used in previous studies they were visually examined so that the areas of variation could be avoided resulting in subjective selection of scaffold samples for analysis.⁸ However, an improved lyophilisation technique was later developed which incorporated a constant cooling rate which controlled the formation and growth of ice-crystals thus resulting in CG scaffolds with homogenous pore structures.¹³ The traditional final temperature of freezing used to produce these scaffolds is −40°C; however, further modifications to the lyophilisation process demonstrated that by changing the final temperature of freezing, it is possible to tailor the mean pore size in the scaffolds. This study showed that by varying the temperature of freezing from −40 to −10°C it was possible to produce homogenous CG scaffolds with mean pore sizes ranging from 96–151 µm.⁶A cellular solid is one made up of an interconnecting porous network and cellular solids modeling techniques can be used to describe both mechanical and microstructural (i.e., specific surface area) properties of scaffolds. A cellular solids model utilizing a tetrakaidecahedral unit cell (a 14-sided polyhedron that packs to fill space) was used to determine the effect of mean pore size on specific surface area. Specific surface area can be related to the relative density of a scaffold and using a tetrakaidecahedral unit cell it was possible to model the geometry of the CG scaffolds.^5,6,14 As a result the specific surface area (SA) per unit volume (V) available for cell adhesion in each of the scaffolds with different mean pore sizes (d) was estimated as: SA/V = 0.718/d(1)This relationship demonstrates that the specific surface area is inversely proportional to the mean pore size. The authors then carried out a simple experiment and seeded the scaffold range with osteoblasts and monitored initial cell adhesion up to 48 h post-seeding. Cell adhesion is the binding of cells to their extracellular environment via specific ligand-integrin interactions. The results demonstrated that cell adhesion decreased with increasing pore size and that the highest levels of cell attachment were found on the scaffolds with the smallest pore size (96 µm). The rationale for this result, as suggested by the authors, was the effect of specific surface area on cell adhesion due to the scaffolds with larger pores having less available specific surface area and thus a lower ligand density for initial cell attachment.^5,6The results of this study conflicted with other studies within the literature which demonstrate a need for larger pores. The relationship between scaffold pore size and cell activity is not fully understood and as a result, over the years there have been conflicting reports on the optimal pore size required for bone tissue engineering. Pores ranging from 20–1,500 µm have been used in bone tissue engineering applications.^15–18 Initial studies demonstrated that the minimum pore size for significant bone growth is 75–100 µm with an optimal range of 100–135 µm.^15,19 Since this early work it has been reported that pores greater than ∼300 µm are essential for vascularisation of constructs and bone ingrowth, while pores smaller than ∼300 µm can encourage osteochondral ossification.^20–22In a very recent study in our laboratory, which utilized improved technical capability of our freeze-drying system and introduced a novel annealing step during lyophilisation, we have been able to further expand the range of mean pore sizes produced in the CG scaffolds from 96–151 µm up to 85–325 µm.²³ We then investigated the effect of this new expanded range of scaffolds on initial cell attachment followed by migration and proliferation by monitoring cellular activity up to 7 days post-seeding (as opposed to 48 h in the earlier study⁶) to see whether the pattern of specific surface area affecting initial cell adhesion as seen in the previous studies would continue as cells proliferated.²⁴The results provide a possible insight into why there are conflicting reports in the literature on the optimal scaffold pore size for bone tissue engineering. A non-linear effect of pore size was seen on cell proliferation over the 7 day incubation period. Scaffolds with the largest pore size of 325 µm facilitated higher cell number at all time points in comparison to the other scaffold types. However, within the lower range of pore sizes there was a small peak in cell number at 24 h and 48 h post-seeding in scaffolds with a mean pore size of 120 µm. This peak disappeared by day 7 (Fig. 1). This peak is consistent with that seen in the earlier study⁶ and can therefore be explained by the effect of pore surface area on cell attachment. Collagen, a natural component of bone ECM, contains binding sites (ligands) that are recognized by specific cell surface receptors (integrins), the main collagen integrins being α₁β₁ and α₂β₁. Based on the interactions between integrins and their corresponding ligands, cells can detect subtle changes in ECM that can influence cell attachment and consequently determine cell proliferation, speed and migration. Our results reflected this within the smaller pore range (85–190 µm) when cell number was presented as a percentage of the cells seeded onto the scaffolds,²⁴ indicating that high specific surface area in scaffolds is important for optimal cell attachment. However, when this range of pore sizes was expanded (85–325 µm) the linear relationship between mean pore size and specific surface area was no longer applicable (Fig. 1) and scaffolds with the largest pores showed the highest cell numbers even though the surface area is lower than that for the other scaffold variants. We propose that the effect of specific surface area is overcome in larger pores by the improved potential for cell migration and proliferation as was seen histologically in scaffolds with 325 µm.Open in a separate windowFigure 1Effect of mean pore size on cell number at each time point. Cell number increases to a small peak 24 h post seeding in scaffolds with a pore size of 120 µm. This peak declines at later time points. Cell number significantly peaks in scaffolds with a mean pore size of 325 µm. *p < 0.001 (reviewed in ref. ²⁴).When seeding three-dimensional scaffolds it is desirable that the cells infiltrate and colonize the scaffold laying down their own ECM. The CG scaffolds are highly porous (∼99%)⁵ and it has previously been shown that cell migration behavior decreases with increasing pore size.²⁶ However, similarly to other studies,⁶ these results were based on limited range of mean pore sizes incubated for less than 48 h. In this study, migration of cells was assessed histologically after 7 days incubation. Cells were observed lining the pores in all scaffolds. However, cell aggregations were seen along the edges of the scaffolds with smaller pore sizes of 85 µm–120 µm limiting the number of cells infiltrating the scaffold (Fig. 2A). Cell aggregations form a “skin” around the outer surface of the scaffold which restricts the diffusion of nutrients and removal of waste from the cells colonizing the center of the scaffold. As the mean pore size increased, cells migrated further away from the edges and in towards the center of the scaffold until cells were seen colonizing the center of the scaffolds with the largest mean pore size of 325 µm (Fig. 2B). An increase in cell number was seen in 120 µm pore size, but the aggregations seen on the surface of these scaffolds compound the hypothesis that this peak was related to initial cell adhesion and the advantages of this pore size were lost with subsequent cell proliferation and migration.Open in a separate windowFigure 2Effect of mean pore size on cell infiltration and distribution CG scaffolds after 7 days. Scaffolds were stained with H&E: (A) 85 µm pore size at x40 magnification, (B) 325 µm pore size at ×40 magnification. Collagen scaffold is stained pink and cell nuclei a deep purple. The arrow indicates cell aggregations along the edges of the scaffold. Aggregations disappeared and cell migration increased with increasing pore size (reviewed in ref. ²⁴).The study²⁴ had a number of limitations. It was not possible to determine the upper pore size limit for cell activity within a CG scaffold. If the pores become too large the mechanical properties of the scaffold will be compromised due to void volume⁷ and as pore size increases further, the specific surface area will eventually reduce to a level that will limit cell adhesion. Furthermore, this study has determined the optimal pore size for MC3T3-E1 pre-osteoblast activity. It has been hypothesised that the optimal pore size will vary with different cell types⁶ and another recent study from our laboratory has demonstrated that mesenchymal stem cells seeded on the smaller range of CG scaffolds and maintained in osteogenic culture for 3 weeks showed improved osteogenesis on the scaffolds with bigger pores²⁵. For this reason it is important to repeat this study with different cell types. However, regardless of these limitations, this paper has demonstrated that mean pore size does affect cell behavior within a scaffold and that subtle changes in pore size can have a significant effect on cell behavior. We also provide an insight into why the literature reports conflicting results on the optimal pore size required for bone tissue engineering, whereby increased specific surface area provided by scaffolds with small pores has a benefi- cial effect on initial cell attachment, but this is overcome by the improved cellular infiltration provided by scaffolds with larger pores suggesting that these scaffolds might be optimal for longer term in vitro culture with the aim of facilitating bone tissue repair.     

STEREOLOGICAL ANALYSIS OF MAMMALIAN SKELETAL MUSCLE : I. Soleus Muscle of the Adult Guinea Pig

A quantitative analysis of the volumes, surface areas, and dimensions of the ultrastructural components in the soleus muscle fibers of the guinea pig was made by using point counting methods of stereology. Muscle fibers have structural orientation (anisotropy) and have spatial gradients of the structures within the fiber; therefore the standard stereological methods were modified where necessary. The entire analysis was repeated at two section orientations to test the modifications and identical results obtained from both. The volume of lipid droplets was 0.20 ± 0.06% (mean ± standard error, n = 5 animals) and the nuclei volume was 0.86 ± 0.20% of the fiber volume. The total mitochondrial volume was 4.85 ± 0.66% of the fiber volume with about one-third being found in an annulus within 1 µm of the sarcolemma. The mitochondrial volume in the remaining core of the fiber was 3.6 ± 0.4%. The T system has a volume of 0.14 ± 0.01% and a surface area of 0.064 ± 0.005 µm²/µm³ of the fiber volume. The surface area of the sarcolemma is 0.116 ± 0.013 µm²/µm³ which is twice the T system surface area. The volume of the entire sarcoplasmic reticulum is 3.52 ± 0.33% and the surface area is 0.97 ± 0.09 µm²/µm³. The sarcoplasmic reticulum is composed of the terminal cisternae whose volume is 1.04 ± 0.19% and surface area is 0.24 ± 0.05 µm²/µm³. The tubules of the sarcoplasmic reticulum in the I band and A band have volumes of 1.97 ± 0.24% and 0.51 ± 0.08%, and the surface areas of the I and A band reticulum are 0.56 ± 0.07 µm²/µm³ and 0.16 ± 0.04 µm²/µm³, respectively. The Z line width, myofibril and fiber diameters were measured.     

Morphology,Classification, and Distribution of the Projection Neurons in the Dorsal Lateral Geniculate Nucleus of the Rat

The morphology of confirmed projection neurons in the dorsal lateral geniculate nucleus (dLGN) of the rat was examined by filling these cells retrogradely with biotinylated dextran amine (BDA) injected into the visual cortex. BDA-labeled projection neurons varied widely in the shape and size of their cell somas, with mean cross-sectional areas ranging from 60–340 µm². Labeled projection neurons supported 7–55 dendrites that spanned up to 300 µm in length and formed dendritic arbors with cross-sectional areas of up to 7.0×10⁴ µm². Primary dendrites emerged from cell somas in three broad patterns. In some dLGN projection neurons, primary dendrites arise from the cell soma at two poles spaced approximately 180° apart. In other projection neurons, dendrites emerge principally from one side of the cell soma, while in a third group of projection neurons primary dendrites emerge from the entire perimeter of the cell soma. Based on these three distinct patterns in the distribution of primary dendrites from cell somas, we have grouped dLGN projection neurons into three classes: bipolar cells, basket cells and radial cells, respectively. The appendages seen on dendrites also can be grouped into three classes according to differences in their structure. Short “tufted” appendages arise mainly from the distal branches of dendrites; “spine-like” appendages, fine stalks with ovoid heads, typically are seen along the middle segments of dendrites; and “grape-like” appendages, short stalks that terminate in a cluster of ovoid bulbs, appear most often along the proximal segments of secondary dendrites of neurons with medium or large cell somas. While morphologically diverse dLGN projection neurons are intermingled uniformly throughout the nucleus, the caudal pole of the dLGN contains more small projection neurons of all classes than the rostral pole.     

Kinetics of thiamin cleavage by sulphite

Results are presented on the rate of thiamin cleavage by sulphite in aqueous solutions as affected by temperature (20–70°), pH(2·5–7·0), and variation of the concentration of either thiamin (1–20μm) or sulphite (10–5000μm as sulphur dioxide). Plots of the logarithm of percentage of residual thiamin against time were found to be linear and cleavage thus was first-order with respect to thiamin. At pH5 the rate was also found to be proportional to the sulphite concentration. In the pH region 2·5–7·0 at 25° the rate constant was 50m⁻¹hr.⁻¹ at pH5·5–6·0, and decreased at higher or lower pH values. The rate of reaction increased between 20° and 70°, indicating a heat of activation of 13·6kcal./mole.     

A MORPHOMETRIC STUDY ON THE NEXUS OF RAT CARDIAC MUSCLE

A morphometric study of the nexus of rat cardiac muscle was carried out. The nexus surface of one intercalated disk of one 15 µm thick fiber is found to range between 47 µm² and 94 µm², the latter value taking into account the maximal underestimation caused by tangential sectioning. Dividing the lower, minimal value by the surface of the observed subunits (90 Å periodicity), one obtains for one intercalated disk 6.7 x 10⁵ subunits, each of them assumed to be permeated by a central pore. These pores are thought to be equivalent to intercellular channels in a recently proposed model. Taking our morphometric and recently reported physiological values, this model is examined for its consistency with a low resistance pathway between cardiac muscle cells.     

Nitrate Transport and Not Photoinhibition Limits Growth of the Freshwater Cyanobacterium Synechococcus Species PCC 6301 at Low Temperature

The effect of low temperature on cell growth, photosynthesis, photoinhibition, and nitrate assimilation was examined in the cyanobacterium Synechococcus sp. PCC 6301 to determine the factor that limits growth. Synechococcus sp. PCC 6301 grew exponentially between 20°C and 38°C, the growth rate decreased with decreasing temperature, and growth ceased at 15°C. The rate of photosynthetic oxygen evolution decreased more slowly with temperature than the growth rate, and more than 20% of the activity at 38°C remained at 15°C. Oxygen evolution was rapidly inactivated at high light intensity (3 mE m⁻² s⁻¹) at 15°C. Little or no loss of oxygen evolution was observed under the normal light intensity (250 μE m⁻² s⁻¹) for growth at 15°C. The decrease in the rate of nitrate consumption by cells as a function of temperature was similar to the decrease in the growth rate. Cells could not actively take up nitrate or nitrite at 15°C, although nitrate reductase and nitrite reductase were still active. These data demonstrate that growth at low temperature is not limited by a decrease in the rate of photosynthetic electron transport or by photoinhibition, but that inactivation of the nitrate/nitrite transporter limits growth at low temperature.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

Elemental Composition (C, N, P) and Cell Volume of Exponentially Growing and Nutrient-Limited Bacterioplankton

Marine bacterioplankton were isolated and grown in batch cultures until their growth became limited by organic carbon (C), nitrogen (N), or phosphorus (P). Samples were taken from the cultures at both the exponential and stationary phases. The elemental composition of individual bacterial cells was analyzed by X-ray microanalysis with an electron microscope. The cell size was also measured. The elemental content was highest in exponentially growing cells (149 ± 8 fg of C cell⁻¹, 35 ± 2 fg of N cell⁻¹, and 12 ± 1 fg of P cell⁻¹; average of all isolates ± standard error). The lowest C content was found in C-limited cells (39 ± 3 fg of C cell⁻¹), the lowest N content in C- and P-limited cells (12 ± 1 and 12 ± 2 fg of N cell⁻¹, respectively), and the lowest P content in P-limited cells (2.3 ± 0.6 fg of P cell⁻¹). The atomic C:N ratios varied among treatments between 3.8 ± 0.1 and 9.5 ± 1.0 (average ± standard error), the C:P ratios between 35 ± 2 and 178 ± 28, and the N:P ratios between 6.7 ± 0.3 and 18 ± 3. The carbon-volume ratios showed large variation among isolates due to different types of nutrient limitation (from 51± 4 to 241 ± 38 fg of C μm⁻¹; average of individual isolates and treatments ± standard error). The results show that different growth conditions and differences in the bacterial community may explain some of the variability of previously reported elemental and carbon-volume ratios.     

QUANTITATIVE AUTORADIOGRAPHIC STUDY OF LABELED RNA IN RABBIT OPTIC NERVE AFTER INTRAOCULAR INJECTION OF [3H]URIDINE

The distribution of labeled RNA in the optic nerve of the rabbit was studied by quantitative ultrastructural autoradiography after the intraocular injection of [³H]uridine. The highest density of silver grains related to [³H]RNA (27–40 grains/100 µm²) was found in glial cell perikarya; a slightly lower density was present in the glial nuclei (19–20 grains/100 µm²). Axons (4–5 grains/100 µm²) and myelin (2–3 grains/100 µm²) had the lowest grain densities. 74–83% of all counted grains were located outside the axons. By comparing the grain density distribution over the axon with that expected in the case of an exclusive labeling of the surrounding myelin and glial cell processes, it was concluded that the axons contained a number of grains representing [³H]RNA significantly higher than that expected to scatter from myelin and glial processes. Most of these grains were concentrated at the periphery of the axon and were not related to axonal mitochondria.     

Ion permeation and block of the gating pore in the voltage sensor of NaV1.4 channels with hypokalemic periodic paralysis mutations

Hypokalemic periodic paralysis and normokalemic periodic paralysis are caused by mutations of the gating charge–carrying arginine residues in skeletal muscle Na_V1.4 channels, which induce gating pore current through the mutant voltage sensor domains. Inward sodium currents through the gating pore of mutant R666G are only ∼1% of central pore current, but substitution of guanidine for sodium in the extracellular solution increases their size by 13- ± 2-fold. Ethylguanidine is permeant through the R666G gating pore at physiological membrane potentials but blocks the gating pore at hyperpolarized potentials. Guanidine is also highly permeant through the proton-selective gating pore formed by the mutant R666H. Gating pore current conducted by the R666G mutant is blocked by divalent cations such as Ba²⁺ and Zn²⁺ in a voltage-dependent manner. The affinity for voltage-dependent block of gating pore current by Ba²⁺ and Zn²⁺ is increased at more negative holding potentials. The apparent dissociation constant (K_d) values for Zn²⁺ block for test pulses to −160 mV are 650 ± 150 µM, 360 ± 70 µM, and 95.6 ± 11 µM at holding potentials of 0 mV, −80 mV, and −120 mV, respectively. Gating pore current is blocked by trivalent cations, but in a nearly voltage-independent manner, with an apparent K_d for Gd³⁺ of 238 ± 14 µM at −80 mV. To test whether these periodic paralyses might be treated by blocking gating pore current, we screened several aromatic and aliphatic guanidine derivatives and found that 1-(2,4-xylyl)guanidinium can block gating pore current in the millimolar concentration range without affecting normal Na_V1.4 channel function. Together, our results demonstrate unique permeability of guanidine through Na_V1.4 gating pores, define voltage-dependent and voltage-independent block by divalent and trivalent cations, respectively, and provide initial support for the concept that guanidine-based gating pore blockers could be therapeutically useful.     

Correlation Spectroscopy and Molecular Dynamics Simulations to Study the Structural Features of Proteins

In this work, we used a combination of fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulation methodologies to acquire structural information on pH-induced unfolding of the maltotriose-binding protein from Thermus thermophilus (MalE2). FCS has emerged as a powerful technique for characterizing the dynamics of molecules and it is, in fact, used to study molecular diffusion on timescale of microsecond and longer. Our results showed that keeping temperature constant, the protein diffusion coefficient decreased from 84±4 µm²/s to 44±3 µm²/s when pH was changed from 7.0 to 4.0. An even more marked decrease of the MalE2 diffusion coefficient (31±3 µm²/s) was registered when pH was raised from 7.0 to 10.0. According to the size of MalE2 (a monomeric protein with a molecular weight of 43 kDa) as well as of its globular native shape, the values of 44 µm²/s and 31 µm²/s could be ascribed to deformations of the protein structure, which enhances its propensity to form aggregates at extreme pH values. The obtained fluorescence correlation data, corroborated by circular dichroism, fluorescence emission and light-scattering experiments, are discussed together with the MD simulations results.     

DNA bending induced by DNA (cytosine-5) methyltransferases

DNA bending induced by six DNA (cytosine-5) methyltransferases was studied using circular permutation gel mobility shift assay. The following bend angles were obtained: M.BspRI (GG^m5CC), 46–50°; M.HaeIII (GG^m5CC), 40–43°; M.SinI (GGW^m5CC), 34–37°; M.Sau96I (GGN^m5CC), 52–57°; M.HpaII (C^m5CGG), 30°; and M.HhaI (G^m5CGC), 13°. M.HaeIII was also tested with fragments carrying a methylated binding site, and it was found to induce a 32° bend. A phase-sensitive gel mobility shift assay, using a set of DNA fragments with a sequence-directed bend and a single methyltransferase binding site, indicated that M.HaeIII and M.BspRI bend DNA toward the minor groove. The DNA curvature induced by M.HaeIII contrasts with the lack of DNA bend observed for a covalent M.HaeIII–DNA complex in an earlier X-ray study. Our results and data from other laboratories show a correlation between the bending properties and the recognition specificities of (cytosine-5) methyltransferases: enzymes recognizing a cytosine 3′ to the target cytosine tend to induce greater bends than enzymes with guanine in this position. We suggest that the observed differences indicate different mechanisms employed by (cytosine-5) methyltransferases to stabilize the helix after the target base has flipped out.     

Inhibition of purine phosphoribosyltransferases of Ehrlich ascites-tumour cells by 6-mercaptopurine

1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 4·7 μm) and hypoxanthine phosphoribosyltransferase (K_i 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of K_i for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.     

Convergent Evolution in the Assembly of Polyubiquitin Degradation Signals by the Shigella flexneri IpaH9.8 Ligase

The human pathogen Shigella flexneri subverts host function and defenses by deploying a cohort of effector proteins via a type III secretion system. The IpaH family of 10 such effectors mimics ubiquitin ligases but bears no sequence or structural homology to their eukaryotic counterpoints. Using rates of ¹²⁵I-polyubiquitin chain formation as a functional read out, IpaH9.8 displays V-type positive cooperativity with respect to varying concentrations of its Ubc5B∼¹²⁵I-ubiquitin thioester co-substrate in the nanomolar range ([S]_½ = 140 ± 32 nm; n = 1.8 ± 0.1) and cooperative substrate inhibition at micromolar concentrations ([S]_½ = 740 ± 240 nm; n = 1.7 ± 0.2), requiring ordered binding to two functionally distinct sites per subunit. The isosteric substrate analog Ubc5BC85S-ubiquitin oxyester acts as a competitive inhibitor of wild-type Ubc5B∼¹²⁵I-ubiquitin thioester (K_i = 117 ± 29 nm), whereas a Ubc5BC85A product analog shows noncompetitive inhibition (K_i = 2.2 ± 0.5 μm), consistent with the two-site model. Re-evaluation of a related IpaH3 crystal structure (PDB entry 3CVR) identifies a symmetric dimer consistent with the observed cooperativity. Genetic disruption of the predicted IpaH9.8 dimer interface reduces the solution molecular weight and significantly ablates the k_cat but not [S]_½ for polyubiquitin chain formation. Other studies demonstrate that cooperativity requires the N-terminal leucine-rich repeat-targeting domain and is transduced through Phe³⁹⁵. Additionally, these mechanistic features are conserved in a distantly related SspH2 Salmonella enterica ligase. Kinetic parallels between IpaH9.8 and the recently revised mechanism for E6AP/UBE3A (Ronchi, V. P., Klein, J. M., and Haas, A. L. (2013) E6AP/UBE3A ubiquitin ligase harbors two E2∼ubiquitin binding sites. J. Biol. Chem. 288, 10349–10360) suggest convergent evolution of the catalytic mechanisms for prokaryotic and eukaryotic ligases.     

Voltage-Dependent Modulation of Cardiac Ryanodine Receptors (RyR2) by Protamine

It has been reported that protamine (>10 µg/ml) blocks single skeletal RyR1 channels and inhibits RyR1-mediated Ca²⁺ release from sarcoplasmic reticulum microsomes. We extended these studies to cardiac RyR2 reconstituted into planar lipid bilayers. We found that protamine (0.02–20 µg/ml) added to the cytosolic surface of fully activated RyR2 affected channel activity in a voltage-dependent manner. At membrane voltage (V_m; SR lumen - cytosol) = 0 mV, protamine induced conductance transitions to several intermediate states (substates) as well as full block of RyR2. At V_m>10 mV, the substate with the highest level of conductance was predominant. Increasing V_m from 0 to +80 mV, decreased the number of transitions and residence of the channel in this substate. The drop in current amplitude (full opening to substate) had the same magnitude at 0 and +80 mV despite the ∼3-fold increase in amplitude of the full opening. This is more similar to rectification of channel conductance induced by other polycations than to the action of selective conductance modifiers (ryanoids, imperatoxin). A distinctive effect of protamine (which might be shared with polylysines and histones but not with non-peptidic polycations) is the activation of RyR2 in the presence of nanomolar cytosolic Ca²⁺ and millimolar Mg²⁺ levels. Our results suggest that RyRs would be subject to dual modulation (activation and block) by polycationic domains of neighboring proteins via electrostatic interactions. Understanding these interactions could be important as such anomalies may be associated with the increased RyR2-mediated Ca²⁺ leak observed in cardiac diseases.     

Anterograde Degeneration along the Visual Pathway after Optic Nerve Injury

Purpose

To investigate anterograde degenerative changes along the visual pathway in a rat model of optic nerve axotomy.

Methods

Optic nerve transection was performed in adult Sprague-Dawley rats. Animals were sacrificed at regular time intervals and tissues harvested. Immunoblotting followed by densitometric analysis was used to determine the phosphorylation profile of Akt in the dorsal lateral geniculate nucleus (dLGN) and the primary visual cortex (V1). The neuronal cell size and cell density were measured in the dLGN and the V1 using Nissl staining. The prevalence of apoptosis was characterized by terminal deoxynucleotidyl-transferase-mediated biotin-dUTP nick end labelling (TUNEL) histochemistry. Caspase-3 antibodies were also used to identify apoptotic cells. Neurons and astrocytes were detected using NeuN and glial fibrillary acidic protein (GFAP), respectively.

Results

An early and sustained loss of Akt phosphorylation was observed after optic nerve transection in both dLGN and V1. At week one, a decrease in the neuronal cell size (50.5±4.9 vs 60.3±5.0 µm², P = 0.042) and an increase of TUNEL positive cells (7.9±0.6 vs 1.4±0.5 ×10² cells/mm², P<0.001) were evident in the dLGN but not in V1. A significant decline in neuronal cell number (14.5±0.1 vs 17.4±1.3 ×10² cells/mm², P = 0.048), cell size (42.5±4.3 vs 62.1±4.7 µm², P = 0.001) and an increase in apoptotic cells (5.6±0.5 vs 2.0±0.4 ×10² cells/mm², P<0.001) appeared in V1 initially at one month post-transection. The changes in the visual pathway continued through two months. Both neuronal cells and GFAP-positive glial cells were affected in this anterograde degeneration along the visual pathway.

Conclusions

Anterograde degeneration along the visual pathway takes place in target relay (LGN) and visual cortex following the optic nerve injury. Apoptosis was observed in both neural and adjacent glial cells. Reduction of Akt phosphorylation preceded cellular and apoptotic changes.