Characterization of the role of melanoma growth stimulatory activity (MGSA) in the growth of normal melanocytes, nevocytes, and malignant melanocytes

Melanoma growth stimulatory activity (MGSA) was originally described as an endogenous growth factor for human melanoma cells. To test the hypothesis that an MGSA autocrine loop is responsible for the partial freedom from growth control observed in nevocytes and melanoma cells, MGSA growth response and MGSA mRNA/protein levels were examined in these cells compared with normal melanocytes. As a single agent, or in combination with other factors, MGSA stimulated the growth of normal human epidermal melanocytes as well as other growth promoters for melanocytes. Nevocytes were not as responsive to exogenous MGSA as melanocytes. MGSA mRNA was minimal or not detected in cultured normal melanocytes, although the protein was present when the cells were cultured in the presence of serum/growth factors and absent when serum/growth factors were omitted. In contrast, MGSA mRNA was constitutively expressed in the absence of exogenous growth factors in cultures established from benign intradermal and dysplastic nevi and melanoma lesions in different stages of tumor progression. Nevus cultures contained immunoreactive MGSA protein in the presence of serum but were negative or only faintly positive in the absence of serum. Melanoma cell lines were positive for MGSA protein in both the presence and the absence of serum. Thus, continued expression of both MGSA mRNA and MGSA protein in the absence of exogenous hormones or serum factors may correlate with partial freedom from growth control exhibited by malignant melanocytes.     

Disruption of baker's yeast by a new bead mill

Summary A continuous high-speed bead mill of novel design (Sulzer Annu Mill 01) was tested for cell disruption of baker's yeast as a model system. The efficiency of cell disruption was evaluated for the relative amount of released protein. The effects of rotation speed, cell concentration and flow rate of cell suspension on the cell disruption were investigated. The maximum yield of released protein was found to be 2.62 kg protein/L.h. This novel design appears to be more effective than existing commercially available mills.Notations Cs cell concentration (g packed yeast/L) - F flow rate of suspension, mL/min - F_R cumulative residence time distribution - N rotation speed of the rotor (rpm) - P number of passes of suspension through mill - R amount of protein released from cell, mg/g packed yeast - R_m maximum amount of protein released, mg/g packed yeast - t time, s - mean residence time, s     

Models of epidermal wound healing

The spreading of cells across the surface of an epidermal wound enables epidermal migration to be studied independently of the wound contraction that occurs in deeper wounds. In particular, the stimulus for the increase in epidermal mitosis during would healing is uncertain. Our modelling suggests that biochemical regulation of mitosis is fundamental to the process, and that a single chemical with a simple regulatory effect can account for the healing of circular epidermal wounds. The model results compare well with experimental data.     

Three-dimensional reconstruction of maltoporin from electron microscopy and image processing.

Two dimensional crystals of maltoporin (or phage lambda receptor) were obtained by reconstitution of purified maltoporin trimers and Escherichia coli phospholipids by detergent dialysis. Two different trimer packing forms were observed. One was hexagonal (a = 7.8 nm) and one rectangular (a = 7.8 nm, b = 13.6 nm). In this paper we describe the three-dimensional structure of maltoporin, deduced from the study of the rectangular form by electron microscopy and image processing. At a resolution of approximately 2.5 nm, maltoporin trimers form aqueous channel triplets which appear to merge into a single outlet at the periplasmic surface of the outer membrane. The pore defined by maltoporin has a similar structure to that outlined by the matrix protein. From the results of functional studies by conductance measurement, it is concluded that the three channels defined by maltoporin act, contrary to those formed by the porin (OmpF protein), as a single conducting unit. A tentative outline of the maltoporin promoter is given. Maltoporin appears to be constituted by three different domains: a major rod-like domain spanning the membrane, a minor domain located near the periplasmic surface of the membrane and finally a central domain responsible for the splitting of the channel.     

Partial purification and some properties of rat brain inositol 1,4,5-trisphosphate 3-kinase.

An enzyme which catalyses the ATP-dependent phosphorylation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was purified approx. 180-fold from rat brain cytosol by (NH4)2SO4 precipitation, chromatography through hydroxyapatite, anion-exchange fast protein liquid chromatography and gel-filtration chromatography. Gel filtration on Sepharose 4B CL gives an Mr of 200 x 10(3) for the native enzyme. The inositol tetrakisphosphate (InsP4) produced by the enzyme has the chromatographic, chemical and metabolic properties of Ins(1,3,4,5)P4. Ins(1,4,5)P3 3-kinase displays simple Michaelis-Menten kinetics for both its substrates, having Km values of 460 microM and 0.44 microM for ATP and Ins(1,4,5)P3 respectively. When many of the inositol phosphates known to occur in cells were tested, only Ins(1,4,5)P3 was a substrate for the enzyme; the 2,4,5-trisphosphate was not phosphorylated. Inositol 4,5-bisphosphate and glycerophosphoinositol 4,5-bisphosphate were phosphorylated much more slowly than Ins(1,4,5)P3. CTP, GTP and adenosine 5'-[gamma-thio]triphosphate were unable to substitute for ATP. When assayed under conditions of first-order kinetics, Ins(1,4,5)P3 kinase activity decreased by about 40% as the [Ca2+] was increased over the physiologically relevant range. This effect was insensitive to the presence of calmodulin and appeared to be the result of an increase in the Km of the enzyme for Ins(1,4,5)P3. Preincubation with ATP and the purified catalytic subunit of cyclic AMP-dependent protein kinase did not affect the rate of phosphorylation of Ins(1,4,5)P3 when the enzyme was assayed at saturating concentrations of Ins(1,4,5)P3 or at concentrations close to its Km for this substrate.     

The eccentric cell of theLimulus lateral eye: encoder of circadian changes in visual responses

Efferent fibers from a central circadian clock innervate both photoreceptor cells and second-order neurons (eccentric cells) in the lateral compound eye ofLimulus, and release octopamine when activated. We have used intracellular microelectrodes to study the modulation of eccentric cell function by efferent optic-nerve activity, octopamine agonists, and a K⁺-channel blocker, TEA.

Interesterification selectivity in lipase catalyzed reactions of low molecular weight triglycerides

Summary In the absence of an organic solvent, a buffer to enzyme weight ratio of 1 gives maximum selectivity to interesterification over hydrolysis in a lipase-catalyzed mixture of triacetin and tributyrin. Addition of hexane enhances interesterification as does a reversed micelle configuration.     

Estimation of oxygen penetration depth in immobilized cells

Summary A simple method is proposed for calculating oxygen pentration depth in immobilized cells by assuming zero order kinetics in the presence of several external oxygen transport resistances. Calculations indicate that typical penetration depths of oxygen for immobilized microbial cells are in the range of 50–200 and those for immobilized or encapsulated animal and plant tissue culture are about 500–1000 . Based on calculations, oxygen transport in microencapsulation and microcarriers for tissue cultures are not transport-limited, but a slight limitation is expected for those in a hollow fiber reactor.Nomenclature a_s specific area of a support (cm) - Bi Biot number - dimensionless - C_b oxygen concentration in the bulk liquid (mM) - C _b C _b ^* -C_cr (mM) - C _b ^* bulk oxygen concentration in equilibrium with air (mM) - C_cr critical oxygen concentration (mM) - C_s oxygen concentration in the solid phase (mM) - d_p diameter or thickness of a support (cm) - D_eff effective diffusivity of oxygen in the solid phase (cm²/s) - k_m membrane permeability of oxygen (cm/s) - k _m ^* D_eff/_m - k_La_L liquid phase mass transfer rate coefficient (1/s) - k_sa_s solid phase mass transfer rate coefficient (1/s) - (OUR)_v volumetric oxygen uptake rate (mmol O₂/l) - p geometry parameter, p=0 for slab, p=1 for cylinder, p=2 for sphere - P_d oxygen penetration depth (cm) - P _d oxygen penetration depth in the absence of external diffusion limitation (cm) - Q volumetric oxygen uptake rate, (mmol O₂/l·h) - specific oxygen uptake rate (mmol O₂gm biomass (dry)·h) - r length coordinate (cm) - r_c oxygen penetration depth for sphere (cm) - r _c r_c in the absence of external diffusion limitation (cm) - r _c ^* oxygen penetration depth for cylinder (cm) - r _c ^* r _c ^* in the absence of external diffusion limitation (cm) - r_com combined mass transfer rate resistance (s) - r_d location where C_s becomes zero or C_cr (cm) - r_i radius of cylinder or sphere, half thickness of slab (cm) - U_sg superficial gas velocity (cm/s) - X cell concentration (g/l) Greek letters Thiele modulus, dimensionless - _L, _s liquid and solid phase volume fraction, respectively, dimensionless - effectiveness factor On sabbatical leave from KAIST, Seoul, Korea