Complexation of C6-Ceramide with Cholesteryl Phosphocholine – A Potent Solvent-Free Ceramide Delivery Formulation for Cells in Culture

Ceramides are potent bioactive molecules in cells. However, they are very hydrophobic molecules, and difficult to deliver efficiently to cells. We have made fluid bilayers from a short-chain D-erythro-ceramide (C6-Cer) and cholesteryl phosphocholine (CholPC), and have used this as a formulation to deliver ceramide to cells. C6-Cer complexed with CholPC led to much larger biological effects in cultured cells (rat thyroid FRTL-5 and human HeLa cells in culture) compared to C6-Cer dissolved in dimethyl sulfoxide (DMSO). Inhibition of cell proliferation and induction of apoptosis was significantly more efficient by C6-Cer/CholPC compared to C6-Cer dissolved in DMSO. C6-Cer/CholPC also permeated cell membranes and caused mitochondrial Ca²⁺ influx more efficiently than C6-Cer in DMSO. Even though CholPC was taken up by cells to some extent (from C6-Cer/CholPC bilayers), and was partially hydrolyzed to free cholesterol (about 9%), none of the antiproliferative effects were due to CholPC or excess cholesterol. The ceramide effect was not limited to D-erythro-C6-Cer, since L-erythro-C6-Cer and D-erythro-C6-dihydroCer also inhibited cell priolifereation and affected Ca²⁺ homeostasis. We conclude that C6-Cer complexed to CholPC increased the bioavailability of the short-chain ceramide for cells, and potentiated its effects in comparison to solvent-dissolved C6-Cer. This new ceramide formulation appears to be superior to previous solvent delivery approaches, and may even be useful with longer-chain ceramides.     

DNA Methylation in Eukaryotes: Kinetics of Demethylation and De Novo Methylation during the Life Cycle

We present a model for the kinetics of methylation and demethylation of eukaryotic DNA; the model incorporates values for de novo methylation and the error rate of maintenance methylation. From the equations, an equilibrium is reached such that the proportion of sites which are newly methylated equals the proportion of sites which become demethylated in a cell generation. This equilibrium is empirically determined as the level of maintenance methylation. We then chose reasonable values for the parameters using maize and mice as model species. In general, if the genome is either hypermethylated or hypomethylated it will approach the equilibrium level of maintenance methylation asymptotically over time; events occurring just once per life cycle to suppress methylation can maintain a relatively hypomethylated state. Although the equations developed are used here as framework for evaluating events in the whole genome, they can also be used to evaluate the rates of methylation and demethylation in specific sites over time.     

Strukturanalyse der methyl-2,3,6-tridesoxy-2-C-[2-hydroxy-1,1-(Äthylendithio)Äthyl]-α-l-threo-hexopyranosid-4-ulo-22,4-pyranose

Methyl 2,3,6-trideoxy-2-C-[2-hydroxy-1,1-(ethylenedithio)ethyl]-α-l-threo-hexopyranosid-4-ulo-2²,4-pyranose (1) crystallizes in a rhombic space group P2₁2₁2₁ with four molecules in the elementary unit. The structure was refined to an R-value of 0.057. The aldopyranose ring adopts a ¹C₄ conformation with an axial side-chain forming a hemiacetal ring to the keto group at C-4. Both six-membered rings connected in the 2,7-dioxabicyclo[3.3.1]nonane system differ only slightly from the ¹C₄ chair conformation. The spirocyclic dithiolane ring adopts a nearly ideal envelope form with a deviation of C-21 from the plane S-1-C-7-S-2-C-22. The dihedral angle O-5-C-1 O-1-C-11 of 59.1° is an agreement with the exo-anomeric effect.     

A dye mixture (Neurobiotin and Alexa 488) reveals extensive dye-coupling among neurons in leeches; physiology confirms the connections

Although the neuronal circuits that generate leech movements have been studied for over 30 years, the list of interneurons (INs) in these circuits remains incomplete. Previous studies showed that some motor neurons (MNs) are electrically coupled to swim-related INs, e.g., rectifying junctions connect IN 28 to MN DI-1 (dorsal inhibitor), so we searched for additional neurons in these behavioral circuits by co-injecting Neurobiotin and Alexa Fluor 488 into segmental MNs DI–1, VI–2, DE–3 and VE–4. The high molecular weight Alexa dye is confined to the injected cell, whereas the smaller Neurobiotin molecules diffuse through gap junctions to reveal electrical coupling. We found that MNs were each dye-coupled to approximately 25 neurons, about half of which are likely to be INs. We also found that (1) dye-coupling was reliably correlated with physiologically confirmed electrical connections, (2) dye-coupling is unidirectional between MNs that are linked by rectifying connections, and (3) there are novel electrical connections between excitatory and inhibitory MNs, e.g. between excitatory MN VE-4 and inhibitory MN DI-1. The INs found in this study provide a pool of novel candidate neurons for future studies of behavioral circuits, including those underlying swimming, crawling, shortening, and bending movements.     

Gelatin-induced Reversion of Protoplasts of Bacillus subtilis to the Bacillary Form: Electron-microscopic and Physical Study

Protoplasts of Bacillus subtilis plated on SD medium form L colonies in quantitative yield and propagate in the L form indefinitely. L bodies or protoplasts placed in 25% gelatin medium form bacillary colonies. Details of the reversion of naked bodies to the walled form are reported. In 25% gelatin medium, reversion begins earlier (about 50% reversion in 4 hr) than the multiplication of bacilli. Thus, virtually all the observed bacillary forms are themselves revertants and not the offspring of a few growing clones. The optimal temperature for reversion is 26 C in 25% gelatin. When cells reverting at 26 C are warmed to 40 C for 3 min, reversion is delayed markedly, whereas viability is unaffected. For electron microscopy, a dense protoplast inoculum was placed on a gelatin surface, incubated, and then fixed in situ. There was no multiplication, but crowding delayed reversion markedly. Successive events of reversion are as follows. The loose nucleoid of the protoplasts condenses in response to the gelatin medium and condenses further and further as reversion proceeds. A thin coat of wall develops around the bodies of various sizes and shapes and then increases uniformly in thickness until a wall of normal aspect is formed. Rod-shaped cells grow out from these bodies-sometimes in several directions at once. A few mesosomes begin to appear only after a thin coat of wall has been formed. These are dense, atypical structures compartmented by membranes. They are located at the cell periphery and do not seem to be in contact with the nucleoids. Quantitative estimates showed that only 20 to 25% of revertant cells or cells grown on gelatin contain even a single mesosome. The others have no mesosome at all. Mesosomes thus do not appear to play a significant role in reversion, and normal mesosome functions must presumably be performed elsewhere in the cell in gelatin-grown bacilli. The role of cell wall, its synthesis, and its chemical nature in successive steps in reversion are discussed.     

In vitro reconstitution of a trimeric complex of DivIB, DivIC and FtsL, and their transient co-localization at the division site in Streptococcus pneumoniae

DivIB, DivIC and FtsL are bacterial proteins essential for cell division, which show interdependencies for their stabilities and localization. We have reconstituted in vitro a trimeric complex consisting of the recombinant extracellular domains of the three proteins from Streptococcus pneumoniae. The extracellular domain of DivIB was found to associate with a heterodimer of those of DivIC and FtsL. The heterodimerization of DivIC and FtsL was artificially constrained by fusion with interacting coiled-coils. Immunofluorescence experiments showed that DivIC is always localized at mid-cell, in contrast to DivIB and FtsL, which are co-localized with DivIC only during septation. Taken together, our data suggest that assembly of the trimeric complex DivIB/DivIC/FtsL is regulated during the cell cycle through controlled formation of the DivIC/FtsL heterodimer.