Naphthalene association and uptake in Pseudomonas putida.

Two methods for bacterial membrane transport, filtration and flow dialysis, were used to study cellular association of Pseudomonas putida with naphthalene. It is not technically possible to determine the exact cellular or vesicular location of the naphthalene, and because it is hydrophobic, it could be at the membrane(s) rather than inside the cells. As an index of naphthalene having crossed the inner membrane we used the intracellular formation of its first catabolite. An energized membrane or ATP was not essential for association or movement into the cell. Evidence for a nonspecific association and a movement into cells by simple diffusion are the lack of saturation of association, an absence of inhibition of association by protein inhibitors and structural analogs, and the passage of naphthalene through cell membranes in the presence of iodoacetamide. Specific naphthalene metabolism gene expression was not required for association.     

Binding of sulfosuccinimidyl fatty acids to adipocyte membrane proteins: Isolation and ammo-terminal sequence of an 88-kD protein implicated in transport of long-chain fatty acids

Summary We recently reported (Harmon et al., J. Membrane Biol. 124:261–268, 1991) that sulfo-N-succinimidyl derivatives of long-chain fatty acids (SS-FA) specifically inhibited transport of oleate by rat adipocytes. These compounds bound to an 85–90 kD membrane protein which was also labeled by another inhibitor of FA transport [³H]DIDS (4,4-diisothiocyanostilbene-2-2-sulfonate). These results indicated that the protein was a strong candidate as the transporter for long-chain fatty acids. In this report we determined that the apparent size of the protein is 88 kD and its isoelectric point is 6.9. We used [³H]SS-oleate (SSO), which specifically labels the 88-kD protein, to isolate it from rat adipocyte plasma membranes. Identification of 15 amino acids at the N-terminus region revealed strong sequence homology with two previously described membrane glycoproteins: CD36, a ubiquitous protein originally identified in platelets and PAS IV, a protein that is enriched in the apical membranes of lipidsecreting mammary cells during lactation. Antibody against PAS IV cross-reacted with the adipocyte protein. This, together with the N-terminal sequence homology, suggested that the adipocyte protein belongs to a family of related intrinsic membrane proteins which include CD36 and PAS IV.     

Presence of methylated adenine in GATC sequences in chromosomal DNAs from Campylobacter species.

We digested chromosomal DNAs from 12 Campylobacter strains (C. jejuni, 4 strains; C. coli, 2 strains; C. fetus subsp. fetus, 2 strains; C. hyointestinalis, 2 strains; and C. upsaliensis, 2 strains) and from 4 Helicobacter strains (H. pylori, 2 strains; and H. mustelae, 2 strains) with HindIII, SstI, BamHI, DpnI, MboI, and Sau3AI. Restriction fragments were then separated by electrophoresis in 1% agarose or 10% polyacrylamide gels. Only DNAs from three Campylobacter species (C. jejuni, C. coli, and C. upsaliensis) were digested with DpnI (an enzyme that recognizes only methylated adenine in GATC sequences). We used MboI and Sau3AI to confirm these findings.     

Pattern generation in the lobster (Panulirus) stomatogastric ganglion

There are a number of perspectives gained from a quantitative analysis of the pyloric system which may be applicable to other simple pattern generators: 1. The system is organized around a dominant, endogenously-bursting neuron group, and its properties are tailored to that dominance. In particular, synaptic strengths and firing frequencies of that group appear just sufficient to suppress postsynaptic "follower" cells if the latter are not too highly excited. 2. Repetitive firing properties of follower neurons are such as to facilitate their switch-like mode of activity. This includes pacemaker response nonlinearities, rebound properties, and "burstiness" properties. 3. Proper sequencing of follower cells may be controlled by particular synaptic strengths and time-courses, feedback on the oscillator cells, and functional cellular properties of follower neurons (e.g., rebound; see also next paper). All such properties interact and must be tuned to each other for proper patterns to result.     

Neurohormonal alteration of integrative properties of the cardiac ganglion of the lobster Homarus americanus.

The spontaneous burst discharges of isolated lobster (Homarus americanus) cardiac ganglia were recorded with a spaced array of electrodes. Small regions (less than 1 mm) of the ganglion were exposed to the cardioexcitor neurohormone in extracts of pericardial organs (XPO) or to 10(-5) M 5-hydroxytryptamine (5HT). All axons were excited (increased mean firing frequency, f) by both substances, but only by applications in the region between the soma (but excluding it) and proximal site of impulse initiation. Units not so exposed changed their f relatively little despite f increases of as much as threefold in exposed units and changes in burst rate and overall length. Regularity and grouping of all impulse activity into bursts was never disturbed. 5HT increases burst rate at any point of application. The increases are larger if small cells are affected than if only large cells are exposed. Burst length decreases except when the pacemaker is affected. In contrast, XPO affects neither burst rate or length unless small cells are affected. Length is increased if non-pacemaker small cells are affected; both rate and length increase if the pacemaker is affected. The pacemaker usually exhibits an f of intermediate value. Rate changes are not simply related to its f. A small cell can "burst" in the absence of impulses from any other cells. XPO may enhance endogenous "driver potentials," while 5HT may excite by depolarizing at limited sites.     

Corrections for space-clamp errors in measured parameters of voltage-dependent conductances in a cylindrical neurite

The ability to correct parameters of voltage-gated conductances measured under poor spatial control by point voltage clamp could rescue much flawed experimental data. We explore a strategy for correcting errors in experiments that employs a full-trace approach to parameter determination. Simulated soma voltage-clamp runs are made on a model neuron with a single voltage-gated, Hodgkin-Huxley channel type distributed uniformly along an elongate process. Estimates for both kinetic and I(V) parameters are obtained by fitting a form of the Hodgkin-Huxley equations to the complete time course of leak-subtracted current curves. The fitted parameters are used to determine how much correction in each parameter is needed to regenerate the set actually belonging to the channel. Corrections are generated for a range of neurite lengths, conductance densities, and channel characteristics.     

Tumor-infiltrating dendritic cell precursors recruited by a beta-defensin contribute to vasculogenesis under the influence of Vegf-A

The involvement of immune mechanisms in tumor angiogenesis is unclear. Here we describe a new mechanism of tumor vasculogenesis mediated by dendritic cell (DC) precursors through the cooperation of beta-defensins and vascular endothelial growth factor-A (Vegf-A). Expression of mouse beta-defensin-29 recruited DC precursors to tumors and enhanced tumor vascularization and growth in the presence of increased Vegf-A expression. A new leukocyte population expressing DC and endothelial markers was uncovered in mouse and human ovarian carcinomas coexpressing Vegf-A and beta-defensins. Tumor-infiltrating DCs migrated to tumor vessels and independently assembled neovasculature in vivo. Bone marrow-derived DCs underwent endothelial-like differentiation ex vivo, migrated to blood vessels and promoted the growth of tumors expressing high levels of Vegf-A. We show that beta-defensins and Vegf-A cooperate to promote tumor vasculogenesis by carrying out distinct tasks: beta-defensins chemoattract DC precursors through CCR6, whereas Vegf-A primarily induces their endothelial-like specialization and migration to vessels, which is mediated by Vegf receptor-2.     

Simulations of voltage clamping poorly space-clamped voltage-dependent conductances in a uniform cylindrical neurite

Significant error is made by using a point voltage clamp to measure active ionic current properties in poorly space-clamped cells. This can even occur when there are no obvious signs of poor spatial control. We evaluated this error for experiments that employ an isochronal I(V) approach to analyzing clamp currents. Simulated voltage clamp experiments were run on a model neuron having a uniform distribution of a single voltage-gated inactivating ionic current channel along an elongate, but electrotonically compact, process. Isochronal Boltzmann I(V) and kinetic parameter values obtained by fitting the Hodgkin-Huxley equations to the clamp currents were compared with the values originally set in the model. Good fits were obtained for both inward and outward currents for moderate channel densities. Most parameter errors increased with conductance density. The activation rate parameters were more sensitive to poor space clamp than the I(V) parameters. Large errors can occur despite normal-looking clamp curves.     

Human cytomegalovirus UL97 Kinase is required for the normal intranuclear distribution of pp65 and virion morphogenesis

Recombinant human cytomegaloviruses that do not express UL97 kinase activity exhibit a distinctive plaque morphology characterized by the formation of highly refractile bodies late in infection. These structures were also observed in infected cells treated with the UL97 kinase inhibitor maribavir. Nuclear inclusions were purified to near homogeneity, and the constituent proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. This analysis demonstrated that the aggregates were formed principally of the tegument proteins pp65 and ppUL25 but also contained additional virion structural proteins including the major capsid protein. Immunoblotting experiments confirmed these results and identified a number of additional viral proteins present in the purified tegument aggregates. Interestingly, the formation of these structures appeared to be dependent on pp65, since it was not induced in cells infected with a recombinant virus with this open reading frame deleted. Morphologically similar aggregates could be reproduced in nuclei of uninfected cells by overexpressing pp65, and their formation was prevented by coexpressing the UL97 kinase. Inhibition of UL97 kinase activity with maribavir or mutation of an essential amino acid in the kinase abolished its ability to prevent aggregate formation. These data taken together suggest that the UL97 kinase impacts the aggregation of pp65 in the nuclei of infected cells. We propose that the kinase plays an important role in the acquisition of tegument during virion morphogenesis in the nucleus and that this activity represents an important step in the production of mature virus particles.     

Synthesis and antiviral activity of certain second generation methylenecyclopropane nucleosides

A second-generation series of substituted methylenecyclopropane nucleosides (MCPNs) has been synthesized and evaluated for antiviral activity against a panel of human herpesviruses, and for cytotoxicity. Although alkylated 2,6-diaminopurine analogs showed little antiviral activity, the compounds containing ether and thioether substituents at the 6-position of the purine did demonstrate potent and selective antiviral activity against several different human herpesviruses. In the 6-alkoxy series, antiviral activity depended on the length of the ether carbon chain, with the optimum chain length being about four carbon units long. For the corresponding thioethers, compounds containing secondary thioethers were more potent than those with primary thioethers.