Recognition and clearance of methoxypoly(ethyleneglycol)2000-grafted liposomes by macrophages with enhanced phagocytic capacity. Implications in experimental and clinical oncology.

Intravenous injection of an endotoxin-free solution of poloxamine-908 to rats can enhance the phagocytic clearance capacity of tissue macrophages, particularly those of the liver and the spleen. Such stimulated cells were able to clear a significant portion of intravenously injected methoxypoly(ethyleneglycol)2000 liposomes (mean size of 87 nm), labelled with technetium-99m via the N-hydroxysuccinimidyl hydrazine nicotinate hydrochloride derivative of distearoyl phosphatidylethanolamine, within 4 h post administration. These liposomes, otherwise, exhibit long circulatory behaviour in control animals, with poor localization to the liver and spleen. We suggest that such technetium-99m-labelled engineered vesicles may be of aid for detection of the liver and spleen macrophages with enhanced phagocytic clearance capacity by gamma scintigraphy. Alterations in the phagocytic activity of liver and spleen macrophages is known to occur during cancer. Therefore, such diagnostic procedures may prove useful for patient selection or for monitoring the progress of treatment with long circulating nanoparticles carrying anti-cancer agents, thus minimizing damage to this important line of body's defence cells, and are discussed.     

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.     

Clinical applications of the subgaleal fascia

The anatomic boundaries and vascular supply of the subgaleal fascia have been described previously. The thin and malleable subgaleal fascia was selected for difficult reconstructive problems in seven patients. This flap has been based on either the supraorbital or the superficial temporal vascular leash. The subgaleal fascia is readily dissected from superficial galea and deep periosteum, leaving behind a well-vascularized scalp and a skin-graftable calvarium. The flap conforms to a cartilage framework for ear reconstruction. It takes a skin graft well. The subgaleal fascia can patch dural defects and fill sinus dead space. It has been used to augment facial contour. Free vascularized transfer of the subgaleal fascia has included the temporoparietal fascia, which was partially split from the subgaleal fascia for bilobed flap resurfacing of the hand. The subgaleal fascial flap should be considered when ultrathin, vascularized coverage is needed.     

Cell wall-DNA association in Bacillus subtilis.

Autolysis of cell walls of Bacillus subtilis 168 resulted in solubilization of wall-associated DNA. Most of the DNA was solubilized only in the later stages of autolysis. Solubilization of up to 70% of the wall by autolysins resulted in only 25 to 30% solubilization of wall-associated DNA. When the wall fragments remaining after 70% autolysis were analyzed by electron microscopy, it was observed that the preparations were highly enriched for completed septa, or poles. Partial autolysis at pH 5.2 or pH 8.6, both of which reflect hydrogen ion levels that permit either N-acetylglucosaminidase or N-acetylmuramyl-L-alanine amidase, but not both, to act, gave rise to enrichment of cell poles. When walls were incubated with subtilisin, DNase, or RNase, release of DNA (or DNA fragments) was accelerated. Density gradient centrifugation patterns of lysates of cells pulse-labeled with N-[3H]acetylglucosamine and then chased revealed that a small, but significant, proportion of the radioactivity sedimented to a density position equivalent to that of DNA-membrane complexes. Because the pulse-chase sequence enriched for radioactivity in cell poles, the results suggest that at least some molecules from polar cell walls have an affinity for DNA-membrane complexes. We suggest that DNA binds strongly, possibly via a DNA-membrane complex, to cell poles of B. subtilis. The results provide support for a view offered previously (Koch et al., FEMS Microbiol. Lett. 12:201-208, 1981) that some special structure in or very near the poles of gram-positive bacilli is involved in the segregation of DNA during cell division.     

Characterization of the genome of Pseudomonas aeruginosa bacteriophage phi PLS27 with particular reference to the ends of the DNA.

The DNA of Pseudomonas aeruginosa rough-specific bacteriophage phi PLS27 was studied. The genome size as determined by summing the sizes of restriction fragments was 42.7 kilobase pairs. Of particular interest was the fact that the DNA was insensitive to certain common restriction endonucleases including EcoRI, BamHI, and HindIII. The ends of the phage DNA were cloned and sequenced, revealing direct repeats of 318 nucleotides. The left end of the genome when cloned into the promoter selection vector pKK232-8 exhibited promoter activity in Escherichia coli. Two promoters bearing greater than 70% sequence homology to the plasmid pNM74 TOL operon and PAK pilin promoters were identified.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

The stimulatory effect of chronic lithium treatment on basal thyrotropin secretion in rats: In vivo antagonism by methylparaben

Chronic treatment of rats with lithium chloride was examined in order to determine its effect on hypothalamic monoamine and metabolite content, basal thyrotropin (TSH) secretion and thyroid function. The hypothalamic concentrations of noradrenaline (NA), dopamine (DA) and its metabolites, dihydroxyphenylacetic acid. (DOPAC) and homovanillic acid (HVA) in the lithium treated rats remained unaltered when compared to control levels. NA turnover and the NA metabolite, 3-methoxy-4-hydroxyphenylglycol (total MHPG), were significantly lower (p<0.01), whereas both serotonin (5-HT) and its metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), were significantly higher (p<0.01 and p<0.02, respectively) in the lithium treated rat hypothalami than in controls. Chronic lithium treatment significantly elevated basal TSH levels (p<0.05). This effect was antagonized by methylp-hydroxybenzoate (methylparaben, p<0.01), which did not itself affect basal TSH levels. Free serum T₃ and T₄ levels were not significantly affected by chronic lithium treatment, although T₄ tended to be slightly lower than control levels. The monoamine changes observed in the hypothalamus of lithium treated rats did not appear to account for the elevated TSH levels observed in these rats since NA activity which is generally regarded as stimulatory was decreased and 5-HT which has an inhibitory effect on TSH secretion, was increased. The elevated TSH levels may have been due to a reduced negative feedback inhibition of TSH release by the mildly reduced circulating T₄ levels caused by chronic lithium treatment. A further possibility is that the pituitary cGMP (and hence TSH) response to TRH may have been enhanced by chronic lithium treatment and methylparaben may have antagonized this effect.     

Absence of an effect of the lithium-induced increase in cyclic GMP on the cyclic GMP-stimulated phosphodiesterase (PDE II). Evidence for cyclic AMP-specific hydrolysis

Chronic treatment of rats with LiCl is known to induce a decrease in cAMP, while this decrease has also been found to occur together with both a simultaneous increase in total cortical phosphodiesterase (PDE; EC 3.1.4.17) activity and a concomitant increase in cGMP. These studies have implicated an involvement of PDE in lithium (Li⁺) action and it has been suggested that cGMP and the cGMP-stimulated PDE may be instrumental in the observed effects of Li⁺ on cAMP. In this study, three isozymes of PDE were isolated and identified from rat cortex and their activity determined, together with simultaneous measurement of cAMP and cGMP, after chronic treatment with oral LiCl (0.35% m/m). Li⁺ treatment exerted profound effects on cyclic nucleotides in the cortex, inducing significant suppression of cAMP while increasing cGMP levels. However, the ion only induced a slight but insignificant increase in the activities of the three PDE isozymes. To confirm these observations, methylparaben (MPB), a drug demonstrating both an ability to induce a selective stimulation of cAMP-specific PDE and also to lower intracellular levels of cGMP, was co-administration orally (0.4% m/m) with Li⁺ over the same period. This combination emphasized certain actions of Li⁺ not noted with Li⁺ alone. MPB inhibited the Li⁺-induced increase in cGMP, yet did not prevent the ion from decreasing cAMP. However, the combination of Li⁺ and MPB engendered a synergistic 100% increase in the activity of the membrane-bound, cAMP-specific PDE, PDE IV. This combination also produced a significant suppression of cAMP, while no reduction in cGMP was observed. The data is indicative that Li⁺-induced suppression of cAMP does not appear to be related to an effect on the cGMP-dependent PDE II, and that the increases in cGMP and PDE induced by Li⁺ observed previously and in the present study are two unrelated events. Instead, the synergistic response of Li⁺ plus MPB on PDE IV, and the associated reduction of cAMP, indicate that Li⁺ may promote selective cAMP hydrolysis via an effect on membrane-bound forms of PDE. This effect of Li⁺ on PDE IV, as well as the reciprocal effects on cyclic nucleotide balance, may have important implications in explaining the antipsychotic actions of the ion.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Molecular Cloning and Physical Mapping of Restriction Endonuclease Fragments of Autographa californica Nuclear Polyhedrosis Virus DNA

A restriction fragment library containing Autographa californica nuclear polyhedrosis virus (AcNPV) DNA was constructed by using the pBR322 plasmid as a vector. The library, which is representative of more than 95% of the viral genome, consists of 2 of the 7 BamHI fragments, 12 of the 24 HindIII fragments, and 23 of the 24 EcoRI fragments. The cloned fragments were characterized and used to generate physical maps of the genome by hybridizing nick-translated recombinant plasmid to Southern blots of AcNPV DNA digested with SmaI, BamHI, XhoI, PstI, HindIII, and EcoRI restriction endonucleases. This information was used to define our strain of AcNPV (HR3) with respect to other strains for which physical maps have been previously published. The hybridization data also indicate that reiteration of DNA sequences occurs at the HindIII-L and -Q regions of the genome.