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The paper investigates the accuracy of the coherent gravity method by using measurements reported in a large database of full-scale instrumented walls that was not available at the time the original method was developed. The new database includes data for bar mat, welded wire and steel strip soil reinforced walls. Measured reinforcement loads under operational conditions are compared with predicted values for bar mat and steel strip reinforced walls. The accuracy of the coherent gravity method as presented in the BS 8006 design standard is quantified by computing the mean and coefficient of variation of the ratio (bias) of measured to predicted loads. The paper shows that for steel strip walls the coherent gravity method is reasonably accurate for soils with friction angles less than 45°. For granular soils with higher friction angles and bar mat walls, the current coherent gravity method is shown to be less accurate and, on average, non-conservative for design. Modifications to the method as currently described in BS 8006 are proposed to improve the accuracy of the method for bar mat reinforced soil walls and steel strip reinforced soil walls with high friction angle backfill soils.

Targeted Delivery of Antisense Oligodeoxynucleotides In Vivo by Means of Coated Cationic LipoplexesEarlier we reported on the massive uptake of liposomes surface-modified with negatively charged aconitylated albumin (Aco-HSA) by liver endothelial cells (EC) in vivo. In the present work we apply this principle for in vivo delivery of antisense oligodeoxynucleotides (ODN) to these cells by means of coated cationic lipoplexes (CCL) (). CCL were prepared by complexing ODN with the cationic lipid DOTAP and subsequent coating of the complex by neutral lipids including a lipid-anchored poly(ethylene glycol). Aco-HSA was covalently coupled.The Aco-HSA-CCLs were 160 nm in size, contained 1.03 ± 0.35 nmol ODN and 54 ± 18 µg Aco-HSA per µ mol total lipid. The Aco-HSA-CCLs were rapidly eliminated from plasma, 60% of the injected dose being recovered in the liver after 30 m. Within the liver, the EC accounted for two thirds of total liver uptake. Non-targeted CCLs were eliminated very slowly: after 30 m >90% of the pa...

Background Children with 22q11.2 deletion syndrome (22q11DS) are at risk for social-behavioural and neurocognitive sequelae throughout development. The current study examined the impact of family environmental characteristics on social-behavioural and cognitive outcomes in this paediatric population. Method Guardians of children with 22q11DS were recruited through two medical genetics clinics. Consenting guardians were asked to complete several questionnaires regarding their child's social, emotional and behavioural functioning, as well as family social environment and parenting styles. Children with 22q11DS were asked to undergo a cognitive assessment, including IQ and achievement testing, and measures of attention, executive function and memory. Results Modest associations were found between aspects of the family social environment and parenting styles with social-behavioural and cognitive/academic outcomes. Regression models indicated that physical punishment, socioeconomic status, parental control and family organisation significantly predicted social-behavioural and cognitive outcomes in children with 22q11DS. Conclusion Characteristics of the family social environment and parenting approaches appear to be associated with functional outcomes of children with 22q11DS. Understanding the impact of environmental variables on developmental outcomes can be useful in determining more effective targets for intervention. This will be important in order to improve the quality of life of individuals affected by 22q11DS.

Infection of rhesus macaques with simian immunodeficiency virus (SIV) is an excellent model system for studying viral adaptation to immune responses. In this review, we discuss how the SIV-infected macaque has provided unequivocal evidence for cytotoxic T-lymphocyte (CTL) selection of viral escape variants. This improved understanding of CTL escape may influence human immunodeficiency virus (HIV) vaccine design as well as our understanding of HIV pathogenesis.

A previous investigation revealed that various 4-aryl-3-arylcarbonyl-1-ethyl-4-piperidinols and related vinylogs were cytotoxic to both murine and human tumour cell lines. In particular, 1a and 2a were identified as useful prototypic molecules. Structural modifications of 1a and 2a were accomplished leading to 1b-e and 2b-d which displayed cytotoxicity towards murine P388 and L1210 leukemic cells as well as human Molt 4/C8 and CEM T-lymphocytes. Among the new compounds, the greatest average potencies against these four cell lines were displayed by 1b and 2b, having approximately one quarter and one half of the potency of the reference drug melphalan, respectively. The synthesis and bioevaluation of three open chain analogues of 1b-d, namely 3a-c, did not reveal unequivocally whether this molecular modification led to increases in cytotoxicity or not. Compounds 2a-d were substantially more active than melphalan using a panel of human tumour cell lines. In addition, several compounds displayed selective toxicity to both colon and leukemic cancer cells. The 4-piperidinol 2d was active in the in vivo hollow fibre assay. This study revealed compounds with greater potency than 1a and 2a and it has confirmed that 1,3,4-trisubstituted-4-piperidinols and related compounds are novel groups of candidate antineoplastic and anticancer agents.

Recent advances in membrane introduction mass spectrometry (MIMS) are reviewed. On-line monitoring is treated by focusing on critical variables, including the nature and dimensions of the membrane, and the analyte vapor pressure, diffusivity, and solubility in the membrane barrier. Sample introduction by MIMS is applied in (i) on-line monitoring of chemical and biological reactors, (ii) analysis of volatile organic compounds in environmental matrices, including air, water and soil, and (iii) in more fundamental studies, such as measurements of thermochemical properties, reaction mechanisms, and kinetics. New semipermeable membranes are discussed, including those consisting of thin polymers, low vapor pressure liquids, and zeolites. These membranes have been used to monitor polar compounds, selectively differentiate compounds through affinity-binding, and provide isomer differentiation based on molecular size. Measurements at high spatial resolution, for example, using silicone-capped hypodermic needle inlets, are also covered, as is electrically driven sampling through microporous membranes. Other variations on the basic MIMS experiment include analyte preconcentration through cryotrapping (CT-MIMS) or trapping in the membrane (trap-and-release), as well as differential thermal release methods and reverse phase (i.e., organic solvent) MIMS. Method limitations center on semivolatile compounds and complex mixture analysis, and novel solutions are discussed. Semivolatile compounds have been monitored with thermally assisted desorption, ultrathin membranes and derivatization techniques. Taking advantage of the differences in time of membrane permeation, mixtures of structurally similar compounds have been differentiated by using sample modulation techniques and by temperature-programmed desorption from a membrane interface. Selective ionization techniques that increase instrument sensitivity towards polar compounds are also described, and comparisons are made with other direct sampling (nonchromatographic) methods that are useful in mixture analysis.

Melanoma is a highly malignant and increasingly common neoplasm. Because metastatic melanoma remains incurable, new treatment approaches are needed. Immunoliposomes have been previously shown to enhance the selective localization of immunoliposome-entrapped drugs to solid tumors with improvements in the therapeutic index of the drugs. Previously, we reported that the synthetic retinoid fenretinide (HPR) is an inducer of apoptosis in neuroblastoma (NB) cells, sharing the neuroectodermal origin with melanoma cells. HPR is a strong inducer of apoptosis also in melanoma cells, although at doses 10-fold higher than those achievable clinically. Thus, our purpose was to investigate the in vitro potentiation of its cytotoxic effect on melanoma cells in combination with long-circulating GD2-targeted immunoliposomes. GD2 is a disialoganglioside extensively expressed on tumors of neuroectodermal origin, including melanoma. Murine anti-GD2 antibody (Ab) 14.G2a and its human/mouse chimeric variant ch14.18 have been ligated to sterically stabilized liposomes by covalent coupling of Ab to the polyethylene glycol (PEG) terminus. Ab-bearing liposomes showed specific, competitive binding to and uptake by various melanoma cell lines compared with liposomes bearing non-specific isotype-matched Abs or Ab-free liposomes. Cytotoxicity was evaluated after 2 hr treatment, followed by extensive washing and 72 hr incubation. This treatment protocol was designed to minimize non-specific adsorption of liposomes to the cells, while allowing for maximum Ab-mediated binding. When melanoma cells were incubated with 30 microM HPR entrapped in anti-GD2 liposomes, a significant reduction in cellular growth was observed compared to free HPR, entrapped HPR in Ab-free liposomes or empty liposomes. Cytotoxicity was not evident in tumor cell lines of other origins that did not express GD2. Growth of NB cells was also inhibited by immunoliposomes with entrapped HPR.

Melanoma is a highly malignant and increasingly common tumour. Since metastatic melanoma remains incurable, new treatment approaches are needed. Previously, we reported that the synthetic retinoid N-(4-hydroxyphenyl)retinamide (fenretinide, HPR) induces apoptosis in neuroblastoma cells, sharing a neuroectodermal origin with melanoma cells. Since no data exist thus far on the effects of HPR on human melanoma tumours, our purpose was to investigate the in vitro modulation of cell growth and apoptosis by HPR in melanoma cells. Ten human melanoma cell lines were exposed in vitro to increasing concentrations of HPR. Dose-dependent growth inhibition and cytotoxicity were observed. According to cytofluorimetric analysis, propidium iodide staining and TUNEL assay, HPR-treated melanoma cells were shown to undergo apoptosis. However, IC50 values ranged from 5 to 28 microM, while IC90 values were between 10 and 45 microM. These last concentrations are approximately 10-fold higher than those achievable in patients given oral HPR. To explore the potential of new delivery strategies, HPR was loaded at high concentrations into immunoliposomes directed to disialoganglioside GD2, a tumour-specific antigen extensively expressed by neuroectoderma-derived tumours. Treatment of melanoma cells for a short time (2 hr) with HPR-containing immunoliposomes followed by culture in drug-free medium gave rise to apoptosis of target cells, whereas cells treated for 2 hr with equivalent concentrations of the free drug survived. The efficacy of immunoliposomal HPR was strongly dependent on the density of GD2 expression in the different cell lines.

The majority of immunogenic CTL epitopes bind to MHC class I molecules with high affinity. However, peptides longer or shorter than the optimal epitope rarely bind with high affinity. Therefore, identification of optimal CTL epitopes from pathogens may ultimately be critical for inducing strong CTL responses and developing epitope-based vaccines. The SIV-infected rhesus macaque is an excellent animal model for HIV infection of humans. Although a number of CTL epitopes have been mapped in SIV-infected rhesus macaques, the optimal epitopes have not been well defined, and their anchor residues are unknown. We have now defined the optimal SIV gag CTL epitope restricted by the rhesus MHC class I molecule Mamu-A*01 and defined a general peptide binding motif for this molecule that is characterized by a dominant position 3 anchor (proline). We used peptide elution and sequencing, peptide binding assays, and bulk and clonal CTL assays to demonstrate that the optimal Mamu-A*01-restricted SIV gag CTL epitope was CTPYDINQM(181-189). Mamu-A*01 is unique in that it is found at a high frequency in rhesus macaques, and all SIV-infected Mamu-A*01-positive rhesus macaques studied to date develop an immunodominant gag-specific CTL response restricted by this molecule. Identification of the optimal SIV gag CTL epitope will be critical for a variety of studies designed to induce CD8+ CTL responses specific for SIV in the rhesus macaque.

A number of 1-aryl-1-penten-3-ones 1 and related Mannich bases 2 were synthesized and converted either directly or indirectly to the corresponding biscarbohydrazones 3 and 4. Reaction of representative unsaturated ketones with thiocarbohydrazide led to the formation of only the monothiocarbohydrazones 5. The cytotoxicity of these compounds against murine P388 and L1210 cells, human T-lymphocytes and human tumour cell lines was undertaken in order to ascertain their bioactivity and review these data in the light of the theory of sequently cytotoxicity whereby the carbohydrazones would be predicted to be more than twice as cytotoxic as the ketones and Mannich bases from which they were derived. In general, conversion of 1 into 3 was accompanied by a lowering of cytotoxicity. On the other hand, in general the compounds in series 4 were significantly more potent cytotoxic agents than the analogues 2; in fact, some compounds displayed activity comparable with or exceeding that of melphalan. Thus the biscarbohydrazones 4 represent a novel group of cytotoxic agents serving as lead molecules for subsequent development.

Liposomal drug delivery systems markedly alter the biodistribution of their associated drugs by delaying drug clearance, retarding drug metabolism, decreasing the volume of distribution, and shifting the distribution in favour of diseased tissues with increased capillary permeability. This increases the therapeutic indices of the associated drugs, by increasing the drug concentration in solid tumours and regions of infection, and reducing the drug concentration in normal tissues. Three liposomal formulations have been approved for clinical use.

Cationic liposomes are considered to be safe vectors for gene transfer, but they are less efficient at delivering DNA to cells when compared with retroviral vectors. Cationic liposomes complexed with DNA were targeted to specific cells in vitro by means of monoclonal antibodies (mAbs) or ligands associated with the liposomes. Significant increases in expression of a beta-galactosidase reporter gene were observed in vitro in mAb-targeted liposomes, compared with non-targeted liposomes, in both an adherent tumor cell line (human adenocarcinoma) and a suspension cell line (human T-lymphoma). Also, use of asialofetuin as a targeting ligand significantly increased expression of the reporter gene in human hepatoma cells. Our results suggest that site-specific targeting of cationic liposomes is a good strategy for increasing both the selectivity and the efficiency of DNA delivery to cells and with further development may lead to targeted DNA delivery in vivo.

Ever since the first report of laser action, it has been recognized that laser ablation (evaporation/volatilization) may provide a useful sampling mechanism for chemical analysis. In particular, laser ablation is rapidly gaining popularity as a method of sample introduction for mass spectrometry. Since its original description, numerous research papers and review articles have appeared on various aspects of laser mass spectrometry. While most laser ablation/mass spectrometry has been performed with fixed frequency lasers operating at relatively high intensities/fluences (≥108 W/cm2, ≥1 J/cm2), there has been some recent interest in the use of low-power tunable lasers to ablate and resonantly ionize selected components in the ablation plume. This process has been termed resonant laser ablation (RLA).1 Potential advantages of RLA include: 1) simplification of the mass spectrum, by enhancement of signal from the analyte of interest; 2) improvement of the absolute detection limits by improving the ionization efficiency, and 3) improvement in relative sensitivity by reduction of spurious signal in the detection channel of interest (due to bleed through from adjascent mass channels or from isobaric interferences).

A number of Mannich bases of alicyclic ketones containing one and two basic centres were prepared in order to evaluate the theory of sequential cytotoxicity and develop structure-activity relationships in these series of compounds. The compounds were evaluated in vitro against murine P388 D1 lymphocytic leukemia cells. The data generated supported the theory of sequential cytotoxicity and in general, compounds containing alicyclic rings of five and six carbon atoms possessed greater activity than the corresponding dodecyl analogues. Those Mannich bases containing dialkylamino groups were associated with greater cytotoxicity than related compounds possessing a basic heterocycle. Calculations of the atomic charges of the enone groups from selected compounds afforded some rationalization for the cytotoxic screening results.

Reaction of 1-(4-bromophenyl)-4,4-dimethyl-5-(1-piperidino)-1-penten-3-one hydrochloride (1f) with sodium 2-mercaptoethanesulphonate (mesna) gave rise to the thiol adduct. 3. Recrystallization of this compound led to the formation of the corresponding zwitterion 4f. A series of analogues of 4f were prepared and the structure of a representative compound was confirmed by X-ray crystallography. In general, the thiol adducts had similar activity towards P388 cells and human tumour cell lines as the precursor enones 1 although greater selectivity to malignant diseases was found with the thiol adducts. A stability study of representative compounds conducted by 1H NMR spectroscopy revealed that the thiol adducts decomposed in solution. In one case regeneration of the ketone was noted while for the other compounds, the decomposition products were not identified.

The preparation of thirteen N-acyl and N-acyloxy analogues of two 3,5-bis(arylidene)-4-piperidones were prepared as potential prodrugs of the parent ketones. Approximately half of the compounds demonstrated antileukemic properties when evaluated against murine L1210 lymphoid leukemia cells. Three of the derivatives were more active than or equipotent with the reference drug melphalan. All of the compounds were examined against approximately 55 human tumours representing eight different neoplastic diseases. Not only were most of the compounds more cytotoxic than melphalan but 60% of the derivatives displayed selective toxicity to leukemia. A stability study of six of the candidate prodrugs using 1H NMR spectroscopy revealed that while some decomposition in solution occurred, the parent amine was not liberated.

The ability of long-circulating liposomes to act as drug sustained release systems or to passively target to solid tumors has resulted in a number of therapeutic applications which are being tested in animal models and in the clinic. Long-circulating liposomes, also called sterically stabilized liposomes (S-liposomes), have a number of properties which make them suitable for these applications (Table 1). This review will cover applications involving passive targeting to solid tumors of S-liposomes entrapping the anthracyline anticancer drugs doxorubicin (S-DOX) and epirubicin (S-EPI), as these formulations are the closest to being approved for use in the clinic.

Cancer chemotherapy is limited by adverse side effects resulting from toxicities to normal tissues. Targeted delivery of drugs to diseased tissues in vivo would help to reduce these side effects. Liposomes containing lipid derivatives of polyethylene glycol have circulation times sufficiently long to allow for effective in vivo drug delivery. Polyethylene glycol liposomes, containing entrapped doxorubicin, targeted to KLN-205 squamous cell carcinoma of the lung by means of specific antibodies attached at the liposome surface were capable of reducing tumor burden to a high degree and eradicating tumor in a significant percentage of mice.

The role of epidermal melanin pigments in the development of skin cancer remains unclear. A new technique for the specific introduction of compartmentalized melanin into nonpigmented human fibroblasts through the use of liposomes and polyethylene glycol (PEG) is presented. The delivery of liposome-encapsulated material to cells was characterized by: (a) high efficiency of delivery through PEG-mediated endocytosis at 37 degrees C; (b) intracellular acidification of liposome entrapped pH-sensitive 8-hydroxypyrene-3,6,8-trisulfonic acid after delivery; (c) similar incorporation and acidification of apolipoprotein E-associated liposomes into fibroblasts via the low-density lipoprotein-receptor pathway; and (d) discharge into the extracellular space after incorporation. Similar experiments were carried out with melanin-containing liposomes that were used to introduce compartmentalized melanin into fibroblasts, through PEG-mediated delivery. These "artificial" melanocytes had functional analogies to genuine melanocytes in that (a) in both cell types melanin compartmentalization was at a lower pH; and (b) liposome contents were later expelled in analogy to the putative biological process of melanin expulsion from the melanocyte. The modified fibroblasts provided potential "mutator" phenotypes with specific melanin pigmentation, and established a new basis for studying the role of melanin pigmentation in cancer development.

Liposome entrapment of doxorubicin has been shown to reduce its cardiotoxicity in vivo and increase its therapeutic index. A further improvement in therapeutic index could be achieved through targeting of liposome-entrapped drug selectively to cancer cells. Monoclonal antibodies against the squamous lung cancer cell line KLN-205 have been ligated to the surface of long-circulating (Stealth) and conventional liposomes. The antibody-bearing liposomes showed specific, competitive uptake by KLN-205 cells as compared to liposomes bearing nonspecific isotype-matched antibodies or antibody-free liposomes. Doxorubicin-containing antibody-liposomes resulted in as much as a 15-fold decrease in the 50% inhibitory concentration for doxorubicin against KLN-205 cells as compared to free doxorubicin or doxorubicin entrapped in antibody-free liposomes.

The synthesis of fourteen N-acyl derivatives of two 3,5-bis(arylidene)-4-piperidones was accomplished and these compounds were evaluated against L1210 leukemia cells in vitro. With one exception, the compounds had IC50 values of less than 10 microM and six of them had IC50 figures in the 0.2-0.6 microM range which were comparable to a reference drug melphalan. Twelve of the sixteen compounds showed specificity for human leukemia cell lines in the NCI in vitro screen. Studies using 1H NMR spectroscopy revealed that solutions of three N-acetylated compounds underwent deamination and possibly other reactions, the deaminated product itself being unstable in the solvent used.

Newly developed liposomes with prolonged circulation half-lives and dose-independent pharmacokinetics (Stealth liposomes) have been tested for their efficacy as a slow release system for the rapidly degraded, schedule-dependent, antineoplastic drug 1-beta-D-arabinofuranosylcytosine (ara-C) in the treatment of murine L1210/C2 leukemia. Mice were given injections of either 10(5) cells or 10(6) cells by either the i.v. or the i.p. routes. Leukemia-bearing mice were treated with either i.v. or i.p. injections of free drug, i.v. or i.p. injections of liposome-entrapped drug, or 24-h i.v. infusions of free drug. Long-circulating liposomes contained, as the stealth component, either monosialoganglioside or polyethylene glycol-distearoylphosphatidylethanolamine. Liposomes lacking the stealth components (non-stealth liposomes) were also injected for comparison. At lower dose ranges, stealth liposomes were superior to non-stealth liposomes in prolonging mean survival times of the mice, and all liposome preparations were superior to injections of the free drug. Drug entrapped in stealth liposomes, when administered at or near the maximum tolerated dose of 100 mg/kg ara-C were considerably superior to 24-h free drug infusions given at the same total drug dose. Therapeutic effect was related to the half-life of leakage of ara-C from the liposome formulations, as well as to circulation half-life, with maximum therapeutic effect achieved with long circulation half-lives and more rapid leakage rates. The therapeutic efficacy of non-stealth liposomes increased with increasing liposome (and drug) dose as a result of saturation of liposome uptake by the mononuclear phagocyte system, which resulted in longer circulation half-lives for these liposomes at higher doses (Michaelis-Menten pharmacokinetics). Liposome entrapment can protect rapidly degraded drugs from breakdown in vivo, with release of the drugs in a therapeutically active form over periods of up to several days. The dose-independent pharmacokinetics and reduced mononuclear phagocyte system uptake of stealth liposomes gives them distinct advantages over non-stealth liposomes.

A series of 3,5-bis-arylidene-1-methyl-4-piperidone methohalides 2 and two related analogues in general demonstrated activity against L 1210 leukemia cells in vitro and bound to a synthetic DNA, poly[d(AT)]. Plots of various physicochemical constants of the aromatic substituents in series 2 versus the IC50 figures revealed correlations between the aryl MR and pi values but not the sigma constants. The delta Tm values of six members of series 2 were correlated with the MR figures of the aryl substituents but not the sigma nor pi values of the aromatic atoms and groups. Some suggestions for future molecular modification with a view to increasing cytotoxicity are presented.

The 5-[1-hydroxy-2-chloro-2-(ethoxycarbonyl)ethyl]-2'-deoxyuridine (7) and 5-[1-hydroxy-2-bromo-2-(ethoxycarbonyl)ethyl]-2'- fluoro-2'-deoxyuridine/uridine nucleosides (8, 9) were synthesized by the regiospecific addition of HOX (X = Br or Cl) to the vinyl substituent of the respective (E)-5-[2-(ethoxycarbonyl)-vinyl]-2'-deoxyuridines (6a-b) and uridine (6c). A related reaction of (E)-5-(2-carboxyvinyl)-2'-deoxyuridines (10a-b) and uridine (10c) with iodine and potassium iodate afforded the 5-(1-hydroxy-2-iodo-2-carboxyethyl) derivatives (11-13). 5-(1-Hydroxy-2-iodoethyl)-arabinouridine (18) was obtained by the reaction of (17) with iodine in the presence of the oxidizing agent iodic acid. Treatment of (18) with methanolic sulfuric acid afforded 5-(1-methoxy-2-iodoethyl)-arabinouridine (19) in 65% yield. Of the newly synthesized compounds, 7, 11 and 12 showed activity in vitro against HSV-1. The most active compound (12, ID50 = 0.1 microgram/ml) was 10 times less active than acyclovir (ID50 = 0.01 microgram/ml) against HSV-1. Compounds 7 and 11 were cytotoxic to L1210 cells in culture, exhibiting an ED50 of 7.2 and 4.7 micrograms/ml respectively, relative to melphalan (ED50 = 0.15 microgram/ml), but were inactive against the KB cell line.

Electron density calculations were undertaken on several atoms in a series of 3-substituted-4-phenyl-3-buten-2-ones in order to gain insight into the molecular features which affect charge densities. The results indicate that substituents at position 3 alter the electron densities of the olefinic group but have little effect on the acetyl function. The compounds were tested against L1210 cells in vitro, and the results suggest that electronic--but not steric--factors are important in affecting cytotoxicity. The most active compound was 3-phenylmethylene-2,4-pentanedione (1c) with an ED50 value of 1.06 x 10(-8) M.

1-(2-Nitrovinyl) derivatives of nuclear substituted 1,4-dihydropyridines (7a-c), and 1-(N-tert-butyl-formiminyl) derivatives of 1,4-dihydropyridines (9a-c) or 2-pyridones (11a-c) were synthesized for evaluation as cytotoxic agents (see Table I for structures). The in vitro cytotoxic activities, determined in the L1210 assay, indicated that the 4-substituent on a 1,4-dihydropyridine ring system was a determinant of activity in both the 1-(2-nitrovinyl) (7) and 1-(N-tert-butylformiminyl) (9) series, the relative activity order being n-BuPhMe. In the 1-(N-tert-butylformiminyl) series, the 1,4-dihydropyridine derivatives (9) were generally more cytotoxic than the 2-pyridone derivatives (11). The most active compound was 1-(N-tert-butylformiminyl)-3-(4,4-dimethyloxazolin-2-yl)-4-n -butyl-1,4- dihydropyridine (9b), but it was 2-3 log units less active than the reference standard melphalan.

Two novel bicyclic diterpenoides, agelasimine-A (9), and agelasimine-B (10), have been isolated from the orange sponge Agelas mauritiana. Also, a new bromine-containing alkaloid, 5-debromomidpacamide (12), along with midpacamide (13) and methyl N-methyl-4,5-dibromopyrrole-2-carboxylate (11), has been isolated. The structures were determined by interpretation of their spectral data. Agelasimine-A and -B exhibit a wide range of interesting biological activities such as cytotoxicity, inhibition of adenosine transfer into rabbit erythrocytes, Ca2+-channel antagonistic action, and α1 adrenergic blockade.

The lipid composition of Sendai virus, propagated in chicken eggs, was analyzed by high performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and gas-liquid chromatography (GLC). Phosphatidylcholine was found to be the dominant phospholipid (37.3%) with phosphatidylethanolamine (26.8%) and phosphatidylserine (12.0%) also present in significant amounts. Analysis of the fatty acid methyl esters revealed that the dominant fatty acids in total phospholipid were: C16:0 (17.6%), C18:0 (15.4%), C18:1 (n-9) (22.0%), and C24:0 (6.0%). Cardiolipin, phosphatidylserine, and sphingomyelin contained higher levels of saturated fatty acids relative to phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine.

The peptides [Glu1,8,Leu11,17] 18A and [Glu4,9,Leu11,17] reverse-18A are 18-residue peptides designed to form amphipathic helices with opposite charge distribution; [Glu1,8,Leu11,17] 18A having positively charged residues at the hydrophobic/hydrophilic interface. Both [Glu1,8,Leu11,17] 18A and [Glu4,9,Leu11,17] reverse-18A strongly disrupt the bilayer structure as indicated by the relatively narrow lipid 1H and 31P NMR peaks. In addition, the 1H chemical shift of the quaternary ammonium methyl groups indicates that [Glu1,8,Leu11,17] 18A forms smaller lipoprotein particles with dimyristoylphosphatidylcholine (DMPC) than does [Glu4,9,Leu11,17] reverse-18A. However, motional properties of the lipid head group indicate that no specific salt bridges are formed between the phospholipid head group and the side chains of polar amino acids of either of the two peptides. In addition, the acyl chain conformation for the DMPC complexes with [Glu1,8,Leu11,17] 18A and with [Glu4,9,Leu11,17] reverse-18A are indistinguishable by the criterion of IR spectroscopy. The 2H linewidth of the solvent 2H2O remains narrower in frozen solutions of the DMPC-[Glu1,8,Leu11,17] 18A complexes suggesting the presence of more unfrozen bound water in this case. The two peptides exhibit many similarities in their interaction with lipids. However, [Glu1,8,Leu11,17] 18A can more readily lyse vesicles and activate lecithin:cholesterol acyltransferase. These differences do not appear to result from differences in specific charge interactions between the lipid and peptide but may be manifested through differences in hydration properties.

The amphipathic helix hypothesis for plasma lipoproteins was investigated using synthetic peptides. The lipid-associating properties of two potentially amphipathic model peptides and two analogs were studied by incubating synthetic peptides with small unilamellar vesicles and protein-lipid association examined by equilibrium density centrifugation, leakage of liposome-entrapped fluorescence compounds, intrinsic tryptophan fluorescence, and circular dichroism spectroscopy. The analog peptides were designed to determine the significance of the number and specific location of the charged residues in amphipathic domains of plasma lipoproteins to protein-lipid association. Based on the four procedures used to examine protein-lipid interactions, the two model peptides (18Aa, 18As) were found to associate strongly with liposomes; the two analog peptides (18As1, 18Asr), differing only with respect to the number and/or position of their charged residues, failed to demonstrate similar lipid binding properties. These findings support the earlier suggestions of the importance of the charged residues, but do not define the precise mechanisms involved. Such amino acids may help initiate the lipid-protein association by electrostatic interactions, contribute to the hydrophobicity of the nonpolar face of the helix by the acyl portion of lysine and arginine, and/or complement the charge distribution in the polar head regions of the phospholipid molecules.

Liposomes containing cortisol palmitate showed greater anti-inflammatory activity than water soluble cortisol succinate, following intravenous injection into rats, as sssessed by inhibition of (a) carrageenan-induced paw swelling, and (b) inflammatory responses in subcutaneous sponge implants impregnated with heat-killedMycobacterium tuberculosis.

Air sampling is a useful tool for controlling, as well as for analyzing, the environment. The equipment and techniques described were designed to facilitate the collection of localized samples directly at the source of potential contamination. Routine use of membrane filters was simplified by the design of a small, inexpensive filter holder, providing advantages over commerically available units, e.g. reduced size and weight, tight sealing against any filter material without the twist-lock action which often damages filter membranes, and parts exposed to contamination are expendable. Techniques for determining sample collection rates directly from vacuum-gauge readings are discribed. A combination alpha - gamma scintillation detector assembly, designed specifically for counting membrane-filter samples, was constructed. Sample preparation techniques for use with internal proportional counters are also mentioned. (auth)

Five types of subunits are postulated to explain the lactate dehydrogenase zymogram patterns of cod tissues. The subunits designated A, B, and C occur predominantly in white skeletal muscle, heart, and liver tissues, respectively. The D subunit is found almost exclusively in the retina. Activity of the E subunit is found in most tissues but does not appear to be predominant in any one tissue. The A subunits are most susceptible to heat treatment. The B subunits are the least reactive when AcPyAD, an NAD analogue, is used in place of NAD. Subbands may be produced in incubating heart or muscle extracts with pyloric caeca extract or with trypsin or chymotrypsin. Subbands normally appearing in tissues zymograms may represent partially synthesized or partially degraded active LDH tetramers normally present in a metabolizing tissue. In a sampling of three Canadian and one European cod populations, four forms of the heart subunit were found. They are designated B, B′, B″, and B‴. Allele frequencies of the heart types for each population sample are compared.

The organ distribution and half-life in blood of liposome-entrapped [14C]sucrose was examined in rats for two compositions of liposomes having widely differing drug release properties as determined by an in vitro assay in the presence of serum at 37 degrees C. The liposomes were composed of 1) egg phosphatidylcholine-cholesterol, 2:1 molar ratio and of 2) bovine brain sphingomyelin-phosphatidylcholine, 4:1 molar ratio. The two types of liposome were determined to have half-lives for release of contents in vitro in the presence of serum of 2.6 and 35 hr, respectively. Organ distribution and blood values were followed with time for small unilamellar, multilamellar and reverse-phase evaporation liposomes of both compositions. Free [14C]sucrose was almost totally eliminated from rats within 0.5 hr of injection. Liposome-entrapment increased the circulation time of sucrose in vivo in the following order: small unilamellar greater than reverse-phase evaporation greater than multilamellar liposomes. For all sizes of liposomes, the composition with slower leakage as determined by in vitro assay (sphingomyelin-phosphatidylcholine, 4:1) resulted in significantly longer half-lives in blood and in whole body. Liposomes containing sphingomyelin were taken up by liver to a lesser extent and by spleen to a greater extent than phosphatidylcholine-cholesterol liposomes of all sizes.

Copper deficiency has been described in premature infants on hyperalimentation. The bony abnormalities are generalized and are usually associated with anemia and neutropenia. These changes present with normal serum levels of iron, ascorbic acid, calcium, phosphorus, and magnesium, as well as with depressed levels of copper and ceruloplasmin. They appear at about three to nine months of age in infants with a low birth weight who are receiving total parenteral nutrition, but can be prevented by greater than normal maintenance levels of copper supplements. Established bone changes improve rapidly after the administration of therapeutic supplements.

5-(1-Hydroxy-2-haloethyl)- (4), 5-(1-methoxy-2-haloethyl)- (5) and 5-(1-hydroxy-2-methoxyethyl)uracils (6) (see Figure 2 for structures) were synthesized to investigate the effect of the C-5 substituents on cytotoxic and antiviral activity. The bromo compounds (4b and 5b) exhibited greater cytotoxic activity than the chloro or iodo analogues in the in vitro L1210 assay. Replacement of the hydroxyl substituent of 4b (bromo) and 4c (iodo) by a methoxyl substituent (5b-c), or substitution of their halogen substituents by methoxyl (providing 6) increased the potency. However, the cytotoxic activity of all the compounds was weak, the most active (6) producing a 45% decrease in cell survival at a concentration of 50 micrograms/ml, as compared with a 97% decrease when the reference standard (melphalan) was tested at 1 microgram/ml. They were inactive antiviral agents against herpes simplex virus type 1 (HSV-1) infected Vero cells at 10 micrograms/ml; in the same test, the reference standard (acyclovir) exhibited an ID50 of 0.01 micrograms/ml.

Enzyme albumin conjugates have been proposed as a means of increasing the efficacy of enzyme use in vivo and decreasing immune response to the enzyme. Particulate drug carriers, however, have a pronounced tendency to localize in the mononuclear phagocyte (reticuloendothelial) system. We have examined in mice the effect on phagocytic index, tissue distribution and organ size of continued administration of conjugates of alpha-glucosidase with either homologous or heterologous albumin. Mice received 10 X 2-mg injections of bovine serum albumin (BSA) or mouse serum albumin (MSA), either free, polymerized or conjugated with alpha-glucosidase. Experiments involving BSA had to be terminated before the end of the experiment because of anaphylaxis, but these reactions were less severe to the polymerized albumin than to free albumin. Free BSA, BSA polymer and BSA-enzyme conjugates all caused a decrease in phagocytic index after six injections. Mice receiving MSA showed no evidence of anaphylaxis, but mice receiving six or more injections of free MSA, MSA polymer or MSA-enzyme conjugate had significantly decreased phagocytic indices as compared to controls. Phagocytic indices had returned to normal by 7 days after the final injection. Tissue distribution of 125-I-labeled albumin preparations was determined in either naive or chronically injected mice.

Nineteen 2-pyridylthio (2), 2-pyridylsulfinyl (3) or 2-pyridylsulfonyl (4) derivatives of (Z)-methyl acrylate were synthesized in order to investigate the effect of the oxidation state of the sulfur atom, and the position and nature of pyridyl substituents on cytotoxic activity. Analogous sulfinyl and sulphonyl derivatives were equipotent, and more potent than analogous thio derivatives, in an vitro L1210 screen. In most cases, incorporation of nuclear trifluoromethyl and chloro substituents at various positions of the pyridyl ring of the sulfinyl derivatives (compounds 3b-3h) decreased activity relative to the unsubstituted sulfinyl 3a (ED50 0.43 micrograms/ml). Compounds 3b, 3d and 3i exhibited weak antineoplastic activity in an in vivo P388 screen at a dose of 5 mg/kg.

Intalipid® was administered intravenously to mice at a level of 2 g kg−1 day−1 for 23 days. No alterations in phagocytic index, liver or spleen size were observed in the chronically injected mice as compared with control mice that received saline injections. Tissue distribution of 0.45 μm multilamellar liposomes of egg phosphatidylcholine:cholesterol (2:1) was similar in mice that had been chronically injected with Intralipid® to that in control mice. Mice chronically given the same total amount of phospholipid in the form of 0.2 μm liposomes of phosphatidylcholine:cholesterol (2:1) rather than as a lipid-triglyceride emulsion showed altered tissue distribution of entrapped label with decreased liver uptake and increased splenic uptake, which is indicative of reticuloendothelial blockade. Tissue distribution of [14C]dipalmitoylphosphatidylcholine Intralipid® was compared with that of [14C]dipalmitoylphosphatidylcholine 0.2 μm MLV of phosphatidylcholine:cholesterol (2:1). Intralipid® was taken up 2- to 3-fold less by liver and 5- to 10-fold less by spleen than liposomes. Blood levels of Intralipid® were higher than those of liposomes. [14C]dipalmitoylphosphatidylcholine Intralipid® was eliminated from the body at a faster rate than [14C]dipalmitoylphosphatidylcholine liposomes. The lack of reticuloendothelial blockade caused by Intralipid® as compared with liposomes appears to be related to its diminished uptake into reticuloendothelial tissues. This diminished uptake may be related to differences in apolipoprotein uptake of Intralipid®, which is primarily in the form of a phospholipid monolayer, and liposomes, which have their phospholipid organized into a bilayer.

The peptides [Glu1,8,Leu11,17] 18A and [Glu4,9,Leu11,17] reverse-18A are 18-residue peptides designed to form amphipathic helices with opposite charge distribution; [Glu1,8,Leu11,17] 18A having positively charged residues at the hydrophobic/hydrophilic interface. Both [Glu1,8,Leu11,17] 18A and [Glu4,9,Leu11,17] reverse-18A strongly disrupt the bilayer structure as indicated by the relatively narrow lipid 1H and 31P NMR peaks. In addition, the 1H chemical shift of the quaternary ammonium methyl groups indicates that [Glu1,8,Leu11,17] 18A forms smaller lipoprotein particles with dimyristoylphosphatidylcholine (DMPC) than does [Glu4,9,Leu11,17] reverse-18A. However, motional properties of the lipid head group indicate that no specific salt bridges are formed between the phospholipid head group and the side chains of polar amino acids of either of the two peptides. In addition, the acyl chain conformation for the DMPC complexes with [Glu1,8,Leu11,17] 18A and with [Glu4,9,Leu11,17] reverse-18A are indistinguishable by the criterion of IR spectroscopy. The 2H linewidth of the solvent 2H2O remains narrower in frozen solutions of the DMPC-[Glu1,8,Leu11,17] 18A complexes suggesting the presence of more unfrozen bound water in this case. The two peptides exhibit many similarities in their interaction with lipids. However, [Glu1,8,Leu11,17] 18A can more readily lyse vesicles and activate lecithin:cholesterol acyltransferase. These differences do not appear to result from differences in specific charge interactions between the lipid and peptide but may be manifested through differences in hydration properties.