Production of membrane whorls in rat liver by some inhibitors of protein synthesis.

Inhibitors of protein synthesis capable of differential effects on nascent peptide synthesis on membrane-bound and free polyribosomes were employed to investigate the structure and function of cellular membranes of liver. The formation of membranous whorls in the cytoplasm and distension of nuclear membranes were induced by inhibitors of protein synthesis (i.e., cycloheximide and emetine) which predominantly interfere with nascent peptide synthesis on membrane-bound polyribosomes in situ. Other inhibitors of protein synthesis such as puromycin and fusidic acid, which inhibit nascent peptide synthesis on both free and membrane-bound polyribosomes, and chloramphenicol, which inhibits mitochondrial protein synthesis, did not induce these alterations. Cycloheximide, puromycin, and chloramphenicol produce some common cellular lesions as reflected by similar alterations in morphology, such as swelling of mitochondria, degranulation of rough endoplasmic reticulum, and aggregation of free ribosomes. The process of whorl formation in the cytoplasm, the incorporation of [(3)H]leucine and of [(3)H]choline into endoplasmic reticulum and the total NADPH-cytochrome c reductase activity of the endoplasmic reticulum were determined. During maximum formation of membranous whorls, [(3)H]leucine incorporation into cytoplasmic membranes was inhibited, while [(3)H]choline incorporation into these structures was increased; maximum inhibition of protein synthesis and stimulation of choline incorporation into endoplasmic reticulum, however, preceded whorl formation. Cycloheximide decreased the activity of NADPH-cytochrome c reductase of rough endoplasmic reticulum, but increased NADPH-cytochrome c reductase activity of smooth endoplasmic reticulum. In addition, cycloheximide decreased the content of hemoprotein in both the microsomal and mitochondrial fractions of rat liver, and the activities of mixed function oxidase and of oxidative phosphorylation were impaired to different degrees. Succinate-stimulated microsomal oxidation was also inhibited. The possible mechanisms involved in the formation of membranous whorls, as well as their functions, are discussed.

Synthetic CD4 peptide derivatives that inhibit HIV infection and cytopathicity.

Synthetic peptide segments of the CD4 molecule were tested for their ability to inhibit infection of CD4+ cells by the human immunodeficiency virus (HIV) and to inhibit HIV-induced cell fusion. A peptide mixture composed of CD4(76-94), and synthesis side products, blocked HIV-induced cell fusion at a nominal concentration of 125 micromolar. Upon high-performance liquid chromatography, the antisyncytial activity of the peptide mixture was found not in the fraction containing the peptide CD4(76-94) itself, but in a side fraction containing derivatized peptide products generated in the automated synthesis. Derivatized deletion and substitution peptides in the region CD4(76-94) were used to demonstrate sequence specificity, a requirement for benzyl derivatization, and a core seven-residue fragment required for antisyncytial activity. A partially purified S-benzyl-CD4(83-94) peptide mixture inhibited HIV-induced cell fusion at a nominal concentration of less than or equal to 32 micromolar. Derivatized CD4 peptides blocked cell fusion induced by several HIV isolates and by the simian immunodeficiency virus, SIV, and blocked infection in vitro by four HIV-1 isolates with widely variant envelope gene sequences. Purified CD4(83-94) dibenzylated at cysteine 86 and glutamate 87 possessed antisyncytial activity at 125 micromolar. Derivatization may specifically alter the conformation of CD4 holoreceptor peptide fragments, increasing their antiviral efficacy.

Monoclonal antibodies in the lymphatics: selective delivery to lymph node metastases of a solid tumor.

After subcutaneous injection, monoclonal antibodies directed against a tumor can enter local lymphatic vessels, pass to the draining lymph nodes, and bind to metastases there. Lymphatic delivery of antibody to early metastases is more efficient than intravenous administration, and the lymphatic route can be used to image smaller metastatic deposits. Perhaps more important, the lymphatic route minimizes binding of antibodies to circulating tumor antigens and to cross-reactive antigens present on normal tissues. Antibodies inappropriate for intravenous use because of binding to normal tissues may therefore be useful against lymph node metastases when injected subcutaneously or directly into lymphatic vessels.

Dynamic interaction of 111indium-labeled monoclonal antibodies with surface antigens of solid tumors visualized in vivo by external scintigraphy.

Two 111indium-labeled murine monoclonal antibodies (MoAb), D3 and 9.2.27, directed to tumor antigens of L-10 hepatocarcinoma and human melanoma, respectively, selectively localized antigen-positive target cells in guinea pigs and nude mice. The fate of MoAb differed in the two antigen-antibody systems after reacting with their corresponding tumor antigens in vivo as reflected by patterns of distribution and turnover in vivo. The 9.2.27 localized in melanoma xenograft in nude mice after intravenous administration with slow loss from tumor but more rapid loss from normal tissues and thus demonstrated optimal imaging of small tumors (approximately equal to 5 mm) between 3 and 6 days after injection of the radiolabeled antibody. In contrast, D3 demonstrated a biphasic localization in guinea pig L-10 hepatocarcinoma with a maximal activity on the 2d day after administration and showed rapid loss from both tumor and normal tissues. Nonspecific localization of antibodies in liver and in kidney was found both in syngeneic (nude mice) and xenogeneic (guinea pig) hosts but was more pronounced in the xenogeneic species. These results indicate that the nature of the antigen-antibody interaction may be of importance in selecting MoAb for both diagnosis and therapy of malignant diseases.

Analysis of the involvement of cells from donor and recipient mice in adoptive transfer of antitumor immunity.

In this study, we attempted to analyze the effector cells for adoptive transfer of protective immunity directed against a P815Ys tumor. The spleen, lymph node, and peritoneal exudate cells obtained from immune mice at Day 7 to Day 10 after last challenge were tested for their in vitro cell-mediated cytotoxicity against P815Ys cells, using a 4-hr 51Cr release assay. The immune spleen lymphocytes (ISL) showed no cytotoxicity, whereas the peritoneal exudate cells exhibited marked cytotoxicity. Unexpectedly, when ISL or peritoneal exudate cells were adoptively transferred i.v. into mice bearing the P815Ys tumor, it was the ISL but not the peritoneal exudate cells that provided the hosts with significant protection. Using alloantibodies for negative depletion of cells in ISL, it was found that, after treatments with anti-Thy 1.2 or anti-Lyt 1 antiserum plus complement but not with anti-Lyt 2 or complement alone, the protective capacity of ISL can be abolished, indicating that the effector cells for conferring protective immunity to the host are Lyt 1-bearing T-cells. Moreover, culture supernatants of ISL with or without mitomycin C-treated P815Ys contain helper factor, interferon, and interleukin 2, which enhanced the in vitro generation of cell-mediated cytotoxicity against P815Ys. Taken together, these results strongly suggest that the donor helper T-cells are the effector cells responsible for adoptive transfer of protective immunity. We next examined the contribution of host cells. Syngeneic mice were made to become either T-cell (with thymectomy and irradiation)- or macrophage (with the administration of silica) depleted and were then subjected to adoptive transfer experiments. Both the thymectomized and the silica-treated mice, after receiving the ISL, showed significantly better survival times than did normal mice. Thus, the data suggest that the elimination of T-cells or inactivation of macrophages, presumably with immunosuppressive activity in the recipients, will allow further improvement of their battle for survival against tumor.

Radiolocalization of xenografted human malignant melanoma by a monoclonal antibody (9.2.27) to a melanoma-associated antigen in nude mice.

A murine monoclonal antibody (9.2.27), directed to a Mr 250,000 glycoprotein-chondroitan sulfate proteoglycan complex, was radiolabeled with 125I and assessed for radiolocalization in tumor and normal tissues of normal and tumor-bearing nude mice. The 125I-9.2.27 localized in vivo preferentially in Mr 250,000 antigen-expressing human melanomas (FMX-Met, SESX) but not in low antigen-expressing tumors (LOX-L) xenografted in nude mice. The imaging index of tumor cells was positively correlated with the antigen density of the various melanoma cell lines as measured by flow cytometry. The nonspecific immunoglobulin RPC-5 of the same IgG2a subclass as 9.2.27 did not specifically localize to xenografts of melanoma. The total amount of 125I-9.2.27 accumulated in the tumor was directly correlated with tumor size. However, the specific radioactivity (cpm/g) in smaller tumors was higher than that in larger tumors. Nonspecific uptake and circulating antibody levels differed between normals and tumor-bearers. The organs of the reticuloendothelial system of normal mice accumulated more labeled antibody than did those of tumor bearers, and conversely, tumor bearers had higher levels of circulating labeled antibody in the blood than normals. The circulating labeled antibody in tumor bearers was still monomeric but had no detectable antigen-binding capacity.

Selective antitumor effect on L10 hepatocarcinoma cells of a potent immunoconjugate composed of the A chain of abrin and a monoclonal antibody to a hepatoma-associated antigen.

The toxic A chain of abrin was isolated by affinity chromatography and was demonstrated to be a potent inhibitor of protein synthesis in a cell-free rabbit reticulocyte system with a complete inhibitory dose at 1 X 10(-9) M. This A chain was coupled by a disulfide linkage to a purified monoclonal antibody directed against a tumor-associated antigen found on the line 10 hepatocarcinoma tumor in strain two guinea pigs. The immunoconjugate retained the functions of the individual components, i.e., antigen binding to the intact cell in vitro and inhibition of its protein synthesis. This conjugate was a selective antineoplastic agent with a cytocidal dose at 5 X 10(-9) M toward antigen-bearing cells in vitro. Several antigen-negative cells were much less susceptible to its cytotoxic effect. The cytotoxicity of the conjugate appeared to be by antibody-mediated delivery of toxic A chain into the target cell. When cells were pretreated with excess free antibody followed by a brief exposure to conjugate, there was a reversal of the cytotoxicity to antigen-positive cells but not to the antigen-negative cells. The therapeutic efficacy of the conjugate was assayed by injecting a single dose s.c. or i.v. into syngeneic guinea pigs bearing established line 10 tumors. These in vivo studies showed that (a) the conjugate was not toxic at a dosage of 60 to 1120 micrograms/guinea pig, (b) the conjugate decreased or abolished the growth of established solid tumors, (c) the conjugate delayed or inhibited tumor metastases to lymph nodes, and (d) 20 to 40% of the animals in selective groups had a long-term complete regression.

Mode of action of the bioreductive alkylating agent, 2,3-bis(chloromethyl)-1,4-naphthoquinone.

The bioreductive alkylating agent, 2,3-bis(chloromethyl)-1,4-napthoquinone (CMNQ), has been shown to inhibit the growth of Sarcoma 180 ascites cells in vivo. Evidence for the reductive activation of this agent via the mitochondrial respiratory chain was provided by CMNQ-induced oxidation of reduced nicotinamide adenine dinucleotide; the interaction was shown to be on the substrate side of the site of rotenone inhibition. Consistent with the concept that reduction of CMNQ to a hydroquinone results in the generation of an alkylating species (i.e., a quinone methide) was the finding that radioactivity from [14C]CMNQ present in Sarcoma 180 ascites cells was associated with DNA, RNA, and protein for a period of up to 72 hr after exposure to tumor-bearing animals to this agent. Inhibition of the incorporation of [3H]thymidine, [3H]uridine, and [14C]leucine into DNA, RNA, and protein, respectively, of Sarcoma 180 ascites cells was produced by this agent, with DNA biosynthesis being the most susceptible. The inhibitory effect of CMNQ on the formation of DNA was, at least in part, the result of a prevention of the conversion of thymidine to its nucleotide forms. This action was due to (a) a drug-induced decrease in intracellular levels of adenosine 5'-triphosphate, presumably resulting from uncoupling of oxidative phosphorylation by CMNQ; and (b) a partial loss of thymidine kinase activity in Sarcoma 180 cells, which did not appear to be due to direct inhibition of the enzyme by the drug. Although the primary event produced by CMNQ at the mitochondrial level appeared to be release of respiratory control, other effects of mitochondrial metabolism occurred. These included inhibition of reduced nicotinamide adenine dinucleotide and succinoxidase activities, as previously demonstrated, and mitochondrial swelling, which suggested interaction of CMNQ with the inner mitochondrial membrane. These findings indicate a variety of biochemical lesions are associated with the antineoplastic activity of CMNQ and demonstrate a relationship between the effects of this drug on mitochondrial respiratory control and DNA biosynthesis.

Localization of 111In- and 125I-labeled monoclonal antibody in guinea pigs bearing line 10 hepatocarcinoma tumors.

A murine monoclonal antibody (D3) with demonstrated specificity for the guinea pig line 10 hepatocarcinoma (L10) was radiolabeled with either 125I or 111In and used to image dermal tumors in vivo. In one set of experiments, L10 tumors were established middorsally in one group of animals, and the similarly derived, antigenically distinct line 1 tumor was established in another group of animals. In spite of background imaging of liver, kidney, and spleen, L10 tumors were visualized clearly. Incorporation of radiolabel was demonstrated to predominate in the L10 tumor. In a separate set of experiments, L10 and line 1 tumors were established in contralateral thighs in the same animals. L10 tumors were visualized clearly, and tissue uptake of radiolabel was demonstrated to reside predominantly in the L10 tumor.

Guinea pig line 10 hepatocarcinoma model: characterization of monoclonal antibody and in vivo effect of unconjugated antibody and antibody conjugated to diphtheria toxin A chain.

Monoclonal antibodies were raised against the guinea pig line 10 (L10) hepatocarcinoma, and an IgG1-producing hybridoma (D3) was selected for further study. D3 is a true monoclonal antibody as demonstrated by two-dimensional gel electrophoresis. Radioimmunoassays on live cells revealed no cross-reactivity with normal tissues or with the line 1 hepatocarcinoma which was used as a control. Membrane immunofluorescence assays demonstrated similar specificity. Immunoperoxidase staining of cryostat sections of tumor and normal tissues of both adult animals and fetuses showed that the D3 monoclonal antibody reacted primarily with the L10 tumor, but some cross-reactivity with smooth muscle, placenta, fetal skeletal muscle, and fetal liver was also demonstrated. Radioimmunoprecipitation of detergent extracts of iodinated L10 cells showed that the antigen is present on the cell surface as a dimer of Mr 290,000 (unit size, Mr 148,000). Therapy studies with unconjugated D3 antibody demonstrated a minor dose-dependent effect on tumor growth. D3 antibody conjugated to the A chain of diphtheria toxin (10(-7) M) was cytotoxic to 100% of L10 cells in vitro. Animals treated with a single 1-mg i.v. injection of this immunoconjugate on Day 7 following the intradermal injection of 10(5) tumor cells demonstrated a highly significant inhibition of tumor growth compared to control animals and those treated with unconjugated antibody.

Antitumor agents. 134. New shiraiachrome-A- and calphostin-C-related perylene derivatives as cytotoxic and antiviral agents and inhibitors of protein kinase C.

Shiraiachrome-A and -B have been isolated from the mycelium of the Chinese bamboo fungus Shiraia bambusicola as the cytotoxic principles. A series of new perylene derivatives (7-27) related to Shiraiachrome-A and -B as well as Calphostin-C have been synthesized and evaluated for their cytotoxicity, antiviral activity, and inhibitory activity against protein kinase C. The results indicated that 11 and 12 are potent cytotoxic agents against HCT-8, RPMI-7951, and TE-671 solid tumor cells, whereas 24 and 26 demonstrated strong antiviral activity against HSV-1 and HSV-2. Compound 10 is an inhibitor of protein kinase C.

Synthetic peptides allow discrimination of structural features of CD4(81-92) important for HIV-1 infection versus HIV-1-induced syncytium formation.

Benzylated peptides with a primary amino acid sequence corresponding to either human CD4(81-92) (#18), or chimpanzee CD4(81-92) (#18C), were equipotent inhibitors of human immunodeficiency virus type 1 (HIV-1) infection of CD4+ cells and high-affinity binding of 125I-gp120 to CD4+ cells. The chimpanzee-based CD4(81-92) peptide, however, which differs from the human peptide by a single amino acid substitution (E for G) at position 87, was considerably less potent than the human CD4(81-92)-based peptide congener to inhibit HIV-1-induced cell-cell fusion. These data suggest that a portion of the CD4 molecule contained within the sequence CD4(81-92) is involved in binding gp120 during both HIV-1 infection and HIV-1-induced syncytium formation in human cells, but that the presence of a glutamic acid at position 87 in this sequence is more critical for the CD4/gp120 interaction leading to syncytium formation than for the CD4/gp120 interaction leading to primary infection of CD4-positive cells. The region CD4(81-92) may critically contribute to CD4-mediated HIV-1 pathogenesis in humans, and its alteration might explain the lack of pathogenic sequelae of HIV-1 infection in chimpanzees.

CD4(81-92)-based peptide derivatives. Structural requirements for blockade of HIV infection, blockade of HIV-induced syncytium formation, and virostatic activity in vitro.

CD4(81-92) peptide block human immunodeficiency virus (HIV) infection, virus-induced cell fusion, and antigen production by HIV-1-infected cells when derivatized on specific amino acid residues. An extensive series of structural variants of 1,4,5-tribenzyl-10-acetyl-CD4(81-92) were tested as anti-viral agents in an attempt to define the sequence and derivatization requirements for antiviral activity, and to maximize potency and stability for use as potential therapeutic agents. Alteration of the primary amino acid sequence of the stem compound 1,4,5-tribenzyl-CD4(81-92) diminished or abolished in parallel all three indices of anti-viral activity in a series of altered sequence compounds. Replacement of d- for l-amino acid residues at positions 1, 2, 3, 4, 5, or 6 but not position 10 decreased anti-viral potency, again with parallel effects on infection, synctium formation, and virostatic activity. Omission of the glutamine residue at position 9 did not affect anti-viral potency, while removal of the glutamic acids at position 11 and 12 resulted in virtually complete loss of biological activity. Changes in the derivatization pattern of the CD4(81-92) peptide backbone also affected anti-viral potency and efficacy. Optimal activity was obtained with benzyl residues at positions 1, 4, and 5, whereas the 1,4,7-tribenzyl-CD4(81-92) compound was without activity in all assays tested. Replacement of one of the benzyl groups with an acetamidomethyl moiety resulted in complete loss of biological activity. The previously reported (Nara et al., Proc Natl Acad Sci USA 86: 7139-7143, 1989) virostatic activity of 1,4,5-tribenzyl-10-acetyl-CD4(81-92) (peptide #18) is apparently due to acetylation, since the desacetyl stem compound shows much less virostatic activity while still possessing full anti-infective and anti-syncytial activity, and acetylation of the N-terminus rather than the lysine of 1,4,5-tribenzyl-CD4(81-92) yields a virostatic compound equipotent to peptide #18. Cyclization of the tribenzyl peptide to further conformationally restrict the molecule resulted in a compound with anti-infection, anti-syncytial, and virostatic activity at submicromolar concentrations.

Peptides derived from the CDR3-homologous domain of the CD4 molecule are specific inhibitors of HIV-1 and SIV infection, virus-induced cell fusion, and postinfection viral transmission in vitro. Implications for the design of small peptide anti-HIV therapeutic agents.

Peptides 12-25 amino acids in length from the V1J1 region of the CD4 molecule (residues 1-120) were synthesized as randomly derivatized, deliberately derivatized, or pure peptide products, and tested for their ability to inhibit HIV-1-induced cell fusion, HIV-1 and SIV infection of CD4-positive human cells, HIV-1 envelope glycoprotein binding to the CD4 molecule, CD4-neutralizing antibody binding to the CD4 holoreceptor, and CD4-dependent cellular immune function in the mixed lymphocyte and cytotoxic T-cell bioassays. Only peptides derived from the complementarity-determining region 3 (CDR3)-homologous domain of CD4, in particular CD4(81-92) and CD4(81-101), were effective antiviral agents. Within the CD4(81-92) series, R-group derivatization of selective amino acid residues was an absolute requirement for biological activity. The prototype compound T1C4E5-tribenzyl-K10-acetyl-TYICEVEDQKEE inhibited HIV-1-induced cell fusion at 32 microM, HIV-1 infection of CEM-SS cells at 10 microM, SIV infection of CEM-174 cells at less than 125 microM, gp120/CD4 binding at 60 microM, and postinfection cell-mediated viral transmission at 10-15 microM. Compounds of identical structure and derivatization, but of altered primary sequence, were substantially less active, or without activity, in these assays. These data indicate that the effect of amino acid derivatization of the CD4(81-92) peptide was most likely restriction of the flexible underivatized peptide backbone to a conformation closely approximating that of the CDR3-homologous gp120 binding site of the native CD4 molecule. Peptide antiviral activity was specific, as judged by lack of cytotoxicity, lack of inhibition of HTLV-1-induced cell fusion, and lack of inhibition of CD4-dependent cellular immune function in vitro. Further derivatization of the prototype compound involving the production of cyclic congeners yielded peptides with submicromolar potency to block HIV-1 infection, strengthening the hypothesis that previous peptide derivations accomplished partial restriction of the conformation of CD4(81-92) to one favorable for interaction with gp120. Concentrations of the original prototype compound T1C4E5-tribenzyl-CD4(81-92) that inhibited infection in vitro more than 50% could be achieved for several hours by intravenous infusion in primates and were well-tolerated at these levels. The peptide was not efficacious to inhibit establishment of viral infection at these doses; however, peptide treatment did lower average viral antigenemia and delay the cumulative time to morbidity relative to the control group.

Age-related changes in vitamin D metabolites, osteocalcin, alkaline phosphatase and parathyrin in normal Chinese women in Taipei.

To evaluate the age-related changes in the bone remodeling rate, the vitamin D status, and parathyroid function of healthy Chinese women, we selected two serum markers of bone turnover, osteocalcin and alkaline phosphatase (ALP). These markers as well as vitamin D metabolites and parathyrin (parathyroid hormone, PTH) were tested in healthy Chinese female volunteers aged 18 to 80 years residing in the Taipei urban area. The results showed no significant change with aging in the 25-hydroxyvitamin D (25(OH)D), 1,25-dihydroxyvitamin D (1,25(OH)2D) and immunoreactive c-terminal PTH (i-cPTH) serum levels. However, there was a trend towards lower 25(OH)D levels at the two extremes of age. The serum levels of 1,25(OH)2D and i-cPTH were comparable with reports of other countries. The serum 25(OH)D levels of our subjects were in general lower than those reported in U.S. white women, but similar to those of European women. The serum osteocalcin levels showed a triphasic change: high in early adulthood, decreasing during the 4th decade of life and then increasing continuously until age 70. After age 70, a decreasing trend was again seen. The serum ALP levels showed a continuous increase from the 3rd to the 8th decade of life. All of the subjects had their bone mineral density (BMD) measured. Linear or polynominal regression analysis as well as multiple regression analysis failed to show a significant correlation between the serum parameters and the BMD measurements at various sites.(ABSTRACT TRUNCATED AT 250 WORDS)