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The passive transfer of antibodies and vaccination procedures against p185, the erbB2/ neu oncoprotein, are approaches being explored for treatment of human breast cancer. We now report the possibility of using the erbB2/ neu gene as an immunogen. This study demonstrates that intramuscular or intradermal injections of rat neuNT full-length DNA into mice generate anti-p185 autoantibodies. Anti-p185 polyclonals were also shown to bind the homologous human receptor ErbB2 and to stain specimens of breast adenocarcinoma from both neu-transgenic mice and humans. Further, in vitro assays demonstrated that anti-p185 IgG (probably dependent on CD4+ Th1) were able to inhibit human SKBR3 tumour cell growth and to mediate their lysis by natural killer cells. The continuous presence of circulating neu autoantibodies in mice did not cause any discernible toxic effects on normal tissues expressing low levels of self-antigen, even after 1 year. [ABSTRACT FROM AUTHOR]

P62/SQSTM1, a multi-domain protein that regulates inflammation, apoptosis, and autophagy, has been linked to age-related pathologies. For example, previously we demonstrated that administration of p62/SQSTM1-encoding plasmid reduced chronic inflammation and alleviated osteoporosis and metabolic syndrome in animal models. Herein, we built upon these findings to investigate effect of the p62-encoding plasmid on an age-related macular degeneration (AMD), a progressive neurodegenerative ocular disease, using spontaneous retinopathy in senescence-accelerated OXYS rats as a model. Overall, the p62DNA decreased the incidence and severity of retinopathy. In retinal pigment epithelium (RPE), p62DNA administration slowed down development of the destructive alterations of RPE cells, including loss of regular hexagonal shape, hypertrophy, and multinucleation. In neuroretina, p62DNA prevented gliosis, retinal thinning, and significantly inhibited microglia/macrophages migration to the outer retina, prohibiting their subretinal accumulation. Taken together, our results suggest that the p62DNA has a strong retinoprotective effect in AMD.

A high-calorie diet (HCD) induces two mutually exacerbating effects contributing to diet-induced obesity (DIO): impaired glucose metabolism and increased food consumption. A link between the metabolic and behavioral manifestations is not well understood yet. We hypothesized that chronic inflammation induced by HCD plays a key role in linking together the two components of diet-induced pathology. Based on this hypothesis, we tested if a plasmid (DNA vaccine) encoding p62 (SQSTM1) would alleviate DIO including its metabolic and/or food consumption abnormalities. Previously we reported that injections of the p62 plasmid reduce chronic inflammation during ovariectomy-induced osteoporosis. Here we found that the p62 plasmid reduced levels of pro-inflammatory cytokines IL-1β, IL-12, and INFγ and increased levels of anti-inflammatory cytokines IL-4, IL-10 and TGFβ in HCD-fed animals. Due to this anti-inflammatory response, we further tested whether the plasmid can alleviate HCD-induced obesity and associated metabolic and feeding impairments. Indeed, p62 plasmid significantly reversed effects of HCD on the body mass index (BMI), levels of glucose, insulin and glycosylated hemoglobin (HbA1c). Furthermore, p62 plasmid partially restored levels of the satiety hormone, serotonin, and tryptophan, simultaneously reducing activity of monoamine oxidase (MAO) in the brain affected by the HCD. Finally, the plasmid partially reversed increased food consumption caused by HCD. Therefore, the administering of p62 plasmid alleviates both metabolic and behavioral components of HCD-induced obesity., Competing Interests: CONFLICTS OF INTEREST Andrey Chursov, Vladimir Gabai, and Alexander Shneider are employees of CureLab Oncology Inc and have financial interest in this company. All other authors have no conflicts of interests.

Elenagen is a plasmid encoding p62/SQSTM1, the first DNA vaccine possessing two mutually complementing mechanisms of action: it elicits immune response against p62 and mitigates systemic chronic inflammation. Previously, Elenagen demonstrated anti-tumor efficacy and safety in rodent tumor models and spontaneous tumors in dogs. This multicenter I/IIa trial evaluated safety and clinical activity of Elenagen in patients with advanced solid tumors. Fifteen patients were treated with escalating doses of Elenagen (1- 5 mg per doses, 5 times weekly) and additional 12 patients received 1 mg dose. Ten patients with breast and ovary cancers that progressed after Elenagen were then treated with conventional chemotherapy. Adverse events (AE) were of Grade 1; no severe AE were observed. Cumulatively twelve patients (44%) with breast, ovary, lung, renal cancer and melanoma achieved stable disease for at least 8 wks, with 4 of them (15%) had tumor control for more than 24 wks, with a maximum of 32 wks. The patients with breast and ovary cancers achieved additional tumor stabilization for 12-28 wks when treated with chemotherapy following Elenagen treatment. Therefore, Elenagen demonstrated good safety profile and antitumor activity in advanced solid tumors. Especially encouraging is its ability to restore tumor sensitivity to chemotherapy., Competing Interests: CONFLICTS OF INTEREST Vita Sherbinina, Andrey Chursov, Vladimir L Gabai, and Alexander M Shneider are employees of CureLab Oncology Inc. All other authors have no conflicts of interests.

Lactococcus lactis is well documented as a promising candidate for development of novel oral live vaccines. It has been broadly engineered for heterologous expression, as well as for plasmid expression vector delivery, directly inside eukaryotic cells, for DNA vaccine, or as therapeutic vehicle. This work describes the characteristics of a new plasmid, pExu (extra chromosomal unit), for DNA delivery using L. lactis and evaluates its functionality both by in vitro and in vivo assays. This plasmid exhibits the following features: (1) a theta origin of replication and (2) an expression cassette containing a multiple cloning site and a eukaryotic promoter, the cytomegalovirus (pCMV). The functionality of pExu: egfp was evaluated by fluorescence microscopy. The L. lactis MG1363 (pExu: egfp ) strains were administered by gavage to Balb/C mice and the eGFP expression was monitored by fluorescence microscopy. The pExu vector has demonstrated an excellent stability either in L. lactis or in Escherichia coli . The eGFP expression at different times in in vitro assay showed that 15.8% of CHO cells were able to express the protein after transfection. The enterocytes of mice showed the expression of eGFP protein. Thus, L. lactis carrying the pExu is a good candidate to deliver genes into eukaryotic cells.

Autophagy plays an important role in neoplastic transformation of cells and in resistance of cancer cells to radio- and chemotherapy. p62 (SQSTM1) is a key component of autophagic machinery which is also involved in signal transduction. Although recent empirical observations demonstrated that p62 is overexpressed in variety of human tumors, a mechanism of p62 overexpression is not known. Here we report that the transformation of normal human mammary epithelial cells with diverse oncogenes (RAS, PIK3CA and Her2) causes marked accumulation of p62. Based on this result, we hypothesized that p62 may be a feasible candidate to be an anti-cancer DNA vaccine. Here we performed a preclinical study of a novel DNA vaccine encoding p62. Intramuscularly administered p62-encoding plasmid induced anti-p62 antibodies and exhibited strong antitumor activity in four models of allogeneic mouse tumors - B16 melanoma, Lewis lung carcinoma (LLC), S37 sarcoma, and Ca755 breast carcinoma. In mice challenged with Ca755 cells, p62 treatment had dual effect: inhibited tumor growth in some mice and prolonged life in those mice which developed tumor size similar to control. P62-encoding plasmid has demonstrated its potency both as a preventive and therapeutic vaccine. Importantly, p62 vaccination drastically suppressed metastasis formation: in B16 melanoma where tumor cells where injected intravenously, and in LLC and S37 sarcoma with spontaneous metastasis. Overall, we conclude that a p62-encoding vector(s) constitute(s) a novel, effective broad-spectrum antitumor and anti-metastatic vaccine feasible for further development and clinical trials.

Fms-like tyrosine-kinase 3 ligand (Flt-3L), is a powerful hematopoyetic growth factor, known to modulate the immune response against delivered antigens by acting either as an adjuvant or tolerogenic stimulus. In this study we evaluated the use of murine Flt-3 ligand plasmid (pFl) in combination with a DNA vaccine encoding rat-p185 oncoprotein extra cellular domain (pECD) in the prevention of mammary carcinogenesis in rat-neu HER-2 mutated (neuT) transgenic mice. We demonstrate that intramuscular (i.m.) co-immunization of pFl inhibits the production of anti-HER-2 antibody elicited by pECD vaccine, resulting in the development of spontaneous carcinomas in all co-immunized mice. The inhibitory effect on antibody production by mFlt3 gene appeared to be: dose-dependent, linked to the injection site and timing, and transient in nature. Additionally, we show that co-administration of pFI and pECD plasmids was unable to trigger cytotoxic T-cell immune response in neuT mice. On the other hand, we found that the combination of pFl with pECD had no impact on the ability of pECD to reject HER-2+ transplantable tumors in parental mice. In summary our results demonstrate that, depending on tumor model, co-administration of pFl gene can produce untoward effects to immune response, and thus its application as a vaccine adjuvant should be carefully evaluated., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Background: We have previously shown that xenogenic DNA vaccines encoding rat neu and melanosomal differentiation Ag induce tumor immunity. Others have developed vaccines targeting tumor neovasculature. Tumor endothelial marker 8 (TEM8) is expressed in the neovasculature of human tumors, and in the mouse melanoma B16, but its expression is limited in normal adult tissues. We describe a DNA vaccine combining xenogeneic tumor Ag and TEM8., Methods: In-situ hybridization was used to detect TEM8 RNA in mouse tumors. Mice transgenic for the rat neu proto-oncogene were immunized with DNA vaccines encoding TEM8 and the extracellular domain of rat neu and challenged with the 233-VSGA1 breast cancer cell line. In parallel experiments, C57BL/6 mice were immunized with TEM8 and human tyrosinase-related protein 1 (hTYRP1/hgp75) and challenged with B16F10 melanoma., Results: TEM8 was expressed in the stroma of transplantable mouse breast and melanoma tumors. In both model systems, TEM8 DNA had no activity as a single agent but significantly enhanced the anit-tumor immunity of neu and hTYRP1/hgp75 DNA vaccines when given in concert. The observed synergy was dependent upon CD8+ T cells, as depletion of this cell population just prior to tumor challenge obviated the effect of the TEM8 vaccine in both tumor models., Discussion: A local immune responses to TEM8 may increase inflammation or tumor necrosis within the tumor, resulting in improved Ag presentation of HER2/neu and hTYRP1/hgp75. Alternatively, TEM8 expression in host APC may act more as an adjuvant than an immunologic target.

The effects of frozen condition on the quality of three sweet corn ears (2038, 2010, 2004) and the pattern (Bonanza), were evaluated. Biometrics characteristics like ear size, ear diameter, row and kernel deep were measured as well as chemical and physical measurement in fresh and frozen states. The corn ears were frozen at -95 degrees C by 7 minutes. The yield and stability of the frozen ears were evaluated at 45 and 90 days of frozen storage (-18 degrees C). The average commercial yield as frozen corn ear for all the hybrids was 54.2%. The industry has a similar value range of 48% to 54%. The ear size average was 21.57 cm, row number was 15, ear diameter 45.54 mm and the kernel corn deep was 8.57 mm. All these measurements were found not different from commercial values found for the industry. All corn samples evaluated showed good stability despites the frozen processing and storage. Hybrid 2038 ranked higher in quality.

The super sweet corns Krispy king, Victor and 324 (sh2 hybrids) were evaluated to determine their adaptabilities to the industrial canning process as whole kernels. All these hybrids and Bonanza (control) were sown in San Joaquín (Carabobo, Venezuela), harvested and canned. After 110 days storage at room temperature they were analyzed to be compared physically, chemically and sensorially with Bonanza hybrid. Results did not show significant differences among most of the physical characteristics, except for percentage of broken kernels which was higher in 324 hybrid. Chemical parameters showed significant differences (P < 0.05) comparing each super sweet hybrid with Bonanza. The super sweet hybrids presented a higher sugar content and soluble solid of the brine than Bonanza, also a lower pH. The super sweet whole kernel presented a lower soluble solids content than Bonanza but they were not significant (Krispy king and 324). Appearance, odor and overall quality were the same for super sweet hybrids and Bonanza (su). Color, flavor and sweetness were better for 324 than all the other hybrids. Super sweet hybrids presented a very good adaptation to the canning process, having as an advantage that doesn't require sugar addition in the brine and a very good texture (firm and crispy).

With the purpose of evaluating adaptability to the freezing process of super sweet corn sh2 hybrids Krispy King, Victor and 324, 100 cobs of each type were frozen at -18 degrees C. After 120 days of storage, their chemical, microbiological and sensorial characteristics were compared with a sweet corn su. Industrial quality of the process of freezing and length and number of rows in cobs were also determined. Results revealed yields above 60% in frozen corns. Length and number of rows in cobs were acceptable. Most of the chemical characteristics of super sweet hybrids were not different from the sweet corn assayed at the 5% significance level. Moisture content and soluble solids of hybrid Victor, as well as total sugars of hybrid 324 were statistically different. All sh2 corns had higher pH values. During freezing, soluble solids concentration, sugars and acids decreased whereas pH increased. Frozen cobs exhibited acceptable microbiological rank, with low activities of mesophiles and total coliforms, absence of psychrophiles and fecal coliforms, and an appreciable amount of molds. In conclusion, sh2 hybrids adapted with no problems to the freezing process, they had lower contents of soluble solids and higher contents of total sugars, which almost doubled the amount of su corn; flavor, texture, sweetness and appearance of kernels were also better. Hybrid Victor was preferred by the evaluating panel and had an outstanding performance due to its yield and sensorial characteristics.

Krispy King, Víctor and 324, super sweet hybrids (sh2) were cultivated in San Joaquín, estado Carabobo, Venezuela. The scheme was stablished to produce refrigerated fresh ears to be commercialized. The chemistry, microbiology and sensorial characteristics were evaluated at 0; 7; 14; 21 and 28 days of storage. One hundred ears of each hybrid were picked at the ripe fresh stage and packed in polystyrene trays covered with polyethylene. The storage temperature was 4 degrees C +/- 1 degree C. The scheme used was well adapted, allowing a good stability of the ears until 28 days of storing. The plastic cover avoid the lost of humidity. The soluble solids, total sugars and pH went down during the storage. The acidity and the microorganisms increased as expected. The sensorial variables kept the same for Krispy king and Víctor, while the hybrid 324 shown the lowest humidity content, the highest count of microorganisms and the poorest sensorial quality.

The Fas ligand (FasL) and its receptor Fas (APO-1 or CD95) are members, respectively, of the tumor necrosis factor family that, upon interaction with each other, play a key role in the initiation of one apoptotic pathway. Faulty regulation of the Fas system has been described in a variety of human tumors with different histogenetic origin. Here, we describe the expression and distribution of Fas receptor and ligand pair antigens in surgical samples collected from a cohort of 186 patients bearing breast neoplasms (45 benign and 141 malignant lesions). Immunoperoxidase staining of formalin-fixed tissues showed that 91.1% of benign lesions expressed Fas, which was present in only 56.7% of malignant tumors. On the other hand, FasL was found positive in 22.2% of benign neoplasms and up-regulated in in situ as well as invasive carcinomas (53.9%). Moreover, in malignant tumors, the expression of receptor and ligand antigens appeared to be inversely related. When these findings were correlated with pathological parameters of prognostic relevance, a significant association was observed between FasL and the presence of metastatic lymph nodes and larger tumor size while Fas expression correlated to node-negative status and smaller tumor size. Patients with Fas positive tumors exhibited longer disease-free survival than those with Fas-negative carcinoma while FasL did not influence patient outcome. These relationships indicate that benign and malignant mammary lesions are characterized by differential cellular expression of Fas and FasL and suggest that a neoplastic Fas negative/FasL positive phenotype may be linked to breast cancer progression., (Copyright 2000 Wiley-Liss, Inc.)

Background: Thymectomy (Tx) is a common therapeutic option to treat myasthenia gravis (MG), but its effects on the immune system are still obscure in humans., Objective: We sought to evaluate long-term immunologic effects of therapeutic Tx in patients with MG., Methods: T- and B-cell subsets and T-cell repertoire were analyzed in 35 patients with MG, 16 with previous Tx (at least 8 years before), 6 with recent (<1 year) Tx, and 13 without Tx, as well as in 32 healthy subjects used as normal control subjects. Serum immunoglobulins and a variety of autoantibodies were also measured. A subsequent 3-year clinical follow-up was performed to verify the possible appearance of systemic autoimmune diseases., Results: The long-term thymectomized (Txd) patients had mild T-cell lymphopenia and an expansion of some Vbeta families among circulating CD4+ and CD8+ T cells. They displayed a normal number of total B and CD5+ B-circulating lymphocytes, but they also displayed a polyclonal increase in serum IgM and IgG associated with the presence of high levels of a variety of organ- and nonorgan-specific autoantibodies, including anti-dsDNA and anticardiolipin, without clinical evidence of autoimmune disease. These serologic abnormalities were not detectable in both non-Txd and recently Txd patients. After 3 years, 2 long-term Txd patients had systemic lupus erythematosus and an undifferentiated connective tissue disease., Conclusions: The association between MG and laboratory findings of systemic autoimmune disease may be in part related to Tx rather than to MG. Tx may represent a risk for the development of systemic autoimmune disorders over years in patients with MG.

erbB2/neu, an overexpressed oncogene product, has been proposed as a human cancer vaccine target. In the present study, transgenic (rat neuNT oncogene) FVB/neu mice, developing metastasizable mammary carcinoma, were immunized with a plasmid DNA encoding are not tolerant to the self antigen and sequences. We report that transgenic tumour-bearing mice, like some breast cancer patients erbB2 + X, develop anti-neu autoimmune responses, which can be boosted and skewed to a Th1 phenotype by DNA immunization. Intramuscular injections of neuNT plasmid drastically reduced (or even prevented in a small number of treated mice) the outgrowth of mammary neoplasms as well as their metastatic penetrance. Furthermore, DNA immunization caused haemorrhagic necrosis of established cancer nests, leaving a greatly reduced portion of the tumour burden for the host to cope with. The antitumour activities we obtained, in this very challenging model for cancer immunotherapy, lay the foundation for DNA-based immunization to control erbB2/neu-overexpressing neoplasms.

Available data on the existence of lentivirus proteins with properties of unconventional Ag for B cells, have been so far restricted to human immunodeficiency virus (i.e. gp-120 of HIV-I). By using biotinylated-MAbs-anti-biotin IgG as readout system, we now report that gag-p24 antigen, either assembled in feline immunodeficiency virus (FIV) particles or expressed as recombinant polypeptide (rec.p24) may bind to nonimmune IgGs purified from mouse or cat sera. Moreover, FACS scanning experiments are consistent with the possibility that rec.p24 interacts with surface-Ig in a sub-population (5-6%) of rodent B cells. We hypothesize that gag-p24 peptide encoded regions may bind to unconventional Ig sites or, alternatively, that they may represent 'public' epitopes for natural polyreactive antibody.

The passive transfer of antibodies and vaccination procedures against p185, the erbB2/neu oncoprotein, are approaches being explored for treatment of human breast cancer. We now report the possibility of using the erbB2/neu gene as an immunogen. This study demonstrates that intramuscular or intradermal injections of rat neuNT full-length DNA into mice generate anti-p185 autoantibodies. Anti-p185 polyclonals were also shown to bind the homologous human receptor ErbB2 and to stain specimens of breast adenocarcinoma from both neu-transgenic mice and humans. Further, in vitro assays demonstrated that anti-p185 IgG (probably dependent on CD4+ Th1) were able to inhibit human SKBR3 tumour cell growth and to mediate their lysis by natural killer cells. The continuous presence of circulating neu autoantibodies in mice did not cause any discernible toxic effects on normal tissues expressing low levels of self-antigen, even after 1 year. The experiments reported here raise the possibility that boosting anti-ErbB2 immunity by DNA vaccination will not induce harmful autoimmunity in humans.

Anti-10 deacetylbaccatin III (DAB) antibodies (IgY) were elicited in hens immunized with a succinyl-DAB/BSA conjugate and extracted from egg yolk. As shown by indirect competitive inhibition enzyme immunoassay (CIEIA), the addition of free-DAB competitively inhibited the binding of affinity purified anti-DAB IgY to DAB/BSA solid phase conjugated antigen. The assay enabled the detection of DAB in concentrations as low as 7.5ng/ml (13.7 nM DAB), whereas anti-DAB IgY did not react with taxol even at a concentration a thousand times higher. The structural requirements of the diterpenoid nucleus for binding to IgY were considered on the basis of the levels of cross-reaction found with 10 authentic taxanes. The results indicate that anti-DAB IgY represents the first high affinity antibody produced capable of recognizing the ring skeleton of taxol precursors.

High percentages of gamma/delta+ T cells in the peripheral blood of a subgroup of patients with primary Sjögren's syndrome (SS) were found. This allowed us to purify and analyze them without their being previously expanded in vitro, and to investigate, therefore, the role of these cells in the pathological immune response which characterizes such systemic autoimmune disorders. The results showed poor proliferation of patient gamma/delta+ T cells in response to anti-CD3, due not to macrophage-dependent suppression but to defective interleukin 2 (IL-2) synthesis. Despite the defective proliferation patient gamma/delta+ cells, unlike those of the normal controls, provided a helper effect in inducing B cells to secrete immunoglobulins (Ig), particularly when they were preincubated with IL-2. The relative increase in a gamma/delta+ T cell subset which, although it secretes low levels of IL-2, is able to provide help for B-cell Ig synthesis, suggests that this T-cell subpopulation may be functional in vivo and may be involved in the pathological immune response encountered in pSS.

Nucleic acids fractions were isolated from pre-Columbian maize seeds and characterized using different approaches such as polyacrylamide gel electrophoresis, anti-DNA antibody binding, HPLC fractionation, molecular hybridization with cloned genes, and DNA amplification by the polymerase chain reaction. The nucleic acids were found to be very depolymerized (less than or equal to 140 base pairs in length) and composed mainly of ribosomal RNA. Despite the very low amount and degree of polymerization of seed DNA, specific maize nuclear Mu1, Mu4, Mu8 and, possibly, Mu5 element components could be detected, thanks to the use of amplification systems as short as 90 bp. The results suggest that evaluation of the relative proportions of Mu-type element components and, possibly, other maize genomic components in single mummified kernels, may offer a new key to the study of ancient maize populations.

Low-molecular-weight peptides are linked to the chromatin DNA of several tissues, from which they can be dissociated by alkaline extraction at pH 9.5. The level of the active peptide fraction ranges between 10 and 35 micrograms/mg DNA. The removal of peptides from DNA causes a relevant amplification of DNA template capacity for prokaryotic and eukaryotic RNA polymerases. Gel filtration on Sephadex G-25 or BioGel P4 shows that the chromatin peptide fraction from purified DNA migrates as a sharp peak with an elution volume corresponding to a molecular weight of about 1000. The chromatin peptides are further purified by Sephadex G-10 and high-performance liquid chromatography. Four active fractions are isolated, one of which shows very high inhibition activity on the RNA synthesis in vitro. The amino acid analysis and the inhibition mechanism of the purified peptides are reported.

Enzymatic breakdown of residual proteins occurs at mild alkaline pH (pH optimum 8.5) as monitored by using radioiodinated, purified genomic DNA from calf thymus. These DNA fibers also possess a differential ability to hydrolyze added exogenous small and linker histones. The results described argue strongly that a putative protease activity, co-purified with DNA, is the source of short chain peptides which inhibit transcription in vitro3. Therefore, we propose that RNA repressor peptides must be of higher molecular weight than previously reported.

A perfused preparation of the hind limb of normal and diabetic rats was used to study the effects of lactic acidosis, alone or associated with hypoinsulinemic diabetes, on the incorporation of glucose and inorganic orthophosphate (Pi) into the skeletal muscle. A well oxygenated perfusate was recirculated for ninety minutes during which the lactic acid accumulated into the medium with the ensuing pH drop. The perfusions were practiced in the hind limb of alloxanized diabetic rats, in the hind limb of diabetic rats with perfusate containing 200 microU of insulin/ml, in the hind limb of 24 hour fasted rats, and on the hind limb of fed rats, and they were compared to similar groups with normalized pH perfusate with a sodium bicarbonate infusion. In the diabetic perfusions with lactic acidemia, it was observed that the addition of insulin increased the uptake of Pi and of glucose, and reduced the release of Pi by the muscular tissues. A smaller release of Pi by the preparations obtained from fed rats was observed when compared to the hind limb preparations of fasted rats. The diabetic preparations showed an increased glucose uptake when the pH was normalized, and a decrease of Pi released by the muscles, even in the absence of insulin, and at the same time, the administration of insulin associated with the normalization of pH increased the uptake of Pi and of glucose, and decreased the Pi released by the muscles. In all the groups, the administration of sodium bicarbonate significantly increased the lactate release into the medium. It was also found that the lactic acidosis reduced the uptake of Pi by the preparations inducing hyperphosphatemia. According to these results, muscular tissue plays a role in the hypophosphatemia that has been reported in the insulin treated diabetic ketoacidosis by increasing the incorporation of Pi and reducing its release by the same tissue.

Isolated perfused fed rat livers spontaneously liberated glucose and orthophosphate to the medium; 24-hr fasted rat livers did not exhibit these phenomena. In perfused fed rat livers, glucagon (2 mug) increased glucose output and promoted orthophosphate incorporation. In perfused fed rat livers, insulin (250 or 500 mU) inhibited the spontaneous liberation of glucose and orthophosphate. Comparable doses of insulin significantly reduced the glucagon (2 mug)-induced increase in glucose output from perfused fed rat liver, but did not affect orthophosphate uptake by the organ.