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The human genome actively transcribes a large amount of non-coding genes, including microRNA and long noncoding RNAs (lncRNAs). Compared to well-known microRNAs, lncRNAs are poorly characterized. However, emerging evidence suggests that lncRNAs may be dysregulated in prostate cancer, but their role in prostate cancer remains elusive. We hypothesize that prostate cancer may exploit this mechanism to promote tumor progression and metastasis, and therapy resistance. Thus, overall goal of this application is to determine whether lncRNAs function as competitive endogenous RNAs (CeRNAs) which are capable of sequestering microRNAs to regulate RNA transcripts. A better understanding of how microRNAs are dysregulated in prostate cancer may help biomarker discovery, ultimately developing a better strategy for prostate cancer treatment., The original document contains color images.

Lung cancer is the leading cause of cancer deaths in the world, and according to the statistics of National Cancer Institute s SEER, 219,000 men and women will be diagnosed and 159,000 patients will die from this disease in 2014. Small cell lung carcinoma (SCLC) is the cause of approximately 20% of lung cancer. Chemo-therapy is the first choice of treatment for this carcinoma because SCLC is often highly malignant and metastasizes to the distant organs even at an early stage. Although SCLC is chemotherapy-sensitive at the initial stage, it becomes ultimately chemo-resistant with worse prognosis. Recent stem cell theory suggests that there is a distinct population of tumor cells that has stem-cell like characteristics as well as ability of resistance to chemo-therapy. Moreover, several lines of evidence indicated that the self renewal ability of cancer stem cells is regulated by microRNAs. Therefore, we hypothesize that drug resistance of SCLC is mediated by microRNA in tumor stem cell. In this project, we planned to identify the specific microRNA in the stem cells from SCLC. We have established a method to isolate cancer stem cells from human SCLC cell line, and our results indicate that miR16 and 21 are strongly expressed in these CSCs. We also identified five microRNAs (including miR16 and 21) that are involved in Cisplatin resistance by microRNA library screening. Our results strongly suggest that miR16 and miR21 play critical roles in chemo-resistance of cancer stem cell derived from SCLC. Therefore, these oncomirs may serve as potential biomarkers and therapeutic targets for chemoresistant SCLC., The original document contains color images.

PTEN is the gene which is down-regulated in the majority of prostate cancer, and PTEN-Akt pathway is considered to play a critical role in radiation resistance. The overall goal of this project is to identify a specific gene that is essential for this important pathway in the hope that targeting such gene will sensitize PTEN-negative tumor stem cells to be radiation-sensitive. To accomplish this goal, we isolated cancer stem cells from prostate tumor cell lines and patient samples followed by preparing PTEN-deficient cells. We then screened genes that play critical roles in the PTEN pathway using a technique called the shRNA library screening, with or without radiation treatment of these cells. We have identified synthetic lethal genes, and we plan to examine the effect of such genes in an animal model to test our hypothesis. During the funding period, we have successfully accomplished Aim 1 and 2 and we identified five synthetic lethal genes. Testing the effect of knockout of these genes on radiation therapy in an in vivo model as planed in Aim 3 is currently in progress. We believe that targeting the synthetic lethal genes that we discovered will lead to the development of a novel therapeutic approach for radiation-resistant prostate cancer., The original document contains color images.

PTEN is the gene which is down-regulated in the majority of prostate cancer, and PTEN-Akt pathway is considered to play a critical role in radiation resistance. The overall goal of this project is to identify a specific gene that is essential for this important pathway in the hope that targeting such gene will sensitize PTEN-negative tumor stem cells to be radiation-sensitive. To accomplish this goal, we will isolate cancer stem cells from patients and prepare PTEN-deficient cells. We will then screen genes that play critical roles in the PTEN pathway using a technique called shRNA library screening, with or without radiation treatment of these cells. When we identify a gene, we will then test the effect of such gene in an animal model to test our hypothesis. We believe that identifying such genes will lead to development of a novel therapeutic approach to radiation-resistant prostate cancer., The original document contains color images.

Lung cancer is the leading cause of cancer deaths in the world, and according to the statistics of National Cancer Institute s SEER, 219,440 men and women will be diagnosed and 159,000 patients will die from this disease in 2010. Small cell lung carcinoma (SCLC) is the cause of approximately 20% of lung cancer. Chemo-therapy is the first choice of treatment for this carcinoma because SCLC is often highly malignant and metastasizes to the distant organs even at an early stage. Although SCLC is chemotherapy-sensitive at the initial stage, it becomes ultimately chemo-resistant with worse prognosis. Recent stem cell theory suggests that there is a distinct population of tumor cells that has stem-like characteristics as well as ability of resistance to chemo-therapy. Moreover, several lines of evidence indicated that the self renewal ability of cancer stem cells is regulated by microRNAs. Therefore, we hypothesize that drug resistance of SCLC is mediated by microRNA in tumor stem cell. In this project, we are proposing to identify the specific microRNA in the stem cells from SCLC. We also plan to characterize these microRNAs so that we can eventually identify specific target molecules to block the chemo-resistance.

Bone is the most common metastatic site for prostate cancer which affects approximately 70% of patients with advanced disease. Despite this clinical importance, the exact molecular mechanism of the bone-specific metastasis has not been clearly defined. The growth of the tumor cells in the bone is generally slow and they often become dormant until an appropriate microenviroment is established for their re-growth. According to the recent cancer stem cell theory, which still remains a hypotheses, recurrent tumor must arise from a dormant tumor stem cell. However, the precise mechanism of dormancy is virtually unknown, and identifying the responsible factors and understanding the underlining mechanism are crucial for developing a novel therapeutic approach. Our recent preliminary data suggest that BMP7 (bone morphogenetic protein 7) which is secreted from bone stromal cells is able to induce senescence to prostate tumor cells and that this induction is mediated by activation of the tumor metastasis suppressor gene, NDRG1 (N-myc downstream regulated gene 1). Therefore, we hypothesize that a prostatic tumor stem cell becomes dormant in the bone through the BMP7-mediated activation of p38 and NDRG1., The original document contains color images.

Prostate cancer (CaP) remains among the most common causes of cancer related deaths in men. Because CaP is characterized by unique prostate associated antigens, it has been considered among prime candidates for immunotherapy. Despite numerous laboratory advances, clinical outcomes have been partial and transient. One plausible reason for the incomplete response is that vaccine cells, prepared under standard tissue culture conditions, can drastically differ in expression of macromolecules in situ, and thus may immunize against a less complete antigen spectrum. The purpose of the proposed studies is to optimize the effectiveness of therapeutic whole cell CaP vaccines by taking into consideration tumor associated hypoxia as a relevant determinant factor of tumor antigenicity., The original document contains color images.

MicroRNAs are non-coding small RNAs that exert their silencing functions at the posttranscriptional level; they play a fundamental role in regulation of diverse cellular pathways. In addition to microRNAs, the cell also expresses abundantly other non-coding RNAs such as long non-coding RNAs (200 bases) (lncRNAs), which could account for 4-9% of transcripts in human genome. In this application we proposed to test whether lncRNAs are dysregulated in prostate cancer. We demonstrated that lncRNA GAS5 is a direct target for miR-21. Furthermore, GAS5 can also suppress miR-21. In addition to GAS5, we identified that PCGEM1 is upregulated in prostate cancer, and it interacted with androgen receptor (AR). More importantly, both AR and PCGEM1 were co-localized in prostate cancer cells as well as in the clinical specimens, especially in high grade prostate tumors, suggesting that their interaction may contribute to aggressiveness of prostate cancer and castration resistance. Together, our study suggests that lncRNAs are important players in prostate cancer biology, and further characterization of these lncRNAs will provide a better understanding of prostate cancer biology. As a result, lncRNAs may serve as biomarkers for prostate cancer., The original document contains color images.

Prostate cancer (CaP) remains among the most common causes of cancer related deaths in men. Because CaP is characterized by unique prostate associated antigens, it has been considered among prime candidates for immunotherapy. Despite numerous laboratory advances, clinical outcomes have been partial and transient. One plausible reason for the incomplete response is that vaccine cells, prepared under standard tissue culture conditions, can drastically differ in expression of macromolecules in situ, and thus may immunize against a less complete antigen spectrum. The purpose of the proposed studies is to optimize the effectiveness of therapeutic whole cell CaP vaccines by taking into consideration tumor associated hypoxia as a relevant determinant factor of tumor antigenicity. We hypothesize that hypoxically cultured CaP cells are more similar in their antigen landscape to CaP cells in situ than are normoxically cultured CaP cells. The following Tasks were defined in the approved statement of work; Task 1. Identify oxygen tension responsive genes and proteins in the cells comprising a clinical grade CaP cellular vaccine. This first task was accomplished at the end of the first period. Task 2. Validate differentially expressed molecules in CaP that are associated with tissue hypoxia. This last task has been partially executed and is reported in the present progress report. If the proposed studies demonstrate that CaP cells grown under low oxygen tension (pO2) are more antigenically similar to cells in situ, this will justify the evaluation of their therapeutic value in a preclinical model., The original document contains color images.

Although surgical resection with adjuvant chemotherapy and/or radiotherapy are used to treat breast tumors, normal tissue tolerance, development of metastases, and inherent tumor resistance to radiation or chemotherapy can hinder a successful outcome. Therefore, it is necessary to consider alternative targeted therapeutic approaches for adjuvant therapy that would significantly reduce undesired side effects in normal tissues. This proposal describes a thermally responsive polypeptide (CPP-ELP-H1) that inhibits breast cancer cell proliferation by blocking the activity of the oncogenic protein c-Myc. The objective of the proposed research is to demonstrate that these genetically engineered polypeptides can be targeted to the tumor site by applying local hyperthermia and can inhibit tumor growth in an animal model. During the course of this training program, we have used fluorescently labeled polypeptides to demonstrate that thermal targeting can, in fact, be used to enhance the accumulation of an ELP-based c-Myc inhibitory polypeptide at the tumor site. We have defined the plasma clearance and biodistribution of the Bac-ELP1-H1 polypeptide, and we have used in vivo imaging to demonstrate tumor deposition and thermal targeting of the polypeptide in real time. We have also collected tumor reduction data using a lead c-Myc inhibitory polypeptide, and determined that treatment of mice with this polypeptide combined with thermal targeting resulted in a 70% inhibition of tumor volumes., The original document contains color images.

Although surgical resection with adjuvant chemotherapy and/or radiotherapy are used to treat breast tumors, normal tissue tolerance, development of metastases, and inherent tumor resistance to radiation or chemotherapy can hinder a successful outcome. Therefore, it is necessary to consider alternative targeted therapeutic approaches for adjuvant therapy that would significantly reduce undesired side effects in normal tissues. This proposal describes a thermally responsive polypeptide (CPP-ELP-H1) that inhibits breast cancer cell proliferation by blocking the activity of the oncogenic protein c-Myc. The objective of the proposed research is to demonstrate that these genetically engineered polypeptides can be targeted to the tumor site by applying local hyperthermia and can inhibit tumor growth in an animal model. During the first year of this training program, we have used radio-labeled polypeptides to demonstrate that thermal targeting can, in fact, be used to enhance the accumulation of an ELP-based c-Myc inhibitory polypeptide at the tumor site, and we have collected promising tumor reduction data using a lead c-Myc inhibitory polypeptide. Several modifications have been made to the research plan, including altering the way the polypeptide is labeled and adding an additional animal model, and future experiments will complete the proposed aims of determining the optimum CPP for thermal targeting and demonstrating tumor reduction efficacy with the lead CPP-ELP-H1 polypeptide., The original document contains color images.

The object of this project was the folding and association of Cys-rich recombinant and native silk proteins from aquatic larvae of the midge, Chironomus tentans. Specific aims include. (A) Learn which Cys residues are critical to the pathway for intramolecular disulfide bond formation in rCAS, a recombinant repeat from silk protein, spIa. (B) Determine if intramolecular disulfide bonds stabilize the higher-order structure of rCAS. (C) Determine the conditions and which Cys participate in formation of intermolecular disulfide bonds. (D) Learn how and where other native silk proteins interact with spIs. In addition, we also chose to do the following. (E) Determine if N-linked glycosylation sites in ssp160 are evolutionarily conserved. (F) Acquire consensus repeat sequences for Cys-containing motifs in sp185/sp220 homologs and sp195. The final results of this project indicate: (1) Aquatic silk proteins can be expressed and purified from bacteria in large quantities. (2) However, fibrous proteins may prove difficult to analyze: CD and FTTR basis spectra lack fibrous protein standards and proteins with Cys may refold into multiple conformers. (3) The abundance and conservation of glycosylation sites on some aquatic silk proteins suggest such post-translational modification merits study before biotechnological applications are considered. (4) Aquatic silk proteins contain more Cys motifs than previously thought.

Protein-protein and intraprotein interactions were studied in natural, recombinant and synthetic domains of proteins that form aquatic silk. Subsets of natural proteins were found to associate into discrete high molecular mass complexes thought to be the non-covalent precursors to insoluble silk. A recombinant protein mimicking a single core repeat" from a 1000-kDa silk protein reversibly formed two intramolecular disulfide bonds; however, more than one comformation existed. A synthetic peptide encompassing all four cysteines also formed intramolecular disulfide bonds. The results obtained suggested new steps in the fiber formation pathway including properties of the recombinant protein that may render it useful as a degradable biomolecular material.

A variety of natural silk secretions (from spiders, silkworms and aquatic insect larvae), and also reconstituted silk solutions, are able to form a nematic liquid crystalline phase. The anisotropic structures that self- assemble in this phase are formed from the isotropic phase by aggregation of molecules, rather than by individual molecules undergoing a conformational change to a rod-like form. This enables the molecules to retain their solubility in water while, simultaneously, the viscosity of the solution is reduced. The liquid crystalline phase is stable under a wide range of equilibrium conditions, but its ability to form is sensitive to the rate at which the initially isotropic solution is allowed to dry. The kinetics of phase transitions exhibited by solutions of silk proteins must be taken into account if solutions of silk fibroin are to be successfully processed in vitro

The overall goal of this project has been to learn about the structure, developmentally regulated synthesis and assembly of a family of secretory proteins (SPs) that are synthesized in salivary glands of an aquatic insect and assembled into an insoluble biopolymer of silk-like threads. Molecular biological and immunological techniques were used to ascertain in the structure of two additional SPs and the developmental regulation of the expression of their genes. A 185/220-kDal Cys-rich SP homologue has been identified in three species of Chironomus. Biochemical, biophysical and electron microscopic techniques were used to study SP disassembly/reassembly in vitro and measure the higher order structure of 1000-kDal SPs and their repeated domains in the form of synthetic peptides. This domain approach to protein structure was extended to insect chorions. Eukaryotic genes, secretory polypeptides, tandemly repeated amino acid sequences, in vitro assembly/disassembly of polypeptide fibers, protein-protein interactions

The overall goal of this project has been to learn about the structure, developmentally regulated synthesis and assembly of a family of secretory proteins (SPs) that are synthesized in salivary glands of an aquatic insect and assembled into an insoluble biopolymer of silk-like threads. Molecular biological and immunological techniques were used to ascertain the structure of two additional SPs and the developmental regulation of the expression of their genes. A 185/220-kDal Cys-rick SP homologue has been identified in three species of Chironomus. Biochemical, biophysical and electron microscopic techniques were used to study SP disassembly/reassembly in vitro and measure the higher order structure of 1000-kDal SPs and their repeated domains in the form of synthetic peptides. This domain approach to protein structure was extended to insect chorions.

The possibility of unusual toxicity due to interaction of toxic chemicals upon environmental or occupational exposures to two or more chemicals, particularly when exposures involve levels ordinarily considered harmless individually is an important toxicological concern. Progress in this area of environmental toxicology has suffered for want of a model where the two interactants are individually nontoxic. Models, where toxic doses of chemicals are employed are not very representative of low level, environmental exposure. Prior exposure to nontoxic levels of the pesticide Kepone (chlordecone, CD) results in a 67-fold amplification of CCI4 lethality in experimental animals. This propensity for chlordecone to potentiate hepatotoxicity of halomethanes such as CCI4, CHCI3 and BrCCI3 has been the subject of this intense inquiry to unravel the underlying mechanism. The biological effects of this interaction include extensive hepatotoxicity characterized by histological alterations, hepatic dysfunction, and perturbation of related biochemical parameters.

A model to study the toxic effect for hydrogen cyanide (HCN) by inhalation and to evaluate the efficiencies and relative potencies of possible antidotes has been established. Several chambers and accompanying mechanics of administration of HCN were designed and evaluated. This model is especially designed to be used to study the effects of HCN at different concentrations and to evaluate the effectiveness of the antidotes with pre- and post-exposure of mice and rats to HCN. In addition, pulmonary and cardiovascular changes in the rat can be monitored. This allows more discrete determination of the efficacy of antidotes and monitoring of toxic effects other than lethality. During exposure, HCN concentrations in the chamber were continuously monitored by detector tubes, electrochemical sensor, and chemical colorimetric methods. Mice were placed in a cage which was quickly inserted and removed from the chamber. Rats were placed in a rat hold (tube) for insertion of nose-only exposure.

The goal of this project is to learn the structure and assembly characteristics of a family of secretory proteins (SP's). SP's are synthesized in salivary glands of aquatic larvae of the midge, Chironomus. Larvae construct underwater tubes for filter feeding and pupation using silk fibers composed of SP's. During the past year we made progress in two areas. (1) A 1-kb cDNA clone was sequenced that represents a portion of a 4.8-kb poly(A) + RNA. Though it lacks sequences found in other SP mRNA's, its abundance in salivary glands is equal to other SP mRNA's. Antipeptide antibodies are being raised to identify the protein encoded by this mRNA. (2) Infrared and circular dichroism spectrometry have been used to study the structure of synthetic peptides representing the alternating 'constant' and 'subrepeat' domains of SP's. These 1000-kDal proteins appear to consist of alternating domains of alpha- and POLY(Gly)II-helices. (KT)

There is a significant interest in the possibility of unusual toxicity due to interaction of toxic chemicals upon environmental or occupational exposures even though such exposures may involve levels ordinarily considered harmless individually. While many laboratory and experimental models exist for such interactions, progress in this area of toxicology has suffered for want of a model where the two interactants are individually nontoxic. We developed such a model where prior exposure to nontoxic levels of the pesticide Kepone (chlordecone) results in a 67-fold amplification of carbon tetrachloride (CC14) lethality in experimental animals. The mechanism(s) by which chlordecone amplifies the hepatotoxicity of halomethanes such as CC14, chloroform (CHC13), and bromotrichloro (BrCC13) has been a subject of intense study. The biological effects of this interaction include extensive hepatotoxicity characterized by histopathological alterations, hepatic dysfunction, and perturbation of related biochemical parameters. Close structural analogs of chlordecone such as mirex and photomirex do not share the propensity of chlordecone to potentiate halomethane toxicity., This article is from Proceedings of the Conference on Toxicology (18th) Held in Dayton, Ohio on 1-3 November 1988, AD-A235 912, p134-164.

The purpose of this project was to investigate the effectiveness of pretreatment with potential cyanide antagonists in protecting against the lethal effects of cyanide. The primary methodology used was LD50 studies in mice. Some studies involving intravenous infusion were done using rats. The data indicate that alpha-ketoglutaric acid is the most potent single chemoprophylactic agent reported in the literature. When a combination of alpha-ketoglutaric acid and sodium thiosulfate was administered to animals prior to injection of cyanide, the LD50 increased 20 fold compared to that in animals given saline pretreatment. This combination of alpha-ketoglutaric acid and sodium thiosulfate produced a 2.9-fold greater increase in LD50 than did the classic combination of sodium nitrite and sodium thiosulfate. Keywords: Chemotherapeutic agents; Toxins and antitoxins.

The purpose of this project is primarily to establish a model system for exposing animals (mice and rats) to hydrogen cyanide (HCN). A chamber has been designed, constructed and tested for the nose-only dynamic exposure of rats to HCN. A chamber of two compartments is available for exposure of mice to HCN after allowing the chamber to equilibrate. Analytical methods (microdiffusion and gas chromatography) for the determination of cyanide (CN) levels in blood and chamber air are established and confirmed. A method, employing selective ion electrode, is being used to monitor the test solutions fo cyanide salts. Subject terms: Cyanide; Exposure chamber; Antidotes; Alpha-ketoglutaric acid.

This report contains the Appendices to the First Annual report on Basal Ganglia Dopamine-Gamma-Aminobutyric Acid-Acetylcholine Interaction in Organophosphate Induced Neurotoxicity. The appendices include information in reference to the following: Variation of Commercial Diisopropyl Fluorophosphate Preparations in Toxicological Studies; Assessment of Diisopropyl-Fluorophosphate (DFP) Toxicity; The Effect of Acute and Chronic Cholinesterase Inhibition with Diisopropylfluorophosphate On Muscarinic, Dopamine and GABA Receptors of the Rat Striatum; Diisopropylfluorophosphate and GABA Synaptic Function: Effect on Levels, Enzymes, Release and Uptake in the Rat Striatum., See also ADB091642.

The tritiated muscarinic cholinergic antagonist quinuclidinyl benzilate ((3H)QNB) was used to characterize the muscarinic receptors associated with membranes prepared from swine tracheal smooth muscle. As judged from receptor-binding assays, the membrane homogenate had specific high-affinity receptors for (3H)QNB. Specific binding was saturable with respect to (3H)QNB and tissue concentrations and was time- and pH-dependent. The association of (3H)QNB (180 pM) with the muscarinic receptors reached equilibrium much sooner at 37 C than 25 C (30 min and 2 hrs, respectively). Equilibrium at both temperatures was attained within 5 min at a high concentration of (3H)QNB, 1800 pM. All other experiments were performed at 37 C. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (K sub D) of 51 + or - 20 pM and a maximum receptor density (B max) of 2.17 + or - 0.27 pmole/mg protein. The Hill coefficient for (3H)QNB binding was 1.07 + or - 0.16. The association and dissociation rate constants were determined to be (5.51 + or - 0.16) x 100, 000,000/M/min and (1.41 + or - 0.18) x .01/min, respectively. The muscarinic antagonists atropine and scopolamine competitively inhibited (3H)QNB binding to the homogenate, with a Hill coefficient of 0.9 and inhibition constant of 1.8 NM. Muscarinic agonists and the nicotinic antagonist d-tubocurare inhibited (3H) QNB binding in micromolar concentrations. Tolerance to daily diisopropylfluoro phosphonate (DFP) injection was observed in these animals., Presented at Society of Toxicology Regional Meeting, Oxford, MS, 2 Nov 1984.

The aim of this project is to learn about the structure, developmentally regulated synthesis and assembly of a family of secretory proteins (SPs) into an insoluble polymer of silk-like threads. SPs are exclusively synthesized in salivary glands of aquatic larvae of the Dipteran, Chironomus. All SPs studied to date are composed of tandemly repeated amino acid sequences. Recombinant cDNA probes are used to map SP-coding genes on polytene chromosomes, identify their mRNAs on Northern blots and derive the amino acid sequence of their encoded protein. This experimental system provides a unique opportunity to study how naturally occurring soluble proteins can assemble into an insoluble fiber that functions in an aqueous environment. Keywords: Eukaryotic genes, Secretory proteins, Tandemly repeated amino acid sequences, In vitro assembly/disassembly of polypeptide fibers, Protein-protein interactions.

The aim of this project is to learn about the structure, developmentally regulated synthesis and assembly of a family of secretory polypeptides (SPs) into an insoluble polymer of silk-like threads. SPs are exclusively synthesized in salivary glands of aquatic larvae of the Dipteran, Chironomus. All Sps studied to date are composed of tandemly repeated amino acid sequences. Recombinant cDNA probes are used to map SP-coding genes on polytene chromosomes, identify their mRNAs on Northern blots and derive the amino acid sequence of their encoded polypeptide. cDNA probes and anti-SP antibodies are used to study the level at which SP gene regulation occurs during normal larval development and under conditions of galactose-induced alterations in gene expression. SP assembly in vitro is being studied by a combination of physical, electron microscopic and biochemical methods. We hope to learn which SPs interact with each other and what is the chemical nature of these interactions. We eventually plan to determine the spatial distribution of SPs within assembled complexes by making three-dimensional tomographci reconstructions from immunoelectron micrographs. This experimental system provides a unique opportunity to study how naturally occurring soluble polypeptides can assemble into an insoluble fiber that functions in an aqueous environment.

The purpose of this project has been to investigate the development of an antidotal and/or prophylactic agent to antagonize the toxic effects of cyanide (CN). The work centered around carbonyl containing chemicals - alpha-keto acids. Alpha-ketobutyric (alpha-KB), glutamic, alpha-ketoglutaric (alpha-KG), B-ketoglutaric (B-KG), dehydroascorbic and pyruvic (PRY) acids were studied. Alpha-Ketoacids were shown to bind cyanide in vitro. Evidence of this binding is the decreased concentration of alpha-keto acids and the decreased quantities of cyanide released from mixtures of potassium cyanide and alpha-keto acids both prophylactically and antidotally by intraperitoneal administration. Alpha-KG showed the least toxicity (up to 4.0 grams/kg) although other keto acids were slightly more effective as antidotes. Studies into the mechanism have been done, alpha-KG does prevent the inhibition of cytochrome oxidase by cyanide and has no effect on the transulfurase, rhodanese. Alpha-KG does not enhance the formation of methemoglobin or thiocyanate. In studies involving the dogs, nine cardiovascular and thirteen blood gas parameters were measured and evaluated for the antagonistic effects of alph-KG on cyanide toxicity. From these studies, alpha-KG is effective in antagonizing administered dose of CN of five times the lethal dose before the toxic effects are irreversible.

Chronic reduction in acetylcholinesterase activity leads to accumulation of acetylcholine at synapses. The continual presence of acetylcholine induces changes in sensitivity of tissues to acetylcholine, in part due to down-regulation (decreased number) of muscarinic receptors. The following report describes experiments designed to determine whether tracheal muscle muscarinic receptors and the tissue response to activation of these receptors change with subacute exposure to diisopropylfluorophosphaonate (DFP). Muscarinic receptor binding characteristics on control and DFP-treated animals, and isometric tension developed in response to various agents (acetylcholine, DFP, Bethanechol, K+) and membrane potential of muscle cells were determined in control and subacutely animals treated with DFP. Displacement of Tritium quinuclidinyl benzilate Tritium QNB binding by agonists and antagonists suggests that the tracheal muscle receptors are of the M2 subtype. Agonists induce a state change in the receptor to a lower affinity form. Subacute treatment with DFP results in a decrease in receptor number of 70% after 7 days and 95% decrease in acetylcholinesterase activity. The dose-response relationships for contraction induced by acetylcholine or bethanechol show several shifts in animals treated for up to 14 days with DFP. In vitro treatment with DFP in control tissue causes a spontaneous development of tension which can be clocked by atropine and pirenzepine and marginally by mecamylamine, but is unaffected by tetrodotoxin (TTX).

Dogs with varying hematocrit ratios were subjected to cardiovascular and respiratory insults designed to determine what cell to plasma ratio was most effective in carrying oxygen to the tissues of the body. It was found that dogs with initial hematocrit ratios of from 35 to 39 were able to carry more oxygen to their tissues after a massive hemorrhage than those dogs with hematocrit ratios outside this range. Furthermore, since the hematocrit ratio tends to increase during hemorrhage, the optimal value for the hematocrit ratio is 40. Normal dogs with this optimal value were subjected to high altitude studies and it was found that these dogs could survive and be active at altitudes lethal to animals with hematocrit ratios outside the optimal range. The optimal range is indicated by the dual nature of the hematocrit ratio as it affects both oxygen carrying capacity and blood viscosity. With low cell content, the viscosity is low but the oxygen carrying capacity is also low. As the cell content of the blood increases, so does both oxygen carrying capacity and viscosity. The inflection of the curve has been at 40 for all types of experiments of this nature. Acclimatized dogs show an optimum at 46, indicating that high altitude polycythemia is detrimental. A mathematical analysis, and a discussion of these facts are included.

Dogs with Dirofilaria immitis microfilaremia were obtained from three geographically distinct areas to determine whether divergent strains of the parasite might develop differently in the mosquito Aedes triseriatus Say. Because no significant differences were noted, results of these experiments were combined to give an overall view of development of D. immitis in Aedes triseriatus. An average of twenty microfilariae was found in the midgut and malpighian tubules of the mosquitoes 24 hours following a blood meal on an infected dog. Over a 15 day period the average number of first and second stage larvae found in the tubules was 6.5. Third stage (infective) larvae were found in the tubules commencing on the 11th day following ingestion of the blood meal. The average number was 2.7/mosquito. These larvae varied in length from 776-865 microns. Mortality of A. triseriatus utilized during these experiments was not a critical factor. (Author)