Sherry F. Queener

Vitamin-D-Dependent Rickets Type II: Resistance of Target Organs to 1,25-Dihydroxyvitamin D

Studies were done to determine the cause for hypocalcemia, secondary hyperparathyroidism, osteomalacia and osteitis fibrosa cystica in a 22-year-old black woman. The patient had normal serum 25-hydroxyvitamin D (14 ng per milliliter) and markedly elevated serum 1,25-dihydroxyvitamin D (137 pg per milliliter). Vitamin D3, 4000 units per day for four weeks, increased the serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D to as high as 29 and 297 pg per milliliter, respectively, and corrected the hypocalcemia and secondary hyperparathyroidism. The results suggest that the disorder results from impaired end-organ response to 1,25-dihydroxyvitamin D. We propose that the entity be called vitamin-D-dependent rickets Type II.

Control of gene expression in carbohydrate, pyrimidine and DNA metabolism.

Summary The patterns of behavior of key enzymes in carbohydrate and nucleic acid metabolism were investigated under conditions involving modulation of gene expression. 1. The changes in hepatic cellularity and DNA content were examined in the liver during post-natal differentiation. The cellularity was 650 million cells per g wet weight in the liver of the newborn rat and it decreased during differentiation to 220 million in the adult. The total liver cellularity was 168 million in newborn rats and it increased 14-fold to reach a cell population of over 2 billion in the adult. The DNA content per g liver wet weight and per total liver underwent a similar order of magnitude of change. Thus, the DNA content per cell remained unaltered during differentiation. It was suggested that the enzymatic and metabolic changes in differentiation entail sequential alterations in the read-out of nuclear genes and in their selective transcription and translation. These processes then determine the phenotypic pattern of metabolism in liver cells in different age groups. 2. During post-natal differentiation the key enzymes of liver carbohydrate metabolism follow a characteristic pattern of behavior that is in good agreement with predictions that can be deduced from the Functional Genic Unit Concept. After birth, the activities of the key gluconeogenic enzymes are high whereas the key glycolytic ones are low. During subsequent development for a period of about 30 to 40 days the key gluconeogenic enzymes decrease and the key glycolytic ones increase to achieve the activity levels observed in the adult rat. 3. The opposing enzymes of gluconeogenesis and glycolysis reach a steady state balance in the adult; however, as it has been shown it is possible to markedly modulate gene expression by altering the endocrine status of the organism. When rats were made alloxan diabetic, the liver glucose-6-phosphatase activity increased and the pyruvate kinase activity decreased. Administration of insulin caused the activity of the gluconeogenic enzyme to decrease and that of the glycolytic one to increase to the normal levels of the liver of the adult rat. Thus, insulin coordinates hepatic metabolism, in part, through causing an antagonistic behavior for the opposing gluconeogenic and glycolytic enzymes. 4. In neoplasia a characteristic behavior pattern is also seen which agrees with the predictions of the Functional Genic Unit Concept. In the spectrum of hepatomas of different growth rates, in parallel with the rise in tumor proliferation rate, the key glycolytic enzymes increased, whereas the key gluconeogenic ones decreased. The ratios of the opposing key enzymes, e.g., hexokinase/glucose-6-phosphatase, correlate closely with hepatoma growth rate. Thus, the expression of genes for the key enzymes of carbohydrate metabolism is linked with the expression of the potential for cell proliferation rate in the different lines of hepatomas. 5. The examination of the pattern of gene expression and its regulation revealed that the behavior of the key gluconeogenic and glycolytic enzymes exhibits an antagonistic pattern during differentiation, under hormonal regulatory conditions and in neoplasia. The behavior of the activities of the overall pathways of gluconeogenesis and glycolysis is in close accord with that of the key enzymes of these pathways under the same conditions of gene regulation. 6. In normal liver in UMP metabolism the pathway of catabolism is more active than the synthetic pathway that produces UMP. In the spectrum of hepatomas it was observed that the key enzymes involved in the synthesis of UMP increased whereas dihydrouracil dehydrogenase and the overall degradative pathway decreased in parallel with the increase in hepatoma growth rate. Thus, gene expression in pyrimidine metabolism is also coordinated in terms of the antagonistic behavior of opposing pathways and the enzymes involved. 7. The activities of the enzymes of pyrimidine and DNA metabolism were recalculated from the literature and expressed in a uniform fashion for comparison in a Table. The activities of enzymes in the synthetic processes were very low, especially those that play a role at strategic parts of the pathway, e.g., DNA polymerase, ribonucleotide reductase, dTMP synthase and dTMP kinase. In contrast, the activities of enzymes that function in the degradation of uridine and thymidine were several magnitudes higher than those of the synthetic enzymes. 8. In hepatic thymidine metabolism the behavior of the activity of the synthetic utilization (TdR to DNA) contrasts with that of the degradative pathway (TdR to CO 2 ). In the newborn rat the degradative pathway is active and during development the activity increases 2-fold to the high levels of the adult. The activity of TdR to DNA is very high in the liver of the newborn rat and during differentiation it decreases to less than 10% of this value in the adult. In the regenerating liver the synthetic pathway is markedly increased and the catabolic pathway is decreased. In differentiation and in regeneration the antagonistic behavior of the two opposing pathways of thymidine is closely reflected in that of the activities of the enzymes involved. These results suggest the operation of a coordinated behavior pattern of gene expression manifesting opposing changes in the activities of antagonistic pathways and enzymes in thymidine metabolism. 9. In the hepatomas the TdR to DNA pathway increased and concurrently the TdR to CO 2 pathway decreased in parallel with tumor growth rate. Consequently, there was a 140,000-fold increase in the ratio of the two pathways in the rapidly growing hepatomas. In the neoplastic cells the behavior of the opposing pathways of thymidine utilization and the enzymes involved exhibits a mirror picture. 10. The pattern of the activities of the opposing pathways in carbohydrate, pyrimidine and DNA metabolism suggests a close linkage in the expression of the replicative potential of the genome with the extent of the progressive imbalance in these metabolic pathways. These observations provide further support for the validity of the Molecular Correlation Concept in the hepatoma system. The applicability of this Concept has also been shown for kidney tumors, mammary neoplasms and other tumors of different growth rates indicating that the approaches of the Molecular Correlation Concept are valid not only for the hepatomas, but also for various other types of neoplastic cells.

Activity of clindamycin with primaquine against Pneumocystis carinii in vitro and in vivo.

The combination of primaquine with clindamycin is effective in both in vitro and in vivo models of Pneumocystis infection. Primaquine alone at concentrations from 10 to 300 micrograms/ml reduced the numbers of organisms in cultures to less than 7% of control. Significant inhibition was observed down to 0.1 microgram/ml. Clindamycin at 5 micrograms/ml was ineffective alone. Combinations of clindamycin and primaquine in culture at various concentrations were effective, but there was no evidence of true synergy. In rats with established Pneumocystis pneumonia, clindamycin alone at 5 or 225 mg/kg was ineffective. Primaquine alone at 0.5 or 2 mg/kg did not significantly affect the numbers of organisms remaining. The combination of 0.5 mg of primaquine per kg and 225 mg of clindamycin per kg was effective for therapy, lowering the numbers of organisms in the lungs by about 90%. The combination of 2 mg of primaquine per kg and 225 mg of clindamycin per kg was more effective, lowering the numbers of organisms by almost 98%. In the in vivo prophylaxis model, primaquine at 0.1 or 0.2 mg/kg did not prevent the development of Pneumocystis pneumonia in immune-suppressed rats. Clindamycin at 50 mg/kg had a modest effect alone, but at 5 mg/kg all animals became heavily infected. At 0.5 mg/kg, primaquine alone reduced the severity of infection, but seven of eight rats were still infected. In contrast, the combination of 5 mg of clindamycin per kg and 0.5 mg of primaquine per kg prevented infection in 8 of 10 rats; 2 rats had minimal infection. These studies suggest that the combination of clindamycin and primaquine should be tested in therapy or prophylaxis of Pneumocystis infections in humans. Images

Phosphorylation of eukaryotic initiation factor-2α promotes the extracellular survival of obligate intracellular parasite Toxoplasma gondii

While seeking a new host cell, obligate intracellular parasites, such as the protozoan Toxoplasma gondii, must be able to endure the stress of an extracellular environment. The mechanisms Toxoplasma use to remain viable while deprived of a host cell are not understood. We have previously shown that phosphorylation of Toxoplasma eukaryotic initiation factor-2α (TgIF2α) is a conserved response to stress. Here we report the characterization of Toxoplasma harboring a point mutation (S71A) in TgIF2α that prevents phosphorylation. Results show that TgIF2α phosphorylation is critical for parasite viability because the TgIF2α-S71A mutants are ill-equipped to cope with life outside the host cell. The TgIF2α-S71A mutants also showed a significant delay in producing acute toxoplasmosis in vivo. We conclude that the phosphorylation of TgIF2α plays a crucial role during the lytic cycle by ameliorating the stress of the extracellular environment while the parasite searches for a new host cell.

Novel triazine JPC-2067-B inhibits Toxoplasma gondii in vitro and in vivo

Background and Methodology Toxoplasma gondii causes substantial morbidity, mortality, and costs for healthcare in the developed and developing world. Current medicines are not well tolerated and cause hypersensitivity reactions. The dihydrotriazine JPC-2067-B (4, 6-diamino-1, 2-dihydro-2, 2-dimethyl-1-(3′(2-chloro-, 4-trifluoromethoxyphenoxy)propyloxy)-1, 3, 5-triazine), which inhibits dihydrofolate reductase (DHFR), is highly effective against Plasmodium falciparum, Plasmodium vivax, and apicomplexans related to T. gondii. JPC-2067-B is the primary metabolite of the orally active biguanide JPC-2056 1-(3′-(2-chloro-4-trifluoromethoxyphenyloxy)propyl oxy)- 5-isopropylbiguanide, which is being advanced to clinical trials for malaria. Efficacy of the prodrug JPC-2056 and the active metabolite JPC-2067-B against T. gondii and T. gondii DHFR as well as toxicity toward mammalian cells were tested. Principal Findings and Conclusions Herein, we found that JPC-2067-B is highly effective against T. gondii. We demonstrate that JPC-2067-B inhibits T. gondii growth in culture (IC50 20 nM), inhibits the purified enzyme (IC50 6.5 nM), is more efficacious than pyrimethamine, and is cidal in vitro. JPC-2067-B administered parenterally and the orally administered pro-drug (JPC-2056) are also effective against T. gondii tachyzoites in vivo. A molecular model of T. gondii DHFR-TS complexed with JPC-2067-B was developed. We found that the three main parasite clonal types and isolates from South and Central America, the United States, Canada, China, and Sri Lanka have the same amino acid sequences preserving key binding sites for the triazine. Significance JPC-2056/JPC-2067-B have potential to be more effective and possibly less toxic treatments for toxoplasmosis than currently available medicines.

Activity of lipid-soluble inhibitors of dihydrofolate reductase against Pneumocystis carinii in culture and in a rat model of infection.

Trimetrexate and BW301U (piritrexim isethionate), lipid-soluble inhibitors of dihydrofolate reductase, are potent inhibitors of the growth of Pneumocystis carinii in culture with WI-38 cells. Inhibition was observed with 0.1 microgram of trimetrexate or BW301U per ml. Trimethoprim is ineffective at 100 micrograms/ml in this culture system. Both trimetrexate and BW301U were effective as prophylactic agents against P. carinii pneumonia in rats; trimetrexate at 7.5 mg/kg protected 9 of 10 rats, and BW301U at 5 mg/kg protected 4 of 10.

Changes in renal phospholipid fatty acids in diabetes mellitus: Correlation with changes in adenylate cyclase activity

Male Sprague-Dawley rats made diabetic with alloxan (37.5 mg/kg) or streptozotocin (65 mg/kg) were killed after 3–6 weeks of disease; renal tissues were studied for phospholipid content and for fatty acid composition of the phospholipids. No consistent change was noted in total phospholipid content nor in the proportion of various phospholipids in diabetics. However, diabetic animals showed a consistent reduction of arachidonic acid content in phosphatidylcholine (PC) and phosphatidylethanolamine in whole renal cortex, plasma membranes purified from renal cortex, and in isolated glomeruli. Associated with the fall in arachidonic acid was a rise in linoleic acid in the samples studied. Insulin therapy returned the fatty acid profiles to normal. These results are similar to patterns observed in other diabetic tissues and suggest that diabetes is associated with generalized changes in cell membranes. That these structural changes may have functional significance is suggested by demonstrated alterations in the temperature-dependence of adenylate cyclase in renal plasma membranes of diabetic animals. Adenylate cyclase is thought to be intimately associated with PC in plasma membranes, a phospholipid showing significant changes in fatty acid content in diabetes (unsaturation index 165±2 for normals, 147±5 for diabetics). Na+,K au+-ATPase which is thought to be primarily associated in vivo with phosphatidylinositol (PI), shows no change in apparent energy of activation in diabetes. The fatty acid content of PI is minimally altered in diabetes, and the unsaturation index is unchanged.

Synthesis and DHFR inhibitory activity of a series of 6-substituted-2,4-diaminothieno[2,3-d]pyrimidines

A series of 6-aralkyl substituted 2,4-diaminothieno[2,3-d]pyrimidines in which the 6-aryl group is separated from the thieno[2,3-d]pyrimidine ring by two to five methylene groups were synthesized and studied as inhibitors of dihydrofolate reductase from Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium, and rat liver. Compounds in which the thieno[2,3-d]pyrimidine ring is separated from the 6-aryl substituent by three methylene groups were the most potent inhibitors of the series (with IC(50) values ranging from 0.24 and 11.0 microM) but those with two methylene groups between the aromatic rings were the most selective agents.

Pneumocystis carinii is resistant to imidazole antifungal agents.

Because Pneumocystis carinii is closely related to fungi, drugs useful for treating mycoses have been considered for use in the treatment of P. carinii pneumonia. Six antifungal imidazole drugs were tested for their activities against P. carinii in a short-term culture screen and in animals. None of the imidazoles tested was effective in inoculated infected rats, and only miconazole showed slight effects in culture at the high concentration of 10 micrograms/ml. Analysis of cell membranes from culture-grown P. carinii showed that ergosterol, the target for this class of antifungal agents, was absent, so that the lack of effect of these agents is rational.

Synthesis and anti-Pneumocystis carinii activity of conformationally restricted analogues of pentamidine

Abstract Aseries of conformationally restricted analogues of pentamidine in which the flexible central bridge has been replaced by trans-cyclopropyl, phenyl, pyridinyl, piperazinyl or homopiperazinyl groups as conformationally restricted linkers have been synthesized. The anti-Pneumocystis carinii activity of these compounds was evaluated in a cell culture model and the DNA binding affinity was determined by thermal denaturation measurements. At 1 μM, compounds 2, 3, 5, 7, 9 and pentamidine were highly effective and caused total inhibition of P. carinii growth in culture. At 0.1 μM, compounds 2, 5, 7 and 10 were more active than pentamidine with N, N′-bis(4-amidinophenyl)piperazine 7 being approximately 15-fold more effective than pentamidine. The most active compounds, 7 and 10, showed strong binding affinities for calf thymus DNA and poly(dA-dT); however, a clear correlation between DNA binding affinity and the in vitro anti-P. carinii activity of these compounds was not observed. The results suggest that the nature of the central linker influences the biological actions of these compounds.