Simultaneous stability of D = 0 and D ≠ 0 for multiplicative viabilities at two loci: An analytical study

The phenomenon of simultaneous stability of D = 0 and D ≠ 0 in a discrete generation, deterministic, two locus, two allele model with multiplicative viabilities and overdominance is studied using analytical techniques based on bifurcation theory. It is shown that in any model without symmetry, this phenomenon will occur. The importance of the phenomenon increases as the asymmetry increases, and as the range of recombination values over which D = 0 is stable increases. Evolutionary implications of the results are discussed. Other applications of the analytical techniques used are indicated.     

Differential voltage-sensitivity of D2-like dopamine receptors

Agonist potency at some neurotransmitter receptors has been shown to be regulated by transmembrane voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by autoreceptors and in synaptic plasticity. We have recently described the voltage-sensitivity of the dopamine D_2L receptor and we now extend our studies to include the other members of the D₂-like receptor subfamily; the D_2S, D₃, and D₄ dopamine receptors. Electrophysiological recordings were performed on Xenopus oocytes coexpressing human dopamine D_2S, D₃, or D₄ receptors with G protein-coupled potassium (GIRK) channels. Comparison of concentration-response relationships at −80 mV and at 0 mV for dopamine-mediated GIRK activation revealed significant rightward shifts for both D_2S and D₄ upon depolarization. In contrast, the concentration-response relationships for D₃-mediated GIRK activation were not appreciably different at the two voltages. Our findings provide new insight into the functional differences of these closely related receptors.     

Metabolism and binding properties of 24,24-difluoro-25-hydroxyvitamin D3

To evaluate possible functional roles for 24,25-dihydroxyvitamin D₃, 24,24-difluoro-25-hydroxyvitamin D₃ has been synthesized and shown to be equally as active as 25-hydroxyvitamin D₃ in all known functions of vitamin D. The use of the difluoro compound for this purpose is based on the assumption that the C-F bonds are stable in vivo and that the fluorine atom does not act as hydroxyl in biological systems. No 24,25-dihydroxyvitamin D₃ was detected in the serum obtained from vitamin D-deficient rats that had been given 24,24-difluoro-25-hydroxyvitamin D₃, while large amounts were found when 25-hydroxyvitamin D₃ was given. Incubation of the 24,24-difluoro compound with kidney homogenate prepared from vitamin D-replete chickens failed to produce 24,25-dihydroxyvitamin D₃, while the same preparations produced large amounts of 24,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. Kidney homogenate prepared from vitamin D-deficient chickens produced 24,24-difluoro-1,25-dihydroxyvitamin D₃ from 24,24-difluoro-25-hydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃. In binding to the plasma transport protein for vitamin D compounds, 24,24-difluoro-25-hydroxyvitamin D₃ is less active than 25-hydroxyvitamin D₃ and 24R,25-dihydroxyvitamin D₃. In binding to the chick intestinal cytosol receptor, 24,24-difluoro-25-hydroxyvitamin D₃ is more active than 25-hydroxyvitamin D₃ which is itself more active than 24R,25-dihydroxyvitamin D₃. The 24,24-difluoro-1,25-dihydroxyvitamin D₃ is equal to 1,25-dihydroxyvitamin D₃, and both are 10 times more active than 1,24R,25-trihydroxyvitamin D₃ in this system. These results provide strong evidence that the C-24 carbon of 24,24-difluoro-25-hydroxyvitamin D₃ cannot be hydroxylated in vivo, and, further, the 24-F substitution acts similar to H and not to OH in discriminating binding systems for vitamin D compounds.     

Some notes on the distribution and certain modifications of Mahalanobis' generalized distance (D2)

Different modifications of Mahalanobis' generalized distance (D²) utilized in anthropological research are given, and it is shown that they all derive from the distribution of D² when the distances between the populations investigated are real. To investigate whether observed distances are real, testing of D² for significance is stressed. It is demonstrated that D² ought to be tested for significance according to its relation to the F-distribution, but that it may be related to the chi-squared distribution as an approximation when the numbers of individuals in the samples are very large. The great disadvantages of utilizing this approximation for smaller numbers of individuals in the samples are demonstrated, as well as the dependence of D² on sample size and the number of measurements used when testing for significance.     

Packaging of coliphage lambda DNA. II. The role of the gene D protein

The gene D protein (pD) of coliphage λ is normally an essential component of the virus capsid. It acts during packaging of concatemeric λ DNA into the phage prohead and is necessary for cutting the concatemers at the cohesive end site (cos). In this report we show that cos cutting and phage production occur without pD in λ deletion mutants whose DNA content is less than 82% that of λ wild type. D-independence appears to result directly from DNA loss rather than from inactivation (or activation) of a phage gene. (1) In cells mixedly infected with undeleted λ and a deletion mutant, particles of the deletion mutant alone are efficiently produced in the absence of pD; and (2) D-independence cannot be attributed to loss of a specific segment of the phage genome. pD-deficient phage resemble pD-containing phage in head size and DNA ends; they differ in their extreme sensitivity to EDTA, greater density, and ability to accept pD.pD appears to act by stabilizing the head against disruption by overfilling with DNA rather than by changing the capacity of the head for DNA. This is shown by the observation that the amount of DNA packaged by a “headful” mechanism, normally in excess of the wild-type chromosome size, is not reduced in the absence of pD. In fact, pD is required for packaging headfuls of DNA. This implies that a mechanism exists for preventing the entry of excess DNA into the head during packaging of concatemers formed by deletion mutants, and we suggest that this is accomplished by binding of cos sites to the head.The above results show that pD is not an essential component of the nuclease that cuts λ concatemers at cos during packaging, and they imply that 82% of a wild-type chromosome length can enter the prohead in the absence of pD. Yet, pD is needed for the formation of cohesive ends after infection with undeleted phage. We propose two models to account for these observations. In the first, cos cutting is assumed to occur early during packaging. The absence of pD leads to release of packaged DNA and the loss of cohesive ends by end-joining. In the second, cos cutting is assumed to occur as a terminal event in packaging. pD promotes cos cutting indirectly through its effect on head stability. We favor the second model because it better explains the asymmetry observed in the packaging of the chromosomes of cos duplication mutants (Emmons, 1974).     

Functional expression of the murine D2, D3 and D4 dopamine receptors in Xenopus laevis oocytes

The different murine D2-type dopamine receptors (D_2L, D_2S, D_3L, D_3S, and D₄) were expressed in Xenopus laevis oocytes. The D2-type receptors were all similarly and efficiently expressed in Xenopus oocytes and were shown to bind the D2 antagonist [¹²⁵I]sulpride. They were all shown to activate Cl⁻ influx upon agonist stimulation. Using the diagnostic inhibitor bumetanide, we were able to separate the Na⁺/K⁺/2Cl⁻ cotransporter component of the Cl⁻ influx from the total unidirectional Cl⁻ influx. The D_3L subtype was found to operate exclusively through the bumetanide-insensitive Cl⁻ influx whereas the other D2-type receptors acted on the Na⁺/K⁺/2Cl⁻ cotransporter as well. The pertussis toxin sensitivity of the receptor-activated chloride influx via the Na⁺/K⁺/2Cl⁻ cotransporter varied between the various D2-type receptors showing that they may couple to different G proteins, and activate different second messenger systems.     

25,26-dihydroxyvitamin D3 is not a major intermediate in 25-hydroxyvitamin D3-26,23-lactone formation

Structural similarities between 25S,26-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃-26,23-lactone and their concomitant multifold increase in the plasma of animals treated with pharmacological doses of vitamin D₃ suggest a precursor-product relationship. However, a single dose of 25S,26-[³H]dihydroxyvitamin D₃ given to rats treated chronically with pharmacological amounts of vitamin D₃ did not result in detectable plasma 25-[³H]hydroxyvitamin D₃-26,23-lactone. Multiple doses of synthetic 25S,26-dihydroxyvitamin D₃ given to vitamin D₃-deficient rats treated chronically with pharmacological amounts of vitamin D₂ also did not result in detectable plasma 25-hydroxyvitamin D₃-26,23-lactone. Furthermore, homogenates prepared from vitamin d-deficient chickens, dosed with 1,25-dihydroxyvitamin D₃, converted 25-[³H]hydroxyvitamin D₃ to 25-[³H]hydroxyvitamin D₃-26,23-lactone. But these same homogenates did not convert 25S,26-[³H]dihydroxyvitamin D₃ to 25-[³H]hydroxyvitamin D₃-26,23-lactone. These data indicate that 25,26-dihydroxyvitamin D₃ is not an intermediate in 25-hydroxyvitamin D₃26, 23-lactone formation.     

25-Hydroxyvitamin D3 is an agonistic vitamin D receptor ligand

25-Hydroxyvitamin D₃ 1α-hydroxylase encoded by CYP27B1 converts 25-hydroxyvitamin D₃ into 1α,25-dihydroxyvitamin D₃, a vitamin D receptor ligand. 25-Hydroxyvitamin D₃ has been regarded as a prohormone. Using Cyp27b1 knockout cells and a 1α-hydroxylase-specific inhibitor we provide in four cellular systems, primary mouse kidney, skin, prostate cells and human MCF-7 breast cancer cells, evidence that 25-hydroxyvitamin D₃ has direct gene regulatory properties. The high expression of megalin, involved in 25-hydroxyvitamin D₃ internalisation, in Cyp27b1^?/? cells explains their higher sensitivity to 25-hydroxyvitamin D₃. 25-Hydroxyvitamin D₃ action depends on the vitamin D receptor signalling supported by the unresponsiveness of the vitamin D receptor knockout cells. Molecular dynamics simulations show the identical binding mode for both 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ with the larger volume of the ligand-binding pocket for 25-hydroxyvitamin D₃. Furthermore, we demonstrate direct anti-proliferative effects of 25-hydroxyvitamin D₃ in human LNCaP prostate cancer cells. The synergistic effect of 25-hydroxyvitamin D₃ with 1α,25-dihydroxyvitamin D₃ in Cyp27b1^?/? cells further demonstrates the agonistic action of 25-hydroxyvitamin D₃ and suggests that a synergism between 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ might be physiologically important. In conclusion, 25-hydroxyvitamin D₃ is an agonistic vitamin D receptor ligand with gene regulatory and anti-proliferative properties.     

Elevated expression of the V-ATPase D2 subunit triggers increased energy metabolite levels in KrasG12D-driven cancer cells

Mutations of the Ras oncogene are frequently detected in human cancers. Among Ras-mediated tumorigenesis, Kras-driven cancers are the most dominant mutation types. Here, we investigated molecular markers related to the Kras mutation, which is involved in energy metabolism in Kras mutant-driven cancer. We first generated a knock-in Kras^G12D cell line as a model. The genotype and phenotype of the Kras ^G12D-driven cells were first confirmed. Dramatically elevated metabolite characterization was observed in Kras ^G12D-driven cells compared with wild-type cells. Analysis of mitochondrial metabolite-related genes showed that two of the 84 genes in Kras ^G12D-driven cells differed from control cells by at least twofold. The messenger RNA and protein levels of ATP6V0D2 were significantly upregulated in Kras ^G12D-driven cells. Knockdown of ATP6V0D2 expression inhibited motility and invasion but did not affect the proliferation of Kras ^G12D-driven cells. We further investigated ATP6V0D2 expression in tumor tissue microarrays. ATP6V0D2 overexpression was observed in most carcinoma tissues, such as melanoma, pancreas, and kidney. Thus, we suggest that ATP6V0D2, as one of the V-ATPase (vacuolar-type H ⁺-ATPase) subunit isoforms, may be a potential therapeutic target for Kras mutation cancer.     

Evidence for the metabolism of 24R-hydroxy-25-fluorovitamin D3 and 1alpha-hydroxy-25-fluorovitamin D3 to 1alpha,24R-dihydroxy-25-fluorovitamin D3.

High-pressure liquid chromatography capable of resolving all known vitamin D metabolites and a sensitive competitive binding protein assay specific for 1α,25-dihydroxyvitamin D₃ were used to assay the blood of rats dosed with ethanol, 1α-hydroxyvitamin D₃, 24R-hydroxy-25-fluorovitamin D₃, or 1α-hydroxy-25-fluorovitamin D₃. Compared to the ethanoldosed animals, the blood of rats dosed with 1α-hydroxyvitamin D₃ had increased levels of 1α,25-dihydroxyvitamin D₃; but those dosed with the fluorinated vitamins did not. Instead, their blood contained a compound that cochromatographs with 1α,24R-dihydroxyvitamin D₃ on high-pressure liquid chromatography and binds to the 1,25-dihydroxyvitamin D₃ receptor proteins. 1α,24R-Dihydroxyvitamin D₃ binds as well as 1α, 25-dihydroxyvitamin D₃ to the chick-intestinal cytosol receptor protein for 1α,25-dihydroxyvitamin D₃; whereas 1α,24S-dihydroxyvitamin D₃ binds only one-tenth as well as 1α,25-dihydroxyvitamin D₃. Thus it appears that in vivo, the fluorinated vitamin D compounds are converted to a compound likely to be 1α,24R-dihydroxy-25-fluorovitamin D₃ and that may rival the potency of 1α,25-dihydroxyvitamin D₃.     

Arabidopsis snc2-1D Activates Receptor-Like Protein-Mediated Immunity Transduced through WRKY70

Plant immune receptors belonging to the receptor-like protein (RLP) family contain extracellular leucine-rich repeats (LRRs) and a short cytoplasmic tail linked by a single transmembrane motif. Here, we report the identification of snc2-1D (for suppressor of npr1-1, constitutive 2), a semidominant Arabidopsis thaliana mutant with constitutively activated defense responses. Map-based cloning of snc2-1D showed that it encodes an RLP. The point mutation in snc2-1D leads to substitution of the second Gly for Arg in the conserved GXXXG motif of the transmembrane helix, suggesting that this residue is important for negative regulation of the protein. Epistasis analysis revealed that the snc2-1D mutant phenotype is not affected by mutations in genes known to be required for the nucleotide binding (NB)-LRR Resistance (R) protein signaling. A suppressor screen of snc2-1D was performed, and map-based cloning of one suppressor revealed that mutations in WRKY70 suppress the constitutive defense responses in snc2-1D, suggesting that WRKY70 functions downstream of snc2-1D. The identification of snc2-1D provides us with a unique system for genetic analysis of resistance pathways downstream of RLPs, which may be distinct from those downstream of NB-LRR type R proteins.     

Voltage-sensitivity at the human dopamine D2S receptor is agonist-specific

Recently, we and others have shown that agonist potencies at some, but not all, G protein-coupled receptors are voltage-sensitive. Several of those studies employed electrophysiology assays in Xenopus oocytes with G protein-coupled potassium channels as a readout. Using this assay, we have now obtained evidence that voltage-sensitivity at the dopamine D_2S receptor is agonist-specific. Whereas the potency of dopamine at the D_2S receptor is decreased by depolarization, the potencies of β-phenethylamine, p- and m-tyramine are voltage-insensitive. Furthermore, both monohydroxylated and non-hydroxylated N,N-dipropyl-2-aminotetralin compounds are voltage-sensitive. Differential activation of G protein subtypes or differential ratios between effector and active G protein do not underlie this agonist-selective voltage-sensitivity. This is the first demonstration of voltage-sensitive and voltage-insensitive behaviour of different agonists acting via the same receptor.     

Vitamin D in lactation. I. The localization, specific binding and biological effect of 1,25-dihydroxyvitamin D3 in mammary tissue of lactating rats

Radiolabelled 1, 25-dihydroxyvitamin D₃ was found to accumulate in mammary tissue of lactating rats, to bind to a specific high affinity binding component in mammary cytosol and to associate with chromatin in vivo. 1,25-dihydroxyvitamin D₃ was also shown to have a direct effect on milk calcium concentration in vivo. The cytosolic binding protein was found to sediment at 3.2S on sucrose gradients and to have a dissociation constant of 2.5 × 10^?10 M. Localization of 1, 25-dihydroxyvitamin D₃ in mammary gland and other tissues of lactating rats was compared. These results provide evidence that the lactating mammary gland is a target tissue for 1,25-dihydroxyvitamin D₃.     

Molecular origin of the pH dependence of tyrosine D oxidation kinetics and radical stability in photosystem II

A role for redox-active tyrosines has been demonstrated in many important biological processes, including water oxidation carried out by photosystem II (PSII) of oxygenic photosynthesis. The rates of tyrosine oxidation and reduction and the Tyr/Tyr reduction potential are undoubtedly controlled by the immediate environment of the tyrosine, with the coupling of electron and proton transfer, a critical component of the kinetic and redox behavior. It has been demonstrated by Faller et al. that the rate of oxidation of tyrosine D (Tyr_D) at room temperature and the extent of Tyr_D oxidation at cryogenic temperatures, following flash excitation, dramatically increase as a function of pH with a pK_a of ≈ 7.6 [Faller et al. 2001 Proc. Natl. Acad. Sci. USA 98, 14368-14373; Faller et al. 2001 Biochemistry 41, 12914-12920]. In this work, we investigated, using FTIR difference spectroscopy, the mechanistic reasons behind this large pH dependence. These studies were carried out on Mn-depleted PSII core complexes isolated from Synechocystis sp. PCC 6803, WT unlabeled and labeled with ¹³C₆-, or ¹³C₁(4)-labeled tyrosine, as well as on the D2-Gln164Glu mutant. The main conclusions of this work are that the pH-induced changes involve the reduced Tyr_D state and not the oxidized Tyr_D state and that Tyr_D does not exist in the tyrosinate form between pH 6 and 10. We can also exclude a change in the protonation state of D2-His189 as being responsible for the large pH dependence of Tyr_D oxidation. Indeed, our data are consistent with D2-His189 being neutral both in the Tyr_D and Tyr_D states in the whole pH6-10 range. We show that the interactions between reduced Tyr_D and D2-His189 are modulated by the pH. At pH greater than 7.5, the ν(CO) mode frequency of Tyr_D indicates that Tyr_D is involved in a strong hydrogen bond, as a hydrogen bond donor only, in a fraction of the PSII centers. At pH below 7.5, the hydrogen-bonding interaction formed by Tyr_D is weaker and Tyr_D could be also involved as a hydrogen bond acceptor, according to calculations performed by Takahashi and Noguchi [J. Phys. Chem. B 2007 111, 13833-13844]. The involvement of Tyr_D in this strong hydrogen-bonding interaction correlates with the ability to oxidize Tyr_D at cryogenic temperatures and rapidly at room temperature. A strong hydrogen-bonding interaction is also observed at pH 6 in the D2-Gln164Glu mutant, showing that the residue at position D2-164 regulates the properties of Tyr_D. The IR data point to the role of a protonatable group(s) (with a pK_a of ≈ 7) other than D2-His189 and Tyr_D, in modifying the characteristics of the Tyr_D hydrogen-bonding interactions, and hence its oxidation properties. It remains to be determined whether the strong hydrogen-bonding interaction involves D2-His189 and if Tyr_D oxidation involves the same proton transfer route at low and at high pH.     

Specific binding protein for 1,25-dihydroxyvitamin D3 in bovine mammary gland

Specific binding proteins for 1,25-dihydroxyvitamin D₃ were identified in bovine mammary tissue obtained from lactating and non-lactating mammary glands by sucrose density gradient centrifugation. The macromolecules had characteristic sedimentation coefficients of 3.5-3.7 S. The interaction of l,25-dihydroxy[³H]vitamin D₃ with the macromolecule of the mammary gland cytosol occurred at low concentrations, was saturable, and was a high affinity interaction (K_d = 4.2 × 10^?10M at 25 °C). Binding was reversed by excess unlabeled 1,25-dihydroxyvitamin D₃, was destroyed by heat and/or incubation with trypsin. It is thus inferred that this macromolecule is protein as it is not destroyed by ribonuclease or deoxyribonuclease. 25-hydroxyvitamin D₃, 24,25-dihydroxyvitamin D₃, and vitamin D₃ did not effectively compete with 1,25-dihydroxyvitamin D₃ for binding to cytosol of mammary tissue at near physiological concentrations of these analogs, thus demonstrating the specificity of the binding protein for 1,25-dihydroxyvitamin D₃. In vitro subcellular distribution of 1,25-dihydroxy[³H]vitamin D₃ demonstrated a time- and temperature-dependent movement of the hormone from the cytoplasm to the nucleus. By 90 min at 25 °C 72% of the 1,25-dihydroxy[³H]vitamin D₃ was associated with the nucleus. In addition a 5–6 S macromolecule which binds 25-hydroxy[³H]vitamin D₃ was demonstrated in mammary tissue. Finally, it is possible that the receptor-hormone complex present in mammary tissue may function in a manner analogous to intestinal tissue, resulting in the control of calcium transport by 1,25-dihydroxyvitamin D₃ in this tissue.     

Analysis of the primary structure of collagen for the origins of molecular packing

The amino acid sequence in the triplet region of the α1 chain of collagen was analyzed for complementary relationships that would explain the stagger of multiples of 670 Å between the rod-like molecules in the fibril. The analysis was done by moving the sequence of 1011 amino acids past itself and scoring for complementarity between opposing amino acids allowing a range of ±2 to 3 residues. It was found that interactions between amino acids of opposite charge and between large hydrophobic amino acids in the overlapping region between two chains are maximal when the chains are staggered by 0D, 1D, 2D, 3D and 4D, where D = 234 ± 1 residues. The residue repeat derived from this value is 2.86 ± 0.02 Å. The existence of a D separation between interacting residues was shown to be reflected in the actual distribution of large hydrophobic amino acids. Surprisingly, the distribution approximates the pattern ($\text{2D}\text{11}$)₅($\text{D}\text{11}$) repeated over 4.4D intervals. The regularity may arise from structural constraints imposed by super-coiling. The distribution of charged residues is less regular and does not show a well-defined periodicity. However, positively-charged residues tend to be near negatively-charged residues, allowing intramolecular charge neutralization as well as strong intermolecular charge interactions at 0D.     

Metabolism of vitamin D3 in the chick embryo

In agreement with previous reports, chick intestinal calcium-binding protein does not appear in the chick embryo until 1 day after hatching while intestinal alkaline phosphatase begins to appear at 19–20 days of embryonic life. The ability of chick embryo to metabolize vitamin D₃ to 25-hydroxyvitamin D₃, 1,25-dihydroxyvitamin D₃, and 24,25-dihydroxyvitamin D₃ is present at least by day 18 of embryonic life as demonstrated by in vivo and in vitro techniques. It also illustrates that metabolism of vitamin D₃ was not the limiting factor in the appearance of calcium-binding protein and alkaline phosphatase in intestine. Instead, the uptake of 1,25-dihydroxyvitamin D₃ by the duodenum was very low prior to hatching, even though significant amounts were present in the yolk sac. Injection of a physiological dose of 1,25-dihydroxyvitamin D₃ to chick embryo at 9 days failed to stimulate appearance of calcium binding protein by 18 days of embryonic life. Thus, it appears that either the normal mechanism for transport of 1,25-dihydroxyvitamin D₃ to intestine or its receptors in intestine may not be present prior to day 18–19.A large fraction of radioactive vitamin D₃ injected into the yolk sac was found esterified especially in the embryonic liver. The significance of this is not yet understood.Injection of 1,25-dihydroxyvitamin D₃ at 325 pmoles/per egg at 9 days resulted in 70% mortality of embryos while a 32-pmole dose resulted in no significant increase in mortality. The basis for this toxicity is not yet understood.     

Metabolism of 25-hydroxyvitamin D3 by rat kidney cells in culture: Isolation and identification of cis- and trans-19-nor-10-oxo-25-hydroxyvitamin D3

A primary confluent culture of epithelial cells from rat kidney has been developed. These cells possess a 3.2–3.4 S high-affinity, low-capacity binding protein for 1,25-dihydroxyvitamin D₃. They metabolize 25-hydroxyvitamin D₃ to at least five metabolites. Two have been identified as 1,25-dihydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃. Two others have been identified by means of physical data and cochromatography as trans 19-nor-10-oxo-25-hydroxyvitamin D₃ and the other as its cis isomer. These two “metabolites” have not been observed in vivo, but one of them (cis) comigrates with 1,25-dihydroxyvitamin D₃ on straight-phase high-performance liquid chromatography. Thus, mere cochromatography on high-performance liquid chromatography is not sufficient to identify critical vitamin D metabolites.