Biomedical Applications of Synthetic Melanotropins

Alpha-melanotropin (alpha-melanocyte stimulating hormone, alpha-MSH) is a hormone produced by the pituitary gland of most vertebrate animals. This melanotropic peptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2, regulates melanin pigmentation of the skin of some mammals. Although MSH may be absent from the human pituitary gland, this peptide can stimulate pigment formation in human skin. We have synthesized several analogues of alpha-MSH, which are superpotent, prolonged-acting, and resistant to inactivation by serum enzymes. One such analogue, [NLe⁴, D-Phe⁷]alpha-MSH, has proven particularly useful in a number of physiological studies. In addition, some [Nle⁴, D-Phe⁷]-substituted fragment analogues of MSH are even more active than the native hormone, alpha-MSH. For example, these analogues are 100–1,000 times more active than alpha-MSH in stimulating S-91 mouse melanoma tyrosinase activity in vitro. We have successfully labeled one such peptide to high specific activity; this melanotropin, [³H]-Ac-[Nle⁴, D-Phe⁷]alpha-MSH_4–11NH₂, has been shown by others to bind to B16 melanoma cells. We have also conjugated several ligands (fluorescein and biotin) to [Nle⁴, D-Phe⁷]alpha-MSH. These melanotropin conjugates might prove useful for melanotropin receptor studies and for the clinical localization of metastatic melanoma. We have demonstrated that [Nle⁴, D-Phe⁴]alpha-MSH can be topically applied and transdermally delivered across the skin of mice and humans in vitro, as determined by bioassay and RIA. Initial toxicologic studies indicate that the analogue is nontoxic to mice and is not mutagenic. Studies are underway to determine whether this analogue may prove useful as a “tanning hormone” for increasing the pigmentation of light-skinned individuals or possibly even for treating people with certain hypopigmentary disorders.     

Benzothiadiazole (BTH) activates sterol pathway and affects vitamin D3 metabolism in Solanum malacoxylon cell cultures

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a particularly efficient inducer of systemic acquired resistance (SAR), was developed as an immunizing agent to sensitize various crop species against pathogen infections. Recent works highlighted its activating effect on different metabolic pathways, concerning both primary and secondary metabolites. In this study, we investigated the effect of BTH treatment on sterol levels and vitamin D₃ metabolism in Solanum malacoxylon cultures. Calli of S. malacoxylon were incubated in Gamborg B5 liquid medium alone or added with 50 μM BTH for different times (one, two or three cycles of light). Histocytochemical investigations performed on our experimental system using 3,3′-diaminobenzidine (DAB) for hydrogen peroxide (H₂O₂) detection and phloroglucinol for lignin staining showed that BTH causes H₂O₂ accumulation and lignin deposition in treated calli. Gas chromatographic analysis of principal cell membrane sterols (β-sitosterol, campesterol, stigmasterol) showed that BTH transiently increases their cellular levels. Callus cultures were found to contain also cholesterol, 7-dehydrocholesterol, the putative precursor of vitamin D₃, and the hydroxylated metabolites 25-hydroxyvitamin D₃ [25(OH)D₃] and 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃]. BTH treatment enhanced 7-dehydrocholesterol while reduced cholesterol. HPLC analysis of sample extracts showed that BTH does not affect the cell content of vitamin D₃, though results of ELISA tests highlighted that this elicitor moderately enhances the levels of 25(OH)D₃ and 1α,25(OH)₂D₃ metabolites. In conclusion, BTH treatment not only causes cell wall strengthening, a typical plant defence response, as just described in other experimental models, but in the same time increases the cellular level of the main sterols and 7-dehydrocholesterol.     

Separation of D-forms and L-forms of DL-aspartic acid derivatives by the enzyme subtilisin DY

The ability of the enzyme subtilisin DY for the synthesis of derivatives of DL-aspartic acid which are differently N and C-terminal protected and semiproducts of the peptide synthesis was investigated. The enzyme reaction was characterized by high yields and a comparatively short reaction time. Two of the substrates, Z-D,L-Asp-(OMe)₂ and PhAc-D,L-Asp-(OMe)₂, were hydrolyzed for about 15 min; the reaction time for Boc-D,L-Asp-(OMe)₂ was 2.5 h. The values for the MICHAELIS constants obtained for Z-D,L-Asp-(OMe)₂ (K_m = 0.576 mM) and PhAc-D,L-Asp-(OMe)₂ (K_m = 0.300 mM) showed a high affinity of the enzyme to the substrates. For Boc-D,L-Asp-(OMe)₂ the affinity of the enzyme is considerable lower (K_m = 14.07 mM). The results of these investigations can be effectively used for the separation of N-protected derivatives of D,L-aspartic acid and with a high probability also for other amino and racemic forms.     

Stomatal responses to changes in vapor pressure deficit reflect tissue‐specific differences in hydraulic conductance

The vapor pressure deficit (D) of the atmosphere can negatively affect plant growth as plants reduce stomatal conductance to water vapor (g_wv) in response to increasing D, limiting the ability of plants to assimilate carbon. The sensitivity of g_wv to changes in D varies among species and has been correlated with the hydraulic conductance of leaves (K_leaf), but the hydraulic conductance of other tissues has also been implicated in plant responses to changing D. Among the 19 grass species, we found that K_leaf was correlated with the hydraulic conductance of large longitudinal veins (K_lv, r² = 0.81), but was not related to K_root (r² = 0.01). Stomatal sensitivity to D was correlated with K_leaf relative to total leaf area (r² = 0.50), and did not differ between C₃ and C₄ species. Transpiration (E) increased in response to D, but 8 of the 19 plants showed a decline in E at high D, indicative of an ‘apparent feedforward’ response. For these individuals, E began to decline at lower values of D in plants with low K_root (r² = 0.72). These results show the significance of both leaf and root hydraulic conductance as drivers of plant responses to evaporative demand.     

Static and dynamic light scattering from dilute insulin solutions

Static and dynamic light-scattering measurements are reported on zinc-insulin at room temperature (21 ± l°C) and pH = 6.88 in 0.1M NaCl aqueous solution. The experiments were performed at very low concentration, in the range 0.12 × 10^?4 to 0.90 × 10^?4 g cm^?3. Within experimental error, we find no evidence for a critical micellar concentration in this system. The aggregation phenomenon starts immediately after preparation of the solutions, and takes several days to come to stable equilibrium. The concentration dependence of the diffusion coefficients, D _z, = D_o (1 — k_DC), is negative, and k_D was observed to decrease as a function of time, while the aggregate size was found to increase. The equivalent concentration coefficient, ?2BM _W, obtained from static light scattering, showed a similar behavior, and, within experimental error, was found to be numerically equal to k_D. From the relation found between the diffusion coefficient at infinite dilution and the molecular weight of the aggregates, log D₀ = ?0.240 log M _w ? 5.077, we deduce that the insulin aggregates are compact structures with a characteristic radius of 0.71 Å/(dalton)^1/3, surrounded by a hydration layer of a thickness of 8.0 Å. The equilibrium aggregation number is approximately 10.     

Optimality of Some Class of Incomplete Block Designs

A class of incomplete block designs called C-design, was considered by Caliński (1971), Saha (1976), Ceranka (1983) and Ceranka , Kozłowska (1983, 1984). In this paper we extend the theory of block designs having the C-property. We consider optimality of C-designs with respect to any criterion of a described form. Das and Kageyama (1991) considered a class of E-optimal proper efficiency balanced designs (strictly speaking, Das and Kageyama considered E_R-optimality of some class of block designs). Hence we consider E_R, A_R, D_R optimality of C-designs.     

Arylpiperazinylalkyl derivatives of 8-amino-1,3-dimethylpurine-2,6-dione as novel multitarget 5-HT/D receptor agents with potential antipsychotic activity

A series of new 7-arylpiperazinylalkyl-1,3-dimethyl-purine-2,6-dione derivatives with diversified 8-amino substituent in 8 position was synthesized and their 5-HT_1A, 5-HT_2A, 5-HT₆, 5-HT₇, and D₂ receptor affinities were determined. The binding study allowed identifying some potent 5-HT_1A/5-HT_2A/5-HT₇/D₂ ligands. The most interesting because of their multireceptor profile were 8-piperidine (30–35) and 8-dipropylamine (45–47) analogs with four and five carbon aliphatic linkers. The selected compounds 24, 31, 34, 39, 41, 43, 45, and 46 in the functional in vitro evaluation for all targeted receptors showed significant partial D₂ agonist, partial 5-HT_1A agonist, and 5-HT_2A antagonist properties. The advantageous in vitro affinity of compound 34 for 5-HT_1A and D₂ receptors has been explained by means of molecular modeling, taking into consideration its partial agonist activity towards the latter one. In behavioral studies, compounds 32 and 34 revealed antipsychotic-like properties, significantly decreasing d-amphetamine-induced hyperactivity in mice.     

Transformation of vitamin D3 to 1α,25-dihydroxyvitamin D3 via 25-hydroxyvitamin D3 using Amycolata sp. strains

To enzymatically synthesize active metabolites of vitamin D₃, we screened about 500 bacterial strains and 450 fungal strains, of which 12 strains were able to convert vitamin D₃ to 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] via 25-hyroxyvitamin D₃ [25(OH)D₃]. The conversion activity was only detected in strains belonging to the genus Amycolata among all the organisms tested. A preparative-scale conversion of vitamin D₃ to 25(OH)D₃ and 1,25(OH)₂D₃ in a 200-1 tank fermentor using A. autotrophica FERM BP-1573 was accomplished, yielding 8.3 mg 25(OH)D₃/l culture and 0.17 mg 1,25(OH)₂D₃/l culture. A related compound, vitamin D₂, could be also converted to 25-hydroxyvitamin D₂ and 1,25-dihydroxyvitamin D₂ using the same strain. The cytochrome P-450 of FERM BP-1573 was detected by reduced CO difference spectra in whole-cell suspensions. Vitamin D₃ in the culture induced cytochrome P-450 and the conversion activity simultaneously, suggesting that the hydroxylation at C-25 of vitamin D₃ and at C-1 of 25(OH)D₃ originates from cytochrome P-450.Correspondence to: J. Sasaki     

Characterization of 1,25-dihydroxyvitamin D3-dependent calcium uptake in isolated chick duodenal cells

Summary Thein vivo andin vitro effects of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) on calcium uptake by isolated chick duodenal cells were studied.In vivo, 1,25-(OH)₂D₃ given orally to vitamin D-deficient chicks increased the initial rate of calcium uptake by cells prepared 1 hr after administration of the hormone. The rate was stimulated approximately 100%, 17 to 24 hr after repletion.In vitro, pre-incubation of 1,25-(OH)₂D₃ with cells from D-deficient chicks increased the cellular rate of calcium uptake in a concentration-dependent relationship. Enhancement was found with 10^–15 m, was maximal at 10^–13 m, and was diminished at higher (10^–11 m) concentrations. Stimulation was observed after a pre-incubation period as brief as 1 hr. The potency order for vitamin D₃ analogs was 1,25-(OH)₂D₃=1-(OH)D₃>25-(OH)D₃>1,24,25-(OH)₃D₃>24,25-(OH)₂D₃>D₃. The maximal enhancement in calcium uptake induced by the analogs was the same, only the concentration at which the cell responded was different. The effectiveness of 1,25-(OH)₂D₃ was five orders of magnitude greater than D₃. Kinetically, 1,25-(OH)₂D₃ increased theV _max of calcium uptake; the affinity for calcium (K _m=0.54mm) was unchanged. The enhanced uptake found after the cells were pre-incubated for 2 hr with the hormone was completely blocked by inhibitors of protein synthesis. 1,25-(OH)₂D₃,in vitro, also increased calcium uptake in cells isolated from D-replete chicks. The maximal rates of uptake were the same in cells from D-deficient and D-replete animals. The hormone had no effect of calcium efflux from cells. Calcium uptake in microvillar brush-border membrane vesicles was increased by 1,25-(OH)₂D₃. These findings suggest that thein vitro cell system described in this paper represents an appropriate model to examine the temporal relationships between 1,25-(OH)₂D₃ induction of calcium transport and specific biochemical correlates.     

Osmoregulatory capacity,health status and growth as functions of moult stages from various weight classes in marron (Cherax cainii) and yabbies (Cherax destructor)

The study investigated the relationship between five moult stages and the osmoregulatory capacities (OC), moisture content of hepatopancreas (HM%), hepatosomatic indices (HI_wet, HI_dry) and growth of 6 weight classes of 235 marron (Cherax cainii) and 235 yabbies (Cherax destructor) under laboratory conditions. In each moult stage, OC increased linearly with the increase in wet body weight. Intermoult stage C showed the highest OC significantly lower than premoult stages D₀, D₁, D₂ and postmoult stage AB in every weight class in both crayfish. HM% decreased from AB to D₂, while both HI_wet and HI_dry reduced from C to D₂. Percentage dry matter of whole body carcass was highest in stage C and lowest during the AB stage with a significant difference between the two species. Larger than 15-g marron and yabbies differ with each other in OC, HI_wet, and HI_dry, moult intervals and growth rates.     

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.     

A simple model of the relationship between asymmetry and developmental stability

The relationship between developmental stability and morphological asymmetry is derived under the standard view that structures on each side of an individual develop independently and are normally distributed. I use developmental variance of sizes of parts, V_D, as the converse of developmental stability, and assume that V_D follows a gamma distribution. Repeatability of asymmetry, a measure of how informative asymmetry is about V_D, is quite insensitive to the variance in V_D, for example only reaching 20% when the coefficient of variation of V_D is 100%. The coefficient of variation of asymmetry, CV_FA, also increases very slowly with increasing population variation in V_D. CV_FA values from empirical data are sometimes over 100%, implying that developmental stability is sometimes more variable than any previously studied type of trait. This result suggests that alternatives to this model may be needed.     

Tree size frequency distributions, plant density, age and community disturbance

We show that explicit mathematical and biological relationships exist among the scaling exponents and the allometric constants (α and β, respectively) of log–log linear tree‐community size frequency distributions, plant density N_T, and minimum, maximum and average stem diameters (D_min, D_max, and , respectively). As individuals grow in size and D_max increases, N_T is predicted to decrease as reflected by a decrease in the numerical value of α and an increase in the value of β. Our derivations further show that N_T decreases as increases even if D_min or D_max remain unchanged. Because D_max and the age of the largest individuals in a community are correlated, albeit weakly, we argue that the interdependent relationships among the numerical values of α, β, N_T, and shed light on the extent to which communities have experienced recent global disturbance. These predicted relationships receive strong statistical support using two large datasets spanning a broad spectrum of tree‐dominated communities.     

1α,25-dihydroxy-24-oxo-16-ene vitamin D3, a metabolite of a synthetic vitamin D3 analog, 1α,25-dihydroxy-16-ene vitamin D3, is equipotent to its parent in modulating growth and differentiation of human leukemic cells

1α,25(OH)₂-16-ene-D₃, a synthetic analog of the steroid hormone, 1α,25(OH)₂D₃, has great potential to become a drug in the treatment of leukemia and other proliferative disorders, because of its minimal in vivo calcemic activity associated with a potent inhibitory effect on cell growth. However, at present, the mechanisms through which 1α,25(OH)₂-16-ene-D₃ expresses its biological activities are still not completely understood. Our previous in vitro study in a perfused rat kidney indicated for the first time that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently. 1α,25(OH)₂-24-oxo-16-ene-D₃, an intermediary metabolite of 1α,25(OH)₂-16-ene-D₃ formed through the C-24 oxidation pathway, accumulated significantly in the perfusate when compared to 1α,25(OH)₂-24-oxo-D₃, the corresponding intermediary metabolite of 1α,25(OH)₂D₃. In a subsequent in vivo study, we also reported that 1α,25(OH)₂-24-oxo-16-ene-D₃ exerted immunosuppressive activity equal to its parent, without causing significant hypercalcemia. In order to establish further the critical role of 1α,25(OH)₂-24-oxo-16-ene-D₃, in generating some of the key biological activities ascribed to its parent, we performed the present in vitro study using a human myeloid leukemic cell line (RWLeu-4) as a model. Comparative target tissue metabolism studies indicated that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently in RWLeu-4 cells, and the differences were similar to the ones we previously observed in the rat kidney. The significant finding was the accumulation of 1α,25(OH)₂-24-oxo-16-ene-D₃ in RWLeu-4 cells because of its resistance to further metabolism. Biological activity studies indicated that both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite produced growth inhibition and promoted differentiation of RWLeu-4 cells to the same extent, and these activities were several fold higher than those exerted by 1α,25(OH)₂D₃. In addition, the genomic action of each vitamin D compound was assessed in a rat osteosarcoma cell line (ROS 17/2.8) by measuring its ability to transactivate a gene construct containing the vitamin D response element of the osteocalcin gene linked to the growth hormone reporter gene. In these studies, both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite exerted similar but potent transactivation activity which was several fold greater than that exerted by 1α,25(OH)₂D₃ itself. In summary, our results indicate that the production and slow clearance of the bioactive intermediary metabolite, 1α,25(OH)₂-24-oxo-16-ene-D₃, in RWLeu-4 cells contributes significantly to the final expression of the enhanced biological activities ascribed to its parent analog, 1α,25(OH)₂-16-ene-D₃.     

The diffusion-solubility of oxygen in lipid bilayers

The product, D_oα, of the oxygen diffusion coefficient, D_o, and the oxygen solubility, α, is determined in phosphatidylcholine bilayers at temperatures above the lipid phase transitions from ESR spin-exchange measurements. The resulting values of D_oα are in good agreement with those obtained from fluorescence-quenching experiments. The use of fatty acid spin labels makes it possible to measure D_oα as a function of the coordinate perpendicular to the bilayer surface. The results indicate that D_oα is a strong function of this coordinate; it is greatest in the bilayer center and least near the bilayer head groups.     

Carbohydrate uptake in Advenella mimigardefordensis strain DPN7T is mediated by periplasmic sugar oxidation and a TRAP‐transport system

In this study, we investigated an SBP (DctP_Am) of a tripartite ATP‐independent periplasmic transport system (TRAP) in Advenella mimigardefordensis strain DPN7^T. Deletion of dctP_Am as well as of the two transmembrane compounds of the tripartite transporter, dctQ and dctM, impaired growth of A. mimigardefordensis strain DPN7^T, if cultivated on mineral salt medium supplemented with d ‐glucose, d ‐galactose, l ‐arabinose, d ‐fucose, d ‐xylose or d ‐gluconic acid, respectively. The wild type phenotype was restored during complementation studies of A. mimigardefordensis ΔdctP_Am using the broad host vector pBBR1MCS‐5::dctP_Am. Furthermore, an uptake assay with radiolabeled [¹⁴C(U)]‐d ‐glucose clearly showed that the deletion of dctP_Am, dctQ and dctM, respectively, disabled the uptake of this aldoses in cells of either mutant strain. Determination of K_D performing thermal shift assays showed a shift in the melting temperature of DctP_Am in the presence of d ‐gluconic acid (K_D 11.76 ± 1.3 µM) and the corresponding aldonic acids to the above‐mentioned carbohydrates d ‐galactonate (K_D 10.72 ± 1.4 µM), d ‐fuconic acid (K_D 13.50 ± 1.6 µM) and d ‐xylonic acid (K_D 8.44 ± 1.0 µM). The sugar (glucose) dehydrogenase activity (E.C.1.1.5.2) in the membrane fraction was shown for all relevant sugars, proving oxidation of the molecules in the periplasm, prior to transport.     

Analysis of theD-region products ofH-2444using monoclonal antibodies reveals the expression of a new class I-like molecule

To analyze how many D-region-encoded molecules could be detected inH-2 ^q , we produced a panel of nine monoclonal antibodies from AKR (K^kD^k) anti-AKR.M (K^kD^q) immunizations. All of the D_q region antibodies cross-reacted on D^d and/or L^d, and all except one cross-reacted on D^b, confirming the previously observed serologic and amino acid sequence homology between theD-region products ofH-2 ^d ,H-2 ^b , andH-2 ^q . All of these monoclonal antibodies precipitated 46 000 dalton molecules from both cell-surface-labeled and biosynthetically labeled BIO.AKM spleen cells, indicating that all were reactive with class I-like molecules. Sequential immunoprecipitation analysis with one of these antibodies, 66-3-5, reveals the presence of a previously unidentified class I-like molecule. Tryptic peptide map analysis reveals that this molecule may be the product of a newly describedH-2D ^q -region gene.     

Antifungal Activity of <Emphasis Type="Italic">Pv</Emphasis>D1 Defensin Involves Plasma Membrane Permeabilization,Inhibition of Medium Acidification,and Induction of ROS in Fungi Cells

In recent years, studies have demonstrated the function of many antimicrobial peptides against an extensive number of microorganisms that have been isolated from different plant species and that have been used as models for the study of various cellular processes linked to these peptides’ activities. Recently, a new defensin from Phaseolus vulgaris (L.) seeds, named PvD_1, was isolated and characterized. PvD₁ was purified through anion exchange and phase-reverse chromatography. PvD₁’s antifungal activity was tested. A SYTOX Green uptake assay revealed that the defensin PvD₁ is capable of causing membrane permeabilization in the filamentous fungi Fusarium oxysporum, Fusarium solani, and Fusarium laterithium and in yeast strains Candida parapsilosis, Pichia membranifaciens, Candida tropicalis, Candida albicans, Kluyveromyces marxiannus, and Saccharomyces cerevisiae at a concentration of 100 μg/ml. Ultrastructural analysis of C. albicans and C. guilliermondii cells treated with this defensin revealed disorganization of both cytoplasmic content and the plasma membrane. PvD₁ is also able to inhibit glucose-stimulated acidification of the medium by yeast cells and filamentous fungi, as well as to induce the production of reactive oxygen species and nitric oxide in C. albicans and F. oxysporum cells.     

The titratable joint phenomenon in ΦW-14 DNA

Dynamic light scattering (DLS) studies are carried out on native ΦW-14 DNA, which has putrescines covalently attached at the methyl groups of half its thymines, and on a chemically modified form of the same DNA in which the ammonium groups of its putrescines are almost completely acetylated. From neutrality to pH 9.6, both forms of ΦW-14 DNA exhibit the same curve of D_app vs K² over the range K² = 0.5 × 10¹⁰ to K² = 20 × 10¹⁰ cm^?2, and this coincides with curves that we have observed for other DNAs. (D_app, apparent diffusion coefficient; K, scattering vector). However, when the pH is raised to pH 10.0–10.2, native ΦW-14 exhibits a spectacular decrease in D_app at large wave vector, whereas the acetylated form shows no sign of such behavior. It is inferred that bound ammonium groups make an essential contribution to the stabilization of titratable joints. Comparing the pH profiles of the absorbance (A₂₆₀) for these two DNAs gives some evidence that base unstacking may be involved in titratable joint formation.