Epididymal genomics and the search for a male contraceptive.

This report represents the joint efforts of three laboratories, one with a primary interest in understanding regulatory processes in the epididymal epithelium (TTT) and two with a primary interest in identifying and characterizing new contraceptive targets (DSJ and SAJ). We have developed a highly refined mouse epididymal transcriptome and have used it as a starting point for determining genes in the human epididymis, which may serve as targets for male contraceptives. Our database represents gene expression information for approximately 39,000 transcripts, of which over 17,000 are significantly expressed in at least one segment of the mouse epididymis. Over 2000 of these transcripts are up- or down-regulated by at least four-fold between at least two segments. In addition, human databases have been queried to determine expression of orthologs in the human epididymis and the specificity of their expression in the epididymis. Genes highly regulated in the human epididymis and showing high tissue specificity are potential targets for male contraceptives.

A calorimetric study of the thermotropic behaviour of mixtures of brain cerebrosides with other brain lipids.

We have used a computer-controlled differential scanning calorimeter to determine the phases present in mixtures of the brain galactocerebrosides with other representative brain lipids. There are two types of brain galactocerebroside, those which possess an alpha-hydroxy substituent on the acyl chain (HFA) and those that do not (NFA). In the liquid crystalline state both cerebrosides were miscible with all the lipids studied, but in the gel state they were immiscible with cholesterol and the brain phosphatidylcholines. However, cholesterol mixtures in which the cholesterol mole fraction exceeded one third formed homogeneous metastable gel states on cooling from above the melting point of the cerebroside. Relaxation to the stable two phase state took place slowly over several hours. The solubilities of the galactocerebrosides in the other main brain sphingolipid, sphingomyelin, were much higher. Only in the case of the NFA galactocerebroside and at low mole fractions of sphingomyelin was immiscibility detected. Ternary mixtures of the two cerebrosides with sphingomyelin/cholesterol and phosphatidylcholine/cholesterol (PC/Chol) showed different miscibility characteristics. On cooling from 80 degrees C all mixtures formed homogeneous gel states. However, on standing the cerebrosides separated into discrete gel phases in all mixtures but one, that in which HFA galactocerebrosides were mixed with sphingomyelin and cholesterol. The cerebroside in the mixture with the composition closest to that of myelin, HFA/PC/Chol, melted at 38 degrees C. On scanning guinea pig CNS myelin which had been equilibrated at 5 degrees C a transition was detected with Tmax 33 degrees C. On the basis of comparison with the HFA/PC/Chol mixture we propose that the transition in myelin at this temperature is due to the melting of a galactocerebroside gel phase.

The properties of brain galactocerebroside monolayers.

Using a Langmuir film balance we have compared the properties of films of the brain galactocerebrosides at 37 degrees C. There are two types of cerebroside in brain, those with an alpha-hydroxy substituent on the acyl chain (HFA) and those without (NFA). At equivalent pressures the areas of both cerebroside films are significantly less than the areas of films of the brain glycerolipids, the choline and ethanolamine phosphatides. The isotherm of NFA galactocerebrosides has two discontinuities, one at low and one at high film pressure, while the isotherm of HFA galactocerebrosides is a smooth curve at all film pressures. Below the high-pressure transition the area of the NFA film is significantly larger than the area of the HFA film. When compressed beyond the high-pressure transition there is a marked hysteresis between compression and expansion isotherms of the NFA galactocerebrosides. The pressures of both films continue to rise steeply when they are compressed into areas which are too small for them to exist as simple monolayers. We conclude that under compression cerebroside films form bilayer structures; that bilayer formation starts at low pressure and occurs progressively as the HFA cerebroside monolayer is compressed, but occurs more abruptly in the NFA cerebroside monolayer at the high-pressure-transition region of the isotherm. A study of pure cerebrosides with a single defined acyl chain shows that there is a correlation between the relative volumes of the hydrophobic and hydrophilic parts of the molecule and the ease of bilayer formation. The larger the relative volume of the hydrophilic group the more readily the cerebroside forms a bilayer film. Other brain lipids added to cerebroside monolayers have sharply differing effects on their areas. The areas of films containing cholesterol are less than the areas calculated by adding the areas of the pure components multiplied by their mole fractions. On the other hand, the area of phosphatidylcholine-containing films is much larger than calculated.

A Langmuir film balance study of the interactions of ionic and polar solutes with glycolipid monolayers.

Using a Langmuir film balance experiments have been conducted to discover if dissolved salts or carbohydrates interact with glycolipid monolayers. Two types of glycolipid were studied, simple glycosides made by ether linking monosaccharides to fatty alcohols and cerebrosides extracted from natural sources. It was found that salts or carbohydrates in the subphase expanded glycolipid monolayers. That is, a monolayer spread on a solution occupied a greater area at a given pressure than it would have spread on pure water. Of the carbohydrates galactose and glucose, galactose caused a markedly greater expansion of monolayers than glucose. However, the magnitude of the expansions measured for stearyl glucoside, mannoside and galactoside films on solutions of a particular sugar were not significantly different, demonstrating that this phenomenon is independent of the glycolipid sugar residue. As with carbohydrates, salts also have differing effects on glycolipid monolayers. Although the effect an individual ion has on a monolayer cannot be directly measured, comparisons between salts indicate that there is a correlation between the size of an ion and the extent of the monolayer expansion it causes. To explain these observations two different mechanisms are proposed. In the case of salts it is suggested that large ions which have a low charge density disrupt water structure in such a way that monolayers spread on the surface of their solutions are expanded. The ability of carbohydrates to expand monolayers is explained in terms of the carbohydrate replacing water molecules bound to the polar groups of the monolayer and in so doing increasing the effective area of the lipid molecules. It is suggested that the molecular mechanisms involved in the interactions of ions and carbohydrates with glycolipid monolayers may also operate in the interactions of glycolipids and glycoproteins with extracellular agents and surfaces.

The optical activity and circular dichroic spectra of diacetylenic phospholipid polymers.

Phospholipids (phosphatidylcholines) which contain a diacetylene group in a single acyl chain and within both acyl chains have been synthesized. Upon irradiation with ultraviolet light, both types of lipid crosslink via the diacetylene groups to produce coloured polymers. The colour arises from the conjugated double and triple bonds which make up the polymer backbone. These phospholipid polymers can exhibit optical activity, as shown by their circular dichroic spectra. The optical activity is thought to stem from asymmetric packing of the polydiacetylene chains, a packing of one particular screw sense being favoured by the chiral glycerol moiety of the lipid. The presence of an intrinsic membrane protein within the liposome structure affects the CD spectra of polymer produced by irradiation.

Differential scanning calorimetric and Fourier transform infrared spectroscopic investigations of cerebroside polymorphism.

Calorimetric and Fourier transform infrared (FTIR) spectroscopic studies have been made of the polymorphism exhibited by bovine brain cerebroside-water systems, and the effect of cholesterol and dipalmitoylphosphatidylcholine (DPPC) upon this polymorphism was investigated. The conversion of the cerebroside from the thermodynamically stable to the metastable form is found to be accompanied by spectral changes, indicating a decrease in cerebroside headgroup hydration and a rearrangement of the hydrogen-bond network. The incorporation of low concentrations of cholesterol and DPPC into cerebroside bilayers broadens the thermal transitions associated with the cerebroside as a result of the disruption of cerebroside-cerebroside interactions. This disruption is evident in the spectra of cerebroside/cholesterol mixtures.

Stable biomembrane surfaces formed by phospholipid polymers.

Phospholipids (phosphatidylcholines) with diacetylene in each acyl chain have been deposited in Langmuir-Blodgett multilayers on a variety of substrates. Upon irradiation the diacetylene groups polymerise and link the phospholipid molecules together with a conjugated chain made up of alternating single, double and triple bonds. Advantage has been taken of this polymerisation process to increase the stability of these lipids layers and to produce stable biomembrane hydrophilic surface. These surfaces may be useful for studies of blood coagulation and protein adsorption. In addition they could also have medical application.

Phospholipid polymers--synthesis and spectral characteristics.

A new approach has been developed for the study of model and natural biomembranes. This involves the cross-linking of diacetylene groups after ultraviolet irradiation. For the study of model biomembranes, pure phospholipids (phosphatidylcholines) have been synthesized containing diacetylene groups in each acyl chain. The physical properties of these lipids have been examined and the conditions under which they polymerise have been determined. Polymerisation occurs when the lipid is in a crystalline phase, either compressed in KBr, dispersed in water (liposomes) or deposited on a suitable support (multilayers). The resultant polymer contains a conjugated backbone and is coloured. The visible spectrum of the phospholipid polymer is sensitive to its environment. Preliminary experiments show that similar polymerisation can be induced in Acholeplasma laidlawii cells grown on diacetylenic fatty acid.

The formation of polymeric model biomembranes from diacetylenic fatty acids and phospholipids.

Diacetylenic fatty acid monolayers at the air/water interface and multilayers on suitable supports polymerise when exposed to ultraviolet radiation. It has been found that polymerisation still occurs when monolayers are diluted with cholesterol or gramicidin. The rigid, crystalline nature of the films formed makes them useful biomembrane models. Phospholipids made from the fatty acids were less reactive. Multilayers deposited on hydrophobic supports would polymerise but not monolayers on water.

Germ cell transplantation and the study of testicular function.

Spermatogonial stem cell transplantation is a novel technique in which donor testicular cells are transferred into recipient testes. A population of germ cells from a transgenic or mutant donor is introduced into the seminiferous tubules of recipient testes by microinjection. Following injections, spermatogonial stem cells can colonize the recipient testis, initiate spermatogenesis and produce sperm capable of fertilization. This technique will allow scientists to: (1) investigate fundamental aspects of spermatogenesis; (2) provide a method to regenerate spermatogenesis in infertile individuals; and (3) genetically manipulate spermatogonial stem cells to develop transgenic animals.

Murine germ cells do not require functional androgen receptors to complete spermatogenesis following spermatogonial stem cell transplantation.

The spermatogonial stem cell transplantation technique was employed to determine if murine germ cells require functional androgen receptors to complete qualitatively normal spermatogenesis. Testicular cells from testicular feminized mice were injected into the seminiferous tubules of azoospermic mice expressing functional androgen receptors. Recipient testes were analyzed between 110 and 200 days following transplantation. Multiple colonies of complete and qualitatively normal donor-derived spermatogenesis were seen within the seminiferous tubules of each recipient testis, demonstrating that murine germ cells do not require functional androgen receptors to complete spermatogenesis.

Polymerized liposomes as stable oxygen-carriers.

We have produced a surrogate erythrocyte ('hemosomes') by encapsulating human hemoglobin in polymerized vesicles composed of diacetylenic phospholipids plus or minus cholesterol. Hemoglobin (in the presence or absence of allosteric effectors) was encapsulated by a freeze-thaw method in large, unilamellar vesicles composed of monomeric lipids. Entrapment was demonstrated by molecular-sieve chromatography. Brief irradiation with ultraviolet light produced polymeric hemosomes with polymerization kinetics and conversions similar to liposomes in the absence of protein. Photo-induced oxidation of the heme was eliminated or severely limited by a combination of prior ligation with CO and the maintenance of high intravesicular hemoglobin concentrations (5-10 mM internal hemoglobin). The inclusion of allosteric effectors within polymerized hemosomes facilitated near-quantitative conversion to the oxy-HbA form. Gas permeability of monomeric and polymeric hemosomes was demonstrated by spectroscopic methods. Reversible spectral shifts, corresponding to oxygenation-deoxygenation, were obtained after brief evacuation and exposure to oxygen or nitrogen. The gas permeability of polymerized hemosomes appears sufficient for the vesicles to act as oxygen carriers in vivo, a notion that is strengthened by their apparent hemocompatibility.

Mobilization of iron by chiral and achiral anionic 3-hydroxypyrid-4-ones.

In the search for 3-hydroxypyrid-4-ones with enhanced iron-mobilizing ability, seven chiral, anionic amino acid derivatives of maltol (3-hydroxy-2-methyl-4-pyrone) have been synthesized, utilizing L-methionine, L-serine, L-leucine, L-phenylalanine, L-glutamic acid, and the D- and L-isomers of alanine. Two achiral, aromatic compounds were also synthesized and compared with the phenylalanine derivative. The biliary iron excretion following iv injection and the urinary iron excretion following po administration were measured using female Sprague-Dawley rats and compared to that of the standard, 1,2-dimethyl-3-hydroxypyrid-4-one (L1). While none of the compounds was as effective as L1 in enhancing the urinary excretion of iron, all monoanionic chelators increased excretion relative to the controls. All monoanionic compounds were at least equivalent to L1 in enhancing the biliary excretion of iron, with the methionine, leucine, and benzoate derivatives surpassing the standard and the other aromatic compounds also showing strong activity. The dianionic glutamate derivative showed low activity relative to the controls for both urinary and biliary iron excretion. No significant difference in iron excretion was observed due to variation in chirality; molecular weight and the number of negative charges appeared to have the greatest influence on the ability of the various derivatives to enhance iron excretion. In order to evaluate the relative purity of the stereoisomers, the alanine derivatives were analyzed by circular dichroism. Further characterization was provided by UV/vis spectroscopy for all compounds and X-ray crystallography for the novel dianionic derivative.

Polymeric phospholipids as new biomaterials.

Phospholipid polymers form a new class of biomaterials with many potential applications in medicine and research. The development of these compounds is based upon the mimicry of cell surfaces and reflects our current understanding of the properties of membrane lipids. Physicochemical characterization of the monomeric, diacetylenic phospholipids illustrates the similarities to naturally occurring lipids, similarities that are confirmed by the capacity to enrich the membranes of A. laidlawii to the level of 90% diacetylenic lipid. Polymerization of diacetylenic phospholipids is easily attained by irradiation and produces a stable, crystalline array. The ability to link membrane lipids covalently permits the isothermal restriction in their motion, and is useful in basic studies of biomembranes. The thromboresistance of polymeric phosphatidylcholines in vitro may be a consequence of the inability of phosphatidylcholines to participate in coagulation. The restricted lateral diffusion of proteins along a polymeric lattice will also inhibit the formation of coagulation complexes. Existing polymers may be altered by a coating of polymeric lipid obtained by the Langmuir-Blodgett method. Polymerized vesicles display significant reductions in permeability and aggregation. Entrapment of soluble materials and reconstitution of membrane proteins may be exploited in controlled and site-directed drug delivery. Polymerization of cells in situ produces "cellular capsules" with entrapped membrane and cellular components. Polymeric hemosomes are capable of gas transport and may function as red cell surrogates. The hybrid qualities of biomembranes (polar surfaces, nonthrombogenic, low antigenic potential, and low permeability) and synthetic polymers (chemical and physical stability) suggest that polymeric phosphatidylcholines may serve as models for biomaterials design.

Monomeric ferric heme peptide derivatives: model systems for hemoproteins.

Electron paramagnetic resonance spectra of a number of ferric heme peptide derivatives, in aqueous-detergent and various aqueous-alcohol solvent mixtures, have been obtained using samples in the concentration range 0.1-1.0 mM. Some of these were clearly monomeric, homogeneous, mixed-ligand adducts, entirely suitable for use as model systems for hemoprotein spectroscopic studies. As anticipated, the measured EPR parameters were largely independent of solvent environment. Surprisingly, micellar preparations of ferric heme undecapeptide in mildly alkaline solution showed no evidence for the formation of a hydroxide adduct, contrary to a previous report [S. Mazumdar, O. K. Medhi and S. Mitra, Inorg. Chem. 30 700 (1991)].

The elimination of miR-23a in heat-stressed cells promotes NOXA-induced cell death and is prevented by HSP70.

Protein-damaging stress stimulates cell destruction through apoptosis; however, non-lethal proteotoxic stress induces an adaptive response leading to the increased synthesis of heat shock proteins, which inhibit apoptosis. In this study, we sought to determine the mechanism responsible for the accumulation of the BH3-only protein NOXA in heat-stressed cells and its prevention by the heat shock protein HSP70. Analysis of transcript levels by RT-qPCR revealed that miR-23a levels decreased in heat-stressed cells and that this was correlated with an increased abundance of NOXA mRNA, which contains a miR-23a binding site in its 3' untranslated region. Cells overexpressing HSP70 had higher levels of miR-23a, maintained these levels after heat shock and accumulated lower levels of NOXA mRNA and protein. The enhanced abundance of mir-23a in these HSP70-expressing cells is primarily due to its increased stability although higher levels of pri/pre-miR-23a expression, nuclear export and maturation were also contributing factors. Stable overexpression of miR-23a in the acute lymphoblastic T-cell line PEER resulted in reduced basal and heat-induced levels of NOXA mRNA and significantly inhibited heat-induced apoptosis. Additionally, stable overexpression of an shRNA targeting miR-23a in U937 lymphoma cells produced stable knockdown of miR-23a and resulted in increased NOXA mRNA and an increased sensitivity to heat-induced apoptosis. These results demonstrate the novel finding that hyperthermia affects the abundance of a microRNA that targets the expression of a pro-apoptotic protein and that HSP70 protects cells from heat-induced apoptosis by regulating the abundance of this microRNA. We speculate that the inhibition of miRNA transcription in heat-stressed cells could represent a general mechanism for apoptosis induction that is regulated by the molecular chaperone protein HSP70. Furthermore, we propose that HSP70 could be beneficial to tumor cells by helping to maintain the expression of oncogenic miRNAs under conditions of cellular stress.

Refined solution structure of 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 opposite CpA in the complementary strand of an oligodeoxynucleotide duplex as determined by 1H NMR.

The solution structure of d(CCATCAFBGATCC).d(GGATCAGATGG), containing the 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct, was refined using molecular dynamics restrained by NOE data obtained from 1H NMR. The modified guanosine was positioned opposite cytosine, while the aflatoxin moiety was positioned opposite adenosine in the complementary strand. Sequential 1H NOEs were interrupted between C5 and AFBG6, but intrastrand NOEs were traced through the aflatoxin moiety, via H6a of aflatoxin and H8 of the modified guanine. Opposite the lesion, the NOE between A16 H1' and G17 H8 was weak. A total of 43 NOEs were observed between DNA protons and aflatoxin protons. Molecular dynamics calculations restrained with 259 experimental and empirical distances, and using sp2 hybridization at AFBG6 N7, refined structures with pairwise rms differences < 0.85 A, excluding terminal base pairs. Relaxation matrix calculations yielded a sixth root rms difference between refined structures and NOE intensity data of 7.3 x 10(-2). The aflatoxin moiety intercalated on the 5'-face of the modified guanine. The extra adenine A16 was inserted between base pair AFBG6.C15 and the aflatoxin moiety. A 36 degree bending between the plane of base pair AFBG6.C15 and the plane of the aflatoxin moiety was predicted. The aflatoxin moiety stacked below the top domain of the oligodeoxynucleotide, which consisted of base pairs C1.G21, C2.G20, A3.T19, T4.A18, and C5.G17. The bottom domain consisted of base pairs AFBG6.C15, A7.T14, T8.A13, C9.G12, and C10.G11. The average winding angle between base pair C5.G17, the intercalated aflatoxin moiety, A16, and base pair AFBG6.C15 was reduced to 10 degrees. The preponderance of base pair substitutions in the aflatoxin B1 mutational spectrum, particularly G-->T transversions, suggests that the stability of this modified oligodeoxynucleotide, which models a templated +1 addition mutation, does not reliably predict the frequency of frame shifts.

Replication of a site-specific trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B(1) adduct by the exonuclease deficient klenow fragment of DNA polymerase I.

A 19-mer oligodeoxynucleotide containing a site-specific trans-8, 9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B(1) adduct was prepared and purified. This was used as a template for replication with DNA polymerase I exo(-) (Klenow exo(-)) in vitro. The chemical stability of the modified template strand containing the cationic aflatoxin B(1) adduct was monitored by mass spectrometry. Under the conditions used in these assays, the cationic aflatoxin B(1) adduct remained intact; quantitative conversion to the corresponding formamidopyrimidine adduct was not observed. The results revealed that the cationic guanine AFB(1) N7 adduct blocked translesional DNA synthesis at the adducted site and one nucleotide 3' to the adducted site. Correct incorporation of cytosine opposite the lesion led to blockage, while incorrect incorporation of adenine allowed full-length extension. The in vitro experiments with polymerase I yielded base pair substitutions at the lesion site but not the 5'-neighbor substitutions observed in vivo [Bailey, E. A., Iyer, R. S., Stone, M. P., Harris, T. M., and Essigmann, J. M. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 1535-1539].

Site-specific synthesis of aflatoxin B(1) adducts within an oligodeoxyribonucleotide containing the human p53 codon 249 sequence.

This work describes the preparation of the cationic trans-8, 9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B(1) ((AFB)G) adducts at the positions corresponding to G(746) or G(747), within the oligodeoxyribonucleotide d(GGAGGCCT) containing the codon 249 sequence (underlined) of the p53 gene, using DNA triplexes to target adduction at the desired site. This approach enabled the successful preparation and purification of sufficient quantities of d(GGAG(AFB)GCCT) for NMR structural studies, using only standard phosphoramidites. The presence of multiple guanines in this oligodeoxynucleotide precluded the direct reaction of d(GGAGGCCT). d(AGGCCTCC) with aflatoxin epoxide as a method for producing large quantities of site-specific adducts for physical studies. Of the multiple potential alkylation sites at guanine N7 in d(GGAGGCCT). d(AGGCCTCC), it was found that sites G(2) and G(5) exhibited approximately equal reactivity with aflatoxin B(1)-exo-8,9-epoxide; the reactivity at site G(4) was reduced by approximately a factor of 2 as compared to that at G(2) or G(5). To successfully prepare the site-specific adducts, the p53 oligodeoxyribonucleotide was annealed with either the blocking strand d(CTCCATTTTCCT) or d(CCTCCATTTTCCTC) to form the corresponding partial triplexes which targeted AFB(1) adduction either to G(4) or to G(5). Piperidine cleavage, followed by heating, confirmed that in each instance, the product corresponded to the lone guanine not protected from adduction by the partial DNA triplex. The adducted oligodeoxyribonucleotides were examined with regard to purity by capillary electrophoresis. The primary advantage of this modified triple helix methodology is that it requires only standard phosphoramidites; thus, it is applicable to large-scale preparations that are necessary for NMR structural studies or other physical measurements.