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.

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.

Identification of a novel retrovirus expressed in rat Sertoli cells and granulosa cells.

Differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) was used to identify a novel retrovirus, designated SC1, that is expressed at high levels in rat granulosa cells and prepubertal Sertoli cells. The initial DDRT-PCR screen was performed using RNA from cultured prepubertal rat Sertoli cell, liver, and brain samples. SC1 was detected in the prepubertal rat Sertoli cell samples but not in those from liver and brain. SC1 cDNA was 6 kilobases in length and contained regions encoding for the gag, pol, and env retroviral proteins. Northern blot analysis failed to detect expression of the SC1 gene in total RNA isolated from adult brain, heart, spleen, lung, liver, skeletal muscle, kidney, prostate, and epididymis. Similarly, Northern blot analysis of testes from rats at various ages of development showed that high-level expression of the SC1 gene was limited to prepubertal testis samples. In situ hybridization analysis localized the SC1 mRNA to the seminiferous tubules of prepubertal testes and at a much lower level in Sertoli cells of adult testes. Northern blot analysis of total RNA isolated from Sertoli cells from 20-, 27-, and 35-day-old rat Sertoli cells and type A spermatogonia, pachytene spermatocytes, and round spermatids showed expression of the SC1 gene to be restricted to 20- and 27-day-old Sertoli cells, with no expression detected in germ cells. Furthermore, Northern blot analysis also showed expression of the SC1 gene in rat ovaries, and the level of expression was affected during eCG/hCG-induced ovulation. Expression of SC1 mRNA was localized by in situ hybridization of eCG-treated ovaries to the granulosa cell layer in developing follicles. Southern blot analysis showed SC1 to be endogenous in the rat and absent in mouse and human cell genomes. Transient transfection assays using the SC1 promoter region showed high promoter activity in MSC-1 and cultured prepubertal rat Sertoli cells, and no activity in 3T3 or MCF-7 cell lines.

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.

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.

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].

The effects of prolonged administration of 5-bromodeoxyuridine on cells of the immune system.

We have determined the in vivo effect of 5-bromodeoxyuridine (BrdU) administered to mice in the drinking water for various lengths of time on the performance of T and B lymphocytes in a number of experimental protocols. Young mice continuously exposed to BrdU fail to gain weight, and the lymphocytes recovered after a prolonged period of exposure are fewer in number than in control mice. The recovery of normal levels of T and B lymphocytes after irradiation is severely impaired. Ag-specific cells responding to Ag in an adoptive transfer model fail to expand as much in the presence of BrdU as in the absence, and the Ag-specific effectors produced in the presence of BrdU are less able to secrete cytokines upon restimulation in vitro. Polarized populations of Tc1 and Tc2 effectors generated in vitro proliferate less in the presence of BrdU, and the resulting effectors make less cytokines per cell upon restimulation. Thus, the incorporation of BrdU into T or B lymphocytes can, under some circumstances, seriously impair the performance of the labeled cells, and these findings raise a note of caution in the interpretation of studies that make use of long-term exposure to BrdU.

Advances in spermatogonial stem cell transplantation.

Spermatogonial stem cell transplantation was first reported by Ralph Brinster's laboratory in 1994. It has proven to be a technological breakthrough in the study of both stem cells and Sertoli cell-germ cell interactions. This technique can be used to transfer testicular stem cells successfully from one animal to another of the same species (referred to as syngeneic transplants) and sometimes to an animal of a different species (xenogeneic transplants). This transfer technique, combined with developments in cryopreservation, long-term culture, and the enrichment of stem cell populations makes more significant breakthroughs likely in the near future. Ultimately, the application of spermatogonial stem cell transfer will allow transplantation of cultured stem cells manipulated genetically in vitro to give rise to functional male gametes with an altered genotype. This achievement will have applications in basic science, human medicine, and domestic and wild animal reproduction. Although progress toward this goal has been swift, potentially significant barriers, such as the stable incorporation of genetic material into stem cells and immunological responses to the introduced germ cells, remain to be overcome. This article is a review of the scientific advances made since the initial report of successful transplantation in 1994.

Juvenile spermatogonial depletion (jsd) mutant seminiferous tubules are capable of supporting transplanted spermatogenesis.

In mice, the juvenile spermatogonial depletion (jsd) mutation results in a single wave of spermatogenesis followed by failure of type A spermatogonial stem cells to repopulate the testis, rendering male animals sterile. It is not clear whether the defect in jsd resides in a failure of the somatic component to support spermatogenesis or in a failure that is intrinsic to the mutant's germ cells. To determine if the jsd intratesticular environment is capable of supporting spermatogenesis, germ cell transplantation experiments were performed in which C57BL/6 ROSA germ cells were transplanted into jsd recipients. To determine if jsd spermatogonia are able to develop in a permissive seminiferous environment, jsd germ cells were transplanted into W/W(v) and busulfan-treated C57BL/6 animals. The data demonstrate that up to 7 mo after transplantation of normal germ cells, jsd seminiferous tubules are capable of supporting spermatogenesis. In contrast, when jsd germ cells were transplanted into busulfan-treated C57BL/6 testis, or into testis of W/W(v) mice, no jsd-derived spermatogenesis was observed. The data support the hypothesis that the jsd phenotype is due to a defect in the germ cells themselves, and not in the intratubular environment.

Identification of INSL6, a new member of the insulin family that is expressed in the testis of the human and rat.

A new member of the insulin gene family (INSL6) was identified from an Expressed Sequence Tag database through a search for proteins containing the insulin family B-chain cysteine motif. Human and rat INSL6 encoded polypeptides of 213 and 188 amino acids, respectively. These orthologous sequences contained the B-chain, C-peptide, and A-chain motif found in other members of the insulin family. Human INSL6 was 43% identical to human relaxin H2 in the B- and A-chain regions. As with other family members, human and rat INSL6 had predicted dibasic sequences at the junction of the C-peptide and A-chain. Human INSL6 sequence had an additional dibasic site near the C-terminus of the A-chain. The presence of a single basic residue at the predicted junction of the B-chain and C-peptide suggests that multiple prohormone convertases are required to produce the fully mature hormone. INSL6 was found to be expressed at high levels in the testis as determined by Northern blot analysis and specifically within the seminiferous tubules in spermatocytes and round spermatids as detected by in situ hybridization analysis. Radiation hybrid mapping placed the human INSL6 locus at chromosome 9p24 near the placenta insulin-like homologue INSL4 and the autosomal testis-determining factor (TDFA) locus.

Phosphorylcholine-containing polyurethanes for the control of protein adsorption and cell attachment via photoimmobilized laminin oligopeptides.

In this study, we synthesized a biomaterial whose surface inhibits non-specific protein and cell attachment. The polymer was designed to mimic the external cell plasma membrane properties through the introduction of particular chemical constituents of the cell membrane: phospholipid polar headgroups. This was done by copolymerizing phosphorylcholine (PC) groups into a polyurethane polymer backbone (PCPUR). Peptides known to induce specific cell attachment were subsequently bound to the surface of this copolymer in a photoadressible manner to obtain surfaces that allowed the attachment of cells in a specific pattern. Two polymers with different phosphorylcholine concentrations were synthesized and their bulk and surface properties were characterized through differential scanning calorimetry, wettability measurements, angle-resolved X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry. Protein and lipid adsorption investigation using optical waveguide light mode spectroscopy showed that the irreversible adsorption of both proteins and lipids is drastically reduced as a result of simultaneous contributions of the PC groups, molecular mobility and strong hydrophilicity of the polymers. Consequently, this leads to a marked reduction in the cellular attachment response, which further decreases with increasing PC concentration. Finally, when the polymer surface was photo-derivatized, attachment of the neural NG108-15 cell line occurred only on the areas of the PCPUR where the laminin CDPGYIGSR peptide sequence was photoimmobilized. Cell attachment was nevertheless found to be non-specific with respect to the peptide sequence used and reasons for such results are therefore discussed.

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.

Refined structure of the doubly intercalated d(TATAFBGCATA)2 aflatoxin B1 adduct.

The refined solution structure for the 8, 9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct was refined from the oligodeoxynucleotide duplex d(TATAFBGCATA)2 using a molecular dynamics protocol restrained by NOE data obtained from 1H NMR and compared with the refined structure of the unmodified oligomer, d(TATGCATA)2. The two aflatoxin B1 (AFB1) moieties were symmetry related by the pseudodyad axis of the self-complementary oligodeoxynucleotide. Each AFB1 intercalated into the helix above the 5'-face of the modified guanine, corroborating NMR spectroscopic data [Gopalakrishnan, S., Harris, T. M., and Stone, M. P. (1990) Intercalation of Aflatoxin B1 in Two Oligodeoxynucleotide Adducts: Comparative 1H NMR Analysis of d(ATCAFBGAT).d(ATCGAT) and d(ATAFBGCAT)2 Biochemistry 29, 10438-10448]. Molecular dynamics calculations restrained with 292 experimentally and empirically derived distances refined a family of structures characterized by pairwise root mean square differences of <1.3 A. Complete relaxation matrix calculations yielded a sixth root residual of 11 x 10(-2). Comparison of the refined structure with that of the corresponding unmodified oligodeoxynucleotide suggested that the two AFB1 adducts introduced a perturbation of the DNA localized at the two sites of adduction. The calculations predicted that each adduct introduced a "kink" into the DNA helical axis. However, the pseudodyad symmetry relating the two intercalation sites resulted in no net bending of the DNA. The results suggest the possibility that AFB1 lesions at adjacent guanines in the 5'-GC-3' sequence may be recognized or processed differently than are isolated AFB1 lesions.

Murine sperm-zona binding, a fucosyl residue is required for a high affinity sperm-binding ligand. A second site on sperm binds a nonfucosylated, beta-galactosyl-capped oligosaccharide.

An essential initial step in murine fertilization is the binding of acrosome-intact sperm to specific O-linked oligosaccharides on zona pellucida glycoprotein 3. While there is agreement on the primary role of O-linked glycans in this process, there is a lack of consensus on both the terminal monosaccharide(s) required for a functional sperm binding site and the corresponding protein on the sperm cell surface that recognizes this ligand. Much current debate centers on an essential role for either a terminal N-acetylglucosaminyl or, alternatively, a terminal alpha-galactosyl residue. To gain insight into the terminal saccharides required to form a functional sperm-binding ligand, dose-response curves were generated for a series of related tri- and tetrasaccharides to evaluate their relative effectiveness to competitively inhibit the in vitro binding of murine sperm to zona pellucida-enclosed eggs. A GlcNAc-capped trisaccharide, GlcNAc beta 1,4GlcNAc beta 1,4GlcNAc,was inactive (1-72 microM range). In contrast, a beta 4-galactosyl-capped trisaccharide (Gal beta 1,4GlcNAc beta 1, 4GlcNAc) and an alpha 3-galactosyl-capped trisaccharide (Gal alpha 1,3Gal beta 1,4 GlcNAc) inhibited sperm-zona binding with low or moderate affinity (ED50 = 42 microM and 5.3 microM, respectively). The addition of an alpha 3-fucosyl residue to each of these two competitive inhibitors, forming Gal beta 1,4[Fuc alpha 1,3] GlcNAc beta 1,4GlcNAc or Gal alpha 1,3Gal beta 1, 4[Fuc alpha 1,3]Glc NAc, resulted in ligands with 85- and 12-fold higher affinities for sperm, respectively (ED50 = 500 and 430 nM). Thus, the presence of a fucosyl residue appears to be obligatory for an oligosaccharide to bind sperm with high affinity. Last, mixing experiments with pairs of competitive inhibitors suggest that murine sperm-zona binding is mediated by two independent oligosaccharide-binding sites on sperm. The first (apparently high affinity) site binds both the alpha 3-galactosyl-capped trisaccharide and the two fucosylated tetrasaccharides. The second (apparently low affinity) site binds a nonfucosylated beta-galactosyl-capped trisaccharide.

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)].

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.

The gene encoding murine alpha 1,3-galactosyltransferase is expressed in female germ cells but not in male germ cells.

An essential step in murine fertilization is the binding of acrosome-intact sperm to specific O-linked glycans on zona pellucida glycoprotein 3 (ZP3). While there is agreement on the primary role of O-linked glycans in sperm-ZP3 binding, there is a striking lack of consensus on both the terminal monosaccharide(s) required for a functional binding site and the cognate protein on the sperm cell surface that recognizes this glycan. Much current debate centers on the essential role of nonreducing terminal N-acetyl-glucosaminyl or alternatively, alpha-galactosyl residues, to form a functional sperm binding ligand. Relevant to this debate, we demonstrated that alpha 1,3-galactosyltransferase (alpha 3-GT), which adds nonreducing terminal alpha-galactosyl residues to glycans, is not expressed in murine spermatocytes or spermatids. The objectives of this study were to determine whether alpha 3-GT is expressed in female germ cells and to compare the pattern of expression of two other terminal glycosyltransferases, beta 1,4-galactosyltransferase (beta 4-GT) and alpha 2,6-sialyltransferase (alpha 6-ST), between male and female germ cells. Total RNA was isolated from growing oocytes obtained from 15-day-old animals, fully grown oocytes, and eggs as well as spermatogonia, spermatocytes, and spermatids. The presence of alpha 3-GT, beta 4-GT, and alpha 6-ST mRNAs was analyzed by an RT-PCR-based assay. Our data demonstrate that the alpha 3-GT gene is expressed in female germ cells, but not in male germ cells. In contrast, both beta 4-GT and alpha 6-ST are expressed during oogenesis and spermatogenesis. This differential expression of alpha 3-GT in female germ cells is consistent with the model of sperm-egg binding in which a nonreducing terminal alpha-galactosyl residue is required for a functional determinant on ZP3 and with our hypothesis that the biological significance for the suppression of alpha 3-GT expression in male germ cells is to prevent sperm-sperm aggregation.

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.

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.