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The venom-containing barb attached to their 'whip-like' tail provides stingrays a defensive mechanism for evading predators such as sharks. From human encounters, dermal stingray envenomation is characterized by intense pain often followed by tissue necrosis occurring over several days to weeks. The bioactive components in stingray venoms (SRVs) and their molecular targets and mechanisms that mediate these complex responses are not well understood. Given the utility of venom-derived proteins from other venomous species for biomedical and pharmaceutical applications, we set out to characterize the bioactivity of SRV extracts from three local species that belong to the Dasyatoidea 'whiptail' superfamily. Multiple cell-based assays were used to quantify and compare the in vitro effects of these SRVs on different cell lines. All three SRVs demonstrated concentration-dependent growth-inhibitory effects on three different human cell lines tested. In contrast, a mouse fibrosarcoma cell line was markedly resistant to all three SRVs, indicating the molecular target(s) for mediating the SRV effects are not expressed on these cells. The multifunctional SRV responses were characterized by an acute disruption of cell adhesion leading to apoptosis. These findings aim to guide future investigations of individual SRV proteins and their molecular targets for potential use in biomedical applications.

Elasmobranchs represent a distinct group of cartilaginous fishes that harbor a remarkable ability to heal wounds rapidly and without infection. To date very little work has addressed this phenomenon although it is suggested that antibiotic capabilities associated with epidermal surfaces may be a factor. The study of benefits derived from mutualistic interactions between unicellular and multicellular organisms is a rapidly growing area of research. Here we survey and identify bacterial associates of three ray and one skate species in order to assess the potential for antibiotic production from elasmobranch associated bacteria as a novel source for new antibiotics.

Representatives of Subclass Elasmobranchii are cartilaginous fish whose members include sharks, skates, and rays. Because of their unique phylogenetic position of being the most primitive group of vertebrates to possess all the components necessary for an adaptive immune system, the immune regulatory compounds they possess may represent the earliest evolutionary forms of novel compounds with the potential for innovative therapeutic applications. Conditioned medium, generated from short term culture of cells from the epigonal organ of bonnethead sharks (Sphyrna tiburo), has been shown to have potent reproducible cytotoxic activity against a variety of human tumor cell lines in vitro. Existing data suggest that epigonal conditioned medium (ECM) exerts this cytotoxic activity through induction of apoptosis in target cells. This manuscript describes apoptosis induction in a representative tumor cell line, Jurkat E6-1, in response to treatment with ECM at concentrations of 1 and 2 mg/mL. Data indicate that ECM exposure initiates the mitochondrial pathway of apoptosis through activation of caspase enzymes. Future purification of ECM components may result in the isolation of an immune-regulatory compound with potential therapeutic benefit for treatment of human cancer.

Ampullary organ electroreceptors excited by weak cathodal electric fields are used for hunting by both cartilaginous and non-teleost bony fishes. Despite similarities of neurophysiology and innervation, their embryonic origins remain controversial: bony fish ampullary organs are derived from lateral line placodes, whereas a neural crest origin has been proposed for cartilaginous fish electroreceptors. This calls into question the homology of electroreceptors and ampullary organs in the two lineages of jawed vertebrates. Here, we test the hypothesis that lateral line placodes form electroreceptors in cartilaginous fishes by undertaking the first long-term in vivo fate-mapping study in any cartilaginous fish. Using DiI tracing for up to 70 days in the little skate, Leucoraja erinacea, we show that lateral line placodes form both ampullary electroreceptors and mechanosensory neuromasts. These data confirm the homology of electroreceptors and ampullary organs in cartilaginous and non-teleost bony fishes, and indicate that jawed vertebrates primitively possessed a lateral line placode-derived system of electrosensory ampullary organs and mechanosensory neuromasts.

The effects of ionising radiation on the peripheral blood, spleen, and epigonal and Leydig organs of cartilaginous fishes were investigated using juvenile clearnose skates, Raja eglanteria. Skates (N = 80) were sacrificed 12 days after exposure to 0-75 Gy of X-radiation, and morphometrics (body mass, disc width, total length), mass of spleens and epigonal organs, and peripheral blood leucocyte (PBL) counts were compared to controls using ANOVA. Spleen and epigonal organ mass and PBL counts declined logarithmically as a function of radiation dose. To assess recovery from X-radiation, skates (N = 40) were exposed to 0, 9 or 18 Gy and sacrificed when moribund or on days 10, 20, 30 and 40 post-irradiation. Partial recovery of Leydig organ and splenic red pulp was evident after 40 days in skates exposed to 9 Gy, but no indication of recovery was apparent at higher doses. Median lethal dose by 30 days (LD50/30) was calculated to be 9-18 Gy, similar to that determined for other fishes.

SYNOPSIS: Reports that elasmobranchs (sharks, skates, and rays) may have a low incidence of disease have stimulated interest in understanding the role of their immune system in this apparent resistance. Although research in this area may potentially translate into applications for human health, a basic understanding of the elasmobranch immune system components and how they function is essential. As in higher vertebrates, elasmobranch fishes possess thymus and spleen, but in the absence of bone marrow and lymph nodes, these fish have evolved unique lymphomyeloid tissues, namely epigonal and Leydig organs. As conditions for short-term culture of elasmobranch immune cells have become better understood, the opportunity to examine functional activity of cytokine-like factors derived from conditioned culture medium has resulted in the identification of growth inhibitory activity against a variety of tumor cell lines. Specifically, the medium enriched by short term culture of bonnethead shark (Sphyrna tiburo) epigonal cells (epigonal conditioned medium, ECM) has been shown to inhibit the growth of mammalian tumor cell lines, including fibrosarcoma (WEHI-164), melanoma (A375.S2), B-cell lymphoma (Daudi), T-cell leukemia (Jurkat), pancreatic cancer (PANC-1), ovarian cancer (NIH:OVCAR-3), and three breast carcinoma cell lines (MCF7, HCC38, Hs578T). Of the cell lines tested, WEHI-164, A375.S2, Daudi, and Jurkat cells were among the most sensitive to growth inhibitory activity of ECM whereas PANC-1 and NIH:OVCAR-3 cells were among the least sensitive. In addition, ECM demonstrated preferential growth inhibition of malignant cells in assays against two different malignant/non-malignant cell line pairs (HCC38/HCC38 BL and Hs 578T/Hs 578Bst). Separation of protein components of ECM using SDS-PAGE resulted in a very reproducible pattern of three major bands corresponding to molecular sizes of approximately 40-42 kD, 24 kD, and 17 kD. Activity is lost after heating at 75 degrees C for 30 min, and can be diminished by treatment with proteinase K and protease. Activity is not affected by treating with trypsin, DNase I or RNase A.

Reactive nitrogen intermediates, such as nitric oxide (NO), are important immunomodulators in vertebrate immune systems, but have yet to be identified as mediators of host defence in any member of class Chondrichthyes, the cartilaginous fishes. In the present study, production of NO by nurse shark (Ginglymostoma cirratum) peripheral blood leucocytes (PBL) stimulated with bacterial cell wall lipopolysaccharide (LPS) was investigated. PBL were cultured for 24 to 96 h following stimulation with LPS at concentrations ranging from 0 to 25 microg ml(-1), in both serum-supplemented and serum-free culture conditions. Production of NO was measured indirectly using the Griess reaction, with maximal NO production occurring after 72 h using 10% FBS and 10 microg LPS ml(-1). Application of these culture conditions to PBL from another cartilaginous fish (clearnose skate, Raja eglanteria) resulted in a similar NO response. Addition of a specific inhibitor of inducible nitric oxide synthase (iNOS), L-N(6)-(1-iminoethyl)lysine (L-NIL), resulted in a significant decrease in the production of NO by PBL from both species.

The health of many Florida manatees (Trichechus manatus latirostris) is adversely affected each year by exposure to cold weather or harmful algal blooms (red tide; Karenia brevis). Exposures can be sublethal, resulting in stressed animals that are rescued and taken to authorized facilities for rehabilitation, or lethal if exposures are prolonged or unusually severe. To investigate whether sublethal environmental exposures can impair immune function in manatees, rendering animals vulnerable to disease or death, mitogen-induced proliferation was assessed in lymphocytes from manatees exposed to cold temperatures (N=20) or red tide (N=19) in the wild, and compared to lymphocyte responses from healthy free-ranging manatees (N=32). All animals sampled for this study were adults. Lymphocytes were stimulated in vitro with either concanavalin A (ConA) or phytohemagglutinin (PHA) and proliferation was assessed after 96 h using incorporation of the thymidine analog, bromodeoxyuridine (BrdU), into newly synthesized DNA. Proliferation of lymphocytes from manatees rescued from exposure to red tide or cold-stress was approximately one-third that of lymphocytes from healthy free-ranging manatees. To examine the direct effects of red tide toxins on lymphocyte function, mitogen-induced proliferation was assessed following co-culture of lymphocytes with K. brevis toxin extracts. Stimulation indices decreased with increasing toxin concentration, with a significant decrease in proliferation occurring in the presence of 400 ng red tide toxins/ml. When lymphocytes from cold-stressed manatees were co-cultured with red tide toxin extracts, proliferative responses were reduced even further, suggesting multiple stressors may have synergistic effects on immune function in manatees.

To investigate for the first time the effects of ionizing radiation on thymus of a representative cartilaginous fish, juvenile clearnose skates, Raja eglanteria, were exposed to 0-75 Gy of X-radiation and sacrificed after 12 days. Morphometrics (weight, disc width and total length) and thymus and thymic cyst area were compared to controls using ANOVA. Thymus area declined logarithmically and medullary cysts increased as a function of dose (P < or = 0.05). To assess thymic recovery, skates were exposed to 0, 9, 13.5 or 18 Gy of X-radiation and sacrificed when moribund or on days 10, 20, 30 and 40 post-irradiation. Complete restoration of the thymus was not achieved during the 40-day observation period, although repopulation with pro-thymocytes and partial recovery of thymic architecture were evident histologically. The observed high radiosensitivity of R. eglanteria thymocytes was similar to responses of other vertebrates, but recovery time was prolonged.

Humoral immunity is effected through the rearrangement of immunoglobulin (Ig) genes in individual somatic cells committed to the B lymphocyte lineage. Haplotype or allelic exclusion restricts B lymphocytes to the expression of a single Ig receptor that can sustain further somatic modification. In most species, a specific Ig chain is encoded at a single genetic locus. However, in cartilaginous fish, hundreds of independent Ig heavy- (IgH) and Ig light-chain (IgL) gene loci are present, many of which are joined in the germ line. Ig gene transcripts have been amplified from single peripheral blood lymphocytes isolated from the clearnose skate (Raja eglanteria) using reverse-transcription PCR, and a single productive IgH transcript was detected in the majority of cells analyzed. Similarly, only a single IgL transcript was detected in over half of the individual cells. Taken together, these findings suggest that a mechanism for haplotype exclusion arose early in the evolution of antibody diversity and is independent of a single genetic locus., (Copyright 2004 Wiley-VCH Verlag GmbH & Co.)

The evolutionary origins of lymphocytes can be traced by phylogenetic comparisons of key features. Homologs of rearranging TCR and Ig (B cell receptor) genes are present in jawed vertebrates, but have not been identified in other animal groups. In contrast, most of the transcription factors that are essential for the development of mammalian T and B lymphocytes belong to multigene families that are represented by members in the majority of the metazoans, providing a potential bridge to prevertebrate ancestral roles. This work investigates the structure and regulation of homologs of specific transcription factors known to regulate mammalian T and B cell development in a representative of the earliest diverging jawed vertebrates, the clearnose skate (Raja eglanteria). Skate orthologs of mammalian GATA-3, GATA-1, EBF-1, Pax-5, Pax-6, Runx2, and Runx3 have been characterized. GATA-3, Pax-5, Runx3, EBF-1, Spi-C, and most members of the Ikaros family are shown throughout ontogeny to be 1) coregulated with TCR or Ig expression, and 2) coexpressed with each other in combinations that for the most part correspond to known mouse T and B cell patterns, supporting conservation of function. These results indicate that multiple components of the gene regulatory networks that operate in mammalian T cell and B cell development were present in the common ancestor of the mammals and the cartilaginous fish. However, certain factors relevant to the B lineage differ in their tissue-specific expression patterns from their mouse counterparts, suggesting expanded or divergent B lineage characteristics or tissue specificity in these animals.

Juvenile clearnose skates (Raja eglanteria) were injected intramuscularly with dexamethasone-21-phosphate at 50, 75, and 100mg/kg body weight. After 24h, skates were sacrificed and lymphomyeloid tissues (thymus, spleen, Leydig organ, and epigonal organ) were removed and whole blood was sampled. Tissues were used fresh for imprints or prepared for histology by solvent fixation or freezing in liquid nitrogen. Apoptosis in fixed tissues was assessed by transmission electron microscopy. Frozen sections and cytospin preparations of peripheral blood leukocytes (PBL) were evaluated by the TUNEL reaction to detect DNA strand breaks. Dexamethasone treatment increased apoptotic activity in all lymphomyeloid tissues as well as in PBL. These studies demonstrate that immune cells of elasmobranchs have the capacity for glucocorticoid-driven apoptosis, and that programmed cell death as a mechanism to regulate immune cell production appears to have been conserved during vertebrate evolution.

Cartilaginous fish express canonical B and T cell recognition genes, but their lymphoid organs and lymphocyte development have been poorly defined. Here, the expression of Ig, TCR, recombination-activating gene (Rag)-1 and terminal deoxynucleosidase (TdT) genes has been used to identify roles of various lymphoid tissues throughout development in the cartilaginous fish, Raja eglanteria (clearnose skate). In embryogenesis, Ig and TCR genes are sharply up-regulated at 8 weeks of development. At this stage TCR and TdT expression is limited to the thymus; later, TCR gene expression appears in peripheral sites in hatchlings and adults, suggesting that the thymus is a source of T cells as in mammals. B cell gene expression indicates more complex roles for the spleen and two special organs of cartilaginous fish-the Leydig and epigonal (gonad-associated) organs. In the adult, the Leydig organ is the site of the highest IgM and IgX expression. However, the spleen is the first site of IgM expression, while IgX is expressed first in gonad, liver, Leydig and even thymus. Distinctive spatiotemporal patterns of Ig light chain gene expression also are seen. A subset of Ig genes is pre-rearranged in the germline of the cartilaginous fish, making expression possible without rearrangement. To assess whether this allows differential developmental regulation, IgM and IgX heavy chain cDNA sequences from specific tissues and developmental stages have been compared with known germline-joined genomic sequences. Both non-productively rearranged genes and germline-joined genes are transcribed in the embryo and hatchling, but not in the adult.

Serum steroid hormones in the peripheral circulation of the clearnose skate, Raja eglanteria, were measured at the time of capture and at various times throughout the year while the animals were maintained as a captive breeding population. These analyses demonstrate interesting correlations between changes in hormone concentrations and annual reproductive events. Animals were sampled once (78 females, 20 males) or multiple times (15 females). For both groups of females, 17beta-estradiol was detected throughout the year with significant elevations occurring during October and November when ovarian follicles begin to mature (as determined through necropsy examinations), and January and February when maximum mating activity is observed and egg laying begins. Testosterone and dihydrotestosterone concentrations were significantly elevated in females only during January and February. Testosterone elevations were synchronous with longer-term elevations in 17beta-estradiol in females sampled either once or repetitively. Testosterone concentrations in males were significantly elevated during times of maximum breeding activity compared to periods of sexual inactivity. Data from females sampled during five stages of the egg laying process, as defined by the position of palpable egg capsules within the reproductive tract, revealed that 17beta-estradiol was highest when egg capsules were forming in the nidamental gland (stage 2) or uterus (stage 3); testosterone and dihydrotestosterone were maximal when eggs were in the uterus (stage 3) or cloaca (stage 4); and progesterone was significantly elevated immediately after oviposition (stage 5), suggesting a possible role for progesterone in the regulation of sequential laying of egg pairs. J. Exp. Zool. 284:575-585, 1999., (Copyright 1999 Wiley-Liss, Inc.)

Differential screening has been used to identify cDNAs encoding a long form of IgX in Raja eglanteria (clearnose skate). Comparisons of the IgX long form with the previously described short-form IgX cDNAs and the genomic IgX locus indicate that the V and two 5' C regions of the short and long forms of IgX are >90% identical at the nucleotide level. Differences between the V sequences of the long- and short-form IgX genes are concentrated in complementarity determining regions, suggesting that these forms are derived through alternative splicing of the same genomic loci or transcription of highly related loci. The extreme conservation of nucleotide sequence, including third position codons, among different cDNAs as well as the near identity of nucleotide sequence in the intervening sequences of germline IgX, IgX short-form sterile transcripts and IgX long-form sterile transcripts indicate that the multiple IgX loci are recently diverged from one another and/or are under intense gene correction. Phylogenetic analyses of the known cartilaginous fish immunoglobulin loci demonstrate that the long form of IgX is orthologous to IgW/IgNARC (NARC) and is most consistent with: 1) the divergence of the IgX/IgW/NARC and IgM-like loci from a common ancestral locus prior to the divergence of the cartilaginous/bony fish lineages and 2) the divergence of the NAR locus from the IgX/IgW/NARC gene(s) after the cartilaginous/bony fish split but prior to the shark/skate split, approximately 220 million years ago.

This study examined the response properties of skate electrosensory primary afferent neurons of pre-hatch embryo (8-11 weeks), post-hatch juvenile (1-8 months), and adult (> 2 year) clearnose skates (Raja eglanteria) to determine whether encoding of electrosensory information changes with age, and if the electrosense is adapted to encode natural bioelectric stimuli across life history stages. During ontogeny, electrosensory primary afferents increase resting discharge rate, spike regularity, and sensitivity at best frequency. Best frequency was at 1-2 Hz for embryos, showed an upwards shift to 5 Hz in juveniles, and a downward shift to 2-3 HZ in adults. Encapsulated embryos exhibit ventilatory movements that are interrupted by a "freeze response" when presented with weak uniform fields at 0.5 and 1 Hz. This phasic electric stimulus contains spectral information found in potentials produced by natural fish predators, and therefore indicates that the embryo electrosense can efficiently mediate predator detection and avoidance. In contrast, reproductively active adult clearnose skates discharge their electric organs at rates near the peak frequency sensitivity of the adult electrosensory system, which; facilitates electric communication during social behavior. We suggest that life-history-dependent functions such as these may shape the evolution of the low-frequency response properties for the elasmobranch electrosensory system.

Volume expansion of cardiac cells from a wide variety of species stimulates the efflux of the beta-amino acid taurine through an osmolyte channel. Previous studies have suggested that the osmolyte channel in epithelial cells is a swelling-activated anion channel (pICln). In skate heart, a 37-kDa protein is present which is recognized by a specific antibody to a protein characterized in MDCK cells as pICln. This protein is present predominantly in the cytosol (only 10% in the membrane fraction) of heart incubated under isotonic conditions. After transfer to hypotonic medium (one-half osmolarity), the distribution of this protein is markedly altered and significant amounts of the protein are found in the membrane fraction. After hypotonic exposure, the amount of the protein in the membrane fraction rises to 38 +/- 11% (range 18-53, n = 3). The translocation to the membrane fraction suggests that this protein may play a role in the taurine efflux in this tissue stimulated by hypotonic stress.

The clearnose skate (Raja eglanteria) develops in an almost opaque eggcase and lays its eggs in pairs. One sibling from each of eight pairs of skates was removed from its eggcase during embryonic development, while the other sibling developed inside the eggcase. The refractive development of the eyes at hatching was examined to see if ambient light exposure during embryonic development could influence the refractive states of hatchlings. Measurements included refractive states, ocular dimensions and lens focal properties. The differences in measurements between the two groups were not significant, which would indicate that environmental light does not influence the refractive development of the embryonic skate eye.

The olfactory organ of juvenile clearnose skates (Raja eglanteria) was studied with the light and electron microscopes. The organ is ovoid in shape, and its free surface is complicated by the presence of some 20 lamellae. Each lamella has a folded surface lined by a typical neurosensory olfactory epithelium. Bipolar olfactory receptor neurons, ciliated sustentacular cells, and basal cells are the pre-eminent cellular components of the epithelium. Two types of receptor neurons, both bearing microvilli but not cilia, were identified. The type 1 neuron is similar to that previously described in other fishes. The type 2 neuron has a characteristic morphology justifying a separate description. Its dendritic knob is larger than that of type 1, and its microvilli, which are shorter and thicker, are straight and regularly arranged. Tight bundles of filaments provide a skeleton to each microvillus, and these filament bundles reach more than 5 microns down into the dendrite. Type 2 receptor neurons have a well-developed Golgi complex and sparse rough endoplasmic reticulum (rER), whereas type 1 receptor neurons have a less well-developed Golgi complex and a conspicuous system of rER lamellae. The mucous layer on the epithelial surface is provided by the secretion of goblet cells that are situated mostly in the peripheral regions of each lamella. Secretory granules in the sustentacular cells and glands in the lamina propria were not observed.

Embryos of the clearnose skate, Raja eglanteria, develop in sea water at 20-22 degrees C, hatching after 82 +/- 4 days (Luer and Gilbert, Environ. Biol. Fishes, 13:161-171, 1985). Eyes develop as steadily enlarging spheres whose corneas have the same radius of curvature as the sclera. The cornea begins development as a 2-cell thick epithelium beneath which by Day 12 there is only a basal lamina and a wispy matrix separating it from the underlying lens. This matrix, modified by Day 16, is displaced on Day 22 by a few orthogonal plies of fibrillar primary stroma. Ply number increases to at least 13 by Day 30, reaching the final number of 20 +/- 2 by Day 42. Stromal fibroblasts (keratocytes) appear at the corneal periphery by Day 22, and in increased numbers by Day 30, a time at which no keratocytes are seen in the central stroma. However, by Day 40, many fibroblasts are present at the corneal periphery, invading the primary stroma between plies, occasionally reaching even the central cornea. By Day 53, keratocytes are present between all plies, from corneal periphery to center. Thickness of each ply in this secondary stroma increases, but the number of plies remains the same as in the primary stroma. Bowman's layer, non-invaded matrix beneath the epithelial basal lamina, is not evident until Day 53. Sutural fibers, first seen on Day 22, originate in the corneal epithelial basal lamina, traversing perpendicularly the plies of the primary stroma. Sutural fibers persist throughout development of the secondary stroma and into adulthood. In contrast to chicks, skate corneas remain transparent throughout development, and never form an endothelium.

Morphogenesis of the clearnose skate, Raja eglanteria, was not significantly inhibited as a result of 7 days of exposure to 1-2 mM selenate in the sea water during Days 59-69 of embryonic development (hatching would normally have occurred at 82 +/- 4 days of incubation). Although corneal transparency appeared normal in the eye, preliminary measurements of the thickness of Bowman's layer of the cornea suggested that it was significantly thinner in the corneas of embryos exposed to 1-2 mM selenate. Selenate is an ion reported to inhibit sulfation of glycosaminoglycans in connective tissue.

The shape and focal properties (spherical aberration) of the ocular lens of hatchling, yearling and adult clearnose skates (Raja eglanteria) were examined. In contrast to the spherical shape of the typical teleost lens, the lens of the clearnose skate is slightly aspherical; equatorial diameter being greater than axial diameter. The asphericity is approximately equal, in absolute measures, at all ages and is thus more obvious in younger skates. Lens spherical aberration is well corrected at all three levels of development. Yearling and adult lenses have relative focal lengths (Matthiessen's Ratio) which are close to the representative value of 2.55:1 expected for the teleost lens. However, the relative focal length of lenses from hatchling lenses is higher (about 2.75:1). This value approaches the upper limit of reports dealing with teleost lens focal characteristics. The difference between hatchling lenses and those of adult and yearling lenses may be due to the absence of extensive compression of central lens tissue by peripheral growth at early stages in development.

1. A 14.5 kDa fatty acid binding protein was isolated from the liver of the nurse shark, Ginglymostoma cirratum. 2. Purified shark liver FABP (pI = 5.4) bound oleic acid at a single site with an affinity similar to that of mammalian FABP. 3. The apparent size, pI and amino acid composition of shark liver FABP indicate a close structural relationship between this protein and mammalian heart FABP.

The conformation of Escherichia coli ribosomal protein L7/L12 in solution has been studied using spectroscopic and hydrodynamic methods. Circular dichroism studies in the near-ultraviolet region reveal two bands at 262 and 268 nm originating from the tertiary conformational environment of the phenylalanyl residues. Additional characterization of the phenylalanine environment includes an intrinsic fluorescence emission spectrum arising from the phenylalanine fluorophores. Computer analysis of the far-ultraviolet circular dichroism spectrum suggests that L7/L12 contains as much as approximately 76% alpha helix. Hydrodynamic properties of L7/L12, measured with the purpose of providing relevant shape information, include the frictional coefficient ratio (1.84 +/- 0.03) and intrinsic viscosity (28 +/- 0.4 mL/g). The experimentally determined frictional coefficient (6.15 +/- 0.15 X 10(-8) has been compared with theoretical calculations of the same value employing two independent methods and assuming various dimensions for the L7/L12 dimer. Combining the experimental results from this work with those available from the literature, and using conformation predictive methods of Chou & Fasman [P. Y. Chou & G. D. Fasman (1974) Biochemistry 13, 211-222, 222-245] and of Maxfield & Scheraga (F. R. Maxfield & H. A. Scheraga (1976) Biochemistry 15, 5138-5153), several possible molecular models of the L7/L12 dimer have been constructed and critically examined. A model which is consistent with all of the available data is proposed.

The effects of pH, temperature, and guanidinium chloride on the conformation of ribosomal protein L7/L12 have been investigated in order to understand the stability of this protein dimer. The results indicate that many of the molecular forces stabilizing the conformation of the dimer are disrupted at low pH or high temperature. These acid- and thermal-denatured states, however, still retain considerable secondary structure. Approximately half of the alpha-helical content present in the native protein remains intact at pH below 2 and at temperatures above 90 degrees C. Further denaturation of the acid-denatured protein by 6 M guanidinium chloride results in a state which still contains approximately 20% alpha helix. Similar amounts of residual conformation remain when the native L7/L12 dimer is denatured with guanidinium chloride. Thermodynamic analysis of the conformational transitions studied indicates that none is compatible with a simple two-state process. The complexity of these denaturation data and the structural characterizations of the various denatured states are consistent with the possible existence of structural domains in the protein molecule possessing different conformational stabilities.

1. Liver postmitochondrial supernatant preparations of calf, clearnose skate, and nurse shark were able to metabolize the fungal toxin aflatoxin B1 to various metabolites. 2. Calf liver produced aflatoxin M1 and Q1 as the major chloroform soluble metabolites, with small amounts of aflatoxicol formed during incubation. 3. Liver preparations of the elasmobranchs, however, produced aflatoxicol as the major chloroform soluble metabolite with no other metabolite being detected. 4. The water soluble metabolite profiles for the three species were also quite different with the tris diol adduct being produced to a much greater extent in calf liver preparations. 5. Aflatoxicol production by the elasmobranch liver homogenates was reversible with the skate reconverting a large amount (30%) of aflatoxicol to AFB1. The nurse shark, however, appeared to convert a portion of aflatoxicol to an unknown metabolite more polar than AFB1. 6. Calf liver DNA bound approximately 3 x more 3H-AFB1 than shark liver DNA.

The effects of pH and temperature on the structure of human erythrocyte membranes were studied by circular dichroism (CD). The results obtained demonstrate that the membrane CD spectra undergo significant changes when the pH of the solution deviates from its native pH range of 7 to 8. Spectral changes in the acidic pH region include drastic reductions and slight shifts in the CD signal which may reflect a decrease in alpha-helical content of the proteins and/or an increase in optical artifacts, both of which are irreversible. In the alkaline pH region, dramatic increases in ellipticity and blue-shifts in the spectra are observed between pH 8 and 10. In addition, the spectra more closely resemble those reported for membrane samples where the spectral distortions have been removed. The changes in the alkaline region are demonstrated to be only partially reversible and may be due to conformational alterations in the membrane proteins and/or to a reduction in optical distortions. Thermal stability studies reinforce the irreversible behavior of the membrane samples.

Liver microsome preparations of the elasmobranchs Ginglymostoma cirratum (nurse shark) and Raja eglanteria (clearnose skate) were examined for monooxygenase activity using aflatoxin B1 as substrate. At equiprotein concentrations, elasmobranch microsomes were less than 20% as active as calf liver in producing mutagenic metabolites of aflatoxin B1.