Evidence for independent recruitment of zeta-crystallin/quinone reductase (CRYZ) as a crystallin in camelids and hystricomorph rodents

Zeta-crystallin/quinone reductase (CRYZ) is an NADPH oxidoreductase expressed at very high levels in the lenses of two groups of mammals: camelids and some hystricomorph rodents. It is also expressed at very low levels in all other species tested. Comparative analysis of the mechanisms mediating the high expression of this enzyme/crystallin in the lens of the Ilama (Lama guanacoe) and the guinea pig (Cavia porcellus) provided evidence for independent recruitment of this enzyme as a lens crystallin in both species and allowed us to elucidate for the first time the mechanism of lens recruitment of an enzyme- crystallin. The data presented here show that in both species such recruitment most likely occurred through the generation of new lens promoters from nonfunctional intron sequences by the accumulation of point mutations and/or small deletions and insertions. These results further support the idea that recruitment of CRYZ resulted from an adaptive process in which the high expression of CRYZ in the lens provides some selective advantage rather than from a purely neutral evolutionary process.      

Evidence of independent gene duplications during the evolution of archaeal and eukaryotic family B DNA polymerases

Eukaryotes and archaea both possess multiple genes coding for family B DNA polymerases. In animals and fungi, three family B DNA polymerases, alpha, delta, and epsilon, are responsible for replication of nuclear DNA. We used a PCR-based approach to amplify and sequence phylogenetically conserved regions of these three DNA polymerases from Giardia intestinalis and Trichomonas vaginalis, representatives of early-diverging eukaryotic lineages. Phylogenetic analysis of eukaryotic and archaeal paralogs suggests that the gene duplications that gave rise to the three replicative paralogs occurred before the divergence of the earliest eukaryotic lineages, and that all eukaryotes are likely to possess these paralogs. One eukaryotic paralog, epsilon, consistently branches within archaeal sequences to the exclusion of other eukaryotic paralogs, suggesting that an epsilon-like family B DNA polymerase was ancestral to both archaea and eukaryotes. Because crenarchaeote and euryarchaeote paralogs do not form monophyletic groups in phylogenetic analysis, it is possible that archaeal family B paralogs themselves evolved by a series of gene duplications independent of the gene duplications that gave rise to eukaryotic paralogs.      

Severe hepatotoxicity caused by the tropical cyanobacterium (blue-green alga) Cylindrospermopsis raciborskii (Woloszynska) Seenaya and Subba Raju isolated from a domestic water supply reservoir.

Cylindrospermopsis raciborskii, a tropical blooming species of cyanobacterium (blue-green alga), was isolated from the domestic water supply reservoir on Palm Island, a continental island off the tropical northeast coast of Australia. This species, not previously known to be toxic, was shown to be severely hepatotoxic for mice. The 50% lethal dose at 24 h after injection was found to be 64 +/- 5 mg of freeze-dried culture per kg of mouse. The principal lesion produced was centrilobular to massive hepatocyte necrosis, but various degrees of injury were also seen in the kidneys, adrenal glands, lungs, and intestine. The possible implication of this finding in relation to an incident of hepatoenteritis in humans living on the island is discussed.     

Inhibition by low concentrations of ouabain of prolactin-induced lactogenesis in rabbit mammary-gland explants

1. Explants of mammary tissue from pseudopregnant rabbits were cultured at 37 degrees C in air for 24-48h in Medium 199 buffered with 20mm-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid]. The medium contained insulin and corticosterone, or insulin, corticosterone and sheep prolactin in the presence or absence of ouabain, an inhibitor of Na(+)/K(+)-dependent adenosine triphosphatase. The responses of explants were assessed histologically, by measuring the tissue concentration of K(+), and by rates of synthesis of RNA, protein and fatty acids. The effect of ouabain on Na(+) and K(+) concentrations in slices of lactating rabbit mammary-gland tissue incubated for 1h at 37 degrees C in Krebs bicarbonate buffer was also studied. 2. Prolactin increased the concentration of K(+) in mammary explants, an effect prevented by ouabain. In slices of lactating tissue, there was a linear relationship between the log dose of ouabain (from 0.1 to 10mum) and increased Na(+) and decreased K(+) concentrations in the tissue. 3. Ouabain at concentrations up to 1mum did not affect the rate of synthesis of RNA, protein or fatty acids by explants cultured with insulin and corticosterone. By contrast, the stimulatory effect of prolactin on protein synthesis was diminished and the induction of medium-chain fatty acid synthesis by prolactin was almost abolished. RNA synthesis was unaffected. Histological examination showed no tissue damage by 1mum-ouabain. 4. Explants cultured in the presence of 2mum-ouabain for 24h retained their ability to respond to prolactin when the ouabain was removed from the culture medium. Between 24 and 48h they showed responses to prolactin of a magnitude similar to those of explants never exposed to ouabain. 5. These results show that a fully functional Na(+)/K(+)-dependent adenosine triphosphatase system is necessary for prolactin to promote secretory activity in rabbit mammary gland.     

Studies on the congenitally goitrous sheep. The iodinated compounds of serum, and circulating thyroid-stimulating hormone

1. A group of normal and congenitally goitrous Merino sheep were investigated to identify the metabolic defect present in the abnormal animals. 2. Protein-bound iodine concentrations of serum from goitrous animals (average 5·7μg./100ml.) were higher than normal (average 4·2μg./100ml.; P 0·001), but the hormonal iodine measured as butanol-extractable ¹³¹I was low in the serum of goitrous (average 40·3% of protein-bound ¹³¹I) compared with that of normal (84·2%; P 0·02) sheep. The non-hormonal iodine of the serum of goitrous sheep appeared to include iodotyrosines and iodinated protein. 3. Starch-gel-electrophoretic separations of sera from normal and goitrous sheep after ¹³¹I injection (100–500μc) showed no qualitative differences in the radioactivity of protein components. No significant differences in thyroxine-binding in vitro by serum proteins of normal and goitrous sheep were observed. 4. The clearance rates of ¹³¹I-labelled iodotyrosines (t_½ 1·2–2·9hr.) and iodothyronines (t_½ 33·5–47·4hr.) were similar in normal and goitrous sheep. 5. The concentration of circulating thyroid-stimulating hormone was significantly higher (P<0·01 in three sheep, P<0·05 in one sheep) in goitrous sheep. 6. The congenital goitre appears to be due to compensatory hypertrophy of the gland resulting from an inability to synthesize an adequate supply of thyroid hormone.     

Applications of isothermal titration calorimetry – the research and technical developments from 2011 to 2015

Isothermal titration calorimetry is a widely used biophysical technique for studying the formation or dissociation of molecular complexes. Over the last 5 years, much work has been published on the interpretation of isothermal titration calorimetry (ITC) data for single binding and multiple binding sites. As over 80% of ITC papers are on macromolecules of biological origin, this interpretation is challenging. Some researchers have attempted to link the thermodynamics constants to events at the molecular level. This review highlights work carried out using binding sites characterized using x‐ray crystallography techniques that allow speculation about individual bond formation and the displacement of individual water molecules during ligand binding and link these events to the thermodynamic constants for binding. The review also considers research conducted with synthetic binding partners where specific binding events like anion‐π and π‐π interactions were studied. The revival of assays that enable both thermodynamic and kinetic information to be collected from ITC data is highlighted. Lastly, published criticism of ITC research from a physical chemistry perspective is appraised and practical advice provided for researchers unfamiliar with thermodynamics and its interpretation. Copyright © 2016 John Wiley & Sons, Ltd.     

Variations in lymphokine generation by individual lymph nodes draining human malignant tumors

Summary Individual lymph nodes draining tumors vary in their degree of immunological activity. Cell suspensions from tumor-free nodes located relatively near to tumors are spontaneously less reactive and respond poorly to exogenous stimulation by mitogens and lymphokines. Diminished spontaneous uptake of tritiated thymidine by lymph node cells not exposed to exogenous stimulation suggests that tumor-proximate immune suppression exists in vivo and is not purely a laboratory artefact. The present study was undertaken to explore that possibility further. Fluid in which cell suspensions from tumor-free nodes were prepared, and supernatants from short-term cultures of nodes located at different distances from tumors were compared for their capacity to inhibit the in vitro migration of the human lymphoblastoid cell line QIMR-WIL. Inhibitory activity of fluids from individual nodes was related to their position relative to the tumor and their immune competence, assessed by the responses to mitogens of cell suspensions prepared from them. Cell suspension fluids from 92/111 nodes (83%) significantly inhibited the migration of QIMR-WIL, at a level similar (44±14%) to that induced by the supernatants of mixed lymphocyte cultures (43±17%). Fluids from the nodes of melanoma patients were more inhibitory than those from breast cancer patients (49±12% and 37±13%, respectively,P = 0.003). The inhibitory activity of the different nodes of individual node groups varied significantly in 25 of 33 patients (76%), the node nearest the tumor generating least inhibitory activity (indexing the greatest immune suppression) in 20 of these 25 patients (80%). The strength of migration-inhibitory activity was concordant with the responsiveness to mitogen stimulation in up to 14 of 18 patients (78%). Studies of molecular size and heat stability indicated that the inhibitory factors had characteristics consistent with common migration-inhibitory lymphokines such as leukocyte-migration-inhibitory factor, macrophage-inhibitory factor and interleukin-2. Our findings further support the hypothesis that lymph nodes nearest to tumors are relatively immune-suppressed in vivo.Supported by grants CA 29938 and CA 43658, awarded by the National Cancer Institute, DHHS and a grant from the Candle Foundation, Los Angeles