Functional consequences of ligand-linked dissociation in hemoglobin from the sea cucumber molpadia arenicola

It has been established that Molpadia hemoglobin tends to dissociate into subunits as oxygen is bound. The kinetics and equilibria of carbon monoxide and ehtylisocyanide binding reported here show a dependence on protein concentration that supports the conclusions that the aggregated hemoglobin has a lower ligand affinity than the dissociated subunits. This is true for the isolated D-chain as well as for the unfractionated hemolysate that contains four distinct polypeptide chains (A-D). This indicates that even homopolymers of Molpadia hemoglobin have lower ligand affinity than the dissociated subunits. At high protein concentration hemolysates of Molpadia hemoglobin show slight cooperativity. The time course of ligand binding to the deoxy D-chain also suggests cooperative interactions, The low affinity of the aggregated state may have a different molecular explanation than in human hemoglobin were tetramers of identical subunits (as in Hb H) show high oxygen affinity. The absence of tyrosine and histidine at the C-tremini of the Molpadia D-chains also suggests a different stabilization of the low affinity deoxy state. An additional functional difference between Molpadia hemoglobin and human hemoglobin is that organic phosphate do not alter the ligand affinity of the sea cucumber hemoglobin.     

Testosterone and photoperiod interact to regulate locomotor activity in male hamsters

Testicular size, plasma testosterone levels, copulatory behavior, and daily locomotor activity are reduced in male hamsters after 10 weeks of exposure to short days. The role of testosterone in the short day-induced decline in locomotor activity was investigated, determining whether or not photoperiod could alter the effect of testosterone on activity. Castrated adult hamsters were allowed to acclimate to running wheels (wired to digital counters) and then were kept on either long (L:D 14:10) or short (L:D 6:18) days for 60 days. On Day 60, half of the animals on each light cycle were implanted with 12-mm-long testosterone-filled Silastic capsules; half received empty capsules. Digital counting of wheel-running activity continued for another 140 days. Blood samples taken on Day 200 confirmed L:D 14:10 and L:D 6:18 testosterone-treated hamsters had equivalent plasma testosterone levels. After an initial decline in activity, L:D 14:10 animals exhibited a progressive rise in mean running activity (from ~2000 to ~5000 wheel revolutions per day) through 100 days after the initiation of testosterone treatment. In contrast, activity levels in testosterone-treated L:D 6:18 animals remained uniform (~2000 wheel revolutions per day) during this time, indicating exposure to short days rendered the hamsters less sensitive to the stimulatory effect of testosterone on activity. Of further interest was a marked increase in activity after 160–200 short days in animals treated with either testosterone-filled or empty capsules. It appears the total amount of daily locomotor activity in the hamster is modulated by circulating testosterone levels in a manner which is dependent upon the environmental photoperiod.     

The effects of salinity,and calcium and potassium variations on the growth of two estuarine red algae

The effects of varying salinity and calcium and potassium concentrations on the growth of two species of estuarine Rhodophyta, Bostrychia radicans Montagne and Caloglossa lepricurii (Montagne) J. Agardh, were examined in unialgal culture. Inocula of settled tetraspores on glass coverslips were incubated in six concentrations of potassium (0. 100, 200, 300, 400 and 500 mg/l) at five salinities (0, 5, 15, 25, and 35%) in the presence of calcium. Growth responses of each alga were determined from the average cell number of 75 tetrasporelings after 4 days in Ott's synthetic sea-water medium. The concentrations of dissolved potassium and calcium in sea water along a salinity gradient in an estuary were determined with atomic absorption spectrophotometry. Four-day-old tetrasporeling of Bostrychia and Caloglossa demonstrated growth over wide ranges of potassium concentrations with growth maxima at 400 500 and 200 400 mg/l, at the optimal salinities for growth of 25 and 15%, respectively. These studies indicate also that the presence of calcium is essential for appreciable growth of both species at each salinity and the effects of variations in potassium are dependent upon the presence of calcium. The abundance of both species in the Mullica River estuary. New Jersey, appears to be a response to a total osmotic effect along the salinity gradient because sufficient levels of the major cations are present. However, the upper limit of both species towards the head of the Mullica River estuary may be determined by combinations of low salinity and low levels of dissolved calcium and potassium rather than by low salinity alone.     

The histology and fine structure of the olfactory organ of the squid Lolliguncula brevis Blainville.

The olfactory organ of the squid has a thick, pseudostratified epithelium containing five morphological types of ciliated receptors. In the simplest receptors the cilia originate separately in the distal pole of the cell. All other receptors have some type of cilia filled cavity, varying from a simple pocket of cilia at the surface to a completely closed vesicle filled with cilia in cells deep in the epithelium. The receptors are compared to cells in the rhinophore of Nautilus and the olfactory organs of coleoid cephalopods. Possible functions of the olfactory organ, based on its morphology, are discussed.     

The different effects on courtship of volatile compounds from mated and virgin Drosophila females

Virgin Drosophila melanogaster females, which are courted vigorously, emit pheromones which stimulate males to court each other (Tompkinset al., 1980). Females which have recently copulated are courted less vigrously, and volatile compounds produced by mated females stimulate less courtship between males. Analysis of these compounds from fertilized females by gas chromatography and behavioral assays indicates that mated females emit less of the sex attractant made by virgins and may also produce material which inhibits courtship. These changes in pheromone production are initiated after the first few minutes of copulation.     

Intestinal protein dynamics in the stonefly, Pteronarcys californica

Characteristics of the intestinal proteins of a detritus-eating aquatic insect, Pteronarcys californica were assessed to determine their possible role as a nutrient source during metamorphosis. Total soluble intestinal protein was significantly correlated to larval weitht, averaging 3.9% of dry weight for both males and females. Proteinase activity in the gut was separated into several bands by electrophoresis. Electrophoresis patterns of total intestinal proteins of P. californica larvae of differing instar, sex, and diet were remarkably similar. Larvae incubated with tritium-labelled amino acids produced labelled intestinal proteins. When early instar larvae were removed from label, there was no significant loss of tritium from the intestine after 2 months. Penultimate instars labelled in the same manner however, showed depuration of tritium from the gut according to first order kinetics. This loss was correlated to the 6–8 wk non-feeding stage of metamorphosis, just prior to adult emergence. These results indicate that proteins are accumulated in the intestinal tract of immature P. californica, then are reabsorbed from the gut during metamorphosis.     

Villin is a major protein of the microvillus cystoskeleton which binds both G and F actin in a calcium-dependent manner

The cores of the microvilli present on intestinal epithelial cells are currently the only microfilament arrangement which can be isolated ultrastructurally intact and in sufficient quantities for biochemical analysis. We have isolated and characterized villin, a major protein of the microvillus core. Using villin's ability to bind very tightly to immobilized monomeric actin in a calcium-dependent manner, we have developed a method for its rapid purification by affinity chromatography on G actin, which itself was bound to immobilized pancreatic deoxyribonuclease I (DNAase I). The villin-G actin complex on DNAase I is resistant to high ionic strength, and villin, but not actin, is released when the calcium concentration is less than 10⁶ M. Purified villin behaves as a globular monomeric protein of molecular weight 95,000, and is free of carbohydrate. Villin also interacts with F actin. In the absence of calcium, villin cross-links F actin having the properties of an F actin bundling or gelation factor. In the presence of calcium (>10^?7 M), villin apparently restricts the polymerization of actin to short filaments which cannot be readily sedimented. The properties of villin are not compatible with its previously suggested role as the cross-filament between the microvillus microfilament core and the plasma membrane, but rather indicate a function as a calcium-dependent F actin-bundling protein. The role of villin is discussed in terms of the other protein components of the microvillus core and in relation to recently described calcium-dependent gelation factors.     

Crystallographic analysis of the phytoagglutinin from Abrus precatorius by X-ray diffraction and electron microscopy

The lectin from the seeds of Abrus precatorius has been crystallized and the crystals subjected to study by X-ray diffraction and electron microscopy. Three closely related crystal forms were obtained, of orthorhombic space group P2₁2₁2₁ with $\text{a = 138}\text{A}\text{?}$, $\text{b = 142}\text{A}\text{?}$, and $\text{c = 173}\text{A}\text{?}$, of tetragonal space group P4₁2₁2 with $\text{a = b = 136}\text{A}\text{?}$, $\text{c = 176}\text{A}\text{?}$, and a twinned intermediate of the first two. From electron microscopy and two-dimensional spatial filtering of electron micrographs of the crystals, the molecule appears to consist of four similar domains grouped in a roughly planar diamond-shaped arrangement having a local intramolecular dyad axis. The average diameter of the Abrus lectin molecule is 50 to 60 Å and the individual domains appear to have a diameter of about 25 Å.     

RNA synthesis in imaginal disks of Galleria mellonella: Effects of alpha- and beta-ecdysone and fat body in vitro

The effects of alpha-ecdysone (α-E), beta-ecdysone (β-E), and larval fat body on morphogenesis and total RNA synthesis in wing imaginal disks of Galleria mellonella were studied. Both ecdysones induce morphogenesis of disks in vitro. Alpha-ecdysone and β-E (0·3–3·0 μg/ml) stimulate RNA synthesis 30 and 60 per cent above control levels, respectively. While less α-E (0·3 μg/ml) is required to increase RNA synthesis than to induce morphogenesis, the reverse is true for β-E. Morphogenesis (i.e. tracheole migration and evagination) can proceed in the presence of concentrations of β-E (0·03 μg/ml) that are subthreshold for the induction of RNA synthesis (0·3 μg/ml). We conclude, therefore, that the increase in total RNA (presumbly ribosomal) is unrelated to and not a prerequisite for tracheole migration or evagination. If morphogenically active preconditioned medium (i.e. medium in which α-E and fat body have been incubated for 48 hr and the fat body then removed) is added to disk cultures, RNA synthesis is not stimulated. Apparently, increases in total RNA caused by both ecdysones may not be necessary for early in vitro disk development. The independent nature of some ecdysone-induced events and implications of our conclusions are discussed.     

Age-associated changes in plasma testosterone levels in male mice and their relation to social dominance or subordinance

Plasma testosterone (T) levels were determined in male mice of the CD-1 (ICR) strain from 30 to 680 days of age. The mean plasma T was 5.2 ± 1.0 ng/ml for adult male mice (70–400 days old, 102 mice) and 1.8 ± 0.9 ng/ml for old males (450–680 days old, 11 mice). Through 140 determinations, the highest T value obtained was 52.3 ng/ml in an 80-day-old individual while the lowest was 0.08 ng/ml in a 680-day-old animal. It was found that plasma T was significantly decreased in the old age, although individual variation in T levels was considerable. On the other hand, social dominance or subordinance was examined among male mice in each cage and its relation to plasma T was investigated. Dominance or subordinance was verified by frequency of chases or attacks delivered or received, or number of fights won or lost. It was observed that dominant-subordinate relationships were present in 6 out of 10 cages and that the dominant individual had a significantly higher T level than the subordinate male; the mean T level was 10.5 ± 2.5 ng/ml for the dominant individuals vs 2.2 ± 0.8 ng/ml for the subordinate ones. Marked individual variation in plasma T titers in male mice seems to be related to the dominance/subordinance rank within a group.     

Deficiency of immunoreactive somatostatin in the median eminence of Snell dwarf mice

Snell dwarf mice (dw/dw) are characterized by a genetically determined, congenital lack of pituitary GH, TSH and prolactin. Given that hypothalamic somatostatin is involved in the regulation of pituitary GH and TSH release, it was decided to investigate the content of immunoreactive somatostatin (IRS) in the median eminence of dw/dw and phenotypically normal mice of the same strain. The content of IRS in the pyloric antrum and pineal gland of these animals was also examined. The effects of ovariectomy and of hyperprolactinemia (induced by a pituitary graft under the kidney capsule) on the median eminence content of IRS were also studied in both normal and dwarf mice. Median eminence IRS content was significantly lower in the dw/dw (23.6 +/- 1.8 ng) than in normal mice (57.4 +/- 7.1 ng); no difference was found in the pyloric IRS content of dw/dw (16.9 +/- 1.6 ng/mg of protein) and normal animals (13.8 +/- 1.9 ng/mg of protein), nor in the pineal content of IRS (639.4 +/- 64.4 pg/gland in the dw/dw; 732 +/- 265 pg/gland in normals). Neither ovariectomy nor hyperprolactinemia were found to affect the IRS content in the tissues studied in normal or dwarf mice. Treatment of an additional group of 9 dwarf mice with L-thyroxine (L-T4 2 micrograms/48 h. s.c. for 2 weeks) significantly increased the animals weight (10.2 +/- 0.4 g versus 7.4 +/- 0.3 g) and produced maturation of facial features; however, it did not change the IRS content in any of the tissues studied. It is concluded that the content of IRS in the median eminence of mice with a congenital lack of GH, TSH and prolactin is significantly reduced and that this is unlikely to be related to the deficiency of thyroid hormones in these animals.     

Worldwide distribution of the conjugative Clostridium perfringens tetracycline resistance plasmid, pCW3

The aim of this study was to test the hypothesis that all conjugative R-plasmids of Clostridium perfringens are closely related to the previously characterized tetracycline resistance plasmid, pCW3. Fourteen conjugative R-plasmids derived from 11 C. perfringens strains isolated in Australia, the United States, France, Belgium, and Japan were analyzed. Eleven of the plasmids encoded tetracycline resistance while three carried both tetracycline and chloramphenicol resistance. Each of these plasmids was compared, by restriction analysis, to the reference plasmid, pCW3. Seven of the tetracycline resistance plasmids had EcoRI, XbaI, and ClaI restriction profiles that were identical to those of the corresponding pCW3 digests. The seven remaining R-plasmids were different from pCW3. Comparison of partial restriction maps of these plasmids with a complete map of pCW3 indicated that they contained at least 17 kb of DNA that also was present in pCW3. Hybridization analysis confirmed that these plasmids shared substantial homology with pCW3. The three tetracycline and chloramphenicol resistance plasmids frequently lost a 6-kb chloramphenicol resistance segment during conjugation. Cloning experiments showed that the chloramphenicol resistance determinant was expressed in Escherichia coli and that the chloramphenicol resistance gene of one of these plasmids, pIP401, was contained within a 1.5-kb region of the 6-kb deletion segment. Hybridization analysis indicated that the deletion segment of pIP401 was related to those of the other two chloramphenicol resistance plasmids. During the course of this study, conjugative R-plasmids which appear to be identical to pCW3 or closely related to pCW3 were identified from C. perfringens strains from human, animal and environmental sources in five countries. It is concluded that C. perfringens strains in humans and animals throughout the world have overlapping gene pools and that all the conjugative C. perfringens R-plasmids examined probably evolved from a pCW3-like element.     

A new theory on the origin and the nature of viruses

The hypothetical model presented herein concerns the origin and nature of viruses. It advances the possibility of the appearance and existence of an organism lacking a cohesive morphological structure, that is: its subsystems are not in structural continuity. An attempt to delimit the concepts of life and organism and to integrate the viruses into this framework is made. Viruses are presented as organisms which pass in their ontogenetic cycle through two distinctive phenotypic phases: (1) the vegetative phase and (2) the phase of viral particle or nucleic acid. In the vegetative phase, considered herein to be the ontogenetically mature phase of viruses, their component molecules are dispersed within the host cell. In this phase the virus shows the major physiological properties of other organisms: metabolism, growth, and reproduction. Therefore, life is an effective presence. It is shown also, that in this phase so called "DNA viruses" have both nucleic acids: RNA as well as DNA. The virions are considered to be "spores" or reproductive forms of the virus, possessing life only as a potential property.     

H-NMR of G-U-C and G-U-C-C in D2O: assignment of nonexchangeable protons and analysis of solution conformation

The ribonucleotide oligomers G-U-C and G-U-C-C have been synthesized enzymatically. These oligomers are cognates of the m⁷G⁴⁶-U⁴⁷-C⁴⁸-m⁵C⁴⁹ sequence found in the variable loop of t-RNA^phe. The ¹H-NMR chemical shifts of the base and ribose Hl; protons as well as the couplings. J_1'–2' of the ribose protons have been examined as a function of temperature. Assignments for these resonances have been completed, and used in the analysis of solution conformation for these oligomers. The results are consistent with the basic features of the A-RNA structure and suggest the absence of alternative ordered solution structures.     

A spectral shift in cytochrome a induced by calcium ions

Ca²⁺ induces a red shift in the absorption spectrum of ferrocytochrome a when added to uncoupled mitochondria, sub-mitochondrial particles or isolated cytochrome aa₃. The shift is identical within experimental error to the previously reported energy-linked shift in intact mitochondria (Wikström, M. K. F. (1972), Biochim. Biophys. Acta 283, 385–390). One mol of calcium produces the shift in one mol of cytochrome a, the K_D being approx. 20–30 μM. The calcium-induced shift is readily reversed by chelating agents such as EDTA, ethyleneglycol-bis-(μ-aminoethyl ether)N,N′-tetraacetic acid (EGTA) and ATP and is insensitive to uncoupling agents and inhibitors of calcium transport (La³⁺ and ruthenium red). It is shown that the binding site for calcium that is responsible for the spectral shift is located on the outside of the permeability barrier of the mitochondrial cristae membrane.It is proposed that calcium simulates the energy-linked shift in cytochrome a by binding to a site of cytochrome aa₃ that is occupied by protons in energized mitochondria and that is located at the external surface of the mitochondrial membrane.     

Development of the chick embryo mesoblast. Formation of the embryonic axis and establishment of the metameric pattern

The mesoblast of the primary organizer region of the developing chick embryo at the early head process stage was examined with the scanning electron microscope. It was found that the mesoblast layer is patterned from its inception at the primitive streak. Viewed dorsally, the mesoblast region most recently traversed by Hensen's node is metameric. Each segment consists of two 175-μm-diameter circular buttons of paraxial mesoblast (somitomeres) and an enclosed axial region. These tripartite segments are stacked tandemly and mark precisely, in the ectoderm above, the limit of neural plate formation. Viewed ventrally, the metameric pattern of the mesoblast is most closely mimicked by underlying endoblast, which shows corresponding radially arranged wedge-shaped cells in somitomere-sized circular patches. At this stage of development, each paraxial somitomere is a slightly hollowed, squat cylinder, composed of tapering mesenchymal cells whose long axes are directed toward the core center. Closely timed with neurulation, somitomeres undergo morphogenesis, being first converted to triangular wedges and, finally, condensed into cubes. Anteriorly, somitomeres participate in branchiomeric development, while posteriorly, they develop into somites. Examination of segmental plates shows that they consist of about 11 tandem somitomeres not visible by light microscopy. The most mature somitomeres, closest to the emerging somites, are delineated from one another by cellular orientations and the progressive buildup of fibrous extracellular matrix. The least mature somitomeres are not as well defined, but appear initially just posterior to Hensen's node and merge medially with the notochordal process. The observations suggest that the emergence of somitomeres from the paraxial mesoblast of the primitive streak is the result of its association with nodal cells. Further, this combined association of the mesoblast heralds primary induction and establishes the metameric pattern of the basic body plan.     

Interaction of poly(l-lysine) and Ca2+ with stearic acid monolayers

Interaction of poly(l-lysine) and Ca²⁺ with stearic acid monolayers is studied at pH 9.1, 9.9 and 10.7. The competition between the condensation effect of Ca²⁺ and the expansion effect of the protein on the monolayer is seen to depend on surface pressure as well as pH. Ca²⁺ is much less effective in the competition when the poly(l-lysine) penetration into the monolayer is stabilized by electrostatic interactions.     

Incorporation of specifically labeled cysteine into Escherichia coli protein

Protein obtained from several strains of Escherichia coli grown in the presence of [3,3′-¹⁴C]cystine contained the radiolabel in nearly all the other amino acids, suggesting catabolism of cysteine to pyruvic acid. Utilization in amino acid synthesis of the pyruvate thus generated can be blocked by growing the bacteria in a medium specifically enriched with most of the naturally occurring amino acids. Cysteine that is incorporated intact is diluted by de novo synthesis at low cystine concentrations; also, it was found that E. coli can use the sulfur of methionine for cysteine biosynthesis. Both of these latter two processes can be prevented by supplying an excess of exogenous cystine. This regiment leads to protein that is highly specifically labeled in the cysteine residues, with a minor amount (20–25%) of the label also appearing in alanine residues. Although this strategy was developed expressly for cysteine, it may be useful for incorporating other labeled amino acids that are also readily catabolized.     

Maintenance of the shore-level size gradient in the marine snail Tegula funebralis (A. Adams): Importance of behavioral responses to light and sea star predators

The intertidal gastropod, Tegula funebralis (A. Adams) exhibits a shore-level size gradient with mean shell size increasing in a down-shore direction. Snails transferred to zones where they do not usually occur migrated back towards their original zone, thus re-establishing a size gradient and implying differential movement among size classes. Both large (≥2.1 cm shell width) and small (≤ 1.77 cm) snails were photonegative on a horizontal surface and geonegative in the laboratory; there were no statistical differences between size classes. Light, however, inhibited upward, or caused downward, movement of large snails on vertical surfaces. Small snails were unaffected, ranging higher on illuminated vertical surfaces than large snails. Both sizes exhibited similar distributions in the dark. In an experimental chamber providing both emersed and immersed surfaces, T. funebralis established vertical size gradients when the chamber was illuminated from above. It is suggested that light is an important factor in the formation and maintenance of Tegula's shore-level size gradient.In response to water-borne chemicals derived from the sea star Pisaster ochraceus (Brandt), large snails moved up vertical surfaces in greater proportion than small. In response to contact with the predator, large snails moved away faster than small and individuals collected from crevices in the field moved away slower than those collected from open rock faces. Although predation may select for a size gradient in Tegulafunebralis, it is unlikely that responses to predatory sea stars directly and proximally cause or maintain them over the short term.