Magnetic Resonance of Isotope Hybrid Flavoprotein <Superscript>2</Superscript>H-Flavoprotein (<Superscript>1</Superscript>-Flavin Mononucleotide)

FLAVOPROTEINS lend themselves particularly well to study by the combined techniques of magnetic resonance and isotope substitution^1–3. Recent electron spin resonance (ESR) and electron-nuclear double resonance (ENDOR) investigations have already indicated the utility of this approach to the study of the structure and function of flavin and flavoproteins^4–8. We have now prepared an unusual isotope hybrid flavoprotein that gives promise of great value for magnetic resonance studies. This substance is obtained by the introduction (by exchange) of an ¹H-flavin mononucleotide (¹H-FMN) prosthetic group into (fully deuterated) ²H-flavoprotein that can be isolated from the blue-green thermophilic alga Synechococcus lividus. This isotope hybrid protein makes it possible to apply in a unique way proton magnetic resonance (PMR) and ESR to the chemistry of protein-bound FMN.     

Externally Suppressive +1 “Glycine” Frameshift: Possible Quadruplet Isomers for Glycine and Proline

WE have reported our original finding of frameshift suppression in Salmonella^1,2. The frameshift we studied initially was induced in the histidinol dehydrogenase (HDH) gene with the intercalating agent ICR-191 (ref. 3.) It is a +1 type most likely containing an extra C in an mRNA repeat of C residues². External suppressors are efficiently induced by ICR-191 (ref. 1). The suppressors restore small amounts of HDH with the normal amino-acid sequence to the mutant cell⁴. We have hypothesized a proline suppressor tRNA with a quadruplet (+G) anticodon or its functional equivalent^2,4. Prompted by our findings, Riddle and Roth showed that most frameshifts tentatively classified as +1 types by genetic criteria are externally suppressible. Almost all were induced with ICR-191 (ref. 5). Two classes of suppressible frameshift were found, each with a set of mutually exclusive suppressors⁵. Judging from the demonstrated capacity of ICR compounds to produce + 1 additions in DNA repeats of GC pairs, we have further suggested to Riddle and Roth that these two frameshift-suppressor systems represent +1 additions in RNA repeats of C residues (proline codons, glycine anticodons) and in RNA repeats of G residues (glycine codons, proline anticodons)⁴ (personal communication to J. R. Roth, Histidine Workshop, 1970); that is, the two types of +1 frameshift are genetic “isomers”, the one involving proline and the other glycine (Fig. 1). The recent demonstration by Riddle and Roth of altered proline tRNA and glycine tRNA in appropriate suppressed strains⁶ is consistent with this suggestion. Further characterization of frameshifts of the type originally investigated has implicated a proline mRNA quadruplet, CCCg, as a sufficient if not necessary condition for suppression^7,8. A requirement for neighbouring sequences, particularly chain terminating codons, cannot be completely ruled out, however⁸. I have now examined a suppressible frameshift of the second type and present evidence that it contains a +1 addition in or near a glycine codon (Fig. 2). Oddly enough, this mRNA site is followed by an extensive nucleotide sequence reminiscent of two out of three +1 “proline” sequences examined (Fig. 2)⁸. The ICR compounds seem to have a marked bias for inducing suppressible +1 frameshifts in this extensive sequence. Whether some property of this extensive sequence is crucial to suppression is not yet clear.     

Resistance of Calves to Reinfection with Eimeria bovis

SYNOPSIS. An immunity to reinfection with E. bovis was demonstrated in 3 experiments involving 60 calves. This immunity develops rapidly, as indicated by resistance to a challenge given 14 days after the immunizing inoculation. In 3 groups of 3 to 6 young calves each, immunity was still present to a moderate degree 2 to 3 months after inoculation; in one group of 5 animals about a year old there was apparently a high degree of immunity about 7 months after the last inoculation. In one experiment an immunizing inoculum of 10,000 oöcysts did not produce as much immunity as 50,000 oöcysts. In 2 experiments there appeared to be little difference in the immunity produced by a single inoculation of 50,000 as compared with 100,000 oöcysts, but inoculation with 100,000 oöcysts, resulted in substantially longer and more severe illness than 50,000 oöcysts. There appeared to be no appreciable difference in clinical symptoms or development of immunity between calves given a single immunizing inoculum and those given the same number of oöcysts in 5 equal inocula on successive days. Treatment with sulfamethazine and sulfamerazine (Merameth) 13 to 15 days after inoculation alleviated the clinical symptoms of coccidiosis without interfering appreciably with the development of immunity. In one experiment with 7 calves, no beneficial effect was noted from 1 or 2 transfusions of 500 ml. of plasma and leucocytes from immune calves into 4 calves 1 and 12 days or 11 days after a challenge inoculation.     

Effects of Osmotic Pressure on the Oxidative Metabolism of Leishmania major Promastigotes

Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer. The rate of oxidation of ¹⁴C-labeled substrates was then measured as a function of osmolality. An acute decrease in osmolality (achieved by adding H₂O to the cell suspension) caused an increase in the rates of ¹⁴CO₂ production from [6-¹⁴C]glucose and, to a lesser extent, from [1, (3)-¹⁴C]glycerol. An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of ¹⁴CO₂ production from [1-: ¹⁴C]alanine, [1-¹⁴C]glutamate, and [1, (3)-¹⁴C]glycerol. The rates of ¹⁴CO₂ formation from [1-¹⁴C]laurate, [1-¹⁴C]acetate, and [2-¹⁴C]glucose (all of which form [1-¹⁴C]acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality. These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress. Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.     

Lake restoration by biomanipulation: Round Lake, Minnesota, the first two years

SUMMARY. 1. Rotenone was applied to Round Lake in the autumn of 1980 in order to eliminate predominantly planktivorous and benthivorous fish. The lake was subsequently restocked with a higher population density of piscivores. The effect of this biomanipulation on the phytoplankton and zooplankton communities and on total nutrient concentrations was monitored at fortnightly intervals during the summers, from May 1980 to September 1982.
2. The abundance of phytoplankton was much lower after biomanipulation and was consistent with observed changes in Secchi disc transparency, total attenuation coefficient and chlorophyll a concentration. Zooplankters were also less abundant in 1981 and 1982 but the decrease in numbers was more than offset by the large increase in the mean sizes of the zooplankters present, so that the estimated grazing pressures in 1981 and 1982 were at least double the 1980 value.
3. Daphnia , rare in 1980, became the dominant genus in 1981 and 1982, and a shift to progressively larger-bodied Daphnia species was observed.
4. Although total nitrogen and total phosphorus levels were generally lower after biomanipulation, their decline could not explain the reduction in phytoplankton abundance which was attributed to the increased grazing pressure. Possible causes of the observed declines in nutrient concentrations are discussed.     

Sperm Incorporation Independent of Fertilization Cone Formation in the Danio Egg

The responses of the egg to insemination in a modified Fish Ringer's solution (FRS) were examined in eggs of the zebrafish ( Brachydanio rerio ) primarily by scanning electron microscopy. FRS is a physiological saline which temporarily inhibits parthenogenetic activation of the egg for 5–8 min. Spermatozoa were collected in a small volume of water and pipetted over eggs in FRS. Eggs inseminated in FRS typically incorporated the fertilizing sperm within 3–4 min. Inseminated cells showed an absence of a fertilization cone and no cortical granule exocytosis. The deep conical depression in the egg surface beneath the micropyle remained unaltered. Control eggs inseminated in tank water developed a large fertilization cone during sperm incorporation. Occasionally, eggs inseminated in water were observed to incorporate the entire sperm head prior to egg activation. Our results corroborate earlier findings showing that in the zebrafish, cortical granule exocytosis, fertilization cone formation and elevation of the sperm entry site are not triggered by the fertilizing sperm in experimental conditions (18, 19). Furthermore, sperm incorporation requires neither egg activation nor formation of a fertilization cone in this fish.     

Temporal patterns of geographic parthenogenesis in a freshwater snail

Geographic parthenogenesis describes the observation that parthenogenetic organisms tend to occupy environments different from those of their close, sexually reproducing relatives. These environments are often described as extreme or disturbed habitats. We examined whether patterns of geographical parthenogenesis persist over time, by conducting a 3-year life-history survey and comparing two very proximate habitats of the freshwater snail Melanoides tuberculata : Nahal Arugot, a desert stream naturally disturbed by flash floods, and Or Ilan, a stable freshwater pond. Both sites occur in a xeric environment and are subject to otherwise similar biotic (e.g. parasites, predators) and climatic conditions. In the stable habitat, male frequencies and snail densities were significantly higher than in the disturbed one, whereas infection levels, mean embryo counts, and water temperatures were similar at both sites. Additionally, male frequencies declined after density decreased, thereby providing evidence for geographical parthenogenesis via reproductive assurance. Infection prevalence was very low regardless of reproduction mode. Although further genetic work is required, the apparent metapopulation structure of M. tuberculata in the Judean desert may be suitable for evaluating other possible explanations of geographical parthenogenesis.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 91 , 711–718.     

Land clearing reduces gene flow in the granite outcrop‐dwelling lizard,Ctenophorus ornatus

An important question for the conservation of species dwelling in fragmented habitats is whether changes to the intervening landscape create a barrier to gene flow. Here, we make use of the spatial distribution of the granite outcrop‐dwelling lizard, Ctenophorus ornatus, to compare inferred levels of gene flow between outcrops in a nature reserve with that between outcrops in the adjacent agricultural land. Genetic variation, relatedness and subdivision were compared within groups of individuals from different outcrops similar in size and distance apart at each site. In the agricultural land, we found significantly lower genetic variation within outcrops and greater genetic differentiation between outcrops than in the reserve. Further, the rate at which genetic divergence between outcrops increased over geographical distance was significantly greater in the agricultural land than in the reserve. We also found that individuals were more closely related within outcrops but more distantly related between outcrops in the cleared land. These effects occur over a small spatial scale with an average distance between outcrops of less than five kilometres. Thus, even though land clearing around the outcrops leaves outcrop size unchanged, it restricts gene flow, reducing genetic variation and increasing population structure, with potentially negative consequences for the long‐term persistence of the lizards on these outcrops.     

Changes in the Shape of Leishmania major Promastigotes in Response to Hexoses, Proline, and Hypo-osmotic Stress

Leishmania major promastigotes in late-log phase are generally long and slender, and remain so during a 1 h incubation in buffer without exogenous substrate. When glucose, 2-deoxyglucose, fructose, mannose, or proline are added, the cells become shorter and more rounded. The shape change in response to glucose is complete within 20 min and is reversible upon incubating the cells without substrate. Galactose, 3-O-methylglucose, 6-deoxyglucose, sucrose, maltose, ribose, glycerol, alanine, glutamate or aspartate do not cause the shape change. Decreasing the osmolarity of the medium causes a rounding of the cells similar to that observed in the presence of glucose, and increasing the osmolarity inhibits the shape change in response to glucose. Inhibitors of glucose transport and 2nd messenger analogs do not affect the shape change.