M?ssbauer spectroscopic studies of the cores of human, limpet and bacterial ferritins

Ferritin cores from human spleen, limpet (Patella vulgata) haemolymph and bacterial (Pseudomonas aeruginosa) cells have been investigated using 57Fe M?ssbauer spectroscopy. The M?ssbauer spectra were recorded over a range of temperatures from 1.3 to 78 K, all the spectra are quadrupole-split doublets with similar quadrupole splittings and isomer shifts, characteristic of iron(III), while at sufficiently low temperatures the spectra of all the samples show well-resolved magnetic splitting. At intermediate temperatures, the spectra from the human ferritin exhibit typical superparamagnetic behaviour, while those from the bacterial ferritin show behaviour corresponding to a transition from a magnetically ordered to a paramagnetic state. The spectra from the limpet ferritin show a complex combination of the two effects. The results are discussed in terms of the magnetic behaviour of small particles. The data are consistent with magnetic ordering temperatures of about 3 and 30 K for the bacterial and limpet ferritin cores, respectively, while the data indicate that the magnetic ordering temperature for the human ferritin cores must be above 50 K. These differences are interpreted as being related to different densities of iron in the cores and to variations in the composition of the cores. The human ferritin cores are observed to have a mean superparamagnetic blocking temperature of about 40 K, while that of the limpet ferritin cores is about 25 K. This difference is interpreted as being due not only to different mean numbers of iron atoms in the two types of core but also to the higher degree of crystallinity in the cores of the human ferritin.     

Comparison of glucokinase in C3H/He and C58 mice that differ in their hepatic activity

Partially purified preparations of the hepatic glucokinase from C3H/He and C58 inbred mice have been used to explore the molecular basis for the observed twofold difference in activity between the strains. The single codominant gene that appears to regulate activity, the alleles of which are designated Gk^a and Gk^b, respectively, for the two strains, could represent a structural gene change. This now seems unlikely because the mouse enzyme, although showing small differences from rat glucokinase, appeared to be identical in the two strains with respect to thermal stability, electrophoretic mobility in agarose gels, and kinetic properties such as the apparent K _m values for MgATP^2– and glucose and the unique cooperative interaction with the latter substrate. The enzymes also reacted identically in a range of immunological tests (double-diffusion, immunoelectrophoresis, immune precipitation and immune inhibition assays) and ELISA immune inhibition assays indicated that the twofold difference in activity was due to a similar difference in antigenically active enzyme. Genetic control over the physiologically significant regulation of enzyme amount is therefore probable.This work has been supported in part by a grant from the British Diabetic Association and a Training Studentship to PAJ from the Medical Research Council (U.K.).     

Porphyra nereocystis: A dual-daylength seaweed

Conchospores from the perennial conchocelis phase of the annual, epiphytic, marine red alga Porphyra nereocystis Anderson, which in nature lives on the large annual kelp Nereocystis luetkeana (Mertens) Postels et Ruprecht, are released in culture only in response to a short-day photoperiod treatment followed by a long-day treatment. Each treatment requires a minimum of three to four weeks and is enhanced by lower temperature during the second photoperiod treatment. To our knowledge P. nereocystis is the first known dualdaylength seaweed and requires a short-day-longday treatment for completion of its life cycle. This stringent environmental control of its reproduction appears to be an adaptation to coordinate conchospore production with the seasonal availability of its host kelp Nereocystis.Abbreviations LD long day - SD short day - SLD shortlong day     

Early stimulation of ATP turnover induced by growth factors : Synergistic effect of EGF and insulin and correlation with DNA synthesis

Addition of a mixture of EGF + insulin to quiescent cell cultures synergistically stimulates the cells to reinitiate DNA synthesis and cell division. We have previously demonstrated that this mixture rapidly increases ATP turnover in quiescent cells. The present work shows that each of the two growth factors, EGF and insulin, when added separately to quiescent cells was able to stimulate the phosphorylation of the organic acid-soluble compounds (Po) pool and ATP turnover. The stimulation of ATP turnover was closely correlated with the increase in phosphorylation of the Po pool which suggests that Po labelling reflects the ATP turnover. In many experiments, the synergy between the two growth factors on the early increase in phosphorylation of the Po pool was clearly shown. Doubling the concentration of EGF (12-24 ng/ml) or insulin (50-100 ng/ml) did not increase early stimulation of phosphorylation of the Po pool, whereas simultaneous addition of the two growth factors induced a greater stimulation than that of each growth factor separately added. The augmentation in Po labelling after addition of EGF or insulin alone was transient. The synergistic effect of the two growth factors was more significant when determined 150 or 300 min after growth-factor addition. In our experimental conditions, each of the two growth factors, EGF and insulin, was able to induce a stimulation of DNA synthesis. However, the best stimulatory effect was observed with the mixture of the two which synergistically increased DNA synthesis determined between 6 and 24 h after growth-factor addition. The comparison between DNA replication and Po labelling suggests a correlation between the increase in DNA replication and in the total ATP synthesized in the first 5 h after cell stimulation by growth factors added separately or in combination.     

Extracellular and intracellular acid-base status following strenuous activity in the sea raven (Hemitripterus americanus)

Summary Exhausting activity in the sea raven resulted in a pronounced extracellular acidosis, which consisted of a large, short-lived respiratory component and a small, longer-lived metabolic component. Thi disturbance had been corrected by 12 h. White muscle experienced a pronounced intracellular acidosis of chiefly metabolic origin, with pH_i dropping from a resting value of 7.51 to a low of 7.10 immediately post-activity. The recovery of pH_i was associated with a reduction in muscle lactate. Despite the large increase in , cardiac muscle pH_i remained constant postactivity, actually showing an alkalosis at 30 min into recovery. Maintenance of cardiac muscle pH_i was achieved by an accumulation of HCO ₃ ^– intracellularly.     

Phosphopeptide analysis of phenylalanine hydroxylase isolated from liver cells exposed to hormonal stimuli.

Hormonal control of the phosphorylation of phenylalanine hydroxylase was studied by using rat liver cells incubated with [32P]Pi. After immunoprecipitation from cell extracts, the hydroxylase was subjected to proteinase digestion and subsequent sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. V8-proteinase digestion yielded one major 32P-labelled fragment, of approx. 9 kDa. Chymotrypsin digestion gave five 32P-labelled fragments ranging from approx. 39 kDa to approx. 10 kDa. Noradrenaline (10 microM) and glucagon (0.1 microM) enhanced the 32P content of all peptide fragments uniformly. Phorbol ester, in contrast with ionophore A23187, did not stimulate enzyme phosphorylation or enhance phenylalanine metabolism in liver cells. These results are discussed in relation to the nature of the protein kinase(s) that mediate phosphorylation of phenylalanine hydroxylase in liver cells.     

Interaction of genotype and parental environment in the adaptation of wild House mice Mus musculus to cold

Wild House mice, Mus musculus, were bred in two laboratory environments, one warm (controls) and one cold (Eskimo). At the seventh generation, mice of both stocks were cross-fostered at birth in both environments. In the warm environment, differences in both genotype and nest environment influenced growth: (1) Eskimo reared by Eskimo females were the heaviest of the four classes of fostered young; and (2) control foster parentage retarded growth. There was, however, no good evidence of differences in the reproductive performance of the four classes of fostered mice. In the cold environment, the effects of both genetical differences and of fostering were greater. Both the superior growth of Eskimo reared by Eskimo and the retarding effect of control foster parentage were more marked. Moreover, adult males with control foster parents had less fat than had those with Eskimo foster parents. Reproductive performance was also affected: (1) the young of the pairs with Eskimo genotype were heavier than the young of control pairs; (2) the litters of mice with Eskimo foster parents were larger than those of mice with control foster parents, and their young were heavier. Differences among the young of fostered mice represent a grandmother effect. Evidently, selection in a cold environment had led, not only to adaptive genetical changes in the ability to respond directly to cold, but also to changes in parental performance; and the latter enhanced the fitness, in the cold environment, of their offspring and grandoffspring.     

Biochemical Identification of the Two Races of Radopholus similis by Starch Gel Electrophoresis

Analysis of genetic variation between the banana and the citrus races of Radopholus similis by starch gel eleclrophoresis demonstrated that 7 of 16 enzyme-encoding loci could be used for their diagnostic separation. The two races are closely related arid share approximately 75% of the enzymes evaluated. The level of dissimilarities o1 inherited bands indicates that no gene flow occurs between the races. Aldolase, α + β esterase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, and phosphoglucose isomerase are diagnostic markers of the races.     

Sequence and expression of the mouse mammary tumour virus env gene

We have determined the DNA sequence of the envelope gene region of the GR strain of mouse mammary tumour virus. The sequence extends for 3012 nucleotides from the single EcoRI site to beyond the PstI site in the 3' long terminal repeat (LTR) of the provirus. There is a major open reading frame from nucleotides 752 to 2818 which encompasses the entire env gene. This reading frame extends through a polypurine tract and into the LTR. There is another open reading frame from the first nucleotide to position 803, presumably corresponding to the end of the pol gene. The splice acceptor site which generates env mRNA has been mapped experimentally to nucleotide 750. The env gene products, gp52 and gp36, have been positioned on the sequence using the directly determined amino acid sequences of the amino terminus of gp52; and both the amino and carboxyl termini of gp36. The start of gp52 is preceded by a series of 19 uncharged amino acids which could function as a typical signal sequence, but this sequence is only part of a much longer leader peptide. The tetrad Arg-Ala-Lys-Arg is the presumed cleavage site in the gPr73env precursor, and occurs just before the gp36 amino terminus. There are five potential asparagine-linked glycosylation sites which agrees with previous experimental results. The gp36 has two long hydrophobic regions at its amino and carboxy termini, these are suggested to act as a fusion peptide and the trans-membrane anchor, respectively.     

Pyruvate Kinase of Streptococcus lactis

The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5′-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent V_max values and decreased the K_m values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5′-triphosphate also caused inhibition. The K_m values for phosphoenolpyruvate and adenosine 5′-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5′-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5′-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.