Stimulation of hexose transport by human polymorphonuclear leukocytes: a possible role for protein kinase C

The protein C kinase activators 1-O-oleoyl, 2-O-acetylglycerol, 12-O-tetradecanoyl phorbol-13-acetate, and mezerein, stimulated deoxyglucose uptake in human neutrophils. The responses were stimulus specific since no effect was noted with the diether analogues 1-O-hexadecyl-2-O-ethylglycerol, 1-O-palmitoyl-2-O-acetyl or 1-O-palmitoyl-3-O-acetyl diesters of propanediol, or with 1,2-diolein. Stimulation of deoxyglucose uptake had the characteristics of carrier facilitated hexose transport. Stimulated uptake of deoxy-glucose was inhibited by trifluoperazine (10-30 microM). Activation of protein kinase C therefore appears to trigger events involved in hexose transport.     

Regulation of the oxidative response of human granulocytes to chemoattractants. No evidence for stimulated traffic of redox enzymes between endo and plasma membranes

Experiments were performed to probe the role of exocytotic and endocytotic processes in the regulation of the human granulocyte O-2-generating system. Analytical subcellular fractionation studies indicated that 25-30% of the total cellular b-cytochrome and 8-10% of the flavin co-sedimented with plasma membrane markers, irrespective of stimulation of the cells by the chemoattractants N-formyl-Met-Leu-Phe (FMLP) or C5a. Phorbol myristate acetate stimulation resulted in significant translocation of b-cytochrome but not flavin from the specific granule/Golgi to the plasma membrane-enriched fractions. These results indicated that approximately 3.1 X 10(5) flavin and 0.8-1 X 10(6) b-cytochrome molecules are present in the plasma membrane of an isolated unstimulated human granulocyte and that these levels are invariant upon stimulation with chemoattractants. Maximal instantaneous rates of O-2 generation by cells in these preparations, however, were equivalent for all the stimuli. Since stimulation of granulocytes by phorbol myristate acetate, FMLP, or C5a results in exocytosis and/or endocytosis, then the role of these processes in regulating stimulated O-2 production by controlling the content of plasma membrane redox enzymes is questionable. This conclusion was supported by observations made with cytoplasts, which do not have an intracellular reserve of granules. Cytoplasts prepared from granulocytes produced O-2 at equivalent rates as their parent cells on a per unit surface area basis. These results suggest: 1) that stimulation of granulocytes with chemotactic peptides leads to full generation of O-2 at the cell surface without exocytotic recruitment of additional b-cytochrome and flavoprotein from the cytoplasmic compartment; 2) that these redox enzymes are not internalized along with chemoattractant receptors; and 3) that traffic of these redox enzymes between endo- and plasma membranes is not involved in the regulation of O-2 production in suspensions of human granulocytes stimulated by chemoattractants.     

Immunoregulation in disseminated murine histoplasmosis: disturbances in the production of interleukins 1 and 2

Production of IL 1 and IL 2 by splenocytes from C57BL/6 mice was measured at wk 1, 3, 8, and 14 after i.v. inoculation with 6 X 10(5) Histoplasma capsulatum (Hc) yeasts. As compared with age-matched controls, IL 1 production by splenocytes from Hc-infected mice was reduced severely at wk 1 and 3 of infection, greater than normal at wk 8, and within normal range at wk 14. IL 2 production was also reduced at wk 1 and 3 of infection; it was normal at wk 8 and was elevated at wk 14. Indomethacin and catalase failed to restore IL 1 production by splenocytes from infected mice, and exogenous IL 1 did not augment IL 2 production by these cells. A factor capable of suppressing the activity of IL 2 was detected in supernatants of concanavalin A-stimulated splenocytes from infected animals at wk 1 and 3 of infection, respectively. No factor capable of suppressing IL 1 activity was detected. Thus, the deficits of cell-mediated immunity in mice with systemic Hc infection may derive, in part, from impaired amplification of the immune response consequent to abnormal generation of IL 1 and IL 2.     

Structure of complex flagellar filaments in Rhizobium meliloti

The complex flagella of Rhizobium meliloti 2011 and MVII-1 were analyzed with regard to serology, fine structure, subunits, and amino acid composition. The serological identities of flagellar filaments of the two strains were demonstrated by double immunodiffusion with antiflagellin antiserum. The filaments had a diameter of 16 nm. Their morphology was dominated by the prominent undulations of an external three-start helix running at a 10-nm axial distance and at an angle of 32 degrees. Faint nearly axial striations indicated the presence of a tubular core of a different helical order. The complex filaments consisted of 40,000-dalton flagellin monomers. Typically, the amino acid composition was 3 to 4% higher in nonpolar residues and 5 to 7% lower in aspartic and glutamic acids (and their amides) than that of plain flagellar proteins. There were no immunochemical relationships among Pseudomonas rhodos, Rhizobium lupini, and R. meliloti complex flagella, suggesting that the latter represent a new class.     

Population Dynamics of Heterodera glycines Life Stages on Soybean

Population fluctuations of Heterodera glycines differ in fields with high and low initial population densities. In a field with low initial numbers of nematodes, the numbers of cysts and eggs in soil remained low through 100 days from planting then increased during the remainder of the growing season. In a field with high initial nematode populations, numbers increased at 30 days, decreased to low numbers at 100 days, and then resurged to maximum populations at harvest. Numbers of juveniles were greatest at 100 days in the low initial population density field and at planting in the high initial population density field. The initial numbers of eggs in the soil gave the best correlation to soil and root nematode populations 15 and 30 days later. Juveniles in the soil at planting gave the largest correlation coefficients with nematode populations in the roots at 15 days in the field with the low initial population density. Eggs and juveniles in the soil at harvest were poorly related to numbers that overwintered.     

Definition of three resolvase binding sites at the res loci of Tn21 and Tn1721.

The dual functions of resolvase, site-specific recombination and the regulation of its own expression from tnpR, both require the interaction of this protein with the DNA sequence at res, but the specificity of this interaction differs between groups of Tn3-like elements. In this study, DNA fragments that contained res from Tn21 or Tn1721 were subjected to either cleavage by DNase I or methylation by dimethyl sulphate in the presence of the purified resolvase from Tn21 or Tn1721. These experiments showed that each resolvase bound to the same three sites (I, II and III) within res from Tn1721 and to an equivalent series of three sites on Tn21: the differences in the amino acid sequences of the two proteins did not affect their interaction with either DNA. The DNA sequences at each site had some similarities and, in conjunction with data from the related transposon Tn501, a consensus was established. However, the three sites are functionally distinct: site I (tnpR-distal) spans the recombination cross-over point and sites II and III (tnpR-proximal) overlap the promoter of tnpR. The binding sites on these transposons were compared with those in the gamma delta/Tn3 system: the similarities between the two groups of transposons revealed some general features of resolvase-DNA interactions while the differences in fine structure elucidated the specificity of each resolvase.     

Inhibition of calf thymus type II DNA topoisomerase by poly(ADP-ribosylation).

The effect of poly(ADP-ribosylation) on calf thymus topoisomerase type II reactions has been investigated. Unknotting of phage P4 head DNA, and relaxation and catenation of supercoiled PM2 DNA are inhibited. We conclude that the inhibition results from poly(ADP-ribosylation) on the following grounds. Firstly, the enzyme poly(ADP-ribose) (PADPR) synthetase and NAD are required, secondly, the competitive synthetase inhibitor nicotinamide abolishes topoisomerase inhibition, and thirdly, the polymer alone is not inhibitory. The mechanism of inhibition appears to be disruption of the strand cleavage reaction. A topoisomerase-DNA complex can be formed that upon treatment with protein denaturant at low ionic strength results in strand cleavage. The amount of DNA present in such a cleavable-complex progressively decreased following pretreatment of topoisomerase type II with PADPR synthetase and increasing concentrations of NAD. Treatment of the pre-formed complex with NAD and PADPR synthetase had no effect on its salt-induced dissociation. This suggests that either poly(ADP-ribosylation) has no influence on dissociation of topoisomerase, in contrast to association, or topoisomerase is not accessible to the synthetase when bound to DNA. Similar data were obtained with calf thymus type I topoisomerase.     

Biofeedback control of migraine headaches: A comparison of two approaches

In order to assess the relative effectiveness of finger warming and temporal blood volume pulse reduction biofeedback in the treatment of migraine, 22 female migraine patients were assigned to one of three experimental conditions: temporal artery constriction feedback, finger temperature feedback, or waiting list. Biofeedback training consisted of 12 sessions over a 6-week period. All patients completed 5 weeks of daily self-monitoring of headache activity (frequency, duration, and intensity) and medication before and after treatment. Treatment credibility was assessed at the end of Sessions 1, 6, and 12. Results showed that temporal constriction and finger temperature biofeedback were equally effective in controlling migraine headaches and produced greater benefits than the waiting list condition. Power analyses indicated that very large sample sizes would have been required to detect any significant differences between the two treatment groups. No significant relationships were found between levels of therapeutic gains and levels of thermal or blood volume pulse self-regulation skills. Likewise, treatment outcome was not found to be related to treatment credibility. Further analyses revealed that changes in headache activity and medication were associated with changes in vasomotor variability. Because blood volume pulse variability was not significantly affected by biofeedback training, questions about its role in the therapeutic mechanism are raised.This research was supported in part by grants from the Quebec Ministry of Education and the Quebec Ministery of Social Affairs to the first author, and an award from the Medical Research Council of Canada to the second author. The authors are indebted to Drs. Frank Andrasik, Howard Barbaree, Edward Blanchard, Martin Ford, and Patrick McGrath, as well as to two anonymous reviewers, for their helpful comments on an earlier draft of this paper.     

Circadian Rhythms in Competitive Sabre Fencers: Internal Desynchronization and Performance

During a 7-10 day span, circadian rhythms of sleep-wake, self-rated fatigue and mood, oral temperature, eye-hand skill and right and left hand grip strength were investigated in eight subjects: five males (21-28 years of age), members of the French sabre fencing team selected for the 1984 Olympic Games in Los Angeles, and three females (19-26 years of age) practicing fleuret (foil) fencing as a sports activity. On the average six measurements/day/variable/subject were performed. The single cosinor method showed that a circadian rhythm was detectable for only 26 out of the 56 time series (46.4%). Power spectrum analysis gave almost the same figure (19 out of 48: 39.5%) with regard to rhythms with τ=24hr indicating that with one exception (subject JFL) rhythms were internally desynchronized including differences τ between right and left hand grip strength rhythms for three subjects. Results suggest: (a) a physiologic synchronization of circadian rhythms may be a predictor of good performance; (b) however, internal desynchronization as shown previously may be a trivial phenomenon and thus does not imply in itself alterations of either health or performance; (c) chronobiologic methods should be recommended for a better understanding of changes in performance by those participating in competitive and other sports.     

Lymphokine regulation of human lymphocyte proliferation: Formation of resting G0 cells by removal of interteukin 2 in cultures of proliferating T lymphocytes

The question of whether lymphocytes which have once been activated and have completed one or several cell cycle(s) can return to the G₀ phase and stay ready for a new activation (G₀-G₁ transition), rather than simply die, was investigated. To do so interleukin 2 (IL-2) was removed from cultures of continuously proliferating human T lymphocytes and the formation of resting (G₀) cells was measured. Kinetic analyses in freshly prepared peripheral blood lymphocytes (PBL) revealed that the onset of detectable RNA synthesis and the appearance of structures binding the anti-Tac antibody occurred simultaneously. This allowed the expansion of the definition of G₀ T lymphocytes as cells having a low RNA (and DNA) content, and no Tac antigen. When cultured human T cells proliferating continuously by means of IL-2 were characterized in terms of their distribution in the cell cycle, 7 days after the initial PHA stimulation, it could be demonstrated that very few cells were in the G₀ phase, supporting the concept of direct S/G₂/M-G₁ transition. However, when IL-2 was removed from the cultures, the [³H]thymidine incorporation per 10⁴ cells and correspondingly the number of cells in the S/G₂/M and G₁ phases were reduced drastically and during the following 72-hr period, the number of G₀ cells increased markedly. Restimulation of such in vitro formed G₀ cells, under conditions permitting observation of their shift from the G₀ to G₀ phase, demonstrated that most cells could respond normally. Based on these observations, it was concluded that IL-2 not only ensures T-lymphocyte survival and proliferation, but IL-2 starvation induces many continuously proliferating T lymphocytes to stop cycling and to return to the G₀ phase of the cell cycle where they remain functional.