Influence of humic, fulvic and hydrophilic acids on the growth, photosynthesis and respiration of the dinoflagellate Prorocentrum minimum (Pavillard) Schiller

Blooms of the dinoflagellate Prorocentrum minimum often occur in coastal regions characterized by variable salinity and elevated concentrations of terrestrially derived dissolved organic carbon (DOC). Humic, fulvic and hydrophilic acid fractions of DOC were isolated from runoff entering lower Narragansett Bay immediately after a rainfall event and the influence of these fractions upon P. minimum growth, cell yield, photosynthesis and respiration was examined. All organic fractions stimulated growth rates and cell yields compared with controls (no organic additions), but the extent of stimulation varied with the fraction and its molecular weight. Greatest stimulations were observed with humic and fulvic acids additions; cell yields were more than 2.5 and 3.5 times higher than with hydrophilic acid additions while growth rates were 21 and 44% higher, respectively. Responses to additions of different molecular weight fractions of each DOC fraction suggest that growth rate effects were attributable to specific molecular weight fractions: the >10,000 fraction of humic acids, both the >10,000 and <500 fractions of fulvic acids and the <10,000 fraction of hydrophilic acids. The form and concentration of nitrogen (as NO₃⁻ or NH₄⁺) present also influenced P. minimum response to DOC; 10–20 μg ml⁻¹ additions of fulvic acid had no effect upon growth rates in the presence of NH₄⁺ but significantly increased growth rates in the presence of NO₃⁻, a relationship probably related to fulvic acid effects upon trace metal bioavailability and subsequent regulation of the biosynthesis of enzymes required for NO₃⁻ assimilation. The influence of DOC additions on P. minimum respiration and production rates also varied with the organic fraction and its concentration. Production rates ranged from 1.1 to 3.4 pg O₂ cell⁻¹ h⁻¹, with highest rates observed upon exposure to fulvic and hydrophilic acid concentrations of >10 μm ml⁻¹. Low concentrations (5–10 μg ml⁻¹) of humic acid had no statistically significant effect upon production, but exposure to concentrations >25 μg ml⁻¹ resulted in a 30% decrease in O₂ evolution, probably due to light attenuation by the highly colored humic acid fraction. Respiration rates ranged from 1.2 to 2.7 pg O₂ cell⁻¹ h⁻¹ and were elevated upon exposure to both fulvic and hydrophilic acids, but not to humic acid. These results demonstrate that terrestrially derived DOC fractions play an active role in stimulation of P. minimum growth via direct effects upon growth, yield and photosynthesis as well as via indirect influences such as interactions with nitrogen and effects upon light attenuation.     

Validation of an Instrument to Measure Older Adults' Expectations Regarding Movement (ERM)

Background

Many individuals with Parkinson''s disease are not diagnosed and treated. Attitudes about aging and related help-seeking may affect the timely diagnosis of Parkinson''s disease. Our objectives were to develop measures of older adults'' expectations regarding movement with aging, specifically related to parkinsonism, and their beliefs about seeking healthcare for the diagnosis and treatment of parkinsonism.

Methods

We established content and face validity from interviews with experts, review of the literature, and pre-testing with key informants. Two 9-item instruments resulted: Expectations Regarding Movement (ERM) and Healthcare Seeking Beliefs for parkinsonism (HSB). These instruments were administered to 210 older adults at senior centers to investigate internal consistency and construct validity.

Results

192 (91%) of the older adults completed more than 90% of the survey. The mean age was 76; 17 (9%) reported parkinsonism. Both scales demonstrated good internal consistency (α = 0.90). Factor analysis supported construct validity of the ERM and HSB scores. Older age, lower education, worse self-reported health and African American race each were associated with lower ERM scores, but not HSB scores.

Conclusion

The ERM, a brief measure of expectations regarding movement with aging, shows reliability and validity. This scale may be useful in identifying older adults at increased risk for under-identification of Parkinson''s disease. Further work is needed to measure healthcare seeking for parkinsonism.     

CD4+ T Cells Support Production of Simian Immunodeficiency Virus Env Antibodies That Enforce CD4-Dependent Entry and Shape Tropism In Vivo

CD4⁺ T cells rather than macrophages are the principal cells infected by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) in vivo. Macrophage tropism has been linked to the ability to enter cells through CCR5 in conjunction with limiting CD4 levels, which are much lower on macrophages than on T cells. We recently reported that rhesus macaques (RM) experimentally depleted of CD4⁺ T cells before SIV infection exhibit extensive macrophage infection as well as high chronic viral loads and rapid progression to AIDS. Here we show that early-time-point and control Envs were strictly CD4 dependent but that, by day 42 postinfection, plasma virus of CD4⁺ T cell-depleted RM was dominated by Envs that mediate efficient infection using RM CCR5 independently of CD4. Early-time-point and control RM Envs were resistant to neutralization by SIV-positive (SIV⁺) plasma but became sensitive if preincubated with sCD4. In contrast, CD4-independent Envs were highly sensitive to SIV⁺ plasma neutralization. However, plasma from SIV-infected CD4⁺ T cell-depleted animals lacked this CD4-inducible neutralizing activity and failed to neutralize any Envs regardless of sCD4 pre-exposure status. Enhanced sensitivity of CD4-independent Envs from day 42 CD4⁺ T cell-depleted RM was also seen with monoclonal antibodies that target both known CD4-inducible and other Env epitopes. CD4 independence and neutralization sensitivity were both conferred by Env amino acid changes E84K and D470N that arose independently in multiple animals, with the latter introducing a potential N-linked glycosylation site within a predicted CD4-binding pocket of gp120. Thus, the absence of CD4 T cells results in failure to produce antibodies that neutralize CD4-independent Envs and CD4-pretriggered control Envs. In the absence of this constraint and with a relative paucity of CD4⁺ target cells, widespread macrophage infection occurs in vivo accompanied by emergence of variants carrying structural changes that enable entry independently of CD4.     

Neil3 and NEIL1 DNA Glycosylases Remove Oxidative Damages from Quadruplex DNA and Exhibit Preferences for Lesions in the Telomeric Sequence Context

The telomeric DNA of vertebrates consists of d(TTAGGG)_n tandem repeats, which can form quadruplex DNA structures in vitro and likely in vivo. Despite the fact that the G-rich telomeric DNA is susceptible to oxidation, few biochemical studies of base excision repair in telomeric DNA and quadruplex structures have been done. Here, we show that telomeric DNA containing thymine glycol (Tg), 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh), or spiroiminodihydantoin (Sp) can form quadruplex DNA structures in vitro. We have tested the base excision activities of five mammalian DNA glycosylases (NEIL1, NEIL2, mNeil3, NTH1, and OGG1) on these lesion-containing quadruplex substrates and found that only mNeil3 had excision activity on Tg in quadruplex DNA and that the glycosylase exhibited a strong preference for Tg in the telomeric sequence context. Although Sp and Gh in quadruplex DNA were good substrates for mNeil3 and NEIL1, none of the glycosylases had activity on quadruplex DNA containing 8-oxoG. In addition, NEIL1 but not mNeil3 showed enhanced glycosylase activity on Gh in the telomeric sequence context. These data suggest that one role for Neil3 and NEIL1 is to repair DNA base damages in telomeres in vivo and that Neil3 and Neil1 may function in quadruplex-mediated cellular events, such as gene regulation via removal of damaged bases from quadruplex DNA.     

A structural limitation on enzyme activity: the case of HMG-CoA synthase

Recent structural studies of the HMG-CoA synthase members of the thiolase superfamily have shown that the catalytic loop containing the nucleophilic cysteine follows the phi and psi angle pattern of a II' beta turn. However, the i + 1 residue is conserved as an alanine, which is quite unusual in this position as it must adopt a strained positive phi angle to accommodate the geometry of the turn. To assess the effect of the conserved strain in the catalytic loop, alanine 110 of Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was mutated to a glycine. Subsequent enzymatic studies showed that the overall reaction rate of the enzyme was increased 140-fold. An X-ray crystallographic study of the Ala110Gly mutant enzyme demonstrated unanticipated adjustments in the active site that resulted in additional stabilization of all three steps of the reaction pathway. The rates of acetylation and hydrolysis of the mutant enzyme increased because the amide nitrogen of Ser308 shifts 0.4 A toward the catalytic cysteine residue. This motion positions the nitrogen to better stabilize the intermediate negative charge that develops on the carbonyl oxygen of the acetyl group during both the formation of the acyl-enzyme intermediate and its hydrolysis. In addition, the hydroxyl of Ser308 rotates 120 degrees to a position where it is able to stabilize the carbanion intermediate formed by the methyl group of the acetyl-S-enzyme during its condensation with acetoacetyl-CoA.     

Echolocating bats use a nearly time-optimal strategy to intercept prey

Acquisition of food in many animal species depends on the pursuit and capture of moving prey. Among modern humans, the pursuit and interception of moving targets plays a central role in a variety of sports, such as tennis, football, Frisbee, and baseball. Studies of target pursuit in animals, ranging from dragonflies to fish and dogs to humans, have suggested that they all use a constant bearing (CB) strategy to pursue prey or other moving targets. CB is best known as the interception strategy employed by baseball outfielders to catch ballistic fly balls. CB is a time-optimal solution to catch targets moving along a straight line, or in a predictable fashion--such as a ballistic baseball, or a piece of food sinking in water. Many animals, however, have to capture prey that may make evasive and unpredictable maneuvers. Is CB an optimum solution to pursuing erratically moving targets? Do animals faced with such erratic prey also use CB? In this paper, we address these questions by studying prey capture in an insectivorous echolocating bat. Echolocating bats rely on sonar to pursue and capture flying insects. The bat's prey may emerge from foliage for a brief time, fly in erratic three-dimensional paths before returning to cover. Bats typically take less than one second to detect, localize and capture such insects. We used high speed stereo infra-red videography to study the three dimensional flight paths of the big brown bat, Eptesicus fuscus, as it chased erratically moving insects in a dark laboratory flight room. We quantified the bat's complex pursuit trajectories using a simple delay differential equation. Our analysis of the pursuit trajectories suggests that bats use a constant absolute target direction strategy during pursuit. We show mathematically that, unlike CB, this approach minimizes the time it takes for a pursuer to intercept an unpredictably moving target. Interestingly, the bat's behavior is similar to the interception strategy implemented in some guided missiles. We suggest that the time-optimal strategy adopted by the bat is in response to the evolutionary pressures of having to capture erratic and fast moving insects.     

Floral resources and risk of exposure to pesticides for Melipona quadrifasciata anthidioides Lepeletier 1836 in a Cerrado of São Paulo (Brazil)

Honey and bee bread samples from storage pots of Melipona quadrifasciata anthidioides were collected monthly from April 2015 to May 2016 in the Mogi Guaçu Biological Reserve (22º 10? S, 47º 11? W). The flora in the site is characteristic of the Atlantic Forest with preserved areas of savanna-like vegetation surrounded by commercial forests, orchards and various crops of exotic and native plants. Samples were analysed with the use of melissopalynological methodology and 46 pollen types from 38 genera and 30 families were identified in 25 honey samples. Fabaceae, Asteraceae, Myrtaceae, Sapindaceae showed the greatest pollen richness in honey. Predominant nectariferous pollen types were Anadenanthera, Cordia, Eucalyptus, Mimosa scabrella, Schefflera, Sida, Serjania and Vernonia. Twenty-eight types of pollen from 21 genera and 19 families were identified in 22 bee bread samples. Fabaceae, Asteraceae and Myrtaceae showed the highest pollen richness. Anadenanthera, Cecropia, Eucalyptus, Melastomataceae, Mimosa scabrella, Mimosa verrucosa and Myrcia were the most frequent polliniferous pollen types. Principal component analysis (PCA) demonstrated that honey and pollen samples formed two main groups of similarity, mainly due to Eucalyptus’ nectar and pollen of Melastomataceae, respectively. Melipona quadrifasciata anthidioides collected nectar and pollen from the preserved areas as well as in the secondary and ‘ruderal’ vegetation and in cultivated forests/fields, suggesting their importance as pollinators both of native flora and exotic species. The use of trophic resources of plants grown with pesticides is a concern for the conservation of these species of bee and should be better studied.     

Protein kinase B/Akt mediates cAMP- and cell swelling-stimulated Na+/taurocholate cotransport and Ntcp translocation

Cyclic AMP and cell swelling stimulate hepatic Na+/TC cotransport and Ntcp translocation via the phosphoinositide 3-kinase signaling pathway. To determine the downstream target of the phosphoinositide 3-kinase action, we examined the role of protein kinase B (PKB)/Akt using SB203580 in hepatocytes as well as by transfection with a dominant negative (DN-PKB) or a constitutively active (CA-PKB) form of PKB in HuH-Ntcp cells. Both cAMP and cell swelling stimulated p38 mitogen-activated protein (MAP) kinase as well as PKB activity. Although 100 microm SB203580 inhibited cell swelling- and 8-chlorophenylthio-cAMP-induced activation of both p38 MAP kinase and PKB, 1 microm SB203580 inhibited activation of p38 MAP kinase, but not of PKB, in hepatocytes. 100 microm, but not 1 microm SB203580, inhibited cell swelling- and cAMP-induced increases in taurocholate (TC) uptake and Ntcp translocation in hepatocytes. TC uptake in HuH-Ntcp cells was more than 90% dependent on extracellular Na+. Cyclic AMP and cell swelling increased TC uptake by 50-100% and PKB activity 2-4-fold in HuH-Ntcp cells transfected with the empty vector and failed to increase PKB activity, TC uptake, and Ntcp translocation in DN-PKB-transfected HuH-Ntcp cells. Transfection with CA-PKB increased PKB activity, TC uptake, and Ntcp translocation in HuH-Ntcp cells compared with cells transfected with the empty vector. In contrast, transfection with DN-PKB did not affect basal PKB activity, TC uptake, or Ntcp translocation. Taken together, these results strongly suggest that cell swelling and cAMP-mediated stimulation of hepatic Na+/TC cotransport and Ntcp translocation requires activation of PKB and is mediated at least in part via a phosphoinositide 3-kinase/PKB-signaling pathway.