Studies of the ferredoxin from Thermus thermophilus

The soluble ferredoxin from Thermus thermophilus was examined by M?ssbauer and EPR spectroscopies and by reductive titrations. These studies demonstrate the presence of one 3Fe center, responsible for the characteristic g = 2.02 EPR signal in the oxidized protein, and one [4Fe-4S] center which is responsible for the rhombic EPR spectrum of the fully reduced protein. These assignments should replace those made by Ohnishi et al. (Ohnishi, T., Blum, H., Sato, S., Nakazawa, K., Hon-nami, K., and Oshima, T. (1980) J. Biol. Chem. 255, 345-348) prior to the discovery of the 3Fe clusters. The amino acid composition was determined and is discussed with reference to recent structural studies of 7Fe ferredoxins.     

Enhanced skeletal muscle contraction with myosin light chain phosphorylation by a calmodulin-sensing kinase

Repetitive low frequency stimulation results in potentiation of twitch force development in fast-twitch skeletal muscle due to myosin regulatory light chain (RLC) phosphorylation by Ca(2+)/calmodulin (CaM)-dependent skeletal muscle myosin light chain kinase (skMLCK). We generated transgenic mice that express an skMLCK CaM biosensor in skeletal muscle to determine whether skMLCK or CaM is limiting to twitch force potentiation. Three transgenic mouse lines exhibited up to 22-fold increases in skMLCK protein expression in fast-twitch extensor digitorum longus muscle containing type IIa and IIb fibers, with comparable expressions in slow-twitch soleus muscle containing type I and IIa fibers. The high expressing lines showed a more rapid RLC phosphorylation and force potentiation in extensor digitorum longus muscle with low frequency electrical stimulation. Surprisingly, overexpression of skMLCK in soleus muscle did not recapitulate the fast-twitch potentiation response despite marked enhancement of both fast-twitch and slow-twitch RLC phosphorylation. Analysis of calmodulin binding to the biosensor showed a frequency-dependent activation to a maximal extent of 60%. Because skMLCK transgene expression is 22-fold greater than the wild-type kinase, skMLCK rather than calmodulin is normally limiting for RLC phosphorylation and twitch force potentiation. The kinase activation rate (10.6 s(-1)) was only 3.6-fold slower than the contraction rate, whereas the inactivation rate (2.8 s(-1)) was 12-fold slower than relaxation. The slower rate of kinase inactivation in vivo with repetitive contractions provides a biochemical memory via RLC phosphorylation. Importantly, RLC phosphorylation plays a prominent role in skeletal muscle force potentiation of fast-twitch type IIb but not type I or IIa fibers.     

5,7-Dodecadien-1-ol: Candidate for the Sex Pheromone of the Pine Moth

Ferredoxin-nitrite reductase (EC 1.7.7.1), an enzyme which catalyzes the 6-electron reduction of nitrite to ammonia, has been isolated from Spinacia oleracea. The isolated enzyme was homogeneous by disc electrophoresis with polyacrylamide gel. The molecular weight of the enzyme was estimated to be 86,000 by Ultrogel AcA 34 gel filtration. In the oxidized form, the enzyme had absorption maxima at 278, 388 (Soret band), 573 (α band) and 690 nm, indicating that siroheme is directly involved in the catalysis of nitrite reduction. This absorption spectrum was modified by sulfite, hydroxylamine and cyanide. The enzyme exhibited electron paramagnetic resonance signals with g values of 6.9 and 5.2, which are characteristic of a high spin Fe³⁺ -siroheme in the molecule. These signals disappeared upon the addition of dithionite or nitrite. This isolated enzyme also contained four moles of labile sulfide and 7 g-atoms of iron per 86,000 g of protein.     

Impact of Pregnancy-Associated Malaria on Infant Malaria Infection in Southern Benin

Background

Infants of mothers with placental Plasmodium falciparum infections at delivery are themselves more susceptible to malaria attacks or to infection in early life.

Methodology/ Principal Findings

To assess the impact of either the timing or the number of pregnancy-associated malaria (PAM) infections on the incidence of parasitemia or malaria attacks in infancy, we followed 218 mothers through pregnancy (monthly visits) up to delivery and their infants from birth to 12 months of age (fortnightly visits), collecting detailed clinical and parasitological data. After adjustment on location, mother’s age, birth season, bed net use, and placental malaria, infants born to a mother with PAM during the third trimester of pregnancy had a significantly increased risk of infection (OR [95% CI]: 4.2 [1.6; 10.5], p = 0.003) or of malaria attack (4.6 [1.7; 12.5], p = 0.003). PAM during the first and second trimesters had no such impact. Similarly significant results were found for the effect of the overall number of PAM episodes on the time to first parasitemia and first malaria attack (HR [95% CI]: 2.95 [1.58; 5.50], p = 0.001 and 3.19 [1.59; 6.38], p = 0.001) respectively.

Conclusions/ Significance

This study highlights the importance of protecting newborns by preventing repeated episodes of PAM in their mothers.     

The active centers of adenylylsulfate reductase from Desulfovibrio gigas. Characterization and spectroscopic studies

In order to utilize sulfate as the terminal electron acceptor, sulfate-reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO4) reductase plays one of the major roles, reducing AdoPSO4 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovibrio gigas. The protein is composed of two non-identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (approximately 400 kDa). It is an iron-sulfur, flavin-containing protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa. Low-temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g = 2.02 and only detectable below 20 K. This signal represented a minor species (0.10-0.25 spins/mol) and showed line broadening in the enzyme isolated from 57Fe-grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe-S center I) observable below 30 K and a concomitant decrease in intensity of the g = 2.02 signal were detected. Effects of chemical reductants (ascorbate, H2/hydrogenase-reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g = 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin-spin-coupled nature (observable below 20 K), indicating the presence of at least two iron-sulfur centers, (centers I and II). M?ssbauer studies on 57Fe-enriched D. gigas AdoPSO4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential less than or equal to 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin-type [4Fe-4S] cluster. Center I exhibits spectra with atypical M?ssbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin-type [4Fe-4S] cluster, suggesting a different structure and/or a distinct cluster-ligand environment.     

Effect of hypercapnia on intracellular pH regulation in a rainbow trout hepatoma cell line,RTH 149

Fish exposed to elevated water CO₂ experience a rapid increase in blood CO₂ levels (hypercapnia), resulting in acidification of both intra- and extra-cellular compartments. While the mechanisms associated with extracellular pH regulation have been well explored, much less is known about intracellular pH (pH_i) regulation. There is great interest in developing non-animal models for research. One such model is the rainbow trout hepatoma cell line (RTH 149), which has been used to study a wide range of topics; however, no studies have investigated its potential use in pH_i regulation. Employing the pH-sensitive fluoroprobe BCECF, the present study examined pH_i regulation in RTH 149 under normocapnia and during extracellular acidification induced by either elevated CO₂ or 1 M HCl. During exposure to hypercapnia, RTH 149 cells were acidified without recovery as long as the elevated CO₂ was maintained. In addition, rates of pH_i recovery from NH₄Cl-induced acidosis were significantly lower in cells exposed to hypercapnia or HCl compared to that in normocapnic cells, indicating that elevated CO₂ indirectly impeded pH_i recovery through a reduction in pH_e and/or pH_i. Moreover, pH_i regulation in RTH 149 was EIPA-sensitive, suggesting that an NHE may be involved. Overall, RTH 149 may have the potential for identifying transporters likely to play a role in pH_i regulation in fish. However, it should not be used as a complete replacement for in vivo studies, especially to quantify acid–base regulatory ability at whole animal level, since RTH 149 appeared to have enhanced pH_i recovery rates relative to primary hepatocytes.     

Insights into phytase-containing transgenic <Emphasis Type="Italic">Lemna minor</Emphasis> (L.) as a novel feed additive

This study assessed the effect of supplementation of novel transgenic phytase on growth performance and bone mineralization in Korean native broiler chickens. The experiment was designed using four dietary groups: those with a diet supplemented with (A) recombinant phytase, (B) transgenic phytase from the plant Lemna minor, (C) or wild-type L. minor as well as (D) a control group that was supplemented with commercially available feed. Three hundred 1-day-old Korean native broiler chicks were used and divided into these four dietary treatment groups having three replicates of 25 birds each (n?=?75). The results showed increases in growth performance and bone mineralization in Groups B and C; compared with Groups A and D. Hematological analyses revealed notable contrasts in erythrocyte sedimentation rate, red blood cell count, and hemoglobin levels among the experimental groups, whereas no impacts of dietary treatment were observed on total eosinophil, lymphocyte, heterophil, monocyte, and basophil levels. The relative expression profiling of candidate genes showed that the genes involved in growth response, meat quality, and P–Ca metabolism were significantly highly expressed in the phytase-supplemented groups. Hence, it is suggested that dietary supplementation with transgenic phytase plant L. minor for enhancing growth performance is a promising new approach in the broiler feed industry. To the best of our knowledge, we report here the most comprehensive analysis using a broiler model that provides a workable platform for further research on the cost-effective production of feed with different compositions that might be beneficial in the livestock feed industry.     

Historical and Contemporary DNA Indicate Fisher Decline and Isolation Occurred Prior to the European Settlement of California

Establishing if species contractions were the result of natural phenomena or human induced landscape changes is essential for managing natural populations. Fishers (Martes pennanti) in California occur in two geographically and genetically isolated populations in the northwestern mountains and southern Sierra Nevada. Their isolation is hypothesized to have resulted from a decline in abundance and distribution associated with European settlement in the 1800s. However, there is little evidence to establish that fisher occupied the area between the two extant populations at that time. We analyzed 10 microsatellite loci from 275 contemporary and 21 historical fisher samples (1880–1920) to evaluate the demographic history of fisher in California. We did not find any evidence of a recent (post-European) bottleneck in the northwestern population. In the southern Sierra Nevada, genetic subdivision within the population strongly influenced bottleneck tests. After accounting for genetic subdivision, we found a bottleneck signal only in the northern and central portions of the southern Sierra Nevada, indicating that the southernmost tip of these mountains may have acted as a refugium for fisher during the anthropogenic changes of the late 19^th and early 20^th centuries. Using a coalescent-based Bayesian analysis, we detected a 90% decline in effective population size and dated the time of decline to over a thousand years ago. We hypothesize that fisher distribution in California contracted to the two current population areas pre-European settlement, and that portions of the southern Sierra Nevada subsequently experienced another more recent bottleneck post-European settlement.     

Impact of participant and interventionist race concordance on weight loss outcomes

Objective:

We have previously shown that racial composition of behavioral intervention groups does not affect achieved weight loss. However, it is unclear if the race of the interventionist affects intervention outcomes. The objective of this analysis is to estimate the impact of race concordance between participant and interventionist on weight change in the initial weight loss phase (phase I) of the Weight Loss Maintenance trial (WLM).

Design and Methods:

A total of 1,685 overweight or obese adults (BMI 25‐45 kg/m²) who were taking medication for hypertension and/or dyslipidemia participated in phase I of the WLM trial. All participants received a 6‐month intensive behavioral intervention in groups of 15‐20 facilitated by a trained interventionist. The main outcome is change in weight at 6 months.

Results:

Participants were on average 55 years of age, 67% female and 44% African American (AA). Three of seventeen interventionists were AA, 14 were non‐AA. Seventy‐three percent of participants shared race concordance with the interventionist. There was a small but statistically significant difference in weight change of participants who were the same race as the interventionist (?5.84 kg, s.e. 0.17) as compared with those who were not race concordant (?5.04 kg, s.e. 0.33), a difference of 0.8 kg, (P = 0.04). The impact of concordance on weight change differed by race (i.e., interaction of race and concordance was significant, P = 0.02).

Conclusions:

In a post hoc analysis of a group‐based behavioral intervention, race concordance for non‐AA participants was associated with slightly greater weight loss. Race concordance was not associated with weight loss for AA participants.