Expression of CYP4F2 in human liver and kidney: assessment using targeted peptide antibodies

P450 enzymes comprising the human CYP4F gene subfamily are catalysts of eicosanoid (e.g., 20-HETE and leukotriene B4) formation and degradation, although the role that individual CYP4F proteins play in these metabolic processes is not well defined. Thus, we developed antibodies to assess the tissue-specific expression and function of CYP4F2, one of four CYP4F P450s found in human liver and kidney. Peptide antibodies elicited in rabbits to CYP4F2 amino acid residues 61-74 (WGHQGMVNPTEEG) and 65-77 (GMVNPTEEGMRVL) recognized on immunoblots only CYP4F2 and not CYP4F3b, CYP4F11 or CYP4F12. Immunoquantitation with anti-CYP4F2 peptide IgG showed highly variable CYP4F2 expression in liver (16.4+/-18.6pmol/mg microsomal protein; n=29) and kidney cortex (3.9+/-3.8 pmol/mg; n=10), with two subjects lacking the hepatic or renal enzyme entirely. CYP4F2 content in liver microsomes was significantly correlated (r> or =0.63; p<0.05) with leukotriene B4 and arachidonate omega-hydroxylase activities, which are both CYP4F2-catalyzed. Our study provides the first example of a peptide antibody that recognizes a single CYP4F P450 expressed in human liver and kidney, namely CYP4F2. Immunoquantitation and correlation analyses performed with this antibody suggest that CYP4F2 functions as a predominant LTB4 and arachidonate omega-hydroxylase in human liver.     

X-ray crystal structure of galabiose, O-alpha-D-galactopyranosyl-(1---4)-D-galactopyranose

O-alpha-D-Galactopyranosyl-(1---4)-D-galactopyranose, C12H22O11, Mr = 342.30, crystallises in the orthorhombic space group P2(1)2(1)2(1), and has alpha = 5.826(1), b = 13.904(3), c = 17.772(4) A, Z = 4, and Dx = 1.579 g.cm-3. Intensity data were collected with a CAD4 diffractometer. The structure was solved by direct methods and refined to R = 0.063 and Rw = 0.084 for 2758 independent reflections. The glycosidic linkage is of the type 1-axial-4-axial with torsion angles phi O-5' (O-5'-C-1'-O-1'-C-4) = 98.1(2) degrees, psi C-3 (C-3-C-4-O-1'-C-1') = -81.9(3) degrees, phi H (H-1'-C-1'-O-1'-C-4) = -18 degrees, and psi H (H-4-C-4-O-1'-C-1') = 35 degrees. The conformation is stabilised by an O-3 . . . O-5' intramolecular hydrogen-bond with length 2.787(3) A and O-3-H . . . O-5' = 162 degrees. The glycosidic linkage causes a folding of the molecule with an angle of 117 degrees between the least-square planes through the pyranosidic rings. The crystal investigated contained 56(1)% of alpha- and 44(1)% of beta-galabiose as well as approximately 70% of the gauche-trans and approximately 30% of the trans-gauche conformers about the exocyclic C-5'-C-6' and C-5-C-6 bonds. The crystal packing is governed by hydrogen bonding that engages all oxygen atoms except the intramolecular acceptor O-5' and the glycosidic O-1' oxygen atoms.     

Uterine Microbiology and Antimicrobial Susceptibility in Isolated Bacteria from Mares with Fertility Problems

Uterine microbiology and antimicrobial susceptibility was investigated in 239 mares with fertility problems in a prospective study in Sweden. Uterine swab samples were collected with double guarded swabs and transported overnight before being cultured. The Minimum Inhibitory Concentrations (MIC) was determined for a panel of antimicrobials. From 152 of the 239 mares at least one bacterial species was isolated, most frequently E. coli (104 isolates), β -haemolytic streptococci (31) and fungi (16). β -haemolytic streptococci were more frequently (p < 0.01) associated with clinical endometritis than with repeat breeding. The opposite was true for E. coli (p < 0.01). Among β -haemolytic streptococcal isolates some resistance was noted for 4 of 11 tested antibiotics, however, all isolates were susceptible to the widely used penicillin G. Among E. coli isolates enrofloxacin was the only of the 10 tested antibiotics for which no resistance was noted. Resistance was most commonly noted to cephalothin (39% of the isolates), streptomycin (22%), trimethoprim/sulphamethoxazole (15%) and ampicillin (11%). In conclusion, we show that both E. coli and β -haemolytic streptococci are frequently associated with fertility problems in mares and that antimicrobial resistance is a common feature of E. coli but also recognised for β -haemolytic streptococcal uterine isolates.     

Thyrotropin stimulation of cultured thyroid cells increases steady state levels of the messenger ribonucleic acid for thyroid peroxidase

We have examined the effect of TSH on thyroid peroxidase (TPO) mRNA levels in dog thyroid cell primary cultures. Freshly dispersed dog thyroid cells were cultured for up to 5 days in the absence or presence of 5 mU/ml bovine TSH. At the outset of culture, and at daily intervals thereafter, total cytoplasmic RNA was extracted and applied to Nytran paper using a slot-blot apparatus. A nick-translated cDNA fragment of the porcine TPO gene was used to probe these filters. Autoradiographs were quantified by densitometry. Nonspecific binding was negligible as determined using a pUC18 probe. During the first 2 days of culture, TPO mRNA levels declined irrespective of whether or not TSH was present in the medium. TSH did not affect this decline. Between 3 and 5 days of culture, TPO mRNA levels in control (no TSH) cells increased to 3 times the initial level (expressed relative to cellular DNA). However, during the same period TSH stimulated TPO mRNA levels 8-fold above the initial level. To confirm that the signal with the cDNA probe was actually that of dog TPO mRNA, cellular RNA (day 4 of culture) was subjected to Northern blot analysis using the same cDNA probe. Specific bands of 2.9 kilobases were detected corresponding to the known size of TPO mRNA in pig thyroid tissue. The signal of this 2.9 kilobase species was enhanced by TSH. In conclusion, the data indicate that chronic TSH stimulation raises steady state levels of TPO mRNA and provide an explanation, at least in part, for the mechanism by which TSH enhances TPO bioactivity in thyroid tissue.     

Partitioning seasonal time: interactions among size, foraging activity and diet in leaf-litter frogs

This study investigates hypotheses about partitioning of food resources among all species and several size classes in an assemblage of diurnal leaf-litter frogs in central Amazonia. All species in this assemblage change the type and size of prey as they grow. An ordination of diet composition was significantly associated with frog size and species-specific behaviour. However, a partial Mantel analysis indicated that species explained about 1.5 times more of the variation in diet overlap between individuals than frog size. Diet and foraging activity are correlated in juveniles, but not in adults, and this result holds whether species are considered as statistically independent observations or whether relationships are analysed using phylogenetically independent contrasts. This study showed that the partitioning of food resources between species changes with the population size structures. Thus, intraspecific and interspecific changes in diet, coupled with different patterns of juvenile recruitment, cause diet segregation among species due to temporal segregation of equivalent size classes. Received: 25 August 1997 / Accepted: 1 April 1998     

A new non-equilibrium enzyme linked immunosorbent assay for a glycogen-derived urinary tetrasaccharide

The tetrasaccharide, Glc1-6Glc1-4Glc1-4Glc, denoted (Glc)₄, is a limit dextrin formed by amylolytic degradation of glycogen. In order to evaluate the possible clinical importance of (Glc)₄ excretion as an indicator of certain physiological and pathological conditions, we have developed a new rapid and inexpensive immunoassay using a monoclonal antibody raised against (Glc)₄ glycosidically-linked to a carrier protein. As the antibody is highly specific, it can be used to measure native (Glc)₄ directly, without the chemical reduction step required in previous methods. A new type of non-equilibrium ELISA inhibition test was developed based on the capacity of free (Glc)₄ to decrease initial rates of antibody binding to (Glc)₄-coated microtiter wells. The method is highly reproducible and is as sensitive and accurate as the gas chromatography method or radioimmunoassay used previously.Abbreviations (Glc)₄ Glc1-6Glc1-4Glc1-4Glc - KLH keyhole limpet hemacyanin - BSA bovine serum albumin - PEG polyethylene glycol     

Transmembrane topology of FRO2, a ferric chelate reductase from Arabidopsis thaliana

Iron uptake in Arabidopsis thaliana is mediated by ferric chelate reductase FRO2, a transmembrane protein belonging to the flavocytochrome b family. There is no high resolution structural information available for any member of this family. We have determined the transmembrane topology of FRO2 experimentally using the alkaline phosphatase fusion method. The resulting topology is different from that obtained by theoretical predictions and contains 8 transmembrane helices, 4 of which build up the highly conserved core of the protein. This core is present in the entire flavocytochrome b family. The large water soluble domain of FRO2, which contains NADPH, FAD and oxidoreductase sequence motifs, was located on the inside of the membrane.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at This work was supported by grants from the Swedish Research Council (VR) to S. Al-Karadaghi and Hans Hebert.     

Psychosocial Working Conditions and Cognitive Complaints among Swedish Employees

Background

Cognitive complaints involving problems with concentration, memory, decision-making and thinking are relatively common in the work force. The sensitivity of both subjective and objective cognitive functioning to common psychiatric conditions, stress levels and to cognitive load makes it plausible that psychosocial working conditions play a role in cognitive complaints. Thus, this study aimed to test the associations between psychosocial work factors and cognitive complaints in nationally representative samples of the Swedish work force. Cross-sectional (n = 9751) and prospective (n = 3644; two time points two years apart) sequential multiple regression analyses were run, adjusting for general confounders, depressive- and sleeping problems. Additional prospective analyses were run adjusting for baseline cognitive complaints.

Cross-sectional results

High quantitative demands, information and communication technology (ICT) demands, underqualification and conflicts were positively associated with cognitive complaints, while social support, good resources at work and overqualification were negatively associated with cognitive complaints in all models. Skill discretion and decision authority were weakly associated with cognitive complaints. Conflicts were more strongly associated with cognitive complaints in women than in men, after adjustment for general confounders.

Prospective results

Quantitative job demands, ICT demands and underqualification were positively associated with future cognitive complaints in all models, including when adjusted for baseline cognitive complaints. Decision authority was weakly positively associated with future cognitive complaints, only after adjustment for depressive- and sleeping problems respectively. Social support was negatively associated with future cognitive complaints after adjustment for general confounders and baseline cognitive complaints. Skill discretion and resources were negatively associated with future cognitive complaints after adjustment for general confounders. The associations between quantitative demands and future cognitive complaints were stronger in women.

Discussion/Conclusions

The findings indicate that psychosocial working conditions should be taken into account when considering cognitive complaints among employees.     

Habitual loading results in tendon hypertrophy and increased stiffness of the human patellar tendon.

The purpose of this study was to examine patellar tendon (PT) size and mechanical properties in subjects with a side-to-side strength difference of >/=15% due to sport-induced loading. Seven elite fencers and badminton players were included. Cross-sectional area (CSA) of the PT obtained from MRI and ultrasonography-based measurement of tibial and patellar movement together with PT force during isometric contractions were used to estimate mechanical properties of the PT bilaterally. We found that distal tendon and PT, but not mid-tendon, CSA were greater on the lead extremity compared with the nonlead extremity (distal: 139 +/- 11 vs. 116 +/- 7 mm(2); mid-tendon: 85 +/- 5 vs. 77 +/- 3 mm(2); proximal: 106 +/- 7 vs. 83 +/- 4 mm(2); P < 0.05). Distal tendon CSA was greater than proximal and mid-tendon CSA on both the lead and nonlead extremity (P < 0.05). For a given common force, stress was lower on the lead extremity (52.9 +/- 4.8 MPa) compared with the nonlead extremity (66.0 +/- 8.0 MPa; P < 0.05). PT stiffness was also higher in the lead extremity (4,766 +/- 716 N/mm) compared with the nonlead extremity (3,494 +/- 446 N/mm) (P < 0.05), whereas the modulus did not differ (lead 2.27 +/- 0.27 GPa vs. nonlead 2.16 +/- 0.28 GPa) at a common force. These data show that a habitual loading is associated with a significant increase in PT size and mechanical properties.     

Anthropogenic Landscape in Southeastern Amazonia: Contemporary Impacts of Low-Intensity Harvesting and Dispersal of Brazil Nuts by the Kayapó Indigenous People

Brazil nut, the Bertholletia excelsa seed, is one of the most important non-timber forest products in the Amazon Forest and the livelihoods of thousands of traditional Amazonian families depend on its commercialization. B. excelsa has been frequently cited as an indicator of anthropogenic forests and there is strong evidence that past human management has significantly contributed to its present distribution across the Amazon, suggesting that low levels of harvesting may play a positive role in B. excelsa recruitment. Here, we evaluate the effects of Brazil nut harvesting by the Kayapó Indigenous people of southeastern Amazonia on seedling recruitment in 20 B. excelsa groves subjected to different harvesting intensities, and investigated if management by harvesters influences patterns of B. excelsa distribution. The number of years of low-intensity Brazil nut harvesting by the Kayapó over the past two decades was positively related to B. excelsa seedling density in groves. One of the mechanisms behind the higher seedling density in harvested sites seems to be seed dispersal by harvesters along trails. The Kayapó also intentionally plant B. excelsa seeds and seedlings across their territories. Our results show not only that low-intensity Brazil nut harvesting by the Kayapó people does not reduce recruitment of seedlings, but that harvesting and/or associated activities conducted by traditional harvesters may benefit B. excelsa beyond grove borders. Our study supports the hypothesis that B. excelsa dispersal throughout the Amazon was, at least in part, influenced by indigenous groups, and strongly suggests that current human management contributes to the maintenance and formation of B. excelsa groves. We suggest that changes in Brazil nut management practices by traditional people to prevent harvesting impacts may be unnecessary and even counterproductive in many areas, and should be carefully evaluated before implementation.