Different roles of N‐ and C‐ termini in the functional activity of FGF21

Fibroblast growth factor 21 is a member of endocrine FGFs subfamily, along with FGF19 and FGF23. It is emerging as a novel regulator with beneficial effects on a variety of metabolic parameters, including glucose and lipid control. FGF21 activity depends on membrane protein βKlotho that physically complexes with various FGF receptors, thus conferring them the ability to bind FGF21 and activate downstream signaling pathways. FGF21, like other FGFs, folds to a β‐trefoil‐like core region, with disordered N‐ and C‐termini. In order to investigate their role in the activity of FGF21, we have constructed a series of deletion mutants and tested them for their ability to (1) bind βKlotho, analyzed by surface plasmon resonance spectroscopy (2) signal through MAPK phosphorylation and inhibit apoptosis in 3T3‐L1/βKlotho fibroblasts (3) stimulate GLUT1 mRNA upregulation and glucose uptake in 3T3‐L1 adipocytes. Binding studies with βKlotho revealed that the interaction with the co‐receptor involves the C‐terminus, as progressive removal of amino acids from the carboxy end decreased affinity for βKlotho. By contrast, removal of up to 17 amino acids from the N‐terminus had no effect on the interaction with βKlotho. Terminal deletions had greater effect on function, as deletions of six amino acids from the amino‐terminus and only four from the carboxy‐terminus each significantly impacted activity (10‐fold). Of the extreme terminal truncations, with no detectable activity, ΔN17 acted as competitive antagonist while ΔC20 did not. Our structure/function studies show that the C‐terminus is important for βKlotho interaction whereas the N‐terminus likely interacts directly with FGF receptors. J. Cell. Physiol. 219: 227–234, 2009. © 2008 Wiley‐Liss, Inc.     

Assessment of an enterovirus sewage surveillance system by comparison of clinical isolates with sewage isolates from milwaukee, wisconsin, collected august 1994 to december 2002

The quantity and serotypes of enteroviruses (EVs) in the influent of a local sewage treatment plant were compared to local clinical EV cases to determine if testing of sewage is adequate for an EV surveillance system. The study was carried out from August 1994 to December 2002. Monthly influent specimens were processed by organic flocculation, and dilutions of concentrate were inoculated onto a number of different cell types for virus isolation. EVs were detected in 88 of 100 monthly influent samples. Sewage EV titers were calculated by using software provided by the U.S. Environmental Protection Agency for most-probable-number determination. All 1,068 sewage EV isolates were further grouped (echovirus, coxsackievirus B, coxsackievirus A, or poliovirus) by cell culture host range analysis (growth pattern of isolates on passage to seven cell lines), and 39.0% of the 1,022 EV isolates categorized as non-poliovirus EVs were specifically serotyped. For clinical cases, primary virus isolation tests were performed on specimens submitted by local hospitals and EV isolates submitted by hospitals were serotyped. Clinical EVs were documented for 81 of the 100 months studied. In all, 694 EV isolates from clinical cases were serotyped. Annually, between 4 and 11 different serotypes of non-poliovirus EVs were identified in sewage and from 9 to 19 different non-poliovirus EV serotypes were identified from clinical specimens. Usually, the most commonly detected sewage EV serotypes were similar to the most commonly detected clinical serotypes; e.g., for 1997, echovirus 6 accounted for 53.1% of the typed sewage isolates and 39.4% of the clinical infections, while in 1998, echovirus 30 accounted for 50.0 and 46.1%, respectively. In 1999, 60.3% of the EVs from clinical cases and 79.7% of the sewage isolates were echovirus 11; in 2000, 33.3% of the EVs from clinical cases and 40.7% of the sewage isolates were coxsackievirus B5; and in 2001, 44.1% of the EVs from clinical cases and 36.2% of the sewage isolates were echovirus 13. Annual peaks of both sewage EV titers and clinical cases occurred in late summer or early fall. In some years, early spring sewage EVs portended some of the EVs that would predominate clinically during the following summer.     

A behavioral role for dendritic integration: HCN1 channels constrain spatial memory and plasticity at inputs to distal dendrites of CA1 pyramidal neurons

The importance of long-term synaptic plasticity as a cellular substrate for learning and memory is well established. By contrast, little is known about how learning and memory are regulated by voltage-gated ion channels that integrate synaptic information. We investigated this question using mice with general or forebrain-restricted knockout of the HCN1 gene, which we find encodes a major component of the hyperpolarization-activated inward current (Ih) and is an important determinant of dendritic integration in hippocampal CA1 pyramidal cells. Deletion of HCN1 from forebrain neurons enhances hippocampal-dependent learning and memory, augments the power of theta oscillations, and enhances long-term potentiation (LTP) at the direct perforant path input to the distal dendrites of CA1 pyramidal neurons, but has little effect on LTP at the more proximal Schaffer collateral inputs. We suggest that HCN1 channels constrain learning and memory by regulating dendritic integration of distal synaptic inputs to pyramidal cells.     

Simple miniaturized gel system for DNA sequence analysis.

A simple miniaturized gel system suitable for DNA sequencing is described. Small ultrathin polyacrylamide gels are cast, eight or more at a time, using standard microscope slides. Gels, ready to use, can be stored for approximately 2 weeks. Gels are run horizontally in a standard mini-agarose gel apparatus. Typical run times are 6-8 min. A novel sample loading system permits volumes of standard sequencing reactions as small as 0.1 microl to be analyzed. Sequencing ladders were visualized using 35S-labeled DNA by autoradiography and by colorimetric detection. Band resolution compares favorably with that of large gels. The methods introduced here serve as a step toward the miniaturization of DNA sequencing and are amenable to automated sample loading and detection.     

Mutants and Intersexual Heterokaryons of Blakeslea trispora for Production of β-Carotene and Lycopene

The industrial production of β-carotene with the zygomycete Blakeslea trispora involves the joint cultivation of mycelia of opposite sex in the presence of β-ionone and other chemical activators. We have obtained improved strains by mutation and heterokaryosis. We chose wild strains on the basis of their growth and carotene content in single and mated cultures. Following exposure of their spores to N-methyl-N′-nitro-N-nitrosoguanidine, we obtained high-carotene mutants, which were more productive than their parents but similar to them in having β-carotene as the main product. Further increases in carotene content were obtained after a new round of mutagenesis in one of the mutants. The production was shifted to lycopene in cultures incubated in the presence of nicotine and in lycopene-rich mutants derived from the wild strains. The highest production levels were achieved in intersexual heterokaryons, which contained mutant nuclei of opposite sex. These contained up to 39 mg of β-carotene or 15 mg of lycopene per g (dry mass) under standard laboratory conditions in which the original wild strains contained about 0.3 mg of β-carotene per g (dry mass). β-Ionone did not increase the carotene content of these strains. Not all wild strains lent themselves to these improvements, either because they produced few mutants or because they did not increase their carotene production in mated cultures.     

Comparative analysis of GDF 8 (myostatin) in Bos indicus and Bos taurus.

We report the myostatin gene sequence of Bos indicus cattle in comparison to Bos taurus. B. indicus genomic sequence was obtained by overlapping PCR amplification of genomic DNA. Exon splice sites were confirmed by mRNA sequencing. There were 5 exonic single nucleotide polymorphisms (SNP) only one of which was a non-synonymous mutation that resulted in a serine to asparagine (S214N) amino acid substitution. The B. indicus gene has two insertions of 16 and 12 bases in the first intron. In addition, SNPs in the 3' UTR and intronic regions are also reported.     

Utilization of alkaline phosphatase fusions to identify secreted proteins, including potential efflux proteins and virulence factors from Helicobacter pylori

The targeted genomic strategy of random fusions to a partial gene encoding a signal sequence-deficient fragment of bacterial alkaline phosphatase was utilized to screen for secreted proteins in Helicobacter pylori. The rationale for targeting extracytoplasmic proteins was based on the hypothesis that most virulence factors and vaccine candidates are secreted or exported proteins. In addition, extracytosolic proteins represent good potential targets for drug intervention since they are in general more accessible to drugs than are cytoplasmically localized proteins. The application of this strategy to H. pylori allowed the identification of putative virulence factors and novel targets for drug intervention including four putative antibiotic efflux genes. The strategy used here is rapid and technically simple, relatively inexpensive, adaptable to a wide variety of microbes and genetic systems, and selects for expressed and accessible proteins.