Fatigue responses of human triceps surae muscles during repetitive maximal isometric contractions.

Nine healthy men (22-45 yr) completed 100 repetitive maximal isometric contractions of the ankle plantar flexor muscles in two knee positions of full extension (K0) and flexion at 90 degrees (K90), positions that varied the contribution of the gastrocnemii. Electromyographic activity was recorded from the medial and lateral gastrocnemii and soleus muscles by using surface electrodes. Plantar flexion torque in K0 was greater and decreased more rapidly than in K90. The electromyographic amplitude decreased over time, and there were no significant differences between muscles and knee joint positions. The level of voluntary effort, assessed by a supramaximal electrical stimulation during every 10th contraction, decreased from 96 to 70% (P < 0.05) with no difference between K0 and K90. It was suggested that a decrease in plantar flexion torque was attributable to both central and peripheral fatigue and that greater fatigability in K0 than in K90 would result from a greater contribution and hence more pronounced fatigue of the gastrocnemius muscle. Further support for this possibility was provided from changes in twitch torque.     

Interaction of a nuclear factor-1-like protein with the regulatory region of the human polyomavirus JC virus

We have initiated a study to identify host proteins which interact with the regulatory region of the human polyomavirus JC (JCV), which is associated with the demyelinating disease, progressive multifocal leukoencephalopathy. We examined the interaction of nuclear proteins prepared from different cell lines with the JCV regulatory region by DNA binding gel retardation assays. Binding was detected with nuclear extracts prepared from human fetal glial cells, glioma cells, and HeLa cells. Little or no binding was detected with nuclear extracts prepared from human embryonic kidney cells. Competitive binding assays suggest that the nuclear factor(s) which interacted with the JCV regulatory region was different from those which interacted with the regulatory region of the closely related polyomavirus SV40. We found three areas in the JCV regulatory region protected from DNase I digestion: site A, located just upstream from the TATA sequence in the first 98-base pair (bp) repeat; site B, located upstream from the TATA sequence in the second 98-bp repeat; and site C, located just following the second 98-bp repeat. There were some differences in the ability of the nuclear factor(s) from the two brain cell lines and HeLa cells to completely protect the nucleotides within the footprint region. The results from the DNase I protective studies and competitive DNA binding studies with specific oligonucleotides, suggest that nuclear factor-1 or a nuclear factor-1-like factor is interacting with all three sites in the JCV regulatory region. In addition, the results suggest that the nuclear factor which interacts with the JCV regulatory region from human brain cell lines is different from the factor found in HeLa cells.     

The compactness of ribonuclease A and reduced ribonuclease A

The compactness of ribonuclease A with intact disulfide bonds and reduced ribonuclease A was investigated by synchrotron small-angle X-ray scattering. The R_g values and the Kratky plots showed that non-reduced ribonuclease A maintain a compact shape with a R_g value of about 17.3 Å in 8 M urea. The reduced ribonuclease A is more expanded, its R_g value is about 20 Å in 50 mM Tris-HCl buffer at pH 8.1 containing 20 mM DTT. Further expansions of reduced ribonuclease A were observed in the presence of high concentrations of denaturants, indicating that reduced ribonuclease A is more expanded and is in neither a random coil [A. Noppert et al., FEBS Lett. 380 (1996) 179–182] nor a compact denatured state [T.R. Sosnick and J. Trewhella, Biochemistry 31 (1992) 8329–8335]. The four disulfide bonds keep ribonuclease A in a compact state in the presence of high concentrations of urea.     

Tuatara (Sphenodon) genomics: BAC library construction, sequence survey, and application to the DMRT gene family

The tuatara (Sphenodon punctatus) is of "extraordinary biological interest" as the most distinctive surviving reptilian lineage (Rhyncocephalia) in the world. To provide a genomic resource for an understanding of genome evolution in reptiles, and as part of a larger project to produce genomic resources for various reptiles (evogen.jgi.doe.gov/second_levels/BACs/our_libraries.html), a large-insert bacterial artificial chromosome (BAC) library from a male tuatara was constructed. The library consists of 215 424 individual clones whose average insert size was empirically determined to be 145 kb, yielding a genomic coverage of approximately 6.3x. A BAC-end sequencing analysis of 121 420 bp of sequence revealed a genomic GC content of 46.8%, among the highest observed thus far for vertebrates, and identified several short interspersed repetitive elements (mammalian interspersed repeat-type repeats) and long interspersed repetitive elements, including chicken repeat 1 element. Finally, as a quality control measure the arrayed library was screened with probes corresponding to 2 conserved noncoding regions of the candidate sex-determining gene DMRT1 and the DM domain of the related DMRT2 gene. A deep coverage contig spanning nearly 300 kb was generated, supporting the deep coverage and utility of the library for exploring tuatara genomics.     

Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model

Yersinia pestis is the etiologic agent of plague that has killed more than 200 million people throughout the recorded history of mankind. Antibiotics may provide little immediate relief to patients who have a high bacteremia or to patients infected with an antibiotic resistant strain of plague. Two virulent factors of Y. pestis are the capsid F1 protein and the low-calcium response (Lcr) V-protein or V-antigen that have been proven to be the targets for both active and passive immunization. There are mouse monoclonal antibodies (mAbs) against the F1- and V-antigens that can passively protect mice in a murine model of plague; however, there are no anti-Yersinia pestis monoclonal antibodies available for prophylactic or therapeutic treatment in humans. We identified one anti-F1-specific human mAb (m252) and two anti-V-specific human mAb (m253, m254) by panning a naïve phage-displayed Fab library against the F1- and V-antigens. The Fabs were converted to IgG1s and their binding and protective activities were evaluated. M252 bound weakly to peptides located at the F1 N-terminus where a protective mouse anti-F1 mAb also binds. M253 bound strongly to a V-antigen peptide indicating a linear epitope; m254 did not bind to any peptide from a panel of 53 peptides suggesting that its epitope may be conformational. M252 showed better protection than m253 and m254 against a Y, pestis challenge in a plague mouse model. A synergistic effect was observed when the three antibodies were combined. Incomplete to complete protection was achieved when m252 was given at different times post-challenge. These antibodies can be further studied to determine their potential as therapeutics or prophylactics in Y. pestis infection in humans.     

Skeletogenic potential of induced secondary mesenchyme cells derived from the presumptive ectoderm in echinoid embryos

 During the normal development of echinoids, an animal cap consisting of 8 mesomeres in a 16-cell stage embryo differentiates exclusively into ectoderm. Micromeres in an embryo at the same stage differentiate into primary mesenchyme cells (PMC) and coelomic pouch constituents. An animal cap and a quartet of micromeres were isolated from a 16-cell stage embryo and recombined to make a chimeric embryo devoid of presumptive endoderm and secondary mesenchyme cells (SMC). The PMC in the chimeric embryo were completely removed at the mesenchyme blastula stage. The PMC-depleted chimeric embryos formed an archenteron derived from the mesomeres. Some secondary mesenchyme-like cells (induced SMC) were released from the archenteron tip. A considerable fraction of the induced SMC formed the typical mesenchyme pattern after migrating into the vegetal region, synthesized skeletogenic mesenchyme cell-surface protein (msp130) and produced the larval skeleton. These findings indicate that induced SMC derived from the presumptive ectoderm have the same nature as natural SMC in both the timing of their release and their skeletogenic potential expressed in the absence of PMC. Received: 14 November 1996 / Accepted: 30 December 1996     

Met-145 is a key residue in the dark adaptation of bacteriorhodopsin homologs.

Composition of retinal isomers in three proton pumps (bacteriorhodopsin, archaerhodopsin-1, and archaerhodopsin-2) was determined by high performance liquid chromatography in their light-adapted and dark-adapted states. In the light-adapted state, more than 95% of the retinal in all three proton pumps were in the all-trans configuration. In the dark-adapted state, there were only two retinal isomers, all-trans and 13-cis, in the ratio of all-trans: 13-cis = 1:2 for bacteriorhodopsin, 1:1 for archaerhodopsin-1, and 3:1 for archaerhodopsin-2. The difference in the final isomer ratios in the dark-adapted bacteriorhodopsin and archaerhodopsin-2 was ascribed to the methionine-145 in bacteriorhodopsin. This is the only amino acid in the retinal pocket that is substituted by phenylalanine in archaerhodopsin-2. The bacteriorhodopsin point-mutated at this position to phenylalanine dramatically altered the final isomer ratio from 1:2 to 3:1 in the dark-adapted state. This point mutation also caused a 10 nm blue-shift of the adsorption spectrum, which is similar to the shift of archaerhodopsin-2 relative to the spectra of bacteriorhodopsin and archaerhodopsin-1.     

Micromere-derived signal regulates larval left-right polarity during sea urchin development

The micromeres (Mics) lineage functions as a morphogenetic signaling center in early embryos of sea urchins. The Mics lineage releases signals that regulate the specification of cell fates along the animal-vegetal and oral-aboral axes. We tested whether the Mics lineage might also be responsible for differentiation of the left-right (LR) axis by observing of the placement of the adult rudiment, which normally forms only on the left side of the larvae, after removal of the Mics lineage. When all of the Mics lineage were removed from embryos of the regular sea urchin Hemicentrotus pulcherrimus between the 16- and 64-cell stages, the LR placement of the rudiment became randomized. However, the immediate retransplantation of the Mics rescued the normal LR placement of the rudiment, indicating that the Mics lineage releases a signal that specifies LR polarity. Additionally, we investigated whether the specification of LR polarity of whole embryos in the indirect-developing sea urchin H. pulcherrimus is affected by LiCl exposure, which disturbs the establishment of LR asymmetry in a direct-developing sea urchin. Larvae derived from normal animal caps combined with LiCl-exposed Mics descendants were defective in normal LR placement of the rudiment, suggesting that LiCl interferes with the Mics-derived signal. In contrast, embryos of two sand dollar species (Scaphechinus mirabilis and Astriclypeus manni) were resistant to alteration of LR placement of the rudiment by either removal of the Mics lineage or LiCl exposure. These results indicate that the Mics lineage is involved in specification of LR polarity in the regular sea urchin H. pulcherrimus, and suggest that LiCl impairs the normal LR patterning by affecting Mics-derived signaling.