Role of FGFs in skeletal muscle and limb development

Fibroblast growth factors (FGFs) are a family of nine proteins that bind to three distinct types of cell surface molecules: (i) FGF receptor tyrosine kinases (FGFR-1 through FGFR-4); (ii) a cysteine-rich FGF receptor (CFR); and (iii) heparan sulfate proteoglycans (HSPGs). Signaling by FGFs requires participation of at least two of these receptors: the FGFRs and HSPGs form a signaling complex. The length and sulfation pattern of the heparan sulfate chain determines both the activity of the signaling complex and, in part, the ligand specificity for FGFR-1. Thus, the heparan sulfate proteoglycans are likely to play an essential role in signaling. We have recently identified a role for FGF in limb bud development in vivo. In the chick limb bud, ectopic expression of the 18 kDa form of FGF-2 or FGF-2 fused to an artificial signal peptide at its amino terminus causes skeletal duplications. These data, and the observations that FGF-2 is localized to the subjacent mesoderm and the apical ectodermal ridge in the early developing limb, suggest that FGF-2 plays an important role in limb outgrowth. We propose that FGF-2 is an apical ectodermal ridgederived factor that participates in limb outgrowth and patterning. © 1994 Wiley-Liss, Inc.     

MONOPHYLY OF ANEUPLOID ASTRAGALUS (FABACEAE): EVIDENCE FROM NUCLEAR RIBOSOMAL DNA INTERNAL TRANSCRIBED SPACER SEQUENCES

Evolutionary relationships within Astragalus L. (Fabaceae) were inferred from nucleotide sequence variation in nuclear ribosomal DNA of both New World and Old World species. The internal transcribed spacer regions (ITS) of 18S–26S nuclear ribosomal DNA from representatives of 26 species of Astragalus, three species of Oxytropis DC., and two outgroup taxa were analyzed by polymerase chain reaction amplification and direct DNA sequencing. The length of the ITS 1 region within these taxa varied from 221 to 231 bp, while ITS 2 varied in length from 207 to 217 bp. Of the aligned, unambiguous positions, approximately 34% were variable in each spacer region. In pairwise comparisons among Astragalus species and outgroup taxa, sequence divergence at these sites ranged from 0 to 18.8% in ITS 1 and from 0 to 21.7% in ITS 2. Parsimony analyses of these sequences resulted in a well-resolved phylogeny that is highly concordant with previous cytogenetic and chloroplast DNA evidence for a major phylogenetic division in the genus. These data suggest that the New World aneuploid species of Astragalus form a monophyletic but morphologically cryptic group derived from euploid species of Old World (Eurasian) origin, which are consequently paraphyletic.     

Approximation and characterization of molecular surfaces

The representation and characterization of molecular surfaces are important in many areas of molecular modeling. Parametric representations of protein molecular surfaces are a compact way to describe a surface, and are useful for the evaluation of surface properties such as the normal vector, principal curvatures, and principal curvature directions. Simplified representations of molecular surfaces are useful for efficient rendering and for the display of large-scale surface features. Several techniques for representing surfaces by expansions of spherical harmonic functions have been reported, but these techniques require that the radius function is single valued, that is, each ray from an origin inside the surface intersects the surface at one and only one point. A new technique is described that removes this limitation and can be used to compute surface shape properties. © 1993 John Wiley & Sons, Inc.     

Modification of supported lipid membranes by atomic force microscopy

The atomic force microscope (AFM) was used to structurally modify supported lipid bilayers in a controlled quantitative manner. By increasing the force applied by the AFM tip, lipid was removed from the scanned area, leaving a cut through the lipid bilayer. Cuts were repaired with the AFM by scanning the region with a controlled force and driving lipid back into the cut. A slow self-annealing of cuts was also observed.     

Temperature preferences and tolerances of three fish species inhabiting hyperthermal ponds on mangrove islands

The fish species Cyprinidon artifrons, Floridichthys carpio, and Gambusia yucatana inhabit shallow mangrove ponds off the coast of Belize. Portions of these ponds experience a diurnal temperature change from 26 °C at night to 40 °C and above during midday. Repeated field observations indicate Cyprinidon prefer the warmer (and much larger) portions of the ponds whereas the other two species stay in the cooler areas.The hypothesis that temperature is serving as a cue for partitioning within the ponds was supported by laboratory thermal gradient tests in which Cyprinidon preferred temperatures clearly higher than the other two species.The critical thermal maximum (CTM) was determined for the three species using members that had been acclimated to either a daily cycling temperature similar to that for the ponds, or to the mean of the 24-hour cycle (30 °C). Cyprinidon acclimated to the cycling temperature had a CTM of 45.5 °C, which apparently sets a new record for fish CTM. Acclimation to a constant 30 °C lowered the CTM to 43.7 °C. Floridichthys and Gambusia acclimated to the cycled temperature had CTMs of 43.9 and 43.3 °C respectively, and 42.5 and 42.6 °C for those acclimated to 30 °C.All three species appear to have the ability to tolerate the high temperatures throughout the ponds but only Cyprinidon utilize the whole pond during the day. This may help to explain the large populations of Cyprinodon found in these mangrove ponds compared to the other species.     

Nucleotide substitutions at the -6 position in the promoter region of the factor IX gene result in different severity of hemophilia B Leyden: consequences for genetic counseling

Mutations in the promoter region of the factor IX gene result in hemophilia B Leyden, which is characterized by considerable improvement in the disease after puberty. We have found that distinct nucleotide substitutions at the -6 position in the Leyden-specific (LS) region are associated with a different severity of hemophilia B. The proband (aged 2) from one family is a severe hemophiliac with factor IX activity (F.IXC) and antigen (F.IXAg) levels less than 1.0U/dl. F.IXC and F.IXAg levels in two affected uncles are approximately 30% of normal levels. The LS region was targeted for analysis because the phenotypes suggested the inheritance of a factor IX Leyden gene. An abnormal TaqI digestion pattern was found in amplified DNA from the proband, and sequencing showed a G (-6) to C transversion that was linked to the disease in the family. In another family, two brothers (aged 8 and 9) suffer from mild hemophilia with F.IXC ranging from 7 to 10 U/dl and F.IXAg from 3 to 4 U/dl. They are the only documented members of the family with a bleeding tendency. Denaturing gradient gel electrophoresis on amplified fragments from one of the patient's genomic DNA corresponding to the 8 exons and flanking sequences of the factor IX gene suggested a defect only in a segment from the 5 region. This segment showed an altered TaqI digestion pattern, and sequencing demonstrated a G(-6) to A transition that was traced to the patients's mother and a grandmother. The different phenotypes associated with the G (-6) to A purine nucleotide transition compared with a G(-6) to C transversion provide evidence that this area is directly involved in the regulation of the human factor IX gene expression in vivo by binding of regulatory factors. The ability to predict that the conditions of a hemophilia B patient will improve with age has important implications for genetic counseling of the family. Therefore, the LS region should always be included when scanning the factor IX gene for mutations.     

Enhancement of Antigenic Site Detection with Gold Labeled Secondary and Tertiary Antibodies Using the Immunogold-Silver Staining Method

We report a modification of the immunogold-silver staining method (IGSS) for localizing hepatic phosphoenolpyruvate carboxykinase (PEPCK) in tissue sections, and we compare the efficacy of localizing the primary antibody with either a 5 nm gold labeled secondary antibody or 5 nm gold labeled secondary and tertiary antibodies. Light microscope examination of 10 μm frozen sections demonstrated that the use of combined secondary and tertiary gold labeled antibodies was superior to using a secondary gold labeled antibody alone. The increased labeling density (number of colloidal gold particles/antigenic site/cell) achieved by combined gold labeled antibodies was confirmed by electron microscopy. The increased labeling density resulted in a two-thirds reduction in the time needed for the IGSS physical development of the silver shells and less background. We achieved intense specific staining of hepatocytes expressing PEPCK while minimizing background staining. The use of combined secondary and tertiary gold labeled antibodies enhances the signal-to-noise ratio, achieves high resolution and is a suitable method for use in both light and electron microscopy.     

Mapping of ben genes of Pseudomonas aeruginosa

Abstract Four ben genes responsible for the conversion of benzoate to catechol in Pseudomonas aeruginosa PAO have been mapped to a 4.6 kb Kpn I fragment. ben -1 and ben -4 were known to be separate genes but now ben-1508 has been found to be different from ben-2 . The two genes were distinguished by Tn 5 mutagenesis of a cosmid clone and deletion mapping. It is likely that the four genes mapped ( ben-4, ben-2, ben-1508 and ben-1 ) correspond to the previously characterized benR (regulatory gene) and benABC (benzoate dioxygenase) respectively.     

Congruent evolution between whiteflies (Homoptera: Aleyrodidae) and their bacterial endosymbionts based on respective 18S and 16S rDNAs

Whiteflies (family Aleyrodidae) possess heritable eubacterial endosymbionts sustained in specialized organ-like structures called mycetomes. Comparisons of distances between the ash whitefly,Siphoninus phillyreae, and two biotypes (A and B) of the sweetpotato whitefly,Bemisia tabaci, based on sequence analysis of genes for 18S rRNAs (rDNAs), were equivalent to the distances represented by the 16S rDNAs of their respective endosymbionts. This finding indicates that evolutionary divergence in whitefly hosts and their endosymbionts is congruent. The nucleotide sequences of the 18S rDNAs and endosymbiont 16S rDNAs indicate the two biotypes ofB. tabaci are the same species.