Interferometric Studies of Growth Kinetics and Surface Morphology in Macromolecular Crystal Growth: Canavalin, Thaumatin, and Turnip Yellow Mosaic Virus

In situ laser Michelson interferometry was utilized to investigate mechanisms of growth and surface morphology in protein and virus crystallization, These included plant proteins canavalin and thaumatin and turnip yellow mosaic virus. The experimental apparatus allowed us to obtain interferometric patterns and investigate growth kinetics from growing macromolecular crystals as small as 20 μm. For the crystallization of canavalin, dislocations are the sources of growth steps on the surfaces. Supersaturation and time dependencies of the normal growth rates, tangential growth step velocities, and the slopes of the dislocation hillocks were measured. The kinetic coefficient β (rate of incorporation of protein molecules into the growing crystal) was estimated for canavalin to be 9 × 10^-4 cm/sec. This is among the first estimates of such fundamental kinetic parameters for macromolecular crystallization. The change in the activities of dislocation sources under different growth conditions was also analyzed. Michelson interferometry was clearly demonstrated to be a useful tool for quantitative studies of macromolecular crystal growth.     

Use of Genetic and Physical Mapping to Locate the Spinal Muscular Atrophy Locus between Two New Highly Polymorphic DNA Markers

The gene for autosomal recessive forms of spinal muscular atrophy (SMA) has recently been mapped to chromosome 5ql3, within a 4-cM region between the blocks D5S465/D5S125 and MAP-1B/D5S112. We identified two new highly polymorphic microsatellite DNA markers—namely, AFM265wf5 (D5S629) and AFM281yh9 (D5S637)—which are the closest markers to the SMA locus. Multilocus analysis by the location-score method was used to establish the best estimate of the SMA gene location. Our data suggest that the most likely location for SMA is between locus D5S629 and the block D5S637/D5S351/MAP-1B/D5S112/D5S357. Genetic analysis of inbred SMA families, based on homozygosity by descent and physical mapping using mega-YACs, gave additional information for the loci order as follows: cen–D5S6–D5S125/D5S465–D5S435–D5S629–SMA–D5S637–D5S351–MAP–1B/D5S112–D5S357–D5S39–tel. These data give the direction for bidirectional walking in order to clone this interval and isolate the SMA gene.     

Light quality growth promotion of Spiraea nipponica: the influence of a low photon fluence rate and transfer time to a higher fluence rate

Combinations of different light quality and fluence exposure times were investigated for their effects on in vitro growth of the woody plant Spiraea nipponica. An interaction was demonstrated between different levels of benzyladenine (BA) used for in vitro propagation and the specific light regimes investigated. This relationship was affected by the length of exposure to either white or red/FR light and the time of transfer from one fluence rate to another. In all instances exposure to red/FR light resulted in more extensive growth than under white light. Thus explants cultured under 0.25 and 0.4 mg l^-1 of BA exhibited high shoot proliferation rates when transferred, after 4 weeks of low photon fluence red/FR light, to higher fluence white light for a further week. The proliferation rates obtained were higher than any white light treatment including that with the highest BA level of 0.5 mg l^-1. In addition, the combination of red/FR light exposure with a white light stage of higher fluence improved proliferation at lower exogenous BA levels.     

Sequence and structural homology among membrane-associated domains of CFTR and certain transporter proteins

A sequence comparison of the two membrane-associated (MA) domains of the cystic fibrosis transmembrane conductance regulator (CFTR), multidrug resistance transporter (MDR), and -factor pheromone export system (STE6) proteins, each of which are believed to contain a total of 12 transmembrane (TM) segments, reveals significant amino acid homology and length conservation in the loop regions that connect individual TM sequences. Similar structural homology is observed between these proteins, hemolysin B (HLYB) and the major histocompatibility-linked peptide transporter, HAM1, the latter two which contain a single MA domain composed of six TM segments. In addition, there are specific sequences that are conserved within the TM segments of the five different membrane proteins. This observation suggests that the folding topologies of the MA domains of MDR, STE6, and CFTR in the plasma membrane are likely to be very similar. The sequence analysis also reveals that there are three characteristic motifs (a pair of aromatic residues, LTLXXXXXXP and GXXL) that are conserved in MDR, STE6, HLYB, HAM1, but not in CFTR. We propose that although CFTR may be evolutionarily related to these other membrane proteins, it belongs to a separate subclass.     

Diagnostic dilatation and curettage: is it used appropriately?

OBJECTIVE--To determine patterns of use of dilatation and curettage in Britain as compared with those in the United States; to examine variations in utilisation rates within one regional health authority. DESIGN--Analysis of routinely collected hospital inpatient statistics. SETTING--Statistics for England, Scotland, and the United States; local statistics for Oxford region. SUBJECTS--All inpatient episodes in which dilatation and curettage was performed but excluding those related to pregnancy. RESULTS--Dilatation and curettage rates remained stable in Britain between 1977 and 1990, whereas in the United States they declined dramatically. In 1989-90 the rate was 71.1 per 10,000 women in England as compared with only 10.8 per 10,000 in America. In 1989, 6936 women underwent diagnostic dilatation and curettage in the Oxford region, making it the most common elective operation. A total of 2726 (39%) of these women were under 40. There was a more than twofold variation in usage of the procedure among district health authorities within the region and even greater variation in rates in women under 40. The proportion of patients treated as day cases in the district general hospitals ranged from 22% to 82%. CONCLUSIONS--Dilatation and curettage may frequently be used inappropriately. The considerable variations in usage of dilatation and curettage internationally and nationally indicate differences in clinical perception of its appropriateness. This makes it suitable for audit. In developing guidelines it will be important to agree on the most appropriate patients and the relative merits of alternative methods of endometrial sampling. Probably this could result in considerable cost savings at no risk and possibly some benefit to patients.     

A highly conserved nuclear gene for low-level phylogenetics: elongation factor-1 alpha recovers morphology-based tree for heliothine moths

Molecular systematists need increased access to nuclear genes. Highly conserved, low copy number protein-encoding nuclear genes have attractive features for phylogenetic inference but have heretofore been applied mostly to very ancient divergences. By virtue of their synonymous substitutions, such genes should contain a wealth of information about lower-level taxonomic relationships as well, with the advantage that amino acid conservatism makes both alignment and primer definition straightforward. We tested this postulate for the elongation factor-1 alpha (EF-1 alpha) gene in the noctuid moth subfamily Heliothinae, which has probably diversified since the middle Tertiary. We sequenced 1,240 bp in 18 taxa representing heliothine groupings strongly supported by previous morphological and allozyme studies. The single most parsimonious gene tree and the neighbor-joining tree for all nucleotides show almost complete concordance with the morphological tree. Homoplasy and pairwise divergence levels are low, transition/transversion ratios are high, and phylogenetic information is spread evenly across gene regions. The EF-1 alpha gene and presumably other highly conserved genes hold much promise for phylogenetics of Tertiary age eukaryote groups.      

Mesodern-inducing factors and mesodermal patterning

Identification of the signalling molecules involved in mesoderm formation in amphibian embryos still presents problems. None of the original candidates, such as activin, have been definitively ruled out, and the new factors, such as the nodal-related genes, have come on to the scene. Of the original candidates, activin has been definitively shown to act as a morphogen, whereas bone morphogenetic protein (BMP)-4 has emerged as a ventral inducer and an inhibitor of neural differentiation. The effects of BMP-4 are antagonized by chordin, a molecule related to the product of the Drosophila gene short gastrulation.     

Crystallization of alpha 1-acid glycoprotein

A possible link between cellular cyclic AMP content and Na+K+ATPase activity was investigated in homogenates of rat kidney. Enzyme kinetics of Mg2+ and Na+K+ATPase were run in the presence of cyclic AMP, dibutyryl cAMP and compounds expected to elevate cyclic AMP levels such as forskolin, a potent adenylate cyclase activator, IBMX, an inhibitor of phosphodiesterases, and the beta-agonist isoproterenol. Medullary Na+K+ATPase is strongly inhibited by cyclic AMP whereas cortical Na+K+ATPase was stimulated in the same conditions. The correlation between ATPase activity and cellular cyclic AMP content supports the concept of a possible regulation of the enzyme by cyclic AMP.