The competitive interaction of actin and PIP2 with actophorin is based on overlapping target sites: design of a gain-of-function mutant.

We studied the effect of mutations in an alpha-helical region of actophorin (residues 91-108) on F-actin and PIP(2) binding. As in cofilin, residues in the NH(2)-terminal half of this region are involved in F-actin binding. We show here also that basic residues in the COOH-terminal half of the region participate in this interaction whereby we extend the previously defined actin binding interface [Lappalainen, P., et al. (1997) EMBO J. 16, 5520-5530]. In addition, we demonstrate that some of the lysines in this alpha-helical region in actophorin are implicated in PIP(2) binding. This indicates that the binding sites of F-actin and PIP(2) on actophorin overlap, explaining the mutually exclusive binding of these ligands. The Ca(2+)-dependent F-actin binding of a number of actophorin mutants (carrying a lysine to glutamic acid substitution at the COOH-terminal positions of the actin binding helical region) may mimic the behavior of members of the gelsolin family. In addition, we show that PIP(2) binding, but not actin binding, of actophorin is strongly enhanced by a point mutation that leads to a reinforcement of the positive electrostatic potential of the studied alpha-helical region.

The impact of fetal compromise on outcome at the border of viability.

OBJECTIVE: Our goal was to evaluate the impact of fetal compromise on the outcome of borderline viable babies. STUDY DESIGN: All 142 babies born in our hospital from 1990 to 1995 with a gestational age of 23 to 25 weeks were included. Fetal compromise was considered present if one of the following was documented: a major anomaly, congenital sepsis, chronic intrauterine infection, intrauterine drug exposure, congenital anemia, severe growth restriction, fetal acidosis, or cardiorespiratory and neurologic depression in the delivery room. RESULTS: The 43 babies who had at least one cause of fetal compromise had a lower birth weight (p < 0.001), but there were no other differences in demographics or complications of prematurity. The survival rate was significantly better for babies free of fetal compromise (75% vs 33%, p < 0.001), particularly for babies born at 23 weeks of gestation (75% vs 6%, p < 0.001). For surviving babies free of fetal compromise, the outcome at 23 weeks was comparable to that at 24 to 25 weeks for major causes of long-term neurologic morbidity. CONCLUSIONS: Like advancing gestational age and increasing birth weight, the absence of fetal compromise has a major beneficial impact on the outcome of borderline viable babies that might be important when decisions are made about the appropriate level of support.

A phage display technique for a fast, sensitive, and systematic investigation of protein-protein interactions.

Phage display is a technique in which a foreign protein or peptide is presented at the surface of a (filamentous) bacteriophage. This system, developed by Smith [(1985), Science 228, 1315-1317], was originally used to create large libraries of antibodies for the purpose of selecting those that strongly bound a particular antigen. More recently it was also employed to present peptides, domains of proteins, or intact proteins at the surface of phages, again to identify high-affinity interactions with ligands. Here we want to illustrate the use of phage display, in combination with PCR saturation mutagenesis, for the study of protein-protein interactions. Rather than selecting for mutants having high affinity, we systematically investigate the binding of every variant with its natural ligand. Via a modified ELISA we can calculate a relative affinity. As a model system we chose to display thymosin beta 4 on the phage surface in order to study its interaction with actin.

Analogous F-actin binding by cofilin and gelsolin segment 2 substantiates their structural relationship.

Cofilin is representative for a family of low molecular weight actin filament binding and depolymerizing proteins. Recently the three-dimensional structure of yeast cofilin and of the cofilin homologs destrin and actophorin were resolved, and a striking similarity to segments of gelsolin and related proteins was observed (Hatanaka, H., Ogura, K., Moriyama, K., Ichikawa, S., Yahara, I., and Inagaka, F. (1996) Cell 85, 1047-1055; Fedorov, A. A., Lappalainen, P., Fedorov, E. V., Drubin, D. G., and Almo, S. C. (1997) Nat. Struct. Biol. 4, 366-369; Leonard, S. A., Gittis, A. G., Petrella, E. C., Pollard, T. D., and Lattman, E. E. (1997) Nat. Struct. Biol. 4, 369-373). Using peptide mimetics, we show that the actin binding site stretches over the entire cofilin alpha-helix 112-128. In addition, we demonstrate that cofilin and its actin binding peptide compete with gelsolin segments 2-3 for binding to actin filaments. Based on these competition data, we propose that cofilin and segment 2 of gelsolin use a common structural topology to bind to actin and probably share a similar target site on the filament. This adds a functional dimension to their reported structural homology, and this F-actin binding mode provides a basis to further enlighten the effect of members of the cofilin family on actin filament dynamics.

Evidence for an actin binding helix in gelsolin segment 2; have homologous sequences in segments 1 and 2 of gelsolin evolved to divergent actin binding functions?

Gelsolin is built up of six homologous segments that perform different functions on actin. Segments 1 and 2, which are suggested to be highly similar in their overall folds, bind monomeric and filamentous actin respectively. A long alpha-helix in segment 1 forms the major contact site of this segment with actin. We show that sequence 197-226 of segment 2, equivalent to the region around the actin binding helix in segment 1, contains F-actin binding activity. Consequently, positionally similar parts of segment 1 and 2 are implicated in the actin contact and solvent exposed residues in these parts must have evolved differentially to meet their different actin binding properties.

The actin binding site of thymosin beta 4 mapped by mutational analysis.

We characterized in detail the actin binding site of the small actin-sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full-length T beta 4 variants. The N-terminal part (residues 1-16) and a hexapeptide motif (residues 17-22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross-linking, complex formation in native gels and actin-sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N-terminal part needs to adopt an alpha-helix for actin binding and interacts through a patch of hydrophobic residues (6M-I-F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N-terminal alpha-helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta-thymosin family and in addition to this we identify a similar pattern in the C-terminal headpiece of villin and dematin.

Current gestational age-related incidence of major intraventricular hemorrhage.

The overall incidence of germinal matrix intraventricular hemorrhage (GMIVH) for an inborn population of 250 very low birth weight infants from 1991 through 1993 was 20%; the incidence of major GMIVH was 5.2%. Of infants < or = 25 weeks, 60% survived and 94% of the survivors were free of major GMIVH, whereas 94% of infants > or = 25 weeks survived and 99% had no major GMIVH.

Preparation of plant DNA for separation by pulsed field gel electrophoresis.

A method was developed for the preparation of completely intact plant DNA embedded in agarose, and suitable for restriction enzyme digestion. Digestion with restriction enzyme was carried out according to modified protocols of Anand and Kenwrick et al. The new method of DNA isolation allows the separation of high molecular weight plant DNA by pulsed field gel electrophoresis.

Incidence and severity of intraventricular hemorrhage: 1981-1984.

We have examined the trend in the incidence and mortality of intraventricular hemorrhage (IVH) in low birthweight infants from 1981 through 1984. During this time we admitted 407 infants in the first week of life with a birthweight less than or equal to 1500 gm in whom a cranial ultrasonogram or autopsy had been performed. Though the mean birthweight and gestational age, proportion of infants who were inborn, and percentage of infants requiring mechanical ventilation did not change over the 4 years, cesarean deliveries were performed more frequently (P less than .001). The overall incidence of IVH was 62% in 1981, 56% in 1982, 49% in 1983, and 58% in 1984, thus no significant trend was evident. Although the incidence of minor hemorrhages (grades I and II) remained relatively constant, there was a decrease in the incidence of grade III IVH (1981, 11%; 1984, 2%, P = .01). The incidence of grade IV hemorrhage did not change during the 4 years and ranged from 7 to 9%. Mortality rate for all infants weighing less than or equal to 1500 gm and for infants with a minor hemorrhage remained unchanged; however, the mortality rate for infants with a major hemorrhage (grade III or IV) tended to decrease (P = .07). We conclude that although some minor changes in the incidence and mortality have occurred, IVH continues to be a major problem in very-low-birthweight infants at our institution.