Evolution of vertebrate steroid receptors from an ancestral estrogen receptor by ligand exploitation and serial genome expansions.

The evolution of novelty in tightly integrated biological systems, such as hormones and their receptors, seems to challenge the theory of natural selection: it has not been clear how a new function for any one part (such as a ligand) can be selected for unless the other members of the system (e.g., a receptor) are already present. Here I show-based on identification and phylogenetic analysis of steroid receptors in basal vertebrates and reconstruction of the sequences and functional attributes of ancestral proteins-that the first steroid receptor was an estrogen receptor, followed by a progesterone receptor. Genome mapping and phylogenetic analyses indicate that the full complement of mammalian steroid receptors evolved from these ancient receptors by two large-scale genome expansions, one before the advent of jawed vertebrates and one after. Specific regulation of physiological processes by androgens and corticoids are relatively recent innovations that emerged after these duplications. These findings support a model of ligand exploitation in which the terminal ligand in a biosynthetic pathway is the first for which a receptor evolves; selection for this hormone also selects for the synthesis of intermediates despite the absence of receptors, and duplicated receptors then evolve affinity for these substances. In this way, novel hormone-receptor pairs are created, and an integrated system of increasing complexity elaborated. This model suggests that ligands for some "orphan" receptors may be found among intermediates in the synthesis of ligands for phylogenetically related receptors.

Identification of a third distinct estrogen receptor and reclassification of estrogen receptors in teleosts.

This paper describes three distinct estrogen receptor (ER) subtypes: ERalpha, ERbeta, and a unique type, ERgamma, cloned from a teleost fish, the Atlantic croaker Micropogonias undulatus; the first identification of a third type of classical ER in vertebrate species. Phylogenetic analysis shows that ERgamma arose through gene duplication from ERbeta early in the teleost lineage and indicates that ERgamma is present in other teleosts, although it has not been recognized as such. The Atlantic croaker ERgamma shows amino acid differences in regions important for ligand binding and receptor activation that are conserved in all other ERgammas. The three ER subtypes are genetically distinct and have different distribution patterns in Atlantic croaker tissues. In addition, ERbeta and ERgamma fusion proteins can each bind estradiol-17beta with high affinity. The presence of three functional ERs in one species expands the role of ER multiplicity in estrogen signaling systems and provides a unique opportunity to investigate the dynamics and mechanisms of ER evolution.

A new method to localize and test the significance of incongruence: detecting domain shuffling in the nuclear receptor superfamily.

When a data set is partitioned, the resulting subsets may contain phylogenetically conflicting signals if they have different evolutionary histories. In a data set with many taxa, a single taxon that contains multiple phylogenetic histories may result in global incongruence, but no methods are available in a parsimony framework to localize incongruence to specific clades in a phylogeny or to test the significance of incongruence on a local scale. Here we present a new method to quantify the conflict between data partitions for any clade in a phylogeny and to test the statistical significance of that conflict by using a metric called the local incongruence length difference. We apply this method to the evolutionary history of the nuclear receptor superfamily, a large group of transcriptional regulators that play essential roles in metazoan development and physiology. All nuclear receptors are composed of several discrete domains, including one that binds to DNA response elements on specific target genes and another that binds to the appropriate ligand. We have performed combined and separate phylogenetic analyses of these two domains and have tested the hypothesis that nuclear receptors evolved by a simple process of lineage splitting and divergence, without domain shuffling or other forms of sequence transfer between proteins. Our analysis indicates that significant conflict exists between the partitined domains at a few nodes on the tree, suggesting that several groups of receptors are "hybrid proteins" formed by domain shuffling or other forms of sequence transfer between more ancient nuclear receptors.

Gene family evolution and homology: genomics meets phylogenetics.

With the advent of high-throughput DNA sequencing and whole-genome analysis, it has become clear that the coding portions of the genome are organized hierarchically in gene families and superfamilies. Because the hierarchy of genes, like that of living organisms, reflects an ancient and continuing process of gene duplication and divergence, many of the conceptual and analytical tools used in phylogenetic systematics can and should be used in comparative genomics. Phylogenetic principles and techniques for assessing homology, inferring relationships among genes, and reconstructing evolutionary events provide a powerful way to interpret the ever increasing body of sequence data. In this review, we outline the application of phylogenetic approaches to comparative genomics, beginning with the inference of phylogeny and the assessment of gene orthology and paralogy. We also show how the phylogenetic approach makes possible novel kinds of comparative analysis, including detection of domain shuffling and lateral gene transfer, reconstruction of the evolutionary diversification of gene families, tracing of evolutionary change in protein function at the amino acid level, and prediction of structure-function relationships. A marriage of the principles of phylogenetic systematics with the copious data generated by genomics promises unprecedented insights into the nature of biological organization and the historical processes that created it.

Evolution of the androgen receptor: structure-function implications.

To shed light on the nature and evolution of structure-function relations in the androgen receptor (AR), we have undertaken a comparative analysis of all available AR and other steroid receptor sequences. We have identified a group of amino acids that "diagnose" the clade of androgen receptors--residues that are strictly conserved among the ARs but not shared with other receptors. We hypothesize that these amino acids, clustered in a few regions of the protein, confer upon the androgen receptor its unique functions, including recognition of specific DNA response elements and affinity for androgens, rather than other steroid hormones. The four domains of the AR display markedly different rates of evolutionary divergence; conserved portions of the sequence, including small stable stretches within otherwise divergent regions, may be essential to receptor function. Current data from experimental, crystallographic, and clinical studies support these hypotheses, which can now be further tested in the laboratory.

Growth and adherence of xeroderma pigmentosum keratinocytes in vitro.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disease in which cells are unable to repair ultraviolet light-induced DNA defects. Keratinocytes were obtained from the right medial leg of a 16-year-old girl with XP. Two donor sites were harvested: One was an obviously damaged area; the second was an area that had been harvested five years previously and now grossly appeared normal. The cells were plated on untreated plastic tissue culture dishes. All XP keratinocytes grew much faster and spread more rapidly than non-XP controls. There was a 1.5 to 2.5 times increase in the growth rate of XP cells from the obviously damaged area over XP cells from the normal-appearing area. The XP keratinocytes also showed an in vitro expressed alteration in adhesion compared with control keratinocytes, which appears to be independent of the cell growth rate.

In vitro and in vivo testing of a collagen sheet to support keratinocyte growth for use as a burn wound covering.

Neonatal Fischer 344 rat keratinocytes were cultured on a collagen sheet to create a 'quasi-skin.' The cells were grown in either control medium or a low-calcium supplemented medium to enhance proliferation. The rate of 3H-thymidine incorporation into DNA was assessed at days 3, 7, 10, and 20 to determine the effectiveness of the medium supplements. The cells grown in supplemented medium did have an elevated rate of incorporation. On days 7 and 14 post culture, the quasi-skin was used to cover full-thickness defects on the dorsum of adult male rats. The covered defects were biopsied 45 and 60 days postoperatively. Light microscopy showed the wounds to be completely re-epithelialized. Barr bodies were present at the wound site, confirming the presence of quasi-skin keratinocytes up to 60 days postoperatively.

The temporal island scalp flap for management of facial burn scars.

Facial burn scars are difficult to conceal and often preclude an aesthetic rehabilitation of the patient. Multistaged scalp and neck flaps have been described to provide hair-bearing skin to resurface burn scars in men. We have been resurfacing the upper lip and cheek in a one-stage procedure using a temporal artery island scalp flap. The temporoparietal fascia has been well described in recent years, and the understanding of this anatomy has facilitated the use of the island scalp flap for more distal transfers.

Studies on the endogenous flora of the human breast.

Identification of the endogenous microbiological flora of the human breast and its role in breast infections following subglandular augmentation or reduction mammaplasty was undertaken. A total of 231 cultures were performed on 59 breasts in 30 patients. Patients were followed for 12 months. No fungus was cultured from any specimen. Of the breasts cultured 53% were positive for coagulase-negative staphylococcus. Other aerobes found included diphtheroids, lactobacillus, D-enterococcus, micrococcus, and alpha-hemolytic streptococcus. Propionibacterium acne was the most frequent anaerobic bacteria cultured. Other anaerobes included peptococcus and clostridium sporogenes. There was no correlation with respect to the type of bacterium and the depth within the breast where the culture specimens were taken. Postoperative wound infections developed in 2 of 19 patients undergoing reduction mammaplasty. Bacteria identical to those cultured at the time of surgery were again cultured from the wound. Twenty subglandular augmentation mammaplasties were performed with a 25% capsular rate at one year. Two capsules were associated with no bacterial growth at the time of mammaplasty surgery, whereas three were associated with coagulase-negative staphylococcus, Propionibacterium acne, and diphtheroids, respectively. Of the 15 breasts with no capsular contracture after one year, operative culture revealed coagulase-negative staphylococcus in 8 and no bacterial growth in 7. Even breast tissue located deep within the gland away from the nipple contains a flora that is similar to that of normal skin. Cases of infection in which the endogenous bacteria were correlated with later infection was documented.

Osteoradionecrosis of the olecranon: treatment by radial forearm flap.

Osteoradionecrosis of the olecranon is an unusual pathologic entity, treated best by debridement and wound closure using vascularized tissue. Local skin is often unavailable for flap design and transposition. The radial forearm flap can be isolated on a proximal vascular pedicle and transposed to cover the wound. In the case presented, healing was brisk and complete, allowing early elbow mobilization. Although the donor site is not easily concealed, no functional impairment results from flap elevation and all full-thickness wounds are confined to the involved extremity.

Rapid expansion: experimental and clinical experience.

One of the drawbacks of clinical expansion is delay of the final reconstruction and patient inconvenience. The authors have explored the physiologic response to rapid expansion in an animal model and present initial clinical experiences with three patients.

Expanded myocutaneous flaps: their clinical use.

In particularly large defects the best available flap may not provide sufficient tissue for satisfactory correction. Pre-expansion of an overlying musculocutaneous flap allows transposition of a larger flap to reconstruct such defects. The authors present their experience in four patients to illustrate the efficacy of this modality in select patients.

Burn management: the role of tissue expansion.

Tissue expansion has facilitated reconstruction in selected burn patients. For the past 5 years the authors have used this modality in 45 patients, reconstructing the head and neck, trunk, and extremities.

Doppler blood flow measurement in atherosclerotic posterior tibial artery.

An important element in successful free tissue transfer is an intact recipient artery. This vessel must be anatomically accessible, structurally suitable for anastomosis, and able to transmit an adequate blood volume. In the case presented, there was radiographic evidence of recipient artery disease, with a presumed decrease in flow. However, Doppler blood flow studies predicted near normal flow. This information was useful in making intraoperative decisions regarding the suitable vessel for anastomosis.

Cardiovascular reserve in idiopathic dilated cardiomyopathy as determined by exercise response during cardiac catheterization.

Simultaneous right- and left-sided cardiac high-fidelity hemodynamic measurements were obtained at rest and supine exercise during cardiac catheterization in 27 patients (mean age 32 +/- 10 years) with idiopathic dilated cardiomyopathy to investigate the hemodynamic exercise response and possible mechanisms for the wide variation in exercise tolerance observed clinically. There were no significant differences in other rest hemodynamic variables between group 1, patients with a normal exercise factor (more than 600, n = 10), and group 2, patients with an abnormal exercise factor (less than 600, n = 17). A greater increase in stoke volume index (12 +/- 6 vs 2 +/- 8 ml/beats/m2, mean +/- standard deviation) and a greater decrease in systemic vascular resistance with exercise occurred in group 1 than in group 2 (-614 +/- 304 vs -406 +/- 291 dynes cm-5). Elevation of right ventricular end-diastolic pressure with exercise was significantly greater in group 2 than in group 1 (7 +/- 5 vs 1 +/- 4 mm Hg, respectively, p less than 0.05). A maintained cardiac reserve in patients with idiopathic dilated cardiomyopathy appears to be largely dependent on 2 primary factors: preservation of normal right ventricular function during exercise; and preservation of systemic vasodilator capability sufficient to produce a significant degree of afterload reduction during exercise.

Zeta potential of synthetic and biological skin substitutes: effects on initial adherence.

Early adherence of a skin substitute to the wound surface is paramount if it is to function as a skin equivalent. A surface electrical property (the zeta potential) was evaluated, and a positive correlation was found in which 5-hour adherence properties increased as the zeta potential became more positive. The following materials were tested: nylon-silicone composite (Z = -24.8 mV), Biobrane (Z = -15.2 mV), fresh-frozen porcine skin (Z = +12.5 mV), Opsite (Z = +14.9 mV), human amnion (Z = +18.2 mV), and human skin (Z = +23.0 mV). This order was also followed for increasing adherence values at 5 hours, which ranged from a low of 48.9 gm/cm2 for the nylon-silicone composite to a high of 88 gm/cm2 for human skin. Also determined was that both adherence and zeta potential decreased as increasing concentrations of glutaraldehyde were used to cross-link fresh-frozen porcine skin. Values ranged from a maximum of 85.5 gm/cm2 (0% glutaraldehyde; Z = +12.5 mV) to a minimum of 42.5 gm/cm2 (10% glutaraldehyde; Z = -26.4 mV). Additionally, deliberate biochemical modifications of porcine skin were undertaken in an attempt to increase zeta potential and adherence.

Covert splenic abscess: a continuing challenge.

Although splenic abscess is a rare cause of intra-abdominal sepsis, the mortality rate remains high especially in patients with silent or covert lesions. The clinical presentation and course of five patients with overt splenic abscess and seven patients with covert splenic abscess seen during a thirty year period were analyzed. Average age of patients with overt lesions was 44.5 years. Direct extension from a contiguous source, hematogenous spread from a distant site (metastatic) and trauma comprised the known etiologies. Clinical features of localized left upper quadrant sepsis were commonly present but only one patient exhibited multiple organ failure. The clinical diagnosis was established preoperatively in four patients (80%) and all underwent splenectomy without mortality. All resected spleens contained solitary abscesses. In contrast, patients with covert lesions tended to be older (average age 56.1 years), uniformly exhibited multiple organ failure and rarely demonstrated local clinical findings of left upper quadrant sepsis. Trauma was a less common etiology than metastatic infection and direct extension. Four patients died without operation. Three patients underwent exploration for unrelated reasons, but the diagnosis of splenic abscess was made intraoperatively in only one patient. Mortality among patients with covert lesions was 86%. Multiple splenic abscesses were demonstrated in all patients with covert lesions. Splenic abscess presents as a spectrum of clinical disease. Solitary lesions can be readily diagnosed and treated by splenectomy. Multiple abscesses are usually covert, associated with multiple organ failure and highly lethal. The role of splenectomy in patients with covert lesions remains unknown.

Predicting helplessness in human subjects.

A group of 340 college students was administered a 140-item "helplessness" inventory. From this group, 90 were selected as subjects: 30 with extreme high scores, 30 with extreme low scores, and 30 scoring at the mean. Prior to Task 1, they were assigned to one of three experimental conditions: in Condition 1 they received a series of eight solvable Wechsler block designs; in Condition 2 they received a similar task, except that the designs were unsolvable; in Condition 3 they were given an unrelated article to read for the duration of Task 1. Following Task 1, all subjects were transferred to a second task which involved the learning of a 10-choice point-stylus maze. Significance was attained for personality type, experimental treatment, and blocks of Task 2 trials.

Epidermal growth factor in the healing of second degree burns: a controlled animal study.

The effects of the topical application of epidermal growth factor (EGF) on the healing of partial-thickness scald burns in rats was evaluated. Time to complete healing, the rate of healing, daily percentage weight change, and cell growth were the measured parameters. We were able to demonstrate an insignificant healing advantage with the topical application of EGF, by itself or in combination with silver sulphadiazine. This insignificant healing advantage does not warrant clinical evaluation.