Mouth development in the Senegal bichir Polypterus senegalus does not involve the oropharyngeal membrane: possible implications for the ecto‐endoderm boundary and tooth initiation

Polypterid fishes are considered the basal‐most group of extant actinopterygians and thus may serve as a direct link to understand the evolution of the first bony fishes. Embryonic and larval specimens, however, are extremely scarce, making it difficult to study their developmental patterns and processes. During the past few years we collected many embryos and larvae of the Senegal bichir Polypterus senegalus and in this paper we describe some novel observations concerning the supposed ecto‐endoderm border in the mouth and consecutive initiation of dental development. The mouth of the Senegal bichir does not develop via a classical oropharyngeal membrane but instead, opening the mouth occurs via a separation of the upper and lower jaws that are connected by epithelial bridges. These structures are bilaterally symmetrical and are found invariantly at places of the earliest tooth bud development. It is suggested that the epithelial bridges may represent the ecto‐endoderm bordering zone, are both structurally and functionally homologous to the oropharyngeal membrane and consequently it is hypothesized that the epithelial bridges and the developmental factors producing them play a key role in initiation and early distribution of the particular dental domains.     

Decrease of calmodulin and actin in the plasma membrane of rat liver cells during proliferative activation

After proliferative activation of rat liver cells in vivo by a partial hepatectomy a decrease of the calmodulin content in the three plasma membrane domains (blood sinusoidal, canalicular and lateral) was observed. At 24 hours after partial hepatectomy calmodulin was found to be 3 fold lower in the sinusoidal and lateral fractions whereas a 2 fold decrease was detected in the canalicular domain. Decreases on the actin levels have been also detected at 24 hours after a partial hepatectomy. Since at this time after surgery increases on nuclear actin and calmodulin have been reported, these results suggest the possibility that the actin and calmodulin dissociated from the plasma membrane after a partial hepatectomy could subsequently be translocated into the nuclei.     

The <Emphasis Type="Italic">Leiognathus aureus</Emphasis> complex (Perciformes: Leiognathidae) with the description of a new species

Taxonomic analysis of a group of morphologically similar ponyfishes (Perciformes: Leiognathidae) establishes a complex comprising three valid species: Leiognathus aureus Abe and Haneda, 1972, widely distributed in the western Pacific Ocean (Taiwan, Philippines, Thailand, Singapore, Indonesia, and northern Australia); L. hataii Abe and Haneda, 1972, currently known only from Ambon, Indonesia; and L. panayensis sp. nov. Kimura and Dunlap, currently known only from Panay Island, the Philippines. The L. aureus complex can be defined by the following combination of characters: mouth protruding forward, not downward; small but sharp conical teeth uniserially on jaws; a black line between lower margin of eye and lower jaw articulation; and lateral line incomplete, ending below posterior part of dorsal fin base or on anterior caudal peduncle. Leiognathus hataii differs from both L. aureus and L. panayensis in having a large dark blotch below the spinous dorsal fin base and fewer counts of scales (lateral line scales 50–58 vs. 64–85 in the latter two species; scales above lateral line 7–10 vs. 12–18; scales below lateral line 22–26 vs. 30–41). Leiognathus panayensis is distinguished from L. aureus in having a deeper body (41–51% SL vs. 35–45% SL in the latter), long posterior limb of maxilla (21–25% HL vs. 15–23% HL), wholly scaled belly (vs. naked along preanal median keel), and a dark blotch on nape (vs. absent).     

Expression of a<Emphasis Type="Italic"> SoxB</Emphasis> and a<Emphasis Type="Italic"> Wnt2/13</Emphasis> gene during the development of the mollusc<Emphasis Type="Italic"> Patella vulgata</Emphasis>

We cloned and analysed the expression of a SoxB gene (PvuSoxB) in the marine mollusc, Patella vulgata. Like its orthologues in deuterostomes, after an early broad ectodermal distribution, PvuSoxB expression only persists in cells competent to form neural structures. In the post-gastrulation larva, PvuSoxB is expressed in the prospective neuroectoderm in the head and in the trunk. No expression can be seen dorsally, around the mouth and the anus, or along the ventral midline. We also report the expression of a Wnt2/13 orthologue (PvuWnt2) in Patella. After gastrulation, PvuWnt2 is expressed in the posterior part of the mouth, along the ventral midline and around the anus. This expression seems to be complementary to that of PvuSoxB in the trunk. We suggest the existence of a fundamental subdivision of the Patella trunk ectoderm into midline (mouth, midline, anus) and more lateral structures.Edited by D. Tautz     

Evolution of deuterostomy – and origin of the chordates

The chordates are usually characterized as bilaterians showing deuterostomy, i.e. the mouth developing as a new opening between the archenteron and the ectoderm, serial gill pores/slits, and the complex of chorda and neural tube. Both numerous molecular studies and studies of morphology and embryology demonstrate that the neural tube must be considered homologous to the ventral nerve cord(s) of the protostomes, but the origin of the ‘new’ mouth of the deuterostomes has remained enigmatic. However, deuterostomy is known to occur in several protostomian groups, such as the chaetognaths and representatives of annelids, molluscs, arthropods and priapulans. This raises the question whether the deuterostomian mouth is in fact homologous with that of the protostomes, viz. the anterior opening of the ancestral blastopore divided through lateral blastopore fusion, i.e. amphistomy. A few studies of gene expression show identical expression patterns around mouth and anus in protostomes and deuterostomes. Closer studies of the embryology of ascidians and vertebrates show that the mouth/stomodaeum differentiates from the anterior edge of the neural plate. Together this indicates that the chordate mouth has moved to the anterior edge of the blastopore, so that the anterior loop of the ancestral circumblastoporal nerve cord, which is narrow in the protostomes, has become indistinguishable. In the vertebrates, the mouth has moved further around the anterior pole to the ‘ventral’ side. The conclusion must be that the chordate mouth (and that of the deuterostomes in general) is homologous to the protostomian mouth and that the latest common ancestor of protostomes and deuterostomes developed through amphistomy, as suggested by the trochaea theory.     

Expression of γ‐tocopherol methyltransferase in chloroplasts results in massive proliferation of the inner envelope membrane and decreases susceptibility to salt and metal‐induced oxidative stresses by reducing reactive oxygen species

The γ‐tocopherol methyltransferase (γ‐TMT) is an important enzyme regulating synthesis of four tocopherols (α, γ, β and δ). In this report, we investigated the role of γ‐TMT in regulating abiotic stress within chloroplasts. The At γ‐tmt overexpressed via the tobacco chloroplast genome accumulated up to 7.7% of the total leaf protein, resulting in massive proliferation of the inner envelope membrane (IEM, up to eight layers). Such high‐level expression of γ‐TMT converted most of γ‐tocopherol to α‐tocopherol in transplastomic seeds (~10‐fold higher) in the absence of abiotic stress. When grown in 400 mm NaCl, α‐tocopherol content in transplastomic TMT leaves increased up to 8.2‐fold and 2.4‐fold higher than wild‐type leaves. Likewise, under heavy metal stress, α‐tocopherol content in the TMT leaves increased up to 7.5‐fold, twice higher than in the wild type. Under extreme salt stress, the wild type accumulated higher starch and total soluble sugars, but TMT plants were able to regulate sugar transport. Hydrogen peroxide and superoxide content in wild type increased up to 3‐fold within 48 h of NaCl stress when compared to TMT plants. The ion leakage from TMT leaves was significantly less than wild‐type plants under abiotic stress and with less malondialdehyde, indicating lower lipid peroxidation. Taken together, these studies show that α‐tocopherol plays a crucial role in the alleviation of salt and heavy metal stresses by decreasing ROS, lipid peroxidation and ion leakage, in addition to enhancing vitamin E conversion. Increased proliferation of the IEM should facilitate studies on retrograde signalling from chloroplast to the nucleus.     

The role of the columellar strut in rhinoplasty: indications and rationale

There are a variety of techniques that can be used to enhance or improve the nasal tip. These techniques often use suture techniques and invisible grafts to achieve the desired result. The former methods have been well described throughout the literature. Among the latter techniques, the columellar strut remains a popular and effective form of an invisible graft in rhinoplasty. The purpose of this article is to define the role of the columellar strut graft, describe how to perform it correctly in rhinoplasty, provide a clinical algorithm for its application, and detail a 15-year retrospective analysis of the senior author's (R.J.R.) experience. Previous references to the importance of the columellar strut graft in rhinoplasty have been described; however, none has formally defined its singular importance in both primary and secondary open rhinoplasty. This article details the role of the columellar strut and its relationship to nasal tip projection and lower lateral cartilage symmetry with an explanation of methods for improving each. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.     

Prokaryotic expression and antimicrobial mechanism of XPF‐St7‐derived α‐helical peptides

XPF‐St7 (GLLSNVAGLLKQFAKGGVNAVLNPK) is an antimicrobial peptide isolated from Silurana tropicalis. We developed an α‐helical segment of XPF‐St7 termed as XPF2. Using the XPF2 as a framework, we increased the positive net charge of XPF2 by amino acid substitutions, and thus obtained two novel antimicrobial peptides XPF4 and XPF6. These were each fused with an ubiquitin tag and successfully expressed in Escherichia coli. This ubiquitin fusion system may present a viable alternative for industrial production of antimicrobial peptides. XPF4 and XPF6 showed much better overall antimicrobial activity against both Gram‐negative and Gram‐positive bacteria than XPF2. The therapeutic index of XPF4 and XPF6 was 5.6‐fold and 6.7‐fold of XPF2, respectively. Bacterial cell membrane permeabilization and genomic DNA interaction assays were utilized to explore the mechanism of action of XPF serial peptides. The results revealed that the target of these antimicrobial peptides was the bacterial cytoplasmic membrane. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Morphology of the mechanosensory lateral line system in the Atlantic Stingray,Dasyatissabina: The mechanotactile hypothesis

The biological function of anatomical specializations in the mechanosensory lateral line of elasmobranch fishes is essentially unknown. The gross and histological features of the lateral line in the Atlantic stingray, Dasyatis sabina, were examined with special reference to its role in the localization and capture of natural invertebrate prey. Superficial neuromasts are arranged in bilateral rows near the dorsal midline from the spiracle to the posterior body disk and in a lateral position along the entire length of the tail. All dorsal lateral line canals are pored, contain sensory neuromasts, and have accessory lateral tubules that most likely function to increase their receptive field. The pored ventral canal system consists of the lateral hyomandibular canal along the disk margin and the short, separate mandibular canal on the lower jaw. The extensive nonpored and relatively compliant ventral infraorbital, supraorbital, and medial hyomandibular canals form a continuous complex on the snout, around the mouth, and along the abdomen. Vesicles of Savi are small mechanosensory subdermal pouches that occur in bilateral rows only along the ventral midline of the rostrum. Superficial neuromasts are best positioned to detect water movements along the transverse body axis such as those produced by tidal currents, conspecifics, or predators. The pored dorsal canal system is positioned to detect water movements created by conspecifics, predators, or possibly distortions in the flow field during swimming. Based upon the stingray lateral line morphology and feeding behavior, we propose the Mechanotactile Hypothesis, which states that the ventral nonpored canals and vesicles of Savi function as specialized tactile mechanoreceptors that facilitate the detection and capture of small benthic invertebrate prey. J. Morphol. 238:1–22, 1998. © 1998 Wiley-Liss, Inc.     

A modified Goldman nasal tip procedure for the drooping nasal tip

A modification of Irving Goldman's nasal tip procedure that borrows from the lateral crus to augment the height of the medial crus is described. Goldman's procedure has been modified by not including the vestibular skin with the segment of the lateral crus that is rolled medially to increase nasal tip projection, by adding a nasal septal cartilage strut between the medial crura for support when the medial crura are weak, and by maintaining a small separation caudally of the repositioned lateral crura at the new nasal dome to simulate a double nasal dome. This modified Goldman nasal tip procedure allows the surgeon to reshape the lower lateral nasal cartilage to increase nasal tip projection as an alternative to the use of a shield-type nasal tip graft, and at the same time it narrows the nasal tip with minimal resection of the lateral crus of the lower lateral nasal cartilage.     

Functional morphology of the mouth tube of a lernaeopodid Pseudocharopinus narcinae (Pillai, 1962) (Copepoda: Siphonostomatoida)

A detailed account of the structure and musculature of the mouth tube and mode of feeding in P. narcinae is presented. The double-walled mouth tube is formed by a close fitting of the lateral, longitudinal ridges of the labium into the longitudinal groove of the inner, lateral edges of the labrum. The musculature of the mouth tube consists of two pairs of retractores oris, four pairs of compressores labri and two pairs of levatores labii. The host tissues are scraped off by the mandibles repeating a very characteristic movement brought about by roto adductor mandibulae and roto abductor mandibulae pair of muscles. The tissue particles are forced into the buccal cavity with the help of labral muscles. The suction is broken by the contraction of levatores labii group of muscles.     

Desmoglein‐2 during pregnancy and its role in the evolution of viviparity in a marsupial (Sminthopsis crassicaudata; Dasyuridae)

Attachment of the blastocyst and formation of the placenta during pregnancy is dependent on structural and cellular changes occurring in the uterine epithelium and in particular to the plasma membrane of these uterine cells. Desmosome expression decreases during pregnancy in eutherians and some squamates, presumably allowing for remodeling of the uterine epithelium and invasion of the trophoblast during implantation. Marsupials are a distinct mammalian amniote lineage of viviparity, with a short implantation or attachment period and varying levels of invasive placentation. To test the generality of changes to the uterine epithelium during pregnancy across mammals, we characterized the distribution of desmosomes in the uterine epithelial cells of a marsupial, Sminthopsis crassicaudata, using electron microscopy and immunohistochemistry. The absolute number of desmosomes along the lateral plasma membrane decreases during pregnancy and desmosomes are redistributed towards the apical region of the lateral plasma membrane as pregnancy proceeds, similar to what occurs during pregnancy in eutherian mammals. Despite the lower level of maternal investment in pregnancy and the noninvasive structure of fetal membranes in marsupials there are similarities in number and redistribution of desmosomes along the plasma membrane and changes to the morphology of the uterine epithelial cells suggesting that similar plasma membrane changes occur across all lineages of amniote vertebrates. J. Morphol. 276:261–272, 2015. © 2014 Wiley Periodicals, Inc.     

Seasonal and spatial variability of dissolved organic matter composition in the lower Amazon River

We analyzed the molecular composition of dissolved organic matter (DOM) in the lower Amazon River (ca. 850 km from Óbidos to the mouth) using ultrahigh-resolution mass spectrometry and geochemical tracers. Changes in DOM composition along this lower reach suggest a transition from higher plant-derived DOM to more algal/microbial-derived DOM. This result was likely due to a combination of autochthonous production, alteration of terrigenous DOM as it transits down the river, and increased algal inputs from floodplain lakes and clearwater tributaries during high discharge conditions. Spatial gradients in dissolved organic carbon (DOC) concentrations varied with discharge. Maximal DOC concentrations were observed near the mouth during high water, highlighting the importance of lateral inputs of DOM along the lower river. The majority of DOM molecular formulae did not change within the time it takes the water in the mainstem to be transported through the lower reach. This is indicative of molecules representing a mixture of compounds that are resistant to rapid alteration and reactive compounds that are continuously replenished by the lateral input of terrestrial organic matter from the landscape, tributaries, and floodplains. River water incubations revealed that photo- and bio-transformation alter at most 30% of the DOM molecular formulae. River discharge at the mouth differed from the sum of discharge measurements made at Óbidos and the main gauged tributaries in the lower Amazon. This indicates that changes in hydrology and associated variations in the source waters along the lower reach affected the molecular composition of the DOM that is being transported from the Amazon River to the coastal ocean.     

Mixed reverse micelles facilitated downstream processing of lipase involving water‐oil‐water liquid emulsion membrane

Our earlier work for the first time demonstrated that liquid emulsion membrane (LEM) containing reverse micelles could be successfully used for the downstream processing of lipase from Aspergillus niger. In the present work, we have attempted to increase the extraction and purification fold of lipase by using mixed reverse micelles (MRM) consisting of cationic and nonionic surfactants in LEM. It was basically prepared by addition of the internal aqueous phase solution to the organic phase followed by the redispersion of the emulsion in the feed phase containing enzyme, which resulted in globules of water‐oil‐water (WOW) emulsion for the extraction of lipase. The optimum conditions for maximum lipase recovery (100%) and purification fold (17.0‐fold) were CTAB concentration 0.075 M, Tween 80 concentration 0.012 M, at stirring speed of 500 rpm, contact time 15 min, internal aqueous phase pH 7, feed pH 9, KCl concentration 1 M, NaCl concentration 0.1 M, and ratio of membrane emulsion to feed volume 1:1. Incorporation of the nonionic surfactant (e.g., Tween 80) resulted in remarkable improvement in the purification fold (3.1–17.0) of the lipase. LEM containing a mixture of nonionic and cationic surfactants can be successfully used for the enhancement in the activity recovery and purification fold during downstream processing of enzymes/proteins. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1084–1092, 2014     

Comparative morphology and ultrastructure of the mouthparts in unfed larvae of <Emphasis Type="Italic">Platytrombidium fasciatum</Emphasis> and <Emphasis Type="Italic">Camerotrombidium pexatum</Emphasis> (Acariformes: Microtrombidiidae)

The mouthparts of unfed larvae of Platytrombidium fasciatum (C. L. Koch, 1836) and Camerotrombidium pexatum (C. L. Koch, 1837) (Acariformes: Microtrombidiidae) were studied using both light optical (whole-mounted specimens, toluidine blue stained semi-thin sections) and electron microscope (TEM, SEM) methods. The mouth apparatus incorporated within the gnathosoma occupies an axial position and is covered from above by the arched dorsal shield, or scutum. The chelicerae are comparatively long and separated, and the lateral lips form a permanent apomorphic sucker provided with an internal cuticular sclerite. The pharynx is extremely wide and totally fused with the bottom of the infracapitulum. The pharyngeal dilators originate on the posterior portions of the cervix (epistome) and on the capitular apodemes and run nearly parallel to the cervix to the dorsal pharyngeal wall. Comparatively short sigmoid pieces serve as origin of the muscles—cheliceral levators inserting on the posterior wall of the basal cheliceral segments. There are two sets of the extrinsic gnathosomal muscles originating on the posterior portion of the scutum: retractors of chelicerae inserting on the posterior portions of the basal cheliceral segments, and retractors of the gnathosoma inserting on the very posterior parts of the capitular apodemes. The labrum and the cervix delimit the pharynx and the subcheliceral space. The labrum and the cervix for the most part are weakly sclerotized cuticular plates and do not have own muscles. The larval mouth apparatus, in comparison with that of adult microtrombidiid mites, is simply organized and more specialized for ingestion of large masses of liquid food.     

Defining the fold space of membrane proteins: the CAMPS database

Recent progress in structure determination techniques has led to a significant growth in the number of known membrane protein structures, and the first structural genomics projects focusing on membrane proteins have been initiated, warranting an investigation of appropriate bioinformatics strategies for optimal structural target selection for these molecules. What determines a membrane protein fold? How many membrane structures need to be solved to provide sufficient structural coverage of the membrane protein sequence space? We present the CAMPS database (Computational Analysis of the Membrane Protein Space) containing almost 45,000 proteins with three or more predicted transmembrane helices (TMH) from 120 bacterial species. This large set of membrane proteins was subjected to single‐linkage clustering using only sequence alignments covering at least 40% of the TMH present in a given family. This process yielded 266 sequence clusters with at least 15 members, roughly corresponding to membrane structural folds, sufficiently structurally homogeneous in terms of the variation of TMH number between individual sequences. These clusters were further subdivided into functionally homogeneous subclusters according to the COG (Clusters of Orthologous Groups) system as well as more stringently defined families sharing at least 30% identity. The CAMPS sequence clusters are thus designed to reflect three main levels of interest for structural genomics: fold, function, and modeling distance. We present a library of Hidden Markov Models (HMM) derived from sequence alignments of TMH at these three levels of sequence similarity. Given that 24 out of 266 clusters corresponding to membrane folds already have associated known structures, we estimate that 242 additional new structures, one for each remaining cluster, would provide structural coverage at the fold level of roughly 70% of prokaryotic membrane proteins belonging to the currently most populated families. Proteins 2006. © 2006 Wiley‐Liss, Inc.     

Structural characterization of the NAP; the major adhesion complex of the human pathogen Mycoplasma genitalium

Mycoplasma genitalium, the causative agent of non‐gonococcal urethritis and pelvic inflammatory disease in humans, is a small eubacterium that lacks a peptidoglycan cell wall. On the surface of its plasma membrane is the major surface adhesion complex, known as NAP that is essential for adhesion and gliding motility of the organism. Here, we have performed cryo‐electron tomography of intact cells and detergent permeabilized M. genitalium cell aggregates, providing sub‐tomogram averages of free and cell‐attached NAPs respectively, revealing a tetrameric complex with two‐fold rotational (C2) symmetry. Each NAP has two pairs of globular lobes (named α and β lobes), arranged as a dimer of heterodimers with each lobe connected by a stalk to the cell membrane. The β lobes are larger than the α lobes by 20%. Classification of NAPs showed that the complex can tilt with respect to the cell membrane. A protein complex containing exclusively the proteins P140 and P110, was purified from M. genitalium and was structurally characterized by negative‐stain single particle EM reconstruction. The close structural similarity found between intact NAPs and the isolated P140/P110 complexes, shows that dimers of P140/P110 heterodimers are the only components of the extracellular region of intact NAPs in M. genitalium.     

Release of sperm clusters in spheres by the black coral <Emphasis Type="Italic">Cupressopathes pumila</Emphasis> (Anthozoa,Antipatharia)

Fecund polyps from a bottlebrush-shaped colony of black coral Cupressopathes pumila (Brook 1889) from Ambon Island (Maluku Archipelago, Indonesia) were studied at the structural and ultrastructural levels. Five fragments, each 5 cm long and containing about 60 polyps, were cut from a single parental colony. The fragments underwent different treatments: two were fixed in 70% alcohol for species identification; the remaining three were reared in aerated aquaria and then processed for anatomic studies by light and transmission electron microscopy. Some polyps from one of these reared fragments displayed spheres of various sizes protruding from the mouth. Four hours after sampling, spheres settled on the bottom of the aquarium. Comparative analysis of the fecund polyps showed: (i) the differentiation of spermatocysts inside the transverse primary mesenteries that separate the lateral tentacles from one another; (ii) spermatocysts entering the gastric cavity after breakage of the mesentery wall and (iii) location of spermatocysts close to the basal part of the pharynx. Analysis of the largest spheres, both close to the external surface of the mouth and settled on the bottom of the small aerated aquaria, showed that they were irregularly bordered with residual mesentery tissue and contained clusters of spermatocysts. Gametes presented various phases of differentiation up to the final sperm, as it occurs in the spermatocysts developing inside the mesenteries of the fecund polyps. In consideration of the particular distribution of the colonies of C. pumila, which grow very far apart, the release of buoyant spheres is a particular modality of spawning which can be viewed as a strategy for successful fertilisation over long distances.     

Biochemical maturation of Spam1 (PH‐20) during epididymal transit of mouse sperm involves modifications of N‐linked oligosaccharides

Indirect immunofluorescence of mouse caput and caudal sperm shows distinctly different distributions of Spam1 protein, which is associated with structural and functional differences of the molecule. Spam1 is uniformly distributed over the surface of the head of caput sperm while in caudal sperm, light and confocal microscopy demonstrate that it is localized to the anterior and posterior regions. The hyaluronidase activity of Spam1 in acrosome‐intact caput sperm was significantly lower (4.3‐fold; P < 0.0001) than that of caudal sperm. The increase in enzymatic activity in caudal sperm is accompanied by a reduction in the molecular weight (MW): in extracts from caput sperm there was a major band at ∼74 kDa and a minor band at ∼67 kDa; while for the cauda there was a major band at ∼67 kDa and minor bands at ∼70 and ∼56 kDa. Additionally, the bands from caput sperm were 4.9 to 7.7‐fold less intense than those from caudal sperm. This decreased affinity for the polyclonal anti‐Spam1 suggests the presence of different surface characteristics of the molecule from the two epididymal regions. Computer analysis of the protein structure from Spam1 cDNA sequence reveals four putative N‐linked glycosylation sites, and enzymatic deglycosylation suggests that all sites are functional. After endoglycosidase activity of extracts from caput and caudal sperm, both show a major band with a MW of ∼56 kDa, the size of the membrane‐anchored polypeptide backbone. Based on the difference in size and intensity of the Spam1 bands and hyaluronidase activities from caput and caudal sperm, the data suggest that the activation of Spam1 during epididymal maturation is regulated by deglycosylation. Mol. Reprod. Dev. 52:196–206, 1999. © 1999 Wiley‐Liss, Inc.