Multiple origin of diurnality in geckos: evidence from eye lens crystallins.

The large lizard family Gekkonidae comprises about 90 genera (1000 species). While most geckos are nocturnal, the members of about 15 genera are diurnal. All of these species are 'tertiarily' diurnal, i.e. they are descended from 'secondarily' nocturnal ancestors. They have adapted to a diurnal lifestyle in quite different ways, as can be deduced by the crystallin proteins in their lenses. Evaluation of the heterogeneous lens crystallin compositions of diurnal geckos reveals that there are at least three lineages that regained diurnality independently.

Retina of Bouton's skink (Reptilia, Scincidae): visual cells, fovea, and ecological constraints.

Bouton's skink, Cryptoblepharus boutonii africanus, is a small, diurnal lizard living on outcrops along the coast of East Africa under high ambient light intensities. It is characterized by relatively large eyes (maximal diameter about 2 mm), with immovable eyelids forming a transparent spectacle and with a virtually constant pupil diameter. The single fovea in the central retina is well developed, with a clearly defined pit, which is relatively deep but not funnel-shaped. The foveal pit is not devoid of the outer nuclear and outer plexiform layers; only the main part of the inner nuclear layer is displaced laterally, resulting in a pit with gradual sloping towards its edges. Thus, the fovea appears to be concaviclivate, as in the eyes of lacertids, varanids, and gekkonids. The central position of the foveae in these laterally placed scincid eyes corresponds with monocular fixation, e.g., of detected prey. C. boutonii has a pure-cone retina containing single and double visual cells. The latter consist of two cells of unequal sizes. Yellowish oil droplets are present in single cones and the minor members of the double cones in all retinal regions. The visual cells of the different retinal regions do not differ in the ultrastructure of their components but differ considerably in size. The outer segments of the foveal cones are twice as long as those of the peripheral cones. Except for the pedicles, the diameters of the components of the visual cells decrease towards the fovea, resulting in an increase in visual acuity.

Evolution of the aldose reductase-related gecko eye lens protein rhoB-crystallin: a sheep in wolf's clothing.

rhoB-crystallin (AJ245805) is a major protein component (20%) in the eye lens of the gecko Lepidodactylus lugubris. Limited peptide sequence analysis earlier revealed that it belongs to the aldo-keto reductase superfamily, as does the frog lens rho-crystallin. We have now determined the complete cDNA sequence of rhoB-crystallin and established that it is more closely related to the aldose reductase branch of the superfamily than to frog rho-crystallin. These gecko and frog lens proteins have thus independently been recruited from the same enzyme superfamily. Aldose reductase is implicated in the development of diabetic cataract in mammals, and, if active, rhoB-crystallin might be a potential risk for the gecko lens. Apart from a replacement 298 Cys --> Tyr, rhoB-crystallin possesses all amino acid residues thought to be required for catalytic activity of the aldose reductases. However, modeling studies of the rhoB-crystallin structure indicate that substrate specificity and nicotinamide cofactor affinity might be affected. Indeed, neither recombinant rhoB-crystallin nor the reverse mutant 298 Tyr --> Cys showed noticeable activity toward aliphatic and aromatic substrates, although cofactor binding was retained. Various other oxidoreductases are known to be recruited as abundant lens proteins in many vertebrate species; rhoB-crystallin demonstrates that an aldose reductase-related enzyme also can be modified to this end.

Gecko vision--retinal organization, foveae and implications for binocular vision.

Geckos comprise both nocturnal and diurnal genera, and between these categories there are several transitions. As their retinae have definitely to be classified as pure cone retinae, they provide an especially attractive model for comparison of organization and regional specializations adapted to very different photic environments. While the visual cells themselves show clear adaptations to nocturnal or diurnal lifestyles, the overall retinal organization is more related to that of diurnal vertebrates. Nocturnal geckos have lost any foveae of their diurnal ancestors, but they have retained a low convergence ratio and a high visual cell density. To enhance visual sensitivity, they exploit binocular - but not necessarily stereoscopic - vision. Diurnal species have retained binocular vision. Most diurnal species have developed new foveae, which are consequently located not in the central but in the temporal region of the retina.

Characterization of retinal oil droplets in diurnal geckos (reptilia, gekkonidae).

Retinal oil droplets have been documented in the retinae of four genera of diurnal geckos (Phelsuma, Gonatodes, Quedenfeldtia, and Pristurus), while other large diurnal genera (Sphaerodactylus and Lygodactylus) lack oil droplets. Where they occur, droplets are found only in the minor members of double cones of type B of the extrafoveal and peripheral regions, whereas in the foveal cones droplets could not be detected. Oil droplets in gecko retinae have neither an internal structure nor an own membrane; this is typical for oil droplets of amphibians and sauropsids. The droplets are nonfluorescent and definitely transparent. Thus, they do not function as filters modifying the spectral composition of the light reaching the outer segments. The constant relationship between the diameters of the oil droplets and those of the outer segment bases might still suggest an optical function of the droplets (e.g., as microlenses focusing light on the outer segments). However, as the ecologically very similar genera Phelsuma, Gonatodes, and Lygodactylus differ in the presence or absence of oil droplets, this potential function seems to be not of physiological significance.

Carotenoid and retinoid--two pigments in a gecko eye lens.

The eye lenses of the Moroccan day gecko Quedenfeldtia trachyblepharus contain two different pigments: a retinoid (minor pigment) and a carotenoid (major pigment). The retinoid, all-trans 3, 4-didehydroretinol, is bound to iota-crystallin, which comprises only 2% of the total amount of crystallins. The carotenoid is associated to gammas-crystallin - comprising about 10% of total amount of crystallins--and causes the dark yellow colour of the lens. The absorption spectrum of the isolated carotenoid shows a major, triple-peaked band at 372, 392, and 416 nm and two minor peaks at 284 and 294 nm. This spectrum reminds of that of galloxanthin, a carotenoid found in oil droplets of some avian retinae. The absorption spectrum of the carotenoid-gammas-crystallin complex is shifted 6-8 nm bathochromically. In the lens, this complex absorbs ultraviolet and shortwave blue radiation, supposedly improving the optical quality of the dioptric apparatus and protecting the retina against photodamage. Both the retinoid and the carotenoid are present in eye cups. The lenticular carotenoid of Quedenfeldtia is the first example of a carotenoid in the lens of a terrestrial vertebrate with a sufficiently high concentration to be physiologically effective as a UV-filter. Additionally, it is unique in being the first example of a carotenoid associated with gammas-crystallin.

Gecko iota-crystallin: how cellular retinol-binding protein became an eye lens ultraviolet filter.

Eye lenses of various diurnal geckos contain up to 12% iota-crystallin. This protein is related to cellular retinol-binding protein type I (CRBP I) but has 3,4-didehydroretinol, rather than retinol, as a ligand. The 3,4-didehydroretinol gives the lens a yellow color, thus protecting the retina by absorbing short-wave radiation. iota-Crystallin could be either the gecko's housekeeping CRBP I, recruited for an additional function in the lens, or the specialized product of a duplicated CRBP I gene. The finding of the same CRBP I-like sequence in lens and liver cDNA of the gecko Lygodactylus picturatus now supports the former option. Comparison with iota-crystallin of a distantly related gecko, Gonatodes vittatus, and with mammalian CRBP I, suggests that acquiring the additional lens function is associated with increased amino acid changes. Compared with the rat CRBP I structure, the iota-crystallin model shows reduced negative surface charge, which might facilitate the required tight protein packing in the lens. Other changes may provide increased stability, advantageous for a long-living lens protein, without frustrating its role as retinol transporter outside the lens. Despite a number of replacements in the ligand pocket, recombinant iota-crystallin binds 3,4-didehydroretinol and retinol with similar and high affinity (approximately 1.6 nM). Availability of ligand thus determines whether it binds 3,4-didehydroretinol, as in the lens, or retinol, in other tissues. iota-Crystallin presents a striking example of exploiting the potential of an existing gene without prior duplication.

Multiregional evaluation of the SimPlate heterotrophic plate count method compared to the standard plate count agar pour plate method in water.

A new SimPlate heterotrophic plate count (HPC) method (IDEXX Laboratories, Westbrook, Maine) was compared with the pour plate method at 35 degrees C for 48 h. Six laboratories tested a total of 632 water samples. The SimPlate HPC method was found to be equivalent to the pour plate method by regression analysis (r = 0. 95; y = 0.99X + 0.06).

Gecko vision-visual cells, evolution, and ecological constraints.

Geckos comprise both nocturnal and diurnal genera, and between these categories there are several transitions. As all geckos depend on their visual sense for prey capture, they are promising subjects for comparison of morphological modifications of visual cells adapted to very different photic environments. Retinae of 22 species belonging to 15 genera with different activity periods are examined electron microscopically. Scotopic and photopic vision in geckos is not divided between "classical" rods and cones, respectively; both are performed by one basic visual cell type. Independent of the activity periods of the individual species, the visual cells of geckos exhibit characteristics of cones at all levels of their ultrastructure. Thus, gecko retinae have to be classified as cone retinae. Only the large size and the shape of the photoreceptor outer segments in nocturnal geckos are reminiscent of rods; the outer segments are up to 60 microm in length and up to 10 microm in diameter. The visual cells of diurnal geckos have considerably smaller outer segments with lengths ranging from 6 to 12 microm and diameters ranging from 1.3 to 2.1 microm. Nocturnal and diurnal species differ in the structure of their ellipsoids. One type of visual cell in nocturnal geckos has modified mitochondria with either rudimentary cristae or no cristae at all, and one type of visual cell in diurnal geckos possesses an oil droplet. The visual cells of Phelsuma guentheri and Rhoptropus barnardi are intermediate between those of nocturnal and diurnal species.

Somatotypes of 7- to 16-year-old boys in Saskatchewan, Canada.

The purpose of this study was to examine the stability of somatotypes of 63 boys in Saskatoon, Canada who were followed from 7 to 16 years of age. Somatotype photos were taken annually and rated by a criterion rater (BH-R). Comparisons were made longitudinally across all years using repeated-measures ANOVAs of the whole somatotype (S), somatotype attitudinal means (SAM), analysis of categories, separate components (endomorphy, mesomorphy, ectomorphy), and partial correlations. In the first year, the means were age = 7.1 yr, height = 121.0 cm, mass = 22.8 kg, S = 2.9-3.6-1.6, and SAM = 1.1. In the last year, the means were age = 16.7 yr, height = 172.6 cm, mass = 59.9 kg, S = 2.5-4.0-3.7, and SAM = 1.4. Mean somatotypes across years were different [F(9,558) = 67.9, P < .01], with the largest differences between 7-10 yr and 14-16 yr. These differences were largely due to significant increases in mesomorphy (F = 24.6, P < .01) and ectomorphy (F = 159.9, P < .01). Partial correlations between ages for each component, with the other two held constant, revealed poor predictions for three or more years apart (r2 < .35). Thus, both group and individual somatotypes changed between 7 and 16 years of age. The overall pattern was from endo-mesomorph through central to mesomorph-ectomorph somatotypes. The trends are similar to those observed in comparable samples from other countries. Am. J. Hum. Biol. 9:257-272, 1997. © 1997 Wiley-Liss, Inc.

Vitamin A2 bound to cellular retinol-binding protein as ultraviolet filter in the eye lens of the gecko Lygodactylus picturatus.

The yellow eye lenses of the diurnal gecko Lygodactylus picturatus contain, in addition to the usual crystallins, a monomeric protein with a molecular mass of 16kDa. It comprises 6-8% of the total water-soluble lens proteins. We here identify it as a novel type of crystallin, most closely related with cellular retinol-binding protein I (CRBP I). Because of its tiny size, we designate it as iota-crystallin. The typical endogenous ligand of CRBP is all-trans-retinol. In the gecko lens, however, the ligand of iota-crystallin turns out to be 3-dehydroretinol (vitamin A2), which causes the yellow color of this lens. The iota-crystallin.3-dehydroretinol complex absorbs shortwave radiation, supposedly improving the optical quality of the dioptric apparatus and protecting the retina against ultraviolet damage. Whereas other crystallins have been recruited from stress proteins and metabolic enzymes, iota-crystallin represents a completely new class of taxon-specific lens proteins. Also, its ligand 3-dehydroretinol represents a novel type of lens pigment.

Rho B-crystallin, an aldose reductase-like lens protein in the gecko Lepidodactylus lugubris.

The ocular lenses of the diurno-nocturnal gecko Lepidodactylus lugubris contain a monomeric 38-kDa protein at a level of 20 to 22% of the total water-soluble protein. Amino acid sequences of peptides from this protein are most similar--up to 72% identity--to mammalian aldose reductase, an NADPH-dependent reductase which normally occurs at house-keeping levels in the eye lens, and which is involved in the development of diabetic cataract. The sequences show 56% identity with rho-crystallin from lenses of the frog genus Rana. It is concluded that different genes from the same superfamily of NADPH-dependent reductases have been recruited to become highly expressed as lens proteins in at least two different evolutionary lineages. To reflect the relationship with frog rho-crystallin, the gecko lens protein is designated as rho B-crystallin. As for frog rho-crystallin, no enzymatic activity could be established for rho B-crystallin in the gecko lens. Up to now, rho B-crystallin has not been detected in lenses of other reptiles or amphibians.

Detection of Legionella species in reclaimed water and air with the EnviroAmp Legionella PCR kit and direct fluorescent antibody staining.

Reclaimed water is an important resource for areas with inadequate water supplies. However, there have been few studies on the variety of microorganisms found in this type of water, since typically reclaimed water is examined only for the presence of coliform bacteria. Many microorganisms, including the legionellae, are known to be more resistant to chlorine than are coliform bacteria. Previously, we detected > 10(3) Legionella cells per ml in primary and secondary sewage effluents and observed no significant reduction in population numbers throughout the treatment process. In this study, we detected Legionella spp. in chlorinated effluent by using an EnviroAmp Legionella PCR kit and direct fluorescent antibody (DFA) staining. However, we were not able to isolate Legionella spp. from either natural or seeded reclaimed water samples. This suggests that the Legionella spp. detected by the PCR and DFA methods may be injured or viable but nonculturable after exposure to the high residual chlorine levels typically found in this type of water source. The numbers of coliform bacteria were low (< 2 cells per 100 ml) in most reclaimed water samples and were not correlated with the presence or absence of Legionella spp. We also collected air samples from above a secondary aeration basin and analyzed them by using the PCR, DFA, and plate culture methods. Legionella spp. were detected in the air obtained from above the secondary basin with all three methods. We concluded that the PCR was superior to the culture and DFA methods for detecting Legionella spp. in environmental water samples.

Phosphorylation of microtubule-associated proteins MAP2a,b and MAP2c at Ser136 by proline-directed kinases in vivo and in vitro.

The microtubule-associated protein 2 (MAP2) and its juvenile splicing variant MAP2c contain a phosphorylation site at Ser136 which is part of a Ser-Pro motif. This site lies within the N-terminal region common to MAP2b and MAP2c. It has been mapped by site-directed mutagenesis of recombinant MAP2c and by a monoclonal antibody AP18 whose epitope contains the phosphorylated Ser136. In vitro this site is phosphorylated by proline-directed kinases such as MAP kinase, GSK-3, or members of the cdk family, but not by other kinases such as PKA, PKC, or CaMK-II. MAP2a,b or MAP2c isolated from brain is found to be endogenously phosphorylated at Ser136. After microinjection into several cell lines dephosphorylated MAP2 isoforms or recombinant MAP2c become also phosphorylated at Ser136 in vivo. Injection of MAP2a,b or MAP2c into living cells causes reorganization of microtubules, including bundle formation. This effect is independent of the phosphorylation at Ser136. The specificity of the phosphorylation reaction provides a tool for analyzing the role and posttranslational processing of MAP2 in nerve cell development.

Supporting another view.

Baroness Cox has been treated very badly. It is terrible that militant groups of homosexuals and socialists can threaten to boycott a conference of which she is co-chairman and cause her to withdraw. Their bluff should have been called. They are all holding us to ransom.