Limiting similarity and species assemblages in the short-tailed fruit bats

We analyzed the morphometrics of seven taxa of short-tailed fruit bats Chiroptera : Phyllostomidae : Carollia , ( Carollia benkeithi , Carollia brevicauda , Carollia castanea , Carollia perspicillata azteca , Carollia perspicillata perspicillata , Carollia sowelli and Carollia subrufa ) to assess within-taxon variation and to address the possibility that morphological distinctions facilitate sympatry within this genus of ecologically similar bats. Whereas morphological similarities were found among species in certain combinations of allopatric taxa, members of sympatric assemblages differ significantly, indicating that there is a consistent representation of morphotypes within various assemblages of Carollia despite changes in species composition across the range of the genus. Furthermore, the differences in the skull size and shape among taxa correspond to potential differences in food-handling abilities and point to dietary differentiation among species as a factor in the facilitation of coexistence.     

Vitamin A transport: in vitro models for the study of RBP secretion

Retinol-binding protein (RBP) is the specific plasma carrier of retinol, encharged of the vitamin transport from the liver to target cells. Ligand binding influences the RBP affinity for transthyretin (TTR), a homotetrameric protein involved in the RBP/TTR circulating complex, and the secretion rate of RBP. In fact, in vitamin A deficiency, the RBP release from the hepatocytes dramatically decreases and the protein accumulates in the cells, until retinol is available again. The mechanism is still not clear and new cellular models are needed to understand in detail how the soluble RBP can be retained inside the cell. In fish, a vitamin A transport system similar to that of higher vertebrates is emerging, although with significant differences.     

Study on the mechanism of interference of 3,4,3',4'-tetrachlorobiphenyl with the plasma retinol-binding proteins in rodents

The mechanism of plasma retinol reduction in rodents by 3,4,3',4'-tetrachlorobiphenyl (TCB) was investigated by radioimmunochemical analysis of the amounts of circulating and hepatic retinol-binding protein (RBP) and transthyretin (TTR) in exposed and control animals. Plasma RBP concentrations were markedly reduced in C57BL/Rij mice (50%) at 4 days, in DBA/2 mice (37-41%) at 4 and 8 days, and in Sprague-Dawley rats (58%) at 2 days after exposure to TCB. These reductions paralleled the time course of reduction of plasma retinol after exposure to TCB. Hepatic RBP concentrations were somewhat increased in TCB-treated animals, especially in the C57BL/Rij mouse and Sprague-Dawley rat. However, the release of hepatic RBP into the circulation was not blocked by TCB treatment, as analysed in vitamin A deficient rats. In addition, the amount of plasma TTR was in the normal range in TCB-treated rats. The dissociation constants of the RBP-TTR complex as analysed by polarization of fluorescence appeared to be significantly increased (from 0.5 x 10(-7) M-1 to 2.4 x 10(-7) M-1) in the presence of a TCB metabolite, isolated from plasma of TCB-treated rats. In addition, the estimated number of binding sites for RBP on the TTR molecule was reduced (from 2.8 to 1.7 sites) upon treatment of TTR with the TCB metabolite. These data support the hypothesis that plasma retinol reduction by TCB might result from a weakening of the RBP-TTR complex, in the presence of the TCB metabolite bound to the TTR.     

Serum alpha- and gamma-tocopherols, retinol, retinyl palmitate, and carotenoid concentrations in captive and free-ranging bottlenose dolphins (Tursiops truncatus)

Concentrations of retinol, retinyl palmitate, beta-carotene, alpha-carotene, cryptoxanthin, lutein, lycopene, alpha-tocopherol, and gamma-tocopherol were measured in blood samples collected from 15 captive and 55 free-ranging bottlenose dolphins (Tursiops truncatus). From June 1991 to June 1994, blood samples were collected from captive animals residing at two locations; at Seven Seas (Brookfield Zoo, Brookfield, IL) and Hawk's Cay (Marathon Key, FL). Blood samples were collected from free-ranging animals from June 1991 to June 1996. Retinol levels were not significantly different between captive dolphin groups. However, Seven Seas animals had higher (P < 0.01) serum retinol concentrations compared to free-ranging animals (0.061 vs 0.041 microgram/ml). Retinyl palmitate was not detected in the serum of captive or free-ranging dolphins. Alpha-tocopherol levels were significantly (P < 0.05) higher for Seven Seas dolphins (16.4 micrograms/ml) than for Hawk's Cay (13.0 micrograms/ml) and free-ranging dolphins (12.5 micrograms/ml). Gamma-tocopherol concentrations were similar among captive and free-ranging dolphins. Free-ranging dolphins showed levels of circulating carotenoids (lutein and beta-carotene) while the captive animals did not. Additional carotenoids (lycopene, alpha-carotene and cryptoxanthin) were analyzed but not detected in any samples. Serum vitamin differences between captive and free-ranging dolphins may reflect the natural diet or indicate some potential biological or nutritional status significance.     

Plasma levels of vitamin A and its protein vectors (RBP and PA), carotene, total cholesterol and cholesterol-HDL in patients with dysplasia of the uterine cervix

In this study plasma levels of vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol were determined in 19 female subjects with cervical dysplasia and compared to those of the healthy female subjects described in our previous research. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion and HDL- and total cholesterol by enzymatic colorimetry. Plasma mean values of vitamin A and HDL-cholesterol were lower (P less than 0.05 and P less than 0.01, respectively) than in the control group. On the contrary total cholesterol was higher (P less than 0.01) in the patients than in the control group. Vitamin A plasma levels were significantly related (P less than 0.01) to RBP and PA. No significant statistical correlation was found between the severity of the dysplasia and vitamin A plasma levels.     

Structure-assisted discovery of the first non-retinoid ligands for Retinol-Binding Protein 4

Retinol-Binding Protein 4 (RBP4) is a plasma protein that transports retinol (vitamin A) from the liver to peripheral tissues. This Letter highlights our efforts in discovering the first, to our knowledge, non-retinoid small molecules that bind to RBP4 at the retinol site and reduce serum RBP4 levels in mice, by disrupting the interaction between RBP4 and transthyretin (TTR), a plasma protein that binds RBP4 and protects it from renal excretion. Potent compounds were discovered and optimized quickly from high-throughput screen (HTS) hits utilizing a structure-based approach. Inhibitor co-crystal X-ray structures revealed unique disruptions of RBP4–TTR interactions by our compounds through induced loop conformational changes instead of steric hindrance exemplified by fenretinide. When administered to mice, A1120, a representative compound in the series, showed concentration-dependent retinol and RBP4 lowering.     

Plasma levels of vitamin A and its protein vectors (RBP and PA), carotene, total cholesterol and HDL-cholesterol in patients with carcinoma of the cervix

In this study plasma levels of vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol of 40 subjects with carcinoma of cervix uteri were determined and compared to those of the healthy female subjects described in our previous research. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion, and HDL-and total cholesterol by enzymatic colorimetry. Plasma mean values of carotene, vitamin A, RBP and HDL-cholesterol were lower (P less than 0.01) than in the control group, and the same applies to the PA mean plasma level (P less than 0.05). On the contrary, total cholesterol mean value of the patients resulted to be higher (P less than 0.01) than in the control group. Vitamin A plasma levels were significantly related (P less than 0.01) to RBP and PA. No significant statistical correlation was found between the clinical stage and the histological grading and vitamin A plasma levels.     

Plasma values of vitamin A and its binding proteins (retinol binding proteins and prealbumin), carotene, total cholesterol and HDL-cholesterol in subjects with breast carcinoma

In this study plasma levels of vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol were determined in 33 subjects with breast cancer and compared to those of a group of healthy subjects previously described. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion, and HDL-and total cholesterol by enzymatic colorimetry. Mean plasma values of vitamin A, carotene and HDL-cholesterol were lower (P less than 0.01) than in the control group, the same applies to the RBP and PA mean levels (P less than 0.05). On the contrary, the mean value of total cholesterol was higher (P less than 0.01) in the patients than in the control group. Vitamin A plasma levels were significantly related to RBP and PA. No significant statistical correlation was found between clinical stage and vitamin A plasma levels.     

Plasma levels of vitamin A and its protein vectors (RBP and PA), carotene, total cholesterol and HDL-cholesterol in healthy adults of the female sex

Plasma vitamin A, carotenoids, retinol binding protein (RBP), prealbumin (PA), HDL-and total cholesterol were examined in healthy adult females. Plasma levels of vitamin A and carotene were determined by a spectrophotometric method using trifluoroacetic acid, plasma RBP and PA by single radial immunodiffusion, and HDL-and total cholesterol by enzymatic colorimetry. Vitamin A and carotene mean values resulted as 43.0 +/- 8.2 micrograms/100 ml and 231.9 +/- 69.0 micrograms/100 ml, respectively. RBP and PA values averaged as 4.2 +/- 1.1 mg/100 ml and 29.4 +/- 6.1 mg/100 ml, respectively; whereas HDL-and total cholesterol were 179 +/- 16 mg/100 ml and 57 +/- 8 mg/100 ml. Vitamin A plasma levels were shown to be significantly related (P less than 0.01) to RBP and PA, but not to the other parameters examined (carotene, HDL-and total cholesterol).     

Assay of human transthyretin-bound holo-retinol-binding protein with reversed-phase high-performance liquid chromatography

We describe a reversed-phase high-performance liquid chromatographic method for the determination of vitamin A-transporting (holo) transthyretin-bound (TTR) retinol-binding protein (RBP) concentrations in serum or plasma. Holo-TTR—RBP and free retinol derived primarily from free RBP are consistently observed with this chromatographic method. Holo-TTR—RBP concentrations determined by this method are highly correlated to holo-TTR—RBP concentrations measured by chromatography. This method has the advantage of using less expensive columns and having peak areas which are more proportional to their true concentrations in plasma, as determined by comparison to purified protein spectrophotometry and radial immunodiffusion. The percentage of RBP circulating as holo-TTR—RBP decreased significantly as the total concentration of RBP or retinol increased. Because purified holo-TTR—RBP did not dissociate under these chromatographic conditions, this suggests that more vitamin A circulates as holo-free RBP or free retinol in the blood of people with high serum RBP.     

Comparative phylogeography of short-tailed bats (Carollia: Phyllostomidae)

This is the first study of comparative phylogeography involving closely related species of Neotropical bats of the family Phyllostomidae. We compared patterns of geographical variation within the five species of fruit-eating bats currently recognized in the genus Carollia using the complete mitochondrial cytochrome-b gene. Our results suggest that the combined effect of the uplift of the Andes and the Panamanian land bridge has been as important for bats as for terrestrial mammals in shaping present-day biodiversity in the New World tropics. Species in this genus can be arranged in two highly supported clades, with a deep subdivision within each that corresponds well to differences across the Andes. We found three congruent phylogeographical patterns across species in this genus. First, the closer relationship between samples from western Ecuador and those from Central America, compared with populations east of the Andes in C. brevicauda, C. castanea and C. perspicillata. Second, the likelihood of a similar timing in South America for the arrival and diversification of C. brevicauda and C. perspicillata from their Central America ancestors. Third, the expansion of C. perspicillata and C. sowelli into northwestern Central America in the relatively recent past. Using a molecular clock, with rates ranging from 2.3 to 5% per 10(6) years, diversification within Carollia would have occurred over the last 1-4.5 Myr. These estimates agree well with the last rise of the Northern Andes and the Panama isthmus.     

Evidence for Exploitative Competition: Comparative Foraging Behavior and Roosting Ecology of Short-Tailed Fruit Bats (Phyllostomidae)

Chestnut short-tailed bats, Carollia castanea , and Seba's short-tailed bats, C. perspicillata (Phyllostomidae), were radio-tracked ( N = 1593 positions) in lowland rain forest at Tiputini Biodiversity Station, Orellana Province, Ecuador. For 11 C . castanea , mean home range was 6.8 ± 2.2 ha, mean core-use area was 1.7 ± 0.8 ha, and mean long axis across home range was 438 ± 106 m. For three C . perspicillata , mean home range was 5.5 ± 1.7 ha, mean core-use area was 1.3 ± 0.6 ha, and mean long axis was 493 ± 172 m. Groups of less than five C. castanea occupied day-roosts in earthen cavities that undercut banks the Tiputini River. Carollia perspicillata used tree hollows and buildings as day-roosts. Interspecific and intraspecific overlap among short-tailed bats occurred in core-use areas associated with clumps of fruiting Piper hispidum (peppers ) and Cecropia sciadophylla . Piper hispidum seeds were present in 80 percent of the fecal samples from C . castanea and 56 percent of samples from C . perspicillata . Carollia perspicillata handled pepper fruits significantly faster than C . castanea ; however, C . castanea commenced foraging before C . perspicillata emerged from day-roosts. Evidence for exploitative competition between C . castanea and C . perspicillata is suggested by our observations that 95 percent of ripe P . hispidum fruits available at sunset disappear before sunrise ( N = 74 marked fruits). Piper hispidum plants produced zero to 12 ripe infructescences per plant each night during peak production. Few ripe infructescences of P . hispidum were available during the dry season; however, ripe infructescences of C . sciadophylla , remained abundant.     

Nutrition or detoxification: why bats visit mineral licks of the Amazonian rainforest

Many animals in the tropics of Africa, Asia and South America regularly visit so-called salt or mineral licks to consume clay or drink clay-saturated water. Whether this behavior is used to supplement diets with locally limited nutrients or to buffer the effects of toxic secondary plant compounds remains unclear. In the Amazonian rainforest, pregnant and lactating bats are frequently observed and captured at mineral licks. We measured the nitrogen isotope ratio in wing tissue of omnivorous short-tailed fruit bats, Carollia perspicillata, and in an obligate fruit-eating bat, Artibeus obscurus, captured at mineral licks and at control sites in the rainforest. Carollia perspicillata with a plant-dominated diet were more often captured at mineral licks than individuals with an insect-dominated diet, although insects were more mineral depleted than fruits. In contrast, nitrogen isotope ratios of A. obscurus did not differ between individuals captured at mineral lick versus control sites. We conclude that pregnant and lactating fruit-eating bats do not visit mineral licks principally for minerals, but instead to buffer the effects of secondary plant compounds that they ingest in large quantities during periods of high energy demand. These findings have potential implications for the role of mineral licks for mammals in general, including humans.     

Interactions of Transthyretin (TTR) and Retinol-Binding Protein (RBP) in the Uptake of Retinol by Primary Rat Hepatocytes

The mechanism by which cells take up retinol from retinol-binding protein (RBP) and the role of the RBP–transthyretin (TTR) complex remain unclear. Here we report on retinol uptake through the RBP–TTR complex by primary cultured rat hepatocytes (parenchymal cells, PC) and nonparenchymal cells (NPC) following incubation with [³H]retinol–RBP or the [³H]retinol–RBP–TTR complex under several conditions. The cellular accumulation of retinol was time and temperature dependent in both PC and NPC. Analysis by HPLC showed that the incorporated [³H]retinol in NPC was mainly converted to retinyl ester, although in PC it remained mainly as unesterified retinol. However, the amount of retinol taken up from the RBP–TTR complex was nearly twofold greater than that from RBP alone. The uptake of [³H]retinol from protein-bound retinol was inhibited by an excess of either retinol–RBP or retinol–RBP–TTR complex. Moreover, retinol uptake through the RBP–TTR complex was inhibited by an excess of free TTR. From these results we postulate that TTR may take part as a positive regulator in the delivery of RBP-bound retinol from plasma, possibly by a membrane receptor, and that retinol uptake takes place preferentially from the RBP–TTR complex into both PC and NPC. The uptake of [³H]retinol (2 μM) by PC was saturated, whereas uptake by NPC was not. These results indicate that the physiological importance of TTR in retinol delivery may be especially important to vitamin A-storing stellate (Ito) cells in the NPC fraction.     

The structure of human retinol-binding protein (RBP) with its carrier protein transthyretin reveals an interaction with the carboxy terminus of RBP

Whether ultimately utilized as retinoic acid, retinal, or retinol, vitamin A is transported to the target cells as all-trans-retinol bound to retinol-binding protein (RBP). Circulating in the plasma, RBP itself is bound to transthyretin (TTR, previously referred to as thyroxine-binding prealbumin). In vitro one tetramer of TTR can bind two molecules of retinol-binding protein. However, the concentration of RBP in the plasma is limiting, and the complex isolated from serum is composed of TTR and RBP in a 1 to 1 stoichiometry. We report here the crystallographic structure at 3.2 A of the protein-protein complex of human RBP and TTR. RBP binds at a 2-fold axis of symmetry in the TTR tetramer, and consequently the recognition site itself has 2-fold symmetry: Four TTR amino acids (Arg-21, Val-20, Leu-82, and Ile-84) are contributed by two monomers. Amino acids Trp-67, Phe-96, and Leu-63 and -97 from RBP are flanked by the symmetry-related side chains from TTR. In addition, the structure reveals an interaction of the carboxy terminus of RBP at the protein-protein recognition interface. This interaction, which involves Leu-182 and Leu-183 of RBP, is consistent with the observation that naturally occurring truncated forms of the protein are more readily cleared from plasma than full-length RBP. Complex formation prevents extensive loss of RBP through glomerular filtration, and the loss of Leu-182 and Leu-183 would result in a decreased affinity of RBP for TTR.     

Parotid salivary gland ultrastructure in an omnivorous neotropical bat: evolutionary diversity at the cellular level

The parotid salivary gland in an omnivorous neotropical bat, Carollia perspicillata (L.), was studied by transmission electron microscopy and compared to the parotid gland in other species of bats and in mammals in general. The parotid acinar secretory granules were found to be unique among mammals: they consist of a finely punctate matrix containing a variety of electron dense inclusions ranging from punctate densities to complex cage-like geodesic structures. The parotid acinar product in Carollia perspicillata is intermediate in morphology between that formed by homologous secretory cells in insectivorous/carnivorous bats and that in frugivorous bats. Both the intercalated and striated ducts probably release additional secretory products into the saliva.     

Application of an allosteric model to describe the interactions among retinol binding protein 4, transthyretin, and small molecule retinol binding protein 4 ligands

Retinol binding protein 4 (RBP4) is a serum protein that serves as the major transport protein for retinol (vitamin A). Recent reports suggest that elevated levels of RBP4 are associated with insulin resistance and that insulin sensitivity may be improved by reducing serum RBP4 levels. This can be accomplished by administration of small molecules, such as fenretinide, that compete with retinol for binding to RBP4 and disrupt the protein-protein interaction between RBP4 and transthyretin (TTR), another serum protein that protects RBP4 from renal clearance. We developed a fluorescence resonance energy transfer (FRET) assay that measures the interaction between RBP4 and TTR and can be used to determine the binding affinities of RBP4 ligands. We present an allosteric model that describes the pharmacology of interaction among RBP4, TTR, retinol, and fenretinide, and we show data that support the model. We show that retinol increases the affinity of RBP4 for TTR by a factor of 4 and determine the affinity constants of fenretinide and retinyl acetate. The assay may be useful for characterizing small molecule ligands that bind to RBP4 and disrupt its interaction with TTR. In addition, such a model could be used to describe other protein-protein interactions that are modulated by small molecules.     

Serum concentrations of vitamin D metabolites, vitamins A and E, and carotenoids in six canid and four ursid species at four zoos

Nutritional status for six captive canid species (n=34) and four captive ursid species (n=18) were analyzed. The species analyzed included: African wild dog (Lycaon pictus), arctic fox (Alopex lagopus), gray wolf (Canis lupus), maned wolf (Chrysocyon brachyurus), Mexican wolf (Canis lupus baleiyi), red wolf (Canis rufus), brown bear (Ursus arctos), polar bear (Ursus maritimus), spectacled bear (Tremarctos ornatus), and sun bear (Ursus malayanus). Diet information was collected for these animals from each participating zoo (Brookfield Zoo, Fort Worth Zoo, Lincoln Park Zoological Gardens, and North Carolina Zoological Park). The nutritional composition of the diet for each species at each institution met probable dietary requirements. Blood samples were collected from each animal and analyzed for vitamin D metabolites 25(OH)D and 1,25(OH)(2)D, vitamin A (retinol, retinyl stearate, retinyl palmitate), vitamin E (alpha-tocopherol and gamma-tocopherol) and selected carotenoids. Family differences were found for 25(OH)D, retinol, retinyl stearate, retinyl palmitate and gamma-tocopherol. Species differences were found for all detectable measurements. Carotenoids were not detected in any species. The large number of animals contributing to these data, provides a substantial base for comparing the nutritional status of healthy animals and the differences among them.     

Retinol-binding protein and transthyretin expressed in HeLa cells form a complex in the endoplasmic reticulum in both the absence and the presence of retinol

To establish a suitable experimental system for studies of the interaction of retinol-binding protein (RBP) with transthyretin (TTR) we have expressed the corresponding cDNAs in HeLa cells. To investigate whether complex formation might occur already in the endoplasmic reticulum (ER), the C-terminal ER retention signal, KDEL, was attached to TTR. The tetrameric TTR-KDEL fusion protein was retained in the ER of HeLa cells. When RBP was co-expressed with TTR-KDEL, RBP was retained intracellularly. A cDNA-encoding purpurin, a protein which is 50% identical to RBP, was then expressed together with TTR-KDEL. Purpurin was not retained intracellularly and did not bind to TTR coupled to Sepharose. The effect of the vitamin A status on the secretion of TTR and RBP was examined. While TTR expressed alone was not retained intracellularly, TTR was retained in vitamin A-deficient cells when co-expressed with RBP. Addition of retinol stimulated rapid secretion of both proteins. These results demonstrate that TTR can form a complex with RBP in the ER. The data suggest that RBP and TTR are secreted as a complex.     

Biochemical basis for retinol deficiency induced by the I41N and G75D mutations in human plasma retinol-binding protein

Retinol-binding protein (RBP) is the retinol-specific carrier protein present in plasma, where it circulates almost entirely bound to thyroxine-binding transthyretin (TTR). Recently, depressed plasma retinol and RBP levels in carriers of the I41N and G75D RBP point mutations have been reported. We show here that although recombinant human N41 and D75 RBPs can form complexes with retinol and TTR in vitro, the retinol-mutated RBP complexes are significantly less stable than human normal holo-RBP, as revealed by the markedly facilitated retinol release by mutated holo-RBPs to phospholipid membranes, in accordance with the location of mutated residues inside the RBP retinol-binding cavity. Taken together, the data are consistent with the I41N and G75D point mutations being the cause of an altered interaction of retinol with RBP, resulting in a remarkably reduced stability of the retinol-RBP complex, which in turn can lead to the lowering of plasma retinol and RBP levels.