Herschel/HIFI observations of the circumstellar ammonia lines in IRC+10216.

CONTEXT: A discrepancy exists between the abundance of ammonia (NH3) derived previously for the circumstellar envelope (CSE) of IRC+10216 from far-IR submillimeter rotational lines and that inferred from radio inversion or mid-infrared (MIR) absorption transitions. AIMS: To address the discrepancy described above, new high-resolution far-infrared (FIR) observations of both ortho- and para-NH3 transitions toward IRC+10216 were obtained with Herschel, with the goal of determining the ammonia abundance and constraining the distribution of NH3 in the envelope of IRC+10216. METHODS: We used the Heterodyne Instrument for the Far Infrared (HIFI) on board Herschel to observe all rotational transitions up to the J = 3 level (three ortho- and six para-NH3 lines). We conducted non-LTE multilevel radiative transfer modelling, including the effects of near-infrared (NIR) radiative pumping through vibrational transitions. The computed emission line profiles are compared with the new HIFI data, the radio inversion transitions, and the MIR absorption lines in the ν2 band taken from the literature. RESULTS: We found that NIR pumping is of key importance for understanding the excitation of rotational levels of NH3. The derived NH3 abundances relative to molecular hydrogen were (2.8 ± 0.5) × 10-8 for ortho-NH3 and [Formula: see text] for para-NH3, consistent with an ortho/para ratio of 1. These values are in a rough agreement with abundances derived from the inversion transitions, as well as with the total abundance of NH3 inferred from the MIR absorption lines. To explain the observed rotational transitions, ammonia must be formed near to the central star at a radius close to the end of the wind acceleration region, but no larger than about 20 stellar radii (1σ confidence level).

Discovery of water vapour around IRC+10216 as evidence for comets orbiting another star.

Since 1995, planets with masses comparable to that of Jupiter have been discovered around approximately 60 stars. These planets have not been seen directly, but their presence has been inferred from the small reflex motions that they gravitationally induce on the star they orbit; these motions result in small periodic wavelength shifts in the stellar spectrum. The presence of analogues of the smaller bodies in our Solar System cannot, however, be determined using this technique, because the induced reflex motions are too small-so an alternative approach is needed. Here we report the observation of circumstellar water vapour around the ageing carbon star IRC+10216; water is not expected in measurable quantities around such a star. The only plausible explanation for this water is that the recent evolution of IRC+10216, which has been accompanied by a prodigious increase in its luminosity, is causing the vaporization of a collection of orbiting icy bodies-a process considered in an earlier theoretical study.

Fluorescent bone viewed through toenails of living animals: a method to observe bone regrowth.

We have developed a new method to observe bone and to document growth in living animals. The technique involves injecting calcein, a fluorescent calcium deposition marker, waiting approximately 4 hr for it to clear the vascular system, and observing bone directly through the toenails of lightly anesthetized living animals. Bone regrowth can be monitored in situ by amputating the digit through the nail plate, waiting the desired number of days, and injecting a second fluorescent label, alizarin red. Bone that has regrown since the amputation appears as a red area distal to the green calcein label on toes of lightly anesthetized animals when viewed under FITC fluorescence. This method has been used to demonstrate blocked bone synthesis and to quantitate significant differences in bone growth in control and experimental toes of individual animals. Advantages of this method include its simplicity, the use of fewer animals to collect sequential data, and increased reliability of repeated microscopic measurements using the same animal.

Denervation retards but does not prevent toetip regeneration.

Toetips of mammals regrow after amputation by a process similar, but not identical, to that which occurs during regeneration of a newt limb. Nerve is needed as a mitotic stimulant for newt limb regeneration but the requirement for nerve during rodent digit-tip regeneration is not known. Nerve dependence in rats was tested by severing the sciatic nerve in one hindlimb, amputating digit-tips from the central digits of both hind feet, and comparing the amount of regrowth in innervated and denervated digits. Denervation delayed soft-tissue wound healing. However, denervation did not significantly affect bone regrowth when animals were examined at one month. Because we suspected delayed bone regrowth, we used a new method that we developed to follow bone growth at several time points in each animal. Termed visible bone fluorescence through nail, this technique used serial injections of fluorescent calcium-deposition markers and observation through the toenails to observe bone growth in living animals. Using this method it was possible to detect retarded bone regrowth in denervated digits. Thus, although denervation of rodent tips delayed both soft tissue healing and bone regrowth, it did not prevent ultimate restitution of the amputated part. This suggests that neurotrophic stimulation in the mammalian digit-tip is not identical to that documented during newt limb regeneration, and that growth stimulation may be provided by tissues other than nerve.

Bone growth is induced by nail transplantation in amputated proximal phalanges.

Mammals are able to regrow the tips of amputated fingers and toes. However, regrowth is limited to regions covered by, and is dependent upon, the presence of the nail organ. If the nail organ is responsible for bone growth in digit-tips, we reasoned that transplanted nail organ might also be able to induce outgrowth from other levels of the digit. Partial nail organ has been transplanted to amputated proximal phalanges of young rats. To date, six transplants have successfully produced outgrowth of nail. New bone growth, not seen in control amputated digits, was documented by x-ray and by alizarin red and calcein injections to be directed toward implanted nail organ. These results support an inductive role for nail organ epithelium in bone growth after amputation and provide encouragement for attempts to enhance a positive outcome after appendage amputation.

Use of enzyme overlay membranes to survey proteinase activity in frozen sections: cathepsin-like and plasmin-like activity in regenerating newt limbs.

We present a method that permits extremely simple and rapid screening of proteolytic enzyme activity in sectioned tissues. Enzyme overlay membranes (EOMs) are custom-made membranes designed to fluoresce at sites of specific proteolytic enzyme activity after separation of proteins by gel electrophoresis. EOMs, selected to detect either plasmin-like or cathepsin B-like activity, have been used in a novel way to document the distribution of enzyme activity in frozen sectioned tissues. When moistened membranes were placed in contact with sectioned regenerating newt limbs, a fluorescent pattern of enzyme activity was generated. In limbs at 3 hr post amputation, cathepsin B-like activity was prominent across the amputation site but plasmin-like activity was distributed in dermal and deeper proximal tissues, suggesting different roles for these two classes of enzymes. EOM enzymology in situ (EEI) on frozen sectioned tissues may be a widely useful technique to display distribution and level of activity of proteolytic enzymes in various systems.

Perspective: a suggested role for basement membrane structures during newt limb regeneration.

BACKGROUND: Interactions between epithelium and mesenchyme, which occur across a basement membrane (BM) zone, are essential to generate a growth bud, or blastema, from which a new limb regenerates. An intact BM at that interface is believed to inhibit regeneration, but that mechanism of inhibition is not understood. METHODS: Interference contrast microscopy and antibodies to laminin have been used to describe reformation of the BM and the basal lamina (BL) and their relationships to wound epithelium and mesenchyme in successive stages of blastema formation. RESULTS: The BL is initially absent from the amputation surface and is reestablished to continuity by the late bud stage of regeneration. It forms generally from base to apex, precedes reticular lamina (RL) formation, and is absent beneath most of the wound epithelium. Our inability to correlate mesenchymal cell accumulation exclusively with the area lacking BL apically and postaxially prompted rethinking of the significance of the BL. CONCLUSIONS: Consistent with these and other observations, we suggest that the BL, when it forms during blastema formation, appears to function as in other developing systems to stabilize the phenotype of adjacent cells. Thus, epithelium becomes epidermis and adjacent mesenchyme synthesizes RL and becomes dermis. Accordingly, the feature that distinguishes regenerating from nonregenerating appendages is the ability of regenerating appendages to delay BL closure until after a critical mass of mesenchymal cells has accumulated.

Stabilizing role of the basement membrane and dermal fibers during newt limb regeneration.

BACKGROUND: Following amputation of a newt limb, tissues at the amputation site undergo histolysis to give rise to a growth bud, or blastema, but they also provide a base on which the regenerate is constructed. Studies suggest that dermal tissues may differentially resist histolysis. METHODS AND RESULTS: To examine stability of tissues at the amputation site, more than 80 preblastemal staged regenerating limbs were examined histologically. Initially, all soft tissues not attached to bone retracted and were covered by migrating epithelium. The dermis was seen to be stable during the first week postamputation. Muscle dedifferentiated and was heavily stained with anti-tenascin antibodies, but the intact overlying dermis was unstained. Fiber bundles, revealed by staining with phosphotungstic acid hematoxylin, isolated the dermis from dedifferentiating deeper tissues during the first week postamputation, but partially broke down during the second week. However, the basement membrane (BM) remained as the distalmost intact structure at the amputation site in all limbs examined. The BM was the foundation for new BM synthesis which preceded dermis synthesis in the base of the blastema during the second week, even while undifferentiated cells were accumulating centrally. CONCLUSIONS: We suggest that the dermis resists histolysis long enough for new BM to form in continuity with that of the stump. Dermis formation (dermogenesis) distal to the amputation plane begins early as in mammalian healing but is not completed until after blastema formation. Thus, factors that inhibit dermal closure appear to distinguish regenerating from non-regenerating appendages.

Bone regrowth after digit tip amputation in mice is equivalent in adults and neonates.

Despite clinical and experimental reports of digit tip regrowth, bone regrowth after amputation through terminal phalanges has not been methodically documented. We have examined bone regrowth in mice after amputation through the terminal phalanx to determine how the level of amputation affects the response and whether the response varies between adults and neonates. Digit tips were amputated, and, at selected intervals greater than 5 weeks after amputation, digits were photographed and processed for whole mount staining with alizarin red stain. Amputations within the distal 40% of the terminal phalanx yielded grossly normal digits within 5 weeks and bone regrowth which usually attained or surpassed the original length. Amputations through the proximal 20% of the distal phalanx precluded nail plate regrowth, and bone grew minimally or regressed. Amputation through the intermediate 40% of the distal phalanx produced digit tips in which bone regrowth was correlated with nail regrowth and in which bone regrowth did not reach original levels. The response in adults and neonates was similar. The correlation between nail regrowth and bone regrowth may help to predict phalangeal bone regrowth after amputation in adults and juveniles.

Bone regrowth in young mice stimulated by nail organ.

Recent studies of postamputational repair following digit-tip amputation revealed an unexpected correlation between nail regrowth and bone regrowth in mice. To examine putative effects of nail on bone regrowth, phalangeal tips were amputated such that nail was artificially removed from distal levels or retained following proximal level amputations. In the absence of nail, bone did not regrow at distal levels. Conversely, when nail was surgically retained bone regrew from proximal levels. The nail organ profoundly influences bone regrowth.

Phalangeal regrowth in rodents: postamputational bone regrowth depends upon the level of amputation.

Conflicting reports of distal phalangeal regrowth prompted a reexamination of bone growth following phalangeal amputation in mammals. Digits of neonatal and adult mice and rats were amputated at various levels. The short-term response was examined on histological sections, and long-term growth was documented by alizarin red-staining of KOH-digested digits. Three patterns of response were seen to correspond to three general levels of amputation. Complete bone regeneration occurred frequently by five weeks following amputation through the distal one-quarter of the distal phalanx. Amputation through the central region of the distal phalanx yielded substantial bone growth, but the form of the regrowth was imperfect even three months after amputation. Amputation through more proximal levels of the digit yielded no significant elongation. To investigate why the response varies in relation to the level of amputation, we are conducting both in vivo and in vitro experiments. We have learned that simple avulsion of the nail plate provokes substantial remodeling of the distal phalanx. We are further exploring the trophic influence of nail organ on bone structure and growth in vivo. We have also recently determined that entire digits may be kept alive in vitro when cultured in DMEM:F-12:BGJb medium supplemented with insulin, EGF and FGF. This system sufficiently replicates in vivo conditions such that osteogenesis occurs both endosteally and distal to the amputation plane in vitro. The effects of growth factors, retinoic acid, and the presence or absence of nail organ components on amputational bone growth at all three levels are currently being studied in vitro. The goal of these studies is to determine why bone fails to grow, undergoes hyperplasia, or regenerates following amputation at different levels in mammals.

Epidermis, basement membrane, and connective-tissue healing after amputation of mouse digits: implications for mammalian appendage regeneration.

Soft tissues from amputation sites of mice were examined at both light and electron microscope levels to determine whether features of growth buds (blastemas), which are necessary for amphibian limb regeneration, exist in nonregenerating mice. Several such features were found. A small area of the wound bed was covered by wound epithelium which, as in regenerating newt limbs, initially lacked an underlying basement membrane. Serially sectioned digits revealed blastemalike growth in the subdermal layer surrounding periosteal chondrogenic cells. Mesenchymelike cells were seen among the fibroblasts and leucocytes within the proliferating tissues. However, no evidence of dedifferentiation was seen in the dermis, which persisted as an apparent intact obstruction to growth bud formation. Existence of the essential ingredients of growth buds and soft-tissue proliferation adjacent to chondrogenic cells proximally suggest that the tissues of mammalian healing may differ quantitatively rather than qualitatively from tissues of appendage regeneration. This premise is encouraging for efforts at growth enhancement in mammals.

Association of mesenchyme with attenuated basement membranes during morphogenetic stages of newt limb regeneration.

The interface between epithelium and mesenchyme may be involved in inductive interactions which occur during development. This interface within the growth bud, or blastema, of a regenerating limb has been examined to determine whether changes in basement-membrane structures are visible in regions of putative epithelial-mesenchymal inductive interaction. Regenerating forelimbs of adult newts were fixed by perfusion with osmotically balanced aldehydes. Late-bulb to early-digit stage regenerates were collected and processed either for light and transmission electron microscopy or for scanning electron microscopy. Light microscopy confirmed that regions characterized by increased numbers of subepithelial mesenchymal cells were covered by a diffusely stained basement membrane. Transmission electron microscopy of these regions revealed two structural components of the basement membrane. The thin basal lamina was continuous in all regions of all stages examined, but it was attenuated apically in areas of mesenchymal cell accumulation. The thicker underlying reticular lamina was markedly attenuated in these regions near the blastemal apex. Scanning electron microscopy of de-epithelialized blastemas revealed that, apically, the reticular lamina formed only a delicate lacelike network. On the base of the blastema, it formed a dense fibrillar meshwork which was further organized into a geometric pattern in the adjacent stump skin. Cumulatively, these observations suggest that physical contact between epithelial and mesenchymal cells is not essential at these stages, but that regions of putative epithelial-mesenchymal interaction are characterized by a distinctly diminished reticular lamina. Structural changes in basement-membrane components may be related to termination of local inductive events.

Bone healing after amputation of mouse digits and newt limbs: implications for induced regeneration in mammals.

Postamputational healing was compared in nonregenerating and regenerating animals to determine whether bone healing might interfere with a regenerative response in mice. More than 150 mouse toes and 100 newt limbs were examined at the light microscope level. Stages of normal bone healing with approximate times of occurrence were established. Major differences in healing of these two species were seen. The periosteum produced hyaline cartilage, woven bone, and chondroid bone in mice, but only hyaline cartilage in newts. The endosteum produced woven bone in mice but no new growth in newts. Dead bone persisted in mice but was removed in newts. The marrow cavity became sealed in mice but remained open in newts. Despite these differences both animals produced skeletal tissue distal to the amputation plane. Woven bone formed distal to the amputation plane of mice. Cartilage formed distal to the amputation plane of newts, but cartilage was never seen distal to the plane of mice. Results of previous studies reveal that cartilage can be formed distal to the amputation plane of experimentally treated mice. Thus, although it does not regenerate, mouse bone is capable of producing, distal to the amputation plane, the type of skeletal tissue which appears at that location during an epimorphic regenerative response. This observation, in combination with other experimental results, indicates that both skeletal and soft tissues at the amputation site of treated mammals can resemble comparable tissues of newt limbs at an early stage of regeneration.

Persistent left superior vena cava and associated structural and functional considerations.

Potentially significant associations are presented between anomalous systemic venous return (including both left superior vena cava and left hepatic venous drainage to the coronary sinus), a history of atrial fibrillation and a forme fruste of cor triatriatum in an elderly woman. Lack of associated structural defects or functional deficits makes it difficult to assess the frequency of occurrence in the general population of bilateral superior venae cavae in association with a persistent left hepatic vein draining into the coronary sinus. However, the potential for these systemic venous anomalies needs to be considered when unexplained arrhythmias are encountered. More specifically, the size of the coronary sinus needs to be assessed in patients with arrhythmia.

Partial blastema formation after amputation in adult mice.

The amputation surface of toes of adult mice was treated by repeated skin removal and exposure to a saturated solution of NaCl. Histological examination of treated appendages revealed the presence of wound epithelium, numerous irregularly oriented subepithelial cells, many of which were mitotically active as indicated by [3H] thymidine uptake, and a remodeled viable bone adjacent to subepithelial cells. Thus, tissues at the amputation site of mammals can resemble comparable tissues of newt limb stumps at an early stage of regeneration. Complete blastema formation was not achieved, however.