Water in the terrestrial planet-forming zone of the PDS 70 disk.

Terrestrial and sub-Neptune planets are expected to form in the inner (less than 10 AU) regions of protoplanetary disks1. Water plays a key role in their formation2-4, although it is yet unclear whether water molecules are formed in situ or transported from the outer disk5,6. So far Spitzer Space Telescope observations have only provided water luminosity upper limits for dust-depleted inner disks7, similar to PDS 70, the first system with direct confirmation of protoplanet presence8,9. Here we report JWST observations of PDS 70, a benchmark target to search for water in a disk hosting a large (approximately 54 AU) planet-carved gap separating an inner and outer disk10,11. Our findings show water in the inner disk of PDS 70. This implies that potential terrestrial planets forming therein have access to a water reservoir. The column densities of water vapour suggest in-situ formation via a reaction sequence involving O, H2 and/or OH, and survival through water self-shielding5. This is also supported by the presence of CO2 emission, another molecule sensitive to ultraviolet photodissociation. Dust shielding, and replenishment of both gas and small dust from the outer disk, may also play a role in sustaining the water reservoir12. Our observations also reveal a strong variability of the mid-infrared spectral energy distribution, pointing to a change of inner disk geometry.

A novel whole "Joint-in-Motion" device reveals a permissive effect of high glucose levels and mechanical stress on joint destruction.

OBJECTIVE: Osteoarthritis (OA) has recently been suggested to be associated with diabetes. However, this association often disappears when accounting for body mass index (BMI), suggesting that mechanical stress may be a confounding factor. We investigated the combined influence of glucose level and loading stress on OA progression using a novel whole joint-in-motion (JM) culture system. DESIGN: Whole mouse knee joints were placed in an enclosed chamber with culture media and actuated to recapitulate leg movement, with a dynamic stress regimen of 0.5 Hz, 8 h/day for 7 days. These joints were treated with varying levels of glucose and controlled for osmolarity and diffusion. Joint movement and joint space were examined by X-ray fluoroscopy and microCT. Cartilage matrix levels were quantified by blinded Mankin scoring and immunohistochemistry. RESULTS: Culturing in the JM device facilitated proper leg extension and flexion movements, and adequate mass transport for analyzing the effect of glucose on cartilage. Treatment with higher levels of glucose either via media supplementation or intra-articular injection caused a significant decrease in levels of glycosaminoglycan (GAG) and an increase in aggrecan neoepitope in articular cartilage, but only under dynamic stress. Additionally, collagen II level was slightly reduced by high glucose levels. CONCLUSIONS: High levels of glucose and dynamic stress have permissive effects on articular cartilage GAG loss and aggrecan degradation, implicating that mechanical stress confounds the association of diabetes with OA. The JM device supports novel investigation of mechanical stress on the integrity of an intact living mouse joint to provide insights into OA pathogenesis.

Antinociceptive action of intrathecally administered IGF-I and the expression of its receptor in rat spinal cord.

mRNA transcripts for insulin-like growth factor I (IGF-I) and its receptor are expressed in the lumbar region of the spinal cord. Accordingly, we examined the involvement of IGF-I in nociceptive transmission. An intrathecal injection of IGF-I (200-1000 ng) produced a dose-dependent elevation in nociceptive threshold as indicated by tail flick/withdrawal latency. In contrast, comparable doses of insulin had no significant effect. The time-response curve (15-75 min) revealed that the peak for IGF-I's antinociceptive effect is attained at 30 min. Our data provide evidence that the IGF-I system within the spinal cord may serve as a target for novel analgesics.

Distribution of the integral plasma membrane glycoprotein CE9 (MRC OX-47) among rat tissues and its induction by diverse stimuli of metabolic activation.

We have compared the levels of the integral plasma membrane glycoprotein CE9 (MRC OX-47) in different tissues of the rat and have ascertained that the levels of CE9 protein and mRNA in selected tissues and cells exhibit moderate increases in response to diverse stimuli of metabolic activation. When normalized on the basis of total protein, the level of CE9 detected in the different tissues was found to vary over a 50-fold range. In addition, the apparent molecular mass of CE9 was observed to vary from 40 kDa to 68 kDa as a consequence of tissue-specific glycosylation. The highest level of CE9 was detected in brown adipose tissue, where the protein was found to be localized to the plasma membranes of the adipocytes. The metabolic activation of brown adipose tissue that occurs upon exposure of rats to the cold was found to be accompanied by 3.0 +/- 0.4-fold and 1.7 +/- 0.2-fold increases in the levels of CE9 mRNA and protein respectively. An intermediate level of CE9 was detected in the liver, where the protein is known to be expressed within the basolateral domain of the hepatocyte plasma membrane. The metabolic activation of hepatocytes that occurs upon administration of thyroid hormone to euthyroid rats was found to be accompanied by 2.2 +/- 0.3-fold and 1.9 +/- 0.3-fold increases in the levels of CE9 mRNA and protein respectively. A low level of CE9 was detected in the lymphoid organs, such as thymus and spleen. The metabolic activation of isolated rat splenocytes that occurs upon concanavalin A-mediated blast transformation in culture was found to be accompanied by 2.1 +/- 0.2-fold and 1.6 +/- 0.2-fold increases in the levels of CE9 mRNA and protein respectively. On the basis of these and other observations, we suggest that the level, and possibly also the localization, of the integral plasma membrane glycoprotein CE9 may be correlated in a positive fashion with metabolic activity in a diverse array of cell types.

Breaching the diffusion barrier that compartmentalizes the transmembrane glycoprotein CE9 to the posterior-tail plasma membrane domain of the rat spermatozoon.

CE9 is a posterior-tail domain-specific integral plasma membrane glycoprotein of the rat testicular spermatozoon. During epididymal maturation, CE9 undergoes endoproteolytic processing and then redistributes into the anterior-tail plasma membrane domain of the spermatozoon (Petruszak, J. A. M., C. L. Nehme, and J. R. Bartles. 1991. J. Cell. Biol. 114:917-927). We have determined the sequence of CE9 and found it to be a Type Ia transmembrane protein identical to the MRC OX-47 T-cell activation antigen, a member of the immunoglobulin superfamily predicted to have two immunoglobulin-related loops and three asparagine-linked glycans disposed extracellularly. Although encoded by a single gene and mRNA in the rat, the majority of spermatozoal CE9 is of smaller apparent molecular mass than its hepatocytic counterpart due to the under-utilization of sites for asparagine-linked glycosylation. By fluorescence recovery after photobleaching, CE9 was determined to be mobile within the posterior-tail plasma membrane domain of the living rat testicular spermatozoon, thus implying the existence of a regional barrier to lateral diffusion that is presumed to operate at the level of the annulus. Through the development of an in vitro system, the modification of this diffusion barrier to allow for the subsequent redistribution of CE9 into the anterior-tail domain was found to be a time-, temperature-, and energy-dependent process.

Endoproteolytic cleavage in the extracellular domain of the integral plasma membrane protein CE9 precedes its redistribution from the posterior to the anterior tail of the rat spermatozoon during epididymal maturation.

Originally identified as a basolateral domain-specific integral plasma membrane protein of the rat hepatocyte, CE9 mRNA and protein were also detected at high levels in the testis of the rat by Northern and Western blotting and immunoprecipitation. CE9 proved to be a domain-specific integral plasma membrane protein of the rat spermatozoon: on testicular spermatozoa, it was concentrated within the posterior tail domain of the plasma membrane, whereas on vas deferens spermatozoa, CE9 was concentrated within the anterior tail domain. This change in the localization of CE9 was observed to take place in a offgressive fashion during the passage of the spermatozoa from the caput epididymidis to the cauda epididymidis and was preceded by the specific endoproteolytic cleavage of CE9 in the proximal portion of the caput epididymidis. Amino-terminal amino acid microsequencing of CE9 immunoaffinity purified from epididymis suggested that the cleavage occurred on the carboxy-terminal side of arginine-74 in the primary sequence of CE9, resulting in the loss of approximately 40% of the amino acids in the extra-cellular domain of this transmembrane glycoprotein.

Decreases in the relative concentrations of specific hepatocyte plasma membrane proteins during liver regeneration: down-regulation or dilution?

Antibodies were used to quantify seven domain-specific integral proteins of the rat hepatocyte plasma membrane during rat liver regeneration in response to two-thirds hepatectomy. Quantitative immunoblotting revealed that a subset of the plasma membrane proteins exhibited transient 30-70% decreases in relative concentration during the period of hepatocyte proliferation. The list of affected proteins included at least one representative from each of the plasma membrane domains: the apical protein HA 4, the lateral protein HA 321, and the basolateral receptors for epidermal growth factor and asialoglycoproteins. In contrast, the relative concentrations of three other plasma membrane proteins, the basolateral protein CE 9 and the two apical proteins dipeptidylpeptidase IV and aminopeptidase N, remained unchanged throughout liver regeneration. The decreases in the relative concentrations of the plasma membrane proteins were observed even when the synthesis of hepatocyte DNA was blocked by hydroxyurea, suggesting that the signalling for these two delayed consequences of two-thirds hepatectomy occurred along parallel, dependent pathways. Pulse and pulse-chase metabolic radiolabeling studies revealed that the decreases in the concentrations of the PM proteins were accomplished through protein-selective decreases in the rates of synthesis of the high-mannose precursors of the affected proteins, but not through the accelerated degradation of the mature plasma membrane proteins.

Expression and compartmentalization of integral plasma membrane proteins by hepatocytes and their progenitors in the rat pancreas.

A combination of Western blotting, Northern blotting and immunofluorescence was used to examine the expression and compartmentalization of plasma membrane proteins by those hepatocyte-like cells that arise in the pancreases of rats subjected to sequential dietary copper depletion and repletion. The pancreatic hepatocytes were found to: (1) express several integral membrane proteins known to be concentrated within the apical, lateral or basolateral domains of the plasma membranes of hepatocytes in liver; and (2) compartmentalize the membrane proteins to equivalent plasma membrane domains, despite the organization of these cells into clusters instead of highly vascularized plates. The apical plasma membrane proteins dipeptidylpeptidase IV and HA 4 were found to line bile canaliculus-like openings between adjacent pancreatic hepatocytes; these openings were shown to be continuous with the pancreatic exocrine duct by India ink infusion. In contrast, the basolateral plasma membrane protein rat hepatic lectin-1 and lateral plasma membrane protein HA 321 were detected elsewhere about the surfaces of the pancreatic hepatocytes: by analogy to their respective localizations on hepatocytes in liver, rat hepatic lectin-1 was concentrated on those surfaces exposed to the pancreatic matrix at the periphery of the hepatocyte clusters (the basal surface equivalent), whereas HA 321 was concentrated on those surfaces exposed to adjacent hepatocytes within the clusters. The hepatocyte plasma membrane proteins were found to be expressed in the pancreas at different times during the copper depletion/repletion protocol: for example, rat hepatic lectin-1 and the bulk of the HA 4 were expressed relatively late in the protocol, only after large numbers of pancreatic hepatocytes had appeared; whereas dipeptidylpeptidase IV was induced greater than 10-fold early in the protocol and proved to be an apical-specific marker for those ductular epithelial cells that are believed to be the progenitors of the pancreatic hepatocytes.

Peroxisome proliferator-induced alterations in the expression and modification of rat hepatocyte plasma membrane proteins.

Rats were fed the peroxisome proliferator ciprofibrate (0.025%), and the effects on the expression, modification, and localization of seven domain-specific integral proteins of the rat hepatocyte plasma membrane were assessed using a combination of immunoblotting, -precipitation, and -fluorescence. Ciprofibrate caused the down-regulation of five of the plasma membrane proteins (the epidermal growth factor receptor, the asialoglycoprotein receptor, HA 321, HA 4, and dipeptidylpeptidase IV) and induced the expression of a more basic, lower-Mr isoform of the basolateral plasma membrane protein CE 9. Pulse labeling, chemical deglycosylation, and 125I-wheat germ lectin blotting suggested that the ciprofibrate-induced isoform of CE 9 differed in the posttranslational modification of its oligosaccharides and contained more sialic acid. These changes in hepatocyte surface differentiation were first observed between Days 1 and 5 on the ciprofibrate-containing diet, coincident with other aspects of the pleiotropic response of the hepatocyte to peroxisome proliferators, e.g., the induction of the Mr 78,000 peroxisome proliferation-associated protein. The effects were reversed within 2-3 weeks upon removal of ciprofibrate. The three other peroxisome proliferators tested, di(2-ethylhexyl)phthalate, clofibrate, and Wy-14,643, were found to exert most of these same effects on the expression and modification of the hepatocyte plasma membrane proteins, but the compounds differed in relative potency. The ciprofibrate-induced decreases in the concentrations of the epidermal growth factor receptor, the asialoglycoprotein receptor, HA 321, and HA 4 were similar to the selective down-regulation of these proteins observed transiently during the period of hepatocyte proliferation following two-thirds hepatectomy. Other compounds frequently used in studies of liver enzyme induction and carcinogenesis, the antioxidants ethoxyquin and butylated hydroxyanisole and the liver tumor promoter phenobarbital, were not as effective as ciprofibrate or two-thirds hepatectomy at causing the down-regulation of these proteins. The induction of the lower-Mr isoform of the basolateral plasma membrane protein CE 9 was not observed following two-thirds hepatectomy or upon the feeding of the antioxidants or phenobarbital but was specific to the feeding of the peroxisome proliferators.