Reduced peripheral activity leading to hepato-preferential action of basal insulin peglispro compared with insulin glargine in patients with type 1 diabetes.

AIMS: Basal insulin peglispro (BIL), a novel PEGylated basal insulin with a large hydrodynamic size, has a delayed absorption and reduced clearance that prolongs the duration of action. The current study compared the effects of BIL and insulin glargine (GL) on endogenous glucose production (EGP), glucose disposal rate (GDR) and lipolysis in patients with type 1 diabetes. MATERIALS AND METHODS: This was a randomized, open-label, four-period, crossover study. Patients received intravenous infusions of BIL and GL, each at two dose levels selected for partial and maximal suppression of EGP, during an 8 to 10 h euglycemic clamp procedure with d-[3-3 H] glucose. RESULTS: Following correction for equivalent human insulin concentrations (EHIC), low-dose GL infusion resulted in similar EGP at the end of the clamp compared to low-dose BIL infusion (GL/BIL ratio of 1.03) but a higher GDR (GL/BIL ratio of 2.42), indicating similar hepatic activity but attenuated peripheral activity of BIL. Consistent with this, the EHIC-corrected GDR/EGP at the end of the clamp was 1.72-fold greater for GL than BIL following low-dose administration. At the lower dose of BIL and GL (concentrations in the therapeutic range), BIL produced less suppression of lipolysis compared with GL as indicated by free fatty acid and glycerol levels at the end of the clamp. CONCLUSIONS: Compared with GL, BIL restored the hepato-peripheral insulin action gradient seen in normal physiology via its peripherally restricted action on target tissues related to carbohydrate and lipid metabolism.

Regulation of receptor tyrosine kinase signaling by endocytic trafficking.

Activated receptor tyrosine kinase (RTK) receptors are rapidly internalized and eventually delivered to the lysosomes. Although ligand-induced endocytosis was originally thought to be a mechanism of receptor inactivation, many studies suggest that receptors remain active within endosomes. This review discusses the role that internalized signaling complexes may play in different RTK systems including recent data on how ubiquitination may regulate this process. In general, it appears that some receptor systems have evolved to enhance endosomal signaling, as is the case for TrkA and NGF. In contrast, the insulin receptor system appears to limit the extent of endosomal signaling. The EGFR system is the intermediate example. In this case, some signals are specifically generated from the cell surface while others appear to be generated from within endosomes. This may act as a mechanism to produce ligand-specific signals. Thus, trafficking could play diverse roles in receptor signaling, depending on the specific cell and tissue type.

Human mammary epithelial cells rapidly exchange empty EGFR between surface and intracellular pools.

Binding of ligand to the epidermal growth factor receptor (EGFR) initiates a series of processes including activation of the intrinsic EGFR tyrosine kinase, receptor autophosphorylation, and the assembly of active signaling complexes at the plasma membrane. Concomitantly, receptor trafficking is initiated, and the receptor is ultimately delivered to the lysosome, where it is degraded. Virtually all studies on EGFR trafficking have used fibroblasts and transformed cells. Because EGFR exerts a potent effect on the physiology of epithelial cells, we examined the regulation of EGFR activity and trafficking in nontransformed human mammary epithelial cells (HMEC). We found that HMEC that displayed a luminal phenotype were largely unresponsive to EGF and maintained a majority of their EGFR at the cell surface. In contrast, HMEC with a basal phenotype were highly responsive to EGF and, at steady state in the absence of exogenous ligand, distributed empty EGFR into intracellular pools. Maintenance of the intracellular pools was a direct consequence of specific and rapid endocytosis of the empty EGFR. The trafficking pattern was EGFR specific, used coated pits, and did not require receptor tyrosine kinase activity. Such an mechanism redistributes EGFR signaling potential among different membrane domains and into vesicles with unique biochemical microenviroments. In addition, our data show that EGFR endocytosis can be regulated in the absence of ligand binding and receptor activation in a cell-type-specific manner.

Removal of the membrane-anchoring domain of epidermal growth factor leads to intracrine signaling and disruption of mammary epithelial cell organization.

Autocrine EGF-receptor (EGFR) ligands are normally made as membrane-anchored precursors that are proteolytically processed to yield mature, soluble peptides. To explore the function of the membrane-anchoring domain of EGF, we expressed artificial EGF genes either with or without this structure in human mammary epithelial cells (HMEC). These cells require activation of the EGFR for cell proliferation. We found that HMEC expressing high levels of membrane- anchored EGF grew at a maximal rate that was not increased by exogenous EGF, but could be inhibited by anti-EGFR antibodies. In contrast, when cells expressed EGF lacking the membrane-anchoring domain (sEGF), their proliferation rate, growth at clonal densities, and receptor substrate phosphorylation were not affected by anti-EGFR antibodies. The sEGF was found to be colocalized with the EGFR within small cytoplasmic vesicles. It thus appears that removal of the membrane-anchoring domain converts autocrine to intracrine signaling. Significantly, sEGF inhibited the organization of HMEC on Matrigel, suggesting that spatial restriction of EGF access to its receptor is necessary for organization. Our results indicate that an important role of the membrane-anchoring domain of EGFR ligands is to restrict the cellular compartments in which the receptor is activated.

Structure of a protein photocycle intermediate by millisecond time-resolved crystallography.

The blue-light photoreceptor photoactive yellow protein (PYP) undergoes a self-contained light cycle. The atomic structure of the bleached signaling intermediate in the light cycle of PYP was determined by millisecond time-resolved, multiwavelength Laue crystallography and simultaneous optical spectroscopy. Light-induced trans-to-cis isomerization of the 4-hydroxycinnamyl chromophore and coupled protein rearrangements produce a new set of active-site hydrogen bonds. An arginine gateway opens, allowing solvent exposure and protonation of the chromophore's phenolic oxygen. Resulting changes in shape, hydrogen bonding, and electrostatic potential at the protein surface form a likely basis for signal transduction. The structural results suggest a general framework for the interpretation of protein photocycles.

Endocytosis and lysosomal targeting of epidermal growth factor receptors are mediated by distinct sequences independent of the tyrosine kinase domain.

Ligand-induced internalization of the epidermal growth factor receptor (EGFR) leads to accelerated receptor degradation. Two models have been proposed to explain this. In the first model, induced internalization expands the intracellular pool of receptors, leading to enhanced lysosomal targeting. The second model proposes that activation of intrinsic receptor kinase activity induces inward vesiculation of endosomes, thus interrupting receptor recycling. To test these models, we created EGFR mutants that lack the conserved tyrosine kinase domain, but retain different parts of the distal carboxyl terminus regulatory region. Mutants lacking all distal regulatory sequences underwent slow internalization (0.02 min-1) and turnover (t1/2 approximately 24 h), similar to unoccupied, holo-EGFR. Mutant receptors that lacked the kinase domain, but retained the entire distal regulatory domain, were constitutively internalized and targeted to lysosomes, even in the absence of EGF. The turnover of these receptors (t1/2 approximately 11 h) was similar to that of occupied, kinase-active holo-EGFR (t1/2 approximately 9.5 h). These results show that receptor tyrosine kinase activity is not required for the targeting of EGFR to lysosomes. Receptor mutants which expressed previously identified endocytic sequences underwent rapid internalization. Unexpectedly, enhanced turnover of EGFR mutants required additional sequences located between residues 945 and 991 in the holo-EGFR. Thus, internalization and lysosomal targeting of EGFR are separate processes mediated by distinct sequences. Our results indicate that induced internalization is necessary, but not sufficient, for enhanced EGFR degradation. Instead, down-regulation requires exposure of previously cryptic internalization and lysosomal targeting sequences. Occupied EGFR thus appear to be handled by the endocytic machinery in the same fashion as other constitutively internalized or lysosomally targeted receptors.

Complete chemical structure of photoactive yellow protein: novel thioester-linked 4-hydroxycinnamyl chromophore and photocycle chemistry.

The unique ability of photoactive proteins to capture and use energy from a photon of light depends on the chromophore, its linkage to the protein, and the surrounding protein environment. To understand the molecular mechanisms by which a chromophore and protein interact to undergo a light cycle, we are studying photoactive yellow protein (PYP), a 14-kDa water-soluble photoreceptor from Ectothiorhodospira halophila with a photocycle similar to that of sensory rhodopsin. Here, we report the cloning and sequencing of the pyp gene and the chemical identification of both the chromophore and its covalent linkage to the protein. Elemental composition data from high-resolution mass spectrometry of a proteolytically derived chromopeptide, pH titrations and UV-visible spectroscopy of the protein-bound and chemically released chromophore, and fragmentation mass spectrometry of the liberated chromophore amide were combined with results from the 1.4-A-resolution protein crystal structure to identify the chromophore in PYP as a 4-hydroxycinnamyl group covalently bound to the sole cysteine residue via a thioester linkage. While 4-hydroxycinnamate is a metabolic product of the phenylpropanoid pathway and a key molecule in plant stress response, this is the first report of covalent modification of a protein by this group. In the dark (yellow) state of PYP, the protein stabilizes the chromophore as the deprotonated phenolate anion. By combining our biochemical characterization of the chromophore with other published observations, we propose a chemical basis for the photocycle: following the initial absorption of a photon, the photocycle of PYP involves protonation of the chromophore to a neutral phenol form corresponding to the observed photobleached intermediate.

Maternal psychiatric disorders in pediatric inflammatory bowel disease and cystic fibrosis.

The mothers of 72 children and adolescents with inflammatory bowel disease (IBD) and 44 mothers of children and adolescents with cystic fibrosis (CF) were given A-SADS interviews. Fifty-one percent of IBD mothers and 41% of cystic fibrosis mothers had a lifetime history of depression. More IBD than CF mothers had a history of suicide gestures or attempts, and were more likely to have a history of obsessive compulsive disorder. However, CF mothers were more likely to have experienced panic attacks.

Correlates of depression in new onset pediatric inflammatory bowel disease.

Of thirty six children with new-onset inflammatory bowel disease given a Kiddie-SADS interview, five children were depressed and ten had some depressive symptoms. Depressed children had less severe illness, and were more likely to have a maternal history of depression, more life events, and families characterized by less cohesion and more conflict.

Ruptured abdominal aortic aneurysm: a community experience.

A retrospective review of a community experience with ruptured abdominal aortic aneurysm (AAA) identified 117 cases from five community hospitals over a 12-year period. There were 90 men (77%) and 27 women (23%), ranging in age from 49 to 87 years. In the initial 6 years, the majority of operations were performed by general surgeons, with a survival rate of 35%. In the latter 6 years, most operations were performed by vascular surgeons, with a survival rate of 61%. For the entire period, patient survival with vascular surgeons was significantly greater than that with general surgeons (P < 0.05). Preoperative risk factors assessed in terms of their impact on survival were similar to those of previously reported series. Patients in the latter half of the study were older (72.8 versus 69.9 years), with survivors requiring a longer length of hospital stay than those in the initial 6-year period (34.5 versus 22.5 days), indicative of the changing patient population and having diagnostic-related group implications. There was also increased reliance by emergency room physicians on computed tomography for diagnosis, with increased survival rates. Misdiagnosis (patient sent home or admitted with incorrect diagnosis) occurred in 12 patients with no deleterious impact on survival. Some 15 patients with known AAA who either refused surgery for medical reasons or were considered to be at excessive risk for elective resection had a survival rate of 33%, indicating a definite potential for survival. It is concluded that there is continuing evolution of care for patients with ruptured AAA in the community setting.(ABSTRACT TRUNCATED AT 250 WORDS)

Parent and peer attachment in early adolescent depression.

Insecure attachment relations have been theorized to play a significant role in the development of depressogenic modes of adaptation and to thus form a vulnerability factor for the emergence of depressive disorder in children. This study examined security of parent and peer attachment among four groups of early adolescents: clinically depressed, nondepressed psychiatric controls, nonpsychiatric controls, and adolescents with resolved depression. Depressed adolescents reported significantly less secure parent attachment than either of the control groups, and less secure peer attachment than the nonpsychiatric control group. Attachment security of adolescents with resolved depression was on a par with the nonpsychiatric control group. Among all psychiatric patients, security of attachment to parents was negatively correlated with severity of depression according to interview and self-report ratings. Less secure attachment to parents, but generally not to peers, was also related to more maladaptive attributional styles, presence of separation anxiety disorder, and history of suicidal ideation.

Fluoride accumulation and bone strength in wild black-crowned night-herons.

Fluoride was measured in femurs of black-crowned night-herons (Nycticorax nycticorax) living adjacent to a phosphate processing complex near Pocatello, Idaho. Fluoride (ash wt.) in femurs ranged from 540 micrograms/g to 11,000 micrograms/g and increased (P = 0.0001) with age, but with no difference (P = 0.80) between sexes. Adult males (greater than or equal to 4 years) contained 5,409 micrograms/g compared to 6,042 micrograms/g for adult females. The tibiotarsus (= tibiae in text) increased in diameter with age (P = 0.015) in this study; fluoride was nearly related (P = 0.065) to the increase. As the diameter increased with age, wall thickness decreased (P = 0.011) suggesting excessive internal bone resorption, but fluoride concentrations were not implicated in the relationship (p = 0.64). The apparent increase in diameter and decrease in wall thickness may have partially neutralized each other's effects on strength. Although significantly higher concentrations of fluoride were present in adults than in Third Year herons, no significant change in bone strength (maximum load or modulus of rupture) was detected between the two age classes, but three of the four comparisons showed adults with less strength (i.e., a hint of diminished strength with age). The tibiae of Hatch Year birds were significantly weaker than documented in older age classes, but incomplete growth was thought responsible. The strong relationship between age and fluoride concentrations reduced our ability to separate a "fluoride effect" from an "age effect." Other authors believed fluoride was responsible for an increase in bone diameter and the fluoride residues encountered in adults were within the range indicative of poisoning in cattle.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimal grafting methods for the small abdominal aorta.

Aortobifemoral bypass procedures were performed on 42 patients with the hypoplastic aortoiliac syndrome over an eight year period. During the first five years, 22 patients had either a 13 X 6.5 mm or a 14 X 7 mm knitted Dacron graft and served as historic controls for a second group of 20 others who had 14 X 7 mm grafts of expanded polytetrafluoroethylene (PTFE), during the last three years of the study. Although the two groups were neither concurrent nor randomized, they were nonetheless well matched with regard to age, sex, risk factors, indications for operations, distribution and severity of distal occlusive disease, and type of distal anastomosis. There was only one early graft limb occlusion in the PTFE group after a median follow-up of 14 months, compared to nine occlusions and one amputation in the Dacron group after a median follow-up of 26.2 months. The cumulative patency of the PTFE grafts was 95% at four years compared to only 52% for Dacron (p = 0.11). If the single postoperative occlusion which occurred in each group is excluded, then the difference in patency rates bordered on statistical significance (p = 0.06). Inclusion of the profunda femoris orifice in the distal anastomosis was also found to be an important factor contributing to long-term patency which approached statistical significance (p = 0.075). The configuration of the proximal aortic anastomosis (end to end vs. end to side), or the concomitant use of lumbar sympathectomy or the postoperative infusion of Dextran did not have a statistically significant influence on patency.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between serum and salivary cortisol levels in depressed and nondepressed children and adolescents.

Serum and saliva cortisol levels were determined in 34 depressed and nondepressed children and adolescents. The serum and salivary measures were highly correlated. The measurement of salivary cortisol may be a useful noninvasive technique in the study of cortisol secretion patterns in young subjects.

Complications after use of the Angelchik antireflux prosthesis.

We report two cases of severe complications after insertion of an Angelchik antireflux prosthesis. In one case, the prosthesis migrated into the pelvis. In the other, severe dysphagia resulted from its insertion. In both instances, surgical removal was required. The high incidence of complications following the use of this prosthesis would suggest that other safer antireflux operations should be the preferred form of therapy.

Manic psychosis in childhood. Report of two cases.

Two cases of acute manic psychosis in prepubertal children are described. Mania in adolescence has been described with increasing frequency, but the existence of childhood-onset mania remains controversial. That may be attributed to the presence of schizophreniform features in mania in young individuals. The management and prognosis of juvenile-onset mania are discussed.

Swallowing and the organization of sucking in the human newborn.

The effect of varying amount and concentration of a sucrose solution on swallowing and sucking activity of newborns was studied. Swallowing activity increased relative to the number of sucks, and sucking rate slowed as amount and concentration of sucrose was increased. Sucking interresponse times associated with swallowing were approximately 200 msec longer than interresponse times not associated with swallows. The results are discussed in relation to previous studies of sucking in the newborn.