Ondansetron with propofol reduces the incidence of emesis in children following tonsillectomy.

PURPOSE: This study tested the hypothesis that the antiemetic effects of a combination of ondansetron and propofol were superior to propofol alone in children undergoing tonsillectomy surgery. METHODS: A prospective, randomized, double-blind, placebo-controlled study design was employed. Young children underwent mask induction with halothane, nitrous oxide and oxygen and then had i.v. access established: older children had i.v. induction with propofol. All patients received 0.3 mg x kg(-1) mivacurium and 2-4 microg x kg(-1) fentanyl i.v. and 30 mg x kg(-1) acetaminophen pr to a maximum dose of 650 mg. Following induction, patients received either 100 microg x kg(-1) ondansetron or placebo. Anaesthesia was maintained with 120-140 microg x kg(-1) x min(-1) propofol, nitrous oxide and oxygen to maintain vital signs within 20% of baseline. After surgery, in all patients the tracheas were extubated in the operating room without use of neuromuscular reversing agents. Episodes of emesis were recorded by PACU nurses for four to six hours. A telephone interview on the following day was also used for data recovery. Groups were compared in relation to age using the Mann-Whitney test, and with respect to sex and number of episodes of vomiting using the Fisher Exact Test. RESULTS: Three of the 45 patients who received ondansetron vomited (6.7%), whereas 10 of the 45 patients who received placebo vomited (22.2%). (P = 0.035) CONCLUSION: Ondansetron in a dose of 100 microg x kg(-1), when combined with propofol for children undergoing tonsillectomy reduced the incidence of postoperative vomiting to very low levels.

The absolute neutrophil count: is it the best indicator for occult bacteremia in infants?

Occult bacteremia affects approximately 5% of febrile children ages 2 to 36 months. Many physicians empirically treat children who have a temperature higher than 39 degrees C, a white blood cell (WBC) count of more than 15.0 x 10(9)/L, and no focus of infection with antibiotics. We undertook this investigation to better define predictive indicators for bacteremia. Specifically we were concerned with determining whether the absolute neutrophil count (ANC) is a better diagnostic indicator than the total WBC count and whether the manual differential (which includes a band cell count) is necessary or helpful. Three separate groups of patients aged 2 to 36 months were assessed retrospectively. Group A consisted of febrile children (temperature, > 39 degrees C) who had positive blood cultures (50 patients). Group B included febrile children (temperature, > 39 degrees C) who had negative blood cultures (59 patients). Group C, nonfebrile children admitted to the hospital was the control group (61 patients). The ANC and the total WBC count were significantly higher in group A than in group B. Although they were equally sensitive, the ANC was more specific than the total WBC count. Band cell counts of greater than 10% and the percentage of total neutrophils also were greater in group A than in group B. The values for group C were not significantly different from those for group B. Although a total WBC count of 15.0 x 10(9)/L is currently used to identify children at risk for occult bacteremia, the ANC seems to be as sensitive an indicator and may be more specific. Our study demonstrated that (1) the WBC count is a good indicator of occult bacteremia, (2) the ANC is as sensitive as the WBC count and may be more specific, (3) automated ANCs are comparable to manual ANCs, and (4) the band cell count is insensitive as an indicator and does not add any predictive value.

The incidence of acute and remote seizures in children with intraventricular hemorrhage.

Seizures are a well-known complication of intraventricular hemorrhage (IVH) in premature infants; however, the rate at which they occur is not known. The authors decided, therefore, to investigate both the incidence of acute and remote seizures in infants with IVH and the association with the grade of hemorrhage. One hundred and three infants with IVH were identified and their records were reviewed for acute seizures, remote seizures, and associated morbidity and mortality. The average gestational age of these infants was 29 weeks (range, 23-40 weeks). Of the 103 infants, 32 (31%) developed grade 4 IVH; 19 (18%), grade 3 IVH; and 52 (50%), grades 1 and 2 IVH. Seventeen (17%) patients had acute seizures during their first month of life. Six of the 61 patients (10%) who survived the neonatal period and for whom follow-up data were available had remote seizures. Infants with grade 4 IVH had significantly more acute seizures than infants with grades 1 and 2. In this cohort, only infants with grades 3 and 4 IVH developed remote seizures. Furthermore, among infants with grade 4 IVH acute seizures were a significant risk factor for development of remote seizures. The use of long-term antiepileptic drug therapy in neonates with a history of acute seizures is not established. These results suggest that antiepileptic drug therapy beyond the neonatal period should be reserved for infants with grade 4 IVH with history of acute seizures.

Localization of procollagen I in the lysosome/endosome system of human fibroblasts.

A significant amount of newly synthesized collagen is degraded intracellularly rather than secreted, but there is controversy about whether this process occurs in the lysosomes. We addressed this problem using confocal microscopy and immunofluorescence imaging to study the distribution of procollagen I in the Golgi and the lysosome/endosome system of cultured human fibroblasts. Cells were incubated under basal conditions and then permeabilized and exposed to fluorescently tagged probes for procollagen, Golgi markers (Helix pomatia binding protein or beta-coatamer protein), and lysosome/endosome markers (cathepsin B or LAMP-2). Strong signals for procollagen codistributed with the Golgi and lysosome/endosome markers. Of note, many structures were positive for procollagen and lysosome/endosome markers but not for Golgi markers. When cells were incubated with the proline analog cis-hydroxyproline, which inhibits correct triple helix formation and increases intracellular degradation, the amount of procollagen codistributing with the lysosome/endosome markers increased greatly. Similar results were obtained in I-cells, which do not have functioning lysosomal hydrolases. These findings strongly indicate that the lysosome/endosome system participates in the intracellular degradation of newly synthesized procollagen and that trafficking of procollagen to the lysosome/endosome system does not depend on the cells having active lysosomal hydrolases. We present a model that integrates our findings with other work and resolves inconsistencies in the literature. This model postulates the existence of three separate degradation paths for newly synthesized procollagen. In addition to the endosome/lysosome system, degradation also takes place in the proximal region of the secretory pathway such as the endoplasmic reticulum, cis-Golgi network, or cis-Golgi and in a distal region of the secretory pathway such as the trans-Golgi or trans-Golgi network.

Predictors of weight loss in children with attention deficit hyperactivity disorder treated with stimulant medication.

OBJECTIVE: A retrospective study was undertaken to examine predictors of weight loss in children with attention deficit hyperactivity disorder (ADHD) treated with stimulant medication. DESIGN: Children diagnosed with ADHD and treated with methylphenidate hydrochloride (MPH) or dextroamphetamine sulfate (DEX) for at least 5 months were identified by retrospective chart review. Analyses were performed on 32 children (29 boys) treated with MPH and 28 children (25 boys) treated with DEX. Variables examined included pretreatment weight, pretreatment body mass index (BMI), duration of treatment, total daily dose, and weight-adjusted dose. RESULTS: The MPH-treated group was initially seen at a mean age (+/- standard deviation) of 7.5 years (+/- 2.1). Mean duration of follow-up was 11.2 months (+/- 3.8). Mean total daily dose was 25.5 mg (+/- 8.2) and mean weight-adjusted dose was 1.0 mg/kg/day (+/- .5). Mean change in weight as expressed in z scores was -0.4 SD (+/- .7). The DEX-treated group was initially seen at a mean age of 9.3 years (+/- 2.7). Mean duration of follow-up was 10.8 months (+/- 4.1). Mean total daily dose was 14.9 mg (+/- 8.2) and mean weight-adjusted dose was .5 mg/kg/day (+/- .3). Mean change in weight was -0.6 SD (+/- .6). Although the MPH group was younger than the DEX group, there were no between-group differences in weight or height either before treatment or at follow-up when adjusted for age and gender. Using linear regression analysis, significant correlations were found between pretreatment weight and change in weight for both the MPH-treated group (r = .63) and the DEX-treated group (r = .47). Stepwise multiple-regression analysis indicated that pretreatment age, weight-adjusted dose, and duration of follow-up did not significantly contribute to the variance in change of weight for either the MPH- or DEX-treated groups. To adjust for height, changes in BMI were also analyzed. For both medication groups, no differences were found between the heavier (BMI > or = 50%) and thinner (BMI < 50%) children in duration of follow-up, total daily dose, or weight-adjusted dose. When the medication groups were combined, a greater proportion of heavier children experienced a decrease relative to their predicted BMI compared with thinner children (80% vs 52%). Comparisons of mean BMI slopes revealed that the heavier group experienced an absolute decrease in their BMI (-.139) in contrast to the thinner group (+.014) (t = 2.70). CONCLUSIONS: Pretreatment weight, adjusted for age, gender, and height, is a significant predictor of weight loss in children with ADHD treated with either MPH or DEX. In contrast, pretreatment age, duration of treatment, and weight-adjusted dose were not found to be significant predictors.

The value of brain imaging in children with headaches.

OBJECTIVE: To determine the value of performing computed tomography (CT) on magnetic resonance imaging (MRI) studies in children with chronic headaches. BACKGROUND: Headache is a common complaint in children. With the proliferation of brain imaging centers and the increasing patient demand for CT or MRI studies, brain imaging has become widely used to evaluate headaches. METHODS: A retrospective chart review was conducted of all children referred to the pediatric neurology clinic for evaluation of headaches over a 2-year period. Charts were reviewed for headache characteristics, clinical indications for performing CT and MRI studies, and imaging results. Particular attention was paid to evidence of brain tumors, vascular anomalies, or hydrocephalus. RESULTS: A total of 133 records were studied. Subjects ranged in age from 3 to 18 years. Most patients were diagnosed as having either vascular migrainous headaches (52%) or chronic tension headaches (21%). Other headache diagnoses were mixed tension-migraine, psychogenic, and post-traumatic. Headaches were unclassified in 25 patients (19%). Seventy-eight patients (59%) had brain imaging: 45 had MRI, 27 had CT, and 6 patients had both. In most cases, brain imaging studies were performed in patients with atypical headache pattern, presence of neurologic abnormalities during the headache, general symptoms (ie, weight loss or fatigue), or because of parents' or doctors' concerns about brain tumors. Cerebral abnormalities were found on brain imaging in four patients, but none indicated the presence of a treatable disease and all were deemed unrelated to the presenting complaint. Our findings of no relevant abnormalities in a series of 78 brain imaging studies indicate that the maximal rate at which such abnormalities might appear in this population is 3.8%. CONCLUSIONS: These results indicate that brain imaging studies have very limited value in evaluating headaches in pediatric patients without clinical evidence of an underlying structural lesion.

Control of collagen deposition in mammalian lung.

This review considers the mechanisms controlling collagen deposition in mammalian lung in five different states: normal development, fibrosis, erosion, pneumonectomy, and the steady state. Deposition is the net result of positive and negative processes. The major positive processes are control of cell number and type, regulation of transcription and translation, post-translational modifications, fibril formation, and covalent cross-linking. The negative mechanisms are intracellular degradation, collagenase-mediated degradation, and phagocytosis, and they are integral to the life cycle of collagen. Cytokines and growth factors have many and complex effects on all the processes that constitute collagen metabolism. Interleukin-1 and tumor necrosis factor-alpha can either stimulate or inhibit collagen accumulation, presumably depending on the immediate environment. Interleukin-6 inhibits collagen degradation, and gamma-interferon inhibits collagen production. Platelet derived growth factor and fibroblast growth factor have powerful mitogenic effects on connective tissue cells in lung, and can also affect collagen production directly. Transforming growth factor-beta activates a battery of processes that uniformly contribute to accumulation of collagen. Transforming growth factor-beta may be the "master switch" for a fibrotic program in lung. Therapeutic approaches to controlling lung fibrosis by manipulating cytokine levels are promising. Prostaglandin E has uniformly negative effects on net collagen accumulation and may play a central role in an erosion program.

An audit of adverse events in children sedated with chloral hydrate or propofol during imaging studies.

We examined records of sedations provided by the paediatric anaesthesiology staff for 455 children (ages 1 mo-17 yr) undergoing MRI or CT scans at our institution over a twelve-month period with regard to the monitoring of adverse events: excessive sedation, agitation, vomiting, hypoxaemia, and major airway compromise. One hundred-and-thirty-one patients (29%) received chloral hydrate; 324 patients (71%) received propofol. All patients were monitored with continuous noninvasive pulse oximetry and received supplemental oxygen via nasal cannulae. Of the patients who received chloral hydrate, 64 (49%) were over one year of age; of the patients who received propofol, 318 (98%) were one year of age or older. In the chloral hydrate group, 23 patients (19%) were deemed excessively sedated and four patients (3%) were agitated; no patients in the propofol group experienced any of the adverse outcomes reviewed. Furthermore, no patients in either group had significant airway compromise and none was admitted to the hospital as a result of the sedation.

Propofol reduces the incidence of vomiting after tonsillectomy in children.

We compared the effect of a propofol-based anaesthetic to an isoflurane-based anaesthetic on the incidence of postoperative vomiting in children following tonsillectomy. Thirty-nine children were enrolled in the study and randomized to receive one of the proposed anaesthetics. All patients underwent a mask induction with halothane, nitrous oxide, and oxygen. Intravenous access was established and all children received fentanyl (2-4 micrograms.kg-1) i.v., mivacurium (0.3 mg.kg-1) i.v. and acetaminophen (10-15 mg.kg-1) p.r. Following tracheal intubation, patients received either isoflurane (0.8-1.6%) or propofol (120-180 micrograms.kg-1 min-1) i.v. with nitrous oxide 70%/oxygen 30% for maintenance of anaesthesia. Vital signs were maintained within 20% of baseline. All patients were extubated in the operating room. PACU nursing staff recorded episodes of vomiting for 4-6 h prior to discharge. A telephone interview the following day was also used for data recovery. Age, sex, and duration of the procedure were not significantly different between the two study groups. Of 19 patients who received propofol, four vomited (21%); in contrast, of the 20 patients who received isoflurane, 11 vomited (55%). This difference is significant (P = 0.048 two-tailed Fisher's Exact Test). These data suggest that using propofol for anaesthesia can diminish the incidence of vomiting following tonsillectomy.

Taurine prevents glucose-induced lipid peroxidation and increased collagen production in cultured rat mesangial cells.

Hyperglycemia is directly involved in the development of diabetic nephropathy. A high glucose concentration promotes membrane lipid peroxidation and stimulates collagen production in a variety of cultured cells. Taurine, a sulfur amino acid, is an endogenous antioxidant and antifibrotic agent. We tested whether taurine ameliorates the above effects of elevated ambient glucose on renal cells in vitro. Raising glucose concentration from 5.6 to 33.3 mM enhanced lipid peroxidation in rat mesangial cells, as assessed by malondialdehyde and conjugated diene content, and increased collagen production by 59%. Taurine prevented both glucose-induced effects in mesangial cells. In contrast, neither high glucose nor taurine, alone or in combination, affected lipid peroxidation or collagen production in MDCK or LLC-PK1 cells, derived from renal tubular epithelium. These results indicate that taurine may be a useful therapeutic agent to attenuate diabetic glomerulosclerosis.

Brefeldin A inhibits degradation as well as production and secretion of collagen in human lung fibroblasts.

Brefeldin A (BFA) inhibits protein secretion, collapses the Golgi complex into the endoplasmic reticulum (ER), causes redistribution of processing enzymes normally resident in the Golgi to the ER, and uncouples the proximal and distal regions of the secretory pathway. We used BFA to determine where intracellular degradation of newly synthesized collagen degradation occurs. In normal human fetal lung fibroblasts, BFA (50 ng/ml) completely blocked collagen secretion and reduced collagen production by two-thirds. In cells synthesizing collagen under normal conditions, intracellular degradation was about 16%; BFA (50 ng/ml) reduced degradation to less than 5%. In cells induced to synthesize structurally abnormal collagen (by incubation with the proline analog cis-hydroxyproline), degradation was approximately 33%; BFA reduced this level to less than 10%. When the y axes were scaled appropriately, the dose-response curves for collagen degradation +/- cis-hydroxyproline versus BFA concentration coincided. A pulse-chase experiment demonstrated that BFA did not inhibit hydroxylation of prolyl residues, a major posttranslational modification of collagen that occurs in the ER, and that inhibition of degradation was independent of inhibition of collagen synthesis. Immunofluorescence examination revealed that BFA redistributed Golgi glycoproteins to the ER. At the ultrastructural level, Golgi complex could not be found in fibroblasts exposed to BFA for 1 h; however, clusters of small vesicles were observed. A different structure, comprising one or two lamellae and resembling a partial Golgi complex, was observed in cells incubated with BFA for 6 h. This structure was adjacent to ER but far from the nucleus. In addition, the ER was devoid of ribosomes. The inhibition of intracellular collagen degradation by BFA indicates that collagen degradation does not occur in the ER. Rather, it suggests that collagen degradation occurs beyond the BFA block, perhaps in the trans-Golgi network.

Organization of intestinal epithelial cells into multicellular structures requires laminin and functional actin microfilaments.

Epithelial cell organization into multicellular structures is a critical biological process required for both organogenesis and repair following injury. The basement membrane and the cytoskeleton have important roles in this process; however, the functions of individual components of basement membrane and cytoskeleton are poorly understood. We used IEC-6 cells, a rat intestinal crypt cell line, grown on a three-dimensional gel of reconstituted basement membrane as a model system to determine which extracellular matrix and cytoskeletal components mediate intestinal epithelial cell organization. The cells entered the gel and formed hollow, tubular structures that resembled intestinal crypts. These structures were characterized by a single layer of polarized cells with apical tight junctions and microvilli on the luminal surface. Antiserum to laminin and the pentapeptide Tyr-Ile-Gly-Ser-Arg (which prevents cell attachment to laminin) inhibited this organization, but a control pentapeptide (Tyr-Tyr-Gly-Asp-Ala) and antiserum to collagen IV did not. Cytochalasin B, which interferes with actin microfilament polymerization, also inhibited organization of cells into multicellular structures, but vinblastine and Colcemid, which disrupt microtubules, and cycloheximide, which inhibits protein synthesis, did not. We conclude that organization of intestinal epithelial cells on a basement membrane into multicellular structures results from specific interactions between cells and laminin and requires intact actin microfilaments.

Collagen degradation in I-cells is normal.

There is evidence that lysosomal proteases mediate the intracellular degradation of structurally abnormal collagen. I-Cell disease (Mucolipidosis II) is characterized by marked deficiency of many lysosomal hydrolases, including the collagenolytic enzyme cathepsin B. The experiments reported here tested the hypothesis that degradation of abnormal collagen would be severely impaired in I-cells. Skin fibroblasts from 3 patients with I-cell disease were incubated with and without cis-hydroxyproline, a proline analog that causes structural abnormalities in collagen, and [14C]proline. The amount of [14C]hydroxyproline in a low molecular weight fraction relative to total [14C]hydroxyproline was used as a measure of intracellular collagen degradation. Levels of degradation were significantly higher in I-cells exposed to cis-hydroxyproline than in cells incubated without the analog. Similar data were obtained for normal human fetal lung fibroblasts incubated under the same conditions. Degradation of [125I]-epidermal growth factor was used to assess the functionality of the lysosomal pathway for protein degradation, and it was much lower in I-cells than in normal cells. It can be concluded that a completely functional complement of lysosomal enzymes is not necessary for structurally abnormal collagen to be degraded intracellularly; the data suggest that a nonlysosomal pathway exists.

Ammonium chloride inhibits basal degradation of newly synthesized collagen in human fetal lung fibroblasts.

The objective of this work was to characterize basal degradation of newly synthesized collagen in human fetal lung fibroblasts. Analysis of 22 separate determinations showed that in cells incubated under normal conditions, the level of intracellular degradation was normally distributed with a mean of 15.2% and a standard deviation of 2.6%. Within each experiment, however, the uncertainty (standard deviation) in determining degradation was very small, usually less than 1.5%. Consideration of the large variation between experiments and the ability of our analytic technique to detect small, but "statistically significant," differences between groups within the same experiment led us to formulate two criteria for determining whether degradation measured in cultures exposed to some agent differs in a "biologically significant" way from degradation measured in control cultures. These criteria were used to evaluate the effects of the following proteinase inhibitors on basal degradation: NH4Cl, which increases the pH of subcellular compartments that are normally acidic; and leupeptin and Na-p-tosyl-L-lysine chloromethyl ketone (TLCK), which are inhibitors of lysosomal cathepsins (B and L) that degrade collagen. NH4Cl (16 mM) lowered degradation to an extent that was both statistically and biologically significant, but neither leupeptin nor TLCK affected degradation. The effect of NH4Cl on degradation was independent of its inhibitory effects on production of collagen, protein, and ATP. These results suggest that basal degradation occurs in, or beyond, an acidic (i.e., NH4Cl-sensitive) but nonlysosomal compartment of the cell, and that NH4Cl inhibits processing within, or transport to, that compartment. This is the first report of an agent that inhibits basal degradation of newly synthesized collagen in soft tissue fibroblasts.

Effects of puromycin and hydroxynorvaline on net production and intracellular degradation of collagen in human fetal lung fibroblasts.

Amino acid substitutions in collagen that impair folding of the triple helix result in significant increases in intracellular degradation of newly synthesized collagen. We have studied the effects of agents that cause other kinds of defects in collagen: hydroxynorvaline, a threonine analog that interferes with association of pro-alpha chains; and puromycin, an antibiotic that causes premature release of nascent polypeptides. cis-Hydroxyproline and cycloheximide, whose effects on collagen synthesis and degradation have already been studied and reported, were employed as reference compounds. Human fetal lung fibroblasts were used in these experiments. All the agents inhibited total protein production, and all except cycloheximide inhibited percentage collagen production. Intracellular collagen degradation was increased in cultures exposed to puromycin, hydroxynorvaline, and cis-hydroxyproline, but not in cultures exposed to cycloheximide. These results suggest that pro-alpha chains that were either unassociated (due to hydroxynorvaline) or shortened (due to puromycin) were recognized as abnormal and degraded to the same extent as chains that contained cis-hydroxyproline. However, the increases in degradation could not account completely for the decreases in collagen production (except when cis-hydroxyproline was used at low concentrations). These findings indicate that, in addition to rendering newly synthesized procollagen molecules or partial polypeptide chains more susceptible to intracellular degradation, puromycin, hydroxynorvaline, and cis-hydroxyproline significantly inhibited collagen synthesis.

Intracellular degradation of newly synthesized collagen.

This chapter reviews recent work on intracellular degradation of newly synthesized collagen, the most abundant protein in the mammalian body. Approximately 10-20% of collagen synthesized by human fibroblasts under normal culture conditions is broken down rather than secreted; this is referred to as the basal level of degradation. It is not known where basal degradation occurs, but indirect evidence suggests a compartment of the secretory pathway, perhaps the Golgi complex. Intracellular degradation is increased when cells are induced to synthesize structurally abnormal collagen, either by incubation in the presence of amino acid analogs or because of mutations in collagen genes. There is evidence that lysosomal proteases mediate degradation of structurally abnormal collagen, however, recent work indicates that I-cells, which are genetically deficient in several lysosomal enzymes, can also degrade abnormal collagen. Modulation of the level of intracellular degradation can effect a change in net collagen production independently of changes in the rate of synthesis. Intracellular degradation of newly synthesized collagen has been observed in vivo, but it is not clear whether decreasing degradation contributes to increasing collagen production in fibrosis. This chapter concludes by relating collagen degradation to the more general phenomenon of intracellular degradation of newly synthesized secretory proteins, and by indicating directions for future work.

Effects of prostaglandin E1 on collagen production and degradation in human fetal lung fibroblasts.

We examined the effects of prostaglandin E1 on the production and degradation of collagen in human fetal lung fibroblasts. Percentage collagen production was determined by incubating confluent cultures for 6 h with [3H]proline and either [14C]glycine or [14C]leucine and measuring the relative amounts of radioactivity incorporated into collagenase-sensitive and collagenase-insensitive material. Percentage collagen degradation was determined by measuring hydroxy[14C]proline in a low-molecular-weight fraction relative to total hydroxy[14C]proline. Prostaglandin E1, when present at a concentration as low as 0.25 micrograms/ml, reduced net collagen production by a factor of one-half, from 8 +/- 2 to 4 +/- 1% (P less than 0.05). In contrast, the change in percentage degradation was relatively gradual, rising steadily from the control value of 15 +/- 2 to 33 +/- 2% at 4 micrograms/ml (P less than 0.05). The increase in degradation, while significant, could not account for the total decrease in collagen production. We conclude that prostaglandin E1 exerts its inhibitory effect on collagen production in two essentially independent ways: lowering the rate of synthesis and increasing intracellular degradation. However, the decrease in synthesis is greater than the increase in degradation.

Fetal type 2 pneumocytes form alveolar-like structures and maintain long-term differentiation on extracellular matrix.

We investigated the effects of reconstituted basement membrane (a crude extract of the Engelbreth-Holm-Swarm tumor) on type 2 pneumocyte differentiation during long-term culture. Cells were derived from mature 29 d fetal rabbits. Morphology was studied by light and electron microscopy. On thin gel, the cells initially segregated into clumps; they were cuboidal with apical microvilli and contained lamellar bodies, but dedifferentiated by 8 d. On thick gel, epithelial cells associated into spherical clusters surrounding a central lumen. These alveolarlike structures persisted at least 22 d. The cells were cuboidal and had lamellar bodies and intercellular tight junctions; they exhibited polarity, with apical microvilli facing the lumen, basally located nuclei, and gel matrix abutting the basal surface. In contrast, cells cultured on plastic formed colonies, then a monolayer, but dedifferentiated 5-7 d after plating. [14C]Acetate was used to label newly synthesized phospholipids. The amount of disaturated phosphatidylcholine (DSPC), expressed as a percentage of total phosphatidylcholine (PC), was used as an indicator of surfactant lipid production; percentage DSPC synthesized by cells cultured on thick gel did not change significantly, from 55 +/- 3 at 3 d, to 63 +/- 2 at 22 d in culture. DSPC synthesized by cells cultured on plastic decreased from 57 +/- 1% at 3 d to 45 +/- 2% at 22 d (p less than 0.001), which is consistent with the morphologic evidence of dedifferentiation. Synthesis of total PC compared with total phospholipid did not vary with either time in culture or substrate. This study emphasizes the importance of a complex extracellular matrix for maintenance of type 2 pneumocyte differentiation. The system should prove useful for studying the interaction of these cells with basement membrane, including the role of events occurring at the cell surface in modulating expression of a differentiated phenotype.

Kinetics of intracellular degradation of newly synthesized collagen.

The objective of this work was to determine the time dependence of the basal component of intracellular degradation of newly synthesized collagen. Chick embryo tendon fibroblasts were incubated with [14C]proline, and degradation was quantified by measuring hydroxy[14C]proline in a low molecular weight fraction. When cultures were pulse labeled for 15 min and then incubated under chase conditions for 105 min, the amount of degraded collagen attained a value equal to approximately 20% of the amount synthesized during the labeling period; the data were fit with a simple exponential function that had a 40-min rise time and a 12-min lag time. In continuously labeled cultures, the rates of collagen synthesis and secretion reached constant values within 15 and 45 min, respectively. Degradation products were first detected 6-9 min after collagen synthesis began and were transported out of the cells more rapidly than intact collagenous molecules; however, percent degradation increased slowly and did not reach a constant value even after 240 min of incubation. Since collagen degradation lags collagen synthesis, it follows that degradation is a posttranslational, rather than a cotranslational, process, and since degradation and secretion are kinetically distinguishable, it follows that they occur in parallel pathways. A simple nonlinear model for posttranslational processing of collagen is proposed.