Inhibition of prolidase activity by non-steroid antiinflammatory drugs in cultured human skin fibroblasts

201-Thallium (Tl) and (99m)Tc-sestamibi (SM) present different biological properties and kinetics, suggesting a complementary evaluation of mitochondrial and cell membrane functions with subsequent implications regarding myocardial injury mapping. To verify the usefulness of a dual isotopic approach in Q infarcted patients, 30 subjects were submitted at rest, within 5 days, to SM imaging, 4h-delayed Tl scans and echocardiography (ECHO). Left ventricle segmental uptake and wall motion were graded on a 3 points scale (0=absent to 2=normal) and compared on the basis of an 11 segments model. RESULTS AND DISCUSSION: 1) The analysis of SM normal segments demonstrated a strong concordance (97%) with Tl and ECHO, suggesting that both mitochondrial and cell membrane functions are preserved; 2) 49% of SM graded 0 segments were scored 1 by Tl and ECHO, suggesting a worse impairment of mitochondrial function with respect to cell membrane function; 3) approximately 55% of segments showing a reduced MIBI uptake were found normal using Tl, then an impaired mitochondrial but a normal cell membrane function could be hypothesized. 4) Tl provided a better estimation of the effective infarction size with respect to SM. CONCLUSIONS: The SM and Tl dual approach, allowing scintigraphic mapping of myocardial injury, seems to provide a useful tool for a complete evaluation of infarcted patients.     

Respiratory activity and growth of human skin derma fibroblasts

Symptom control is the goal of palliative irradiation. Approximately 1 month is required before symptomatic relief is accomplished with radiotherapy. However, many patients with cancer-related pain do not receive adequate analgesics, and opioids are often not prescribed until patients fail to respond to palliative irradiation. The presenting symptoms of 108 patients who were referred to a multidisciplinary clinic for bone metastases were evaluated with the Wisconsin Brief Pain Inventory (BPI). This validated instrument evaluates the severity of pain using a 0-10 scale; 10 represents the worst pain imaginable. The population comprised 65 men (60%) and 43 women whose ages ranged from 33 years to 81 years; median age was 55 years, and 69% of patients were less than 65 years of age. Despite the presence of metastatic disease, 21% of patients were working full-time outside the home, and 6% were employed part-time outside the home; 13% were homemakers. Only 17 patients (16%) were unemployed. The time since diagnosis ranged from 2 weeks to 23 years; the median time since diagnosis was 22 months, and 30% of patients had been diagnosed with the past 6 months. Pain was a presenting symptom in 74% (N = 80) of patients at diagnosis. At its worst, the pain was rated as severe (levels 7-10) by 78% and intolerable (level 10) in 22% of the patients in the 24 hr prior to the clinic appointment. On average, the pain was rated moderate to severe (levels 4-10) in 79% and severe in 23% of patients. Only 45% of patients experienced good relief from the prescribed analgesics, and 23% of patients indicated that the prescribed analgesics were ineffective. This survey demonstrates that bone metastases incur significant pain that is often undertreated with analgesics before antineoplastic therapy is administered.     

Insulin-like growth factor binding protein-3 is overexpressed in senescent and quiescent human fibroblasts

Cellular insulin-like growth factor binding protein-3 (IGFBP-3) mRNA and IGFBP-3 levels in conditioned medium were consistently higher in cultures of late passage normal (old) fibroblasts and prematurely senescent fibroblasts derived from Werner syndrome (WS) during quiescence induced by serum depletion and during the renewed growth ensuing after serum repletion, compared to cultures of early passage normal (young) fibroblasts. Molar ratios of IGFBP-3/IGF-II were always higher in senescent cultures and maintained a hierarchy of old > WS > young human diploid fibroblasts. Transfection into fibroblasts of the normal full-length IGFBP-3 cDNA in an expression vector resulted in a significant reduction in colony formation compared to cells transfected with an empty expression vector (no cDNA) or with IGFBP-3 cDNA altered by a 273 base pair (bp) deletion. Addition to old and young cultures of recombinant human IGFBP-3 and IGF-I at 1:1 or 5:1 molar ratios inhibited IGF-I-mediated DNA synthesis by approximately 70-80%. These data indicate that IGFBP-3 may play an important role in the quiescent and senescent growth arrest of HDF.     

Alkaline phosphatase activity in cultured skin fibroblasts from fibrodysplasia ossificans progressiva

Alkaline phosphatase activity in four strains of cultured skin fibroblasts obtained from a patient with fibrodysplasia ossificans progressiva was at the low normal range. The enzyme activity in normal fibroblasts significantly increased at late confluency. It appears that the high levels of alkaline phosphatase activity reported in biopsies of lesions are not genetically determined but are secondary events of local tissue reaction.     

Repeated mild heat shock delays ageing in cultured human skin fibroblasts

We used functional magnetic resonance imaging (fMRI) to monitor stimulus-selective responses of the human fusiform face area (FFA) and parahippocampal place area (PPA) during binocular rivalry in which a face and a house stimulus were presented to different eyes. Though retinal stimulation remained constant, subjects perceived changes from house to face that were accompanied by increasing FFA and decreasing PPA activity; perceived changes from face to house led to the opposite pattern of responses. These responses during rivalry were equal in magnitude to those evoked by nonrivalrous stimulus alternation, suggesting that activity in the FFA and PPA reflects the perceived rather than the retinal stimulus, and that neural competition during binocular rivalry has been resolved by these stages of visual processing.     

Action spectrum for UV-induced lipid peroxidation in cultured human skin fibroblasts

Lipid peroxidation was measured by release of thiobarbituric acid-reactive substances (TBARS) into the supernatant of cultured human skin fibroblasts. This process is triggered by ultraviolet A (UVA) and ultraviolet B (UVB) radiations. For UVA irradiances and irradiation times up to 40 W.m-2 and 90 min, respectively, the peroxidation response is linear and obeys the reciprocity law. Corresponding values for UVB are 12 W.m-2 and 30 min, respectively. The action spectrum of the peroxidation process shows a continuously increasing response from about 425 to 275 nm. Whereas the UVB to UVA effectiveness ratio lies in the range of 10(3) to 10(4) for most in vitro or in vivo UV-induced responses, the ratio is only 10 to 100 for the peroxidation process. Given the solar spectral distribution, solar UVA radiation is by far the most effective in triggering the peroxidation response.     

Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.     

Growth properties and growth factor responsiveness in skin fibroblasts from centenarians

Human fibroblast cultures, which have a finite replicative lifespan in vitro, are the most widely used model for the study of senescence at the cellular level. An inverse relationship between replicative capability and donor age has been reported in human fibroblast strains. We studied the growth capacity of fibroblast primary cultures derived from people whose lifespan was as closer as possible to the expected maximum human lifespan, i.e. people over one hundred. Our data suggest that outgrowth of fibroblasts from biopsies, growth kinetics at different population doubling levels, capability to respond to a classical mitogenic stimulus (such as 20% serum) and a variety of growth factors, were remarkably similar in fibroblasts from centenarians and young controls. On the whole, our data challenge the tenet of a simple and strict relationship between in vivo aging and in vitro proliferative capability of human fibroblasts, at least at the individual level.     

Insulin-like growth factor binding protein production in human follicular thyroid carcinoma cells

OBJECTIVE: The study investigated the value of using national or regional data bases to examine care in a specific hospital. DATA SOURCES: The following data sources were included: (1) the results of the 1992 HCFA analysis of the index hospital for patients hospitalized in fiscal year 1990; (2) the 1989 Medicare Provider Analysis and Review (MEDPAR) file; and (3) clinical information from bypass surgery patients in Wisconsin and from the index hospital. PRINCIPAL FINDINGS: The assessment of the mortality rates in the index hospital for all conditions combined and for CABG patients differed depending on what data base was used and how the data were analysed. The national data were most useful in establishing that the coding practices for all patients and the mortality rate for intra-aortic balloon patients differed between the index hospital and other hospitals. The regional clinical data base for bypass surgery patients was used to establish that the high mortality rates for intra-aortic balloon patients were due to patient selection. CONCLUSIONS: National claims data must be analysed carefully before applying results to an individual hospital. Even a careful analysis is more for raising questions about care at a specific hospital rather than for reaching definitive conclusions.     

Insulin-like growth factor I stimulates proliferation and Fas-mediated apoptosis of human osteoblasts

alpha-Tectorin is one of the major noncollagenous components of the mammalian tectorial membrane in the inner ear. We have mapped the gene encoding alpha-tectorin to mouse chromosome 9 and human chromosome 11 in a known region of conserved synteny. Human YAC clones containing alpha-tectorin have been identified, demonstrating physical linkage to the anonymous marker D11S925. This places alpha-tectorin within the genetic interval that contains both the human nonsyndromic autosomal dominant deafness DFNA12 and the proximal limit of a subset of deletions within Jacobsen syndrome. Thus both DFNA12 and the hearing loss in some cases of Jacobsen syndrome may be due to haploinsufficiency for TECTA.     

Prolidase deficiency in cultured human fibroblasts: biochemical pathology and iminodipeptide-enhanced growth

Prolidase deficiency is a rare autosomal recessive disorder characterized by iminodipeptiduria, severe skin ulcers, recurrent infections, and mental retardation. The enzyme prolidase hydrolyzes dipeptides containing C-terminal proline or hydroxyproline. We investigated the metabolic abnormality caused by prolidase deficiency in human cultured skin fibroblasts. These studies were undertaken to test biochemical hypotheses regarding the metabolic origins of the skin lesion occurring in this disease. Our results indicate that prolidase plays a major role in the recycling of dipeptide-bound proline. Control fibroblasts were able to use iminodipeptides in lieu of proline to sustain normal growth, whereas cells homozygous for the prolidase deficiency mutation were not. Proline derived from iminodipeptides diluted incorporation of radiolabeled extracellular proline into cellular protein in normal cells but not in mutant cells. Substitution of a prolidase-free medium for FCS did not affect the growth rate of control cell lines but increased the doubling time of prolidase-deficient cells by 19% (28% in the presence of iminodipeptides). Iminodipeptides added to control and mutant cells maintained in serum-free medium showed no adverse effects on protein synthesis. These results are consistent with a mechanism of biochemical pathology in which proline deprivation caused by the enzyme deficit is a primary cause of damage to skin cells. Prolidase regulation by product and substrate was studied. A 44% decrease in activity was observed in fibroblasts grown for 3 wk in proline-containing medium relative to proline-free medium. However, cells grown in medium in which iminodipeptides replaced proline showed no significant difference in prolidase activity.     

Human epidermal growth factor and the proliferation of human fibroblasts

The majority of the mRNA molecules in HeLa cells contain 1-2 residue(s) of m6Ap and one blocked, methylated 5' terminal "cap" structure. The hnRNA, which is longer than mRNA, contains both m6Ap and caps but 4-6 times as many m6Ap residues per chain. In addition, nuclear molecules contain T2 RNA ase-resistant, methyl-labeled oligonucleotides ("di-" and "tri-" nucleotides) which are not found in mRNA. Some of the dinucleotides may be precursors to the 2'-0-methylated nucleotides in the cap structures. These results are compatible with internal methylation of hnRNA molecules (both m6Ap and 2'-0-methyl) followed by hnRNA cleavage and the addition of the cap structure to generate at least some of the HeLa cell mRNA. It also appears that some hnRNA molecules, which are longer than most mRNA molecules, contain cap structures suggesting the derivation of some mRNA molecules from the 5' regions of hnRNA.     

All-trans-retinoic acid down-regulates elastin promoter activity elevated by ultraviolet B irradiation in cultured skin fibroblasts

OBJECTIVE: Little research has evaluated maternal experience with fetal pulse oximetry for fetal surveillance. The purpose of this study was to compare maternal perceptions of labor with intrapartal cardiotocography with or without fetal pulse oximetry in a research setting. METHODS: One hundred women with vaginal, vertex deliveries and uncomplicated fetal outcomes were enrolled. The study group was a subset of 50 mothers who had participated in a pulse oximetry trial. The control group of 50 mothers was monitored by cardiotocography only. Both groups were matched for age, parity, weeks of gestation, epidural anesthesia use, and duration of labor. A global measure of maternal perception of labor was established by experience with labor, general attitude toward monitoring devices, satisfaction with monitoring, nursing and medical care, and anxiety, each of which was evaluated separately. The mothers in the study group were also interviewed about aspects related to the fetal pulse oximetry research setting, such as information, movement restriction, discomfort, care, privacy, and safety. The questionnaires were based on a standardized rating scale model, and the interviews were conducted two to four days after delivery. The results were analyzed by chi-squared, paired t test, and ANOVA. RESULTS: No significant differences were observed between the study and control participants in any parameter concerning the maternal perception of labor. Mothers' experiences with pulse oximetry as assessed by interview was overwhelmingly positive. CONCLUSIONS: Fetal monitoring by pulse oximetry in a research setting did not affect maternal perceptions of labor. Mothers' experiences with pulse oximetry were highly positive, suggesting that research in fetal pulse oximetry need not compromise maternal perceptions of labor.     

Expression of growth factor mRNAs by human Tenon''s capsule fibroblasts

We determined the nucleotide sequences of Norwalk-like viruses in 10 PCR products from stool or oyster specimens obtained from four outbreaks of gastroenteritis in which shellfish was suspected as the cause in Shizuoka prefecture in Japan between 1987-94. The sequences were determined from nucleotide positions 4561-4852 (292 bp) in the polymerase region. Two types of sequences were detected. One (genotype 1) had 87% sequence homology with the prototype Norwalk virus, and the other (genotype 2) had 59% sequence homology. The sequences from isolates belonging to the same genotype were almost the same regardless of the year of isolation. Because sequences of 2 genotypes were detected in 2 of the 4 outbreaks, nested PCR was performed with genotype-specific primers to detect the presence of 2 genotypes in the same specimen. In 5 of 10 specimens, PCR bands were detected with both genotype-specific primers, indicating the coexistence of 2 genotypes in 1 specimen. We also detected two genotypes of Norwalk-like virus in an oyster from a sample implicated in one of the outbreaks which may provide direct evidence of oysters as the cause of the gastroenteritis.     

Insulin-like growth factor receptors and binding proteins

The insulin-like growth factor receptors are integral membrane proteins and demonstrate separate, but important effects on the regulation of cellular processes. The IGF-I receptor signals multiple cascades via its inherent tyrosine kinase activity. The IGF-II/M-6-P receptor on the other hand is primarily involved in targeting of enzymes to various subcellular compartments. In contrast, the insulin-like binding proteins are secreted by the cells and accumulate in the extracellular matrix or on the external surface of the cell. They are also involved in regulating cellular processes more indirectly. They modulate the interactions of the IGFs with their receptors, and in addition, may have some IGF-independent effects probably by direct interaction with integrin and other cell membrane receptor proteins. The recent studies, as outlined in this review, strongly suggest an important, if not essential role for the IGF system in normal physiology and disease states. The challenge now is to define the mechanisms involved in these effects. More studies are required to fully understand the post-receptor mechanism involved in IGF-I receptor signal transduction and the mechanisms whereby the IGFBPs exert their interesting effects. Understanding these mechanisms will enable investigators to create new therapeutic modalities for diseases that are affected by the IGF system.