High-speed gel-permeation chromatography of glycosaminoglycans: Its application to the analysis of heparan sulfate of embryonic carcinoma and its degradation products by tumor cell-derived heparanase

A high-speed gel-permeation chromatographic system for analyzing glycosaminoglycans which uses two 0.7 X 75-cm stainless-steel columns containing Fractogel (Toyopearl) TSK HW-55(S), was developed. Glycosaminoglycans were applied and eluted with a 0.2 M sodium chloride solution and monitored by ultraviolet absorption at 210 nm or radioactivity. The best resolution of glycans was obtained at 55 degrees C at a flow rate of 1.0 ml/min. Acidic and neutral glycans in the molecular weight (Mr) range 600-60,000 eluted within 45 min. A linear relationship was found between retention time and molecular weight using standard glycosaminoglycans, chitin oligosaccharides, and a porcine thyroglobulin glycoprotide. This system was used to analyze the heparan sulfate synthesized by PYS-2 embryonic carcinoma cells and the degradation products produced by incubating it with extracted glycosidases from metastatic B16 melanoma cells. The results indicated that B16 melanoma cells contain at least two different heparan sulfate degradative activities, one of which appears to be an endoglycosidase.     

Protochlorophyll forms in roots of dark-grown plants

Protochlorophyll was found in roots of dark-grown plants of seven species investigated. It was identified by absorbance and fluorescence spectra of acetone and ether extracts. Chlorophyll was also found in roots of one pea species. The concentration of protochlorophyll was usually highest in young root tips and decreased upwards along the roots. The maxima of the in vivo absorbance spectra of the species studied varied between 634 and 638 nm. Low temperature in vivo fluorescence emission spectra had two maxima, one at ca 633 and the other at ca 642 nm, when the wavelengths of the excitation light were 440 and 460 nm, respectively. In vivo fluorescence excitation spectra displayed a shift of the excitation maximum from 438 to 445 nm, when emission varied from 620 to 647.5 nm. Deconvolution of these three types of spectra into Gaussian components made it possible to identify two spectral forms of protochlorophyll: protochlorophyll_629–633 and protochlorophyll_638–642.     

Retinoic acid-induced modifications in the growth and cell surface components of a human carcinoma (HeLa) cell line

The addition of retinoic acid to cultures of HeLa-S3 cells caused a reduction in cell proliferation rate which became apparent after 72 h and was linearly dependent on retinoic acid concentration in the range 10⁻⁹–10⁻⁵ M. After 72 h of exposure to retinoic acid, the cells assumed a flattened appearance and no longer formed multilayers. These changes were reversed within 48 h after removal of retinoic acid from the medium. Structural analogs of retinoic acid with a free ---COOH group at C-15 were usually more potent in growth inhibition than compounds with an alcohol, aldehyde, ether or ester group. A cellular retinoic acid-binding protein was detected in cell homogenates, and the binding of [³H]retinoic acid to the binding protein was inhibited by most, but not all, analogs possessing a free terminal ---COOH group. For example, the 4-oxo analog of retinoic acid, while capable of inhibiting cellular proliferation, failed to bind to the retinoic acid-binding protein. Analysis of cell surface and cellular glycoproteins by lactoperoxidase-catalysed ¹²⁵I iodination and by metabolic labeling with [³H]glucosamine revealed that a 190000 D glycoprotein which was labeled by both methods and a 230000 D glycoprotein which was labeled only with [³H]glucosamine were labeled more intensely in retinoic acid-treated cells compared with untreated cells. The electrophoretic mobility of the 230000 D glycoprotein could be modified by treatment of intact cells with either neuraminidase or proteolytic enzymes, suggesting that this glycoprotein is also exposed on the cell surface. The cell surface alterations were detected much earlier than the onset of growth inhibition and appeared as early as 24 h after exposure to retinoic acid. The possible relationship between retinoic acid-induced changes in cell membrane structure, cell morphology, and cell proliferation is discussed.     

Studies on the turnover of protein and glycoprotein components in rabbit kidney brush borders

The kinetics of incorporation of [³H]lysine and [¹⁴C]glucosamine into kidney brush borders were studied in vivo. The patterns of incorporation and loss of radioactivity from the brush borders were similar for both radioactively labelled precursors. Maximal labelling occurred 15–20h after injection of the precursors, and then the radioactivity declined rapidly until 50h. The radioactivity of brush borders then remained constant until 120h and thereafter declined slowly. These results are interpreted as indicating that two processes contribute to the turnover of brush-border components, pinocytosis and a slower turnover of the components of the microvilli. Studies of the distribution of radioactivity among glycoprotein components of the brush borders, separated by polyacrylamide-gel electrophoresis, indicated that the membrane components turned over in unison.     

Activity of partially inhibited serine palmitoyltransferase is sufficient for normal sphingolipid metabolism and viability of HSN1 patient cells

Hereditary sensory neuropathy type I (HSN1) is a common degenerative disorder of peripheral sensory neurons. HSN1 is caused by mutations in the gene, encoding the long chain base 1 of serine palmitoyltransferase (SPT) [Nat. Genet. 27 (2001) 309]. Here, we show a 44% reduction of SPT activity in transformed lymphocytes from HSN1 patients with mutation T399G in the SPTLC1 gene. However, the decrease in SPT activity had no effect on de novo sphingolipid biosynthesis, cellular sphingolipid content, cell proliferation and death (apoptosis and necrosis). The removal of extracellular sphingolipids did not affect viability of HSN1 cells. We also found no significant difference in whole blood counts, viability, and permeability to Triton X-100 of primary lymphocytes from HSN1 patients. These results suggest that, despite the inhibition of mutant allele, the activity of nonmutant allele of STP may be sufficient for adequate sphingolipid biosynthesis and cell viability. Therefore, the neurodegeneration in HSN1 is likely to be caused by subtler and rather long-term effect(s) of these mutations such as loss of a cell-type selective facet of sphingolipid metabolism and/or function, or perhaps accumulation of toxic species, including abnormal protein(s) as in other neurodegenerations.     

Incidence of earlobe ptosis and pseudoptosis in patients seeking facial rejuvenation surgery and effects of aging

The authors have previously described a classification system for earlobe ptosis and have established a criterion for earlobe pseudoptosis. Earlobe heights were characterized based on anatomic landmarks, including the intertragal notch, the otobasion inferius (the most caudal anterior attachment of the earlobe to the cheek skin), and the subaurale (the most caudal extension of the earlobe free margin). The classification system was derived from earlobe height preferences as determined by a survey of North American Caucasians, and it identified the ideal free caudal lobule height range to measure 1 to 5 mm from otobasion inferius to subaurale (grade I ptosis). Also, earlobe pseudoptosis was defined by the attached cephalic lobule height measuring an intertragal notch to otobasion inferius distance greater than 15 mm. In this study, the preoperative earlobe height measurements of 44 patients seeking facial rejuvenation were evaluated. The average attached cephalic segment (intertragal notch to otobasion inferius distance) of patient earlobes measured 11.10 +/- 0.46 mm, and the average free caudal segment (otobasion inferius to subaurale distance) of patient earlobes measured 7.15 +/- 0.49 mm. Assessment of patient groups based on single-decade age differences demonstrated an increase in the free caudal segment (otobasion inferius to subaurale distance) with increasing age (p = 0.003). Assessment of patient groups based on single-decade age differences demonstrated no increase in the attached cephalic segment (intertragal notch to otobasion inferius distances) with increasing age (p = 0.281). When evaluating for the ideal otobasion inferius to subaurale distance, only 22.2 percent of earlobes demonstrated an ideal free caudal earlobe height (grade I ptosis). Moreover, pseudoptosis was detected in 12.3 percent of earlobes. Finally, a majority of earlobes demonstrated intrapatient variability, with only 16.2 percent of patients demonstrating identical attached cephalic segment (intertragal notch to otobasion inferius distances) and 37.8 percent demonstrating identical free caudal segment (otobasion inferius to subaurale distances) when compared with their contralateral ear. Plastic surgeons should be aware that a significant number of patients (77.8 percent of earlobes) may not possess an ideal free caudal segment and that 12.3 percent of earlobes may present with pseudoptosis. Therefore, earlobe height assessment should be an essential aspect of evaluation in patients desiring facial rejuvenation surgery. Evaluation of both ears should be performed independently due to intrapatient earlobe height variations. Finally, patients should be counseled with regard to the ideal earlobe parameters and aging patterns (stable attached cephalic segment versus increasing free caudal segment). With the natural progression of both facial rhytides and caudal segment earlobe ptosis (increasing free lobule segment) with increasing age, independent and accurate assessment of earlobe height is indicated so that the aging ear may be addressed concurrently with the aging face.     

GSK3 involvement in amylin signaling in isolated rat soleus muscle

Amylin can evoke insulin resistance by antagonizing insulin in a non-competitive manner. Here, we investigated the glycogenolytic effect of amylin in isolated skeletal muscle and compared it to the effects of a calcitonin gene-related peptide (CGRP). Amylin alone had no statistically significant effect on glucose transport. However, amylin decreased insulin-stimulated glucose transport by about 30%. The involvement of cAMP could not be detected at the concentrations shown to promote glycogenolysis. Previously, it has been shown that increased glycogen synthase kinase 3 (GSK3) activity plays a role in insulin resistance. Here, the ratio of GSK3 :β isoforms in rat soleus was found to be 1.2:1. We found that amylin increased GSK3 activity, which in turn led to increased phosphorylation of glycogen synthase and decreased glycogen synthesis de novo.