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Hexa(methoxymethyl)melamine (HMMM) is commonly used as a cross-linking agent in coatings and as a vulcaniser in tyre production to increase the durability of tyres. Early reports of elevated aquatic concentrations of HMMM and a range of co-occurring cyclic amines have been linked to toxicity and mortality events of aquatic organisms. There are currently only few studies reporting environmental concentrations of HMMM and the co-occurring cyclic amines, and this study reports the first environmental assessment in Australian surface waters. Archive passive water samples from 40 rivers, creeks and lakes in South East Queensland, Australia, and covering five years of biannual sampling, were analysed to determine spatial and temporal trends. Concentrations of HMMM and cyclic amines in Australian surface waters (<5-46 and

Purpose: Blood vessel networks within the retina are crucial for maintaining tissue perfusion and therefore good vision. Their complexity and unique patterns often require a steep learning curve for humans to identify trends and changes in the shape and topology of the networks, even though there exists much information important to identifying disease within them., Methods: Through image processing, the vasculature is isolated from other features of the fundus images, forcing the viewer to focus on the complex vascular feature. This article explores an approach using a grammar based on shape to describe retinal vasculature and to generate realistic and increasingly unrealistic artificial vascular networks that are then reviewed by ophthalmologists via digital survey. The ophthalmologists are asked whether these artificial vascular networks appeared realistic or unrealistic., Results: With only three rules (initiate, branch, and curve), the grammar accomplishes these goals. Networks are generated by adding noise to rule parameters present in existing networks. Via the survey of synthetic networks generated with different noise parameters, a correlation between noise in the branch rule and realistic association is revealed., Conclusions: By creating a language to describe retinal vasculature, this article allows for the potential of new insight into such an important but less understood feature of the retina, which in the future may play a role in diagnosing or helping to predict types of ocular disease., Translational Relevance: Applying shape grammar to describe retinal vasculature permits new understanding, which in turn provides the potential for new diagnostic tools., Competing Interests: Disclosure: R.Y. Yeh, None; K.K. Nischal, None; P. LeDuc, None; J. Cagan, None, (Copyright 2020 The Authors.)

This review of literature published in 2017 focuses on waste related to chemical and allied products. The topics cover waste management, aerobic treatment, anaerobic treatment, pharmaceutical wastewater, dye wastewater, agricultural wastewater, detergent wastewater, cosmetic wastewater, pigment wastewater.

This review of literature published in 2016 focuses on waste related to chemical and allied products. The topics covered include waste management, aerobic waste treatment, anaerobic waste treatment, air emission, biological waste treatment, groundwater remediation, water recycle, water analysis, water reuse, soil remediation, sorption, physicochemical treatment, ozonation, and fertilizer waste. Future topics include the pollution effect on the environment, especially, to the ecosystem and human as well as improved treatment processes to alleviate the detrimental effects.

This review of literature published in 2015 focuses on waste related to chemical and allied products. The topics cover the waste management, physicochemical treatment, aerobic granular, aerobic waste treatment, anaerobic granular, anaerobic waste treatment, chemical waste, chemical wastewater, fertilizer waste, fertilizer wastewater, pesticide wastewater, pharmaceutical wastewater, ozonation. cosmetics waste, groundwater remediation, nutrient removal, nitrification denitrification, membrane biological reactor, and pesticide waste.

This review of literature published in 2014 focuses on waste related to chemical and allied products. The topics cover the waste management practices, hospital waste, pesticide waste, chemical wastewater, pesticide wastewater and pharmaceutical wastewater. The other topics include aerobic treatment, anaerobic treatment, sorption and ozonation.

Pool water disinfection is vital to prevent microbial pathogens. However, potentially hazardous disinfection by-products (DBP) are formed from the reaction between disinfectants and organic/inorganic precursors. The aim of this study was to evaluate the presence of DBPs in various swimming pool types in Brisbane, Australia, including outdoor, indoor and baby pools, and the dynamics after a complete water renewal. Chemical analysis of 36 regulated and commonly found DBPs and total adsorbable organic halogens as well as in vitro bioassays targeting cytotoxicity, oxidative stress and genotoxicity were used to evaluate swimming pool water quality. Dichloroacetic acid and trichloroacetic acid dominated in the pool water samples with higher levels (up to 2600 μg/L) than the health guideline values set by the Australian Drinking Water Guidelines (100 μg/L). Chlorinated DBPs occurred at higher concentrations compared to tap water, while brominated DBPs decreased gradually with increasing pool water age. Biological effects were expressed as chloroacetic acid equivalent concentrations and compared to predicted effects from chemical analysis and biological characterisation of haloacetic acids. The quantified haloacetic acids explained 35-118% of the absorbable organic halogens but less than 4% of the observed non-specific toxicity (cytotoxicity), and less than 1% of the observed oxidative stress response and genotoxicity. While the DBP concentrations in Australian pools found in this study are not likely to cause any adverse health effect, they are higher than in other countries and could be reduced by better hygiene of pool users, such as thorough showering prior to entering the pool and avoiding urination during swimming., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: To describe how to approach eyes with phakic or pseudophakic bullous keratopathy that have an anterior chamber intraocular lens (AC IOL) using thin Descemet-stripping endothelial keratoplasty (thin-DSEK) or Descemet membrane endothelial keratoplasty (DMEK) with or without AC IOL removal., Setting: Tertiary referral center., Design: Comparative case series., Methods: Descemet membrane endothelial keratoplasty or thin-DSEK was performed in pseudophakic eyes with iris-claw AC IOLs (Group 1) or in phakic eyes with angle-supported AC IOLs (Group 2). In both groups, DMEK was routinely performed except in eyes with insufficient corneal transparency or a high risk for graft detachment. Preoperative surgical considerations, postoperative corrected distance visual acuity (CDVA), endothelial cell density, and complications were documented., Results: In Group 1, all AC IOLs were left in situ. In Group 2, AC IOLs were removed in 90% of cases. At 6 months, the CDVA was 20/40 (≥0.5 decimal) or better in 36% of eyes in Group 1 and 90% in Group 2. Graft detachment occurred in 20% of eyes and de novo or glaucoma exacerbation in 29%., Conclusions: Bullous keratopathy treatment in eyes with an AC IOL was feasible with DMEK. Intraocular lens removal may be required if postoperative complications are anticipated, but not to facilitate surgery. Overall, the surgical approach may aim to minimize postoperative complications; that is, thin-DSEK in eyes with low visual potential and/or concomitant pathology and DMEK in eyes with a phakic AC IOL and normal visual potential., (Copyright © 2013 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

Purpose: To determine the various causes of unexpected incomplete visual rehabilitation at 6 months postoperative after Descemet membrane endothelial keratoplasty (DMEK)., Design: Retrospective study of prospectively collected data at a tertiary referral center., Methods: From a larger group of 400 consecutive DMEK surgeries, the last 200 consecutive eyes were reviewed for visual discomfort despite a best-corrected visual acuity (BCVA) of ≥20/25 (≥0.8) or unexpected subnormal BCVA (≤20/28; ≤0.7) at 6 months after DMEK. Biomicroscopy, funduscopy, Pentacam imaging, noncontact specular microscopy, anterior segment optical coherence tomography, and surgical videos were used to determine the causes of incomplete visual rehabilitation., Results: A total of 69 eyes out of 178 eyes that were included in the analysis (38.8%) presented with incomplete visual rehabilitation after DMEK, further categorized as "primarily patient-related" in 40 of 178 (22.5%), "primarily graft-related" in 21 of 178 (11.8%), and a combination of "patient-/graft-related" in 8 of 178 cases (4.5%). Unrecognized pre-existing ocular pathology and/or posterior segment disease in 19 of 178 eyes (10.7%), clinically significant corneal irregularities and/or central corneal scarring often secondary to long-standing preoperative corneal edema in 14 of 178 eyes (7.9%), or (partial) graft detachment in 20 of 178 eyes (11.2%) were the main causes of unexpected incomplete visual rehabilitation. Transient or persistent monocular ghost images or diplopia occurred in 10 of 178 eyes (5.6%), sometimes requiring contact lens fitting., Conclusions: In contrast to earlier endothelial keratoplasty techniques that may frequently be associated with undefined transplant-related subnormal visual outcomes, incomplete visual rehabilitation after DMEK may virtually always be explained by concomitant ocular pathology or evident graft failure., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Purpose: To report the outcomes of Descemet membrane endothelial keratoplasty (DMEK) using a large diameter graft in the management of endothelial decompensation in buphthalmic eyes., Methods: Four eyes of 4 adults (1 man, 3 women) with bullous keratopathy and buphthalmos secondary to congenital glaucoma were treated with DMEK using posterior lamellar grafts with diameters ranging from 10 to 12 mm. The mean age was 31 (±9) years (range, 20-38 years). Mean follow-up time was 13.5 (±7.5) months (range, 6-24 months). Main outcome measures were best-corrected visual acuity (BCVA), intraocular pressure (IOP), pachymetry, endothelial cell density, and complications after surgery., Results: In all eyes, there was improved corneal clarity with decrease in pachymetry. The final postoperative BCVA improved in most eyes. There was no significant change in IOP, with 3 eyes needing additional antiglaucoma medication. Endothelial cell loss ranged from 37% to 42%. Postoperative complications were early partial graft detachment in 2 eyes, one resolving spontaneously without intervention and the other requiring a rebubbling, and cataract formation requiring phacoemulsification in 1 eye., Conclusions: DMEK using a large or even full diameter Descemet membrane graft may be an effective treatment for bullous keratopathy in buphthalmic eyes. Partial graft detachment after surgery may be the main complication. Postoperative IOP control is mandatory, and BCVA may vary with ocular comorbidity unrelated to the transplanted cornea.

Purpose: To evaluate the predictive value of early anterior segment optical coherence tomography (AS-OCT) on graft adherence or detachment after Descemet's membrane endothelial keratoplasty (DMEK)., Design: Retrospective study of prospectively collected data at a tertiary referral center., Participants: A total of 87 eyes of 87 patients of a consecutive series of 142 DMEK surgeries., Methods: Anterior segment OCT was performed within the first hour after DMEK and at 1 week, 1 month, 3 months, and 6 months, and for each time interval detachments were classified as "none," ≤ 1/3 detachment, >1/3 detachment of the total graft surface area, or "complete" detachment. Throughout the study, no rebubbling procedures were performed., Main Outcome Measures: Graft adherence at various postoperative time intervals., Results: One-hour AS-OCT scans were more accurate at predicting the final 6-month graft adherence status than those at 1 week or 1 month. Grafts showing complete attachment or <1/3 detachment at 1 hour remained stable or improved in 73% of the cases at 1 week, 82% at 1 month, 86% at 3 months, and 90% at 6 months. All grafts attached at 1 week remained attached at 6 months. Graft detachments of >1/3 at 1 hour showed reattachment at 6 months in 25% of the cases, whereas 67.5% of the cases showed a persistent detachment of >1/3 at 6 months and 12.5% showed a complete detachment., Conclusions: The 1-hour AS-OCT scan showed the best predictive value on 6-month graft adherence status. The combined information of the 1-hour and 1-week AS-OCT scans may facilitate decision making about surgical reintervention after DMEK., (Copyright © 2013 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: To describe corneal clearance after re-endothelialization of the recipient posterior stroma through Descemet membrane endothelial transfer (DMET) (ie, a "free-floating" donor Descemet graft in the recipient anterior chamber after descemetorhexis), in managing corneal endothelial disorders., Design: Nonrandomized prospective study at a tertiary referral center., Methods: Twelve eyes enrolled in our study, 7 suffering from Fuchs endothelial dystrophy and 5 with bullous keratopathy. The clinical outcome was monitored by biomicroscopy, optical coherence tomography, confocal microscopy, endothelial cell density, and pachymetry measurements., Results: All eyes operated on for Fuchs endothelial dystrophy showed corneal clearance, with pachymetry values returning to normal (533 ±47 μm). The denuded recipient stroma re-endothelialized with an average endothelial cell density of 797 (± 743) cells/mm(2) at 6 months after surgery. In contrast, none of the bullous keratopathy eyes showed any improvement throughout the follow-up period., Conclusion: DMET may be effective in the management of Fuchs endothelial dystrophy (primarily a Descemet membrane disorder), but not in bullous keratopathy (primarily an endothelial depletion). Apparently, the indication for surgery (ie, a "dystrophy" vs a "depletion" of recipient endothelial cells) relates to the capacity of the cornea to clear. This suggests that the remaining rim of recipient endothelium (after descemetorhexis) is involved in the re-endothelialization of the recipient posterior stroma after DMET., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: The periodontal status and effects of non-surgical periodontal treatment in patients with type 2 diabetes mellitus and periodontal disease are assessed., Methods: One-hundred patients with type 2 diabetes (mean ± SD hemoglobin (Hb)A1c level: 7.3% ± 0.94%) and periodontal disease were recruited for this study. The group with moderate-to-severe periodontal disease included patients with >1 tooth with a probing depth (PD) ≥5 mm and >2 teeth with a clinical attachment loss (AL) ≥ 6mm, and the group with mild periodontal disease included patients with <1 affected tooth, and >2 affected with a clinical AL ≥ 6mm. Patients (28 patients in the mild group and 72 patients in the moderate-to-severe group) underwent non-surgical periodontal treatments. We analyzed differences in serum concentrations of metabolic parameters (glycated hemoglobin and low-density lipoprotein), inflammatory parameters (interleukin [IL]-1β and C-reactive protein [CRP]), and periodontal parameters between the two groups before treatment and at 3, 6, 9, and 12 months post-therapy., Results: Seventy-five patients with diabetes (21 patients in the mild group and 54 patients in the moderate-to-severe group) completed the study. Significant differences in the plaque index (PI), gingival index (GI), PD, and clinical AL at examination times were observed in the whole cohort (P <0.05). We observed significant differences in the PI, GI, and PD in the moderate-to-severe group (P <0.05), whereas there was only a significant difference in PD in the mild group (P <0.05) between baseline and 12 months post-treatment. Both groups experienced improved glycemic control, but the difference was insignificant. CRP and IL-1β levels were significantly different at examination times for the whole cohort (P <0.05). No significant positive association among metabolic and inflammatory parameters at 12 months post-therapy were found., Conclusion: Non-surgical periodontal treatment improved and maintained the periodontal health of patients with well-controlled diabetes, but no significant reduction of metabolic parameters was observed over a 1-year period.

The traditional burning process is used to recover copper from scrapped PC board (printed circuit board) but it causes serious environmental problems. In this research a new process was developed which not only prevents pollution problems, but also maximizes the utility of all the materials on the waste board. First, the scrapped PC board was crushed and grounded, then placed in the NH3/NH5CO3 solution with aeration in order to dissolve copper. After distilling the copper NH3/NH5CO3 solution and then heating the distilled residue of copper carbonate, pure copper oxide was obtained with particle size of about 0.2 microm and the shape elliptical. The remaining solid residue after copper removal was then leached with 6N hydrochloric acid to remove tin and lead. The last residue was used as a filler in PVC plastics. The PVC plastics with PC board powder as filling material was found to have the same tensile strength as unfilled plastics, but had higher elastic modulus, higher abrasion resistance, and was cheaper.

Effects of essential oils and hot-water extracts isolated from leaf and twig of stout camphor tree, Cinnamomum kanehirae on antibacterial activity to pathogen of fish, abalone, marine fish and freshwater prawn, and the white shrimp, Litopenaeus vannamei immunity and disease resistance to Vibrio alginolyticus were carried out in this study. A better antibacterial activity against nine selected pathogen bacteria was recorded in twig essential oil, and the selected pathogens of both Gram-positive bacteria and Gram-negative bacteria were sensitive to the leaf and twig essential oils in the present study. No antibacterial activity was recorded in the hot-water extracts of leaf and twig. In challenge trial, a significant decrease of sensitivity to V. alginolyticus (1 x 10(6) cfu shrimp(-1)) was found in that of shrimp received hot-water extract from twig at the levels of 2 microg g shrimp(-1) compared to control. In addition, the how-water extract of twig in vitro showed greater enhanced effects on phenoloxidase activity, respiratory burst and phagocytosis of white shrimp compared to the hot-water extract of leaf. It is considered that the extracts of stout camphor tree could be a candidate to replace the chemo-therapeutants through the inhibitory effects against the growth of pathogens, and enhanced effects on shrimp immunity and disease resistance.

The mitogen-activated protein (MAP) kinase families of ERK and JNK participate in numerous intracellular signaling pathways and are abundantly expressed in the CNS. Activation of ERK and JNK during reperfusion of ischemic tissue is implicated in promoting cell death, insofar as inhibition of either pathway reduces neuronal cell death. However, ERK or JNK activation provides protection in other neuronal injury models. In this study, we monitored the concurrent modulation of ERK and JNK activity in the hippocampus, neocortex, and striatum during ischemia and immediately upon reperfusion in a rat model of transient global ischemia. All three regions incur a similar reduction in blood flow during occlusion but show different extents and temporal patterns of injury following reperfusion. ERK and JNK were active in the normal rat forebrain, and phosphorylation was reduced by ischemia. Upon reperfusion, ERK was rapidly activated in the hippocampus, neocortex, and striatum, whereas JNK phosphorylation increased in the hippocampus and striatum but not in the neocortex. The response of JNK vs. ERK more closely reflects the susceptibility of these regions. JNK1 was the predominant phosphorylated isoform. A minor pool of phosphorylated JNK3 increased above the control level after reperfusion in hippocampal but not in neocortical particulate fractions. In addition, a novel 32-35-kDa c-Jun kinase activity was detected in the hippocampus, neocortex, and striatum. The results show that ERK and JNK activities are rapidly, but not identically, modulated by ischemia and reperfusion and indicate that the MAP kinase pathways contribute to regulating the response to acute CNS injury., (Copyright 2006 Wiley-Liss, Inc.)

The extracellular signal-regulated kinases (ERK) participate in numerous signaling pathways and are abundantly expressed in the CNS. It has been proposed that ERK activation promotes survival in models of neuronal injury. Inhibition of MEK, the upstream kinase that activates ERK, however, leads to neuroprotection in models of cerebral ischemia and trauma, suggesting that in this context ERK activation contributes to cellular damage. The effect of ischemia and reperfusion on activity and expression of ERK was investigated using a reversible model of rabbit spinal cord ischemia. Active ERK was observed in nai;ve animals, which decreased during 15 to 60 min of ischemia. Upon reperfusion, a robust activation of ERK was observed in animals occluded for 60 min that remained permanently paraplegic. Immunohistochemical analyses revealed increased staining of phosphorylated ERK (pERK) in glial cells and faint nuclear staining in motor neurons of animals occluded for 60 min and reperfused for 18 h. In contrast ERK activity did not increase in animals occluded for 15 min that regained motor function. No evidence of increased pERK immunoreactivity in motor neurons or nuclear translocation was noted in these animals. ERK1 was demonstrated to be identical to a p46 c-Jun/ATF-2 kinase previously shown to be activated by reperfusion after a 60-min occlusion. The results suggest that activation of ERK during reperfusion of ischemic spinal cord participates in the cellular pathways leading to neuronal damage.

Activation of the c-Jun N-terminal (JNK) or stress-activated protein kinases (SAPK) is associated with a wide range of disparate cellular responses to extracellular stimuli, including either induction of or protection from apoptosis. This study investigates the effect of ischemia and reperfusion on JNK isoform activities using a reversible rabbit spinal cord ischemia model. High basal JNK activity, attributed to the p46 JNK1 isoform, was expressed in the CNS of untreated rabbits. JNK activity decreased in the lumbar spinal cord of rabbits occluded for 15-60 min. During reperfusion animals occluded for 15 min recovered neurological function and JNK activity returned to normal levels. In contrast animals occluded for 60 min remained permanently paraplegic and JNK activity was half the control activity after 18 h of reperfusion. In these animals proteolytic fragments of JNK1 and JNK3 were observed and protein levels, but not activity, of JNK isoforms increased in a detergent-insoluble fraction. Two novel c-Jun (and ATF-2) kinase activities increased during reperfusion of animals occluded for 60 min. An activity designated p46(slow) was similar in M(r) to a JNK2 isoform induced in these animals. A second 30-kDa activity associated with the detergent-insoluble fraction co-migrated with a JNK3 N-terminal fragment. The results show that JNK1 is active in the normal CNS and increased activity is not associated with durations of ischemia and reperfusion that induce cell death. However, specific JNK isoform activation may participate in the cell death pathways as increased activity of novel c-Jun (ATF-2) kinase activities was observed in paraplegic animals.

The effect of transient cerebral ischemia on phosphorylation of the microtubule-associated protein (MAP) tau was investigated using the rat four-vessel occlusion model. Phosphorylation of tau is proposed to regulate its binding to microtubules, influencing the dynamics of microtubule assembly necessary for axonal growth and neurite plasticity. In this study, tau was rapidly dephosphorylated during ischemia in the hippocampus, neocortex, and striatum. Dephosphorylation of tau was observed within 5 min of occlusion and increased after 15 min in all three brain regions, regardless of their relative vulnerability to the insult. Thus, dephosphorylation of tau is an early marker of ischemia and precedes the occlusion time required to cause extensive neuronal cell death in this model. On restoration of blood flow for a little as 15 min, tau was phosphorylated at a site(s) that causes a reduction in its electrophoretic mobility. The dephosphorylation/phosphorylation of tau may alter its distribution between axon and cell body, and affect its susceptibility to proteolysis. These changes would be expected to influence microtubule stability, possibly contributing to disruption of axonal transport, but also allowing neurite remodeling in a regenerative response.

The effects of cerebral ischemia on calcium/calmodulin-dependent kinase II (CaM kinase II) were investigated using the rat four-vessel occlusion model. In agreement with previous results using rat or gerbil models of cerebral ischemia or a rabbit model of spinal cord ischemia, this report demonstrates that transient forebrain ischemia leads to a reduction in CaM kinase II activity within 5 min of occlusion onset. Loss of activity from the cytosol fractions of homogenates from the neocortex, striatum, and hippocampus correlated with a decrease in the amount of CaM kinase alpha and beta isoforms detected by immunoblotting. In contrast, there was an apparent increase in the amount of CaM kinase alpha and beta in the particulate fractions. The decrease in the amount of CaM kinase isoforms from the cytosol but not the particulate fractions was confirmed by autophosphorylation of CaM kinase II after denaturation and renaturation in situ of the blotted proteins. These results indicate that ischemia causes a rapid inhibition of CaM kinase II activity and a change in the partitioning of the enzyme between the cytosol and particulate fractions. CaM kinase II is a multifunctional protein kinase, and the loss of activity may play a critical role in initiating the changes leading to ischemia-induced cell death. To identify a structural basis for the decrease in enzyme activity, tryptic peptide maps of CaM kinase II phosphorylated in vitro were compared. Phosphopeptide maps of CaM kinase alpha from particulate fractions of control and ischemic samples revealed not only reduced incorporation of phosphate into the protein but also the absence of a limited number of peptides in the ischemic samples. This suggested that certain sites are inaccessible, possibly due to a conformational change, a covalent modification of CaM kinase II, or steric hindrance by an associated molecule. Verifying one of these possibilities should help to elucidate the mechanism of ischemia-induced modulation of CaM kinase II.

Reversible spinal cord ischemia in rabbits induced a rapid loss of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) activity measured as incorporation of phosphate into exogenous substrates. About 70% of the activity was lost from the cytosolic fraction of spinal cord homogenates after 15 min of ischemia preceding irreversible paraplegia, which takes 25 min in this model. The loss of enzyme activity correlated with a loss of in situ renaturable autophosphorylation activity and a loss of CaM kinase II alpha and beta subunits in the cytosol detected by immunoblotting. CaM kinase II activity in the particulate fraction also decreased but the protein levels of the alpha and beta subunits increased. Thus ischemia resulted in an inactivation of CaM kinase II and a sequential or concurrent subcellular redistribution of the enzyme. However, denaturation and renaturation in situ of the CaM kinase subunits immobilized on membranes partly reversed the apparent inactivation of the enzyme in the particulate fraction. CaM kinase II activity was restored after reperfusion following short (< or = 25 min) durations of ischemia but not after longer durations (60 min) that result in irreversible paraplegia. The ischemia-induced inactivation of CaM kinase II, which phosphorylates proteins regulating many cellular processes, may be important in the cascade of events leading to delayed neuronal cell death.

We previously reported that I-cell disease lymphoblasts maintain normal or near-normal intracellular levels of lysosomal enzymes, even though N-acetylglucosamine-1-phosphotransferase activity is severely depressed or absent (Little et al., Biochem. J., 248, 151-159, 1987). The present study, employing subcellular fractionation on colloidal silica gradients, indicates that both light and heavy lysosomes isolated from I-cell disease and pseudo-Hurler polydystrophy lymphoblasts possess normal specific activity levels of N-acetyl-beta-D-hexosaminidase, alpha-D-mannosidase and beta-D-glucuronidase. These current findings are in contrast to those of cultured fibroblasts from the same patients, where decreased intralysosomal enzyme activities are found. Column chromatography on Ricinus communis revealed that N-acetyl-beta-D-hexosaminidase in both heavy and light I-cell disease lysosomal fractions from lymphoblasts possesses an increased number of accessible galactose residues (30-50%) as compared to the enzyme from the corresponding normal controls. Endo-beta-N-acetylglucosaminidase H treatment of N-acetyl-beta-D-hexosaminidase from the I-cell lysosomal fractions suggests that the majority of newly synthesized high-mannose-type oligosaccharide chains are modified to complex-type carbohydrates prior to being transported to lysosomes. This result from lymphoblasts differs from previous findings with fibroblasts, where N-acetyl-beta-D-hexosaminidase from I-cell disease and pseudo-Hurler polydystrophy lysosomes exhibited properties associated with predominantly high-mannose-type oligosaccharide chains.(ABSTRACT TRUNCATED AT 250 WORDS)

alpha-D-Mannosidase and beta-D-glucosidase from Dictyostelium discoideum are efficiently endocytosed into mutant human fibroblasts through a saturable, mannose 6-phosphate (Man-6-P)-inhibitable uptake system (Freeze, H. H., Kaplan, A., and Miller, A. L. (1980) J. Biol. Chem. 255, 11081-11084). We have extended this study using both of these active, purified enzymes and 125I-labeled beta-glucosidase for uptake into normal human fibroblasts. The pH optimum of uptake is 6.0 for both enzymes and greater than 95% is inhibited by 2 mM Man-6-P (Ki = 5 X 10(-5) M). A variety of mono-and diesterified mannans or mannan derivatives also inhibited uptake of the enzymes. Both enzymes compete with each other for uptake (Ki, 2.0 X 10(-9) M) and have Kuptake of 1.0-2.2 X 10(-9) M and a Vmax of 0.35-0.48 pmol/mg of cell protein/h. The specific binding of 125I-beta-glucosidase to fibroblasts was measured at 0-4 degrees C and found to have a Kd of 1.0 X 10(-9) M with approximately 15,900 +/- 900 receptors/cell. The receptors could be internalized every 5-7 min at saturating concentrations of enzyme at 37 degrees C. The 125I-beta-glucosidase previously bound to the cells at 4 degrees C could be released by continued incubation at 4 degrees C in the presence of Man-6-P, however, after brief warming to 37 degrees C followed by reincubation at 4 degrees C, Man-6-P could no longer release the ligand. Chloroquine inhibited 95% of the uptake of 125I-beta-glucosidase at 50 microM. Following internalization of the enzyme, it is degraded to trichloroacetic acid-soluble fragments with a half-life of approximately 6.5 h. These data suggest that the slime mold enzymes are bound to the same receptors which function in the uptake of mammalian lysosomal enzymes and make the slime mold lysosomal enzymes useful models to study uptake involving this receptor in normal human fibroblasts.

Chediak-Higashi-syndrome cultured skin fibroblasts were used to study the possible involvement of lysosomal enzymes and lysosomal dysfunction in this disorder. Our evidence indicated that Chediak-Higashi fibroblasts displayed a significant decrease in the specific activity of the acidic alpha-D-mannosidase (pH 4.2) compared with normal controls. Additional studies revealed a small, but significant, decrease in the rate of degradation of 125I-labelled beta-D-glucosidase that had been endocytosed into Chediak-Higashi cells.

Human lymphoblast and fibroblast cell lines from a patient with I-cell disease and normal individuals were characterized with respect to certain properties of UDP-N-acetylglucosamine:lysosomal enzyme precursor N-acetylglucosamine phosphotransferase. The enzyme isolated from normal lymphoblast and fibroblast cell lines expressed similar kinetic properties, substrate specificities and subcellular localizations. Coincident with the severe reduction of N-acetylglucosamine phosphotransferase activity in both I-cell fibroblast and lymphoblast cell lines, there was an increased secretion of several lysosomal enzymes compared to normal controls. Subsequent examination of N-acetyl-beta-D-hexosaminidase secreted by the I-cell lymphoblasts demonstrated a significant increase in adsorption of the I-cell enzyme to Ricinus communis agglutinin, a galactose-specific lectin. However, the I-cell lymphoblasts did not exhibit the significant decrease in intracellular lysosomal activities seen in I-cell fibroblasts. Our results suggest that lymphoblasts not only represent an excellent source for the purification of N-acetylglucosamine phosphotransferase, but in addition, represent a unique system for studying alternate mechanisms involved in the targeting of lysosomal enzymes.