Detection, production, modification, and application of arylsulfatases.

Arylsulfatase is a subset of sulfatase which catalyzes the hydrolysis of aryl sulfate ester. Arylsulfatase is widely distributed among microorganisms, mammals and green algae, but the arylsulfatase-encoding gene has not yet been found in the genomes of higher plants so far. Arylsulfatase plays an important role in the sulfur flows between nature and organisms. In this review, we present the maturation and catalytic mechanism of arylsulfatase, and the recent literature on the expression and production of arylsulfatase in wild-type and engineered microorganisms, as well as the modification of arylsulfatase by genetic engineering are summarized. We focus on arylsulfatases from microbial origin and give an overview of different assays and substrates used to determine the arylsulfatase activity. Furthermore, the researches about arylsulfatase application on the field of agar desulfation, soil sulfur cycle and soil evaluation are also discussed. Finally, the perspectives concerning the future research on arylsulfatase are prospected.

The arylsulfatase- and phospholipase-rich venom of the plutoniumid centipede Theatops posticus.

Research on centipede venoms has led to the discovery of a diverse array of novel proteins and peptides, including those with homology to previously discovered toxin families (e.g., phospholipase A2s and pM12a metalloproteases) and novel toxin families not previously detected in venoms (e.g., ß-pore forming toxins and scoloptoxins). Most of this research has focused on centipedes in the order Scolopendromorpha, particularly those in the families Scolopendridae, Cryptopidae, and Scolopocryptopidae. To generate the first high-throughput venom characterization for a centipede in the scolopendromorph family Plutoniumidae, we performed venom-gland transcriptomics and venom proteomics on two Theatops posticus. We identified a total of 64 venom toxins, 60 of which were detected in both the venom-gland transcriptome and venom proteome and four of which were only detected transcriptomically. We detected a single highly abundant arylsulfatase B (ARSB) toxin, the first ARSB toxin identified from centipede venoms. As ARSBs have been detected in other venomous species (e.g., scorpions), ARSBs in T. posticus highlights a new case of convergent evolution across venoms. Theatops posticus venom also contained a much higher abundance and diversity of phospholipase A2 toxins compared to other characterized centipede venoms. Conversely, we detected other common centipedes toxins, such as CAPs and scoloptoxins, at relatively low abundances and diversities. Our observation of a diverse set of toxins from T. posticus venom, including those from novel toxin families, emphasizes the importance of studying unexplored centipede taxonomic groups and the continued potential of centipede venoms for novel toxin discovery and unraveling the molecular mechanisms underlying trait evolution.

Transcription level and phylogeny analyses of Chlamydomonas reinhardtii arylsulfatases.

Sulfur is a required macroelement for all organisms, and sulfate deficiency causes growth and developmental defects. Arylsulfatases (ARS) hydrolyze sulfate from sulfate esters and make sulfate bioavailable for plant uptake. These enzymes are found in microorganisms and animals; however, plant genomes do not encode any ARS gene. Our database searches found nineteen ARS genes in the genome of Chlamydomonas reinhardtii. Among these, ARS1 and ARS2 were studied in the literature; however, the remaining seventeen gene models were not studied. Our results show that putative polypeptide sequences of the ARS gene models all have the sulfatase domain and sulfatase motifs found in known ARSs. Phylogenetic analyses show that C. reinhardtii proteins are in close branches with Volvox carterii proteins while they were clustered in a separate group from Homo sapiens and bacterial species (Pseudomonas aeruginosa and Rhodopirellula baltica SH1), except human Sulf1, Sulf2, and GNS are clustered with algal ARSs. RT-PCR analyses showed that transcription of ARS6, ARS7, ARS11, ARS12, ARS13, ARS17, and ARS19 increased under sulfate deficiency. However, this increase was not as high as the increase seen in ARS2. Since plant genomes do not encode any ARS gene, our results highlight the importance of microbial ARS genes.

Silkworm arylsulfatase in the midgut content is expressed in the silk gland and fed via smearing on the food from the spinneret.

We found the activity of arylsulfatase in the midgut contents of the silkworm, Bombyx mori. We identified a 60-kDa protein that comigrates with the activity on a column chromatography following ammonium sulfate precipitation. Based on its partial amino acid sequence, we searched for its coding gene using Basic Local Alignment Search Tool (BLAST) and identified KWMTBOMO05106. Transcriptional data suggest a specific expression of the gene in middle silk glands. The majority (80%) of arylsulfatase activity was found in the silk glands, concurring the specific transcription in the silk gland. Observing the feeding behaviour of the silkworm, we found that silkworms smear a mucus secretes from the spinneret on the food pellet as they feed on. Arylsulfatase activity was also detected in the food pellet bitten by the silkworm as well as in the gut content. Furthermore, arylsulfatase activity was not detected either in the food pellet and in the gut content when silkworms had obstructed the spinneret. These results suggest that arylsulfatase is secreted from the silk glands and may contribute to digestive function.

Development and validation of a CRISPR interference system for gene regulation in Campylobacter jejuni.

BACKGROUND: Campylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuni responsible for 80% of these infections. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which in turn constrains our ability to interrogate the function of essential and conditionally essential genes. We have addressed this by developing and utilising a CRISPR-based interference system known as CRISPRi in C. jejuni to control gene expression. To achieve this, a catalytically inactive ("dead") cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. jejuni-derived promoters of predetermined expression activities to develop a CRISPRi-based repression tool in C. jejuni strains M1Cam and 81-176. RESULTS: The CRISPRi tool was validated through successful repression of the arylsulphatase-encoding gene astA using a range of sgRNA target sequences spanning the astA gene. The tool was also applied to target astA in an M1Cam CRISPR-Cas9 deletion strain, which showed that the presence of an endogenous CRISPR-Cas9 system did not affect the activity of the CRISPRi-based repression tool. The tool was further validated against the hippicurase-encoding gene hipO. Following this, the flagella genes flgR, flaA, flaB and both flaA and flaB were targeted for CRISPRi-based repression, which resulted in varying levels of motility reduction and flagella phenotypes as determined by phenotypical assays and transmission electron microscopy (TEM). CONCLUSIONS: This is the first report of a CRISPRi-based tool in C. jejuni, which will provide a valuable resource to the Campylobacter community.

Engineering Pseudomonas aeruginosa arylsulfatase for hydrolysis of α-configured steroid sulfates.

Steroid sulfate esters are important metabolites for anti-doping efforts in sports, pathology and research. Analysis of these metabolites is facilitated by hydrolysis using either acid or enzymatic catalysis. Although enzymatic hydrolysis is preferred for operating at neutral pH, no known enzyme is capable of hydrolyzing all steroid sulfate metabolites. Pseudomonas aeruginosa arylsulfatase (PaS) is ideal for the hydrolysis of ß-configured steroid sulfates but like other known class I sulfatases it is inefficient at hydrolyzing α-configured steroid sulfates. We have used directed evolution with liquid chromatography mass spectrometry screening to find variants capable of hydrolyzing a α-configured steroid sulfate: etiocholanolone sulfate (ECS). After targeting two regions of PaS, four residues were identified and optimized to yield a final variant with a total of seven mutations (DRN-PaS) capable of hydrolyzing ECS ~80 times faster than the best PaS variant previously available. This DRN-PaS also shows improved activity for other α-configured steroid sulfates. Simultaneous mutagenesis was essential to obtain DRN-PaS due to complementarity between targeted residues.

The lysosomal proteome of senescent cells contributes to the senescence secretome.

Senescent cells accumulate in tissues over time, favoring the onset and progression of multiple age-related diseases. Senescent cells present a remarkable increase in lysosomal mass and elevated autophagic activity. Here, we report that two main autophagic pathways macroautophagy (MA) and chaperone-mediated autophagy (CMA) are constitutively upregulated in senescent cells. Proteomic analyses of the subpopulations of lysosomes preferentially engaged in each of these types of autophagy revealed profound quantitative and qualitative changes in senescent cells, affecting both lysosomal resident proteins and cargo proteins delivered to lysosomes for degradation. These studies have led us to identify resident lysosomal proteins that are highly augmented in senescent cells and can be used as novel markers of senescence, such as arylsulfatase ARSA. The abundant secretome of senescent cells, known as SASP, is considered their main pathological mediator; however, little is known about the mechanisms of SASP secretion. Some secretory cells, including melanocytes, use the small GTPase RAB27A to perform lysosomal secretion. We found that this process is exacerbated in the case of senescent melanoma cells, as revealed by the exposure of lysosomal membrane integral proteins LAMP1 and LAMP2 in their plasma membrane. Interestingly, a subset of SASP components, including cytokines CCL2, CCL3, CXCL12, cathepsin CTSD, or the protease inhibitor SERPINE1, are secreted in a RAB27A-dependent manner in senescent melanoma cells. Finally, proteins previously identified as plasma biomarkers of aging are highly enriched in the lysosomes of senescent cells, including CTSD. We conclude that the lysosomal proteome of senescent cells is profoundly reconfigured, and that some senescent cells can be highly active in lysosomal exocytosis.

Characterization and substrate-accelerated thermal inactivation kinetics of a new serine-type arylsulfatase.

Arylsulfatase is useful in industrial agar processing by removing sulfate groups. A full-length arylsulfatase gene, designated ArySMA1, was obtained from marine bacteria Serratia sp. SM1. The ArySMA1 gene encoded a novel serine-type arylsulfatase and the enzymatic properties were characterized. The enzyme presented notable capacity of removing sulfate groups from natural algae substrates. Kinetic study suggested that the microscopic thermal inactivation rate of ArySMA1 in free form was smaller than that of the enzyme-substrate complex. The presence of substrate could unexpectedly accelerate ArySMA1 to deactivate at high temperature. Such phenomenon was opposite to many findings that substrate could stabilize enzymes against heat. Molecular dynamics simulation and ANS fluorescent assay indicated the substrate led the hydrophobic regions of the active site more flexible and the sulfate group of the substrate could retard the processivity of ArySMA1 catalysis. This study provides guidance for agar desulfation and down-stream processing industry.

ARSD, a novel ERα downstream target gene, inhibits proliferation and migration of breast cancer cells via activating Hippo/YAP pathway.

Advanced breast cancer (BC), especially basal like triple-negative BC (TNBC), is a highly malignant tumor without viable treatment option, highlighting the urgent need to seek novel therapeutic targets. Arylsulfatase D (ARSD), localized at Xp22.3, is a female-biased gene due to its escaping from X chromosome inactivation (XCI). Unfortunately, no systematic investigation of ARSD on BC has been reported. In this study, we observed that ARSD expression was positively related to ERα status either in BC cells or tissue specimens, which were associated with good prognosis. Furthermore, we found a set of hormone-responsive lineage-specific transcription factors, FOXA1, GATA3, ERα, directly drove high expression of ARSD through chromatin looping in luminal subtype BC cells. Opposingly, ARSD still subjected to XCI in TNBC cells mediated by Xist, CpG islands methylation, and inhibitory histone modification. Unexpectedly, we also found that ectopic ARSD overexpression could inhibit proliferation and migration of TNBC cells by activating Hippo/YAP pathway, indicating that ARSD may be a molecule brake on ERα signaling pathway, which restricted ERα to be an uncontrolled active status. Combined with other peoples' researches that Hippo signaling maintained ER expression and ER + BC growth, we believed that there should exist a regulative feedback loop formation among ERα, ARSD, and Hippo/YAP pathway. Collectively, our findings will help filling the knowledge gap about the influence of ARSD on BC and providing evidence that ARSD may serve as a potential marker to predict prognosis and as a therapeutic target.

Decoding the consecutive lysosomal degradation of 3-O-sulfate containing heparan sulfate by Arylsulfatase G (ARSG).

The lysosomal degradation of heparan sulfate is mediated by the concerted action of nine different enzymes. Within this degradation pathway, Arylsulfatase G (ARSG) is critical for removing 3-O-sulfate from glucosamine, and mutations in ARSG are causative for Usher syndrome type IV. We developed a specific ARSG enzyme assay using sulfated monosaccharide substrates, which reflect derivatives of its natural substrates. These sulfated compounds were incubated with ARSG, and resulting products were analyzed by reversed-phase HPLC after chemical addition of the fluorescent dyes 2-aminoacridone or 2-aminobenzoic acid, respectively. We applied the assay to further characterize ARSG regarding its hydrolytic specificity against 3-O-sulfated monosaccharides containing additional sulfate-groups and N-acetylation. The application of recombinant ARSG and cells overexpressing ARSG as well as isolated lysosomes from wild-type and Arsg knockout mice validated the utility of our assay. We further exploited the assay to determine the sequential action of the different sulfatases involved in the lysosomal catabolism of 3-O-sulfated glucosamine residues of heparan sulfate. Our results confirm and extend the characterization of the substrate specificity of ARSG and help to determine the sequential order of the lysosomal catabolic breakdown of (3-O-)sulfated heparan sulfate.

New clinical and molecular evidence linking mutations in ARSG to Usher syndrome type IV.

In murine and canine animal models, mutations in the Arylsulfatase G gene (ARSG) cause a particular lysosomal storage disorder characterized by neurological phenotypes. Recently, two variants in the same gene were found to be associated with an atypical form of Usher syndrome in humans, leading to visual and auditory impairment without the involvement of the central nervous system. In this study, we identified three novel pathogenic variants in ARSG, which segregated recessively with the disease in two families from Portugal. The probands were affected with retinitis pigmentosa and sensorineural hearing loss, generally with an onset of symptoms in their fourth decade of life. Functional experiments showed that these pathogenic variants abolish the sulfatase activity of the Arylsulfatase G enzyme and impede the appropriate lysosomal localization of the protein product, which appears to be retained in the endoplasmic reticulum. Our data enable to definitely confirm that different biallelic variants in ARSG cause a specific deaf-blindness syndrome, by abolishing the activity of the enzyme it encodes.

Evaluation of Arylsulfatase D (ARSD) and long noncoding RNA ARSD-AS1 gene expression in breast cancer patients and their association with oncogenic transcription factors.

PURPOSE: Long non-coding RNAs (LncRNAs) are thought as tumorigenic factors in cancer progression. We investigated the clinical significance of arylsulfatase D (ARSD) and ARSD antisense in breast cancer patients. METHODS: Eighty breast cancer tumors were obtained from the Tumor Bank of Cancer Institute, Imam Khomeini Hospital. The expression level of ARSD and ARSD-AS1 were examined in breast tumors in comparison to the margin of normal tissues using quantitative real-time PCR. Demographic information and the clinicopathologic characteristics including tumor grade, presence of cell receptors, lymph node and vascular invasion were also evaluated. Bioinformatics databases were used for identification of ARSD and ARSD-AS1 molecular targets and their association with cancer. RESULTS: Significant up-regulation of ARSD was observed in tumor tissues in comparison with its antisense (p<0.05). Both ARSD and ARSD-AS1 expression in tumor specimens were notably lower than those in adjacent normal tissue. High expression of ARSD was associated to lower tumor grade (p<0.05). Bioinformatics results revealed the interaction of ARSD with STS and SUMF1 proteins was attributed to the inhibiting of sulfates activity. Also, ARSD co-expressed genes were associated with oncogenic transcription factors, MAF and GATA. TP53 transcription factor site was identified as a target of ARSD-AS1 mRNA. The interaction of this antisense with microRNA (miR-618) could explain its participation in tumor cell proliferation. CONCLUSION: Low expression of ARSD was associated with higher tumor grade. The evidence from this study enhance our understanding of ARSD and ARSD-AS1 function in cancer gene therapy. Accordingly, they could be introduced as great potential targets for breast cancer treatment.

Arylsulfatase K inactivation causes mucopolysaccharidosis due to deficient glucuronate desulfation of heparan and chondroitin sulfate.

Mucopolysaccharidoses comprise a group of rare metabolic diseases, in which the lysosomal degradation of glycosaminoglycans (GAGs) is impaired due to genetically inherited defects of lysosomal enzymes involved in GAG catabolism. The resulting intralysosomal accumulation of GAG-derived metabolites consequently manifests in neurological symptoms and also peripheral abnormalities in various tissues like liver, kidney, spleen and bone. As each GAG consists of differently sulfated disaccharide units, it needs a specific, but also partly overlapping set of lysosomal enzymes to accomplish their complete degradation. Recently, we identified and characterized the lysosomal enzyme arylsulfatase K (Arsk) exhibiting glucuronate-2-sulfatase activity as needed for the degradation of heparan sulfate (HS), chondroitin sulfate (CS) and dermatan sulfate (DS). In the present study, we investigated the physiological relevance of Arsk by means of a constitutive Arsk knockout mouse model. A complete lack of glucuronate desulfation was demonstrated by a specific enzyme activity assay. Arsk-deficient mice show, in an organ-specific manner, a moderate accumulation of HS and CS metabolites characterized by 2-O-sulfated glucuronate moieties at their non-reducing ends. Pathophysiological studies reflect a rather mild phenotype including behavioral changes. Interestingly, no prominent lysosomal storage pathology like bone abnormalities were detected. Our results from the Arsk mouse model suggest a new although mild form of mucopolysacharidose (MPS), which we designate MPS type IIB.

Discovery of the First Human Arylsulfatase A Reversible Inhibitor Impairing Mouse Oocyte Fertilization.

Arylsulfatase A (ARSA) plays a crucial role in the reproduction of mammals due to its involvement in the specific gamete interaction preceding sperm and egg fusion leading to fertilization. Recently, it has been shown that zona pellucida (ZP) sperm binding and in vivo fertilization in mice are markedly hampered by using a specific anti-ARSA antibody. Herein, the design and discovery of the first ARSA small molecule inhibitor based on a coumarin-containing polycycle are presented. Through a structure-based approach applied on our in-house library, compound 1r was identified as an ARSA reversible inhibitor (ARSAi); then its activity was validated through both surface plasmon resonance and biochemical inhibition experiments, the first providing a KD value of 21 µM and the latter an IC50 value of 13.2 µM. Further investigations highlighted that compound 1r induced 20% sperm death at 25 µM and also impaired sperm motility; nevertheless both the effects were mediated by ROS production, since they were rescued by the cotreatment of 1r and N-acetyl cysteine (NAC). Interestingly, while 1r was not able to hamper the ZP/sperm binding, it markedly decreased the in vitro oocyte fertilization by mouse sperm up to 60%. Notably, this effect was not hampered by 1r/NAC coadministration, hence allowing the ruling out of an ROS-dependent mechanism. In conclusion, herein is reported the first ever hit of ARSAi as a chemical tool that will enable better exploration of ARSA's biological role in fertilization as well as provide a starting point for developing 1r structure optimization aimed at increasing enzyme inhibition potency but also providing a deeper understanding of the involvement of ARSA in the fertilization pathway mechanism.

A mutant of Pseudoalteromonas carrageenovora arylsulfatase with enhanced enzyme activity and its potential application in improvement of the agar quality.

Enzymatic desulfation using arylsulfatase provides an attractive approach to improve agar quality. We have previously characterized a functional arylsulfatase from Pseudoalteromonas carrageenovora. To further improve its enzymatic performance, we isolated a mutant arylsulfatase of K253Q with improved enzyme activity from a random mutant library. Compared to wild-type arylsulfatase (WT), K253Q showed 33% increase in enzyme activity, with optimal temperature and pH of 55 °C and 8.0, respectively. K253Q demonstrated better substrate binding ability with lower Km value. Structure analysis indicated that a combination of the additional hydrogen bond and the enhanced substrate binding affinity could account for the improved enzyme activity of K253Q. K253Q exhibited about 54% sulfate removal against agar, resulting in additional 8% increase in 3,6-AG content and 20% increase in gel strength compared to WT. Scanning electron microscopy showed that K253Q treatment led to a stronger crosslinking structure of agar.

Implementation of an Affordable Method for MPS Diagnosis from Urine Screening to Enzymatic Confirmation: Results of a Pilot Study in Morocco.

BACKGROUND: Rapid and accurate diagnosis of mucopolysaccharidoses (MPS) is still a challenge due to poor access to screening and diagnostic methods and to their extensive clinical heterogeneity. The aim of this work is to perform laboratory biochemical testing for confirming the diagnosis of mucopolysaccharidosis (MPS) for the first time in Morocco. METHODS: Over a period of twelve months, 88 patients suspected of having Mucopolysaccharidosis (MPS) were referred to our laboratory. Quantitative and qualitative urine glycosaminoglycan (GAG) analyses were performed, and enzyme activity was assayed on dried blood spots (DBS) using fluorogenic substrates. Enzyme activity was measured as normal, low, or undetectable. RESULTS: Of the 88 patients studied, 26 were confirmed to have MPS; 19 MPS I (Hurler syndrome; OMIM #607014/Hurler-Scheie syndrome; OMIM #607015), 2 MPS II (Hunter syndrome; OMIM #309900), 2 MPS IIIA (Sanfilippo syndrome; OMIM #252900), 1 MPS IIIB (Sanfilippo syndrome; OMIM #252920) and 2 MPS VI (Maroteaux-Lamy syndrome; OMIM #253200). Parental consanguinity was present in 80.76% of cases. Qualitative urinary glycosaminoglycan (uGAGs) assays showed abnormal profiles in 31 cases, and further quantitative urinary GAG evaluation and Thin Layer Chromatography (TLC) provided important additional information about the likely MPS diagnosis. The final diagnosis was confirmed by specific enzyme activity analysis in the DBS samples. CONCLUSIONS: The present study shows that the adoption of combined urinary substrate analysis and enzyme assays using dried blood spots can facilitate such diagnosis, offer an important tool for an appropriate supporting care, and a specific therapy, when available.

Alteration of enzyme activities and functional diversity of a soil contaminated with copper and arsenic.

Copper (Cu) mining has to address a critical environmental issue related to the disposal of heavy metals and metalloids (HMs). Due to their deleterious effects on living organisms, Cu and arsenic (As) have gained global attention, and thus their monitoring in the environment is an important task. The aims of this study were: 1) to evaluate the alteration of soil enzyme activities (EAs) and soil microbial functional diversity with Cu/As contamination, and 2) to select the most reliable biochemical indicators of Cu/As contamination. A twelve-week soil experiment was performed with four increasing levels of Cu, As, and Cu/As from 150/15 to 1000/100 mg Cu/As kg-1. Soil enzyme activities and soil community-level physiological profile (CLPP) using MicroResp™ were measured during the experiment. Results showed reduced EAs over time with increasing Cu and Cu/As levels. The most Cu-sensitive EAs were dehydrogenase, acid phosphatase, and arylsulfatase, while arginine ammonification might be related to the resilience of soil microbial communities due to its increased activity in the last experimental times. There was no consistent response to As contamination with reduced individual EAs at specific sampling times, being urease the only EA negatively affected by As. MicroResp™ showed reduced carbon (C) substrate utilization with increasing Cu levels indicating a community shift in C acquisition. These results support the use of specific EAs to assess the environmental impact of specific HMs, being also the first assessment of EAs and the use of CLPP (MicroResp™) to study the environmental impact in Cu/As contaminated soils.

Proteoglycan desulfation: a critical step in oncogenesis?

Proteoglycans are essential constituents of tissue- and organ microenvironments, modulating both the structural scaffolds that surround cells as well as signaling cues that determine cellular phenotype. An important modification of proteoglycans is the sulfation of the monosaccharides that comprise them. Sulfates are added by sulfotransferases and desulfation occurs through the action of sulfatases. In this essay, we examine the biochemistry of a conserved family of desulfating enzymes known as arylsulfatases. A subset of these enzymes mediates the desulfation of proteoglycans. We review the consequences of their aberrant expression in the light of carcinogenesis and carcinomatosis: the dissemination of cancer cells. A closer understanding of their cellular-molecular roles reveals their promise for future strategies for cancer therapy.

Effect of reclamation treatments on microbial activity and phytotoxicity of soil degraded by the sulphur mining industry.

The aim of the work was to determine the trend, intensity and changes of selected microbial and phytotoxic parameters of degraded soil in the area of former sulphur mine reclaimed by post-flotation lime (PFL), sewage sludge (SS), mineral wool (MW- mixed with soil, MWP-pad) and mineral fertilizer (NPK). The following parameters: number of proteolytic bacteria and fungi, ammonification, nitrification, activities of alkaline phosphatase and arylsulphatase Lepidium sativum growth index (GI) and phenolic compounds were analysed in the soil in second and third year of the experiment. The addition of the SS separately or in combination with other remediation agents was found to be the most valuable for the number of microorganisms, intensification of nitrification process and enzymatic activities. In objects where other materials were added without sewage sludge, the inhibition of fungal growth as well as alkaline phosphatase and arylsulphatase activities was observed, however the inhibitory effect declined with time. The observed increase of GI shows the long-term, positive effect of treatments on soil properties concerning plant growth. The use of lime and lime together with sewage sludge contributed to the decrease in the content of phenolic compounds in the reclaimed soil.

Three-dimensional morphology of the Golgi apparatus in osteoclasts: NADPase and arylsulfatase cytochemistry, and scanning electron microscopy using osmium maceration.

This study was designed to observe osteoclasts in the rat femora by light and electron microscopic cytochemistry for nicotinamide adenine dinucleotide phosphatase (NADPase) and arylsulfatase, and scanning electron microscopy using osmium maceration to assess the three-dimensional morphology of the Golgi apparatus in osteoclasts. The Golgi apparatus showed strong NADPase activity and surrounded each nucleus with the cis-side facing the nucleus. The Golgi apparatus could be often traced for a length of 20 µm or longer. Observations of serial semi-thin sections confirmed that a single line of reaction products (=lead precipitates) intervened somewhere between any two neighboring nuclei. The nuclear membrane showed strong arylsulfatase activity as well as rough endoplasmic reticulum and lysosomes. Scanning electron microscopy showed that the Golgi apparatus covered the nucleus in a porous sheet-like configuration. Under magnification, the cis-most saccule showed a sieve-like configuration with fine fenestrations. The saccules decreased fenestration numbers toward the trans-side and displayed a more plate-like appearance. The above findings indicate the following. (1) The Golgi saccules of osteoclasts have a three-dimensional structure comparable with that generally seen in other cell types. (2) The Golgi apparatus forms a porous multi-spherical structure around nuclei. Within the structure, in most cases a Golgi stack partitions the room into several compartments in each of which a nucleus fits. (3) The nuclear membrane synthesizes some kinds of proteins more stably and sufficiently than the rough endoplasmic reticulum. Consequently, the Golgi apparatus accumulates around nuclei with the cis-side facing the nucleus.