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Cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates the translation of numerous mRNAs. We previously showed that AU‐rich binding factor 1 (AUF1) regulates Cpeb1 expression through the 3’ untranslated region (3’UTR). To investigate the molecular basis of the regulatory potential of the Cpeb1 3’UTR, here we performed reporter analyses that examined expression levels of Gfp reporter mRNA containing the Cpeb1 3’UTR. Our findings indicate that CPEB1 represses the translation of Cpeb1 mRNA and that miR‐145a‐5p and let‐7b‐5p are involved in the reduction in Cpeb1 expression in the absence of AUF1. These results suggest that Cpeb1 expression is post‐transcriptionally regulated by AUF1, CPEB1, and microRNAs., We have previously shown that loss of interaction between AREs in the Cpeb1 3’UTR and AUF1, an mRNA decay factor, increased Cpeb1 mRNA levels but reduced the protein levels (Oe S. et al. 2021, Biochem Biophys Res Commun 534, 491‐497). In this study, we revealed that Cpeb1 expression is post‐transcriptionally regulated by multiple inhibitory machineries that are regulated by AUF1, CPEB1, and microRNAs.

Lamellar corpuscles function as mechanoreceptors in the skin, composed of axon terminals and lamellae constructed by terminal Schwann cells. They are classified into Pacinian, Meissner, and simple corpuscles based on histological criteria. Lamellar corpuscles in rat dermal papilla cells have been reported; however, the morphological aspects have yet to be thoroughly investigated. In the present study, we analyzed the enzyme activity, distribution, fine structure, and three-dimensional innervation of lamellar corpuscles in rat plantar skin. The lamellar corpuscles exhibiting non-specific cholinesterase were densely distributed in rat footpads, evident as notable skin elevations, especially at the apex, the highest portion of the ridges in each footpad. In contrast, only a few lamellar corpuscles were found in other plantar skin areas. Lamellar corpuscle was considered composed of a flat axon terminal Schwann cell lamellae, which were roughly concentrically arranged in the dermal papilla. These histological characteristics correspond to those of the simple corpuscle. Moreover, the axon tracing method revealed that one trunk axon innervated several simple corpuscles. The territory of the trunk axons overlapped with each other. Finally, the animals' footprints were analyzed. During the pausing and walking phases, footpads are often in contact with the floor. These results demonstrate that the type of lamellar corpuscles in the dermal papillae of rat plantar skin is a simple corpuscle and implies that their distribution pattern in the plantar skin is convenient for efficient sensing and transmission of mechanical stimuli from the ground.

Urodele amphibians have exceptional regeneration ability in various organs. Among these, the Iberian ribbed newt (Pleurodeles waltl) has emerged as a useful model organism for investigating the mechanisms underlying regeneration. Neural stem cells (NSCs) are an important source of regeneration in the central nervous system (CNS) and their culture method in vitro has been well established. NSCs form spherical cell aggregates called neurospheres and their formation has been demonstrated in various vertebrates, including some urodele species, but not in P. waltl. In this study, we reported neurosphere formation in brain- and spinal cord-derived cells of post-metamorphic P. waltl. These neurospheres showed proliferative activity and similar expression of marker proteins. However, the surface morphology was found to vary according to their origin, implying that the characteristics of the neurospheres generated from the brain and spinal cord could be similar but not identical. Subsequent in vitro differentiation analysis demonstrated that spinal cord-derived neurospheres gave rise to neurons and glial cells. We also found that cells in neurospheres from P. waltl differentiated to oligodendrocytes, whereas those from axolotls were reported not to differentiate to this cell type under standard culture conditions. Based on our findings, implantation of genetically modified neurospheres together with associated technical advantages in P. waltl could reveal pivotal gene(s) and/or signaling pathway(s) essential for the complete spinal cord regeneration ability in the future.

Scaffold attachment factor (SAFB) 1 and its homologue SAFB2 are multifunctional proteins that are involved in various cellular mechanisms, including chromatin organization and transcriptional regulation, and are also corepressors of estrogen receptor alpha (ERα). Both SAFBs are expressed at high levels in the brain. However, the distributions of SAFB1 and SAFB2 have yet to be characterized in detail and it is unclear whether both proteins interact with ERα in the brain. In this study, we investigated the expression and distribution of both SAFBs and their interaction with ERα in adult male rat brain. Immunohistochemical staining showed that SAFB1 and SAFB2 have a similar distribution pattern and are widely expressed throughout the brain. Double-fluorescence immunohistochemical and immunocytochemical analyses in primary cultures showed that the two SAFB proteins are localized in nuclei of neurons, astrocytes, and oligodendrocytes. Of note, SAFB2 was also found in cytoplasmic regions in these cell lineages. Both SAFB proteins were also expressed in ERα-positive cells in the medial preoptic area (MPOA) and arcuate and ventromedial hypothalamic nuclei. Co-immunoprecipitation experiments revealed that both SAFB proteins from the MPOA reciprocally interact with endogenous ERα. These results indicate that, in addition to a role in basal cellular function in the brain, the SAFB proteins may serve as ERα corepressors in hormone-sensitive regions.

Diverse molecular species of sulfatide with differences in FA lengths, unsaturation degrees, and hydroxylation statuses are expressed in the kidneys. However, the physiological functions of specific sulfatide species in the kidneys are unclear. Here, we evaluated the distribution of specific sulfatide species in the kidneys and their physiological functions. Electron microscopic analysis of kidneys of Cst-deficient mice lacking sulfatide showed vacuolar accumulation in the cytoplasm of intercalated cells in the collecting duct, whereas the proximal and distal tubules were unchanged. Immunohistochemical analysis revealed that vacuolar H

Injured optic nerves induce death in almost all retinal ganglion cells (RGC) and cause a loss of axons. To date, we have studied injured RGC axon regeneration by using a traumatic optic nerve injury (TONI) rodent model, and we revealed that axonal regeneration is induced by the graft of an autologous peripheral nerve. The efficient approach to the regeneration of axons thus needs an environmental adjustment of RGC. However, the RGC environment induced by TONI remains unknown. Here, we analyzed female and male C57BL/6 mouse retinal tissue alterations in detail after TONI and focused on the major phospholipid species that are enriched in the whole retina. Reactive astrocyte accumulation, glia scar formation, and demyelination were observed in the injured optic nerve area, while RGC cell death, astrocyte accumulation, and Glial fibrillary acidic protein (GFAP) positive Müller cell increases were detected in the retinal layer. Furthermore, phosphatidylinositol (PI) 18:0/20:4 was localized to three nuclear layer structures: the ganglion cell layer (GCL), the inner nuclear layer (INL), and the outer nuclear layer (ONL) in control retina; however, the localization of 18:0/20:4 PI in TONI was disturbed. Meanwhile, phosphatidylserine (PS) 18:0/22:6 showed that the expression was specifically in the inner plexiform layer (IPL) with similar signal intensity in both cases. Other PS species and phosphatidylethanolamine (PE) were differentially localized in the retinal layer; however, the expressions of PE including docosahexaenoic acid (DHA) were affected by TONI. These results suggest that not only GCL but also other retinal layers were influenced by TONI.

In the dorsal root ganglia (DRG), two types of glial cells (Schwann cells and satellite glial cells) have been identified based on cell morphology and expression of specific markers. In the present study, we observed unknown glial cells that were positive for p75 neurotrophin receptor (p75NTR), and therefore were immunohistochemically and ultrastructurally characterized for the first time. These cells exhibited stronger immunoreactivity against an anti-p75NTR antibody than the DRG neurons (hereafter referred to as p75NTR++ cells). Moreover, these cells covered the glial cells surrounding proximal process of the large-diameter DRG neurons. The proximal process is called "dendro-axon." The p75NTR++ cells were predominantly distributed where the first myelinating Schwann cells appear. The p75NTR++ cells were also positive for the pan-glial cell markers S100, nestin, and Sox10, but negative for fibroblast and macrophage markers. Moreover, they were negative for a satellite glial cell marker, inwardly rectifying potassium channel Kir4.1, as well as a nonmyelinating Schwann cell marker, glial fibrillary acidic protein. In addition, their morphological features were distinct from those of the myelinating Schwann cells. To investigate the three-dimensional ultrastructure of the p75NTR++ cells, we used array tomography combined with correlative light and electron microscopic observation. Three-dimensional ultrastructural observation revealed that the p75NTR++ cells loosely covered glial cells around the dendro-axons with highly ramified processes. Glial cells with these morphological features have not been reported before, indicating that the p75NTR++ glial cells are a new glial cell type in the DRG. Our results will give new insights into cell-cell relationships.

The hypocretin/orexin neuropeptide system coordinates the regulation of various physiological processes. Our previous study reported that a reduction in the expression of pleomorphic adenoma gene-like 1 (Plagl1), which encodes a C2H2 zinc-finger transcription factor, occurs in hypocretin neuron-ablated transgenic mice, suggesting that PLAGL1 is co-expressed in hypocretin neurons and regulates hypocretin transcription. The present study examined whether canonical prepro-hypocretin transcription is functionally modulated by PLAGL1. Double immunostaining indicated that the majority of hypocretin neurons were positive for PLAGL1 immunore-activity in the nucleus. Notably, PLAGL1 immunoreactivity in hypocretin neurons was altered in response to several conditions affecting hypocretin function. An uneven localization of PLAGL1 was detected in the nuclei of hypocretin neurons following sleep deprivation. Chromatin immunoprecipitation revealed that endogenous PLAGL1 may bind to a putative PLAGL1-binding site in the proximal region of the hypocretin gene, in the murine hypothalamus. In addition, electroporation of the PLAGL1 expression vector into the fetal hypothalamus promoted hypothalamic hypocretin transcription. These results suggested that PLAGL1 may regulate hypothalamic hypocretin transcription.

Highlights • This work demonstrates the expression patterns for lamin subtypes in neural cells in the adult rat cerebral cortex. • This work demonstrates suitable fixative conditions of multiple immunostaining systems for lamins and cell-type specific marker proteins. • Neural cells in the adult cerebral cortex showed heterogeneous lamin antibody labeling patterns., Lamins are type V intermediate filament proteins that are located beneath the inner nuclear membrane. In mammalian somatic cells, LMNB1 and LMNB2 encode somatic lamins B1 and B2, respectively, and the LMNA gene is alternatively spliced to generate somatic lamins A and C. Mutations in lamin genes have been linked to many human hereditary diseases, including neurodegenerative disorders. Knowledge about lamins in the nervous system has been accumulated recently, but a precise analysis of lamin subtypes in glial cells has not yet been reported. In this study we investigated the composition of lamin subtypes in neurons, astrocytes, oligodendrocyte-lineage cells, and microglia in the adult rat cerebral cortex using an immunohistochemical staining method. Lamin A was not observed in neurons and glial cells. Lamin C was observed in astrocytes, mature oligodendrocytes and neurons, but not observed in oligodendrocyte progenitor cells. Microglia also did not stain positive for lamin C which differed from macrophages, with lamin C positive. Lamin B1 and B2 were observed in all glial cells and neurons. Lamin B1 was intensely positive in oligodendrocyte progenitor cells compared with other glial cells and neurons. Lamin B2 was weakly positive in all glial cells compared to neurons. Our current study might provide useful information to reveal how the onset mechanisms of human neurodegenerative diseases are associated with mutations in genes for nuclear lamin proteins.

Cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates polyadenylation and subsequent translation of CPE-containing mRNAs involved in various physiological and pathological phenomena. Although the significance of CPEB1-mediated translational regulation has recently been reported, the detailed regulatory mechanism of Cpeb1 expression remains unclear. To elucidate the post-transcriptional regulatory mechanisms of Cpeb1 expression, we constructed reporter plasmids containing various deletions or mutations in the Cpeb1 mRNA 3' untranslated region (3'UTR). We investigated their expression levels in Neuro2a neuroblastoma cells. We found that Cpeb1 expression is regulated through an AU-rich element in its 3'UTR. Furthermore, the mRNA decay factor AU-rich binding factor 1 (AUF1) regulates Cpeb1 expression, and knockdown of AUF1 upregulates Cpeb1 mRNA expression but results in a decrease in CPEB1 protein levels. These findings indicate that AUF1 has a discordant role in the expression of Cpeb1.

Sox2 is a transcriptional factor expressed in neural stem cells. It is known that Sox2 regulates cell differentiation, proliferation and survival of the neural stem cells. Our previous study showed that Sox2 is expressed in all satellite glial cells of the adult rat dorsal root ganglion. In this study, to examine the role of Sox2 in satellite glial cells, we establish a satellite glial cell-enriched culture system. Our culture method succeeded in harvesting satellite glial cells with the somata of neurons in the dorsal root ganglion. Using this culture system, Sox2 was downregulated by siRNA against Sox2. The knockdown of Sox2 downregulated ErbB2 and ErbB3 mRNA at 2 and 4 days after siRNA treatment. MAPK phosphorylation, downstream of ErbB, was also inhibited by Sox2 knockdown. Because ErbB2 and ErbB3 are receptors that support the survival of glial cells in the peripheral nervous system, apoptotic cells were also counted. TUNEL-positive cells increased at 5 days after siRNA treatment. These results suggest that Sox2 promotes satellite glial cell survival through the MAPK pathway via ErbB receptors.

Sex-determining region Y (SRY)-box 2 (Sox2) is a member of the Sox family transcription factors. In the central nervous system, Sox2 is expressed in neural stem cells from neurogenic regions, and regulates stem cell proliferation and differentiation. In the peripheral nervous system, Sox2 is found only in the immature and dedifferentiated Schwann cells, and is involved in myelination inhibition or N-cadherin redistribution. In the present immunohistochemical study, we found that Sox2 is also expressed in other cells of the adult rat peripheral nervous system. Nuclear Sox2 was observed in all satellite glial cells, non-myelinating Schwann cells, and the majority of terminal Schwann cells that form lamellar corpuscles and longitudinal lanceolate endings. Sox2 was not found in myelinating Schwann cells and terminal Schwann cells of subepidermal free nerve endings. Satellite glial cells exhibit strong Sox2 immunoreactivity, whereas non-myelinating Schwann cells show weak immunoreactivity. RT-PCR confirmed the presence of Sox2 mRNA, indicating that the cells are likely Sox2 expressors. Our findings suggest that the role of Sox2 in the peripheral nervous system may be cell-type-dependent.

Dramatic changes in the cytoskeleton and the morphology of oligodendrocytes (OLs) occur during various stages of the myelination process. OLs in culture produce large membrane sheets containing cytoskeletal veins of microtubules and actin filaments. We recently showed that estrogen receptors (ER) related to ERα/β were expressed in the membrane sheets of mature OLs in culture. Ligation of these or other membrane ERs in OLs with both 17β- and 17α-estradiol mediated rapid non-genomic signaling. Here, we show that estrogens also mediate rapid non-genomic remodeling of the cytoskeleton in mature OLs in culture. 17β-Estradiol caused a rapid loss of microtubules and the actin cytoskeleton in the OL membrane sheets. It also increased phosphorylation of the actin filament-severing protein cofilin, thus inactivating it. Staining for actin barbed ends with rhodamine-actin showed that it decreased the amount of actin barbed ends. 17α-Estradiol, on the other hand, increased the percentage of cells with abundant staining of actin filaments and actin barbed ends, suggesting that it stabilized and/or increased the dynamics of the actin cytoskeleton. The specific ERα and ERβ agonists, 4,4',4″-(4-propyl-(1H)-pyrazole-1,3,5-triyl) trisphenol (PPT) and diarylpropionitrile 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN), respectively, also caused the rapid phosphorylation of cofilin. Estrogen-induced phosphorylation of cofilin was inhibited by Y-27632, a specific inhibitor of the Rho-associated protein serine/threonine kinase (ROCK). The Rho/ROCK/cofilin pathway is therefore implicated in actin rearrangement via estrogen ligation of membrane ERs, which may include forms of ERα and ERβ. These results indicate a role for estrogens in modulation of the cytoskeleton in mature OLs, and thus in various processes required for myelinogenesis.

Lamins are type V intermediate filament proteins that support nuclear membranes. They are divided into A-type lamins, which include lamin A and C, and B-type lamins, which include lamin B1 and B2. In the rat brain, lamin A and C are expressed in relatively equal amounts, while the expressions of lamin B1 and B2 vary depending on the cell type. Lamins play important roles in normal morphogenesis and function. In the nervous system, their abnormal expression causes several neurodegenerative diseases such as peripheral neuropathy, leukodystrophy and lissencephaly. The retina belongs to the central nervous system (CNS) and has widely been used as a source of CNS neurons. We investigated the expression patterns of lamin subtypes in the adult rat retina by immunohistochemistry and found that the staining patterns differed when compared with the brain. All retinal neurons expressed lamin B1 and B2 in relatively equal amounts. In addition, horizontal cells and a subpopulation of retinal ganglion cells expressed lamin A and C, while photoreceptor cells expressed neither lamin A nor C, and all other retinal neurons expressed lamin C only. This differential expression pattern of lamins in retinal neurons suggests that they may be involved in cellular differentiation and expression of cell-specific genes in individual retinal neurons.

Glycosphingolipids (GSLs) can interact with each other by homotypic or heterotypic trans carbohydrate-carbohydrate interactions across apposed membranes, resulting in cell-cell adhesion. This interaction can also provide an extracellular signal which is transmitted to the cytosolic side, thus forming a glycosynapse between two cells. The two major GSLs of myelin, galactosylceramide (GalC) and its sulfated form, galactosylceramide I(3)-sulfate (SGC), are an example of a pair of GSLs which can participate in these trans carbohydrate-carbohydrate interactions and trigger transmembrane signaling. These GSLs could interact across apposed oligodendrocyte membranes at high cell density or when a membranous process of a cell contacts itself as it wraps around the axon. GalC and SGC also face each other in the apposed extracellular surfaces of the multilayered myelin sheath. Communication between the myelin sheath and the axon regulates both axonal and myelin function and is necessary to prevent neurodegeneration. Participation of transient GalC and SGC interactions in glycosynapses between the apposed extracellular surfaces of mature myelin might allow transmission of signals throughout the myelin sheath and thus facilitate myelin-axonal communication.

Galactosylceramide (GalC) and its sulfated analogue, sulfatide, are major galactosphingolipid components of myelin and oligodendrocyte plasma membranes in the nervous system. We previously hypothesized that these galactolipids play functional roles in the regulation of oligodendrocyte terminal differentiation by acting as sensors/transmitters of environmental information. Evidence strongly supports this idea. First, these molecules are initially expressed on the cell surface at the interface at which oligodendrocyte progenitors first enter terminal differentiation. Second, exposure of oligodendrocyte progenitors to anti-GalC/-sulfatide (RmAb) or antisulfatide (O4), but not anti-GalC (O1), antibodies leads to the reversible arrest of oligodendrocyte lineage progression at this interface. Third, in cerebroside galactosyl transferase-null mice (Cgt−/−) that are unable to synthesize either GalC or sulfatide, terminal differentiation and morphological maturation of oligodendrocytes are enhanced. In the present study, we examined oligodendrocytes differentiation in cerebroside sulfotransferase-null mice (Cst−/−) that lack sulfatide but express GalC. We show that cerebroside sulfotransferase mRNA expression begins already in the embryonic spinal cord and progressively increases with age, that the late progenitor marker POA is not synthesized in the absence of this enzyme, and that, most notably, there is a two- to threefold enhancement in the number of terminally differentiated oligodendrocytes both in culture and in vivo, similar to that in mice lacking both GalC and sulfatide. We conclude that primarily sulfatide, rather than GalC, is a key molecule for the negative regulation of oligodendrocyte terminal differentiation. © 2003 Wiley-Liss, Inc.

Mononuclear cells infiltrating the interstitium are involved in renal tubulointerstitial injury. The unilateral ureteral obstruction (UUO) is an established experimental model of renal interstitial inflammation. In our previous study, we postulated that L-selectin on monocytes is involved in their infiltration into the interstitium by UUO and that a sulfated glycolipid, sulfatide, is the physiological L-selectin ligand in the kidney. Here we tested the above hypothesis using sulfatide- and L-selectin-deficient mice. Sulfatide-deficient mice were generated by gene targeting of the cerebroside sulfotransferase (Cst) gene. Although the L-selectin-IgG chimera protein specifically bound to sulfatide fraction in acidic lipids from wild-type kidney, it did not show such binding in fractions of Cst(-/-) mice kidney, indicating that sulfatide is the major L-selectin-binding glycolipid in the kidney. The distribution of L-selectin ligand in wild-type mice changed after UUO; sulfatide was relocated from the distal tubules to the peritubular capillaries where monocytes infiltrate, suggesting that sulfatide relocated to the endothelium after UUO interacted with L-selectin on monocytes. In contrast, L-selectin ligand was not detected in Cst(-/-) mice irrespective of UUO treatment. Compared with wild-type mice, Cst(-/-) mice showed a considerable reduction in the number of monocytes/macrophages that infiltrated the interstitium after UUO. The number of monocytes/macrophages was also reduced to a similar extent in L-selectin(-/-) mice. Our results suggest that sulfatide is a major L-selectin-binding molecule in the kidney and that the interaction between L-selectin and sulfatide plays a critical role in monocyte infiltration into the kidney interstitium.

Myelinated axons are divided into four distinct regions: the node of Ranvier, paranode, juxtaparanode, and internode, each of which is characterized by a specific set of axonal proteins. Voltage-gated Na(+) channels are clustered at high densities at the nodes, whereas shaker-type K(+) channels are concentrated at juxtaparanodal regions. These channels are separated by the paranodal regions, where septate-like junctions are formed between the axon and the myelinating glial cells. Although oligodendrocytes and myelin sheaths are believed to play an instructive role in the local differentiation of the axon to distinct domains, the molecular mechanisms involved are poorly understood. In the present study, we have examined the distribution of axonal components in mice incapable of synthesizing sulfatide by disruption of the galactosylceramide sulfotransferase gene. These mice displayed abnormal paranodal junctions in the CNS and PNS, whereas their compact myelin was preserved. Immunohistochemical analysis demonstrated a decrease in Na(+) and K(+) channel clusters, altered nodal length, abnormal localization of K(+)channel clusters appearing primarily in the presumptive paranodal regions, and diffuse distribution of contactin-associated protein along the internode. Similar abnormalities have been reported previously in mice lacking both galactocerebroside and sulfatide. Interestingly, although no demyelination was observed, these channel clusters decreased markedly with age. The initial timing and the number of Na(+) channel clusters formed were normal during development. These results indicate a critical role for sulfatide in proper localization and maintenance of ion channels clusters, whereas they do not appear to be essential for initial cluster formation of Na(+) channels.

Chondroitin 4-sulfotransferase, which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of N-acetylgalactosamine in chondroitin, was purified 1900-fold to apparent homogeneity with 6.1% yield from the serum-free culture medium of rat chondrosarcoma cells by affinity chromatography on heparin-Sepharose CL-6B, Matrex gel red A-agarose, 3',5'-ADP-agarose, and the second heparin-Sepharose CL-6B. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands. Molecular masses of these protein were 60 and 64 kDa under reducing conditions and 50 and 54 kDa under nonreducing conditions. Both the protein bands coeluted with chondroitin 4-sulfotransferase activity from Toyopearl HW-55 around the position of 50 kDa, indicating that the active form of chondroitin 4-sulfotransferase is a monomer. Dithiothreitol activated the purified chondroitin 4-sulfotransferase. The purified enzyme transferred sulfate to chondroitin and desulfated dermatan sulfate. Chondroitin sulfate A and chondroitin sulfate C were poor acceptors. Chondroitin sulfate E from squid cartilage, dermatan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin hardly served as acceptors of the sulfotransferase. The transfer of sulfate to the desulfated dermatan sulfate occurred preferentially at position 4 of the N-acetylgalactosamine residues flanked with glucuronic acid residues on both reducing and nonreducing sides.

We have isolated a cDNA clone encoding human 3′-phosphoadenylylsulfate:galactosylceramide 3′-sulfotransferase (EC 2.8.2.11). Degenerate oligonucleotides, based on amino acid sequence data for the purified enzyme, were used as primers to amplify fragments of the gene from human renal cancer cell cDNA by the polymerase chain reaction method. The amplified cDNA fragment was then used as probe to screen a human renal cancer cell cDNA library. The isolated cDNA clone contained an open reading frame encoding 423 amino acids including all of the peptides that were sequenced. The deduced amino acid sequence predicts a type II transmembrane topology and contains two potential N-glycosylation sites. There is no significant homology between this sequence and either the sulfotransferases cloned to date or other known proteins. Northern blot analysis demonstrated that a 1.9-kilobase mRNA was unique to renal cancer cells. When the cDNA was inserted into the expression vector pSVK3 and transfected into COS-1 cells, galactosylceramide sulfotransferase activity in the transfected cells increased from 8- to 16-fold over that of controls, and the enzyme product, sulfatide, was expressed on the transformed cells.