Human acid ceramidase ((AC) N-acylsphingosine amidohydrolase, EC 3.5.1.23) hydrolyzes the sphingolipid ceramide into sphingosine and free fatty acid. Ceramide is an essential component of all sphingolipids and an important cell-signaling molecule. Moreover, an inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease. Human AC was purified from urine, and 117 amino acid residues were determined by microsequencing. Degenerative oligonucleotide probes were then constructed and used to screen for human fibroblast and pituitary cDNA libraries. Several partial cDNA clones were obtained, and two of these were combined to construct a full-length cDNA containing a 17-base pair (bp) 5-untranslated sequence, a 1185-bp open reading frame encoding 395 amino acids, a 1110-bp 3-untranslated sequence, and an 18-bp poly(A) tail. Transient expression of the full-length cDNA in COS-1 cells led to a 10-fold increase in AC activity. In addition, biosynthetic studies carried out in the transfected cells demonstrated that 13-kDa (␣) and 40-kDa (␤) AC subunits were derived from a common 55-kDa precursor encoded by the full-length cDNA. This protein pattern was identical to that seen in normal human skin fibroblasts. A homoallelic point mutation (T222K) was also identified in the AC gene of a patient suffering from Farber disease, further confirming the authenticity of the full-length cDNA.Human acid ceramidase ((AC) 1 N-acylsphingosine amidohydrolase, EC 3.5.1.23) catalyzes the hydrolysis of ceramide to free fatty acid and sphingosine (1). An inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease (FD), also called Farber lipogranulomatosis (2). Patients with FD accumulate ceramide in most tissues, leading to painful swelling of the joints and tendons, pulmonary insufficiency, and a shortened life-span. The clinical diagnosis of FD is usually confirmed by biochemical methods, including the determination of lysosomal ceramide accumulation and/or the deficiency of AC activity. To date, seven FD subtypes have been described with varying degrees of clinical involvement; notably, a direct correlation between the amount of ceramide accumulation and the clinical severity of FD patients has recently been demonstrated (3).In addition to its central role in disease pathogenesis, sphingolipid biosynthesis, and membrane formation, ceramide is an important cell-signaling molecule involved in a variety of diverse processes such as neurite growth, monocyte differentiation, and Fas (Apo1/CD95)-induced apoptosis (for reviews, see Refs. 4 and 5). Moreover, sphingosine (the catabolic product of ceramide degradation) has been shown to inhibit protein kinase C activity and can exert a variety of effects on cell growth and differentiation (6 -8). Since ceramide degradation is the only catabolic source of intracellular sphingosine (9, 10), AC activity may be the rate-limiting step in determining the intracellular levels of this compound.About 2 years ago, AC was purified...

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Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human urine and 12 tryptic peptides were microsequenced (128 residues). Based on regions of minimal codon redundancy, four oligonucleotide mixtures were synthesized and oligonucleotide mixture 1 (20mer; 256 mix) was used to screen 3 X 10(6) independent recombinants from a human fibroblast cDNA library. Putative positive clones (92) were purified and analyzed by Southern hybridization with oligonucleotide mixtures 2‐4. These studies revealed two groups of clones; group 1 (80 clones; inserts ranging from approximately 1.2 to 1.6 kb) hybridized with oligonucleotides mixtures 1‐4, while group II (12 clones; inserts ranging from approximately 1.2 to 1.4 kb) hybridized with oligonucleotide mixtures 1‐3. Several group II clones had larger inserts than those in group I, but did not hybridize with oligonucleotide mixture 4. Screening of a human placental cDNA library with a 450 bp group I fragment, also resulted in the isolation of group I and II clones. Representative clones from group I (pASM‐1) and group II (pASM‐2) were sequenced. pASM‐1 contained a 1879 bp insert which was colinear with 96 microsequenced amino acids, while the pASM‐2 1382 bp insert was colinear with 78 microsequenced residues. Notably, pASM‐2 did not have an internal 172 bp sequence encoding 57 amino acid residues, but had instead an in‐frame 40 bp sequence encoding 13 amino acids which was not present in pASM‐1. These findings demonstrate the presence of two distinct acid sphingomyelinase transcripts in human fibroblasts and placenta and suggest the occurrence of alternative processing of the mRNA encoding this lysosomal hydrolase.

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Abstract— A UV‐dosimeter has been developed for routine measurements which mainly weights the various components of the spectrum in relation to their damaging effects on a microorganism. For this purpose a biofilm was constructed, comprising dried spores of Bacillus subtilis (wild‐type or DNA repair defective strain), immobilized on transparent polyester plastic sheets. After irradiation, the biofilm was incubated in a growth medium. The proteins, synthesized by the immobilized microorganisms after spore germination and several cell divisions, were stained and determined by photometry, giving a measure of the biological activity. The ”biologically effective dose“ was determined from a calibration curve. It reflects the dose equivalent to that of the calibration source producing the same effect. The UV‐response of the biofilm is additive and follows the reciprocity law in the range of fluence rates investigated. The response is nearly independent of temperature within the range of ‐20°C to +70°C as well as of humidity between about 37 and 80% relative humidity. Storage for up to 9 months at <70% relative humidity and room temperature does not significantly influence the viability of the spores.