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Telomerase is a specialized reverse transcriptase consisting of both RNA and protein components. Previous characterization of yeast telomerase function in vivo identified four EST (for ever shorter telomeres) genes that, when mutated, result in the phenotypes expected for a defect in telomerase. Consistent with this genetic prediction, the EST2 gene has recently been shown to encode the catalytic component of telomerase. Using an in vitro assay, we show here that telomerase activity is present in extracts prepared from yeast strains carrying est1-Delta, est3-Delta, and cdc13-2(est) mutations. Therefore, while these three genes are necessary for telomerase function in vivo, they do not encode components essential for core catalytic activity. When Est2p, the one EST gene product found to be essential for catalytic activity, was immunoprecipitated from extracts, the telomerase RNA subunit was also specifically precipitated, supporting the conclusion that these two components are in a stable complex.

Soft-tissue tumors with a predilection to involve the head and neck region include spindle cell lipoma, pleomorphic lipoma, and nuchal fibroma. We have recently studied three patients with distinctive soft-tissue fibrocartilaginous masses in the posterior aspect of the base of the neck, at the junction of the nuchal ligament and the deep cervical fascia. Two of the patients were women (ages 37 and 40) and one a man (age 53). All three had sustained neck injuries in automobile accidents in the past (27 years, 20 years, and 2 months before surgery, respectively). Each patient presented with a soft-tissue nodule overlying the spinous process of one of the lower cervical vertebrae. Two were painful. Computed tomography and magnetic resonance imaging performed in two patients showed focal thickening of the ligamentum nuchae at the C4-5 and C5-6 levels. All three masses were completely excised. They ranged in size from 1.0 to 1.5 cm. The three lesions were histologically identical, and each consisted of a poorly defined, moderately cellular fibrocartilage nodule located within the nuchal ligament at its junction with the deep cervical fascia. Atypia or mitotic activity was not present. The ligamentous tissue adjacent to the mass was irregular and degenerated. None of the masses have recurred in follow-up periods of 3 to 6 months, and all patients are currently asymptomatic. The lesion we describe is a distinctive soft-tissue pseudotumor that occurs in the mid-line of the lower cervical spine within the nuchal ligament. It is likely non-neoplastic and probably develops as a reaction to soft-tissue injury, in an analogous manner to fibrocartilage metaplasia seen in degenerated tendoligamentous structures. We propose the term "nuchal fibrocartilaginous pseudotumor" for these lesions.

Telomerase is a ribonucleoprotein enzyme essential for the replication of chromosome termini in most eukaryotes. Telomerase RNA components have been identified from many organisms, but no protein component has been demonstrated to catalyze telomeric DNA extension. Telomerase was purified from Euplotes aediculatus, a ciliated protozoan, and one of its proteins was partially sequenced by nanoelectrospray tandem mass spectrometry. Cloning and sequence analysis of the corresponding gene revealed that this 123-kilodalton protein (p123) contains reverse transcriptase motifs. A yeast (Saccharomyces cerevisiae) homolog was found and subsequently identified as EST2 (ever shorter telomeres), deletion of which had independently been shown to produce telomere defects. Introduction of single amino acid substitutions within the reverse transcriptase motifs of Est2 protein led to telomere shortening and senescence in yeast, indicating that these motifs are important for catalysis of telomere elongation in vivo. In vitro telomeric DNA extension occurred with extracts from wild-type yeast but not from est2 mutants or mutants deficient in telomerase RNA. Thus, the reverse transcriptase protein fold, previously known to be involved in retroviral replication and retrotransposition, is essential for normal chromosome telomere replication in diverse eukaryotes.

We have recently completed a large mutant screen designed to identify new mutants of Saccharomyces cerevisiae with a telomerase-like defect. From this screen; 22 mutants were identified that mapped to three genes, called EST1, EST2 and EST3, as well as a novel EST-like mutation in a fourth gene, previously identified as CDC13. Mutations in each of these genes give rise to phenotypes that are indistinguishable from those observed when TLC1, encoding the yeast telomerase RNA, is deleted. In addition, genetic analysis indicates that all four genes function in the same pathway for telomere replication as defined by TLC1, the one known component of telomerase. This indicates that these genes encode factors that are essential in vivo for telomerase function. Genetic and biochemical analyses have shown that EST1 and CDC13 encode single-stranded telomeric DNA-binding proteins, suggesting that these two proteins may function to mediate access of telomerase to the end of the telomere.

The CDC13 gene has previously been implicated in the maintenance of telomere integrity in Saccharomyces cerevisiae. With the use of two classes of mutations, here it is shown that CDC13 has two discrete roles at the telomere. The cdc13-2est mutation perturbs a function required in vivo for telomerase regulation but not in vitro for enzyme activity, whereas cdc13-1ts defines a separate essential role at the telomere. In vitro, purified Cdc13p binds to single-strand yeast telomeric DNA. Therefore, Cdc13p is a telomere-binding protein required to protect the telomere and mediate access of telomerase to the chromosomal terminus.

The regulation of macrophage lipoprotein lipase (LPL) by cytokines is of potentially crucial importance in the pathogenesis of atherosclerosis and in the responses to endotoxin challenge. However, the precise mechanisms by which different cytokines modulate the expression of macrophage LPL activity are poorly understood. The action of six cytokines and bacterial lipopolysaccharide (LPS) on LPL function using the murine J774.2 cell line as a model system has, therefore, been studied. Although exposure to LPS, interleukin 11 (IL-11), tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma) and IL-1, over the physiological range of concentrations, resulted in a decrease in the heparin-releasable LPL activity, LPL-mRNA levels and LPL-protein content of the cells, stimulation with IL-6 and leukaemia inhibitory factor (LIF) had no effect. The maximum suppression of LPL activity and mRNA levels in the cells by IFN-gamma (60%) was lower than that produced by LPS, IL-11, TNF-alpha and IL-1 (78-97%). Each cytokine displayed a characteristic dose-dependent pattern for the suppression of LPL activity and mRNA levels with IL-11/TNF-alpha being more potent than IFN-gamma/IL-1. More than 80% of the decrease in the LPL activity, at all doses of IL-11, TNF-alpha, IFN-gamma and IL-1, was due to a corresponding reduction in the mRNA levels. The time course of responses to LPS, IL-11, TNF-alpha, IFN-gamma and IL-1 were similar, with the time required to achieve half maximal suppression of LPL activity being between 7 and 9.5 h in each case. These results indicate that LPL in J774.2 macrophages is regulated differentially by various cytokines and that the major control responsible for the reduction of LPL activity by IL-11, TNF-alpha, IFN-gamma and IL-1 is exerted at the level of mRNA metabolism (decreased transcription or RNA stability). The responses identified also displayed several differences to those described previously for adipocytes (e.g. 3T3-L1 cell line), thereby suggesting the existence of potential cell-specific mechanisms for the regulation of LPL by cytokines.

In order to search for additional regulatory elements in the human CYP17 (steroid 17 alpha-hydroxylase) gene and to compare it with potential regulatory elements in bovine CYP17 genes, 3.5 kb of 5' flanking region of CYP17 was cloned and analyzed. The newly acquired sequence was shown to be a highly defective copy of the human endogenous retrovirus HERV-K family. This retroviral sequence was itself interrupted by a novel element, a low copy number repeat occurring about 20 times in the human genome, including a known copy in the human catechol-O-methyltransferase gene. A reanalysis of the entire 5' flanking region of human CYP17 indicates that only the 300 bp immediately distal to the promoter is of unique sequence; other regulatory sequences, including any that are similar to the upstream region of the bovine genes, are unlikely to occur within 5.5 kb of the promoter.

Human CCRF-CEM cells were shown to contain energy-dependent and unidirectional efflux systems for the extrusion of methotrexate and cholate. Efflux activity was sensitive to temperature and to energy deprivation by treatment with antimycin A and to various other compounds which do not affect energy metabolism. A comparison of inhibitor sensitivities revealed that methotrexate and cholate efflux exhibit substantial differences in half-maximal inhibition (IC50) by indomethacin, reserpine, ethacrynic acid, ketoprofen, probenecid, and bromosulfophthalein, although these systems could not be distinguished by their responses to prostaglandin A1, meclofenamic acid, indoprofen, and biphenylacetic acid. Comparisons between cell lines showed that methotrexate efflux in CCRF-CEM cells exhibits an inhibitor response comparable to a secondary efflux system for methotrexate in L1210 cells (system II), whereas the inhibitor response of cholate efflux in CCRF-CEM cells resembles efflux system I in L1210 cells, the primary efflux route for both methotrexate and cholate. These results indicate that CCRF-CEM cells contain similar but separate systems for the efflux of methotrexate and cholate. CCRF-CEM cells thus differ from L1210 cells in that the latter mediate the efflux of methotrexate and cholate primarily via a single system.

1-Chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid were examined for the ability to inhibit unidirectional efflux routes in L1210 cells that extrude both methotrexate and cholate (system I) and methotrexate alone (system II). These electrophiles were selected for study because of their known ability to undergo rapid intracellular conversion to glutathione conjugates. CDNB produced typical inhibitor kinetics and was a moderate inhibitor of both system I (IC50 = 4.8 microM) and system II (IC50 = 7.5 microM) with methotrexate as the substrate. However, a complex response was observed when cholate was employed as an alternative substrate for system I. Cholate efflux was stimulated initially at low levels of CDNB, but then slowed to a net inhibition as CDNB concentrations exceeded 10 microM. The latter characteristics for CDNB were not observed with ethacrynic acid, which produced a comparable inhibition of efflux system I regardless of the substrate employed (IC50 = 4.6 microM). Efflux measurements in an L1210/C7 variant which lacks system I confirmed that CDNB stimulates the activity of a substantial and unique efflux activity for cholate (system III). The inhibition of system I and II by CDNB and ethacrynic acid was not reversed by a wash step but required inhibitor removal and subsequent incubation at 37 degrees C. This slow reversal was attributed to a time-dependent clearance of inhibitory glutathione conjugates. A correlation between efflux systems for anions and anionic glutathione conjugates was demonstrated further by the ability of prostaglandin A1 and indomethacin, two potent inhibitors of methotrexate and cholate efflux, to inhibit the efflux of 2,4-dinitrophenyl-S-glutathione. These results support the hypothesis that efflux systems for methotrexate and cholate in L1210 cells are part of a family of efflux pumps which function in vivo to extrude various anions and anionic glutathione conjugates.

CD59, a potent inhibitor of the human membrane attack complex (MAC) of complement, is present on many different tissues throughout the body. Recently we identified and characterized the rat analogue of CD59 and produced a number of monoclonal antibodies (mAb). We have now used these antibodies to demonstrate, by immunofluorescence microscopy, that rat CD59 was widely expressed in the rat kidney. Staining of renal sections revealed the presence of rat CD59 in abundance in the glomerulus, collecting ducts and distal tubules. Staining was abolished by treatment of sections with phosphatidylinositol-specific phospholipase C (PIPLC). Rat mesangial cells also stained strongly for rat CD59, giving an intensely granular staining pattern. In complement attack experiments the cultured mesangial cells were rendered more susceptible to lysis by rat or human complement after preincubation with the anti-inhibitor mAb. The results demonstrate that the rat analogue of CD59 is present and functionally active in rat renal tissue. The protective effect of CD59 against MAC-mediated tissue injury can now be examined in rat models of human renal disease.

The specificity and function of two unidirectional anion-efflux pumps in mouse L1210 cells were evaluated using a variant cell line selected for growth in the presence of cholate and bromosulfophthalein. Transport analysis revealed that cholate efflux in the variant L1210/C7 cell line had declined 8-fold, due to the loss of a bromosulfophthalein-sensitive efflux system, the major extrusion route for cholate in parental cells. Efflux measurements showed further that a bromosulfophthalein-sensitive efflux system for methotrexate was also absent in L1210/C7 cells. Total unidirectional efflux of methotrexate, however, was similar in the variant and parental cells, since the loss in the bromosulfophthalein-sensitive system was compensated by a rise in a second probenecid-sensitive route. The latter was identified from inhibitor studies to be the same system which acts as a minor efflux route for methotrexate in parental cells. These results support the hypothesis that L1210 cells contain a bromosulfophthalein-sensitive efflux system which mediates the unidirectional extrusion of either methotrexate or cholate, and a second probenecid-sensitive route which differs from the bromosulfophthalein-sensitive system in inhibitor specificity and also in its ability to transport methotrexate but not cholate.

The membrane attack complex (MAC) of complement in humans is regulated by several membrane-bound proteins; however, no such proteins have so far been described in other species. Here we report the isolation and characterization of a rat erythrocyte membrane glycoprotein of molecular mass 21 kDa which inserts into cell membranes and is a potent inhibitor of the rat MAC. This protein, here called rat inhibitory protein (RIP), was first partially purified by column chromatography from a butanol extract of rat erythrocyte membranes. Monoclonal antibodies (Mabs) were raised against RIP and used for its affinity purification. Affinity-purified RIP was shown to inhibit in a dose-dependent manner the cobra venom factor (CVF)-mediated 'reactive' lysis of guinea pig erythrocytes by rat complement. Conversely, the anti-RIP MAbs 6D1 and TH9 were shown to markedly enhance the CVF-mediated lysis of rat erythrocytes by rat complement. RIP acted late in the assembly of the MAC (at or after the C5b-8 stage) and was releasable from the membranes of rat erythrocytes by phosphatidylinositol-specific phospholipase C. These features, together with its size, deglycosylation pattern and N-terminal amino acid sequence, lead us to conclude that RIP is the rat homologue of the human MAC-inhibitory protein CD59 antigen.