Plasma levels of ß2-microglobulin are associated with atherosclerosis in patients with systemic lupus erythematosus: a cross-sectional cohort study.

Objective The objective of this paper is to examine the association between plasma levels of ß2-microglobulin (ß2MG), a protein previously associated with atherosclerosis, and the presence of carotid plaque (CP) or coronary artery calcium (CAC) in a cross-sectional cohort study of patients with systemic lupus erythematosus (SLE). Methods Patients with SLE were enrolled between June 2013 and May 2014. The presence of CP and CAC was assessed with ultrasonography and computed tomography scan, respectively. The presence of CP or CAC in the SLE patients was analyzed with respect to plasma levels of ß2MG and renal function expressed as the estimated glomerular filtration rate (eGFR). Results The study cohort consisted of 147 patients, 89% women and 95% Caucasians. The median age was 46 (range: 21-75) years with a median disease duration of 14 years. CP and CAC was observed in 29 (20%) and 57 (39%) of patients, respectively. CP or CAC was seen in 62 (42%) patients and was associated with the highest quartile of plasma ß2MG in patients with eGFR ≥ 90 ml/min/1.73 m2; OR = 18 (95% CI: 1.7-181). ß2MG adjusted for eGFR was also associated with presence of CP or CAC in the total cohort. The exclusion of 25 patients with a prior history of cardiovascular disease did not change the observed associations. Conclusion In this study, we found significant associations between imaging markers of atherosclerosis and high plasma levels of plasma ß2MG. These data suggest that ß2MG is a candidate for further study as a biomarker for atherosclerosis in SLE.

Expression patterns of DLK1 and INSL3 identify stages of Leydig cell differentiation during normal development and in testicular pathologies, including testicular cancer and Klinefelter syndrome.

STUDY QUESTION: What is the differentiation stage of human testicular interstitial cells, in particular Leydig cells (LC), within micronodules found in patients with infertility, testicular cancer and Klinefelter syndrome? SUMMARY ANSWER: The Leydig- and peritubular-cell populations in testes with dysgenesis contain an increased proportion of undifferentiated cells when compared with control samples, as demonstrated by increased delta-like homolog 1 (DLK1) and decreased insulin-like peptide 3 (INSL3) expression. WHAT IS KNOWN ALREADY: Normal LC function is essential for male development and reproduction. Signs of LC failure, including LC micronodules, are often observed in patients with reproductive disorders. STUDY DESIGN, SIZE, PARTICIPANTS: In this retrospective study, a panel of markers and factors linked to the differentiation of LCs was investigated in 33 fetal and prepubertal human specimens and in 58 adult testis samples from patients with testicular germ cell tumours, including precursor carcinoma in situ (CIS), infertility or Klinefelter syndrome. PARTICIPANTS/MATERIALS, SETTING, METHODS: The expression patterns of DLK1, INSL3, chicken ovalbumin upstream promoter transcription factor 2 (COUP-TFII), cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) and smooth muscle actin (SMA) were investigated by immunohistochemistry and quantitative RT-PCR. The percentage of positive LCs was estimated and correlated to total LC numbers and serum levels of reproductive hormones. MAIN RESULTS AND THE ROLE OF CHANCE: DLK1, INSL3 and COUP-TFII expression changed during normal development and was linked to different stages of LC differentiation: DLK1 was expressed in all fetal LCs, but only in spindle-shaped progenitor cells and in a small subset of polygonal LCs in the normal adult testis; INSL3 was expressed in a subset of fetal LCs, but in the majority of adult LCs; and COUP-TFII was expressed in peritubular and mesenchymal stroma cells at all ages, in fetal LCs early in gestation and in a subset of adult LCs. CYP11A1 was expressed in the majority of LCs regardless of age and pathology and was the best general LC marker examined here. SMA was weakly expressed in peritubular cells in the fetal and infantile testis, but strongly expressed in the adult testis. In pathological testes, the numbers of DLK1-positive interstitial cells were increased. The proportion of DLK1-positive LCs correlated with total LC numbers (R = 0.53; P < 0.001) and was higher in testis with enlargement of the peritubular layers (P < 0.01), which was also highly associated with DLK1 expression in the peritubular compartment (P < 0.001). INSL3 expression was absent in some, but not all LC micronodules, and in the majority of LCs, it was mutually exclusive of DLK1. LIMITATIONS, REASONS FOR CAUTION: The number of samples was relatively small and no true normal adult controls were available. True stereology was not used for LC counting, instead LCs were counted in three fields of 0.5 µm(2) surface for each sample. WIDER IMPLICATIONS OF THE FINDINGS: The population of LCs, especially those clustered in large nodules, are heterogeneous and comprise cells at different stages of differentiation. The study demonstrated that the differentiation and function of LCs, and possibly also peritubular cells, are impaired in adult men with testicular pathologies including testis cancer and Klinefelter syndrome. STUDY FUNDING/COMPETING INTERESTS: This work was funded by Rigshospitalet's research funds, the Danish Cancer Society and Kirsten and Freddy Johansen's foundation. The authors have no conflicts of interest.

Paracetamol (acetaminophen), aspirin (acetylsalicylic acid) and indomethacin are anti-androgenic in the rat foetal testis.

More than half the pregnant women in the Western world report taking mild analgesics. These pharmaceutical compounds have been associated with congenital cryptorchidism in humans, the best-known risk factor for low semen quality and testicular germ cell cancer. Furthermore, some of these mild analgesics exert potent anti-androgenic effects in the male rat and several endocrine-disrupting compounds, known to alter masculinization, have also been shown to be potent inhibitors of prostaglandin (PG) synthesis similar to mild analgesics. Using a 3-day ex vivo organotypic model system based on gestational day 14.5 rat testes, we herein show that testosterone production was inhibited by paracetamol, at doses of 0.1 µm to 100 µm. Similar results were obtained for aspirin (1-100 µm) and indomethacin (10 µm). The production of the other Leydig cell hormone, Insl3, was not disrupted by exposure to paracetamol. Investigations of the gross anatomy of the testis as well as Leydig cells number and rate of gonocyte apoptosis after the 3 days of ex vivo differentiation showed no significant effect of the analgesics tested compared with controls. These data indicate therefore that mild analgesics specifically inhibit testosterone production in rat foetal testes in vitro and that these compounds had no effect on gonocyte survival. Parallel determinations of prostaglandin D2 (PGD2) production indicated that the effects of paracetamol and aspirin on PGD2 and testosterone were not connected, whereas the effects of indomethacin were correlated. We conclude that mild analgesics exert direct and specific anti-androgenic effects in rat foetal testis in our experimental setup and that the mechanism of action is probably uncoupled from the inhibition of PG synthesis.

Expression of the vitamin D metabolizing enzyme CYP24A1 at the annulus of human spermatozoa may serve as a novel marker of semen quality.

Vitamin D (VD) is important for male reproduction in mammals and the VD receptor (VDR) and VD-metabolizing enzymes are expressed in human spermatozoa. The VD-inactivating enzyme CYP24A1 titrates the cellular responsiveness to VD, is transcriptionally regulated by VD, and has a distinct expression at the sperm annulus. Here, we investigated if CYP24A1 expression serves as a marker for VD metabolism in spermatozoa, and whether CYP24A1 expression was associated with semen quality. We included 130 men (53 healthy young volunteers and 77 subfertile men) for semen analysis and immunocytochemical (ICC) detection of CYP24A1. Another 40 men (22 young, 18 subfertile) were tested for in vitro effects of 1,25(OH)(2)D(3) on intracellular calcium concentration ([Ca(2+)](i)) and sperm motility. Double ICC staining showed that CYP24A1 and VDR were either concomitantly expressed or absent in 80% of the spermatozoa from young men. The median number of CYP24A1-expressing spermatozoa was 1% in subfertile men and thus significantly (p < 0.0005) lower than 25% in spermatozoa from young men. Moreover, CYP24A1 expression correlated positively with total sperm count, -concentration, -motility and -morphology (all p < 0.004), and the percentage of CYP24A1-positive spermatozoa increased (15 vs. 41%, p < 0.0005) after percoll-gradient-centrifugation. We noticed that the presence of >3% CYP24A1-positive spermatozoa distinguished young men from subfertile men with a sensitivity of 66.0%, a specificity of 77.9% and a positive predictive value of 98.3%. Functional studies revealed that 1,25(OH)(2)D(3) increased [Ca(2+)](i) and sperm motility in young healthy men, while 1,25(OH)(2)D(3) was unable to increase motility in subfertile patients. In conclusion, we suggest that CYP24A1 expression at the annulus may serve as a novel marker of semen quality and an objective proxy for sperm function.

A novel double staining strategy for improved detection of testicular carcinoma in situ cells in human semen samples.

Prompted by the recently reported expression of POU5F1 (OCT3/4) in epididymis, a panel of markers for carcinoma in situ (CIS) testis and testicular germ cell tumours (TGCT), including AP-2γ(TFAP2C), NANOG, OCT3/4, KIT, placental-like alkaline phosphatase (PLAP), M2A/PDPN and MAGE-A4 were examined by immunohistochemistry or in situ hybridisation in urogenital epithelia, which may interfere with detection of CIS cells in semen. In addition to OCT3/4, the expression of AP-2γ and NANOG or their variants was detected in urogenital epithelia, while other CIS markers, including PLAP/alkaline phosphatase were absent. A combination of immunocytological staining for AP-2γ or OCT3/4 and rapid cytochemical alkaline phosphatase reaction was subsequently developed. This approach was tested in 22 patients with TGCT. In 14 patients (63.6%), double stained cells were found and thus the method was proven suitable for the detection of CIS cells in semen. In conclusion, transcription factors related to pluripotency and undifferentiated state of cells, which most likely have several variants or modifications, are unexpectedly detected using currently available antibodies in urogenital epithelial cells which may be shed into semen. Combining the immunohistochemical nuclear markers with a rapid cytochemical alkaline phosphatase reaction for detection of CIS cells in ejaculates may provide a more reliable diagnostic method.

Lipoprotein lipase and endothelial lipase in human testis and in germ cell neoplasms.

The aim of this study was to investigate endothelial lipase (EL, LIPG) and lipoprotein lipase (LPL) mRNA and protein expression in normal human testis and testicular germ cell tumours (GCT). Both EL and LPL were expressed in normal seminiferous tubules and in the interstitial compartment. EL mRNA and protein were found in all germ cells as well as in Sertoli and Leydig cells. EL mRNA was abundant in pre-invasive carcinoma in situ (CIS) cells and GCTs, and EL protein was present in the cytoplasm of these cells. LPL mRNA was also relatively abundant in germ cells, Sertoli cells, CIS cells and GCTs. The LPL protein, however, was restricted to the cell membranes of pachytene spermatocytes and spermatids in normal tubules, absent from CIS cells and scarcely represented in tumours. The distribution of LPL protein in non-seminomas resembled the distribution of OCT3/4, a marker of embryonal carcinoma. The results suggest that both EL and LPL participate in the supply of nutrients and steroidogenesis in the testes, and that especially EL may be important for the supply of cholesterol for testosterone production in the Leydig cells. The partial cellular separation of the expression of the two lipases in normal testis suggests the existence of distinct biological roles, perhaps developmentally regulated, as indicated by the LPL expression in GCTs with embryonic features. A high expression of EL and abundance of lipid in tubules with CIS may have a diagnostic value.

Hypothesis: exposure to endocrine-disrupting chemicals may interfere with timing of puberty.

A recent decline in onset of puberty - especially among girls - has been observed, first in the US in the mid-1990s and now also in Europe. The development of breast tissue in girls occurs at a much younger age and the incidence of precocious puberty (PP) is increasing. Genetic factors and increasing prevalence of adiposity may contribute, but environmental factors are also likely to be involved. In particular, the widespread presence of endocrine-disrupting chemicals (EDCs) is suspected to contribute to the trend of earlier pubertal onset. The factors regulating the physiological onset of normal puberty are poorly understood. This hampers investigation of the possible role of environmental influences. There are many types of EDCs. One chemical may have more than one mode of action and the effects may depend on dose and duration of the exposure, as well as the developmental stage of the exposed individual. There may also be a wide range of genetic susceptibility to EDCs. Human exposure scenarios are complex and our knowledge about effects of mixtures of EDCs is limited. Importantly, the consequences of an exposure may not be apparent at the actual time of exposure, but may manifest later in life. Most known EDCs have oestrogenic and/or anti-androgenic actions and only few have androgenic or anti-oestrogenic effects. Thus, it appears plausible that they interfere with normal onset of puberty. The age at menarche has only declined by a few months whereas the age at breast development has declined by 1 year; thus, the time span from initiation of breast development to menarche has increased. This may indicate an oestrogen-like effect without concomitant central activation of the hypothalamic-pituitary axis. The effects may differ between boys and girls, as there are sex differences in age at onset of puberty, hormonal profiles and prevalence of precocius puberty.

Association of the polymorphism of the CAG repeat in the mitochondrial DNA polymerase gamma gene (POLG) with testicular germ-cell cancer.

BACKGROUND: A possible association between the polymorphic CAG repeat in the DNA polymerase gamma (POLG) gene and the risk of testicular germ-cell tumours (TGCT) was investigated in this study. The hypothesis was prompted by an earlier preliminary study proposing an association of the absence of the common 10-CAG-long POLG allele with testicular cancer as well as previously reported in some European populations' association with male subfertility, which is a condition carrying an increased risk of TGCT. PATIENTS AND METHODS: The number of CAG repeats in both POLG alleles was established in 243 patients with TGCT and in 869 controls by the analysis of the genomic DNA fragment. RESULTS: A significantly higher proportion of men homozygous allele of other than the common 10 CAG repeats was found among the patients with TGCT in comparison to the controls (4.9% versus 1.3%, respectively, P = 0.001). The vast majority of the homozygous patients had a seminoma (11 of 12; 97%), despite that only about half (55%) of the studied patients had this tumour type. CONCLUSIONS: The findings indicate that the POLG polymorphism may be a contributing factor in the pathogenesis of TGCT particularly in seminoma, but the mechanisms remain to be elucidated.

Adverse trends in male reproductive health: we may have reached a crucial 'tipping point'.

Healthy men produce an enormous number of sperms, far more than necessary for conception. However, several studies suggest that semen samples where the concentration of sperms is below 40 mill/mL may be associated with longer time to pregnancy or even subfertility, and specimens where the concentration of sperms is below 15 mill/mL may carry a high risk of infertility. Historic data from the 1940s show that the bulk of young men at that time had sperm counts far above 40 mill/mL with averages higher than 100 mill/mL. However, recent surveillance studies of young men from the general populations of young men in Northern Europe show that semen quality is much poorer. In Denmark approximately 40 percent of the men have now sperm counts below 40 mill/mL. A simulation assuming that average sperm count had declined from 100 mill/mL in 'old times' to a current level close to 40 mill/mL indicated that the first decline in average sperm number of 20-40 mill/mL might not have had much effect on pregnancy rates, as the majority of men would still have had counts far above the threshold value. However, due to the assumed decline in semen quality, the sperm counts of the majority of 20 year old European men are now so low that we may be close to the crucial tipping point of 40 mill/mL spermatozoa. Consequently, we must face the possibility of more infertile couples and lower fertility rates in the future.

The SRP9/14 subunit of the signal recognition particle (SRP) is present in more than 20-fold excess over SRP in primate cells and exists primarily free but also in complex with small cytoplasmic Alu RNAs.

The heterodimeric protein SRP9/14 bound to the Alu sequences of SRP RNA is essential for the translational control function of the signal recognition particle (SRP). The Alu RNAs of primate cells are believed to be derived from SRP RNA and have been shown to bind to an SRP14-related protein in vitro. We have used antibodies to characterize SRP9/14 and examine its association with small RNAs in vivo. Although SRP9 proteins are the same size in both rodent and primate cells, SRP14 subunits are generally larger in primate cells. An additional alanine-rich domain at the C-terminus accounts for the larger size of one human isoform. Although the other four SRP proteins are largely assembled into SRP in both rodent and primate cells, we found that the heterodimer SRP9/14 is present in 20-fold excess over SRP in primate cells. An increased synthesis rate of both proteins may contribute to their accumulation. The majority of the excess SRP9/14 is cytoplasmic and does not appear to be bound to any small RNAs; however, a significant fraction of a small cytoplasmic Alu RNA is complexed with SRP9/14 in a 8.5 S particle. Our findings that there is a large excess of SRP9/14 in primate cells and that Alu RNAs are bound to SRP9/14 in vivo suggest that this heterodimeric protein may play additional roles in the translational control of gene expression and/or Alu transcript metabolism.

Increased expression of alpha- and beta-globin mRNAs at the pituitary following exposure to estrogen during the critical period of neonatal sex differentiation in the rat.

Deterioration of reproductive health in human and wildlife species during the past decades has drawn considerable attention to the potential adverse effects of exposure to xenosteroids during sensitive periods of sex development. The hypothalamic-pituitary (HP) unit is a key element in the neuroendocrine system controlling development and function of the reproductive axis; the HP unit being highly sensitive to the organizing effects of endogenous and exogenous sex steroids. To gain knowledge on the molecular mode of action and potential biomarkers of exposure to estrogenic compounds at the HP unit, we screened for differentially expressed genes at the pituitary and hypothalamus of rats after neonatal exposure to estradiol benzoate. Our analyses identified persistent up-regulation of alpha- and beta-globin mRNAs at the pituitary following neonatal estrogenization. This finding was confirmed by combination of RT-PCR analyses and in situ hybridization. Induction of alpha- and beta-globin mRNA expression at the pituitary by neonatal exposure to estrogen was demonstrated as dose-dependent and it was persistently detected up to puberty. In contrast, durable up-regulation of alpha- and beta-globin genes was not detected at the hypothalamus, cortex, cerebellum, liver and testis. Finally, enhanced levels of alpha- and beta-globin mRNAs at the pituitary were also demonstrated after neonatal administration of the anti-androgen flutamide. In summary, alpha- and beta-globin genes may prove as sensitive, pituitary-specific biomarkers of exposure to estrogenic (and/or anti-androgenic) compounds at critical periods of sex development, whose potential in the assessment of endocrine disrupting events at the HP unit merits further investigation.

Identification of two human nuclear proteins that recognise the cytosine-rich strand of human telomeres in vitro.

Most studies on the structure of DNA in telomeres have been dedicated to the double-stranded region or the guanosine-rich strand and consequently little is known about the factors that may bind to the telomere cytosine-rich (C-rich) strand. This led us to investigate whether proteins exist that can recognise C-rich sequences. We have isolated several nuclear factors from human cell extracts that specifically bind the C-rich strand of vertebrate telomeres [namely a d(CCCTAA)(n)repeat] with high affinity and bind double-stranded telomeric DNA with a 100xreduced affinity. A biochemical assay allowed us to characterise four proteins of apparent molecular weights 66-64, 45 and 35 kDa, respectively. To identify these polypeptides we screened alambdagt11-based cDNA expression library, obtained from human HeLa cells using a radiolabelled telomeric oligonucleotide as a probe. Two clones were purified and sequenced: the first corresponded to the hnRNP K protein and the second to the ASF/SF2 splicing factor. Confirmation of the screening results was obtained with recombinant proteins, both of which bind to the human telomeric C-rich strand in vitro.

Effect of prenatal exposure to bisphenol A on the serum testosterone concentration of rats at birth.

In the rat, testosterone (T) in the neonatal period plays an important role in sexual differentiation and there is a serum T surge in male rats 2 hours after birth. Pregnant female rats were exposed to various doses of bisphenol A (BPA) from gestational day 1 (GD1) through 2 hours after parturition. About half of the BPA-exposed and control dams were subjected to cesarean section on GD22. The male fetuses on GD22 were immediately sacrificed and blood was collected. The other half of the BPA-treated and control dams delivered at GD23 (parturition day). The male pups were sacrificed 2 hours after birth. Serum T concentration was determined by radioimmunoassay (RIA). The BPA concentration in the fetal serum on GD22 increased inversely to the T levels in the serum. The T concentration in the pups' serum 2 hours after birth decreased inversely to the BPA concentration in the serum. However, there were no differences in the serum T concentration among the various doses of BPA. These results suggest that exposure to BPA in utero inhibits the T surge in the neonatal period and we speculate that exposure to BPA in utero disrupts the endocrine environment in the neonatal male.

Sequence, organization, transcription and evolution of RNA polymerase subunit genes from the archaebacterial extreme halophiles Halobacterium halobium and Halococcus morrhuae.

The genes for the four largest subunits, A, B', B" and C, of the DNA-dependent RNA polymerase were cloned from the extreme halophile Halobacterium halobium and sequenced and their transcription was analyzed. The downstream half of this gene cluster from another extreme halophile Halococcus morrhuae was also cloned, sequenced and its transcription products characterized. The H. halobium genes were transcribed into a common transcript from an upstream promoter in the order B", B', A and C. They are flanked by, and co-transcribed with, two smaller genes coding for 75 and 139 amino acid residues, respectively. Immediately downstream from these genes were two open reading frames that are homologous to ribosomal proteins S12 and S7 from Escherichia coli. In both extreme halophiles these genes were transcribed from their own promoter, but in Hc. morrhuae there was also considerable read-through from the RNA polymerase genes. Sequence alignment studies showed that the combined B" + B' subunits are equivalent to the B subunits of the eukaryotic polymerases I and II and to the eubacterial beta subunit, while the combined A + C subunits correspond to the A subunits of eukaryotic RNA polymerases I, II and III and to the eubacterial beta' subunit. The sequence similarity to the eukaryotic subunits was always much higher than to the eubacterial subunits. Conserved sequence regions within the individual subunits were located which are likely to constitute functionally important domains; they include sites associated with rifampicin and alpha-amanitin binding and two possible zinc binding fingers. Phylogenetic analyses based on sequence alignments confirmed that the extreme halophiles belong to the archaebacterial kingdom.

Domain VI of Escherichia coli 23 S ribosomal RNA. Structure, assembly and function.

Domain VI at the 3' end of the 23 S ribosomal RNA from Escherichia coli was prepared using the in vitro T7 RNA polymerase system. Its structure was examined by probing with ribonucleases and chemical reagents, including a psoralen derivative, of various nucleotide specificities, using a reverse transcriptase procedure for analysis. The data provided support for the most recent secondary structure derived from phylogenetic sequence comparisons and for additional structuring that was inferred from earlier experimental data. Moreover, the structure was essentially the same in the free domain, in renatured 23 S RNA and in 50 S subunits. Protein L3 bound to the isolated domain and its binding site was located at a long-range double helix containing a large internal loop. This structure is unusual for a protein-RNA binding site and it may characterize a new (third) class of site. Protein L3 has been implicated, together with L24, in initiating assembly of the 50 S subunit and it shares the exceptional property with L24 that it binds adjacent to the junction of two RNA domains from where it can maximally influence RNA folding. Protein L6 also assembled to domain VI and, in a control experiment, protein L2 bound to isolated domain IV. Domain VI was largely inaccessible in the 50 S subunit and the few accessible RNA sites occurred mainly within conserved sequence regions that constitute potential functional sites. alpha-Sarcin inactivates ribosomes by cutting at one of these sites in 50 S subunits; it also recognized the same site in the free 23 S RNA and in the free domain. Both the EF-Tu ternary complex, and the EF-G ternary complex stabilized by fusidic acid or by a non-hydrolyzable GTP derivative, inhibited alpha-sarcin attack while non-enzymatically bound tRNA did not, thus providing evidence, more direct than before, for the involvement of the RNA region in a common elongation factor binding site.

Structure and accessibility of domain I of Escherichia coli 23 S RNA in free RNA, in the L24-RNA complex and in 50 S subunits. Implications for ribosomal assembly.

Domain I of 23 S RNA of Escherichia coli was probed in renatured RNA, in the protein L24-RNA complex and in 50 S subunits with ribonucleases specific for single- and double-stranded regions and with chemical reagents specific for guanosines (N-1 and N-2), adenosines (N-1, N-7 and N-6), cytidines (N-3) and uridines (N-3). Reactive sites were detected by a reverse transcriptase procedure. The results support most new features of the latest version of the Santa Cruz/Urbana model of the secondary structure, which is based on evidence from sequence comparison. Most double-helical segments were reactive to cobra venom ribonuclease to some degree; the exceptions were the five "long-range" helices that are probably compactly folded within the structure. The data provide evidence for the occurrence of A(syn) X G(anti) pairings in internal loops and at the ends of some helices; they also support the existence of extensive higher-order structuring, especially within the interhelical regions, and are compatible with two of three tertiary interactions in the free RNA that were predicted from comparative sequence studies. Protein L24 is the only primary binding protein that associates with domain I and it strongly protects two sites against ribonuclease and chemical activity. Site A has the properties of a classic protein binding site and we conclude from four lines of evidence that it is the primary attachment site. Site B is rich in highly conserved, unpaired adenosine residues and lies in a potentially critical region of the structure adjoining a group of long-range helices; we infer that L24 binding here is related to the important role of L24 in initiating ribosomal assembly; the existence of both sites is supported, independently, by genetic experiments. L24-induced enhanced reactivities were detected throughout the domain and are consistent with a general "tuning" of the RNA structure. The RNA domain in the 50 S subunits is almost completely resistant to ribonucleases and only a few sites, mainly interhelical, are accessible to chemical reagents. The appearance of several newly reactive nucleotides in the subunit RNA and the enhancement of some others suggest that some minor conformational changes occur on assembly. Nevertheless, the minimal secondary structure of the renatured RNA appears to be retained. We draw the general conclusion that domain I is a highly structured domain that is important for initiating assembly and for the subsequent organization of the ribosome.

Evolutionary relationships amongst archaebacteria. A comparative study of 23 S ribosomal RNAs of a sulphur-dependent extreme thermophile, an extreme halophile and a thermophilic methanogen.

The 23 S RNA genes representative of each of the main archaebacterial subkingdoms, Desulfurococcus mobilis an extreme thermophile, Halococcus morrhuae an extreme halophile and Methanobacterium thermoautotrophicum a thermophilic methanogen, were cloned and sequenced. The inferred RNA sequences were aligned with all the available 23 S-like RNAs of other archaebacteria, eubacteria/chloroplasts and the cytoplasm of eukaryotes. Universal secondary structural models containing six major structural domains were refined, and extended, using the sequence comparison approach. Much of the present structure was confirmed but six new helices were added, including one that also exists in the eukaryotic 5.8 S RNA, and extensions were made to several existing helices. The data throw doubt on whether the 5' and 3' ends of the 23 S RNA interact, since no stable helix can form in either the extreme thermophile or the methanogen RNA. A few secondary structural features, specific to the archaebacterial RNAs were identified; two of these were supported by a comparison of the archaebacterial RNA sequences, and experimentally, using chemical and ribonuclease probes. Seven tertiary structural interactions, common to all 23 S-like RNAs, were predicted within unpaired regions of the secondary structural model on the basis of co-variation of nucleotide pairs; two lie in the region of the 23 S RNA corresponding to 5.8 S RNA but they are not conserved in the latter. The flanking sequences of each of the RNAs could base-pair to form long RNA processing stems. They were not conserved in sequence but each exhibited a secondary structural feature that is common to all the archaebacterial stems for both 16 S and 23 S RNAs and constitutes a processing site. Kingdom-specific nucleotides have been identified that are associated with antibiotic binding sites at functional centres in 23 S-like RNAs: in the peptidyl transferase centre (erythromycin-domain V) the archaebacterial RNAs classify with the eukaryotic RNAs; at the elongation factor-dependent GTPase centre (thiostrepton-domain II) they fall with the eubacteria, and at the putative amino acyl tRNA site (alpha-sarcin-domain VI) they resemble eukaryotes. Two of the proposed tertiary interactions offer a structural explanation for how functional coupling of domains II and V occurs at the peptidyl transferase centre. Phylogenetic trees were constructed for the archaebacterial kingdom, and for the other two kingdoms, on the basis of the aligned 23 S-like RNA sequences.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular cloning and expression of the transformation sensitive epithelial marker stratifin. A member of a protein family that has been involved in the protein kinase C signalling pathway.

We have identified a family of abundant acidic human keratinocyte proteins with apparent molecular masses ranging between 30,000 and 31,100 (isoelectric focussing sample spot proteins 9109 (epithelial marker stratifin), 9124, 9125, 9126 and 9231 in the master two-dimensional gel database of human keratinocyte proteins) that share peptide sequences with each other, with protein 14-3-3 and with the kinase C inhibitory protein. Immunofluorescence staining of keratinocytes showed that two of these proteins (IEF SSPs 9124 and 9126) localize to the Golgi apparatus, while stratifin is distributed diffusely in the cytoplasm. Significant levels of stratifin, and in smaller amount the sample spot proteins 9124, 9125 and 9126, were detected in the medium of cultured human keratinocytes suggesting that they are partially secreted by these cells. Two-dimensional gel analysis of proteins from cultured human cells and fetal tissues showed that polypeptides comigrating with proteins 9124, 9125 and 9126 are ubiquitous and highly expressed in the brain. Stratifin, however, was present only in cultured epithelial cells and was most abundant in fetal and adult human tissues enriched in stratified squamous keratinising epithelium. We have cloned and sequenced cDNAs coding for members of this family. The complete identity of the sequenced peptides from stratifin with the amino acid sequence translated from the stratifin cDNA clone indicated that this cDNA codes for stratifin. The identity of clones 1054, HS1 and AS1 is less clear as, with few exceptions, none of the individual peptide sequences fits the predicted protein sequences. The polypeptides synthesized by clones 1054 and HS1 in the vaccinia expression system, on the other hand, comigrate with proteins 9126 and 9124, suggesting cell-type-specific expression of members of the protein family. Database searches indicated that clone HS1 corresponds to a human T-cell cDNA 14-3-3 clone, while the high level of similarity of clones 1054 and AS1 with the 14-3-3 beta and eta sequences respectively, suggested that they code for the human equivalent of the two bovine proteins. Microsequence data indicated that IEF SSP 9124 corresponds to the human homolog of bovine 14-3-3 gamma.

Identification, molecular cloning, expression and chromosome mapping of a family of transformation upregulated hnRNP-K proteins derived by alternative splicing.

Acidic nuclear proteins (M(r) between 64,000 and 66,000; pI 4.9 to 5.5) that are highly upregulated in transformed cells and that belong to the hnRNP-K family have been identified using a monoclonal antibody (mAB B4B6) that distinguish between quiescent and proliferating human keratinocytes. The family, which is composed of four major proteins (hnRNPs-K A, B, C and D) and their modified forms, is present in similar overall levels in quiescent and proliferating normal keratinocytes although clear differences were observed in the levels of some of the individual variants. Immunofluorescence staining of proliferating normal keratinocytes with mAB B4B6 showed that about 40% of the keratinocytes, corresponding mainly to G1 and to half of the cells in S-phase, reacted with the antibody depicting a dotted, nucleoplasmic staining that excluded the nucleolus. Only 3 to 4% of the quiescent keratinocytes reacted with the antibody while simian virus 40 (SV40) transformed keratinocytes (K14) stained constitutively throughout the cell cycle. Using mAB B4B6 as a probe we cloned a cDNA coding for one member of the family (hnRNP-K B) and this was used to screen for additional family members. Sequencing of the positive clones revealed four different cDNAs, all resulting from alternative splicing of a common primary transcript of a gene that mapped to chromosome 9. Expression of the cDNAs in the vaccinia virus system confirmed their identity as hnRNPs-K A, B, C and D and showed that their modified forms are phosphorylated. All four hnRNPs bound poly(rC) on NorthWestern blots, although the more acidic of the phosphorylated forms, did so at a much reduced level. hnRNP-K has been implicated in pre-mRNA metabolism of transcripts containing cytidine-rich sequences and our results point towards a role during cell cycle progression.

Should we watch what we eat and drink? Report on the International Workshop on Hormones and Endocrine Disrupters in Food and Water: possible impact on human health, Copenhagen, Denmark, 27-30 May 2000.

The aim of this Workshop was to bring together scientists with different backgrounds, including clinical endocrinologists, basic researchers and epidemiologists, to discuss the complex and controversial topic of endocrine disrupters, and their impact on human health. Nearly 250 scientists attended the Workshop, and 50 lectures and 90 posters were presented and discussed. The most important scientific findings and reviews are to be published in Human Reproduction and Human Reproduction Update. Some of the highlights are presented here.