Splice variants of the relaxin and INSL3 receptors reveal unanticipated molecular complexity

LGR7 and LGR8 are G protein-coupled receptors that belong to the leucine-rich repeat-containing G-protein coupled receptor (LGR) family, including the thyroid-stimulating hormone (TSH), LH and FSH receptors. LGR7 and LGR8 stimulate cAMP production upon binding of the cognate ligands, relaxin and insulin-like peptide 3 (INSL3), respectively. We cloned several novel splice variants of both LGR7 and LGR8 and analysed the function of four variants. LGR7.1 is a truncated receptor, including only the N-terminal region of the receptor and two leucine rich repeats. In contrast, LGR7.2, LGR7.10 and LGR 8.1 all contain an intact seven transmembrane domain and most of the extracellular region, lacking only one or two exons in the ectodomain. Our analysis demonstrates that although LGR7.10 and LGR8.1 are expressed at the cell surface, LGR7.2 is predominantly retained within cells and LGR7.1 is partially secreted. mRNA expression analysis revealed that several variants are co-expressed in various tissues. None of these variants were able to stimulate cAMP production following relaxin or INSL3 treatment. Unexpectedly, we did not detect any direct specific relaxin or INSL3 binding on any of the splice variants. The large number of receptor splice variants identified suggests an unforeseen complexity in the physiology of this novel hormone-receptor system.     

Investigation of the simian polyomavirus SV40 as a potential causative agent of human neurological disorders in AIDS patients

Neurological diseases and a variety of neoplasms frequently occur in AIDS patients. Human JC and BK polyomaviruses have been associated with neurological disorders in such patients. SV40 polyomavirus sequences have been detected in human brain tumours, other neoplasms and normal tissues. JCV, BKV and SV40 DNA sequences were investigated in cerebrospinal fluid (CSF) samples from 12 AIDS patients affected by different neurological disorders, by PCR assay and filter hybridisation with specific internal oligoprobes, and DNA sequencing. Three of the 12 CSF samples were positive for JCV (one sample) or SV40 (one) DNA, or both (one). No sample was positive for BKV DNA. JCV- and SV40-specific genomic regions were confirmed by DNA sequencing. CSF samples from the two patients diagnosed clinically as having progressive multifocal leukoencephalopathy (PML) contained either JCV (one sample) or SV40 (one) DNA. The CSF found to contain both JCV and SV40 DNA originated from a patient with a cerebral mass lesion of unknown aetiology. These results suggest that SV40 may be involved in the aetiology of PML in AIDS patients, and raise the possibility that SV40 and JCV may act synergically in vivo to enhance their pathogenicity.     

High circulating levels of biologically inactive IL-6/SIL-6 receptor complexes in systemic juvenile idiopathic arthritis: evidence for serum factors interfering with the binding to gp130

We previously demonstrated that high levels of IL-6/sIL-6R complexes are present in sera of patients with systemic juvenile idiopathic arthritis (s-JIA) and that the amount of IL-6 estimated in the IL-6/sIL-6R complexes is markedly higher than that measured by the B9 assay. Here, we show that two additional bioassays, employing human myeloma XG-1 cells and human hepatoma Hep3B cells, detected serum IL-6 levels similar to those measured by the B9 assay and approximately 10-fold lower than the IL-6 levels estimated to be present in the IL-6/sIL-6R complex. Using an assay for the measurement of the amount of circulating IL-6 complexed with the sIL-6R and available for binding to gp130 (gp130 binding activity), we show that the IL-6/gp130 binding activity is similar to that detected by the bioassays and again significantly lower than that estimated to be present in the IL-6/sIL-6R complex. Addition of recombinant human IL-6 (rhIL-6) to sera of patients or controls results in a markedly lower increase in the gp130 binding activity in patients than in controls. Moreover, sera from s-JIA patients inhibited in a dose dependent manner the gp130 binding activity assay. These results show that sera from patients with s-JIA contain a factor, or factors, that inhibit(s) the binding of the IL-6/sIL-6R complex to gp130. This inhibitory activity does not appear to be due to soluble gp130, C-reactive protein or autoantibodies to IL-6.     

Detecting pre-ovulatory luteinizing hormone surges in urine

The study objectives were to determine (i) if pre-ovulatory luteinizing hormone (LH) surges, undetected in urine by two immunoradiometric assays (IRMA), were detectable by an ultrasensitive immunofluorometric assay (IFMA) and (ii) the influence of creatinine adjustment on the detection and timing of the urinary LH surges. Daily urine specimens were contributed by healthy 25-36 year old volunteers during 14 ovulatory menstrual cycles for an epidemiological study conducted in 1983-1985. Specimens were selected as having been previously assayed by two IRMA without consistently detecting LH surges. These urine specimens were remeasured using an IFMA and adjusted for creatinine concentration. IFMA measurements revealed unambiguous LH surges in all cycles. Adjusting IRMA urinary LH values for creatinine concentrations revealed previously undetected LH surges in four of eight cycles. Creatinine adjustment also altered the timing of IRMA and IFMA LH surges by 1-5 days. These results demonstrate an IFMA that detects pre- ovulatory LH surges in unpreserved, frozen urine from cycles where such surges were previously undetectable. Further, creatinine adjustment can markedly affect detection and timing of the onset and peak of the urinary LH surge. While our analysis suggests that this adjustment improves the validity of the LH measure, this requires further investigation.      

Analysis of the steroidogenic acute regulatory protein (StAR) gene in Japanese patients with congenital lipoid adrenal hyperplasia

Genomic DNA from 19 Japanese patients with congenital lipoid adrenal hyperplasia (lipoid CAH) representing 16 different families was examined to identify the genetic alterations of steroidogenic acute regulatory protein (StAR). Ten of 19 patients had a 46,XX karyotype and nine had a 46,XY karyotype. Six of the 46,XX patients have experienced spontaneous pubertal changes including breast development and irregular menstruation whereas none of the 46,XY subjects displayed pubertal changes. Eight different mutations were identified. Sixteen patients were either homozygotes or compound heterozygotes for the Q258X mutation. The seven other mutations identified were 189delG, 246insG, 564del13bp, 838delA, Q212X, A218V and M225T. The 189delG, 246insG, 546del13bp and Q212X mutants encode truncated proteins. COS-1 cells transfected with expression vectors encoding cDNAs for the mutant StAR proteins which affect the C-terminus, 838delA, A218V and Q258X, exhibited no steroidogenesis enhancing activity. However, the M225T mutant retained some steroidogenic activity. The patient with the M225T mutation had late onset of this disorder and some capacity to secrete testosterone in response to hCG. These findings suggest: (i) that the Q258X mutation can be used as a genetic marker for the screening of Japanese for lipoid CAH, (ii) that the C-terminus of StAR plays an important role in the protein's activity and (iii) that there are differences in the extent of functional impairment of the testis and ovaries in lipoid CAH.      

Improved oocyte quality is obtained with follicle stimulating hormone alone than with follicle stimulating hormone/human menopausal gonadotrophin combination

The aim of this study was to compare the efficacy of pure follicle stimulating hormone (FSH) with that of FSH/human menopausal gonadotrophin (HMG) combination in downregulated cycles. A total of 357 patients was evaluated retrospectively. Sixty percent of patients in the FSH group and 55% in the FSH/HMG group were new; the others were repeat patients. Ovulation was suppressed with leuprolide acetate in all patients, followed by either FSH (n = 218) or FSH/HMG (n = 119). There was no difference in patients' age, infertility factors, number of ampoules used, length of stimulation, oestradiol levels on day of human chorionic gonadotrophin (HCG) administration, number of oocytes recovered or the number of embryos transferred. Also, nuclear maturity at aspiration and fertilization rates were not different between the two groups. FSH stimulation resulted in a significantly higher percentage of mature oocytes that showed the typical 'mature' morphological characteristics (P < 0.0001). The clinical pregnancy rates per transfer were 40 and 28% in patients stimulated with pure FSH and FSH/HMG respectively (P < 0.05). The significantly higher number of immature oocytes matured in vitro in the FSH/HMG group (P = 0.001) suggests a possible effect on in-vitro maturation, due to luteinizing hormone present in HMG. The difference in mature oocyte quality may be an important determinant in the higher pregnancy rates for the FSH- stimulated patients.      

Veränderte Aktivitäten der Enzyme des Energiestoffwechsels in Thrombocyten von Patienten mit Lebercirrhose und Splenomegalie

Zusammenfassung 1. Die Aktivitäten von 28 Enzymen aus verschiedenen Abschnitten des Energiestoffwechsels wurden in isolierten Thrombocyten von 17 gesunden Menschen und 15 Patienten mit gesicherter Lebercirrhose gemessen.2. Die für Gewebe bekannte Proportionskonstanz der Enzymaktivitäten des zentralen Segmentes der Glykolyse (TIM, GAPDH, PGK, GPM und EN) findet sich auch in den Thrombocyten als Ordnungsprinzip. Die mitochondrial lokalisierten Enzyme NAD-spezifische Isocitratdehydrogenase und Glycerophosphatoxydase wurden erstmalig in Thrombocyten mit relativ hoher Aktivität nachgewiesen.3. Die Plättchen von Patienten mit Lebercirrhose und Milzvergrößerung infolge portaler Hypertension zeigten signifikant erhöhte Enzymaktivitäten in allen untersuchten Stoffwechselwegen, insbesondere der mitochondrial lokalisierten Enzyme. Funktionell bedeutsam erscheinen der ausgeprägte Anstieg der Mg⁺⁺-aktivierbaren ATPase, die gleichbleibende Aktivität der Fructose-6-Phosphatkinase und eine Verminderung der Lactatdehydrogenase. Bei der Patientengruppe mit Cirrhose ohne Milzvergrößerung lagen fast alle gemessenen Aktivitäten der Thrombocyten im Normbereich.4. Es wird ein Patient mit Lebercirrhose ohne Milzvergrößerung beschrieben, dessen Thrombocyten um den Faktor 8 höhere Aktivitäten fast aller gemessenen Enzyme aufwiesen. Auch in diesen Plättchen zeigte die Lactatdehydrogenase eine Aktivitätsverminderung gegenüber der Norm.5. Ein Enzymdefekt der Plättchen wie bei Thrombasthenie wurde bei Lebercirrhose nicht gefunden. Nach unseren Kenntnissen über die Enzymausstattung von Blutzellen handelt es sich bei den Thrombocyten mit höherer Enzymaktivität um eine jugendliche Zellpopulation. Die Pathogenese wird besonders hinsichtlich der Rolle der vergrößerten Milz diskutiert.

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Summary 1. The activities of 28 enzymes from different pathways of energy producing metabolism were measured in the isolated platelets of 17 normal persons and 15 patients with proven cirrhosis of the liver.2. In mammalian tissues a constant proportion between the enzymes of the central segment of the glycolytic pathway (TIM, GAPDH, PGK, GPM, EN) has been described. This constant proportion has been demonstrated also in the platelets. The mitochondrially localized enzymes NAD-specific Isocitratdehydrogenase and Glycerophosphatoxidase have been measured for the first time in platelets with a high activity.3. The platelets of patients with liver cirrhosis and splenomegaly following portal hypertension showed significant higher enzyme activities in all investigated pathways, mainly in the citric acid cycle. Functionally important could be the marked increase of the Mg⁺⁺ activated ATPase, the unaltered activity of the Phosphofructokinase and a lowered activity of the Lactatdehydrogenase in these platelets. The patients with cirrhosis but without a large spleen had normal enzyme activities of the platelets.4. One patient is described with cirrhosis without splenomegaly who had an elevation of nearly all measured enzymes by a factor 8 in his platelets. The LDH showed a decreased activity.5. No enzyme defect in the platelets of cirrhotic patients as in thrombasthenia was found. Basing on our knowledge of the enzyme equipment of blood cells the conclusion is drawn that the platelets with higher enzyme content represent a young cell population. The pathogenesis is discussed with special reference to the role of the enlarged spleen.

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Teilergebnisse wurden vorgetragen auf dem 2. Symposium der European Association for the Study of the Liver, Göteborg, 1967 und der 24. Tagung der Deutschen Gesellschaft für Verdauungs- und Stoffwechselkrankheiten, Hamburg 1967.     

Veränderungen von α-Ketoglutarsäure,Brenztraubensäure und Diphosphopyridinnucleotid bei chronischen Leberkranken

Zusammenfassung Bei 41 Kranken mit Lebercirrhose wurden die -Ketoglutarsäure, die Brenztraubensäure und das Diphosphopyridinnucleotid enzymatisch bestimmt. — Als Normalwerte wurden gefunden für -KGS=0,14 mg-%±0,05, für BTS=0,77 mg-%±0,19 und für DPN=1,57 mg-%±0,14.Die Leberkranken wurden nach ihrer klinischen Symptomatologie in Schweregrade eingeteilt und eine Zuordnung von klinischem Schweregrad und biochemischen Werten vorgenommen.Dabei zeigt sich, daß die Werte für -KGS gut der klinischen Symptomatologie entsprechen. Diese Werte betrugen für Grad 0=0,21 mg-%±0,08, für Grad 1=0,37 mg-%±0,09, für Grad 2=0,48 mg-%±0,13 und für Grad 3=0,61 mg-%±0,27.Die Werte für BTS zeigten keine so gute Übereinstimmung mit der klinischen Symptomatologie.Die Werte für DPN lagen für alle klinischen Grade mit wenigen Ausnahmen im Normalbereich.Auszugsweise vorgetragen auf der Tagung der Deutschen Gesellschaft für Physiologische Chemie, Hamburg, 28. 9. 56²⁷ und (G. A. M.) auf dem Jahrestreffen der American Association for the Study of the Liver, Chicago, 8. 11. 56³².In dieser Arbeit wurden folgende Abkürzungen benutzt: -Ketoglutarsäure (-KGS); Brenztraubensäure (BTS); Diphosphopyridinnucleotid (DPN); Glutaminsäuredehydrogenase (GluDH); Milchsäuredehydrogenase (MDH); Alkoholdehydrogenase (ADH).     

Early detachment of titanium particles from various different surfaces of endosseous dental implants

Titanium (Ti) endosseous dental screws with different surfaces (smooth titanium--STi, titanium plasma-sprayed-TPS, alumina oxide sandblasted and acid-etched--Al-SLA, zirconium oxide sandblasted and acid etched--Zr-SLA) were implanted in femura and tibiae of sheep to investigate the biological evolution of the peri-implant tissues and detachment of Ti debris from the implant surfaces in early healing. Implants were not loaded. Sections of the screws and the peri-implant tissues obtained by sawing and grinding were analysed by light microscopy immediately after implantation (time 0) and after 14 days. All samples showed new bone trabeculae and vascularised medullary spaces in those areas where gaps between the implants and host bone were visible. In contrast, no osteogenesis was induced in the areas where the implants were initially positioned in close contact with the host bone. Chips of the pre-existing bone inducing new peri-implant neo-osteogenesis were surrounded by new bone trabeculae. The threads of some screws appeared to be deformed where the host bone showed fractures. Ti granules of 3-60 microm were detectable only in the peri-implant tissues of TPS implants both immediately after surgery and after 14 days, thus suggesting that this phenomenon may be related to the friction of the TPS coating during surgical insertion.