RNA1 is sufficient to mediate plasmid ColE1 incompatibility in vivo.

The multicopy plasmid ColE1 specifies a small RNA designated RNA1 that has been implicated in copy number control and incompatibility. We have inserted a 148 base-pair ColE1 DNA fragment containing a promoter-less RNA1 gene into a plasmid vector downstream from the tryptophan promoter of Serratia marcesens . The ColE1 RNA1 produced by this plasmid is not functional in vivo due to the presence of 49 nucleotides appended to the 5'-terminus of the wild-type RNA1 sequence. Deletions of these sequences by Bal3l nuclease in vitro and genetic selection for ColE1 incompatibility function in vivo permitted isolation of a plasmid expressing wild-type ColE1 RNA1 initiated properly from the S. marcesens trp promoter. These experiments demonstrate that RNA1 is sufficient to mediate ColE1 incompatibility in vivo. In addition, several plasmids were isolated that contain altered RNA1 genes. These alterations consist of additions or deletions of sequences at the 5'-terminus of RNA1. Analysis of the ability of these altered RNA1 molecules to express incompatibility in vivo suggests that the 5'-terminal region of RNA1 is crucial for its function.

Isolation and characterization of mutants affecting functional domains of ColE1 RNAI.

The control of DNA replication initiation in the plasmid ColE1 is mediated by RNAI, a 108 nucleotide plasmid-encoded RNA that is entirely complementary to the 5'-terminal region of the replication primer RNA. RNAI acts in trans to inhibit primer maturation. Previously, we constructed a plasmid in which the ColE1 RNAI was separated from the primer and placed under transcriptional control of the Serratia marcesens tryptophan promoter. This plasmid provides RNAI in trans in vivo and mediates ColE1-type incompatibility. To determine the critical structural and functional domains of RNAI, we have undertaken a mutational analysis of the RNAI gene carried by this plasmid. We have selected mutants that no longer mediate ColE1-type incompatibility in trans. From the DNA sequences of 18 mutants we have identified mutations at nine new sites in RNAI. In addition, we have determined the secondary structural features of several mutant RNAI species and compared them to wild-type RNAI. Analysis of these mutations has revealed several key features of RNAI secondary structure and function. The domains of RNAI identified in this work which are essential for its function are: the single-stranded loop regions; the integrity of the double-stranded stems; and the single-stranded 5' terminus.

Signal transduction by GABA(B) receptor heterodimers.

GABA(B) receptors are G-protein-coupled receptors that mediate inhibition throughout the central and peripheral nervous systems. A single cloned receptor, GABA(B)R1, which has at least three alternatively spliced forms, appears to account for the vast majority of binding sites in the brain for high-affinity antagonists. In heterologous expression systems GABA(B)R1 is poorly expressed on the plasma membrane and largely fails to couple to ion channels. A second gene, GABA(B)R2, which exhibits moderately low homology to GABA(B)R1, permits surface expression of GABA(B)R1 and the appearance of baclofen-sensitive K(+) and Ca(+1) currents. We review the data that supports a model of the native GABA(B) receptor as a heterodimer composed of GABA(B)R1 and GABA(B)R2 proteins. New data from mutagenesis experiments are presented that point to amino acid residues on GABA(B)R1 critical for ligand activation of the heterodimer. The possible role of GABA(B)R2 in signal transduction is also discussed. The interdependent nature of the two subunits for receptor function makes the GABA(B) receptor a useful model to explore the larger significance of GPCR dimerization for G-protein activation.

A single point mutation increases the affinity of serotonin 5-HT1D alpha, 5-HT1D beta, 5-HT1E and 5-HT1F receptors for beta-adrenergic antagonists.

The serotonin 5-HT1B and 5-HT1A receptors bind certain beta-adrenergic antagonists, such as propranolol and pindolol, with high affinity. Other 5-HT1 receptors that display very low affinity for beta-adrenergic antagonists, have either a threonine (T) (5-HT1D alpha, 5-HT1D beta and 5-HT1E) or an alanine (A) (5-HT1F) residue in the homologous position in the seventh transmembrane domain. In the case of the human 5-HT1D beta receptor, replacement of this T with asparagine (N), dramatically increases its ability to bind beta-adrenergic antagonists. To assess whether other 5-HT1 receptors would behave similarly, we have used site-directed mutagenesis to replace the T or A in 5-HT1D alpha, 5-HT1E and 5-HT1F receptors with N. Both the wild-type and mutant genes were expressed transiently in COS-7 cells and radioligand binding studies were performed by using [3H]5-HT and [125I]iodocyanopindolol. Using [3H]5-HT, we found that the affinities of all the mutant receptors for propranolol and pindolol were significantly increased by 100-1000 fold, 5-HT1D alpha and 5-HT1F receptors showing the highest and the 5-HT1E receptor displaying the lowest affinity. On the other hand, the affinities for 5-HT were essentially unchanged as compared to the wild-type receptors. All mutant receptors bound [125I]iodocyanopindolol with high affinity, KD values ranging between 0.04 nM (mutant 5-HT1D alpha) and 0.57 nM (mutant 5-HT1E), whereas the wild-type receptors failed to show any specific binding with this radioligand in the same concentration range used for the mutant receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple radioimmunoassay for the measurement of testosterone glucosiduronate in unextracted urine.

A simple, reliable and rapid radioimmunoassay (RIA) for the determination of testosterone glucosiduronate (TG) in crude urine is described. Two protein-TG complexes were investigated in raising antibodies: a) Bovine serum albumin (BSA)-TG and b) human plasma Cohn's fraction IV-4 (CF)-TG. In rabbits, high titers of antibodies were obtained after the injection of CF-TG. The specificity of the antiserum was sufficiently high (cross reaction with free testosterone 27%, with 5alpha-dihydrotestosterone-glucosiduronate 20%). TG was estimated in small aliquots of male and female urine after evaporation overnight at 50 degrees C in order to eliminate interfering material. The intraassay coefficient of variation (CV) was found to be 6% and the interassay CV 11%. TB has been determined in 40 samples of urine simultaneously by "direct" RIA and by a "classical" RIA following hydrolysis with beta-glucuronidase. The coefficient of correlation was found to be 0.89. The mean excretion of TG in the urine of 26 healthy men amounted to 164+/-51 mug/24 hours with a range from 97 to 346 mug/24 hours. In a group of 16 women a mean urinary excretion of TG of 24+/-10 mug/24 hours was determined. The method allows a technician to assay 40 samples per day.

Judicial murder.

It is catastrophic when a judicial error results in the implementation of the death penalty. The western system of courts of appeal diminishes the risk of the condemnation of innocent persons but does not rule it out. Intrinsic influences of psychological effects are just as detrimental as preconceived notions as was the case, for instance, when Socrates' life ended unnaturally. This happened despite the fact that he was able to establish persuasively that the court had considered false evidence and had therefrom made unfair and untruthful deductions. This article uses the example in psychosomatic medicine of a member of a family who becomes aggressive because of envy and jealousy and this results in often tragic consequences for the 'victim' of his offensive behaviour. The principle is the same. The only solution is the intercession of an unbiased influence to work out the problem from a point of view that is unaffected by such turmoils which are often inherent in the system. If this is not done any organization or society can make deadly mistakes.

Materialism and spiritualism: the dualistic way of Western thinking.

Ever since the beginning of Western philosophy in ancient Greece there have been two fundamental approaches to life in general: materialism and spiritualism. In the Renaissance this basic question came into discussion again. In the 19th century the real fight began. On the one hand we have had the materialistic 'leftists', namely Marx, with the political consequence of the Russian revolution. On the other hand we had Kierkegaard, Bergson, etc. searching for the fundamental facts in human life, enabling us to survive in dignity. The moment has come, where we must see that human emotion is by far more important in the question of life and death, namely in psychosomatic illness, than all the riches of the world.

Characterization of the ColE1 primer-RNA1 complex: analysis of a domain of ColE1 RNA1 necessary for its interaction with primer RNA.

RNA1 is a small, plasmid-encoded transcript involved in the replication control of the plasmid ColE1. RNA1 blocks replication by preventing processing of the primer RNA necessary for the initiation of replication. It has been proposed that inhibition by RNA1 involves a direct interaction between RNA1 and primer RNA. Here we describe an in vitro system that allows the detection and characterization of the RNA1-primer complex. Using this system, we have demonstrated that the association between ColE1 RNA1 and primer results in the formation of an RNA X RNA hybrid between RNA1 and the 5' end of primer RNA. Furthermore, analysis of mutant RNA1 molecules containing sequence alterations in the 5' single-stranded region of the molecule has revealed that this portion of RNA1 is necessary for the formation of the RNA1-primer complex. These experiments indicate that, in addition to the three single-stranded loops of RNA1, the 5' single-stranded region is a functional domain of the molecule.

Spontaneous fractures of the neck of the femur occurring in pregnancy in the absence of pregnancy osteoporosis.

Case report on a patient suffering from spontaneous fractures of the neck of the femur which occured during her first pregnancy at the age of 23 in the left leg and during another pregnancy seven years later in the right leg. Both fractures occured during the eighth month of pregnancy. Special attention is drawn to the fact that local or general osteoporosis which, according to literature, should be obligatory for the occurence of spontaneous necrosis of the femoral head and/or fractures of the neck of the femur, could be demonstrated neither during the first nor the second pregnancy.

Structural analysis of RNA molecules involved in plasmid copy number control.

DNA replication of the plasmid ColE1 and its relatives is controlled mainly by a small plasmid-encoded transcript known as RNA11. We have studied the conformation of RNA1 molecules from two related but compatible plasmids, ColE1 and RSF1030, using T1 ribonuclease, S1 nuclease, cobra venom nuclease, and diethyl pyrocarbonate modification as probes for secondary structure. Both RNA1 molecules contain three double-stranded stems, three single-stranded loops, and an exposed 5' tail. All loops in both molecules show similar sensitivities to the probes used, as do stems 1 and 3. The region comprising stem 2 of each RNA1 molecule contains the most sequence differences as well as the most structural changes of any region in the two molecules. The structure of the RNA1 molecule encoded by a recessive high copy number mutant of ColE1 was also investigated. Structural alterations involving the first stem and loop of the molecule are proposed to be responsible for the inability of the mutant RNA1 to function in vivo.

Unconjugated 5 alpha-androstan-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol in human plasma as measured by radioimmunoassay without chromatography.

A radioimmunoassay (RIA) without chromatography for the determination of 5 alpha-androstan-3 alpha, 17 beta-diol and of 5 alpha-androstane-3 beta, 17 beta-diol in human plasma by using highly specific antisera against the 15 beta-carboxyethylmercapto-bovine serum albumin conjugates is described. It could be shown that the 13% cross reaction of the 5 alpha-androstane-3 beta, 17 beta-diol antiserum with pure androst-5-ene-3 beta, 17 beta-diol was negligibly low in plasma extracts. Sensitivity, accuracy, precision, and linearity revealed results which comply with the requisites of a reliable RIA. The plasma levels obtained were as follows (mean +/- SD): 5 alpha-androstan-3 alpha, 17 beta-diol: Normal males (N = 27) 0.86 +/- 0.22 nmol/l; normal females (N = 10) 0.31 +/- 0.07 nmol/l; hirsute females (N = 25) 0.60 +/- 0.24 nmol/l; 5 alpha-androstane-3 beta, 17 beta-diol: Normal males 1.47 +/- 0.43 nmol/l; normal females 0.49 +/- 0.10 nmol/l; hirsute females 0.91 +/- 0.32 nmol/l.

Separation of interstitial and tubular cells of human testis by means of an anti-substance P-antiserum. Androstane-3 beta, 17 beta-diol is exclusively formed by seminiferous tubules.

Leydig cells and seminiferous tubules of human testicular tissue were successfully separated by means of an anti-substance P-antiserum. On incubation with (14C)-testosterone or (14C)-dihydrotestosterone (DHT) it could be shown that besides DHT and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) also 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) originated in the tubular compartment. The ratio 3 alpha/3 beta-diol was found to be higher (0.7) here compared to incubation with whole testicular tissue (0.2), indicating that the testicular interstitium influences the metabolism of DHT by the seminiferous tubules.

Construction of Co1E1 RNA1 mutants and analysis of their function in vivo.

We have carried out experiments designed to investigate the relationship between structure and function for the Co1E1 RNA1 species. RNA1 is a small RNA (108 nucleotides) that has been implicated in copy number control of the multicopy plasmid Co1E1. In vitro, RNA1 inhibits the processing of the primer precursor required for initiation of DNA replication. The RNA1 gene is entirely complementary to the 5'-terminal region of the primer. We have functionally separated these 2 RNA species by cloning the RNA1 gene downstream from the S. marcescens trp promoter. When cloned in a Co1E1-compatible plasmid, a trp-RNA1 fusion has been shown to mediate Co1E1-type incompatibility in vivo. The construction scheme described here also generates mutant RNA1 species with altered sequences at the 5' terminus of RNA1 which have been assayed for function in vivo. These experiments have indicated that sequences at the 5' terminus play a critical role in RNA1 function.

Ultrafiltrable plasma testosterone and 5 alpha-dihydrotestosterone as measured across the human testes.

It has been confirmed that within the human plexus pampiniformis a venous-arterial transfer of testosterone (T) (5.95%) and of dihydrotestosterone (DHT) (16.58%) occurs. It could be established that in the testicular artery the percentage of ultrafiltrable DHT was significantly lower compared with the venous compartment. It is assumed that by this means a biological recycling of the active androgen DHT into the testis is achieved.

The effect of infusions of 5 alpha-dihydrotestosterone or estradiol-17 beta on the concentration of some steroids in the human testicular vein and artery.

The concentrations of testosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstan-3 alpha, 17 beta-diol, 5 alpha-androstane-3 beta, 17 beta-diol, estradiol-17 beta and testosterone-glucosiduronate were measured in the plasma of the testicular vein and artery simultaneously with the estimation in peripheral venous and arterial plasma 60 min after an infusion of 3000 micrograms dihydrotestosterone (DHT) or estradiol (E2), respectively, in patients undergoing orchiectomy for prostatic cancer. The results were as follows; following infusion of DHT or E2, both steroids were completely metabolized by the testes. After DHT the testicular secretion of E2 was significantly reduced. In peripheral plasma 3 alpha-diol concentration was increased. Following E2 a transient elevation of testosterone in the spermatic vein was observed, whereas a slight decrease of DHT and an increase especially of 3 beta-diol levels occurred. It is assumed that DHT as well as E2 plays a role as intratesticular regulator of steroid synthesis and metabolism.

The effect of short term treatment with cyproterone acetate or flutamide on the metabolism of 5 alpha-dihydrotestosterone in human testicular tissue.

Testicular tissue obtained from ten patients orchiectomized for prostatic cancer was incubated with [3H]5 alpha-dihydrotestosterone (DHT) in order to study the metabolic transformation into 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol). Throughout 5 days before surgery four subjects were treated with cyproterone acetate (CA). To three patients flutamide (F) was administered for the same period of time. Three subjects remained untreated. Compared to the control group the administration of CA decreased the formation of 3 beta-diol whereas that of 3 alpha-diol increased. Treatment with F lead to an elevated formation of both diols. However, the 3 alpha/3 beta ratio did not change. As 3 beta-diol is considered to be an index of tubular function in the human testis it is concluded that CA has a direct inhibitory effect upon this testicular compartment whereas F has none.

[Extended diagnosis for lesions of the capsule and the ligament of the knee joint (author's transl)]. / Erweiterte Diagnostik der Kapselbandverletzungen am Kniegelenk.

X-rays taken under mechanical positioning of the joint with standardized conditions are helpful aids in the diagnosis of injuries of the knee capsule and ligaments. In comparison to the non-affected knee the extent of instability of the joint can be exactly demonstrated. Arthrography is of little help in fresh injuries to the knee capsule and ligaments, but can be useful to assess the residuals of old damage to the joint. Arthroscopy has its place mainly in the diagnosis of old injuries and hardly any in fresh lesions. In general, however, arthroscopy is a very valuable diagnostic procedure.