Clastogenic activity of urethane in mice.

Single intraperitoneal (i.p.) treatment of male and female BDF1 (C57B1 x DBA2) mice with urethane (0.5 or 1.0 g/kg) caused a significant increase in micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after 24 h. The clastogenic effect observed was dose-, sex- and age-dependent, the male and younger (6-8 weeks old) animals being more susceptible than the female and older (6 months of age) mice. 3-week oral treatment of female Balb/c mice with urethane (3 g/l added to the drinking water) caused an up to 4-fold increase in the number of micronucleated normochromatic erythrocytes (MNNCE) in mouse peripheral blood. In a month after the carcinogen treatment was stopped, the number of MNNCE dropped to the control values. In addition, a single i.p. treatment of pregnant BDF1 mice on day 17 of gestation with urethane (1.0 g/kg) caused a 514.3% (p less than 0.001) elevation of MNPCE in mouse fetal liver after 24 h as well as a 154.4% (p less than 0.05) increase in MNPCE frequency in the fetal peripheral blood. At this time point, the clastogenic response in mouse fetal liver erythroblasts was less pronounced than that detected in the maternal bone marrow cells. Urethane is a strong clastogen in mice when administered either intraperitoneally or orally and the micronucleus test applied to adult and fetal erythroblasts is a convenient method of choice for studying the acute and subchronic clastogenicity of this carcinogen, its transplacental effects as well as the influence of modifying factors on these processes.


Long-term results of nervous tissue alterations caused by epineurial electrode application: an experimental study in rat sciatic nerve.

In order to evaluate the long-term effects of epineurial electrode application for functional electrical stimulation (FES) the left sciatic nerve of seven rats was exposed. Four ring-shaped stainless steel wire electrodes were sutured to the epineurium of each nerve in the same manner as performed clinically for carrousel stimulation in man. The nerves were reexposed 1 year after implantation and the stimulation threshold to obtain a tetanic contraction in the lower limb was determined for each electrode. Afterwards the animals were sacrificed. The electrodes were excised and cross sections of the sciatic nerve directly at site of the electrodes, 2-mm proximal and 2-mm distal to them were harvested for histologic and planimetric assessment of nerve lesions. The area of damaged neural tissue was expressed as a percentage of the total cross-sectional area within the perineural sheath. The sciatic nerves of the right side served as controls. The values for the stimulation thresholds ranged between 0.1 and 1.0 mA (mean 0.43 mA). By morphometric examination five of seven nerves were seen altered, the altered areas captured between 1% and 4.8% of the total cross-sectional area of the nerves within the perineural sheath. Besides two specimens, all altered nerve segments exhibited distinct signs of nerve fiber regeneration. The clinical implications of the results for long-term electrical stimulation, such as phrenic pacing, are discussed.


Atrial latency in a dual chambered pacing system causing inappropriate sequence of cardiac chamber activation.

A transvenous atrioventricular pacemaker was implanted in a patient in whom junctional bradycardia and recurrent ventricular tachycardia had occurred after coronary artery bypass surgery. The period between delivery of the atrial stimulus and resultant atrial excitation was prolonged by up to 0.22 seconds and exceeded the programmed delay between atrial and ventricular stimulation. Consequently, ventricular stimulation preceded atrial excitation. Cardiac failure resulted: it was resolved by reprogramming the pacemaker to the atrial inhibited mode.


Prolonged asystole during head-up tilt table testing after beta blockade.

Neurally mediated vasodepressor syncope is a common clinical problem. The diagnosis is generally associated with a benign prognosis, however, a less common "malignant" form has been identified. Head-up tilt table testing is helpful in the confirmation of the diagnosis of neurally mediated vasodepressor syncope and may be useful in the selection of therapy. One form of therapy commonly used is beta blockade. In this case report we describe a patient with neurally mediated vasodepressor syncope who developed asystole during head-up tilt table testing after treatment with a beta blocker.


Follow-up of a respiratory rate modulated pacemaker.

The efficacy of 27 respiration sensitive rate modulated pacemakers (Biorate RDP-3 Biotec) implanted in the left pectoral area was evaluated every 3 months during a mean follow-up period of 29 months (range 10-50 months). Rate modulation function was unchanged other than for three patients in whom the auxiliary leads became displaced. Two implants lost ventricular sensing in this nonprogrammable model. In all but the three patients, Holter monitoring demonstrated pacing rate variation corresponding to daily activity. Stress test duration increased from 8.2 +/- 1.5 minutes (in fixed rate VVI rate) to 12.83 +/- 2.0 minutes (in the VVIR mode) (P less than 0.05). Right arm movement increased the pacing rate by 5 +/- 3 beats/min (NS), while the left arm movement increase was 30 +/- 5 beats/min (P less than 0.05). Mental, arithmetic, and nifedipine tests did not change the rate modulated pacing rate. The system responded to a change in respiratory rate by an increase in stimulation rate. A satisfactory response in sensitivity and velocity was present only with medium-high workloads. Interference with rate modulation occurred with movement of the arm ipsilateral to the implanted pulse generator.


Impedance monitoring during radiofrequency catheter ablation in humans.

Radiofrequency catheter ablation of accessory pathways and the atrioventricular junction often requires multiple applications of energy. The inability to determine the effects of any given application on the underlying tissue may contribute to this problem. In the present study, impedance was monitored in 20 patients undergoing radiofrequency catheter ablation, and the relationship between an initial decrease in impedance and subsequent effects were examined. An initial fall in impedance of more than 10 omega was 78% sensitive and 88% specific for predicting subsequent evidence of tissue heating (interruption of conduction or an abrupt rise in impedance due to coagulum formation). In contrast, initial values of voltage, current, or impedance did not distinguish between effective and ineffective applications of radiofrequency energy. Continuous monitoring of impedance may facilitate radiofrequency catheter ablation.


Double ventricular responses to a single atrial depolarization in a patient with dual AV nodal pathways.

Electrophysiological study was performed in a patient with atrioventricular nodal reentrant tachycardia (AVNRT). Double ventricular responses through dual AV nodal pathways were observed by atrial extrastimulus technique followed by initiation of AVNRT. The difference in conduction time between the slow and fast AV nodal pathways was longer than 320 msec. A ventricular extrastimulus delivered during sinus rhythm, which was not followed by ventriculoatrial conduction, also induced AVNRT. These findings indicated the presence of an antegrade critical delay and retrograde block in the slow AV nodal pathway, criteria necessary for the occurrence of a double ventricular response.


The use of implantable sensors for the control of pacemaker mediated tachycardias: a comparative evaluation between minute ventilation sensing and acceleration sensing dual chamber rate adaptive pacemakers.

The role of implantable sensors to control pacemaker mediated tachycardias was investigated in 16 patients with two different dual chamber rate adaptive (DDDR) pacemakers, which sensed eiter minute ventilation (DDDR-Meta, nine patients) or body acceleration (Relay, seven patients). Successive atrial sensed events beyond a programmable rate occurring in the absence of detection of exercise by the sensors were considered to represent retrograde conduction or atrial arrhythmias, and the pacemakers responded by either a mode shift from DDDR to ventricular rate adaptive (VVIR) pacing (DDDR-Meta) or by tracking at an interim rate, the so-called conditional ventricular tracking limit (CVTL, Relay). In the unipolar atrial sensing mode, myopotential sensing (MPI) and external chest wall stimulations (CWS) at 250 beats/min were induced to be preferentially sensed by the atrial channel to simulate the conditions of atrial arrhythmias. In the DDD mode, these maneuvers resulted in ventricular responses of 88 +/- 3 beats/min and 110 +/- 3 beats/min for MPI and CWS, respectively. The pacing rate was significantly reduced in the DDDR mode with the sensors correctly detecting and responding to the sensed abnormal atrial signals (68 +/- 5 beats/min during MPI and 71 +/- 5 beats/min during CWS, P less than 0.005 compared with the corresponding DDD rate). One patient with a Relay pacemaker developed spontaneous atrial flutter and the ventricular tracking responses were 140 and 85 beats/min in the DDD and DDDR pacing modes, respectively. Thus MPI and CWS are useful bedside testing methods to assess pacemaker response during atrial arrhythmias. The use of implantable sensors to judge the appropriateness of atrial rate is a new approach to the management of pacemaker mediated tachycardias.


Simultaneous unipolar and bipolar recording of cardiac electrical activity.

An analog mapping system using a true bipolar left ventricular balloon electrode array is described, which enables simultaneous unipolar and bipolar recordings. It is an adaptation of a previous clinical analog mapping system used in the investigation of ventricular arrhythmias. The bipolar balloon array consists of 112 electrode pairs, each having a 2-mm separation. The signals from the electrodes are sensed in parallel by separate unipolar and bipolar amplifier units, which then drive a common multiplexer bus. The bipolar recording unit consists of high quality instrumentation amplifiers with adjustable gain and exhibits a full bandwidth minimum common mode rejection of 78 dB. Using this combination, it is possible to record local cardiac micropotentials while still retaining the advantages of unipolar electrograms to track overall cardiac activation.


Myocardial rupture after pulling out a tined atrial electrode with continuous traction.

Transvenous traction for the removal of retained pacemaker electrodes is common practice with few reported complications. This case reports a patient with a DDD pacemaker with extruded electrodes, who died of a myocardial rupture at the atrial lead site after prolonged (i.e., after 24 hours) traction was applied.


Nd:YAG laser-photocoagulation: acute electrophysiological, hemodynamic, and morphological effects in large irradiated areas.

Laser-photocoagulation (LPC) of arrhythmogenic myocardium has been reported to successfully ablate ventricular tachycardia. The purpose of this study was to investigate the acute hemodynamic and electrophysiological effect of continuous laser energy (Nd:YAG, 1060 nm) applied via a 0.4-mm quartz fiberoptic on the epicardial surface of the heart in nine dogs. A total of 51 +/- 2.3 pulses was delivered in each animal to induce homogeneous tissue necrosis. Applied energy was 12.3 +/- 2.7 J/mm2, irradiated surface measured 12.6 +/- 3.0 cm2, lesion depth was 6.3 +/- 1.2 mm (range: 5.0-8.1 mm), lesion volume was 8.1 +/- 2.8 cm3 (6.8% of left ventricular [LV] mass). After LPC, epicardial stimulation threshold significantly rose from 1.0 +/- 0.3 to 10.2 +/- 4.9 mA in the border zone to nontreated tissue and from 0.9 +/- 0.4 to 32 +/- 15.7 mA in the center of the lesions. Loss of epicardial activation in the irradiated areas could be demonstrated by epicardial mapping. Ventricular extrasystoles during LPC were seen in all dogs, ventricular tachycardia in seven, and ventricular fibrillation in two dogs. After LPC, cardiac output and LV dP/dtmax significantly decreased by 14.2% and 11.2%. LPC induced predictable homogeneous tissue edema, eosinophilic staining, contraction band necrosis, and sharp demarcated hemorrhagic border zones with a sharp electrical border zone to nontreated tissue and loss of epicardial activation. During LPC, various arrhythmogenic effects could be observed. However, no persistent arrhythmic activity developed after LPC. The results confirm the feasibility of epicardial LPC of the myocardium. Although not rested in this study, LPC of arrhythmogenic tissue may also be feasible as a treatment modality of ventricular tachycardia.


Analysis of deaths in patients with an implantable cardioverter defibrillator.

The cause of death and clinical characteristics of 26 patients that died after implantable cardioverter defibrillator placement were reviewed and compared to the 145 patients still living after a mean follow-up of 17 months. Operative mortality was 4% (7/171) and resulted from postoperative ventricular arrhythmias (four patients), heart failure (two patients), and respiratory failure (one patient). Operative mortality was significantly higher (1.7% vs 9.6%, P less than 0.05) following concomitant surgical procedures. Total late mortality was 11% (18/171). Thirteen deaths (75%) occurred in-hospital from progressive deterioration of left ventricular function (nine patients), arrhythmia (two patients), and noncardiac causes (two patients). Outpatient mortality was 3.5% (6/171) and resulted from presumed sudden cardiac death in five of six patients; two of the five had devices that were inactive, one had high defibrillation thresholds, and two had suspected bradyarrhythmic deaths. One postoperative death and one late in-hospital death were also considered sudden cardiac deaths for a total of seven patients with defibrillation system failures. By multivariant analysis, preoperative clinical characteristics associated with a worse prognosis following defibrillator implantation were identified: presentation as ventricular tachycardia (P less than 0.02), induction of sustained monomorphic ventricular tachycardia (P less than 0.05), poor left ventricular performance (P less than 0.01), poor functional status (P less than 0.001), and the use of diuretics (P less than 0.01). Frequent device discharges (P less than 0.001) and concomitant antitachycardia pacing systems (P less than 0.001) were markers for greater arrhythmia recurrence and were potent predictors of a worse prognosis and particularly sudden death.


Atrial vulnerability and electrophysiology determined in patients with and without paroxysmal atrial fibrillation.

For elucidation of atrial electrophysiology and vulnerability an electrophysiological study was performed in 45 patients with documented paroxysmal atrial fibrillation and a control group (n = 46). Atrial vulnerability was assessed by programmed atrial stimulation with up to two extrastimuli during sinus rhythm and paced cycle lengths of 600 msec, 430 msec and 330 msec. Sustained atrial fibrillation or flutter was induced in 37/45 patients with paroxysmal atrial fibrillation in contrast to 9/46 patients in the control group (P less than 0.001). Left atrial diameter (M-mode echocardiogram), P wave duration, sinus cycle length, sinus node recovery time, and the effective refractory period of the right atrium were not significantly different between the two study groups. Intraatrial conduction time from the high right atrium (HRA) to the basal right atrium (A) and the functional refractory period of the right atrium were significantly longer in patients with paroxysmal atrial fibrillation.


The effect of magnetic resonance imagers on implanted neurostimulators.

This in-vitro study was designed to investigate the safety of various implanted neurostimulators in magnetic resonance (MR) imagers. The effects of the static and changing magnetic fields and the radio frequency (RF) electromagnetic field generated by 0.35 and 1.5 T MR imagers on the voltage output of four models of implantable passive neurostimulators and two models of implantable self-powered neurostimulators was studied. The neurostimulators were mounted on a support and placed in the imagers. An oscilloscope monitored the voltages at the outputs of the neurostimulators. For an Avery single-channel stimulator, located at the isocenter, the amplitude of the output pulses induced by the 0.35 T imager was 6V; from a 1.5 T imager, it was 12 V. These amplitudes can cause discomfort and possible harm to a patient if the typical therapeutic value is 1-5 V. The amplitude of the stimulator receiver's output decreased to relatively safe values beyond 40 cm from the isocenter. By contrast, there was no significant voltage output from the Medtronic SE-4 receiver. For two models of self-powered neurostimulators, the Medtronic Itrel and the Cordis MK II, the programmed stimulus parameters were not affected by the pulsed magnetic fields of the MR imagers. However, the RF fields at the isocenter heated the metal case of the stimulators. The rotational and linear forces produced by the fixed magnet on the Cordis MK II were judged to be too strong for a patient with this implant to be scanned. The study showed that patients with certain types of implanted neurostimulators can be scanned safely under certain conditions.


Serum creatine kinase activity and sensing characteristics after intraoperative arrhythmia induction using implantable defibrillator rate sensing leads.

In 29 patients (24 men, 5 woman, mean age 57 +/- 14 years) we evaluated the effect of intraoperative arrhythmia induction during implantable defibrillator (ICD) placement using alternating current (AC) applied through the epicardial rate sensing leads on acute and chronic pacing thresholds, electrogram amplitudes, slew rates and serum creatine kinase levels. In 15 patients undergoing new ICD implantation, pacing thresholds, electrogram amplitudes, slew rates, and resistances were measured before and following at least three inductions of ventricular fibrillation (VF) using AC applied through the epicardial rate sensing leads. Fourteen patients who underwent VF induction using AC through the epicardial leads during initial implant (mean time of 31 months previously) underwent ICD pulse generator replacement only with parameters measured as above before and after at least two inductions, and these compared to the values at initial implant. In all 29 patients serum creatine kinase levels were obtained before, immediately following, and at 8, 16, and 24 hours after surgery. No significant change in acute pacing threshold, electrogram amplitude, slew rate or resistance occurred. Chronically there was an expected 154% increase in pacing threshold but no significant change in electrogram amplitude or resistance. Serial serum creatine kinase and MB isoenzyme determinations demonstrated no evidence of myocardial necrosis. We conclude that intraoperative arrhythmia induction during ICD implantation using AC applied through the rate sensing leads is a safe and effective technique.


Argyrophilic nucleolar organizer regions in benign hyperplastic and cancerous human prostates.

Argyrophilic nucleolar organizer regions (AgNORs) were examined histologically in cells of benign hyperplastic and cancerous human prostates. Individual dots of AgNORs inside nucleus that were stained as separate granules or as parts of clusters were counted as one, and the average number of dots per cell was obtained by counting 100 nuclei. The number in epithelial cells was similar to that in stromal cells of hyperplastic prostates. In cancerous prostates, the number was larger than in hyperplastic prostates and increased along with upgrading. The number in incidental cancers was smaller than in clinical cancers as compared with cells of the same Gleason pattern. Number correlated with T factor, but not with N and M factors. Response to treatment and cause-specific survival in stage D2 patients receiving endocrine therapy did not correlate with number, although a relationship between Gleason pattern and survival was shown in these patients. It was concluded that AgNORs might not be an indicator to predict prognosis after endocrine therapy, since a number of AgNORs did not influence response to the therapy.


Effects of extracellular matrix components and dihydrotestosterone on the structure and function of human prostate cancer cells.

The extracellular matrix (ECM) has been shown to play a major role in cell structure and function. Several studies have demonstrated that the ECM can alter cell morphology and effect DNA synthesis and gene expression. The ECM also interacts with growth hormones which have been shown to be located in or near the ECM where they are believed to effect cell structure and function. In the nontransformed cell, these ECM and hormone-mediated effects appear to be tightly regulated and this is believed to be accomplished through cell receptor-tissue matrix interactions. We, therefore, undertook a study to determine the effects of a variety of ECM components and the adrogenic hormone dihydrotestosterone (DHT) on the structure and function of the human prostate cancer cell line, LNCaP. The effects of individual matrix components in the presence and absence of 1 nM DHT on the static and dynamic morphology, growth rate, and PSA production of the LNCaP cell line were studied. We determine that the ECM and DHT interact in complex ways to effect cell structure and function. DHT produced alterations in cytoplasmic structure that increased cell size and decreased the nuclear area/cytoplasmic area ratio. Dynamic cell structure as measured by cell motility was very sensitive to the ECM components and the presence of DHT. PSA and growth could be regulated by substratum and DHT and there was an inverse relationship between PSA production and growth rate. These data exemplify the complex interactions which occur between prostate cancer cells, ECM components, and exogenous DHT that are reflected in cell structure and function.


Activation of Src-like protein-tyrosine kinase Lyn and its association with phosphatidylinositol 3-kinase upon B-cell antigen receptor-mediated signaling.

Crosslinking of membrane-bound immunoglobulins, which are B-cell antigen receptors, causes proliferation and differentiation of B cells or the inhibition of their growth. The receptor-mediated signaling involves tyrosine phosphorylation of cellular proteins. The Src-like protein-tyrosine kinase Lyn is expressed preferentially in B cells and is an intracytoplasmic constituent of the B-cell antigen receptor complex. Crosslinking of membrane-bound immunoglobulin M with antibody induced rapid increases in the kinase activities of Lyn and Lyn-associated phosphatidylinositol 3-kinase. Crosslinking of B-cell antigen receptor also induced association of Lyn with an 85-kDa noncatalytic subunit of phosphatidylinositol 3-kinase. Thus, Lyn is functionally associated with membrane-bound immunoglobulin M and seems likely to participate in B-cell antigen receptor-mediated signaling.


Onset of cell-specific gene expression in the developing mouse pancreas.

A central question in developmental biology has been the initiation of cell-specific gene expression and its temporal relationship to morphogenesis. We have coupled embryo microdissection with the exquisite sensitivity of the polymerase chain reaction to define the onset of cell-specific gene expression during pancreatic organogenesis. Using the precise assignment of gestational age by the number of somites in each embryo, we determined the onset of transcription of major genes of the endocrine and exocrine pancreas during mouse development to within 2-3 hr. Somatostatin mRNA was detected at the 10-somite stage throughout the foregut, consistent with the presence of somatostatin-producing cells throughout the adult gut. Mature mRNA for insulin and glucagon first appears surprisingly early, at the 20-somite stage in the wall of the embryonic foregut and is restricted to only the area of the duodenum from which the pancreas will arise 10-12 hr later. In contrast, exocrine gene transcription begins 24 hr after formation of the pancreatic diverticulum. Thus cell-specific gene expression in the endocrine pancreas begins in a "pre-morphogenetic phase." This early expression of insulin and glucagon could reflect the initiation of an endocrine cell lineage.


Heterogeneous nuclear ribonucleoprotein A1 catalyzes RNA.RNA annealing.

Within the nucleus, pre-mRNA molecules are complexed with a set of proteins to form heterogeneous nuclear ribonucleoprotein complexes. A1, an abundant RNA binding protein present in these complexes, has been shown to bind selectively to single-stranded RNAs and destabilize base-pairing interactions. In this study.A1 is shown to promote the rate of annealing of complementary RNA strands greater than 300-fold under a wide range of salt concentration and temperature. Maximal annealing is observed under saturating or near saturating concentrations of protein, but annealing decreases sharply at both higher and lower concentrations of A1. Kinetic analysis shows that the rate of annealing is not strictly first or second order with respect to RNA at a ratio of protein/RNA that gives optimal rates of annealing. This result suggests that A1 protein may affect more than one step in the annealing reaction. Two polypeptides representing different domains of A1 were also examined for annealing activity. UP1, a proteolytic fragment that represents the N-terminal two-thirds of A1, displays very limited annealing activity. In contrast, a peptide consisting of 48 amino acid residues from the glycine-rich C-terminal region promotes annealing at a rate almost one-quarter that observed with intact A1. The RNA.RNA annealing activity of A1 may play a role in pre-mRNA splicing and other aspects of nuclear mRNA metabolism.


Host-cell-phenotype-dependent control of the BCR2/BWR1 promoter complex regulates the expression of Epstein-Barr virus nuclear antigens 2-6.

Epstein-Barr virus nuclear antigens (EBNAs) are expressed in a cell-phenotype-dependent manner. EBNA 1 is regularly expressed in all Epstein-Barr virus-carrying cells, whereas EBNAs 2-6 are only expressed in Epstein-Barr virus-carrying cells with a lymphoblastoid phenotype including group III Burkitt lymphoma (BL) lines positive for B-cell activation markers. Transcripts are initiated at the BCR2 or exceptionally at one BWR1 promoter in lymphoblastoid cell lines and group III BL lines. In group I BL lines, nasopharyngeal carcinoma, and the somatic cell hybrids, where EBNAs 2-6 are downregulated, the BCR2/BWR1 promoter complex is inactive or switched off. Upregulation of EBNAs 2-6 in group III BL cells and in 5-azacytidine-treated group I BL cells accompanies the activation of the silent BCR2/BWR1 promoters. Activation of BCR2 parallels demethylation of at least one CpG pair in the same promoter region. The activity of BCR2/BWR1 promoter complex depends on a particular B-cell phenotype. EBNA 1 transcription must be initiated at another promoter in cells that express only EBNA 1.


Extensive size polymorphism of the human keratin 10 chain resides in the C-terminal V2 subdomain due to variable numbers and sizes of glycine loops.

Existing data suggest that the human keratin 10 intermediate filament protein is polymorphic in amino acid sequence and in size. To precisely define the nature of the polymorphism, we have used PCR amplification and sequence analyses on DNA from several individuals including five with documented size variations of the keratin 10 protein. We found no variation in the N-terminal or rod domain sequences. However, we observed many variations in the V2 subdomain near the C terminus in glycine-rich sequences with a variation of as much as 114 base pairs (38 amino acids), but all individuals had either one or two variants. Our results show that (i) the keratin 10 system is far more polymorphic than previously realized, (ii) the polymorphism is restricted to insertions and deletions of the glycine-rich quasipeptide repeats that form the glycine-loop motif in the C-terminal domain, (iii) the polymorphism can be accounted for by simple allelic variations that segregate by normal Mendelian mechanisms, and (iv) the differently sized PCR products most likely represent different alleles of a single-copy gene per haploid genome.


Double-stranded RNA-dependent RNase activity associated with human immunodeficiency virus type 1 reverse transcriptase.

Early events in the retroviral replication cycle include the conversion of viral genomic RNA into linear double-stranded DNA. This process is mediated by the reverse transcriptase (RT), a multifunctional enzyme that possesses RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities. In the course of studies of a recombinant RT of human immunodeficiency virus type 1 (HIV-1), we observed an additional, unexpected activity of the enzyme. The purified RT catalyzes a specific cleavage in HIV-1 RNA hybridized to tRNALys, the primer for HIV-1 reverse transcription. The cleavage at the primer binding site (PBS) of HIV RNA is dependent on the double-stranded structure of the HIV RNA-tRNALys complex. This RNase activity appears to be distinct from the RNase H activity of HIV-1 RT, as the substrate specificity and the products of the two activities are different. Moreover, Escherichia coli RNase H and avian myeloblastosis virus RT are unable to cleave the HIV RNA-tRNALys complex. We refer to this unusual activity as RNase D. Two lines of evidence indicate that the specific RNase D activity is an integral part of recombinant HIV RT. The specific RNase D activity comigrates with the other RT activities, DNA polymerase, and RNase H upon filtration on a Superose 6 gel column or chromatography on a phosphocellulose column. Moreover, three recombinant HIV-1 RT preparations expressed and purified in different laboratories by various procedures exhibit RNase D activity. Sequence analysis indicated that RNase D activity cleaves the substrate HIV-1 RNA-tRNALys at two distinct sites within the PBS sequence 5'-UGGCGCCCGA decreases ACAG decreases GGAC-3'. The sequence specificity of RNase D activity suggests that it might be involved in two stages during the reverse transcription process: displacement of the PBS to enable copying of tRNALys sequences into plus-strand DNA or to facilitate the second template switch, which was postulated to occur at the PBS sequence.


Regulation of adenylyl cyclase from Paramecium by an intrinsic potassium conductance.

Hyperpolarization of the cell membrane of Paramecium stimulates adenosine 3',5'-monophosphate (cAMP) formation. Manipulations of the K+ resting conductance of the ciliate by adaptation in different buffers affected excitability of the cAMP generating system. Blockade of K+ channels inhibited hyperpolarization-stimulated cAMP formation. A mutant of Paramecium that is unable to control its K+ resting conductance had a defect in cAMP formation. Purified adenylyl cyclase, when incorporated into an artificial lipid bilayer membrane, revealed properties of a voltage-independent K+ channel. This indicates that the adenylyl cyclase of Paramecium has a secondary function as carrier of the K+ resting conductance. A hyperpolarization-activated K+ efflux appears to directly regulate adenylyl cyclase activity in vivo.


[Acute urinary obstruction].

Acute urinary obstruction is a painful occurrence well known to every physician. It is a matter of a reflexogenic closure of the bladder neck. As the nerves, regulating the neck as well as the motoricity of the bladder, extend directly over the capsule of the prostate, functional disorders during growth of the prostate are possible at any time, independent of the prostatic volume. Since in case of prostatic hyperplasia the smooth muscles of the bladder neck are partly transformed into fibrous tissue, drug treatment in case of acute urinary obstruction is impossible. Therefore catheterisation is applied now as before.


[Ultrasonography of the prostate].

Suprapubic sonography of the prostate is a comfortable and painless examination which requires not much effort and yields the relevant information about size, form and residual urine for the daily clinical routine. For the assessment of the echo structure, particularly in case of carcinoma of the prostate, transrectal sonography is superior to suprapubic examination. However, transrectal ultrasound examination in case of prostatic carcinoma or suspicion of prostatic carcinoma is not often indicated, since in the majority of the cases the diagnosis as well as the therapeutic concept can be clearly determined, based on rectal palpation, determination of prostate specific antigen (PSA) and prostatic biopsy. As screening investigation in the early diagnosis of prostatic carcinomas, transrectal sonography is an inappropriate measure due to poor sensibility as well as specificity.


Comparison of Bay K 8644, nitrendipine and atropine on spontaneous and pelvic-nerve-induced bladder contractions on rat bladder in vivo.

The effects of the dihydropyridine-type calcium antagonist (nitrendipine) and agonist (Bay K 8644) in comparison to atropine have been studied after intravenous administration on spontaneous and pelvic-nerve-induced contraction of rat urinary bladder. Bay K 8644 increased the basal internal bladder pressure as well as the amplitude of the spontaneous bladder contractions in a dose-dependent manner. In addition, an increase in systemic arterial blood pressure was noted for a period of about 20 min. In the presence of atropine the effects of Bay K 8644 on the urinary bladder were almost completely antagonized. Both nitrendipine and atropine reduced in a dose-dependent manner the amplitude of spontaneous and nerve-induced bladder contraction. The spontaneous and nerve-induced bladder contractions were significantly reduced by atropine or nitrendipine. Only nitrendipine caused a reduction of the spontaneous bladder contraction frequency. The systemic blood pressure was decreased significantly by nitrendipine but not after atropine administration. We suggest that both calcium antagonist and agonist can change the tension of the urinary bladder in vivo. As a side-effect the systemic blood pressure is altered. Atropine can antagonize the effect of BayK 8644 on the urinary bladder and reduces spontaneous and nerve-induced bladder contractions more specifically than nitrendipine.


Receptor function studies in specimens from the proximal human urethra obtained by transurethral resection.

During transurethral resection (TUR) for prostatic hyperplasia, specimens were taken from the proximal urethra. Muscle strips thus obtained were mounted in an organ bath and muscle contraction was induced by adding increasing concentrations of noradrenaline (NA), methoxamine (alpha 1-agonist) and clonidine (alpha 2-agonist). NA and methoxamine induced a dose-dependent muscle contraction, but clonidine had no effect. The influence of prazosin (alpha 1-antagonist) and yohimbine (alpha 2-antagonist) on the NA-induced muscle contraction was also evaluated. Both antagonists had an inhibitory effect, which was much more potent with prazosin. The specimens taken during TUR were found to be suitable for in vitro receptor function studies. The alpha-adrenergic receptor function in the proximal human urethra was found to be mainly of the alpha-type.


Fine specificity of the B-cell epitopes recognized in HIV-1 NEF by human sera.

We have previously used partially overlapping synthetic nonapeptides to characterize the human natural antibody response against HIV-1 negative regulatory factor (NEF), and identified nine 5 to 13 amino acid long regions that were recognized by sera of HIV-1-infected individuals. In this report we define the minimal size of these epitopes with the use of shorter, from 3 to 8 amino acid long partially overlapping peptides covering the complete sequence of the previously identified reacting regions and the N- and C-terminal flanking sequences. We also introduce a new method for the analysis of the reactivities obtained with peptides of different lengths. In six of the antigenic regions the epitopes were found to be noncontiguous and to consist of multiple, down to three amino acid long separate reactive stretches (epitope 1: WSK, VGW, TVRERMRR; epitope 3A: PLRPM, SHFLK; epitope 3B: SQRRQD, DLW; epitope 3C: IYHT, QGYFPDWQN; epitope 4: SLL, VSL; epitope 5: EVLEWRFDSR, VAR). Three epitopes were clearly linear (epitope 2: CAWLE; epitope 3D: LTFGWC; epitope 6: PEYF). Interestingly, five of the minimized B-cell epitopes (1, 3A, 3C, 3D, 5) recognized by human sera overlap totally or partly with the previously identified T-cell epitopes in HIV-1 NEF. Also, only three of the epitopes (3C, 3D, 5) were in a computer-based homology search shown to contain strictly NEF-specific sequences.


Neutralizing epitopes on herpes simplex virus-1-expressed rotavirus VP7 are dependent on coexpression of other rotavirus proteins.

We constructed a recombinant thymidine kinase-negative herpes simplex virus type 1 (HSV-1) that expressed the rotavirus major outer capsid glycoprotein, VP7. In the recombinant HSV-1, a promoter from the 5' noncoding region of the HSV-1 glycoprotein B locus regulated the expression of VP7 as a HSV-1 gamma 1 gene product. HSV-1-expressed VP7 resembled rotavirus-expressed VP7 in its SDS-PAGE mobility, high mannose-type glycosylation, disulfide bonding, perinuclear to cytoplasmic localization, intracellular retention, and reactivity with polyclonal antisera and nonneutralizing antibodies. Unlike rotavirus-expressed VP7, HSV-1-expressed VP7 lacked several neutralizing epitopes by immuno-histochemical staining and by ELISA. One neutralizing epitope identified on HSV-1-expressed VP7 by ELISA was masked by paraformaldehyde fixation of recombinant HSV-1- but not rotavirus-infected cells. Neutralizing epitopes were restored to HSV-1-expressed VP7 by coinfection of cells with the HSV-1 recombinant and a heterologous rotavirus that lack the neutralizing epitopes. The recovered neutralizing epitopes were detected on double-shelled rotavirus particles produced in the coinfected cells. This study indicates that the formation of several neutralizing epitopes on rotavirus VP7 requires interaction of VP7 with other rotavirus proteins. In addition, HSV-1 was a useful vector for studying the localization, processing, and antigenicity of an RNA virus glycoprotein.


Characterization of a major neutralization domain of Ross river virus using anti-viral and anti-peptide antibodies.

The E2 glycoprotein of the alphavirus Ross River virus (RRV) contains three defined neutralization epitopes (a, b1 and b2) with determinants located between amino acids 216 and 251 in the linear sequence (Vrati et al., 1988, Virology 162, 346-353). The antigenic structure of this region has been examined using hyperimmune mouse antiserum against RRV and antiserum against four synthetic peptides representing linear amino acid sequences in the neutralization region of E2. In plaque reduction neutralization tests using hyperimmune antiserum to RRV, an RRV mutant altered at all three neutralization epitopes was markedly more resistant than the parental virus; variants altered at single epitopes could not be distinguished in these tests. Sera from mice immunized with synthetic RRV E2 peptides conjugated to keyhole limpet haemocyanin reacted, in a direct ELISA, with the specific region of RRV represented by the peptide. The same sera did not neutralize or immunoprecipitate RRV in solution or bind to RRV in a capture ELISA. The RRV peptides did not prime mice to react to a subimmunogenic dose of RRV; they did not bind monoclonal or polyclonal antibodies to RRV. We conclude that a significant proportion of the neutralizing antibody response in mice is elicited by epitopes a, b1, and b2 of RRV E2 and that the sites to which neutralizing antibodies bind are formed by complex folding.


Epitope-mapped monoclonal antibodies against the HPV16E1--E4 protein.

The human papillomavirus (HPV) E1--E4 protein is the only nonstructural late protein encoded by the virus. We have isolated three hybridomas producing monoclonal antibodies to the E1--E4 protein of HPV16, which is the HPV type most frequently associated with cervical cancer. The three antibodies (TVG 401, 402, and 403) detect adjacent epitopes within the major seroreactive region of the molecule and show no reactivity against the E4 proteins of HPV1, HPV2, HPV4, or HPV6. The E1--E4 protein migrates as a 10K species on SDS-gel electrophoresis and forms cytoplasmic inclusion granules in infected cells in vitro similar in appearance to those produced by HPV1 in benign warts. In naturally occurring HPV16-induced tumors the E1--E4 protein was detected in the cytoplasm of cells in the upper layers of the lesion in areas in which HPV16 DNA replication was occurring, as determined by in situ hybridization. Although the epitopes recognized by these monoclonal antibodies survive brief fixation in 5% formaldehyde, reactivity was destroyed by prolonged fixation. These monoclonal antibodies represent the first against HPV16 E1--E4 and should complement those already available to E7 and L1 for the screening of frozen sections of clinical biopsies and will be of value in monitoring the progression of HPV infection from benign lesions to invasive cancer.


Localization of the antigenic sites and intrinsic protein kinase domain within a 300 amino acid segment of the ribonucleotide reductase large subunit from herpes simplex virus type 2.

The 140-kDa ribonucleotide reductase (RR1) protein of herpes simplex virus type 2 (HSV-2) functions as the large subunit of virus-specified RR1 and exhibits an intrinsic protein kinase (PK) activity at its unique NH2-terminal region. The N-terminal half of RR1 contains the protein and DNA functions of the morphological transforming region III (mtrIII) of HSV-2. In the present study, we have expressed a number of truncated RR1 derivatives in a mammalian expression vector containing NH2-terminal RR1 gene fragments and amber mutants generated by site-specific mutagenesis. These derivatives, synthesized in transient expression assays, were used as test antigens to localize the epitopes of a panel of HSV-2 RR1-reactive monoclonal antibodies and to fine-map the PK catalytic domain. Our data show that the epitope for HSV-2-specific monoclonal antibody 6A-6 is located in a region of RR1 protein spanning aa 72-350. The epitopes for cross-reactive antibodies to HSV RR1, i.e., 48S and 51S, are formed predominantly by a stretch of amino acid residues specified by aa 350-376 of the RR1 molecule. The 6A-6 antibody utilized in conjunction with the RR1 amber mutants has allowed us to define a 278 aa domain within the NH2-terminal half of the 140-kDa RR1 (aa 72-350) that is sufficient for PK activity.


Mechanism of inhibition of HIV-1 infection in vitro by purified extract of Prunella vulgaris.

Crude extracts of four Chinese herbs, Arctium lappa, Astragalus membranaceus, Andrographis paniculata, and Prunella vulgaris, were assessed in several tissue culture lines for anti-HIV activity and for cytotoxicity. One extract, obtained from P. vulgaris, was able to significantly inhibit HIV-1 replication with relatively low cytotoxicity. The active factor was purified using sequential precipitations with ethanol and n-butanol, followed by reverse-phase and gel permeation high-performance liquid chromatographic separations. The active component was anionic with a molecular weight of approximately 10 kDa. The purified extract inhibited HIV-1 replication in the lymphoid cell line MT-4, in the monocytoid cell line U937, and in peripheral blood mononuclear cells at effective concentrations of 6, 30, and 12.5 micrograms/ml, respectively. Pretreatment of uninfected cells with the extract prior to viral exposure did not prevent HIV-1 infection. By contrast, preincubation of HIV-1 with the purified extract dramatically decreased infectiousness. The purified extract was also able to block cell-to-cell transmission of HIV-1, prevented syncytium formation, and interfered with the ability of both HIV-1 and purified gp120 to bind to CD4. PCR analysis confirmed the absence of HIV-1 proviral DNA in cells exposed to virus in the presence of the extract. These results suggest that the purified extract antagonizes HIV-1 infection of susceptible cells by preventing viral attachment to the CD4 receptor.


Activation of human immunodeficiency virus type 1 provirus in T-cells and macrophages is associated with induction of inducer-specific NF-kappa B binding proteins.

We have analyzed the limiting factors involved in the induction of human immunodeficiency virus type 1 (HIV-1) provirus expression by tumor necrosis factor-alpha (TNF-alpha), phorbol-12-myristate-13-acetate (PMA), and bryostatin-1 in T-cells (ACH-2) and monocytes (U1). We have demonstrated that, while there is a correlation among the increase of 9.2-kilodalton (kDa) HIV-1 RNA, the increase of viral proteins (p24) in the cells, and the release of HIV-1 virions into the medium, there is no direct correlation between the levels of induced NF-kappa B binding proteins and the expression of HIV-1 provirus. The presence of nuclear NF-kappa B-specific proteins appears to be essential only for the initiation of viral replication, since the HIV-1 transcripts could be detected in TNF-alpha or bryostatin-1-stimulated cells also at later times postinduction, times when no NF-kappa B proteins could be detected in the nucleus. The uv crosslinking of DNA and proteins has shown that TNF-alpha, PMA, and bryostatin-1 induce different sets of NF-kappa B binding proteins with distinct kinetics of binding.


Ontogenic differences in the nutritional regulation of circulating IGF binding proteins in sheep plasma.

Well-fed castrated male sheep (N = 3) and 125 days gestation pregnant ewes (N = 6) with chronically catheterized fetuses were fasted for 72 h. Insulin-like growth factor-binding protein (IGFBP) levels in fed and starved fetal, maternal and castrated male sheep plasma were measured using ligand blot analysis. IGFBPs in adult and fetal sheep differed in distribution both before and after 72 h starvation. IGFBP-3 was the major postnatal binding protein, while in the fetus IGFBP-2, IGFBP-3 and the circulating IGF type 2 receptor fragment each contributed 25-30% of total IGF binding capacity. After starvation, total IGF binding capacity and IGFBP-3 fell in plasma of maternal and castrated male sheep (p less than 0.05). Total IGF binding capacity rose with starvation in fetal plasma (p less than 0.05) as a result of an increase in IGFBP-1 (p less than 0.01) and IGFBP-2 (p less than 0.05). The different nutritional control of the IGFBPs in the fetus and the adult may reflect ontogenic differences in the regulation and function of circulating IGFs and their binding proteins.


Functional expression of human myometrial endothelin receptors in Xenopus laevis oocytes.

We demonstrate the existence of functional endothelin receptors in human uterine myometrium using the Xenopus oocyte expression system. Fifty nanograms of poly(A)+RNA from myometrium was injected into Xenopus laevis oocytes and incubated for 70-80 h. The membrane potential of the oocyte was clamped at -60 mV and membrane current was measured during and after endothelin stimulation. Endothelin-1 elicited a large inward membrane current in the oocytes injected with poly(A)+RNA; endothelin-2 elicited a small current; while endothelin-3 did not induce any membrane current. These results indicate the existence of messenger RNA encoding functional endothelin-1 receptors in human uterine myometrium.


Polarized efflux of iodide in porcine thyrocytes occurs via a cAMP-regulated iodide channel in the apical plasma membrane.

The intracellular regulation of thyrotropin-stimulated iodide efflux was studied in polarized porcine thyrocytes grown as a continuous, tight monolayer in bicameral culture chambers. From a previous study using this system we know that thyrotropin rapidly increases iodide efflux in the apical but not basal direction of the polarized epithelium. [125I]-iodide efflux in apical direction was stimulated by thyrotropin in a concentration-dependent manner (1-10 U/l), whereas efflux in basal direction was unchanged at any thyrotropin dose. Thyrotropin-induced elevation of intracellular cAMP showed a corresponding concentration dependence. The selective stimulation of apical efflux by thyrotropin was evident also when re-uptake of iodide released in basal direction was blocked by perchlorate. The effect of thyrotropin on apical efflux was mimicked by 8-bromo-cAMP and forskolin, whereas agents known to activate the Ca2+/phosphatidylinositol cascade (epidermal growth factor) and protein kinase C (phorbol ester) or increase cytosolic [Ca2+] (A23187) were inactive. We conclude that the selective stimulation by thyrotropin of apical iodide efflux, corresponding to efflux in luminal direction in intact follicles, occurs via cAMP-regulated iodide channels present in the apical domain of the plasma membrane.


Preservation of endothelium-dependent vasodilation in the spastic segment of the human epicardial coronary artery by substance P.

The objective of this study was to determine if endothelium-dependent vasodilation is preserved in the spastic segment of the epicardial coronary artery. Segmental responses of the coronary artery to substance P were examined by the use of a quantitative angiographic technique in 21 patients with variant angina. Coronary diameter at the basal state did not differ between the spastic and the nonspastic segments (2.3 +/- 0.2 mm, 2.3 +/- 0.4 mm, p greater than 0.05). Changes in coronary diameter in response to substance P did not differ between segments with ergonovine-induced spasm and nonspastic segments. Maximal dilation averaged 27.1 +/- 9.5% in the spastic segment and 24.4 +/- 9.6% in the nonspastic segment (expressed as a percent increase over the value before drug administration). It appears that both the potential of the endothelium to release endothelium-dependent relaxing factor and the dilating response of the smooth muscle to endothelium-dependent relaxing factor are preserved, even in the spastic segment.


The validity of continuing developmental follow-up of high-risk infants to age 5 years.

We conducted a study to determine whether performance on developmental tests at age 5 years could predict academic achievement at age 8 years. As part of a longitudinal developmental surveillance project, 179 children at risk due to perinatal complications who had passed developmental screening through age 2 1/2 years and 50 comparison children underwent an extensive prekindergarten psychoeducational test battery at age 5 years and took the Iowa Tests of Basic Skills at age 8 years, if they had reached the third grade. The mean Iowa Tests of Basic Skills score was significantly lower for those children who were "flagged" on the prekindergarten psychoeducational test battery (t = 5.39). Preacademic, rather than developmental, items appeared to be the best predictors. However, the prekindergarten psychoeducational test battery correctly predicted low achievement or grade retention in only 58% of cases. Its sensitivity was 0.45 and its specificity was 0.85. No significant difference was noted between group Iowa Tests of Basic Skills mean scores for the high-risk or comparison group. When low achievement and failure to reach the third grade were combined, prevalence of "failure" was higher for the high-risk group (31% vs 24%). The only perinatal variable predictive of low achievement was neonatal seizures. In summary, because the ability to predict future academic achievement at age 5 years is limited, routine developmental testing for symptom-free preschool children is not warranted. High-risk infant follow-up programs should focus on the first several years of life.


Mechanism of action of foscarnet against viral polymerases.

Foscarnet is a pyrophosphate analogue with activity against herpesviruses, human immunodeficiency virus (HIV), and other RNA and DNA viruses. Foscarnet and its analogues achieve their antiviral effects via inhibition of viral polymerases, with such inhibition not being dependent on activation or phosphorylation of the compounds by viral or cellular proteins. Current evidence indicates that foscarnet interferes with exchange of pyrophosphate from deoxynucleoside triphosphate during viral replication by binding to a site on the herpesvirus DNA polymerase or HIV reverse transcriptase. Reviewed herein are basic findings regarding the mechanism of action and antiviral activity of foscarnet and the related compound phosphonoacetic acid (PAA), as well as findings regarding potential mechanisms of viral resistance and interactions with other antiviral agents.


Clinical pharmacology: foscarnet.

Foscarnet exerts its antiviral effects via reversible inhibition of viral polymerases. Pharmacodynamic data indicate that herpesvirus and human immunodeficiency virus replication is inhibited by therapeutically achievable concentrations of foscarnet; however, the concentrations of foscarnet required for such inhibition have been found to vary widely. Pharmacokinetic data indicate that foscarnet is eliminated via the renal route, undergoes negligible metabolism, and appears to be distributed widely from the circulation. However, the available data indicate that the pharmacokinetics of the drug varies among patients and within the individual patient, particularly with regard to plasma drug levels; furthermore, such factors as the intracellular kinetics of the drug have yet to be well characterized. It is thus difficult to formulate optimal dosing regimens on the basis of what is known of foscarnet pharmacodynamics and pharmacokinetics. Nevertheless, dosages that produce clear-cut therapeutic benefits without unacceptable toxicity have been identified in clinical trials of foscarnet in acquired immunodeficiency syndrome (AIDS) patients with cytomegalovirus (CMV) retinitis.


In vitro effects of interleukin-2 and gamma globulin on immune dysfunction in a patient with severe mucocutaneous herpes simplex virus infection.

A previously healthy woman developed severe, recurrent mucocutaneous herpes simplex virus (HSV) infection at 21 years of age. Immunologic assessment over the past 2 years has revealed persistent T-cell and natural killer cell dysfunction despite normal numbers of these cells as measured by flow cytometry. We studied the effect of recombinant interleukin 2 (rIL-2) and gamma globulin on the patient's mononuclear cells in 18-hour 51Cr release assays using HSV-infected and uninfected target cells. Both gamma globulin and rIL-2 significantly enhanced target cell lysis of HSV-infected target cells (P less than .001), but did not increase lysis of uninfected target cells. Addition of the patient's serum had no effect on HSV-infected target cell lysis despite a high HSV IgG titer, indicating a possible specific abnormality in production of antibody-dependent cellular cytotoxicity antibody.


Exercise-induced allergies: the role of histamine release.

Exercise is a physical cause of allergic reactions, including exercise-induced anaphylaxis (EIAna), exercise-induced urticaria (EIU), exercise-induced asthma (EIA), and exercise-induced rhinitis (EIR). Since its first description in 1979, EIAna has been reported with variable clinical manifestations, with exercise alone, and in combination with food ingestion. Elevated serum histamine levels and cutaneous mast cell degranulation have been noted. Exercise-induced urticaria appears as small, punctate lesions that differ from the classic coalescent type seen with EIAna. Variant forms of EIAna with cholinergic urticarial lesions manifesting systemic collapse and/or respiratory distress have been studied. Exercise-induced urticaria and cold-induced urticaria may cause elevated plasma histamine levels coincident with the onset of pruritus and hives. Theories accounting for EIA include respiratory heat loss, water loss, and mast cell activation. Although some studies have shown increased plasma histamine with EIA, others have not. Recently, bronchoalveolar lavage in atopic subjects with EIA has been evaluated preexercise and postexercise, with no significant differences in histamine or tryptase, suggesting a pathogenesis of EIA independent of the mast cell. Exercise-induced rhinitis, with varying degrees of rhinorrhea, congestion, and sneezing, has been increasingly recognized in athletes who run, cycle, and ski. Cold-air-induced rhinorrhea in laboratory challenges displays a mediator release pattern similar to that produced by allergen-induced nasal challenges. Therapeutically, H1 antihistamines are recommended for EIAna both as pretreatment and acute therapy. H1 antihistamines may be helpful in EIU, but are recommended for EIAna both as pretreatment and acute therapy.(ABSTRACT TRUNCATED AT 250 WORDS)


Endoprosthetic replacement for bony metastases.

A series of 38 patients with long bone metastases treated at the Birmingham Bone Tumour Treatment Service with resection of the metastatic lesion and replacement of the bone defect with an endoprosthesis was reviewed. The majority of cases had pathological fractures due to a massive destructive lesion. Two-thirds of the patients had a solitary metastasis. Metastases from hypernephroma and breast carcinoma accounted for the majority of cases. All the patients were independently mobile after the endoprosthetic replacement and were pain free. The average survival rate after the endoprosthetic replacement was 14.7 months and this varies with the primary tumour. The indications for endoprosthetic replacement for the treatment of long bone metastases are outlined and the results and complications are discussed. It is concluded that endoprosthetic replacement for bony metastases is an effective palliative procedure for a selected group of patients.


Differential effects of human recombinant interferons on the expression of two early gene products of Epstein-Barr virus.

Two human Burkitt's lymphoma (BL) cell lines, Raji and Daudi, have been previously characterized as resistant and sensitive, respectively, to the anti-Epstein-Barr virus (EBV) effects of human leukocyte interferon. These cells are equally susceptible to P3HR-1 EBV superinfection as determined by EBV early antigen (EA) expression. The cell lines were pretreated with human recombinant interferons alpha 2, beta, or gamma and subsequently superinfected with P3HR-1 EBV. Their expression of two distinct EBV early gene products was evaluated by fluorescence microscopy. Monoclonal antibodies to the diffuse (EA-D) and restricted (EA-R) components of the EA complex were used to determine the number of cells expressing each of these antigens in the treated cell lines. As previously described with human leukocyte interferon, EA-D expression in Raji cells was relatively resistant to interferon-alpha 2 pretreatment. Also, EA-D expression in Daudi cells was relatively sensitive. However, interferon alpha 2 pretreatment produced an opposite pattern with respect to the expression of EA-R in these two cell lines; Raji cells were sensitive and Daudi cells relatively resistant. Interferon beta had the most uniformly effective anti-EBV activity on both cell lines; less than 15 U/ml produced 50% inhibition of both antigens in both cell lines. EA-D expression in both cell lines was sensitive to interferon-gamma pretreatment and EA-R was resistant. These data suggest that different gene products of EBV are independently regulated by interferons based on at least three factors: (1) the host cell, (2) the type of interferon and (3) the affected gene product.


[Clinical approach to infection in patients with hematologic malignancy].

Patients with hematologic malignancy are susceptible to infection because of the disease process and its treatment. Profoundly granulocytopenic patients are at increased risk of developing Pseudomonas aeruginosa bacteremia, often with a fatal outcome. Therapy with one or two anti-pseudomonal beta-lactam antibiotics and an aminoglycoside in combination that were effective in vitro against the infecting organism proved to be superior, by one-week survival, to therapy with either one in vitro effective beta-lactam or aminoglycoside or inadequate drugs. On the other hand, treatment with granulocyte colony-stimulating factor had no significant association with longer survival, although a favorable outcome was well correlated with an increase in the granulocyte count during therapy. An active mycobacteriosis was documented in 2% of all patients with hematologic malignancy. Dissemination occurred in half of them. The prognosis of tuberculosis was depended mainly on early diagnosis and treatment, while that for the atypical variety was largely influenced by the underlying disease. The frequency of deep fungal infection in patients with acute leukemia at autopsy increased progressively from 10% in 1970-1974 to 38% in 1983-1986, but it decreased somewhat to 29% in 1987-1989 after the introduction of empiric amphotericin B therapy in 1986. Early empiric antifungal therapy should therefore be started in granulocytopenic patients with fever refractory to antibacterial therapy, because of unreliability of the current serodiagnosis. A total protective isolation for patients undergoing bone marrow transplantation (BMT) was associated with a reduced incidence of pneumonia, especially due to Aspergillus, and to a lesser extent, bacteremia. Cytomegalovirus pneumonia complicating BMT continues to have a poor prognosis, although the frequency has gradually been decreasing with the introduction of effective preventive measures. An early diagnosis and treatment as well as preventive measures is thus necessary for infection control in patients with hematologic malignancy.


[Respiratory infections associated with lung cancer].

We have analyzed the clinical significance of secondary infections associated with lung cancer patients. The incidence of secondary infections was 51.4% in 214 in-patients with lung cancer admitted to our institution in 1988 and 1989, and almost all of them had respiratory tract infections. The incidence was high in patients with cell types other than adenocarcinoma, and in those with hypoproteinemia, impaired cellular immunity and obstruction of the airway. The prognosis in patients with infection was much poorer than that in patients without infection. Major causative pathogens were Staphylococcus aureus including methicillin-resistant S. aureus (MRSA), Haemophilus influenzae, Klebsiella spp. and Pseudomonas aeruginosa. These pathogens except for H. influenzae were isolated at the terminal stage, in cases with airway obstruction and in post cancer-chemotherapeutic phase. The efficacy rate of 194 chemotherapeutic regimens against infection was 57.7%. Although the efficacy rate in 1988 and 1989 exceeded that in the 1970s, there was no significant difference in the efficacy rate between monotherapy (57.1%) and combined therapy (59.3%). The effectiveness was very poor for infections caused by P. aeruginosa and MRSA, or for cases with airway obstruction and marked impairment of pulmonary blood flow. The above results showed that a new combined therapy as well as the measures to improve the general condition of compromised hosts are required in the treatment of secondary infections in these patients.


[High-dose thio-TEPA with escalating doses of epirubicin and autologous hematopoietic stem cell transplant for refractory cancers].

We studied high-dose chemotherapy with autologous hematopoietic stem cell transplant for patients (pts) with non-Hodgkin's lymphoma (NHL) and breast cancer (BC) refractory to conventional therapies. The conditioning regimen consisted of thio-TEPA 6 mg/kg/day for 3 consecutive days with escalating doses of epirubicin (EPI) in dose steps of 120, 150, 180 and 210 mg/m2 on day 1. Mucositis was dose limiting toxicity at 210 mg/m2 on this regimen, and the recommended dose of EPI was judged to be 180 mg/m2. No cardiotoxicities were observed. There were 3 with complete responses (CR), one partial response (PR) in pts with NHL, 3CR and 5PR in pts with BC. The median duration of response was 8 months (mos) and 4 mos, respectively. Hematological recovery was significantly earlier in the pts receiving both autologous bone marrow transplant (ABMT) and peripheral blood stem cell transplant (PBSCT) than ABMT alone. This approach made it possible to overcome the prolonged PLT recovery, which was one of the major problems on ABMT.


[Fundamental study of subrenal capsule assay by measuring specific activity of succinate dehydrogenase].

It may not show accurate results if subrenal capsule assay (SRCA) is made only by measuring tumor size, because of infiltration of host inflammation cells resulted from host immune reaction. We developed a new method which make possible an accurate determination of chemosensitivity by measuring specific activity of succinate dehydrogenase (SD) of the tumor cells implanted in the subrenal capsular space. With reference to SDI test, the assay condition for measuring specific activity of SD was determined. A comparative study was carried out in which malignant tumors of the oral cavity serially transplanted in nude mice were tested with SRCA and subcutaneous transplantation assay in nude mice. Chemosensitivity to peplomycin (PEP), CDDP and 5-fluorouracil (5-FU) evaluated SSDI method and nude mouse assay showed a high correlation than those evaluated by TGIR method and nude mouse assay. The overall predictive accuracy compared with nude mouse assay was 72.2% by TGIR method and 88.9% by SSDI method. SSDI method seemed to be a useful method to evaluate the chemosensitivity in SRCA.


[A case of diffuse large cell lymphoma treated with combination chemotherapy of CHOP (cyclophosphamide, doxorubicin, vincristine, prednisolone) and BEMP (bleomycin, etoposide, mitoxantrone, procarbazine) with complete remission].

A 73-year-old man with diffuse large cell lymphoma was treated with noncross resistant alternating combination chemotherapy of CHOP and BEMP, consisting of cyclophosphamide (1,000 mg/body, i.v., day 1), doxorubicin (60 mg/body, i.v., day 1), vincristine (2 mg/body, i.v., day 1), prednisolone (100 mg/body, p.o., day 1-5), bleomycin (30 mg/body, i.v., day 22), etoposide (80 mg/body, i.v., day 22-24), mitoxantrone (6 mg/body, i.v., day 22), and procarbazine (100 mg/body, p.o., day 22-26). Following the three courses' administration of CHOP and BEMP, complete remission was obtained with a remission duration of over ten months. Leukocytopenia was a dose-limiting factor. It is concluded that noncross resistant alternating combination chemotherapy of CHOP and BEMP is effective for diffuse large cell lymphoma.


Binding of transforming growth factor-beta 1 to methylamine-modified alpha 2-macroglobulin and to binary and ternary alpha 2-macroglobulin-proteinase complexes.

The binding of 125I-labelled transforming growth factor-beta 1 (TGF-beta 1) to human alpha 2-macroglobulin (alpha 2M) was studied by native PAGE and autoradiography. TGF-beta 1 bound preferentially to alpha 2M-methylamine and minimally, if at all, to native alpha 2M. Preparations of alpha 2M-proteinase complex were generated by incubating a standard concentration of alpha 2M (0.4 microM) with different concentrations of trypsin, chymotrypsin or neutrophil elastase (0.04-2.0 microM). The 125I-TGF-beta 1-binding activity depended on the initial ratio of active proteinase to alpha 2M, or r value, used to form the alpha 2M-proteinase complex. With all three proteinases, r values of 2 or greater yielded preparations with unchanged or decreased TGF-beta 1-binding activity relative to native alpha 2M. By contrast, r values near 1 yielded preparations with significantly increased TGF-beta 1-binding activity. The results of [3H]thymidine-incorporation studies performed in mouse keratinocytes were consistent with the 125I-TGF-beta-binding experiments. alpha 2M-trypsin and alpha 2M-chymotrypsin prepared at an r value of 1.0 counteracted the activity of TGF-beta 1, whereas the equivalent complexes prepared at an r value of 3.0 had no effect. As determined by SDS/PAGE, 125I-TGF-beta 1 binding to alpha 2M-methylamine was at least 80% non-covalent. Reaction of alpha 2M-methylamine with iodoacetamide or 5,5'-dithiobis-(2-nitrobenzoic acid) decreased the percentage of covalent binding but had no effect on total binding. Neuraminidase treatment had no effect on the binding of 125I-TGF-beta 1 to alpha 2M-methylamine. Cleavage of the 'bait regions' in alpha 2M-methylamine by prolonged treatment with trypsin also had no effect. These studies suggest that TGF-beta 1 binding to alpha 2M is enhanced by conformational change in the proteinase inhibitor resulting from reaction with proteinase or amine. If both proteinase-binding sites in a single alpha 2M molecule are occupied, TGF-beta 1-binding activity is decreased or perhaps eliminated.


Isolation of cDNA including a reverse transcriptase-like sequence transcribed from the long interspersed repetitive DNA sequence of rat.

The mammalian genome congains long interspersed repetitive sequences, but the role of these repetitive sequence is not clear. A cDNA clone has been isolated that contains part of the L1 sequences from a cDNA library of rat liver. The DNA sequence analysis showed the homology of cDNA to several reverse transcriptases. The homology between the amino acid sequences predicted from L1 consensus sequences and reverse transcriptases has been reported previously. However, this is the first isolation of a cDNA clone containing a reverse transcriptase-like sequence.


Purification and properties of gamma-glutamyltranspeptidase from Bacillus subtilis (natto).

To understand the mechanism by which gamma-polyglutamic acid (gamma-PGA) in the sticky material of natto was synthesized, we purified the gamma-glutamyltranspeptidase (gamma-GTP) (EC 2.3.2.2) from the culture broth of Bacillus subtilis (natto) to homogeneity. gamma-GTP was composed of two subunits with molecular weight of 45,000 and 22,000. The N-terminal amino acid sequence of light subunit was homologous with that of gamma-GTP from Escherichia coli. The optimum pH and temperature of activity were 8.5 and 60 degrees C. The enzyme was inactivated by incubation for 15 min at pH 8.0 and 55 degrees C, but little loss of the activity was detected at 40 degrees C. gamma-GTP used glutamine as a gamma-glutamyl donor and acceptor for gamma-PGA synthesis. Dipeptides were better gamma-glutamyl acceptors than free amino acids.


A sensitive viral capture assay for detection of plasma viremia in HIV-infected individuals.

Human immunodeficiency virus was detected in the serum/plasma of individuals infected with human immunodeficiency virus (HIV) after capture of virions on microparticles coated with monoclonal antibodies to external and transmembrane proteins of HIV-1. We analyzed serial samples obtained from 6 individuals who seroconverted, 18 asymptomatic, and 12 AIDS patients. HIV-1 RNA was detected in all (29/29) seropositive samples and in 6 seronegative samples immediately preceding seroconversion. In contrast, HIV antigen was detected in 13/29 (45%) of seropositive samples. HIV-1 RNA was also detected in 3 antigen-negative samples from one individual 8-5 months prior to seroconversion and in one sample from another person 2 days before antigen positivity. The intensity of the polymerase chain reaction (PCR) signal paralleled the concentration of HIV antigen. Conversely, seropositive HIV antigen-negative samples gave a weaker PCR signal. HIV-1 RNA was detected in 10/18 (60%) samples from asymptomatic, HIV antigen-negative, individuals and in 11/12 (92%) specimens obtained from AIDS patients. The viral capture method may provide a sensitive, specific, and semiquantitative means of detecting circulating HIV at all stages of infection.


Transfer of several phytopathogenic Pseudomonas species to Acidovorax as Acidovorax avenae subsp. avenae subsp. nov., comb. nov., Acidovorax avenae subsp. citrulli, Acidovorax avenae subsp. cattleyae, and Acidovorax konjaci.

DNA-rRNA hybridizations, DNA-DNA hybridizations, polyacrylamide gel electrophoresis of whole-cell proteins, and a numerical analysis of carbon assimilation tests were carried out to determine the relationships among the phylogenetically misnamed phytopathogenic taxa Pseudomonas avenae, Pseudomonas rubrilineans, "Pseudomonas setariae," Pseudomonas cattleyae, Pseudomonas pseudoalcaligenes subsp. citrulli, and Pseudomonas pseudoalcaligenes subsp. konjaci. These organisms are all members of the family Comamonadaceae, within which they constitute a separate rRNA branch. Only P. pseudoalcaligenes subsp. konjaci is situated on the lower part of this rRNA branch; all of the other taxa cluster very closely around the type strain of P. avenae. When they are compared phenotypically, all of the members of this rRNA branch can be differentiated from each other, and they are, as a group, most closely related to the genus Acidovorax. DNA-DNA hybridization experiments showed that these organisms constitute two genotypic groups. We propose that the generically misnamed phytopathogenic Pseudomonas species should be transferred to the genus Acidovorax as Acidovorax avenae and Acidovorax konjaci. Within Acidovorax avenae we distinguished the following three subspecies: Acidovorax avenae subsp. avenae, Acidovorax avenae subsp. cattleyae, and Acidovorax avenae subsp. citrulli. Emended descriptions of the new taxa are presented.


Piscirickettsia salmonis gen. nov., sp. nov., the causative agent of an epizootic disease in salmonid fishes.

A novel intracellular pathogen morphologically similar to the ehrlichiae has been isolated in cell culture and identified as the cause of an epizootic disease of salmonid fish. Like the ehrlichiae, the salmonid pathogen, designated strain LF-89, replicates within membrane-bound cytoplasmic vacuoles in host cells. This agent is the first with characteristics of this type to be isolated from a fish. Analysis of the LF-89 16S rRNA indicated that, unlike the ehrlichiae, LF-89 is a gamma proteobacterium distantly related to Coxiella burnetii and perhaps Wolbachia persica. A new genus and species (Piscirickettsia salmonis gen. nov., sp. nov.) are proposed for this organism, with ATCC(R) VR 1361 as the type strain.


Marine star-shaped-aggregate-forming bacteria: Agrobacterium atlanticum sp. nov.; Agrobacterium meteori sp. nov.; Agrobacterium ferrugineum sp. nov., nom. rev.; Agrobacterium gelatinovorum sp. nov., nom. rev.; and Agrobacterium stellulatum sp. nov., nom. rev.

Two new species of aerobic, gram-negative, peritrichously flagellated or nonmotile marine bacteria usually forming star-shaped aggregates were isolated from northeastern Atlantic Ocean bottom sediments. These organisms resembled eight star-shaped-aggregate-forming bacterial species from the Baltic Sea originally ascribed to the genus Agrobacterium but not included on the Approved Lists of Bacterial Names because of their questionable relationships to true agrobacteria. These two sets of star-shaped-aggregate-forming bacteria were compared by means of phenotypic data, DNA base compositions, DNA-DNA relatedness, and one-dimensional electrophoretic analysis of low-molecular-weight RNAs (5S rRNA and tRNA). According to the results of genotyping, the northeastern Atlantic Ocean isolates and three of the Baltic Sea species formed a group of closely related bacteria that could not be excluded from the genus Agrobacterium with certainty. Until more genotypic data are available, these five marine species are regarded as a distinct subdivision of the genus Agrobacterium consisting of Agrobacterium atlanticum sp. nov. (type strain, 1480T = DSM 5823T), A. meteori sp. nov. (type strain, 1513T = DSM 5824T), A. ferrugineum sp. nov. nom. rev. emend. (type strain, ATCC 25652T), A. gelatinovorum sp. nov. nom. rev. emend. (type strain, ATCC 25655T), and A. stellulatum sp. nov. nom. rev. emend. (type strain, ATCC 15215T). "A. aggregatum" proved to be a later subjective synonym of A. stellulatum, which had priority. The remaining four Baltic Sea species, "A. agile," "A. kieliense," "A. luteum," and "A. sanguineum," could not be placed in the new subdivision of Agrobacterium.


Taxonomic status of Kitasatosporia, and proposed unification with Streptomyces on the basis of phenotypic and 16S rRNA analysis and emendation of Streptomyces Waksman and Henrici 1943, 339AL.

Species classified within the genus Kitasatosporia share many of the phenotypic characteristics typical of streptomycetes. By using a probabilistic identification scheme, they were identified with Streptomyces exfoliatus cluster 5, a species group within Streptomyces. The four species studied hybridized with a 16S rRNA genus probe for Streptomyces spp., indicating a close relationship between the two genera. The kitasatosporias were resistant to selected polyvalent streptomycete phages tested. Quantitative analysis showed that meso-diaminopimelic acid varied from 49 to 89% in Kitasatosporia species and from 1 to 16% in Streptomyces species depending on growth conditions. On the basis of 16S rRNA analysis, it is proposed to reduce Kitasatosporia to synonymy with Streptomyces. As a result, the new names proposed are Streptomyces mediocidicus comb. nov., Streptomyces phosalacineus comb. nov., Streptomyces setae comb. nov., and Streptomyces griseolosporeus comb. nov., nom. nov.


Phylogenetic evidence for the transfer of Eubacterium suis to the genus Actinomyces as Actinomyces suis comb. nov.

The 16S rRNA primary structures of Eubacterium suis DSM 20639T (T = type strain) and Bifidobacterium bifidum DSM 20456T were determined by sequencing in vitro amplified rDNA. Sequence comparisons indicated that B. bifidum is moderately related to representatives of the genera Actinomyces and Mobiluncus. The closest relative of E. suis is Actinomyces pyogenes. E. suis and A. pyogenes are more closely related phylogenetically to one another than to the other Actinomyces species that have been investigated by using comparative 16S rRNA analysis. Therefore, we propose that E. suis should be transferred to the genus Actinomyces as Actinomyces suis comb. nov.


How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity.

16S rRNA (genes coding for rRNA) sequence comparisons were conducted with the following three psychrophilic strains: Bacillus globisporus W25T (T = type strain) and Bacillus psychrophilus W16AT, and W5. These strains exhibited more than 99.5% sequence identity and within experimental uncertainty could be regarded as identical. Their close taxonomic relationship was further documented by phenotypic similarities. In contrast, previously published DNA-DNA hybridization results have convincingly established that these strains do not belong to the same species if current standards are used. These results emphasize the important point that effective identity of 16S rRNA sequences is not necessarily a sufficient criterion to guarantee species identity. Thus, although 16S rRNA sequences can be used routinely to distinguish and establish relationships between genera and well-resolved species, very recently diverged species may not be recognizable.


Identification of xenobiotic-degrading isolates from the beta subclass of the Proteobacteria by a polyphasic approach including 16S rRNA partial sequencing.

Nineteen gram-negative, aerobic, biodegradative isolates were identified by using a polyphasic taxonomic approach. The presence of the specific polyamine 2-hydroxyputrescine and the presence of a ubiquinone with eight isoprenoid units in the side chain (ubiquinone Q-8) allowed allocation of these organisms to the beta subclass of the Proteobacteria. On the basis of the results of additional characterization experiments (i.e., API 20NE tests, determinations of soluble protein patterns, and DNA-DNA hybridization experiments), we classified six isolates as either Comamonas testosteroni, Comamonas acidovorans, or Alcaligenes xylosoxidans subsp. denitrificans. By using the same criteria we allocated two additional isolates to the genus Alcaligenes. A comparison of a 16S rRNA fragment (positions 1220 to 1377; Escherichia coli nomenclature) indicated that the remaining isolates should be allocated as follows: one is a member of C. testosteroni and one is a member of Acidovorax facilis, as confirmed by the results of additional DNA-DNA hybridizations; two others probably belong to the family Alcaligenaceae; six are related to "Alcaligenes eutrophus"; and one, strain NRRL 12228, occupies an isolated position.


Reinterpretation of the taxonomic position of Xanthomonas maltophilia and taxonomic criteria in this genus. Request for an opinion.

The inclusion of "Pseudomonas maltophilia" Hugh 1981 in the genus Xanthomonas as Xanthomonas maltophilia (Hugh 1981) Swings et al. 1983 is questioned in view of the significant differences between these two taxa. This reclassification is not acceptable if practical means of differentiation in this genus are considered. The proposed alteration of the description of the genus Xanthomonas is also questionable because of the implications for everyday phytobacteriology. In view of the natural similarities, as well as the profound differences, between X. maltophilia and the genus Xanthomonas, we propose that a new genus should be created for X. maltophilia, which could be placed together with the genus Xanthomonas in a separate natural group.


Towards a phylogeny of the genus Vibrio based on 16S rRNA sequences.

The inter- and intrageneric relationships of the genus Vibrio were investigated by performing a comparative analysis of the 16S rRNAs of 10 species, including four pathogenic representatives. The results of immunological and 5S rRNA studies were confirmed in that the genus is a neighboring taxon of the family Enterobacteriaceae. With regard to the intrageneric structure, Vibrio alginolyticus, Vibrio campbellii, Vibrio natriegens, Vibrio harveyi, Vibrio proteolyticus, Vibrio parahaemolyticus, and Vibrio vulnificus form the core of the genus, while Vibrio (Listonella) anguillarum, Vibrio diazotrophicus, and Vibrio hollisae are placed on the outskirts of the genus. Variable regions around positions 80, 180, and 450 could be used as target sites for genus- and species-specific oligonucleotide probes and polymerase chain reaction primers to be used in molecular identification.


Phylogenetic analysis of Alloiococcus otitis gen. nov., sp. nov., an organism from human middle ear fluid.

The partial 16S rRNA sequence of an unknown bacterium that was originally isolated from middle ear fluids of children with persistent otitis media was determined by reverse transcription. A comparison of this sequence with sequences from other gram-positive species having low guanine-plus-cytosine contents revealed that this bacterium represents a new line of descent, for which the name Alloiococcus otitis gen. nov., sp. nov., is proposed. The type strain is strain NCFB 2890.


Phylogeny of fast-growing soybean-nodulating rhizobia support synonymy of Sinorhizobium and Rhizobium and assignment to Rhizobium fredii.

We determined the sequences for a 260-base segment amplified by the polymerase chain reaction (corresponding to positions 44 to 337 in the Escherichia coli 16S rRNA sequence) from seven strains of fast-growing soybean-nodulating rhizobia (including the type strains of Rhizobium fredii chemovar fredii, Rhizobium fredii chemovar siensis, Sinorhizobium fredii, and Sinorhizobium xinjiangensis) and broad-host-range Rhizobium sp. strain NGR 234. These sequences were compared with the corresponding previously published sequences of Rhizobium leguminosarum, Rhizobium meliloti, Agrobacterium tumefaciens, Azorhizobium caulinodans, and Bradyrhizobium japonicum. All of the sequences of the fast-growing soybean rhizobia, including strain NGR 234, were identical to the sequence of R. meliloti and similar to the sequence of R. leguminosarum. These results are discussed in relation to previous findings; we concluded that the fast-growing soybean-nodulating rhizobia belong in the genus Rhizobium and should be called Rhizobium fredii.


Staphylococcus muscae, a new species isolated from flies.

A new coagulase-negative species of the genus Staphylococcus, Staphylococcus muscae, is described on the basis of the results of a study of four strains that were isolated from flies. 16S rRNA sequences of the type strains of S. muscae, Staphylococcus schleiferi, and Staphylococcus sciuri were determined and used, together with the corresponding sequences of Staphylococcus aureus and Staphylococcus epidermidis, for a comparative analysis. The new species is characterized taxonomically; this species is differentiated from the other novobiocin-susceptible staphylococci by its physiological and biochemical activities, cell wall composition, and levels of genetic relatedness. The type strain of this species is strain MB4 (= CCM 4175).


The use of discriminant analysis to guide palliative treatment for lung cancer patients.

The aim of this study was to develop a prognostic index for patients with inoperable non-small cell lung cancer which could predict survival to 3 months. This would enable less radiation dose to be given to patients where prognosis is limited by occult metastases, giving rise to less treatment morbidity and raising the therapeutic ratio. Data on 18 known prognostic factors were collected on 96 patients. Performance status, lymphocyte count, weight loss and extent of disease were the most predictive factors and were combined into an index. Logistic discriminant analysis was employed to give a numerical score of likelihood of survival to 3 months, ranging from 0 (not likely) to 1 (certain). In this first set of 96 patients, 16 deaths were observed before 3 months, of which 6 were predicted. There was one false positive prediction. Overall accuracy of prediction was therefore 89% with 99% specificity. The same 4 prognostic factors were measured on a second set of 80 patients. Nineteen died before 3 months, of which 5 were predicted with 2 false positives, giving an overall accuracy of 80% and 97% specificity. A probability of survival of less than or equal to 0.2, although highly specific, was only applicable to 9% of patients and this was the limiting factor in the clinical usefulness of the test. A 16-branch tree diagram allows any patient to be assigned a risk factor based on the four predictive factors at the first clinic attendance. Use of the index could encourage more rational prescribing of radiation dose.


Factors affecting treatment patterns of radiation oncologists in the United States in the palliative treatment of cancer.

A questionnaire was sent to 488 radiation oncologists in the United States and 268 replied. Each was given a brief account of three hypothetical patients (one with brain metastases, one with locally advanced lung cancer and one with bone metastases) and asked how they would approach the problems posed. Younger radiation oncologists (less than 46 years) treated patients with brain metastases with a smaller number of fractions and were more likely to view the case of locally advanced lung cancer as palliative. The aim of the radiation oncologist was related to the treatment pattern chosen, with the aim to extend life frequently related to higher total dose and number of fractions. When the amount of private funding was compared with dose, number of treatments, and whether the case was called palliative or not, no relationship was found.


The observed inhibitory potency of 3'-azido-3'-deoxythymidine 5'-triphosphate for HIV-1 reverse transcriptase depends on the length of the poly(rA) region of the template.

The inhibitory potency of 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP) against HIV-1 reverse transcriptase (HIV-1 RT) has been further evaluated. The results indicate that the previously reported low Ki values for AZTTP against HIV-1 RT (2.35 nM) are due neither to the to the direct tight binding of AZTTP to HIV-1 RT nor to the interaction of the enzyme with AZTMP moiety terminated primer-templates, but instead they are an artifact of the use of a homotemplate-primer [poly(rA).oligo(dT)]. With a set of RNAs of defined sequence as templates, we demonstrate that the observed Ki value for AZTTP depends on the length of the poly(rA) region following the primer in the RNA template. The more adenosyl residues in the RNA template that are available for processive incorporation of TMP moieties, the lower is the observed Ki value for AZTTP. Since the potencies of new inhibitors of HIV-1 RT are usually compared with that for AZTTP, these results have important consequences for the process of discovery of new HIV inhibitors that are of potential use in AIDS therapy.


NMR analysis of helix I from the 5S RNA of Escherichia coli.

The structure of helix I of the 5S rRNA from Escherichia coli has been determined using a nucleolytic digest fragment of the intact molecule. The fragment analyzed, which corresponds to bases (-1)-11 and 108-120 of intact 5S rRNA, contains a G-U pair and has unpaired bases at its termini. Its proton resonances were assigned by two-dimensional NMR methods, and both NOE distance and coupling constant information have been used to calculate structural models for it using the full relaxation matrix algorithm of the molecular dynamics program XPLOR. Helix I has A-type helical geometry, as expected. Its most striking departure from regular helical geometry occurs at its G-U, which stacks on the base pair to the 5' side of its G but not on the base pair to its 3' side. This stacking pattern maximizes interstrand guanine-guanine interactions and explains why the G-U in question fails to give imino proton NOE's to the base pair to 5' side of its G. These results are consistent with the crystal structures that have been obtained for wobble base pairs in tRNAPhe [Mizuno, H., & Sundaralingam, M. (1978) Nucleic Acids Res. 5, 4451-4461] and A-form DNA [Rabbinovich, D., Haran, T., Eisenstein, M., & Shakked, Z. (1988) J. Mol. Biol. 200, 151-161]. The conformations of the terminal residues of helix I, which corresponds to bases (-1)-11 and 108-120 of native 5S RNA, are less well-determined, and their sugar puckers are intermediate between C2' and C3'-endo, on average.


Type-specific antibodies to the platelet-derived growth factor receptors: role in elucidating the structural and functional characteristics of receptor types.

Two types of platelet-derived growth factor receptors have been cloned and sequenced. Both are glycoproteins with similar molecular weights. We have earlier established the ligand binding specificity, ligand-induced dimerization, and kinase activation of these two receptor types [Bishayee et al. (1989) J. Biol. Chem. 264, 11699-11705; Kanakaraj et al. (1991) Biochemistry 30, 1761-1767]. In the present studies, we have investigated the biosynthesis, processing, and glycosylation of the alpha-receptor and compared its structural and functional characteristics to those of the beta-receptor. Unlike an anti-peptide antibody, AbP2 (amino acid residues 964-979), to the human beta-receptor which detects a phosphorylation-specific conformation of the receptor, an antibody, AbP alpha 2 (amino acid residues 956-971), to the corresponding region of the human alpha-receptor failed to do so. However, our studies revealed that the stability of the alpha-receptor is comparable to that of the beta-receptor. In addition, N-linked glycosylation of the alpha-receptor, like that of the beta-receptor, is not important in kinase activation. We have exploited the lack of an effect of N-linked oligosaccharides on the functioning of the alpha-receptor to develop a simple and rapid method for direct demonstration of ligand-induced noncovalently linked alpha-beta-receptor heterodimer formation. This method is based on the interaction between functionally active short and the long forms of two receptor types which can be resolved by denaturing gel electrophoresis.


Segment spanning residues 727-768 of the complement C3 sequence contains a neoantigenic site and accommodates the binding of CR1, factor H, and factor B.

CR1, CR2, DAF, MCP, factor H, C4bp, factor B, and C3 are members of a family of structurally related molecules, the majority of which belong to the complement system. Several of these molecules also share functional features such as cofactor and decay/dissociation activity and compete with one another in binding to C3b. Since factor H appears to bind to multiple sites in C3, we investigated the relationship between the factor H- and CR1-binding sites in C3b. Factor H binding to C3b is inhibited by either the C3c or C3d fragments, and addition of both fragments together augments this inhibition. One monoclonal anti-C3c antibody, anti-C3-9, which recognizes a neoantigenic epitope expressed upon cleavage to C3 to C3b, inhibited both factor H and CR1 binding to EC3b cells. This monoclonal antibody (MoAb) also inhibited factor B binding to EC3b. Two observations further supported our hypothesis that these molecules bind to proximal sites in C3b. First, a synthetic peptide spanning this region of C3b (C3(727-768)) inhibited factor H binding. Second, antibodies raised against this peptide inhibited binding to CR1, factor H, and factor B to C3b. These data show that H binds to at least two sites in C3b: the site in the C3c fragment is within the identified CR1-binding domain while the site in the C3d fragment surrounds the CR2-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)


Purification and partial characterization of two types of growth-inhibitory protein latently present in rabbit serum.

Normal rabbit serum contained two kinds of growth-inhibitory protein, GI-I and GI-II, in latent forms. These latent inhibitors were activated by incubation at 37 degrees C for 12 h, and their activation was lowered by inhibitors for serine, cysteine and metalloproteinases. Both growth inhibitors were highly purified in active forms by successive column chromatographies. GI-I showed a major protein band with an Mr of 18,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while GI-II showed a major protein band with an Mr of 36,000. GI-I and GI-II half-inhibited the growth of rat tumorigenic cell line (RSV-BRL) at concentrations of 0.5 ng/ml and 10 ng/ml, excess concentrations. Of the 15 cell lines tested, GI-I specifically inhibited the growth of rodent and lagomorph cells, whereas GI-II nonspecifically inhibited the growth of all cell lines tested. Specificities for cell type and malignancy were not observed with either inhibitor. These growth inhibitors were stable to a reducing reagent and proteinase inhibitors, but labile to urea, acid, organic solvents, trypsin, plasmin and heating at 95 degrees C for 5 min. These properties suggested that both growth inhibitors might be distinct from known growth-inhibitory factors.


Molecular interaction between HIV-1 major envelope glycoprotein and dextran sulfate.

We investigated at the molecular level the interaction between, HIV-1 recombinant gp160 (rgp160) and low-molecular-weight dextran sulfate. We demonstrate the occurrence of a specific interaction between rgp160 and sulfated dextran beads, which is saturable, pH-dependent and inhibitable by soluble dextran sulfate but not by soluble dextran. This specific interaction has a low affinity, with an estimated Kd in the 10(-4) M range. In addition, the binding of rgp160 to soluble recombinant CD4 (sT4) can only be inhibited by the preincubation of rgp160, but not of sT4, with dextran sulfate. Taken together, these results demonstrate the occurrence of a low affinity, but specific interaction between dextran sulfate and rgp160. This may account, at least in part, for the anti-HIV-1 activity of dextran sulfate.


CSF amine metabolites in depression.

The amine metabolites, namely homovanillic acid (HVA) and 5-hydroxy indoleacetic acid (5-HIAA) were measured in cerebrospinal fluid (CSF) of depressives (n = 30) and controls (n = 30). Depressed patients had significantly lower HVA levels than controls. No significant differences were noted between the two groups in 5-HIAA levels. However, the differences between the groups for the CSF HVA/5-HIAA ratio were larger than those for the CSF HVA alone (p less than 0.01 versus p less than 0.025, respectively). HVA levels correlated positively with monoamine oxidase activity and adenosine deaminase activity.


Selection of benign primitive hematopoietic progenitors in chronic myelogenous leukemia on the basis of HLA-DR antigen expression.

Chronic myelogenous leukemia (CML) is a lethal malignancy of the human hematopoietic stem cell. Here we report that coexistent benign, primitive hematopoietic progenitors can be distinguished from their malignant counterparts in CML bone marrow by differences in cell surface antigen expression. Selection of bone marrow cells expressing the CD34 antigen but lacking the HLA-DR antigen results in recovery of small lymphocyte-like blasts, which initiate and sustain production of myeloid clonogenic progeny in vitro. Secondary clonogenic cells derived at week 1, 5, and 8 from long-term bone marrow cultures (LTBMCs) initiated with primitive progenitors, which lack HLA-DR antigens, exhibit neither the Philadelphia chromosome (Ph1) nor the corresponding bcr/abl mRNA characteristic of CML. In contrast, clonogenic cells recovered at week 1, 5, and 8 from LTBMCs initiated with the CML HLA-DR+ population contain Ph1 and express bcr/abl mRNA. This observation indicates that it may be possible to select a population of viable, exclusively benign hematopoietic stem cells from CML bone marrow capable of repopulating the hematopoietic compartment following autologous bone marrow transplantation.