Formation of heterophile antibodies by human tonsilar lymphocytes: II. In vitro stimulation with allogeneic cells

Short-term cultures of human tonsilar lymphocytes (HTL), 5 × 10⁶ cells/culture, in medium RPMI 1640 supplemented with human group AB serum were studied for the production of plaque-forming cells (PFC) against sheep (SRBC) and bovine (BRBC) red blood cells following in vitro stimulation by various allogeneic lymphoid cells. Of 55 HTL specimens examined, 48 produced a significant number (50–300/culture) of PFC against SRBC and/or BRBC following the in vitro stimulation. The optimal doses of the stimulator HTL and peripheral blood lymphocytes (PBL) were 10⁷ and 5 × 10⁶/culture, respectively. After the stimulation, PFC appeared in significant numbers on the third day, reached the peak number on the sixth day, and decreased sharply in number thereafter. Removal of E-rosetting cells from both stimulator and responder populations abolished the PFC formation. PFC formation against SRBC was inhibited by solubilized Forssman antigen, while PFC formation against BRBC was inhibited strongly by Hanganutziu-Deicher antigen, hardly by Paul-Bunnell antigen and not at all by Forssman antigen. Supernatants of mixed lymphocyte culture of PBL were shown to enhance PFC formation of HTL cultures stimulated by allogeneic lymphocytes. The results of this study indicated that in vivo primed B cells of the HTL were triggered in vitro by allogeneic stimulation for the heterophile antibody formation. Since these antibodies are apparently directed against Forssman and Hanganutziu-Deicher antigens, the “allo” nature of these antigens as well as their relationship to the previously described heterophile transplantation antigens have to be clarified.     

Co-oligopeptides containing two aromatic residues spaced by glycyl residues. X. Proton magnetic resonance study of co-oligopeptides of tryptophan and glycine

The ¹H-nmr spectra of co-oligopeptides of tryptophan and glycine with structure H-Gly-Trp-(Gly)_n-Trp-Gly-OH (n = 0–2) and those of several di- and tripeptides have been recorded at 360 MHz with CD₃OD solutions containing 0.1N NaOD. The assignment of resonance signals was generally possible by comparing the spectra of structurally related peptides with each other. In order to solve the remaining ambiguities in the assignment, H-(αL,βS)(α,β-d₂)Trp-OH, H-Trp-(αL,βS)(α,β-d₂)Trp-OH, and H-Trp-(δ¹,ε²,ζ²,ζ³,η²-d₅)Trp-OH have been prepared and their spectra compared with those of the undeuterated compounds. The distribution of rotamers around the χ₁ and (in two cases) χ₂ torsion angles of the side chains has been obtained from the vicinal coupling constants ³J and from the long-range coupling constants ⁴J. These data and an analysis of the chemical shifts of the Gly-C^α protons suggest that the orientation of the aromatic side chain is influenced by the following order of decreasing interaction with the functional groups at N- and C-side: -NH₂ > –NHCO– > –CONH–> –COO^?. This rule does not hold for the second Trp residue of di- and tripeptides containing the -Trp-Trp- sequence, which has tentatively been attributed to steric effects.     

On the synthesis of viral ribonucleic acids and ribonucleoproteins in the submitochondrial system completely free of interfering cytoplasmic contaminations

Summary The synthesis of virus-specific macromolecules was studied in the reconstituted system containing inner membrane-matrix fraction from rat liver mitochondria and infectious RNA of Venezuelian equine encephalomyelitis (VEE) virus. In a series of preliminary experiments it was shown that isolated submitochondrial fraction was completely free of interfering cytoplasmic contaminations and particularly, of cytoplasmic 80S ribosomes. VEE RNA when added to submitochondrial system caused significant stimulation of RNA and protein synthesis. These processes were resistant to actinomycin D which inhibited profoundly the synthesis of proper mitochondrial macromolecules. The stimulating effect of VEE RNA in experiments with submitochondrial system was about three times higher than that with intact mitochondria. The stimulation of¹⁴C-amino acid incorporation increased as a function of incubation time; a certain lag-period being observed. The newly formed virus-specific RNA's and ribonucleoproteins were identified with the aid of sedimentation analysis. In particular, radioactive RNA's with sedimentation coefficients 40S and 26-18S were isolated from the incubated system. These RNA's are similar respectively to VEE genome RNA and doublestranded VEE replicative RNA. In double labelling experiments with³H-uridine and¹⁴Camino acids it was shown that VEE RNA induced synthesis of ribonucleoproteins containing newly formed RNA and protein. These RNP possessed sedimentation coefficients 60-80S, 140S and 300S in sucrose gradient and buoyant densities 1.32 and 1.50 g/cm³ in cesium chloride gradients. These properties of ribonucleoproteins synthesized de novo in submitochondrial system are close to those of RNP intermediates of VEE virus reproduction in the infected cells. We concluded that viral RNA could program virus-specific synthesis in the submitochondrial system under conditions that eliminated the contribution of cytoplasmic ribosomes.     

The effect of several diphosphonates on acid phosphohydrolases and other lysosomal enzymes.

Diphosphonates are known to inhibit bone resorption in tissue culture and in experimental animals. This effect may be due to their ability to inhibit the dissolution of hydroxyapatite crystals, but other mechanisms may be important. Since lysosomal enzymes have implicated in the process of bone resorption, we have examined the effect of several phosphonates and of a polyphosphate (P20,2) on lysosomal hydrolases derived from rat liver and rat bone. Dichloromethylene diphosphonate strongly inhibited acid beta-glycerophosphatase (EC 3.1.3.2) and acid p-nitrophenyl phosphatase (EC 3.1.3.2) and to a lesser degree (in descending order) acid pyrophosphatase (EC 3.1.3.-), arylsulfatase A (EC 3.1.6.1), deoxyribonuclease II(EC 3.1.4.6) and phosphoprotein phosphatase (EC 3.1.3.16) of rat liver. Inhibition of acid p-nitrophenyl phosphatase and arylsulfatase A was competitive. Ethane-1-hydroxy-1, 1-diphosphonate did not inhibit any of these enzymes, except at high concentrations. Neither dichloromethylene diphosphonate nor ethane-1-hydroxy-1, 1-diphosphonate had any effect on beta-glucuronidase (EC 3.2.1.31), arylesterase (EC 3.1.1.2) and cathepsin D (EC 3.4.23.5). Of several other phosphonates tested only undec-10-ene-1-hydroxy-1, 1-diphosphonic acid inhibited acid p-nitrophenyl phosphatase strongly, the polyphosphate (P20, I) had little effect. Acid p-nitrophenyl phosphatase in rat calvaria extract behaved in the same way as the liver enzyme and was also strongly inhibited by dichloromethylene diphosphonate, but not by ethane-1-hydroxy-1, 1-diphosphonate. It is suggested that the inhibition of bone resorption by dichloromethylene diphosphonate might be due in part to a direct effect of this diphosphonate on lysosomal hydrolases.     

Existence in fetal and adult rat of several forms of material reacting with anti-insulin antibody (IRI).

The double antibody procedure detects 3 peaks in the elution fractions of adult or fetal rat sera, after passage on Sephadex G50 or G100 columns. Peak A (apparent MW 6000) contains insulin monomer; peak B (apparent MW 10-12000) is tentatively attributed to proinsulin (or proinsulin like substances); peak C (apparent MW 50-100000) is similar to the so called "big big" insulin. During intravenously induced hyperglycemia, the 3 peaks show parallel increases, but, after the disappearance of peaks A and B in streptozotocin treated rats, peak C remains unaltered. Pancreatic extracts and secreta present a very minor and inconstant peak C, the bulk of their immunoreactive material belonging to peaks A and B. A companion paper further discusses the nature of peaks B and C materials.     

Microbial oxidation of methane and methanol: crystallization and properties of methanol dehydrogenase from Methylosinus sporium.

Obligate methylotrophs are divisible into two types on the basis of ultrastructural biochemical characteristics. Both groups possess a soluble phenazine methosulfate (PMS)-dependent methanol dehydrogenase. In addition, particulate PMS-dependent methanol dehydrogenase and PMS-independent methanol oxidase have been found in the type I membrane group. A procedure was developed for the crystallization of methanol dehydrogenase from the soluble fraction of the type II obligate methylotroph Methylosinus sporium. This is the first report of a crystalline methanol dehydrogenase from a methylotrophic bacterium. The crystallized enzyme is homogeneous as judged by ultracentrifugation and by acrylamide gel electrophoresis. In the presence of an electron acceptor (phenazine or phenazinium compound) and an activator (ammonium compound), the crystallized enzyme catalyzed the oxidation of primary alcohols and formaldehyde. Secondary, tertiary, and aromatic alcohols were not oxidized. The molecular weight of the enzyme as estimated by gel filtration is approximately 60,000, and as estimated by sedimentation equilibrium analysis it is 62,000. The sedimentation constant (S20,W) is 2.9. The subunit size determined by sodium dodecyl sulfate-gel electrophoresis is approximately 60,000. The amino acid composition and spectral properties of the enzyme are also presented. Antisera prepared against the crystalline enzyme are nonspecific, they cross-reacted and inhibited isofunctional enzymes from other obligate methylotrophic bacteria.     

Pyronaridine–artesunate real-world safety,tolerability, and effectiveness in malaria patients in 5 African countries: A single-arm,open-label,cohort event monitoring study

BackgroundIn Phase II/III randomized controlled clinical trials for the treatment of acute uncomplicated malaria, pyronaridine–artesunate demonstrated high efficacy and a safety profile consistent with that of comparators, except that asymptomatic, mainly mild-to-moderate transient increases in liver aminotransferases were reported for some patients. Hepatic safety, tolerability, and effectiveness have not been previously assessed under real-world conditions in Africa.Methods and findingsThis single-arm, open-label, cohort event monitoring study was conducted at 6 health centers in Cameroon, Democratic Republic of Congo, Gabon, Ivory Coast, and Republic of Congo between June 2017 and April 2019. The trial protocol as closely as possible resembled real-world clinical practice for the treatment of malaria at the centers. Eligible patients were adults or children of either sex, weighing at least 5 kg, with acute uncomplicated malaria who did not have contraindications for pyronaridine–artesunate treatment as per the summary of product characteristics. Patients received fixed-dose pyronaridine–artesunate once daily for 3 days, dosed by body weight, without regard to food intake. A tablet formulation was used in adults and adolescents and a pediatric granule formulation in children and infants under 20 kg body weight. The primary outcome was the hepatic event incidence, defined as the appearance of the clinical signs and symptoms of hepatotoxicity confirmed by a >2× rise in alanine aminotransferase/aspartate aminotransferase (ALT/AST) versus baseline in patients with baseline ALT/AST >2× the upper limit of normal (ULN). As a secondary outcome, this was assessed in patients with ALT/AST >2× ULN prior to treatment versus a matched cohort of patients with normal baseline ALT/AST. The safety population comprised 7,154 patients, of mean age 13.9 years (standard deviation (SD) 14.6), around half of whom were male (3,569 [49.9%]). Patients experienced 8,560 malaria episodes; 158 occurred in patients with baseline ALT/AST elevations >2×ULN. No protocol-defined hepatic events occurred following pyronaridine–artesunate treatment of malaria patients with or without baseline hepatic dysfunction. Thus, no cohort comparison could be undertaken. Also, as postbaseline clinical chemistry was only performed where clinically indicated, postbaseline ALT/AST levels were not systematically assessed for all patients. Adverse events of any cause occurred in 20.8% (1,490/7,154) of patients, most frequently pyrexia (5.1% [366/7,154]) and vomiting (4.2% [303/7,154]). Adjusting for Plasmodium falciparum reinfection, clinical effectiveness at day 28 was 98.6% ([7,369/7,746] 95% confidence interval (CI) 98.3 to 98.9) in the per-protocol population. There was no indication that comorbidities or malnutrition adversely affected outcomes. The key study limitation was that postbaseline clinical biochemistry was only evaluated when clinically indicated.ConclusionsPyronaridine–artesunate had good tolerability and effectiveness in a representative African population under conditions similar to everyday clinical practice. These findings support pyronaridine–artesunate as an operationally useful addition to the management of acute uncomplicated malaria.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03201770","term_id":"NCT03201770"}}NCT03201770.

Gaston Tona Lutete and co-workers report on safety and effectiveness of the antimalarial drug pyronaridine-artesunate in African countries.     

Absence of repetitive DNA sequences associated with sex chromosomes in natural populations of the Trinidad guppy (Poecilia reticulata)

The genus Poecilia has been widely studied as a model for the evolution of sex chromosomes. In the course of molecular studies on population genetic structure and sexual selection in the Trinidad guppy, we examined our preparations for male-linked, repetitive DNA polymorphisms. We have not obtained any evidence of male-specific polymorphisms, in contrast to an earlier study. Our results have significant implications for theories on the evolution of sex chromosomes.Correspondence to: F. Breden