Construction of immunogens for synthetic malaria vaccines

The immunogenicity of a peptide consisting of eight repeats of the tetrapeptide sequence NANP (Asn-Ala-Asn-Pro) contained in the circumsporozoite protein of Plasmodium falciparum was investigated in mice under different modes of presentation. This peptide was able to produce biologically active antibodies when administered with adjuvant and linked to a protein carrier. However, a (NANP) peptide polymerized by carbodiimide was found to be immunogenic in the absence of protein carrier in H-2b mice. In contrast, the (NANP)8 peptide polymerized by glutaraldehyde was not immunogenic in the same strain. Furthermore, the efficacy of murabutide in saline, as an immunological adjuvant, was compared to the efficacy of Freund's complete adjuvant.     

Protection against yellow fever in monkeys by immunization with yellow fever virus nonstructural protein NS1.

Immunization of monkeys with yellow fever virus-specified nonstructural protein NS1 resulted in protection against fatal hepatitis as well as marked reduction in the magnitude of viremia after subcutaneous challenge with yellow fever virus. The results may be relevant to the design of possible subunit or recombinant flavivirus vaccines.     

The dynamic state of heat shock proteins in chicken embryo fibroblasts

Subcellular fractionation and immunofluorescence microscopy have been used to study the intracellular distributions of the major heat shock proteins, hsp 89, hsp 70, and hsp 24, in chicken embryo fibroblasts stressed by heat shock, allowed to recover and then restressed. Hsp 89 was localized primarily to the cytoplasm except during the restress when a portion of this protein concentrated in the nuclear region. Under all conditions, hsp 89 was readily extracted from cells by detergent. During stress and restress, significant amounts of hsp 70 moved to the nucleus and became resistant to detergent extraction. Some of this hsp 70 was released from the insoluble form in an ATP-dependent reaction. Hsp 24 was confined to the cytoplasm and, during restress, aggregated to detergent-insoluble perinuclear phase-dense granules. These granules dissociated during recovery and hsp 24 could be solubilized by detergent. The nuclear hsps reappeared in the cytoplasm in cells allowed to recover at normal temperatures. Sodium arsenite also induces hsps and their distributions were similar to that observed after a heat shock, except for hsp 89, which remained cytoplasmic. We also examined by immunofluorescence the cytoskeletal systems of chicken embryo fibroblasts subjected to heat shock and found no gross morphological changes in cytoplasmic microfilaments or microtubules. However, the intermediate filament network was very sensitive and collapsed around the nucleus very shortly after a heat shock. The normal intermediate filament morphology reformed when cells were allowed to recover from the stress. Inclusion of actinomycin D during the heat shock--a condition that prevents synthesis of the hsps--did not affect the intermediate filament collapse, but recovery of the normal morphology did not occur. We suggest that an hsp(s) may aid in the formation of the intermediate filament network after stress.     

Common and distinct regions of defective-interfering RNAs of Sindbis virus

Defective-interfering (DI) particles are helper-dependent deletion mutants which interfere specifically with the replication of the homologous standard virus. Serial passaging of alphaviruses in cultured cells leads to the accumulation of DI particles whose genomic RNAs are heterogeneous in size and sequence composition. In an effort to examine the sequence organization of an individual DI RNA species generated from Sindbis virus, we isolated and sequenced a representative cDNA clone derived from a Sindbis DI RNA population. Our data showed that: (i) the 3' end of the DI RNA template was identical to the 50 nucleotides at the 3' end of the standard RNA; (ii) the majority (75%) of the DI RNA template was derived from the 1,200 5'-terminal nucleotides of the standard RNA and included repeats of these sequences; and (iii) the 5' end of the DI RNA template was not derived from the standard RNA, but is nearly identical to a cellular tRNAAsp (S. S. Monroe and S. Schlesinger, Proc. Natl. Acad. Sci. U.S.A. 80:3279-3283, 1983). We have also utilized restriction fragments from cloned DNAs to probe by blot hybridization for the presence of conserved sequences in several independently derived DI RNA populations. These studies indicated that: (i) a 51-nucleotide conserved sequence located close to the 5' end of several alphavirus RNAs was most likely retained in the DI RNAs; (ii) the junction region containing the 5' end of the subgenomic 26S mRNA was deleted from the DI RNAs; and (iii) the presence of tRNAAsp sequences was a common occurrence in Sindbis virus DI RNAs derived by passaging in chicken embryo fibroblasts.     

The action of α-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acids

α-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of α-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of α-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10–20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by α-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.     

Inhibition of Growth of Escherichia coli and of Homoserine O-Transsuccinylase by α-Methylmethionine

The methionine analogue, alpha-methylmethionine, inhibits bacterial growth, but its action is overcome by methionine, homocysteine, and cystathionine. The effect of the analogue on growth is attributed to its ability to mimic methionine as a feed-back inhibitor of the first enzyme specific to methionine biosynthesis. This conclusion is based on the findings that (i) alpha-methylmethionine inhibits excretion of O-succinylhomoserine, the product of the first enzyme, by a methionine auxotroph unable to convert succinylhomoserine to cystahionine, and that (ii) the enzyme homoserine O-transsuccinylase is inhibited by alpha-methylmethionine in extracts of Escherichia coli. alpha-Methylmethionine also inhibits methionyl-ribonucleic acid synthetase in extracts, but this inhibition probably does not affect growth.     

Informationstheoretisches lernmodell

Summary The formation of the interconnection stimulus-response in a learning system is analysed. The system, a technical or a biological one establishes this correspondence by processing the information fed back from the medium during the learning process. This information has two aspects: a quantitative one related to the probability of the events, and a qualitative one related to the utility of the events in view of a goal. Both aspects are taken into consideration; by successive experiences the system eliminates the double uncertainty concerning the probabilistic dependence: stimulu — sresponse — outcome and its utility. Learning implies then a system for evaluating the utilities of different outcomes in view of a goal, a memory to record them and a decision system for selecting the corresponding responses upon a given criterion.

---

---

Der Beitrag stellt einen Teil der Untersuchungen dar, die zur Ausarbeitung der Doktor-Dissertation am Polytechnischen Institut Gheorghe Gheorghiu-Dej in Bukarest unternommen wurden.     

Phagocytosis of Mycobacterium tuberculosis is mediated by human monocyte complement receptors and complement component C3

We have examined the receptor-ligand interactions and the method of phagocytosis of virulent Mycobacterium tuberculosis by human monocytes. mAb against complement receptors (CR) inhibit adherence and phagocytosis of M. tuberculosis in fresh nonimmune serum. A mAb against the type 1 CR (CR1) inhibits adherence of M. tuberculosis by 40 +/- 5%, and three different mAb against the type 3 CR (CR3) each inhibit adherence by 39 +/- 5% to 47 +/- 4%. A mAb against CR1 used in combination with one of the three mAb against CR3 inhibits adherence by up to 64 +/- 7%. Most strikingly, two mAb used in combination against CR3 inhibit adherence by up to 81 +/- 2%. mAb against other monocyte surface Ag do not significantly influence adherence. In like fashion, mAb against CR but not other monocyte surface Ag inhibit adherence of preopsonized M. tuberculosis in the presence of heat-inactivated serum. By electron microscopy, monocytes ingest all M. tuberculosis that adhere in the presence of nonimmune serum; mAb against CR3 markedly inhibit ingestion. In contrast to CR, the FcR and the beta-glucan-inhibitable receptor for zymosan play little or no role in mediating M. tuberculosis adherence or ingestion. Adherence of M. tuberculosis is serum-dependent, requiring greater than or equal to 2.5% serum for optimal adherence. Heat inactivation of serum markedly reduces adherence of M. tuberculosis (75.5 +/- 7%) and preopsonization of bacteria enhances adherence by 2.9 +/- 0.4-fold. Adherence is also markedly reduced in C3- or factor B-depleted serum; repletion with C3 or factor B increases adherence by 2.1 +/- 0.4-fold and 1.86 +/- 0.05-fold, respectively. Fab anti-C3 IgG markedly inhibits monocyte adherence of preopsonized M. tuberculosis (71 +/- 1%). C component C3 is fixed to M. tuberculosis by the alternative C pathway as determined by a whole bacterial cell ELISA. Human monocytes ingest M. tuberculosis by conventional phagocytosis as viewed by electron microscopy. This study demonstrates that human monocyte CR1 and CR3 mediate phagocytosis of M. tuberculosis and C component C3 in serum is acting as the major bacterium-bound ligand.     

Heat Shock RNA Levels in Brain and Other Tissues After Hyperthermia and Transient Ischemia

A number of studies have demonstrated increased synthesis of heat shock proteins in brain following hyperthermia or transient ischemia. In the present experiments we have characterized the time course of heat shock RNA induction in gerbil brain after ischemia, and in several mouse tissues after hyperthermia, using probes for RNAs of the 70-kilodalton heat shock protein (hsp70) family, as well as ubiquitin. A synthetic oligonucleotide selective for inducible hsp70 sequences proved to be the most sensitive indicator of the stress response whereas a related rat cDNA detected both induced RNAs and constitutively expressed sequences that were not strongly inducible in brain. Considerable polymorphism of ubiquitin sequences was evident in the outbred mouse and gerbil strains used in these studies when probed with a chicken ubiquitin cDNA. Brief hyperthermic exposure resulted in striking induction of hsp70 and several-fold increases in ubiquitin RNAs in mouse liver and kidney peaking 3 h after return to room temperature. The oligonucleotide selective for hsp70 showed equivalent induction in brain that was more rapid and transient than observed in liver, whereas minimal induction was seen with the ubiquitin and hsp70-related cDNA probes. Transient ischemia resulted in 5- to 10-fold increases in hsp70 sequences in gerbil brain which peaked at 6 h recirculation and remained above control levels at 24 h, whereas a modest 70% increase in ubiquitin sequences was noted at 6 h. These results demonstrate significant temporal and quantitative differences in heat shock RNA expression between brain and other tissues following hyperthermia in vivo, and indicate that hsp70 provides a more sensitive index of the stress response in brain than does ubiquitin after both hyperthermia and ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)