Frequency of cytokine-producing CD4-CD8- peripheral blood mononuclear cells in patients with Plasmodium falciparum malaria.

The frequency of cytokine-producing CD4-/CD8- mononuclear cells was assessed in patients of different age groups (29 infants, aged 1-5 years; 30 schoolchildren, aged 6-14 years, 26 adults, aged > 15 years) with acute Plasmodium falciparum malaria, from Gabon. Fifteen patients were followed up before antimalarial treatment (day 0), during parasite clearance (day 3) and after resolution of parasitemia (day 10). By using flow cytometry for intracellular detection of cytokines, a striking expansion of CD4-/CD8- cells producing the type 1 cytokines interleukin (IL)-2-/interferon (IFN)-gamma+, IL-2+/IFN-gamma+ and IL-2+/IFN-gamma- was observed in adults as compared with children. Type 2 cytokine expression (IL-4+/IFN-gamma-, IL-13+/IFN-gamma-) and type 0 cells (IL-4+/IFN-gamma+, IL-13+/IFN-gamma+) were not significantly different between the three age groups. Patients with severe malaria had a significantly increased frequency of type 2 cytokine-producing CD4-/CD8- cells. Drug-induced clearance of parasitemia was characterized by a decrease of IL-2+/IFN-gamma- and type 2 cytokine expressing CD4-/CD8- cells and by a gradual increase of IL-10+/IFN-gamma- expression. The type 1/type 2 dichotomy observed within the CD4-/CD8- cell population is likely to be of significance in the host response against P. falciparum malaria.     

Characterization of the state of differentiation of six newly established human non-small-cell lung cancer cell lines

Six new non-small-cell lung cancer (NSCLC) cell lines were established directly from human tissue or indirectly via nude mouse xenografts in serum-supplemented media with success rates of 8% and 13%, respectively. They comprised one adenocarcinoma (ADLC-5M2), two squamous cell carcinomas (EPLC-32M1, EPLC-65H), two large cell carcinomas (LCLC-97TM1, LCLC-103H), and one malignant biphasic mesothelioma (MSTO-211H). All cell lines grew adherent to culture vessels with population doubling times (PDT) of 16-40 h, formed colonies in soft agarose with efficiencies of 0.1%-5.1%, and all grew in athymic nude mice. Xenograft histologies appeared as follows: (a) undifferentiated carcinomas with feeble resemblance to the original tumors in the case of adenocarcinomas and squamous cell carcinomas; (b) large cell carcinoma with high resemblance to the original tumor; (c) an undifferentiated tumor with predominance of large epithelial cells and few fibrous cells in the case of mesothelioma. Human chorionic gonadotropin (HCG) was found by radioimmunoassay and high-affinity binding sites for epidermal growth factor (EGF) by radio-receptor assay in 4/4 cell lines. A very low activity of L-DOPA decarboxylase (DDC) was detectable only in the adenocarcinoma cell line. All cell lines overexpressed the c-myc protooncogene, and no gene rearrangement or amplification was observed. Chromosome analysis revealed modal chromosome numbers of 70-73 in ADLC-5M2, EPLC-32M1, EPLC-65H, and MSTO-211H. Cell lines derived from large cell carcinoma had modal values of 65 and 170 and a wider chromosome distribution than all other cell lines. A NSCLC specific chromosomal aberration has been undetectable until now. These cell lines may aid in elucidating the biology of NSCLC and its interrelationship to other lung tumors.     

Models of the geometry of the human ankle bone pulley: two series of geometric models for demonstrating the biomechanics of the ankle joint

2 series of models demonstrate the geometrical shape of the human trochlea tali. We have changed step by step the shape of the 2 flanking articular facets of the trochlea, the course of the edges of the trochlea, and the length of their radii, and so we have found a model responding to the biomechanic conditions of the trochlea tali. The convex surface of this model (corresponding to the superior articular surface, i.e. the facies superior trochleae tali) is a torse, the medial flanking facet (corresponding to the medial articular facet of the trochlea, i.e. the facies malleolaris medialis) is a flat cone, the lateral flanking facet (corresponding to the lateral articular facet of the trochlea, i.e. the facies malleolaris lateralis) is a screwed (helicoidal) face. The resulting model shows the 2 completely different phases of motion in the ankle joint: During dorsiflexion (motion setting out from the neutral position towards the final position of dorsiflexion), the internal malleolus leads the talus, whereas the external malleolus is pushed outwards by the screwed lateral articular facet of the trocheal. The trochlea is moved like a hinge. In the final position of dorsiflexion, the malleoli tightly embrace the 2 flanking facets of the trochlea, whilst an obvious cleft appears dorsally and medially between the superior articular surface of the trochlea and the tibial roof (i.e. the facies articularis inferior tibiae). During plantarflexion (motion setting out from the neutral position towards the final position of plantarflexion), the external malleolus leads the talus, whereas the medial articular facet of the trochlea withdraws from the internal malleolus. The trochlea is moved like a screw. In the final position of plantarflexion, the superior articular surface of the trochlea closely contacts the tibial roof, whilst an obvious cleft appears between the medial articular facet of the trochlea and the internal malleolus.     

Genetic analysis of thirty-three platelet polypeptides detected in two-dimensional polyacrylamide gels.

Two-dimensional gel electrophoresis followed by silver-staining was utilized to visualize platelet polypeptides for genetic analysis. A subset of 33 polypeptides that were most suited for scoring was selected. Families consisting of father-mother-child trios were studied. Thirty-six polypeptides of a total of 1,413 scored in children's gels exhibited the combination of a normal and a variant polypeptide. The observed index of heterozygosity of 2.55% is comparable to our previously reported findings for red cell proteins.     

Frequency of polymorphisms for alleles encoding for liver proteins of domesticated mice

Three different inbred strains of mice have been crossed with a lethal albino line (cch/c3H) and the liver polypeptides of the parents and offspring examined by two-dimensional polyacrylamide gel electrophoresis for evidences of protein polymorphisms, different alleles of which have gone to fixation in different strains. In the battery of polypeptides considered most favorable for scoring, 3.3 +/- 1.6 percent of the battery exhibited paired variants and 1.6 +/- 1.2 percent, unpaired. An adjustment for the fact the same allele of a biallelic polymorphism may go to fixation in two inbred lines of common ancestry leads to the suggestion that in the stock from which these inbred lines were ultimately derived, there were some 11.0 percent paired and 5.3 percent unpaired polymorphisms in the average mouse. This is about half the frequency of polymorphisms observed in wild European Mus musculus musculus and Mus musculus domesticus with one-dimensional electrophoresis of blood plasma and erythrocyte proteins. Three explanations were considered for the lower estimated frequency for liver protein polymorphisms: the difference is real, the apparent difference is due to the lower resolving power of two-dimensional gels, or the mouse strains from which the present inbred lines were drawn had already, lost through inbreeding, a considerable amount of their genetic variation before the inbreeding leading to the present strains commenced.     

Analysis of Staphylococcal Enterotoxin B by the Polyacrylamide Electrophoresis Technique

The electrophoretic mobility of enterotoxin B was investigated through the use of the disc electrophoresis technique. Ideal patterns were developed with a 7.5% acrylamide gel system (pH 4.3). The toxin can be separated and identified from other complex proteins such as serum or suspect samples of foods by this technique. The technique can be used as an assay method for the toxin as well as to elucidate physical changes in the toxin due to temperature. The method should not be considered exclusive for enterotoxin B.     

Elicitation of volatile compounds in photomixotrophic cell culture of Petroselinum crispum

Photomixotrophic cells of Petroselinum crispum accumulated >500 mg chlorophyll per kg wet weight and grew well in a broad range of phytoeffector conditions. Autoclaved fungal cells were lethal for photoheterotrophic cells, but induced in photomixotrophic cells the formation of volatile n-alkanes, phthalides, coumarins, and elemicine. Most of the compounds elicited reached a concentration maximum between 20 and 30 h after addition of the mycelium, whereas the group of n-alkanes increased steadily during the 90 h monitored. Maximum concentrations were: 12 mg of graveolone, 1 mg of bergapten, 0.5 mg of sedanenolide, and 0.5 mg of n-tetradecane per 1 nutrient medium. A dose/effect relationship was found; 10 to 25 g of fungal wet weight per 1 culture medium resulted in maximum accumulation of volatiles. The formation of volatiles by photomixotrophic in vitro cells is discussed as an integral part of plant responses to ecological stress.     

Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein.

We recently identified Vav, the product of the vav proto-oncogene, as a guanine nucleotide exchange factor (GEF) for Ras. Vav is enzymatically activated by lymphocyte antigen receptor-coupled protein tyrosine kinases or independently by diglycerides. To further evaluate the physiological role of Vav, we assessed its GDP-GTP exchange activity against several Ras-related proteins in vitro and determined whether Vav activation in transfected NIH 3T3 fibroblasts correlates with the activity status of Ras and mitogen-activated protein (MAP) kinases. In vitro translated purified Vav activated by phorbol myristate acetate (PMA) or phosphorylation with recombinant p56lck displayed GEF activity against Ras but not against recombinant RacI, RacII, Ral, or RhoA proteins. Expression of vav or proto-vav in stably transfected NIH 3T3 cells led to a approximately 10-fold increase in basal or PMA-stimulated Ras exchange activity, respectively, in total-cell lysates and Vav immunoprecipitates. Elevated GEF activity was paralleled in each case by a significant increase in the proportion of active, GTP-bound Ras. PMA had a minimal effect on the low Ras. GTP level in untransfected control fibroblasts but increased it from 20 to 37% in proto-vav-transfected cells. vav-transfected cells displayed a constitutively elevated Ras. GTP level (35%), which was not increased further by PMA treatment. MAP kinases, known downstream intermediates in Ras-dependent signaling pathways, similarly exhibited increased basal or PMA-stimulated activity in Vav-expressing cells by comparison with normal NIH 3T3 cells. These results demonstrate a physiologic interaction between Vav and its target, Ras, leading to MAP kinase activation.     

Origin of the main class of repetitive DNA within selected Pennisetum species

In an attempt to identify relationships among genomes of the allotetraploid Pennisetum purpureum Schumach and closely related Pennisetum species with which it can be successfully hybridized, repetitive DNA sequences were examined. Digestion with KpnI revealed two highly repetitive fragments of 140 by and 160 bp. The possibility that these sequences could be used as genome markers was investigated. Average sequences were determined for the 140 by and 160 by KpnI families from P. purpureum and P. squamulatum Fresen. Average sequences (based upon four or five repeats) were determined for the P. glaucum (L.) R. Br. 140 by KpnI family and the diploid P. hohenackeri Hochst. ex Steud. 160 bp KpnI family. The average sequences of the 160 by KpnI families in P. purpureum and P. squamulatum differ by only nine bases. The 140 by KpnI families of the three related species, P. purpureum, P. squamulantum, and P. glaucum are nearly identical, and thus likely represent a recent divergence from a common progenitor or a common genome. Each repetitive sequence may contain internal duplications, which probably diverged following amplification of the original sequence. The 140 by KpnI repeat probably evolved from the 160 by KpnI repeat since the missing 18 by segment is part of the internal duplication that is otherwise conserved in the subrepeats. Tandemly arrayed repetitive sequences in plants are likely to be composed of subrepeats which have been duplicated and amplified.Florida Aqricultural Experiment Station series #R-02758