Differential requirement of protein tyrosine kinase and protein kinase C in the generation of IL-2-induced LAK cell and αCD3-induced CD3-AK cell responses

This study examined the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the signal transduction pathways for lymphocyte activation through IL-2R to generate LAK cells and through TCR—CD3 to generate CD3-AK cells. Two PTK inhibitors [herbimycin A and genistein (PTK-I)] and two PKC inhibitors [calphositin C and staurosporine (PKC-I)] were used in the experiments. It was found that the primary activation pathway through IL-2R was PTK-dependent; that is, generation of both the IL-2-induced proliferative and the cytotoxic responses was completely abrogated by PTK-I and not by PKC-I. Quite different results were obtained with the αCD3-induced CD3-AK cell response. First, the αCD3-induced proliferation was only partially inhibited by PTK-I or PKC-I alone. Second, generation of CD3-AK cytotoxic response was primarily PKC-dependent; that is, only PKC-I induced significant inhibition. Genistein was found to reduce protein tyrosine phosphorylation in both LAK cells and CD3-AK cells, indicating that CD3-AK cells were also susceptible to PTK-I treatment. Further studies showed that PTK-I and not PKC-I suppressed perforin mRNA expression and N-2-benzyoxycarbonyl-l-lysine thiobeneylester esterase production in LAK cells, and the opposite was true for CD3-AK cells. These results indicate that different pathways were employed in lymphocyte activation through IL-2R and TCR—CD3. The former pathway is primarily PTK-dependent. Activation through TCR—CD3 is a more complex event. Induction of a proliferative response can employ either a PTK- or a PKC-dependent pathway, whereas induction of a cytotoxic response is primarily PKC-dependent. Furthermore, it appears that a PTK-independent pathway exists for the induction of a CD3-AK response and thus suggests that activation of the second messenger PKC may not necessarily be preceded by PTK activation.     

Translation of tyrosine aminotransferase mRNA in a modified reticulocyte system.

Tyrosine aminotransferase messenger RNA has been translated in a cell-free system derived from rabbit reticulocytes. Cytoplasmic poly(A)-containing RNA from rat liver was used as the source of the messenger RNA. The newly synthesized subunits of the enzyme were isolated by immunoprecipitation and identified and quantitated using polyacrylamide gel electrophoresis. These procedures were used to demonstrate that glucocorticoid induction is associated with increased cytoplasmic levels of functional tyrosine aminotransferase messenger RNA.     

IL-4 regulation of a protein kinase C independent pathway for the generation of alpha CD3-induced activated killer cells.

alpha CD3 induced the generation of activated killer cells from resting T cells. Pretreatment of the splenic responders with PMA, a phorbol ester, depleted protein kinase C and induced unresponsiveness to the generation of alpha CD3-induced activated killer (CD3-AK) cells. Addition of exogenous IL-4 (1 U/ml) restored the cytotoxic response, with the maximal effect achieved with 30 to 100 U/ml. The phenotypes of CD3-AK cells maintained in IL-2 or in IL-4, with or without PMA, were the same: Thy1+ and CD8+. These results were reproduced with purified T cells and purified CD8+ cells, indicating that both the effectors and precursors were CD8+ cells and IL-4 had a selective effect to upregulate the CD8+ cells. Similar results were obtained by using SSP (staurosporine), another PKC inhibitor. At 2 days prior to testing, switching the lymphokine added to 2-week PMA- and IL-2-maintained CD3-AK cells reversed their cytolytic activity: switching from IL-2 to IL-4 restored cytolytic activity, and switching from IL-4 to IL-2 reduced cytolytic activity. The cytolytic activity of these CD3-AK cells correlated with their ability to produce BLT-esterase. In the absence of PMA, CD3-AK cells cultured in either IL-2 or IL-4 were cytolytic and contained high levels of BLT-esterase. In contrast, in the presence of PMA, only the IL-4-maintained CD3-AK cells were cytolytic and produced significant amounts of BLT-esterase. The effect of IL-4 was abrogated by the alpha IL-4 antibody 11B11, which reduced the cytolytic activity of CD3-AK and the ability to produce BLT-esterase. The requirement of IL-2 was less stringent and its major role appeared to be maintaining the cell growth. These findings indicate that IL-4 may participate in the regulation of a PKC-independent pathway for the generation of CD3-AK cells by regulating the production of cytolytic granules.     

New concepts in the law of the sea

Abstract

Current ocean law negotiations reflect conflicts between two old and competing approaches: the view that the coastal state should control activities in any large adjacent ocean area, and the view that most of the ocean should be left open to the free use of all nations. Both approaches are laissez‐faire, leave the distribution of benefits to arbitrary factors, and are based on national exclusivity. In the negotiations this conflict is exhibited in competing claims regarding navigation, mineral resources, fishing, environmental protection, and strategic uses. A possible resolution has emerged in the concept of the whole ocean as a common resource of humankind, according to which no individual state has a right to benefit from the ocean except pursuant to arrangements sanctioned by the community, and rights to benefit are determined not arbitrarily but by membership in the community. The regime now likeliest to be produced by such an approach includes (1) a narrow territorial sea and various navigation guarantees, (2) a wide coastal band coupling coastal state managerial functions with permanent international prerogatives, and (3) purely international manage‐ment of the deep seabed.     

Immunolocalization of Non-Symbiotic Hemoglobins During Somatic Embryogenesis in Chicory

Hemoglobins are ancient O₂-binding proteins, ubiquitously found in eukaryotes. They have been categorized as symbiotic, nonsymbiotic and truncated hemoglobins. We have investigated the cellular localization of nonsymbiotic hemoglobin proteins during somatic embryogenesis in Cichorium hybrid leaves (Cichorium intybus L. var. sativum × C. endivia var. latifolia) using immunolocalization technique. These proteins were detected during the two steps of culture: induction and expression. In leaves, hemoglobins colocalised with plastids, which were dispersed in the parietal cytoplasm as well as in the two guard cells of a stomata, but not in epidermis cells. Upon induction of embryogenesis, in the dark, this pattern disappeared. During the induction phase, where competent cells reinitiate the cell cycle and prepare for mitosis, hemoglobins appeared initially near chloroplasts, and then in the vicinity of vascular vessels especially in the phloem and in cells surrounding the xylem vessels. When leaf fragments were transferred to another medium for the expression phase, hemoglobins were observed in the majority of the leaf blade cells and in small young embryos but not in the older ones. Hemoglobins were also detected in other leaves cells or tissues all along the process. The role of these nonsymbiotic hemoglobins during somatic embryogenesis is discussed.Key Words: chicory, immunolocalization, nonsymbiotic hemoglobin, somatic embryogenesis     

Crystal structure of a nonsymbiotic plant hemoglobin

BACKGROUND: Nonsymbiotic hemoglobins (nsHbs) form a new class of plant proteins that is distinct genetically and structurally from leghemoglobins. They are found ubiquitously in plants and are expressed in low concentrations in a variety of tissues including roots and leaves. Their function involves a biochemical response to growth under limited O(2) conditions. RESULTS: The first X-ray crystal structure of a member of this class of proteins, riceHb1, has been determined to 2.4 A resolution using a combination of phasing techniques. The active site of ferric riceHb1 differs significantly from those of traditional hemoglobins and myoglobins. The proximal and distal histidine sidechains coordinate directly to the heme iron, forming a hemichrome with spectral properties similar to those of cytochrome b(5). The crystal structure also shows that riceHb1 is a dimer with a novel interface formed by close contacts between the G helix and the region between the B and C helices of the partner subunit. CONCLUSIONS: The bis-histidyl heme coordination found in riceHb1 is unusual for a protein that binds O(2) reversibly. However, the distal His73 is rapidly displaced by ferrous ligands, and the overall O(2) affinity is ultra-high (K(D) approximately 1 nM). Our crystallographic model suggests that ligand binding occurs by an upward and outward movement of the E helix, concomitant dissociation of the distal histidine, possible repacking of the CD corner and folding of the D helix. Although the functional relevance of quaternary structure in nsHbs is unclear, the role of two conserved residues in stabilizing the dimer interface has been identified.     

Mesenchymal stem cells and collagen patches for anterior cruciate ligament repair

AIM: To investigate collagen patches seeded with mesenchymal stem cells(MSCs) and/or tenocytes(TCs) with regards to their suitability for anterior cruciate ligament(ACL) repair. METHODS: Dynamic intraligamentary stabilization utilizes a dynamic screw system to keep ACL remnants in place and promote biological healing, supplemented by collagen patches. How these scaffolds interact with cells and what type of benefit they provide has not yet been investigated in detail. Primary ACL-derived TCs and human bone marrow derived MSCs were seeded onto two different types of 3D collagen scaffolds, Chondro-Gide?(CG) and Novocart?(NC). Cells were seeded onto the scaffolds and cultured for 7 d either as a pure populations or as "premix" containing a 1:1 ratio of TCs to MSCs. Additionally, as controls, cells were seeded in monolayers and in co-cultures on both sides of porous high-density membrane inserts(0.4 μm). We analyzed the patches by real time polymerase chain reaction, glycosaminoglycan(GAG), DNA and hydroxyproline(HYP) content. To determine cell spreading and adherence in the scaffolds microscopic imaging techniques, i.e., confocal laser scanning microscopy(c LSM) and scanning electron microscopy(SEM), were applied.RESULTS: CLSM and SEM imaging analysis confirmed cell adherence onto scaffolds. The metabolic cell activity revealed that patches promote adherence and proliferation of cells. The most dramatic increase in absolute metabolic cell activity was measured for CG samples seeded with tenocytes or a 1:1 cell premix. Analysis of DNA content and c LSM imaging also indicated MSCs were not proliferating as nicely as tenocytes on CG. The HYP to GAG ratio significantly changed for the premix group, resulting from a slightly lower GAG content, demonstrating that the cells are modifying the underlying matrix. Real-time quantitativepolymerase chain reaction data indicated that MSCs showed a trend of differentiation towards a more tenogenic-like phenotype after 7 d.CONCLUSION: CG and NC are both cyto-compatible with primary MSCs and TCs; TCs seemed to perform better on these collagen patches than MSCs.     

Using Tournament Angler Data to Rapidly Assess the Invasion Status of Alien Sport Fishes (Micropterus spp.) in Southern Africa

Fishes are one of the most commonly introduced aquatic taxa worldwide, and invasive fish species pose threats to biodiversity and ecosystem function in recipient waters. Considerable research efforts have focused on predicting the invasibility of different fish taxa; however, accurate records detailing the establishment and spread of invasive fishes are lacking for large numbers of fish around the globe. In response to these data limitations, a low-cost method of cataloging and quantifying the temporal and spatial status of fish invasions was explored. Specifically, angler catch data derived from competitive bass angling tournaments was used to document the distribution of 66 non-native populations of black bass (Micropterus spp.) in southern Africa. Additionally, catch data from standardized tournament events were used to assess the abundance and growth of non-native bass populations in southern Africa relative to their native distribution (southern and eastern United States). Differences in metrics of catch per unit effort (average number of fish retained per angler per day), daily bag weights (the average weight of fish retained per angler), and average fish weight were assessed using catch data from 14,890 angler days of tournament fishing (11,045 days from South Africa and Zimbabwe; 3,845 days from the United States). No significant differences were found between catch rates, average daily bag weight, or the average fish weight between countries, suggesting that bass populations in southern Africa reach comparable sizes and numbers relative to waters in their native distribution. Given the minimal cost associated with data collection (i.e. records are collected by tournament organizers), the standardized nature of the events, and consistent bias (i.e. selection for the biggest fish in a population), the use of angler catch data represents a novel approach to infer the status and distribution of invasive sport fish.     

Modelling optimal timing and frequency of insecticide sprays for eradication or knockdown of closed populations of tsetse flies Glossina spp. (Diptera: Glossinidae)

A population model for tsetse species was used to assess the optimal number and spacing of airborne sprays to reduce or eradicate a tsetse population. It was found that the optimal spray spacing was determined by the time (days) from adult emergence to the first larviposition and, for safety, spacing was assigned to that duration minus 2 days. If sprays killed all adults, then the number of sprays required for eradication is determined by a simple formula. If spray efficiency is less than 100% kill per spray, then a simulation was used to determine the optimal number, which was strongly affected by spray efficiency, mean daily temperature, pupal duration, age to first larviposition and the acceptance threshold for control, rather than eradication. For eradication, it is necessary to have a spray efficiency of greater than 99.9% to avoid requiring an excessive number of sprays. Output from the simulation was compared with the results of two aerial spraying campaigns against tsetse and a least squares analysis estimated that, in both cases, the kill efficiency of the sprays was not significantly less than 100%.