Ordered assembly of the V(D)J synaptic complex ensures accurate recombination

Recombination of gene segments at the immunoglobulin and T-cell receptor loci requires that the RAG1 and RAG2 proteins bring together DNA signal sequences (RSSs) with 12- and 23-bp spacers into a synaptic complex and cleave the DNA. A RAG1/2 multimer that can cleave both signals is shown to assemble on an isolated RSS, and the complementary RSS enters this complex as naked DNA. When RAG1/2 is allowed to bind 12 and 23 RSSs separately prior to their mixing, synaptic complex assembly and cleavage activity are greatly reduced, indicating that only a complex initially assembled on a single RSS leads to productive cleavage. RAG1/2 complexes assembled on 12 RSSs will only incorporate 23 partners, while complexes assembled on 23 RSSs show a 5- to 6-fold preference for 12 partners. Thus, initial assembly on a 12 RSS most accurately reflects the strict 12/23 coupled cleavage observed in the cell. Additional cellular factors such as chromatin may ensure that RAG1/2 first assembles on a 12 RSS, and then a free 23 RSS enters to activate cleavage.     

Reduced synthesis of basement membrane heparan sulfate proteoglycan in streptozotocin-induced diabetic mice

In diabetes, certain basement membranes become thicker yet more porous than normal. To identify possible changes in the basement membrane, we have grown the Engelbreth-Holm-Swarm tumor, a tissue that produces quantities of basement membrane in normal mice and in streptozotocin-treated, insulin-deficient, diabetic mice. The level of laminin, a basement membrane-specific glycoprotein, and the level of total protein were slightly elevated in the diabetic tissue. In contrast, the level of the basement membrane specific heparan sulfate proteoglycan was only 20% of control. The synthesis of this proteoglycan was also reduced in the diabetic animals, while the synthesis of other proteoglycans by tissues such as cartilage was normal. The synthesis of the heparan sulfate proteoglycan in diabetic animals was inversely related to plasma glucose levels showing an abrupt decrease above the normal range of plasma glucose. Insulin restored synthesis to normal but this required doses of insulin that maintained plasma glucose at normal levels for several hours. Since the heparan sulfate proteoglycan in the basement membrane restricts passage of proteins, its absence could account for the increased porosity of basement membrane in diabetes. A compensatory synthesis of other components could lead to their increased deposition and the accumulation of basement membrane in diabetes.     

Characterization of the interleukin-1-induced tyrosine phosphorylation of a 41-kDa plasma membrane protein of the human tumor cell line K 562

A 41-kDa protein, which was specifically phosphorylated upon incubation with natural purified murine interleukin 1, was recently identified by us [Martin, M., Lovett, D. H. and Resch, K. (1986) Immunobiology 171, 165-169] in highly purified plasma membranes from the human tumor cell line K 562. An in vitro assay was used to investigate and characterize the phosphorylation induced by interleukin 1, possibly involved in signal transduction and generation. Plasma membranes were incubated with radiolabeled ATP in the presence of purified natural murine interleukin 1, or recombinant human interleukin 1 alpha and the pattern of phosphoproteins was studied after separation by SDS/PAGE and subsequent autoradiography. A 41-kDa protein (pp41) was specifically phosphorylated on a tyrosine residue in the presence of interleukin 1 in a dose- and time-dependent manner. The protein showed a weak background phosphorylation in the absence of monokine. Phosphorylation took place very efficiently at 0 degrees C, whereas phosphatases were not active at that temperature. At 37 degrees C, a rapid dephosphorylation was observed which was inhibited specifically by Zn2+ and vanadate. The interleukin-1-specific induction of the phosphorylation could also be observed after detergent solubilization of the plasma membranes. Affinity labeling with an ATP analogue revealed an ATP-binding and cleaving site at 41 kDa. Interleukin 1 did not induce the phosphorylation of p41 in plasma membranes obtained from a subclone of K 562, which did not respond to interleukin 1 with growth inhibition, as was reported recently for the K 562 mother line [Lovett, D. H., Kozan, B., Hadam, M., Resch, K. and Gemsa, D. (1986) J. Immunol. 136, 340-347]. These data suggest that the interleukin-1 receptor is functionally linked to a protein-tyrosine kinase, which is implicated in its biological function.     

Flavobacterium meningosepticum peptide:N-glycosidase: influence of ionic strength on enzymatic activity.

Flavobacterium meningosepticum peptide:N-glycosidase-mediated deglycosylation of N-linked glycan strands of glycoproteins has been found to be strongly influenced by the ionic strength of the assay medium. By use of a modification of a previously published assay procedure for quantitative analysis of glycan release we have been able to improve reproducibility and thus to compare the extent of deglycosylation achieved under a variety of conditions of ionic strength. We have observed that enzyme activity is adversely affected by high ionic strength buffers such as those recommended for deglycosylation of various glycoproteins and recommend the use of low ionic strength buffers for routine use.     

Ontogenetic shift in diet of a large elapid snake is facilitated by allometric change in skull morphology

Evolutionary Ecology - As snakes are limbless, gape-limited predators, their skull is the main feeding structure involved in prey handling, manipulation and feeding. Ontogenetic changes in prey...     

State of art and limitations in genetic engineering to induce stable chondrogenic phenotype

Current protocols for chondrocyte expansion and chondrogenic differentiation of stem cells fail to reduce phenotypic loss and to mitigate hypertrophic tendency. To this end, cell genetic manipulation is gaining pace as a means of generating cells with stable chondrocyte phenotype. Herein, we provide an overview of candidate genes that either induce cartilage regeneration or inhibit cartilage degeneration. We further discuss in vitro, ex vivo and in vivo viral transduction and non-viral transfection strategies for targeted cells (chondrocytes, mesenchymal stem cells, induced pluripotent stem cells and synovial cells), along with the most representative results obtained in pre-clinical models and in clinical trials. We highlight current challenges and associated risks that slowdown clinical acceptance and commercialisation of gene transfer technologies.     

Environmental correlates of the Late Quaternary regional extinctions of large and small Palaearctic mammals

Most studies of mammal extinctions during the Pleistocene–Holocene transition explore the relative effects of climate change vs human impacts on these extinctions, but the relative importance of the different environmental factors involved remains poorly understood. Moreover, these studies are strongly biased towards megafauna, which may have been more influenced by human hunting than species of small body size. We examined the potential environmental causes of Pleistocene–Holocene mammal extinctions by linking regional environmental characteristics with the regional extinction rates of large and small mammals in 14 Palaearctic regions. We found that regional extinction rates were larger for megafauna, but extinction patterns across regions were similar for both size groups, emphasizing the importance of environmental change as an extinction factor as opposed to hunting. Still, the bias towards megafauna extinctions was larger in southern Europe and smaller in central Eurasia. The loss of suitable habitats, low macroclimatic heterogeneity within regions and an increase in precipitation were identified as the strongest predictors of regional extinction rates. Suitable habitats for many species of the Last Glacial fauna were grassland and desert, but not tundra or forest. The low‐extinction regions identified in central Eurasia are characterized by the continuous presence of grasslands and deserts until the present. In contrast, forest expansion associated with an increase in precipitation and temperature was likely the main factor causing habitat loss in the high‐extinction regions. The shift of grassland into tundra also contributed to the loss of suitable habitats in northern Eurasia. Habitat loss was more strongly related to the extinctions of megafauna than of small mammals. Ungulate species with low tolerance to deep snow were more likely to go regionally extinct. Thus, the increase in precipitation at the Pleistocene–Holocene transition may have also directly contributed to the extinctions by creating deep snow cover which decreases forage availability in winter.     

Direct Observation of Surface Plasmon Polariton Propagation and Interference by Time-Resolved Imaging in Normal-Incidence Two Photon Photoemission Microscopy

Plasmonics - Time-resolved imaging of the propagation and interference of isolated ultrashort surface plasmon polariton wave packets is demonstrated using two photon photoemission microscopy. The...     

Small-scale field evaluation of the efficacy and residual effect of Fludora® Fusion (mixture of clothianidin and deltamethrin) against susceptible and resistant Anopheles gambiae populations from Benin,West Africa

The emergence of mosquitoes that can avoid indoor-deployed interventions, such as treated bed nets and indoor residual spraying, threatens the mainstay of malaria control in Zambia. Furthermore, the requirement for high coverage of these tools poses operational challenges. Spatial repellents are being assessed to supplement these vector control tools, but limitations exist in the residual effect of the repellent and the need for external power or heat for diffusion of the volatiles. A semi-field evaluation of a novel controlled release spatial repellent device (CRD) was conducted in Macha, Zambia. These devices emanate metofluthrin with no need for external power. Devices were deployed in huts within the semi-field system (SFS). Female Anopheles gambiae sensu stricto released within the SFS were trapped overnight by light traps and collected by aspiration the next morning inside and outside of huts to determine the extent of mosquito repellency and the impact on host-seeking and survival. Experiments studied the impact of number of devices as well as the presence of hut occupants. The study was complemented with numerical methods based on computational fluid dynamics to simulate spatial distribution of metofluthrin. Presence of CRDs was associated with significant reductions in indoor counts of mosquitoes, regardless of whether huts were occupied or not. Repellency ranged from 15 to 60% compared to huts with no devices. Reducing the number of devices from 16 to 4 had little impact on repellency. When huts were occupied, indoor mosquito host-seeking was higher in the presence of CRDs, whilst survival was significantly reduced. This study demonstrated that deployment of as few as four CRDs within a hut was associated with reduced indoor mosquito densities. As would be expected, presence of occupants within huts, resulted in greater indoor catches (both with and without devices). The increased indoor mosquito host-seeking and mortality in huts when devices were present may be explained by the excito-repellency activity of metofluthrin. These semi-field experiments provide preliminary data on the utility of CRD spatial repellents to reduce indoor densities of An. gambiae mosquitoes. Studies will further investigate the impact of CRDs on mosquito behaviour as well as epidemiological protective efficacy.     

Elevated Prolactin during Pregnancy Drives a Phenotypic Switch in Mouse Hypothalamic Dopaminergic Neurons

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