Three recombinant serine proteinase inhibitors expressed from the coding region of the thrombin inhibitor dipetalogastin

Dipetalogastin is a potent thrombin inhibitor from Dipetalogaster maximus. The cDNA of dipetalogastin codes for a large protein which consists of six Kazal-type domains. There are three tandem, homologous regions each including two domains. Three biologically active recombinant proteins rDI, rDII and rDIII each corresponding to one region of the dipetalogastin cDNA were expressed, purified and investigated with regard to their biological activities. rDI and rDII with molecular masses of 12,660 and 12,911 Da, respectively, proved to be potent thrombin inhibitors. The investigation of their influences on amidolytic activities of different serine proteases showed no inhibition of factor Xa (FXa) and alpha-chymotrypsin. At a large molar excess of rDI and rDII over the enzymes only low effects on the activities of trypsin and plasmin were observed. rDIII differs much from the both others. An inhibition of thrombin was found only at a molar excess of rDIII over the enzyme. Furthermore, an inhibition of trypsin and low effects on plasmin were detected at a molar excess of inhibitor over these enzymes. These results indicate that rDIII is active against thrombin, trypsin and plasmin, and finally possesses no specificity for only one serine proteinase.     

Induction of tumor cell apoptosis in vivo increases tumor antigen cross-presentation,cross-priming rather than cross-tolerizing host tumor-specific CD8 T cells

Cross-presentation of cell-bound Ags from established, solid tumors to CD8 cells is efficient and likely to have a role in determining host response to tumor. A number of investigators have predicted that when tumor Ags are derived from apoptotic cells either no response, due to Ag "sequestration," or CD8 cross-tolerance would ensue. Because the crucial issue of whether this happens in vivo has never been addressed, we induced apoptosis of established hemagglutinin (HA)-transfected AB1 tumors in BALB/c mice using the apoptosis-inducing reagent gemcitabine. This shrank the tumor by approximately 80%. This induction of apoptosis increased cross-presentation of HA to CD8 cells yet neither gross deletion nor functional tolerance of HA-specific CD8 cells were observed, based on tetramer analysis, proliferation of specific CD8 T cells, and in vivo CTL activity. Interestingly, apoptosis primed the host for a strong antitumor response to a second, virus-generated HA-specific signal in that administration of an HA-expressing virus after gemcitabine administration markedly decreased tumor growth compared with viral administration without gemcitabine. Thus tumor cell apoptosis in vivo neither sequesters tumor Ags nor cross-tolerizes tumor-specific CD8 cells. This observation has fundamental consequences for the development of tumor immunotherapy protocols and for understanding T cell reactivity to tumors and the in vivo immune responses to apoptotic cells.     

Are Mojave Desert annual species equal? Resource acquisition and allocation for the invasive grass Bromus madritensis subsp. rubens (Poaceae) and two native species

Abundance of invasive plants is often attributed to their ability ot outcompete native species. We compared resource acquisition and allocation of the invasive annual grass Bromus madritensis subsp. rubens with that of two native Mojave Desert annuals, Vulpia octoflora and Descurainia pinnata, in a glasshouse experiment. Each species was grown in monoculture at two densities and two levels of N availability to compare how these annuals capture resources and to understand their relative sensitivities to environmental change. During >4 mo of growth, Bromus used water more rapidly and had greater biomass and N content than the natives, partly because of its greater root-surface area and its exploitation of deep soils. Bromus also had greater N uptake, net assimilation and transpiration rates, and canopy area than Vulpia. Resource use by Bromus was less sensitive to changes in N availability or density than were the natives. The two native species in this study produced numerous small seeds that tended to remain dormant, thus ensuring escape of offspring from unfavorable germination conditions; Bromus produced fewer but larger seeds that readily germinated. Collectively, these traits give Bromus the potential to rapidly establish in diverse habitats of the Mojave Desert, thereby gaining an advantage over coexisting native species.     

Compromised influenza virus-specific CD8(+)-T-cell memory in CD4(+)-T-cell-deficient mice

The primary influenza A virus-specific CD8(+)-T-cell responses measured by tetramer staining of spleen, lymph node, and bronchoalveolar lavage (BAL) lymphocyte populations were similar in magnitude for conventional I-A(b+/+) and CD4(+)-T-cell-deficient I-A(b-/-) mice. Comparable levels of virus-specific cytotoxic-T-lymphocyte activity were detected in the inflammatory exudate recovered by BAL following challenge. However, both the size of the memory T-cell pool and the magnitude of the recall response in the lymphoid tissues (but not the BAL specimens) were significantly diminished in mice lacking the CD4(+) subset. Also, the rate of virus elimination from the infected respiratory tract slowed at low virus loads following challenge of na?ve and previously immunized I-A(b-/-) mice. Thus, though the capacity to mediate the CD8(+)-T-cell effector function is broadly preserved in the absence of concurrent CD4(+)-T-cell help, both the maintenance and recall of memory are compromised and the clearance of residual virus is delayed. These findings are consistent with mathematical models that predict virus-host dynamics in this, and other, models of infection.     

The C-terminal domain of the epsilon subunit of the chloroplast ATP synthase is not required for ATP synthesis

The epsilon subunit of the ATP synthases from chloroplasts and Escherichia coli regulates the activity of the enzyme and is required for ATP synthesis. The epsilon subunit is not required for the binding of the catalytic portion of the chloroplast ATP synthase (CF1) to the membrane-embedded part (CFo). Thylakoid membranes reconstituted with CF1 lacking its epsilon subunit (CF1-epsilon) have high ATPase activity and no ATP synthesis activity, at least in part because the membranes are very leaky to protons. Either native or recombinant epsilon subunit inhibits ATPase activity and restores low proton permeability and ATP synthesis. In this paper we show that recombinant epsilon subunit from which 45 amino acids were deleted from the C-terminus is as active as full-length epsilon subunit in restoring ATP synthesis to membranes containing CF1-epsilon. However, the truncated form of the epsilon subunit was significantly less effective as an inhibitor of the ATPase activity of CF1-epsilon, both in solution and bound to thylakoid membranes. Thus, the C-terminus of the epsilon subunit is more involved in regulation of activity, by inhibiting ATP hydrolysis, than in ATP synthesis.     

Effect of hypothermia on insulin-receptor interaction in different rat tissues

Experimental hypothermia caused extensive changes in the number of both classes of insulin receptors in different rat tissues. In the liver, the number of high affinity insulin receptors (HAIRs) decreased by 50% (from 25.3 to 12.6 fmol/mg membrane protein), whereas number of low affinity insulin receptors (LAIRs) was almost unchanged in comparison to normothermic animals (5.63 and 4.39 pmol/mg, respectively). In the adipose tissue, number of both classes was reduced--HAIRs by 81% (from 24.0 to 4.50 fmol/mg) and LAIRs by 92% (from 16.0 to 1.29 pmol/mg). In the skeletal muscle, capacity of HAIRs was not changed (16.2 and 19.3 fmol/mg in normo- and hypothermic animals, respectively), whereas number of LAIRs increased by 150% (from 6.65 to 16.6 pmol/mg). Hypothermic rats also showed lower amount (by 85%) of LAIRs in the heart muscle (9.37 and 1.43 pmol/mg in control and experimental animals, respectively). Simultaneously, no significant changes were found in HAIRs (16.3 and 11.9 fmol/mg, respectively) and LAIRs (4.43 and 3.88 pmol/mg, respectively) in the brain. These differences in insulin receptors responses to hypothermia may reflect different physiological role of insulin in the regulation of target cell metabolism and/or the differences in tissue distribution of the insulin receptor isoforms.     

Effects of metal ions on activity of plasmin

The effects of some metal ions on amidolytic and fibrinogenolytic activities of highly purified human plasmin were investigated in vitro. In the presence of Zn²⁺, Cu²⁺, Cd²⁺, and Au⁺ in the incubation mixture at the concentrations of 1×10⁻⁵−1×10⁻³ M, the anidolytic plasmin activity was strongly inhibited, whereas Ca²⁺ and Mg²⁺ at the same concentrations were not effective. The analysis of the kinetic study has shown that Zn²⁺ or Cu²⁺ acts as mixed-type inhibitors of plasmin activity. The inhibition of amidolytic plasmin activity by Zn²⁺ and Cu²⁺ was reduced in the presence of EDTA, histidine, or albumin. Incubation of plasmin with Zn²⁺ or Cu²⁺ (at the concentration of 5×10⁻⁴ M) resulted in complete loss of its proteolytic action on fibrinogen, whereas Cd²⁺ and Au⁺ under the same conditions only partially inhibited this process.     

Population Reduction and Social Disorganization in Alouatta pigra Following a Hurricane

The opportunity to study the effects of a powerful hurricane on monkey populations, diet, and behavior via pre- and post-hurricane data was presented when hurricane Iris virtually destroyed the forest along Monkey River in southern Belize on October 8, 2001, including a 52-ha area where black howlers have been subjects since 1999. Before the hurricane, 8 social groups, averaging 6.37 members, had been stable in both group composition and range for q 2 years. The hurricane, which levelled much of the forest, resulted in the complete loss of the forest canopy. The trees that remained standing lost most or all branches and were 100% defoliated. The monkey population in the study area was reduced by 42% and survivers experienced a period of extended social disorganization involving transient individuals, high numbers of solitary monkeys, and small fragmentary social groups. The period of disorganization lasted 12 weeks, after which the number of solitaries reduced and stability of the large groups increased. Within the study area, 5 social groups have been more or less stable since ca. week 15; however, home ranges had yet to stabilize at week 35. The social and ranging effects are similar to what has been described for translocated primates. Post-hurricane diet was limited to fruit and leaves remaining in the deadfall for the first 2 weeks and to new leaves and leaf buds for many weeks after that. Normal fruit consumption in April and May was prevented by the failure of surviving trees to produce fruit. With the loss of forest canopy there has been increased use of low foliage and ground travel, and with the reduction in population density there has been a reduction in vocalization frequency.