Problemas que se presentan en sistemas de enfriamiento

A drop in temperature has long been known to play a role in differentiation from short stumpy to procyclic cells. For this reason it is frequently used to induce differentiation

in vitro_{, together with citrate / cis-aconitate (Ziegelbauer} et al._{, 1990; Rolin} et al._{, 1993;}

Matthews and Gull, 1994; Vassella and Boshart, 1996). The mechanism of cold shock sensing in trypanosomes was established by recent studies from this lab. Engstler and Boshart (2004) could show that cold shock (37°C->20°C) induces the expression of the insect stage specific procyclin in BSF cells by a postranscriptional mechanism. Furthermore, although cold shock alone is not sufficient to induce differentiation, it dramatically increases the sensitivity of the cells towards the differentiation stimuli cis- aconitate / citrate. Thus, there is strong evidence for the participation of cold shock in the differentiation from short stumpy to procyclic cells.

We measured in vivo _{activity of} T. brucei _{PKA-like kinase at different temperatures with}

the aid of the VASP reporter substrate and found a significant increase in activity at all temperatures below 37°C. Thereby the increase in kinase activity was nearly proportional to the decrease in temperature. Kinase activity reached its maximum at 12°C and then remained equally high at lower temperatures. Conclusively, the activity of T. brucei _PKA

To our knowledge, T. brucei _{PKA-like kinase is the first PKA homologue that has been}

shown to be activated by changes in temperature. Although temperature sensing has been described for many organisms and a large body of information is available on cold shock induced (mainly prokaryotic) genes (reviewed in Yamanaka et al._{, 1999; Los} et al._,

1999), surprisingly little is known about the upstream factors of a temperature sensing signaling pathway.

In bacteria it was shown that membranes can sense temperature changes (reviewed in Rock and Cronan, 1996) and transmit signals as a consequence of changes in their membrane phase state and microdomain organization (Vigh et al._{, 1998; Hoppe} et al._,

2000; Suzuki et al._{, 2000). A two component-signal transduction pathway, consisting}

of a sensor kinase and a response regulator, has been shown to transmit cold shock signals across the bacterial plasma membrane (Aguilar et al._{, 2001). For plants, a similar}

cold shock sensing mechanism has been proposed. There is evidence that membrane structures of plants change after cold shock and that these membrane changes cause an influx in Ca2+ _{and subsequent activation of cold shock dependent MAP kinases} (Sangwan et al._{, 2002a; Gimalov} et al._{, 2003; Jonak} et al._{, 1996). Using cell free extracts}

it was shown that a heat shock activated MAP kinase is even able to sense temperature shifts directly (Sangwan et al._{, 2002b). The involvement of MAP kinases in cold and}

heat shock response is not restricted to plants but has also been shown in yeast (Soto

et al._{, 2002). Most interestingly, heat shock response in} Leishmania _{was found to be}

accompanied by an Ca2+ _{influx, both from internal pools and from the outside milieu} (Sarkar et al._{, 1995).}

It is tempting to speculate that T. brucei _{PKA-like kinase participates in the upstream}

signaling events in response to cold shock. It has already been shown that a temperature drop below 26°C results in major changes in the T. brucei _{plasma membrane (Ter Kuile} et al._{, 1992).}

4.3.1.3. pH value

transformation of short stumpy cells into the insect stage form even in the absence of citrate/cis-aconitate, although very inefficiently (Rolin et al._{, 1998). It still remains unclear}

whether mild acid stress has any relevance for in vivo _{differentiation, since the pH value}

in the alimentary tracts of the tsetse fly is alkaline rather than acid (J.Van Den Abbeele, unpublished). However, mild acid stress might occur at some stage on the way from the mammalian blood to the intestine of the insect vector and there act as differentiation stimulus.

We measured in vivo _{activity of} T. brucei _{PKA-like kinase of cells in both acid and alkaline}

medium and found a linear dependency between kinase activity and the extracellular pH values. Low pH values resulted in kinase activation and high pH values in kinase inhibition. The tested pH values ranged from 5.5 to 9.5 and had no effect on the viability and motility of the cells during the time of observation, as judged by phase microscopy. Furthermore, changes in kinase activity were reversible. Conclusively, the activity of T. brucei _{PKA-like kinase is depending on the pH value of the environment.}

The signaling events that underlie mild acid stress response in T. brucei _{were studied by}

Rolin et al. _{(1996). The authors found an increase in adenylate cyclase activity at pH 5.5.}

With an artificial acidification of the cytosol by a protonophore they observed the same effect even at neutral extracellular pH. This suggests that adenylate cyclase is activated due to a slight acidification of the cytosol. Interestingly, the activation of adenylate cyclase after mild acid stress was restricted to LS trypanosomes, but absent from the insect stage (Rolin et al._{, 1996) and the cell cycle arrested fly-preadapted short stumpy stage (Nolan} et al._{, 2000).}

Whether the observed activation of PKA-like kinase at mild acid stress is dependent on the activation of adenylate cyclases remains to be shown. Like the observed activation of PKA-like kinase with PDE inhibitors, this would be contradictory to the fact that T. brucei

PKA-like kinase is inhibited by cAMP.

4.3.1.4. Life cycle stage dependent differences in expression and posttranslational