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The lac operon

Posted by star on 2018-11-06 18:37:48
Hits:27

    After the lac operon was sequenced, it eventually became clear that it has two additional operators. The original operator with its center of symmetry at position + 11 is now designated lacO1. Auxiliary operators lacO2 and lacO3 have their centers of symmetry at positions+412 and-82, respectively. Thus, lacO3 is upstream of the lac promoter while lacO2 is located in lacZ.
    The discoveries of lacO2 and lacO3 led investigators to ask whether these auxiliary operators play a role in lac operon regulation. Millier-Hill and coworkers performed a series of genetic experiments that answered this question. Their approach was to alter one or more lac operators and then determine the alteration's effect on repression. Point mutations in taco, caused a 5-to 50-fold decrease in repression, but some repression was still observed. Destruction of either lacO2 or lacO3 caused a twofold decrease in repression and destruction of both auxiliary operators caused a 70-fold decrease in repression. The ability to repress the lac system was completely lost in cells in which lacO1, and either of the auxiliary opera-tors were destroyed. These re X indicate that lacO1 plays a major role in repression but the two auxiliary operators also make important contributions. We will now examine the structure of the Lac repressor to see how it interacts with the lac operators.



A new method for blocking cancer immunosuppression

Posted by star on 2018-11-06 17:52:10
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    In a new study, Belgian researchers elucidated the three-dimensional structure of a protein complex that suppresses immune responses on the cell surface. They also discovered how antibodies can block this protein complex and its downstream induced immunosuppression. The antibodies may be used to activate the immune response against tumor cells in cancer patients, thereby triggering immune cells to destroy the tumor. The results of the study were published in Science on October 25, 2018, entitled "Structure based on potential TGF-β1 expression and GARP activation of human regulatory T cells."

    Regulatory T cells (Tregs) are immunosuppressive cells that, under normal conditions, resist excessive immune responses in the body to prevent autoimmune diseases. However, in cancer patients, they play a detrimental role by inhibiting the immune response against tumor cells. Treg cells exert their effects by producing a messenger protein called TGF-β. This messenger transmits an inhibitory signal to nearby immune cells, especially those that should destroy tumors in cancer patients. The way which Treg cells produce TGF-β is complex and finely regulated because TGF-β is very efficient and must be tightly controlled.
    3 years ago, the researchers found that TGF-β is released from a protein called GARP on the surface of Treg cells. It is possible to block the release of TGF-β from GARP using specific antibodies.

    The researchers resolved the three-dimensional structure of a protein complex assembled by GARP and TGF-β. The structure of GARP is similar to horseshoe, and TGF-β straddles this horseshoe structure. The two molecules are assembled so complexly that TGF-β itself contributes to the formation of the horseshoe-shaped structure of GARP. In this protein complex, this blocking antibody binds to GARP and TGF-β. It seems to bind the t......

The Lac repressor binds to the lac operatsr in vitro

Posted by star on 2018-11-05 22:22:40
Hits:26

    An important step in proving the principal hypothesis of the operon model was the isolation of the Lac repressor and the demonstration of its expected properties. Walter Gilbert and Benno Mullier-Hill succeeded in isolating the Lac repressor from E. coli extracts.They fractionated proteins by standard techniques and then assayed individual fractions for their ability to bind [14C]IPTG (one of the gratuitous inducers). Binding was detected by equilibrium dialysis.
    The Lac repressor is a homotetramer with a molecular mass of 154 kDa. Each subunit, which is made of 360 amino acids, can bind one molecule of IPTG. Crude cell extracts bind about 20 to 40 molecules of IPTG per cell, so there are roughly 5 to 10 repressor molecules per cell. Support for the idea that the IPTG-binding protein is the lac repressor comes from the observation IPTG-binding protein is absent in extracts of ldcl- mutants. Still stronger support comes from the observation that lacI mustants, which introduce amino acid substitutions in lac repressor, alter the repressor's affinity for IPTG.
    Because the number of repressor molecules is extremely small, these molecules must be translated from no more than one or two repressor mRNA molecules transcribed per generation time. The number of mRNA molecules is so small that either repressor synthesis itself is regulated or the mRNA is transcribed from a weak promoter. Both mechanisms have been observed for regulation of repressor synthesis in other operons, but for the Lac repressor the second explanation is correct, that is, repressor mRNA is transcribed constitutively from a weak promoter. The reason for the small number of repressor molecules is made clear from the properties of several mutants in which the weak lacI promoter is converted to a strong promoter. These mutants are noninducible because it is not possible to fill a cell with enough inducer to overcome repression.......

Reveal a new inflammation control mechanism

Posted by star on 2018-11-05 17:50:40
Hits:27

    In the event of infection or tissue damage, an inflammatory immune response attacks the infection and repairs the damaged tissue. However, sometimes excessive inflammation has the opposite effect: in a process called immunopathology. This will increase tissue damage. In a new study, researchers from CNIC have discovered a new mechanism of inflammation control that demonstrates how to control the tissue damage caused by inflammatory immune response. The results of the study were published in Science on October 19, 2018 , entitled "DNGR-1 in dendritic cells limits tissue damage by inhibiting neutrophil recruitment."
    The first immune cells that reach the site of infection or inflammation are neutrophils, and the task of these cells is to eliminate the root cause of this problem. However, neutrophils are very destructive, not only acting on infectious pathogens, but also destroying damaged tissues. This tissue damage caused by our own defense system is called an immunopathological response. This new study confirms that neutrophil infiltration into tissues is controlled by dendritic cells. It is well known that these dendritic cells play an important role in guiding the specific response of T lymphocytes. This new study suggests that dendritic cells regulate neutrophil infiltration to help avoid excessive tissue damage.
    Dendritic cells attract neutrophils into inflammatory focus by releasing factors such as chemokine Mip-2. At the same time, these dendritic cells also express the surface receptor DNGR-1. The cell surface molecules detect tissue damage by recognizing cellular components that are only accessible when the cells are damaged or "ruptured". When DNGR-1 detects damaged tissue, it reduces the ability of dendritic cells to produce Mip-2, thereby limiting neutrophil infiltration into damaged organs. This mechanism prevents the expansion of tissue damage that may be life-threatening.


The inducer of the lactose operon

Posted by star on 2018-11-05 00:06:08
Hits:51

    Two related problems became evident as the operon model was tested. First, inducers must enter a cell if they are to bind to repressor molecules, yet lactose transport requires permease, and permease synthesis requires induction. Thus, we must explain how the inducer gets into a cell in the first place. Second, the isolated Lac repressor does not bind lactose (4-O-β-D-galactopyranosyl-D-glucose) but does bind a lactose isomer called allolactose (6-O-β-D-galactopyra-nosyl-D-glucose). Remarkably, β-galactosidase, the enzyme that catalyzes lactose hydrolysis, also converts a small proportion of lactose to allolactose. Therefore, induction of the synthesis of β-galactosidase by lactose requires that β-galactosidase be present.


    Both problems are solved in the same way in the uninduced state, a small amount of lac mRNA is synthesized (roughly one mRNA molecule per cell per generation). This synthesis, called basal synthesis, occurs because the binding of repressor to the operator is never infinitely strong. Thus, even though the repressor binds very strongly to the operator, it occasionally comes off and an RNA polymerase molecule can initiate transcription during the instant that the operator is free.
    We can now describe in molecular terms the sequence of events following addition of a small amount of lactose to a growing Lac+ culture. Consider bacteria growing in a medium in which the carbon source is glycerol. Each bacterium contains one or two molecules of β-galactosidase and of lactose permease. Lactose is then added. The few permease moIecules transport a few lactose molecules into the cell and the few β-galactosidase molecules convert some of these lactose molecules into allolactose. An allolactose......

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