RSS

Complement in a Nutshell

Complement is an ancient component of our innate immune system that was initially discovered in the 19th century and named for its ability to complement antibody in the lysis of cells.

In the simplest of terms, complement is triggered by one of three mechanisms (Antibody Triggers the Classical Pathway; Carbohydrates Trigger the Lectin Pathway; The Alternative Pathway is triggered spontaneously.) Once triggered, a cascade of events leads to the assembly of a C3 Convertase, which breaks C3 into the soluble anaphylatoxin C3a and the insoluble C3b, which precipitates onto the surface of the cell and forms a component of additional C3 convertase, thus amplifying the reaction, and also a C5 convertase.Screen Shot 2020-02-17 at 1.53.41 PM

C5 is then digested into an additional, more powerful soluble anaphylatoxin, C5a, and the insoluble C5b, which cooperates with other components to lead to the formation of the Membrane Attack Complex (MAC) comprised of C9 molecules inserted into the plasma membrane.

The anaphylatoxins, C3a, C5a (and, to a lesser extent, C4a), function to induce contraction of the smooth muscle and then an increase in vascular permeability of the capillaries. They further increase the expression of adhesion molecules on these same vascular epithelial cells so as to recruit immune cells to the location. Finally, they promote receptor-mediated chemoattraction of leukocytes.

In summary, complement can be activated through three somewhat distinct pathways, each one converging at a C3 Convertase. Complement will lead to direct cell death via pore formation (MAC complex formation), it will recruit leukocytes to the area of infection via chemotaxis, and will facilitate phagocytosis of pathogens via complement receptor-mediated endocytosis.

As a last note, it is relevant to bring up the presence of ‘natural antibody.’ These (IgM) antibodies are made by a special group of innate (B-1a) B Cells found most prevalently in young organisms and at lower concentrations later in life. Interestingly, these antibodies are produced prior to antigen exposure and have a pre-defined array of specificities including targets such as phosphorylcholine and certain carbohydrates common on bacteria. As such, they are distinctly innate mediators of immunity and are specifically capable of fixing complement on their bacterial targets.

Comparison-of-chemotaxis-of-eosinophils-mediated-by-C3a-and-C5a-Anaphylatoxin-mediated

Comparison of chemotaxis of eosinophils mediated by C3a and C5a.Discipio 1999

 
1 Comment

Posted by on February 18, 2020 in B-1 B Cells, complement, immunology

 

The Curse of Knowledge

There are several simple problems that are often at the root of communication failures. Sometimes, this may be because the speaker is not thinking clearly. Sometimes this might be because the speaker really does not understand what they are talking about at all. Sometimes, it might even be that the speaker knows what they are talking about too well. In his recent book, The Sense of Style: The Thinking Person’s Guide to Writing in the 21st Century, Harvard Psychologist, Steven Pinker, argues that the curse of knowledge is often to blame. Check out this Inc. article by Glenn Leibowitz on the topic.

 
Leave a comment

Posted by on February 12, 2020 in Uncategorized

 

The Wuhan CoronaVirus

A graphic and a short film on the Wuhan Coronavirus.

The reproduction number is a valuable piece of information that enables epidemiologists to predict how rapidly a virus (or other infectious organism) will spread within a population. For comparison, Measles has a reproduction number of 12-18, which is extraordinarily high, the 1918 influenza that was responsible for infecting some 500 million people worldwide had a reproduction number of 2-3.

The reproduction number tells us nothing about the lethality of the virus, but it does tell us how quickly we can expect it to spread.

Screen Shot 2020-01-30 at 8.57.54 AM

Here is a short film showing the impact that the Wuhan virus is having in one small community in China.

 
Leave a comment

Posted by on January 30, 2020 in Uncategorized

 

Regulating the Regulator of Heat Shock Response in Bacteria

This week in my Molecular class we discussed the role of sigma (σ) factors in regulating transcription. Sigma factors are proteins which bind to the DNA-Dependent RNA polymerase and mediate that enzyme’s binding to the promoters of prokaryotic genes. In this way, we can consider the core polymerase enzyme to be agnostic to which genes are transcribed, it is only the mediation of the sigma factor that guides the polymerase to one gene or another.

Under resting conditions, σ70 is expressed highly and guides the polymerase to constitutively express ‘normal’, housekeeping genes. However, under conditions of stress, such as heat shock, another sigma factor, σ24, becomes active, leading to the transcription (and subsequent translation) of a third factor, σ32. This third sigma factor then leads the polymerase to translate a number of heat shock response proteins such as chaparones, which help prevent protein denaturation and aggregation.

The question is, how does σ24 know when to become active and initiate this heat shock cascade?

Before answering this, we should ask ourselves what to expect.

How can a cell respond to heat shock before the heat shock proteins are made? The answer must be that somehow the heat shock itself leads to a difference in the sigma factors being utilized. I speculated in class that one possible mechanism would be that either the polymerase or σ24 might partially denature in a way that their association was favored over that of the polymerase and σ70.

Although that could work, it turns out not to be the case.

Boldrin et al. examine this question in the bacteria Mycobacterium tuberculosis and suggest that an anti-sigma factor, regulator of sigma E factor A (RseA), binds σ24 (which they call σE – however, I will continue calling it σ24 for consistency) and sequesters it under resting conditions. They continue, “[i]f [RseA] acted as a σ24-specific anti-sigma factor, we would expect to detect an upregulation of those genes whose expression is regulated by σ24 when [RseA] is absent.” One such gene regulated by σ24 is sigB.

To demonstrate that σ24 does regulate sigB, cells missing σ24 were generated. Indeed, the expression of sigB was repressed about 10 times in these cells compared to the wild type (Figure 4). Similarly, when RseA was overexpressed, sigB was also repressed(Figure 5). Genes not regulated by σ24 were unaffected by the deletion of σ24 or the overexpression of RseA (data not shown)Screen Shot 2019-10-18 at 10.13.59 AM.png

Taken together these data indicate that the absence of RseA specifically increases the activity of σ24.Screen Shot 2019-10-17 at 4.35.45 PM.png

De Las Peñas et al. confirm that RseA is predicted to be an inner membrane protein, and the purified cytoplasmic domain binds to and inhibits σ24.

 

 

 

Of course the rabbit hole continues to deepen as we ask how RseA knows to release . It turns out that (cellular) envelope stress promotes RseA degradation, which occurs by a proteolytic cascade initiated by DegS, but that’s as far as we’ll go here. I hope this helps!

Screen Shot 2019-10-18 at 10.14.44 AM.png

 

Assessing the role of Rv1222 (RseA) as an anti-sigma factor of the Mycobacterium tuberculosis extracytoplasmic sigma factor SigE Francesca Boldrin, Laura Cioetto Mazzabò, Saber Anoosheh, Giorgio Palù, Luc Gaudreau, Riccardo Manganelli & Roberta Provvedi  Scientific Reports volume 9, Article number: 4513 (2019)

The σE‐mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of σE Alejandro De Las Peñas, Lynn Connolly, and Carol A. Gross 31 October 2003

See also:

The Single Extracytoplasmic-Function Sigma Factor of Xylella fastidiosa Is Involved in the Heat Shock Response and Presents an Unusual Regulatory Mechanism José F. da Silva Neto, Tie Koide, Suely L. Gomes, Marilis V. Marques Journal of Bacteriology Dec 2006, 189 (2) 551-560

 
1 Comment

Posted by on October 18, 2019 in Uncategorized

 

Ocean

I didn’t know the song Ocean, by the Velvet Underground, despite owning Unloaded for years, until I hear a cover version by MGMT off their Late Night Tales album.

I wasn’t very impressed. in fact, as the song neared its end, I was struck by the realization that it had a bizarre ripoff of Alive and Kicking‘s signature riff. I was a little angry that MGMT had pulled off this larceny and was determined to listen more closely to see if I was right.

Unfortunately, it was as if the song didn’t exist – I kept googling “MGMT ocean” to find a youtube version to play side-by-side against the Simple Minds’ song, but I kept coming up short. Until I added the album name, ‘Late Night Tales‘ to my search and found the Underground version. And then I was stuck.

Alive and Kicking came out in 1985,  Ocean came out on the Velvet Underground’s VU album the same year. So, neither was a copy of the other, just two bands hitting similar riffs at the same time. However,  Ocean had been recorded for MGM studio years earlier, in 1969, and was simply shelved until its release on VU.

Except that there was another version re-recorded by the Underground, but released on Lou Reed’s eponymous album in 1972. So it is possible that Simple Minds had heard this and was influenced by it.

Or maybe I’m just hearing things that aren’t there.

And if I’m really interested in hearing an ocean song, it’s probably this one:

 
Leave a comment

Posted by on March 23, 2019 in Uncategorized

 

Basic Gene Structure

It’s tempting to think of genes as simply a series of nucleotides beginning with a START codon and ending with one of the three STOP codons. However, there are a number of additional regulatory elements that must be present in order for a gene to be transstopstartcribed and translated appropriately.

Transcription is regulated by signals for the DNA-dependent RNA Polymerase ( or simply ‘RNA Polymerase’) to attach and detatch from the DNA in the nucleus.

The attachment point is known as the promoter, and ‘Initiation’ of transcription is characterized by the recruitment of the RNA polymerase to the DNA upstream of the coding sequence. Polymerase engagement unwinds the DNA allowing for recruitment of ribonucleotides and the start of RNA synthesis (Elongation). There may be a number of false starts until a sufficiently long RNA is made to stabilize the enzyme, and even after elongation begins, it may stall and restart until the polymerase reaches a termination signal. There are a number of different kinds of termination signals, but they all occur downstream of the stop codon and serve to disengage the polymerase from the DNA (Termination) so that it is free to recycle back to the promoter.

There are some key differences between the ways that prokaryotes and eukaryotes perform these operations, but all the above elements occur in each system. The key to understanding this clearly is that transcription must occur before translation, and therefore, the transcribed region must have all the translated region within it. This sounds obvious, but can be helpful in order to envision the gene correctly as a physical object.BasicGeneStructure

 
Leave a comment

Posted by on September 26, 2018 in Uncategorized

 

Directional Cloning: a quick description

PrintImagine that you are a scientist interested in cloning gene A. You’ve just amplified the entire gene A including some flanking sequence by PCR and run the resulting amplicon onto a 2% gel with a 100 bp ladder. See the results in Figure 1.

figure1Happy with your result, you clone the DNA into a cloning vector, pCR2.1 where you can make up tons of DNA to work with. These cloning vectors are great for making lots of DNA, but they do not express any of the genes as proteins (i.e. the DNA is replicated, but not transcribed and translated.) Because you do want to express protein, you need to subclone your gene from the cloning vector into an expression vector. To complicate matters, the gene needs to go into the expression vector with the promoter upstream of the gene and the poly A signal downstream of the gene (See Figure 2). The promoter is the location that the RNA polymerase binds to transcribe the gene, the polyA site is what signals the polymerase to add a polyA tail to the mRNA.

Figure2In order to clone your gene into the expression vector, you decide to determine the direction that your gene has inserted into pCR2.1. To do this, you take advantage of the fact that there is a NotI site off-center in the insert and also one in the plasmid (See Figure 3). Gene A is just under 900bp long in total, the Not I site is located at position 800bp.Figure3

You cut the plasmid with NotI expecting either an ~800bp band or a ~100bp band depending upon the orientation of the insert. The results of your digest are seen in figure 4, leading you to believe that your insert is in the direction seen in figure 5.

Figure 4Figure 5In order to subclone from the cloning vector into the expression vector (figure 6), you cut the gene out of pCR2.1 with SpeI and NsiI and isolate the ~900 bp fragment. The same two enzymes can be used to open the expression vector and isolate the linear plasmid. The two fragments can then be combined in the presence of DNA ligase to complete the subcloning.

Figure6

 
Leave a comment

Posted by on September 17, 2018 in Uncategorized