Botulinum: from Toxin to Treatment tool

Botulinum is a highly poisonous exotoxin secreted mainly by the bacterium Clostridium botulinum. There exist 7 serotypes of botulinum toxins, from toxin A to toxin G each differing in their potency and receptors to which they bind. Structurally, these comprise of 2 different protein chains- the A chain and the B chain which are joined together by a disulfide bond. These toxins act at the neuro-muscular junctions and are hence called neurotoxins. They prevent the release and uptake of the neurotransmitter acetylcholine, which is responsible for muscle contraction. As a result of this, the person suffers from flaccid paralysis. Functionally, the neurotoxin can be said to comprise of three domains - a receptor binding domain, a translocation domain and a catalytic domain. The B chain (or the binding domain) attaches to the receptors on the neurons facilitating the entry of the A or active domain into the cell by cleavage of the disulfide bond.. Although it still not clear how this is achieved, it has been presumed that the translocation domain forms a channel to facilitate the entry of the A chain into the cell.

When administered in very small quantities,the toxin effect is reversible and largely restricted to the affected area. It may take anywhere between 8 weeks to 6 months for the effect of the toxin to subside. Due to these factors it has increasingly found use as a treatment tool in various disorders and afflictions. The most famous of all would definitely be BOTOX!

It was in 1989, that BOTOX was approved by FDA to treat disorders like strabismus (squint eye), blepharospasm (abnormal twitching of eyelid), and hemifacial spasm (involuntary muscle contractions on one side of face). The botulinum injection is given at the site of affected muscle. In delicate places, the process is aided by electromyography. The machine detects the point of increased electrical activity thus indicating the site where the injection must be given. Another disorder where BOTOX is used is cervical dystonia (involuntary movement of the neck). Along with BOTOX, Dysport is another marketed botulinum toxin that can be used to treat cervical dystonia. Both these preparations are made from botulinum toxin A. However, some people may develop antibodies against toxin A and then they may be given the botulinum toxin B preparation marketed as MyoBloc (in US) and NeuroBloc (in Europe).

It was only in the year 2002 that BOTOX was approved by FDA for cosmetic treatments with which it is today synonymous. In addition, botulinum toxin A is also used as a treatment tool for axillary hyperhidrosis (excessive sweating) and detrusor overactivity. The botulinum toxins are also under experimentation for hypersalivation, wound healing, chronic musculoskeletal pain , just to name a few. Efforts are also underway to engineer novel recombinant botulinum toxins with improved efficiency.


References:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2856357/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3496996/#!po=6.25000

Colors Galore: Microbial pigments and virulence

Staphylococcus aureus

 Chromobacterium violaceum

Don't the above pictures look pretty? Specially the hue of colors. These are various bacterial cultures which form colored colonies. Though the colors look pleasing to the eye, the actual function of the pigments responsible for these colors is not so pleasant- Many of these pigments are responsible for the virulence possessed by the bacteria.

Take the case of Staphylococcus aureus- the left most picture. The pigment carotenoid moves through a pathway producing the golden colored staphyloxanthin. Staphyloxanthin comprises of alternate single and double carbon bonds in its backbone which help absorb the reactive oxygen species (ROS). In case there is a mutation and the ability to produce staphyloxanthin is lost, the bacteria forms colorless colonies. These colorless staph are more susceptible to killing  by superoxides , oxygen radicals, hydrogen peroxide. It is hence not difficult to assume that the golden color is the "armor" against all these weapons.

Another pigment, Melanin, is produced by the fungus Cryptococcus neoformans. The strains producing the dark brown-black melanin were found to be more adept at resisting phagocytic invasions from the host cells as compared to the lightly or non-pigmented forms. In addition, they were also found to be more resistant to antimicrobial drugs. The melanin has also been said to have the ability to stabilize ROS.

Pseudomonas aeruginosa produces the greenish colored pyocyanin. In contrast to others, it actually promotes formation of reactive oxygen species which cause damage to the host cells. It is a zwitter ion and can easily penetrate the cell membrane. It acts as an electron acceptor thus interfering with the respiratory chain and increasing oxidative stress in the cell.

Chromobacterium violaceum gets its name because it forms purple colored colonies- courtesy the pigment violacein. Experiments with violacein have demonstrated that it brings about apoptosis in leukocyte cell lines. It is believed that this property could play a role in immune evasion during severe human infections. In addition, violacein has shown antimicrobial activities against some bacteria and protozoan and may help the bacterium survive in the environment.

Thus, apart from imparting virulence, protection may be another important function of microbial pigments. These pigments may protect the organism from UV light, oxidative species, other micro-organisms and extremes of heat and cold among others.


References:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2743764/

Why does the Bobtail Squid glow?

Yes, it does glow. And so does a jellyfish, many other marine organisms, some mushrooms, bacteria and of course, the fire flies. We have all seen the fire flies glow in the dark of the night. But why do these organisms give out light and how do they manage it?
           
This "glowing" phenomenon is termed as Bioluminescence. In simple terms, Bioluminescence is the light produced by living organisms. They may have their own reasons for producing it and also different means to get it accomplished.

WHAT MAKES THEM GLOW?
           
Lets take the story of the bobtail squid first. The new born squids are born with a light organ. When small, they have to scour the sea waters to find one special bacterium species, the Vibrio fischeri, and offer them the use of their light organ as a permanent residence. Only when the light organ is colonized by a monoculture of these bacteria does it mature and develop to become fully functional. The maturation can be seen in the various physical changes of the organ such as the swelling of its epithelial cells. These Vibrio fischeri residing in the light producing organ of the squid are responsible for emitting a blue-green glow.
  However if the squid does not harbor a wild type of the bacterium it would show reduced luminescence or no physical changes to its light organ. Thus it can be said that the actual role played by the bacterium is in the development of the light organ in exchange for their home and food from the squid. The luminescence is a product of the lux regulon which in the presence of an inducer(oxygen) emits light from the luciferin by the action of luciferase enzyme. 

The jelly fish on the other hand does not need the help of a bacterium to glow. It follows a chemical mechanism catalysed by the calcium ion. The photoprotein aquoerin gives out a blue light upon reaction with calcium ions either in the presence or absence of oxygen. But this is not the end of it. This blue light emitted is then absorbed by a second protein which now emits a green light. This second protein the green fluorescent protein has been responsible to bring about a change in the biotechnology field in the way assays are designed and genetically modified organisms detected.

             Even the miniscule dinoflagellates shine bright. They have the distinction of being the only photosynthetic organisms capable of bioluminescence. The bioluminescence apparatus is present in scintillons and comprises of the substrate luciferin, the enzyme luciferase and a protein that binds to luciferin. Tetrapyrrole, a pigment related to chlorophyll also plays some role. 

         

WHY DO THEY GLOW?

 We now know some ways in which bioluminescence may be produced but it still is not clear why does the bobtail squid, or for that matter why does any of these organisms glow? How does Bioluminescence help them?

           For starters, it might help in food hunting. In the deep, dark depths of the ocean, bioluminescence can play the role of a flashlight helping the owner locate its food. Alternatively, it might be a trick on the part of the light producer to fool the prey into thinking that there is food for it when actually the prey is going to be the one who’s eaten. A great example is the viperfish. A modified fin ray extends forth from its mouth and has a luminescent glow at its tip. The prey thinks it is food and when it pounces, it is instead impaled on the fangs of the viperfish.               Bioluminescence may play a role in protection of the organism. In this case, bioluminescence may be used as an offensive tactic or a defensive tactic. When on the offensive it may be used to stun the predator or call for help from someone larger than the predator. The deep sea shrimp saves itself by blinding its attacker by shining brightly in its face and then zooming off. Camouflaging is one brilliant defensive strategy. Take the case of the hatchetfish. It has photophores(light emitting organs) on its belly which give out a blue light. This light matches the sunlight being filtered from above thus hiding the fish in a blur.

            Lastly, Bioluminescence may be employed as a mating tool by the deep sea organisms. 

References:

http://jb.asm.org/content/182/16/4578.full

http://www.amjbot.org/content/91/10/1523.full

http://www.mbari.org/earth/mar_tech/eits/biolumen.pdf

More reading:

http://www.smithsonianmag.com/science-nature/Bioluminescence-Light-is-Much-Better-Down-Where-its-Wetter-192132481.html?c=y&page=1

http://www.popularmechanics.com/science/environment/6-bright-ideas-for-bioluminescence-tech#slide-5

What are Isozymes?

Definition: Isozymes may be defined as multiple forms of the same enzymes having similar, if not identical enzymatic properties due to amino acid substitutions in their structure or slight differences in the tertiary or quarternary structures.

How are isozymes generated:

•        Multiple alleles at the same locus.

The enzymes produced due to such alleles are  known as Allozymes. The number of alleles present at a particular locus is dependent upon ploidy number of the organism and its genetic makeup (homozygous or heterozygous). The number of allozymes present depend upon the number of alleles present. Each allozyme codes for a different polypeptide chain. If the enzyme is monomeric and the individual is homozygous, simple band patterns of the allozymes will be seen. However the complexity of these patterns will increase if the individual is heterozygous and the enzyme under consideration is a multimeric protein.

• Single or multiple alleles at multiple loci   

This is another way in which isozymes may be generated. Multiple alleles at different loci generally result in formation of isozymes that may be expressed in different tissues or maybe compartmentalized in a cell. Such compartmentalization or differential expression means they can be tightly regulated and may be directed towards different metabolic functions at different periods of time in different tissues under slightly varying conditions. Malate dehydrogenase isozymes are the best example of this.

                       Also such isozymes may prove to be a useful tool in detecting metabolic anomalies or diseases since under a particular condition only one type of isozyme may be affected while the other is not, leading to a different band pattern being observed as compared to the normal band pattern. Therefore detection of isozymes is an important diagnostic tool. eg: salivary amylase isozymes are detected to confirm whether the person suffers from pancreatic disorders.

            However the band patterns generated by such isozymes are even more complex and hence difficult to interpret.

• Secondary Isozymes

   Post translational changes occurring in an enzyme structure may also lead to isozyme formation; such isozymes being termed as secondary isozymes. These enzymes are first synthesized normally(primary enzymes) which then may undergo changes in vivo or in vitro.

In vivo changes include secondary steps like methylation, acetylation, phosphorylation, sialation, cleavage by proteolytic enzymes, loss of amide groups and addition of carbohydrate side chains to reactive residues.

Isozymes may be generated in vitro because of storage, improper handling of the specimens or degradative reactions.

Conformational isozymes, having same chemical structure but different 3 dimensional conformations, are also included under secondary isozymes. These are believed to be interconvertible by chemical means but till date no definite examples have been found.

Detection of isozymes can be done by methods like zone electrophoresis, gel filtration chromatography, PAGE to name a few. All these techniques are used for separation of the isozymes. Detection involves activity staining or zymogen staining procedures. The stains used are specific for specific enzymes; some maybe enzyme specific while some maybe group specific. However only active enzymes are detected by these techniques. This may be perceived as a limitation of the techniques. Another disadvantage is that Only isozymes having a considerable difference in charge or conformation can be detected. However, this may not always be the case and hence many of the isozymes may remain undetected.

Isozyme studies have found use in detecting and differentiating unknown organisms and plant pathogens . They can be used in detecting the homozygosity or heterozygosity of individuals as well as classifying them in the appropriate taxonomic group.

References:

Isozymes: Methods and Applications; J. A. Micales and M. R. Bonde

Isozymes; D.A.Hopkinson; journal of clinical pathology, vol 8 1974 pg 122-127