Saturday, 28 February 2009

pharmacology - Why does Penicillin only affect bacterial cell walls

Bacteria have a mesh-like structure surrounding their plasma membrane called a cell wall. The cell wall is made up of peptidoglycan polymers that form a rigid crystalline structure that helps protect the osmotic pressure of the bacterial cytoplasm.



Penicillin and other β-lactams work by inhibiting the final step of peptidoglycan synthesis, which prevents transpeptidation (crosslinking) of the peptidoglycan molecules. This leads to the death of the bacterium by osmotic pressure due to the loss of the cell wall.



This drug doesn't affect human cells because they lack a cell wall surrounding their plasma membrane.

Wednesday, 25 February 2009

protein binding - Phagemid display

Experimental protocol



Standard cloning procedures, determination of colony-forming units and
plaque-forming units, and immunoblot after PAGE were carried out
according to Sambrook et al.



Construction of the packaging cell line.



DH5alpha/pIII
[M13KO7DeltapIII]. The M13 pIII gene fused to promoter P/A1/04/03
(ref. 17) was inserted into the multiple cloning site of the
lambdaattP plasmid pLDR9 (ref. 18). The origin of replication and the
kanamycin resistance gene were cut out and the remaining
lambdaattP-pIII nonreplicative DNA was religated. Escherichia coli
DH5alpha containing plasmid pLDR8 (ref. 18) were transformed with the
lambdaattP-pIII nonreplicative DNA and plated on agar plates
containing 25 mug/ml ampicillin. After incubation overnight at 42°C,
the colonies obtained were replica-plated and ampicillin-resistant
clones were identified, named E. coli DH5alpha/pIII. They were
subsequently transformed with M13KO7DeltapIII, resulting in the
packaging cell line E. coli DH5alpha/pIII [M13KO7DeltapIII].



Production of phage.



To obtain M13KO7DeltapIII helper phage,
M13KO7DeltapIII DNA was electrotransfected into E. coli K802
containing pNDI (ref. 15). To obtain hyperphage, M13KO7DeltapIII DNA
was electrotransfected into E. coli DH5alpha/pIII. Phage were produced
by cultivation in the presence of 0.5 mM
isopropyl-beta-D-thiogalactoside (IPTG) at 30°C for 24 h with shaking
at 200 r.p.m. Escherichia coli Top10F' or E. coli Xl1blue carrying the
pSEXphOx(Yol) phagemid or a scFv library in pSEX81, respectively, were
infected with a helper phage preparation at a OD600 of 0.1 at a
multiplicity of infection of 20 as described.



Phage quantification by ELISA.



Dilution series of phage were coated in
100 mM NaHCO3, pH 8.6 in MaxiSorb ELISA plates (Life Technologies,
Karsruhe, Germany) for 16 h at 4°C. Blocking was done with 2% skim
milk powder in PBS (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1 mM KH2PO4,
pH 7.3) for 2 h at room temperature. Phage were detected with the
monoclonal mouse antibody B62-FE2 (Progen, Heidelberg, Germany)
specifically binding an epitope on the pVIII major coat protein of the
M13 phage. M13KO7 phage of known colony-forming units were used for
standardization.



Immunoblot.



Approximately 1010 phage were applied per lane on a 10%
polyacrylamide gel. Blocking was done with 2% skim milk powder in PBS
for 2 h at room temperature. Immunostaining was done with the mouse
mAb anti-g3p (MoBiTec, Göttingen, Germany) recognizing the pIII coat
protein of M13KO7, visualized with 3,3', 5,5'-tetramethylbenzidene
(TMB) substrate (Promega, Madison, WI).



Antigen-binding phage ELISA.



Dilution series of BSA-phOx antigen in
100 mM NaHCO3, pH 8.6, were coated in MaxiSorb ELISA plates (Life
Technologies) for 16 h at 4°C. After blocking of unspecific binding
with 2% skim milk powder in PBS for 2 h at room temperature, 2 times
109 single-chain phage diluted in 2% skim milk powder in PBS were
applied to each well for 1 h at room temperature. After washing six
times with 400 mul PBS per well, the bound phage were detected with
mAb B62-FE2 as described above.



Panning.



A human scFv fragment library in pSEX81 with a calculated
maximal complexity of 2 times 107 was generated from peripheral
lymphocyte preparations as described21. Tetanus toxin obtained from
Virotech (Rüsselsheim, Germany) was coated to six ELISA wells (Life
Technologies) at a concentration of 0.1 mug/ml in 100 mM NaHCO3, pH
8.6, for 16 h at 4°C. The wells were blocked with 2% skim milk in PBS for 2 h at room temperature, after which 1011 phage of the library
were applied to each well and incubated at 4°C overnight. After five
washes with PBS/0.1% Tween and five with PBS, bound phage were eluted
with 1 mug/ml trypsin (Life Technologies) in PBS for 15 min at room
temperature. Eluted phage were used for the infection of 20 ml of E.
coli XL1blue at a OD600 of 0.4. Infected bacteria were grown on
Luria–Bertani agar plates containing glucose and ampicillin, scratched
from the plates, and used for the production of antibody phage for the
next round of panning as previously described.

Tuesday, 24 February 2009

eyes - Why do glial cells of the retina become more abundant the closer they are to the optic nerve?

Glia serve the classic support role in the retina, similar to that served by neuroglia elsewhere in the CNS. That role mostly consists of taking used neurotransmitters and recycling them such as in the glutamate-glutamine cycle. As Preece suggested, glial cells are a heterogenous group, and with a general marker like GFAP you could very well be observing two different types of glial cell, one with a uniform presentation and one specifically in the central retina.



That being said, the most familiar type of glial cell in the retina is the Muller glial cell. It is particularly interesting in zebrafish, where it has been shown to act as a neural progenitor cell upon tissue damage in adult animals As you can see from that article, it fits your requirement of being GFAP expressing, although I couldn't find any good flat mount pictures showing whether it was preferentially expressed in the central retina.



So that gives us two potential roles for glial cells in the zebrafish: as potential multipotent retinal stem cells and as housekeeping neurons present to clean things up. You could come up with reasons either of those might be important in the central retina, but for my money the arguments are all pretty hypothetical: "the central retina is more active and therefore has a higher requirement for removing neurotransmitter" or "the central retina needs to be protected from potential damage more than the peripheral".



One thing I think we have a good basis to rule out is that it's the optic nerve. From a retinal point of view, you refer to the point where the optic nerve passes through the rest of the retina as the optic nerve head or optic disc. Axons from ganglion cells all across the retina stretch out from their soma toward this single point. By the time they reach the optic nerve, the vast majority of axons are far from their axon hillock. That means that they're being propagated from one node of Ranvier to the next. Because that process requires voltage gated ion and not neurotransmitter gated channels, there is no neurotransmitter release associated with that process and consequently no additional reason for glial cells to be present to clean up.



Reference:

Sunday, 15 February 2009

evolution - Are there other mechanisms for mutation besides imperfect DNA replication?

Absolutely. Ionizing (X-rays, neutrons, electrons, heavy ions) and non-ionizing radiation (UV), chemicals, etc. are able to induce DNA Damage, which is then imperfectly repaired. So it's not an issue of imperfect replication, but also of imperfect damage repair.

Thursday, 12 February 2009

evolution - What is the most difficult feature to explain evolutionarily?

To answer your question directly, there are a number of examples which creationists like to bring up, such as woodpecker's tongue which is wrapped around its brain or the archer fish which shoots down insects with water and has to adjust it's aim for refractive difference between water and air. These features supposedly 'couldn't have evolved' but of course one's disbelief doesn't make anything true or untrue. This is what Dawkins calls 'argument from personal incredulity'.



Let me also point out a few evolutionary counter arguments to such reasoning.



The presence of selection radically changes what is 'likely' or 'possible' to happen. For example, you wouldn't argue that it's highly improbable that the stones on the beach are ordered by size, if you know that the action of waves orders them in a certain way. Dawkins' weasel (http://en.wikipedia.org/wiki/Weasel_program) also demonstrates this principle.



Another Dawkins' argument is that, contrary to what creationists claim, complex features such as eye don't have to be completely functional to be useful. In the early history of life, when no organism had eyes, any simple system which could distinguish light from darkness could provide a huge advantage to its owner. The simple system can then evolve to a more complex one to provide a competitive edge etc.



Finally, evolution happens over time scales we cannot grasp. We don't have a good feeling of what billions of years of small changes which are selected for can add up to.

Monday, 9 February 2009

structural biology - How do I change bond colors to something besides a different hue in Coot?

You could try other visual differences, like make what you're interested in ball-and-stick, and everything else wireframe, like in this example:



like on this example.



.... Because, from: http://www.ysbl.york.ac.uk/~lohkamp/coot/doc/coot-python.html




procedure: hilight_binding_site (imol, centre_residue_spec,
hilight_colour, radius): hilight-colour is specified in degrees (round
the colour wheel - starting at yellow (e.g. 230 is purple))




It looks from this like you only get to choose Hue (color_map_rotation) and not Saturation or Lightness, even with the script interface.



However, text can be full RGB




3.28.3 (set-font-colour red green blue)



where:



red is an inexact number green is an inexact number blue is an inexact
number


Saturday, 7 February 2009

human biology - Is it true that newborn babies don't produce tears while crying? Why?

It is not completely true. Babies develop the lacrimal system at 42 days, in utero. The canals and ducts for tears are formed at 60 days [2].




At birth, tear production by the lacrimal gland is minimal. Normal tearing develops several days to 2 weeks after birth.[1]




Some 6% of newborns are born with a tear duct obstruction (in some online articles it stated up to 10%, but I think my source is more reliable). The child still produces tears, but they can well up on the eyelid [2].



Other more serious abnormalities can cause this problem. Duct cysts or hemangiomas can also cause tear duct blockages in more severe cases. Various factors, such as anatomical abnormalities and problems with pumping tears increase the propensity for the disorder in children with Trisomy-21 [2]. As nico has alluded to in the comments, there may be a lag in the development of the physiology of tear production even in non-Trisomy-21 children.




[1] Thomas K. McInerny, MD, FAAP, ed. 2009. American Academy of Pediatrics Textbook of Pediatric Care. Elk Grove Village, IL. American Academy of Pediatrics, p1706.



[2]M. E. Wilson et al. (eds.), Pediatric Ophthalmology, 2009, pp. 276-284. DOI 10.1007/978-3-540-68632-3_1.


Thursday, 5 February 2009

evolution - What are the major evolutionary pressures for Bioluminescence?

In many cases, and in particular in marine invertebrates, the bioluminescence is in fact produced by symbiotic bacteria of the Vibrionaceae family. In most cases the bacteria can sense when they are being hosted by the animal through quorum sensing mechanisms, and start producing light.



The evolutionary pressure for the bacteria to produce light is to be in the protected environment of the animal which often feeds them. On the animal side, there can be several reasons to host the luminescent bacteria. In many cases, the evolutionary pressure is linked to reproduction. In order to successfully reproduce in the immensity of oceans, marine animals need to know that they are among their kind before they release their gametes.



Another example is the one of some deep see fish like the anglerfish which have developed a special "flash light" organ to host bio-luminescent bacteria. Fish can use the bacteria to emit a light to aid in camouflage, hunting, and attracting mates. See http://microbewiki.kenyon.edu/index.php/Deep_sea_fish

Sunday, 1 February 2009

neuroscience - Why have humans evolved much more quickly than other animals?

This question appears to address at least two distinct concepts:



  1. the "speed" of evolution

  2. whether there is some "end goal" that evolution seeks

I will provide an explanation of each separately below:



The Speed of Evolution



The speed at which a species evolves—that is, the speed at which it acquires new heritable characteristics—can be affected by numerous factors. Among the most obvious which come to mind are:



  • existing population size

  • reproductive cycle rate

  • number of offspring

  • offspring survival rate

  • environmental demands

That said, have humans evolved faster than other species?



On the whole, I do not feel that I could say without a doubt that humans have evolved significantly faster than other organisms. Considering the above factors (we have a slow reproductive cycle, few offspring, etc.), it seems unlikely but this is where a zoologist would know better. The answer provided by bobthejoe may provide insight as well.



I would just raise a word of caution before leaping to the conclusion that we have evolved rapidly without looking into it, because while we often study human evolution quite extensively in biology classes and thus are more aware of changes like upright walking and opposable thumbs and increased brain size, that doesn't mean other animals didn't evolve that much as well (we just don't learn about them in as much detail). Being a psychologist and not a zoologist, I do not have the knowledge to say with any surety that other species did not evolve in their own ways to the same magnitude as we have in the last 3-4 million years, so perhaps someone else can help you there.



But you ask whether we have a particular part of our brain that no other animal has; the answer to this is no, but the parts of our brains which we do share with animals are often much larger than as seen in those other species.



The "Goal" of evolution



I would like to point out here that human technology has nothing to do with evolution. Not only have all of our notable technological achievements occurred in the last couple hundreds years when human evolution was at its slowest (early human evolution as we know it today dates back at least 5-7 million years, and it was during this several million year period when all the genetic evolution you speak of happened), I would argue that human spaceships and computers are no more sophisticated to us than a mushroom garden and ventilation system are to a termite. I could be wrong here but it seems as if you are under the impression that evolution is striving towards something... That humans are somehow intrinsically better than lions or sharks or beetles. On the contrary, each of these creatures is not significantly more or less adapted to their environments as we are to our own, and in many ways these creatures possess features which are beyond our own.



I shall leave you with one of my favorite quotes:




"We need a wiser and perhaps a more mystical concept of animals. We
patronize them for their incompleteness, for their tragic fate having
taken form so far below ourselves. And therein we err, and greatly
err. For the animal shall not be measured by man. In a world older and
more complete than ours, they move finished and complete, gifted with
extensions of the senses we have lost or never attained, living by
voices we shall never hear. They are not brethren, they are not
underlings. They are other nations caught with ourselves in the net of
life and time, fellow prisoners of the splendor and travail of the
earth."




–Henry Beston, in The Outermost House



I don't have a dog in this fight, and I could very well be falsely assuming that you think this particular way when you in fact don't (you only wrote 4 lines after all, it's hard to glean much from that), but I wanted to make sure that these concepts were clear either way, particularly for any future visitors who might get the wrong impression. :)