Posts by Steve Lolait

There is loads of info about agarose gel electrpohoresis found by a simple search on-line, including the Wikipedia entry. I will say that the resolution of DNA bands are affected by a number of factors other than agarose concentration (and there are different types of agarose!), such as the amount of DNA loaded and the comb width.

As a general point Yujin, when we watch a person perform a lab technique with which we are unfamiliar we should ask questions at the time (or subsequently) to make sure we understand what is going on (e.g., “what is in that solution?”). Your question can probably be answered by a quick search on-line, even the Wikipedia entry on 'DNA extraction' will help you. Briefly, genomic DNA is soluble in most DNA genotyping-extraction buffers and you need to precipitate it out of solution, which subsequently requires centrifugation to isolate the DNA (and perhaps other material that can be reduced/removed if required).

The classical Hershey-Chase experiment is something a little different, involving incorporation of radiolabelled ‘DNA’ by bacteriophage into bacteria (which were pelleted by centrifugation - note that bacteria will settle to the bottom of a test tube slowly even without centrifugation)

see - Hershey AD and Chase M (1952). Independent functions of viral protein and nucleic acid in growth of bacteriophage. J Gen Physiol 36: 39-56 (this can be downloaded - not many scientific articles from this era can be!).

Also there will be differences between and within populations - there are hundreds of human olfactory receptors (exhibiting a range of polymorphisms) and hundreds of volatile chemicals found in food. Ajna, in your type of experiment do you obtain the same result(s) when you take into account shape and texture - e.g., if the products are emulsified?

EBV is able to induce a T-cell independent immunoglobulin response by B cells, i.e., antibodies can be made to some EBV-encoded proteins without T-cell involvement. Another example of a T-cell independent antigen would be bacterial lipopolysaccharide (LPS), and if you search on google for 'T-cell independent antibody responses to viruses' you will come up with other examples.

That does not mean that EBV can't infect T-cells, or that there no T-cell responses to EBV infection.

I can only offer generalities here! You can certainly make antibodies (which are proteins) that can bind (and may block function) to all three of these surface receptors (see - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3539746/), but you would have to do some reading to establish whether one antibody or vaccine could be/has been produced against all three - e.g., is there enough similarity between the antigenic epitopes of the three receptors?

There may also be antibacterials, not necessarily proteins, already in existence that recognise IsdA/B/H to interfere with heme binding and/or transport-function, although you would not classify many of these drugs (such as porphyrins - see http://www.ncbi.nlm.nih.gov/pubmed/11178343) as true receptor ‘antagonists’ (e.g., they may bind to Isd’s to ‘piggy-back’ into the cell, mimicking the binding properties of the natural ligand, and there exert antimicrobial activity).

As to receptor ‘antagonists’ in general there are protein antagonists that can block the activity of any number of receptors particularly in a closely-related receptor family, if there is sufficient structural similarity in the antagonist binding domains. A pan-specific receptor antagonist may often have differing affinities between receptors.

Designing such an antagonist has traditionally been based on identifying a candidate from a screen of ‘lead’ compounds that might bind to a number of receptors. It is then usually chemically refined to increase its affinity and specificity to one receptor (often to reduce off-target effects). As the 3D structures of more and more receptors are being elucidated a lot of effort is being invested in computer-aided molecular modelling (‘locking and docking’) of receptor ligands. This is mostly the remit of highly specialised labs.

see also - https://en.wikipedia.org/wiki/Antimicrobial_peptides

In addition, this article - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2732559/ - gives references that highlight substratum effects on microbe growth in a biofilm (e.g., sometimes better attachment on nonpolar surfaces), and discusses some variables including type and hydrodynamics of the bug-growing medium and type of microbe (encompassing cell surface charge).

Here is also a quite good, brief summary that backs up what Andy is saying - http://www.ncbi.nlm.nih.gov/books/NBK11117/

Note that this talks about postsynaptic effects. GABA is the major inhibitory major neurotransmitter in the brain, which it can do by activating GABAA channels (composed of a variety of subunits) on, I think, mostly post-synaptic membranes, but also at pre-synaptic and ‘extra-synaptic’ sites. There is another type of GABA receptor, the ‘metabotropic’ G protein coupled GABAB, which can also mediate pre- and post-synaptic inhibition.

Excitatory and inhibitory neurons can synapse on the same neuron - this is not uncommon. And there is a lot of input, e.g., from neuropeptides and other substances like a major inhibitory transmitter such as endocannabinoids (e.g., activating the cannabinoid CB1 receptor in the brain, one of the most abundant central G protein-coupled receptors), that modulate either excitatory or inhibitory synapses.

Quite often you have to look at synapses in the brain as part of a network, e.g., inhibitory input onto an excitatory neurone (e.g., GABA onto a glutamate neurone), excitatory input onto an inhibitory neurone, inhibitory input onto an inhibitory neurone, etc.

Dopamine receptors are members of the G protein-coupled receptor (GPCR) superfamily. GPCRs have a dynamic structure that can assume a spectrum of conformational states. Upon ligand binding there are changes in the GPCR that lead to structural re-arrangements that facilitate interaction with intracellular effectors (as David points out) such as G proteins, kinases and arrestins that leads to the activation of a plethora of signalling molecules and cascades. These structural changes have been determined by biochemical, biophysical and mutagenesis studies and validated more recently by X-ray crystallography of a number of GPCRs.

Dopamine receptors comprise 5 structurally-related members and fall into 2 classes; the D1-like (D1 and D5) and D2-like (D2, D3 and D4). The genes for the former are intronless while the D2-like receptor genes have introns (interrupting their protein-coding sequences) so they can have alternatively-spliced forms that can confer different pharmacological properties. Subleties in the orientation of the 7 transmembrane helices of each dopamine receptor as well as differences in intracellular and extracellular domains confer the pharmacological and biochemical properties that make each dopamine receptor unique. Broadly speaking D1-like receptors tend to bind Gs protein to activate adenyl cyclase (raising cyclic AMP) whereas D2-like receptors bind to Gi/o proteins to inhibit adenyl cyclase, but the D2's can also bind to other G proteins such as Gq (which often leads to intracellular calcium mobilization).

Where dopamine receptors 'live' is obviously integral to their function. They may reside on different tissues or brain regions, on separate cells, or be located pre- or post-synaptically. Some dopamine receptors may be co-expressed in the same neuron and may interact at the physical level (e.g., by receptor dimerization) or cross-talk via converging intracellular pathways. e.g., D1 and D2 receptors have opposing effects on intracellular sodium in striatal neurons in the basal ganglia (http://www.ncbi.nlm.nih.gov/pubmed/10700253). So the final functional output of a cell may be the result of the coordinate activation of dopamine receptors (expressed on the same or different cells) and in collaboration with many other receptors and intracellular signalling pathways (bare in mind that the average neuron expresses hundreds of receptors!).

There are many good references on dopamine receptors (e.g., see - http://pharmrev.aspetjournals.org/conte … 182.full). There is also a good general database on all GPCRs (http://www.guidetopharmacology.org/) that summarizes details on the 5 dopamine receptors.

FYI Ece, although not the same as molecularly engineering a construct to insert bacterial DNA into our genomes, there is evidence for 'natural' lateral gene transfer of viral DNA into the human genome - see
http://journals.plos.org/ploscompbiol/a … bi.1003107 and
http://journals.plos.org/plosgenetics/a … en.1003877

You may also want to look at this very recent commentary on the non-medical uses of brain stimulation in Nature - http://www.nature.com/news/brain-power-1.19475

Interesting question Kaushik, and very ‘specific’ that can probably be answered by your lecturer/tutor (assuming that you are a University student) or by an on-line search! As you likely know the MAPK pathway has been reasonably well-conserved across evolution. According to a recent computer simulation study (see - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC41288) the 3-tiered module is both robust and adaptable (for “random mutations to generate phenotypic variation during evolution”). In terms of the tight control subserved by dual phosphorylation, the same article suggests that a distributive phosphorylation system is good at amplifying input signals (e.g., see also an article on activation mechanism of ERK2 by dual phosphorylation - http://www.cell.com/abstract/S0092-8674 … 980351-7).

To add, another popular use of sequence-specific oligos is PCR (either end-point or 'quantitative' as in qPCR). We often use various oligonucleotide-design programs and DNA databases to check oligo specificity for a single gene product; i.e., using these programs you can usually determine whether your oligo has the potential to 'cross-react' with genes other than the one you are interested in. This is aided by the fact that the sequencing of some genomes (e.g., human) has essentially been completed. We use the word 'stringency' to describe the conditions under which the oligo will bind to a specific DNA (or RNA) strand - a sequence-specific oligo can often be used at 'high stringency' (typically low salt concentration and/or high temperature used in your experiment) so that cross-reaction can be minimised, although on occasion you may want to detect a sequence that is common to a family of genes.

Yes, for humans the amount of purine bases equals the amount of pyrimidine bases, so A + G (or C + T) is about 50%. As far as I am aware there is some overall nucleotide bias in a variety of genomes, where e.g., there are high (greater than 50%) and low G/C content bacteria, and the human genome is less than 50% G/C. It is not necessarily a 1:1:1:1 ratio of A:C:G:T (or 1:1 for A:G; or 1:1 for C:T).

[for a basic reference see Table 2.2, p43 “Genetics: Analysis of Genes and Genomes 7th Ed, Hartl & Jones, 2009]

There are some excellent questions here! The first thing to say is that in general fusing cells is a relatively inefficient process and that the detailed mechanism(s) underlying it are unknown. From some of the earlier experiments polyethylene glycol (PEG)-induced cell fusion involves dehydration of membrane surfaces forcing close contact between membranes, and it appears that the plasma membrane AND then cytoplasmic compartments become one (http://jcb.rupress.org/content/96/1/151.long; http://www.tandfonline.com/doi/pdf/10.1080/096876899294508), and I would imagine that this includes the ER.

The ES-somatic cell hybrids that result from fusion (using PEG, for example) appear to adopt the transcriptional profile of ESCs with apparent silencing of many (but not all) somatic genes (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4030652/). I think that in some cell fusion hybrids there may be some evidence for the organelles of one cell adopting the ‘shape’ and polarity of the other, which may involve alterations in the cytoskeleton - I don’t know if this happens in ES-somatic cells. PEG itself induces cytoskeletal reorganisation in many cells (e.g., see - http://ncbi.nlm.nih.gov/pmc/articles/PMC3736723/). I also don’t know how the various organelles ‘find’ each other - perhaps there is a high level of ‘intermixing’ which leads to unviable hybrids, and the ‘specificity’ is based on physical/chemical interactions that underpins viability/growth. This article - http://www.sciencedirect.com/science/ar … 5492900028 - shows the ultrastructural changes during PEG fusion of two cells (including a nice figure (3) showing intermediate nuclear fusion and possible mitochondria-nucleus fusion).<cite></cite>

I would favour (1) and (2) - as far as I am aware there are also 'no native' koalas in the NT or WA.

P.S. The wikipedia entry on Koalas states that there is fossil evidence for koalas in WA, but that 'they were likely driven to extinction in these areas by environmental changes and hunting by indigenous Australians'.

There are a few interesting relevant comments about species differences in NMJs (see 'Intracellular communication in the Nervous System' (2010), Robert C. Malenka; p186 observed on-line googling ‘neuromuscular junction folds in birds’) re. folds increasing surface area and a possible reciprocal relationship between the quanta of Ach released at the NMJ and the intensity of postsynaptic folding (the more Ach released the fewer folds expressing nicotinic Ach receptors required to ‘amplify’ its effects, as in some fish and birds, and I imagine that this would be muscle-dependent).

It's a bit difficult to decipher these figures and know what the author is referring to - my guess (but I may be completely wrong) would be some type(s) of 'coated pits' or endocytotic vesicles, membrane blebs/invaginations that are involved in such things as phagocytosis, pinocytosis (cell 'drinking') and/or receptor-mediated endocytosis/receptor recycling. If they represented these structures then we do know something about what they do! I would contact the author and see if he can provide further details!

If you search 'plasmid DNA' in the upper right-hand side search box you will find a number of posts that address this question.

David, I've often wondered about this sort of statement - 'it matters little what undergrad degree you do in the broad area of biology..' in the context of applying for post-graduate degrees. Don't you think that it might be advantageous to some degree if, e.g., you wanted to apply for a post-grad qualification in something like psychology, that you had studied psychology at some level as an undergraduate? (at the very least it would give the impression of some on-going interest in the field?).

I’m not sure that I quite understand your question! If we take dopamine as an example, this catecholamine binds to 5 subtypes of receptors, D1-5. These 5 receptors are structurally very similar but have different pharmacological profiles, i.e., they are all activated by the endogenous ligand dopamine but they bind different exogenous antagonists and agonists with different affinities. Subtype-specific antagonists may bind to different places in the D receptor(s). The D receptors also have different tissue distributions, although these may overlap in various places, e.g., in a brain region involved in reward called the striatum. As far as I am aware no D receptor is restricted to one tissue (there are few genes that are truly tissue-specific), so in theory a general antagonist to D1-5 would block dopamine function in multiple tissues, as would a specific antagonist against one D receptor subtype. This can give rise to side-effects in one or more tissues that may be detrimental in terms of therapeutic treatment, or may be well-tolerated. Side-effects may also arise, e.g., if the D receptors are functionally complexed to different proteins that have different (non-dopamine) functions, or if the D receptors are an intermediate in a (non-dopamine) physiological pathway. Please post again if you need further clarification.

Just to add, you should be able to find PhD opportunities and/or projects quite easily at most UK universities, e.g. at Bristol you could start at - http://www.bristol.ac.uk/study/postgraduate/. You may have to search individual Faculties/Schools to find what they offer re. postgraduate study and what PhD studentships may be available, or perhaps you might be interested in a particularly field and you can search that as well. Many applicants have not done a year in industry. A first at under-graduate level is obviously desirable to make you more competitive, but if that doesn't happen it is not the end of the world (you might have to take and do well in a Masters degree which can have a research component). Please post again if you have any other questions.

If I am reading this right, I don’t quite agree with you Reetika! If we are talking about the sodium-potassium-ATPase pump for example, it does appear to get phosphorylated via PKA sites - see http://www.sciencedirect.com/science/ar … 9310003054

Also see the Wkipedia entry on this ATPase.

Here is a cautionary note about 'touch DNA' being used in forensic medicine - http://www.nature.com/news/forensic-dna … le-1.18654

G'Day Lauren: Have a look at this very easy to read article - http://www.nature.com/scitable/topicpag … -14397318; and this one (especially the Intro) - http://elifesciences.org/content/3/e02009, which emphasises the possible role of diffusion.