transcription - Why does the T7 RNA Polymerase require a reducing environment ie. DTT

A quick search on T7 cysteines gave some clues:

Bacteriophage T7-induced DNA polymerase is composed of a 1: 1
complex of phage-induced gene 5 protein and Escherichia coli
thioredoxin. Preparation of active subunits in the absence of
sulfhydryl reagents indicates the reduced form of thioredoxin is
sufficient for formation of the active holoenzyme. The oxidized
form of thioredoxin, thioredoxin modified at one active site
sulfhydryl by iodoacetate or methyl iodide, or thioredoxin modified
at both active site sulfhydryls by N-ethylmaleimide, are all
inactive, being defective in complex formation with gene 5
protein.

Adler and Modrich, J Biol Chem 258:6956 (1983)

There's a more recent paper (Aguirre et al, Inorganic Chemistry 48:4425 (2009)) that mentions the "the enzyme critical sulfhydryl cysteine groups", but unfortunately I only have access to the abstract.

Update: It seems to be an old finding, rather than a rationale concerning the cytoplasmic redox state. According to Chamberlin and Ring, JBC 248:2235 (1973),

General Requirements-The general requirements for T7 RNA
synthesis directed by T7 DNA polymerase are shown in Table
I. As expected for a template directed polymerase, RNA
synthesis shows an absolute requirement for DNA, the 4
ribonucleoside triphosphates and Mg++.

(no surprises there ;)

The activity of the
enzyme is reduced significantly if a sulfhydryl reducing
agent such as b-mercapto-ethanol is omitted from the
reaction. The addition of 10^-5 M p-hydroxymercuribenzoate to
the assay system in the absence of b-mercaptoethanol
abolished all activity, indicating that the enzyme contains a
sulfhydryl group necessary for activity.

However, if you see the table I, the remaining activity after removing bme is still 74%

There seems to be 7 exposed cysteines (Mukherjee et al, Cell 110:81 (2002)), but I could not find any paper discussing their roles.

biochemistry - What does the human body use oxygen for besides the final electron acceptor in the electron transport chain?

Another small addition

---

There is class of oxidoreductases called oxygenases which incorporate molecular oxygen into the substrates and not just use it as an electron acceptor like in oxidases (note that the terminal enzyme in ETC is an oxidase and there are other such oxidases). In other words, oxygen is not a cofactor but a co-substrate. Oxygenases are further classified into dioxygenases and monooxygenases which incorporate two oxygen atoms and one oxygen atom respectively. Examples:

• Cytochrome P450 family (monooxygenease): involved in detoxification of xenobiotics


• Cyclooxygenase (dioxygenase): involved in production of prostaglandins which are involved in pain and inflammation. Many NSAID painkillers like aspirin, paracetamol and ibuprofen target cyclooxygenase-2 (COX2)


• Lipoxygenase (dioxygenase): Involved in production of leukotrienes which are involved in inflammation.


• Monoamine oxidase (monooxygenase): Involved in catabolism of neurotransmitters such as epinephrine, norepinephrine and dopamine.

Does oxygen deprivation result in death just due to the halting of ATP
production, or is there some other reason as well?

Death predominantly occurs because of halt in ATP production. Some cells such as neurons (and also perhaps cardiac muscles) are highly sensitive to loss of oxygen (for energy requirements) and clinical death because of hypoxia usually occurs because of loss basic brain function.

What percentage of the oxygen we take in through respiration is
expelled later through the breath as carbon dioxide?

As already mentioned, it is said that there is a rough 1:1 ratio of CO₂ production and O₂ consumption. However, as indicated in a comment by CurtF, O₂ does not form CO₂; it forms water in the last reaction of ETC. CO₂ is produced in other reactions of Krebs cycle.

Glycolysis produces 32 molecules of ATP for 1 molecule of glucose via ETC (see here). There are three complexes in ETC and the third is dependent on oxygen; so you can assume that 1/2 a molecule of O₂ consumed for production of 3 ATP molecules. Therefore 32 molecules of ATP would consume 4 molecules of O₂. Seems like there is a 1:1 ratio of CO₂ production and O₂ consumption.

We can see it like this:

FADH₂ enters ETC at the second complex whereas NADH enters at the first. We can say that as long as NADH is present FADH₂ would not require an extra oxygen.

An NADH or a FADH₂ molecule would require 1/2 molecule of O₂. There are 8 molecules of NADH and 2 molecules of FADH₂ produced during glycolysis+krebs cycle which would require 10/2 = 5 molecules of O₂. Glycolysis produces 4 molecules of CO₂ during krebs cycle.

However, 2 cytosolic NADH molecules require 2 ATPs (in other words another NADH molecule) to be transported to mitochondria. So the net effect may be actually close to 1:1 O₂:CO₂.

Another factor to keep in consideration is that the three complexes do not actually produce ATP; they just pump proton to create a chemical potential. The F₀F₁-ATP synthase would probably work only after a threshold of H⁺ potential is established. The 1 ATP molecule per complex is most likely to be the mean value and not exactly what really happens per reaction.

physiology - Below which temperature human muscles don't work?

If you dissect striated muscle out of most-any organism, the actual contractile apparatus works over a wide range of temperatures. So that's at the single-muscle-fiber scale. The muscle itself continues to work at all (thawed) temperatures below body temperature -- the problem comes with its regulation.

The shivering response -- a centrally controlled involuntary contractile pattern -- overwhelms voluntary muscle control. So the mechanism behind loss of muscle coordination in hypothermia is the same as the shivering mechanism. When the core temperature drops, the midbrain starts over-riding voluntary control of the muscles. When the core temperature drops far enough; around 32C, the shivering often slows down or stops. Voluntary movement becomes compromised probably because the brain simply isn't working; neuron firing rates are so slow that sensation, processing, and motor responses are all critically impaired.

The feeling of numbness does not actually directly accompany a loss of muscle contractility. You can walk pretty much indefinitely on frozen feet if you can keep your balance (and you keep your core temperature up.) Lots of people survive severe frostbite of their feet (their feet do not often survive, however.) The reason why it seems your hands don't work when they get cold is that you can't feel what you're doing (note; your hands can be much colder than your core body temperature.) But the muscles themselves work right up until they freeze solid.

UPDATE:
Here's a paper that directly addresses the scenario posed by OP -- the decrease in grip strength with temperature. Figure 1 of that paper illustrates their experimental setup; they measure the contractile strength of the index finger while manipulating the temperature of the rest of the hand. They show that contractile function is impaired with temperature and look at temperatures as low as 12C.

They measure as much as a 50% impairment on twitch tension upon cooling to 12C. It's interesting that they review results suggesting that some of this effect is intrinsic to the muscle fiber (not neurological), showing that I should refine what is meant by "continuing to work" in my opening paragraph. (I meant having an ability to generate contractile force when equilibrated in solution containing sufficient ATP and Ca$^{2+}$, not the ability to contract optimally.) For fun, I linearly extrapolated the final arm of Figure 5 and found that the 'voluntary tension' approached 25% at 5C. This suggests that total failure of the voluntary contraction happens somewhere below the freezing point of water (muscle would freeze at a temperature lower than 0C because of colligative effects.)

meiosis - How do you see the stage of the second meiotic arrest in oogenesis in the given video?

I seem to understand the thing now.

The video is utterly simplified for animal cell meiosis I and II.
In oogenesis, you get after every anaphase one cell with very little cytoplasm, polar body, and another cell with much cytoplasm. In the video, the amount of cytoplasm is equal so the thing is idealized.

The video is better to explain male gametogenesis: spermatogenesis, since the amount of cytoplasm does not differ after each anaphase.

The video is too going too fast to teach you oogenesis, since in reality the things are arrested rather long time. The second meiotic arrest in oogenesis can lasts 12 - 50 years starting after puberty. The rupturing secondary oocytes lasts then again less in the second meiotic arrest of oogenesis, since they start to develop into ovum after the monthly release.

human biology - What is the purpose of the adrenal medulla?

The adrenal medulla is less of a 'real' endocrine organ like the others in the endocrine system and much more an extension of the sympathetic nervous system. In fact, its chromaffin cells are modified neurons by descent and secrete adrenalin and some noradrenalin upon stimulation by sympathetic preganglionic fibres, effectively turning the medulla into a sort of 'endocrine ganglion' with the whole cardiovascular system as its 'postganglionic fibres'.

Adrenalin in the circulation seems to have pretty much the same effect as all the adrenergic neurons of the sympathetic nervous system (which innervates all blood vessels, organs etc.): arteriole constriction, cardiac output increase, breath rate increase, pupil dilation, glucagon secretion and insulin inhibition, stimulation of glycolysis and glycogenolysis etc.

So what is the purpose of the adrenal medulla, if all of the effects which the endocrine adrenalin produces are essentially the same as those produced by the sympathetic mass response?

physiology - Are human fetuses more likely to be male?

The Fisher's principle is not applicable to the fetuses because it has been formulated for parental expenditure and basically states that the ratio of male to female parents (implying that both parties have reached the age of fertility) will tend to 1:1.

There are several mechanisms that we can use and that are mentioned in the canonical paper by James (2007):

(a) there are equal numbers of X and Y chromosomes in mammalian sperms
(b) X and Y stand equal chance of achieving conception
(c)
therefore equal number of male and female zygotes are formed, and that
(d) therefore any variation of sex ratio at birth is due to sex
selection between conception and birth.

James brings much evidence that none of these conventional beliefs is true. Rather, they are dependent upon many factors: exposure to stress during pregnancy, glucose level etc. He reports that there is an excess of males at birth in almost all human populations, and the natural sex ratio at birth is usually between 1.02 and 1.08. However, the ratio may deviate significantly from this range for natural reasons. (I really recommend reading the paper I linked, it is available for free).

Branum et al (2009) analyze birth statistics in the US taking into account many factors like ethnicity, gestational age and plurality and show that the ratio can increase even more with growing gestational age and has different values among different races.

So, taking everything together, I can say thay YES, the chances for human fetuses to be male are indeed higher.

biochemistry - Negative feedback in the fructose metabolism in liver

The main reaction of fructose in liver is phosphorylation, catalyzed by ketohexokinase (UniProt P50053). In one paper with rats it was shown that fructose load can deplete the liver of nucleotide triphosphates (ATP, GTP) although ADP and GDP are inhibitors of the enzyme. This shows there is no effective regulation.

M. I. Phillips, D. R. Davies: The mechanism of guanosine triphosphate depletion in the liver after a fructose load. The role of fructokinase. In: The Biochemical journal. Vol 228, Nr 3, Jun 1985, p. 667–671, {{ISSN|0264-6021}}. PMID 2992452. PMC 1145036.