The Serine Proteases

Rajat Das Gupta

Chymotrypsin, trypsin, and elastase are three enzymes that cleave protein chains.

• Each has its own selectivity: that is, they cleave proteins at different structure points
• They have very strong structural similarity
• They use the same mechanism for the cleavage, a mechanism that very much resembles the mechanism we would write for the acid-catalyzed hydrolysis of an amide

First, the structural similarity:

Superposition of Trypsin (yellow), Elastase (green), and Chymotrypsin (blue) Backbones

The three amino acids picked out in red are the three that actually do the catalysis.
Here are sequence alignments for chymotrypsin (5cha) and trysin (5ptp), and elastase (1est) and trypsin (1tld) produced by the Swiss PDB Viewer:

Chymotrypsin vs Trypsin	Elastase vs Trypsin

• The asterisks mark identical residues, and the dots mark very similar side chains, such as Leu and Ile, or Phe and Tyr
• Deep View calculates an RMS difference between chymotrypsin and trypsin of only 0.94 A, and 1.14 A for the elastase-trypsin pair, reflecting the obvious matches in the graphics above

What we see here is a clear example of divergent evolution. All are derived from a common ancestral serine protease, and are described as homologous. Taking a step further back, one finds that some non-mammalian serine proteases have 20-50% sequence identity with mammalian ones, suggesting a common ancestral protease an evolutionary step further back.
Evolution can converge on functionality also. The first crystal structure of a bacterial serine protease, subtilisin, from B. amyloliquefaciens, shows a thoroughly different construction from the mammalian ones, and essentially no sequence homology:

Subtilisin (B. amyloliquefaciens)
The orange balls are Ca++, providing thermal stability

But the enzymes are functionally identical; subtilisin uses the same three catalytic residues, shown in red: Asp32, His64, and Ser221. The mechanism of catalysis is the same, including the positioning of substrate by hydrogen bonding. This appears to be a case of convergent evolution: Mother Nature found a good idea a second time.
As we have seen in several pictures now, the catalytic work of the proteases is done by the so-called catalytic triad, Asp102, His57, and Ser195:



Here is the triad picked out from the crystal structure of chymotrypsin (the extra fine black lines are artifacts of my effort to hide the rest of the enzyme). Remember that X-rays can't see hydrogen atoms; we have to infer their positions.



The mechanism outlined below applies to all of the serine proteases, with small variations. We start with the binding, in cartoon form.



The next step appears to be His57 removing a proton from the the Ser105 OH, while the O does a nucleophilic attack on the peptide carbonyl:

• The shift of negative charge onto the carbonyl oxygen of the peptide is facilitated by hydrogen bonding of the oxygen to the backbone NH groups of Ser195 and Gly193
• These form what is called "the oxyanion hole"
• We now have a structure equivalent to the tetrahedral intermediate in the non-enzymatic mechanism
• The biggest difference is that the two protons that are part of the OH groups are merely hydrogen-bonded in the enzyme

Whether a tetrahedral intermediate is formed was a point of considerable contention in the early investigations of protease mechanisms; biochemists tended to draw nucleophilic acyl substitutions as if they were S_N2 reactions - all one step.
This ignored a fundamental difference between the two kinds of reaction.

• Nucleophilic substitution, in frontier orbital terms, involves an interaction between the HOMO of the nucleophile and the LUMO of the electrophile (substrate)
• Alkyl substrates, which undergo S_N2 reactions have much different LUMOs than acyl substrates, which form tetrahedral intermediates.

To illustrate, here's the LUMO of ethyl chloride, CH₃CH₂Cl:

LUMO of Ethyl Chloride

You can see that it involves chiefly the C-Cl bond, and has a considerable back lobe where the nucleophile interacts. Now here is the LUMO of acetyl chloride, CH₃(C=O)Cl:

LUMO of Acetyl Chloride

This LUMO is almost exclusively on the C=O, and hence there is no way an attacking nucleophile can break the C-Cl bond directly. In short, a tetrahedral intermediate MUST form because the orbital construction of the substrate won't permit any other pathway. End of story.
OK, so we've got the tetrahedral intermediate. Where next? Here's the tetrahedral intermediate, bound to Ser195 with the former carbonyl oxygen in the "oxyanion hole".



The next step is the reconstruction of the carbonyl double bond, with expulsion of the leaving group - in this case, the rest of the protein. This is the stage at which the protein chain actually is cleaved, and it produces an "acyl enzyme": the acyl part of the peptide that was cleaved, bound as an ester to Ser195.



OK, now we've got to cleave the acyl enzyme; enzyme are catalysts, and are not permanently altered in the reaction. To do this, we need a molecule of water.
From here on out, we're writing the mechanism for hydrolysis of an ester:



Restore the carbonyl double bond:



This releases the other end of the original protein, and restores the catalytic triad to its beginning state:



Dissociation of the second protein fragment leaves the enzyme ready to go again.

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Neonatal sepsis

Rajat Das Gupta

Neonatal sepsis is a blood infection that occurs in an infant younger than 90 days old. Early-onset sepsis is seen in the first week of life. Late-onset sepsis occurs between days 8 and 89.

Causes, incidence, and risk factors

A number of different bacteria, including Escherichia coli (E.coli), Listeria, and certain strains of streptococcus, may cause neonatal sepsis.
Early-onset neonatal sepsis most often appears within 24 hours of birth. The baby gets the infection from the mother before or during delivery. The following increases an infant's risk of early-onset sepsis:

• Group B streptococcus (group b strep) infection during pregnancy
• Preterm delivery
• Rupture of membranes (placenta tissue) that lasts longer than 24 hours
• Infection of the placenta tissues and amniotic fluid (chorioamnionitis)

Babies with late-onset neonatal sepsis get infected after delivery. The following increase an infant's risk of sepsis after delivery:

• Having a catheter in a blood vessel for a long time
• Staying in the hospital for an extended period of time

Symptoms

Infants with neonatal sepsis may have the following symptoms:

• Body temperature changes
• Breathing problems
• Diarrhea
• Low blood sugar
• Reduced movements
• Reduced sucking
• Seizures
• Slow heart rate
• Swollen belly area
• Vomiting
• Yellow skin and whites of the eyes (jaundice)

Signs and tests

Laboratory tests can help diagnose neonatal sepsis and identify the bacteria that is causing the infection. Blood tests may include:

• Blood culture
• C-reactive protein
• Complete blood count (CBC)

A lumbar puncture (spinal tap) will be done to examine the cerebrospinal fluid for bacteria.
If the baby has a cough or problems breathing, a chest x-ray will be taken.
Urine culture tests are done in babies older than several days.

Treatment

Babies in the hospital and those younger than 4 weeks old are started on antibiotics before lab results are back. (Lab results may take 24-72 hours.) This practice has saved many lives.
Older babies may not be given antibiotics if all lab results are within normal limits. Instead, the child may be followed closely on an outpatient basis.
Babies who do require treatment will be admitted to the hospital for monitoring.

Expectations (prognosis)

With prompt treatment, many babies with these bacterial infections will recover completely with no remaining problems. Nevertheless, neonatal sepsis is a leading cause of infant death. The more quickly an infant receives treatment, the better the outcome.

Complications

• Disability
• Death

Calling your health care provider

Seek immediate medical help if your infant shows symptoms of neonatal sepsis.

Prevention

Preventative antibiotics may be given to pregnant women who have a Group B Streptococcus infection or who have previously given birth to an infant with sepsis due to the bacteria.
Preventing and treating infections in mothers, providing a clean birth environment, and delivering the baby within 24 hours of rupture of membranes, where possible, can all help lower the chance of neonatal sepsis.

Flying Dutchman

Rajat Das Gupta

The most famous of the phantom vessels, supposedly seem in stormy weather off the Cape of Good Hope but now and then reported in other latitudes.

The Flying Dutchman by Albert Pinkham Ryder c.1887 (Smithsonian American Art Museum)

The term 'Flying Dutchman' actually refers to the captain, not his ship. Legend has it that this maniacal Dutch sea captain was struggling to round the Cape of Good Hope in the teeth of a terrible gale that threatened to sink his ship and all aboard. The sailors warned him to turn around, the passengers pleaded, but the captain, either mad or drunk, refused to change course. Instead, he pressed on, singing loud and obscene songs, before going below to his cabin to drink beer and smoke his pipe. Monstrous waves pummeled the sides of the ship, howling winds bent the masts and tore at the sails, but still the captain held his course, challenging the wrath of God Almighty by swearing a blasphemous oath.      
Finally, there was a mutiny on board; the crew and passengers attemptedto take control of the ship, but the captain, roused from his drunkenstupor, killed the leader of the rebellion and threw him overboard. Themoment the body hit the water, the clouds parted, and a shadowy figurematerialized on the quarterdeck. "You're a very stubborn man," the shadow said, and the captain answered him with an cussword. "I never asked for a peaceful passage," the captain went on. "I never asked for anything. So clear off before I shoot you, too."
But the figure didn't move. Drawing his pistol, the captain tried to fire, but the gun exploded in his hand. Now the figure spoke again, and told the captain he was accursed.
"As a result of your actions you are condemned to sail the oceans for eternity with a ghostly crew of dead men, bringing death to all who sight your spectral ship, and to never make port or know a moment's peace," the shadow said. "Furthermore, gall shall be your drink, and red hot iron your meat." The captain, reckless to the last, cried, "Amen to that!"
And so, for centuries from then on, the Flying Dutchman was seen piloting his spectral vessel, its canvas spread, its masts creaking in a fearful wind. Sometimes, it was said, he led other ships astray, onto rocky shoals and hidden reefs. Also he was said to be responsible for turning sailors' food supplies sour. His ship, looking innocent enough, would sometimes draw alongside another vessel and send letters aboard. But if the letters were opened and read, the ship would founder. Those who saw the captain himself claimed that he was bareheaded and repentant, clasping the wheel on the quarterdeck, pleading the heavens for mercy at last. In the rigging of his ship, some said, they could see a crew of skeletons, grinning miserably as they put on ever more sail. The tale of the Flying Dutchman has been elaborated by many writers, but it is more than a piece of fiction. The phantom ship has been seen many times — and there have been reports even in the 20th century, including the crew of a German submarine boat during World War II.

One of the first recorded sightings was by the captain and crew of a British ship in 1835. They recorded that they saw the phantom ship approaching in the blanket of a terrible storm. It came so close that the British crew feared the two ships might run into each other, but then the ghost ship suddenly vanished.
On 11 July 1881, the Royal Navy ship H.M.S. Bacchante was rounding the tip of Africa, when they sighted The Flying Dutchman. The midshipman, a prince who later became King George V, recorded that the lookout man and the officer of the watch had seen the Flying Dutchman:

"A strange red light as of a phantom ship all aglow, in the midst of which light the mast, spars and sails of a brig 200 yards distant stood out in strong relief."
It was a misfortune that the lookout saw the Flying Dutchman, for soon after on the same trip, he accidentally fell from a mast and died. Fortunately for the English royal family, the young midshipman survived the curse.
As recently as March, 1939, the ghost ship was seen off the coast of South Africa by dozens of bathers who supplied detailed descriptions of the ship, although most had probably never seen a 17th century merchant vessel. The British South Africa Annual of 1939 included the story, derived from newspaper reports:
"With uncanny volition, the ship sailed steadily on as the Glencairn beach folk stood about keenly discussing the whys and wherefores of the vessel. Just as the excitement reached its climax, however, the mystery ship vanished into thin air as strangely as it had come."
The last recorded sighting was in 1942 off the coast of Cape Town. Four witnesses saw the Dutchman sail into Table Bay... and vanish.
Many authorities have argued that the story of the Flying Dutchman has its origin in a real event, though there is very little agreement about what the event was. Further confusion is brought into the matter by the fact that are many versions of the tale — in which the ship's skipper is variously named Vanderdecken, Van Demien, Van Straaten, Van der Decken, or Van something else.
Another version of the legend that allegedly originated the whole affair is said to have happened in 1641, when a Dutch ship sank off the coast of the Cape of Good Hope. The story goes that, as the ship approached the tip of Africa, the captain thought that he should make a proposition to the Dutch East India Company (his employers) to start a settlement at the Cape on the tip of Africa, thereby providing a welcome repose to ships at sea.
He was so deep in thought that he did not notice the dark clouds looming and only when he heard the lookout scream out in terror, did he realize that they had sailed straight into a fierce storm. The captain and his crew battled for hours to get out of the storm and at one stage it looked like they would make it. Then they heard a terrible crunch — the ship had crashed into treacherous rocks and started to sink. As the ship plunged downwards, Captain Van der Decken (or whatever) knew that death was approaching. He was not ready to die and screamed out a curse: "I WILL round this Cape even if I have to keep sailing until doomsday!"
And, like in every version of the tale, this one also claims that even today whenever a storm brews off the Cape of Good Hope, if you look into the eye of the storm, you will be able to see the ship and it's captain — The Flying Dutchman. Don't look too carefully, for the old folk claim that whoever sights the ship will die a terrible death.
On yet another version, this one placed in the year 1729 (others say 1680), the captain this time swears at the Devil, who then condemns him to sail the spectral seas forever. The Devil left him just one small hope; that only through the love of a woman could he be released from his curse.
So the unfortunate Dutch captain returns to land every seven years in a hopeless search for salvation, because the Dutchman can only find eternal peace in the arms of a faithful woman. Wagner's opera, "Der fliegende Hollander," is loosely based on this version of the legend.


 Explanations as an optical illusion:
 Probably the most credible explanation might be a superior mirage or Fata Morgana seen at sea.

 Book illustration showing superior mirages of two boats

The news soon spread through the vessel that a phantom-ship with aghostly crew was sailing in the air over a phantom-ocean, and that itwas a bad omen, and meant that not one of them should ever see landagain. The captain was told the wonderful tale, and coming on deck, heexplained to the sailors that this strange appearance was caused by thereflection of some ship that was sailing on the water below this image,but at such a distance they could not see it. There were certainconditions of the atmosphere, he said, when the sun's rays could form aperfect picture in the air of objects on the earth, like the images onesees in glass or water, but they were not generally upright, as in thecase of this ship, but reversed—turned bottom upwards. This appearancein the air is called a mirage. He told a sailor to go up to the foretopand look beyond the phantom-ship.The man obeyed, and reported that hecould see on the water, below the ship in the air, one precisely likeit. Just then another ship was seen in the air, only this one was asteamship, and was bottom-upwards, as the captain had said thesemirages generally appeared. Soon after, the steamship itself came insight. The sailors were now convinced, and never afterwards believed inphantom-ships   

.Another optical effect, known as looming,occurs when rays of light are bent across different refractive indices.This could make a ship just off the horizon appear hoisted in the air.

Antimicrobial resistance: no action today, no cure tomorrow

Rajat Das Gupta

 
Antimicrobial resistance is not a new problem but one that is becoming more dangerous; urgent and consolidated efforts are needed to avoid regressing to the pre-antibiotic era.
For World Health Day 2011, WHO is introducing a six-point policy package to combat the spread of antimicrobial resistance.
 

 

Challenges facing the health sector in Bangladesh

The present elected government, since its assumption of office, has established a large number of community clinics across the country. But are the preponderant rural people of the country getting significantly any extra healthcare from their establishment? Marginally, perhaps, for the same remain understaffed. A large number of doctors stay away from their places of duties in such public healthcare facilities, in an uninterrupted sequence with the past, for promoting their careers in private hospitals or in private practice in cities. They are unconscientiously taking their salaries from the government. The government has not been able to stop this unacceptable practice. Under the present government, thus, infrastructural developments have outpaced the availability of the people who should run the system effectively.

In this backdrop, The World Health Day was observed in Bangladesh, as elsewhere abroad on Thursday amid different kinds of functions that were organized to mark the event and to reiterate commitment to making sustained efforts for accomplishing the long cherished national task of ensuring the availability of healthcare facilities to all citizens. But the same can do little to hide the fact that healthcare in the country remains in a state which is far from being described as even reasonably satisfactory. While speaking as the chief guest in a function to mark the World Health Day, the health adviser said regulatory reforms on health sector is needed very much as the country now does not need mainly investment in infrastructure, rather it needs skilled manpower. He was pointing perhaps to both not getting adequate services from the existing employed manpower in the publicly-run healthcare system as well as the dearth of trained manpower in it.

Bangladesh, as the report said, has a staggering shortage of over 60,000 doctors, nearly 0.3 million nurses and 0.5 million technologists. There are only about five physicians, two nurses and half technologist per 10000 people. So, a basic challenge to be overcome in the public healthcare system would be getting dedicated service from those already engaged in it and to recruit a large number to fill the vacant slots as well as ensuring that they are also obliged to discharge services sincerely.

There are also other aspects to be addressed and cured. Everyone in Bangladesh knows that most of the medical doctors in public hospitals either own or have a contractual relationship with private clinics. For these doctors, public service is a false identity. Their true identity is making money through their private practice or business. They appoint public hospital staff members as brokers to bring clients to private clinics, instead of properly treating them in public hospitals. Even if people are treated in a public hospital, they are forced to visit private clinics for a diagnosis so that doctors can earn "commissions."

In public hospitals, from the time patients arrive for treatment, the staff encourage them and their relatives to move to a private clinic to seek "better treatment." Simultaneously, doctors neither seriously listen to the patients nor explain their health problems and discuss possible treatments, including the diagnosis and medication. As a result, patients and their relatives always remain confused about the role of the doctors as well as the possibilities for treatment for alleviating their suffering. The greed on the part of a section of doctors is also reflected in their relationship with other healthcare professionals. They rarely delegate responsibilities to other medical specialists, such as physiotherapists, psychologists, counselors, nurses, speech therapists and so on, who are often looked down upon as lower class members of the health care profession in Bangladesh.

Moreover, medical equipment in public hospitals, as the reports in the media from the countryside from time to time do amply suggest, is intentionally kept "out of order" for years, in anticipation of increasing the business of private clinics and earning commissions. Patients in many cases cannot expect simple pathological or radiological tests in public hospitals. Ultimately, they are forced to go to private clinics and spend money far beyond their financial capacities. This culture has established a new group of millionaires in Bangladesh -- the owners of private clinics and diagnostic centres. In addition, many thousands of people who can afford to do so -- rich and middle-class patients -- go to hospitals abroad, to avoid even the undeveloped locally available privately offered health services, while a lack of governmental monitoring helps sustain such systems at home.