Biotechnology and Biochemistry

Virology: Introduction to Swine Flu and Avian Flu

Viruses are submicroscopic, obligate intracellular parasites. Clearly, it is not a problem to differentiate viruses from higher macroscopic organisms. Even within a broad definition of microbiology encompassing prokaryotic organisms and microscopic eukaryotes such as algae, protozoa, and fungi, in most cases it will suffice. A few groups of prokaryotic organisms, however, have specialized intracellular parasitic life cycles and confound the above definition.

These are the Rickettsiae and Chlamydiae—obligate intracellular parasitic bacteria
which have evolved to be so cell-associated that they can exist outside the cells of their hosts for only a short period of time before losing viability. Therefore, it is necessary to add further clauses to the definition of what constitutes a virus.

 Virus particles are produced from the assembly of preformed components,
whereas other agents grow from an increase in the integrated sum of their components
and reproduce by division.
 Virus particles (virions) themselves do not grow or undergo division.
 Viruses lack the genetic information that encodes apparatus necessary for the
generation of metabolic energy or for protein synthesis (ribosomes).

No known virus has the biochemical or genetic potential to generate the energy
necessary to drive all biological processes (e.g., macromolecular synthesis).They are
therefore absolutely dependent on the host cell for this function. It is often asked
whether viruses are alive or not. One view is that inside the host cell viruses are
alive, whereas outside it they are merely complex assemblages of metabolically inert
chemicals. That is not to say that chemical changes do not occur in extracellular
virus particles, as will be explained elsewhere, but these are in no sense the ‘growth’
of a living organism.

Living Host Systems
In 1881, Louis Pasteur began to study rabies in animals. Over several years, he
developed methods of producing attenuated virus preparations by progressively
drying the spinal cords of rabbits experimentally infected with rabies which,
when inoculated into other animals, would protect from challenge with virulent
rabies virus. In 1885, he inoculated a child, Joseph Meister, with this, the first artificially
produced virus vaccine (as the ancient practice of variolation and Jenner’s
use of cowpox virus for vaccination relied on naturally occurring viruses).Whole
plants have been used to study the effects of plant viruses after infection ever since
tobacco mosaic virus was first discovered by Iwanowski. Usually such studies
involve rubbing preparations containing virus particles into the leaves or stem of
the plant.

During the Spanish–American War of the late nineteenth century and the subsequent
building of the Panama Canal, the number of American deaths due to yellow fever was colossal. The disease also appeared to be spreading slowly northward into the continental United States. In 1990, through experimental transmission to mice,Walter Reed demonstrated that yellow fever was caused by a virus spread by mosquitoes. This discovery eventually enabled Max Theiler in 1937 to propagate the virus in chick embryos and to produce an attenuated vaccine—the 17D strain—which is still in use today.The success of this approach led many other investigators from the 1930s to the 1950s to develop animal systems to identify and propagate pathogenic viruses.

Eukaryotic cells can be grown in vitro (tissue culture) and viruses can be propagated
in these cultures, but these techniques are expensive and technically quite
demanding. Some viruses will replicate in the living tissues of developing embryonated
hens eggs, such as influenza virus. Egg-adapted strains of influenza virus
replicate well in eggs and very high virus titres can be obtained. Embryonated
hens eggs were first used to propagate viruses in the early decades of the twentieth
century. This method has proved to be highly effective for the isolation and
culture of many viruses, particularly strains of influenza virus and various poxviruses
(e.g., vaccinia virus). Counting the ‘pocks’ on the chorioallantoic membrane of eggs
produced by the replication of vaccinia virus was the first quantitative assay for any
virus. Animal host systems still have their uses in virology:

 To produce viruses that cannot be effectively studied in vitro (e.g., hepatitis B
virus)
 To study the pathogenesis of virus infections (e.g., coxsackieviruses)
 To test vaccine safety (e.g., oral poliovirus vaccine)
Nevertheless, they are increasingly being discarded for the following reasons:
 Breeding and maintenance of animals infected with pathogenic viruses is
expensive.
 Whole animals are complex systems in which it is sometimes difficult to discern
events.
 Results obtained are not always reproducible due to host variation.
 Unnecessary or wasteful use of experimental animals is morally repugnant.
 They are rapidly being overtaken by ‘modern science’—cell culture and molecular
biology.

source: Cann AJ. 2005. Principles of Molecular Virology 4th Edition. New York: Elsevier Academic Press.

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Biocatalysys and biopolimer as biotechnology applications

The most important components of living cell, proteins, carbohydrates, and nucleic acids are polymers. Nature uses polymers as constructive elements and parts of complicated cell machinery. The salient feature of functional biopolymers is their all-or-nothing or at least highly nonlinear response to external stimuli. Small changes happen in response to varying parameters until the critical point is reached; then a transition occurs in the narrow range of the varied parameter, and after the transition is completed, there is no significant further response of the system.

Recent decades witnessed the appearance of synthetic functional polymers, which respond in some desired way to a change in temperature, pH, electric or magnetic fields, or some other parameters. These polymers were nicknamed stimuli-responsive. The name “smart polymers” was coined because of the similarity of the stimuli-responsive polymers to biopolymers.

Applications of polymers in biotechnology and medicine are discussed in this article. The highly nonlinear response of smart polymers to small changes in the external medium is of critical importance for the successful functioning of a system. Most applications of polymers in biotechnology and medicine include biorecognition and/or biocatalysis, which take place principally in aqueous solutions. Thus, only water-compatible smart polymers are considered; smart polymers in organic solvents or water/organic solvent mixtures are beyond the scope of the article.

One could define smart polymers used in biotechnology and medicine as macromolecules that undergo fast and reversible changes from hydrophilic to hydrophobic microstructure triggered by small changes in their environments. These microscopic changes are apparent at the macroscopic level as precipitate formation in solutions of smart polymers or changes in the wettability of a surface to which a smart polymer is grafted. The changes are reversible, and the system returns to its initial state when the trigger is removed.

note: for further information, please read Encyclopedia of Polimer Science and Technology. John Wiley & Sons, Inc. (2005)

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Molecular Methods

The analysis and measurement of variation in molecular biology are important to be known. For example, molecular techniques that measure DNA variation are genetic fingerprinting and polymerase chain reaction (PCR). These techniques are continually being developed, and thus properly prepared and stored samples may be useful for analysis by these new methods [1].

Simply, molecular method is began with some basic steps. Basic steps in molecular method are:
1. Sample source for DNA
In this section, we should preserve any samples are used, so can be utilized. After ready to be analyzed, they must be extraxted by extracting DNA. DNA extraction used to get pure DNA with some essential methods extraction.

2. Enzyme used in molecular biology
Molecular methods need some enzymes to manipulate and modify DNA. Some of them are restriction enzyme (e.g: EcoRI, BamHI, PstI, HaeIII, Sau3A), polymerase enzyme (e.g: Taq polimerase, Pfu polimerase, T7, T3, SP6), ligase enzyme (e.g: T4 DNA ligase), and modifying enzime (e.g: phosphatases, kinases, proteases).

3. Separating restriction fragments by electrophoresis
This step includes gel properties, electrophoresis buffers, and running an agarose gel. Additional, in agarose preparation, we must prepare it so containing variable amounts of sulphated polysaccharide chains.

4. Detecting DNA
DNA is detected by using gels and some solutions. Silver staining and ethidium bromide are the most common stains for detecting DNA. Beside that, we can detect DNA with radioactive analysis.

5. Blotting
Blotting is a trasfering process of DNA fragments from the gel to the membrane hile faithfully retaininng their relative positions.

6. Fixing
The aim of this step is to protect DNA during subsequent hybridization steps. Subsequent hybridization procedures used baking in vacuum and washing that will cause damage DNA’s if they weren’t protected. Fixing, may be use different UV-cross-linking and alkaline fixation protocols.

7. Hbridization
This technique is to study gene structure and function. But, today was increasingly being applied for the diagnosis of heritable diseases, detection of viral and bacterial pathogens, and the analysis of genetic variation in ecology and evolution.

Hybridization includes probes, labels, and protocols. Beside those, it is also includes stabiliting, deprobing, detecting, and measuring matter in hybridizations. In this posting, we will not study these steps.

8. DNA sequensing
Two methods of sequencing DNA are used by molecular biologists. First, involves specific chemical cleavage of DNA chains [2]. Second, the synthesis of a DNA template that is specially terminated at individual residues [3]

9. Other recombinant DNA technologies
To express some specific function in molecular methods, it is often necessary to grow and maintain the bacteria harbouring the recombinant DNA under rigorously controlled selection conditions.

10. Safety
A molecular biology laboratory can pose hazards to human health and the environment. It because in molecular methods used some substances poisonous, for examples: acrylamide (neurotoxin), ethidium bromide (carcinogens), acids/bases (make chemical burns as an fenol).

Finish.

[1] Royston E. Carter. 2000. General Molecular Biology in Molecular Metods in Ecology. Allan J. Baker, Ed. London: Blackwell Science.
[2] Maxam & Gilbert. 1977. A new method for sequencing DNA. Proceedings of The National Academy of Sciences USA. [include in 1].
[3] Sanger et al. 1977. DNA sequencing with chain terminating inhibitors. Proceedings of The National Academy of Sciences USA. [include in 1].

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Methods for sample storage

This posting is to describe the method to store a sample in molecular techique. There are four steps. I have taken it from General Molecular Biology (Royston 2000).

1. Frozen whole blood Whole blood is transferred to a sterile Eppendorf tube and froze as soon as possible (preferably at – 80°C, or – 20°C is suffice for most instances). Please pay your attention that if the samples are repeatedly thawed and re-frozen, some of DNA integruty will be loss.

2. Storage in extraction buffer If possible, appropriate-sized aliquots of fresh whole blood (routinely 15 µL of avian blood or 1-2 mL of mammalian blood) are resuspended in 500 µL (5 mL if mammalian) of 1xSET extraction buffer (sodium chloride/EDTA/Tris). Then, frozen at – 80°C. Individual tubes can then be removed and processed without thawing the stock sample.

3. Storage in lysis buffer It resuspended for long-term storage at ambient temperature. It contains SDS, which lyses cells, and EDTA, which chelates Ca2+ ions and therefore prevents nuclease activity.

4. Storage in ethanol An absolute ehtanol is a powerful dehydrating agent. If small volume of fresh whole blood are suspended, individual erythrocytes remain intact and separate. Nucleases and other degradative enzymes remain inactive ease. Usually, one drop (100 µL) of avian blood is suspended into 1 mL of ehtanol.

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