The conversion of α helices into β sheets feature in the formation of scrape prion protein.

A research held at University of California, San Francisco to study the conversion of α helices into β sheets feature in the formation of scrape prion protein.

Prions are infectious particles that are known to cause neurodegenerative disease in humans and animals. Scrapie Prion Proteins (PrPsc) were previously thought to be derived from PrPc via post-translational modification but since attempts to indentify such chemical modifications were unsuccessful this study was designed to examine the possibility that the formation of PrPsc involves conversion of α helices in PrPc to β sheets.
For the sake of this study a PrPc purification protocol was utilized. All chemicals utilized were of the highest grade. Purification of PrPc was modified accordingly to avoid denaturation of PrPc . PrPc, PrPsc and PrP27-30 were purified from Scrapie infected Syrian hamster brains. Purified samples were observed under electron microscope and FTIR spectroscopy was performed on each sample.
FTIR spectroscopy of PrPc showed characteristics features suggestive of high α helix content, on the other hand FTIR spectroscopy of PrPsc showed characteristics of high β sheets content. Meanwhile the secondary structures of PrPc, PrPsc and PrP27-30 were predicted using neural network algorithm. The results obtained were consistent with the hypothesis that it is the conformational change from α helix to β sheets that is responsible for the formation of PrPsc from PrPc.
The study successfully concluded that the formation of PrPsc involves the conversion of α-helices in PrPc into β sheets but the possibility of any other chemical modification cannot be eliminated. The study helped us to recognize that the conversion of α helices on PrPc into β sheets is likely to be the primary lesion in neurodegenerative diseases. Although protein folding is commonly thought to be controlled by thermodynamic preferences, it has been concluded that kinetic issues can alter the folding landscape. In a study done by V. Baskakov, Giuseppe Legname, Stanley B. Prusiner and Fred E. Cohen it was proved that the folding of native PrPC is under kinetic control.  So, If a β-rich amyloid competent structure is an intrinsic preference especially at a high protein concentration, then compartmentalization of partially folded intermediates and proteins that mediate unfolding and clearance of misfolded proteins play critical roles in cellular health. In the future we might be able to halt the disease progression by stopping these conformational changes.

Safe and effective dose of ozone therapy in the treatment of abortions

An experiment was conducted at the Department of Obstetrics and Gynecology, Medical Academy, Nizhny, Novgorod and Central Research Laboratory, Medical Academy, Nizhny, Novgorod, Russia and Russian Association of Ozone Therapy Jordan to study the safe and effective dose of ozone therapy in the treatment of abortions.

In this experiment, blood from 45 women was drawn, these women were belonging to 3 distinct groups

1. 15 subjects were having threatened abortion.
2. 15 subjects were healthy, non pregnant women.
3. 15 subjects were pregnant without any abnormality.

20ml blood was drawn from each women and was further divided in 4 equal parts.

1. One part was left intact to be used as control.
2. Other 3 were treated with 0.4ml ozonated saline solution with concentrations of 400mcg/l, 800mcg/l,1200Mcg/l respectively.

Results:

The criteria for safety and efficiency of ozone therapy was considered to be intensification of antioxidant defense mechanism,that protects against free radical damage, and a decrease in products of free radical injury (lipid peroxidation) measured by presence of certain products of lipid per oxidation known as Conjugated dienes.

• Products of free radical injury which were measured to be 2.78nmol/mg in intact untreated blood showed a slight improvement at 400mcg/l and 800mcg/l and became 2.23mcg/l and 2.22mcg/l respectively but at concentration of 1200mcg/l it showed a significant worsening of condition and became 3.05nmol/l showing significant free radical injury.
• Products of free radical injury in patients with threatened abortion which were 6.72nmol/mg in intact untreated blood improved and became 5.51nmol/mg at 400mcg/l but keep on worsening at higher concentrations of 800mcg/l and 1200mcg/l and became 9.81nmol/mg and 11.22nmol/mg respectively.

Similarly antioxidant activity showed following trend:

• Antioxidant activity in healthy pregnant women which was 0.15 rel.U in intact untreated blood showed improvement and became 1.64 rel.U and 0.16rel.U at 400mcg/l and 800mcg/l respectivly. The levels of antioxidant activity at high concentrations ozone solutions did not show any significant change and became 0.154 rel.U.
• Antioxidant activity in healthy non pregnant women which was 0.12 rel.U in intact untreated blood showed improvement and became 1.74 rel.U and 0.18rel.U at 400mcg/l and 800mcg/l respectively. The levels of antioxidant activity at high concentrations ozone solutions did not show any significant change and became 0.12 rel.U.
• Antioxidant activity in patients with threatened abortion which was 0.065 rel.U in intact untreated blood showed significant improvement and became 1.74 rel.U while no significant change was seen at 800mcg/l and 1200mcg/l.

Conclusions:

These experiments proved applicability of ozone therapy in treatment of various complications of pregnancy and fetal diseases but within a narrow range of 400mcg/l and 800mcg/l concentrations.

Efficiency of topical ozone treatment in patients of old diabetes with diabetic ulcers of lower limbs.

A study was held at the Division of Plastic and reconstructive Surgery, Banai-Zon Medical Center, Haifa to assess the efficiency of topical ozone treatment along with other conventionally used treatment modalities in patients of old diabetes with diabetic ulcers of lower limbs.

For the sake of this study, 183 diabetic patients with lower limb diabetic ulcers were given topical ozone treatment along with their normal conventional treatments and were followed over a period of 4 years to see if there would be any benefit in adding ozone therapy to the conventional treatments.

Treatment that patients received:

• Topical and systemic antibiotics.
• Periodic wound cleaning.
• Changing dressings when needed.
• Topical Ozone application. 3 x per week
• Taking wound cultures for detection of bacterial infections.

Results:

• Prior to the usage of topical ozone as treatment, the average healing time of the wound was 14.1 months which after the treatment became only 49 days or 1.5 months.
• Out of all the patients that were followed, 84.8% were fully healed.
• Remaining patients were either discontinued from the trials;skins grafted or were diagnosed as non-responsive patients.
• A very small number of patients (4.3%) had to undergo limb amputation.

This study concluded that when topical ozone is used along with other modalities of treatment for diabetic ulcers of lower limbs is very beneficial.

This study concluded that when ozone was added to the normal conventional protocol, the wounds healed 9.5 times faster then without ozone topical treatment. This is the measurable what is the most important part of all

Efficiency of Ozone therapy as treatment of patients with Diabetic foot.

A study conducted at The University of Havana, Cuba and Ozone research center, Cuba. In collaboration with University of Milan, Italy and Institute of Angiology and Vascular Surgery, Cuba to prove the efficiency of Ozone as treatment of patients with Diabetic foot.

In this study a total of 100 diabetic patients were selected which were all having ulcers of feet and toes. The patients were divided into 2 groups:

1. A group of 49 patients which were treated with antibiotics only.
2. Another group of 51 patients which were treated with Ozone, administered via rectal and topical routes in 20 different sessions.

Parameters considered were:

• Appearance of wound before and after the treatment in both study groups
• Blood glucose levels at the start and end of the study.
• Antioxidant and enzyme levels before and after therapy.

Results:

• The average blood glucose levels in patients of both groups before study were 10.11(+-4.2)mM.
• After giving treatment allocated to both the groups, improvement in average blood glucose levels was seen in 84% patients belonging to the group that received ozone treatment, and only 40% patients of the group receiving antibiotics.
• After the treatment,the blood glucose levels of ozone treated patients were within the normal range I.e. less than 7.8mM while the Blood glucose levels of patients given antibiotics were still higher than the normal range.
• The wounds of the patients that received ozone treatment were healed two times faster as compared to the wounds of the patients which were given antibiotics.
• The duration of hospitalization was also significantly reduced in the patients of ozone group as compared to antibiotic group.
• Oxidant levels, which were approximately 142µM before the treatment were brought back to normal i.e. 103.7(+-17.7)µM in Ozone group, while no decrease in oxidant levels were seen in antibiotic group.
• Other enzyme parameters were also improved significantly in ozone treated patients as compared to patients treated with antibiotics

Conclusion:

The study proved that diabetic patients with foot ulcers which were treated with ozone not only had 25% less expenditure on treatment but also showed a very significant and quick improvement of sign and symptom and biochemical parameters as compared to patients that received only antibiotics.

Practical applications of DNA fingerprinting

1. Paternity and Maternity 
Because a person inherits his or her VNTRs from his or her parents, VNTR patterns can be used to establish paternity and maternity. The patterns are so specific that a parental VNTR pattern can be reconstructed even if only the children's VNTR patterns are known (the more children produced, the more reliable the reconstruction). Parent-child VNTR pattern analysis has been used to solve standard father-identification cases as well as more complicated cases of confirming legal nationality and, in instances of adoption, biological parenthood.
2. Criminal Identification and Forensics 
DNA isolated from blood, hair, skin cells, or other genetic evidence left at the scene of a crime can be compared, through VNTR patterns, with the DNA of a criminal suspect to determine guilt or innocence. VNTR patterns are also useful in establishing the identity of a homicide victim, either from DNA found as evidence or from the body itself.

3. Personal Identification 
The notion of using DNA fingerprints as a sort of genetic bar code to identify individuals has been discussed, but this is not likely to happen anytime in the foreseeable future. The technology required to isolate, keep on file, and then analyze millions of very specified VNTR patterns is both expensive and impractical. Social security numbers, picture ID, and other more mundane methods are much more likely to remain the prevalent ways to establish personal identification.

Meselson and Stalh experiment

Meselson and Stalh used two different isotopes of nitrogen in order to give two different densities to cell growth. . The DNA of the cells grown in ¹⁵N medium had a higher density than cells grown in normal ¹⁴N medium.  After that, E. coli cells with only ¹⁵N in their DNA were transferred to a ¹⁴N medium and were allowed to divide; the progress of cell division was monitored by microscopic cell counts and by colony assay.

DNA was extracted periodically and was compared to pure ¹⁴N DNA and¹⁵N DNA. After one replication, the DNA was found to have intermediate density. Since conservative replication would result in equal amounts of DNA of the higher and lower densities (but no DNA of an intermediate density), conservative replication was excluded. 

Conclusion of experiment

• Results show that after one generation, the double stranded DNA is 1/2 heavy (from the parent) and 1/2 light (newly synthesized). This means that 100% of the strands are of intermediate density.
• After a second generation, one half of the new daughter strands are light (using ¹⁴N DNA as template and synthesizing ¹⁴N NA) and one half are intermediate density (using ¹⁵N DNA as a template and ¹⁴N DNA for synthesis). This result is predicted by semiconservative replication.
• Conclusion- as predicted by Watson and Crick, DNA strands serve as templates for their own replication.

Result of experiment

 After one replication, the DNA was found to have intermediate density. Since conservative replication would result in equal amounts of DNA of the higher and lower densities (but no DNA of an intermediate density), conservative replication was excluded. However, this result was consistent with both semiconservative and dispersive replication. Semiconservative replication would result in double-stranded DNA with one strand of ¹⁵N DNA, and one of ¹⁴N DNA, while dispersive replication would result in double-stranded DNA with both strands having mixtures of ¹⁵N and ¹⁴N DNA, either of which would have appeared as DNA of an intermediate density.

Characteristics of genetic code

Characteristics of genetic code

Following are the characteristics of genetic code accepted world wide

1. Only 61 triplets or codons code for amino acids
   3 stop codons (aka nonsense codons or terminator codons) UUA UAG UGA.
    
2. The code is a degenerative code
   Several codons code for the same amino acid.
   The first two letters seem to be the most important the third one tends to be interchangeable
3. The is no punctuation between each codon.
   The reading frame is set at the beginning of the gene. Frame shift mutations can be caused by the ADDITION or DELETION of only one or two bases. Everything downstream is misread.
    
4. The reading of mRNA is always in the same direction 5’ to 3’ (the same way as transcription and replication).
   The polypeptide chain is constructed from the amino end to the carboxyl end.
    
5. The code is universal for all organisms. So it is very ancient.
    
6. Similar amino acids have similar codons.
   Example
   Aspartic acid codons GAU and GAC. Glutamic acid codons GAA and GAG. Both are acidic amino acids.
    
7. Some amino acids are chemically altered AFTER translation.
   e.g. In collogen proline is converted to hydroxyproline.