The teen gene: Switching on puberty

DID you stare into the mirror every morning, desperate for signs that your body was starting to develop like those of your classmates? Or perhaps you were the one who started sprouting breasts or facial hair years before your peers, and got teased just as much as the late developers.
Though it happens to practically everyone, the start of puberty is highly unpredictable: it can happen as naturally to a girl of 8 as to one aged 13. The range is just as wide in boys,....Read More.....................

Love (Addicted to love,Sexual chemistry,Love evolution)

The word love appears in many contexts: there’s maternal love, familial love, romantic love, sexual love, a wider love for fellow humans and religious love for God, to name but........(Read more...)

Who's the daddy? US sperm banks must be better regulated

CAREFUL, exacting, high-tech - that's the image US sperm banks like to project to their customers. They run limited genetic tests on their donors and trace medical records back three generations. They also screen for chemical exposure, drug use and even do complex psychological profiling. (Read More.........)

How to build a stem cell line

Alcoholism : A Widespread Problem

Excessive habitual consumption of alcoholic beverages despite physical, mental, social, or economic harm (e.g., cirrhosis, drunk driving and accidents, family strife, frequently missing work). It is usually considered an addiction and a disease. The causes are unclear, but there may be a genetic predisposition. Alcoholism occurs in about 4.2% of adults in the U.S. It is more common in men, but women are more likely to hide it. Treatment may be physiological (with drugs that cause vomiting and a feeling of panic when alcohol is consumed; not an effective long-term treatment), psychological (with therapy and rehabilitation), and/or social (with group therapies). Group therapies such as Alcoholics Anonymous are the most effective treatments. Suddenly stopstopping heavy drinking can lead to withdrawal symptoms, incl. delirium tremens.

Status of the World AIDS Epidemic, End of 2006

People newly infected with HIV in 2006 :-
Total 4.3 million
Adults 3.8 million
Children under 15 years 530,000
Number of people living with HIV/AIDS :-
Total 39.5 million
Adults 37.2 million
Children under 15 years 2.3 million
AIDS deaths in 2006 :-
Total 2.9 million
Adults 2.6 million
Children under 15 years 380,000

MY PRIDE "MY VILLAGE"

LOCATION OF MY VILLEGE

DARAM KHOLA

Utility of Streptomyces spp. In health

22nd Dec 2006

Gautam, Sujan

Seminar presentation Abstract

This presentation discusses streptomycetes utility in human health and their antibiotic producing capabilities. Streptomyces is a major genus among actinomycetes, which are major group gram-positive mycelial prokaryotes predominantly found in soil. Antibiotic are the major therapeutic drugs for bacterial diseases and, hence, are related directly to the human health. Amino glycosides (e.g., streptomycin, neomycin, or gentamycin) and macrolides (e.g., erythromycin) target bacterial ribosomes while quinolones (e.g.; ciprofloxacin) attack bacterial DNA gyrase (1). Mitomycin has been shown to be an anticancer or antitumor agent that halts the cell in G2 phase, resulting in cell death (2). Unlike other anticancer agents, FK317 does not induce weight loss and is more effective than MC (3,4). FK506; a macrolide, is a potent anti viral agent (5). NP-06, a 21-mer oligopeptide antiviral compound, appears to block early stage of HIV-1 infection (6) while nystatin has antifungal properties (1).

References
1. Pelczar MJ, Chan ECS and Krieg NR, 2004.Antibiotics and Other Chemotherapeutic Agents In: Microbiology, TATA McGraw-HILL publishing Co. Ltd, New Delhi, 5th ed, pp:510-539.

2. Martin TW, Dauter Z, Devedjiev Y, Sheffield P, Jelen F, He M, Sherman DH, Otlewski J and Derewenda U, 2002. Molecular Basis of Mitomycin C Resistance in Streptomyces: Structure and Function of the MRD Protein. Structure 10: 933-942.

3. Naoe Y, Kawamura I, Inami M, Tsujimoto S, Manda T and Shimomura K, 1998. Anti-cachetic Effect of FK317, a Novel Anti-cancer Agent, in Colon26 and LX-1 Models in Mice. Jpn. J. Cancer Res. 89: 1318-1325.

4. Naoe Y, Inami M, Matsumoto S, Takagaki S, Fujiwara T, Yamazaki S, Kawamura I, Nishigaki F, Tsujimoto S, Manda Tand Shimomura K, 1998. FK317, a Novel Subsituted Dihydrobenzoxazine, Exhibits Potent Antitumor Activity against Human Tumor Xenografts in Nude Mice. Jpn. J. Cancer Res. 89: 1306-1317

5. Reis S.A, Moussatche N, and Damaso C.R, 2006. FK506, a secondary metabolite produced by Streptomyces presents a novel antiviral activity against Orthopoxvirus infection in cell culture. J. Applied Micorbiology 100: 1373-1380.

6. Chokelijchai S, Anderson S, Nomizu M, Machida M and Roller P.P, 1995. NP-06: a Novel Anti-Human Immunodeficiency Virus Polypeptide Produced by a Streptomyces Species. Antimicrobial Agents and Chemotherapy 39:2345-2347.

Natural Scavenger

In flight

Vultures are scavenging birds, feeding mostly the caracus of dead animals. Three species of asian vultures has fallen by 97 % in 12 years.

Why South Asia Vultures are on the Brink of Extinction?

In short, the reason is because the farmers in Katmandu, Nepal feed their cow’s diclofenac, a drug used to relieve fever and lameness. The scavenging vultures are one step up the food chain. Once the cow dies the scavenger eats the dead cow's carcass and becomes poisoned. The drug causes acute kidney failure in the vultures after dining on the animals treated with diclofenac. The American Chemical Association states that we should not be alarmed when chemicals show up in the cord blood of your newborns. But we'd like to ask you to decide for yourself: "Do chemicals in our blood matter?" In 15 years the population of the vultures in Nepal have gone from 50,000 nesting pairs to only 500 nesting pairs.
Vultures are natural scavengers in nature and remember, "it's not nice to fool Mother Nature." So how are conservationists in Nepal trying to remedy this problem? After all they need their vultures. They have opened a "restaurant" for the scavengers sixty miles outside of Katmandu. At least some of the vultures are being given a chance to eat chemical-free food. Sick and dying cows who have never been fed diclofenac are brought in and when they die, their carcasses are left for the vultures. While the use of diclofenic is prohibited in Nepal and India, the ban is widely ignored.(click here to learn more)

PRIDE OF NEPAL

Mount Everest

Naming

The Tibetan name for Mount Everest is Chomolungma or Qomolangma (meaning "Mother of the Universe). In the 1960s, the Government of Nepal gave the mountain an official Nepali name: Sagarmatha (सगरमाथा), meaning "Head of the Sky".

Messurement

Radhanath Sikdar, an Indian mathematician and surveyor from Bengal, was the first to identify Everest as the world's highest peak in 1852, using trigonometric calculations based on measurements of "Peak XV" (as it was then known) made with theodolites from 240 km (150 miles) away in India. Measurement could not be made from closer due to a lack of access to Nepal.

More recently, the mountain has been found to be 8,848 m (29,028 feet) high, although there is some variation in the measurements. The elevation of 8,848 m (29,029 ft) was first determined by an Indian survey in 1955, made closer to the mountain, also using theodolites. It was subsequently reaffirmed by a 1975 Chinese measurement. In both cases the snow cap, not the rock head, was measured. In May 1999 an American Everest Expedition, directed by Bradford Washburn, anchored a GPS unit into the highest bedrock. A rock head elevation of 8,850 m (29,035 ft), and a snow/ice elevation 1 m (3 ft) higher, were obtained via this device. Although it has not been officially recognized by Nepal, this figure is widely quoted. Geoid uncertainty casts doubt upon the accuracy claimed by both the 1999 and 2005 surveys.

YARSA GUMBA

Yarsa Gumba

Scientific Name:- Cordycep sinensis

Common Name:- Himalayan Viagra

What it is?

Yarsa gumba is a unique combination of a yellow caterpillar and a mushroom. Yarsa gumba in Tibetan means ‘summer grass, winter insect’. Just prior to the rainy season, spores of cordyceps mushroom lands on these Himalayan caterpillars that live on moist grass and hollow ground. After the fungus buries itself in the caterpillar’s body, it works its way out through the insect’s head. The parasite gets the energy from the caterpillar which eventually dies. As temperature increases and the snow melts -yarsa gumba emerges and is harvested at this time. During monsoon, the yarsa gumba is swept away.

It is between 3,300m - 4,000m in Bhutan, India, Nepal and Tibet

Government of Nepal:- Policy

Nepalese government banned its collection in 1996 and allowed it to be collected after 2001. The reason for the ban was on an adhoc basis and trial and error method – it was proposed that if harvesting were discontinued – population of yarsa gumba could increase. Today, it is recognized that yarsa gumba is to be harvested annually.
As this region is devoid of trees and little vegetation, the local communities thrive through such collection. This year about a thousand people had gathered to collect yarsa gumba and two people have died of bad weather. As the revenue for collection is exorbitantly high with the government charging a royalty of US$ 271.11 per kg. Local collectors avoid paying royalty for their collection however they do pay fraction of their collection and this has promoted illegal trade.

Exported countries

The major countries involved in its import are China, Hong Kong, Taiwan and Korea where it is used extensively for oriental medicine. India is one of the leading importers of yarsa gumba compared to other countries of the world.

Amoeba vaccine!

When Spaniards invaded and captured Mexico in the early 16th century, they face an unexpected form of resistance: life threatening dysentery, which they called “Montezuma’s revenge”. Amoebic disease today claims the lives of thousands years and afflicts millions and more, mainly in impoverished communities.
Unfortunately, the means to fight amoebic disease remain very limited. Because affected populations usually reside on poor countries, pharmaceutical company does not have sufficient economic incentive to invest in developing new therapies. Now a team at the weizmann institute of science, Israel have succeeded in engineering an amoeba that could became the basis for a pioneering vaccines against amoebic disease.
Prof. Carlos gitter discovered that amoeba kills human cells by infecting a small protein into their membranes. He called this protein an amoeba pore; and the phenomenon was termed, “the amoeba Kiss of death”. He hoped to produce antibiotics that would be used against the amoebapore. However the antibiotics proved ineffective because they could not reach the amoebapore, which passed directly from the amoebas into the intestinal cells with out being exposed.
David Mirelamn and team members Rivka Bracha and Yeael Nachamawtz isolated the gene encoding the amoebapore, made a copy of the gene encoding the amoebapore, and reversed the orientation of components. They then introduced the reversed gene into the amoeba, creating an organism that carries both the original (“Sense”) amoebapore gene and the antisense gene. When the original amoebapore gene starts getting expressed, the anti-sense gene does the same. The resulting messenger RNAs fit together perfectly, clinging to reach other like two sides of a zipper . As a result neither of the messenger RNA molecules is available for producing the amoebapore protein.
Using this technique, the scientists have managed to block about 60% of amoebapore production in the amoeba. The engineer amoeba was much less aggressive than their original counterparts. In the follow up research they managed to completely block the gene that encodes the lethal protein in effect developing a new breed of “Silenced” amoebae incapable of making amoebapore .Now the scientists are experimenting an whether the silenced amoebae can be used as a vaccine against aggressive amoebae.
It successful, this will herald a possibility of the first vaccine of it’s kind.

Where do DNAs go?

“Where have all the birds gone” used to be the plaintive cry of the environmentalists. Now neurobiologists are stunned by the mystery of the disappearing DNA. Many cells in the average brain may be missing huge chunks of genome say scientists and these emissive might decide our risk of disease.
Cells are generally assumed to need a full set of DNA to run with out major flow. In fact, a third set of dividing cells in one region of the adults mouse brain have gained or lost at least one chromosome, the same goes for 15% of the adult neuron these cells produce, biologists have discovered. This hints that every person’s brain may be a mosaic of cells with different genetic make-ups. The scientists working at university of California said they were stunned cells that gain or loss chromosomes could predispose of protect from certain disease, as speculated. Cells lacking chromosomes might also be prone to from tumors. Other scientists speculated that an increase risk of developing Alzheimer’s disease might arise in otherwise healthy people who carry a subset of brain cells with an extra copy of chromosome z1.
According to the scientists the cellular phenomenon thought to arise when chromosomes are divided up inaccurately at cell division must serve some biological purpose in the brain. Immune cells and blood cells appear not to show the same effect, so “it doesn’t seem to be mistake”. However, losing genes “changes what a nerve cell do”, they say perhaps slowing the speed at which they communicate. Some bacteria, for example, suffle genomes when they are in uncomfortable conditions, to create a new mutant that can survive. It is interesting that cells lacking the correct number of chromosomes in the growing embryo are carefully eliminated from the body’s tissue and embryo with chromosomes anomalies are often aborted spontaneously. Embryo and placenta sort from the some ball of cells yet according to placental diagnostic testing around 2% of placenta but not the embryos, they naturally contains a mixture of chromosomally normal and abnormal cells. At first normal any aberrant cells are mixed but about a third of the way through gestation they selected against and many commit suicide or be sorted into the placenta. The case appears to be a tangled or with tantalization clues, which will no doubt be solved in time. Till then one can only wonder at the mysteries of nature.

Poem

THE NEW BEGNNING
The day will come for our departure,
We’ll cry
But we will come across a new beginning.
The laughter, the quarrels we had
We will remain near by
With in the vision of our mind
The happiness and sorrow we had
Was not enough but..
We’ll meet again with a new beginning.

RESARCH PROJECT

A ResearchProposal
On
BIOCHEMICAL STUDY OF SERUM PROTEINS
OF BUFFALO (Bubalus bubalis) and
IT’S COMMERCIAL UTILIZATIOM

Submitted by:
Sujan Gautam
Universal Science College, Maitidevi plaza,
Kathmandu Nepal

Submitted To:
Department of Biochemistry, Universal
Science
College, Maitidevi plaza,
Kathmandu, NEPAL

Principal investigator: Sujan Gautam

co-investigator: Vijayendra agrawal

Introduction

Blood and serum are frequently used as samples for proteomic analysis. Serum is a rich source of biochemical products that act as biomarkers of disease or reflect physiological status of a patient. Proteomic technologies are being used to discover and identify disease-associated biomarkers. The application of current proteomic technologies in the search for potential diagnostic/prognostic indicators in the serum of patients is limited by highly abundant albumin and immunoglobulins that constitute more than 60% of the total serum protein. Removal of abundant serum proteins will help in the discovery and detection of less abundant proteins that may prove to be informative disease markers (Chen et al., 2005). The concentration of plasma proteins is a useful clinical diagnostic tool and is often indicative of metabolic disorder.
Albumin is the most abundant protein in serum and is synthesized by the liver to maintain homostatic levels when protein intake is sufficient. Decreased albumin concentration, which is typically correlated with decreased total protein concentration, indicates protein malnutrition. Depending on the composition of the diet, decreased albumin may indicate energy malnutrition whereby proteins are catabolized for gluconeogenesis.
Immunoglobulin G (IgG) is the second most abundant protein in plasma and is synthesized by B-lymphocytes in response to the sum of immunogenic stimulation. The levels of IgG are decreased by (protein) malnutrition and elevated by infectious disease.
Acute phase proteins (APP) are an ensemble of plasma proteins synthesized by the liver in response to tissue damage and inflammation associated with traumatic and/or infectious disease. Transferrin (Tf) and fibrinogen (Fb) are the most abundant acute proteins that can be accurately measured. There are additional proteins that become detectable when transferrin and fibrinogen levels are increased. Increased IgG and acute phase proteins indicate an infection with concurrent tissue damage. Increased IgG without increased fibrinogen indicates immunological response to infection without concomitant decrease in tissue function (Acute phase protein, 15 June 2007).
Asian buffalo, Bubalus bubalis, has been heavily domesticated and thus is now widespread. The suspected native domain of B. bubalis was from central India to southern Nepal in the west to Vietnam and Malasiya in the east. In the wild, water buffalo are found in tropical and subtropical forests as well as wet grasslands (Water buffalo, 17 th June 2007). The buffalo population in Nepal can be broadly classified into three groups based on their breed characteristics- Hill buffalo, Terai buffalo and Indian breeds (Rasali, 2000). Lime, Parkote and Gaddi are the three breeds of Hill buffalo reported in the literature (Rasali and Crow, 1999).
Forward motility-promoting protein isolated from buffalo blood serum has the potential for the treatment of human infertility (due to low sperm motility) a great social curse in all human races. The product isolated by the present invention has also the potential for improving farm animal breeding with special reference to buffalo, a milch animal of great economic importance in many countries including India (Majumder et al., 2003).
This study is directed towards the isolation and purification of albumin from serum for its commercial utilization. In addition, study for the identification of some biomarker protein will be done to differentiate normal and diseased buffalo. As far as my knowledge, such a study hasn’t been conducted so far in Nepal.

Objectives:

. General objective:
· Study of serum protein from Buffalo.

ii. Specific Objectives:
· To isolate serum from blood sample.
· To isolate albumin from blood serum

a. By (NH4)2SO4 precipitation
b. By TCA/ acetone (or ethanol/ methanol/ DMSO) method
· To perform size exclusion chromatography to purify the albumin fraction.
· To check the purity of protein and also compare the pattern of proteins isolated from diseased as well as non-diseased serum samples by SDS-PAGE and find some biomarkers for the identification of disease one.
Materials and methods:
Chemicals and Reagents: Acetic acid, TCA, Acetone, Methanol, DMSO, Acrylamide, Bis-acrylamide, Ammonium persulphate, Ammonium sulphate, bis-Acrylamide, b-mercaptoethanol, Bromophenol blue, CBB-G 250 (Coomassie brilliant blue), CBB-R 250, Ethanol, Sucrose, Glycerol, Glycine, Phosphoric acid, Potassium dihydrogen phosphate, di-Sodium hydrogen phosphate, Sodium dodecyl sulphate, Hydrochloric acid, Bovine Serum Albumin (BSA), TEMED (Tetramethylene ethylenediamine), Tris (tris-hydroxymethyl aminomethane), Potassium chloride, Sephadex G-100.

Equipments: Analytical balance, Cold centrifuge, Fraction collector, Magnetic stirrer, Micropippetes, pH meter, Protein electrophoretic set, Spectrophotometer, Vaccum filter, Chromatographic column

I. Isolation of blood serum

· Buffalo blood will be collected both by venepuncture and by sacrifice of buffalo into plain collection tubes.
· The blood samples will be allowed to clot at room temperature for 30 minutes and then centrifugation will be done at 5000 rpm for 15 min to collect the serum.
· The amount of protein present in serum will be determined by Bradford method and SDS-PAGE will be done according to Laemmli.
· For the purification of albumin from serum, two approaches will be adopted: (NH4)2SO4 precipitation and TCA precipitation.

II. Purification of serum albumin by (NH4)2SO4 precipitation(Little and Little, 1994)

· Proteins present in serum sample will be precipitated using different percent saturation of ammonium sulfate. Precipitation will be done slowly over a period of 30-45 minutes with gentle stirring with the help of a magnetic stirrer to get the desired % saturation.

Take the serum
↓
Dilute the serum with 0.1 M phosphate buffer, pH 7.0 to get 1 mg /ml protein concentrati.


*The number of grams of ammonium sulfate to be added to 1 liter of solution at 20°C to get a desired saturation= 533(S2-S1)/100-0.3S2, where S1 is the initial % saturation and S2 is the final % saturation.
The ammonium sulphate precipitation containing albumin as a major fraction will be subjected to size exclusion chromatography for further purification.

III. Purification of serum albumin by 10% TCA in acetone/ ethanol/ methanol and DMSO (Chen et al., 2005).
Serum albumin will be precipitated by 10% TCA in different solvents viz. acetone, ethanol, methanol and DMSO and the purity of albumin fractions obtained will be accessed by SDS-PAGE. The fraction of maximum purity will be used for further purification of albumin by size exclusion chromatography.


Take 20 ml sample of buffalo serum and add four volumes of ice-cold acetone (or ethanol/ methanol/ DMSO) containing 10% w/v TCA.


Mix by gentle vortexing



Incubate the mixture at -200C for 90 min and centrifuge at 15 000 rpm, 4 0 C, for 10 min


Store the supernatant (a)


Then, add 1 mL of ice-cold acetone (or ethanol/ methanol/ DMSO) to wash the precipitate

Incubate the sample on ice for 15 min and then
Centrifuge at 15000 rpm/10min

Store the supernatant (b)


Mix supernatant (a) and (b) and add 1 ml of ice-cold acetone (or ethanol/ methanol/ DMSO) to the supernatant mixture to completely precipitate the proteins in the supernatant. Dissolve the protein precipitate in minimum volume of 0.1 M Phosphate buffer, pH 7.0.

IV. Purification of serum albumin by size exclusion chromatography (Andrews, 1965)

1. Prepare slurry of Sephadex G100 in 0.15 M KCl to get a bed volume of 50 ml. Mix required amount of gel beads with KCl and let the beads swell for 24 h.
2. Slowly pour the swelled beads in the column with outlet closed. Let the beads settle and then pass the KCl solution through the column at a flow rate of 0.5 ml/ min. Percolate the column with the same KCl solution 1-2 times so that column is well packed.
3. Equilibrate the column with 0.05 M Tris.Cl, pH 7.5 containing 0.1 M KCl.
4. Apply the protein sample on top of the column.
5. Elute the protein at a flow rate of 0.5 ml/ minute and collect 3 ml sample per tube.
6. Quantitate each eluted fraction by taking absorbance at 280 nm and pool the fractions showing peak value.
7. Dialyse the pooled fraction against 0.1 M Phosphate buffer, pH 7.0 containing 50% glycerol for 12 h with 2 changes of buffer.
8. Quantitate the protein content and do SDS-PAGE to access the purity of albumin purified.

V. Protein Quantitation by Bradford method (Bradford, 1976)

Mix Protein solution (0.1ml) with 4.9 ml of Bradford reagent. Vortex and incubate for 2 minutes. Measure absorbance at 595nm. Use BSA as a standard and Bradford reagent as blank.
(Bradford reagent: Dissolve 0.1 gm of CBB-G 250 in 50 ml of 95% C2H5OH and add 100 ml of 85% w/v H3PO4. Dilute the solution to 1L. Filter before use.)
VI. SDS-PAGE (Laemmli, 1970)
SDS-PAGE will be performed to check the purity of isolated albumin sample. It will also be used to compare the protein profile of normal and diseased buffalo serum so as to find some biomarker for the identification of diseased buffalo.
Expected outcome:
Albumin will be isolated and purified from the serum of buffalo blood. In addition, may be, some biomarker for the differentiation of normal and diseased buffalo can be found among the myriad of proteins present in serum.
References:
Acute phase protein. (15 June 2007) http://en.wikipedia.org/wiki/Acute_phase_protein

Andrews P (1965) The Gel-Filtration Behaviour of Proteins Related to their Molecular Weights over a Wide Range. Biochem. J. 96: 595

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254

Chen YY, Lin SY, Yeh YY, Hsiao HH, Wu CY, Chen ST, Wang AH (2005) A modified protein precipitation procedure for efficient removal of albumin from serum. Electrophoresis 26: 2117-2127

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the Head of Bacteriophage T4. Nature 227: 680-685

Little SE, Little JT (1994) Rapid Protease Screening Using Ammonium Sulfate Precipitations and the AvantiTM High Performance Centrifuge. Application Information A-1796A. Fullerton, CA, Beckman Instruments, Inc.
http://www.beckman.com/literature/Bioresearch/1796A(A).pdf

Majumder GC, Mandal M, Banerjee S (2003) Process for the purification of a new motility-promoting protein from buffalo serum: a slaughter house waste. http://www.freepatentsonline.com/6613737.html

Rasali DP (2000) Recent Trends in Buffalo Production in Nepal. In Buffalo Newsletter http://www.buffaloreview.20m.com/Leadarticle.html, pp 6-10

Rasali DP, Crow GH (1999) Production of buffaloes (Bubalus bubalis) in the mountains and hills of Nepal: Constraints and opportunities. In http://www.buffaloreview.20m.com/commentary.htm.

Water buffalo. (17 th June 2007) http://animaldiversity.ummz.umich.edu/site/accounts/information/Bubalus_bubalis.html


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