ALS - Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS), also known as motor neuron disease (MND), or Lou Gehrig's disease, is a specific disease which causes the death of neurons controlling voluntary muscles. ALS is characterized by stiff muscles, muscle twitching and gradually worsening weakness due to muscles decreasing in size. This results in difficulty speaking, swallowing and eventually breathing.

SIGNS AND SYMPTOMS:

• The disorder causes muscle weakness, atrophy and muscle spasms throughout the body due to the degeneration of the upper motor and lower motor neurons.
• Individuals affected by the disorder may ultimately lose the ability to initiate and control all voluntary movement, although bladder and bowel function and the muscles responsible for eye movement are usually spared until the final stages of the disorder.
• Cognitive or behavioral dysfunction is present in 30-50% of individuals with ALS.
• Around half of the people with ALS will experience mild changes in cognition and behavior, and 10-15% will show signs of frontotemporal dementia.
• Repeating phrases or gestures, apathy and lose of inhibition are frequently reported behavioral features of ALS.
• Language dysfunction, executive dysfunction and troubles with social cognition and verbal memory are the most commonly reported cognitive symptoms in ALS.
• About half of the people who have ALS experience emotional lability, in which they cry or laugh for no reason.
• Sensory nerves and the autonomic nervous system are generally unaffected, meaning the majority of people with ALS maintain heavy sight, touch, smell and taste.
• Although the order and rate of symptoms vary from person to person, the disease eventually spread to unaffected regions and the affected regions become more affected.
• Most people eventually are not able to walk or use their hands and arms, lose ability to apeak and swallow food and their own saliva, and begin to lose the ability to cough and to breathe.
• The rate of progression can be measured using an outcome measure called the 'ALS Functional Rating Scale Revised (ALSFRS-R)', a 12 item instrument administered as a clinical interview or self-reported questionare that produces a score between 48 (normal function) and 0 (severe disability).
• Difficulty in chewing and swallowing makes eating very difficult and increase the risk of choking or of aspirating food into the lungs. As the diaphragm and intercoastal muscles of the ribcage that support breathing weaken, measure of lung function such as vital capacity and inspiration pressure diminish. Most people with ALS die of respiratory failure or pneumonia.

CAUSES:

Though the exact cause of ALS is unknown, genetic factors and environmental factors are thought to be roughly equal important. The genetic factors are better understood than the environmental factor; no specific environmental. factors has been definitively shown to cause ALS.

GENETICS:

• ALS can be classified as familial or sporadic, depending on whether or not there is a family history of the disease.
• The strictest definition of familial ALS is that a person with ALS must have two or more first degree relatives (children, siblings or parents) who also have ALS. or a person with ALS must have at least one second degree relative (grandparents, uncles, aunts or nephews) who also have ALS.
• Familial ALS is usually said to account for 10% of all cases of ALS.
• In sporadic ALS, there is no family history of the disease. Sporadic ALS and familial ALS appear identical clinically and pathological and are similar genetically.
• More than 20 genes have been associated with familial ALS, of which four account for the majority of cases: C9orf72 (40%), SOD1 (20%), FUS (1-5%) and TARDBP (1-5%).

ENVIRONMENTAL FACTORS:

Where no family history of the disease is present possible evidence of this disease include smoking, various proposed factors include exposure to environmental toxins, as well as alcohol and tobacco use. Chronic exposure to lead or exposure to other heavy metals, beta-carotene intake, head injury, omega-three fatty acid intake, exposure to extremely low frequency electromagnetic fields, pesticides and serum uric acid levels.

MECHANISM:

ALS attacks motor neurons in the brain and spinal cord. The progressive degeneration of the motor neurons in ALS eventually leads to their death. When the motor neurons die, the ability of the brain to initiate and control muscle development or movement is lost. With voluntary muscle action progressively affected, patients in the later stages of the disease may become totally paralyzed.

Other cell types in the CNS that support motor neurons called glia, including astrocytes and oligodendrocytes are also involved in disease. 

Following are the some known disease mechanisms:

Axon structure and dynamics: Transport of materials up and down the length of the motor neuron is an important cellular process that may play role into the damage seen in ALS. There are proteins within the nerve fibers that maintain the motor neurons.

Mitochondria: In ALS, evidence is building that actions on or originating in the mitochondria may be an important part of the disease. Mitochondria show damage early in the ALS disease process.

Glutamate: It can be a destructive factor in ALS. Investigators are working to find out its role and process.

Inflammation: Neuroinflammation accompanies the death of motor neurons in ALS.

TREATMENT:

Treatment depends on stage. Medications and therapies that can slow ALS and reduce discomfort, are:

Two medications are currently approved by the Food And Drug Administration for the treatment of ALS are : Riluzole (Rilutek) and Edaravone (Radicava).

Breathing core: The patient may choose mechanical ventilation to help breathe. Doctors also insert a tube surgically (tracheostomy) which is connected to respirator.

Physical therapy: Regular exercise can help patient to improve sense of well-being. Appropriate stretching can help prevent pain and help patients muscles function.

Speech therapy:  Speech therapist help the patient to explore other methods of communication.

Nutritional Support: ALS affected people should eat foods that are easier to swallow and meet their nutritional requirement.

Physiological and Social Support

PREVENTION:

• At this time, medical science can offer no means for preventing ALS because the causes are unknown. Research into molecular and biochemical factors may lead to earlier diagnosis and prevention of ALS.
• ALS can be prevented by eating plenty of vegetables and fruits.
• A higher total carotenoid intake was associated with decrease risk of ALS.
• Diets high in carotene and lutein commonly present in dark green vegetables had a decreased risk of ALS. 

Different DNA binding motifs

HELIX-TURN-HELIX MOTIF;

The helix turn helix motif is present in many prokaryotes and eukaryotic DNA binding protein. The HTH motif was the first DNA binding structure to be identified. The motif is made up  of alpha-helices separated by beta-turn, made up of four amino acid, the second of which is usually glycine. This turn, in conjunction with the first alpha-helix, position. The second helix on the surface of the protein in an orientation that unable it to fit inside the major groove of DNA molecule. This second alpha-helix is known as recognition helix. The HTH is usually 20 or 50 amino acids in length as is just a small part of the protein as a whole. 

The homeodomain is an extended HTH motif passed by protein encoded by homeotic gene. It is made up of highly conserved domain of 60 amino acids encoded by 180 bp DNA sequence called homeobox. It has three alpha helices and helices 2 and 3 separated by beta-turn. Helices 3, which is 17 amino acid residue long act as recognition helix and bind to major groove of DNA. Helix 1 make contact with the minor groove.

HELIX-LOOP-HELIX MOTIF:

The helix-loop-helix (HLH) motif is involved in both DNA binding and protein dimerization. The HLH motif is composed of two region of alpha-helix of 15 to 16 residue separated by region of variable length, which form loop between two helices. Length of connecting loop varies from 12 to 28 amino acid residues. This motif is quite similar to leucine zipper motif.

LEUCINE ZIPPER MOTIF:

Leucine zipper motif (Lzip) mediates both DNA binding and dimerization. Two distinct right handed alpha-helices participate in the formation of homo or hetero dimer structure. N-terminal DNA binding domain is rich in positively charged amino acid residues. C-terminal dimerization domain is 30-40 amino acids long with leucine at every seventh position. The leucine zipper domain is necessary for protein dimerization. Two alpha-helices in parallel wind around each other to form a coiled coil structure. The leucine residue end up with their R group protruding from the helical domain in which the leucine residue reside. The protruding R-group are thought to interdigitate with leucine R group of another leucine zipper domain, thus stablishing homo- or hetero- dimerization.

ZINC FINGER:

Zinc-coordinated DNA binding motif are called zinc-finger motif. There are several type of zinc finger motif. The most widely occurring zinc finger motif in eukaryotes is the Lys2-His2 (C2-H2) zinc finger, which was first discovered in a transcription factor called TFIIA that regulate the transcription of gene for 5S rRNA. This class of zinc finger occur in cluster. Each finger is 130 amino acids long and is characterized by pair of Cys and His that always occur at the same relative portion.

It form compact, globular domain in which a zinc ion is coordinated tetrahedrally by Cys and His residue. The zinc is essential for correct folding and DNA binding. NMR and X-ray studies shown that C2H2 finger is a compact globular domain composed of 12 residue alpha-helix packed against irregular beta-sheet, with the zinc in between. The alpha-helix of each finger lie in major groove and contact three base pairs.

Explain the structure and mechanism of Nitrate Reductase.

Nitrate Reductase (NR) shows cell-specific expression. For e.g., at low nitrate conc. NR is primarily found in the root epidermal while at higher external nitrate conc. NR activity is more widespread. Most higher plant NRs use NADH or NADPH. NR is described as a molybdoflavoheme- containing protein. It requires three cofactors that provide the redox center that facilitates the movement of electron transfer, the molybdenum (MoCo), the FAD and the heme iron cofactor.  The MoCo domain is at the N-terminal region, heme iron domain is in middle and FAD domain at C-terminal region of NR. These three functional regions are connected by two hinges.
NR forms homodimers with a binding site for nitrate and for NAD(P)H. Each NR subunit is 1000 amino acids long. Partial proteolysis of the NR protein show discrete fragments having different enzyme activities. One fragment binds FAD and can use NADH to reduce the artificial electron acceptor ferricyanide. A different fragment contains the MoCo and heme iron domain and can reduce nitrate in the presence of the artificial electron donor methyl viologen.
The MoCo region is 360-370 amino acids long and belongs to the special class of MoCo-binding proteins. These include Xanthine oxidase, biotin sulfoxide reductase etc. The central heme domain is 75-80 amino acid residues long and similar to cytochrome b5 family. The FAD binding region is 260-265 amino acids long and similar to ferredoxin-NADP+ oxidoreductase family. It consist of two domains, each forming a lobe that is separated by a cleft. The N-terminal lobe binds FAD, while the C-terminal lobe binds the substrate NAD(P)H. There is a cysteine in the C-terminal lobe that provides a thiol group which interacts with NAD(P)H. Thus, the NR enzyme can be regarded as mini electron transport chain that uses NAD(P)H as its source of electron and NO3- as its terminal electron acceptor.

NR is a substrate-inducible enzyme i.e., NR levels increase in response to the conc. of nitrate in plants. Regulation is at level pf transcription i.e., NR mRNA accumulation within minutes when exposed to nitrate. Light or photosynthetic production of sucrose also induces NR transcription. Up-regulating NR transcription potentially increases nitrite production.
There are two hinge regions, one connecting the MoCo and heme region (I) and the other connecting FAD and heme region (II). The phosphorylated serine residues (at hinge I) is alone efficient to direct effect the NR activity i.e., phosphoNR. An additional inhibitor protein, or NIP (NR inhibitor protein) is required to inactivate NR. The binding of this 14-3-3 NIP directly to the phosphoNR in the presence of Mg2+ causes a conformational change leading to the movement of the hinge and blocking electron flow between the Heme-Fe cytochrome and Mo cofactor. This results in a completely inactive NR form that cannot transfer electron from NAD(P)H to nitrate. A low Mg2+ conc. promotes the dissociation of 14-3-3 complex. Depending on  the external conditions, NR exists in three states: free NR (active); phosphorylated NR (phosphoNR, active) and phosphoNR:NIP complex (inactive). NR phosphorylation and NIP binding are also involved in the control of the NR degradation. All forms of NR are also capable of catalyzing the production of nitric oxide from nitrite, required for germination, stomatal regulation and pathogen responses.

Differences between group I self-splicing introns and group II self splicing introns:

GROUP I SELF SPLICING INTRONS:

1. Group-I is found in bacteriophage; mRNA; tRNA, rRNA of lower eukaryotes prevently in higher plants and sporadically in tRNA, rRNA of bacteria.
2. Group I of ORF codes for a homing endonuclease, that is responsible for its mobility.
3. In the splicing mechanism group I requires GTP.
4. Guanine nucleoside cofactor is used whose 3-OH makes a nucleophilic attack on the phospho and forms 3,5, phosphodiester bond. The released 3-exon end now attacks the 3- intron end in a similar nucleophilic attack and the intron is excised and exons ligated.
5. Group I has the core structure with a paired region (P1-P9) that forms two domains,  P4-P6 domain (P5, P4, P6, P6a) and P3-P9 domain (P3, P8, P7, P9 helices) with tertiary structure stabilizing it.

GROUP II SELF SPLICING INTRONS:

1. Group II is found in mobile genetic elements in bacteria, organellar elements of archaea and eukaryotes.
2. Group II ORF has multifunctional: Reverse transcriptase, mutases activity.
3. Group II is independent of GTP.
4. 2-OH group within the intron makes a nucleophilic attack on the 5- splice site and forms a lariat like structure. This lariat like structure acts as an intermediate. 3-end of the exon makes a nucleophilic attack on the 3-end of the intron and lariat shaped intron is excised and the exons are ligated.
5. Group II forms 6 typical stem- loop structure D1- linked by tertiary interaction.

Differences between translational intiation in prokaryotes and eukaryotes:

Translational initiation in prokaryotes:

1. In the translational initiation in prokaryotes, the association of the small subunit with the mRNA is mediated by base- pairing interaction between the ribosome binding site and the 16S rRNA.
2. Initiating amino acid (met) needs to be formylated, (therefore, two tRNAs for methionine i.e., tRNA fmet and tRNA met are found).
3. Ribosomes enter the mRNA at AUG codon or at nearby Shine-Dalgarno sequence.
4. No initiation factor or co-factors are required for initial contact between ribosome and mRNA.
5. Small ribosomal subunit (30S) can engage mRNA before binding of initiator met-tRNA fmet.
6. At the end of initiation, the 70S initiation complex are found.

Translational initiation in eukaryotes:

1. In the translational initiation in eukaryotes, the small subunit is already associated with an initiator tRNA when it is recruited to the capped 5end of the mRNA.
2. Initiating amino acid (methionine) is not formylated: (only one tRNA for met i.e., tRNAmet is found)
3. Ribosomes enter at the capped 5-end of mRNA and then advance to AUG codon by linear scanning.
4. ATP and a number of protein factors are needed for ribosomes to engage the mRNA.
5. At the end of initiation the 80S initiation complex are found.
6. Small ribosomal subunit (43S) binds stably to mRNA only after initiator met-tRNA has bound.

Polycystic Ovarian Syndrome (PCOS)

• PCOS is heterogenous disorder of uncertain cause. Some evidence shows it is a genetic disease (autosomal dominant) with high genetic penetrance but variable expressivity in females. It is a health problem that affects 1 in 10 women. It is the most common endocrine disorder which is caused by an imbalance of reproductive hormones which creates problems in ovary. In PCOS, the egg from the ovaries may not develop as it should or it may not be released during ovulation. This causes missed or irregular menstrual periods. Irregular periods can lead to : (a) Infertility (b) development of cysts (small fluid-filled sacs) in the ovaries.
• PCOS encompasses hyperandrogenism which has internal effects on ovarian function and metabolism and external manifestation on skin. Ovarian dysfunction is associated with erratic menses and anovulation.
•  Cysts are a symptom instead of the cause. Cysts are the immature follicles whose development has arrested at early antral stage. Polycystic ovaries develop when the ovaries are stimulated to produce excessive amounts of androgenic hormones, mainly testosterone.
• Women with PCOS experience an increased frequency of hypothalamic GnRH pulses, which in turn results in an increase in LH/FSH ratio. Hyperinsulinemia amplifies hyperandrogenism . Majority of women are obese and insulin-resistant. Hyperinsulinemia increases GnRH pulses frequencies. LH over FSH dominance increased ovarian androgen production, decreased follicular maturation.  Adipose tissue possesses aromatase which convert androstenedione to estrone and testosterone. Hence, obese women will have high androgen production.

CAUSES: 

(a) Excess insulin = when cells become resistant to the action of insulin, blood sugar as well as insulin levels arises. This leads to increase in androgen production.

(b) Low grade inflammation = this stimulates polycystic ovaries to produce androgen in PCOS women.

(c) Other causes are hypothyroidism, Cushing syndrome and hyperprolactinemia.

MECHANISM:

(a) Androgen control the development of male traits. Women with PCOS have more androgens than normal. High levels prevent the ovaries from releasing an egg (ovulation) and cause extra hair growth and acne (signs of PCOS).

(b) The action of insulin on the liver leads to decrease production of sex hormone binding globulin which results in increased free testosterone.

SIGNS AND SYMPTOMS:

1. Chronic anovulation 
2. Reduced menstrual bleeding
3. Absence of menstrual bleeding (Amenorrhea)
4. Hirsutism (increased facial and body hair)
5. Alopecia (baldness)
6. Obesity (high waist to hip ratio)
7. Infertility or recurrent miscarriages.
8. Anxiety\
9. Oily skin, severe and late-onset acne
10. pelvic pain
11. patches of thickened, dark, velvety skin- a condition called as Acanthosis nigricans 

DIAGNOSIS:

1. Physical examination : BP, BMI, WHR, extra hair on body, acne, skin discoloration, enlarged thyroid gland.
2. Pelvic exam: if ovaries are enlarged or swollen.
3. Ultrasound: cysts in ovaries or check the endometrium lining of uterus
4. Blood tests: androgen level, cholesterol level etc.
5. Anti-Mullerian hormone (AMH) is increased in PCOS, and become a best diagnostic criteria.

TREATMENT:

1. Medical:  Metformin is used to treat type II diabetes. It improves insulin levels and bring back the normal cycle. Clomiphene is ovulation inducer and improves fertility. Oral contraceptives decrease the action of insulin and hence increase the production of sex hormone binding globulin which leads to low levels of free testosterone.
2. Taking estrogen and progestin daily regulate ovulation, relieve symptoms and protect against cancer.
3. Lifestyle changes: exercise, eating healthy and avoid junk.

Differences between diabetic coma and insulin shock.

DIABETIC COMA:

1. It is more fatal.
2. It occurs to the patient of high blood sugar- hyperglycemia.
3. Causes are- ketoacidosis, hyperosmolar syndrome
4. Experienced by diabetic patients generally.
5. Symptoms: dry mouth, frequent urination, sweating, anxiety, shakiness.

INSULIN SHOCK:

1. Comparatively less fatal.
2. It is the body reaction to too little sugar- hypoglycemia
3. It occurs due to the result of too much insulin.
4. Even people without diabetes can also experience.
5. Symptoms: fast breathing, rapid pulse, dizziness, numbness and hunger.