Functions of a healthy tear film
- Optical clarity and refractive power
- Ocular surface comfort; lubrication
- Environment for corneal epithelium
Definition of Dry Eye
- multifactorial disease of tears and ocular surface
- Symptoms: discomfort, visual disturbance, tear instability, and potential damage to ocular surface
- Increased osmolarity of tear film
- inflammation of the ocular surface
Progression of Dry Eye
- Increase in the osmotic gradient between the tear film and the ocular surface
- Increases conjunctival cell desquamation.
- Disease first affects the conjunctival morphology while the corneal morphology initially remains normal.
Further Dry Eye Progression
- attachments between corneal cells loosen
- increase in corneal desquamation
- severe corneal epithelial changes: glycoprotein loss and destabilization of the cornea-tear interface
Artificial Tears' goal
- replenish eyes' tears
- delay evaporation of patients' tears
Components of Artificial Tears
- Demulcent (hydrogels)
- Buffer (oil gelling agent ions)
- does not contain growth factor or anti-inflamatory components
Artificial Tears Summary
- viscosity of polymers minimizes thinning of tear film
- Provide temporary palliative relief
- avoid repeated use of banzalkonium chloride (BAK), which is toxic with multiple doses per day
- fail to break the cyclic inflammatory nature of dry eye
- lack proteins and nutrients found in normal, healthy tears
- Prescribed in pulsated, tapered therapeutic modality (fancy word for "treatment")
- start 3-4 times a day and taper to 2x/week
- mild steroid is Loteprednol (Lotemax by Bausch and Lomb)
- cyclosporine opthalmic elulsion 0.05%
- topical anti-inflammatory
- for moderate to severe dry eye
- Side effects: burning, conjunctival heperemia, discharge, epiphora (overflow of tears), eye pain, foreign body sensation, pruritus (itch), stinging, and blurring
- Function: prevents T cells from releasing cytokines
- Dosing: one drop twice/day
- Takes 3-4 months for relief to start
- amino acids are fundamental building blocks
- created from peptide bonds
- 3 divisions: Peptides (molecular weight below 10,000), Proteins (molecular weight above 10,000), Polypeptides (basically another name for protein)
Role of proteins in eye
- Mechanical support
- Clarity of cornea and refraction properties
- Electrical signaling
- Protecting eye through lysis of bacteria (tears)
Classification of Aminoacids
- Acidic (more than one carboxylic group; ex: aspartic acid, glutamic acid)
- Basic (more than one amino group; ex: lysine, arginine)
- Neutral (equal number of amino and carboxylic acid groups; ex: alanine, glycine)
Amino Acid Types
- Glycine (can give tighter "twist" to proteins such as collagen)
- impart hydrophobic characteristics to proteins
Dicarboxylic amino acids
- Aspartic (aspartate)
- Glutamic (glutamate)
- Impart negative charges to proteins due to ionization of one carboxylate.
Diamino amino acids
- Impart positive charges to proteins due to ionization of one amine
Amido amino acids
- derived from aspartic and glutamic; hydrophilic character
Hydroxy amino acids
- hydroxy groups give them hydrophilic character
Aromatic amino acids
- Tyrosine (less hydrophobic due to OH group)
- impart hydrophobic characteristics to proteins
Sulfur amino acids
- Cysteine (forms disulfide bonds between polypeptide chains and is hydrophilic
- Methionine (hydrophobic)
Cyclic amino acids
- nonaromatic and is found in collagen, elastin, and mucin
What amino acid groups are hydrophilic?
- Cystine in Sulfur group
- Important in pH control
Which amino acid groups are Hydrophobic?
Specialized properties of amino acids
- Metal chelation (ex: histidine in hemoglobin)
- Chain binding (ex: cystene in ribonuclease)
- Tight helical turns (ex: glycine in collagen)
- function of protein dependent on number of times each amino acid occurs
Structural Classifications of Proteins
- alpha-helical domains
- beta-helical domains
- alpha/beta domains which consist of "beta-alpha-beta" structural units or motifs that form parallel beta-sheats
- alpha and beta domains formed by independent alpha helices and mainly antiparallel beta-sheetsmulti-domain proteins
- membrane and cell surface proteins and peptides
- "small" proteins
- coiled-coil proteins
- low-resolution protein structures
- peptides and fragments
- designed proteins of non-natural sequence
Amino acid classification based on solubility
- Water soluble (albumin)
- Lipid soluble (rhodopsin)
- Insoluble (collagen)
protein that possesses its unique biological ability
Protein that has lost its property
Primary Protein Structure
normal sequence of amino acids
Secondary Protein Structures (beta)
- beta-sheets are sequences of primary structure connected by hydrogen bonds (ex: crystallins)
- beta-turns connect beta-sheets
- random coils are haphazard forms that connect secondary structures
Secondary Protein Structures (alpha)
- alpha-hlix (ex: rhodopsin)
- alpha-helix defined as 1.2-angstrom rise for 100 degree rotation
- stabilized by hydrogen bonds
Structural or functional part of a chain containing secondary structures or a part of tertiary structure
a simplified version of domain (contains few parts of domain; ex: helix-turn-helix)
Tertiary Protein structure
entire shape or configuration of one polypeptide chain constitutes tertiary structure
two or more polypeptide chains connected
What are crystallins and where are they found?
Group of proteins found in epithelium and in the fiber cells of ocular lens. Primarily eye protein but found other places as well. They help maintain shape of lens fibers and thus the structure of the lens.
beta and gamma crystallins same family on functional classification.
- further division:
- α-crystallins has αA and αB
- β-crystallins has βH and βL (H = heavy, L = light)
- γ-crystallins have six subtypes γA to γF
What is the function of α-crystallins?
- Molecular chaperones- that is help to maintain normal molecular confirmation of other lens crystallins
- prevents crystallin aggregation- process in formation of senile cataract (age related cataract).
α-crystallins are the only crystallin in lens epithelium
Which part of the lens is a layer of epithelial cells, has mitochondria, and has high metabolic rates, uses glucose and o2?
The lens epithelium
The lens epithelium uses glucose and 02, where does it get these primarily?
A) Blood vessels of ciliary body via lens zonules
B) Vitreous active transport
C) Aqueous via diffusion
D) Unknown to science
Glucose in aqueous higher than lens hence glucose diffuses readily
Glycolytic enzymes, TCA enzymes and its intermediates and ATP are in high concentration in lens epithelium, why?
What do you know about lens fibers?
They're the bulk of lens cortex and nucleus, high in number, take up minimal space (why?).
The ocular lens is made up of 65% what and 35% what?
65% water and 35% organic matter (33% is structural proteins)
Where are the soluble ocular lens proteins located? Where are the insoluble lens protein located?
- Soluble lens - cortex
- Insoluble lens - nucleus
- the nucleus is more dehydrated than the cortex
the water equilibrium is of prime importance, why?
What is the proper electrolyte balance of the lens?
Low Na+ and Cl- and water
If sodium moved into the lens that would increase the solutes and the water content will increase (osmotically)
What is the water electrolyte balance of the concentration gradient?
The electrical gradient?
And the water electrolyte balance is maintained by what?
Concentration gradient: Aqueous has more Na+ and low K+
Electrical gradient: lens is electronegative inside
Electrolyte balance maintained by the sodium pump
What do you know about the sodium pump?
operates in the epithelium
for every 3Na+ removed, 2K+ are allowed
it requires energy
What effect does the aging process have on the ocular lens?
- In general increase in phosphorylated chains of α-crystallins in lens fiber
- Disulfide bond formation (crosslinking)between different crystallins increases
- Peptide bond disruption in both β and γ crystallins
what do you know about cataract formation?
- General effect is change in amount of soluble and insoluble lens protein
- Lens grows with age hence the protein content also increases with age.
- The process of change of soluble to insoluble is by cross linking
HIgh molecular (HM) weight
- HM1, HM2, HM3 and HM4
- HM1 and HM2 soluble kind
- HM3 and HM4 insoluble and found in cataractous lens
- HM4 occurs exclusively in nuclear cataract
Senile Cataract Hypothesis
HM fractions grow into the cell cytoplasm
Radiation of what range has potential for being cataractogenic?
What absorbs this wavelength and protects the retina from damage?
range: 300-400 nm
the lens absorbs these wavelengths
Increased intracellular calcium has what effect on cataracts?
What about hypocalcemia?
- increased calcium
- affects glucose metabolism,
- inhibition of protein synthesis,
- induction of HM aggregates
- Direct loss of transparency
- Inhibit sodium pump
- Hypocalcemiarelated to the dependency of the membrane permeability to levels of calcium
- Marked electrolyte and water imbalance in low calcium environment
Diabetic and galactosemic
- Complications of diabetes and glactosemia
- Conversion of excess glucose or galactose to respective sugar alcohols via sorbitol pathway.
- Sorbitol cannot readily diffuse out of intracellular compartment.
- Osmotic gradient favors influx of H2O
- Sugar alcohols and sugar itself will lead to protein aggregation
- Neonates and premature infants at greatest risk
- Remember ocular lens needs energy
Ionizing radiation and microwave cataract
- X-ray radiation (some humans as low as 200 rads)
- Microwave animal experiments shown to cause cataract.
- Mechanism for microwave cataract is ----?
Effects of Radiation
- Radiation retinopathy requires 3000-3500 rads
- 1-2 years after therapy
- Endothelial cell DNA damage of blood vessels
- Patients with diabetes grater risk
What is Rhodopsin and where is it found?
It is a intrinsic membrane protein found in discs and to some extent the plasma membrane or rod outer segment
Rod outer segment
- Rhodopsin is represented as egg shaped with hook.
- Rhodopsin more prevalent in discs
- Rhodopsin has vitamin A
- Vitamin-A is bound to protein at amino acid lysine #296
- Vitamin A is present rhodopsin as an aldehyde (retinal) and consists of 11-cis-form
Light absorption properties
Not a part of visual transduction process per se
Called bleaching of rhodopsin
- Alpha peak- 11 cis retinal
- Beta peak- All trans retinal
- Gamma peak- opsin
Beer's Law or Beer-Lamberts
- A= Absorbance
- b=light pathlength
- c= concentration
- Retina: the point of contact between radiated energy, light and nervous system
- Rod cells:
Scotopic (dim-light) vision
Less sensitive than rods,
- but they mediate
- differentiation of colors
Foveal vs Peripheral Cells
- FOVEA: cone cells have “private line communication”; no divergence
- PERIPHERY: mostly rod cells greatly outnumber the ganglion cells, convergence is the rule
High sensitivity, but low acuity
Additive and Subtractive Colors
Additive Primary Colors
- Blue (435 nm), green (545 nm), and red (700 nm)
- Add up to produce white
Subtractive Primary Colors
- Yellow, magenta-red, and cyan-blue
- From the mixing of any 2/3s of the spectrum
- Subtractive color mixing: the mixing of color pigments or dyes to produce full spectrum of colors
- Add up to produce black
What are the three pigments?
- Erythrolabe (red, long)
- Chlorolabe (green, medium)
- Cyanolabe (cyan, short)
- result of three independent comparisons of rates of photon absorption by the three cone types
- dynamic output relates only to the rate of photon capture
Anomalies of Color Vision
- Congenital: inherited, non-progressive nature
- Acquired: not inherited, secondary to disease (blue-yellow)
a person missing one of three photopigments
person missing chlorolabe (green)
person missing erythrolabe (red)
person missing cyanolabe (cyan)
have photopigment but absorption spectrum of one of the pigments is displaced
Color Vision abnormalities
- 3-8% in males
- females less frequent
- color vision deficiency, can be a problem in certain jobs
X-Chrome contact lenses
- worn on only one eye
- reddish brown tint
- patients may suppress the eye
- proteins to which short chains of sugars are bound
- Roles of glycoprotein:
- structural orientation
- immunological recognition
- biological lubrication
- Rhodopsin is a glycoprotein
Mucin layer of tear film
- made of 55% carbohydrates
- similar to mucins in the GI tract, nasal mucosa, and trachea
- secreted by goblet cells of conjunctiva
- stabilizes tear film by increased viscosity
- biological lubricant
- prevent mechanical shock
- rapid tear film break-up time when amount is low
Pathology that decrease mucins
- Vitamin A deficiency
- Ocular pemphigoid (conjunctival ulcerations)
- Steven-Johnsons syndrome
- Alkali burns
Vitamin A deficiency related to mucin
- Xerosis (dryness of conjunctiva in the interpalpebral zone with loss of goblet cells, squamous metaplasia, and keratinization)
- Bitot spots (triangular patches of foamy keratinizes epithelium in the interpalpebral zone thought to be caused by infection w/ Corynebacterium xerosis)
- twice as common as acid burns
- common alkalis are ammonia, sodium hydroxide, and lime
- penetrate deeper than burns because acids coagulate surface proteins
- life-threatening condition affecting the skin in which epidermis separates from the dermis due to cell death
- caused by infections
- hypersensitivity reactions to medication
Ocular cicatricial pemphigoid
- autoimmune mucocutaneous blistering disease
- keratinization of the caruncle
- adhesion between the bulbar and palpebral conjunctiva
- hyperaemia and oedema
- chronic conjunctivitis
- extracellular, insoluble molecular complex
- 80-90% of bulk of eye
- 12 different types in eye
- protein complex basic unit consists of triple helices (tropocollagens)
- supporting members or fibers - maintain tissue, repair tissue
- scaffolding - platform on which basement membrane is constructed
- anchoring devices - holds cells onto non cellular area
- semiliquid gel in eye (vitreous body)
Synthesis of collagen
disulfide bonds that form at the c-terminal causes the chain to wrap around each other
- units are associated hydrophobically by covalent bonds
- microfibril: 5 rows of cross-linked tropocollagen
Types of collagen fibers found in Descement's membrane
Types of collagen fibers found in Corneal stroma
Types of collagen fibers found in Cornea
Types of collagen fibers found in Sclera
Types of collagen found in Vitreous
Types of collagen found in Blood Vessels?
Types of collagen found in Iris?
Types of collagen found in eye lids?
Types of collagen found in lens capsule?
Type of collagen found in endothelium?
Type of collagen found in Anchoring fibrils of Bowman's membrane?
What are lamellae?
Ocular structural roles-corneal stroma - what are the percentages of each type?
- 70% is type I
- 15% approx type V
- 15% approx type VI
What is the important role of type v collagen?
maintain the optical properties of the cornea
what limits the diameter of type 1 collagen?
Type V collagen oblique running lamellae in stroma
Type VI collagen
- beaded form
- stabilizing molecule
the vitreous body
made of parallel fibers
what is attached at ora serrate and macula?
the vitreous body
what is the principal vitreous collagen type 2 called?
vitreous gel is what percentage water?
what types of collagen hybrids are associated with type 2?
types IX and V-XI
type V-XI limits diameter of type II
The collagen found in what membrance is type 4 (including bowman's membrane, lens capsule, and blood vessels)
What is a basement membrane?
They are thin structures that occur extracellularly.
What are the functions of a basement membrane?
support and separation from the other cells
What membrane is the exception to the rule of collagen of basement membrane?
descement's membrane is what type and what pattern?
type VIII and a very geometric pattern
What may affect the synthesis of anchoring fibrils?
a diabetic state
what types of collagen are in anchoring fibrils?
type VII and type I (more randomly oriented)
what is characterized by blue sclera and findings of megalocornea and keratoconus?
what are the factors of osteogenesis associated with type 1 osteogenesis imperfecta?
- Decreased synthesis of type 1 collagen
- Clinically – fracture of long bones specially prior to puberty
- Blue sclera
what are the factors of osteogenesis associated with type 2 osteogenesis imperfecta?
- Point mutations and rearrangements in triple helical regions
- Malformed and soft fragile bones and perinatal lethality
type IV of osteogenesis imperfecta is associated with what scleral color?
almost normal scleral color
how many subtypes of ehlers-danlos syndrome are there?
what are type 6 and type 4 of ehlers-danlos syndrome known for?
- type 6 - associated with ocular issues
- type 4 - rare
what are symptoms of ehlers-danlos syndrome?
skin is hyperelastic, bruise easily, slow to heal, and joints are hypermobile
what are common ocular features of ehlers-danlos?
Ocular fragility with increased vulnerability to mild trauma, high myopia, retinal detachment and keratoconus
what are uncommon features of ehlers-danlos?
Epicanthic folds, microcornea, blue sclera, ectopia lentis and angioid streaks.
*ectopia lentis displaced lens from its normal position
these are the result of crack-like dehiscences in thickened, calcified and abnormally brittle collagenous and elastic portions of Bruch's membrance
what are enzymes?
proteins that have the ability to optimize rates of biochemical reaction
what is activation energy?
energy required for a reaction to occur
what effect do enzymes have on activation energy?
they decrease the amount of activation energy required
The reaction is A to B. Which is the substrate and which is the product?
Depending on which direction the reaction goes a biochemical substance can be substrate or a product.
an enzyme involved in the principal driving reaction is called ______.
a rate limiting enzyme
what does principal driving reaction mean?
a series of enzyme driven reactions, reaction tends to proceed in a given direction regardless of the substrate and product concentration
uncatalyzed reactions are driven by what?
heat of molecular collision
what kind of reaction has an active site? the enzyme may change but will eventually return to its original state.
what are kinds of classifications of enzymes are there?
kinetic basis, michaelis-menten, and allosteric
kinetic and funcational properties described by michealis and menten in 1913
example = aldose reductase
change is concentration over time
- Vmax is maximum velocity of enzyme
- Km is Michealis-Menten constant
michaelis-menten constant formula
the lower the Km value, the greater the affinity of the substrate
When the Km is NOT greater than the affinity of the enzyme and substrate, what is used to indicate this?
- "other site"
- kinetics influenced by substrates that bind to enzymes at locations other than active sites
- quaternary protein structure (more than one polypeptide chain)
- each chain has its active site
- few active sites occupied = low affinity of enzyme and substrate
Allosteric enzyme active sites increased in occupation
- conformational change in the protein
- increased binding of substrate
- velocity of enzyme is increased
Allosteric enzymes: second method kinetics influenced:
- activator substances occupy sites on enzyme away from active sites
- change in form T to R form
- Biological way of jumpstart reactions at low concentration of substrate
Competitive enzyme inhibition
inhibitor replaces or competes with substrate for active site
Non-competitive enzyme inhibition
inhibitor binds close to active site and prevents reaction despite the binding of the substrate to the active site
Uncompetitive enzyme inhibition
- two substrates are required for the reaction
- inhibitor binds close to the active site after the attachment of the first substrate to the site; the inhibitor prevents second substrate from binding
- Example: aldose reductase
- enzyme of tear film
- destroys gram positive bacteria by breaking glycan of peptidoglycan
- levels of lysozyme reflect tear film dysfunction
- aqueous deficiency
- 1.3 mg of lysozyme found /ml of tears
- described by Alexander Flemming in 1992
- first located in nasal mucosa
Micrococcus agar diffusion assay
- tear film on tissue paper
- dish w/ 5x107 microorganisms
- 24 hours at 37 degrees C
- zone of lysis (zone of clearing) is measured and converted to enzyme activity
- an enzyme located in plasma membrane of cells
- ocular functions:
- control of corneal hydration
- production of aqueous humor
- good allosteric enzyme example
Endothelium of Cornea
- single layer of polygonal cells covering posterior surface of the cornea
- cell density declines with age
- newborn cell density = more than 5500 cells/ square millimeter
- adult cell density = 2500-3000 cells/square millimeter
- minimum cell density = 400-700 cells/mm2
- active in transport, synthesis, and secretory functions
- large nucleus
- numerous mitochondria
- prominent endoplasmic reticulum
- Golgi apparatus
Endothelial Cell-Cell Adhesion: Tight Junctions
- (macula occludens)
- function as a barrier
- do not completely encircle cell like zonula occludens
- they are a leaky barrier between aqueous and stroma!
Endothelial Cell-Cell Adhesion: Gap Junctions
- function is intercellular communication
- mainly in lateral membranes
- do not contribute to the endothelial barrier
Two factors that contribute to prevent stromal swelling
- barrier function of endothelium
- pump function of endothelium
- Water is maintained at 78%
When endothelium is disrupted, the cornea swells at what rate?
When metabolic pump is disrupted, cornea swells at what rate?
In cool temperatures, cornea will... ?
Endothelial Transport NA+ K+ ATPase Pump
- located in the basolateral membrane
- about 1.5x106 pump sites/cell
Ouabain (drug) and Digoxin
- inhibits Na+ K+ ATPase pump
- stops sodium transport
- causes corneal swelling
- prevents temperature reversal
- eliminates the transendothelial potential difference
- increases pump site density
- greater capacity for the pump to counteract the leak
Inflamed, Edematous cornea
decreased pump site density despite increased permeability
Na, K-ATPase pump function
- Na ions are transported by pump into channels between endothelial cells
- this creates osmotic pressure and water follows
- directionality is helped by density of sodium ions in Descemets membrane
- functional unit for production of aqueous humor and its secretion
- made of capillaries, stroma, and pigmented and non-pigmented epithelia (deep to superficial)
- Carefully controlled filtrate of blood
- produced in ciliary body
- antioxidant source for the corneal endothelium and the lens
- carries oxygen
- helps maintain shape of globe
- serves as shock absorber
- carbohydrate metabolism (aerobic and anaerobic)
- aerobic is most efficient process
- anaerobic requires more sugar molecules but produces energy quickly
Lactate dehydrogenase (LDH) subunits
- H - heart; aerobic
- M - muscle; anaerobic
- K - cancer cells, photoreceptor cells; anaerobic
- all called "isozymes"
Anaerobic sugar metabolism of corneal epithelium
- closed eye conditions, contact lens wear, or similar unfavorable environments
- low partial pressure of oxygen
- can support cells survival with partial pressures of oxygen of 15-20 mmHg
Anaerobic sugar metabolism of crystalline lens
- energy demand is low
- lack of blood vessels
- deep fibers lack subcellular organelles
Anaerobic sugar metabolism of Retina/Photoreceptors
- energy demand high
- aerobic tissue (blood supply present)
- Warburg effect: production of lactate coupled with high consumption of glucose and oxygen
- small organic non-protein molecules that carry chemical groups between enzymes
- sometimes called "co-substrates"
Vitamin B3 deficiency
Pellagra (dermatitis, depression, and diarrhea)
Vitamin B2 deficiency
Skin gland malfunction, dryness/scaling of mouth, photophobia
Vitamin B1 deficiency
Berberi (muscle loss, gatigue, depression, loss of eye coordination
Vitamin B6 deficiency
similar to others
Vitamin B12 deficiency
Pernicious anemia and brain demyelination
Vitamin C deficiency (ascorbic acid)
Scurvy (incomplete formation of collagen)