Chemicals have properties. Biochemicals have both properties and ways of interacting with other chemicals eg. Fats vs. Carbohydrates. Fats form monolayers in water. Cell parts and Cells are the basic unit of life. They have attributes: Reproduction, metabolism, storage, transport, movement. We will look at a Prokaryotic cell from the outside in with an eye toward relating cell part properties to the biochemicals which compose them.
1. External Structures:
a. Glycocalyx or Capsule (computer slides)
1. Slime formation prevents dehydration.
2. allows for colony stability under adverse circumstances. Can be a big problem with biofilm development in catheters or industrial pipes. Dangerous source of infection in hospitals which is not readily removed by disinfection.
3. Non pathogenic organisms also have a capsule. Alcaligenes viscolactis is one of them.
4. Capsule can interfere with immune defenses of the host. It can interfere with phagocytosis. It swells during an infection, in many cases (Quellung reaction base of serological testing).
5. Streptococcus mutans sticks to teeth due to a capsule. Plaque becomes tartar if not removed.
6. Alcaligenes Viscolactis in milk.
b. Flagella--protein = flagellin. Anchored through the cell wall to a series of rings which rotate. Flagella is rigid and works more like an outboard motor than a whip. Must rotate thousands of times a minute in order to push cells with all that surface through water. 4mm to 6 mm per minute in E.coli. Flagella can rotate at 2500 rpm. All spirilla have flagella (axial filaments or endoflagella) about 1/2 of the bacilli and a few cocci.
5. Runs and tumbles. Taxis, chemo, photo, thermo, magneto.
When a stimulus is present tumbles are inhibited and the flagella turns counterclockwise constantly toward the stimulus. This is positive taxis. When a substance initiates tumbles the cell's flagella go clockwise and the cell tumbles, resuming its run in another direction.
c. cell walls (exceptions--Mycobacterium, mycoplasma, archaebacteria, L-forms, spheroplasts, protoplasts.
1. Surrounding, encasing molecule is peptidoglycan. Visual.
NAM-NAG-NAM-NAG-NAM-NAG-NAM-NAG. This is the Glycan portion of peptidoglycan. Held together by beta linkages. Each Glycan strand held together by chain of amino acids to another strand. "Chain Link Fence" analogous.
2. Examine differences between Gram + and Gram - cell walls.
To the left is a gram stain of Staphylococcus epidermisdone in our lab. The spherical cocci are tiny and are gram positive because they retain the primary stain crystal violet.
Below is a gram stain of a mixed culture of Micrococcus luteus (box like arrangements of cocci) with Escherishia coli. Micrococcus is gram positive while E.coli is gram negative. E. coli consists of very small delicate rods seen pink because they lose the primary stain as they are gram negative.
The gram negative cell wall of E. coli is much thinner than the wall of Micrococcus. Coli's cell wall also contains less peptidoglycan and more lipopolysaccharide.
3. Explain LPS and its role as an endotoxin and fever producer.
4. Also mention Spheroplasts and Protoplasts, L forms. Chlamydia and Rickettsia.
d. pili and fimbriae: Structures that allow sticking to surfaces---important in pathogenesis eg. UTI'"s, biofilms, teeth, glass, rocks (Pellicle formation in culture). Some Pili specialized. Sex pilus. 1946 experiment with minimal media showed genes transfered requiring contact.Define: auxotroph, prototroph, mutation rate. Minimal media (Glucose salts media). Mating types. F+ and F- (movie) e. other structures:
1. Chromatin Body--Long cirucular piece of DNA. Couple of thousand genes. It is haploid and relatively easy to manipulate eg...stick other genes into using bioengineering techniques.
2. Plasmids---small cirucular pieces of DNA (F factor is on a plasmid, Antibiotic resistance carried on plasmids). They are easily transferred from one cell to another. During sexual transfer recipient becomes F+.
I. Structure supports function. Function is physiology and metabolism. Metabolism is the sum of the biochemical reactions inside a cell. Lets take a closer look at how biochemical reactions or just ordinary chemical reactions occur.
A. Collision theory: This makes sense as an explanation of how two molecules may interact to form bonds. One bangs into another. If the collision is energetic enough--we see this as the reaction mixture being hot enough---or as Activation Energy---and if the vulnerable parts of the molecules are the ones that get hit, we get bonds breaking and new bonds forming.
H2O2 + I- ----> IO- + H2OIO-
+ H2O2 ----> O2 + H2O + I-
Two points should be made about the above reaction. It takes two steps to happen. I- is not used up in the reaction. The reaction occurs due to contact with I- where bonds are broken and new bonds formed. What bond is broken and what new bond forms? Also, I- is an inorganic small ion. Small inorganic speciesthat speed reactions are called catalysts.
ENERGY DIAGRAM WITH AND WITHOUT A CATALYST
Define:Activiation Energy, catalyst (two examples), exergonic reaction, endergonic reaction. Show hydrogen peroxide and Iodide catalyst and catalase reaction. Explain how catalase identifies organisms Staph and Strep.
ENZYME ATTRIBUTES
1. Large protein molecules at approx 40,000 amu, Glucose substrate about 180 amu.
2. Denatured by heat--cooked potato vs. raw potato. Catalyst versus enzyme.
3. Lowers Activation energy by substrate binding at special place on the enzyme called the "Active Site"! There are other important sites on the enzyme but the active site is most important for catalysis.
4. Cofactors which bind and release from the Active site are often required (not always)...Vitamins and minerals act as cofactors. B vitamins often needed for energy reactions.
5 Efficient and able to process thousands of substrates per second. So only a small amount of enzyme needed to do reactions.
6. Can be interfered with. Example: Penicillin combines with an enzyme which is needed to put peptide cross links in cell wall material (Why is penicillin more active against gram positives?)
7. Delicate in that environmental conditions for use are narrow---pH, Temperature, Osmotic pressure, concentrations of substrates must be withing proper limits. Extremes can denature and kill cells.
FACTORS THE AFFECT THE ACTIVITY OF ENZYMES
1. TEMPERATURE
2. PH
3. CONCENTRATION OF SUBSTRATE
4. CONCENTRATION OF ENZYME
GRAPH THE AFOREMENTIONED VARIABLES AGAINST REACTION RATE..
ENZYME INHIBITION
1. Competitive Inhibition: Show succinic to fumaric acid and malonic acid inhibitor.
Succinic Acid Ž Fumaric Acid
Malonic Acid Inhibits the above reaction
2.Show structure of folic acid and PABA:
All inhibitors act at the active site. The inhibition is reversible by adding more substrate. New drugs are made which retain the properties of the inhibitor but which have new properties, like remaining in the urine longer etcetera.
When an inhibitor "looks like" the substrate of an enzyme it can inhibit it either reversibly or irreversibly. In irreversible inhibition the contact usually occurs in a place by covalent bonding and not just by stearic reactions.
Feedback Inhibition, Allosteric Inhibition, Endproduct Inhibition. Precursor Activation, Energy link control. Positive and Negative Feedback. All involve ligands and receptor.
INHIBITION AT THE LEVEL OF THE GENE.
FIRST DISCOVERED WAS THE LACTOSE ARRAY OF GENES THAT E.COLI HAS.
CALLED THE LAC OPERON--JACOB AND MONOD WORKED OUT THE MODEL: Wavy line made at R is repressor protein from R = repressor gene. I is the Incucer gene and a, b, c, and d are structural genes (make enzymes for lactose fermentation. Inducer can bind with repressor protein and "shut off".
Ancients felt the world filled with invisible spirits which would explain things we couldn't understand.
a. Death and Disease, Disability (there has to be a reason) WE STILL STRUGGLE WITH THESE THINGS IDEAS TODAY.
Greeks had anthropomorphic gods who interacted with them and could cause disease. Later Greeks lost faith in their gods and formulated other ideas. They were noted thinkers.
Example: Hippocrates--disease comes from an imbalance of intrinsic factors (nutrition) and extrinsic factors--air, exercise, etc.
Four elements of importance to balance: blood, phlegm, yellow bile, black bile. When these get out of balance problems occur. Bleeding to intervene. Today we infuse (add) blood with different ends in mind.
Hebrews and Egyptians believed in God and an afterlife. Some biblical accounts indicate that there was a vague notion of contagion developing --"Don't sleep in the House of a Leper". But also could get leprosy by angering the Lord. Angry Jewish God changed in Christianity. Jewish God brought plagues famines and disease to Egyptians, for example.
Aristotle and others believed in abiogenesis. Life came from inanimate material. Myths among many ancient people talk of the origin of man, even, from decaying corn. Shakespeare wrote about crockadiles coming from the mud of the Nile.
Van Helmont wrote recipe for mice---dirty underwear, corn, in a vessel---mice come out fully formed. Solves another inexplicable problem: The origin of life.
Until this is properly understood we could never understand contagion. 1546
Fracastorius of Verona wrote of contagium vivum--immersed in a syphilis epidemic at the time. Other terms "Seminaria morbi". Described transmission through inanimate objects--fomites--Through air--"ad distans" and through direct contact. Work is ignored, no evidence for any of this. It just made sense to him.
1609 Janssen and Galileo grind lenses to produce low resolution microscopes---microorganisms below the resolution achieved. But improvements were to come.
Hooke--Discovers cells with an improved microscope.
Schleiden and Schwann discover all plants and animals are composed of cells.
1650's Leewenhoek--Delft Holland in the textile industry and part time lens grinder. Got good resolution to allow about 3 or 4 hundred X useful magnification. He put hay and pepper into water and then looked at it through his microscope. He saw microbes in an infusion as seen below.
Leewenhoek saw bacteria, protozoa, yeasts and described all the microbial forms we now know, except for viruses. Although he is not mentioned in the science literature as observing his animalcules divide, nevertheless he believed spontaneous generation was untrue and in his original papers called the idea a "bad joke" as related by Dr. Moll at the University of Amsterdam.
Left Image depicts a hay infusion. Hay is placed in pure water for a few days and protozoa, bacteria, fungi, and algae develop. The larger cells on the left are ciliate protozoa. The small rod shaped organisms in single and pairs are bacteria. The image was made black and white at Northwestern to save space. This is likely very close to what Leewenhoek saw in the 17th Centrury.
Francesco Redi, in the 1700's did a simple experiment to show flies needed parents. Used cheesecloth screening and meat.
Spallanzani, a monk, in the first part of the 18th Century boiled and sealed broths. When he was careful no microbes developed. His work was criticized by Needham, a Welchman of the Royal Society in that other factors, excluded by Spallanzani, were needed for Sp. Gen. Notably air. Needham performed similar experiments, sloppily executed, in which microbes grew from contamination.
Spallanzani died before he could clearly disprove Needham.
Louis Pasteur, a noted chemist, took up the challance and utilized broths allowing air but disallowing microbes. Grew broths at different altitudes and in a dusty cellar. Used broths with cotton to show the germs accumulated on cotton. Did the Swan neck tube experiment.
Ignaz Semmelweis, an eastern european physician working in a ViennaHospital, noticed the wards where delivery occurred by midwives had 10X less Puerperal Fever and deaths than those tended to by doctors. He showed he could dramatically decrease "Childbed Fever" by having doctors wash hands in chlorine water after dissection of cadavers and between patients. Was fired for blaming deaths on doctors who didn't wash hands.
Oliver Wendel Holmes wrote on the Contagiousness of Puerperal Fever". Author, Physician, and Anatomy Professor. Late 19th Century.
Lister used antiseptics on wounds and during surgery. He showed they healed much faster with the antiseptic treatment. Was also the first to isolate a pure culture by serial dilution: Bacterium lactis.
Pasteur wanted to isolate a bacterium in pure culture that caused disease. Began working with Anthrax.
Robert Koch in Germany did too. He worked with various preparations to provide solid culture media. A woman working in his lab tried agar, recommended by a cook who used it to solidify puddings because it stayed solid at warm temps. Koch was using sterile potato slices for media.
Koch isolated anthrax and formulated his Postulates.
Pasteur went to work on chicken cholera and discovered one could attenuate cultures and produce artifical vaccines.
Pasteur solved the riddle of rancid wines in France's vinyards. Recommended sterile technique and Pasteurization (applied to milk and became a central method for controling TB, Diptheria, and other diseases).
Weinogradski and others showed soil bacteria recycle nutrients.
Chamberland developed a bacterial filter. Resulted in:
a. discovery of viruses
b. discovery of toxins
Pasteur produced attenuated rabies virus and rabies vaccination procedure. Tried it on Joseph Meister. It worked.
During the 20th Century we have:
1. Development of viral culture techniques and attenuation
2. Development of the electron microscope.
3. Discovery of antibiotics (Fleming and Dubos)
4. Discovery of Prions (Pruissner)
5. Bioengineering--removal and replication of genes--incorporating them into microbes and switching them on.
8. Translation of the entire genome of some microorganisms e.g. yeast.
All not rosy---reemergence of infectious diseases a constant problem. St Louis Encephalitis, West Nile Virus, Lyme disease, AIDS, Hanta virus Sin Nombre,Ebola outbreaks, E. coli, Antibiotic resistant bacterial strains and so on.
