Beta-glucuronidases are members of the glycosidase family of enzymes that catalyze breakdown of complex carbohydrates. Human β-glucuronidase is a type of glucuronidase that catalyzes hydrolysis of β-D-glucuronic acid residues from the non-reducing end of mucopolysaccharides such as heparan sulfate. Human β-glucuronidase is located in the lysosome. In the gut, brush border β-glucuronidase converts conjugated bilirubin to the unconjugated form for reabsorption. Beta-glucuronidase is present in breast milk, which contributes to neonatal jaundice; the protein is encoded by the GUSB gene. Human β-glucuronidase is synthesized as an 80 kDa monomer before proteolysis removes 18 amino acids from the C-terminal end to form a 78 kDa monomer. Beta-glucuronidase exists as a 332 kDa homotetramer. Beta-glucuronidase contains several notable structural formations, including a type of beta barrel known as a jelly roll barrel and a TIM barrel. Human β-glucuronidase is homologous to the Escherichia coli enzyme β-galactosidase.
This homologous relationship, along with the knowledge that glycosidases perform hydrolysis catalyzed by two acidic residues, enabled the development of a mechanistic hypothesis. This hypothesis proposes that the two glutamic acid residues Glu540 and Glu451 are the nucleophilic and acidic residues and that the tyrosine residue Tyr504 is involved in catalysis. In support of this hypothesis, experimental mutations in any of these three residues result in large decreases of enzymatic activity. Increased activity of an E451A mutant enzyme after addition of azide is consistent with Glu451 as the acid/base residue. Using analysis of labeled β-glucuronidase peptides after hydrolysis of a substrate that enters a stable intermediate stage, researchers have determined that Glu540 is the nucleophilic residue. Though the particular type of nucleophilic substitution employed by β-glucuronidase is unclear, evidence for the mechanisms of their homologues in the glycosidase family suggests that these reactions are qualitatively SN2 reactions.
The reactions proceed through a transition state with oxocarbenium ion characteristics. These mechanisms, because of this oxocarbenium characteristic of the transition state, were suggested to be SN1 reactions proceeding through a discrete oxocarbenium ion intermediate. However, more recent evidence suggests that these oxocarbenium ion states have lifetimes of 10 femtoseconds - 0.1 nanoseconds. These lifetimes are too short to assign to a reaction intermediate. From this evidence, it appears that these reactions, while having an SN1 appearance due to the oxocarbenium ion characteristics of their transition states, must be qualitatively SN2 reactions; the specific activity of Tyr504 in the catalytic mechanism is unclear. Through comparison to the structural data of the homologous enzyme xylanase, it has been suggested that Tyr504 of β-glucuronidase might stabilize the leaving nucleophile or modulate its activity. In addition to these residues, a conserved asparagine residue has been suggested to stabilize the substrate through the action of a hydrogen bond at the 2-hydroxyl group of the sugar substrate.
Deficiencies in β-glucuronidase result in the autosomal recessive inherited metabolic disease known as Sly syndrome or Mucopolysaccharidosis VII. A deficiency in this enzyme results in the build-up of non-hydrolyzed mucopolysaccharides in the patient; this disease can be debilitating for the patient or can result in hydrops fetalis prior to birth. In addition, mental retardation, short stature, coarse facial features, spinal abnormalities, enlargement of liver and spleen are observed in surviving patients; this disease has been modeled in a strain of mice as well as a family of dogs. More researchers have discovered a feline family that exhibits deficiencies in β-glucuronidase activity; the source of this reduction of activity has been identified as an E351K mutation. Glu351 is conserved in mammalian species. Examination of the human X-ray crystal structure suggests that this residue, buried deep within the TIM barrel domain, may be important for stabilization of the tertiary structure of the enzyme.
In the crystal structure, it appears that Arg216, a member of the jelly roll domain of the protein, forms a salt bridge with Glu352. In molecular biology, β-glucuronidase is used as a reporter gene to monitor gene expression in mammalian and plant cells. Monitoring β-glucuronidase activity through the use of a GUS assay allows determination of the spatial and temporal expression of the gene in question. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing and Informatics at the University of California, San Francisco. Alpha-glucuronidase Glucuronosyl-disulfoglucosamine glucuronidase Glycyrrhizinate beta-glucuronidase Glucuronidase at the US National Library of Medicine Medical Subject Headings Updated research on reporter glucuronidase and other reporters from Reportergene Database of Catalytic Mechanism Research and other information on beta-glucuronidase
Cambridge is a university city and the county town of Cambridgeshire, England, on the River Cam 50 miles north of London. At the United Kingdom Census 2011, its population was 123,867 including 24,506 students. Cambridge became an important trading centre during the Roman and Viking ages, there is archaeological evidence of settlement in the area as early as the Bronze Age; the first town charters were granted in the 12th century, although modern city status was not conferred until 1951. The world-renowned University of Cambridge was founded in 1209; the buildings of the university include King's College Chapel, Cavendish Laboratory, the Cambridge University Library, one of the largest legal deposit libraries in the world. The city's skyline is dominated by several college buildings, along with the spire of the Our Lady and the English Martyrs Church, the chimney of Addenbrooke's Hospital and St John's College Chapel tower. Anglia Ruskin University, which evolved from the Cambridge School of Art and the Cambridgeshire College of Arts and Technology has its main campus in the city.
Cambridge is at the heart of the high-technology Silicon Fen with industries such as software and bioscience and many start-up companies born out of the university. More than 40% of the workforce have a higher education qualification, more than twice the national average; the Cambridge Biomedical Campus, one of the largest biomedical research clusters in the world, is soon to house premises of AstraZeneca, a hotel and the relocated Papworth Hospital. The first game of association football took place at Parker's Piece; the Strawberry Fair music and arts festival and Midsummer Fair are held on Midsummer Common, the annual Cambridge Beer Festival takes place on Jesus Green. The city is adjacent to the A14 roads. Cambridge station is less than an hour from London King's Cross railway station. Settlements have existed around the Cambridge area since prehistoric times; the earliest clear evidence of occupation is the remains of a 3,500-year-old farmstead discovered at the site of Fitzwilliam College.
Archaeological evidence of occupation through the Iron Age is a settlement on Castle Hill from the 1st century BC relating to wider cultural changes occurring in southeastern Britain linked to the arrival of the Belgae. The principal Roman site is a small fort Duroliponte on Castle Hill, just northwest of the city centre around the location of the earlier British village; the fort was bounded on two sides by the lines formed by the present Mount Pleasant, continuing across Huntingdon Road into Clare Street. The eastern side followed Magrath Avenue, with the southern side running near to Chesterton Lane and Kettle's Yard before turning northwest at Honey Hill, it was converted to civilian use around 50 years later. Evidence of more widespread Roman settlement has been discovered including numerous farmsteads and a village in the Cambridge district of Newnham. Following the Roman withdrawal from Britain around 410, the location may have been abandoned by the Britons, although the site is identified as Cair Grauth listed among the 28 cities of Britain by the History of the Britons.
Evidence exists that the invading Anglo-Saxons had begun occupying the area by the end of the century. Their settlement – on and around Castle Hill – became known as Grantebrycge. Anglo-Saxon grave goods have been found in the area. During this period, Cambridge benefited from good trade links across the hard-to-travel fenlands. By the 7th century, the town was less significant and described by Bede as a "little ruined city" containing the burial site of Etheldreda. Cambridge was on the border between the East and Middle Anglian kingdoms and the settlement expanded on both sides of the river; the arrival of the Vikings was recorded in the Anglo-Saxon Chronicle in 875. Viking rule, the Danelaw, had been imposed by 878 Their vigorous trading habits caused the town to grow rapidly. During this period the centre of the town shifted from Castle Hill on the left bank of the river to the area now known as the Quayside on the right bank. After the Viking period, the Saxons enjoyed a return to power, building churches such as St Bene't's Church, merchant houses and a mint, which produced coins with the town's name abbreviated to "Grant".
In 1068, two years after his conquest of England, William of Normandy built a castle on Castle Hill. Like the rest of the newly conquered kingdom, Cambridge fell under the control of the King and his deputies; the first town charter was granted by Henry I between 1120 and 1131. It recognised the borough court; the distinctive Round Church dates from this period. In 1209, Cambridge University was founded by students escaping from hostile townspeople in Oxford; the oldest existing college, was founded in 1284. In 1349 Cambridge was affected by the Black Death. Few records survive; the town north of the river was affected being wiped out. Following further depopulation after a second national epidemic in 1361, a letter from the Bishop of Ely suggested that two parishes in Cambridge be merged as there were not enough people to fill one church. With more than a third of English clergy dying in the Black Death, four new colleges were established at the university over the following years to train new clergymen, namely Gonville Hall, Trinity Hall, Corpus Christi and Clare.
In 1382 a revised town charter effects a "diminution of the liberties that the community had enjoyed", due to Cambridge's pa
Histology microanatomy, is the branch of biology which studies the tissues of animals and plants using microscopy. It is studied using a light microscope or electron microscope, the specimen having been sectioned and mounted on a microscope slide. Histological studies may be conducted using tissue culture, where live animal cells are isolated and maintained in an artificial environment for various research projects; the ability to visualize or differentially identify microscopic structures is enhanced through the use of staining. Histology is one of the major preclinical subjects in medical school. Medical students are expected to be familiar with the morphological features and function of all cells and tissues of the human body from an early stage of their studies, so histology stretches over several semesters. Histopathology, the microscopic study of diseased tissue, is an important tool in anatomical pathology, since accurate diagnosis of cancer and other diseases requires histopathological examination of samples.
Trained physicians licensed pathologists, are the personnel who perform histopathological examination and provide diagnostic information based on their observations. The trained personnel who prepare histological specimens for examination are histotechnicians, histotechnologists, histology technicians, histology technologists, medical scientists, medical laboratory technicians, or biomedical scientists, their support workers, their field of study is called histotechnology. In the 17th century, Italian Marcello Malpighi invented one of the first microscopes for studying tiny biological entities. Malpighi analysed several parts of the organs of bats and other animals under the microscope. Malpighi, while studying the structure of the lung, noticed its membranous alveoli and the hair-like connections between veins and arteries, which he named capillaries, his discovery established how the oxygen enters the blood stream and serves the body. In the 19th century, histology was an academic discipline in its own right.
The French anatomist Bichat introduced the concept of tissue in anatomy in 1801, the term "histology" first appeared in a book of Karl Meyer in 1819. Bichat described twenty-one human tissues, which can be subsumed under the four categories accepted by histologists; the usage of illustrations in histology, deemed as useless by Bichat, was promoted by Jean Cruveilhier. During the 19th century, many fixation techniques were developed by Adolph Hannover, Franz Schulze and Max Schultze, Alexander Butlerov and Benedikt Stilling. In the early 1830, Purkynĕ invented a microtome with high precision. Mounting techniques were developed by Rudolf Heidenhain, Salomon Stricker, Andrew Pritchard and Edwin Klebs. Koelliker's laboratory developed haematoxylin staining, in 1870s, Vysockij introduced eosin as a double or counter staining; the 1906 Nobel Prize in Physiology or Medicine was awarded to histologists Camillo Golgi and Santiago Ramon y Cajal. They had conflicting interpretations of the neural structure of the brain based on differing interpretations of the same images.
Cajal won the prize for his correct theory, Golgi for the silver staining technique he invented to make it possible. There are four basic types of animal tissues: muscle tissue, nervous tissue, connective tissue, epithelial tissue. All tissue types are subtypes of these four basic tissue types. Epithelium: the lining of glands, bowel and some organs like the liver and kidney Endothelium: the lining of blood and lymphatic vessels Mesothelium: the lining of pleural and pericardial spaces Mesenchyme: the cells filling the spaces between the organs, including fat, bone and tendon cells Blood cells: the red and white blood cells, including those found in lymph nodes and spleen Neurons: any of the conducting cells of the nervous system Germ cells: reproductive cells Placenta: an organ characteristic of true mammals during pregnancy, joining mother and offspring, providing endocrine secretion and selective exchange of soluble, but not particulate, blood-borne substances through an apposition of uterine and trophoblastic vascularised parts Stem cells: cells with the ability to develop into different cell typesThe tissues from plants and microorganisms can be examined histologically.
Their structure is different from animal tissues. For plants, the study of their tissues is more called as plant anatomy, with the following main types: Dermal tissue Vascular tissue Ground tissue Meristematic tissue Chemical fixatives are used to preserve tissue from degradation, to maintain the structure of the cell and of sub-cellular components such as cell organelles; the most common fixative for light microscopy is 10% neutral buffered formalin. For electron microscopy, the most used fixative is glutaraldehyde as a 2.5% solution in phosphate buffered saline. These fixatives preserve tissues or cells by irreversibly cross-linking proteins; the main action of these aldehyde fixatives is to cross-link amino groups in proteins through the formation of methylene bridges, in the case of formaldehyde, or by C5H10 cross-links in the case of glutaraldehyde. This process, while preserving the structural integrity of the cells and tissue can damage the biological functionality of proteins enzymes, and
Bacteria are a type of biological cell. They constitute a large domain of prokaryotic microorganisms. A few micrometres in length, bacteria have a number of shapes, ranging from spheres to rods and spirals. Bacteria were among the first life forms to appear on Earth, are present in most of its habitats. Bacteria inhabit soil, acidic hot springs, radioactive waste, the deep portions of Earth's crust. Bacteria live in symbiotic and parasitic relationships with plants and animals. Most bacteria have not been characterised, only about half of the bacterial phyla have species that can be grown in the laboratory; the study of bacteria is known as a branch of microbiology. There are 40 million bacterial cells in a gram of soil and a million bacterial cells in a millilitre of fresh water. There are 5×1030 bacteria on Earth, forming a biomass which exceeds that of all plants and animals. Bacteria are vital in many stages of the nutrient cycle by recycling nutrients such as the fixation of nitrogen from the atmosphere.
The nutrient cycle includes the decomposition of dead bodies. In the biological communities surrounding hydrothermal vents and cold seeps, extremophile bacteria provide the nutrients needed to sustain life by converting dissolved compounds, such as hydrogen sulphide and methane, to energy. Data reported by researchers in October 2012 and published in March 2013 suggested that bacteria thrive in the Mariana Trench, with a depth of up to 11 kilometres, is the deepest known part of the oceans. Other researchers reported related studies that microbes thrive inside rocks up to 580 metres below the sea floor under 2.6 kilometres of ocean off the coast of the northwestern United States. According to one of the researchers, "You can find microbes everywhere—they're adaptable to conditions, survive wherever they are."The famous notion that bacterial cells in the human body outnumber human cells by a factor of 10:1 has been debunked. There are 39 trillion bacterial cells in the human microbiota as personified by a "reference" 70 kg male 170 cm tall, whereas there are 30 trillion human cells in the body.
This means that although they do have the upper hand in actual numbers, it is only by 30%, not 900%. The largest number exist in the gut flora, a large number on the skin; the vast majority of the bacteria in the body are rendered harmless by the protective effects of the immune system, though many are beneficial in the gut flora. However several species of bacteria are pathogenic and cause infectious diseases, including cholera, anthrax and bubonic plague; the most common fatal bacterial diseases are respiratory infections, with tuberculosis alone killing about 2 million people per year in sub-Saharan Africa. In developed countries, antibiotics are used to treat bacterial infections and are used in farming, making antibiotic resistance a growing problem. In industry, bacteria are important in sewage treatment and the breakdown of oil spills, the production of cheese and yogurt through fermentation, the recovery of gold, palladium and other metals in the mining sector, as well as in biotechnology, the manufacture of antibiotics and other chemicals.
Once regarded as plants constituting the class Schizomycetes, bacteria are now classified as prokaryotes. Unlike cells of animals and other eukaryotes, bacterial cells do not contain a nucleus and harbour membrane-bound organelles. Although the term bacteria traditionally included all prokaryotes, the scientific classification changed after the discovery in the 1990s that prokaryotes consist of two different groups of organisms that evolved from an ancient common ancestor; these evolutionary domains are called Archaea. The word bacteria is the plural of the New Latin bacterium, the latinisation of the Greek βακτήριον, the diminutive of βακτηρία, meaning "staff, cane", because the first ones to be discovered were rod-shaped; the ancestors of modern bacteria were unicellular microorganisms that were the first forms of life to appear on Earth, about 4 billion years ago. For about 3 billion years, most organisms were microscopic, bacteria and archaea were the dominant forms of life. Although bacterial fossils exist, such as stromatolites, their lack of distinctive morphology prevents them from being used to examine the history of bacterial evolution, or to date the time of origin of a particular bacterial species.
However, gene sequences can be used to reconstruct the bacterial phylogeny, these studies indicate that bacteria diverged first from the archaeal/eukaryotic lineage. The most recent common ancestor of bacteria and archaea was a hyperthermophile that lived about 2.5 billion–3.2 billion years ago. Bacteria were involved in the second great evolutionary divergence, that of the archaea and eukaryotes. Here, eukaryotes resulted from the entering of ancient bacteria into endosymbiotic associations with the ancestors of eukaryotic cells, which were themselves related to the Archaea; this involved the engulfment by proto-eukaryotic cells of alphaproteobacterial symbionts to form either mitochondria or hydrogenosomes, which are still found in all known Eukarya. Some eukaryotes that contained mitochondria engulfed cyanobacteria-like organisms, leading to the formation of chloroplasts in algae and plants; this is known as primary endosymbiosis. Bacteria display a wide diversity of sizes, called morphologies.
Bacterial cells are about one-tenth the size of eukaryotic cells
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria; the GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm, in the lower green portion of the visible spectrum; the fluorescence quantum yield of GFP is 0.79. The GFP from the sea pansy has a single major excitation peak at 498 nm. GFP makes for an excellent tool in many forms of biology due to its ability to form internal chromophore without requiring any accessory cofactors, gene products, or enzymes / substrates other than molecular oxygen. In cell and molecular biology, the GFP gene is used as a reporter of expression, it has been used in modified forms to make biosensors, many animals have been created that express GFP, which demonstrates a proof of concept that a gene can be expressed throughout a given organism, in selected organs, or in cells of interest.
GFP can be introduced into animals or other species through transgenic techniques, maintained in their genome and that of their offspring. To date, GFP has been expressed in many species, including bacteria, fungi and mammals, including in human cells. Scientists Roger Y. Tsien, Osamu Shimomura, Martin Chalfie were awarded the 2008 Nobel Prize in Chemistry on 10 October 2008 for their discovery and development of the green fluorescent protein. In the 1960s and 1970s, GFP, along with the separate luminescent protein aequorin, was first purified from Aequorea victoria and its properties studied by Osamu Shimomura. In A. victoria, GFP fluorescence occurs when aequorin interacts with Ca2+ ions, inducing a blue glow. Some of this luminescent energy is transferred to the GFP. However, its utility as a tool for molecular biologists did not begin to be realized until 1992 when Douglas Prasher reported the cloning and nucleotide sequence of wtGFP in Gene; the funding for this project had run out, so Prasher sent cDNA samples to several labs.
The lab of Martin Chalfie expressed the coding sequence of wtGFP, with the first few amino acids deleted, in heterologous cells of E. coli and C. elegans, publishing the results in Science in 1994. Frederick Tsuji's lab independently reported the expression of the recombinant protein one month later. Remarkably, the GFP molecule folded and was fluorescent at room temperature, without the need for exogenous cofactors specific to the jellyfish. Although this near-wtGFP was fluorescent, it had several drawbacks, including dual peaked excitation spectra, pH sensitivity, chloride sensitivity, poor fluorescence quantum yield, poor photostability and poor folding at 37 °C; the first reported crystal structure of a GFP was that of the S65T mutant by the Remington group in Science in 1996. One month the Phillips group independently reported the wild-type GFP structure in Nature Biotechnology; these crystal structures provided vital background on chromophore formation and neighboring residue interactions.
Researchers have modified these residues by directed and random mutagenesis to produce the wide variety of GFP derivatives in use today. Further research into GFP has shown, resistant to detergents, guanidinium chloride treatments, drastic temperature changes. Due to the potential for widespread usage and the evolving needs of researchers, many different mutants of GFP have been engineered; the first major improvement was a single point mutation reported in 1995 in Nature by Roger Tsien. This mutation improved the spectral characteristics of GFP, resulting in increased fluorescence, a shift of the major excitation peak to 488 nm, with the peak emission kept at 509 nm; this matched the spectral characteristics of available FITC filter sets, increasing the practicality of use by the general researcher. A 37 °C folding efficiency point mutant to this scaffold, yielding enhanced GFP, was discovered in 1995 by the laboratories of Thastrup and Falkow. EGFP allowed the practical use of GFPs in mammalian cells.
EGFP has an extinction coefficient of 55,000 M−1cm−1. The fluorescence quantum yield of EGFP is 0.60. The relative brightness, expressed as ε•QY, is 33,000 M−1cm−1. Superfolder GFP, a series of mutations that allow GFP to fold and mature when fused to poorly folding peptides, was reported in 2006. Many other mutations have been made, including color mutants. BFP derivatives contain the Y66H substitution, they exhibit a broad absorption band in the ultraviolet centered close to 380 nanometers and an emission maximum at 448 nanometers. A green fluorescent protein mutant that preferentially binds Cu has been developed. BFPms1 have several important mutations including and the BFP chromophore,Y145F for higher quantum yield, H148G for creating a hole into the beta-barrel and several other mutations that increase solubility. Zn binding increases fluorescence intensity, while Cu binding quenches fluorescence and shifts the absorbance maximum from 379 to 444 nm. Therefore, they can be used as Zn biosensor.
Chromophore binding. The critical mutation in cyan derivatives is the Y66W substitution, which causes