Medical brain notes 43

  USING APOPTOSIS TO TREAT CANCER Scientists have been trying to induce death in cancer cells for many years, usually with radiation or chemotherapy. The drawback to most current therapies is that they are nonspecific. They kill noncancerous cells as well as cancer cells. However, every mammalian cell contains the genetic information to undergo apoptosis—even cancer cells, although they have overridden the normal controls. Recently, scientists have focused on deliberately inducing apoptosis in cancer cells. One strategy is to induce the mitochondrial apoptotic pathway. A high level of Bcl-2 protects cells from apoptosis. So if the amount of Bcl-2 were reduced in cancer cells, perhaps they would become more sensitive to apoptosis. (Mice with the  Bcl-2  gene deleted die from too much apoptosis, particularly in the lymphoid tissue, where immune B cells mature.) The synthesis of Bcl-2 protein can be reduced by using a single-stranded antisense DNA of 18 nucleotides. Its sequ...

  PROGRAMMED CELL DEATH IN BACTERIA Although apoptosis has not been seen in single-celled organisms, a genetic system that kills  Escherichia coli  when under extreme stress does exist. Morphologically, the death does not resemble apoptosis, but like apoptosis, the death system is genetically encoded. In  E. coli  , an addiction module of two genes controls the death-inducing system ( Fig. 20.22 ). One gene encodes a toxin, MazF, which is quite stable. The second gene encodes the antitoxin, MazE, which prevents the toxin from killing the bacteria. The antitoxin is unstable and degrades very fast after translation. If its transcription or translation is stopped or slowed in any way, the level of antitoxin plummets and the toxin kills the bacteria. The MazF toxin is a specific endoribonuclease that degrades messenger RNA. It recognizes the sequence ACA and cleaves to the 5′-side. Such enzymes have been named mRNA interferases and have now been found in a variety o...

  Bacterial Infections INTRODUCTION Infections of humankind as well as of animals and plants are caused by a diverse assortment of microorganisms, including viruses, bacteria, and various single-celled eukaryotes. The mechanisms of infection range from the simple approach of certain filamentous fungi that merely grow on unprotected organic matter to the highly sophisticated schemes for invasion and survival of specialized pathogens such as bubonic plague or malaria. Here we are concerned with applying modern molecular biology to understand and combat infection. The molecular mechanisms used by infectious microorganisms are best understood for pathogenic bacteria, especially those closely related to the molecular biologist’s model organism,  Escherichia coli  . While most strains of  E. coli  are harmless, a few virulent strains exist that illustrate many of the principles of infection at the molecular level. Although viruses have smaller genomes than bacteria, t...

  MOLECULAR APPROACHES TO DIAGNOSIS A major contribution of molecular biology has been the development of improved methods for diagnosis. In practice  diagnosis  usually means identifying the agent of disease, whether a bacterium, virus, or protozoan. Some pathogenic bacteria grow slowly or not at all when cultured outside their host organisms. Viruses are obligate parasites and can only be grown in the laboratory by infecting appropriate cultured host cells. Furthermore, different microorganisms require different culture media and culture conditions. All these factors make traditional methods of identification laborious. In contrast, molecular approaches analyze macromolecules such as DNA, RNA, or protein rather than attempting to grow the disease agents. Some new methods involve the use of antibody technology and are dealt . Here we will consider nucleic acid–based approaches. Molecular methods usually start with extraction of DNA from either cultured pathogens or an in...

  VIRULENCE GENES ARE OFTEN FOUND ON MOBILE SEGMENTS OF DNA Many infectious diseases are caused by bacterial invasion of the human body. Some pathogenic bacteria penetrate the interior of host cells, whereas others remain outside and inhabit extracellular spaces. The molecular mechanisms involved in infectious disease vary greatly in detail. Nonetheless, invading microorganisms face similar problems and so share many of the same general abilities. Properties that allow microorganisms to cause infections are called virulence factors and may be subdivided into three main groups: those required for invasion of the host, those for survival inside the host, and those for aggression against the host. The blocks of DNA encoding virulence factors are often mobile. In some cases (e.g., anthrax, bubonic plague) they are borne on virulence plasmids . In other cases (e.g., cholera toxin, diphtheria toxin), they may be carried on lysogenic bacteriophage and inserted into the chromosomes of cert...

  ATTACHMENT AND ENTRY OF PATHOGENIC BACTERIA The first step in many infections is the binding of bacteria to the surface of cells of the host animal. This is mediated by proteins known as adhesins that usually bind to sugar residues of glycoproteins or glycolipids on the animal cell surface. There are two major types of adhesins, fimbrial adhesins and nonfimbrial adhesins ( Fig. 21.3 ). Pili (singular,  pilus  ) or fimbriae (singular,  fimbria  ) are thin filaments that protrude from the surface of bacteria ( Fig. 21.4 ). The shaft is composed of helically arranged subunits of protein ( pilin ). Several specialized proteins, including adhesins , are carried at the very tip. Nonfimbrial adhesins are found on the surface of bacterial cells. In many cases, pili make first contact with the host cell and the nonfimbrial adhesins are responsible for a later and closer stage of binding. A second common step of infection is entering an animal cell. Not all bacteria tha...

  IRON ACQUISITION BY PATHOGENIC BACTERIA Almost all bacteria need iron because it is a cofactor for many enzymes, especially those of the respiratory chain. However, the concentration of free iron in the body, including the bloodstream, is kept low by a variety of specialized proteins that bind iron very tightly. Surplus iron is bound by transferrin and lactoferrin , which act as iron transporters, or by ferritin , which is an iron storage protein. Bacteria use iron chelators, known as siderophores , to bind iron and, if necessary, extract it from host proteins. Siderophores are excreted by the bacteria, bind iron, and are then taken back into the bacterial cell by specialized transport systems. Most bacteria have a variety of suchiron transport systems for use under different conditions. Enterochelin (or enterobactin) is a cyclic trimer of 2,3-dihydroxybenzoylserine (DHBS) and is perhaps the best known siderophore. It is made by  E. coli  and many enteric bacteria. The ...

  BACTERIAL TOXINS In addition to invasion and survival, many pathogenic bacteria also mount aggressive attacks against eukaryotic cells by making toxins. In its broadest sense, the term  toxin  includes any molecule that damages eukaryotic cells. Some toxic effects may be regarded as “accidental,” whereas others are deliberate. For example, bacterial endotoxin is actually the lipid A component of lipopolysaccharide (LPS) that forms part of the outer membrane of gramnegative bacteria. If such bacteria are killed by the immune system, their cell walls will disintegrate and release LPS. The CD14 receptor on immune cells binds the LPS, and this triggers the release of cytokines. Simultaneous destruction of many bacteria will release large amounts of LPS and may result in septic shock. Nonetheless, most pathogenic bacteria make one or more toxins that are designed to deliberately kill or damage the cells of their host. Because these are secreted from still-living bacteria, th...

  ADP-RIBOSYLATING TOXINS A large family of toxins hydrolyzes the cofactor NAD to nicotinamide and ADP-ribose. The toxin then transfers the ADP-ribose fragment to an acceptor molecule, usually a protein that binds GTP. The target protein is locked into its GTP-binding conformation and cannot perform its normal role ( Fig. 21.6 ). Both cholera toxin and diphtheria toxin work by ADP-ribosylation , although the targets are different. Cholera toxin inactivates the G protein that controls adenylate cyclase in animal cells, whereas diphtheria toxin attacks elongation factor EF-2, a translation factor required for protein synthesis in eukaryotic cells. It was originally thought that the toxic effect of such NAD-using toxins was merely due to destruction of NAD. Although high levels of toxin will indeed hydrolyze all the NAD in the cell, the true lethal effect occurs at much lower toxin levels and is due to the ADP-ribosylation of target proteins. The genes for cholera toxin and diphtheria...

  CHOLERA TOXIN The cholera bacterium,  Vibrio cholerae  , does not invade the tissues of the host. It attaches to the exterior of cells lining the small intestine and stays there. The damage is due to the secretion of cholera toxin. This attacks intestinal epithelial cells, causing them to lose sodium ions and then water into the intestinal tract. The clinical  symptoms of cholera are loss of body fluids by massive diarrhea and subsequent death by dehydration. The virulence proteins of  Vibrio cholerae  include cholera toxin as well as both pilus-borne and cell-surface adhesins for binding to intestinal cells. The genes for cholera toxin are carried by the CTXphi filamentous bacteriophage that lysogenizes  Vibrio cholerae.  Synthesis of both adhesins and toxin is co-regulated by the ToxR protein found in the bacterial inner membrane. This senses whether the bacterium is inside an animal intestine (temperature, pH, and bile salts are all involved)...

  ANTHRAX TOXIN Anthrax is caused by the gram-positive bacterium  Bacillus anthracis  , the first bacterium proven to be the cause of a disease. In 1877, Robert Koch grew this organism in pure culture, demonstrated its ability to form spores, and produced anthrax experimentally by injecting it into animals. Virulence factors of anthrax include the exotoxins and the capsule, both plasmid-borne. There are two plasmids: pXO1 carries the regulatory and structural genes for the exotoxins and pXO2 carries the genes for the capsule. The capsule is made from poly- D -glutamic acid and protects against attack by cells of the immune system. Chromosomal sequencing has shown that, apart from its virulence plasmids,  Bacillus anthracis  is remarkably similar to other “species” of  Bacillus  , such as  Bacillus cereus  (a common soil bacterium) and  Bacillus thuringiensis  (well known for making the insecticidal toxins used in transgenic plant en...

  ANTITOXIN THERAPY Even if infections occur and toxins are secreted by the invading bacteria, it may be possible to protect the patient against the toxins. Traditional antitoxin treatment has relied on antibodies against bacterial toxins. However, new geneoriented approaches are emerging. One approach relies on dominant-negative mutations in the binding subunit of the toxin. Defective mutations typically result in proteins that are inactive. However, occasional mutations give rise to proteins that not only are inactive themselves, but interfere with the functional version of the protein. The presence of such a mutation in the same cell as the wild-type version of the gene results in absence of activity—hence the term  dominant-negative  . The mechanism usually involves the binding of a defective protein subunit to functional subunits resulting in a complex that is inactive overall. Consequently, most dominantnegative mutations affect proteins with multiple subunits. The ...

  VIRAL INFECTIONS AND ANTIVIRAL AGENTS Many human diseases are due to viruses. These are less well understood than bacterial diseases, to a large extent because viruses cannot be grown alone in culture but depend on a host cell. Until recently, protection against virus diseases relied on public health measures and vaccination. Only recently have a significant number of specific antiviral agents become available. Pathogenic bacteria contain many unique components not found in eukaryotic cells, whichcan be targeted by antibiotics. In contrast, because viruses rely on the host cell for almost all of their metabolic reactions, they usually have few unique components apart from the structural proteins of the virus particle. Consequently, most chemical agents that prevent virus metabolism are also toxic to the host cells. Like pathogenic bacteria, viruses must also attach to and invade host cells. Recognition proteins on the surface of the virus capsid bind to specific receptors on the ...

  INTERFERONS COORDINATE THE ANTIVIRAL RESPONSE Interferons are a class of proteins induced in animal cells in response to virus infection. Clinical treatment with interferons is used to treat viral infections in a few cases (e.g., against hepatitis B and hepatitis C infections). Interferons  α   and  β  ( INF  α   and INF  β  ) block the spread of viruses by interfering with virus replication. (Although interferon  γ  shares the same name, it is quite distinct and is not induced directly by virus infection. It has a regulatory role in response to intracellular pathogens.) Interferons  α  and  β  are secreted in response to double-stranded RNA, which is symptomatic of the replication of most RNA viruses. They bind to the interferon receptors of both the infected cell itself and its neighbors. Locally, this triggers a phosphorelay signal pathway that activates several genes involved in opposing virus infection ( F...

  INFLUENZA IS A NEGATIVE-STRAND RNA VIRUS Influenza virus , an orthomyxovirus , is an example of a negative-strand single-stranded RNA virus. In other words, the virus genome is present in the virus particle as noncoding (= antisense = negative-strand) RNA. The flu virus particle contains a segmented genome consisting of eight separate pieces of single-stranded RNA ranging from 890 to 2341 nucleotides long. These are each packed into an inner nucleocapsid and are surrounded by an outer envelope ( Fig. 22.3 ). Although the outer membrane is derived from host-cell material, it contains virus-encoded proteins such as neuraminidase, hemagglutinin, and ion channels. These viral proteins are made on the ribosomes of the infected host cell and are involved in virus recognition and entry into successive host cells. The hemagglutinin and neuraminidase of influenza differ slightly but significantly between strains of flu. The variants are designated by H and N numbers. Thus the Spanish flu ...