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C. Kalesch, M.A.S., M.D.

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Neutron activation analysis is an excellent method for determining many of these trace elements. Determinations of trace elements in foodstuffs should be made routinely if we are to understand their role. Synthetic sexual lures, combined with chemical sterilizers usually contain fluorine, phosphorus, sulfur, and bromine atoms in their molecules, fluorine and phosphorus being predominant. Analysis by neutron activation makes it possible to determine phosphorus and bromine in tiny amounts of agro-chemical residues which may be in food products. There are many nuclear reactors in nearly every country of the world capable of doing these analyses. It may be possible to tag agricultural chemicals with rarely occurring but high nuclear cross-section materials so that pesticide residues could be determined easily by activation analysis. It would, of course, be necessary to determine that correlations between pesticides and the added element were constant as a function of time. For this reason it would probably be necessary to have the tagging material an integral part of the molecule. This technique was suggested by scientists working in forensic analysis some time ago, and has been applied in many cases. The amount of tagging material being added would be very low and therefore probably non-toxic although this would have to be proven. Altshuller, is the Director, Division of Chemistry and Physics, Air Pollution Control Office, Environmental Protection Agency. Since 1955 he has held various research assignments in the air pollution program of the Public Health Service in Cincinnati. He has pubHshed about 100 papers related to spectrophotometric analysis, gas chromatography, coulometric analysis, infrared spectrophotometry, photochemistry and kinetics, solution and statistical thermodynamics, thermochemistry, and various aspects of atmospheric chemistry. Chambers Award for outstanding achievement from the Air Pollution Control Association (1970). Altshuller Director, Division of Chemistry and Physics Research and-Monitoring Office Environmental Protection Agency Research Triangle Park, North Carolina 27711 the role of measuring techniques in activities ranging latory activities of the Environmental Protection Agency from research to reguwill be discussed. Field and laboratory instruments and manual techniques wiir receive consideration. Chemical and physical transformations contribute to ments will new or modified pollutant species. Air quality measurements must be capable of properly following such transformations. Especial emphasis needs to be given to the conversion of gaseous into various submicron particulate species. The mass, particle-size distribution and details of chemical composition of these particles is required. Examples will be given of new and improved types of air quality measurements, stationary source and mobile source emission measurements which may be needed to meet air quality and the formation of emission standards. Keywords: Air pollution; air quality instrumentation; air quality measurements; anion analysis; Clean Air Act; elemental analysis; mea- surement of atmospheric gases and vapors; measurement of mospheric particles. Introduction A this small program concerned with development of air pollution new or improved techniques for measuring particularly has existed for quite some time, but from the inception of Federal activities in 1955. Until 1967, program was primarily concerned with support of research and moni- toring needs. Considerable research was done on development of colorimetric and chromatographic techniques. These techniques were applied to air quality measurement and to motor vehicle emissions research. During this period a small number of air quality instruments, particularly those for oxidants and hydrocarbons, were evaluated and improved. Most of the colorimetric procedures used in air quality instruments were developed or improved during this period. Gas chromatographic techniques were well enough developed to justify their use in monitoring instrumentation, but the necessary resources were not available. The costs involved in incorporating a laboratory technique into an effective monitoring instrument for routine general use are considerable. The Clean Air Act, as amended in 1967, referred to instrumentation requirements in two sections. Section 104 states: "The Administrator may conduct and accelerate R/D of low cost instrumentation techniques to facilitate determination of quantity and quality of air pollution emis- sions, including, but not limited to , automotive emissions.

Effect on Km: Noncompetitive inhibitors do not interfere with the binding of substrate to enzyme. Therefore, the enzyme shows the same Km in the presence or absence of the noncompetitive inhibitor. Effect of a noncompetitive inhibitor on the reaction velocity versus substrate ([S]) plot. Effect on Lineweaver-Burk plot: Noncompetitive inhibition is readily differentiated from competitive inhibition by plotting 1/vo versus 1/[S] and noting that the apparent Vmax decreases in the presence of a noncompetitive inhibitor, whereas Km is unchanged (see Figure 5. Enzyme inhibitors as drugs At least half of the ten most commonly prescribed drugs in the United States act as enzyme inhibitors. For example, the widely prescribed -lactam antibiotics, such as penicillin and amoxicillin, act by inhibiting enzymes involved in bacterial cell wall synthesis. These drugs, which include captopril, enalapril, and lisinopril, cause vasodilation and, therefore, a reduction in blood pressure. Aspirin, a nonprescription drug, irreversibly inhibits prostaglandin and thromboxane synthesis (see p. The rates of most enzymes are responsive to changes in substrate concentration, because the intracellular level of many substrates is in the range of the Km. Thus, an increase in substrate concentration prompts an increase in reaction rate, which tends to return the concentration of substrate toward normal. In addition, some enzymes with specialized regulatory functions respond to allosteric effectors and/or covalent modification or they show altered rates of enzyme synthesis (or degradation) when physiologic conditions are changed. Regulation of allosteric enzymes Allosteric enzymes are regulated by molecules called effectors that bind noncovalently at a site other than the active site. These enzymes are almost always composed of multiple subunits, and the regulatory (allosteric) site that binds the effector is distinct from the substrate-binding site and may be located on a subunit that is not itself catalytic. Effectors that inhibit enzyme activity are termed negative effectors, whereas those that increase enzyme activity are called positive effectors. Positive and negative effectors can affect the affinity of the enzyme for its substrate (K0. Homotropic effectors: When the substrate itself serves as an effector, the effect is said to be homotropic. In such a case, the presence of a substrate molecule at one site on the enzyme enhances the catalytic properties of the other substrate-binding sites. These enzymes show a sigmoidal curve when reaction velocity (vo) is plotted against substrate concentration ([S]), as shown in Figure 5. This contrasts with the hyperbolic curve characteristic of enzymes following Michaelis-Menten kinetics, as previously discussed. Heterotropic effectors: the effector may be different from the substrate, in which case the effect is said to be heterotropic. The enzyme that converts D to E has an allosteric site that binds the endproduct, G. If the concentration of G increases (for example, because it is not used as rapidly as it is synthesized), the first irreversible step unique to the pathway is typically inhibited. Feedback inhibition provides the cell with appropriate amounts of a product it needs by regulating the flow of substrate molecules through the pathway that synthesizes that product. For example, the glycolytic enzyme phosphofructokinase-1 is allosterically inhibited by citrate, which is not a substrate for the enzyme (see p. Regulation of enzymes by covalent modification Many enzymes are regulated by covalent modification, most often by the addition or removal of phosphate groups from specific serine, threonine, or tyrosine residues of the enzyme. Protein phosphorylation is recognized as one of the primary ways in which cellular processes are regulated. Phosphate groups are cleaved from phosphorylated enzymes by the action of phosphoprotein phosphatases (Figure 5. Response of enzyme to phosphorylation: Depending on the specific enzyme, the phosphorylated form may be more or less active than the unphosphorylated enzyme.

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By blocking replication these compounds slows the division of rapidly growing cancer cells, bacterial cells and viruses. Inhibitors of replication are used as anti-cancer agents, anti-bacterial or anti-biotics and anti-viral agents. Acyclovir It is another nucleoside analog used in viral infection caused by herpes virus. It is an analog of guanosine in which pentose is replaced with three carbon sugar. Replication origins and cancer Large number of replication origins in eukaryotes are puzzling molecular biologists for long time. During G1 phase of cell cycle replication origins are prepared to fire and subsequently activated in S-phase. Therefore, reduction in replication origins alters progression of S-phase, mitosis and chromosomal translocations. Since gross chromosomal rearrangements are associated with cancer development, large number of replication origins are necessary for normal cell division. Thus, large number of replication origins prevents a normal cell turning into cancer cell. In future diagnostic tests that can predict a cell to become cancer cell based on replication origins may be developed. Leishmaniass and trypanosomiasis caused by protozoan parasites leishmania donovani and trypanosoma brucei affects millions of people worldwide. Some examples of topoisomerase inhibitors with potent anti-trypanosomal activity are pentamidine, berenil, samorine etc. Xeroderma pigmentosum this disease is due to deficiency of endonuclease involved in excision repair. Other symptoms are thorny growth of skin, corneal ulceration, scarred eye lids etc. More than one polymerase can transcribe same template at different places simultaneously. However, there can be an error for every 10 4 bases compared to 3 Ч 10 4 bases of replication. The transcription errors are tolerable because of formation of large number of copies. Aflatoxin A fungus that grows on moist ground nut produces aflatoxin, which inhibit transcription. It has nuclease (hydrolytic) activity and ligase 436 Medical Biochemistry or trans esterification activity. Self-nuclease activity removes introns where as transesterification activity joins exons. It is composed of short G-rich randomly repeated sequence, which is extended to form single stranded over hang. Thus, telomere serveas, replicometer which count cell division and ultimately triggers replicative senescence or cell aging. Further, telomeres prevent chromosomal fusion and offer genomic integrity and stability. Telomerase is constitutively expressed in germ cells, which undergo continuous proliferation. However, in somatic cells, telomeres shorten by about 50 base pairs per each cell division due to lack of telomerase activity. Thus, telomerase activity is developmentally connected in cell cycle dependant manner, which involves either telemore shortening, stability or even elongation. Immortal eukaryotic cells or cancer cells or virus transformed cells that pocess unlimited proliferation capacity exhibit high telomerase activity. As a result, cell division continues, cells proliferate and ultimately immortalization of cells occurs, which is characteristic of cancer cells. An oxo-carbenium ion intermediate of ribozyme reaction indicated by kinetic isotope effects.

Another way of overcoming the penicillin-degrading effects of -lactamase is to combine a lactamase-sensitive agent. Other mechanisms of resistance which have been encountered include modification of the binding sites on penicillin-binding proteins (see below), thus reducing their affinity for the penicillin, and decreased cell permeability, leading to reduced uptake of the antibiotic. Strains of Staphylococcus aureus resistant to both methicillin and isoxazolylpenicillins. Cross-linking of the peptidoglycan chains which constitute the bacterial cell wall (see page 473) involves an acyl-D-Ala­D-Ala intermediate, which in its transition state conformation closely resembles the penicillin molecule (Figure 7. As a result, the penicillin occupies the active site of the enzyme, and becomes bound via an active site serine residue, this binding causing irreversible enzyme inhibition, and cessation of cell wall biosynthesis. Growing cells are then killed due to rupture of the cell membrane and loss of cellular contents. This binding reaction between penicillin-binding proteins and penicillins is chemically analogous to the action of -lactamases, but in the latter case penicilloic acid is subsequently released from the -lactamase, which can continue to function. The bacterial cell wall has no counterpart in mammalian cells, and the action is thus very specific. However, a significant proportion of patients can experience allergic responses ranging from a mild rash to fatal anaphylactic shock. Cleavage of the -lactam ring through nucleophilic attack of an amino group in a protein is believed to lead to the formation of antigenic substances causing the allergic response. Alternatively, an acyltransferase enzyme converts isopenicillin N into penicillin G directly, without 6-aminopenicillanic acid actually being released from the enzyme. Ring expansion then occurs, incorporating one of the methyls into the heterocyclic ring, though the mechanism for this is not clearly defined. Cephalosporin C is the acetyl ester of this, whilst a further group of antibiotics termed the cephamycins are characterized by a 7-methoxy group, and are produced by hydroxylation/methylation, and, in the case of cephamycin C, introduction of a carbamate group from carbamoyl phosphate on to the hydroxymethyl function. In contrast to the penicillins, cephalosporin C was stable under acidic conditions and also was not attacked by penicillinase (-lactamase). Antibacterial activity was rather low, however, and the antibiotic was poorly absorbed after oral administration. However, the structure offered considerable scope for side-chain modifications, more so than with the penicillins since it has two side-chains, and this has led to a wide variety of cephalosporin drugs, many of which are currently in clinical use. Removal of this side-chain by suitable microorganisms or enzymes has proved elusive. The ester side-chain at C-3 may be hydrolysed enzymically by fermentation with a yeast, or, alternatively, the acetoxy group is easily displaced by nucleophilic reagents. Note that all the cephalosporin antibiotics begin with the prefix ceph- or cef-, the latter spelling now being preferred, though both spellings are still encountered for some drugs. The classification into generations is based primarily on the antibacterial spectrum displayed by the drugs but it is also more or less related to the year of introduction. However, drugs in the second generation may have been introduced after the third generation of drugs had been established. There is no intention to suggest that third generation drugs automatically supersede second and first generation drugs, and, indeed, agents from all generations are still currently used. They have comparable activity to ampicillin, and are effective against penicillinase-producing Staphylococcus. However, another -lactamase (cephalosporinase) developed that inactivated these agents. Cefalotin, the first modified cephalosporin to be marketed, is poorly absorbed from the gut, and is thus not orally active. However, cefalexin, cefradine, and cefadroxil may be administered orally, a property that appears to be related to the 3-methyl side-chain. Second generation cephalosporins show a broader spectrum of activity, and are more active against aerobic Gram-negative bacteria like Haemophilus influenzae and Neisseria gonorrhoeae. This group of antibiotics includes cefaclor, cefuroxime, and cefamandole (cephamandole), and in general displays better resistance to -lactamases that inactivated first generation cephalosporins. Many of the third generation cephalosporins are characterized by an aminothiazole ring on the amide side-chain, which appears to impart the high activity against Gram-negative bacteria. The Osubstituted oxime group also improves potency, and confers resistance to -lactamases.