ANTIBIOTIC ANAPHYLAXIS

Anaphylactic reactions following the injection of penicillin or other antibiotics have increased greatly during the past several years. These untoward reactions to penicillin were infrequent during the first nine years of penicillin therapy and there can be no doubt that hypersensitivity to these drugs is developing in many people. Therefore promiscuous use of them is to be condemned. It is safer not to use antibiotics parenterally in patients with allergic conditions such as bronchial asthma. The further use of a particular antibiotic should be avoided if the patient has previously shown any hypersensitivity to that drug.A questionnaire was answered by more than 1,000 California physicians who reported that over 300 patients had severe anaphylactic reactions from parenteral penicillin and streptomycin. There were seven deaths.Since the antibiotics should still be used when needed, prevention of anaphylaxis is of fundamental importance. The frequency of these reactions can be greatly reduced by the use of antihistaminic solutions combined with the antibiotics. Treatment of these shock-like reactions demands the prompt administration of epinephrine intramuscularly, antihistaminic solutions intravenously and oxygen.     

T4 polynucleotide kinase: macromolecular crowding increases the efficiency of reaction at DNA termini

The amount of reaction catalyzed by T4 polynucleotide kinase on a variety of its substrates is greatly increased in the presence of polyethylene glycol 8000 (PEG 8000). Both the forward and reverse reactions as well as the exchange reaction can be stimulated. The stimulation is a general effect on T4 polynucleotide kinase reactions involving high molecular weight DNA substrates. The use of PEG 8000 is particularly advantageous for labeling or removing terminal 5'-phosphate groups which are only slowly or incompletely labeled or removed under ordinary conditions, such as those at recessed termini or at "nicks" in duplex DNA, although the reaction on blunt-ended or protruding termini is also increased. It is further advantageous for labeling very low concentrations of substrates.     

Repair, refold, recycle: how bacteria can deal with spontaneous and environmental damage to proteins

Proteins, like DNA, are subject to various forms of damage that can render them non-functional. Conformational changes and covalent chemical alterations occur spontaneously, and the rates of these reactions can be increased by environmental stresses such as heat, oxidative agents, or changes in pH or osmotic conditions. Although affected proteins can be replaced by de novo biosynthesis, cells – especially those subjected to stress or nutrient limitation – have developed mechanisms which can either restore damaged polypeptides to an active state or remove them. Such mechanisms can spare the biosynthetic capacity of the cell and ensure that the presence of non-functional molecules does not disrupt cell physiology. Three major mechanisms, which operate in bacteria as well as eukaryotic organisms, have been described. First, chaperones not only assist in proper de novo folding of proteins but also provide an important means of restoring activity to conformationally damaged proteins. Second, enzymatic‘repair’systems exist to directly reverse certain forms of protein damage, including proline isomerization, methionine oxidation and the formation of isoaspartyl residues. Finally, proteolysis provides a‘last-resort’means of dealing with abnormal proteins which cannot be repaired. Protein maintenance and repair may be of special importance for bacteria preparing to survive extended periods in stationary phase: both constitutive and induced mechanisms are utilized to permit survival despite greatly reduced protein synthesis.     

Studies on lipogenesis in vivo. Effects of starvation and re-feeding, and studies on cholesterol synthesis

1. Studies in vivo have been carried out on hepatic and extrahepatic cholesterol synthesis and also on the effects of starvation and re-feeding on both cholesterol and fatty acid synthesis. 2. In rats and mice fed on a stock diet, extrahepatic tissues accounted for about 4 times as much newly synthesized cholesterol as did the liver. The liver appeared to be somewhat more important in the rat than the mouse. Feeding with cholesterol greatly decreased and cholestyramine greatly increased hepatic cholesterol synthesis without much effect on extrahepatic synthesis. 3. Mice starved for up to 7hr. did not lose any of the ability to convert a [U-(14)C]glucose meal into fat, whereas 18hr. of starvation resulted in an 80% loss of fatty acid synthesis in liver and carcass, an 80% loss in liver cholesterol synthesis and a 65% decrease in carcass cholesterol synthesis; 18hr. of food deprivation also decreased the proportion of counts in epididymal fat pads present as fat and increased the proportion present as glyceride glycerol. 4. Re-feeding for up to 7hr. restored fatty acid synthesis from a [U-(14)C]glucose meal to about 50% of the values for non-starved mice but had no effect on hepatic cholesterol synthesis. The altered distribution of counts in the epididymal fat pads caused by starvation was restored to normal after feeding for 1hr.     

Comparison of Three Phytochrome-mediated Processes in the Hypocotyl of Mustard

Anthocyanin synthesis, hair formation, and the synthesis of ascorbic acid oxidase are all phytochrome-mediated reactions occurring in the hypocotyl of mustard (Sinapis alba L.), controlled by phytochrome actually located in the hypocotyl. A comparison of these three reactions showed that in certain respects they differ greatly in their response to light. The ability of the seedling to respond to light by showing the three responses was strongly influenced by the state of development of the seedling. White light given very early after seed imbibition was unable to evoke any of the three reactions. By 50 hours after imbibition, all systems were fully inducible by light. The addition of actinomycin D to a fully competent seedling coincident with illumination strongly inhibited the development of all three responses. In contrast, the addition of cordycepin at this time inhibited the synthesis of anthocyanin and ascorbic acid oxidase but had no effect on hair formation. Cycloheximide inhibited all three responses when given up to several hours after light. This suggests the necessity for RNA and protein synthesis for light-induced expression of these reactions, and that the RNA species involved in the three reactions may have differing degrees of polyadenylation. The lag period between the onset of light and the first display of the response was 3 hours for anthocyanin and ascorbic acid oxidase synthesis, and about 5 hours for hair formation. Amounts of light sufficient to give large increases in the levels of ascorbic acid oxidase and hair formation gave a much smaller increase in anthocyanin synthesis. Hair formation and ascorbic acid oxidase synthesis showed a much greater sensitivity to induction at early stages of seedling development than did anthocyanin synthesis. Following an inductive light period, anthocyanin synthesis was sensitive to far red light inhibition for a period twice as long as the other two reactions. The differences in the response of the three reactions to light suggest that the phytochrome-mediated reactions which control their development also differ.     

Leishmania, Trypanosoma and monoxenous trypanosomatids as emerging opportunistic agents

Immunosuppression is associated with the occurrence of a large variety of infections, several of them due to opportunistic protozoa. The parasitic protozoa of the family Trypanosomatidae vary greatly in their importance as potential opportunistic pathogens. African trypanosomiasis is no more common nor severe during AIDS. The situation with Chagas' disease, however, is much different. Although the process is not clearly understood, there appears to be a reactivation of Trypanosoma cruzi infection, which can lead to severe meningoencephalitis. In persons with AIDS, leishmaniasis is often exacerbated, particularly Leishmania infantum, which causes visceral leishmaniasis in southern Europe. Since 1990, 1,616 cases of visceral leishmaniasis/HIV co-infection have been reported, mainly from southern Europe, and particularly from Spain, southern France, and Italy. The co-infected patients are primarily young adults and belong to the risk group of intravenous drug users. Isoenzymatic identification of 272 isolates showed 18 different L. infantum zymodemes, of which 10 represent new zymodemes hitherto found only during HIV co-infection. New foci of co-infection are emerging in various parts of the world, including Brazil and East Africa. Moreover, since 1995, non-human monoxenous trypanosomatids have been found in AIDS patients, causing both diffuse cutaneous lesions and visceral infections. In countries where visceral leishmaniasis is endemic, particularly in southern Europe, immunosuppressive treatments for organ transplants or malignant diseases often result either in reactivation of asymptomatic visceral leishmaniasis or in facilitation of new infections.     

Nitrogen Metabolism, Respiration, and Growth of Cultured Plant Tissue

Critical examination of the amino-acid composition of proteinsin fast-growing and slow-growing tissues reveals only very 8maUdifferences, indicating that some factor other than the amino-acidcomplement is responsible for, or reflects, the great increasein the mass of protein in the fast-growing tissues. Increases in fresh weight and total protein are exactly parallel,indicating that water uptake is an active process associatedwith growth. Respiration, on the other hand, increases far morein the fast-growing over the slow-growing tissues than doestotal protein. A given amount of protein in the fast-growingtissue will support a much greater respiration rate than thesame amount in slow-growing tissue. The incorporation of radioactivity into amino-acids of the proteinin4icates that there are two distinct types: those in whichincorporation is increased in fast-growing tissue much morethan the total protein, and to the same ezte as respiration(notably glutarnic acid, aspartic acid, and threonine); andthose in which the increased incorporation is much nafler, slightlyless than total protein (notably proline and hydroxyproline).It is concluded that there are two n protein fractions: the‘active’ moiety, which is undergoing rapid breakdownd resynthesis, giving rise to much of the CO through oxidationof its residues; a the ‘inactive’ moiety, whichonce synthesized is not reutilized or broken down. It is theformer, or ‘active‘ protein whose synthesis is greatlyincreased in the fast. growing tissues, and it is the pace,rather than the kind, of reactions which differ entiates betweenthe fast- and slow-growing tissues. The entire experimental data are discussed with reference toa number of cur rent theories and investigations. A number ofexperimental observations are noted which admit of interpretationalong the lines here developed.     

1-beta-D-arabinofuranosylcytosine stimulates thymidine incorporation into the DNA of contact-inhibited cells

In rapidly proliferating cells l-β-d-arabinofuranosylcytosine (ara-C) is a potent inhibitor of DNA synthesis whose effect can be irreversible and consequently cytocidal. Whereas thymidine incorporation is greatly reduced in rapidly proliferating cells in the presence of ara-C, contact-inhibited cells, similarly treated, show increased thymidine incorporation by as much as 7-fold. This ara-C-induced stimulation appears to result from an influence on thymidine utilization rather than increased DNA synthesis.     

Trp-999 of beta-galactosidase (Escherichia coli) is a key residue for binding,catalysis, and synthesis of allolactose,the natural lac operon inducer

Trp-999 is a key residue for the action of beta-galactosidases (Escherichia coli). Several site specific substitutions (Phe, Gly, Tyr, Leu) for Trp-999 were made. Each substitution caused greatly decreased affinities for substrates and inhibitors that bind in the "shallow" mode, while the affinities of inhibitors that bind in the "deep" mode were not decreased nearly as much. This shows that Trp-999 is important for binding in the shallow mode. The residue is also very important for binding glucose to galactosyl-beta-galactosidase (as a transgalactosidic acceptor). Substitution greatly diminished the affinity for glucose. Substitutions also changed the activation thermodynamics and, subsequently, the rates of the catalytic reactions. The enthalpies of activation of the glycolytic bond cleavage step (galactosylation, k(2)) became less favorable while the entropies of activation of that step became more favorable as a result of the substitutions. Differing magnitudes of these enthalpic and entropic effects with ONPG as compared to PNPG caused the k(2) values for ONPG to decrease but to increase for PNPG. The enthalpies of activation for the common hydrolytic step (degalactosylation, k(3)) increased while the entropies of activation for this step did not change much. As a result, k(3) became small and rate determining for each substituted enzyme. The substitutions caused the rate constant (k(4)) of the transgalactosidic acceptor reactions with glucose (for the formation of allolactose) to become much larger and of the same order of magnitude as the normally large rate constants for transgalactosidic acceptor reactions with small alcohols. This is probably because glucose can approach with less restriction in the absence of Trp-999. However, since glucose binds very poorly to the galactosyl-beta-galactosidases with substitutions for Trp-999, the proportion of lactose molecules converted to allolactose is small. Thus, Trp-999 is also important for ensuring that an appropriate proportion of lactose is converted to allolactose.     

1-β- -Arabinofuranosylcytosine stimulates thymidine incorporation into the DNA of contact-inhibited cells

In rapidly proliferating cells l-β- -arabinofuranosylcytosine (ara-C) is a potent inhibitor of DNA synthesis whose effect can be irreversible and consequently cytocidal. Whereas thymidine incorporation is greatly reduced in rapidly proliferating cells in the presence of ara-C, contact-inhibited cells, similarly treated, show increased thymidine incorporation by as much as 7-fold. This ara-C-induced stimulation appears to result from an influence on thymidine utilization rather than increased DNA synthesis.     

A Reassessment of Prebiotic Organic Synthesis in Neutral Planetary Atmospheres

The action of an electric discharge on reduced gas mixtures such as H₂O, CH₄ and NH₃ (or N₂) results in the production of several biologically important organic compounds including amino acids. However, it is now generally held that the early Earth’s atmosphere was likely not reducing, but was dominated by N₂ and CO₂. The synthesis of organic compounds by the action of electric discharges on neutral gas mixtures has been shown to be much less efficient. We show here that contrary to previous reports, significant amounts of amino acids are produced from neutral gas mixtures. The low yields previously reported appear to be the outcome of oxidation of the organic compounds during hydrolytic workup by nitrite and nitrate produced in the reactions. The yield of amino acids is greatly increased when oxidation inhibitors, such as ferrous iron, are added prior to hydrolysis. Organic synthesis from neutral atmospheres may have depended on the oceanic availability of oxidation inhibitors as well as on the nature of the primitive atmosphere itself. The results reported here suggest that endogenous synthesis from neutral atmospheres may be more important than previously thought. Stanley L. Miller died May 20, 2007.     

Carbon, nitrogen, and phosphorus dynamics during leaf decay in nutrient-enriched stream microecosystems

We investigated the effects of long-term enrichment with nitrate, phosphate, and nitrate+phosphate on the first 5 weeks of leaf detritus processing in laboratory stream microecosystems. Enrichment with nitrate+phosphate accelerated leaf weight loss and increased rates of respiration associated with the leaves. However, whole-system respiration was little changed from that observed in the control stream since respiration in the water was greatly reduced. Enrichment with phosphate alone had little effect except to lower respiration associated with leaf discs. Enrichment with nitrate alone also decreased leaf-disc respiration but resulted in a greatly increased rate of respiration in the water. Net leaching and fragmentation of carbon from the leaves was also increased by nitrate enrichment. Nitrogen and phosphorus levels in leaf material were little affected by enrichment with nitrate or phosphorus alone. Leaves in those streams and in the control stream released nitrogen and phosphorus to the water. In contrast, percent nitrogen and phosphorus increased greatly in the leaves in the stream enriched with both nitrate and phosphate. The leaves in this system immobilized both nitrogen and phosphorus from the water. We also studied the importance of nitrogen fixation as a vector for nitrogen incorporation associated with leaf decomposition in streams. Somewhat surprisingly, fixation by microbes associated with the leaves and by microbes suspended in the water occurred under all three experimental enrichment treatments as well as in the control, casting doubt on the effectiveness of nitrate in inhibiting nitrogenase synthesis in nature. However, N₂-fixation is only a minor source of nitrogen for leaves decaying under the conditions studied.     

Rate-limiting factors in urate synthesis and gluconeogenesis in avian liver

1. Urate synthesis and other metabolic characteristics of isolated chicken hepatocytes were studied. 2. The distinction is made between immediate precursors of the purine ring (glycine, glutamine, aspartate, formyltetrahydrofolate, bicarbonate) and ultimate precursors from which the immediate precursors are formed in the liver. 3. In hepatocytes from well-fed chickens the rate of urate synthesis was not greatly increased by the addition of amino acids or NH₄Cl, but in hepatocytes from 72h-starved chickens the rate was much increased when alanine or asparagine was added as the only substrate. Other amino acids, when added alone, did not affect the rate. The exceptional effect of alanine and asparagine is due to the ready formation of the immediate precursors. 4. Conditions are described under which glutamine, serine, glycine plus formate, ribose and glucose increased the rate of urate synthesis. 5. At 1mm-NH₄Cl (a concentration not much higher than that of blood plasma) the rate of urate synthesis in the presence of lactate was increased, but higher concentrations inhibited urate synthesis in the presence of lactate or alanine; with alanine even 1mm-NH₄Cl was inhibitory. 6. Glucose synthesis from lactate, alanine or dihydroxyacetone was also inhibited by 1mm-NH₄Cl. 7. NH₄Cl inhibition of urate and glucose synthesis was paralleled by an increased rate of glutamine synthesis. Thus in the presence of NH₄Cl the gluconeogenic precursors are diverted from the pathway of gluconeogenesis to that of glutamate and glutamine synthesis. This implies that the synthesis of these amino acids is the primary process in the detoxication of ammonia in the avian liver. 8. Urate synthesis, like urea synthesis, can be looked on as a cyclic process with either phosphoribosyl pyrophosphate or ribose acting as the carrier on which the purine ring is assembled. 9. The energy requirements of urate synthesis depend on whether phosphoribosyl pyrophosphate is regenerated from IMP by pyrophosphorylase or by phosphorylation and pyrophosphorylation of ribose. It is 6 or 9 pyrophosphate bonds of ATP respectively.     

Caffeoyl coenzyme A O-methyltransferase and lignin biosynthesis.

Lignin, a complex phenylpropanoid compound, is polymerized from the monolignols p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. These three monolignols differ only by the 3- and 5-methoxyl groups. Therefore, enzymatic reactions controlling the methylations of the 3- and 5-hydroxyls of monolignol precursors are critical to determine the lignin composition. Recent biochemical and transgenic studies have indicated that the methylation pathways in monolignol biosynthesis are much more complicated than we have previously envisioned. It has been demonstrated that caffeoyl CoA O-methyltransferase plays an essential role in the synthesis of guaiacyl lignin units as well as in the supply of substrates for the synthesis of syringyl lignin units. Caffeic acid O-methyltransferase has been found to essentially control the biosynthesis of syringyl lignin units. These new findings have greatly enriched our knowledge on the methylation pathways in monolignol biosynthesis.     

Reduction in cholesterol and sialic acid content protects cells from the toxic effects of beta-amyloid peptides

beta-Amyloid (Abeta) is the primary protein component of senile plaques associated with Alzheimer's disease and has been implicated in the neurotoxicity associated with the disease. A variety of evidence points to the importance of Abeta-membrane interactions in the mechanism of Abeta neurotoxicity and indicates that cholesterol and gangliosides are particularly important for Abeta aggregation and binding to membranes. We investigated the effects of cholesterol and sialic acid depletion on Abeta-induced GTPase activity in cells, a step implicated in the mechanism of Abeta toxicity, and Abeta-induced cell toxicity. Cholesterol reduction and depletion of membrane-associated sialic acid residues both significantly reduced the Abeta-induced GTPase activity. In addition, cholesterol and membrane-associated sialic acid residue depletion or inhibition of cholesterol and ganglioside synthesis protected PC12 cells from Abeta-induced toxicity. These results indicate the importance of Abeta-membrane interactions in the mechanism of Abeta toxicity. In addition, these results suggest that control of cellular cholesterol and/or ganglioside content may prove useful in the prevention or treatment of Alzheimer's disease.     

Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains.

A procedure is described for intensifying histochemical reactions by amplification of biotinylated sites. This is achieved by deposition of biotinylated tyramine on the tissue through the enzymatic action of horseradish peroxidase (HRP). The amplified biotin sites are subsequently visualized by binding them to avidin, to which a marker is attached. This amplification greatly increases the sensitivity of staining procedures that employ HRP (and/or biotin) in tissue. For neuroanatomical pathway tracing methods, the procedure greatly increases the detectability of the injected tracer. For lectin histochemistry and immunohistochemistry, the amplification requires that the lectin or primary antibody be greatly diluted. This dilution results in less background staining and yet strong signals are produced even when very dilute reagents are used. Alternatively, the amplification permits much shorter incubations in primary antibodies when dilutions are used that would ordinarily be used with conventional bridge techniques. The procedure is also useful for amplifying very weak signals, such as those of immunoreactions in glutaraldehyde-fixed tissue. The amplification procedure, together with the availability of avidin probes labeled with fluorochromes, colloidal gold, or enzyme systems other than HRP, provides a means of greatly increasing the versatility of a variety of histochemical reactions, including those for detecting in situ hybridization probes, in addition to increasing the sensitivity of the reactions.     

Synthesis and in vitro cytotoxicity of cis,cis,trans-diamminedichloridodisuccinatoplatinum(IV)-peptide bioconjugates

The synthesis and characterization of four Pt(IV)-peptide conjugates, containing one or two peptides in the axial position, designed for the purpose of targeted drug delivery to tumor cells, are described. The precursor cis,cis,trans-diamminedichloridodisuccinatoplatinum(IV) was coupled in the last step of standard solid-phase peptide synthesis (SSPS) with an analogue of neurotensin (pseudo-neurotensin = Lys-Lys-Pro-Tyr-Ile-Leu) and with octreotate (D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OH), an analogue of somatostatin, respectively. For all peptides, the SSPS reactions afforded both mono- and diconjugated Pt-peptide species, which were separated and purified by RP-HPLC. The two couples of conjugates, together with the precursor, were tested as cytotoxic agents towards different cancer cell lines. In general all conjugates are good inhibitors of cellular proliferation when compared to a nontargeting platinum(IV) parent compound, so that its relatively low cytotoxicity is greatly improved by addition of the peptides.     

Beta2-amino acids-syntheses, occurrence in natural products, and components of beta-peptides1,2

Although they are less abundant than their alpha-analogues, beta-amino acids occur in nature both in free form and bound to peptides. Oligomers composed exclusively of beta-amino acids (so-called beta-peptides) might be the most thoroughly investigated peptidomimetics. Beside the facts that they are stable to metabolism, exhibit slow microbial degradation, and are inherently stable to proteases and peptidases, they fold into well-ordered secondary structures consisting of helices, turns, and sheets. In this respect, the most intriguing effects have been observed when beta2-amino acids are present in the beta-peptide backbone. This review gives an overview of the occurrence and importance of beta2-amino acids in nature, placing emphasis on the metabolic pathways of beta-aminoisobutyric acid (beta-Aib) and the appearance of beta2-amino acids as secondary metabolites or as components of more complex natural products, such as peptides, depsipeptides, lactones, and alkaloids. In addition, a compilation of the syntheses of both achiral and chiral beta2-amino acids is presented. While there are numerous routes to achiral beta2-amino acids, their EPC synthesis is currently the subject of many investigations. These include the diastereoselective alkylation and Mannich-type reactions of cyclic- or acyclic beta-homoglycine derivatives containing chiral auxiliaries, the Curtius degradation, the employment of transition-metal catalyzed reactions such as enantioselective hydrogenations, reductions, C-H insertions, and Michael-type additions, and the resolution of rac. beta2-amino acids, as well as several miscellaneous methods. In the last part of the review, the importance of beta2-amino acids in the formation of beta-peptide secondary structures is discussed.