Clinical trial of 3-methyl-5-(beta-N'-(N-m-chlorophenylpiperazino)ethyl)-pyrazole dihydrochloride (Mepiprazol) in the therapy of psychovegetative disorders. In a clinical trial of 30 patients suffering from psychovegetative disorders and anxiety neuroses, Mepiprazol (test designation: EMD 16-923; supplier: E. Merck, Darmstadt, FRG) was proven in a treatment period of 14 days to be an effective, well-tolerated medication. Only two patients failed to respond during the course of treatment. Improvements were significant at the 1-percent level between the pretreatment condition and each control during the treatment period; even between the rating times there was a significant increment in treatment success. Mepiprazol had a particular effect on symptoms such as irritability, anxiety, inner unrest and tension. Mepiprazol also exhibited a positive effect in the treatment of psychosomatic complaints.


[Double-blind study of Aprotininum (Trasylol) in the management of alcoholic delirium]. The results of uncontrolled studies, in which the period of the delirium tremens was reduced and the intensity was lowered significantly by aprotininum, could not be verified in our double-blind study. Continuing a preliminary double-blind study another group of 20 male patients was examined again to evaluate the effectiveness of aprotininum given in a modified experimental arrangement. Not only at the beginning of the treatment but also every 6 h an infusion of 200,000 KIE Trasylol was given during the period of the acute alcohol withdrawal syndromes. Time as well as intensity of symptoms (quantitatively measured by needed chlormethiazol) did not differ significantly between the aprotininum and the placebo group.


Psychotherapeutic profile of a new psychoactive drug trazodone (AF-1161). In a systematic clinical study, including four uncontrolled clinical trials with psychiatric patients belonging to four different diagnostic groups, trazodone, the first triazolopyridine compound subjected to clinical investigation, was found to be a therapeutically effective psychoactive drug. Since the most consistent therapeutic action of trazodone was seen to be in the depressive syndrome, i.e., Guilt Feelings, Motor Retardation and Depressive Mood, it was suggested that trazodone has a therapeutic effect on depressive symptoms whether they occur in depression, neurosis, organic brain syndrome or schizophrenia.


The outcome of dothiepin treatment in 1,900 depressed patients. One thousand, nine hundred and sixty-five depressed patients took part in a multicentre study of dothiepin hydrochloride, which involved 392 doctors. The results indicated that a large percentage of patients improved as measured by all the assessments and no serious or adverse effects were encountered. A good correlation was seen between the global scale and the Rockliff depression scale; and also between the final mean Rockliff score and the number of patients stopping therapy due to recovery, and continuing treatment until recovery, at the final assessment.


[Clinical trial of lofepramine versus imipramine]. Lofepramin is a new derivative of imipramine. Its effects and side effects were compared to those of imipramine in a double-blind trial. 49 hospitalized patients, suffering from endogenous depression, were treated for 20 days with 210 mg lofepramin daily and 52 other patients with 150 mg imipramine daily. The results of the repeated psychiatric examinations were documented using the AMP system. Both substances proved to have high antidepressant effects and showed no significant difference in the quality of their effects.


The primary antibacterial defense mechanism of the bladder. We have developed a model in rabbits for quantitatively determining the attachment of microbial organisms to the vesical mucosa. Our data suggest that the bladder has a primary antibacterial defense mechanism that serves to interrupt microbial attachment. This mechanism seems to be secretory, since after destruction with acid, the resistance to microbial binding returns in less than 24 hr.


Electrocardiographic changes following intravenous pyelography. One hundred consecutive patients who underwent i.v. pyelography had ECG performed before, immediately following and 5 min after the injection. ECG changes appeared in 15 patients and were considered to be major in five of them. The incidence of ECG changes was significantly higher in patients with abnormal ECG prior to pyelography. The average age of the patients who developed changes was higher than that of patients in whom no ECG changes were observed. There was no significant difference in the incidence of ECG changes after standard and infusion pyelography.


Immunocytochemical localization of substance P in mammalian intestine. In mammalian intestine immunoreactive Substance P is localized not only in the plexuses of Auerbach and Meissner, as could be anticipated, but also in a number of basally situated, often basigranular, endocrine cells which have been identified tentatively as enterochromaffin. The presence of a neurohormone in cells of this type confirms their close association with the nervous system, noted by Masson (1924), and suggests that their postulated origin from the nervous system (Danisch, 1924) may well be correct.


Localization of substance P-like immunoreactivity in mouse gut. Substance P immunoreactivity has been demonstrated in the mouse gut in two distinctly different locations; in the myenteric plexus of the proximal colon as well as in endocrine-like cells of the duodenal and colonic mucosa.


Immunoperoxidase localisation of hepatitis B antigen (HB) in formalin-paraffin processed liver tissue. The unlabelled antibody-enzyme (PAP) method was used to demonstrate HB in sections of routine formalin fixed paraffin embedded liver biopsies.


The ultra-structural localization of enzyme activity in unfixed sections. A technique is described for using conventional unfixed cryostat sections for localising enzyme activities in the electron microscope. The sections are incubated on the slide in the presence of a stabiliser, and then only fixed and embedded after the reaction is complete. Thin sections can then be cut of the reacted section, for electron microscopy. The present article shows that good ultra-structural morphology is retained during the freezing and cutting stages, and although some loss of detail occurs after the incubation, sub-cellular membranes are still intact, and membrane-associated reaction product can be clearly seen.


Formaldehyde-ozone-induced fluorescence in isolated gastrin granules. In several mammals the antral gastrin cells display characteristic formaldehyde-ozone-induced fluorescence (375/500-520 nm). Such fluorescence is also given by gastrinoma cells. Gastrin granules, isolated from gastrinomas and identified by immunofluorescence and electron microscopy, were found to show intense formaldehyde-ozone-induced fluorescence.


Control of vascular volume in sheep umbilical circulation. Vascular and extravascular volumes were measured using a single-injection, double-indicator technique, in a perfused umbilical circulation in which umbilical artery (Pfa) and umbilical vein (Pfv) pressures could be independently varied. At constant Pfv, when Pfa was raised from 25 to 35 mmHg, vascular volume increased 16.1  plus or minus 6 ml (mean plus or minus SD) from a control value of 113 plus or minus 38.3 ml. Extravascular volume increased 25.2 plus or minus 7.7 ml from 128 plus or minus 53.3 ml. During further increases in Pfa to 65 mmHg, vascular volume increased approximately 10 ml for each 10 mm Hg increment, but no change in extravascular volume occurred. At a constant Pfa when Pfv was raised to 15, 20, and 25 mmHg, vascular volume increased 12.2 plus or minus 3.6, 22.2 plus or minus 5.4, and 28 plus or minus 12.5 ml from the measurements at low Pfv. There was no increase in extravascular volume during elevation of Pfv. At the lower values of Pfa (25-35 mmHg) recruitment of previously unperfused channels and distension are seen. Over the higher ranges of Pfa change and during Pfv elevation there is distension of vascular bed.


Microbiological methods. Methodology for enteropathogenic Escherichia coli. Pathogenic biotypes of Escherichia coli grow poorly at temperatures greatly different from that of the host. Percentages quantitatively recovered at 42.0, 44.0, 44.5, and 45.5 degrees C in lauryl tryptose broth were 100, 76, 76, and 42, respectively. Corresponding values for 175 strains of varied origin were 98, 89, 82, and 65%. Maximal growth temperature is dependent upon medium. Lauryl tryptose and elevated coliform broths were equivalent in the recovery of small inocula (100 cells/ml) at 41.5-44.5 degrees. MacConkey, enteric enrichment, and Gram-negative broths were inhibitory at corresponding values. Growth at elevated temperature in nutrient broth is enhanced by carbohydrate. Standard lactose enrichment media fail to recover slow lactose fermenters. An acidified glutamic acid medium was unsuitable for recovery of E. coli. The data suggest modification of standard temperatures for the recovery of pathogenic biotypes. Previously recommended analytical methods have been simplified and supplemented. The enhancement of motility in indole-nitrite broth at 35 degrees C is recommended. A 4-tube semiquantitative test is offered for tentative identification of somatic and capsular antigens. Inclusion of Alkalescens-Dispar strains is warranted by their pathogenic behavior. Examination in Shigella and Alkalescens-Dispar sera is required to cover the dysentery-like biotypes. Pathogenic potential cannot be inferred from serotype.


Kanner's syndrome or early-onset psychosis: a taxonomic analysis of 142 cases. Case history data on 142 psychotic children were obtained using the Rimland Diagnostic Check List. A particular variety of the method known as numerical taxonomy was used to classify the children into subgroups on the basis of responses on the Check List. A comparison was made between a classification of Kanner's syndrome vs. "no Kanner's syndrome" and a classification using random classes as starting points. The "best" classification of the data was one in which 2 classes were obtained, one containing all the Kanner's syndrome children plus other early-onset, nonrelating psychotic children and the other containing children with later onset and more varied symptoms. Those attributes which significantly differentiated between the classes both in the "best" classification and in the Kanner's syndrome classification were extracted. A distinction between Kanner children and other similar children was found only for 3 specific behaviors: obsessive desire for sameness, islets of special ability, and skillful manipulation of small objects.


Conservation of Salmonella typhimurium deoxyribonucleic acid by chromosomal insertion in a partially diploid Escherichia coli hybrid. A partially diploid Escherichia coli hybrid recovered from mating with a Salmonella typhimurium donor was converted to an Hfr strain, designated WR2080, as a means to examine the manner in which the added Salmonella genetic material was conserved in it. The Salmonella argH-+, metB-+, and RHA-+ alleles contained as supernumerary genes in WR2080 were transferred together to E. coli recipients in interrupted mating experiments approximately 25 min after initial parental contact; transfer of the allelic E. coli genes by a  haploid Hfr of the same transfer orientation occurred between 23.5 min (argH-+) and 25 min (rha-+) after initial contact. Entry of the E. coli ilv-+ marker of WR2080 in these experiments occurred at 29.5 min, 1.5 min later than the entry time of this marker from the haploid E. coli Hfr. When  unselected inheritance of the recessive E. coli argH-minus and rha-minus alleles of WR2080 was examined among ilv-+ selected E. coli recipients in which unselected inheritance of the Salmonella donor genes was shown to be low (8%), inheritance of argH-minus was only 7%, whereas 51% inherited the neighboring rha-minus gene. In a comparative cross employing a haploid E. coli Hfr, in which rha inheritance was similar at 56%, argH inheritance was 41%. It was concluded that the Salmonella genes contained in WR2080 were conserved on a genetic segment about 1.5 min in length chromosomally inserted near the allelic E. coli genes, thus creating a duplication on that region within the hybrid chromosome.


Genetics of resistance to colicins in Escherichia coli K-12: cross-resistance among colicins of group A. By using each of the available colicins, we have isolated a large number of colicin-resistant mutants. They included both receptor and tolerant mutants and each was screened for cross-resistance to all other colicins. On the basis of the cross-resistance of these mutants it was possible to place known colicins into two mutually exclusive groups, group A and group B. Mutants selected as resistant to colicins of group A may or may not be cross-resistant to other colicins of group A, BUT Are never resistant to colicins of group B. The reverse also applies. The mutants isolated as resistant to colicins of group A (A, E1, E2, E3, K, L, N, S4, and X) have been divided into 21 phenotypic classes on the basis of their colicin resistance patterns. These include most of the tolerant and receptor mutants previously isolated, some of which were previously shown to also have an increased sensitivity to certain antibiotics and detergents. Type strains from each of the phenotypic classes were therefore tested for sensitivity to a range of antibiotics, detergents, and surfactants that included all those previously used. With these new data, it has been possible to speculate informatively on the mode of action of the different colicins. We have confirmed the position of previously isolated mutations on the Escherichia coli K-12 genetic map, and located approximately the loci conferring colicin resistance in some of the newly isolated mutants.


dnaB gene of Escherichia coli K-12 affects superinfection inhibition between F' plasmids. F' Escherichia coli K-12 strains bearing the chromosomal mutation dnaB43 offer significantly less resistance to the conjugational introduction of a second F' plasmid than do nonmutant strains. Both the entry exclusion and incompatibility components of superinfection inhibition are altered. This action of dnaB43 occurs regardless of the presence of a recA-minus mutation in matings in liquid cultures and on membrane filters and is not limited to a particular set of F' plasmids. These effects are co-transducible by phage P1 with the temperature sensitivity conferred by dnaB43. The effects also occur with a strain carrying dnaB107. In the double F' strains that arise, the two plasmids exist as units autonomous of one another and the chromosome.


Restoration of F' superinfection inhibition in a DnaB mutant of Escherichia coli upon construction of heterozygous DnaB merodiploids or P1 lysogens carrying a dnaB analogue. F-prime derivatives of the Escherichia coli strain CR34 bearing the thermosensitivity mutation dnaB43 display low levels of plasmid-determined superinfection inhibition in conjugational crosses at 30 C. Salt-mediated phenotypic suppression of this temperature sensitivity fails to restore normal levels of inhibition, indicating its alteration is not a secondary effect of dnaB43 a-tion on growth or deoxyribonucleic acid syntheiss. Superinfection inhibition is fully restored in mutant cells made merodiploid for the dnaB region by introduction of the F' dnaB-+ plasmid F134-1. dnaB43-bearing strains lysogenized with P1 phage contribution dnaB-analogue protein show eight to nine times more superinfection inhibition than do the same cells carrying P1 prophage repressed dnaB-analogue protein production. Taken together, this evidence suggests a direct causal relationship between dnaB43 and the altered superinfection inhibition phenotype.


Genetic and kinetic evidence for different types of postreplication repair in Escherichia coli B. The changes in molecular weight of deoxyribonucleic acid (DNA) synthesized after ultraviolte irradiation of Escherichia coli WP28 uvrA, and strains additionally mutant at polA, exrA, recA, and exrA and polA loci, were examined by alkaline sucrose gradient centrifugation. In a repari=deficient uvrA recA strain, the frequency of breaks in newly synthesized DNA was equal to that for pyrimidine dimers in parental DNA. Measurements of the amounts and rates of postreplication repair of these breaks indicate that (i) repair is two to three times faster when DNA polymerase I is present, although (ii) almost all breaks are repaired regardless of DNA polymerase I activity. (iii) Increased ultraviolet doses lead to an increase in the proportion of breaks remaining unrepaired in uvrA recA, UVRA exrA, and uvrA exrA polA strains. The numbers of unrepaired breaks resemble the numbers expected if repair of one lesion is prevented by proximity of a second lesion.


Effect of uncouplers on "downhill" beta-galactoside transport in energy-depleted cells of Escherichia coli. Galactoside permease-containing cells of Escherichia coli can be depleted of energy reserves so that the "downhill" cellular hydrolysis of o-nitrophenyl-beta-d-galactopyranoside (ONPG) no longer takes place. Treatment of such energy-depleted cells with proton-conducting agents such as carbonylcyanide m-chlorophenylhydrazone results in stimulation of ONPG transport. The same agents lower transport of non-energy-depleted cells towards the same levels that result from stimulation of the energy depleted cells. Of course, these agents prevent "uphill" accumulation against a concentration gradient under all conditions. Since uncouplers allow normal and energy-depleted cells to assume the same facilitated transport capability, these results lend support to the chemiosmotic hypothesis of Mitchell that comigration of charge is necessary for the transport of neutral galactosides. Our results imply that a potential favorable to transport is maintained by metabolism in non-energy-depleted cells, whereas an unfavorable potential is developed in the initial instant of time when energy-depleted cells are given ONPG.


Isolation of a nontransmissible antibiotic resistance plasmid by transductional shortening of R factor RP1. A plasmid segregant carrying tetracycline and carbenicillin resistance markers has been isolated from R factor RP1 by transductional shortening with phage P22. The new plasmid RP1-S2, which has a molecular weight of 23 times 10-6, has lost the transfer, phage sensitivity, and neomycin resistance functions of RP1. It combines readily with a W group plasmid, R388, to form a transmissible carbenicillin and trimethoprim resistance plasmid, RWP1.


Lambda bacteriophage gene produces and X-ray sensitivity of Escherichia coli: comparison of red-dependent and gam-dependent radioresistance. When gene products of lambda bacteriophage are introduced into a cell by transient induction of a lysogen, increased resistance of the cells to X rays results. This phenomenon has been called phage-induced radioresistance. Genetic studies show at least two classes of induced radioresistance. The first type depends on the products of the lambda red genes and is observed in bacteria that are mutated in the recB gene. It is thought that the lambda red products compensate for the missing RecBC nuclease in the repair of X-ray damage. An optimal effect is obtained even when the lambda red products are supplied 1 h after irradiation. The lesions that are affected by the red-dependent process are probably not deoxyribonucleic acid strand breaks because the extent of deoxyribonucleic acid strand rejoining is not altered by the red products. The second type of phage-induced radioresistance requires the gam product of lambda and is observed in wild-type and polA strains. The lambda gam+ gene produce must be present immediately after irradiation to exert its full effect. In its presence, DNA breakdown is decreased, and a greater fraction of DNA is converted back to high molecular weight. Strains carrying lex, recA, or certain other combinations of mutations do not show any detectable phage-induced radioresistance.


Properties of the imidazolylacetolphosphate aminotransferase produced in a mutant demonstrating no apparent genetic involvement of the structural gene. Genetic studies with strain hisBH22 of Salmonella typhimurium indicate it contains a deletion within the histidine operon involving part of the hisH gene and all of the hisB gene, but not extending into the adjacent hisC gene which is adjacent to hisB. However, the specific activity of the hisC product, imidazolylacetolphosphate aminotransferase (EC 2.6.1.9), in this strain is only 10 to 15% of that found in extracts from other mutants with a normal hisC gene. We have examined the rate of aminotransferase synthesis in this mutant and we find that the rate of synthesis of aminotransferase activity is low in mutant hisBH22, but the rate increases as the temperature of growth is lowered from 37 to 23 C. The low rate of enzyme accumulation is not due to holoenzyme instability at 37 C but instead is due to apoenzyme instability at this temperature. By transducing the hisBH22 marker into a pyridoxine auxotroph and derepressing the histidine operon under conditions where the intracellular concentration of pyridoxal phosphate would be expected to be low, we were able to demonstrate significant apoenzyme production only at the lower temperature. We suggest that the explanation for low aminotransferase specific activity at 37 C is due to the presence of reduced numbers of catalytically active units caused by normal production of an unstable mutant apoenzyme with only approximately 15% of the molecules being activated to holoenzyme. The holoenzyme from strain hisBH22 is stable during growth of this strain at 37 C.


Mode of action of alpha-dehydrobiotin, a biotin analogue. Alpha-Dehydrobiotin, like biotin, represses coordinately the 7,8-diaminopelargonic acid aminotransferase and the dethiobiotin synthetase enzymes that are encoded on the l and r strands, respectively, of the bioA operon. The rate of synthesis for both enzymes is inhibited about 80% in the presence of alpha-dehydrobiotin. Homobiotin and alpha-methylbiotin are less effective than alpha-dehydrobiotin in repressing the synthesis of the two enzymes. The selective repression of transcription from l and by alpha-dehydrobiotin and homobiotin, previously reported in hybridization experiments, is not observed at the enzyme level. A combination of equal concentrations of biotin and alpha-dehydrobiotin which was reported to enhance selectively the level of messenger ribonucleic acid transcribed from the l strand does not increase the rate of synthesis of the aminotransferase enzyme. Instead, the enzymes encoded on both strands are essentially completely inhibited as with biotin alone. Strain differences have been ruled out to account for the different results obtained by the two methodologies. Our evidence would suggest that alpha-dehydrobiotin acts like biotin, presumably as the co-repressor, in the repression of the bioA operon. The low rates of enzyme synthesis observed in the presence of the biotin analogue is the result of incomplete repression due to a lower affinity of either the analogue for the repressor or of the co-repressor/repressor complex for the operator. While our evidence would support the concept of a two promoter/operator complex, both would have to respond equally to biotin and its analogues. The evidence, however, does not rule out other possible alternative models for the regulation of the biotin operon.


Amplification of sex repressor function of one fi-+ R-factor during anaerobic growth of Escherichia coli. It has been reported by Mitsuhashi (1965) that transfer of one R-factor was completely inhibited by anaerobic transfer conditions. In contrast, several workers have observed R-factor transfer, although at a reduced rate, in the animal intestines, a largely anaerobic environment. It is shown here that in vitro transfer of the R-factor R1 (F-type pilus, fi-+) in Escherichia coli K-12 is severely depressed, whereas transfer of R64 (I-type pilus, fi-minus) is slightly stimulated by anaerobiosis. Inhibition of R1 fertility is dependent on anaerobic conditions during pregrowth of the donor cells, whereas the oxygen tension during recipient pregrowth, transfer, and plating is of little importance. Anaerobic pregrowth has a less inhibitory effect on the fertility of R1drd19, a mutant of R1 having a defective sex repressor. The fi-+ property of R1 when introduced into F' or Hfr bacteria is amplified during anaerobic growth. These observations strongly indicate that the sex repressor is the mediator of the anaerobic fertility inhibition of the R-factor R1. This hypothesis was supported by studies of the formation of sex pili, the only gene product identified that is controlled by the sex repressor of R1. Using propagation of the F-type pilus-specific phage MS2 as a measure of the degree of sex piliation of a bacterial population, it is shown that in anaerobic cultures sex piliation due to R1 is strongly repressed, whereas piliation due to R1drd19 is repressed to a lesser extent. The possible survival value of the response of R1 towards oxygen tension is discussed.


Alterations of the rate of movement of deoxyribonucleic acid replication forks. Antibiotics that inhibit ribonucleic acid (RNA) or protein synthesis are often used in studies of deoxyribonucleic acid (DNA) synthesis. The experiments presented here demonstrate that the rate of movement of DNA replication forks can be influenced by such antibotics. Addition of either chloramphenicol, which inhibits movement of ribosomes along messenger RNA, or streptolydigin, which inhibits movement of RNA polymerase, leads to a decrease in the rate of fork movement. Rifampin, which inhibits initiation of RNA synthesis, reverses the effects of chloramphenicol or streptolydigin. The slowed movement of DNA replication forks is discussed in terms of obstruction of fork movement by transcription complexes temporarily immobilized on the DNA template.


Variation in expression of sex factor genes in the Proteus-Providencia group relative to Escherichia coli. Several instances of anomalous expression of genes introduced from Escherichia coli K-12 into Proteus mirabilis have been described. It is shown here that control of sex pilus synthesis directed by the F-like R factor R1 and its depressed derivatives R1-16 (O-C) and R1-19 (i-minus) is also anomalous in P. mirabilis. Piliation in cells bearing the depressed plasmids is expressed at a lower level than in E. coli K-12, and repression is absent in R1-carrying cells. Preliminary results show a similar effect in Providencia. In Proteus morganii, a similarly reduced level of piliation in R1-16-+ or R1-19-+ cultures is observed, but an intermediate level of repression occurs in R1-+ cultures. Less extensive data suggest that expression of the sex factor genes of an R factor of the N incompatibility group differs far less between E. coli and P. mirabilis hosts. Possible bases for these effects are discussed.


RP1 properties and fertility inhibition among P, N, W, and X incompatibility group plasmids. Incompatibility group P plasmids demonstrate strong entry exclusion properties. Stringent incompatibility is also observed in the absence of entry exclusion. These observations have been facilitated by the study of a nontransmissible plasmid, RP1-S2, derived from RP1 by transductional shortening. RP1-S2 retains carbenicillin and tetracycline resistances as well as loci that cause either the loss of P plasmids (incp) or a locus specifying susceptibility to curing (sinp) in the presence of a P plasmid. RP1-S2 can be mobilized by an incompatibility group W plasmid, R388, and also freely forms recombinants with R388. P, N, and W incompatibility group plasmids all encode information for the receptor of the cell wall-adsorbing phage PRD1. Based on the premise that the location of this receptor is analogous to entry exclusion factors for F-like plasmids and hence a regulated transfer region determinant, we tested fertility inhibition relationships among these plasmid groups. We detected both reciprocal and nonreciprocal fertility inhibition relationships for bacteria containing various combinations of W, N, and P group plasmids. The nonreciprocal nature of some combinations, we believe, reflects the identity of the point mutation reading to derepression of the plasmid in question. Reciprocal fertility inhibition, on the other hand, may reflect the reconstruction of a fertility inhibition system through complementation. An X incompatibility group plasmid, known to affect the fertility of an N group plasmid, was also shown to inhibit P plasmid fertility. These observations may indicate a possible evolutionary relationship(s) of plasmids unrelated by the criteria of incompatibility, pilus phage specificity, or plasmid host range.


Gentic properties of an R factor carrying resistance to aminolgycoside antibiotics. R factor Rms 151 is an fi+ R factor and belongs to a incompatibility group FII. It carries the genes governing resistance to various aminoglycoside antibiotics, i.e., kanamycin (KM), lividomycin (LV), gentamicin C complex (GM), and 3',4'-dideoxykanamycin B (DKB), in addition to those governing to tetracycline (TC), chloramphenicol (CM), sulfanilamide (SA), and ampicillin (APC). Electron microscopy observation disclosed that the Rms151 deoxyribonucleic acid was a circular form with length of 31.2 mum. A probable circular genetic map of Rms151 was proposed by genetic and biochemical studies, the genes being in the order of -tet-tra-amp-aad-sul-aph-cml-, in which aad and aph confer resistance to KM.GM.DKB by adenylytransferase or resistance to KM.LV by phosphotransferase, respectively.


Colicin tolerance and map location of conjugation-deficient mutants. Con minus mutants, conjugation-defective mutants of Escherichia coli K-12, have been shown to be tolerant to colicins K and L. They map at approximately 14 min on the genetic map. The significance of the colicin tolerance of conjugation-defective mutants is discussed.


Synchronization of cell division in Escherichia coli by amino acid starvation: strain specificity. Synchronization of cell division by amino acid starvation can be induced in strains B and K-12 of Escherichia coli but not in strain B/r.


Quantitation of the loss of the bacteriophage lambda receptor protein from the outer membrane of lipopolysaccharide-deficient strains of Escherichia coli. The recpetor for the phage lambda, a protein component of the outer membrane, is present at decreased levels in strains of Escherichia coli that are deficient in lipopolysaccharide. Loss of the protein was quantitated both by an assay of the phage receptor function and by an assay of antiserum-blocking ability to detect inactive protein. The loss of protein was correlated with the loss of sugar residues and phosphage from the core region of the lipopolysaccharide. Implications for the importance of ionic interactions in the stabilization of the outer membrane are discussed.


Transition of the R factor NR1 and Proteus mirabilis: level of drug resistance of nontransitioned and transitioned cells. When Proteus mirabilis harboring the R factor NR1 is cultured in Penassay broth containing 100 mug of chloramphenicol (CM) per ml, there is an amplification in the number of copies of the r-determinants per cell. Under these conditions, R factors harboring multiple tandem sequences of r-determinants are formed. Autonomous poly-f-determinants consisting of multiple copies of r-determinants are also formed. This phenomenon has been referred to as the "transition". Transitioned cells have considerably higher levels of resistance to CM and streptomycin (SM), but not to tetracycline (TC), than do nontransitioned cells and grow more rapidly in medium containing either CM or SM. There is essentially no difference in growth rates between transitioned and nontransitioned cells in drug-free medium. The higher level of resistance of transitioned cells to SM has made it possible to investigate the mechanism of the transition. Using replica plating, it has been possible to isolate spontaneously occurring transitioned cells from a nontransitioned population which appear to outgrow the nontransitioned cells during growth in medium containing 100 mug of CM per ml. If transiitoned cells are subsequently cultured in drug-free medium, the cells return gradually to the nontransitioned state, which has been referred to as the "back-transition was monitored by examining the level of resisitance of the cells to SM. In both situations the cell populations were found to be heterogeneous, consisting of a mixture of nontransitioned and transitioned cells. Under the conditions of our experiments, the transition appeared to be due to the more rapid growth of a minor fraction of spontaneously occurring transitioned cells which outgrew the remainder of cells in the population. To obtain the transition, the drug resistance gene must reside on the r-determinants component of the R factor. The transition did not take place when the cells were cultured in medium containing high concentrations of TC. This indicates that the TC resistance genes reside on the resistance transfer factor component of the R factor, which is in agreement with physical studies on R factor deoxyribonucleic acid.


Regulation of the Escherichia coli methylgalactoside transport system by gene mglD. Constitutive activity of the methylgalactoside transport system of Escherichia coli K-12 is shown to result from mutation of a genetic locus distinct from the two previously described regulatory loci for this permease. Employing an autoradiographic procedure whereby constitutive and inducible cells can be differentiated, it is demonstrated that this locus, termed mglD, is 20% cotransducible with ptsF by bacteriophage P1. Selection for constitutive mutants among an inducible population yielded cells who mutations mapped in mglD. Cotransduction of mglD with mglB, minus C, and minus A, three genes required for activity of the methylgalactoside transport system, is 95, 88, and 81%, respectively. The results of recombination studies employing three and four factors indicate that the order of genes in this region is ptsF, mglD, B, C, A.


Genetic and physiological analysis of an envB spherelike mutant of Escherichia coli K-12 and characterization of its transductants. The envB1 mutation mediating a distorted cell morphology of Escherichia coliK-12 was cotransducible with strA, aroE, aspB, and argG. The mapping data is consistent with a gene location for envB around 62.5 min. In partial diploids envB1 was recessive to its wild-type allele. The original envB mutant contained a second mutation in a locus denoted sloB close to strA. The following gene order is suggested: sloB-strA-aroE-envB-aspB-argG. The sloB1 mutation caused a marked reduction in the growth rate of both envB and envB+ strains. Moreover, this mutation in the presence of envB1 appears to increase the ratio between deoxyribonucleic acid and protein in cells growing in rich medium. The phenotypic properties of envB1, sloB+ and envB+ transductants were characterized. Cells with envB1, sloB+ genotype were hypersensitive to several penicillins including the beta-lactam compound, amidino penicillin. Penicillin hypersensitivity could not be explained by increased outer membrane penetrability. The original envB mutant (envB1,SLOB1), as well as envB1, sloB1 or envB+, SLOB1 transductants were resistant to amidino penicillin. Resistance was explained by the slow growth rate mediated by the sloB1 mutation. The similarity between envB cells and wild-type cells treated with sublethal concentrations of amidino penicillin was emphasized.


Role of undecaprenyl phosphate in synthesis of polymers containing sialic acid in Escherichia coli. Membrane-associated sialytransferase complexes from Escherichia coli K-235 catalyze the incorporation of N-acetylneuraminic acid (NeuNAc) from cytidine 5-monophospho-N-acetylneuraminic acid (CMP-NeuNAc) into polymeric products and a lipid fraction. Reconstitution of enzyme activity in lipid-depleted membrane complexes had an absolute dependence on a purified phospholipid which was characterized by high resolution mass spectrometry following dephosphorylation as undecaprenol. An identical mass spectrum was obtained on the phosphorylated lipid confirming that the active derivative was undecaprenyl phosphate. This C55-isoprenoid alcohol accounted for 95% of the polyisoprenol and contained 11 isoprene units, each one unsaturated. The remaining 5% was composed of the C60 homologue, dodecaprenol. Ficarprenyl phosphate, an isomer of undecaprenyl phosphate, also restored enzymatic activity although on a molar basis, it was less active than undecaprenyl phosphate. These results provide direct evidence that sialyl polymer synthesis has an obligatory requirement for undecaprenyl phosphate, a membrane-bound lipid coenzyme which functions as an intermediate carrier of glycosyl residues in the biosynthesis of a variety of microbial cell surface polysaccharides. These data extend further the general class of polysaccharides whose synthesis involves undecaprenyl phosphate and are in accord with the hypothesis that this lipid acts as an intermediate carrier of sialyl residues in sialyl polymer synthesis according to the following reaction: CMP-NeuNAc plus P-undecaprenol in equilibrium NeuNAc-P-undecaprenol plus CMP Although the sialylated lipid remains to be characterized, evidence in support of this conclusion was obtained by kinetic analysis of N-acetylneuraminic acid transfer into the lipid soluble fraction and sialyl polymers. These studies showed a rapid incorporation of N-acetylneuraminic acid into the lipid-soluble fraction prior to maximal sialyl polymer formation, an observation consistent with a possible precursortion, an observation consistent with a possible precursor-product relationship. Confirmation that the radioactivity in the lipid-soluble fraction was lipid-linked N-acetylneuraminic acid was provided by the demonstration of the formation of a transitory sialyl-lipid with chromatographic properties expected of sialyl-undecaprenyl phosphate. Three additional lines of investigation implicated a functional role for undecaprenyl phosphate in sialyl polymer synthesis...


Properties of membrane-associated sialyltransferase of Escherichia coli. Membrane-associated sialyltransferase complexes in Escherichia coli K-235 can be dissociated by lipid deletion and reassembled by the addition of undecaprenyl phosphate, a unique membrane-bound lipid coenzyme. Following disruption of the cells by pressure disintegration and centrifugal fractionation, the sialyltransferase activity is assocatied with both a "particulate" and "soluble" complex. Kinetic studies as well as sugar nucleotide, metal ion, pH, ammonium sulfate, and thiol reagent requirements showed these two complexes contained functionally identical enzymatic activities. Isopycnic sucrose density gradient centrifugation studies carried out on unfractionated total membranes established that these sialytransferase activities were associated with membrane hybrids composed of different relative amounts of inner and outer membranes. Enzyme localization studies employing DPNH oxidase, a marker for the inner membrane, and relative phospholipid to protein composition determinations in the two complexes, provided added support for this conclusion. Sialyl polymer synthesis was not dependent on the incorporation of other monosaccharides and had no demonstrable metal ion requirement. Kinetic studies showed that the Km for cytidine 5-monophospho-N-acetylneuraminic acid in intact soluble and particulate enzyme preparations was 8.1 times 10-5M and 9.2 times 105M, respectively. Similarly, both enzyme complexes had nearly identical Vmax values. Following reassembly of delipidated enzyme preparations, however, there was a 10-fold increase in the Km value for the particulate enzyme and a 3-fold increase for the soluble enzyme. This increase was accompanied by an increase of approximately the same magnitude in the Vmax values. Since the lipid coenzyme was limiting in intact enzyme preparations, the increase in Vmax reflected an increase in the concentration of the active lipid in reconstituted complexes. Sialyl polymer synthesis in intact membrane preparations was stimulated by the exogenous addition of lipid. Insertion of the carrier lipid was dependent on temperature. At 37 degrees, a 120% increase in sialytransferase activity was observed while only a 35% increase was observed at 30 percent. At 20 degrees, no stimulation occurred. Fluidity of the lipid phase is apparently required for proper function of this membraneassociated enzyme complex. Thus, at 20 degrees, a temperature below the membrane lipid transition temperature, the lipids are relatively immobile.


Synthesis and turnover of basal level guanosine tetraphosphate in Escherichia coli. Cultures of escherichia coli growing exponentially in Trisacetate medium were subjected to nutritional shift-up and the pool size of guanosine 5'-3'-diphosphate-3'diphosphate (ppGpp) as well as the rates of protein synthesis and net RNA synthesis were determined. In the shift to a rich medium (glucose plus 19 amino acids plus hypoxanthine) the basal level of ppGpp falls immediately with a decay constant suggesting total inhibition of synthesis; ther is no ppGpp detectable above background for 30 to 40 min. The net rate of RNA synthesis starts to increase within 1 min of the shift-up and has reached its definite postshift value well before the pool of ppGGpp rises above background lvel. In a shift-up from Tris-acetate medium to Tris-glucose medium there is a much slower readjustment of the ppGpp pool size without the transient disappearance of the nucleotide. However, in a shift-up to Tris-acetate plus 5 amino acids, a medium which supports the same growth rate as Tris-glucose medium, a dramatic, transient drop in the ppGpp pool level was observed. Relaxed cells exhibit very similar behavior to strigent cells in the same shift-up. Our data argue strongly against an exclusive role for pGpp in regulating RNA synthesis during niutritional shift-up. The kinetic data of [3H]guanosine uptake into GTP and ppGpp pools were analyzed to determine the rate of pGpp synthesis. This rate was found to be similar during expotential growth in either Tris-acetate medium. During a shift-down from Tris-glucose to Tris-acetate medium the rate of ppGpp syntesis fell by a factor of 1.5 to 2 and the turnover rate is reduced 6- to 8-fold, suggesting that the expansion in the ppGp pool during shift-down canot be due to derepression of synthesis.


The subunit structure of phosphoglucose isomerase from bakers' yeast. Bakers' yeast phosphoglucose isomerase was studied by both chemical and physical methods to determine its submit structure. Gel filtration in 6 M guanidine HCl as well as acrylamide gel electrophoresis of sodium dodecyl sulfatedentured phosphoglucose isomerase showed two speices corresponding to one-half and one-fourth of the preparative molecular weight of 119,400 determined by equilibrium centrifugation. Further centrifugation studies showed that the enzyme could be completely dissociated to species of 30,000 molecular weight. Peptide maps of tryptic hydrolysates of denatured and chemically modified enzyme showed that the protein is composed of four identical or nearly identical sub-units. The results of amino acid analysis, except half-cystine content, were compatible with identical subunits. The appearent partial specific volume and extinction coefficient were also determined.


Two photo-cross-linked complexes of isoleucine specific transfer ribonucleic acid with aminoacyl transfer ribonucleic acid synthetases. Escherichia coli Ile-tRNA synthetase and tRNA-Ile have been cross-linked photochemically by the direct action of ultraviolet light. In addition, photo-induced joining of tRNA-Ile E. coli to Val-tRNA synthetase from yeast has also been achieved. This yeast enzyme is known to mischarge E. coli tRNA-Ile with valine. Regions on tRNA-Ile involved in cross-linking have been determined for both complexes. In each case, three distinct parts of the nucleic acid are found cross-linked. Two of these are the same in both complexes and involve the dihydrouridine stem and loop region. The third part is unique for each complex. It involves the 3' terminus in the cognate one and the 3' side of the anticodon in the non-cognate case. When the cross-linked regions are projected onto a model of the three-dimensional structure of tRNA, it is clear that these and other data are consistent with having each enzyme bound in a similar orientation across tRNA-Ile. The enzymes are viewed as spanning the distance from the anticodon to the 3' terminus and making extensive contact with the area in which the two helical branches of the L-shape tRNA structure come together.


Isolation of the alpha and beta subunits of Escherichia coli succinyl coenzyme A synthetase and their recombination into active enzyme. Pure preparations of the alpha and beta subunits of succinyl-CoA synthetase of Escherichia coli have been obtained by preparative gel electrophoresis in the presence of detergent or by gel filtration in acid urea solution. Substantial enzyme activity (50 to 60%) can be recovered following renaturation of an equimolar mixture of the two subunits prepared by gel filtration. The substrate ATP is required for reconstitution of activity, but the presence of our substrates does not appear to influence the renaturation process. Knowing that ATP phosphorylates the alpha subunit in the native enzyme, these data suggest that phosphorylation of this subunit is necessary for correct assembly. Comparison of the amino acid compositions of the stoichiometry of the subunit reassociation in the reconstitution process confirm the alpha2 beta2 structure for the native enzyme.


Leucine transport in Escherichia coli. The resolution of multiple transport systems and their coupling to metabolic energy. The multiple active transport systems mediating L-leucine accumulation in Escherichia coli strain 7 (K12) and ML 308-225 have been examined. In addition to the previously characterized osmotic shock-sensitive LS (L-leucine-specific) and LIV-I (L-leucine; L-isoleucine-, and L-valine-specific) activities, a third system (designated LIV-II) has been detected, confirming a report by Rahmanian et al. (RAHMANIAN, M., CLAUS, D.R., and OXENDER, D. L. (1973) J. Bacteriol. 116, 1258-1266). This third system transports L-leucine, L-isoleucine, and L-valine with a relatively low affinity (apparent transport Km equals 4 muM for L-leucine) and it is resistant to repression by cell growth on L-leucine. Exploitation of these properties and of the differential sensitivity of the three transport activities to inhibition by L-leucine analogues permits estimation of the contribution by each system to the total transport activity under varying conditions. Such experiments show that, unlike systems LS and LIV-I, system LIV-II is resistant to osmotic shock. The L-leucine, L-iosleucine, and L-valine transport activity in membrane vesicles from strain ML 308-225 has the properties of system LIV-II. Although the L-leucine transport activities in strains 7 and ML 308-225 are in all other respects similar, membrane vesicles from strain 7 do not transport L-leucine, L-isoleucine, or L-valine. L-leucine transport under various conditions of energy supply has been measured in strain ML 308-225 and the corresponding Mg-2+-ATP-ASE-DEFICIENT STRAIN, DL-54. These measurements support the view that the osmotic shock-sensitive LS and LIV-I activities depend on the synthesis of ATP, while the osmotic shock-resistant LIV-II activity depends on the energized membrane state generated by electron flow but not on ATP synthesis, per se. This conclusion is not supported by the inhibitory effects of the energy poisons arsenate and 2,4-dinitrophenol, but these compounds may have secondary chemical effects on the transport systems.


Transport in an Escherichia coli fatty acid auxotroph. A novel case of catabolite repression. The transport of alpha-methylglucoside and a variety of amino acids including histidine, leucine, lysine, proline, and threonine are reduced 5- to 50-fold in Escherichia coli K1061, an unsaturated fatty acid auxotroph, when cells are grown in medium containing glucose as a carbon source and supplemented with either cis-vaccenic acid or cis-9, 10-methylenehexadecanoic acid. Cells grown in glucose medium containing oleate, palmitoleate, or cis-7-hexadecenoate do not exhibit this effect and transport alpha-methylglucoside and amino acids at rates comparable to those of a wild type strain. For cis-vaccenate- and cis-9, 10-methylenehexadecanoate-grown cells the capacity to transport amino acids and alpha-methylglucoside is normal when glycerol or succinate is the energy supply or when cyclic adenosine 3':5'-monophosphate is added to cultures growing on glucose. These results suggest that in this strain several transport systems are affected by catabolite repression, but the phenomenon is unique since repression requires not only glucose but specific unsaturated fatty acids, cis-vaccenic acid, or cis-9, 20-methylenehexadecanoic acid.


Tryptophan operon read-through. Isolation and characterization of an abnormally long tryptophan synthetase alpha subunit from a frame-shift mutant of Escherichia coli. A new mutant strain of Escherichia coli, strain ICR-47, contains a frame-shift mutation in the trpA gene, the gene most distal to the operator in the trp operon. Mapping experiments indicate that the lesion is located at a site within 10 to 15% of the end of this gene. The mutation results in "out-of-phase" translation of the distal portion of the trp mRNA; normal translational termination signal(s) are not encountered and a trpA gene product longer than the wild type protein is produced. As with the other enzymes produced from this operon, the in vivo level of the altered protein (the alpha subunit of the tryptophan synthetase enzyme complex) is controlled by exogenous L-tryptophan. The altered alpha subunit from the strain ICR-47 has been isolated and characterized. Molecular weight estimations indicate a molecular weight of approximately 37,000, an increase beyond the wild type enzyme corresponding to an additional 50 to 70 amino acid residues. The protein has a new COOH-terminal amino acid sequence. Results of preliminary hybridization experiments suggest that the ICR-47 mRNA, which is necessarily longer than that needed to code for wild type enzyme, is not detectably different in size from wild type mRNA. The enzymatic properties of the ICR-47 alpha subunit indicates a greatly reduced ability of the mutant subunit to combine functionally with wild type beta2 subunit, the second protein component in the tryptophan synthetase enzyme complex. In contrast, only 40 to 50% of the intrinsic enzymatic activity of the alpha subunit is lost.


Turnover of protein synthetic elongation and initiation factors in Escherichia coli. In an effort to understand the regulation of expression of the factors required for protein biosynthesis, we have measured the turnover of elongation factor G (EF-G) and initiation factor 2 (IF-2). Using a quantitative assay which involves immunoprecipitation and gel electrophoresis, it was found that both of these factors are very stable cellular proteins, but that their rates of accumulation are quite different, indicating a differential rate of synthesis. The effects of amino acid starvation and the relaxed response were also examined. The results showed that under these conditions EF-G and IF-2 are neither synthesized nor degraded.


Termination of transcription in bacteriophage lambda. Heterogeneous, 3'-terminal oligo-adenylate additions and the effects of rho factor. RNA transcripts were synthesized in vitro from a lambda DNA template with purified Escherichia coli RNA polymerase either in the presence or absence of the protein termination factor, rho. The products were initially characterized by electrophoresis on polyacrylamide slab gels, and two of the lower molecular weight discrete species (6 S and 4 S RNA) were further characterized by standard two-dimensional "fingerprint" analysis. Production of the 4 S RNA was strongly affected by the presence of rho, whereas production of the 6 S RNA species was relatively unaffected by rho. 3'-Terminal oligonucleotide fragments were then selectively isolated on columns of dihydroxyboryl-substituted cellulose from these transcripts. Sequence analysis of these oligonucleotide products indicated: (a) that all of the transcripts examined possess similar degrees of 3'-terminal sequence heterogeneity which consisted predominantly of the addition of 1 to 5 adenylate residues to the 3'-terminus of the transcript; and (b) that rho factor-enhanced termination results in a definite structural change in the nucleotide sequence with which an RNA molecule can terminate.


Resolution of D-serine dehydratase by cysteine.  An analytical treatment. A general method is presented for analysis of the resolution of pyridoxal-P-requiring enzymes by carbonyl reagents.  The method is useful for accurately determining the very small equilibrium constants (KP) which characterize the dissociation of cofactor from many pyridoxal-P-requiring enzymes.  The analysis also establishes the minimum number and relative stabilities of distinct enzymic species involved in the resolution process.  Analysis of the resolution of D-serine dehydratase by L-and D-cysteine resulted in the establishment of an enzyme bound thiazolidine derivative as an intermediate in the pathway for resolution.  The over-all equilibrium constant (KR) for the reaction, D-serine dehydratase + cystein in equilibrium KR thiazolidine derivative +D-serine apodehydratase was determined.  At pH 7.80, T/2 0.33, 25 degrees, KR equal to 1.08 times 10-minus 3.  A value of 7.0 nM for the equilibrium constant for the dissociation of D-serine dehydratase to apoenzyme and free pyridoxal-P was determined from the ratio KR/KT, where KT is the equilibrium constant for the formation of a thiazolidine derivative from free pyridoxal-P and cysteine.  An estimate of 14 nM for KP was also obtained from partial resolution of D-serine dehydratase by high dilution.  The difficulties associated with this direct determination of KP from the dependence on the enzyme concentration of the activity of very dilute solutions of enzyme are discussed.


Bacteriophage T7 Deoxyribonucleic acid replication in vitro.  A protein of Escherichia coli required for bacteriophage T7 DNA polymerase activity. In vivo, replication of T7 DNA does not occur after infection of Escherchia coli tsnC mutants (CHAMBERLIN, M. (1974) J. Virol. 14, 509-516). In vitro, extracts of tsnC mutant E. coli infected with T7 hage are incapable of replicating duplex T7 DNA, although extracts of wild type E. coli infected with T7 phage support replication of T7 DNA. In addition, extracts of the infected tsnC mutant are deficient in T7 DNA polymerase activity. Extracts prepared from uninfected E.coli tsnC-+ cells restore the ability of the infected tsnC extracts to replicate duplex T7 DNA, and also restore normal levels of the phage DNA polymerase activity. A 12,000-dalton heat-stable protein responsible for this complementation has been purified to near homogeneity from uninfected tsnC+ extracts and it is designated "TsnC protein."


Bacteriophage T7 deoxyribonucleic acid replication invitro. Bacteriophage T7 DNA polymerase: an an emzyme composed of phage- and host-specific subunits. The DNA polymerase induced after infection of Escherichia coli by phage T7 has been purified 500-fold to near homogeneity as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.  The purified enzyme complements extracts of cells infected with a T7 gene 5 mutant to permit cell-free replication of duplex T7 DNA.  In contrast, purified T4 DNA polymerase or E. coli DNA polymerase I is unable to do so, thus suggesting a specific requirement for the T7 enzyme in the replication of the viral DNA.  E. coli TsnC protein is present in purified T7 DNA polymerase in one-to-one stoichiometry with T7 gene 5 protein, and can be isolated in homogeneous form from heat-denatured enzyme by chromatography on DEAE-cellulose.  The inactive form of T7 gene 5 protein that accumulates in tsnC hosts has been partially purified.  When partially purified gene 5 protein is mixed with purified TsnC protein, DNA polymerase activity is restored, and formation of a one-to-one complex between the two proteins occurs.  These results indicate that the functional form ofT7 DNA polymerase is a complex composed of phage- and host-specified subunits.


Bacteriophage T7 deoxyribonucleic acid replication in vitro.  Purification and properties of the gene 4 protein of bacteriophage T7. The T7 gene 4 protein, a protein known from genetic analysis to participate in phage DNA replication in vivo, has been purified approximately 500-fold with an in vitro complementation assay. The protein, purified from cells infected with a T7 gene 4 temperature-sensitive mutant, is thermolabile, establishing that the complementation activity is in the protein product of the phage gene 4. The purified protein has no detectable nuclease, DNA polymerase, or RNA polymerase activity. However, in addition to stimulating the rate of DNA replication in crude extracts of T7 gene 4 mutant-infected cells, the gene 4 protein effects a marked stimulation of DNA synthesis by the purified T7 DNA polymerase when duplex T7 DNA is used as template. This effect is not observed when denatured T7 DNA is used as template, or when phage T4 DNA polymerase or Escherichia coli DNA polymerase I, II, OR III is substituted for the T4 enzyme. Analysis of the DNA synthesized by the T7 DNA polymerase in the presence of the gene 4 protein indicates that much of the product is in short DNA chains which are not covalently attached to the template. This result suggests a novel mechanism for the initiation of DNA chains in this reaction.


Purification and characterization of homogeneous initiation factor M2A from rabbit reticulocytes. Rabbit reticulocyte initiation factor M2A has been prepared in homogeneous form.  The final preparation was purified 2,300-fold and ran as a single band on polyacrylamide gel electrophoresis in three different buffer systems: alkaline, sodium dodecyl sulfate, and acidic 6.5 M urea.  IF-M2A also ran as a single band in polyacrylamide gel isoelectric focusing experiments with an apparent pI of 6.45.  The molecular weight of IF-M2A was approximately 125,000 based on determinations by low speed equilibrium centrifugation (118,000), sodium dodecyl sulfate gel electrophoresis (130,000) and s20,w combined with Stokes radius (124,000).  The amino acid composition of IF-M2A revealed three unusual features: a) the basic amino acids represented 19.4 mol %; b) glutamicacid (plus glutamine) constituted 18.8 mol%; c) tryptophan and cysteine residues were only 0.4 and 0.7 mol%, respectively.  Homogeneous IF-M2A was tested in several initiation assays using either natural or artificial mRNAs.  In each assay tested, homogeneous IF-M2A fully substituted for cruder preparations and at concentrations commensurate with its increased purity.  IF-M2A was also examined for ribosome-dependent GTP hydrolysis, an assay requiring ribosomes but no other initiation factors.  Analysis of the data yielded a Km for GTP of 10 muM and a Vmax for hydrolysis of 1.20pmol/mug IF-M2A/min.  In addition, IF-M2A mediated GTP hydrolysis required both 40 S and 60 S subunits for maximal activity.  The possibility that IF-M2A is a factor required for the joining of 40 S and 60 S subunits is discussed.


Escherichia coli succinic thiolinase. Stoichiometry of phosphorylation and coenzyme A binding. Equilibrium and covalent binding studies of succinic thiokinase from Escherichia coli indicates that there can be a stoichiometric relationship between coenzyme A binding and the phosphoyrlation capacity of the enzyme. A comparison of homogeneous enzyme preparations has revealed that enzyme of high specific activity exhibits greater binding capacity and that this property is proportional to enzyme activity. Phosphorylation capacity was related to specific activity in highly active enzyme preparations, but leveled off at 1 mol of phosphorus/mol of thiokinase. These studies show that the "dimer of dimers" structure of succinic thiokinase contains the expected two active sites and that this enzyme does not demonstrate "half-the-sites" reactivity. A coenzyme A binding  site of lower affinity can be detected in some enzyme samples of lower specific activity. Binding of coenzyme A to the higher affinity sites may involve positive cooperativity. ATP, unlike ADP, does not bind to phosphorylated enzyme.


Inhibition of thymidylate synthetase and dihydrofolate reductase by naturally occurring oligoglutamate derivatives of folic acid. Naturally occurring oligoglutamate derivatives of folic acid in extracts of Escherichia coli have been isolated on the basis of their inhibitory actions toward thymidylate synthetase and dihydrofolate reductase. The inhibitor of thymidylate synthetase has been identified as N-5-formyl-H4pteroyloligoglutamate (approximately 5 amino acid residues). It is 150-fold more inhibitory than the monoglutamate. Synthetic N-5-formyl derivatives containing 3 to 6 glutamyl residues were prepared and found to be 67- to 200-fold more inhibitory than the monoglutamate. N-5-Formimino-H4pteroyltriglutamate is one-twentieth as inhibitory as the corresponding N-5-formyl derivative. The inhibitor of mouse leukemia dihydrofolate reductase has been identified as N-10-formylpteropentaglutamate. It is approximately 7 times as inhibitory as N-10-formylpteroylmonoglutamate. It is 4,400 times as inhibitory toward mouse leukemia dihydrofolate reductase compared with the enzyme from E. coli. Lysine analogs of N-5-formyl-H4folate containing alpha0glutamyl groups in peptide linkage to the epsilon-amino group of lysine were relatively poor inhibitors of thymidylate synthetase. The inhibitory action of folic acid oligoglutamates on E. coli thymidylate synthetase was subject to reversal with 0.4 M NaCl, an effect that was more marked with various pteroyloligoglutamates than with H4homopteroylmonoglutamate and N-5, N-8-deaza-N-10-methylpteroylmonoglutamate.


Polyadenylate metabolism in the nuclei and cytoplasm of Saccharomyces cerevisiae. A procedure has been designed for the simultaneous isolation, in a single step, of the nuclei and cytoplasm of Saccharomyces cerevisiae alphas288c spheroplasts. We have examined the polyadenylate poly(A)-containing RNA in these fractions and their kinetics of synthesis. Nuclear RNA saturates with [3H] adenine within 10 min. Labeled RNA appears very quickly in the cytoplasm, exceeding the amount of labeled nuclear RNA within 2 min after the addition of [3H] adenine. Nuclear poly(A)-containing RNA is approximately 10% of the total cellular poly(A)-containing RNA obtained from spheroplasts labeled for 15 min. Nuclear poly(A)-containing RNA is not as large as the giant heterogeneous nuclear RNA of animal cells. The distribution of molecular size in nuclear and cytoplasmic populations of poly(A)-containing RNA is very broad with the average size of the nuclear species being moderately larger than the cytoplasmic species. Three distinct size classes of poly(A), with different apparent kinetic properties, are obtained from yeast poly(A)-containing RNA. Their electrophoretic mobility suggests molecular lengths of approximately 20, 40, and 60 nucleotides (Groner, G., Hynes, N., and Phillips, S. (1975) Biochemistry, 13, 5378-5383). Each of these poly(A) classes are present in the mRNA from large and small polyribosomes.


The 30 S ribosomal precursor RNA from Escherichia coli.  A primary transcript containing 23 S, 16 S, and 5 S sequences. The 30 S ribosomal precursor RNA has been prepared from Escherichia coli AB301/105 (RNase III-) labeled with 32-PO4 in the presence of chloramphenicol. Direct nucleotide sequence studies yield the following information. 1. The major 5'-terminal sequence in our precursor preparations is pppA-C-U-G-. 2. Treatment of the precursor RNA with purified ribonuclease III in vitro releases species sedimenting near 23 S and 17 S, neither of which retain the pppA- end, plus a collection of small fragments with chain lengths of less than 400 nucleotides. 3. The RNase III product sedimenting near 17 S (16 SIII) appears identical with the 17 S RNA typically isolated from pulse-labeled or chloramphenicol-treated cells or from several mutants deficient in ribosome assembly: fingerprint analysis reveals the presence of the same additional RNase T1 oligonucleotides and the 5' terminus (pU-G-) previously described for 17 S RNA. A 3'-terminal T1 oligonucleotide (which was not previously identifiable in the case of the 17 S precursor) has been isolated from 16 S III and its sequence determined: C-U-C-A-C-A-C-A. 4. 5 S rRNA sequences are contained in an RNase III-released fragment of approximately 300 nucleotides. This molecule lacks the 5' terminus of the mature 5 S RNA. The implications of these findings with respect to the control of ribosomal RNA synthesis, the pathways of rRNA processing in vivo, and the specificity of RNase III cleavage of natural substrates are discussed.


Stereochemical course of the adenosine triphosphate phosphoribosyltransferase reaction in histidine biosynthesis. The product of the first reaction in histidine biosynthesis is shown by optical rotation measurements on three derivatives to have inverted, beta stereochemistry at the newly formed bond. This is in contrast to alpha linkage expected on the basis of previously observed exchange, specificity, and covalent intermediate phenomena. The postulate double displacement mechanism for adenosine triphosphate phosphoribosyltransferase must be modified to account for the product stereochemistry.


Acetabuloplasty in the treatment of congenital dislocation of the hip. The results in 102 acetabuloplasties have been assessed in eighty-six patients, with a minimum follow-up of five years. Clinical and radiological assessment was carried out according to a score system very similar to that used in previous studies, and the results were expressed as excellent, good, fair and poor according to the score obtained. Any hip requiring further stabilising procedures was automatically grouped as a failure. Subtrochanteric femoral ostestomy was used when femoral anteversion was excessive. The results revealed that 73 per cent of the hips were satifactory (excellent or good) overall, but that a steady deterioration was evident when the five- and fifteen-year groups were compared. Few of the hips were regarded as normal on radiological grounds. Homologous bone bank rib was found to be the most satisfactory graft material. Coxa magna was the most common abnormality that was consistent with a good or excellent result. Avascular necrosis of the femoral head was found in only six hips; it was more commonly present in patients subjected to previous operation. Concentric reduction of the femoral head is essential if acetabuloplasty is to maintain stability of the hip.


Quadrilateral shaped brims made from high-density polyethylene for long leg calipers. Quadrilateral shaped brims made of high-density polyethylene have been used with satisfactory results on more than 120 patients who needed ischial-bearing long leg calipers. At first the fitting technique was the same as that used for thigh amputees. Subsequently it has been possible to fit most patients from a range of pre-formed brims. The properties of high-density polyethylene allow the caliper side irons to be riveted directly to the brim which, with the use of ready-made brims, allows a patient to be fitted rapidly with a comfortable and effective caliper.


Reversible and permanent effects of the carbon sources and various antibiotics on the morphology and metabolic properties of Ustilago cynodontis cells. The effects of various carbon sources and of antibiotics on the morphology of hypha cells of the fungus Ustilago cynodontis is described. Nonfermentable substrates promote readily reversible yeastlike colonies from hypha cells: all the hypha cells spread on these substrates give rise to yeastlike colonies that revert to the mycelial phenotype when transferred to glucose medium. Among the antibiotics tested, chloramphenicol (CAP) is found to promote, under certain circumstances, a long-lasting, even permanent modification on the morphology of the colonies: the colonies developed on CAP-glucose media are yeastlike, and a percentage of them give rise to colonies whose morphology remains yeastlike even on drug-free media: this effect is also obtained with cells cultivated in liquid medium. This permanent morphological modification is accompanied by a change of metabolic properties. Similar permanent effects are obtained with ethidium bromide, suggesting that mitochondrial functioning is involved in these modifications.


Adhesion of cells to surfaces coated with polylysine. Applications to electron microscopy. Cells of many kinds adhere firmly to glass or plastic surfaces which have been pretreated with polylysine. The attachment takes place as soon as the cells make contact with the surfaces, and the flattening of the cells against the surfaces is quite rapid. Cells which do not normally adhere to solid surfaces, such as sea urchin eggs, attach as well as cells which normally do so, such as amebas or mammalian cells in culture. The adhesion is interpreted simply as the interaction between the polyanionic cell surfaces and the polycationic layer of adsorbed polylysine. The attachment of cells to the polylysine-treated surfaces can be exploited for a variety of experimental manipulations. In the preparation of samples for scanning or transmission electron microscopy, the living material may first be attached to a polylysine-coated plate or grid, subjected to some experimental treatment (fertilization of an egg, for example), then transferred rapidly to fixative and further  passed through processing for observation; each step involves only the transfer of the plate or grid from one container to the next. The cells are not detached. The adhesion of the cell may be so firm that the body of the cell may be sheared away, leaving attached a patch of cell surface, face up, for observation of its inner aspect. For example, one may observe secretory vesicles on the inner face of the surface (3) or may study the association of filaments with the inner surface (Fig. 1). Subcellular structures may attach to the polylysine-coated surfaces. So far, we have found this to be the case for nuclei isolated from sea urchin embryos and for the microtubules of flagella, which are well displayed after the membrane has been disrupted by Triton X-100 (Fig. 2).


Cytoplasmic activation of starfish oocytes by sperm and divalent ionophore A-23187. The relationship between onset of the early cytoplasmic stages of oocyte activation (vitelline membrane separation and elevation) and nuclear meiotic maturation was investigated in starfish oocytes after their exposure to divalent ionophore (A-23187) or sperm. Meiotically mature oocytes, isolated in calcium-free seawater, underwent activation in response to sperm or ionophore as previously reported. Large, immature starfish oocytes, arrested in prophase I of meiosis (germinal vesicle stage), underwent vitelline membrane elevation when treated with divalent ionophore A-23187 or starfish sperm. Histological studies demonstrated that cortical granule breakdown in the oocyte cortex was associated with vitelline membrane elevation after these treatments. Activation of oocytes by sperm occurred only in response to starfish sperm. Sea urchin, sand dollar, surf clam, or marine worm sperm did not induce vitelline membrane elevation of either immature or mature starfish oocytes. Sperm- or ionophore-activated immature oocytes underwent nuclear maturation after addition of the meiosis-inducing hormone, l-methyladenine; however, parthenogenetic development did not occur and embryonic development was markedly inhibited. In contrast to previous studies, the present results indicate that cytoplasmic activation can be initiated before and without hormone induction of the nuclear maturation process. Differentiation of the oocyte cell surface or cortex reactivity therefore appears to occur during oogenesis rather than as a consequence of maturation. The data further support the view that divalent ions mediate certain of the early activation responses initiated by sperm at the time of fertilization and that synchronization of fertilization to the meiotic process in the oocyte is important for the occurrence of normal development.


Ultrastructural localization of lectin-binding sites on the zonae pellucidae and plasma membranes of mammalian eggs. Receptors for Ricinus communis agglutinin I (RCAI), concanavalin A (Con A), and wheat germ agglutinin (WGA) were localized on the zonae pellucidae and plasma membranes of hamster, mouse, and rat eggs with ferritin-lectin conjugates. Intact eggs labeled with the ferritin conjugates showed dense concentrations of RCAI and WGA receptors in the outermost regions of their zonae pellucidae and sparse distributions of Con A receptors throughout the zonae. Ferritin-lectin labeling was specific, since inhibitory saccharides effectively blocked labeling. The asymmetric density of RCAI receptors across the zona was confirmed by ferritin-RCAI and fluorescein-RCAI labeling of mechanically isolated zonae pellucidae, indicating that the RCAI-binding sites are more densely distributed in the exterior zona regions. Plasma membranes of rodent eggs contained RCAI, WGA, and Con A receptors. These receptors were found to be more or less randomly distributed on surfaces of aldehyde-fixed eggs or on eggs labeled near 0 degrees C. However, eggs incubated at 25 degrees C showed aggregated WGA- and Con A-binding site distributions on their plasma membranes. This indicates that lectin-induced receptor redistribution occurs at this temperature. The possibility that plasma membrane receptor mobility is a requirement for sperm-egg fusion is discussed.


Preparation and purification of polymerized actin from sea urchin egg extracts. Isotonic extracts of the soluble cytoplasmic proteins of sea urchin eggs, containing sufficient EGTA to reduce the calcium concentration to low levels, form a dense gel on warming to 35-40 degrees C. Although this procedure is similar to that used to polymerize tubulin from mammalian brain, sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows this gel to have actin as a major component and to contain no tubulin. If such extracts are dialyzed against dilute salt solution, they no longer respond to warming, but gelation will occur if they are supplemented with 1 mM ATP and 0.020 M KCl before heating. Gelation is not temperature reversible, but the gelled material can be dissolved in 0.6-1 M KCl and these solutions contain F-actin filaments. These filaments slowly aggregate to microscopic, birefringent fibrils when  1 mM ATP is added to the solution, and this procedure provides a simple method for preparing purified actin. the supernate remaining after actin removal contains the other two components of the gel, proteins of approximately 58,000 and 220,000 mol wt. These two proteins plus actin recombine to form the original gel material when the ionic strength is reduced. This reaction is reversible at 0 degrees C, and no heating is required.


The chondriome of selected trypanosomatids. A three-dimensional study based on serial thick sections and high voltage electron microscopy. The unitary nature of the chondriome of two species of trypanosomatids, Blastocrithidia culicis and Trypanosoma cruzi, has been demonstrated by utilizing serial thick-sectioning techniques combined with high voltage electron microscopy. Profiles of mitochondrial elements seen in thin sections and suspected to be parts of a continuum were confirmed by serial thick sectioning (0.25-0.50 mum thick) and stereopair analysis to be parts of the same mitochondrion. Three-dimensional models obtained from tracings of mitochondrial profiles on cellulose acetate reveal the mitochondrion of B. culicis to consist of a posterior mass with six tubular extensions extending upward and terminating in the anterior apex. The kinetoplast was found suspended between two of the tubular extensions, or less frequently, protuding as a nodule from one of the extensions. A bifurcation of one of the extensions was found in some specimens. The mitochondrion of T. cruzi consists of a triangular-shaped convoluted tubule, the base being the kinetoplast portion while the apex is directed posteriorly. The mitochondrion bifurcates behind the flagellar pocket, lateral to the kinetoplast, sending two entwined extensions into the tenuous anterior apex. Whether the mitochondrion of T. cruzi is unitary in the trypomastigote form was not determined in this study, since only epimastigote forms were used.


Golgi organelle response to the antibiotic X537A. The effects of the ionophoric antibiotic X537A on cell structure were studied with phase-contrast, fluorescence, and electron microscopy. X537A induced selective vacuolation of the Golgi apparatus of vascular and intestinal smooth muscle, epithelium, plasma cells, and cultured chick heart and guinea pig vascular smooth muscle cells. The swelling of the Golgi apparatus induced by X537A was reversible in the systems examined for reversibility: vascular smooth muscle and cultured chick heart. Myelin figures were common in the Golgi apparatus vacuolated by X537A. Fluorescence microscopy of cultured cells incubated with X537A showed the characteristic blue X537A fluorescence associated with lipid globules in the cultured cells. Incubation of cultured chick heart cells with X537A reduced the beating rate and, after 24-72 h, abolished the sarcomere pattern. The swelling of the Golgi membranes produced by X537A in cultured vascular smooth muscle was associated with inhibition of D-[6-3H]glucosamine and [35S]sulfate incorporation into glycosaminoglycans.


Actin-like filaments in bone cells of cultured mouse calvaria as demonstrated by binding to heavy meromyosin. A variety of intracellular filaments (50-70 A in diameter) found in bone cells was shown to bind specifically to HMM. Because of this property, these filaments are probably biochemically similar to muscle actin. in osteoblasts and osteocytes, these reactive filaments were oriented in bundles parallel to the plasma membrane and filling the cell processes. In the osteoclast the filaments along the cell membrane were not so highly organized. In the clear zone, the quiescent part of the cell adjacent to the motile ruffled border, organized filament bundles were oriented perpendicular to the cell membrane and terminated in short processes at the bone surface. These filaments were also reactive with HMM. The possible significance of the filaments with respect to the physiology of bone cells is discussed.


Cytoplasmic induction of changes in the ultrastructure of the Acetabularia nucleus and perinuclear cytoplasm. The ultrasturcture of the cell nucleus and perinuclear cytoplasm in Acetabularia is quite different in young and old cells. When a nucleus of an old cell was implanted into the cytoplasm of a young cell the nucleus assumed the morphology typical of a young cell within less than 10 days. The cytoplasm of an old cell was able to induce the reverse change in an implanted nucleus from a young cell. This cytoplasmic induction of nuclear ageing appeared to proceed more rapidly than rejuvenation.


Proteins of the newt oocyte nucleus: analysis of the nonhistone proteins from lampbrush chromosomes, nucleoli and nuclear sap. An electrophoretic analysis has been carried out on the total protein of lampbrush chromosomes, nucleoli and nuclear sap, obtained from newt oocyte nuclei. In each case, distinctive and heterogeneous banding patterns are observed. The absence of detectable quantities of histones in occyte chromatin is noted. In the case of the lampbrush chromosome preparation, it is concluded that all protein species are derived from the ribonucleoprotein matrix of the lateral loops.


An ultrastructural study of the spermatozoid of the fern, Marsilea vestita. The ultrastructure of the mature spermatozoid of Marsilea vestita was studied after its release from the microspore and prior to its penetration of the egg. The psermatozoid is a pear-shaped cell with a complex spiral structure coiled around the edge in the narrow anterior end. This coil is composed of a large mitochondrion, elongated nucleus with highly condensed chromatin, a ribbon of microtubules, and a dense band of material (flagellated band) into which the flagella are inserted. There are over a hundred flagella protruding from each spermatozoid along the length of the coil. At the anterior tip of the coil is a short multilayered structure. It is not known what maintains the helical shape of the coil. The microtubular ribbon could be involved, but it is also possible that either the flagellated band, the condensed chromatin, or both, are sufficiently rigid to retain their shpaes unaided. When the spermatozoid is first released from the microspore it includes a cytoplasmic vesicle in the posterior region containing plastids, mitochondria, and other organelles. This vesicle is shed, taking the nuclear envelope with it, before the spermatozoid reaches the egg.


Analysis for carbamate insecticides and metabolites. Of the more conventional pesticidal chemicals, the carbamate insecticides pose some unique problems relative to residue analysis. Most of these compounds are unstable under conditions normally used for GLC analysis and require special attention if this technique is to apply to the carbamates. Moreover, the carbamates are commonly metabolized to products which are toxicologically significant and which must be included in any analytical considerations. These and other problems inherent in carbamate residue methodology are discussed in this report along with technique currently utilized or having potential as sound procedures for the analyses of carbamate insecticides.


Analysis of chlorinated insecticides and congeners. A review on the state-of-the-art for analysis of chlorinated insecticides and their congeners is presented. Tables are included that show comparisons among gas chromatographic systems, high speed liquid chromatographic systems and sample preparation methods as well as an overview on sample cleanup.


Cyclophosphamide suppression of established cell-mediated immunity.  Quantitative vs. qualitative changes in lymphocyte populations. The characteristics of cyclophosphamide-induced suppression of established ccll mediated immunity were studied in guinea pigs previously senstized to tuberculin. Cyclophosphamide treatment for 5 days produced a dose-dependent peripheral lymphoctopenia and disproportionatley  greater neutrophenia which was particularly striking at high doses of 20 mg/kg per day(approximaetly 200 mg/kg-2 per day). Lymphoctes remianing in the circulation of cyclophosphamide treeated aniamls showed a doses-dependent reduction to both in vitro proliferactive and macrophage migration inhibitory factor responses to tuberculin compared to lymphocte responses of controls. Proliferative responses to phytohemaggultinin and concanavalin a were not significatly suppressed. Additional studies showed that cyclophosphamide suppressed the porliferactive and migration inhibitroy factor responses to tuberculin of lymph node and splenic as well as cirulating lymphocte populations. These studies showed that relatively short-term cyclophospamide administration produced immunosuppresion by quantitative as well as qualitative changes in lymphocyte populations. Significant suppresion  of lymphocte function, howerver, was achived only with doses of cyclophoshamide which also produced a severe neutropenia.


A comparison of the acute effects of radiation therapy, including or excluding the thymus, on the lymphocyte subpopulations of cancer patients. Radiation therapy to either mediastinum or pelvis causes a rapid decrease in circulating lymphocytes of both B and T types and in addition an impairment in the function of the remaining lyphocytes, as measured by their ability to proliferate in response to mitogens.  The acute depression is short-lived.  Substantial recovery is apparent within 3 wk after cessation of therapy; however, most patients show a modest, chronic depression in both numbers and functional capacities of circulating lymphocytes.  T cells are somewhat more sensitive than B cells, but both are affected.  Irradiation of the thymus per se seems to have little influence on the acute changes which occur, as patients receiving pelvic and mediastinal (including thymic) radiotherapy show a similiar degree of lymphopenia and depression of lymphocyte responsiveness.


Pimaricin and mycostatin for retarding cottage cheese spoilage. Two antifungal agents, pimaricin and mycostatin, added to Cottage cheese through the wash water at concentrations of 20, 50, or 100 mug/ml of wash water or added through the cheese dressing at 1, 2, or 5 mug/g retarded the growth of Aspergillus niger and Saccharomyces cerevisiae and improved the shelf-life of the cheese. In general, cheese with highest concentration of antifungal agent and stored at lowest temperature had best keeping quality. Pimaricin was slightly more effective than mycostatin in inhibiting fungi; inhibition was greater if the antifungal agents were added to the cheese dressing and the cheese was stored at low temperature; and A. niger was more sensitive to the inhibitors than S. Cerevisiae.


Initial mismanagement and delay in diagnosis of oral cancer. A study of 779 patients with epidermoid carcinoma of the oral cavity revealed that 115 (14.8%) had had a temporally significant initial mismanagement or delay in diagnosis. The responsibility for this mismanagement was shared by the dentist, the physician, and the patient. Earlier recognition of oral cancer by the physician and dentist, with earlier treatment, is essential for improving the generally poor five-year survival rate of this disease.


Histological response to ivory implanted in rabbit mandibles. Ivory rod implants were found to be biologically unacceptable in rabbit mandibles. Fibrous connective-tissue capsules containing numerous multinucleated giant cells formed around the implants. Resorption of the ivory was also apparent. In addition, osteoid deposition and ankylosis at the experimental sites were not evident.


Evaluation of isobaric oxygenation for the aged with cognitive impairment: pilot study. The effect of isobaric oxygenation on psychogeriatric subjects, with mild cognitive impairment was assessed in a single-blind controlled pilot study. Three of the 7 subjects were treated with a mixture of 97% oxygen plus 3% carbon dioxide, and 4 treated with compressed air. The oxygen or air was administered during two 1.5 hr sessions daily for fifteen consecutive days. Use of a miner's type mask ensured a closed system which permits the maximal partial pressure of oxygen. Psychologic and clinical observations did not indicate any advantage for the oxygenation therapy. Minimal side effects, which arose only in the oxygen group, may have involved some interactions between CO2 sensitivity and anxiety. Careful differential diagnosis and the control of motivational factors are of major importance in any study of the effects of oxygenation upon symptoms in psychogeriatric patients.


Electroencephalographic and behavioral changes associated with papaverine administration in healthy geriatric subjects. A double-blind cross-over procedure was used to evaluate the effects of papaverine hydrochloride on the electroencephalogram (EEG), cognition, mood and psychologic test performance of 10 healthy geriatric volunteers. During the two-week period, each subject was tested four times: before and after receiving papaverine and before and after receiving placebo. Period analysis revealed that a 300-mg dose of papaverine (Pavabid Plateau Caps) increased EEG alpha activity (8-13 cycles per second) and decreased the beta2 range (27-40 cps). Baseline values for six mood factors, short-term memory, complex problem solving, and attention were not altered significantly by either drug or placebo. Results of the Subject Paced Digit Symbol Substitution Test indicated that papaverine may produce some improvement in simple cognitive functioning. No adverse effects were associated with the use of papaverine.


Medical experimentation in the elderly.

Medical experimentation in the elderly. Participation in human experimental research constitutes a major problem for the geriatric subject. Because there is a high incidence of noncontagious disease in the elderly, they are the group most useful for the study of new therapeutic agents or procedures. However, normal aging processes, often coupled with disease of the central nervous system, render elderly persons less able to comprehend the nature and risks of such studies. These factors permit easy exploitation of geriatric subjects in medical experimentation, with possible exposure to a significant risk of serious drug reactions and unnecessary hospitalization. Recent federal regulations have given "special protections" to children, prisoners, and the mentally infirm in experimental research, to guard against abuse of their human rights. A basic requirement is that informed consent be carefully obtained and documented. Such "special protections" should now be extended to geriatric subjects so that there will be no further exploitation in the course of valid clinical research.


Chemical composition of the washed cells of Streptococcus sanguis (804) and Streptococcus mutans (B-14). Washed cells of Streptococcus saguis (804) and Streptococcus mutans (B14) were analyzed for water, ash, protein, carbohydrate, lipid, and nucleic acid content. Lipids were analyzed by gas chromatography. Ash was subjected to elemental analysis. These data were compared with the published values for the composition of dental plaque.


Deoxyribonucleic acid fiber autoradiography as a technique for studying the replication of the mammalian chromosome. Deoxyribonucleic acid (DNA) fiber autoradiography is a technique that allows analysis of replication events on mammalian chromosomal DNA. 3H-thymidine is used to pulse-label DNA, which is subsequently released from lysed cells and extended linearly over a glass slide. Autoradiograms are produced by exposing the labeled DNA to light-sensitive emulsion. The effects of inhibition of protein synthesis on DNA replication were examined using this technique. Analysis of the autoradiographic patterns has shown that inhibition of protein synthesis with either cycloheximide or puromycin retards the rate of replication fork progression. In addition, these antibiotics after the normal pattern of multifocal initiation on replication units. The spatial interval between active initiation sites is decreased, there is a decay in synchrony of initiation events on subchromosomal clusters of replication units, the decay in synchrony of initiation events on subchromosomal clusters of replication units, the frequency of initiation is inhibited and the frequency of units showing the normal bidirectional mode of replication is decreased. The retarded fork progression and shorter initiation intervals may result either from the continued operation of a subset of replication units resistant to the inhibition of protein synthesis, or be manifestations of the inhibition of protein synthesis on all active sites. The other alterations, decreased synchrony and frequency of initiation and increased unidirectional replication probably occur on all active units when protein synthesis is inhibited.


Recent developments in the detection of deoxyribonucleic acid synthesis by 33258 Hoechst fluorescence. A number of applications of the detection of deoxyribonucleic acid synthesis by fluorescence microscopy are illustrated. These include (a) the analysis of sister chromatid exchanges and sister chromatid segregation at mitosis, (b) the location of chromosome regions containing deoxyribonucleic acid with an asymmetric distribution of thymine residues between polynucleotide chains and (c) the detection of late replicating regions in metaphase chromosomes. The suppression of 33258 Hoechst fluorescence by 5-bromodeoxyuridine incorporated biosynthetically into interphase nuclei is demonstrated both in fixed cytologic preparations and in unfixed cultured cells. Many of the cytologic observations described might form a basis for future biochemical studies.


The macrophage electrophoretic mobility test (MEM) some technical considerations. The following parameters of the cytopherometric test system have been studied. 1. Temperature is less important for the production of macrophage slowing factor (MSF) by lymphocyte-antigen interaction than it is for the activity of MSF on macrophages. 2. Lymphocyte number titration curves allow discrimination between 'weak' and 'strong' antigen responses. 3. Lymphocyte-antigen reaction reaches a plateau after 30 min incubation, whilst maximum MSF-macrophage reaction requires 60 min. 4. Interaction of MSF with macrophages is sensitive to cycloheximide. 5. Irradiation of peritoneal exudate cells (200 rad) is essential in order to obtain a maximal result in the MEM test. In the 'split MEM test, 200 rad to the indicator peritoneal exudate cells produces a greater result, i.e. it in some way amplifies the macrophage response to MSF. 6. Excess of peritoneal exudate cells, especially when non-irradiated, suppresses the test result. This is not due to consumption of MSF produced. Probably macrophages rather than lymphocytes are responsible for the suppression.


A method for the selection of pig lymphocytes bearing surface immunoglobulin. Coupling of IgG anti-immunoglobulin to polystyrene bottles with carbodiimide has been used to bind selectively pig lymphocytes previously treated with anti-immunoglobulin or anti-Ig class-specific fluorescent conjugates. Subsequent recovery of the bound cells produced populations with approximately 80% of cells stained for surface Ig and 90% viability.


Quick and easy production of monolayer plaque assay slides. A fast and easy method for the production of monolayer plaque-assay slides, making use of double adhesive tape, is described.


Long-term maintenance of psoriatic human skin on congenitally athymic (nude) mice. Transplantation of involved psoriatic and nonpsoriatic human skin onto congenitally athymic (nude) mice has been performed successfully. Although biopsies at selected intervals demonstrate that the excess glycogen deposition normally seen in psoriasis is no longer consistently present, the psoriatic grafts did retain the usual characteristic histologic differences throughout the life of the animal, up to 11 weeks. This grafting procedure potentially represents a useful method whereby the study of psoriasis can be made in a nonhuman, living system.


Mitochondria and nuclei of pig and human epidermis: isolation and lipid composition. Pig and human epidermis were mechanically disrupted with a Polytron homogenizer and mitochondria and nuclei were isolated from homogenates in buffered sucrose and 2.5% citric acid solution, respectively, by differential and density-gradient centrifugation. Examination of the mitochondrial preparations by electron microscopy and the assay of several marker enzymes indicated that they consisted mostly of mitochondria with some microsomal contamination. The nuclear preparations were substantially free from contaminants as judged by particle counts with the aid of phase and electron microscopy. The lipid compositions of the mitochondria from both species were characterized by a high concentration of cardiolipin and low concentration of cholesterol and sphingomyelin and an absence of glycosphingolipids as compared with the lipid composition of the whole epidermal cell. The lipid compositions of the nuclei were characterized by a high proportion of phosphatidyl choline and an absence of glycosphingolipids. The neutral lipids accounted for an unusually high proportion of the total lipids especially in human epidermal nuclei. Although the technical difficulties in isolating subcellular organelles from mammalian epidermis limit the yield and quality of the mitochondria, these preparations as well as those of nuclei are superior to preparations reported previously and are pure enough for a valid comparison with other membrane systems of the epidermal cell.


The application of trace element analysis by X-ray fluorescence to human blood serum. The need for a simple, yet fast and accurate method for trace element analysis of human tissues is remarded on. Such a method is the use of x-ray fluorescence in conjunction with one of the new generation of solid-state x-ray detectors. The method is described, together with an example of its application-apilot study of forty-two blood serum samples supplied by the Manitoba Cancer Research Institute.


Measurement of redox potential changes in anaerobic culture media caused by addition of blood. A method has been described whereby the redox potential of unsealed and manipulated samples of solution can be determined. This method includes treating the platinum electrode with strong cleaning solution, adjusting the electrode with a culture of Proteus vulgaris in tryptic soy broth, and doing all manipulations inside a glove box that is continuously flushed with oxygen-free gas. This study has shown that prereduced media are much more reduced than media which have been dispensed only under a partial vacuum. It also has shown that addition of blood to prereduced medium does not eliminate the strongly reducing condition of the medium although there is a slight rise in redox potential.


Cellular immunity to intrinsic factor in pernicious anemia. Cellular immunity to hog intrinsic factor was detected by a modified agarose-leukocyte migration test in 18 patients with pernicious amemia. Lymphocytes from 17 out of 18 patients with pernicious anemia gave positive responses  to a concentrate of hog intrinsic factor; the intrinsic factor present in 1 mg. of this concentrate bound 128 ng. of vatamin B12. Six patients with atrophic gastritis, 7 with regional enteritis, and 9 out of 10 healthy adults did not respond to this preparation. No correlation existed between the presence of serum autoantibodies to intrinsic factor and in vitro lymphocyte responsiveness to intrinsic factor. The results demonstrate that cellular immunity to intrinsic factor concentrates is present in the majority of patients with pernicious anemia.