Transport-defective Escherichia coli ecf mutant permeable to protons and nucleotides.

This paper describes a third type of transport-defective ecf mutant of Escherichia coli which is altered in membrane proton permeability. Mutants of this class also excrete ATP and other nucleotides and form filament-like cells. This lesion requires cell growth at the nonpermissive temperature.


Small stable RNAs from Escherichia coli: evidence for the existence of new molecules and for a new ribonucleoprotein particle containing 6S RNA.

Small stable RNA molecules of Escherichia coli other than 5S (rRNA) and 4S (tRNA) were studied. Two of the molecules corresponded to 4.5S and 6S RNA, which have been reported previously. The third stable RNA molecule, 10S RNA, has not been described before. RNA labeled with (32)P(i) or [(14)C]uracil for a relatively long time, when separated in 5%/12% tandem polyacrylamide gels, displayed three bands corresponding to 10S, 6S, and 4.5S RNA in addition to rRNA and tRNA bands. These RNAs were stable in pulse-chase-labeling experiments. The amount of these RNAs was small, comprising only 0.2 to 0.5% of the total (32)P incorporation. However, this amount represented a large number of molecules; for 6S and 4.5S, it was about 1,000/DNA molecule. These three RNAs were found in the postribosomal supernatant fraction. None of them was found in purified nucleoid fractions in which the tightly coiled DNA molecules were contained. Of these three RNAs, 6S RNA was unique in that it seemed to exist in a ribonucleoprotein particle. All these RNAs, as well as tRNA, were very stable in the cell under various physiological conditions. 5S RNA was less stable. On the other hand, purified 6S RNA was more susceptible than tRNA to cell nucleases when incubated with cell extracts, suggesting that, being in a particle, it is protected from cell nucleases.


Expression of Escherichia coli fol alleles in Salmonella typhimurium.

Studies of the expression of Escherichia coli fol alleles in Salmonella typhimurium indicated that fol regulatory functions are highly conserved between these bacterial species.


Evidence that glucose starvation-sensitive mutants are altered in the relB locus.

Genetic mapping studies indicate that the relaxed-control mutants isolated on the basis of sensitivity to glucose starvation contain lesions in the relB locus. These mutants, which are sensitive to protein synthesis inhibitors such as sulfacetamide, exhibit relaxed control of both RNA and phospholipid syntheses.


Size of the lipid precursor pool in Escherichia coli.

In Escherichia coli the lipid precursor pool (glycerol and acetate) was found to be less than 5,000 molecules per cell.


Plasmid conferring increased sensitivity to mercuric chloride and cobalt chloride found in some laboratory strains of Escherichia coli K-12.

Some lines of the widely used strain CR34 of Escherichia coli K-12 carry a plasmid. The plasmid has a mass of 12 +/- 2 X 10(6) megadaltons and is maintained at a low copy number per cell (1 to 2), and this number is not amplified by growth of cells in the presence of chloramphenicol. The plasmid is designated as pCU3. The presence of pCU3 confers on the strain increased sensitivity to mercuric chloride and cobalt chloride. The plasmid has also been observed to fuse or recombine with the plasmid R64-11.


Changes in cell size and shape in thymine-requiring Escherichia coli associated with growth in low concentrations of thymine.

Thymine auxotrophs of three unrelated strains of Escherichia coli (K-12, B/r, and 15) were grown in media containing various concentrations of thymine. During steady-state growth conditions, cell volume increased as thymine concentration decreased but, in contrast to previous reports, this change was due to an increase in cell length without change in cell diameter.


Effects of high temperature on Escherichia coli F pili.

The effects of high temperatures (46 to 50 degrees C) on the production of F pili by Escherichia coli were studied by electron microscopy. Attached F pili rapidly disappeared at 48 and 50 degrees C but not at 46 degrees C. Free pili were not denatured at these temperatures. The pili that disappeared from the cells at 50 degrees C did not appear as free pili in the culture supernatant fluid, indicating that the pili had retracted to the cell surface or into the cell. The adsorption of either R17 phage or F pili antibody to the sides of pili prevented retraction. The disappearance of pili was accompanied by a loss in the ability to adsorb R17 phage but not M13 phage, suggesting that the tip of a pilus remains exposed after retraction.


Requirements for suppression of a dnaG mutation by an I-type plasmid.

We have confirmed and further characterized the phenomenon of suppression of a dnaG mutation by an I plasmid and determined the requirements for the complete expression of suppressibility. The authentic derepressed mutation of the conjugal fertility system, described earlier, was shown to be one of the requirements. The second requirement is a previously undescribed type of mutation leading to a far higher degree of derepression of this system than the authentic drd mutation. This second mutation leads to extremely high conjugal fertility and pilus production. A third requirement for the complete expression of suppressibility is that the host recA function  remain intact. recA function is required not merely for the fulfillment of the other two requirements described above, but also for an additional step or steps leading to its complete expression. The nature of this step(s) is unknown, but it is not insertion of the plasmid into the host genome.


General screening procedure for RNA modificationless mutants: isolation of Escherichia coli strains with specific defects in RNA methylation.

A general method for the isolation of mutants of Escherichia coli that are defective in RNA modification is described. The method is based on the fact that RNA with specific undermodifications accumulates under nonpermissive growth conditions and that such a defect can be detected by remodification either in vivo at permissive conditions or in vitro. The method provides a means by which to study mutations affecting essential modification reactions. The usefulness of the method was demonstrated by the isolation of two rRNA and two tRNA methylation defective mutants. Both rRNA mutants accept methyl groups into their 23S rRNA in vitro. Analyses of in vitro methylated 23S rRNA from one of the mutants revealed the presence of several methylated nucleosides, of which 6-methyladenosine was the most abundant  (40% of recovered radioactivity). In 23S rRNA from the other mutant, the only product formed in vitro was 5-methylcytidine. The tRNA mutants are characterized in the accompanying paper.


Escherichia coli mutants with defects in the biosynthesis of 5-methylaminomethyl-2-thio-uridine or 1-methylguanosine in their tRNA.

Two tRNA methyltransferase mutants, isolated as described in the accompanying paper (G.R. Björk and K. Kjellin-Stråby, J. Bacteriol. 133:499-207, 1978), are biochemicaaly and genetically characterized. tRNA from mutant IB13 lacks 5-methylaminomethyl-2-thio-uridine in vivo due to a permanently nonfunctional methyltransferase. Thus tRNA from this mutant is a specific substrate for the corresponding tRNA methyltransferase in vitro. In spite of this defect in tRNA, such a mutant is viable. Mutant IB11 is conditionally defective in the biosynthesis of 1-methylguanosine in tRNA due to a temperature-sensitive tRNA (1-methyl-guanosine) methyltransferase. In mutant cells grown at a high temperature, the level of 1-methylguanosine in bulk tRNA is 20% of that of the wild type, demonstrating that in this mutant an 80% deficiency of 1-methylguanosine in tRNA is not lethal. Genetically these two distinct lesions, trmC2, causing 5=methylaminomethyl-2-thio-uridine deficiency, and trmD1, giving a temperature-sensitive tRNA (1-methylguanosine)methyltransferase, are both located between 50 and 61 min on the Escherichia coli chromosome.


Transfer of nitrogen fixation genes from a bacterium with the characteristics of both Rhizobium and Agrobacterium.

Strain T1K, reported to be Rhizobium trifolii strain T1 carrying the drug resistance plasmid RU-1drd, was able to transfer a cluster of nif+ genes to Escherichia coli K-12. Additional genetic material, resembling the gal-chlA region of E. coli, was also transferred from strain T1K. The segregation pattern of these transferred genes suggested that they were on a plasmid. Although strain TIK was able to nodulate red and white clover, it also formed very slow-growing galls on tomato stems and shared many physiological properties with Agrobacterium tumefaciens, to which it seemed more closely related than to R. trifolii. The R. trifolii hybrid T1 (R1-19drd), constructed by conjugation, did not share any of these properties of both A. tumefaciens. Thus, strain T1K appears to be a bacterium with properties of both A. tumefaciens and R. trifolii and with the capacity to transfer nif+ genes and other functions which it may have "cloned" from another bacterium such as Klebsiella.


Isolation of specialized transducing bacteriophages carrying the structural genes of the hexuronate system in Escherichia coli K-12: exu region.

In Escherichia coli HfrH 58, isolated by Shimada et al., a heat-inducible phage has been integrated in a secondary attachment site. We have characterized tha nature of the lambda integration. The exuR regulatory gene is inactivated by prophage integration. Genetic and biochemical analysis indicated a gene order: uxaA-uxaC-exuT-(exuR')-lambdaNRAJ (exuR''). By induction of HfrH 58, one class of deletions extending into the exu region was obtained. Analysis of these deletions confirms the exu region topography and the regulatory mechanism of the hexuronate system previously described. It has been possible to regenerate a functional exuR gene by prophage exision. Various lambda transducing particles plaque-forming and defective transducing phages carrying the left part or the right part of the exu region, have been derived from the secondary site lysogen HfrH 58. A phage carrying the entire exuR region was constructed by a cross between these two types of phage. The construction and characterization of these exu transducing phages are presented.


2-deoxygalactose, a specific substrate of the Salmonella typhiimurium galactose permease: its use for the isolation of galP mutants.

2-Deoxygalactose is a specific substrate of the galactose permease. The apparent Km is about 500 micron, compared to 45 micron for galactose, whereas the maximal rate of uptake is one-half to one-third of that of galactose. None of the other galactose transport systems, including methyl beta-D-thiogalactosides I and II, the beta-methyl-galactoside permease, and both arabinose systems, is able to catalyze transport of 2-deoxygalactose to a significant extent. 2-Deoxygalactose can also be used to isolate mutants defective in galactose permease, since it is bacteriostatic. Colonies that grow with lactate, malate, or succinate as a carbon source in the presence of 0.5 to 2 mM 2-doexygalactose were found to be mostly galP mutants, lacking galactose permease. Spontaneous 2-deoxygalactose-resistant strains arose with a frequency of about 2 X 10(-6). galP mutants have also been derived from pts deletion mutants that require galactose permease for growth on glucose. Revertants have been obtained that have acquired the parental phenotype.


Role of the chlC gene in formation of the formate-nitrate reductase pathway in Escherichia coli.

Five temperature-sensitive chlC mutants were isolated from Escherichia coli by the technique of localized mutagenesis. All of the mutants produced severely reduced levels of both nitrate reductase and formate dehydrogenase when grown at 43 degrees C. In three of the mutants, the nitrate reductase activity produced at the permissive temperature was shown to be thermolabile compared with the activity produced by the parent wild-type strain, both in membrane preparations and in preparations released from the membrane by deoxycholate. In each case, formate dehydrogenase activity was similar to the wild-type activity in its stability to heat. It is concluded that the chlC gene codes for at least one of the polypeptide chains of nitrate reductase and that the chlC mutations affect indirectly the formation of formate dehydrogenase.


Genetic and segregation analysis of Escherichia coli strains containing a tandem duplication of the trpD-purB region of the chromosome.

Genetic and segregation analysis of Escherichia coli strains containing a partial duplication of the trp operon reveal that the 2.5-min-long region trpD-purB is duplicated in tandem in the chromosome. The adjacent loci cysB and fabD are not duplicated. Although one copy of the duplicated region is longer than the maximum size of bacteriophage P1kc transducing fragments, the frequency at which the duplicated segment trpDCBA is transferred by transduction to tonB-trp deletion strains is equal to that observed for transfer of the normal trp operon. This suggests that three-point recombination events believed to account for transduction of long duplications occur as frequently as two-point recombination events believed to account for normal transduction. Cotransduction frequencies of trpDCBA with the duplicated loci tonB, galU, tyrT, and hemA are very similar to those for the trp operon with the same loci. This indicates that normal genetic linkage is maintained during the three-point recombination event. However, purB, which is normally unlinked to trp by transduction, is closely linked to trpDCBA and thus must be near the repeat point of the duplication. Transduction tests with point mutations in the trp operon indicated that the repeat point occurs near the normal boundary between trpE and trpD. Segregation analysis of heterogenotes constructed from tonB-trp deletion strains shows that the frequency at which a marker is lost is approximately proportional to its distance from the repeat point. This finding is consistent with a random, singlesite crossover event during segregation. Several observations indicate that non-reciprocal genetic exchange also occurs between copies of the duplication. Analysis of heterogenotes containing dadR1 and dadR(+) demonstrate that the mutant allele is transdominant.


Utilization of arginine by Klebsiella aerogenes.

Klebsiella aerogenes utilized arginine as the sole source of carbon or nitrogen for growth. Arginine was degraded to 2-ketoglutarate and not to succinate, since a citrate synthaseless mutant grows on arginine as the only nitrogen source. When glucose was the energy source, all four nitrogen atoms of arginine were utilized. Three of them apparently did not pass through ammonia but were transferred by transamination, since a mutant unable to produce glutamate by glutamate synthase or glutamate dehydrogenase utilized three of four nitrogen atoms of arginine. Urea was not involved as intermediate, since a unreaseless mutant did not accumulate urea and grew on arginine as efficiently as the wild-type strain. Ornithine appeared to be an intermediate, because cells grown either on glucose and arginine or arginine alone could convert arginine in the presence of hydroxylamine to ornithine. This indicates that an amidinotransferase is the initiating enzyme of arginine breakdown. In addition, the cells contained a transaminase specific for ornithine. In contrast to the hydroxylamine-dependent reaction, this activity could be demonstrated in extracts. The arginine-utilizing system (aut) is apparently controlled like the enzymes responsible for the degradation of histidine (hut) through induction, catabolite repression, and activation by glutamine synthetase.


Light effects in yeast: inhibition by visible light of growth and transport in Saccharomyces cerevisiae grown at low temperatures.

Growth rate, sugar transport, and amino acid transport of yeast cells grown at 12 degrees C were inhibited by cool-white fluorescent light. At light intensities below 1,250 lx, growth and membrane transport were only slightly inhibited. Above 1,250 lx, there was increasing inhibition of both processes. Transport of histidine was completely inhibited after 3 to 5 days in cultures grown at 12 degrees C under 3,500-lx illumination. Cells grown at 20 degrees C were not inhibited by light intensities that caused complete loss of viability and membrane transport activity in cells grown at 12 degrees C.


Transduction of plasmid determinants in Staphylococcus aureus and Escherichia coli.

Buoyant density analysis of transducing lysates derived from Staphylococcus aureus and Escherichia coli indicated that phage particles bearing plasmid determinants contain a quantity of DNA equivalent to that found in the lytic particles. Transducing particles that bear plasmid determinants smaller than viral DNA must therefore contain a quantity of DNA in excess of a single plasmid genome. In the E. coli P1vir system, a dependence upon host-mediated recombination for the transduction of small plasmids, but not for large R factors or chromosomal genes, was observed. However, no evidence for the involvement of such functions in the transduction of S. aureus plasmids was obtained. Although the origin of the additional DNA in plasmid transducing particles has not been identified, circumstantial evidence has been presented in the staphylococcal system indicating that transducing particles carrying a small tetracycline plasmid are not formed by the wrapping of multiple copies of this plasmid DNA.


Chemotaxis of Salmonella typhimurium to amino acids and some sugars.

Patterns of chemotaxis by Salmonella typhimurium strain LT-2 to l-amino acids and to several sugars were quantitated by the Adler capillary procedure. Competition experiments indicated that LT-2 possesses three predominant receptors, or interacting sets of receptors, for amino acids. These were termed the aspartate, serine, and alanine classes, respectively. Studies with strains carrying point and deletion mutations affecting components of the phosphoenolpyruvate: glycose phosphotransferase system (PTS) made unlikely a role in primary reception of d-glucose by the three soluble PTS components, namely HPr, enzyme I, and factor III. A ptsG mutant defective in membrane-bound enzyme IIB' of the high-affinity glucose transport system was shown to exhibit normal chemotaxis providing pleiotropic effects of the mutation were eliminated by its genotypic combination with other pts mutations or, phenotypically, by addition of cyclic AMP and substrate. A correlation was demonstrated between chemotaxis to glucose and activity of the low-affinity glucose transport complex, membrane-bound enzymes IIB:IIA, and an enzyme IIB:IIA mutant was shown to have a preponderant defect in chemotaxis to glucose and mannose. Of four systems capable of galactose transport, only the beta-methylgalactoside transport system was implicated in chemotaxis to galactose. Some properties of a mutant possibly defective in processing of signals for chemotaxis to sugars is described.


Frequency of replication from alternative origins in the composite R plasmid NR1.

The relative frequency of initiation of DNA replication within the RTF-Tc and r-determinant components of the composite drug resistance plasmid NR1 in Proteus mirabilis was evaluated. Using fractionated radioactively labeled plasmid DNA, analytical procedures that distinguished between the two components of the composite plasmid were carried out. A mixture of uniformly 14C-labeled and 3H-pulse-labeled plasmid DNA (pulse-labeled origin[s] of replication) was used in each of three experiments. First, shear products of the DNA were analyzed using CsCl density gradient centrifugation. Second, fragmented DNA was hybridized to nonradioactive RTF-Tc and r-determinant DNAs immobilized on nitrocellulose filters. Third, the radioactive plasmid DNAs immobilized on nitrocellulose filters. Third, the radioactive plasmid DNA was digested with restriction enzyme (EcoRI), producing a set of RTF-Tc and r-determinant fragments with differing 3H/14C isotpe ratios. The three experiments suggested that under the conditions used to accumulate replicating plasmid DNA molecules (DNA substrate limitation), the r-determinant origin of replication was preferentially utilized in the composite plasmid.


Regulation of the major proline permease gene of Salmonella typhimurium.

The structural gene for the major proline permease is located in a tight cluster with genes coding for the proline degradative enzymes, proline oxidase and pyrroline-5-carboxylic acid dehydrogenase. Expression of the permease is regulated in parallel with the two degradative enzymes, and all three functions are subject to catabolite repression. Regulatory mutants (putC) have constitutively high levels of all three activities, suggesting that all are regulated by a single mechanism.


Cluster of genes controlling proline degradation in Salmonella typhimurium.

A cluster of genes essential for degradation of proline to glutamate (put) is located between the pyrC and pyrD loci at min 22 of the Salmonella chromosome. A series of 25 deletion mutants of this region have been isolated and used to construct a fine-structure map of the put genes. The map includes mutations affecting the proline degradative activities, proline oxidase and pyrroline-5-carboxylic dehydrogenase. Also included are mutations affecting the major proline permease and a regulatory mutation that affects both enzyme and permease production. The two enzymatic activities appear to be encoded by a single gene (putA). The regulatory mutation maps between the putA gene and the proline permease gene (putP).


Synergistic killing of Escherichia coli by near-UV radiation and hydrogen peroxide: distinction between recA-repairable and recA-nonrepairable damage.

Wild-type cells and six DNA repair-deficient mutants (lexA, recA, recB, recA, recB, polA1, and uvrA) of Escherichia coli K-12 were treated with near-ultraviolet radiation plus hydrogen peroxide (H2O2). At low H2O2 concentrations (6 X 10(-6) to 6 X 10(-4) M), synergistic killing occurred in all strains except those containing a mutation in recA. This RecA-repairable damage was absent from stationary-phase cells but increased in logarithmic cells as a function of growth rate. At higher H2O2 concentrations (above 6 X 10(-4) M) plus near-ultraviolet radiation, all strains, including those with a mutation in recA, were synergistically killed; thus, at high H2O2 concentrations, the damage was not RecA repairable.


Promoter- and attenuator-related metabolic regulation of the Salmonella typhimurium histidine operon.

Expression of the histidine (his) operon in Salmonella typhimurium was found to be positively correlated with the intracellular level of guanosine tetraphosphate (ppGpp). Limitation for amino acids other than histidine elicited a histidine-independent metabolic regulation of the operon. In bacteria grown at decreased growth rates, his operon expression was metabolically regulated up to a point, after which further decreases in growth rate no longer resulted in further enhancement of operon expression. Studies using strains carrying various regulatory and deletion mutations indicated that metabolic regulation is achieved predominantly by increased RNA chain initiations at the primary (P1) and internal (P2) promoters. Metabolic regulation ordinarly did not involve changes in RNA chain terminations at the attenuator site of the his operon. A model is proposed that involves ppGpp-induced changes in RNA polymerase initiation specificity at particular promoters. A second, special form of metabolic regulation may operate which also is histidine independent, but does involve relief of attenuation.


Genetic analysis of antibiotic resistance in Streptococcus pyogenes.

The genetics of antibiotic resistance in mutant strains of Streptococcus pyrogenes was studied. Utilizing a type 6 strain (9440) primarily resistant to strepttomycin (Strr), classes of mutant strains were isolated that were resistant to one of the following antibiotics: rifampin (Rifr), erythromycin (Eryr), thiostrepton (Tstr), spiramycin (Sprr), fusidic acid (Fusr), gramicidin (Grcr), ethidium bromide (Ebrr), kanamycin (Kanr), neomycin (Neor), oleandomycin (Oler), gentamicin (Genr), and novobiocin (Novr). Transduction experiments separated antibiotic resistance markers into two distinct groups: transducible markers, including Fusr, Bacr, Ksg+, Spcr, Eryr, Sprr, Rifr, Stlr, and Tstr (Bacr, Ksgr, Spcr, and Stlr refer to resistance to bacitracin, kasugamycin, spectinomycin, and streptolydigan, respectively), and nontransducible markers, including Grcr, Ebrr, Kanr, Neor, Oler, Genr, and Novr. By means of two- and three-point crosses, transducible markers (excluding tst) were located in three separate linkage groups. spr was found to be linked with ery and spc in the order spc-ery-spr, whereas in a separate linkage group the order was determined to be str-fus-bac-ksg. The third linkage group contained the rif and stl markers.


Near-UV mutagenesis: photoreactivation of 365-nm-induced mutational lesions in Escherichia coli WP2s.

Reversion to tryptophan independence induced by 365-nm and 254-nm radiation was studied in Escherichia coli WP2s (B/r trp uvrA). Under aerobic conditions, the mutant frequency responses was of the fluence-square or "two-hit" type at both 365 and 254 nm when revertants were assayed on minimal agar supplemented with 2% nutrient broth (SEM plates). In contrast, when mutants were assayed on minimal agar supplemented with tryptophan only, the revertant yield was reduced to very low values at 365 nm, whereas values substantially greater than with SEM plates were obtained at 254 nm. Premutational lesions induced by both 365-nm and 254-nm radiation were photoreactivated more than 10-fold when assayed on SEM plates, implicating pyrimidine dimers as premutational lesions at both wavelengths. The strong photoreactivation of 365-nm-induced mutagenesis contrasted strikingly with the complete absence of photoreactivation of 365-nm-induced lethality in this strain.


Nucleoid release from Escherichia coli cells.

The time course of morphological changes during lysis of Escherichia coli cells was examined with respect to an undisturbed release of nucleoids. The addition of detergents to plasmolyzed, osmotic sensitive cells resulted in the immediate reversal of plasmolysis followed by the appearance of rod-shaped ghost cells without any detectable spheroplast formation. Electron microscopic examination of the rod-shaped ghost cells revealed a zonal gap in the cell envelope, allowing the free release of the nucleoid. Due to the high ionic strength, a suitable cell lysis was shown to require higher incubation temperatures. However, in the absence of an appropriate control this may result in the sphering and vesiculation of ghost cell envelopes and even the unfolding of released nucleoids. To avoid this unfavorable consequence of lysis at high temperatures, a microscopic examination on the course of rod-shaped ghost formation is suggested.


Expression of the uvrB gene of Escherichia coli: in vitro construction of a pMB9 uvrB plasmid.

Bacteriophage lambdab2att2 [lambdab2cI857intam6(deltabioAB)bioFCD+uvrB+phr+] codes for a function(s) that confers UV resistance (Uvr+) and reactivation of irradiated phage (Hcr+) to an Uvr-Hcr-Escherichia coli strain. It was demonstrated that these functions are expressed under the control of bacterial regulatory elements located on lambdab2att2 DNA. The location of the E. coli uvrB gene on the DNA of this transducing phage was established by heteroduplex and restriction-enzyme analyses. Recombinant DNA molecules were constructed in vitro from plasmid pMB9 (Tcr), as the vector, and an EcoRI fragment (Eco-RI-F) of lambdab2att2 DNA. The resulting plasmid, designated pNP5, has a molecular weight of 5.1 X 10(6) and replicates in a relaxed fashion. Transformation of E. coli uvrB with plasmid pNP5 resulted in clones that are Uvr+ Tcr. Irradiation of bacteria transformed with plasmid pNP5 with low UV doses revealed a complete restoratation of the Uvr+ phenotype by the presence of the cloned EcoRI-F DNA, while only a partial restoration was observed after irradiation with high UV doses. Likewise, the Hcr+ character was also partially restored due to the presence of pNP5. No correlation was found between the acquired Uvr+, Hcr+ properties, and the presence of correndonuclease II activity in an extract of bacteria that harbor plasmid pNP5.


Incomplete flagellar structures in nonflagellate mutants of Salmonella typhimurium.

Incomplete flagellar structures were detected in osmotically shocked cells or membrane-associated fraction of many nonflagellate mutants of Salmonella typhimurium by electron microscopy. The predominant types of these structures in the mutants were cistron specific. The incomplete basal bodies were detected in flaFI, flaFIV, flaFVIII, and flaFIX mutants, the structure homologous to a basal body in flaFV mutants, the polyhook-basal body complex in flaR mutants, and the hook-basal body complex in flaL and flaU mutants. No structures homologous to flagellar bases or their parts were detected in the early-fla group nonflagellate mutants of flaAI, flaAII, flaAIII, flaB, flaC, flaD, flaE, flaFII, flaFIII, flaFVI, flaFVII, flaFX, flaK, and flaM. From these observations, a process of flagellar morphogenesis was postulated. The functions of the early-fla group are essential to the formation of S ring-M ring-rod complexes bound to the membrane. The completion of basal bodies requires succeeding functions of flaFI, flaFIV, flaFVIII, and flaFIX. Next, the formation of hooks attached to basal bodies proceeds by the function of flaFV and by flaR, which controls the hook length. Flagellar filaments appear at the tips of hooks because of the functions of flaL, flaU, and flagellin genes.


Fine cleavage map of a small colicin E1 plasmid carrying genes responsible for replication and colicin E1 immunity.

A small plasmid (pAO2, 1 megadalton) carrying genes responsible for replication and colicin E1 immunity has been constructed from colicin E1 plasmid (A. Oka, K. Sugimoto, and M. Takanami, Proc. Mol. Biol. Jpn., p. 113-115, 1976). pAO2 DNA was cleaved into unique fragments with seven restriction endonucleases (R.HaeII,R.HaeIII,R.HapII,R.HhaI,R.AluI,R.HgaI, and R.HinfI). R.HaeII cleaved pAO2 DNA at two sites, R.HaeIII at four sites, R.HapII at nine sites, R.HhaI at eight sites, R-AluI at nine sites, R.HgaI at two sites, and R.HinfI at four sites, respectively. The order of HaeIII fragments of pAO2 was deduced from the physical map of colicin E1 plasmid previously reported (A. Oka and M. Takanami, Nature (London) 264:193-196, 1976). HapII, HhaI, and AluI fragments of pAO2 were assigned by analyzing overlapping sets of fragments arising upon digestion of individual HaeIII fragments with one of R.HapII, R.HhaI, or R.AluI, and upon their reciprocal digestion. The cleavage sites for R.HaeII, R.HgaI, and R.HinfI were localized on HapII, HhaI, and AluI fragments by combined digestion. On the basis of these data and estimates of the size of each fragment, a fine cleavage map of pAO2 was constructed.


Production of giant cells of Escherichia coli.

Giant cells, with volumes up to 500-fold those of normal cells, have been produced by both genetic and pharmacological means in Escherichia coli K-12. In the genetic approach, an envB or mon mutation (conferring rounded or irregular morphology) was combined with a lon mutation (block of septation after irradiation). UV irradiation and subsequent incubation for 2 to 5 h in a rich medium supplemented with 1% sodium chloride led t; production of polymorphic giant cells. In the pharmacological approach, incubation of several different strains of E. coli K-12 with the drug 6-amidinopenicillanic acid (FL1060) in the same rich medium gave rise to a homogeneous population of smoothly rounded giant cells.


Effects of phospholipids on L-lactate dehydrogenase from membranes of Escherichia coli. Activation and stabilization of the enzyme with phospholipids.

Membrane-bound L-lactate dehydrogenase was freed from the detergent used during purification. The detergent-free enzyme had about one-half the specific activity of the enzyme in 1.0% Tween 80, and was only partially sensitive to the specific antibody. This enzyme was activated about 3-fold with phosphatidylglycerol, cardiolipin, or a mixture of phospholipids. The phospholipid-activated enzyme had a similar Km value for L-lactate to that of the membrane enzyme and was completely inhibited by the specific antibody. On heat treatment, the phospholipid-activated enzyme was more stable than detergent-free enzyme and was as stable as membrane-bound enzyme. The alpha helical content of the enzyme increased 1.7-fold during preincubation with these lipids and the alpha helix became more stable during heat treatment than that of the detergent-free enzyme. These results suggest that the enzyme showed monomolecular dispersion in the lipid bilayer and that its conformation, including its active site and secondary structure, was different from that of the detergent-free enzyme. Phosphatidylethanolamine, dilauroyl lecithin and lecithin from egg yolk had none of the above effects on the activity or the secondary structure of the enzyme. On the other hand, mixtures of each of these lipids and cholate had essentially similar effects to phosphatidylglycerol.


Characterization of two forms of asparaginase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae X2180-1A synthesizes two forms of asparaginase: L-asparaginase I, an internal constitutive enzyme, and asparaginase II, an external enzyme which is secreted in response to nitrogen starvation. The two enzymes are biochemically and genetically distinct. The structural gene for asparaginase I (asp 1) is closely linked to the trp 4 gene on chromosome IV. The gene controlling the synthesis of asparaginase II is not linked to either the trp 4 or asp 1 genes. The rate of biosynthesis of asparaginase II is unaltered in yeast strains carrying the structural gene mutation for asparaginase I. Asparaginase II has been purified approximately 300-fold from crude extracts of Saccharomyces by heat and pH treatment, ethanol fractionation, ammonium sulfate fractionation followed by Sephadex G-25 chromatography, and DEAE-cellulose chromatography. Multiple activity peaks were obtained which, upon gas chromatographic analysis, exhibit varying mannose to protein ratios. Asparaginase I has been purified approximately 100-fold from crude extracts of Saccharomyces by protamine sulfate treatment, ammonium sulfate fractionation, gel permeation chromatography, and DEAE-cellulose chromatography. No carbohydrate component was observed upon gas chromatographic analysis. Comparative kinetic and analytic studies show the two enzymes have little in common except their ability to hydrolyze L-asparagine to L-aspartic acid and ammonia.


Identification of an Escherichia coli nuclease acting on structurally altered transfer RNA molecules.

A nuclease (RNase D) that can recognize structurally altered transfer RNA molecules has been partially purified from Escherichia coli. The enzyme acts poorly on intact tRNA and is inactive with the synthetic polyribonucleotides, poly(A), poly(U), or double-stranded poly(A).poly(U). The enzyme requires Mg2+ for activity and is stimulated by the monovalent cations, K+ and NH4+. The products of the reaction are 5'-mononucleotides. The molecular weight of the protein is about 60,000 as judged by Sephadex G-100 chromatography. The enzyme does not correspond to any known E. coli ribonuclease and may represent an intracellular scavenging mechanism for denatured tRNAs and other inactive RNA molecules.


Differential effect of hydroxyurea on a ribonucleotide reductase system.

Infection of Escherichia coli with phage T4 induces a large increase in ribonucleotide reductase activity. We show that hydroxyurea inhibits T4-induced CDP, ADP, UDP, and GDP reductase activities in vitro. Moreover, there are significant differences in the degree of inhibition of each ribonucleotide reductase activity. The reductase activities for CDP and ADP are more sensitive to hydroxyurea than those for UDP and GDP, particularly at high hydroxyurea molarities. As little as 5 x 10(-4)M hydroxyurea lowers CDP and ADP reductase activities to 25 to 30% whereas as much as 0.5 M hydroxyurea is needed to lower UDP and GDP reductase activities to 50%.


Human skin collagen. Presence of type I and type III at all levels of the dermis.

Human skin was sliced with a dermatome, and the ratio of type I to type III collagens at various depths was assayed by comparing the quantities of peptides of each derived from cyanogen bromide digestion of the cut skin. Although immunofluorescent studies have suggested type III collagen is located predominantly beneath the epidermis and around appendages, biochemical determination demonstrates the same ratio of type I to type III collagen at all levels of the dermis even in the absence of cutaneous appendages.


Biosynthesis of lipid A. Enzymatic incorporation of 3-deoxy-D-mannooctulosonate into a precursor of lipid A in Salmonella typhimurium.

The cell envelope fraction of Salmonella typhimurium contains an enzyme system which catalyzes transfer of 3-deoxyoctulosonate (KDO) from CMP-KDO to an incomplete, KDO-deficient precursor of lipid A. The enzyme system is firmly membrane-bound, but has been solubilized by treatment with nonionic detergent at alkaline pH and partially purified. Both the particulate and partially purified fractions catalyzed formation of a single reaction product containing 2 residues of KDO. Periodate oxidation of the purified product permitted tentative identification of the KDO disaccharide structure as KDO2-4KDO.


A conditional lethal mutant of Escherichia coli which affects the processing of ribosomal RNA.

A temperature-sensitive mutant strain was isolated from an RNase III-(rnc) strain of Escherichia coli. At the permissive temperature it behaves like the parental strain, but at the nonpermissive temperature it fails to produce normal levels of 23 S and 5 S rRNA, while instead the 25 S rRNA species becomes very prominent. (The 25 S molecule appears in rnc cells and contains 23 S rRNA sequences). When an rnc+ mutation was introduced to such a strain, or when the rnc mutation was replaced by an rnc+ allele, the strain remained temperature-sensitive. At the permissive temperature such strains synthesized rRNA like any other E. coli strain, but at the nonpermissive temperature they remained unable to synthesize normal levels of 5 S rRNA, and instead a larger molecule was accumulated. The simplest interpretation of theses findings is that the mutant strain contains a temperature-sensitive processing endoribonuclease, RNase E, which normally introduces a cut in the growing rRNA chain somewhere between the 23 S and the 5 S rRNA cistrons. These findings help also to explain the nature and origin of the various rRNA species observed in RNase III- cells and add to our understanding of processing of ribosomal RNA in normal cells of Escherichia coli.


The clinical use of combined xenografts of bone and autologous red marrow. A preliminary report.

The beneficial effect upon osteogenesis of imprenating bone grafts with autologous red marrow is well documented. The experimental findings reported in a previous paper suggested that prepared xenograft bone might provide a good medium for osteogenesis by marrow cells. This paper is a preliminary report of the first clinical attempt to use xenografts of bone combined with autologous red marrow. Kiel bone, which was found the most suitable, was impregnated with marrow aspirated from the iliac crest and, apart from one case of infection, gave excellent results in twenty-eight patients under conditions covering a wide range of indications for bone grafting. Further trials should allow a more valid assessment.


The osteoninductive property of decalcified bone matrix. An experimental study,.

Demineralised homologous bone-matrix implant was used to bridge a large circumferential osteoperiosteal gap in the diaphysis of the ulna of rabbits. Periodic observations of the graft were made clinically, radiologically, histologically and by tetracycline fluorescence up to forty-two weeks. By the twelfth week after operation 81 per cent of the animals revealed bone formation in the implant and complete bridging of the gap. The new bone was laid on the surface and in the substance of the matrix, suggesting that the inductive principle was acting locally. The bone, once formed, remodelled to the texture of a mature tubular bone and did not undergo absorption during a long follow-up period. Demineralise bone-matrix proved to be a highly osteoinductive and readily osteoconductive material. The graft did not evoke any appreciable local foreign-body or immunogenic reaction. The high degree of success in bridging massive bone defects justifies further serious studies and hopes for a useful substitute for massive autologous bone grafts.


"Osteopetrosis" in the Fairbank Collection.

The "osteopetrosis" section of the Fairbank Collection in the Radiology Museum of the Royal National Orthopaedic Hospital contains radiographs and case notes of twenty-two patients. This material has been reviewed in terms of modern concepts in an attempt to obtain a long-term follow-up and a firm diagnosis in each individual. Nine patients proved to have the classical autosomal dominant form of osteopetrosis, four had the malignant autosomal recessive type, craniometaphyseal dysplasia was present in two kindreds and isolated individuals had pyknodysostosis, atypical craniodiaphyseal dysplasia and craniosclerosis with osteopathia striata. As these conditions differ greatly in their clinical and genetic prognoses, diagnostic categorisation is of practical importance.


Characterization of columnar absorptive epithelial cells isolated from rat jejunum.

The origin and yield of cells isolated from rat jejunum have been assessed; 80-90% of the mature columnar absorptive cells but none of the Paneth, endocrine or regenerating crypt cells were recovered by the methods described. The structural integrity of the isolated cells was tested by phase contrast-, Nomarski interference- and fluorescence light-microscopy; and also by transmission electron microscopy; the isolated cells appeared structurally intact by all microscopic criteria applied. The isolated cells were judged to be functionally intact because they satisfied a number of tests designed to answer three logically related questions, namely: was the cell-surface membrane intact; were the cells able to accumulate selected solutes against a concentration gradient from the exterior medium; and were the cells able to perform biochemical functions which depended upon the integrated activities of several subcellular structures? The isolated cells did retain and accumulate selected solutes, respired at a steady rate on endogenous substrates, showed enhanced respiration on exogenous glucose and glutamine, maintained favourable intracellular proportions of adenine nucleotides (ATP:ADP:AMP), and synthesized cell protein from extracellular amino acids in proportion to the time of incubation. The isolated cells accounted for 80% of the lactic acid produced by intact rat jejunum under similar conditions of incubation.


Rapid Tween 80 hydrolysis test for mycobacteria.

A rapid Tween 80 hydrolysis test for mycobacteria utilizing gas-liquid chromatography is described. The test requires 1 h of incubation followed by simple extraction and chromatography of nonderivatized oleic acid.


Function of the autotransplanted pancreatic segment in the dog.

Autotransplantation of the left lobe of the pancreas to the neck vessels was performed in 13 dogs. The technique employed made it possible to examine pancreatic secretion and arteriovenous difference in enzymes immediately after transplantation. Following functional examination the pancreatic duct was ligated. From 4 to 20 postoperative days the graft was intact only in 3 dogs. Severe autolysis of the graft and/or of the surrounding tissue developed in ten dogs 4 to 6 days after transplantation. Stimulation with secretin elicited immediate secretion response of the graft, the volume of secretion and output of amylase corresponding to normal values. A statistically significant arteriovenous difference was found both for amylase and LDH before and after secretin. Stimulation of exocrine secretion of the graft elicited a marked increase of enzyme concentration in the arterial and particularly in the venous blood of the graft. Cellular damage with subsequent leakage of enzymes into extracellular fluid plays a significant role in the pathogenesis of autolysis of the graft and surrounding tissues.


Growth and amino acid requirements of various strains of group B streptococci.

A chemically defined medium (FMC; B. Terleckyj, N. P. Willett, and G. D. Shockman, Infect. Immun. 11:649-655, 1975) was used to compare the growth and amino acid requirements of 16 strains of group B streptococci, consisting of both laboratory-passaged organisms and fresh clinical isolates from adult and neonatal infections. The 5 standard Lancefield immunizing strains of group B streptococci, 090 (Ia), H36B (Ib), A909 (Ic), 18RS21 (II), and D136C (III), had doubling times in FMC (28 to 36 min) similar to those observed in Todd-Hewitt glucose broth (24 to 30 min). Similar doubling times were obtained with 11 clinical isolates growing in Todd-Hewitt glucose broth and FMC. The optimum buffering capacity of FMC was provided by 0.06 M sodium phosphate, and 1% glucose gave maximum cell yield. The group B streptococci, with minor exceptions, were very homogeneous in their amino acid requirements under both aerobic and anaerobic growth conditions. Phenylalanine, tyrosine, tryptophan, glutamate, arginine, valine, leucine, lysine, methionine, isoleucine, cystine, and histidine were required by all 16 strains under both aerobic and anaerobic growth conditions. In addition, threonine was required by all strains under aerobic growth conditions, whereas only 9 strains required threonine under anaerobic conditions. Serine was required by only 3 type III fresh clinical isolates aerobically, but not anaerobically. A requirement for glycine varied from strain to strain, apparently influenced by the oxidation-reduction potential of the growth medium.


Comparison of specimen collection and laboratory techniques for isolation of Haemophilus ducreyi.

Sixteen patients with clinical chancroid were studied prospectively; different culture media and sampling techniques from genital lesions were evaluated. Technique A was aspiration of a saline wash from the ulcer which was pooled and inoculated into rabbit blood, rabbit blood + vancomycin (5 microgram/ml), and semisolid chocolate agar + vancomycin (3 microgram/ml). Each primary culture medium was subcultured to chocolate agar with 1% IsoVitaleX (CA), CA with vancomycin (3 microgram/ml) plus polymyxin (7.5 microgram/ml; CA + vp). Technique B was the use of a cotton swab, plated directly on CA, CA + v, and CA + vp. Nine strains of Haemophilus ducreyi were obtained. Technique A yielded seven strains, whereas technique B yielded eight strains; with each technique, five strains were isolated only after use of selective antibiotic  media. CA + v medium yielded the largest number of isolates. Direct inoculation by swab to CA + v from chancroidal ulcers is effective as an isolation technique for growth of H. ducreyi.


New centrifugation blood culture device.

A single-tube blood culture device designed for centrifugation in a tabletop centrifuge is described. Reconstruction experiments using 21 different organisms and human donor blood indicate that excellent recovery can be obtained by centrifugation for 30 min at 3,000 X g.


Practical colorimeter for direct measurement of microplates in enzyme immunoassay systems.

A colorimeter capable of measuring results of enzyme-linked immunosorbent assay (ELISA) reactions directly in the wells of a microtiter plate is described. This colorimeter proved to be as accurate as a conventional spectrophotometer in assessing ELISA reactions, but had the advantage of not requiring transfer of the specimen to a separate chamber. With this colorimeter, 96 specimens can be read in approximately 5 min. A practical colorimeter such as this can make the use of ELISA tests more feasible for many laboratories.


Evaluation of a micromethod for determination of Streptococcus mutans and Lactobacillus infection.

A micromethod was developed for quantitative estimation in saliva of Streptococcus mutans and lactobacilli. With a semiautomatic pipette, 25 microliter of diluted saliva was spotted on the surface of an agar plate containing a selective medium. This volume gave a spot with a diameter of about 10 mm in which separate colonies could be counted. The results obtained with the spotting technique showed excellent agreement with those obtained with conventional agar plating. The method is convenient and results in a substantial saving of culture media.


Rapid screening for significant bacteriuria using a Coulter Counter.

A Coulter Counter was evaluated for detecting significant bacteriuria. Simple urinary particle counts showed agreement in 883 of the 956 (92.4%) urine specimens examined compared to a standard culture procedure. They also provided a semiquantitative estimate of bacterial numbers in specimens diagnosed as infected by culture. Sample preparation before counting was minimal, and results were obtained within two to four minutes.


Use of cell cultures as an indicator of pathogenicity of free-living amoebae.

Results comparing the time needed for the development of cytopathic effects in cell cultures with that needed to cause death in mice using inocula of Naegleria and Acanthamoeba are presented. The significance of the source and concentration of the inocula is demonstrated. The use of cell cultures as an indicator of the pathogenicity of free-living amoebae is discussed.


Specific and non-specific resistance to aminoglycosides in Escherichia coli.

The turbidimetric responses of a strain of Escherichia coli K12 to gentamicin and tobramycin were investigated. Both agents showed antibacterial activity below the conventionally measured minimum inhibitory concentration, but exposure to such subinhibitory concentrations of either agent generated a bacterial population which was able to grow in previously inhibitory concentrations at a rate equivalent to that of the parent culture. The increase in resistance was non-specific in that both aminoglycosides were equally affected, and was unstable on multiple passage in drug-free broth. The response to tobramycin was unaffected by the presence of an R factor conferring gentamicin resistance, but exposure of the R factor bearing strain to gentamicin caused a concomitant increase in the resistance to tobramycin, apparently by a non-specific adaptive mechanism similar to that observed with the parent strain. It is suggested that prior treatment of a gentamicin-resistant organism with gentamicin (as may occur during blind therapy) may adversely affect the subsequent response to other aminoglycosides.


Evaluation and application of an improved bacteriocin typing method for Klebsiella aerogenes.

A bacteriocin typing method was evaluated using 200 strains of Klebsiella aerogenes, 93% of which fell into 11 distinct types. The typing technique was successfully applied to the monitoring and control of hospital cross-infection.


Identification of Enterobacteriaceae by the API 20E system.

Since the introduction of the API 20E kit a number of identification schemes have been developed by the manufacturer for use with the kit. We evaluated the success of these various schemes in identifying 206 strains belonging to 34 taxa of the family Enterobacteriaceae. Many of the strains were atypical and only 94% could be identified by our own system of 50 conventional tests and a computer program. The most advanced identification scheme so far developed for the API 20E kit (the Analytical Profile Index and complementary Computer Service) allowed 88% of the 206 strains to be correctly identified, although 2% were incorrectly identified. The tests in the API 20E kit and 52 conventional tests were separately evaluated for their ability to discriminate between the 34 taxa considered in this study. Our results suggest that replacing some of the tests in the present API 20E kit might further improve its diagnostic performance.


An assessment of radial haemolysis in the detection of rubella antibody.

The results obtained by radial haemolysis in the detection of antibodies to rubella virus compared well with those obtained by haemagglutination inhibition. Radial haemolysis is unaffected by non-specific inhibitors. The sera do not therefore require pretreatment and the results are less equivocal. Radial haemolysis appears to be as sensitive as immunofluorescence and floatation centrifugation. It is possible to examine large numbers of sera with a considerable saving of time compared with the traditional haemagglutination technique. Rheumatoid factor may cause interference in radial haemolysis. Immune sera may be recorded as non-immune. This interference can be removed by 2-mercaptoethanol or reduced by heating sera at 60 degrees C for 20 minutes.


Gastric and duodenal mucosa in 'healthy' individuals. An endoscopic and histopathological study of 50 volunteers.

The results of histological and immunohistochemical examination of gastric and duodenal biopsy specimens from 50 volunteers without a clinical history of gastrointestinal disease are reported. Multiple specimens of tissue from standard sites in the stomach and duodenum were carefully orientated, and serially sectioned for examination by light microscopy and for immunohistochemical characterisation of plasma cells within the lamina propria. The antrum and fundus were normal in 32 of the 50 subjects but the other 18 showed histopathological evidence of gastritis in either the antrum or fundus. The latter appeared to be age-related. There was considerable variation in the appearance of the surface epithelium of the duodenum within as well as among individual subjects. Superficial gastric metaplasia in one or more biopsy specimens from the duodenal bulb was found in 64% of individuals. Histopathological examination of the duodenum revealed signs of chronic inflammation in 12% of the subjects. In two individuals there was active inflammation but in only one of these was the diagnosis made on endoscopic appearances. Histological criteria important for the diagnosis of duodenitis are discussed. The number of plasma cells in different biopsy specimens from subjects not showing histological signs of inflammation was variable. The ratio IgA:IgG:IgM producing plasma cells was remarkably constant from subject to subject as well as from specimen to specimen.


A topical treatment program for psoriasis with low anthralin concentrations.

A treatment program for psoriasis in which 0.01--0.05% anthralin was used has been clinically evaluated. In such low concentrations it is possible to use anthralin if one simultaneously avoids exposing the skin to unnecessary external mechanical trauma, including the mechanical removal of the scales from the lesions. Applied in this fashion, anthralin then also becomes most suitable for use in ambulatory therapy. In these low concentrations it does not irritate either the involved or the non-involved psoriatic skin. No discoloration of the clothes, the skin, the hair or the nails was observed.


Clear and dark basal keratinocytes in human epidermis. A stereologic study.

Standardized techniques of stereologic cytology were applied to normal human epidermis, in order to comparatively describe the suprabasal and the dark and clear basal keratinocytes. The results, expressed mainly in surface and volume densities of epidermal components, showed that some important stereologic parameters of dark basal keratinocyte constituents exhibited values which could be interpreted as intermediate between those of the clear basal keratinocytes and those of the suprabasal keratinocytes. Although the degenerative nature of dark cells cannot be ruled out, the stereologic characterization of their cell architecture also could indicate that dark basal cells are already differentiating keratinocytes still remaining in the basal layer.


Effectiveness of temazepam with short-intermediate-, and long-term use: sleep laboratory evaluation.

The effectiveness of 30 mg temazepam (SaH 47-603) for inducing and maintaining sleep was evaluated in the sleep laboratory in six insomniac subjects under conditions of short-, intermediate-, and long-term drug administration. Administration of temazepam had no effect on sleep induction. In addition, effectiveness was not demonstrated for sleep maintenance: wake time after sleep onset was not significantly decreased on any of the three drug conditions, while the number of nightly awakenings was significantly decreased on all three drug conditions. Total wake time was decreased only slightly with short-term drug administration and was similar to baseline with intermediate- and long-term use. The percent REM sleep was essentially unchanged throughout the drug administration period. On the initial set of withdrawal nights, the per cent REM sleep was slightly but no significantly increased over baseline. The per cent slow wave sleep decreased significantly with short-, intermediate-, and long-term drug administration. After withdrawal, the percent slow wave sleep returned to baseline values.


High-dose furosemide in Nigerians with severe chronic renal failure: effect on fecal water content.

The effect of high doses of furosemide, 200 to 800 mg daily, has been studied in 12 Nigerias with edema associated with severe chronic renal failure and hypertension. They all responded well and lost their edema fluid during a period which varied from 10 to 57 days. The fecal water content and the frequency of bowel opening while on furosemide were significantly higher than without furosemide in six of these patients. It is concluded that the gastrointestinal tract is a major contributory site of fluid loss in patients with grossly impaired renal function.


The morphologic relationship between dental resins and etched dentin.

A scanning electron microscopic examination of the interface between etched dentin and several different commercial resins showed that pretreatment with othophosphoric acid enlarged the dentinal tubules facilitating penetration of the resin. The depth of penetration increased from approximately 0.2 mm to 2.0 mm when dentin was dried for 24 hours compared to using the normal clinical protocol. The hydration of dentin and/or the presence of dentinal fluid probably influences the degree of resin penetration. While penetration would provide a means of mechanical retention of resin it would probably do so at the expense of dentin and pulp viability particularly where remaining dentin thickness is less than 1.0 mm. Penetration of toxic resin would account in part for the enhanced inflammatory pulpal response observed following application of acid etch restorative procedures.


New method evaluates coupling agents bonding polymer to tooth mineral.

Polymerized test specimens consist of a Bis-GMA matrix and a filler of synthetic hydroxyapatite previously coated with the coupling agent under investigation. As the concentration of filler increases for a given coupling agent, the specimen's tensile strength decreases. The more effective the coupling agent, the less pronounced is the decrease. Application of the method is proposed for classifying coupling agents as well as providing a means for assessing the weakening effects of environmental exposure.


Prevention of cobblestoning in hair transplantation.

Cobblestoning, a complication of the hair transplant technique, occurs when donor grafts taken by punch sometimes heal above the surrounding skin. A suturing technique whereby cobblestoning may be prevented is described in detail.


Modification of impulsive tempo in learning-disabled pupils.

Thirty-three impulsive learning-disabled males, aged 9-12 years, were assigned to one of three treatment conditions: (a) Modeling, in which subjects were exposed to a videotape of a boy demonstrating reflective problem-solving activities and instructing himself to perform cautiously; (b) Modeling Plus Self-Verbalization, in which subjects observed the same videotape and were additionally required to verbalize similar reflective instructions; and (c) Control, in which subjects were shown a videotape of the task materials used in the modeling videotape, but without the model. Subjects were tested immediately after exposure to treatment, and again 3 weeks later, on different forms of the Matching Familiar Figures test. Both Modeling and Modeling Plus Self-Verbalization conditions were superior to Control in reducing errors on the immediate test, but the three treatments did not differ on the delayed test. No significant differences among the three conditions were found with regard to response latency. Discrepancies between the present results and those of previous related studies were noted, and potential research directions were suggested.


Reflection-impulsivity in normal and behavior-disordered children.

The primary index of reflection-impulsivity is Kagan's Matching Familiar Figures Test (MFF), which yields both a latency and an error score. To evaluate further the construct validity of these measures, both the latency and error scores of 9- and 15-year-old normal and "acting-out" behavior-disordered children were compared. Young behavior-disordered children were found to be more impulsive than the other groups on the MFF error measure. No differences occurred on the MFF latency measure. This result is consistent with previous findings with normal children in supporting the construct validity of the MFF error score and raising questions about the construct validity of the MFF latency score. Findings were inconsistent with Kagan's assertion that normal children become more reflective with age. The older behavior-disordered children were more reflective on the error measure than the younger behavior-disordered children and had equivalent error scores to both age groups of normals. This finding suggested a lag in the development of reflection in behavior-disordered children.


Oral hygiene: a history of tongue scraping and brushing.

Tongue scraping and brushing have been practiced for hundreds of years but are still little appreciated or used by the public. Throughout the centuries, tongue scrapers have been constructed of thin, flexible strips of wood, various meals, ivory, mother-of-pearl, whalebone, celluloid, tortoiseshell, and plastic. Recent scientific evidence has validated the need to practice habitual and thorough tongue brushing as part of daily home oral hygiene procedures.


Congenital absence of teeth: a review with emphasis on inheritance patterns.

Congenital absence of teeth is a heritably phenomenon probably most often passed to each generation by an autosomal dominant pattern with incomplete penetrance and variable expressivity. Correlation of hypodontia with systemic disease leads to the hypothesis that this frequent dental anomaly may in some cases be a microform of systemic ectodermal dysplasia.


Composite resins bonded to porcelain with silane solution.

A system of bonding composite resins to dental porcelain with silane solution produced a reliable bond. The introduction of stresses, particularly thermal and functional, needs to be investigated further and tested clinically. This technique is now being investigated clinically in a number of porcelain-fused-to-gold restorations. It could be an effective method of intraoral repair of fractured or chipped porcelain restorations.


Techniques to improve the seating of castings.

A convergence of 20 degrees for full crowns is the most likely to be seen clinically, as determined by random measurements taken at a dental laboratory. Castings did not seat without cement, by an average of 215 micron at 10 degrees of convergence, or by 99 micron at 20 degrees of convergence. Biting forces will seat a casting approximately 150 micron, but will cause random concentrations of force against tooth structure, thus compressing it. All castings tend to rebound from this position. Zinc phosphate and polycarboxylate cemented crowns seated to 33 micron and 20 micron respectively when relieved, but each were elevated to 112 micron when not relieved. A silicophosphate cement displaced crowns with 20 degrees of convergence 122 micron under "ideal" clinical conditions, even when relieved; and with CBA 9080 cement, more than 500 micron. A die relief method was found to be the most suitable of the three casting compensation techniques. Casting retention, after cementation, was increased by 25%.


Numerical and experimental evaluation of energy inputs, temperature gradients, and thermal stresses during restorative procedures.

Wet cutting in enamel should be used during restorative procedures. According to evidence reported here, dry cutting can induce sufficiently high thermal stresses to fracture the enamel. Temperatures resulting from dry cutting in dentin are high enough to cause biologic pulp damage if the cutting is within 1 to 2 mm of the pulp. Cracks induced in the cavity walls by dry cutting may eventually contribute to marginal failure. A hand-held syringe directing a small, high-velocity stream of water into the cutting region is a more effective cooling technique than an air-water spray.


Dental management of patients receiving hemodialysis and kidney transplants.

The dental management of patients who received hemodialysis or a kidney transplant has been described. Emphasis has been placed on oral findings as well as on management considerations that are necessary for safe outpatient dental treatment. As with most patients who have systemic disease, emphasis must be on frequent recall examinations and strong preventive measures to minimize the need for extensive dental treatment. Close consultation between dentist and physician is essential for safe dental management of these patients. Few segments of the population need dental services as desperately as do patients receiving hemodialysis or kidney transplants. In this ever-increasing group of patients, dental disease may have consequences much more serious than the loss of function, esthetics, and comfort. Instead, dental problems may compromise the patient's general health and hinder medical efforts to either maintain or replace vital kidney functions.


Centrophenoxine: effects on aging mammalian brain.

A study was made of the effects of centrophenoxine on the learning and memory of old mice. The results were correlated with changes in neuronal lipofuscin in the cerebral cortex and hippocampus. Old female mice (11-12 months) were treated with centropheoxine for three months and their learning and memory were tested in a T-maze. The number of trials required to attain the criterion in the 20 treated old mice were compared with those for 20 untreated mice of the same age and for 20 younger untreated mice. The treated animals learned the task with significantly fewer trials, and also exhibited a reduction of neuronal lipofuscin pigment in both the cerebral cortex and the hippocampus. The changes in lipofuscin were demonstrated by study of the characteristic autofluorescence, and by histolchemical and ultrastructural (electron microscope) observations.


Development of variable- focus lenses and a new refractor.

This paper traces the events that led to the development of the variable-focus lens, the variable-power astigmatic lens, and the principle of remote refraction and "phantom lenses." These developments formed the basis for invention of a new subjective refractor-the Vision Analyzer- whose novel design and rapid operation are described.


Diseases of aging untreated virgin female RFM and BALB/c mice.

Diseases of untreated, virgin female barrier-maintained RFM and BALB/c mice used as controls in a large radiation aging experiment were necropsied after natural death. The spectrum and incidence of neoplastic and nonneoplastic diseases were somewhat different in the two strains. Both strains show a high incidence of neoplasma (largely reticulum cell sarcomas and lung tumors) and of glomerulosclerosis. A wide variety of other diseases was noted in much lower incidence. The findings in the RF were briefly compared with those in earlier experiments with that strain in this laboratory.


The impairment index as a symptom-independent parameter of drug efficacy in geriatric psychopharmacology.

A method for the determination of drug efficacy in geriatric psychopharmacology is presented. Sixty patients with mild senile organic  brain syndrome were evaluated by a battery of neuropsychological tests. Subsequently these patients were randomly assigned to either drug or placebo treatment for 90 days. One group received 300 mg per day of Naftidrofuryl (Praxilene) while the other received identical placebo. Upon retest, the data indicated that Naftidrofuryl produced significant improvements in reaction time, short-term memory, and iconic memory. Ten neuropsychological test scores as well as two POMS mood factors were incorporated into an Impairment Index. This index was drug sensitive and proved to be symptom independent.


An immunocytochemical study of a rat pituitary multipotential clone.

The 2A8 clone, a normal diploid rat anterior pituitary cell strain, was investigated by immunocytochemistry to determine the cell types into which the clonal cells differentiated in vivo and in vitro. The in vivo study was carried out by injecting the 2A8 clone preparation either into the hypothalamic region or under the kidney capsule. After thirty days the implants were removed and studied by immunocytochemistry. In vitro, many prolactin cells and a few growth hormone cells were found. In vivo, however, prolactin, growth hormone, ACTH and TSH cells and gonadotrophs were identified. We concluded that the 2A8 clone was multipotential. Since the gonadotrophs of the implants made in the hypothalamic region were larger and more plentiful than those in the kidney implants, and since gonadotrophs were lacking in the in vitro system, it appeared that the hypophysiotrophic environment was the most conducive to gonadotrophic differentiation and maintenance, and that the factor or factors necessary for cyto-differentiation were apparently present in the general circulation of the rat but absent in the growth medium of our culture cells.


Light and myopic refraction in children.

Studies over many years, carried out in different climatic zones of the country, were focussed on the investigation of the role of light in the prophylaxis of development of near-sightedness. It has been found that not only the accommodation reflex but also the pupillary reflex play a role in the enhancement of the optic power of the eye. A scheme of interaction of the mentioned reflexes depending on the level of visible radiation was presented. Provision of optimum illumination is one of the basic conditions for the prevention of eye fatigue and development of myopia.


Investigation of insecticidal properties of aerosols of mixtures of pyrethroids and DDVP.

Systematic studies of the effectivity of aerosols of the mixtures of DDVP and pyrethroids tested on house flies revealed synergism among the mentioned substances the degree of which was determined by the composition of the mixtures. Among the formulae under study, the mixture of DDVP and neopynamin in a ratio 9 : 1 is of greatest interest.


Core region in Proteus mirabilis lipopolysaccharide.

Four R mutants of P. mirabilis were isolated. The composition of their degraded polysaccharides (PS) obtained from the respective lipopolysaccharides (LPS) as well as the composition and properties of the PS-fractions separated by column chromatography were examined. The results were compared with those obtained with PS of the wild type. One of the mutants could be classified as an Ra-type mutant, presenting a complete LPS core. This polysaccharide core contains: galacturonic acid, glucosamine, glucose, D-glycero-D-mannoheptose, L-glycero-D-mannoheptose in a molar ratio of 1 : 1 : 1 : 1 : 2 and 2-keto-3-deoxyoctonate. Taking into consideration the common sugars described previously in the LPS chemotypes of P. hauseri, the composition of the complete core region mentioned above represents the LPS core part of all the chemotypes, containing two different heptoses.


On the possible mechanism of the protective effect of Escherichia.

A model on a HEp-2 cell culture was elaborated, permitting the study of the ability of microbes to be adsorbed an to proliferate on the surface of cells and of the mechanism of their protective effect. The ability of E. coli strains to be adsorbed and to proliferate on the surface of a cell culture was found to differ. It has been demonstrated that the protection of the cell culture from subsequent infection with virulent Shigella can be explained not only by the antagonistic activity of E. coli strains, but also by their ability to be adsorbed and to proliferate on the surface of cells. A similar mechanism of protective effect is supposed in preparations of the Colibacterin type.


Suppression of natural killer cell cytotoxicity by splenocytes from Corynebacterium parvum-injected, bone marrow-tolerant, and infant mice.

Natural killer (NK) cell cytotoxicity to YAC-1 lymphoma was investigated in mice tolerant to bone marrow grafts (BM-tolerant), Corynebacterium parvum- (C. parvum) treated mice, and infant mice. Also the comparison was made between the NK cell and the hemopoietic-resistance effector (HR-E) cells. It was found that the BM-tolerant mice and C. parvum-treated mice showed either no or markedly decreased NK cell cytotoxicity. These mice were also nonresponders to bone marrow grafts in vivo. The lack of or decreased reactivity was apparently caused by the regulatory cell activities of the suppressor cell since the splenocytes from C. parvum-treated and BM-tolerant mice suppressed significantly the cytotoxic activities of otherwise fully functional NK cells. Similar suppressive effect on NK cells was mounted by splenocytes from infant mice, indicating again the suppressor cell regulation of NK cell cytotoxicity.


C57BL/10/CR mice: nonresponders to activation by the lipid a moiety of bacterial lipopolysaccharide.

C57BL/10/CR mice do not respond to the lipid A moiety of LPS. This defect was demonstrated in vivo by a decreased production of the acute phase serum amyloid protein SAA. In addition, the defect was demonstrated in vitro by using cultures of spleen cells and peritoneal cells. No mitogenesis of spleen cells or enhanced glucose utilization by either spleen cells or peritoneal cells was observed when the cells were stimulated by lipid A or phenol-extracted Escherichia coli K235 LPS. The response of these mice to PHA, Con A, Poly I:C, 8BrcGMP, and butanol extraced E. coli K235 LPS was normal. Thus, the inability of lipid A to stimulate B lymphocyte mitogenesis and activate peritoneal cells in vitro may correlate with its inability to induce the acute phase SAA in vivo. B10/CR mice represent another strain, similar to the C3H/HeJ, which is nonresponsive to lipid A and should be useful in eludcidating the mechanism by which bacterial LPS activates cells.


Infection with Bacillus Calmette-Guérin activates murine thymus-independent (B) lymphocytes.

Thymus-independent (B) lymphocytes from BCG-infected mice respond with enhanced or depressed levels of proliferation relative to the nonspecific levels of stimulation induced by tuberculin. The type of response obtained depends on how BCG was administered and the source of the lymphocytes. The proliferation of splenic B lymphocytes was depressed when BCG was given i.v. and unchanged when BCG was given subcutaneously, whereas the lymph node B cell response was enhanced regardless of the route of injecting BCG. BCG was found also to be a mitogen for normal B cells in vitro and to stimulate polyclonal activation of B cells in vivo. Finally, evidence is presented which indicates that the depressed splenic B cell response to tuberculin may be due to the activation of suppressor T cells.


Immunofluorescence studies with 4-acetamido-4'-isothiocyanato stilbene-2,2'-disulphonic acid (SITS).

The application of the fluorochrome 4-acetamido-4'-isothiocyanato stilbene-2,2'-disulphonic acid (SITS) in immunofluorescence was studied. The optimal excitation wave length is 350 nm, and optimal fluorescence is obtained at 420 nm. After purification of the commercial compound, conjugation is performed in a strong buffer at pH 9.0-9.5. SITS conjugates were very satisfactory for immunofluorescence studies of the cytoplasmic antigens of cell preparations, but their blue emission was difficult to distinguish from autofluorescence in sections of human tissues. Good results with immunofluorescence on membrane bound antigens were obtained by using an ultra-violet laser beam as light source. SITS can be used simultaneously with FITC and TRITC conjugates thus making it possible to show three antigens in one preparation.


Quantitative determination of human leukocyte interferon by microfluorometric immunoassay with FITC-labeled anti-interferon immunoglobulin.

The possibility of quantitative determination of human leukocyte interferon using FITC-labeled anti-interferon antibody was studied. Anti-interferon immunoglobulin of high specific activity was obtained as a result of long-term immunization of a donkey with human leukocyte interferon preparation and subsequent fractionation and immunoadsorption steps. This immunoglobulin was labeled with FITC and then used for titration of human leukocyte interferon by the fluorescence inhibition immunoassay. The titres of different preparations of human leukocyte interferon in the immunoassay were comparable with the titres of the same preparations detected by the inhibition of a cytopathic effect of VSV.


Characterisation and separation of human lymphocytes forming mouse red cell rosettes.

Rosette formation between human lymphocytes and mouse red blood cells (MRBC) was examined as a marker for B lymphocytes and as a method of B lymphocyte separation. A small proportion of lymphocytes formed spontaneous rosettes with MRBC (mean value 6%) and the number was considerably increased by pretreating the lymphocytes with neuraminidase (mean value 16%). Double marker tests demonstrated that lymphocytes forming MRBC rosette were immunoglobulin (Ig) bearing cells, with a high proportion of IgM bearing cells, but not all Ig bearing cells formed MRBC rosettes. Lymphocyte populations enriched with T or B lymphocytes, by SRBC rosette sedimentation and nylon column filtration, gave values for MRBC rosettes consistent with a subpopulation of Ig bearing cells. Separation of MRBC rosette-forming cells gave a relatively poor degree of separation and rerosetting with MRBC produced variable results.


A new method for the simultaneous assay of rosette-forming and antibody-secreting cells, utilising immune adhesion reactions in monolayer.

Mixtures of sheep erythrocytes and immune spleen cells from mice were incubated in shallow slide chambers coated with erythrocyte ghosts or anti-mouse Ig, with poly L-lysine as a coupling agent. Antigen-binding cells and erythrocytes surrounding antibody-releasing lymphocytes became bound to the reactive surfaces by immunocytoagglutination and could be readily observed on inversion of the chambers. The sensitivity of the method compares with those currently in use for quantification of antibody-releasing cells, and resolution of rosettes is markedly superior to that obtainable in other assay systems. The advantages and limitations of the method are discussed.


Use of a unique chemiluminescence spectrometer in a study of factors influencing granulocyte light emission.

Factors contributing to variability in chemiluminescence (CL) measurements of phagocytizing granulocytes were identified and controlled. Observed CL was decreased by light quenching caused by red blood cells, hemoglobin, phagocytizable particles, and granulocytes. Increases in CL were achieved by opsonization with heat labile and heat stable (antibody) serum factors, reaction temperatures in the range of 37-40 degrees C, and mixing. The effects of controlled temperature variation and mixing were studied with a chemiluminescence spectrometer uniquely designed for such studies. Features of this spectrometer which make it more suitable than the previously employed scintillation spectrometers for the observation of granulocyte and other chemiluminescent systems include; (1) the ability to measure CL immediately upon reaction initiation; (2) simplicity of photomultiplier tube exchange; and (3) built-in optical filter holders for spectral analysis.


Rapid method for preparing bone marrow cells from small laboratory animals.

A new method is described by which long bones may be removed from small laboratory animals for the preparation of bone marrow cell suspensions for use in immunologic assays. Bones are obtained free of muscle and cartilage, and the procedures is adaptable for use under aseptic experimental conditions.


Cell migration from clotted plasma droplets in vitro: development of a new method for MIF assay.

A new in vitro method for MIF assay has been developed using cells contained in clotted plasma droplets. It gives results concordant with the capillary tube method for H37 Ra Mycobacteria sensitized mice and PPD sensitized healthy persons. The technique is applicable to any migratory cell population and requires only a small number of cells for performance. It is technically simple, not time consuming and practicable both in patients and experimental animals.


Swine influenza A at Fort Dix, New Jersey (January-February 1976). IV. Summary and speculation.

Influenza A/New Jersey/76 virus was detected at Fort Dix from January 19 through February 9, 1976 and infected at least 230 military personnel. Thirteen hospital admissions for acute respiratory disease were associated with influenza A/New Jersey infection, and additional members of index training companies may have been hospitalized with influenza A/New Jersey. This virus was likely introduced into the reception center by an incoming trainee. Although our studies could not eliminate the possibility that influenza A/New jersey strains are inherently less transmissible in humans than H3N2 viruses, the simultaneous transmission of influenza A/Victoria/75 virus and the unusual environment in basic combat training may explain why influenza A/New Jersey did not spread significantly outside of this training population.


Swine influenza virus infections in humans.

Influenza in swine was first recognized as an epizootic disease in 1918. During that same year influenza virus in humans caused the worst pandemic on record. The virus of swine influenza was isolated in 1930. Swine influenza virus was first isolated from humans in 1974. Since then, including the cases at Fort Dix, there have been a total of nine viral isolations from humans in the United States. Serologic evidence of infections with swine influenza virus in humans has also been obtained. Evidence for transmission of swine influenza virus to humans before 1974 is minimal and circumstantial. Recent recognition of infections with swine influenza virus may be the result of better surveillance, increased numbers of susceptible humans, or increased viral infectivity for humans. Nevertheless, the apparent frequency of human infections and the declining levels of antibodies to swine influenza virus in the human population suggest that influenza viruses of swine may be a potential sources of epidemic disease for humans.


Measurement of hemagglutination-inhibiting antibody to influenza virus in the 1976 influenza vaccine program: methods and test reproducibility.

Titers of hemagglutination-inhibiting (HAI) antibody were determined for all sera obtained from participants in the 1976 Influenza Vaccine Test Program. At least eight control sera were included in each test during the vaccine trial period for the purpose of monitoring HAI test reproducibility. Estimates of day-to-day reproducibility were defined as the percentages of duplicate aliquots of the same sera, tested on two separate days, having HAI antibody titers that did not differ by more than one twofold dilution. These reproducibility estimates ranged from 89% to 97% with influenza A/New Jersey/76 and A/Mayo Clinic/74 antigens. In contrast, within-day reproducibility estimates obtained from all sets of control sera ranged from 96% to 98%. Estimates of day-to-day test reproducibility obtained with selected sera taken after vaccination that were titrated on two differen days ranged from 90% to 98%. Geometric mean titers of these sera tested weeks or months apart differed on some occasions during the test period.


Immunogenicity and reactogenicity of influenza A/New Jersey/76 virus vaccines in normal adults.

Inactivated influenza A/New Jersey/76 virus vaccines were administered intramuscularly to 199 normal adults, aged 19-59, in doses of 200, 400, or 800 chick cell-agglutinating units in a double-blind, placebo-controlled trial. Systemic reactions (including fever) were uncommon, were mild, lasted less than 24 hr, and were more frequently associated with the largest dose. Local reactions were common but mild. A single, rapidly reversible, allergic reaction was noted in a volunteer 2 hr after vaccination. There was a trend toward fewer systemic reactions in vaccines who had preexisting hemagglutination-inhibiting (HAI) antibodies to the vaccine virus in their sera as compared with seronegative vaccines. All vaccine preparations at all three dosages evoked serum HAI titers of greater than or equal to 20 to greater than or equal to 40 in a high proportion of seronegative recipients, with significantly greater geometric mean titers at the highest dosage. Vaccines between the ages of 19 and 23 years manifested significantly lower serologic responses than did vaccinees over the age of 23. Thus, normal adults over the age of 23 can be immunized with a single, well-tolerated dose of A/New Jersey/76 vaccines.


Comparative study of reactogenicity and immunogenicity of influenza A/New Jersey/8/76 (Hsw1N1) virus vaccines in normal volunteers.

Local and systemic reactions and antibody responses to three different doses of influenza virus vaccine (A/New Jersey/76 [Hsw1N1]) produced by four different manufacturers were studied. Local and systemic reactions that peaked between 8 and 24 hr after vaccine administration were related to the source of the vaccine more than to the dose. Antibody conversion occurred in almost 90% of subjects over the age of 25 years but in only 61% of subjects 25 years or younger. This low response rate was due almost entirely to the lack of response of volunteers who received split-product vaccine (only 49% of subjects became seropositive). In addition, individuals with symptoms of local reaction had a rate of antibody conversion and magnitude of antibody response that were significantly higher than those observed in subjects who did not have local reactions. These findings suggest that in subjects older than 25 years, all four vaccines produced a high frequency of antibody response with a minimum of side effects. In addition, split-virus vaccine is less immunogenic than whole-virus vaccine in subjects younger than 25 years. Production of a vaccine devoid of local reaction may not be advisable.


Clinical trials of monovalent influenza A/New Jersey/76 virus vaccines in adults: reactogenicity, antibody response, and antibody persistence.

Responses to monovalent influenza A/New Jersey/76 virus vaccines were evaluated in 22-43-year-old, antibody-negative males. Three doses of vaccine (200, 400, and 800 chick cell-agglutinating [CCA] units) from each of four manufacturers and a placebo were given intramuscularly. Mild systemic complaints occurred in 12% of vaccines and moderate reactions in 5%, mostly in recipients of 800 CCA units of the vaccines. Reactogenicities of subvirion vaccines were intermediate, while different preparations of whole-virus vaccine were the most and the least reactogenic. Local reactions to 200-CCA unit doses of vaccines resembled reactions to placebo. Serum antibody responses against influenza A/swine/37 virus and current swine-like strains were similar for recipients of vaccines from three manufacturers. Among recipients of 200 CCA units of the vaccines, 79% developed antibody titers of greater than or equal to 1:20. Increasing antibody response was not uniformly associated with increasing vaccine dose but did correlate with increasing reactogenicity. Antibody titers fell about twofold over six months, and they fell slightly more for recipients of subvirion than for recipients of whole-virus vaccines.


Antibody responses and reactogenicity of graded doses of inactivated influenza A/New Jersey/76 whole-virus vaccine in humans.

Graded doses of inactivated whole influenza A/New Jersey/8/76 virus vaccine were injected into healthy volunteers. Presence of hemagglutination-inhibiting (HAI) antibody was uncommon before vaccination in persons younger than 45 years and most common in those older than 65 years. All vaccine doses (4-61 microgram of hemagglutinin) induced HAI antibody in at least 50% of recipients, although a booster dose was required to induce high titers in those younger than 24 years of age. A tendency for HAI titers to increase with increasing age and dose was noted. A trivalent vaccine (composed of A/New Jersey/76, A/Victoria/75, and B/Hong Kong/73) given to persons 21-44 years old produced HAI antibody titers to A/New Jersey/76 similar to those produced by the same dose of A/New Jersey/76 as a monovalent vaccine and produced higher titers in subjects 65 years of age or older. Increases in neuraminidase-inhibiting antibody were small and infrequent. Local and system symptoms were commonly reported after vaccination but were mostly mild (11% had moderate pain and 19% had muscular aches). Reactions were less common among those with HAI antibody at the time of vaccination and were unacceptably severe (in 20% of recipients) only in seronegative recipients given the 61-microgram dose of hemagglutinin.


Reactogenicity and immunogenicity of parenteral monovalent influenza A/Victoria/3/75 (H3N2) virus vaccine in healthy adults.

Monovalent influenza A/Victoria/3/75 whole-virus vaccines prepared by Merck Sharp and Dohme (West Point, Pa.) and Merrell-National Laboratories (Cincinnati, Ohio) and split-virus vaccines prepared by Parke, Davis and Company (Detroit, Mich.) and Wyeth Laboratories (Philadelphia, Pa.) containing 200, 400, and 800 chick cell-agglutinating units per dose were compared with a placebo in double-blind trials in which 208 adults participated. Titers of hemagglutination-inhibiting antibody of greater than or equal to 1:20 were found in greater than 80% of the volunteers 21 days after vaccination. Seroconversion, defined as a fourfold or greater increase in antibody titer, occurred more frequently among seronegative volunteers than among seropositive volunteers. The geometric mean titers obtained with the whole-virus or split-virus vaccines were not significantly different. Reaction rates had no relation to seroconversion, nor did seronegative subjects have more reactions than seropositive subjects. Local reactions from all vaccines increased with increasing dose. Significantly more overall reactions, "bothersome" reactions, and febrile reactions occurred in the recipients of whole-virus vaccine. Of nine volunteers who reported temperatures of greater than 100 F, one had received split-virus vaccine, seven had received whole-virus vaccine, and one had received the placebo. Most systemic reactions were mild, and all were self-limited.


Serologic responses and systemic reactions in adults after vaccination with bivalent A/Victoria/75-A/New Jersey/76 and monovalent B/Hong Kong/72 influenza vaccines.

Bivalent A/Victoria/75-A/New Jersey/76 and monovalent B/Hong Kong/72 influenza vaccines were given alone or together to adutls, and systemic reactions and antibody responses were determined. The rates of systemic reactivity observed varied among vaccine groups. Disrupted vaccines and whole-virus vaccines containing type B antigen only did not cause significant reactivity. Systemic reactions were observed after administration of the bivalent A whole-virus vaccines, and this reactivity was increased if the B vaccine was also administered. Reactions to the more reactive vaccines were less frequent in older subjects or in younger individuals with evidence of previous exposure to influenza antigens in the vaccine. Antibody responses in this study indicated that individuals older than 25 years responded better to A/New Jersey antigens than did younger subjects. The A/Victoria antigen produced lower antibody levels in older individuals than in younger subjects. The B/Hong Kong antibody responses were similar in all vaccine groups.


Clinical trials of bivalent influenza A/New Jersey/76-A/Victoria/75 vaccines in the elderly.

This placebo-controlled evaluation in elderly persons of inactivated influenza virus vaccines containing 200 or 400 chicke cell-agglutinating units of both A/New Jersey/76 and A/Victoria/75 antigens revealed mild systemic reactions in 7.8% and moderate reactions in 4.9% of vaccinees. These reactions were more common after administration of whole-virus than subvirion vaccines, more frequent in females than males, and more frequent in persons with low initial titers of antibody to A/New Jersey antigen. Local inflammation occurred in 18.7% of vaccinees (predominantly females) and frequently was seen with systemic reactions, but did not correlate with antibody response. Titers of antibody to A/New Jersey antigen were initially high and 99% of vaccinees had titers of greater than 1:20 after vaccination. Titers of antibody to A/Victoria antigen were initially low, and 56% of vaccinees had titers of greater than 1:20 after vaccination. Fourfold or greater increases in titer occurred in about 50% of vaccinees for each virus, most often concomitantly. Antibody responses to subvirion and whole-virus vaccines were similar, and increased responses with higher doses coincided with increased reactogenicity. Later revaccinations because of low titers of A/Victoria antibody produced negligible antibody responses.


Responses of elderly and chronically ill subjects to bivalent influenza A/New Jersey/8/76 (Hsw1N1)-A/Victoria/3/75 (H3N2) vaccines.

Antibody responses and side effects to bivalent influenza A virus vaccines from three different manufacturers, containing 200 or 400 chick cell-agglutinating (CCA) units each of A/New Jersey/8/76 (Hsw1N1) and A/Victoria/3/75 (H3N2) antigens, were evaluated in 234 ambulatory elderly and chronically ill volunteers in a placebo-controlled, doubld-blind study. Systemic reactions did not occur significantly more often among recipients of vaccine than among volunteers who received placebo. Local reactions to vaccines were observed but were mild, transient, and well tolerated. Occurrence of preexisting antibody to each antigen was common. Titers of antibody to A/New Jersey/8/76 (Hsw1N1) antigen of greater than or equal to 1:40 occurred after immunization in 94%-100% of volunteers receiving vaccines and in 66% of the placebo recipients. Titers of antibody to A/Victoria/3/75 (H3N2) antigen of greater than or equal to 1:40 occurred in 53%-85% of vaccine recipients compared with 34% of those in the placebo group. Thus, bivalent split-product and whole-virus influenza I vaccines containing 200/200 or 400/400 CCA units of the two antigens appeared to be nearly equivalent in incidence and severity of side effects and antibody responses in recipients.


Studies of inactivated influenza virus vaccines in hospital employees: reactogenicity and absenteeism.

The impact of a mass influenza vaccination program on the operation of a major medical center was evaluated in adult volunteer employees who were given either ether-split or whole-virus bivalent influenza vaccine in a double-blind manner by jet-injector gun. Each volunteer completed a reaction information form, nurses monitored temperatures, and data on absenteeism of the employees of the Nursing Service and the Building Management Service were collected. The results of this study demonstrated that the mass campaign did not adversely affect absenteeism. However, the whole-virus vaccine caused significantly more local, systemic, and febrile reactions thant the split-virus vaccine. Purified inactivated influenza virus vaccines can be given in a mass campaign to hospital personnel without adversely affecting the operation of the hospital.


Swine influenza virus vaccination in patients with multiple sclerosis.

A double-blind, placebo-controlled study was conducted to evaluate the safety and efficacy of inactivated influenza virus vaccines in patients with multiple sclerosis. The vaccine used contained 200 chick cell-agglutinating (CCA) units of influenza A/New Jersey/76 and 200 CCA units of influenza A/Victoria/75 whole viruses (Merrell-National Laboratories, Cincinnati, Ohio). The frequency of clinical relapses of multiple sclerosis was the same in the vaccine-treated (four of 33 patients) and placebo-treated (four of 33) groups. An untreated control group had a slightly higher rate of relapses (four of 22). Toxic reactions to the vaccine were not a significant problem. The efficacy of the vaccination as measured by titers of hemagglutination-inhibiting antibody was comparable to that reported for the general population. However, patients with preexisting antibody were less responsive to the vaccine than normal controls studied previously. The patients who did not respond to the vaccine tended to be older and more disabled. There were no changes in titers of antibody to rubeola or rubella virus after vaccination or in relation to change in clinical course. It is concluded that the vaccine is safe and effective in patients with multiple sclerosis.


Reactivity and immunogenicity of monovalent A/New Jersey/76 influenza virus vaccines in children.

This paper presents the methodology of the multicenter collaborative clinical trials that evaluated monovalent A/New Jersey/76 influenza virus vaccines in healthy children six months to 18 years of age and describes the experience involving 264 children six months to 10 years of age in Omaha, Nebraska. Results indicated that chidren can be safely and effectively immunized with carefully adjusted two-dose regimens that take into account the differences between split-virus and whole-virus vaccines and between the two whole-virsu vaccines.