The effect of cation ionophores on intraocular pressure.

The ionophores A23187 and X537A, which increase the permeability of cell membranes to calcium and other divalent cations, produced significant elevation of intraocular pressure in rabbits. Topical instillation of these ionophores in concentrations of 1.0 and 0.1 per cent were effective. Aqueous humor protein and facility of outflow were similar in ionophore-treated and control eyes. Pretreatment with indomethacin did not block the intraocular pressure rise induced by A23187. Alterations of intracellular calcium might control cellular processes within the eye as in many other biological systems.


Nonspecific stimulation of a secondary ocular antibody response.

Rabbits injected intravitreally with bovine gamma globulin (BGG) into the right eyes were challenged 3 months later by an intravitreal injection of ovalbumin (OA) into the left eyes. The rabbits were killed 6, 7, and 9 days after challenge and the number of cells producing antibody to both OA and BGG were enumerated. The uveal tract cells of all left eyes produced anti-BGG even though no BGG had been injected into the left eyes. The uveal tract response to OA was less pronounced and typical of a primary response. High anti-BGG titers were found in the aqueous and vitreous humors of the left eyes of many of the rabbits. The reasons for this apparently non-specific stimulation of a secondary response in the absence of specific antigenic challenge are discussed.


Pathological fractures due to metastases.

The authors review the literature and report on the treatment of metastatic pathological fractures, on the basis of their own experience of 51 operations. They review the radiological and pathological aspects of the lesions, and the methods of investigation. They stress the need for close collaboration between oncologists, radiologists and orthopedic surgeons, and emphasize the role of surgical treatment in the restoration of function and the relief of pain, by stable synthesis of the affected part. The methods employed are those used in traumatology, together with arthroprosthetic replacement. The importance of ensuring a stable synthesis, for the patients' limited life expectancy, justifies the use of acrylic cement, which often establishes continuity that otherwise would not be attainable. The indications for operation are not related to prolonging life but only to improvement in the quality of remaining life as measured by absence of pain and recovery of function. The results should be assessed primarily from the patient's point of view, the aim being to restore his mode of living before the fracture occurred. This has psychological as well as functional value.


Non-union of the humerus after internal fixation-treatment by intramedullary nailing and grafting.

The results of fifteen cases of non-union of the humerus following treatment of the fracture by internal fixation are presented. In these cases there are skin, soft tissue and bone problems which complicate the treatment, which is therefore not free of complications. The authors advocate endomedullary nailing accompanied by bone grafting as the simplest and safest procedure.


An air sampling technique for hepatitis B surface antigen.

A filter-rinse technique capable of detecting low levels of airborne hepatitis B surface antigen (HBsAg) was devised and evaluated. Laboratory tests showed the procedure to have an efficiency of 22% with a coefficient of variation of 11% and a capability of detecting as little as 5 x 10(-5) ml of aerosolized antigen positive serum in a single air sample. The technique was field-tested in a hemodialysis center serving a patient population with a high prevalence of HBsAg seropositivity. The antigen was not detected in any of 60 air samples collected under conditions favoring the occurrence of aerosols.


Transferable drug resistance associated with coliforms isolated from hospital and domestic sewage.

The incidence of antibiotic-resistant fecal coliforms in raw and treated hospital and municipal wastes was investigated to determine whether such wastes may serve as reservoirs for the spread of resistant bacteria and resistance transfer factors. Multiple resistance occurred in 87.8% of isolates from hospital and 42.6% of isolates from municipal wastes. Antibiotic resistance was transferable to Escherichia coli and Salmonella cholerae-suis recipient strains from 62.3% of resistant isolates from hospital and 90.9% of resistant isolates from municipal wastes, and from 56.2% of all isolates from hospital and 45.9% of all isolates from municipal wastes. Numbers of multiply-resistant fecal coliforms decreased during passage through a sewage treatment plant, but their proportion did not change appreciably, although proportions exhibiting resistance to 3, 4, 5, 6, and 7 drugs decreased. A study of transfer in sewage indicated that transfer of resistance from donors present in sewage to pathogenic Salmonella strains can occur under appropriate conditions. The data suggest that both raw and treated wastes, and especially those from hospitals, may serve as reservoirs for the spread of antibiotic-resistant bacteria and transferable resistance in the environment.


A patch-graft technique for correction of pulmonic stenosis in dogs.

Pulmonic stenosis in 6 dogs were corrected surgically by means of a patch-graft technique, without using cardiac bypass or venous occlusion. Five of the 6 dogs have remained clinically normal for 4 to 40 months following surgery. The stenosis in 1 dog was inadequately repaired. The technique resulted in minimal blood loss, no mortality, and was easily adapted to the minimal size of several of the affected dogs. When performed correctly, the technique relieved the stenosis wherever it occurred (viz, supravalvular, valvular, subvalvular, infundibular, or any combination). The success of the procedure was determined, in part, by pre- and postoperative pressure measurements at the time of surgery, and by periodic physical, radiographic, and electrocardiographic examinations after surgery.


Tuberculosis in greater kudu.

Four greater kudu (Tragelaphus strepsiceros) died while maintained in captivity at a zoo. Necropsy revealed tuberculous lesions in the lungs, spleen, and thoracic lymph nodes. Histopathologic findings included granulomas with Langhans' giant cells, necrosis, and mineralization. Acid-fast organisms isolated from tissues of each kudu were identified as Mycobacterium bovis.


Salmonella and Aspergillus infections in common loons overwintering in Florida.

During a 5-year period (1970-1975), 190 common loons (Gavia immer) from overwintering populations on the east and west coasts of Florida were examined for evidence of infectious diseases. Salmonella spp (representing 8 serotypes) were isolated from 27 (14%) of the loons, and lesions typical of those produced by Aspergillus fumigatus were found in 34 (18%) of the loons. Seven loons were infected with Salmonella spp and had lesions typical of aspergillus infection. The largest number of loons (124) was obtained during the winter of 1973-1974, in connection with an offshore oil spill. There was no significant difference between the isolation rates of Salmonella spp from oiled vs nonoiled loons, but the occurrence of aspergillosis was higher in nonoiled than in oiled loons.


Genealogy of the words "audiology" and "audiologist".

A chronology of the use of the words Audiology and Audiologist is traced through a number of American professional and trade journals. The coining of the word Audiology has been credited variously to Mayer B. A. Schier, Willard B. Hargrave, Stanley Nowak, Norton Canfield, and Raymond Carhart. Attempts to document published use of the word before 1946 were unsuccessful.


Technical note: an apparatus for exploring dynamic auditory localization.

The purpose was to develop and test an apparatus for objective specification of localization of a moving acoustic target. The system involves computer analysis and graphic display of vectors created from rotational and linear potentiometers that are activated by listeners using a hand-held pointer as they track an acoustic event.


Pulmonary nocardiosis in an orangutan.

Pulmonary nocardiosis was diagnosed in an adult male orangutan (Pongo pygmaeus) being maintained in an outdoor facility. Periodically, the orangutan had had clinical signs of an upper respiratory tract infection, as evidenced by slight nasal discharge and cough. At the terminal stage, the orangutan had acute respiratory embarrassment, with epistaxis, and died before diagnosis could be made and treatment initiated. Gross necropsy findings included fibrosis of the pulmonary parenchyma as well as evidence of air sac infection, extensive pneumonia, and extensive chronic pleuritis. Microscopically, some of the lung sections had alveoli filled with polymorphonuclear cells, with other sections being diffusely fibrotic; the areas of fibrosis contained interspersed foci of polymorphonuclear cell infiltration. Many of the latter areas contained organism colonized in the form of granules that resembled those seen in actinomycosis. These organisms were gram-positive and branched, and measured close to 1 mum in diameter. Nocardia asteroides was isolated from the pulmonary tissue.


Treatment of monocrotophos-poisoned birds of prey with pralidoxime iodide.

Monocrotophos, and organophosphorus insecticide, was sprayed by farmers to control a plague of levant voles in alfalfa fields. Large numbers of birds of prey attracted by the dying and dead voles developed secondary poisoning after eating the voles. Paralysis of skeletal muscles was the principal clinical finding in these birds. Many of the birds died but some were treated with pralidoxime iodide, 100 mg/kg of body weight, given by intramuscular injection. All treated birds recovered, some in a very short time.


Binding of plant lectins to mycoplasma cells and membranes.

The binding of iodinated wheat germ agglutinin, Ricinus communis agglutinin, and concanavalin A to mycoplasma cells and membranes was examined. All mycoplasmas studied specifically bound concanavalin A  or R. communis agglutinin and, to a lesser degree, wheat germ agglutinin. The binding of lectins to whole cells was similar to that recorded for membranes, suggesting that significant binding only occurred on the outer surface of the mycoplasma membrane. Proteolysis of the membrane almost always increased the capacity to bind lectins, which indicates that additional carbohydrate groups on the mycoplasma membrane are masked by a protein layer or protein complexes on the membrane. The observation that carbohydrates are apparently exposed on the surface of mycoplasma membranes should stimulate more concentrated study on the isolation and chemical characterization of these substances since it is quite likely that they are responsible for a variety of reactions between mycoplasmas and host cells.


Regulation of ribonucleic acid synthesis in spheroplasts, cold-shocked cells, and toluene-treated cells of Escherichia coli.

The effects on the stringent control of ribosomal ribonculeic acid synthesis of the removal of cell wall, cold-shock treatment of cells, LiCl treatment of toluene-treated cells, and hypotonic treatment of spheroplasts were examined using Escherichia coli rel+ cells. Neither the removal of cell wall with penicillin or lysozyme nor the cold-shock treatment of the cells had an effect on the stringent control. The control mechanism, however, disappeared after the LiCl treatment of the toluene-treated cells, with the release of some protein component(s), possibly from the cytoplasmic membrane. The hypotonic and other treatments of spheroplasts, which disrupt the cytoplasmic membrane, also led to the abolishment of the control mechanism. These results suggested that the operation of the stringent control of ribosomal ribonucleic acid synthesis requires the cytoplasmic membrane, in which some proteins labile with LiCl treatment are embedded.


Independence of deoxyribonucleic acid replication and initiation from membrane fluidity and the supply of unsaturated fatty acids in Escherichia coli.

Mutant derivatives of the unsaturated fatty acid auxotroph K1062 were employed to investigate whether the supposedly membrane-bound bacterial replication machinery requires for its replicatory functions a fluid membrane environment as is known for several membrane-associated protein functions. Temperatures Tt for fluid reversible nonfluid phase transitions of membrane phospholipids are raised from below 18 to 38 degrees C when mutant cells are supplemented with elaidate instead of with oleate. In this experimental system current or synchroneously initiated new rounds of DNA replication are shown in vivo to continue 8 degrees below Tt, provided appropriate corrections for the concurrent cellular metabolic breakdown are considered. Temperature rate profiles for in vitro deoxyribonucleic acid replication rates measured in lysates of either oleate- or elaidate-supplemented cells yield congruent Arrhenius plots without discontinuities at corresponding Tt positions. We conclude that neither the start nor the propagation of replication forks depends on a fluid membrane. The capacity for the assembly of new replication complexes was studied in replication-aligned cells either shifted from oleate to elaidate (at temperatures below Tt for newly synthesized phospholipids) or starved for oleate. Regardless of whether unsaturated fatty acids are exchanged or completely withheld, new replication complexes can be normally assembled and initiated. These results do not support the conclusions reached by Fralick and Lark (1973) that the availability of unsaturated fatty acids is a prerequisite for the assembly of a functional replication complex.


Prediction of the rate of glucose utilization from cellular levels of glucose 6-phosphate and fructose 1,6-diphosphate in Escherichia coli.

A comprehensive equation, upsilon = VM/[1 + (A0.5/Fru-P2)n] [ 1 + (Glc-6-P/I0.5)], has been proposed to represent the quantitative interrelationships between the rate of glucose utilization and the levels of glucose-6-phosphate and fructose-1,6-diphosphate in the intact Escherichia coli cell. This comprehensive equation was derived from empirical equations that describe the relationship between the rate of glucose utilization and one of these hexose phosphates in metabolic situations where the other hexoses phosphate was not altered. In the experiments described in this report, treatment of nitrogen (NH4+)-starved cultures of E. coli W4597 (K) with various concentrations of sodium azide altered the levels of both hexose phosphates as well as the rate of glucose utilization. In each case the observed rate and the rate predicted by the comprehensive equation agreed closely, substantiating the validity of this comprehensive relationship as a quantitative indicator of metabolic events in the intact cell. The mechanism of metabolic regulation that is represented by this equation is discussed in light of the cellular levels of adenosine 5'-triphosphate and phosphoenolpyruvate observed in these experiments.


Isolation of pseudorevertants of lac oc mutants: selection system for superoperator mutations.

A selection procedure and rapid screening test are described that allow the isolation of rarely occurring pseudorevertants of lac oc mutants. These techniques were developed with the aim of isolating superoperator (os) mutants in which a secondary mutation in the operator would increase the affinity of the repressor of superoperator, thereby overcoming the effect of the oc mutation. The occurrence of superoperator mutants is predicted on the basis of a probable twofold symmetry in the lac repressor-operator recognition. Over 2,300 oc pseudorevertants were isolated and screened. In vitro measurements of the affinity of lac repressor for the operator region indicate that, among these, one oc pseudorevertant probably results from an oc mutation. The selection procedure also yielded other types of regulatory mutants that behave as promoter mutants and as i gene mutants in which the repressor is capable of overcoming an oc mutation.


Recognition properties of the beta subunit of Escherichia coli ribonucleic acid polymerase.

Changes in the phage protein patterns obtained by gel electrophoresis of extracts from phage S13 and phiX174 infection of rifampin-resistant hosts suggest that the beta subunit of ribonucleic acid polymerase of Escherichia coli has a function in the recognition of promoter or terminator sites or both. The altered protein patterns also provide information on the location of some ribonucleic acid polymerase recognition signals in S13 deoxyribonucleic acid. There is a promoter site before gene A, which lies either in gene H or between H and A. There is evidence for a promotor between genes C and D or in gene C. There is either a terminator or a promoter somewhere between the end of gene D and the beginning of gene F.


Ultraviolet light protection, enhancement of ultraviolet light mutagenesis, and mutator effect of plasmid R46 in Salmonella typhimurium.

Plasmid R46 partially protected Salmonella typhimurium, wild type or uvrB or polA, against the lethal effect of ultraviolet (UV) irradiation, but did not protect recA mutants. The plasmid also increased frequency of UV-induced reversion to His+ in all tested his point mutants (wild type for UV sensitivity), including amber, ochre, UGA, missense, and frame-shift mutants. Plasmid R46 also increased UV-induced reversion to His+ in uvrB and polA strains, but no UV mutagenic effect was detected in R- or R46-carrying recA derivatives of a his (amber) mutant. The spontaneous reversion frequency of his nonsense mutants of all classes, and of some his missense mutants, was increased about 10-fold when the strains carried R46, but the plasmid had no effect on the spontaneous reversion frequency of some other his missense mutations or of reversion rate of his frame-shift mutants (except for two uvrB derivatives of one single-base insertion mutant). The plasmid increased the ability of wild-type, polA, and uvrB hosts to support plaque production by UV-irradiated phage, and made strain LT2 hisG46 less sensitive to methyl methane sulfonate and to X rays and more responsive to the mutagenic effect of visible-light irradiation. R46 increased spontaneous reversion frequency of a his (amber) rec+ strain, but had no such effect in its recA sublines. Since the plasmid in the absence of host recA function fails to produce its mutator effect, or to confer UV protection or to enhance UV mutagenesis, these three effects may be produced via some mechanism involved in recA-dependent deoxyribonucleic acid repair, perhaps by an increase in activity of the "error prone" component of the inducible repair pathway.


Characterization of the hybridization between purified 16S and 23S ribosomal ribonucleic acid and ribosomal deoxyribonucleic acid from Escherichia coli.

An assay to distinguish specifically between 16S and 23S ribosomal ribonucleic acid (rRNA) has been developed. The assay involves hybridization of radioactive rRNA to deoxyribonucleic acid (DNA) from lambdailv5 transducing phage, which carries an rRNA transcription unit. Radioactive 16S or 23S rRNA can be specifically and completely competed from hybrids by using highly purified nonradioactive 16S or 23S competitor RNA, respectively. The preparation and purification of 16S rRNA and 23S rRNA are described in detail. The hybridization assay is extremely sensitive and efficient; 65 to 70% or more of the input radioactivity hybridizes to specific DNA in the absence of homologous competitor RNA, and at saturation virtually all of the specific DNA sequences are hybridized to rRNA. The results indicate that: (i) the 16S rRNA and 23S rRNA prepared as described are greater than 99% pure, (ii) 16S RRNA and 23S rRNA hybridize with equal efficiency and in equal molar amounts of lambdailv5 DNA; (iii) at saturation, about one molecule of 16S and one molecule of 23S rRNA are hybridized per genome equivalent of lambda ilv 5 DNA; (iv) essentially no cross-hybridization occurs between 16S and 23S rRNA; and (v) the sequence homology between 16S and 23S rRNA is negligible.


Isolation of specialized transducing bacteriophages carrying deletions of the regulatory region of the Escherichia coli K-12 tryptophan operon.

A lysogen of a wild-type strain of Escherichia coli K-12 carrying a heat-inducible lambda-phi80 hybrid prophage was induced to yield transducing phages carrying all of the structural genes of the tryptophan operon. The presence or absence of elements of the trp regulatory region was determined by examining the effects of lambda genes N and cI on trp gene expression. The phages were further characterized by transduction studies and by examining anthranilate synthetase (EC 4.1.3.27) (TRYPE+D) synthesis in the presence of the lambda cI product. A number of phages deleted for the trp promoter were found. Recombination studies between trpOc bacteria and the transducing phages have yielded information that can be used to order the trp end points of some phages and to provide an estimate of the size of the trp promoter region.


Regulation of purine utilization in bacteria. VII. Involvement of membrane-associated nucleoside phosphorylase in the uptake and the base-mediated loss of the ribose moiety of nucleosides by Salmonella typhimurium membrane vesicles.

Although uridine and adenosine are converted by membrane-associated nucleoside phosphorylases to ribose-1-phosphate (ribose-1-P) and the corresponding bases (uracil and adenine), only ribose -1-P is accumulated within Salmonella typhimurium LT2 membrane vesicles. In accordance with these observations, no uptake is observed when the vesicles are incubated with the bases or nucleosides labeled in their base moieties. The vesicles lack a transport system for ribos-1-P, since excess ribose-1-P does not inhibit the uptake of the ribose moiety of uridine. In addition, there is no exchange with preaccumulatedribose-1-P. Thus, uridine, rather than ribose-1-P, must serve as the initially transported substrate. The uptake of the ribose portion of uridine is coupled to electron transport, and the levels to which ribose-1-P are accumulated may be reduced by adding various bases to the reaction mixtures. The bases appear to inhibit the uridine phosphorylase reaction and/or cause an efflux of ribose-1-P from the vesicles. This loss of ribose-1-P reflects the accumulation of nucleosides in the external medium after being synthesized within the membranes. Synthesis of the nucleosides from intravesicular ribose-1-P and exogenous base proceeds even though the bases are not accumulated by the vesicles. Furthermore, ribose-1-P cannot significantly inhibit uridine phosphorylase activity unless the membranes are disrupted. These observations indicate that the membrane-associated nucleoside phosphorylases may have a transmembranal orientation with their base and ribose-1-P binding sites on opposite sides of the membranes. Such an asymmetric arrangement of these enzymes may facilitate the uptake of the ribosyl moiety of nucleosides by a group translocation mechanism. Thus, nucleosides may be cleaved during the membrane transport process, with the resultant bases delivered to the external environment while ribose-1-P is shunted to the intravesicular space.


Growth response of Escherichia coli to nutritional shift-up: immediate division stimulation in slow-growing cells.

When Escherichia coli 15T- cells growing exponentially at 70- to 80-min doubling times are subjected to a nutritional shift-up via glucose addition, cell division continues at the preshift rate for about 70 min (rate maintenance). The same cells growing at doubling times of 120 min or longer, however, begin to divide at a new faster rate immediately upon glucose addition. In both the rate maintenance and immediate division situations, cell mass, as measured by optical density (OD), begins to increase immediately upon shift-up. Consequently, the OD/cell pattern differs in the two growth-rate transitions. During rate maintenance, the OD/cell ratio increases dramatically for 60 to 70 min, and then slows appreciably and approaches the OD/cell characteristic of the new medium. During immediate division situations, the OD/cell increases only slightly for the first 180 +/- min; then the rate of increase accelerates but does not stop at the OD/cell characteristic of the new medium. Immediate division upon nutritional shift-up apparently depends upon initial doubling times in excess of 115 to 120 min and provision of a readily metabolized carbon source supporting doubling times of about 40 min. Similar immediate division occurs in E. coli B/r and K-12.


Escherichia coli ornithine carbamolytransferase isoenzymes: evolutionary significance and the isolation of lambdaargF and lambdaargI transducing bacteriophages.

Among the Enterobacteriaceae, Escherichia coli K-12 is the only strain known to have two structural genes (argF and argI) for ornithine carbamoyltransferase. The two gene products interact to form a family of four functional isoenzymes, respectively designated FFF, FFI, FII, and III. The FFF and III isoenzymes exhibit nearly identical kinetic parameters in the conditions applied. FFF is more thermolabile than III; this allows the straightforward characterization of new transducing phages carrying either argF or argI. The bearing of the available information regarding ornithine carbamoyltransferase isoenzymes on the evolution of the ancestral E. coli chromosome is reconsidered.


Mutational alteration of a nitrogen-fixing bacterium to sensitivity to infection by bacteriophage Mu: isolation of nif mutations of Klebsiella pneumoniae M5al induced by Mu.

The nitrogen-fixing bacterium Klebsiella pneumoniae M5al is not sensitive to infection by bacteriophage Mu. A mutant of K. pneumoniae that is sensitive to Mu infection was isolated. Several Mu-induced auxotrophic mutations of K. pneumoniae including nif, trp, and rtl were isolated and genetically characterized. Evidence is presented that the Mu-induced mutations of nif arise as the result of insertion of Mu within (or near) the nif operon(s). The rtl locus, which determines the ability to utilize ribitol as a carbon source, was found to be linked to nif loci.


Effect of 1,10-phenanthroline on bacterial conjugation in Escherichia coli K-12: inhibition of maturation from preliminary mates into effective mates.

The addition of 1 mM orthophenanthroline (OP) into a mating mixture drastically reduced the production of recombinants. Examination of the effect of OP on each step of conjugation showed that the effect on the following steps could not account for the up to 500-fold reduction of recombinant formation: (i) preliminary mate formation and (ii) deoxyribonucleic acid transfer and integration. Taking these results and additional experiments together, we conclude that OP inhibits the maturation of preliminary mates into effective mates. Kinetic experiments showed that there were two phases in the maturation of preliminary (OP-sensitive) mates into effective (OP-resistant) mates. The half-time (the time required to reach 50% OP-resistant mates) was 2.5 min for the first phase and 4 min for the second phase, with an overall half-time of 7.5 min. In contrast, only 3 min was required to reach 50% Zn2+-resistant mates. The difference in half-time suggests that there is an intermediate step involved to form an effective mate from a preliminary mate.


Rapid micromethod for the purification of Escherichia coli ribonucleic acid polymerase and the preparation of bacterial extracts active in ribonucleic acid synthesis.

A rapid micromethod is described for the preparation of nucleic acid-free extracts from Escherichia coli that involves precipitation with polyethylene glycol. Extracts can be prepared from growing cells in 75 min by three short, low-speed centrifugations. The extract did not inhibit added purified ribonucleic acid (RNA) polymerase, suggesting that major inhibitors of RNA synthesis had been removed. This extract should be ideal for assessing the properties of mutant RNA polymerases. The rapid chromatography of the extracts with step elution from deoxyribonucleic acid- and diethylaminoethyl-cellulose columns resulted in high yields of substantially pure RNA polymerase. We used this technique to purify 35S-labeled RNA polymerase. This system should find application for the purification of small quantities of other bacterial RNA polymerases that share the general chromatographic properties of E. coli RNA polymerase.


Structural and regulatory mutations allowing utilization of citrulline or carbamoylaspartate as a source of carbamoylphosphate in Escherichia coli K-12.

Escherichia coli mutants lacking carbamoylphosphate synthase require arginine and uracil for growth. It is, however, possible to obtain mutants in which carbamoylphosphate is obtained by phosphorolysis of citrulline or carbamyolaspartate. Citrulline utilizers are argG bradytrophs or strains in which the synthesis of ornithine carbamoyltransferase (either of the F or I type) is specifically depressed by unstable chromosomal rearrangements or stable mutations that presumably affect the operators of those genes. Carbamoylaspartate utilization as a source of carbamoylphosphate appears to require more than one mutation; the best-understood strains are pyrD pyrH or pyrC pyrH mutants in which aspartate carbamoyltransferase activity is high and the pool of cytidine triphosphate (feedback inhibitor of aspartate carbamoyl-transferase) is presumably low and in which channeling of carbamoylaspartate towards pyrimidine biosynthesis is considerably reduced. Selection of enzyme overproducers based on a metabolic dependency for a reversed enzymatic reaction can be regarded as a means for isolating regulatory mutants.


Cysteine and methionine content of the Escherichia coli ribonucleic acid polymerase subunits.

We describe a procedure that allows cysteine and methionine content to be determined on microgram amounts of partially purified protein. The only requirements are that the protein can be obtained as a pure band after electrophoresis on a polyacrylamide gel and that some data on amino acid content be available. This method involves double labeling by growing bacterial cells with [3H]leucine and [35S]SO4 and determining the ratio of these radioisotopes incorporated into the ribonucleic acid polymerase subunits. The relative specific activities of [3H]leucine and [35S]cysteine and methionine are determined from the ratio of these isotopes incorporated into beta-galactosidase, the leucine, cysteine, and methionine contents of which are known. We have used this procedure to determine the sulfur content of the subunits of Escherichia coli ribonucleic acid polymerase. These new data are necessary to quantitate the rates of synthesis of these subunits by in vivo labeling with [35S]SO4.


Characterization of SmSu plasmids by restriction endonuclease cleavage and compatibility testing.

Twelve plasmids carrying genes for streptomycin and sulfonamide resistance were studied for the number and distribution of sites on the plasmid moleucles susceptible to cleavage by the restriction endonuclease EcoRI. Ten of the twelve were found to have a single cut site, one plasmid (R678) had three such sites, and plasmid PB165, which was isolated as three supercoiled deoxyribonucleic acid species with molecular weights 7.4 x 10(6), 14.7 x 10(6), and 21.4 x 10(6) was reduced to a single (linear) species of molecular weight 7.6 x 10(6) after cutting with EcoRI. We conclude that PB165 forms oligomers in Escherichia coli and that the number of copies of these per chromosome is more consistant and that the number of copies of these per chromosome is more consistent with a negative than a positive control mechanism for plasmid replication. Compatibility testing of a positive control mechanism for plasmid replication. Compatibility testing of these plasmids showed they all belong to the same incompatibility group, which we designate IncQ, suggesting that they may have come from a common ancestor.


Septum formation-defective mutant of Escherichia coli.

Mutants of Escherichia coli defective in septum initiation, as well as in septum formation were obtained spontaneously, without mutagenic treatment, by selection of rifampin-tolerant mutants of an antibiotic-permeable strain carrying the envA mutation. The disturbed phenotype was in all mutants aggrevated the low incubation temperatures. One allele, sefA1, was studied in detail. Septum initiation, as well as septum formation, was promoted by high cell densities or by the addition of low concentrations of certain antibiotics, e.g., rifampin and chloramphenicol, to low-density cultures. The observed rifampicin depencence was studied in detail. These experiments indicated that a very modest shift-down situation suppressed the phenotype and enabled constrictions to proceed to cell separation. The rifampicin sensitivity of the partially purified deoxyribonucleic acid polymerase was not affected by the sefA1 allele, which is located close to proA and is thus distinct from envA. Growth parameters during the shift to 25 degrees C were followed in a transductant carrying HE SEFA1 allele. This constriction was characteristically blunt and did not lead to cell separation. At the time of formation of these frozen constrictions, clear zones representing a separation of wall from cytoplasmic membrane appeared. These polar tips did not inhibit expansion of the cell envelope. The phenotype of cells carrying the sefA1 allele suggests a disturbed relationship among protoplasm expansion, envelope growth, and septum formation. It is thought that the blunt constrictions observed are caused by an inability of the two septal peptidoglycan layers to fuse during an early stage of septation.


Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: independence from host rec functions and orientation of insertion.

Insertion of the transposable deoxyribonucleic acid sequence that specifies the TEM beta-lactamase (TnA) occurred in at least 19 sites on the 5.5 x 10(6)-dalton plasmid RSF1010. There was no significant difference in the frequency of transposition or in the distribution of TnA insertion sites for recombinant plasmids isolated from recombination-proficient (rec+) or recombination-deficient (rec-) bacterial host cells. The site and orientation of TnA insertions were determined by both heteroduplex analysis and enzymatic digestion with restriction endonucleases. Insertion in the gene encoding for sulfonamide resistance occurred without circular permutation in one or the other of two distinct orientations. Insertions in orientation P were strongly polar on distal gene expression, whereas insertions in orientation M were mutagenic but not polar. In addition, we have observed that TnA elements from different R plasmids show fine structural heterogeneity, and that TnA insertion at a site adjacent to the origin of replication causes an increase in plasmid copy number.


Light microscopy of basidia, basidiospores, and nuclei in spores and hyphae of Filobasidiella neoformans (Cryptococcus neoformans).

Three hypha-forming strains of Cryptococcus neoformans were induced to form basidia and basidiospores. Light microscopy showed that basidia formed at the ends of terminal hyphal cells and were able to produce from a few to many basidiospores. The morphology of the sexual structures indicated that these strains belonged to the recently described perfect state of C. neoformans, Filobasidiella neoformans. The average dimensions of the basidiospores were 1.9 mum in width by 2.7 mum in length. Giemsa staining revealed that dikaryotic cells were formed in all three strains. Only one strain had both terminal and subterminal dikaryons, indicating functional clamp connections, whereas the two remaining strains had dikaryons restricted to the terminal cells. Basidiospores of two strains were mononucleate, and yeast cell clones derived from single basidiospores of these two strains were able to complete the sexual life cycle, thus indicating their primary homothallic nature.


Molecular cloning of an Escherichia coli plasmid determinant than encodes for the production of heat-stable enterotoxin.

A conjugative plasmid, ESF0041 was isolated from an enterotoxigenic strain of Escherichia coli from calves. ESF0041 was found to be 65 x 10(6) daltons in mass of a member of the F incompatibility complex. Acquisition of ESF0041 by E. coli K-12 was invariably associated with the capacity to produce heat-stable (ST) enterotoxin. ESF0041 and pSC101 deoxyribonucleic acids were cleaved with EcoRI, and the fragments were ligated with polynucleotide ligase. Transformation of E. coli K-12 with the ligation mixture led to the isolation of an ST+ clone. Further analysis of the plasmid deoxyribonucleic acid from this clone showed that a structural gene(s) associated with ST biosynthesis had been isolated as a 5.7 x 10(6)-dalton ESF0041 fragment in pSC101. In turn, 5.7 x 10(6)-dalton fragment was ligated to a multicopy COLE1 derivative, RSF2124, so that toxin synthesis was amplified about threefold.


Relationship of Bdellovibrio elongation and fission to host cell size.

The extent of Bdellovibrio growth, and hence progeny produced in infected cells, appears to depend upon host cell size as determined from the ratio of ultimitate length of Bdellovibrio to host cell area calculated from light microscopy.


Convenient method for detecting 14CO2 in multiple samples: application to rapid screening for mutants.

A procedure is presented for the rapid screening of bacterial colonies to detect mutants unable to produce 14CO2 from a labeled precursor. The method is especially useful for mass screening for mutants that cannot be easily detected by their phenotypic characteristics.


Genetic location of the gene (ush) specifying periplasmic uridine 5'-diphosphate glucose hydrolase (5'-nucleotidase) in Escherichia coli K-12.

Further genetic mapping to two- and three-factor crosses show that the ush gene is closely linked to two other genes hemG and pisA, and that the probable gene order is proC-hemG-plsA-ush-gal.


Regulation of arginine biosynthesis in Saccharomyces cerevisiae: isolation of a cis-dominant, constitutive mutant for ornithine carbamoyltransferase synthesis.

A cis-dominant mutation linked to argF, the structural gene specifying ornithine carbamoyltransferase, and affecting the control of the synthesis of this enzyme has been obtained. The level of ornithine carbamoyltransferase in this mutation is depressed and less repressible by addition of L-arginine than it is in the wild-type strain. Of 38 tetrads analyzed, resulting from a cross of a strain harboring this mutation with a strain carrying an argF- mutation, none was a tetratype or a nonparental ditype. This operator mutation helps to define a negative mode of control of the synthesis of the arginine biosynthetic enzymes, as had been suggested earlier upon the isolation of argRI- (arg80), argRII- (arg81), and argRIII- (arg82) specific regulatory mutations.


Iron-requiring mutant of Escherichia coli carrying a deletion in the aroG-nadA region of the chromosome.

A mutant of Escherichia coli K-12 carrying a deletion in the aroG-nadA region of the genome requires a high concentration of iron for growth. The strain is chromium sensitive, and in the presence of citrate lower concentrations of iron support cell growth. The deletion mutant lost a gene between aroG and nadA that is responsible for the uptake of iron.


Rate of synthesis of polyadenylate-containing ribonucleic acid during the yeast cell cycle.

The rate of synthesis of polyadenylate-containing ribonucleic acid is constant throughout the cell cycle of Saccharomyces cerevisiae.


Thiogalactoside transacetylase of the lactose operon as an enzyme for detoxification.

Thigalactoside transacetylase, the lacA gene product, confers selective advantage to cells of Escherichia coli K-12 growing on beta-galactosides in the presence of non-metabolizable analogues.


Enzyme secretion in Escherichia coli: synthesis of alkaline phosphatase and acid hexose phosphatase in the absence of phospholipid synthesis.

De novo synthesis of two periplasmic enzymes in Escherichia coli, alkaline phosphatase and acid hexose phosphatase, have been studied in the presence and absence of new phospholipid synthesis. Alkaline phosphatase synthesis was initiated by a temperature shift in a strain carrying a phoA amber mutation and a temperature-sensitive suppressor mutation; acid hexose phosphatase was studied after relief of catabolite repression. Glycerol auxotrophs (gpsA) were used to control phospholipid synthesis. Synthesis of both enzymes proceeded at a normal rate for 0.5 to 1.0 generation of growth, although it was then curtailed. It is concluded that secretion of these enzymes is not obligatorily coupled to new net phospholipid synthesis.


Isolation and characterization of mutations creating high-efficiency transcription initiation signals within the trp operon of Escherichia coli.

Two different mutational events generate promoter-active deoxyribonucleic acid sequences within the trp operon of Escherichia coli, probably through single base-pair changes. The mutations, obtained after ethyl methane sulfonate mutagenesis by selecting for elevated lac gene expression in a trp-lac fusion, are cis dominant and trans recessive with respect to their effects on the synthesis of downstream enzymes. One of the mutants (trpD11), obtained repeatedly under the selective conditions employed, prevents the formation of active phosphoribosyltransferase. The second mutation, trp3B, has no effects on any trp enzyme. By deletion mapping, trpD11 was localized near the operator-distal end of trpD, outside the segment of deoxyribonucleic acid that contains the low-efficiency internal promoter trpP2. Reversion to prototrophy of trpD11 was greatly stimulated by 2-aminopurine and ethyl methane sulfonate. Tests with suppressors indicated that trpD11 is a UAA (ochre) nonsense mutation. Under repression conditions, strains harboring either lesion in a normal trp operon synthesize the tryptophan biosynthetic enzymes in a noncoordinate fashion. The products of the operator-distal structural genes trpC, trpB, and trpA are formed at rates approximately 15-fold higher than those of wild type. The enzymes encoded by operator-proximal genes trpE and trpD are low or not detectable. Under derepression conditions, coordinate expression of the operon was observed.


TolF locus in Escherichia coli: chromosomal location and relationship to loci cmlB and tolD.

The tentative map position on the Escherichia coli chromosome of the tolF locus, determining tolerance to colicins A, E2, E3, K, and L, has been confirmed by three-point transduction. It lies between the aroA and pyrD loci at about 21 min on the linkage map of Bachmann et al. (1976). The cmlB locus, determining increased resistance to the antibiotics chloramphenicol and tetracycline, also lies in this region (Reeve, 1966). Phenotypic and genetic comparison of isogenic strains that carry a mutation in either the tolF or cmlB locus makes it likely that these loci are closely related or identical. The tolD locus determining tolerance to colicins E2 and E3 as well as increased resistance to antibiotics has been reported to be located close to the aroA locus as a result of conjugation experiments (Eriksson-Grennberg et al. 1965). However, tolD did not cotransduce with any of several loci in this region, indicating that the mutation is not located within the region of the genetic map corresponding to approximately 19 to 22.5 min.


Repression of alkaline phosphatase in Salmonella typhimurium carrying a phoA+ phoR- episome from Escherichia coli.

Salmonella typhimurium does not produce alkaline phosphatase (nor beta-galactosidase). Nevertheless, it has the function of the phoR+ regulatory gene but lacks the function of the lacI+ regulatory gene. Several periplasmic proteins are derepressed when cells of S. typhimurium are starved for inorganic phosphate. The role of phoR is discussed.


Protoplast formation in Escherichia coli.

A procedure for protoplast formation in Escherichia coli is described. Removal of the cell wall was confirmed by examination of cells in thin-section preparations.


Escherichia coli mutant lacking 4-thiouridine in its transfer ribonucleic acid.

A mutant of Escherichia coli has been isolated that lacks 4-thiouridine, a rare base in transfer ribonucleic acid. The mutant grows at the same rate as wild-type cells. It shows little near-ultraviolet-induced growth delay, thus supporting earlier hypotheses that 4-thiouridine in transfer ribonucleic acid is the chromophore for this growth delay.


New types of Escherichia coli recombination-deficient mutants.

A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency.


Adenosine utilization in cordycepin-sensitive mutants of Saccharomyces cerevisiae.

A purine-requiring, wild-type yeast strain was cordycepin resistant and failed to grow in medium containing adenosine; in contrast, a cordycepin-sensitive mutant (also purine requiring) grew well in medium containing adenosine. The cordycepin-sensitive mutant incorporated [8-14C]adenosine at nine times the wild-type rate, and adenosine completely fulfilled the purine requirement of the cells. Exogenous adenosine rapidly entered the mutant cells, apparently as free nucleoside, and was phosphorylated; uptake displayed concentration-dependent saturation kinetics (Km, 6 mM). Within 10 min 14C radioactivity was being incorporated into nucleic acids.


Aminoacyl transfer RNA formation. Binding of cations to transfer RNA and its role in aminoacyl transfer RNA formation.

The role of cations (polyamines and Mg2+) in isoleucyl-tRNA formation catalyzed by purified isolecuyl-tRNA synthetase [EC 6.1.1.5] from Escherichia coli was studied. It was found that spermine, spermidine, and Mg2+ bind to tRNA and that when bound to these cations, tRNA acts as substrate of aminoacylation without requiring further cations. These findings suggest that the primary function of cations in aminoacyl-tRNA formation is to bind to tRNA to stabilize its structure, not to bind to the enzyme to activate it.


D-Serine dehydratase from Escherichia coli. Essential arginine residue at the pyridoxal 5'-phosphate binding site.

D-Serine apodehydratase from Escherichia coli is rapidly inactivated by butanedione in K+ borate buffer or by phenylglyoxal in K+ phosphate buffer at pH 8, 25 degrees. Pyridoxal-P protects against the inactivation. Modification of the apoenzyme abolishes its ability to bind the cofactor, pyridoxal-P, but the apparent Km for the substrate, D-serine, is not altered. The concentration dependence of the rate of butanedione inactivation in K+ borate buffer indicates that it is a two-step process with one butanedione bound per molecule of apoenzyme to give an inactive complex; half-maximal rate of inactivation is obtained at 37 mM butanedione. Butanedione inactivation is fully reversed following removal of excess reagent and borate. Similar studies with [14C]phenylglyoxal show that in the presence of pyridoxal-P at least 2 arginine residues may be modified without loss of activity. In the absence of pyridoxal-P modification of a single additional arginine residue results in loss of activity. Results with both inactivating reagents thus demonstrate that a critical arginine residue participates in binding of the coenzyme, pyridoxal-P. The stoichiometry of phenylglyoxal incorporation into the enzyme is different in the presence and absence of borate. Under both conditions incorporated phenylglyoxal is slowly lost on dialysis at neutral pH. A possible explanation of these effects is discussed.


Further studies on the isolation and properties of polyriboadenylate polymerase from Escherichia coli PR7 (RNase I-, pnp).

Polyriboadenylate polymerase was isolated from Escherichia coli PR7 (RNase I-, pnp) in good yield and high purity. The enzyme catalyzes the polymerization of ATP and ADP. These polymerizations show an initial lag which can be removed by the addition of poly(A). However, poly(A) does not function as a primer. UDP and CDP can also serve as substrates but with decreased efficiency. The polymerization of CDP is enhanced by the presence of an oligonucleotide which again does not function as a primer. Polymerization of [gamma-32P]ATP or [beta-32P]ADP result in products with no radioactivity. The product formed from [alpha-32P]ATP on hydrolysis with alkali yields labeled pAp and 2',3'-AMP; thus the enzyme synthesizes poly(A) chains de novo. During the polymerization of ATP, no burst of free ADP can be detected and the time course of phosphate release from ATP ro ADP follows very closely the kinetics of polymerization. dATP and dADP are effective inhibitors of poly(A) synthesis from either ATP or ADP. Sulfhydryl reagents inhibit only the polymerization of ATP and the inhibition is fully reversed by dithiothreitol. However, the enzyme can be protected from sulfhydryl reagents by preincubation with either ATP or ADP in the absence of Mg2+ which is required for polymerization. Studies using acrylamide gel electrophoresis indicate that the polymerization activity with either ATP or nucleoside diphosphates resides in the same protein. The enzyme catalyzes the following exchanges: 32Pi into ADP, 32Pi into ATP, and [14C] ADP into ATP in the presence of phosphate. While the enzyme catalyzes the phosphorolysis of its own product, (pAp-(Ap)nA), it fails to cleave the dephosphorylated product, (Ap(Ap)nA), or ribosomal RNA or tRNA in the presence of inorganic phosphate. The differences and similarities between poly(A) polymerase and polynucleotide phosphorylase are discussed. Based on the 32P exchange studies and other properties of poly(A) polymerase, a plausible mechanism for its action is proposed.


Interactions of adenosine diphosphate-ribosylated elongation factor 2 with ribosomes.

The binding of adenosine diphosphate-ribosylated elongation factor 2 (ADPRib-EF-2) to ribosomes was inhibited both in the presence and absence of GTP in proportion to the amounts of unmodified EF-2 added. Concomitant with this inhibition, an increase in the activity of ribosome-bound EF-2 in polyphenylalanine synthesis was observed. On the other hand, the addition of ADPRib-EF-2 reduced the rate of poly(Phe) synthesis observed in the presence of a saturating amount of EF-2 and increased the amount of EF-2 required for the half-maximal rate of poly(Phe) synthesis. Phe-tRNA, nonenzymatically bound to the ribosome in the presence of poly(U), inhibited the subsequent binding of ADPHRib-EF-2. The same ribosomal population appeared to preferentially bind either aminoacyl-tRNA or ADPRib-EF-2. The Scatchard plot of the binding of ADPRib-EF-2 to the ribosome in the presence of GTP revealed the presence of two ribosomal binding sites (or ribosomal populations) with apparent different affinities for the modified factor (K371 degrees d,1 = 6.6 nM and K37 degrees d,2 = 126 nM). At saturating concentrations of ADPRib-EF-2, a maximum of about 1 molecule of the factor was bound per ribosome. The binding of ADPRib-EF-2 to the ribosome was stimulated by GTP. The binding of radioactive GTP to the ribosome was observed concomitantly with the binding of ADPRib-EF-2. One mole of GTP was bound per mole of ADPRib-EF-2. No significant difference could be found in the binding of GTP to ribosome required in the presence of either EF-2 or ADPRib-EF-2. The binding of ADPRib-EF-2 to the ribosome required the presence of Mg2+ and reached a maximum at 5 mM. The binding was greatest at K+ concentrations below 20 mM. ADPRib-EF-2 was bound primarily to the large ribosomal subunit. A slight, but reproducible binding to the 40 S subunit was also observed. The addition of 40 S to 60 S subunits stimulated the binding of ADPRib-EF-2. GTP displayed a stimulatory effect on the binding only in the presence of recombined subunits. Human ADPRib-EF-2 was bound to rat liver ribosomes as efficiently as to human tonsil ribosomes, while the binding to Escherichia coli ribosomes was insignificant.


Sugar transport. Properties of mutant bacteria defective in proteins of the phosphoenolpyruvate: sugar phosphotransferase system.

The bacterial phosphotransferase system (PTS) catalyzes the transfer of the phosphoryl group from phosphoenolpyruvate to its sugar substrates, PTS sugars, concomitant with the translocation of these sugars across the bacterial membrane. The phosphorylation of a given sugar requires four proteins, two general proteins, Enzyme I, and the histidine-containing phosphocarrier protein of the PTS (HPr), used for all sugars, and a pair of proteins specific for that sugar, designated an Enzyme II complex. The phosphotransferase system has been implicated in regulating the induction of synthesis of some catabolic enzyme systems required for the utilization of sugars that are not substrates of the phosphotransferase system, and this and the accompanying reports are concerned with this phenomenon in Salmonell typhimurium and Escherichia coli. Mutants defective in Enzyme I (ptsI), HPr (ptsH), and certain Enzymes II were isolated, and their abilities to ferment and grow on a wide range of sugars and other compounds were determined. The mutants showed the expected properties on PTS sugars, but in addition, ptsH and tight ptsI mutants were unable to utilize certain non-PTS sugars, including maltose, melibiose, glycerol, glycerol-P, mannose-6-P, and, in E. coli, lactose. Leaky Enzyme I mutants could utilize these carbohydrates, but were unable to use them in the presence of a PTS sugar such as methyl alpha-D-glucopyranoside. In accord with the results reported by other laboratories, the inability of the mutants to utilize the non-PTS sugars was explained by the fact that these cells could not be normally induced to synthesize the corresponding catabolic enzyme systmes. This phenomenon is designated PTS-mediated repression. PTS-mediated repression was also observed in wild type cells, but by comparing wild type and leaky pts mutants it was shown that the sensitivity to repression by PTS sugars was greatest in mutants containing the lowest levels of Enzyme I or HPr. Furthermore, ptsI mutants containing a second site mutation in a gene for an Enzyme II were not repressed by the sugar substrate of that Enzyme II, although repression by other PTS sugars was not affected. Transport and other studies further indicated that neither appreciable uptake nor metabolism of the PTS sugars was required for these compounds to effect repression. The ptsH mutants showed the same phenotypic properties as the ptsI mutants with some important exceptions. First, they could ferment and grow on a PTS sugar, fructose. Second, after growth on fructose, (and to a lesser extent on glucose or mannose), such mutants were capable of utilizing other PTS sugars for a few generations. Third, growth of the ptsH mutants on fructose relieved PTS-mediated repression; after growth on fructose, but not on lactate, the mutants could grow for several generations on non-PTS sugars. Preliminary experiments indicated that growth on fructose resulted in the formation of one or more proteins that could substitute for HPr in the utilization of both PTS and non-PTS sugars.


Sugar transport. The crr mutation: its effect on repression of enzyme synthesis.

The accompanying report describes phosphotransferase system-mediated repression in mutants of Salmonella typhimurium and Escherichia coli defective in Enzyme I and histidine-containing phosphate carrier protein (HPr), the general proteins of the phosphotransferase system (PTS). Such repression prevented the cells from synthesizing the catabolic systems required for utilization of the non-PTS compounds glycerol, maltose, melibiose, mannose 6-phosphate, and alpha-glycerol phosphate. This defect can be overcome by introducing a single mutation, designated crr, into the pts mutants. The pts crr double mutants can be induced to synthesize the non-PTS catabolic systems and can therefore grow on the non-PTS sugars. The crr gene is closely linked to but not part of the pts operon, and may be a regulatory gene for the operon. Assay of the PTS proteins in crr mutants showed that the only component detectably affected was a sugar-specific protein of the PTS, Factor IIIG1c, involved in the phsophorylation of glucose (and methyl alpha-glucoside). In some crr mutants Factor IIIG1c was not detected, whereas in others it was present at reduced levels. Thus the crr gene appears to code for or regulate the synthesis of this protein. In addition to the general crr mutants, several classes of sugar-specific crr mutants were isolated. For example, maltose-, melibiose-, and glycerol-specific crr mutants were isolated, each being inducible for the corresponding catabolic enzyme system but not for the others. Unlike the general crr gene, the sugar-specific crr genes do not map near the pts operon.


Sugar transport. 2nducer exclusion and regulation of the melibiose, maltose, glycerol, and lactose transport systems by the phosphoenolpyruvate:sugar phosphotransferase system.

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) can repress the synthesis of certain catabolic enzyme systems in Salmonelly typhimurium and Escherichia coli. The present studies are concerned with an explanation of PTS-mediated repression by studying induction of the catabolic systems required for utilization of the non-PTS sugars glycerol, maltose, melibiose, and lactose. Repression of synthesis of these enzyme systems by various concentrations of PTS sugars was studied in wild type cells, in pts mutants, and in pts crr double mutants described in the accompanying reports (Saier, M. H., Jr., Simoni, R. D., and Roseman, S (1976) J. Biol. Chem. 251, 6584-6597: Saier, M. H., Jr., and Roseman, S. (1976) J. Biol. Chem., 6598-6605). The extent of repression was found to correlate with the degree of inhibition of uptake of the inducing non-PTS sugars. In both uninduced cells and cells fully induced for the respective transport system listed above, PTS sugars inhibited these transport systems. With both induced and uninduced cell types pts mutants were much more sensitive than wild type cells, while pts crr double mutants were completely resistant to the effects of the PTS sugars. Kinetic studies with the fully induced cells indicated that inhibition of transport by PTS sugars was reversible, that it affected the Vmax rather than the Km of entry, and that PTS sugars did not inhibit the efflux of the non-PTS sugars. These and other results indicate that inhibition did not result from competition between the PTS and non-PTS sugars for the transport systems, nor to competition for metabolic energy. Inhibition appears to require interaction of the PTS sugar with its membrane-bound Enzyme II complex, but whether concomitant uptake and phosphorylation of trace quantities of the PTS sugars is also required remains to be determined.


Purification of the carbodiimide-reactive protein component of the ATP energy-transducing system of Escherichia coli.

The ATP-energy transducing system in membranes of Escherichia coli is inhibited by dicyclohexylcarbodiimide. The protein component of this complex with which carbodiimides covalently react to inhibit function was previously identified by labeling wild type and dicyclohexylcarbodiimide-resistant mutants with dicyclohexyl[14C]carbodiimide (Fillingame, R. H. (1975) J. Bacteriol. 124, 870-883). This specific carbodiimide-reactive protein has now been purified. The protein was extracted from the membrane with chloroform:methanol and chromatographed on DEAE-cellulose and hydroxypropyl Spehadex G-50 in this sulvent mixture. The resultant 700-fold purification yielded a protein that was homogeneous on dodecyl sulfate-acrylamide gel electrophoresis and virtually free of phospholipid. It remained soluble in neutral chloroform:methanol throughout the purification procedure. The amino acid composition of the purified protein was extraordinary in that only 16% of the amino acids present could be considered polar. Histidine, serine, cysteine, and tryptophan were not found. Abnormally high contents of methionine, glycine, alanine, and leucine were present. One mole of lysine and threonine were found/mole of dicyclohexyl[14C]carbodiimide bound. The minimum molecular weight based on the amino acid composition was 8400. The specific carbodiimide-reactive protein has also been purified without prior modification by dicyclohexylcarbodiimide. The unmodified protein eluted from DEAE-cellulose at a higher salt concentration than the dicyclohexylcarbodiimide-modified form, which suggested that the reaction with the carbodiimide neutralized the negative charge. Only one-third of the total carbodiimide-reactive protein in the membrane was modified by dicyclohexylcarbodiimide under conditions which maximally inhibited adenosine triphosphatase activity. These results rais the possibility that the carbodiimide-reactive protein may be present as an oligomer in the energy-transducing complex. The purification of the unmodified carbodiimide-reactive protein should permit assessment of tis biological function, particularly its role in the protein-translocation process that is catalyzed by this energy-transducing complex.


Polyamine stimulation and cation requirements of rabbit liver tRNA nucleotidyltransferase.

We have examined the cation requirements of rabbit liver tRNA nucleotidyltransferase. The enzyme had an absolute requirement fo a divalent cation which could be satisfied by Mg2+, Mn2+ or Co2+. In contrast to the Escherichia coli enzyme, we have found no evidence to implicate Zn2+ in the action of rabbit liver tRNA nucleotidyltransferase. We have also identified a second cation requirement which was satisfied by divalent or monovalent cation or polyamines. The polyamines, spermine and spermidine, were most effective leading to 3-fold higher rates of nucleotide incorporation than could be obtained at any concentration of the other cations. However, the polyamines were not an absolute requirement for enzyme activity. The polyamine stimulation of tRNA nucleotidyltransferase was not due to alteration of the pH or ionic strength of the reaction mixture or reactivation of denatured tRNA or enzyme. Polyamines also had no effect on the apparent Km values of the substrates. Spermine increased the specificity of the enzyme for tRNA substrates and also inhibited the reverse action. Our results suggest that polyamines may be the normal counterions for tRNA in vivo, and that they affect the rate-limiting step in tRNA nucleotidyltransferase catalysis.


Effect of lipids on the reconstitution of D-lactate oxidase in Escherichia coli membrane vesicles.

An unsaturated fatty acid auxotroph lacking D-lactate dehydrogenase activity has been isolated from Escherichia coli ML 308-225 dld-3. While NADH oxidase activity in membrane vesicles prepared from the mutant cells grown in a variety of unsaturated fatty acids is comparable to that of previously isolated fatty acid auxotrophs, D-lactate oxidase activity is absent. However, D-lactate oxidase ativity can be restored when vesicles are incubated with a purified preparation of D-lactate dehydrogenase obtained from wild type cells. The effect of altering the fatty acid composition of the membrane on the reconstitution of D-lactate oxidase activity was examined. Binding of purified D-lactate dehydrogenase was not affected by either the lipid composition of the membrane vesicles or the temperature during reconstitution. However, the reconstitution of D-lactate oxidase activity was strongly influenced by the fatty acid composition of the membrane lipids. The temperature dependence of the reconstituted activity was analyzed. Temperature transitions were not observed with membrane vesicles supplemented with oleic or linolenic acid but palmitelaidic acid-enriched vesicles exhibited a transition temperature at 30 degrees. Attempts to reconstitute the elaidic-supplemented vesicles at temperatures below 30 degrees failed to yield active D-lactate oxidase and the vesicles aggregated. At 42 degrees, aggregation did not occur and D-lactate oxidase activity was obtained to a level of 10% of that found with membranes from the parent strain (ML 308-225). These results suggest that although binding of D-lactate dehydrogenase is independent of the physical state of the membrane, reconstitution of the D-lactate oxidase activity in membrane vesicles is dependent upon the fatty acid composition of the phospholipid, hence the physical state of the membrane.


Metabolic fate of 3,4-dihydroxybutyl-1-phosphonate in Escherichia coli.

3,4-Dihydroxy[3-(3)H]butyl-1-phosphonate, and analogue of glycerol 3-phosphate, is incorporated into a very polar lipid material by cultures of Escherichia coli strain 8 and in vitro by CDP-diglyceride:sn-glycerol-3-phosphate phosphatidyltransferase. These labeled lipids have been fractionated by column chromatography on DEAE-cellulose, revealing that only one labeled compound is formed in vitro, while four are synthesized in vivo. The main component of the material formed by intact cells has been shown to be identical with that produced enzymatically. This species has been identified as the phosphonic acid analogue of phosphatidylglycerophosphate [(1,2-diacyl)-sn-glyceryl-D-4'-phosphoryloxy-3'-hydroxybutyl-1'-phosphonate]. Hydrolysis of this novel lipid with phospholipase C resulted in the production of diglyceride and a water-soluble derivative of 3,4-dihydroxybutyl-1-phosphonate and inorganic phosphate in a molar ratio of 1.03/1. Enzymatic analysis of the phosphonate liberated in this manner showed it to be the D enantiomer, thereby confirming the proposed structure of the lipid analogue. The analogue of phosphatidylglycerophosphate did not turn over and appeared to have no precursor-product relationship to the other labeled lipids derived from 3,4-dihydroxy[3-(3)H]butyl-1-phosphonate in vivo. Analysis of the other three labeled products revealed the tritium to be present on glycerol 3-phosphate and not intact phosphonate, indicating some metabolic degradation of the latter. Examination of cell components other than lipids revealed little incorporation of label, while a significant amount of tritium was found to be present in a distillable form, 3H2O. Experiments with mutants of E. coli lacking the known glycerol-3-phosphate dehydrogenases indicated that these enzymes are not responsible for the removal of tritium from from 3,4-dihydroxy[3-(3)H]butyl-1-phosphonate in vivo. Indirect evidence suggests that the inhibition of cell growth by this analogue is not due to its catabolic products.


Aminoacyl-tRNA conformation. Information from steroid and oligonucleotide probes.

The conformations of aminoacyl- and deacylated tRNA Phe (yeast) have been compared by using the steroid progesterone and the tetranucleotides U-C-C-C and C-G-A-A as probes of transfer RNA ordered structure. U-C-C-C is complementary to G18-G19-G20-A21 in the dihydrouridine loop and C-G-A-A is complementary to T54-psi55-C56-G57 in the ribosylthymine loop. None of the probes bound to deacylated tRNA Phe but all three bound to phenylalanyl-tRNA Phe, with molar association constants of the order of 10(4) M-1. The oligonucleotide binding data imply that the tertiary hydrogen bonds between G18 and psi55, G19 and C56, T54 and m1A58, and A21 and the ribose of U8 (Quigley, G. J., Wang, A. H. J., Seeman, N. C., Suddath, F. L., Rich, A., Sussman, J. L., and Kim, S. H., (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4866-4870) are destabilized or broken on aminoacylation, unmasking the sequence T-psi-C-G thought to be involved in ribosome binding of aminoacyl-tRNA. The presumed progesterone binding site is G18-G19-G20, which is part of the binding site for U-C-C-C. Competition was not, however, observed between these two probes; model building has shown that they could, theoretically, bind simultaneously. Since progesterone bound to N-acetyl-Phe-tRNA Phe, the introduction of the additional positive charge on aminoacylation is not sufficient per se to explain the conformational change. The association of progesterone with peptidyl-tRNA Phe was similar to that with aminoacyl-tRNA Phe, implying that no further conformational change takes place in the region of the steroid binding site on formation of a peptide bond.


Microcalorimetric study of the binding of thiodigalactoside to the lactose permease M protein of Escherichia coli.

The energetics of the binding of thiodigalatoside onto vesicles of Escherichia coli containing M protein is described. The Kd determined from equilibrium dialysis was 5-10(-5) M. The enthalpy change (deltaH) was measured by calorimetry. The derived deltaG and deltaH values allowed estimation of the entropic change associated with the binding reaction. The control experiments were made with membranes from cells that were not induced for the lac system. All the experiments were carried out in presence of 10(-2) M sodium azide to prevent any concentration of thiodigalactoside into the vesicles. It was concluded that such membrane vesicles which are in a de-energized state are able to bine thiodigalactoside specifically with a Kd corresponding to the Km of the entry of beta-galactoside measured with intact, active cells.


Isotope labeling of free and aminoacyl transfer RNA synthetase-bound transfer RNA.

The structural organization of the complex of Escherichia coli Ile-tRNA synthetase and tRNA Ile has been studied by isotope labeling of purine units in the tRNA. Free or bound tRNA is incubated in tritiated water for 5 to 15 h at 37 degrees in order to incorporate tritium into the C-8 positions of purine units. (Previous work has shown that the labeling rate of a purine is very sensitive to its microenvironment.) Under conditions where exchange-out does not occur, the nucleic acid is digested with nucleases and purines are subsequently isolated from known locations in the structure. Four purines are substantially perturbed by bound Ile-tRNA synthetase; in each case, the rate of labeling is retarded in the presence of the synthetase. The four purines occur at or near the 3' terminus and at the interface of the dihydrouridine stem and loop. These bases occur in segments of the tRNA that previous photochemical cross-linking studies have identified as important for synthetase-tRNA interactions. It appears that the effects observed on these sites are caused by their direct interaction with or shielding by the bound synthetase. In addition, two other sites, one in the anticodon and one in the amino acid acceptor-T psi C helix, appear to be perturbed (retarded labeling rates) by the bound enzyme. The data also suggest there is no significant conformational change in the tRNA upon binding to the synthetase.


The bone-metal interface following hip nailing.

In order to establish a base line for tissue reaction in relationship to loaded implanted foreign materials that might be used for comparison with new materials, the interface between the trabecular bone in the femoral head and hip nails has been studied in 20 specimens. The interface consists of four distinguishable layers: a cellular layer at the surface, a layer of fibrovascular connective tissue, a layer of collagenous connective tissue and/or fibrocartilage, and a peri-implant bone plate. Even 10 years after insertion of the nail this interface remained cellularly active, the activity presumably being related to the different modulus of elasticity between the metal and the surrounding trabecular bone.


Prevention of venous thromboembolism by administration of hydroxychloroquine. A preliminary report.

A placebo or the antimalarial agent hydroxychloroquine (Plaquenil), which inhibits platelet adhesiveness, and, to a lesser extent, platelet aggregation, was given to 100 patients between forty and ninety-five years old (average age, seventy-four years) who had either fractures or orthopaedic operations involving the skeleton between the knee and the pelvis. Medication was started at the time of admission in a blind, randomized way, fifty patients receiving hydroxychloroquine (200 milligrams three times a day) and fifty, a placebo. No untoward bleeding complications were noted in the treated group. Eight instances of thromboembolism were encountered in the control group as compared with one in the hydroxychloroquine-treated group, a statistically significant difference at the 1.5 per cent level.


Historical notes on computerized axial tomography.

The original experiments on computerized tomography at the Central Research Laboratories of EMI are discussed. Examples trace the history of improvement in definition and process time, leading to results from the first EMI Scanner which was installed in the Atkinson Morley's Hospital in September 1971.


Cell surface saccharides of Trypanosoma lewis i. II. Lectin-mediated agglutination and fine-structure cytochemical detection of lectin-binding sites.

Bloodstream (BSF) and culture forms (CF) of Trypanosoma lewisi were specifically agglutinated with the plant lectins concanavalin A (Con A), soybean agglutinin (SBA), wheat germ agglutinin (WGA), and fucose-binding protein (FBP). Lectin-mediated cell agglutination was inhibited, and reversed in the presence of specific lectin-binding saccharides. Cells were agglutinated randomly with all lectins suggesting a uniform distribution in the trypanosome cell surface of the lectin-binding saccharide ligands. The BSF and CF were not agglutinated with phytohaemagglutinin-M, phytohaemagglutinin-P, or influenza virions. Living trypsinized BSF, which lacked a surface coat, gave agglutination results with the lectins identical to those obtained with living intact BSF. Glutaraldehyde- or formalin-fixed intact and trypsinized BSF gave results similar to those obtained with living cells and SBA, WGA, and FBP. However, intact, fixed BSF gave much lower agglutination levels with Con A than trypsinized-fixed, living intact, or living trypsinized BSF cells. Intact and trypsinized living and fixed CF gave identical agglutination results with each of the lectins. Living and fixed cells treated extensively with the glycoside hydrolases alpha-amylase, dextranase, and neuraminidase gave results with the lectins identical to those obtained with untreated cells. Con A bound at the cell surface was visualized with an iron-dextran (Fe-Dex) conjugate. Dense iron marker particles were distributed randomly in the intact BSF surface coat. The Con A-bound Fe-Dex marker was present on the pellicular and flagellar membrane outer lamina of trypsinized BSF and intact CF cells. Horseradish peroxidase (HRPO)-diaminobenzidine (DAB) coupled reactions also were used to visualize surface-bound Con A. Dense Con A-HRPO-DAB deposits were present uniformly in the BSF surface coat, and on the membranes of trypsinized BSF and intact CF trypanosomes. SBA and WGA were conjugated to HRPO and these used in DAB-coupled reactions at the ultrastructure level. Results obtained with the HRPO-conjugated lectins were similar in surface localization and distribution to those obtained with the Con A-HRPO-DAB preparations. Treatment of BSF and CF with the several glycoside hydrolases produced no apparent enhanced or reduced reactivity for the lectins in any of the fine-structure cytochemistry experiments. The cumulative results indicate that ligands similar or identical to alpha-D-mannose, N-acetylgalactosamine, and N-acetylglucosamine, and alpha-L-fucose are constituents in the extracellular surface coat matrix of T. lewisi BSF. Similar conclusions also pertain to the pellicular and flagellar membrane ligands of the BSF and CF cells. Moreover, results obtained with the glycoside hydrolases and influenza virions suggest that the T. lewisi cell surface ligands are not associated directly with repetitively bonded alpha-I,4- and alpha-I,6-D-glucans or sialic acid moieties.


Spindle birefringence of isolated mitotic apparatus: further evidence for two birefringent spindle components.

We studied sea-urchin zygote mitotic apparatus (MA) isolated in hexylene glycol, transferred immediately to a glycerol-dimethylsulphoxide medium, and stored for 2 weeks at room temperature. Treatment with 0-5 M KC1 caused loss of 45% of the birefringence, but microtubules remained intact (as seen electron microscopically in glutaraldehyde-fixed MA), and tubulin was not extracted (as determined by polyacrylamide gel electrophoresis). These results suggest that a non-tubulin component which is extracted by the KC1 contributes 45% of the MA birefringence. Further evidence for this conclusion came from indirect immunofluorescence experiments. Non-extracted (control) MA were fixed with formaldehyde and reacted with antibody against tubulin; there was intense staining of the spindle fibres and astral rays. Electron microscopically, however, microtubules were not present in formaldehyde-fixed MA. Since formaldehyde fixation caused breakdown of microtubules but the tubulin remained in the MA (as judged by reaction with antibodies) we suggest that after microtubule breakdown the tubulin remains in the MA because it is bound to a peri-microtubule spindle component (which we call 'substance gamma'). When KCl-extracted MA were fixed with formaldehyde and reacted with antibody against tubulin there was very little staining of spindle fibres and astral rays. Electron microscopically, formaldehyde caused microtubule breakdown, and since the tubulin is lost from formaldehydefixed, KC1-extracted MA (as judged by reaction with antibodies), we suggest that the tubulin-binding component, substance gamma, is extracted by the 0-5 M KC1. Pressure treatment caused the asters not to stain with antibody against tubulin, suggesting that the stability of substance gamma is different in different regions of the mitotic apparatus.


Studies on the mechanism of circus movement in dissociated embryonic cells of a teleost, Oryzias latipes: fine-structural observations.

The fine structure of lobopodia in dissociated embryonic cells of the freshwater fish, Oryzias latipes, was observed with the electron microscope in order to understand the mechanism of the circus movements which they display. Dense material (grandular or fibrillar) is present in the zone between the lobopodium and the endoplasm, as well as in the cortical layer around the cell circumference. The direction of lobopodial movement is related to the distribution of this dense material. The band between the lobopodium and the endoplasm is conspicuous and is connected to the cortical dense layer around the cell periphery at the advancing front of the lobopodium, while the dense material is usually almost absent beneath the cell membrane in the anterior region of the lobopodium. The band between lobopodium and endoplasm is blurred or disrupted near the hind end of the lobopodiu, where the peripheral dense layer is well developed. In situ localization of actin/heavy meromyosin complexes in the cell showed that the dense material has actin-like properties. Cytochalasin B(0-5 mug/ml) induced constriction of the neck of the bleb, shrinkage of the endoplasm, and herniation of the endoplasmic contents to the enlarged hemispherical bleb, and thus arrested the circus movement. On the basis of these results, an hypothesis concerning the mechanism of circus movement is proposed and discussed.


Application of glass capillary-column gas chromatography-mass spectrometry to the studies of human diseases.

Open-tubular glass capillary columns have been used in gas chromatography in combination with mass spectrometry (GC-MS) and computer methods to study human diseases. Patients with maple syrup urine disease excrete not only alpha-keto and alpha-hydroxy acids but also six other metabolites which hitherto have been overlooked. The GC-MS methods demonstrated that a group of patients suffering from hereditary progressive loss of hearing have an impaired metabolism of leucine, leading to the accumulation of 3-hydroxyisovaleric acid and 3-methylcrotonylglycine. GC using the capillary columns proved suitable for mapping of the carbohydrate profile of human seminal fluid and for the analyses of organic compounds accumulating in human adipose tissue. The high resolving power and long life of the glass capillary columns suggest that they will be valuable in the diagnosis and study of human disorders.


Gas chromatographic-mass spectrometric-computer methods in the study of drug metabolism.

The computer-assisted interpretation of repetitively scanned mass spectra can be of great value in the analysis of a complex mixture even in the absence of a library of reference spectra. We have written a set of interpretation programs for a PDP11/45 laboratory computer. A file structure is used that combines economy of storage with ease of creation and retrieval. User interaction is by a simple command language implemented by an interpreter. The program structure is such that additional functions can be added easily.


Radioimmunoassay and chromatographic similarity of circulating endogenous gonadotropin releasing hormone and hypothalamic extracts in man.

A highly sensitive radioimmunoassay for the gonadotropin releasing hormone has been developed in order to study its physiological importance in man. In view of the expected low concentrations in peripheral blood, large volumes of human plasma were extracted by two different methods and characteristics of the radioimmunoassayable material compared with those of synthetic decapeptide and extracts of human hypothalami. The results indicate that radioimmunoassayable gonadotropin releasing hormone is present in some human plasmas but the plasma concentration are less than 2.5 pg/ml. Peripheral levels were more consistently measurable in women at midcycle and after the menopause. The hormone was undetectable in the plasma of normel men, human cerebrospinal fluid, and fetal cerebral tissue, but was present in fetal hypothalami.


Semi-microtechnique for the biochemical characterization of anaerobic bacteria.

A semi-micromethod is described for characterizing anaerobic bacteria by substrate utilization. Small volumes of individual substrates were placed in the wells of plastic microtiter trays. When heavy inocula and a colorimetric indicator were used, complete results were available after 30 h of incubation. Microtiter trays containing a range of substrates can be stored at -20 degrees C. The method is accurate, economical, and convenient for use in a hospital microbiology laboratory.


Absence of wide-zone, alpha-hemolytic streptococci in children with pharyngitis.

Wide-zone, alpha-hemolytic streptococci are potential sources of false-positive throat cultures since they can be misinterpreted as beta-hemolytic streptococci. However, quantitative information regarding their occurrence in sick children was not available. Throat swabs from 312 children with pharyngitis were processed conventionally and also with pour-plate methods and examination of the positive cultures under magnification. One-third of all cultures were positive for beta-hemolytic streptococci. There were no significant differences in the number of positive cultures among or between the different methods. Wide-zone, alpha-streptococci were found in none of the cultures. Therefore, they are unlikely to be a common cause of false-positive cultures in children with pharyngitis.


Immunoglobulin specificity for the fluorescent treponemal antibody-absorption test conjugate.

Quantitative indirect immunofluorescence tests have demonstrated the predominance of immunoglobulin G antibody in most stages of syphilis. A class-specific anti-human immunoglobulin G conjugate is recommended to replace the present fluorescent treponemal antibody-absorption test conjugate. This would allow further definition and standardization of the fluorescent treponemal antibody-absorption test.


Examination of various cell culture techniques for co-incubation of virulent Treponema pallidum (Nichols I strain) under anaerobic conditions.

Treponema pallidum (Nichols virulent) was incubated with and without cells in cell culture medium reduced to -275 mV Ecal, pH 7.3, under deoxygenated conditions. Five to ten percent of the treponemes attached to cells and remained motile for at least 120 h in cell-treponeme systems of co-incubation. Virulent treponemes could be detected after 120 to 144 h in the supernatant fluids of cell-treponeme co-incubation cultures and in cell-free tubes containing medium harvested from aerobically cultivated mammalian cells. Medium supplemented with ox serum ultrafiltrate, pyruvate, and sodium thioglycolate and gas mixtures containing H2 and CO2 enhanced treponemal survival. Increases in treponemal numbers were observed using dark-field microscopy but were not substantiated using the rabbit lesion test. Continuous passage of the treponeme was not achieved in vitro.


Improved medium for detecting pigment production by group B streptococci.

The production of pigment which can be used for presumptive identification of group B streptococci is sometimes weak and difficult to observe in Columbia agar stab cultures. A medium which is whiter and affords better visibility of this pigment is described.


Production of antibody in induced granulomas.

A method of producing antibodies in artificially induced granulomas with various microbial antigens is examined.


Effects of meditation on personality and values.

The purpose of this research was to verify the effect of Zen meditation on personality and values. Two groups of Ss were formed, an experimental group of 9 Ss and a control group of 14 Ss. The California Psychological Inventory and the Study of Values were administered to both groups at the beginning and at the end of the experiment. The EG practised Zen meditation for the 5 months between the two testing sessions. While both groups were equivalent at pretest time, significant differences on the Do and Cs scales of the CPI were found at the posttest.


Rational emotive therapy-a study of initial therapy sessions of Albert Ellis.

Because psychotherapy is what a therapist does, and not necessarily what he says he does, it is important to observe the activity of leaders in the field during their sessions. Twenty taped initial psychotherapy sessions by Albert Ellis were selected randomly from 70 recently recorded ones. Typescripts of each session were made, and two raters naive to the purposes were trained to place each of Ellis' statements into 1 of 17 categories. Each category consisted of a therapeutic technique. Some of these were ones that Ellis did during the 20 sessions examined was related very closely to what he has claimed to do, but that he did vary considerably from client to client.


Aggressive behavior and the Rosenzweig Picture-Frustration (P-F) Study.

The current status of the Rosenzweig Picture-Frustration (P-F) Study, a measure of aggressive verbal behavior in response to frustration at the developmental levels of the child, the adolescent and the adult, is described. Conceived as generically or essentially self-assertive, aggression can be viewed as affirmative or negative, constructive or destructive in effect. Six categories, three under direction and three under type, have been conceptualized to define the modes of aggressive response. These interrelated categories combine into nine scoring factors. Norms at the various age levels have been established, and the reliability and validity of the instrument have veen determined. Pragmatic validity has been shown in the investigation of behavior disorders, psychosomatic conditions, delinquency, school adjustment, and a large variety of vocational and social problems. The P-F has been adapted and standardized world-wide (France, Germany, Italy, Sweden, Argentina, Brazil, India, Japan). Its applicability in cross-cultural research has been demonstrated.


Measles inclusion-body encephalitis in a child with treated acute lymphoblastic leukaemia.

A child with acute lymphoblastic leukaemia, being treated in the UKALL II Trial, had while in remission an attack of measles and made a normal recovery. Four months later she developed an acute encephalopathy and died within two weeks. The brain showed mild inflammatory features and widespread inclusion bodies in neurones and glial cells. Immunofluorescence proved an infection with measles virus. Similar cases have been called SSPE; reasons are given for preferring the term "measles inclusion-body encephalitis".


Solid-phase enzyme-immunoassay for detection of hepatitis B surface antigen.

The preliminary results of a solid-phase enzyme-immunoassay (EIA) for the detection of hepatitis B surface antigen (HBsAg) are presented. This method has been compared with the solid-phase radioimmunoassay (RIA) for HBsAg in dilution series of four HBsAg positive sera four national reference panels (The Laboratory Panel of the Central Laboratory of the Blood Transfusion Service of the Netherlands Red Cross, USA BOB Reference Panels Nos 2 and 3, and the 1st Panel of the National Reference Centre for virus Hepatitis at the Institute of Hygiene of the University of Göttingen, West Germany). In addition, the two test methods were compared in a weekly (up to 16 weeks) follow-up of 14 patients with acute viral hepatitis B. It was seen that, both by reading EIA test results with the naked eye and by colorimetric reading, the sensitivity and specificity of this test method compared very favourably with those of the RIA. EIA may have a slightly lower sensitivity than RIA for the subtype ad, while its sensitivity for the subtype ay may be slightly higher than that of RIA. These minor sensitivity differences may be due to the specificity profiles of the antisera used.


Isolation of L-forms by blood culture.

Culture media for the isolation of bacterial L-forms from the blood were studied. The most successful media had an osmolality of more than 1100 mosm/kg and this appeared to be a critical factor in determining success.


Immunological phenomena in the jejunum and serum after reintroduction of dietary gluten in children with treated coeliac disease.

Jejunal mucosal immunoglobulin-containing cells of all three major classes (IgA, IgM, IgG) were increased in coeliac children on gluten-containing diets but only IgM cell numbers were raised in those on gluten-free diets. Patients with subtotal villous atrophy had greater numbers of immunoglobulin-containing cells than patients with normal mucosa. In previously treated patients studied before and after three months on a gluten-containing diet ther was an increase in all three classes of cell, IgM containing cells showing the greatest proportional rise. Basement membrane staining with anti-IgA serum occurred in coeliacs and was most intense in untreated patients. Apart from one patient with very low levels of serum IgA, serum immunoglobulins did not differ from normal. However, after reintroduction of gluten to the diet a significant fall in serum IgM concentrations occurred compared with levels in the same patients while on gluten-free diets. It seems probable that both IgA and IgM systems are important in the immunopathogenesis of the small intestinal lesion of childhood  coeliac disease.


Significance of urinary C3 excretion in glomerulonephritis.

The third component of complement (C3) was measured in the urine of 98 patients with a variety of renal diseases. Renal biopsy was performed on 83 of the patients and examined by light, electron, and immunofluorescence microscopy. Urinary C3 was detected in cases of membranous glomerulonephritis, mesangiocapillary glomerulonephritis, rapidly progressive glomerulonephritis, and renal amuloidosis. It was not detected in minimal lesion glomerulonephritis; in cases of proliferative glomerulonephritis it was detected only in those showing histological evidence of a progressive lesion. Concentrations were low or undetectable in cases of non-immunological renal diseases. There was a good correlation between urinary C3 concentrations and the deposition of C3 in glomerular capillary walls, as seen by immunofluorescence microscopy, and there was no correlation with the degree or selectivity of proteinuria. Urinary C3 excretion appears to be an accurate indicator of continuing activity of disease. It is suggested that the presence of C3 in urine is due to complement fixation by immune complexes in glomerular capillary walls, and that urinary C3 estimations have potential applications in the study of glomerulonephritis.


Immune potential in human uraemia. 1. Relationship of glomerular filtration rate to depression of immune potential.

Aspects of immune potential in uraemic subjects, categorized by glomerular filtration rate, were intercompared and compared with results obtained from a group of normal volunteers. Evidence is presented to show that depression of both cellular and humoral immune potential occurs with progressive reduction of glomerular filtration rate. Lymphocyte transformation testing to the non-specific mitogen PHA revealed a significant elevation of blastogenic response in uraemia after 96 hours of incubation.


Immune potential in human uraemia. 2. Changes after regular haemodialysis therapy.

Parameters of both humoral and cellular immune potential were measured in a group of patients with severe renal failure before and after three months' regular haemodialysis therapy. Evidence is presented of improvement in cellular immune potential and of a tendency of the response of lymphocytes to PHA to return to normal. No improvement in humoral responsiveness was demonstrable, and it is suggested that uraemic patients on regular haemodialysis may have an impaired capacity to establish new immunological memory.


Haemophilus aphrophilus endocarditis in pregnancy.

The clinical and microbiological features of a case of Haemophilus aphrophilus endocarditis in pregnancy are described. The complicating effect of pregnancy on treatment and the difficulties in identifying the organism in the laboratory are discussed.


Effectiveness of bumetanide in nephrotic syndrome: a double-blind crossover study with furosemide.

A double-blind crossover study was undertaken to delineate the renal tubular sites of action of bumetanide and to compare its effects upon electrolyte excretion to that of furosemide in six nephrotic patients with various degrees of renal insufficiency. Bumetanide was found to be a potent oral natriuretic agent without producing changes in either GFR or effective renal plasma flow. The natriuresis was associated with inhibition of proximal tubular transport as evidenced by an increased distal delivery (CH2O+CNA+K) and inhibition of sodium transport in the loop of Henle as shown by a fall in CH2O/CH2O+CC1 during sustained water diuresis and an unchanged TCH2O with continued hydropenia. The increases in free-water clearance induced by furosemide and bumetanide were equivalent, but bumetanide produced significantly higher flow rate, greater solute delivery from the proximal tubule, and a greater natriuresis. The data indicate that at the dosages used, bumetanide has a proportionately greater inhibitory effect than furosemide in both the proximal tubule and the ascending limb of the loop of Henle.


Long-term effects of minoxidil in the treatment of malignant hypertension in chronic renal failure.

Thirteen acutely symptomatic malignant hypertensive patients were treated with minoxidil in combination with a beta-adrenergic blocking agent and diuretics or dialysis. The degree of endorgan involvement varied, with the kidney being the most compromised in 11 of 13 patients. In 12 of 13 patients, MABP (mean arterial blood pressure) fell significantly within 72 hours, P less than 0.005. After 20 months of therapy, all have now had a favorable response, with a mean reduction in MABP of 48 mm Hg, and with no adverse cardiac disturbances. In addition, the patients with mild azotemia had an improvement in renal function as determined by a reduction in serum creatinine. PRA (peripheral renin activity) rose with the addition of minoxidil despite therapeutic concentration of serum propranolol and a reduction in MABP and heart rate. It was also noted that those patients who had been on guanethidine prior to minoxidil had a more pronounced lowering of MABP within 72 hours of minoxidil therapy. It is concluded that minoxidil is a safe, fast, and effective agent to achieve rapid and sustained control of MABP in malignant hypertensive states associated with chronic renal insufficiency.


Diuretic and clinical effects of low-dose furosemide in congestive heart failure patients.

A dose of 20 mg furosemide in congestive heart failure patients produces a significant diuretic and natriuretic effect. The peak effect was observed within 60-120 minutes in most patients. Twenty-four congestive heart failure patients were hospitalized for evaluation and management. Two withdrew from the study, and in three there was a prolonged (several months) remission of manifestations of congestive heart failure. The remaining 19 patients were treated with 40 mg furosemide per day for a four-week period. Six required 80 to 120 mg furosemide per day, and 13 were controlled on 40 mg per day. On 20 mg twice per day, one of the 13 patients dropped out voluntarily, two required a higher dose of furosemide, and 10 were controlled on this regimen for a four-week period. When dosage was reduced to 20 mg per day, five of the ten patients were controlled for an additional four weeks or longer, and five required higher doses of furosemide. Results of this study suggest that in congestive heart failure patients, 20 mg furosemide per day has significant diuretic and natriuretic properties. It also demonstrates that a number of patients with cardiac decompensation can be controlled on a relatively low dosage of furosemide and that periodic reevaluation of clinical status and diuretic requirements for maintenance therapy is of critical importance in management of patients with congestive heart failure.