White spotting in the California vole.

Previously unreported white spotting was found in two subspecies of the California vole, Microtus californicus. The pattern of spots on the ventral coat of the animals differs between the subspecies, and there is variation in the expressivity of the white spots. Expression of white spotting is greatly reduced by the epistatic action of another coat colour gene, the recessive buffy (bf). The incidence of white spotting, its variation in expression, and its inheritance were investigated in this study. The reproductive performance of white spotted voles were also analysed, and effects of fertility and litter size were found associated with the trait.


The genetic control of DS-RNA virus-like particles associated with Saccharomyces cerevisiae killer yeast.

Previous results have indicated that laboratory strains of yeast possessing two types of dsRNA virus-like particles, V1 and V2, are capable of producing a killer toxin which kills sensitive strains. This paper reports on genetic crosses which correlate the presence of these particles both with a host nuclear gene +/-/mak1 and with the production of a killer toxin. The results show that: (i) V1 particles are inherited independently of the +/-/mak1 gene. (ii) V2 particles are present only in combination with those of V1 and the +/- allele of the +/-/mak1 nuclear gene.


Immunofluorescent localization of secretin in pancreatic monolayer culture.

Immunofluorescent cells to synthetic secretin were identified in monolayer culture of neonatal rat pancreas. No cross reaction of anti-secretin was observed with either glucagon, somatostatin or gastrin. The presence of cells containing secretin or a secretin-like peptide adds a new cell type to the three already characterized (insulin, glucagon and somatostatin containing cells) in monolayer culture.


A tissue model for the study of cell proliferation parameters in vivo.

An intact tissue model which can be used for detailed microscopic studies, quantitative cytochemical analysis and biochemical analysis has been explored, using a number of cell proliferation parameters. The preparations consist of mesenterial "windows" from rats dispersed on object slides. A technique for determining, in one and the same preparation, DNA content and mitotic activity of individual, selected cell types, and DNA synthesis in terms of incorporation of tritiated thymidine into DNA is described.


[Malignant nasal granuloma in a 12 year old boy (author's transl)].

Granuloma gangraenescens is placed between Wegener's granulomatosis and reticulum cell sarcoma. It is very difficult to diagnose due to absence of early symptoms as in this reported case, which was diagnosed histologically and which temporally responded to steroids and Imurek. Change histologically to a reticulum cell sarcoma has not been proven.


[Intranasal skin grafting for Osler's disease (author's transl)].

Between 1965-1972, 12 patients with Osler's disease of the nose were treated surgically by a modification of the method suggested by Saunders. The mucosa of the entire nasal septum and entire floor of the nose up to the insertion of the inferior turbinate was removed without interference to the perichondrium or periosteum. The denuded area was covered with split thickness skin graft taken from the thigh. Haemorrhage, which was never severe, occassionally occurred. In all patients, there occurred crusting and foetor, which was controlled by nasal douching. Patients with Osler's disease should be treated as early as possible in order to curtail the work disability.


Shipping fever pneumonia in yearling feedlot cattle.

During each week of 1974, we surveyed, for illnesses and deaths, a continually changing population of yearling feedlot cattle that, for the year, totaled 407,000 animals. About 5.1% of the cattle sickened and, of these, 18.9% died. From the 3,943 fatalities, 1,988 necropsies were made. About 75% of the clinical diagnoses and 64% of the necropsy diagnoses were respiratory tract diseases; of the fatalities from respiratory tract diseases, 75% were attributed to shipping fever pneumonia. Nearly 72% of fatal cases of shipping fever pneumonia occurred during the first 45 days on feed. In the lungs of most cattle with shipping fever pneumonia, bronchiolitis, fibrinous exudate, colonies of microorganisms, lymphatic clots, intravascular clots, thromboses, and foci of necrosis were found. Pasteurella spp, Mycoplasma spp, and infectious bovine rhinotracheitis virus were isolated from pneumonic tissues. It was hypothesized that pathogenic Pasteurella spp and other microorganisms in nasal secretions transfer from the nasopharynx into the lungs by draining along the tracheal floor into ventral bronchi, bronchioles, and alveoli, and that pasteurella endotoxin, formed in infected lobules, thromboses and occludes lymphatics, capillaries, and veins and thereby causes ischemic necrosis.


The in vitro activity of ceftezol (demethylcefazolin) against dense populations of Escherichia coli.

The activity of ceftezol was examined by continuous turbidimetric monitoring of dense populations of Escherichia coli exposed to the drug. Although ceftezol was found to be very active against strains of E. coli, its activity was consistently less than that of the closely related antibiotics, cefazolin. This difference was also found when strains of E. coli were examined in a dynamic system which simulates some of the conditions in which bacteria and drug interact in the treatment of bacterial cystitis. Evidence is presented that the difference in activity between the two cephalosporins resides in a differential ability to induce certain morphological changes in E. coli and in a differential rate of destruction by escherichial beta-lactamases.


A new semisynthetic macrolide antibiotic 3-O-oleandrosyl-5-O-desosaminylerythronolide A oxime.

A new antibiotic, 3-O-oleandrosyl-5-O-desosaminylerythronolide A oxime (3) was produced from erythronolide A oxime (1) by the oleandomycin-producing culture, Streptomyces antibioticus ATCC 11891. The structure of 3 was determined by degradative studies and confirmed by X-ray analysis. Compound 3 was found to be less active, but more stable to acid, then erythromycin A oxime.


Inhibition of coliphage multiplication and R plasmid transfer by desdanine.

Desdanine inhibited the plaque formation of male-specific coliphages but not that of other coliphages tested. Desdanine also suppressed the multiplication of both RNA phage Q beta and filamentous DNA phage f1 at the concentration of 3.13 approximately 6.25 mug/ml which had no influence on the growth of their host cells. However, the inhibitory effect on the phage multiplication was not due to the inactivation of phage particles nor the prevention of phage adsorption and penetration into the host cells. Desdanine also inhibited the transfer of R plasmid, R 100-1, in E. coli at 6.25 approximately 12.5 mug/ml without affecting the viability of donor and recipient cells.


Role of Rec pathways on sensitivity of Escherichia coli to near-ultraviolet and visible light.

In Escherichia coli lack of the RecRC or RecF pathway is found to cause sensitivity to near-ultraviolet and visible light. Resistance to this light is restored in the RecBC-defective strain carrying either the sbcB (Rec+) or xonA (Rec-) mutation. The sensitivity, therefore, is not found to correlate with the degree of recombination proficiency as measured by genetic crosses.


Iron transport in Salmonella typhimurium LT-2: prevention, by ferrichrome, of adsorption of bacteriophages ES18 and ES18.h1 to a common cell envelope receptor.

Ferrichrome prevents adsorption of phages ES18 and ES18.h1 to cells of Salmonella typhimurium. Studies of albomycin-resistant mutants defective in both ferrichrome utilization and ES18.h1 adsorption suggest a sid gene may specify a receptor for both.


Expression and regulation of lactose genes carried by plasmids.

A number of plasmids carrying the lactose character have been studied. All of the plasmids examined so far code for proteins essential for lactose utilization, i.e., beta-galactosidase and galactoside permease. None of them carries enzymatically or immunologically detectable thiogalactoside transacetylase. The expression of the two enzymes is both negatively and positively controlled: they are inducible by different galactosides and are sensitive to catabolite repression. Since the plasmid-coded lactose systems have many features in common with the Escherichia coli lactose operon, it is suggested that the plasmids could have acquired the lactose genes from an E. coli chromosome.


Identification of a genetic locus affecting chromosome stabiltiy and cellular survival in a dnaB mutant.

A mutation has been identified in an Escherichia coli K-12 strain carrying dnaB42. This mutation potentiates both deoxyribonucleic acid degradation and cell death at nonpermissive temperatures. It is located 2 min away from dnaB between malB and metA.


Physiological and genetic regulation of the aldohexuronate transport system in Escherichia coli.

In Escherichia coli K-12, the specificity of the aldohexuronate transport system (THU) is restricted to glucuronate and galacturonate. There is a relatively high basal-level activity in uninduced wild-type or isomeraseless strains. Supplementary activity is obtained with the inducers mannonic amide (five-fold), galacturonate (fourfold), fructuronate (fivefold), and tagaturonate (sevenfold). Specific THU- mutants were selected as strains unable to grow on either aldohexuronate but able to grow on fructuronate or tagaturonate. The remaining transport activity in uninduced and induced THU- starins represents less than 20% of that found in the wild type. Conjugation and transduction experiments indicate that all of the THU- mutations are located in a unique locus, exuT, half-way between the tolC (59 min) and argG (61 min) markers. exuT is closely linked to the uxaC-uxaA operon (60 min) and to the regulatory gene exuR (60 min), which controls the above-mentioned operon and the uxaB operon (45 min). Growth on either aldohexuronate and transport activity are fully recovered when exuT mutants are allowed to revert to exuT+ on galacturonate or glucuronate. Reversion on glucuronate alone may lead to the mutational derepression of the 2-keto-3-deoxygluconate transport system, which is uninducible in the wild type, which also takes up glucuronate, and whose structural gene belongs to the kdg regulon. Such strains, which remain unable to grow on galacturonate, are exuT and kdgR (constitutive allele of the regulatory gene kdgR of the kdg regulon). THU activity is superrepressed in an exuR mutant in which the uxaC-uxaA operon and the uxaB operon are superrepressed; exuR+/exuR merodiploids are also superrepressed. In a thermosensitive exuR mutant in which the above-mentioned operons are constitutive at 42 degrees C, the THU activity is fully derepressed at this temperature. On the basis of these and other results, it is concluded that THU is coded for by the structural gene exuT, which is negatively controlled by the exuR gene product and which probably belongs to an operon distinct from the uxaA-uxaC operon.


Membrane lipoteichoic acid of Streptococcus pyogenes and its stabilized L-form and the effect of two antibiotics upon its cellular content.

Membrane lipoteichoic acid continues to be synthesized by an osmotically fragile, stabilized L-form of Streptococcus pyogenes. Chromatographic and electrophoretic comparisons indicate that the lipid componenent of lipoteichoic acid in this L-form and its parental streptococcus is glycerophosphoryldiglucosyl diglyceride and not phosphatidylkojibiosyl diglyceride. Based upon dry weight determinations, the yield of lipoteichoic acid from the L-form is 0.19%, as compared with 0.97% from the streptococcus. When grown with bacitracin the L-form contains the same amount of teichoic acid as when grown without this antibiotic; however, its lipoteichoic acid content is reduced by 85%. Similarly, the L-form grown with novobiocin for 10 h contains only 17% of the teichoic acid found in control cells.


Nonintegrated plasmid-chromosome complexes in Escherichia coli.

A number of plasmid systems have been examined for the ability of their covalently closed circular deoxyribonucleic acid (CCC DNA) forms to cosediment in neutral sucrose gradients with the folded chromosomes of their respective hosts. Given that cosedimentation of CCC plasmid and chromosomal DNA represents a bound or complexed state between these replicons, our results can be expressed as follows. (i) All plasmid systems complex, on the average, at least one plasmid per chromosomal equivalent. (ii) Stringently controlled plasmids exist predominantly in the bound state, whereas the opposite is true for plasmids that exist in multiple copies or are under relaxed control of replication. (iii) The degree to which a plasmid population binds to host chromosomes appears to be a function of plasmid genotype and not of plasmid size. (iv) For the colicin E1 plasmid the absolute number of plasmids bound per folded chromosome equivalent does increase as the intracellular plasmid/chromosome ratio increases in cells starved for required amino acids or in cells treated with chloramphenicol; however, the ratio of bound to free plasmids remains constant during plasmid copy number amplification.


Envelope-associated folded chromosomes for Escherichia coli: variations under different physiological conditions.

The folded chromosome of Escherichia coli has been investigated under various lysis and physiological conditions. A new gradient system was devised that allows excellent separation between unlysed cells and envelope-associated and envelope-free chromosomes. Isotope incorporation experiments showed that the fraction often called "membrane-bound nucleoids" contains cell wall in addition to nucleic acids, membranes, and proteins. The amount of lysozyme added and the lysozyme digestion time were found to be important when comparing the rate of sedimentation of envelope-associated chromosomes obtained under various physiological conditions. Amino acid-starved cells were found to be much harder to lyse with lysozyme than exponentially grown cells, The difference in sedimentation coefficient of envelope-associated chromosomes described earlier (Ryder and Smith, 1974) was not detected when the latter two types of cells had been given equivalent, but not identical, lysozyme treatment such that detergent-mediated lysis proceeded at the same rate. Analysis of pulse- and uniformly labeled chromosomes from amino acid-starved cultures revealed no preferential labeling of either envelope-associated or -released nucleoids. Nor was there a difference in sedimentation coefficient between uniform and pulse-labeled envelope-associated nucleoids. These results are in disagreement with the models for chromosome replication of Worcel and Burgi (1974) and Ryder and Smith (1974), respectively. Growing cells on carbon sources poorer than glucose demonstrated that the replicating chromosomes sediment faster than the bulk of envelope-associated nucleoids. The slower the growth rate, the greater this difference became. An alternative hypothesis regarding chromosome replication and its association with the cell envelope is presented.


Isolation of a lambda transducing bacteriophage carrying the relA gene of Escherichia coli.

In Escherichia coli the relA and pyrG loci are 99% cotransducible. On the basis of this knowledge, we have isolated lambdacI857S7dpyrG transducing bacteriophages carrying both the pyrG and relA genes. Single lysogens of this bacteriophage show basal levels of ppGpp that are 10-fold higher than normal. Stringent factor is present among the gene products synthesized by lambdadpyrG relA after infection of ultraviolet-killed cells, as analyzed by polyacrylamide gel electrophoresis. The intracellular content of stringent factor, as determined by enzymatic activity, rises 20-fold after induction of a single lysogen of lambdadpyrG relA. As measured by two-dimensional gel electrophoresis, the amount of stringent factor in an exponentially growing strain carrying a pyrG relA plasmid is at least 10-fold greater than in a normal strain. These data constitute strong evidence that stringent factor is the relA gene product.


Genes for the alpha and beta subunits of the phenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli.

The phenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli is a tetramer that contains two different kinds of polypeptide chains. To locate the genes for the two polypeptides, we analyzed temperature-sensitive mutants with defective phenylalanyl-transfer ribonucleic acid synthetases to see which subunit was altered. The method was in vitro complementation; mutant cell extracts were mixed with purified separated alpha or beta subunits of the wild-type enzyme to generate an active hybrid enzyme. With three mutants, enzyme activity appeared when alpha was added, but not when beta was added: these are, therefore, assumed to carry lesions in the gene for the alpha subunit. Two other mutants gave the opposite response and are presumably beta mutants. Enzyme activity is also generated when alpha and beta mutant extracts are mixed, but not when two alpha or two beta mutant extracts are mixed. The inactive mutant enzymes appear to be dissociated, as judged by their sedimentation in sucrose density gradients, but the dissociation may be only partial. The active enzyme generated by complementation occurred in two forms, one that resembled the native wild-type enzyme and one that sedimented more slowly. Both alpha and beta mutants are capable of generating the native form, although alpha mutants require prior urea denaturation of the defective enzyme. With the mutants thus characterized, the genes for the alpha and beta subunits (designated pheS and heT, respectively) were mapped. The gene order, as determined by transduction is aroD-pps-pheT-pheS. The pheS and pheT genes are close together and may be immediately adjacent.


Bacteriophage-resistant mutants of Salmonella typhimurium deficient in two major outer membrane proteins.

Mutants resistant to bacteriophages (P221 and PH105 or PH51) were isolated from a rfa strain of Salmonella typhimurium. They were found deficient in separate 33,000- to 36,000-dalton band proteins (major band proteins). Double mutants derived from both types of mutants were deficient in both of the bands. The growth behavior of all the mutants was normal. The outer membrane of the mutants appeared to be more wrinkled than normal and formed vesicles in many of the mutants. In freeze-fractured cells, changes were seen in the outer membrane (particleless patches in the concave fracture face, the particles themselves being smaller than normal). These changes were more marked in the double mutants.


Method for the isolation of the replication region of a bacterial replicon: construction of a mini-F'kn plasmid.

A purified fragment of deoxyribonucleic acid (DNA) that determines resistance to kanamycin and is incapable of self-replication was used to select a self-replicating fragment from an EcoRI endonuclease digest of the sex factor F'lac. This F'lac fragment, exhibiting a molecular weight of 6 X 10(6), carries the genes essential for maintenance of the F replicon in Escherichia coli cells. The constructed mini-F'km plasmid also retains the incompatibility properties of the parent F'lac plasmid. Large amounts of the kanamycin resistance fragment of a molecular weight of 4.5 X 10(6) with an EcoRI-cleaved, self-replicating derivative of colicinogenic plasmid E1 that has a molecular weight of 2.2 X 10(6), The recombinant plasmid is able to replicate extensively in E. coli in medium containing chloramphenicol, and, therefore, large quantities of this plasmid DNA can be obtained. The substantial difference in size between the two fragments in the recombinant plasmid greatly facilitates their separation by preparative agarose gel electrophoresis.


Electron microscope study of a plasmid chimera containing the replication region of the Escherichia coli F plasmid.

pML31, a plasmid chimera constructed to contain the replication genes of an Flac plasmid, has been studied by electron microscope methods. Heteroduplex analysis shows that the only F sequence present in pML31 is that with corrdinates 40.3-49.3F. This region has previously been identified as essential for plasmid maintenance. The sequence of pML31, which was derived originally from R6-5, carries the km gene(s) and an inverted duplication of a 1.0-kilobase sequence. On the basis of length measurements, the repeated sequence is different from IS1, IS2, IS3, and an inverted repeat associated with the km gene(s) of plasmid JR67.


Specialized transduction of D-serine deaminase genes: formation of lysogens that yield high lambda-d dsd/lambda ratios and formation of a dimeric lambda-d dsd.

We have obtained two classes of double lysogens that on induction yield higher titers of lambda-d dsd transducing phage than of helper phage. One class was obtained by lysogenization of strain EM6116 (dsddelta attlambdadelta HfrC) with lambda-dsd type 2 (dsdC+ dsdO+ dsdA+, head-tail substitution). In the absence of either a normal attlambda or the homology of a chromosomal dsd region, the transducing phage integrated at other sites, at least one of which, in strain EM6177, is near the origin of HfrC. On induction, strain EM6177 yields a phage burst of 20 to 50 with a lambdadsd:lambda ratio of 10(4):1. The asnychronously high yield of lambda dsd is attributed to an efficiency of excision greater than that of lambda. The other class was obtained by lysogenization of strain EM1407 (dsdA attlambda+) with lambda-dsd type 2 (dsdO6 dsdA, partial deletion of dsdC). The DNA of mature lambda-dsd type 2 is a complete dimer. It lacks nearly all the phage late genes and b2 and carries about five bacterial genes. It could not be packaged as a monomer but is just within the packaging size limit as a dimer. Models for the derivation of these lambda dsd phages and the high-yielding lysogens are presented.


Effect of temperature on motility and chemotaxis of Escherichia coli.

The swimming velocity of Escherichia coli at various constant temperatures was found to increase with increasing temperature. The frequency of tumbling had a peak at 34 degrees C and was very low both at 20 and at 39 degrees C. The swimming tracks near the surface of a slide glass showed curves, and the curvature increased the temperature. When the temperature of a bacterial suspension was suddenly changed, a transient change of the tumbling frequency was observed. A temperature drop induced a temporary increase in the tumbling frequency, and a quick rise of temperature, on the other hand, resulted in a temporary suppression of the tumbling. These dynamic responses to sudden changes of temperature was not observed in the smoothly swimming nonchemotactic strains bearing the mutations cheA and cheC and also in a mutant with the metF mutation under a smooth swimming condition.


Outer membrane of Escherichia coli K-12: differentiation of proteins 3A and 3B on acrylamide gels and further characterization of con (tolG) mutants.

Two classes of mutants, con and tolG, that appeared to be very similar in a number of respects have been shown to be identical and cotransducible with pyrD. By diethylaminoethyl-cellulose chromatography of the outer membranes, we have shown that the mutants are missing only protein 3A and retain protein 3B. Using con mutants, we were thus able to identify protein 3B on the pH 7.2 gel system of Maizel where it runs separately from protein 3A if unheated samples are used. tolG mutants were shown to be identical to con mutants in being conjugation defective with most F-like plasmid donors but not with I-like plasmid donors, and in their resistance pattern to bacteriophages and colicins. During the course of this study, it was observed that the bacteriocin produced by Serratia marcescenc JF246 was identical in its activity spectrum to colicin L-398 and is now considered to be a colicin of type L.


Outer membrane of Escherichia coli K-12: isolation of mutants with altered protein 3A by using host range mutants of bacteriophage K3.

A series of mutants has been isolated with alterations to protein 3A of the outer membrane. These mutations map at the previously described con locus as shown by cotransduction with pyrD. Most of them do not have detectable levels of protein 3A but are thought to have low levels of altered protein. These mutants have been detected by screening con mutants, isolated as resistant to bacteriophage K3, for their ability to plaque host range mutants of this bacteriophage. These host range phage mutants have activity spectra on the various con mutants that enable the bacterial mutants to be arranged in an order of increasing resistance to the host range phage mutants, from mutants sensitive to all host range phage to those sensitive to only one class. Likewise, the phage can be arragned in an order of increasing ability to plaque on the con mutants. Some of the mutants resemble the previously described con mutants in being tolerant to colicins K and L, and others resemble them in being highly defective as recipients with the F factor. These properties vary independently, suggesting that protein 3A can be modified to independently affect the three properties of bacteriophage receptor function, involvement in colicin sensitivity, and involvement in conjugation.


Stringent control of peptidoglycan biosynthesis in Escherichia coli K-12.

[3H]Diaminopimelic acid (Dap) was incorporated exclusively into peptidoglycan by Escherichia coli strains auxotrophic for both lysine and Dap. The rate of [3H]Dap incorporation by stringent (rel+) strains was significantly decreased when cells were deprived of required amino acids. The addition of chloramphenicol to amino acid-starved rel+ cultured stimulated both peptidoglycan and ribonucleic acid synthesis. In contrast, a relaxed (relA) derivative incorporated [3H]Dap at comparable rates in the presence or absence of required amino acids. Physiologically significant concentrations of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) inhibited the in vitro synthesis of both carrier lipid-linked intermediate and peptidoglycan catalyzed by a particulate enzyme system. The degree of inhibition was dependent on the concentration of ppGpp in the reaction mixture. Thus, the results of in vivo and in vitro studies indicate that peptidoglycan synthesis is stringently controlled in E. coli.


Enterobacterial common antigen in rfb deletion mutants of Salmonella typhimurium.

The his-rfb deletion series of Salmonella typhimurium mutants characterized previously by Nikaido et al. was examined for the presence of the enterobacterial common antigen (ECA). All deletions not extending further to the left than the genes for cytidine phosphoabequose synthesis were ECA positive, whereas longer deletions (extending to the genes for thymidine diphosphorhamnose synthesis or further) were ECA negative. When these long-his-rfb deletion strains were studied further, it became clear that they (four out of four studied) had accumulated a second mutation, called rff, close to ilv, which prevented the synthesis of ECA. When rff- was replaced by rff+, the recombinants, now having the his-rfb deletion only, produced traces of ECA, showed reduced viability, increased sensitivity to sodium dodecyl sulfate (SDS) and to a lesser extent, to other anionic detergents, and accumulated secondary "suppressor" mutations upon storage. Such suppressor-containing mutants could be isolated by selecting for resistance to 1% SDS. Thirty of 46 SDS-resistant mutants studied had a second mutation, which alone prevented the synthesis of ECA, close to ilv. This ilv-linked mutation was similar to the rff mutation of the strains studied originally. The new rff mutation was similar to previously described rfe mutations in its close linkage to ilv and association with an ECA-negative phenotype. It differed from rfe, however, by not affecting the synthesis of the O antigens (O-6,7) of group C1. In Salmonella group C1, all ECA genes identified thus far are linked to ilv (rfe and/or rff) and none is linked to rfb.


Deoxyribonucleic acid degradation in vivo and in permeabilized Escherichia coli repair-deficient (recA zab lexA) derivatives.

There are three mutations (recA, zab, and lexA) each of which suppresses the expression of the Escherichia coli tif mutation and causes high deoxyribonucleic acid (DNA) repair deficiency (Castellazi et al., 1972). The effect of the zab mutation on DNA stability was investigated. In vivo, a strain carrying the zab-53 mutation shows (i) no spontaneous DNA degradation and (ii) rapid DNA degradation after ultraviolet irradiation, which depends upon the exonuclease V activity coded by recB+/C+genes and which is independent from the correndonuclease II activity coded by uvrA+/B+. Thus, in regard to DNA stability, the zab mutant behaves like lexA and recA (Howard-Flanders and Boyce, 1966), the latter mutant showing in addition spontaneous DNA breakdown. The degradation patterns of these tif-suppressed strains are shown to be remarkably reproducible in bacteria made permeable to metabolites, by toluene or toluene plus Triton X-100. The degradation properties reflect the activity of the same biochemical system that works in vivo, in that degradation depends upon the presence of recA, zab, or lexA, ultraviolet irradiation, and exonuclease V activity. In addition, adenosine 5'-triphosphate (1 mM) is required. This assay with permeabilized cells offers a useful tool for studying degradation under controlled conditions, especially by permitting the dissociation of energy-dependent from energy-independent steps.


Restoration by ribosomal protein S1 of the defective translation in a temperature-sensitive mutant of Escherichia coli K-12: characterization and genetic studies.

A temperature-sensitive mutant of Escherichia coli was isolated that had a temperature-sensitive defect in ribosomal-wash protein(s) required for translation in vitro of E. coli endogenous messenger ribonucleic acid. It was found that 30S ribosomal protein S1 rescued the defect in the ribosomal-wash protein(s) of the mutant and that the complete restoration to the wild-type level was attained when 1 mol of protein S1 was added to 1 mol of 70S ribosome. The mutation, tss, causing such a defect was mapped at 21 min and was closely linked to the pyrD locus, the region of which was entirely different from that of the other genes coding for the many ribosomal proteins of E. coli. These results indicate that the gene specified by this mutation is involved in the function of the 30S ribosomal protein S1.


Mapping of a new genetic locus responsible for ampicillin resistance in Escherichia coli.

The ampicillin resistance locus of three different ampicillin-resistant, temperature-sensitive Escherichia coli mutants was mapped between proC and purE and does not correspond to any of the known genes in this region. The mutant gene responsible for the temperature sensitivity and consequent morphological changes in each mutant strain was not located in the same 5-min region, even though the two mutants characteristics co-reverted at a very high frequency.


Energy cost of galactoside transport to Escherichia coli.

Energy reserves of Escherichia coli can be depleted by our previously reported procedure to a level such that even the "downhill" transport of o-nitrophenyl-beta-D-galactopyranoside (ONPG) is completely dependent upon the exogenous energy supply. The ONPG concentration is high externally to the cells and is low intracellular because of the action of cytoplasmic beta-galactosidase. In the present work, depleted cell suspensions have been infused at low, steady rates with glucose and other energy sources while measurements of transport were being made. Comparing the rate of ONPG transport with the rate of introduction of glucose under conditions where the chosen glucose infusion rate limits transport, we find that 89 molecules of ONPG are transported per molecule of fully oxidized glucose. This transport yield is constant over a 6.5-fold range in rate of glucose addition. This constancy over a range of infusion rates implies that transport is the major cellular function under these special conditions. The yield value if 89  is in the agreement with the predicitions of 76 from Mitchell's chemiosmotic theory and constitutes an independent proff of its validity, since all the other proposed mechanisms of engery coupling predict much smaller yields. The lag from the start of glucose infusion into the reaction cuvette, to the extrapolated time at which a steady rate of transport and concomitant hydrolysis are achieved, is short (approximately 1 min). Similarly, the time after the infusion is stopped until the rate of transport returns to the background rate is also short. The latter implies that the energy metabolism is directed almost entirely to transport and/or other ongoing cellular processes and not to repair or renewal of an energy-independent, facilitated diffusion system.


Second-site mutations in capR (lon) strains of Escherichia coli K-12 that prevent radiation sensitivity and allow bacteriophage lambda to lysogenize.

capR (lon) mutants of Escherichia coli K-12 are mucoid and sensitive to ultraviolet (UV) and X-ray radiation as well as to nitrofurantoin. The mutants form filaments after exposure to these agents. capR mutants are also conditionally lethal since they die when plated on complex medium even without UV treatment; this phenomenon is designated "complex medium-induced killing". Furthermore, capR mutants are poorly lysogenized by bacteriophage lambda. Second-site revertants were isolated by plating on media containing nitrofurantoin. All 17 of the independent revertants studied were still mucoid but resistant to UV radiation. Sixteen of the 17 revertants contained a mutation, sulA, that cotransduced with pyrD (21 min). A second locus, sulB, was also found that cotransduced with leu (2 min). Studies with partial diploids (F'pyrD+ sulA+/pyrD36 sulA17 capR9 (lon) demonstrated that sulA+ is dominant to sulA; thus the indicated partial diploid is UV sensitive, whereas the haploid parent is UV resistant. Furthermore, two other phenotypic traits of capR (lon) mutants were reversed by the sul mutation:complex medium-induced killing and the inability of lambda phage to efficiently lysogenize capR strains. On the basis of these and other results, the following model is suggested to explain capR (lon) and sul gene interactions. capR (lon) is a regulator gene for the structural genes sulA+ and sulB+. Depression of both sul operons results in UV sensitivity and decreased ability of lambda to lysogenize, whereas inactivation of either sul+ protein by mutation to sul prevents these phenomena.


Regulation of branched-chain amino acid transport in Escherichia coli.

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine, biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyl-transfer ribonucleic acid synthetase was also required for repression of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed.


Temperature-sensitive ribonucleic acid polymerase mutant of Salmonella typhimurium with a defect in the beta' subunit.

Localized mutagenes of Salmonella typhimurium followed by a [3H]uridine enrichment procedure yielded a temperature-sensitive strain with a mutation in the rpo region of the chromosome. Ribonucleic acid (RNA) polymerase (EC 2.7.7.6; nucleoside triphosphate: RNA nucleotidyltransferase) purified from this mutant was considerably less active at the nonpermissive temperature than wild-type enzyme. Furthermore, the enzyme from this mutant, unlike RNA polymerase of previously isolated temperature-sensitive mutants, was as thermostable as wild-type enzyme when preincubated at 50 degrees C. Subunit reconstitution experiments have shown that the temperature sensitivity is caused by an alteration in the beta' subunit of the enzyme.


Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system: purification and characterization of the phosphocarrier protein.

The Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system consists of three components: a membrane-bound enzyme II, a soluble enzyme I, and a soluble phosphocarrier protein, HPr. The HPr has been purified to homogeneity by a combination of ammonium sulfate precipitations, gel filtration and diethylaminoethyl, carboxymethyl Bio-Gel A, and hydroxylapatite column chromatography. The purified protein is relatively heat stable (ca. 50% activity survives 30 min of boiling) and has a molecular weight of ca. 10,000 (determined by sodium dodecyl sulfate-gel electrophoresis and amino acid analysis). It contains a single histidine residue per molecule and can be totally inactivated by photooxidation with Rose Bengal dye. Although the mycoplasma HPr is very similar to that of Escherichia coli, it shows no significant association with antiserum produced against E. coli HPr.


Role of deoxyribonucleic acid polymerase III in the repair of single-strand breaks produced in Escherichia coli deoxyribonucleic acid by gamma radiation.

Cell survival, deoxyribonucleic acid (DNA) degradation, and the repair of DNA single-strand breaks were measured for Escherichia coli K-12 pol+, polA1, polC1026(ts), and polA1 polC1026(ts) cells after 137Cs gamma irradiation. The results indicate that DNA polymerase III is required for growth medium-dependent (type III) repair in polA+ or polA cells. In pol+ or polC cells, DNA polymerase I performs type II repair efficiently. The relative deficiencies of each of these strains in DNA repair generally correlate with their relative sensitivities to cell killing and with the extent of DNA degradation observed.


Conjugation in Agrobacterium tumefaciens in the absence of plant tissue.

A general, reliable conjugation system for Agrobacterium tumefaciens in the absence of plant tissue is described in which A. tumefaciens can serve either as the donor or recipient of plasmid deoxyribonucleic acid with reasonable efficiency. Plasmid RP4 was transferred from Escherichia coli to A. tumefaciens and from strain of A. tumefaciens. Both RP4 and the A. tumefaciens virulence-associated plasmids were detected by alkaline sucrose gradients in A. tumefaciens strains A6 and C58 after mating with E. coli J53(RP4). The pathogenicity (tumor foramtion) of strains A6 and C58 and the sensitivity of strain C58 to bacteriocin 84 were unaffected by the acquistion of RP4 by the Agrobacterium strains. Plasmid R1drd-19 was not transferred to A. tumefaciens. Transformation experiments with plasmid deoxyribonucleic acid were unsuccessful, even though, in the case of RP4, conjugation studies showed taht the deoxyribonucleic acid was compatible with that of the recipient strains.


Iron transport of Escherichia coli K-12: involvement of the colicin B receptor and of a citrate-inducible protein.

It was shown that feuB mutants (defective in ferric enterochelin uptake) were unable to adsorb colicin B. In addition, they were missing one of the three outer-membrane proteins which are over produced in strains grown in iron-deficient, extracted medium. Thus this protein (the feuB protein) is probably the receptor for colicin B and functions in enterochelin-mediated iron transport. The feuB gene was located by P1 transduction at approximately 72.5 min on the Escherichia coli K-12 genetic map and thus maps separately from the other genes concerned with the enterochelin system. The outer membranes of various strains grown in the presence of 1 mM citrate contained a high level of a protein which was present in very small amounts when citrate was absent from the growth medium. This protein was most easily observed in feuB mutants grown in the presence of citrate, since on polyacrylamide gels it ran in a similar position to the feuB protein, which is missing in these mutants. The relationship of this citrate-inducible protein to the inducible citrate-dependent iron uptake system is discussed.


Localization of ampicillin-sensitive sites in Escherichia coli by electron microscopy.

Growth of Escherichia coli B/r ATCC 12407 (doubling time, 65 to 70 min) in the presence of 500 mug of ampicillin per ml for 15 to 20 min induces a sphere alongside the cell. The position was determined with respect to the length axis of the cell by electron microscopy. Although spheres may be found anywhere, some prominent sites do occur. In the shortest cells, which have a length of about 1.5 mum, they are found at the presumed new cell pole. In slightly older cells (length, about 1.8 mum), the position of the sphere is not well defined. Later on spheres occur predominantly at the cell center. In dividing cells (average length, 2.5 mum) a sphere may also occur at about one-quarter of the cell length. The position of the spheres bears resemblance to sites where a pulse of 3H-labeled diaminopimelic acid is incorporated into the peptidoglycan, as has been found by others.


Septation deficiency and phosphilipid perturbation in Escherichia coli genetically constitutive for the beta oxidation pathway.

Mutants of Escherichia coli defective in the regulation of the fatty acids beta oxidation pathway show an ultrastructural deficiency in septum formation at high growth rate. Several independent pairs of parent and mutant strains have been analyzed biochemically. Each parent strain displays a well-defined pattern of cellular phospholipids, which varies with the growth conditions. High ratios of phosphatidylglycerol to cardiolipin characterize fast-growth conditions. None of the mutant strains, although they grow in mass nearly as rapidly as their respective parents, can reach these high ratios. The beta oxidation pathway regulatory mutation leads to an increased turnover of the glycerol moieties of these phospholipids in the inner as well as in the outer cell membrane.


Interference with propagation of typing bacteriophages by extrachromosomal elements in Salmonella typhimurium: bacteriophage type 505.

Samonella typhimurium bacteriophage type 505 is the most  frequently encountered phage type in the Netherlands and its neighboring countries.  Phage type 505 was analyzed with regard o the interference with propagation of the typing phages by the prophages and plasmids, present in the type strain S. typhimurium 505...


Novel mutation that causes a structural change in a lipoprotein in the outer membrane of Escherichia coli.

A novel mutation which caused a structural change in a lipoprotein in the outer-membrane has been found in Escherichia coli K-12. The lipoprotein of the wild-type strain is known to have a peculiar amino terminal structure: glycerylcysteine with two fatty acids attached by ester linkages and one fatty acid by an amide linkage. In contrast to the wild-type lipoprotein, the mutant lipoproteins is isolated from the E. coli envelope as a dimer of molecular weight of about 15,000. The dimer can be reduced by mercaptoethanol to the lipoprotein monomer of molecular weight of about 7,500. The monomer has a free thiol group which is susceptible to monoiodacetie mutant lipoprotein is extremely low in comparison with that into the wild-type lipoprotein. These results suggest that the mutant is defective in transferring a glycerol group to the thiol group of the amino terminal cysteine residue of the lipoprotein. The gene responsible for this modification reaction has been located at 36.5 min on the E. coli chromosome.


Missense mutations in the lacZ gene that result in degradation of beta-galactosidase structural protein.

Thirty-two missenese mutations were found among more than 200 independently induced mutations in the lacZ gene of Escherichia coli. Twenty of these missense mutations were induced by nitrosguandine, and 12 were induced by aminopurine. The lacZ structural protein was endogenously degradable in seven of the mutant strains; the mutations in these strains were found to lie at only three sites in the lacZ gene. Five of the seven independent mutations were at a single site, and some heterogeneity in the degradation of the lacZ protein was observed within these mutant strains.


Nearest-neighbor analysis of Escherichia coli outer membrane proteins using cleavable cross-links.

A specific dimer of the 37,000-dalton, major outer membrane protein was demonstrated by chemical cross-linking with cleavable reagents.


Isolation of temperature-sensitive mutants of R plasmid by in vitro mutagenesis with hydroxylamine.

Temperature-sensitive mutants were isolated from a tetracycline resistance plasmid, pSC101, after mutagenesis with hydroxylamine. They were divided into three classes according to their growth characteristics. They include mutants defective in the replication of plasmid deoxyribonucleic acid and strains having a mutation in the drug resistance gene.


Membrane structures in stable L-forms of Escherichia coli.

Microtubular and lamellate membrane structures were observed at the log phase of growth in the stable L-forms of Escherichia coli cultured in the absence of penicillin.


Method for the isolation of Escherichia coli relaxed mutants, utilizing near-ultraviolet irradiation.

Near-ultraviolet radiation (300 to 380 nm) induces a transient growth inhibition in "stringent" (rel+) strains of Escherichia coli, whereas "relaxed" (rel-) strains are only mildly affected. This difference permits rapid isolation of large numbers of relaxed mutants of E. coli.


Genetic map position of the pdxH gene in Escherichia coli.

The gene for pyridoxine phosphate oxidase, pdxH, is located 1.2 min beyond aroD, proximal to trp.


L-Norvaline and L-homoisoleucine formation by Serratia marcescens,.

Two unnatural amino acids were found as by-products in isoleucine fermentation from threonine by Serratia marcescens. These amino acids were identified as L-norvaline (2-aminopentanoic acid) and L-homoisoleucine (2-amino-4-methylhexanoic acid). Formation of L-norvaline and L-homoisoleucine was not observed when L-leucine was added to the medium. The leucine auxotroph derived from the isoleucine accumlator did not produce L-norvaline or L-homoisoleucine even during the accumulation of large amounts of isoleucine. It is suggested that L-norvaline and L-homoisoleucine formation is closely related to leucine biosynthesis.


Biochemical studies of pyrithiamine-resistant mutants of Escherichia coli K12.

The biochemical properties of strains (PT-R101 and PT-R108) of Escherichia coli K12 resistant to growth inhibition by pyrithiamine, and antimetabolite of thiamine, have been studied. Intracellular thiamine pyrophosphate concentration in these resistant strains was slightly but definitely higher than that in the parent strain. Thiamine synthesis from the pyrimidine and thiazole moieties of thiamine by cell suspensions was greater in the resistant strains than the parent strain. The activities of enzymes involved in thiamine biosynthesis in these pyrithiamine-resistant strains were 2-3 times higher than the parent strain (3301), except for thiamine-phosphate kinase, which was indetectable in in vitro assay of the activity. However, other evidence indicates that this enzyme is not defective but is functioning in vivo and, furthermore, that the negligible activity of this enzyme did not affect the growth rate of the mutants. The activities of these enzymes were further enhanced when PT-R101 was grown on 5mM adenine and were reduced almost to zero when the strain was grown on 0.1 muM thiamine in the same way as the parent strain. However, when these resistant strains were grown on a low concentration of thiamine such as 0,05 muM, thiamine synthesis by cell suspensions also decreased, but only to a limited extent compared with the parent strain. These results suggest that PT-R101 and PT-R108 are altered in the mechanisms of regulation of thiamine biosynthesis. Their altered properties might be due to a reduced binding affinity of the repressor protein, which is involved on the regulation of thiamine synthesis, for the corepressor, thiamine pyrophosphate,


Cooperative conformational change in F-actin filament induced by the binding of heavy meromyosin.

The binding of HMM to F-actin containing bound 1, N6-ethenoadenosine diphosphate (epsilon-ADP), a fluorescent analogue of ADP, caused a significant increase in the fluorescence intensity of epsilon-ADP at 410 nm on excitation at 340 nm. This increase is regarded as due to a conformational change in the actin molecule induced by HMM binding. The fluorescence intensity increase was not directly proportional to the amount of bound HMM. This phenomenon suggests that a conformational change in neighbouring actin molecules is induced cooperatively by the conformational change of the actin molecule binding HMM.


Two substrate binding sites on tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli.

Tryptophanyl-tRNA synthetase of Escherichia coli has 1.8 binding sites for L-tryptophan with Kdiss of 12 x 10(-5) M as shown by equilibrium dialysis. The results are in accord with the known structure of the enzyme, and alpha2 dimer of 74,000 molecular weight, and with 2 binding sites for tryptophanyl-ATP ester. Ordinary sucrose density gradient centrifugation reveals a complex composed of one tRNATrp bound per enzyme dimer. When tRNATrp is mixed throughout the gradient at concentrations from 5.4 x 10(-6) M to 2.0 x 10(-5) M, a new peak appears in the position expected for a complex with two tRNATrp molecules bound per enzyme dimer. Sedimentation through gradients lacking tRNATrp favors dissociation of the 1:2 complex but not the 1:1 complex. The data indicate 2 binding sites for tRNATrp on tryptophanyl-tRNA synthetase.


Effect of cations and anions on the steady state kinetics of energy-dependent Ca2+ transport in rat liver mitochondria.

The divalent cation ionophore A23187 has been used to investigate the kinetics of energy-dependent Ca2+ uptake by rat liver mitochondria under steady state conditions. During A23187-induced cyclic Ca2+ flux, the free Ca2+ concentration is adjusted using [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) buffers. The rate of Ca2+ transport, which is inferred from the rate of succinate oxidation, is a function of the free Ca2+ concentration in the medium. The kinetics are sigmoidal with the free Ca2+ concentration at half-maximal respiratory stimulation (K0.5) equal to 3.1 +/- 0.4 muM at 25 degrees. The maximal Ca2+-stimulated respiratory rate (Vmax) is a function of the ionic composition of the medium. Magnesium and Mg2+ plus phosphate produced a parallel stimulation of the maximal respiration rate whether activated by Ca2+ uptake or by the uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP). In the absence of A23187, Ca:O rations of 4.0 were obtained under most experimental conditions. Magnesium is a potent competitive-like inhibitor, increasing the K0.5 for Ca2+ to 30.0 muM at 2.0 mM MgCl2. Magnesium dramatically decreases the apparent affinity for Ca2+ but does not appear to alter the kinetic mechanism. In contrast, the alkali metal cations are weak inhibitors, at most doubling the K0.5 for Ca2+; however, they antagonized Mg2+ inhibition with an order of effectiveness Li+ greater than or equal to Na+ greater than K+ greater than Rb+ =Cs+. Phosphate and acetate increased the Vmax slightly without altering the K0.5 for Ca2+. Phosphate did not influence the inhibitory effects of Mg2+ or Mg2+ plus K+. This study suggests that during steady state conditions, the maximal rate of Ca2+ accumulation is primarily electron transport-limited. The results are also discussed in terms of a possible physiological role for Mg2+ and K+ in the intracellular regulation of energy-dependent mitochondrial Ca2+ transport in liver.


Oligopeptide transport in proline peptidase mutants of Salmonella typhimurium.

Investigations of peptide transport in Salmonella typhimurium are presented. A strain designated proB25, a proline auxotroph, grew on a variety of di, tri-, and tetrapeptides containing proline. In contrast (Pro)6, peptides acylated on the NH2 terminus and Ala-Pro-D-Ala did not satisfy the nutritional requirement of proB25 for proline because they were not transported. A derivative of proB25, strain TN87, deficient in a proline aminopeptidase and an X-Pro dipeptidase, was able to utilize only four of 25 proline-containing peptides investigated. The inability of TN87 to grow on most of these peptides was due to the lack of the requisite peptidase activity. Evidence for a functional dipeptide transport system in this strain is indicated by growth on Pro-Leu and Pro-Ala, and by growth inhibition by certain X-Pro dipeptides. Leu-Pro, Val-Pro, Met-Pro, and Arg-Pro cause temporary growth inhibition of strain TN87 whereas Gly-Pro, Ala-Pro, and Pro-Pro have no effect on growth. Evidence for a functional oligopeptide transport system is indicated by growth on Pro-Val-Gly and Pro-Gly-Gly and by uptake of label from L-methionyl-L-methionyl-L-[14C]methionine and L-alanyl-L-prolyl-[14C]glycine. The presence of multiple oligopeptide transport systems for certain proline-containing peptides was demonstrated using triornithine-resistant mutants (oligopeptide permease deficient) and competition experiments. Finally L-Ala-L-Pro-[14C]Gly is shown to be transported intact into strain TN87.


Purification of intact old yellow enzyme using an affinity matrix for the sole chromatographic step.

Old Yellow Enzyme has been purified in high yield from crude extracts of brewers bottom yeast using an affinity matrix for the sole chromatographic step. The results of sodium dodecyl sulfate gel electrophoresis indicate that the enzyme is nearly homogeneous and consists of identically sized subunits of molecular weight of about 49,000. However, when the enzyme is isolated without the protective presence of a protease inhibitor, the electrophoretic pattern comprises three bands, which suggests limited, perhaps single bond cleavage of the polypeptide chain. This proteolytic cleavage results in weaker binding of ligands to the enzyme. The binding of a large number of compounds to the enzyme has been investigated, particularly with reference to the concomitant appearance of long wavelength absorption bands. The structural requirements for formation of long wavelength absorption are that the ligand must be aromatic in character, with an ionizable hydroxyl or thiol substituent.


Cellular and humoral immune response analysis of bone-allografted rats.

Long-term cellular and humoral immune responses were studied in inbred rats in which bone was allografted. Transplants were made across major histocompatibility barriers. Mixed lymphocyte culture tests and humoral cytotoxicity assays were performed in order to determine degrees of cellular and humoral immunity. Grafts of complete bone and of bone free of marrow elicited both types of immune response and it is proposed that transplantation antigens for both types of reaction exist in the bone tissue itself. A gene-dose effect was found. Allogeneic bone grafts gave a stronger reaction than semiallogeneic grafts, and second-set grafts triggered a hypersensitivity type of humoral immune response. This study suggests that until the host-bone graft immunological relationship is better understood, the graft tissue should be matched to the host for major transplantation antigens to help prevent unexpected failures in massive bone allotransplantation.


The measurement of skin blood flow in peripheral vascular disease by epicutaneous application of Xenon133.

Radioactive xenon133 applied epicutaneously was used to study the skin blood flow below the knee in sixteen normal subjects, in eight patients with peripheral vascular disease not requiring amputation, and in a blind study of twenty-nine patients requiring amputation. Following these twenty-nine amputations, the flow rates were made known and correlated with the rates of healing. When the flow rates were above 1.5 milliliters per minute per 100 grams of tissue (skin), the wounds generally healed. In thirteen other patients, Syme or below-the-knee amputations were performed on the basis of the flow rates, and all of the wounds healed. The test, therefore, is now used routinely prior to amputation for peripheral vascular disease as an adjunct to clinical judgment in the determination of the level of amputation.


Studies on the antigenicity of bone. I. Freeze-dried and deep-frozen bone allografts in rabbits.

The antigenicity of deep-frozen and freeze-dried cortical and corticocancellous bone allografts placed orthotopically in rabbits was studied using a sensitive microcytotoxicity assay. Target cells were phytohemagglutinin-P-stimulated, 51chromium-labeled peripheral blood lymphocytes from the bone donors (Dutch belted rabbits), and sera or peripheral blood lymphocytes from the graft recipients (New Zealand white rabbits) were used as effectors of cytotoxicity. Fresh allografts and deep-frozen corticocancellous bone evoked detectable humoral and cell-mediated immunity,, whereas freeze-dried cortical bone allografts failed to sensitize the recipients and were the least antigenic of the allografts examined.


Cutis arthroplasty of the elbow joint.

Five patients with an average age of thirty-five years had satisfactory restoration of elbow motion by a cutis arthroplasty technique with minimum medial-lateral instability. In three patients marked stiffness or ankylosis had followed severe fractures or dislocations. The fourth patient had rheumatoid arthritis, and the fifth had sustained a birth injury to the brachial plexus. An average follow-up of three and one-half years confirmed the durability of the results.


Contraction of isolated brush borders from the intestinal epithelium.

Brush borders isolated from epithelial cells from the small intestine of neonatal rats are able to contract in the presence of ATP and Mg2+; Ca2+ is not required. Contraction is characterized by a pinching-in of the plasma membrane in the region of the zonula adherens and a subsequent rounding of the brush borders. No movement or consistent shortening of the microvilli is observed. The contraction appears to involve the 5- to 7-nm diameter microfilaments in the terminal web which associate with the zonula adherens. These filaments bind heavy meromyosin as do the actin core filaments of the microvilli. A model for contraction is presented in which, in the intact cell, terminal web filaments and core filaments interact to produce shortening of the microvilli.


Cytoplasmic zone analysis. RNA flow studied by micromanipulation.

A technique is described, cytoplasmic zone analysis, by which it is possible to study the flow of different RNA-containing components in the cytoplasm after their release from the nucleus. After a pulse of RNA precursors, the salivary glands of the insect Chironomus tentans are fixed and microdissected for the isolation of three zones of cytoplasm situated at increasing distances from the nucleus. The RNA from each zone is isolated and analyzed by gel electrophoresis. The three ribosomal RNA components, 18 S, 28 S and 5 S RNA, appear in steep, specific radioactivity gradients (exit gradients) during the time interval 2-3 h after a precursor injection, the 18 S RNA gradient lying 30-50 mum peripheral to that of the 28 S or 5 S RNA gradient. Administration of puromycin led to the complete disappearance of the 28 S RNA and most of the 28 S RNA gradient within 45 min, suggesting that the gradients are caused by an engagement of the ribosomal subunits into polysomes close to the nucleus immediately or soon after their exit from the nucleus. The gradients may offer a unique model for the study of polysome formation and maintenance in vivo.


Galloylglucoses of low molecular weight as mordant in electron microscopy. I. Procedure, and evidence for mordanting effect.

Gallotannin, consisting mainly of low molecular weight esters such as penta- and hexagalloylglucoses (commercially available as tannic acid produced from Turkish nutgall), can be used for increasing and diversifying tissue contrast in electron microscopy. When applied on tissue specimens previously fixed by conventional methods (aldehydes and OsO4), the low molecular weight galloylglucoses (LMGG) penetrate satisfactorily the cells and induce general high contrast with fine delineation of extra- and intracellular structures, especially membranes. In some features, additional details of their intimate configuration are revealed. Various experimental conditions tested indicate that the LMGG display a complex effect on fixed tissues: they act primarily as a mordant between osmium-treated structures and lead, and concomitantly stabilize some tissue components against extraction incurred during dehydration and subsequent processing. Experiments with aldehyde blocking reagents (sodium borohydride and glycine) suggested that the LMGG mordanting effect is not dependent on residual aldehydes groups in tissues.


Galloylglucoses of low molecular weight as mordant in electron microscopy. II. The moiety and functional groups possibly involved in the mordanting effect.

Synthetic pentamonogalloylglucose applied to fixed tissues acts as a mordant, inducing high and diversified contrast similar to that obtained with natural gallotannins of low molecular weight (LMGG). By the separate use of each of the two moieties of the galloylglucose molecule, it was found that gallic acid is the mordanting agent. Glucose may contribute, however, to the effect by increasing the solubility and cross-linking potential of the compound, since the mordanting induced by gallic acid alone is weaker than that produced by its hexose esters. As suggested by results obtained with various phenolics and benzoic acid derivatives, the functional groups required for the mordanting effect of such agents are the carboxyl group, and at least one hydroxyl group concomitantly present on the benzene ring. In the case of galloylglucoses, it is assumed that the effect is due to hydrolysis products (gallic, digallic, or trigallic acids) or to the multiple hydroxyl groups of the intact molecule. Esters of gallic acid (propyl- and methylgallate), as well as pyrogallol, produce a "reversed staining" of all membranes, except for those of communicating (gap) junctions.


Studies on the posterior silk gland of the silkworm Bombyx mori. V. Electron microscope localization of fibroin in the posterior silk gland at the later stage of the fifth instar.

Electron microscope observations of thin sections of epoxy resin-embeded posterior silk gland cells at the later stage of the fifth instar revealed that the Golgi vacuoles and the secretory granules (fibroin globules) in the cytoplasm and the glandular lumen contain fine fibrous materials. In frozen thin sections these structures appear as electron-dense granules and electron-dense blocks, or a column, respectively. Immunoelectron microscopy has shown that ferritin particles or products of the peroxidase reaction are localized on these structures. It was concluded that the fine fibrous materials most probably represent native fibroin molecules or their aggregates.


[Thyroid cancers masked by hyperthyroidism. 12 cases].

The main object of this report was to attract again attention to the fact that obvious hyperthyroidism does not exclude the possibility of associated thyroid carcinoma, although this is an exceptional association. The 12 cases presented here may be added to about 30 cases found in the world literature. They were observed over a period of 13 years by the same surgical team and correspond to 0,3 p. 100 of all thyroid operations, 1 p. 100 of operated cases of hyperthyroidism and 3,6 p. 100 of cases of thyroid carcinoma. Contrary to most published cases, 11 or these cases out of 12, presented, clinically, mainly as hyperthyroidism the carcinotous lesion was either palpabale clinically in the form of a very small nodule (6 cases) or totally latent and discovered operation or even on histology as in 5 cases. Hyperthyroidism produced in 3 cases the classical picture of Graves' disease, in 6 cases that of toxic nodular goiter and, in 2 cases, that of a solitary toxic adenoma. The hyperplasia and the carcinoma were always in anatomically different areas. There is no apparent physiopahtological link and the classical notion of para-neoplastic hyperthyroidism seems debatable in most cases presented here, even in the cases of diffuse hyperfunctional hyperplasia. In any case, whatever the pathogenesis of this association, its possibility should be brought to mind in a case of hyperthyroidism. Surgery should always be advised in cases of toxic adenoma and in Graves' disease when the goiter is irregular and, especially, when a nodule is found. The course and prognosis seem to be mainly dictated by the histological type and the local spread or distant spread of the carcinoma, hyperthyroidism does not aggravate seriously the prognosis.


[Congenital arteriovenous fistula between the left subclavian artery and Pirogoff's venous junction. Surgical closure].

The authors report a case of arterio-venous fistula between the left sub-clavian artery and Pirogoff's venous junction. There was no past history of trauma, and no associated cardiovascular abnormality. The hemodynamic effects were moderate. Catheterisation and supra-sigmoid aortography permitted us to localise precisely the site of the fistula. Surgical cure was simple and carried out be cervico-sternotomy. This route of approach is broad and safe, providing satisfactory control of all the vascular structures.


[Role of gastric functional exploration in the postoperative testing of surgical vagotomies].

Better knowledge of the physiology of gastric acid secretion and especially the effects of insulin on the secretion, permits better definition of the indications for gastric functional investigation after vagotomy. The most reliable estimation of the effects of vagotomy is obtained by determining, before and after, the basal acid secretion, the maximal and post-insulinic secretions. The results should be expressed as hourly output and not in relation to arbitary criteria based on the titratable acid of the gastric juice. This investigation may be used to determine the quality of vagotomies carried out on a group of patients and to compare series of subjects in which different types of vagotomy were carried out. The tests at present available do not, however, determine precisely the effects of vagotomy in a particular patient. Their use for drawing up individual prognosis has not been demonstrated but their value is far from negligible in the diagnosis of recurrence.


A method for the rapid separation and characterization of biological pyridines.

Pyridine compounds of biological origin are separable by 2-dimensional descending paper chromatography. When assayed in situ for a tertiary or quaternary ring nitrogen, each compound gives a charactertistic reaction which is not predictable from the structural configuration. A cell-free extract of Escherichia coli, incubated in the presence of isoniazid, yields an ultraviolet light quenching spot with Rf values different from any tested standard. The compound is hypothesized to be a metabolite of either nicotinic acid or isonazid.


Chromatographic determination of cis-trans monoethylenic unsaturation in fats and oils--A review.

Chromatographic procedures for the determination of geometric and positional monoethylenic fatty-acid isomers in fats and oils have been reviewed. For the determination of these isomers, the procedure involving prep GLC isolation of the monoethylenic ester fractions on polar stationary phases, further separation and determination of geometric isomers by prep Ag+/TLC, and determination of positional isomers by reductive ozonolysis, is considered to be the most suitable for the analysis of a wide range of oils. Although, in this review, emphasis has been placed on monoethylenic isomers, most of the techniques described are also applicable in the study of polyenoic isomers.


[Assessment of the methods of drainage associated with truncular vagotomy].

The authors analyse 398 cases of truncular vagotomy by the abdominal route. They study the method of associated gastric emptying, the immediate and long-term results. They conclude that truncular vagotomy is a very satisfactory operation, that pyloroplasty is a good means of ensuring gastric emptying after vagotomy, and pyloroplasty according to Murat, or Judd-Lagrot, are the most recommended. Judd-Lagrot's operation is mainly indicated in acute cases of perforation or hemorrhage.


Effects of luteinizing hormone releasing hormone (LHRH) upon bioactive and immunoreactive serum LH levels in normal subjects.

The effects of LHRH stimulation upon plasma LH levels measured by bioassay and radioimmunoassay were examined during the normal menstrual cycle, and in normal men and post-menopausal women. After administration of 100 mug LHRH by subcutaneous injection during the early follicular phase, a 2.7-fold rise in bioactive serum LH to 58 +/- 18 mIU/ml at 30-60 min was accompanied by an equivalent rise in immunoreactive LH, with unchanged bio:immuno (B:I) ratio of 0.9 +/ %/- 0.2 (SD). During the late follicular phase, bioactive serum LH rose 8-fold to 258 +/- 120 mIU/ml at 30-180 min, and the B:I ratio was significantly increased from 1.7 to 2.5. During the luteal phase, bioactive LH values rose 8.1-fold to 223 +/- 97 mIU/ml at 30-60 min, with increase in B:I ratio from 1.1 to 1.8. The LHRH-stimulated serum LH levels declined more rapidly in the early follicular phase than during the late follicular and luteal phases. Elevations of circulating LH concentrations following LHRH administration during the normal menstrual cycle were usually accompanied by a significant rise in B:I ratio, except during the early follic ular phase when the LH responses were small and the B:I ratio did not change. After LHRH stimulation of serum LH levels in men and post-menopausal women, relatively small and inconstant elevations of B:I ratio were observed above the basal value of 2.9. Although the B:I ratio was usually close to unity in cycling women, the ratio was from 2 to 3 in men and postmenopausal women, and during LHRH stimulation of normal women at the late follicular and luteal phases of the menstrual cycle. Thus, the increased LH secretion rate of post-menopausal women and LHRH-stimulated cycling women was frequently accompanied by a rise in the B:I ratio. These observations suggest that circulating LH molecules exhibit a relatively higher biological activity during states of increased biosynthesis and release of gonadotropins.


to luteinizing.

The effect of luteinizing hormone-releasing hormone (LHRH) on plasma prolactin (PRL) was studied in eight acromegalic patients. After LHRH injection a significant increase in plasma prolactin levels was obtained in three of them. This non specific prolactin response to LHRH represents an additional proof of the well known abnormalities of pituitary hormone response to various stimuli in acromegaly.


Production of specific antisera for radioimmunoassay of human luteinizing hormone (LH) in the presence of human chorionic gonadotropin (hCG).

A specific radioimmunoassay for LH, which measures plasma LH in the presence of human chorionic gonadotropin (hCG) is described. Rabbits were immunized with highly purified native LH. One of the antisera with a difference in its reactivity against LH and hCG was further purified by affinity chromatography on a column with hCG coupled to Sepharose 4B. The adsorbed antiserum and 125I-LH was used in a double antibody assay. The LH standard (MRC 68/40) efficiently inhibited the binding of 125I-LH, and the standard curve showed a sensitivity of 0.5 ng/ml in the sample. hCG up to 10 000 ng/ml did not inhibit the binding of 125I-LH. The plasma level of LH in pregnant women in the first trimester was low (1.3 +/- 0.1 ng/ml). When LH was measured in fertile or menopausal women with or without stimulation with LH/FSH releasing hormone (LH-RH)X the results agreed to those found with our conventional LH-assay based on antiserum against hCG.


Hemolysis-in-gel and neutralization tests for determination of antibodies to mumps virus.

A hemolysis-in-gel test for the demonstration of antibodies to mumps virus is described. The results were compared with those of neutralization tests using a modified microtechnique. In the neutralization test viral replication was demonstrated by the hemadsorption of guinea pig erythrocytes, the visibility of which could be further enhanced by the use of o-tolidine. Good correlation was found between the results of the two techniques. The hemolysis-in-gel test was simple to perform, rapid, sensitive, and shown to be a useful test for the demonstration of mumps antibodies.


Qualitative evaluation of the reagin screen test.

After a preliminary study of 557 sera used as a procedural training exercise, the Reagin screen test (RST) for the macroscopic detection of reagin (as an aid to detecting syphilis) was qualitatively compared to the rapid plasma Reagin (RPR) (circle) card test and Veneral Disease Research Laboratory (VDRL) slide test on 435 random portions of sera using fluorescent treponemal antibody adsorption (FTA-ABS) results as a comparative base. A comparison of total agreement (positive and negative) with the FTA-ABS results led to the following order. RPRCT (I5.5%), VDRL (79.8%), and RST (74.5%). Of the total samples shown to be reactive by the FTA-ABS procedure, the percentage of these interpreted as nonreactive (i.e., "false negative") by the procedures compared was considerably higher with the RST procedure (29.3%) than with either the RPRCT (10.9%) or the VDRL (7.1%) procedures. Minor problems encountered with procedural techniques are also mentioned.


Improved chamber for the isolation of anaerobic microorganisms.

A small portable chamber for the recovery of anaerobic bacteria is described. This rigid chamber is constructed of clear acrylic with dimensions of 30 inches (ca. 76.2 cm) wide, 18 inches (ca. 44.7 cm) deep, and 18 inches (ca. 44.7 cm) high. Conventional bacteriological techniques can be used inside the chamber to efficiently isolate strict anaerobic organisms. An adapter allows the attachment of a standard anaerobic jar to the outside of the chamber. The jar can be used to store reduced media. Once the jar is attached to the chamber and the media is removed to the interior of the chamber, the jar is available to receive inoculated media. The anaerobic jar can then be removed from the chamber, without contaminating the jar or chamber with oxygen, and be placed in a conventional 37degreesC incubator. This chamber also allows the microbiologist to process cultures without wearing gloves as was necessary with previous anaerobic chambers. Air-tight latex rubber sleeves seal around the microbiologists arms and to the armport flange of the chamber to prevent the introduction of oxygen into the chamber. Anaerobic conditions are maintained by circulating a 80% N2, 10% H2, 10% CO2 gas mixture through alumina pellets coated with palladium. This study indicates that anaerobic conditions obtained in this chamber are sufficient for recovery of obligate anaerobes.


Comparison of bacteriological methods for the isolation of group of B Streptococcus from vaginal cultures.

Three bacteriological techniques for the isolation of group B streptococci in vaginal cultures were compared. A selective broth medium (SBM) containing gentamicin and nalidixic acid was more sensitive for the detection of vaginal isolates (28/76, 36.8%) from 76 women enrolled in a venereal disease clinic than was an identical selective plate medium (SPM) (17/76, 25%). Similarly, SBM allowed identification of positive cultures from college women (82/459, 17.9%) significantly more often than direct inoculation of swabs onto nonselective blood agar medium (43/460, 9.4%; chi2 = 42.2, P = less than 0.001). Failure to isolate group B streptococci detected in SBM occurred in 32.1% cultures by SPM and 49.4% of cultures by nonselective agar medium. Multiple serotypes were detected in a single vaginal culture from approximately 5% of the patients studied. These data support the routine use of SBM for the most accurate identification of women vaginally colonized with group B Streptococcus.


Effects of selective media and atmosphere of incubation on the isolation of group A streptococci.

The effects of selective media and atmosphere of incubation on the isolation of group A beta-hemolytic streptococci were evaluated. Sheep blood agar medium with gentamicin (2.5 or 5.0 mug/ml) was inferior to antibiotic-free sheep blood agar medium. This resulted from the partial restriction of group A streptococcal growth on gentamicin medium. Recovery of beta-hemolytic streptococci from specimens in air, CO2, or anaerobic incubation was evaluated. The isolation of group A streptococci was equivalent in the three incubation atmospheres; however, non-group A beta- hemolytic streptococci were isolated significantly more often from specimens incubated in an anaerobic or CO2 atmosphere than from those incubated in air. Therefore, sheep blood agar medium, stabbed with a wire loop and incubated in air, is recommended for the isolation of group A streptococci from throat swabs.


Simplified serological test for antibodies to Chlamydia trachomatis.

Three-hundred sixty sera from unselected patients attending two London venereal disease clinics were examined by a microimmunofluorescence test. Eleven egg-grown serotypes of Chlamydia trachomatis and the so-called "fast" strain SA2(f) were used as antigens. Of the 360 sera tested, 119 (33%) reacted to a titer of 1:16 or above with at least one antigen. Of these positive sera, over 50% cross-reacted with all 12 serotypes, and 95.5% reacted with SA2(f) in addition to other antigenic types. It is suggested that SA2(f) could be used as a single antigen for preliminary screening of a large number of sera for the presence or absence of chlamydial antibody.


Antigen distribution in a latex suspension and its relationship to test sensitivity.

Specific aggregation of latex particles due to antigen-antibody reaction occurred only when antigen was latex bound, as found in a study using Escherichia coli 0111:B4 extract. Maximum serological sensitivity was achieved when free antigen was present in a narrow range of ratios with the bound antigen.


Estimation of Mycoplasma pneumoniae inoculum size by rate of tetrazolium reduction.

The rate of appearance of red-colored formazan indicating reduction of 2,3,5,-triphenyltetrazolium chloride by Mycoplasma pneumoniae was dependent on the inoculum size. The test permits estimation of M. pneumoniae cells in preparations that cannot be counted by dilution methods.


Effect of amiodarone on serum triiodothyronine, reverse triiodothyronine, thyroxin, and thyrotropin. A drug influencing peripheral metabolism of thyroid hormones.

2-n-Butyl-3-(4'-diethylaminoethoxy-3',5'-diiodobenzoyl)-benzofurane (amiodarone), a drug used in arrythmias and angina pectoris, contains 75 mg of organic iodine/200 mg active substance. Four studies were performed to test its effect on thyroid hormone metabolism: (a) nine male subjects were treated with 400 mg of amiodarone for 28 days; (b) five male subjects received, for the same period of time, 150 mg of iodine in the form of Lugol's solution; (c) five subjects received 300 mug L-thyroxine (T4) for 16 days; from the 10th to the 16th day, 400 mg of amiodarone was added; and (d) five euthyroid subjects received 300 mug L-T4 for 16 days. The changes in serum thyroid-stimulating hormone (TSH), serum total T4, 3,5,3'-triiodothyronine (T3), free T3, and 3,5',3'-triiodothyronine (reverse T3, rT3) were measured, and the pituitary reserve in TSH was evaluated by a thyrotropin-releasing hormone (TRH) test. The results show that amiodarone induced a decrease in serum T3 (28+/-5.1 ng/100 ml, mean+/-SEM, P less than 0.0S and 82.7+/-9.3 ng rT3/100 ml, P less than 0.01). The control study with an equal amount of inorganic iodine did not induce these opposite changes but slightly lowered serum rT3, T3, and T4. In the third study, serum rT3 increased as under amiodarone treatment, thereby proving that these changes were peripheral. It is suggested that amiodarone changes thyroid hormone metabolism, possibly by reducing deiodination of T4 to T3 and inducing a preferential production of rT3. Amiodarone also increased the response of TSH to TRH. The maximal increment of serum TSH above base line was 32+/-4.5 muU/ml under treatment and 20+/-3 muU/ml before treatment (P less than 0.01). During this test, the serum T3 increase was more pronounced than during the control period (83+/-13 and 47+/-7.4 ng/100 ml, P less than 0.05).


Kinetic analysis of lead metabolism in healthy humans.

The steady state kinetics of lead metabolism were studied in five healthy men with stable isotope tracers. Subjects lived in a metabolic unit and ate constant low lead diets. Their intake was supplemented each day with 79--204 mug of enriched lead-204 as nitrate which was ingested with meals for 1--124 days. The concentration and isotopic composition of lead was determined serially in blood, urine, feces, and diet and less commonly in hair, nails, sweat, bone, and alimentary tract secretions by isotopic dilution, mass spectrometric analysis. The data suggest a three compartmental model for lead metabolism. The first compartment encompasses blood and is 1.5--2.2 times larger than the blood mass. It contains approximately 1.7--2.0 mg of lead and has a mean life of 35 days. This pool is in direct communication with ingested lead, urinary lead, and pools two and three. The second compartment is largely composed of soft tissue, contains about 0.3--0.9 mg of lead, and has a mean life of approximately 40 days. This pool gives rise to lead in hair, nails, sweat, and salivary, gastric, pancreatic, and biliary secretions. Pool three resides primarily in the skeleton, contains the vast quantity of body lead, and has a very slow mean life. Bones appear to differ in their rates of lead turnover. Within the relatively small changes in blood lead observed in the present study, the transfer coefficients between the pools remained constant.


The local immune response to Escherichia coli O and K antigen in experimental pyelonephritis.

Although the systemic and local immune response to the O antigen of Escherichia coli has been well characterized, little information is available on the immune response to K anigen. Experimental hematogenous pyelonephritis was produced with Escherichia coli 06 K13 H1 and the serum and local (intrarenal) antibody response to O and K antigens was determined with the enzyme-linked immunosorbent assay. Both local and serum antibody responses to the K antigen were significantly less than that to the O antigen. The K antigen induced low titer IgM and IgG antibody responses in fewer than one-half of the animals and did not induce a local IgA response in any animal. In contrast, the O antigen induced local antibody responses in each of the immunoglobulin classes in all animals from day 9 of infection. Similarly, the serum IgM and IgG antibody titers to the K antigen were significantly less than those evoked in response to the O component of the Escherichia coli. No serum IgA anti-K antibodies were detected. These observations helf clarify the roles of these two antigens in pyelonephritis. Although the K antigen of Escherichia coli functions as a virulence factor in upper urinary tract infections, this antigen does not elicit a significant immune response, whereas the O antigen does induce a significant antibody response which could be of protective or diagnostic benefit.


Effects of ouabain on myocardial oxygen supply and demand in patients with chronic coronary artery disease. A hemodynamic, volumetric, and metabolic study in patients without heart failure.

The effects of digitalis glycosides on myocardial oxygen supply and demand are of particular interest in the presence of obstructive coronary artery disease, but have not been measured previously in man. We assessed the effects of ouabain (0.015 mg/kg body weight) on hemodynamic, volumetric, and metabolic parameters in 11 patients with severe chronic coronary artery disease without clinical congestive heart failure. Because the protocol was long and involved interventions which might affect the determinations, we also studied in nine patients using an identical protocol except that ouabain administration was omitted. Left ventricular end-diastolic pressure and left ventricular end-diastolic volume fell in each patient given ouabain, even though they were initially elevated in only two patients. Left ventricular end-diastolic pressure fell from 11.5+/-1.4 (mean+/-SE) to 5.6+/-0.9 mm Hg (P less than 0.001) and left ventricular end-diastolic volume fell from 100+/-17 to 82+/-12 ml/m2 (P less than 0.01) 1 h after ouabain infusion was completed. The maximum velocity of contractile element shortening increased from 1.68+/-0.11 ml/s to 2.18+/-0.21 muscle-lengths/s (P less than 0.05) and is consistent with an increase in contractility. No significant change in these parameters occurred in the control patients. No significant change in myocardial oxygen consumption occurred after ouabain administration but this may be related to a greater decrease in mean arterial pressure in the ouabain patients than in the control patients. We conclude that in patients with chronic coronary artery disease who are not in clinical congestive heart failure left ventricular end-diastolic volume falls after ouabain administration even when it is initially normal. Though this fall would be associated with a decrease in wall tension, and, therefore, of myocardial oxygen consumption, it may not be of sufficient magnitude to prevent a net increase in myocardial oxygen consumption. Nevertheless, compensatory mechanisms prevent a deterioration of resting myocardial metabolism.


Comparison of the suppressive effects of elevated plasma glucose and free fatty acid levels on glucagon secretion in normal and insulin-dependent diabetic subjects. Evidence for selective alpha-cell insensitivity to glucose in diabetes mellitus.

To examine whether abnormal pancreatic alpha-cell function found in human diabetes mellitus may represent a selective insensitivity to glucose, plasma glucagon responses to hyperglycemia and elevation of plasma free fatty acid levels (both known suppressors of glucagon secretion) were compared in juvenile-onset, insulin-requiring diabetic subjects, and in normal nondiabetic subjects. In the latter, both elevation of plasma free fatty acid levels induced by heparin administration of hyperglycemia produced by intravenous infusion of glucose resulted in a comparable 30--40% suppression of circulating glucagon levels (P less than 0.01). In the diabetic subjects, glucagon suppression by hyperglycemia (less than 20%) was less than that occurring in normal subjects (P less than 0.01), even when accompanied by infusion of supraphysiologic amounts of insulin. However, suppression of glucagon levels by elevation of plasma free fatty acids in the diabetic group was similar to that found in normal subjects and of comparable magnitude to that due to hyperglycemia in the normal subjects. These results thus demonstrate a selective impairment of the diabetic alpha-cell response to glucose and provide further evidence for the presence of an abnormal alpha-cell glucoreceptor in human diabetes mellitus.


The late phase of the immediate wheal and flare skin reaction. Its dependence upon IgE antibodies.

IgE antibodies are usually thought to induce only immediate skin reactions. We have shown that the intradermal injection of a number of different allergens can produce a prolonged inflammatory reaction after the immediate wheal and flare in most sensitive subjects. This late inflammatory response occurs 6-12 h after challenge and is characterized by diffuse edema, erythema, pruritus, and heat. Both immediate and late responses can also be seen after passive sensitization of skin sites in nonatopic subjects. That IgE is involved in inducing the reaction was shown by the abolition of both immediate and late responses by passive transfer tests in the following experiments: (a) heating atopic serum at 56degreesC for 4 h, (b) removing IgE from the atopic serum by a solid phase anti-IgE immunoabsorbent, and (c) competitively inhibiting the binding of IgE antibodies to cells by an IgE myeloma protein. In addition, both responses were induced by affinity chromatography-purified IgE antibody, followed by antigenic challenge. Very similar lesions could also be induced by intradermal injection of Compound 48/80, thus suggesting a central role in the reaction for the mast cell or basophil. Histologically, the late phase is characterized by edema and a mixed cellular infiltration, predominantly lymphocytic but also containing eosinophils, neutrophils and basophils. Direct immunofluorescent staining did not show deposition of immunoglobulins or complement components, except IgM in 2 of 15 and C3 in 1 of 15 patients. This finding indicates that the late phase does not depend on the deposition of immune complexes. The results of the study suggest that IgE-allergen interaction on the surfaces of mast cells or on infiltrating basophils causes both immediate and late cutaneous responses.


Impaired renal conservation of sodium and chloride during sustained correction of systemic acidosis in patients with type 1, classic renal tubular acidosis.

In 10 patients with classic renal tubular acidosis in whom correction of acidosis was sustained with orally administered potassium bicarbonate, renal conservation of sodium was evaluated when dietary intake of sodium was restricted to 9--13 meq/day. In five patients, renal conservation of sodium was impaired by at least one criterion of impairment. In the remaining patients, renal conservation of sodium appeared to be relatively well-maintained, but an impairment could not be excluded. In each of six patients studied during induced water diuresis, including two in whom renal conservation of sodium was not unequivocally impaired, the minimal urinary concentrations of sodium were inappropriately high and the urinary excretion rates of sodium were flow-dependent. These results provide direct evidence that an abnormality in renal transport of sodium can occur in classic renal tubular acidosis, and compel a reconsideration of the pathophysiology of disordered renal transport of sodium in this disorder. The results indicate that in at least some patients with classic renal tubular acidosis impaired renal conservation of sodium is not exclusively a reversible consequence of the renal acidification defect. These findings raise the question whether renal transport of sodium is unimpaired in any patients with classic renal tubular acidosis. In the presently studied patients, the impairment in renal conservation of sodium appeared to be in part the consequence of an impaired ability of the vasopressin-responsive segments of the distal nephron to generate and maintain appropriately steep transepithelial sodium concentration gradients.


Arginine-stimulated acute phase of insulin and glucagon secretion in diabetic subjects.

To determine if both phases of glucagon secretion are excessive in diabetes, arginine was admimistered intravenously as pulses and as infusions to normal subjects, insulin-dependent diabetics, and noninsulin-requiring diabetics. The acute phase of glucagon secretion, in response to arginine pulses at four different doses (submaximal to maximal alpha-cell stimulating), was indistinguishable in terms of timing, peak levels attained, and total increments comparing controls and diabetics. During the first half of the arginine infusion (500 mg/kg over 30 min) the glucagon rise in controls and diabetics was similar (P greater than 0.1), whereas during the last half of the infusion excessive glucagon levels were seen in the diabetics. No difference in the glucagon responses to arginine administered as either a pulse or an infusion was observed between the two types of diabetics. The acute phase responses of insulin to intravenous, maximal stimulating doses of glucose (20 g) and arginine (2.5 g) were measured in five insulin-independent diabetics. Although the acute insulin response to arginine was normal, there was marked attentuation of the early beta-cell response upon stimulation by glucose. From these results we conclude that although in diabetes excessive glucagon levels are observed with chronic arginine stimulation, the acute phase of glucagon secretion in response to arginine is normal. In addition, the beta-cell in noninsulin-requiring diabetics, although acutely hyporesponsive to glucose, remains normally responsive to another stimulus, arginine.


Malignant lymphoma of the cervix uteri: histology and ultrastructure.

Two cases of primary lymphoma of the cervix uteri are described. Both responded to radiotherapy; both were composed at the ultrastructural level of mature macrophages and immature, apparently neoplastic lymphoreticular cells and are classified as reticulum cell lymphoma.


Presumptive identification and antibiotic susceptibility of group B streptococci.

The comparative performance of three presumptive identification tests for group B haemolytic streptococci was investigated, using 371 different clinical isolates of group B streptococci. Hippurate was hydrolysed by 96-1%, the CAMP reaction was positive in 95-0%, and pigment was produced by 97-3%. A combination of any two tests would have detected over 99-8%. On bile esculin agar 99-0% were able to grow, but non hydrolysed esculin; 5-1% were susceptible to bacitracin. The minimum inhibitory concentrations of five antibiotics for 279 group B streptococci were determined. All were susceptible to penicillin G, ampicillin, cephalothin, and erythromycin, while 80% were resistant to tetracycline. The MIC distributions were independent of the results of any identification test.


Variations in the susceptibility of lactating and non-lactating bovine udders to infection when infused with Escherichia coli.

Small numbers of Escherichia coli were infused into both lactating and non-lactating udders. Twelve of the 17 lactating quarters infused became infected, and all 12 showed clinical symptoms of udder disease. The 5 lactating quarters which did not become infected all had pre-infusion somatic cell counts greater than 300000 cells/ml milk, whilst all the quarters which became infected had cell counts less than 300000 cells/ml milk. E. coli was subsequently recovered from only 6 of the 16 non-lactating quarters infused. In only 2 of these quarters did clinical infection follow, both quarters being in a cow infused 2 d before calving. The remaining 4 quarters from which E. coli was recovered were all negative within 5 d of infusion. These differences in susceptibility are discussed, particularly with reference to the frequent occurrence of coliform mastitis at and shortly after calving.


Rennin and pepsin in stomachs of rats (Rattus norvegicus).

The predominant protease in adult rat stomachs has a pH optimum of 2.0 to 2.3 which corresponds almost exactly to that of porcine pepsin. One-day-old rats have a protease with a pH optimum of 3.8 to 4.2 similar to that of calf rennin. The pH optimum of rat stomach proteases decreases with age, and at 28 days it is similar to that of the adult. The pH of stomach contents of rats decreases with age from pH 5.8 to pH 4.6. These data support the contention by other workers that the onset of pepsin secretion is a prerequisite for weaning and that rennin-like enzymes are not unique to ruminant species.


Technique for restoring endodontically treated anterior teeth with precision posts and porcelain-bonded-to-gold crowns.

A full crown with porcelain bonded to gold and a precision post and sleeve are used in a method to restore endodontically treated anterior teeth. Advantages include protection of the tooth from fracture in the future and utilization of the same sleeve later should another crown be needed.


The behavior modification of a  dentally disabled child.

Parents of children afflicted with a dental handicap may experience difficulty in finding a dentist to treat their child. The dentist may be expected to assume the role of family confidant, because of the family's reluctance to seek other professional help. It is important to gain the early confidence and trust of the patient by techniques known to reduce the patient's fear. The patient can now interact more freely with his peers, because of the dental treatment which he has received. His trust, selfesteem, and confidence are much improved.


Evaluation and restoration of endodontically treated posterior teeth.

The increasing acceptance and utilization of endodontic therapy for the preservation of broken-down posterior teeth presents the practitioner with many difficult restoration problems. If 25% or more of the coronal tooth structure is lost, some form of artificial dentin augmentation is required to provide adequate support and retention for the final restoration. Criteria to be used in the prerestorative evaluation of endodontically treated teeth are presented. Various methods and techniques to protect the remaining toth structure from fracture and to provide support and retention for the final restoration are described.