The efficiency of aldehyde fixation for electron microscopy: stabilization of rat brain tissue to withstand osmotic stress.

Rat brains were fixed either with glutaraldehyde (GA) or formaldehyde (FA). After 20 min or 24 h fixation the osmotic sensitivity of the tissue was tested by immersion in (a) distilled water (b) 0.15 M or (c) 0.3 M cacodylate buffer. GA-fixed material retained some sensitivity to osmotic stress after 20 min fixation but was entirely resistant after 24 h fixation. Ultrastructural preservation was good after only 20 min GA-fixation, provided that the subsequent treatment was with isotonic solutions. The fixation with FA was less efficient and slower. Dark neurons and other artifacts were commonly seen after the 20 min fixation with FA. Prolongation of the FA-fixation overnight gave markedly better preservation, but however, never equivalent to that with GA.


Comparative physiology of the high-pressure neurological syndrome--compression rate effects.

The effect of compression rate on onset of high-pressure convulsions has been studied in 14 vertebrate species, as well as in 10 mouse strains and 4 rat strains. Compression rate effects were observed in 9 of the 14 species. They appear to be independent of exposure temperature, correlate only very loosely with phylogenetic position, and appear to reflect species-specific compensatory mechanisms grafted onto an underlying convulsion-producing effect of high hydrostatic pressure. Five vertebrate species distributed among three of the four classes tested failed to show a significant degree of compression rate dependence of high-pressure neurological syndrome (HPNS) convulsion thresholds. The implications of this finding for the formulation of hypotheses regarding the biophysical basis for HPNS convulsions has been discussed. Comparison of intrinsic HPNS susceptibility in different species, in the light of these findings, requires that the comparison be made at a common compression rate. Four of the five lower vertebrate species fall consistently into the category showing high HPNS convulsion threshold pressures regardless of the compression rate employed, whereas the two primates and the one carnivore tested equally consistently fall in the low convulsion threshold pressure category. The data suggest a parallel between the degree of brain development and the relative HPNS susceptibility of a given species and contrast with the inverse relations observed during maturation of newborn mice and rats. The results are compared with data for other convulsants and suggest grouping HPNS and pentylenetetrazole seizures as against electroshock, hyperoxic, flurothyl, strychine, or picrotoxin convulsions.


Isoproterenol and aminophylline reduce lung capillary filtration during high permeability.

Pseudomonas bacteremia in sheep causes a prolonged increase in lung vascular permeability to protein. Isoproterenol and aminophylline could effect lung fluid balance after Pseudomonas by reducing vascular pressures or by blocking release of permeability mediators. We measured vascular pressures, lung lymph flow, and lymph and plasma protein concentrations in unanesthetized sheep under baseline conditions and during steady-state increased permeability after Pseudomonas. Pseudomonas caused pulmonary vascular pressures to rise and lung lymph flow to increase fivefold, but lymph/plasma protein concentration did not change. Pulmonary vascular pressures and lung lymph flow decreased during intravenous infusion of isoproterenol and aminophylline. The decrease in lymph flow after isoproterenol and isoproterenol plus aminophylline was linearly related to the decrease in microvascular pressure (r = 0.71). Lymph/plasma total protein concentration ratios and lymph clearance of proteins with molecular radii 36--96 A remained high during isoproterenol and aminophylline. These drugs can substantially reduce transvascular filtration primarily because they reduce lung vascular pressures.


Respiratory mechanics in normal bonnet and rhesus monkeys.

We measured lung volumes and quasi-static volume-pressure relationships in 22 normal upright bonnet (Macaca radiata) and 12 rhesus (M. mulatta) monkeys. In comparison with interspecies pulmonary function/body weight regressions our monkeys' lung volumes are larger and their lungs are considerably more compliant, but their chest wall compliance is similar to a wide range of mammalian species. However, chest wall compliance of our monkeys was found to be considerably less than that of other more commonly used experimental mammals such as dogs, cats, and rodents. The monkey chest walls were found to be about four times as stiff (3.3 +/- 0.1 (ml/cmH2O)/kg), whereas their lungs were nearly twice as compliant (9.2 +/- 0.7 (ml/cmH2O)/kg) compared to those of supine beagle dogs. Thus, their stiff chest wall sets their functional residual capacity (64.1 +/- 1.2% TLC30) at a much larger percentage of total lung capacity (TLC30) than that of the supine beagle dog (33.8% TLC30). Residual volume (13.2 +/- 1.9% TLC30) equaled the trapped gas volume after bilateral thoracotomy and was set by airway closure. We found more hysteresis area in the chest wall than in the lungs. Our measurements indicate that the static mechanical behavior of the respiratory system of the monkey compares well to man and that the monkey has considerable merit as an animal model for human respiratory function and disease research.


Flow-volume curves and total pulmonary resistance in normal bonnet and rhesus monkeys.

We measured expiratory flow-volume curves and total pulmonary resistance in 15 normal, anesthetized, upright bonnet (Macaca radiata) and 8 rhesus (M. mulatta) monkeys. Absolute maximum flows are about half that found in humans, but, if expressed in vital capacities per second, monkey flows are about four times that of humans. However, irrespective of flow rate, the typical shape of maximum expiratory flow-volume (MEFV) curves of rhesus monkeys is similar to that of normal young adults and that of bonnet monkeys is similar to that of normal older adults. Flow-limiting mechanisms that may determine MEFV curve shape and explain the differences and similarities of monkey and human MEFV curves are discussed. We propose that differences in function between bonnet and rhesus monkeys relates to the concept of dysanaptic lung growth that evolved to subserve behavioral differences between the two species of monkey.


Isolation and structure elucidation of a novel griseorhodin.

Three antibiotics possessing cytotoxic properties were isolated from a strain of Streptomyces griseus (FCRC-57). One was found to be identical with griseorhodin A. A second, FCRC-57-U, was found to be identical to griseorhodin C. FCRC-57-G is a new antibiotic structurally related to griseorhodins A and C, and is active against KB cells in vitro. The structure of this new antibiotic was determined using mass spectrometry, proton and carbon nuclear magnetic resonance spectroscopy and synthesis.


beta-Galactosidase-inhibiting new isoflavonoids produced by actinomycetes.

Six isoflavonoids having beta-galactosidase inhibiting activity were isolated from the culture filtrate of Streptomyces xanthophaeus. Their structures were determined by spectral analyses to be daidzein, daidzein 7-alpha-L-rhamnoside, daidzein 4',7-di-alpha-L-rhamnoside, genistein, genistein 7-alpha-L-rhamnoside and genistein 4',7-di-alpha-L-rhamnoside.


R plasmids R91 and R91a from Pseudomonas aeruginosa share only the gene for carbenicillin resistance.

Plasmid R91a of Pseudomonas aeruginosa strain 9169 is homologous with RP1. Plasmid R91 carried by the same strain is related in the Tn1 region but is otherwise unrelated to R91a.


Lactose metabolism involving phospho-beta-galactosidase in Klebsiella.

Klebsiella strain RE1755A is a Lac- Gal- mutant which has lost both of its lac operons, but possesses a gene specifying beta-galactosidase III, an enzyme which hydrolyzes o-nitrophenyl-beta-D-galactopyranoside but does not hydrolyze lactose. Selective pressure was applied to isolate mutants able to utilize lactose. The lactose-utilizing mutants obtained were shown to possess an unaltered beta-galactosidase III. Lactose utilization was shown to result from a pleiotropic mutation which also (i) permits galactose utilization and (ii) prevents induction of beta-galactosidase III synthesis by lactose. Evidence is presented suggesting that a phospho-beta-galactosidase enzyme is involved in lactose metabolism.


Release of autolytic enzyme from Streptococcus, faecium cell walls by treatment with dilute alkali.

The autolytic enzyme (endo-beta-1,4-N-acetylmuramoylhydrolase) of Streptococcus faecium (S. faecalis ATCC 9790) was released in a soluble form from insoluble cell wall-autolytic enzyme complexes by treatment with dilute NaOH at 0 degree C. Treatment of cell wall-enzyme complexes, obtained from either exponential- or stationary-phase cells, with 0.008 to 0.01 N NaOH gave maximum yields of autolytic enzyme activity. At a fixed concentration of NaOH, the yield of autolysin increased with increasing wall densities and was accompanied by the release of methylpentose and phosphorus in amounts proportional to the autolysin. Since extraction of wall-enzyme complexes with 4.5 M LiCl at 0 degree C also removed methylpentose and phosphorus, release of enzyme with NaOH did not appear to result from hydrolysis of covalent linkages. The autolytic enzyme activity released from intact cells, or cell walls, was predominantly in the later (proteinase activable) form which could be activated by trypsin or a proteinase present in commerical bovine plasma albumin.


Use of chlC-lac fusions to determine regulation of gene chlC in Escherichia coli K-12.

Gene fusions between the lac structural genes and the chlC locus were isolated, and the regulation of lac gene expression was studied. The fused lac genes were induced by nitrate anaerobically and repressed by the presence of oxygen.


Suppressor mutation in Pseudomonas aeruginosa.

Suppressor mutations were identified in Pseudomonas aeruginosa, and a comparison was made with Escherichia coli suppressor systems. A suppressor-sensitive (sus) derivative of a plasmid, RP4 trp, and several Sus mutants of IncP1 plasmid-specific phages, were isolated by using E. coli. Plasmid RP4 trp (sus) was transferred to P. aeruginosa strains carrying trp markers which did not complement RP4 trp(sus), and Trp+ variants were selected. Some, but not all such revertants, could propagate PRD1 Sus phages, and these mutants were found to be supressor positive. Plating efficiencies of various Sus phages on these strains were compared with on E. coli strains carrying known suppressor genes. The results suggested that the Pseudomonas suppressors were probably amber suppressors. In iddition, some Sus phages (PRD1sus-55, PRD1sus-56) were obtained which, although apparently of the amber type for E. coli, were able to propagate equally well on sup+ or sup strains of P. aeruginosa. On the other hand, several mutants of phage PRR1 which were suppressed in E. coli were not suppressed by the P. aeruginosa suppressor. Suppressor-sensitive mutants were also isolated with P. aeruginosa bacteriophages E79 and D3.


Genetic exchange mechanisms in Neisseria gonorrhoeae.

There are two mechanisms for genetic exchange in Neisseria gonorrhoeae. Plasmid deoxyribonucleic acid can be transferred by conjugation, which is dependent on the presence of a 24.5-megadalton plasmid in the donor cell. We have shown that chromosomal deoxyribonucleic acid can be exchanged between all colonial variants by transformation, but not by conjugation. In the nonpiliated variants, however, this exchange was dependent on the presence of the 24.5-megadalton plasmid in the recipient cell.


Genetic basis for colonial variation in Neisseria gonorrhoeae.

When the piliated colony types of Neisseria gonorrhoeae, which predominate in recent isolates, were nonselectively subcultured in vitro, they gave rise to large numbers of nonpiliated, avirulent colonial variants. Evidence is presented to show that most of this variation occurs after active growth has ceased and that the variation is sensitive to the action of deoxyribonuclease. We suggest that this variation is a result of transformation. A second variation in colonial morphology involved differing levels of "colony opacity-associated proteins" in the outer membrane. This variation was also inhibited by the presence of deoxyribonuclease, but the genetic basis for it is not as yet clear.


Biosynthesis of phenazine pigments in mutant and wild-type cultures of Pseudomonas aeruginosa.

Pigmentation mutants of Pseudomonas aeruginosa, selected by observed visual differences in coloration from the wild-type strain, were examined for altered patterns of phenazine synthesis. Three classes of mutants that were incapable of pyocyanine production were identified. Pigmentation patterns that were found to characterize the various mutant classes implicated precursor-product relationships, and a biochemical scheme covering the terminal reactions of pyocyanine biosynthesis is proposed. Among compounds tested as inhibitors of pigmentation, two effectively inhibited pyocyanine production production while allowing cell growth. p-Aminobenzoate inhibited total pigmentation; i.e., no other phenazine accumulated. m-Aminobenzoate inhibited a presumptive methylation step in pyocyanine biosynthesis, abolishing the formation of pyocyanine and aeruginosin pigments but increasing the yields of phenazine 1-carboxylic acid and oxychlororaphin. D-[2,3,4,5(n)-14C]shikimate was most efficiently incorporated into phenazines in the middle to late exponential phase of growth. Label was incorporated predominantly into pyocyanine in the absence of inhibitors and into phenazine 1-carboxylic acid when the organism was grown in the presence of m-aminobenzoate.


Use of 8-hydroxyquinoline to enrich for temperature-sensitive mutants of Tetrahymena.

The effects of the chelating agent 8-hydroxyquinoline (Hq) on Tetrahymena thermophila were examined. Cell division was completely inhibited by 5 micrograms of Hq per ml. At this concentration deoxyribonucleic acid, ribonucleic acid, and protein syntheses were also completely and nonselectively inhibited. The inhibition was reversible after 6 h of Hq treatment. At concentrations above 20 micrograms/ml a 10,000-fold decrease in survival as seen after 2 h in the drug. The sensitivity of Tetrahymena to Hq was found to be dependent upon cell concentration, wild-type strain, medium, and length of time the culture is at 38 degrees C before Hq is added. Mutants of Tetrahymena that are unable to divide at the restrictive temperature, but which continue macromolecular synthesis, were found to be resistant to Hq treatment. Conditions were obtained in which more than a 1,000-fold difference in survival was seen between this class of mutant and the wild type. The effect of Hq on three other classes of temperature-sensitive mutants was examined, and the results are discussed.


Selective association of the chromosome with membrane in a stable L-form of Bacillus subtilis.

A stalbe L-form (Sal-1) of Bacillus subtilis was found to have retained a markedly modified chromosome-membrane association when compared to intact cells. The membrane-deoxyribonucleic acid complex of the L-form was similar to that of its parental strain in quantity and stability. Genetic analysis of the L-form membrane-deoxyribonucleic acid complex revealed enrichment for markers close to the replication origin, but not for internal markers, indicating preferential attachment of the origin of chromosomal replication to the membrane. These results are in close agreement with those found for the parental bacterial form. In contrast, the replication termius region was not preferentially attached to the membrane of the L-form, even though it is enriched in the bacterial form. The association of the chromosome with the membrane at the replication terminus does not appear to be necessary for cell growth and separation, but because the L-form divides aberrantly, it may be one of the factors required for normal deoxyribonucleic acid segregation and septation.


Transient growth inhibition of Escherichia coli K-12 by ion chelators: "in vivo" inhibition of ribonucleic acid synthesis.

The ion chelators picolinic acid, quinaldic acid, 1,10-phenanthroline, and 8-hydroxyquinoline, but not ethylenediaminetetraacetate, ethyleneglycol-bis-(beta-aminoethyl ether)-N,N-tetraacetate, or dipicolinic acid, rapidly but transiently arrest growth of Escherichia coli K-12. Cells adapt and become resistant to growth inhibition by these agents, a process which requires protein synthesis. Mn2+, at low concentrations, decreases the time required for resumption of growth. Proteins synthesized during the lag are quantitatively and qualitatively different from those synthesized during normal growth. Inhibition of growth can explained by an effect on RNA polymerase, a known metalloenzyme.


Deoxyribonucleic acid sequence common to staphylococcal and streptococcal plasmids which specify erythromycin resistance.

Plasmids from erythromycin-resistant Staphylococcus aureus, Streptococcus sanguis, and Streptococcus faecalis show deoxyribonucleic acid sequence homology. The homologous sequences can be localized to specific restriction endonuclease fragments, which in the case of S. aureus plasmid pI258 involves a single fragment from either EcoRI or HindIII digest known to contain the erythromycin resistance determinant. Complementary ribonucleic acid probes prepared from S. aureus plasmid pI258 and S. sanguis plasmid pAM77 also hybridize to specific fragments in restriction endonuclease digests of deoxyribonucleic acid from erythromycin-resistant Streptococcus progenes and Streptococcus pneumoniae. These studies suggest a common origin for a class of erythromycin resistance determinants in unrelated strains of pathogenic bacteria for which exchange of genetic material has not been demonstrated.


Effect of carbamoyl phosphate on nitrogenase in Anabaena cylindrica Lemm.

Carbamoyl phosphate inhibited acetylene reduction by whole cells and cell-free extracts of Anabaena cylindrica. Higher levels of both endogenous carbamoyl phosphate and carbamoyl phosphate synthase activity were present in NH4+-grown cells (in which acetylene reduction was absent) than in N2-grown cells (in which acetylene reduction was present). However, inhibition of acetylene reduction was observed also with cyanate, the main initial decomposition product under the conditions used. It is concluded that carbamoyl phosphate or one of its metabolites may act as a physiological regulator of both nitrogenase activity and synthesis, but caution must be used in interpreting effects observed several hours after the addition of carbamoyl phosphate, because the effects may be due to cyanate.


Sites within gene lacZ of Escherichia coli for formation of active hybrid beta-galactosidase molecules.

We describe the genetic analysis of 21 Escherichia coli strains in which the amino-terminal sequence of beta-galactosidase has been removed and replaced by an amino-terminal sequence from one or another of the proteins involved in maltose transport. Genetic mapping of the lacZ end of these fused genes indicates that only those fusions in which fewer than 41 amino acids are removed from the amino-terminal sequence of beta-galactosidase result in enzymatically active molecules. Within the region between amino acid 17 and amino acid 41 there are at least four or five sites where enzymatically active hybrid proteins can be formed.


Transduction of Pseudomonas aeruginosa with a mutant of bacteriophage E79.

A mutant of the virulent bacteriophage E79 was isolated which mediated generalized transduction in Pseudomonas aeruginosa. Variable recovery of transductants as a result of phage killing was avoided by the use of recipients carrying the IncP-2 plasmid R38, and transduction frequencies of 4 X 10(-6) to 1 X 10(-5) per plaque-forming unit were obtained. Linkage studies have indicated that the coinheritance frequencies are less than would be expected from the published molecular weight of E79 deoxyribonucleic acid (120 X 10(6). By using recipients carrying R38, low-frequency transduction by wild-type E79 and two other virulent phages, F8 and phi 16, was demonstrated.


Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm.

We have employed the technique of gene fusion to fuse the LacZ gene encoding the cytoplasmic enzyme beta-galactosidase with the malE gene encoding the periplasmic maltose binding protein (MBP). Strains were obtained which synthesize malE-lacZ hybrid proteins of various sizes. These proteins have, at their amino terminus, a portion of the MBP and at their carboxyl terminus, enzymatically active beta-galactosidase. When the hybrid protein includes only a small, amino-terminal portion of the MBP, the hybrid protein residues in the cytoplasm. When the hybrid protein contains enough of the MBP to include an intact MBP signal sequence, a significant portion of the hybrid protein is found in the cytoplasmic membrane, suggesting that secretion of the hybrid protein has been initiated. However, in no case is the hybrid protein secreted into the periplasm, even when the hybrid protein includes almost the entire MBP. In the latter case, the synthesis and attempted export of the hybrid protein interferes with the export of at least certain normal envelope proteins, which accumulate in the cell in their precursor forms, and the cell dies. These results suggest that a number of envelope proteins may be exported at a common site, and that there are only a limited number of such sites. Also, these results indicate that it is not sufficient to simply attach an amino-terminal signal sequence to a polypeptide to assure its export.


Three types of mycolic acid from Mycobacterium tuberculosis Brévanne: implications for structure-function relationships in pathogenesis.

Saponification of the chloroform-soluble wax from Mycobacterium tuberculosis Brévanne led to the isolation of three classes of mycolic acid containing characteristic functional groups along the methylene backbone: type alpha (two cyclopropane rings); type beta (methoxyl, methyl, and cyclopropane); and type gamma (ketone, methyl, and cyclopropane). The structures of these acids were elucidated principally by mass spectrometry. The high mass region of the keto mycolate is presented showing the meromycolal and molecular ion regions. This is first time a molecular peak for this mycolic acid has been reported. The structure of the keto mycolate was further substantiated by study of the mass spectral fragmentation of its dithioketal derivative. Within each type of acid, a series of homologs was encountered, varying according to the number of methylene units in the backbone chain. Chromatographic and infrared spectrophotometric evidence is presented for the alkali-induced isomerization of the three types of mycolates.


Gap-filling repair synthesis induced by ultraviolet light in a Bacillus subtilis Uvr- mutant.

Deoxyribonucleic acid repair synthesis was studied in one wild-type and two mutant strains of Bacillus subtilis that are defective in excision of pyrimidine dimers. The cells were irradiated with ultraviolet light, and 6-(p-hydroxyphenyl-azo)-uracil was used to block replicative synthesis, allowing only repair synthesis. One of the mutations (uvs-42) resulted in a severe inhibition of incision, dimer excision, and repair synthesis. In contrast, the other mutant (uvr-1) slowly incised and excised dimers and did repair synthesis in patches which appear to be several-fold longer than those in the wild-type strain, apparently because large gaps are produced at excision sites. The results indicate that the primary defect in uvs-42 cells is in initiation of dimer excision, whereas the uvr-1 mutation appears to be a defect in the exonuclease normally used to complete dimer excision.


Pyrimidine dimer excision in a Bacillus subtilis Uvr- mutant.

A technique which allows the measurement of small numbers of pyrimidine dimers in the deoxyribonucleic acid (DNA) of cells of Bacillus subtilis irradiated with ultraviolet light has been used to show that a strain mutant at the uvr-1 locus is able to excise pyrimidine dimers. Excision repair in this strain was slow, but incision may not be rate limiting because single-strand breaks in DNA accumulate under some conditions. Excision repair probably accounted for a liquid-holding recovery previously reported to occur in this strain. Recombinational exchange of pyrimidine dimers into newly replicated DNA was readily detected in uvr-1 cells, but this exchange did not account for more than a minor fraction of the dimers removed from parental DNA. Excision repair in the uvr-1 strain was inhibited by a drug which complexes DNA polymerase III with DNA gaps. This inhibition may be limited to a number of sites equal to the number of DNA polymerase III molecules, and it is inferred that large gaps are produced by excision of dimers. Because the uvr-1 mutation specifically interferes with excision of dimers at incision sites, it is concluded that the uvr-1 gene product may be an exonuclease which is essential for efficient dimer excision.


Transposition of Tn7 in Pseudomonas aeruginosa and isolation of alk::Tn7 mutations.

Conjugal crosses with Pseudomonas aeruginosa donors carrying the CAM-OCT and RP4::Tn7 plasmids result in transfer of the Tn7 trimethoprim resistance (Tp(r)) determinant independently of RP4 markers. All Tp(r) exconjugants which lack RP4 markers have CAM-OCT genes and therefore must have received CAM-OCT::Tn7 plasmids formed by transposition of Tn7 from RP4::Tn7 to CAM-OCT. Most crosses yield exconjugants carrying mutant CAM-OCT plasmids which no longer determine either camphor or alkane utilization and thus appear to carry Tn7 inserts in the cam or alk loci, respectively. Transduction and reversion experiments indicated that at least 13 alkane-negative, camphor-positive, Tp(r) CAM-OCT::Tn7 plasmids carry an alk::Tn7 mutation. Determination of linkage between the alk mutation and the Tp(r) determinant of Tn7 on these plasmids is complicated by the presence of multiple copies of the Tn7 element in the genome. Generalized transduction will remove Tn7 from a CAM-OCT alk::Tn7 plasmid to yield alk(+) cells which carry no Tp(r) determinant on the CAM-OCT plasmid (as shown by transfer of the plasmid to a second strain). But the transduction to alk(+) does not remove all Tp(r) determinants from the genome of the recipient cell because the alkane-positive transductants remain trimethoprim resistant. Thus, it appears that copies of Tn7 can accumulate in the genome of P. aeruginosa (CAM-OCT alk::Tn7) strains without leaving their original site. This result is consistent with transposition models that involve replication of the transposable element without excision from the original site.


Genetic recombination during transformation in Bacillus subtilis: appearance of a deoxyribonucleic acid methylase.

In Bacillus subtilis the ability to take up deoxyribonucleic acid (DNA) and undergo genetic transformation may coincide with the induction of defective phage(s) and the expression of possibly related cryptic genes. A restriction-modification enzyme system appears to be expressed. Targets of the restriction activity on the DNA can be blocked my methylation catalyzed by the methyl transferase. It is shown that cellular DNA becomes progressively methylated and reaches the maxium level during the peak of competency. Deoxycytidine residues of both incoming donor and resident DNA are methylated. The possible participation of these enzymes in recombination and the general role of cryptic genes in inducible functions are discussed.


Demonstration of an internal fraction plane in cell walls of Streptococcus faecalis and Streptococcus mutans.

The proposed lower internal density of the gram-positive wall was confirmed by observed an internal fracture plane in the walls of Streptococcus faecalis and Streptococcus mutans. However, the granular surfaces produced by this cleavage appeared to be more of a reflection of distortion during preparation than of subunit construction.


Altered membrane proteins in a minicell-producing mutant of Bacillus subtilis.

The Bacillus subtilis minicell-producing mutant divIV-B1 has a membrane protein profile that is strikingly different from that of the other minicell-producing mutant, divIV-A1, or that of wild-type strain CU403.


Introduction of host-controlled modification and restriction systems of Bacillus subtilis IAM1247 into Bacillus subtilis 168.

Bacillus subtilis IAM1247 had two modification and restriction systems (Bsu1247I and Bsu1247II), the former producing an isoschizomer of PstI endonuclease. A transformant clone was isolated which had Bsu 168, BsuR, and Bsu1247I systems coexisting within a genome.


Characteristics of Anabaena variabilis influencing plaque formation by cyanophage N-1.

Phage N-1 grown in Anabaena strain 7120 [N-1 . 7120] forms plaques on A. variabilis about 10(-7) to 10(-6) as efficiently as on Anabaena 7120. By manipulating different characteristics of the interaction between phage and host, it was possible to increase the relative efficiency of plaque formation to 0.38. Growth of A. variabilis at 40 degrees C for at least three generations resulted in an increase in the rate of phage adsorption and a 10-fold increase in the efficiency of plaque formation. The efficiency of plaque formation was further increased about 42-fold, with little or no further increase in rate of adsorption, in a variant strain. A. variabilis strain FD, isolated from a culture of A. variabilis which had grown for more than 30 generations at 40 degrees C. The low relative efficiency of plaque formation by N-1 . 7120 on A. variabilis could be partially accounted for if A. variabilis contains a deoxyribonucleic acid restriction endonuclease which is absent from Anabaena 7120. Indirect evidence for such an endonuclease included the following: (i) phage N-1 grown in A. variabilis (N-1 . Av) had approximately a 7 X 10(3)-fold higher relative efficiency of plaque formation on A. variabilis than had N-1 . 7120; and (ii) the efficiency of plaque formation by N-1 . 7120 on A. variabilis strain FD was increased by up to 146-fold after heating the latter organism at 51 degrees C.


Genetic evidence for possible interaction between a ribonucleic acid polymerase subunit and the spo0C gene product of Bacillus subtilis.

Spontaneous rifampin-resistant mutants (9V Rifr) were isolated from a mutant strain of Bacillus subtilis, 9V, which has a spo0C mutation. Whereas 90% of the 9V Rifr double mutants maintained the Spo0C phenotype (Spo- Abs +/-), the remaining 10% had the Spo0A phenotype (Spo- Abs-). The latter mutants, termed 9V Rifr Spo- Abs-, were revealed to have other Spo0A characters, such as reduced transformability, higher sensitivity to phage phi 2, and reduced frequency of lysogenization by phage phi 105. The rif mutation of these 9V Rifr Spo- Abs- strains was mapped near the cysA locus. The phenotype of the Rifr transformants of strain 9V by deoxyribonucleic acid derived from these 9V Rifr Spo- Abs- strains was Spo0A, and that of the Rifr transformants of strain 168 was Spo+ Abs+. The ribonucleic acid polymerase of the 9V Rifr Spo- Abs- strains was shown to be resistant to rifampin.


Isolation and characterization of two tryptophan biosynthetic enzymes, indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, from Bacillus subtilis.

Two of the enzymes responsible for tryptophan biosynthesis in Bacillus subtilis have been extensively purified. These proteins are indole-3-glycerol phosphate synthase and N-(5'-phosphoribosyl) anthranilate isomerase. By comparison to the non-differentiating enteric bacteria in which these two enzymes are fused into a single polypeptide, the isolation of the indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase from B. subtilis has demonstrated that the two proteins are separate species in this organism. The two enzymes were clearly separable by anion-exchange chromatography without any significant loss of activity. Molecular weights were determined for both enzymes by gel filtration and sodium dodecyl sulfate-slab gel electrophoresis, and indicated that the indoleglycerol phosphate synthase is the slightly larger of the two proteins. The minimum molecular weight for indoleglycerol phosphate synthase was 23,500, and that for phosphoribosyl anthranilate isomerase was 21,800. Both enzymes have been examined as to conditions necessary to achieve maximal activity of their individual functions and to maintain that activity.


Localization of low-molecular-weight basic proteins in Bacillus megaterium spores by cross-linking with ultraviolet light.

Two low-molecular-weight basic proteins, termed A and B proteins, comprise about 15% of the protein of dormant spores of Bacillus megaterium. Irradiation of intact dormant spores with ultraviolet light results in covalent cross-linking of the A and B proteins to other spore macromolecules. The cross-linked A and B proteins are precipitated by ethanol and can be solubilized by treatment with deoxyribonuclease (75%) or ribonuclease (25%). Irradiation of complexes formed in vitro between deoxyribonucleic acid (DNA) or ribonucleic acid and a mixture of the low-molecular-weight basic proteins from spores also resulted in cross-linking of A and B proteins to nucleic acids. The dose-response curves for formation of covalent cross-links were similar for irradiation of both a protein-DNA complex in vitro and intact spores. However, if irradiation was carried out in vitro under conditions where DNA-protein complexes were disrupted, no covalent cross-links were formed. These data suggest that significant amounts of the low-molecular-weight basic proteins unique to bacterial spores are associated with spore DNA in vivo.


Coupling of energy to folate transport in Lactobacillus casei.

Lactobacillus casei cells can accumulate folate to an intracellular concentration in excess of 500 muM and to concentration gradients (relative to the extracellular compartment) of several thousand-fold. Maximum rates of folate transport are achieved rapidly (t(1/2) < 1 min) after the addition of glucose to energy-depleted cells and occur at intracellular adenosine 5'-triphosphate concentrations above 625 muM. The rate of folate transport and the adenosine 5'-triphosphate content of cells are both extremely sensitive to arsenate and decrease in parallel with increasing concentrations of the inhibitor, indicating a requirement for phosphate-bond energy in the transport process. The energy source is not a membrane potential or a pH gradient generated via the membrane-bound adenosine triphosphatase, since dicyclohexylcarbodiimide (an adenosine triphosphatase inhibitor) and carbonyl cyanide m-chlorophenylhydrazone (a proton conductor) have little effect on the uptake process. The K(+)-ionophore, valinomycin, is an inhibitor of folate transport, but does not act via a mechanism involving dissipation of the membrane potential. This can be deduced from the facts that the inhibition by valinomycin is relatively insensitive to pH, is considerably greater in Na(+)- than in K(+)-containing buffers, and is not enhanced by the addition of proton conductors. Folate efflux is not affected by valinomycin, glucose, or various metabolic inhibitors, although a rapid release of the accumulated vitamin can be achieved by the addition of unlabeled folate together with an energy source (glucose). These results suggest that the active transport of folate into L. casei is energized by adenosine 5'-triphosphate or an equivalent energy-rich compound, and that coupling occurs not via the membrane-bound adenosine triphosphatase but by direct interaction of the energy source with a component of the transport system.


Ribonucleic acid polymerase mutation affecting glutamate synthase activity in and sporulation of Bacillus subtilis.

Spore formation of 15 rifampin-resistant (Rifr) mutants of Bacillus subtilis strain 168 was examined. As a pleiotropic effect of a Rifr mutation, glutamate synthase activity was lost in these mutants. Twelve of the 15 mutants examined formed as many spores as the parent, but the remaining 3 formed significantly fewer (1%) spores. One of the latter mutants characterized further (RF301) was blocked in its sporulation process at stage 0. Thus, it was concluded that a certain modification of ribonucleic acid polymerase may affect specifically the gene expression of glutamate synthase and also the sporulation process at the initial stage.


Genetic location of the Bacillus subtilis sup-3 suppressor mutation.

The sup-3 suppressor mutation of Bacillus subtilis has been located between the aroI and mtlB loci by PBS-1 phage transduction.


Increased synthesis of ribonucleotide reductase after deoxyribonucleic acid inhibition in various species of bacteria.

The specific activity of ribonucleotide reductase was found to increase significantly after deoxyribonucleic acid inhibition in seven species of bacteria investigated. This group of bacteria includes species with B12-dependent ribonucleotide reductase as well as some with an Escherichia coli-type ribonucleotide reductase.


Properties of common wheat ferredoxin, and a comparison with ferredoxins from related species of triticum and aegilops.

Wheat ferredoxin was purified from the leaves of common wheat (Triticum aestivum). The absorption spectrum showed maxima at 465, 425, 332, and 278 nm. The absorbance ratio, A425 nm/A278 nm was 0.49, and the millimolar extinction coefficient at 425 nm was 10.8 mM-1. cm-1. The amino acid composition was determined to be Lys5, His2, Arg1, Asp11, Thr5, Ser7, Glu18, Pro5, Gly6, Ala7, Cys5, Val7, Met1, Ile4, Leu7, Tyr4, Phe1, and Trp1. The total number of amino acid residues was 97. The molecular weight was calculated from the amino acid composition to be 10,829, including iron and sulfur atoms. This value was confirmed by other methods, which were based on the contents of non-heme iron and of terminal amino acid. The N-terminal amino acid was alanine, and the C-terminal amino acid sequence was -Glu-Leu-Thr-AlaCOOH. Comparative studies were performed between T. aestivum ferredoxin and ferredoxins isolated from closely related species; these were T. aegilopoides, T. durum, Ae. squarrosa, and Ae. ovata. No significant differences in the properties of these ferredoxins were detected. It was also shown that these ferredoxins are immunologically homologous. It is, therefore, likely that one molecular species of ferredoxin is distributed through two genera of Triticum and Aegilops.


Sulfhydryl groups of pyocin R1. Morphology and activity modified with sulfhydryl reagents.

Electron microscopic observation of pyocin R1 with the negative staining technique demonstrated that pyocin R1 retained its phage tail-like shape of an extended sheath even when it was inactivated by treatment with p-chloromercuribenzoic acid (PCMB) or 4-(p-sulfophenylazo)-2-mercuriphenol (SAMP). Thus it was shown that the contraction and extension of the sheath does not occur reversibly on the modification of sulfhydryl groups accompanying the change of activity. The activity lost under these conditions was restored to the original level by treatment with 2-mercaptoethanol (2-ME). Numbers of sulfhydryl groups in the pyocin R1 particle were determined to be 208 mol and 152 mol per mol (11.8 x 10(6) daltons) by spectrophotometric titration with SAMP and by membrane-filter assay with radioactive PCMB, respectively. Most of these cysteine residues appeared to be localized in the substructure other than the sheath and core. It was also shown that all of these sulfhydryl groups were not necessary for expression of its activity but a part of them were essential for adsorption to the sensitive cells.


Refolding of the immunoglobulin light chain.

The kinetics of the refolding reactions of type lambda Bence Jones proteins from 4 M GuHCl were studied by CD, ultraviolet absorption, and fluorescence spectrophotometry. The kinetics were complex and consisted of at least three phases, an undetectable fast phase, a detectable fast phase, and a slow phase. The slow phase followed first-order kinetics and the three experimental methods used gave similar rate constants for all the Bence Jones proteins (about 3 X 10(-3) s-1). The refolding reaction of VL fragment was too fast to be measured in the present experiments. The refolding process of CL fragment was very similar to those of Bence Jones proteins except that the detectable fast phase was less significant. The rate constant of the slow phase observed for the CL fragment was similar to those of the slow phase observed for Bence Jones proteins. The activation energy of the slow phase was the same for a Bence Jones protein and its CL fragment. These results indicate that the refolding kinetics of the CL domain are very similar to those of isolated CL fragment and that refolding of the VL domain precedes refolding of the CL domain, even though both domains have similar immunoglobulin folds. However, the results of refolding experiments on Bence Jones proteins, and VL and CL fragments in the presence of ANS, as well as the other lines of evidence, indicate that the refolding kinetics of the Bence Jones protein molecule cannot be expressed as simple sum of the refolding reactions of isolated VL and CL fragments.


Taxonomic implications of primate dental tissues.

Histological studies of dental tissues will provide new and important insights into mammalian evolution. From studies of dental histology, hominoid evolution has become much clearer. Data as to the thickness of enamel and enamel prism patterns clearly provide quantitative support concerning theories of hominoid/hominid differentiation. The importance of comparative dental histology has only recently been realized and when extended to other mammalian orders, including both living and fossil forms, a more complete and clearer understanding of mammalian evolution will emerge. Caution must be emphasized, however, for these features are extremely complex structures and without a thorough knowledge of comparative dental histology and the applications of the SEM results may be misleading or incorrect.


Identification of an NADH-linked disulfide reductase from Bacillus megaterium specific for disulfides containing pantethine 4',4''-diphosphate moieties.

Bacillus megaterium contains an NADH-linked disulfide reductase that is specific for disulfides containing pantethine 4',4''-diphosphate moieties. This reductase is at its highest level in cells late in sporulation and in dormant spores, and could be involved in the formation and cleavage of coenzyme A-protein disulfides which take place late in sporulation and early in spore germination, respectively.


Recombination of heavy and light chains of immunoglobulins. Use of specific variable and constant region probes to monitor reassociation reactions.

The reassociation of antibody (IgG)-derived heavy (gamma) and light (L) chains was studied by utilizing two specifically localized fluorescent probes. L chains labeled in either the variable domain with fluorescein (specific antigen) or the constant domain with N-(iodoacetamidoethyl)-5-naphthylamine-1-sulfonic acid were prepared. Reassociation of these chains with isolated, unlabeled, gamma chains was monitored by following an increase in the fluorescence polarization of fluorophores at 4 degrees C. Results obtained indicate that the reassociation reaction could be resolved into two distinct steps; the primary interaction of the reactants occurring rapidly, followed by a slower secondary phase possibly representing conformational changes in the variable domains of the reactants after reassociation.


The relationship between glandular kallikrein and growth factor-processing proteases of mouse submaxillary gland.

Three highly specific trypsin-like proteases from mouse submaxillary gland; nerve growth factor gamma subunit, beta nerve growth factor-endopeptidase, and epidermal growth factor-binding protein were tested for kallikrein activity. Low molecular weight kininogen was purified from mouse plasma and used as substrate for the three enzymes, and the kinin released by the enzymes was assayed by its ability to induce contraction of isolated rat uterus. All three enzymes were found to have significant kininogenase activity, and the most active enzyme, beta nerve growth factor-endopeptidase, has activity comparable to authentic kallikreins from other glandular sources. Essentially all of the kininogenase activity of submaxillary gland co-purifies with beta nerve growth factor-endopeptidase. Hence, beta nerve growth factor-endopeptidase appears to be identical with submaxillary gland kallikrein. Nerve growth factor gamma subunit, epidermal growth factor-binding protein, and beta nerve growth factor-endopeptidase have similar amino acid compositions and molecular weights, and are immunologically similar. Comparison of published partial primary sequence data confirms our conclusion that nerve growth factor gamma subunit, epidermal growth factor-binding protein, and kallikrein are very closely related enzymes. It is postulated that these three enzymes are members of a larger family of similar enzymes, all of which are involved in the processing of precursors to polypeptide hormones and growth factors.


Transport and binding of riboflavin by Bacillus subtilis.

Riboflavine uptake and membrane-associated riboflavin-binding activity has been investigated in Bacillus subtilis. Riboflavin uptake proceeds via a system whose general properties are indicative of a carrier-mediated process: it is inhibited by substrate analogues, exhibits saturation kinetics, and is temperature-dependent. The organism concentrates riboflavin primarily as the phosphorylated cofactors FMN and FAD. Energy is required for uptake but whether the energy demand is required for both uptake and phosphorylation or only for the phosphorylation step is not known. Membrane-associated binding activity for riboflavin has also been demonstrated in membrane vesicles prepared from B. subtilis, and the binding component can be "solubilized" with Triton X-100. Evidence supporting the function of the binding component in riboflavin uptake by the intact cells includes the following. (i) Riboflavin analogues inhibit binding and uptake to nearly the same extent and with similar specificity of action. (ii) The KD for riboflavin-binding and the Km for uptake are in the same range. Similarly the Ki determined for the inhibitory analogue 5-deazariboflavin in the uptake assay and the KD for its interaction with the riboflavin-binding component of membrane vesicles are in the same range. (iii) Uptake in cells and binding in vesicles vary in the same direction with differences in growth conditions.


The primary structure of the alpha subunit of protocatechuate 3,4-dioxygenase. I. Isolation and sequence of the tryptic peptides.

The carboxymethylated alpha subunit of protocatechuate 3,4-dioxygenase was digested with trypsin. The 14 tryptic peptides were isolated by ion exchange chromatography on DEAE-Sephadex and by gel filtration chromatography. Automated Edman degradation and carboxypeptidase Y and B digestion were used to establish the sequence of these peptides. Further fragmentation of two tryptic peptides, T3 and T5, by Staphylococcus aureus protease and cyanogen bromide, respectively, was necessary to complete the sequences. The tryptic peptides accounted for a minimum of 199 residues out of a total of 202 residues predicted by amino acid analysis.


In vivo evaluation of a high-strength, high-ductility stainless steel for use in surgical implants.

A high-strength, high-ductility, austenitic stainless steel has been evaluated for use in surgical implants by performing in vivo tests in rats, rabbits, dogs, and rhesus monkeys. This stainless steel, a TRIP (Transformation Induced Plasticity) steel containing about 4% Mo, was compared with two alloys in current clinical use: Type 316L stainless steel and cast Vitallium. Compared with the other two alloys, cast Vitallium generally had higher resistance to corrosion and superior biocompatibility in all animals. The tests in rats and dogs indicated that the corrosion resistances of the TRIP steel and the Type 316L stainless steel were similar and that the tissue reactions caused by these alloys were also similar. However, in rhesus monkeys, the TRIP steel was shown to be susceptible to stress-corrosion cracking and much more susceptible to crevice corrosion than Type 316L stainless steel. Limited tests in rabbits supported the observation that the TRIP steel is susceptible to stress-corrosion cracking. These inconsistencies in the in vivo tests underline the need for a reevaluation of the popular test techniques and of the animals commonly chosen for assessing the suitability of candidate implant materials. The "worst case" results from the rhesus monkey tests were entirely consistent with previous results obtained from in vitro studies. However, further work must be performed before the behavior of metals in humans, rhesus monkeys, or any other animal, can be predicted with confidence from an in vitro test program.


Tissue response to surface-treated tantalum implants: preliminary observations in primates.

Samples of capacitor grade tantalum were surface-treated by a variety of methods. These surface treatments allowed testing of the same basic material which was mill-finished, metallurgically polished, electrochemically oxidized, sintered with a porous surface, and glow-discharged. Surface characterization was accomplished by contact angle measurements, Scanning Electron Microscopy, energy-dispensed x-ray analysis, and internal reflection spectroscopy. Subsequent to characterization, the material was surgically implanted in the subperiosteal region of the mandible, the buccal mucosa, and the subcutaneous paravertebral region of the back of Macaca speciosa (stumptail monkey). The tissue reaction at intervals of up to three weeks was evaluated morphologically and ultrastructurally. Significant differences in tissue response were noted at the interfaces with glow-discharge-treated versus lower surface energy samples. Adjacent to the glow-discharge-treated implants, two distinct tissue zones were identified. Zone No. 1, nearest the implant, exhibited an increased cellularity. This consisted of 4-5 layers of highly active mesenchymal cells or fibroblast-like cells with spindle-shaped nuclei and prominent cytoplasmic features. At various foci along the interface, hyperchromatic nuclear forms were noted to project into the space left by removal of the implant. These observations, coupled with a predominance of intercellular ground-substance material and less collagen at the interface, may indicate some form of bioadhesion. The deeper Zone No. 2 was 2-3 times as thick consisted of typical fibroblastic cells with a lamellar configuration, bordered by an occasional delicate-lined space. Independent of implantation site or surface texture, all other implants showed occasional multinucleated giant cells and a decrease in the cellular character of Zone No. 1. Both zones were reduced in thickness and composed of more mature fibroblasts. Some specimens exhibited intracytoplasmic vacuolization. It may be concluded, therefore, that surface-free energy of the implanted specimens played a significant role in inducing differential tissue response to otherwise similar pure metal samples.


Influence of methyl methacrylate on quantitative gel diffusion assay of immunoglobulins.

Infection is not a common problem following implant surgery using the bone cement, polymethyl methacrylate. When infection occurs, its often disastrous results makes for major complications. Many methods have been used in an attempt to reduce bacterial contamination at the time of implant surgery, but little attention has been given to the problem of possible effects of implant materials on the immune mechanisms of the host. In the experiments reported here, the in vitro effect of methyl methacrylate monomer on human immunoglobulins was determined using a quantitative gel diffusion technique. When methyl methacrylate monomer was added to serum samples containing immunoglobulin, it caused no statistically significant change in the immunoglobulin concentration of immunoglobulin-G, immunoglobulin-A, or immunoglobulin-M. It is possible that methyl methacrylate could affect other types of antigen antibody reactions which may be important in resisting infection, but the evidence reported here indicates that in low concentrations, methyl methacrylate monomer does not affect the normal reactivity of immunoglobulins of the G, M, or A class.


Spine deformity in neurofibromatosis. A review of one hundred and two patients.

The natural history, associated anomalies, and response to operative and nonoperative treatment were reviewed in 102 patients with neurofibromatosis and spine deformity. Eighty patients were found to have curvatures associated with dystrophic changes in the vertebrae and ribs. The presence of dystrophic changes such as rib penciling, spindling of the transverse processes, vertebral scalloping, severe apical vertebral rotation, foraminal enlargement, and adjacent soft-tissue neurofibromas was found to be highly significant in prognosis and management. Brace treatment of dystrophic curves was unsuccessful. Posterior fusion, with or without internal fixation, was the procedure of choice for problems due purely to scoliosis. Patients with dystrophic kyphoscoliosis required both anterior and posterior fusion to achieve stability. Sixteen patients had compression of the spinal cord or cauda equina.


Cervical spine abnormalities in neurofibromatosis.

Fifty-six patients with neurofibromatosis were examined for abnormalities of the cervical spine and seventeen of them had demonstrated lesions there. Of the thirty-four patients who had scoliosis or kyphoscoliosis, fifteen (44 per cent) had cervical lesions. Many of the patients with those lesions were asymptomatic. To avoid the complications attributable to the cervical spine, we recommend roentgenographic examination in all neurofibromatosis patients who are about to have general anesthesia or skull traction for treatment of scoliosis.


The surgical treatment of heterotopic ossification at the elbow following long-term coma.

Nine cases of heterotopic ossification at the elbow in seven patients who had a cerebral injury and prolonged coma were treated by excision of the extra-articular bone bridge from the humerus to the ulna. The bridge was either anterior or posterior, and did not involve the radius. The articular cartilage in all elbows showed no significant evidence of degeneration in spite of the prolonged immobility of the joints during the period of coma. Two of the patients also had resection of heterotopic bone about the hip joint which resulted in rapid reformation of bone. In all patients there was restoration of satisfactory function in the elbow operated on without reformation of bone. Follow-up was from ten months to 8.7 years.


Comparison of purified anti-actin and fluorescent-heavy meromyosin staining patterns in dividing cells.

We purified actin antibodies by affinity chromatography from the serum of rabbits immunized with glutaraldehyde-fixed chicken gizzard actin filaments and used this anti-actin to localize actin in myofibrils and fixed cultured cells at each stage of the cell cycle. By double immunodiffusion the anti-actin reacted with both smooth and skeletal muscle actin. Several blocking and absorption experiments demonstrated that the antibodies also bound specifically to actin in nonmuscle cells. The same structures stained using either the direct or the indirect fluorescent antibody technique; and, while the indirect method was more sensitive, the direct method was superior because there was no detectable nonspecific staining. As expected, anti-actin stained the I-band of myofibrils. It also stained stress fibers and membrane ruffles in HeLa cells. Some PtK-2 cells have straight stress fibers which stained with anti-actin, but in confluent cultures all PtK-2 cells have, instead, sinuous phase-dense fibers which stained with antibody. At prophase the whole cytoplasm stained uniformly with anti-actin. During metaphase and anaphase, anti-actin staining was concentrated diffusely in the mitotic spindle. In contrast, fluorescent heavy meromyosin stained discrete fine spindle fibers in these fixed cells. During cytokinesis, anti-actin stained the whole cytoplasm uniformly and was not concentrated in the cleavage furrow.


Reversible alteration  in the neuromuscular junctions of Drosophila melanogaster bearing a temperature-sensitive mutation, shibire.

In this study we report a relationship between the ultrastruct of the neuromuscular junctions of tibial muscles and the temperature-induced paralysis in shibire flies. There is a decrease in the number of synaptic vesicles of neuromuscular junctions in flies which are held at or above 29 degrees. Shortly after return to 22 degrees C, the synaptic vesicles are again present in large numbers. Prior treatment with tetrodotoxin or barbiturate protects the junctions from the temperature change in morphology.


A precursor of cytoplasmic actin in cultured Drosophila cells.

In continuous lines of cultured Drosophila cells two forms of cytoplasmic actin, designated II and III, are detected after a 25- to 120-min pulse label with [35S]methionine. However, only one of these, actin II, accumulates in the cell. With the use of a pulse-chase protocol and two-dimensional gel electrophoresis, it has been found that actin III is synthesized as a precursor of the more stable cytoplasmic actin II. The half-life of actin III was estimated to be approximately 50 min.


Effects of hydrostatic pressure in the range 100-300 atmospheres on cell division and protein synthesis in synchronized Tetrahymena pyriformis: a comparison with cycloheximide and emetine.

Heat-synchronized Tetrahymena pyriformis have been subjected to 5-, 15- and 30-minute pulses of hydrostatic pressure in the range 100-300 atm, without being simultaneously subjected to significant heats of compression. The pressure-induced division delays depend on (1) the level of pressure used, (2) the length of pressure pulse and (3) the time after the synchronizing treatment at which the pressure is applied. A pressure-dependent inhibition of 3H-leucine incorporation into protein was also measured. Comparison of the effects of pressure with those of pulse treatments of cycloheximide and emetine on cell division and protein synthesis revealed that the physical agent produced characteristically different responses from those of the chemical agents. Of particular interest was the fact that the division delays induced by pressures of 200 atm and above were greater than those observed after treatments with cyclohexmide and emetine which produced comparable levels of protein synthesis inhibition. Pressure also delayed cells if it was applied at a time when addition of chemical inhibitors had little effect on the first synchronous division. The results show that inhibition of protein synthesis by pressure cannot entirely account for pressure-induced effects on cell division. The possibility that pressure may also directly affect other processes, such as the assembly of proteins into structure required for division, is discussed.


Dual capacity for nutrient uptake in Tetrahymena. V. Utilization of amino acids and proteins.

We investigated the relative contributions of phagocytosis and plasma membrane transport to the uptake of amino acids and a protein (egg albumin) in amounts which allow Tetrahymena thermophila to grow and multiply. We used a mutant capable of indefinite growth without food vacuole formation (phagocytosis) and its wild type (phagocytosis-competent) isogenic parental strain. Our results suggest that phagocytosis is not required for free amino acid uptake, most or all of which can be attributed to carrier-mediated transport systems, apparently located on the plasma membrane. In contrast, phagocytosis is required for utilization of the protein. Proteins can supply required amino acids in amounts sufficient for growth only when food vacuoles are formed. We conclude that Tetrahymena thermophila either possesses no endocytic mechanisms at the cell surface other than food vacuole formation or, if it does, these putative mechanisms are not capable of nutritionally meaningful rates of protein uptake.


Investigation of a corticosteroid-binding globulin-like molecule in human placenta.

The existence of an endogenous corticosteroid-binding globulin (CBG)-like molecule in human placenta has been reported in studies which depended largely on immunological techniques. The experiments reported herein do not support that contention. Using a well characterized antibody to purified human CBG, we were unable to demonstrate CBG in placental cytosol. Although the immunological methods were too insensitive to detect CBG, the presence of small amounts of it were detected in binding experiments with [3H]cortisol. Simultaneous measurement of serum albumin indicated that the presence of CBG in the placental cytosol could be accounted for by contamination of the preparation with plasma.


Inherited abnormal thyroid hormone-binding protein causing selective increase of total serum thyroxine.

A 9-yr-old boy is described in whom increased serum T4 concentration, increased T3 uptake, and increased free T4 index were associated with a euthyroid clinical state with normal total serum T3. T4-binding globulin (TBG), measured by RIA, was decreased. Reverse flow paper electrophoresis of serum proteins after reaction with radioactively labeled T4 demonstrated increased binding of T4 to a protein with electrophoretic mobility corresponding to albumin. Displacement of serum protein-bo-nd [125I]T4 activity by increasing concentrations of T4 revealed the presence of a low affinity, high binding capacity system with an association constant similar to that of T4-binding prealbumin. This low affinity binding protein cochromatographed with TBG on a DEAE-Sephadex column which normally separates TBG from T4-binding prealbumin. At free T4 concentrations equivalent to those present in the plasma of normal individuals, the T4 bound to free ratio is higher in the patient than in normals and the total serum T4 level is increased in the presence of normal free T4 concentrations. The relative affinity of this abnormal T4-binding protein for T3 is low compared to that of TBG. The patient's father had the same abnormal binding protein, which was not found in his mother or fraternal twin brother. These data suggest an autosomal dominant mode of inheritance of an aberration leading to synthesis of a new protein instead of normal TBG. The new protein is different from TBG in electrophoretic mobility, T4 and T3 binding, and antigenic properties.


Hen fluorescein-labeled gonococcal lipopolysaccharide antibody in the delayed fluorescent antibody technique for the confirmation of Neisseria gonorrhoeae.

A fluorescent antibody reagent (termed anti-LPS conjugate) was prepared from sera obtained from hens immunized with gonococcal R-type lipopolysaccharide. The reagent was absorbed with Formalin-treated cells of Neisseria meningitidis. The anti-LPS conjugate gave uniform brilliant staining of Neisseria gonorrhoeae with little background fluorescence, thus making interpretation and reading of fluorescence simple. The conjugate did not significantly stain cultures of N. meningitidis, Neisseria lactamica, nonpathogenic Neisseria species, or other gram-negative bacteria. Several preparations of the conjugate provided the same specificity and reproducibility of staining. The anti-LPS conjugate was compared with Difco Laboratories fluorescent antibody conjugate for staining of N. gonorrhoeae. Both conjugates stained cells of the light and dark variants of gonococcal colony types 1 and 2, as well as cells of colony types 3 and 4. When used for the confirmation of N. gonorrhoeae, the anti-LPS and Difco conjugates stained 426 of 431 (98.8%) and 210 of 213 (98.6%) of the gonococcal cultures, respectively. Absorption of the anti-LPS conjugate with R-type lipopolysaccharide removed the staining of gonococci. However, absorption of Difco conjugate with R-type lipopolysaccharide did not remove the staining of gonococci, suggesting that the majority of fluorescein-labeled antibody present in the Difco conjugate is directed to gonococcal cell surface components other than lipopolysaccharide. The results of this study indicate that fluorescein-labeled gonococcal lipopolysaccharide antibody should be a reliable fluorescent antibody reagent for the confirmation of N. gonorrhoeae.


Standardization of direct susceptibility test for blood cultures.

Insufficient data are available to establish the reliability of direct disk diffusion susceptibility tests performed utilizing positive blood culture broth as inoculum. When Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 were used, 0.03 ml of turbid overnight blood culture broth was found to produce zone diameters closely approximating the size of diameters obtained by a standardized method. Results of direct (0.03 ml of inoculum) and standardized susceptibility tests were then compared for 116 positive blood cultures (1,069 individual disk comparisons). There were 1,011 test agreements (94.6%). There were also 48 (4.5%) minor discrepancies (change between sensitive and intermediate or between intermediate and resistant) and 10 (0.9%) major discrepancies (change between sensitive and resistant). The major discrepancies were randomly distributed among several organisms and antibiotics. Discrepancies occurred most frequently in the more clinically acceptable direction; i.e., in 79.3% the direct test indicted greater resistance than the standardized test. These data establish that 0.03 ml of turbid overnight blood culture broth produces results which compare closely to those obtained with standard methods, and in practice yield direct susceptibility results with a clinically acceptable level of reliability.


Characterization and identification of 95 diphtheroid (group JK) cultures isolated from clinical specimens.

Ninety-five cultures of group JK bacteria isolated from clinical specimens were characterized morphologically and biochemically. The microorganisms were isolated primarily from blood cultures. The bacterial cultures produced positive reactions when tested for catalase, Tween hydrolysis, and carbohydrate fermentation. Glucose and galactose were fermented by more than 90% of the organisms. Gas-liquid chromatography of trimethylsilyl derivatives of whole-cell hydrolysates of some of the group JK cultures yielded nearly identical elution profiles. The group JK microorganisms were susceptible to vancomycin but were resistant to most of the other 17 antimicrobial agents tested. A method is presented for differentiating the group JK microorganisms from other similar bacteria encountered in clinical specimens. Although these bacteria rarely occur in clinical specimens, they are capable of producing fatal infections (endocarditis and sepsis) in humans.


Evaluation of two transport systems for gonorrhea cultures.

The ability of the Transgrow and JEMBEC systems to yield positive cultures when transported to a central laboratory was compared. There were no significant differences in the recovery rates of the two systems. Statistically significant decreases in recovery rate were noted when each system was compared with the traditional plate-candle jar technique.


Rapid and inexpensive microcapillary immunodiffusion assay technique.

A simple, rapid, and inexpensive single-gel immunodiffusion technique requiring but 1 mul of sample for analysis and an incubation period of only 2 h is described.


Use of 2-aminoacetophenone production in identification of Pseudomonas aeruginosa.

A grapelike odor is often of diagnostic importance in detecting the growth of Pseudomonas aeruginosa in culture and in burn wounds. The compound responsible for the odor has been identified as 2-aminoacetophenone by mass spectroscopy. Although the grape odor is sometimes difficult to detect in culture media, gas chromatographic, fluorometric, and colorimetric methods can be utilized to assay 2-aminoacetophenone production in a variety of media. Its synthesis occurs relatively early in the growth cycle. It has proved easy and convenient to detect 2-aminoacetophenone excretion by P. aeruginosa after 24 h of incubation on blood agar plates employing a fluorometric assay of ether extracts of the agar medium.


Nuclease enhancement of specific cell agglutination in a serodiagnostic test for Neisseria gonorrhoeae.

Antiserum to a purified type R lipopolysaccharide antigen isolated from Neisseria gonorrhoeae was used in a slide agglutination test and compared with conventional carbohydrate utilization and fluorescent antibody tests to confirm the identity of laboratory cultures classified as typical or "atypical" N. gonorrhoeae. Cultures of Corynebacterium vaginalis, N. meningitidis, N. catarrhalis, N. sicca, and N. lactamicus were also tested in the slide agglutination procedure. The addition of both deoxyribonuclease and ribonuclease (1 mg/ml) to the cell suspension medium of phosphate-buffered saline improved the sensitivity and specificity of the agglutination reaction for N. gonorrhoeae. Problems relating to the agglutination test as an aid in identification of N. gonorrhoeae are discussed.


A more sensitive plate assay for detection of protease production by Pseudomanas aeruginosa.

A plate assay to measure protease production by Pseudomonas aeruginosa is described. This assay is more sensitive and rapid than established methods.


Serogroup identification of meningococci by a modified antiserum agar method.

Modifications in the antiserum agar method for serogroup identification of meningococci, which reduce the amount of group-specific antisera required and increase long-term storage of prepoured antiserum agar plates, are described.


Selective depression of the xenogeneic cell-mediated lympholysis in systemic lupus erythematosus.

The immunological responsiveness of a panel of 17 patients with systemic lupus erythematosus (SLE) was studied in an in vitro model of xenogeneic sensitization against mouse lymphoid cells. Generation of cytotoxic thymus-derived (T) cells evaluated by a chromium release assay against labeled target cells was found to be drastically impaired in these lupus patients. Such depression was independent of drug therapy at the time of the study, clinical status, and other immunological parameters such as antibodies against native DNA, complement levels, cryoglobulinemia, circulating immune complexes, or T- and bone marrow-derived (B)-cell numbers. In contrast to the cytotoxic response, the proliferative responses to phytohemagglutinin, to allogeneic lymphocytes, and to xenogeneic lymphocytes were not significantly different from those of normal individuals. The latter response was shown to be H-2 restricted with the primed lymphocyte test. These results suggest the presence of a selective defect in the generation or in the expression of killer cells rather than a deficiency in antigen recognition by T cells. The role of serum factor(s) was examined by educating the lymphocytes of normal subjects in the presence of serum from SLE patients. Such manipulation affected both the generation of killer cells and the proliferative response. Finally our observations indicate that depression of cell-mediated immunity in SLE patients may be associated with several mechanisms including a cellular one, specifically affecting the generation of killer T cells, and a humoral one possibly as a result of antilymphocytic antibodies and(or) immune complexes.


Endocrine consequences of continuous antiestrogen therapy with tamoxifen in premenopausal women.

Daily administration of estrogen antagonists to premenopausal women has been incorporated into the adjuvant treatment of breast cancer. We have studied the changes in reproductive hormones, pituitary responses to hypothalamic-releasing hormones, and endometrial histology during treatment with the antiestrogen tamoxifen in five healthy, premenopausal women. These studies were carried out during one menstrual cycle before and during two cycles of antiestrogen treatment. All subjects continued to have regular menses with biphasic basal body temperature records. During treatment, estradiol (E2) levels were increased but followed the usual pattern reflecting follicular maturation and corpus luteum formation. The mean E2 concentration at the midcycle peak and during the luteal phase was twice that observed during the non-treatment cycle. By contrast, the concentrations and secretory patterns of luteinizing hormone and follicle-stimulating hormone were not greatly changed, and the gonadotropin responses to gonadotropin-releasing hormone were not suppressed. Endometrial biopsies obtained during the follicular phase of control and tamoxifen treatment cycles showed no differences whereas biopsies obtained during the luteal phase of tamoxifen cycles uniformly showed a lack of changes attributed to progesterone action with no progression of histologic changes beyond those expected on day 7-8 of the luteal phase. These observations are consistent with maturation of multiple ovarian follicles, a surprising finding considering the normal gonadotropin concentrations. The retarded development of the endometrium in the presence of supranormal serum E2 and progesterone concentrations is a morphologic demonstration of the antiprogestational effect of antiestrogens. The lack of gonadotropin suppression in the presence of hyperestrogenemia suggests a major antiestrogen action on the hypothalmus and pituitary gland.


Experimental arterial thromboembolism in baboons. Mechanism, quantitation, and pharmacologic prevention.

A quantitative primate model of arterial thromboembolism has been characterized with respect to mechanism and usefulness in evaluating modifying variables. The model involved the kinetic measurements of (51)Cr-platelets and (125)I-fibrinogen consumption by femoral arteriovenous cannulae in chaired baboons. Cannula platelet consumption correlated directly with exposed cannular area for irradiated Silastic and polyurethane (correlation coefficients of 0.940 and 0.901, respectively; P < 0.001) and remained steady state for months. Nonirradiated Silastic was only minimally reactive with platelets. Despite increased rates of platelet consumption circulating fibrinogen was not measurably destroyed by any of the cannulae tested. Cannula platelet consumption was independent of cannula flow rate, platelet count, heparin anti-coagulation, and ancrod defibrinogenation.(111)In-platelet imaging of irradiated Silastic cannulae demonstrated luminal accumulation and subsequent embolization of irregular platelet masses. When irradiated Silastic cannulae were inserted as extension segments in the renal arteries of four animals the glomerular vessels became progressively occluded with nonfibrin-containing platelet thromboemboli. Nonirradiated Silastic cannulae in control arteries produced no significant vascular occlusion. Because the survival of platelets from animals with consumptive cannulae was not shortened in normal recipient animals we concluded that platelets were either irreversibly removed through thromboembolic consumption or unaffected in their viability. Oral administration of dipyridamole and sulfinpyrazone decreased cannula platelet consumption in a dose-dependent manner with complete interruption at 20 and 250 mumol/kg body wt per d (in three divided doses), respectively, whereas oral acetylsalicylic acid (10-330 mumol/kg per d) had no measurable effect on cannula platelet consumption. We conclude that this primate model simulates arterial thrombotic processes in man and that this model is suitable for the in vivo evaluation of biomaterials and of drugs that modify platelet behavior.


Sonically guided renal biopsy.

The results of 91 sonically guided renal biopsies are reported. In the 53 cases where renal size and function allowed other localization techniques, renal tissue was obtained in 100 percent and glomeruli in 88.6 percent. In 38 cases no other techniques could have been used because of poor renal function (serum creatinine greater than or equal to 4.0 mg/dl) and/or decreased renal size (less than or equal to 10 cm); in these biopsies renal tissue was obtained in 86.8 percent and glomeruli in 73.7 percent.


The influence of hydration and bladder distension on the sonographic diagnosis of hydronephrosis.

Renal sonography is frequently employed to exclude obstructive uropathy in patients with uremia. Sonographic findings of moderate bilateral hydronephrosis were encountered in a dehydrated patient undergoing rehydration. Subsequent excretory urography and a follow-up renal sonogram were normal. To investigate the possibility that hydration and bladder distension in an otherwise normal and unobstructed collecting system could result in hydronephrosis detectable by ultrasound, 23 apparently normal volunteers underwent renal sonography in the hydrated and basal states. The filled urinary bladder in a patient undergoing hydration frequently results in the sonographic appearance of hydronephrosis.


The sonographic findings of splenic metastases.

The sonographic findings in five patients with metastases to the spleen are demonstrated. Hypoechoic lesions were seen in patients with histiocytic lymphoma, and both echogenic and hypoechoic lesions were seen in patients with melanoma. The clinical significance of these findings is discussed.


A simple optical method for observing medical ultrasound pulse characteristics in vitro.

An optical method is described for studying the spatial and temporal characteristics of broad-band ultrasonic pulses propagating in transparent media. The simplicity of the approach suggests its consideration as a method for calibration and comparison of the transmitted pulse characteristics of medical ultrasound systems.


Sonographic anatomy of the normal placenta.

The normal anatomy of the placenta and the retroplacental area can be well defined with gray scale sonography. The placenta has a characteristic granular echo pattern, with strong echoes emanating from the chorionic plate. After 37 weeks, strong echoes representing calcification may appear; this is a normal physiologic process. The significance of early calcification is unclear. The retroplacental myometrium and venous drainage of the placenta are easily visualized by sonography. Variations in the appearance of the retroplacental myometrium result from different degrees of bladder filling and from uterine contractions. These normal retroplacental structures should not be mistaken for myomas or hematomas.


Genetic amniocentesis in multiple pregnancy.

Eleven cases of twin pregnancy presenting for genetic diagnosis in the second trimester are reported. In all 11 cases, a correct chromosomal diagnosis was made on each of the twins. The technique used required only ultrasound and Evan's blue dye for direction of the amniocentesis. Amniography was not performed. The technique used is described in detail, and its advantages over more standard techniques are described.


An anatomical reinvestigation of the termination of the spinothalamic tract in the monkey.

The projections of the spinothalamic tract in the macaque monkey have been reinvestigated using the Wiitanen modification of the Fink-Heimer technique. In agreement with previous studies in the monkey (mehler, Bowsher, Kerr) it was found that the spinothalamic tract ascends outside the medial lemniscus, enters the thalamus just dorsal to this structure, and terminates in the posterior, intralaminar and ventral regions, as well as in the zona incerta. The posteromedial nucleus (POm) receives a dense spinothalamic projection medially and ventromedially; elsewhere in the POm the projection is more scattered. The fibers to the intralaminar region terminate in the nucleus centralis lateralis (CL) with a distinct pattern of the distribution. The nucleus centralis medialis (CeM) has a minute projection. There was no evidence for somatotopic organization in the projections to the POm or to the intralaminar region. The distribution of the terminal degeneration in the ventral region was more complex. Although present in the whole nucleus ventralis posterolateralis (VPL), the degeneration was unevenly distributed and also extended beyond the VPL. So-called clusters of dense degeneration lay in the outskirts of the forelimb and hindlimb representation areas, namely at its ventral, ventrolateral, dorsolateral, and medial borders. Centrally the degeneration was scattered. Thus, most of the VPL receives only a sparse spinothalamic projection, but a small portion contains dense networks of terminal spinal fibers. A somatotopic pattern was evident, for after low thoracic lesions most of the medial VPL lacked degeneration. Spinothalamic fibers pass beyond the VPL to terminate in a zone of transition (nucleus ventralis intermedius of V.im of Hassler, '59; Mehler, '71) between the rostral pole of the VPL and the nucleus ventralis lateralis (VL). This zone also reportedly receives cerebellar and vestibular afferent fibers. Observations suggesting that the evolution of the spinothalamic tract and the spino-cervico-thalamic pathway in carnivores and primates may be linked are discussed. The spinothalamic clusters in the monkey's VPL appear to be homologous to much of the cervicothalamic tract projection to the VPL in the cat.


Layer I of striate cortex of Tupaia glis and Galago senegalensis: projections from thalamus and claustrum revealed by retrograde transport of horseradish peroxidase.

We have examined the origin of the subcortical projections to the superficial layers of the striate cortex in Tupaia glis and Galago senegalensis by using the retrograde transport of HRP. Crystals of HRP were laid directly on the moist pial surface of the cortex which had been gently pricked with a small glass pipette. The diffusion of HRP was limited to layers I and II by restricting the length of time that the HRP was in contact with the surface. Following the application of HRP to the striate cortex, labeled cells were found in restricted regions of the lateral geniculate body of both species. Layers 4 and 5 of galago and layer 3 of tree shrew contained dense clusters of labeled cells. Labeled neurons were also found in the zones between the layers of the lateral geniculate body in both species and these cells were always in register with the labeled cells within the layers. In galago, curved columns of labeled cells were observed in the inferior and superior subdivisions of the pulvinar nucleus. These columns were arranged in the shape of two arcs, joined at the fiber bundle which separates the two subdivisions. The position of the bands in the pulvinar nucleus varied with the locus of the application in the striate cortex. While no labeled cells were seen in the body of the pulvinar nucleus of tree shrew, small labeled neurons were found in the external medullary lamina forming the capsule of the pulvinar nucleus. These cells were continuous with a larger population of labeled cells in the lateral intermediate nucleus. In both species, labeled cells were also found in the intralaminar nuclei (particularly the paracentral nucleus) and in the dorsal-caudal portion of the claustrum. In the claustrum, few unlabeled neurons were present within the zone containing labeled cells. In conclusion, layer I os striate cortex appears to be the site of convergence of several projection systems originating from principal and intralaminar thalamic nuclei as well as the claustrum. The significance of this overlap is discussed in terms of the total cortical extent of each system.


A quantitative investigation of spine and dendrite development of neurons in visual cortex (area 17) of Macaca nemestrina monkeys.

In a previous Golgi study (Lund et al., '77) which examined the development of the macaque monkey striate cortex (area 17) it was observed that the dendrites of neurons within the visual cortex show a marked increase in the number of spines on their surface during the first eight weeks of postnatal life. The qualitative observation was also made that all neurons then showed a marked decrease in spine numbers by the time the animal was adult. Since these spines are known to be sites of synaptic contact, changes in their numbers may reflect changes in synapse populations on these neurons. This study examines quantitatively spine frequency and total dendritic development of Golgi impregnated neurons in monkeys ranging in age from 145 days gestation to adult. Four cell types were studied: spiny stellate neurons from laminae IVCalpha and IVCbeta and pyramidal neurons with soma in either lamina IIIB or upper lamina VI. After consideration of possible sources of variation in spine numbers several conclusions are made: (1) Dendritic spine development appears to be a tightly controlled process both in terms of actual numbers of spines on a neuron at any one age and in the rate of change of spine frequency. (2) The neurons populations examined all show a gradual increase in spine numbers up to eight weeks of age. (3) At least two different trends are found in spine population maturation after the eight week point: (A)-the spine population may remain constant at the eight week level for same period of time or (B)-there may be a rapid decline in spine numbers following the eight week peak. (4) There is a suggestion that those neurons associated with direct input, or early stages in the relays, from the parvocellular geniculate laminae show trend B, while those associated with magnocellular input, or later order combined relays within the cortex, show trend A. (5) Different parts of a single pyramidal neuron dendrite may show either trend A or trend B, depending on the lamina location of the dendritic segment considered. (6) All neurons show spine population decreases between nine months of age and adult (5-7 years) suggesting continuing long term maturational changes.


Contrasts in visual responsiveness and emotional arousal between rhesus monkeys raised with living and those raised with inanimate substitute mothers.

Rhesus monkeys were raised with dogs or inanimate surrogates in outdoor cages which provided them with complex, highly varied visual surroundings. Visual responsiveness to a variety of colored transparencies was investigated in three experiments, completed when the monkeys were between 18 and 30 mo old. Results indicated that the frequency and duration of looking at slides was significantly higher for dog-raised than for inanimate-surrogate-raised monkeys and that dog-raised monkeys were much more responsive to the novelty, complexity, ansal were obtained during the final experiment. Heart rate, vocalization, and changes in plasma cortisol were higher for monkeys raised with dogs. The frequency of most self-directed behaviors, however, was higher for monkeys raised with inanimate surrogates. Differences between rearing groups can only be the result of contrasts in attributes of the substitute mothers.


The posterior pararenal space: an escape route for retrocrural masses.

In two patients with retrocrural tumors, dissection of the tumors selectively into the posterior pararenal space was observed by computed tomography. Cadaver studies confirmed the presence of potential communications between the retrocrural space and the extrapleural space of the thorax or the posterior pararneal space of the abdomen.


Characterization of groups N and D streptococci isolated from rumen fluids.

Twelve strains of streptococci of serological groups N and D were isolated from rumen fluids of nine cows on four rations although both groups were not isolated from the same animal. The twelve were representative of a larger number of streptococci isolated by enrichment in milk. Generally, the physiological characteristics of the isolates indicated members of the lactic or enterococcus groups; however, several were atypical in one or more of the tolerance tests. Physiologically typical Streptococcus lactis but not S. cremoris, were isolated.


Immunohistochemical localization of exocrine enzymes in normal rat pancreas.

On the basis of morphological and biochemical differences, the exocrine pancreatic tissue has been divided in peri- and teleinsular regions. In the present study the enzymatic profile of these regions has been investigated by the immunofluorescent technique using antibodies against nine pancreatic enzymes (alpha-amylase, lipase, chymotrypsinogen A, trypsinogen, elastase, carboxypeptidase A and B, DNase and RNase A). These antibodies were specific to their antigens without cross reaction. By immunofluorescence, most acinar cells of the normal rat pancreas were positive to the nine enzymes tested. However, an inhomogeneity in the staining pattern was found; specifically, the cells located in the periinsular region of many islets showed a brighter fluorescence than acinar cells in the teleinsular tissue. These data add a new parameter to describe the inhomogeneity of the exocrine pancreas.


Immunofluorescent localization of 25-hydroxyvitamin-D3-1 alpha-hydroxylase ferredoxin in the renal tissue of chick.

The chick renal mitochondrial 25-hydroxyvitamin-D3-1 alpha-hydroxylase is composed of three proteins, namely, cytochrome P-450, iron-sulfur protein (ferredoxin) and flavoprotein. Antibodies were raised in rabbits against homogeneous preparations of the ferredoxin. The antibodies were used in indirect immunofluorescence studies to localize the ferrdoxin along the nephron of renal tissues obtained either from vitamin D3-deficient or vitamin D3-sufficient chicks. The ferredoxin is predominantly localized in the glomerulus and proximal convoluted tubules. These results suggest that, in addition to the mitochondrial localization of the 1-hydroxylase, the enzyme may also be present in renal nuclei. The amount of the ferredoxin in kidney, as evidenced by the intensity of fluorescence, appeared to be independent of the vitamin D status of the chick. This finding indicated that changes in the concentration of the renal ferredoxin is not a major factor in the regulation of the 1-hydroxylase activity.


Spectra of cells in flow cytometry using a vidicon detector.

A TV type vidicon detector was interfaced to a flow cytometer (FCM) to obtain spectra of fluorophores in cells during flow. The normal operations of the FCM are undisturbed. A spectrograph spreads 320 nm of the fluorophore fluorescence emission across the 500 channels of the detector. Spectra of fluorescamine (a surface labeling agent) and of propidium iodide (a nuclear stain) were obtained from Balb 3T3 cells, and the chlorophyll and phycobilin peaks were resolved from flowing blue-green algae in the FCM. Under typical flow conditions, operation of the vidicon in the continuous mode gives for these fluorophores a S/N of several hundred to one in approximately 3 sec. The vidicon was also gated to obtain spectra of single cells and of cells in selected portions of the cell cycle. For example, the spectrum of fluorescamine was obtained from cells in the G1 phase of the growth cycle by using as a gate trigger the FCM discriminator output derived from the propidium iodide signal.


Measurement of endotoxins with the limulus test in burned patients.

High titres of endotoxin as measured by the Limulus test were usually found in burned patients who had raised body temperatures, and were colonized with gram-negative bacteria; also some infected patients showed raised endotoxin without a raised temperature. Patients vaccinated with an antipseudomonas vaccine rarely showed endotoxin in their plasma but occasional plasma samples from vaccinated patients had a high titre of endotoxin which appeared unrelated to infection or to a raised temperature in the patient.


Vaccination against influenza: a five-year study in the Post Office.

An injection of influenza vaccine was offered to approximately 60 000 Postal and Telecommunications staff at the beginning of five successive winters. The sickness absence of this group, which included those who accepted the offer of vaccine as well as those who did not, was compared throughout the winter with that of a similar number of employees who were not offered vaccine. The two groups, ;vaccinated' and control, comprised the staff of nearly 400 Post Office units scattered throughout Great Britain, the units of the two groups being matched as far as practicable for numbers employed, type of work, region and type of location.The proportion who accepted vaccine fell from 42% in the first year (when only 26 000 Telecommunications employees were offered vaccine) to 35% in the second year, and 25% by the fifth year.With the exception of Telecommunications employees in 1972-73, the sickness absence rate of the group offered vaccine was less than that of the group not offered vaccine, and the difference was evident during the winter observation periods both when influenza was prevalent and when it was not. In the last four years of the study the average difference in sickness absence between the ;vaccinated' and control groups was 1.26 days per 100 employees per week during and 1.12 days outside the influenza periods. Moreover, the difference during the influenza periods was greater than could be expected from the acceptance rate of vaccine and the estimated attack rate of influenza. The apparent reduction in sickness absence of the group offered vaccine in comparison with the group not offered vaccine represented an appreciable saving in cost.It is suggested than an annual influenza vaccination campaign in industry may produce financial benefit, but that only a proportion of the benefit is due to an improvement in health.


Modulation of natural cytotoxicity by alloantibodies. I. Alloantisera enhancement of cytotoxicity of mouse spleen cells toward a human myeloid cell line.

Natural killer activity of spleen cells obtained from different strains of mice against the human myeloid leukemia cell line, K562, and two mouse cell lines P815 and L1210 was measured by using the 4-hr chromium release assay. The level of cytotoxic activity of spleen cells against the K562 target was usually less than 4% lysis. However, treatment of the spleen cells with a specific anti-H-2 antiserum resulted in a dose-dependent augmentation of the degree of lysis of K562 cells. The augmentation of cytotoxic activity could be obtained by pretreatment of the spleen cells with antisera or by directly adding the antisera to the cytotox-incubation medium. Anti-thy-1 and anti-immunoglobulin antisera had no enhancing effect under similar conditions. The specific alloantisera-treated spleen cells did not show any increase in cytotoxicity against P815 and L1210 target cells. Spleen cells responsible for the alloantiserum-mediated augmentation of cytotoxicity against K562 cells appear to be different from T or B cells as indicated by their resistance to anti-thy-1 and complement treatment and lack of adherence to nylon wool columns.


Sensitive and convenient quantitation of antibody binding to cellular antigens using glutaraldehyde preserved cells.

As a preliminary step in the identification and isolation of antibodies to human cancers, we have developed a sensitive and convenient assay for antibody binding to cellular antigens. The basis for the method is antibody binding to glutaraldehyde-fixed cells (AbGfC) and quantitation with radioiodinated staphylococcal protein A (SpA). Glutaraldehyde fixation of intact cells, which does not appear to effect the ability to form antigen-antibody complexes, provides a convenient and standard supply of target cells which may be stored at 4 degrees C and used in the assay over a period of several months. The amount of antibody specifically bound to the cells is quantitated by the addition of 125I-labeled SpA. The sensitivity of the method was compared with two complement-dependent cytotoxicity methods (trypan blue exclusion and 51Cr release assays) and tested with two antisera to human lung cancer and one antiserum to a membrane antigen of a murine lymphoma. These comparisons indicated much greater sensitivity when compared with the trypan blue exclusion assay and equivalent sensitivity with greater dose response characteristics when compared with the 51Cr release assay.


Alveolar macrophages. I. A simple technique for the preparation of high numbers of viable alveolar macrophages from small laboratory animals.

A simple technique is described for the collection of substantial numbers of alveolar macrophages by repeated in situ endobronchial lavage. Macrophage recoveries are maximized by the inclusion of the local anaesthetic lignocaine in the lavage fluids. The technique is suitable for mice, rats and guinea pigs, and yields up to 2.4, 6.7 and 25.3 X 10(6) viable macrophages per animal from these 3 species respectively. A one-step adherence procedure yields alveolar macrophages in excess of 95% purity.


Immunoregulation in infection caused by Mycobacterium tuberculosis: the presence of suppressor monocytes and the alteration of subpopulations of T lymphocytes.

This study was designed to characterize qualitative and quantitative alterations, occurring before and during chemotherapy, in the mononuclear cells of patients with infections caused by Mycobacterium tuberculosis. A hemolytic plaque-forming (PFC) assay indicated that the production of antibody to sheep red blood cells by pokeweed mitogen (PWM)-stimulated lymphocytes was suppressed in treated and untreated patients as compared with that in normal adult donors (P less than 0.001). The removal of adherent cells from the suspensions of mononuclear cells significantly enhanced the responses to the PFC assay for both the untreated (P less than 0.01) and treated (P less than 0.05) patients. Mononuclear cells from patients with tuberculosis, however, did not suppress the PFC responses of allogeneic normal mononuclear cells (P greater than 0.02). Thymus-derived (T) lymphocytes were proportionally reduced in untreated subjects (P less than 0.001) but returned to normal levels after four to six weeks of therapy (P greater than 0.2). Both groups of patients had a consistent reduction in the absolute number of circulating T cells. However, untreated patients had a relative increase in the percentage of TG cells (the subpopulation of T cells with receptors for the Fc portion of IgG) (P less than 0.001) and a concomitant decrease in TM cells (the subpopulation with Fc receptors for IgM) (P less than 0.05). These alterations in the subsets of T cells were reversed after four to six weeks of therapy.