Enhancement of leucine-induced insulin secretion by somatostatin antiserum pretreatment. Insulin secretion induced by leucine and leucine plus glucose from rat pancreatic islets was enhanced by prior treatment with somatostatin antiserum as compared with normal rabbit serum in vitro. The results provide evidence that somatostatin plays an important role in insulin secretion in rats.


Menstrual disturbances in chronic renal failure. Twelve female patients undergoing intermittent hemodialysis (HD) and 5 females posttransplantation (PT) were studied. All the HD patients had menstrual disturbances and 5 had galactorrhea. The mean basal LH level was significantly elevated (p less than .05) in patients on HD compared to normal controls, but the mean LH response to luteinizing hormone releasing hormone (LRH) was not significantly different from the control group. Mean basal FSH and the FSH response to LRH was normal. In the PT pateints the LH response to LRH was significantly greater at 120 min when compared to normal females. In the HD group the serum 17B estradiol, progesterone and testosterone levels were significantly lower than in the controls but in the PT group only testosterone levels were significantly lower. These results differ from those previously found in uremic males. Elevated prolactin levels were found in the patients on hemodialysis and correlated well with the presence of galactorrhea. These was no correlation between the elevated prolactin levels and amenorrhea in the patients on hemodialysis but one PT patient with amenorrhea had elevated prolactin levels.


The brown bowel syndrome: a possible smooth muscle mitochondrial myopathy? Two cases of lipofuscinosis of the gastrointestinal tract are described, and a mitochondrial origin of the pigment is proposed. The mechanism of formation of lipofuscin is discussed, with particular reference to the maintenance of structurally and functionally intact mitochondrial membranes. The central role of vitamin E is considered, and its biochemical significance to mitochondrial metabolism is emphasized. Comparisons are drawn between the abnormalities demonstrated within the smooth muscle cells of the two cases described, and the muscle cells observed in cases of skeletal muscle myopathies of mitochondrial origin. It is proposed that lipofuscinosis of the gastrointestinal tract, otherwise known as the 'brown bowel syndrome', may be regarded as a smooth muscle myopathy of mitochondrial origin.


Urban administrator forges links with community. As executive director of New York's Bellevue Hospital Center, Madeline Bohman has a deep commitment to the concept of community. She sees a need for hospitals to assume responsibility for teaching patients and for providing community outreach services.


Hospitals must publicize VE, Rep. Marks says. Representative Marks believes that hospitals should accelerate the VE program and that they should publicize their progress via television.


Soluble di- and aminopeptidases in Escherichia K-12. Dispensible enzymes. As part of a study of the peptidase content of Escherichia coli K-12, two peptidase-deficient amino acid auxotrophs isolated and characterized by Miller as pepD- (strain CM17) and pepD- pepN- pepA- pepB- pepQ- (strain CM89) were examined for the presence of several peptidases previously obtained from strain K-12 in this laboratory. The soluble fraction of each mutant was found to lack the broad-specificity strain K-12 dipeptidase DP and the strain CM89 fraction also lacked activity characteristic of the strain K-12 aminopeptidases AP, L, and OP; like strain CM17, strain CM89 contained the tripeptide-specific aminopeptidase TP. Strain CM89 (but not CM17) appeared to contain little if any activity attributable to the ribosome-bound aminopeptidase I of strain K-12. Whereas loss of DP, AP, OP, and aminopeptidase I activity may be attributed to the pepD-, pepB-, pepN-, and pepA- mutations, respectively, the reason for the loss of L activity remains uncertain. Grown responses of strain CM89 in liquid media containing di- or tripeptides were in accord with absence of enzymes catalyzing rapid hydrolysis of dipeptides. In synthetic liquid media supplemented with the required amino acids per se or with peptone, cultures of both CM strains grew more slowly than strain K-12 and produced smaller cell-yields than those produced by strain K-12.


Protease B from Saccharomyces cerevisiae. Purification and characterization. Protease B has been isolated from Saccharomyces cerevisiae and purified in six steps as follows: autolysis of the yeast cells, ammonium sulfate fractionation, activation of the proteolytic enzymes, chromatography on DEAE-cellulose, chromatography on CM-cellulose and finally, a second chromatography on DEAE-cellulose. The preparation was shown to be homogeneous on polyacrylamide gels in the absence as well as in the presence of sodium dodecylsulfate. Furthermore, the molecular weight (43,000 daltons) and the isoelectric point (5.45) were in good agreement with earlier published values. The amino acid composition is reported. The absence of disulfide bonds in protease B has to be outlined. The amino acid residues of the protein have been found to be folded nearly quantitatively (at least 80%) in a beta-conformation as deduced from a circular dichroism study. Finally, the tryptophan residues (5 mol/mol protein) are largely buried in the hydrophobic core of the enzyme.


The response of E. coli Bs-1 to tritium-beta particles under aerated and anoxic conditions. E. coli Bs-1 cells were exposed to acute doses of tritium-beta particles by suspension in tritiated water for known lengths of time. The resulting survival rate was compared with that obtained for external irradiation with 7 MeV electrons. The o.e.r. measured for tritium-beta s was not significantly different from the value of 2.15 measured for 7 MeV electrons. The r.b.e. of the tritium beta s relative to 7 MeV electrons was 1.21 in both air and nitrogen. These results were compared with existing data for low voltage electron irradiations and with track segment studies of the effect of varying LET on the radiosensitivity of E. coli Bs-1.


The effect of endotoxin-induced intraocular inflammation on the rat lens epithelium. Intraocular inflammation induced by an intravitreal injection of Shigella endotoxin into the rat eye produces early changes in the number of dividing cells of the lens epithelium and affects the organization of the meridional rows. A depression in mitotic activity in the germinative zone is observed during the first 24 hr after injection. At 48 hr, despite the continued mitotic inhibition in the germinative zone, an increase in cell division occurs in the central zone. By 72 hr, the germinative zone mitosis reappears and exceeds control values, whereas the central zone mitotic activity returns to normal. At that time mitotic figures are found in the transitional zone. Disorganization of the meridional rows is seen as early as 12 hr after injection (the first time period observed) and reaches a peak by 48 hr. During the next 5 days, however, the severity of the disorganization diminishes. By the seventh day the rows appear, for the most part, fully recovered, and the mitotic activity reaches normal or near-normal levels in all regions. The details of these observations and their possible relationship to inflammatory cataracta complicata are discussed.


Immunity and male infertility.

Immunity and male infertility. This article has attempted to review evidence which suggests that immune factors may be operative in a small but significant number of infertile men. Although our current understanding of the possible processes by which autosensitization to previously sequestered reproductive antigens occurs is incomplete, there are laboratory assays presently available that indicate, but do not prove, that immune factors may be contributing to the infertile state. Continued research is needed to develop new assays and materials (especially purified sperm antigens) which might enhance our knowledge of the underlying immunoreproductive changes. Until these are available the outline below might be considered when investigating a patient with infertility of suspected immune origin.


Quantitative age related histologic changes in the human vas deferens. Sections of vas deferens from 50 men between 27 and 82 years of age were examined histologically for evidence of changes in muscle area and for changes in per cent of the cross-sectional area of the vas deferens that was occupied by muscle and connective tissue as a function of age. No variation in quantitative measurement of muscle or connective tissue could be correlated with age but a diffuse increase in unmeasurable amounts of connective tissue was seen with increasing age.


Development of the calyceal system in the human fetus. The calyceal system including the papilla, infundibulum, and renal pelvis in human fetuses and neonates was studied histologically and chronologically. There was a considerable discrepancy between the development of the calyceal muscles and the papilla. The protuberant form of the papilla developed much earlier than the calyceal muscle which was observed in the late stage of intrauterine life. The calyceal muscles were differentiated and extended from the renal pelvic muscles through the infundibular muscles after approximately 21 weeks of intrauterine age and were not fully developed by the late stage of intrauterine life. On the other hand, the cup-shaped calyces and the protuberant form of the papilla developed into the form similar to that observed in the adult by the 21st week; their development began in the 16th week. From these observations the etiology of megacalyces was speculated and discussed based on the discrepancy of the muscular development of the calyceal wall and the development of the protuberant papilla.


Autographa californica nuclear polyhedrosis virus structural proteins compared from in vivo and in vitro sources. Polyhedrin obtained from Autographa californica nuclear polyhedrosis virus (AcMNPV) was apparently not modified in terms of primary structure after passage through alternate host systems, both in vivo and in vitro, as investigated by two-dimensional, high-voltage electrophoresis of tryptic digests of this protein as well as amino acid analysis. N-terminal analyses were conducted using an improved technique which showed proline to be the N-terminal amino acid. In addition, passage of enveloped nucleocapsids through alternate hosts as studied by SDS-PAGE showed the polypeptide compositions to be very similar.


Finish lines in fixed prosthodontics.

Finish lines in fixed prosthodontics. Ideally, all finish lines should be placed supragingivally. Due to esthetic and carious considerations, however, subgingival placement of the finish line is preferred. No one type of finish line can be used for all crown preparations. In lower anterior teeth or periodontically-treated teeth, the knife-edge finish line appears to be the treatment of choice. In short teeth and preparations for porcelain and porcelain-gold crowns, the full shoulder bevelled preparation is the treatment of choice. In full gold and acrylic veneered gold crowns, as in endodontically-treated teeth, the chamfer preparation is the treatment of choice.


[Kidney transplantation in the treatment of hypertension]. Renal autotransplantation seems to be of great value as a means of renal revascularization in hypertension of renovascular origin, especially in those cases in which the preceding angioplastic procedure have resulted in failure or in cases with peripheral or difficult stenoses of the renal artery. One case is reported with a coarctation of the abdominal aorta involving both kidney arteries. The patient was treated by bypass operation and kidney autotransplantation. Two years later both kidneys are functioning normally and the patient is normotensive.


Phenobarbital in prophylaxis of neonatal jaundice. A control trial of two regimens. The prevention of neonatal hyperbilirubinemia by phenobarbital is widely used in many countries. However, dose and period of administration vary according to authors, as the dose-effect relation is not known and since little research in pharmacokinetics of phenobarbital has been done so far. In the present study we compared the effect on serum bilirubin of a single injection of 10 mg/kg body weight and of a prolonged administration of 10 mg/kg/day during 3 days in premature and full-term newborn infants. The single injection had the same effect as the prolonged administration, provided it was given within 12 h after birth.


LH-RH and HCG studies in a Turner phenotype male (Noonan's syndrome). A case report. LH-RH and HCG stimulation tests were performed in a prepubertal 13-year-old boy with Noonan's syndrome. The basal plasma LH (0.8 mIU/ml) was normal and FSH (2.5 mIU/ml) high, with an elevated response of both LH (8.3 mIU/ml) and FSH (9.6 mIU/ml) to LH-RH, as seen in primary hypogonadism. However, the patient had a normal testosterone response to HCG (437 ng%). These conflicting test results illustrate the difficulty of predicting potential for fertility in patients with Turner phenotype male.


High dose granulocyte substitution after bone marrow transplantation in a case of aplastic anaemia in childhood. A bone marrow transplantation (BMT) was successfully carried out in a seven-year-old boy with aplastic anaemia. An HLA and MLC-identical brother served as donor. During the first 16 days after BMT between 0.55 and 7.11X10(10) granulocytes were transfused 14 times (an average of 4.03X10(10) per transfusion). The maximum increment in the peripheral blood after transfusion was over 4000 granulocytes per microliter. The course of local infections was taken as parameter for determining the clinical effectiveness of the granulocyte transfusion. Under the substitution therapy the necrotic ulcer of a fingertip and the mucous membrane ulcers in the oral cavity subsided. The presence of transfused granulocytes in the oral cavity ("orogranulocytes") could be established. There was a statistically significant relationship between the number of granulocytes in the peripheral blood and the number of orogranulocytes. It could be demonstrated that in children a temporary normalisation of the number of granulocytes in the peripheral blood can be attained by means of granulocyte substitution and that the transfused granulocytes are clinically effective.


Antibodies to gliadin as a screening test for coeliac disease. A prospective study. The diagnostic value of gliadin antibody determination using the fluorescent immunosorbent test was examined in a prospective study of 57 children with gastrointestinal disease. Antibodies to gliadin were found in all 20 patients with active coeliac disease, whereas 7 of these children (37%) had a normal xylose absorption test despite a flat small gut mucosa. Only 4 (14%) of 28 children with other gastrointestinal conditions had antibodies to gliadin, invariably in low titre. After at least 2 years on a gluten-free diet none of 9 children with coeliac disease in remission had demonstrable gliadin antibodies. The gliadin antibodies disappear slowly, within 6 to 24 months, after withdrawal of gliadin from the diet. 0.8% (5/606) of a healthy control group of children, adolescents and adults (not biopsied) had gliadin antibodies in low titre. Increased mean cow's milk antibody titres were demonstrable in 8 (40%) of 20 patients with active coeliac disease as well as in 9 (32%) of 28 patients with other gastrointestinal lesions. Our studies show that determination of circulating gliadin antibodies is a worthwhile screening test in suspected cases of coeliac disease. In patients so selected there is a definite indication for small intestinal biopsy to confirm the diagnosis.


Immunocytochemical demonstration of 5-hydroxytryptamine in gastrointestinal endocrine cells. We have been able to demonstrate 5-hydroxytryptamine in the enterochromaffin cells of the mammalian gastrointestinal tract, using a highly specific antiserum. Conventional histochemical techniques for identifying amines as cell markers can thus be replaced by more reliable and sensitive immunocytochemical methods. This has been facilitated by the use of p-benzoquinone as fixative which has been shown to preserve the localization and antigenicity of amines, as well as peptides.


Specific immunohistochemical localization of type I collagen in porcine periodontal tissues using the peroxidase-labelled antibody technique. Antibody against Type I collagen was raised in rabbits and purified by immunoadsorption on Sepharose-conjugated Types I and III collagen. The cross-reactivity of purified antibody to Type III collagen was found to be less than 0.5% by passive haemagglutination and less than 1.5% by radioimmunoassay. When paraffin sections of fixed and decalcified pig molars were incubated with purified antibody to Type I collagen, varying degrees of staining were observed in the ligament, gingiva, bone and cementum. The periodontal ligament adjacent to bone was more widely stained than that adjacent to cementum in some regions, whereas in others, no difference in staining could be discerned between the two halves of the ligament. The lamina propria of gingiva was stained, and this appeared to be most intense in the vicinity of the overlying epithelium. The fibrous component in the endosteal spaces, the dentine and the extracellular coronal elements in the pulp were generally stained. The impression obtained from the staining pattern is that Type I collagen is not restricted to particular regions of the periodontal ligament or the lamina propria of the gingiva.


An immunocytochemical study of the distribution of pancreatic endocrine cells in chicks, with special reference to the relationship between pancreatic polypeptide and somatostatin-immunoreactive cells. Araldite sections of formalin-fixed pancreas from chicks at hatching were treated by an indirect immuno-enzyme technique to reveal cells containing APP, somatostatin, glucagon and insulin. APP cells were found scattered in the exocrine parenchyma. A few were associated with insulin-containing B islets and occasional cells occurred in and around glucagon-containing A islets. Somatostatin-immunoreactive cells were distributed peripherally in A and B islets and were dispersed in the exocrine tissue. APP cells were roughly as numerous in the exocrine parenchyma as somatostatin-immunoreactive cells. Since certain published observations point to the possible occurrence of APP and somatostatin in the same cells, consecutive sections were stained for these hormones. In no case did the two peptides occur in the same cell. Sections subjected to double-staining confirmed this result. Therefore it is likely that the described differences between APP and somatostatin-immunoreactive cells are valid.


Constancy and variability in the content of DNA in cerebellar Purkinje cell nuclei. A cytophotometric study. A cytophotometric study of DNA content in Purkinje cells of the cerebellum of rats, cats, chicken and humans (Feulgen staining) revealed that in a certain number of cells the amount of NDA ranged between the diploid and tetraploid level (H2C cells). The incidence of H2C Purkinje cells varied among the species studied. In rats, which were studied most thoroughly, these cells amounted on average to 3%. In some rats, as well as in some cats and chickens H2C Purkinje cells were entirely absent. In the group of animals possesing H2C Purkinje cells, great interindividual differences were observed. In rats for instance, the incidence of these cells varied from 1 to 23 per cent. Topographic analyses carried out in rat and human cerebellum revealed that H2C Purkinje cells occurred more frequently in the hemispheres than in the vermis. No significant differences were found in the number of H2C Purkinje cells in healthy and Kilham-DNA-virus infected rats. Densitometric analysis of the distribution of nuclear chromatin showed that H2C Purkinje cells were richer in condensed chromatin, especially in the region of the nucleolus, which apparently contains the hyperploid surplus of DNA. It is proposed that the phenomenon of DNA hyperdiploidy arises as a result of either incomplete S-phase in some immature Purkinje cell precursors or the amplification of some DNA sequences particularly those localized in the nucleolar region.


A gravimetric system for lyophilized samples in the sub-microgram range. A new quartz fiber balance is described which is evacuated and electrically shielded. This balance minimizes the problems of moisture uptake by the lyophilized sample, air convection inside the balance and static electricity. Besides it offers a wider useful weighing range and a higher handling comfort. It could be shown that in the regular (i.e. not evacuated) balance the moisture uptake varies considerably with the humidity of the air and with the kind of sample analyzed, and that it might be greater than previously reported.


Growth hormone-like immunoreactivity in gastrin cells and gastrinomas. Growth hormone (GH)-immunoreactive material was found to occur in the antral gastrin cells and in scattered cells of the pancreatic islets in several mammalian species, including man. Examination of gastrinomas revealed the majority of tumour cells to display GH-like immunoreactivity.


[Acute and chronic middle ear pathology in mediaeval man (author's transl)]. The variety, extent and consequences of middle ear cleft disease in ancient times is reviewed. The findings of macroscopic radiological and scanning electron microscopic examination of skeletal material from two south German Merovingian populations and two pre-Columbian population groups from the south west of North America are presented. The possibility of false diagnosis was considered. Cases previously described are reviewed, summarized and compared. In both the Frankish-Alemannian populations diseases of the middle ear cleft significantly occur more frequently within the lower social groups. From this it is possible to deduce that external factors were paramount in the pathogenesis of these diseases in early cultures, whereas in the present mid-European populations constitutional factors predominate. In contrast to recent claims (Cockburn, 1977) that diseases of the middle ear were rare amongst inhabitants of the Americas before the arrival of Europeans, earlier literature and the observations presented in this article show that these diseases were not so uncommon. Cases found in ancient Peru show no evidence of surgical treatment, although the standards of surgery, which at that time included trephination, could have made this feasible.


Trimethoprim in combination with a sulfonamide for oral treatment of canine urinary tract infections. Trimethoprim, in combination with sulfadiazine or sulfamethoxazole was administered orally for 7 to 14 days to 84 dogs with urinary tract infections (UTI). The daily dosage of 26.4 mg/kg (12 mg/lb) was divided into 2 equal parts and administered at about 12-hour intervals. Response to treatment, based on negative urine culture during or after therapy, was 37 of 45 (82%) for UTI caused by Escherichia coli, 11 of 15 (73%) UTI caused by Proteus mirabilis, 8 of 12 (67%) UTI caused by Klebsiella pneumoniae, 6 of 6 (100%) UTI caused by Staphylococcus aureus, and 5 of 9 (56%) UTI caused by Streptococcus spp. These 5 species comprised 88% of the bacteria isolated from the dogs in this study.


Stylism in anatomical illustration from the sixteenth to the twentieth centuries. An overview of four centuries of anatomical illustration is presented, citing specific examples to document trends in style and to show the effects of concurrent advances in the technology of reproduction and printing.


[Oral protozoans and diabetes: study in 117 patients]. A study on the presence of oral Entamoeba gingivalis and Trichomonas tenax has been carried out on calculus or dental plaque from 117 diabetic subjects. Statistical analysis of results shows no correlation between diabetes and protozoa. The same frequency was found between diabetic and normal subjects. Nevertheless, a significant relation has been found between the diabetes and the yeasts.


Peroxidase-dependent deactivation of prostacyclin synthetase. A study of the enzymes of the arachidonic acid cascade revealed a high sensitivity of prostacyclin synthetase and a complete resistance of thromboxane A2 synthetase to time-dependent destruction by an oxidant [Ox] released during the peroxidase-catalyzed reduction of hydroperoxy fatty acids. The destructive action of [Ox] derived from prostaglandin G1 (PGG1), 15-hydroperoxy-PGE1, 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid, and 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid upon prostacyclin synthetase was prevented by 2-aminomethyl-4-t-butyl-6-iodophenol. On the other hand, deactivation resulting from PGG2 metabolism was neither time-dependent nor sensitive to 2-aminomethyl-4-t-butyl-6-iodophenol. The possibility that the action of [Ox] may alter the arachidonic acid cascade in favor of thromboxane A2 is discussed in view of its possible implications in inflammatory and other pathological processes.


Opioid peptides derived from food proteins. The exorphins. Peptides with opioid activity are found in pepsin hydrolysates of wheat gluten and alpha-casein. The opioid activity of these peptides was demonstrated by use of the following bioassays: 1) naloxone-reversible inhibition of adenylate cyclase in homogenates of neuroblastoma X-glioma hybrid cells; 2) naloxone-reversible inhibition of electrically stimulated contractions of the mouse vas deferens; 3) displacement of [3H]dihydromorphine and [3H-Tyr, dAla2]met-enkephalin amide from rat brain membranes. Substances which stimulate adenylate cyclase and increase the contractions of the mouse vas deferens but do not bind to opiate receptors are also isolated from gluten hydrolysates. It is suggested that peptides derived from some food proteins may be of physiological importance.


The effects of substituted pyrimidines in DNAs on cleavage by sequence-specific endonucleases. The rates of cleavage of DNAs containing substituents at position 5 of thymine or cytosine have been measured for a variety of sequence-specific endonucleases, so as to determine which features in the DNA sequence are being probed. Phage phi e DNA fully substituted with 5-hydroxymethyluracil is cleaved more slowly by enzymes whose recognition sequences contain A-T base pairs than are DNAs containing thymine, but both types of DNA are cleaved at similar rates by enzymes recognizing sequences composed only of G-C base pairs. Phage PBS2 DNA with uracil completely substituted for thymine is cleaved slowly by several enzymes which recognize sequences containing A-T base pairs (endonucleases Hpa I, HindII, and HindIII), while the rates of cleavage by other enzymes (endonucleases EcoRI and BamHI) are not affected. Phage lambda- and P22 DNAs containing 5-bromouracil are cleaved more slowly by several enzymes (endonucleases HindIII, Hpa I, BamHI) than are thymine-containing DNAs. Enzymes that recognize sequence isomers with the composition G:C:2A:2T (endonucleases EcoRI, Hpa I, HindIII) are not equally affected by substitution at position 5 of thymine, suggesting that they differ in their contacts with A-T base pairs. DNA containing glucosylated 5-hydroxymethylcytosine in place of cytosine is resistant to cleavage by all the endonucleases examined.


Guanosine 5'-diphosphate-3'-diphosphate inhibition of adenylosuccinate synthetase. The mechanism of ppGpp inhibition of adenylosuccinate synthetase (EC 6.3.4.4) was examined. Initial rate kinetic studies demonstrate the ppGpp inhibition is competitive with respect to GTP and noncompetitive with respect to L-aspartate and IMP. This is in contrast to an earlier report (Gallant, J., Irr, J., and Cashel, M. (1971) J. Biol. Chem. 246, 5812-5816), which suggested that ppGpp did not bind at the GTP site. Possible reasons for the discrepancy are discussed. The potency of the ppGpp inhibition is confirmed.


Small angle X-ray scattering of dimeric yeast hexokinase in solution. Small angle x-ray scattering measurements on dimeric yeast hexokinase B at pH 5.5 in acetate buffer yield a radius of gyration of 31.28 +/- 0.23 angstrom. This measured value is comparable to the radius of gyration of 31.5 angstrom calculated from the refined coordinates of the dimer in the BII crystal form. The hexokinase dimer found in the BI crystal form has a radius of gyration of 42 angstrom calculated from the atomic coordinates. Thus, the measured radius of gyration is consistent with the BII dimer being the predominant species in solution and rules out the existence of the BI dimer as a major species under these conditions.


Novel mechanism of post-transcriptional modification of tRNA. Insertion of bases of Q precursors into tRNA by a specific tRNA transglycosylase reaction. The guanine insertion enzyme isolated from Escherichia coli (tRNA transglycosylase) catalyzed the incorporation of bases of Q (queuosine) precursors into E. coli undermodified tRNAAsn and tRNATyr. These bases of Q precursors were inserted in the first position of the anticodon of tRNASn and tRNATyr, replacing guanine originally located in that position. This is a novel type of post-transcriptional modification, inserting a modified base into the polynucleotide chain by cleavage of the N--C glycoside bond without breakage of the phosphodiester bond. One of the bases of Q precursors, 7-(aminomethyl)-7-deazaguanine, was found in the acid-soluble fraction of E. coli cells, supporting the conclusion that formation of Q, 7-(3,4-trans-4,5-cis-dihydroxy-1-cyclopenten-3-ylaminomethyl)-7-deazaguanosine, in tRNA in vivo actually proceeds by the tRNA transglycosylase reaction.


A rapid procedure for isolation of large quantities of Escherichia coli DNA polymerase I utilizing a lambdapolA transducing phage. DNA polymerase I produced by infection of Escherichia coli K12 with the specialized transducing phage lambdapolA has been purified by a simplified procedure and shown to be identical with the enzyme produced by uninfected E. coli in all aspects which have been examined. The abundance of the enzyme in infected cells and the ease with which it may be purified will simplify the study of the enzyme's physical and chemical characteristics. In addition, the enzyme is now much more readily available for use as an analytical tool in nucleic acid sequence and structure studies.


Sequence of the 16 S-23 s spacer region in two ribosomal RNA operons of Escherichia coli. The transducing phages lambdadaroE and lambdadilv5, which carry the Escherichia coli ribosomal RNA operons rrnD and rrnX, respectively, have been mapped with the restriction endonucleases BamHI, EcoRI, HindIII, and Sma I. Using hybridization techniques, we have located the ribosomal RNA genes on these phage DNAs. The DNA sequence of the 437-base-pair 16 S-23 S ribosomal RNA intergenic spacer in the two rRNA operons rrnD and rrnX has been determined. The nucleotides examined exhibit only one base pair change between rrnD and rrnX. Both spacer regions contain the genes for tRNA1Ile and tRNA1BAla; the gene sequences are identical with the previously deduced tRNA sequences and are clustered within the first 60% of the spacer DNA. The most striking feature of the 16 S-23 S intergenic region in these two operons is the disparity in G-C content between the tRNA gene sequences (60% G-C) and the remaining spacer DNA (37% G-C). Spacer sequences are known to be involved in the processing of the ribosomal RNA transcript by RNase III and RNase P. In addition, we report the sequence of the first 108 base pairs of the 23 S rRNA gene.


Activity changes in lac repressor with cysteine oxidation. The effects of prior covalent cysteine modification or nonspecific DNA presence on the reaction of lac repressor protein with N-bromosuccinimide have been investigated. At low excesses, N-bromosuccinimide oxidation causes loss of operator DNA binding activity with simultaneous retention of inducer and nonspecific DNA binding activities. Cysteine and methionine are oxidized under the conditions utilized. Covalent modification of the cysteines of repressor prior to reaction decreased the observed loss of operator DNA binding capacity; the presence of nonspecific DNA partially prevented oxidation of the cysteines by N-bromosuccinimide, and concurrent protection of operator binding ability was observed. Methionine oxidation was observed in the cases where protection of the operator DNA binding capacity of repressor was seen. The region surrounding cysteine 107 was found to be influential in maintaining intact operator DNA binding function in repressor. This observation provides chemical evidence for the contribution of the core region of repressor in determining specificity of the protein in binding the lac operator. The protection from oxidation of cysteine residues in the core region by the presence of nonspecific DNA suggests that this binding influences the core region of the protein.


Tryptic core protein of lactose repressor binds operator DNA. The core protein produced by mild proteolytic digestion of lactose repressor protein has been purified from native repressor by chromatography on phosphocellulose. The core protein isolated in this manner binds to operator DNA with an apparent dissociation constant of 10(-7) M, and the observed binding is decreased by the presence of inducer. Competition studies with nonspecific DNA indicate that the binding species in the core protein preparations is neither intact lactose repressor nor mixed tetramers containing varying numbers of intact NH2-terminal regions. This conclusion is supported by experiments designed to measure the rate of dissociation of the core protein from the operator DNA. Calculations based on the assumption that the isolated core protein binds similarly to the corresponding region in intact repressor protein indicate that the core region contributes approximately 40 to 50% of the energy of binding to operator DNA. Furthermore, the change in operator affinity upon inducer binding to core accounts for a minimum of 60% of the free energy change in binding to operator observed for the native protein. The demonstration that core protein binds to operator DNA requires a re-evaluation of the various models for repressor binding to DNA. A possible model based on the available information is presented.


Glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Purification and properties. Glutamine 5-phosphoribosylamine:pyrophosphate phosphoribosyltransferase (amidophosphoribosyl-transferase) has been purified to homogeneity from Escherichia coli. The molecular weight of the native enzyme was 194,000 by sedimentation equilibrium centrifugation and 224,000 by gel filtration. A subunit Mr = 57,000 was estimated by gel electrophoresis in sodium dodecyl sulfate. Cross-linking experiments gave species of Mr = 57,000, 117,000, and 177,000. A trimer or tetramer of identical subunits is indicated for the native enzyme. Highly active E. coli amidophosphoribosyl-transferase lacks significant nonheme iron. Enzyme activity was not enhanced by addition of iron salts and sulfide. Amidophosphoribosyltransferase exhibited both NH3- and glutamine-dependent activities. Glutaminase activity was detected in the absence of other substrates. Both glutamine- and NH3-dependent activities were subject to end product inhibition by purine 5'-ribonucleotides. AMP and GMP, in combination, gave synergistic inhibition. AMP and GMP exhibited positive cooperativity. In addition, GMP promoted cooperativity for saturation by 5-phosphoribosyl-1-pyrophosphate. Glutamine utilization was inhibited by NH3, suggesting that the amide of glutamine is transferred to the NH3 site prior to amination of 5-phosphoribosyl-1-pyrophosphate. The glutamine-dependent activity was selectively inactivated by the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid and 6-diazo-5-oxo L-norleucine (DON) and by iodoacetamide. Incorporation of 1 eq of DON/subunit (Mr = 57,000) caused complete inactivation of the glutamine-dependent activity, thus providing evidence for one glutamine site per monomer and for the functional identity of the subunits. Following alkylation with iodoacetamide, carboxymethylcysteine was the only modified amino acid isolated from an acid hydrolysate. The glutamine-dependent activity was sensitive to oxidation. Inactivation by exposure to air was reversed by incubation with high concentrations of dithiothreitol.


Proinsulin precursors in catfish pancreatic islets. The purpose of these experiments was to determine whether insulin-related peptides, larger than proinsulin, could be detected in pancreatic islet cells. Catfish pancreatic islets were incubated with radiolabeled amino acids. After 15- to 60-min incubation, two acid-alcohol-extractable peptides, larger than proinsulin, were detected which were approximately of Mr = 12,000 and 11,000 (12 K and 11K, respectively). They migrated as single polypeptide chains by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis under reducing conditions, and were therefore not aggregates of insulin or proinsulin. The 12 K protein had identical mobility with catfish preoproinsulin synthesized in a wheat germ cell-free system. On standard electrophoresis at pH 8.9, the 12 K protein migrated separately from proinsulin and was at least 65% one protein with two to three minor contaminants. The 12 K and 11 K proteins were chemically related to insulin and proinsulin as shown by tryptic peptide analysis, using cation exchange resin chromatography, and by two-dimensional tryptic peptide maps. Analysis of the tryptic digest of the 12 K protein, compared to proinsulin after leucine aminopeptidase treatment, suggested that the NH2 terminus of the larger protein was different from that of proinsulin. These peptides were specifically bound to anti-insulin antibody. The binding was only 5 to 8% of the protein added, but was specific for the 12 K and 11 K proteins when the immunoprecipitates were examined by electrophoresis and not from contaminating proinsulin. During the continuous incubation of the islets with [3H]leucine, 12 K and 11 K proteins were synthesized in the cell before proinsulin. When islets were first incubated with [3H]leucine for 30 min followed by incubation with excess unlabeled leucine, the 12 K and 11 K proteins appeared to show a precursor-product relationship to proinsulin and insulin. Even when total islet protein synthesis was inhibited by cycloheximide (100 microgram/ml), proinsulin continued to be synthesized for up to 2 h. This suggested that the conversion of the proinsulin precursors to proinsulin in the fish is a post-translational event.


Can a small blood vessel prosthesis be derived from heterologous foreign body reactive tissue? This study investigated as a small diameter vascular replacement the tissue formed adjacent to an implanted cylindrical foreign body, heterologously transplanted. Grafts of 4 mm i.d. were grown in rabbits and transplanted to dogs as segmental carotid and femoral artery replacements. These maintained 50% patency after 3 weeks. Variables in the further development of this concept are discussed.


Nuclear volume control by nucleoskeletal DNA, selection for cell volume and cell growth rate, and the solution of the DNA C-value paradox. The 40,000-fold variation in eukaryote haploid DNA content is unrelated to organismic complexity or to the numbers of protein-coding genes. In eukaryote microorganisms, as well as in animals and plants, DNA content is strongly correlated with cell volume and nuclear volume, and with cell cycle length and minimum generation time. These correlations are simply explained by postulating that DNA has 2 major functions unrelated to its protein-coding capacity: (1) the control of cell volume by the number of replicon origins, and (2) the determination of nuclear volume by the overall bulk of the DNA: cell growth rates are determined by the cell volume and by the area of the nuclear envelope available for nucleocytoplasmic transport of RNA, which in turn depends on the nuclear volume and therefore on the DNA content. During evolution nuclear volume, and therefore DNA content, has to be adjusted to the cell volume to allow reasonable growth rates. The great diversity of cell volumes and growth rates, and therefore of DNA contents, among eukaryotes results from a varying balance in different species between r-selection, which favours small cells and rapid growth rates and therefore low DNA C-values, and K-selection which favours large cells and slow growth rates and therefore high DNA C-values. In multicellular organisms cell size needs to vary in different tissues: size differences between somatic cells result from polyteny, endopolyploidy, or the synthesis of nucleoskeletal RNA. Conflict between the need for large ova and small somatic cells explains why lampbrush chromosomes, nurse cells, chromatin diminution and chromosome elimination evolved. Similar evolutionary considerations clarify the nature of polygenes, the significance of the distribution of haploidy, diploidy and dikaryosis in life cycles and of double fertilization in angiosperms, and of heteroploidy despite DNA constancy in cultured cells, and other puzzles in eukaryote chromosome biology. Eukaryote DNA can be divided into genic DNA (G-DNA), which codes for proteins (or serves as recognition sites for proteins involved in transcription, replication and recombination), and nucleoskeletal DNA (S-DNA) which exists only because of its nucleoskeletal role in determining the nuclear volume (which it shares with G-DNA, and performs not only directly, but also indirectly by coding for nucleoskeletal RNA). Mechanistic and evolutionary implications of this are discussed.


Chemical information sources: aids in the review of drug applications. Applications of computerized databases to the "Chemist's Review" of drug applications are presented with emphasis on the critical topics addressed in the controls review process and the time limitations imposed thereon.


Experimental remarks on various types of small vessel anastomoses. Experiments were carried out on small arteries in dogs with end-to-end anastomosis by conventional sutures. Nakayama's rings and methyl-2-Cyanocrylate adhesive. Problems concerning their application for a satisfactory patency of the vessels are discussed. In conclusion, the best results were observed in the second group, while the use of adhesive material, though promising, is not to be advocated till further development of the material.


Abnormal plasma catecholamine responses in acromegalics. Plasma dopamine, norepinephrine, and epinephrine responses to bromocriptine (2.5 mg orally) or to LRH (100 microgram iv) were studied in seven acromegalic patients. In contrast to normal men, in whom plasma concentrations of catecholamines decrease markedly, acromegalic subjects fail to show decreases in plasma dopamine, norepinephrine, or epinephrine in response to bromocriptine (four of four studied) or LRH (three of four studied). Mean basal plasma catecholamine concentrations do not differ in acromegalic patients, normal men, and a group of nonacromegalic endocrine control patients. Since it appears that bromocriptine and LRH suppress plasma catecholamine concentrations through mechanisms at brain catecholamine neurons, these data are compatible with disordered catecholamine neurons in the brain of patients with acromegaly.


The hypothalamic-pituitary-testicular axis in thyrotoxicosis. The hypothalamic-pituitary-testicular axis was evaluated in seven men with thyrotoxicosis due to Graves' disease. Loss of libido and decreased potency were present in 71% and 56%, respectively. All patients had normal testicular volume (25 ml in all) and gynecomastia was detected in two of seven patients. Total sperm counts were less than 40 million in four of the five men tested. There was an inverse correlation between basal serum 17 beta-estradiol (E2) levels and total sperm count (r = -0.87; P less than 0.05). Mean (+/- SE) total testosterone (T) and E2 levels (1008 +/- 104 ng/100 ml and 104 +/- 16 pg/ml) were significantly higher than in normal men (P less than 0.05). Free T (13.6 +/- 2.4 ng/100 ml) was indistinguishable from normal (15.3 +/- 1.5 ng/100 ml). The mean (+/- SE) response of serum T to hCG administration was blunted (80 +/- 40%) compared to controls (193 +/- 19%; P less than 0.02). Basal plasma LH levels (15.5 +/- 1.5 mIU/ml) were significantly higher (P less than 0.05) than in normal men (9.1 +/- 0.6 mIU/ml) and hyperresponded to 100 microgram LRH iv in five of seven patients. Basal plasma FSH levels and the FSH response to LRH were normal. These results suggest that men with hyperthyroidism have 1) partial Leydig cell failure, 2) impairment of spermatogenesis, and 3) blunting of the feedback effects of E2.


Repetitive infusion of gonadotropin-releasing hormone distinguishes hypothalamic from pituitary hypogonadism. Patients who have severe hypogonadotropic hypogonadism caused by presumed hypothalamic disease often have a subnormal LH response to a bolus dose of gonadotropin-releasing hormone (GnRH). To determine if this subnormal response is the result of lack of exposure of the pituitary gonadotroph cells to GnRH, five such men were given daily infusions of 500 microgram GnRH, for 7 days. A standard 250-microgram bolus test dose of GnRH was administered before and again immediately after the week of GnRH infusions. Five men who had severe hypogonadotropic hypogonadism as a result of presumed pituitary disease also received daily GnRH infusions for 1 week. The mean incremental LH responses (+/- SE) to GnRH of the men with presumed hypothalamic disease were 5.0 +/- 1.9 mIU/ml before and 56.9 mIU/ml after the week of infusions. The mean incremental LH responses of the men with presumed pituitary disease were 2.4 +/- 0.7 mIU/ml before and 3.7 +/- 2.9 mIU/ml after the week of infusions. These data suggest that the normal gonadotroph requires prolonged exposure to GnRH for LH responsiveness to become normal, but that the severely damaged gonadotroph cannot be stimulated to release LH normally even by the same prolonged stimulation with GnRH.


Recognition of a new serogroup of Legionnaires disease bacterium. A strain of the Legionnaires disease bacterium (LDB) that was isolated by Joseph E. McDade from a postmortem lung specimen of a patient with fatal atypical pneumonia at the Veterans Administration Hospital in Togus, Maine was serologically different from 16 other strains of LDB that had been isolated previously from patients in other geographic locations. The serological differences of the Togus isolate were shown in results of direct and indirect fluorescent antibody staining and of immunoelectrophoresis with soluble antigen extracts. Seroconversion for the Togus strain of LDB in acute- and convalescent-phase sera from a second patient with atypical pneumonia at the Veterans Administration Hospital in Togus indicated that this patient had been infected with an LDB that was serologically similar or identical to the Togus isolate. The Togus serogroup of LDB should be considered when performing serological tests for Legionnaires disease.


Single-radial hemolysis as a cost-effective determinant of Rubella antibody status. Single-radial hemolysis was examined for sensitivity, reliability, and cost for determination of rubella antibody levels in the general population. Results obtained with single-radial hemolysis plates made in this laboratory, and those under development by a commercial manufacturer, were compared with those obtained by the hemagglutination inhibition method normally used for rubella antibody determinations. The results suggest that single-radial hemolysis is comparable to hemagglutination inhibition with respect to sensitivity and reliability; it is superior to hemagglutination inhibition in terms of ease of manipulation and economy of use.


Improved reliability of the primary plate bacitracin test on throat cultures with sulfamethoxazole-trimethoprim blood agar plates. The primary plate bacitracin differentiation disk susceptibility test identified 85% of group A streptococci from throat cultures on SXT-BA(CO2) plates within 24 h, as compared to only 26% on a conventional sheep blood agar plate.


Routine separation of staphylococci from micrococci based on bacteriolytic activity production. A new method is described by which separation of staphylococci from micrococci can be achieved in routine laboratory use. The basis of this method is that bacteriolytic activity is produced by staphylococci but not by micrococci.


Adaptation of the staphylococcal coagglutination technique for detection of heat-labile enterotoxin of Escherichia coli. Protein A-containing staphylococci coated with specific antiserum were tested for heat-labile enterotoxin of Escherichia coli. The immunological cross-reactivity of E. coli heat-labile enterotoxin with Vibrio cholerae toxin (choleragen) was the basis for sensitizing stabilized suspensions of the Cowan I strain of Staphylococcus aureus with anticholeragen. Unconcentrated culture supernatant fluid containing E. coli heat-labile enterotoxin produced macroscopic agglutination when mixed with sensitized staphylococci in capillary tubes. A total of 15 toxigenic and 61 nontoxigenic isolates were tested by the staphylococcal coagglutination technique in a coded fashion and found to be in agreement with previous results of the Chinese hamster ovary cell assay and the passive immune hemolysis test. The staphylococcal coagglutination technique is simple, relatively inexpensive to perform, and requires the immunoglobulin fraction of anticholeragen as the only specific reagent. The staphylococcal coagglutination technique appears to have potential for routine use in diagnostic microbiology laboratories.


Improved laboratory efficiency and diagnostic accuracy with new double-lumen-protected swab for for endometrial specimens. Intrauterine specimens were obtained from 22 patients with endometritis and 24 control patients following cesarean section by using both a new protected swab and a standard anaerobic swab. The protected swab improved the value of the direct smear and Gram stain, resulted in fewer false-positive cultures, better defined endometrial flora in patients with endometritis, and permitted major savings in laboratory personnel time and materials.


Solid-phase microtiter radioimmunoassay blocking test for detection of antibodies to Escherichia coli heat-labile enterotoxin. The development of a solid-phase microtiter radioimmunoassay blocking test to detect serum antibody to Escherichia coli heat-labile enterotoxin is described. The assay is easy to perform and quantitate, and it is sensitive and specific.


Factors affecting isolation and identification of Haemophilus vaginalis (Corynebacterium vaginale). The rate of isolation of organisms resembling Haemophilus vaginalis (Corynebacterium vaginale) from vaginal specimens was not significantly affected by anaerobic versus carbon dioxide incubation atmospheres or whether specimens were inoculated on isolation media immediately after collection or after a delay of 6 h. Forty-one clinically isolated strains were provisionally divided into 30 H. vaginalis strains and 11 H. vaginalis-like (HVL) strains based on morphological and growth characteristics. The H. vaginalis strains were less reactive in API-20A identification test strips, (Analytab Products, Inc.) using Lombard-Dowell broth, than in a modified basal medium that contained proteose peptone no. 3 (Difco). The numbers and kinds of substrates fermented by 30 clinical and 2 reference strains of H. vaginalis varied among conventional, API, Minitek (Baltimore Biological Laboratory), and rapid buffered substrate fermentation systems. A greater number and variety of carbohydrates were fermented by the 11 HVL strains more consistently in all four test systems. Analysis of volatile and nonvolatile fermentation end products by gas-liquid chromatography did not reveal significant differences between the H. vaginalis and HVL strains. However, the latter group grew in peptone-yeast extract-glucose broth, whereas the H. vaginalis strains did not grow without the addition of starch to peptone-yeast extract-glucose. All of the reference and clinical strains were similar in their susceptibilities to a variety of antimicrobial compounds except sulfonamides, which inhibited the HVL strains and bifidobacteria but not the H. vaginalis strains. Sulfonamide susceptibility or resistance corresponded in part to the H. vaginalis and HVL-bifidobacteria strain reactions on selected conventional fermentation substrates. Susceptibility or resistance to sulfonamides and metronidazole in conjunction with fermentation tests is described to aid in the separation of H. vaginalis from other possibly unrecognized biotypes of H. vaginalis or other vaginal bacteria that presumptively resemble the organism. A human blood medium known as V agar was also of considerable value in distinguishing H. vaginalis from HVL strains, because only the H. vaginalis strains produced diffuse beta-hemolysis on V agar.


Biochemical, enzymatic, and serological differentiation of Peptococcus indolicus (Christiansen) Sørensen from Peptococcus asaccharolyticus (Distaso) Douglas. Peptococcus indolicus (formerly Micrococcus indolicus) is an asaccharolytic anaerobic coccus that is frequently isolated from udder secretions from cases of heifer and dry-cow mastitis (summer mastitis). To facilitate better identification and its differentiation from Peptococcus asaccharolyticus, a variety of biochemical, enzymatic, and serological properties were studied. Seventy-nine strains of P. indolicus of bovine origin and 10 strains of P. asaccharolyticus of human origin were examined using the API 20A and API-ZYM test kit systems. In addition, production of extracellular enzymes by using sensitive substrate-containing agar plate tests, production of peptocoagulase (a plasma-clotting factor), hemolytic properties, metabolic end products by gas chromatography, and serological characteristics with a set of P. indolicus typing antisera were investigated. P. indolicus and P. asaccharolyticus were not satisfactorily differentiated solely by the API 20A system. P. indolicus differed from P. asaccharolyticus in producing H(2)S, reducing nitrate to nitrite, producing peptocoagulase, possessing alkaline phosphatase, and producing large amounts of propionate from lactate. Moreover, none of the strains of P. asaccharolyticus was typable with the P. indolicus typing antisera. The majority (88%) of P. indolicus strains also gave weak hydrolysis of ribonucleic acid, and 6 out of 79 produced deoxyribonuclease. All strains in this study were sensitive to metronidazole (5 mug) by disk diffusion tests.


Problems affecting performance of the fluorescent treponemal antibody-absorption test for syphilis. Immunofluorescent staining of Treponema pallidum was studied to clarify the effect of three factors on the results of the fluorescent treponemal antibody-absorption test: (i) heat inactivation of sera at 56 degrees C for 30 min before testing, (ii) use of multicircle slides, and (iii) tungsten illumination to visualize and assess unstained treponemes on reactive as well as nonreactive smears. It was found that serum inactivation before testing was not necessary for detection of immunoglobin G antibody, but an immunoglobulin M prozone was detected in unheated serum. On multicircle slides, it was demonstrated that a false-positive reaction could be obtained in 30 s at 37 and 25 degrees C if a smear where a nonreactive serum had been placed was crossed by a strongly reactive serum from another circle. Tungsten illumination proved necessary for correct assessment of unstained treponemes on all fluorescent treponemal antibody-aborption test smears, reactive or nonreactive. The possible role of these factors in incorrect fluorescent treponemal antibody-absorption test results is discussed.


Evaluation of colistin and nalidixic acid in Todd-Hewitt broth for selective isolation of group B streptocci. Todd-Hewitt broth (THB) containing colistin and nalidixic acid was compared with four other media with respect to efficacy of isolation of group B streptocci. It was effective as plain THB, THB with gentamicin and nalidixic acid and blood, and THB with colistin, nalidixic acid, and blood. THB with gentamicin and nalidixic acid, but without blood, was inhibitory to group B streptocci. The value of THB with colistin and nalidixic acid lies in its ability to successfully inhibit gram-negative organisms and still promote group B streptococcal growth without the addition of blood. This greatly reduces the time and expense of media preparation and permits early determination of bacterial growth, so that other means of rapidly identifying group B streptocci can be applied at the earliest possible time.


Simplified chromatographic separation of immunoglobulin M from G and its application to toxoplasma indirect immunofluorescence. The indirect immunofluorescent (IIF) antibody technique for the detection of Toxoplasma gondii immunoglobulin M (IgM) often gives false negative results, probably due to the competition between IgG and IgM. We therefore adapted a gel filtration procedure for the separation of IgG and IgM to a routine diagnostic test capable handling at least 10 sera per day and requiring only 50 microliters of serum. The results from 108 sera having positive complement fixation titers for Toxoplasma showed that 17 were IgM positive when the whole serum was tested by IIF compared with 55 positive when the IgM fraction was used. Sera with antideoxyribonucleic acid titers do not give false positive results after fractionation, and the removal of IgG eliminates false positive results due to rheumatoid factor. A prospective study showed that Toxoplasma IgM may persist up to 9 months.


Accuracy and reproducibility of the Oxi/Ferm system in identifying a select group of unusual gram-negative bacilli. The Oxi/Ferm (O/F) identification system was compared in a double-blind study to a conventional test battery for the characterization of 96 reference and clinical strains consisting of 83 nonfermentative and 13 oxidase-producing, fermentative gram-negative bacilli. The O/F tube and supplemental tests correctly identified 84% of the nonfermentative and 77% of the oxidase-producing, fermentative bacilli. However, when the supplemental tests were excluded and the biochemical profiles generated by all nine O/F tube reactions were examined, the profile accuracy reached 95% (79 of 83) for the nonfermentative and 93% (12 of 13) for oxidase-producing, fermentative bacilli. Seven of the nine O/F substrate reactions demonstrated less than or equal to 89% agreement with conventional reactions, whereas the urea and arginine reactions provided 82 and 85% agreement, respectively. Replicate O/F tests with six selected organisms demonstrated 97% identification reproducibility and 84% overall substrate reproducibility. The mean O/F identification time was 2.6 days as compared to 3.3 days for the conventional system. Although this study suggests that the O/F system is a convenient, rapid, and accurate alternative to conventional identification methods, several modifications are recommended.


Evaluation of sulfamethoxazole-trimethoprim blood agar plates for recovery of group A streptocci from throat cultures. We compared the selective blood agar medium of Gunn et al. (J. Clin. Microbiol. 5:650-655, 1977) which contains sulfamethoxazole plus trimethoprim (SXT-BA) to the conventional blood agar surface plate (SBA) and a modified blood agar pour plate plus broth method for the recovery of group A streptococci from throat swabs. The influence of CO(2) and ambient air incubation of the SXT-BA and SBA plates was also evaluated. A total of 696 throat swabs from symptomatic children were cultured simultaneously by the five methods and observed after overnight incubation; 204 positive cultures were detected overall. Recovery rates of each individual method were: SXT-BA (CO(2)), 90.7%; SXT-BA (air), 87.7%; pour plate plus broth, 83.3%; SBA (CO(2)), 79.4%; and SBA (air) 77%. Approximately one-half of the false-negative cultures in the SXT-BA (CO(2)) and SXT-BA (air) methods had colony counts of >/=10 to 100 colonies per plate. In contrast, for the SBA (CO(2)), SBA (air), and pour plate plus broth methods, approximately 70% of the false-negative cultures had colony counts of >/=10 to 100/plate. False-positive cultures obtained by the SXT-BA (CO(2)) and SXT-BA (air) methods were 11 and 12.7%, respectively-one-half as high as the rates obtained by the remaining methods. Beta-hemolytic streptococci, groups C, F, and G, are inhibited on the SXT-BA plates and were the primary cause of the higher false-positive rates on SBA and pour plate plus broth methods. An additional 3% positive cultures were obtained by incubating SXT-BA (CO(2)) plates up to 48 h before discarding as negative. We recommend either the SXT-BA (CO(2)) or the SXT-BA (air) method with up to 48 h of incubation for routine use in throat cultures.


Sensitivity, precision, and accuracy of the Y1 adrenal cell enterotoxin assay. The Y1 adrenal cell assay for heat-labile enterotoxin (LT) was found to be relatively insensitive for filtrates of toxigenic Escherichia coli H10407. When observations were made blindly and subjected to rigorous controls, reliable detection of LT occured only at filtrate dilutions from 1:4 to 1:10. Detection of LT was unreliable when E. coli H10407 was mixed with another enteric organism. Overall, 7.1% of observers' reports from a single assay were imprecise, and 2.6% would have resulted in errors in detection of LT; however, triplicate assays prevented false positive reports of LT detection. Routine testing for LT production in the clinical diagnostic laboratory awaits a simpler, more sensitive, and more direct method of testing stool specimens.


Reproducibility of an indirect immunofluorescent-antibody technique for capsular serotyping of Klebsiella pneumoniae. Reproducibility of capsular serotypes of 55 consecutive clinical isolates of Klebsiella pneumoniae was evaluated by an indirect immunofluorescent-antibody technique previously described by Riser et al. (J. Clin. Pathol. 29: 296-304, 1976) Five colonies per specimen were examined for colony-to-colony variation, day-to-day variation, and reader-to-reader variation. Seventy-two reference strains were tested with each of 18 pools and 72 specific antisera prior to the clinical specimens to determine antiserum specificity and cross-reaction patterns. Lot-to-lot variation was examined with the reference strains. There was minimal lot-to-lot variation among the antisera tested. Ten antiserum pools required supplementation with individual antisera. The patterns of supplementation may vary from lot to lot. Colony-to-colony variation in intensity of immunofluorescence occurred, but there was no variation in serotype. These findings differ from previously reported colonial variation which occurred when API 20E biotypes were determined for individual colonies of K. pneumoniae directly from clinical specimens. Eighteen percent of clinical isolates studied gave cross-reactions when tested with the indicated specific antiserum. All but one of the cross-reactions were resolved with further dilutions. Day-to-day and reader-to-reader variations were minimal. The immunofluorescent-antibody technique is a reliable and reproducible method for capsular serotype determination. Capsular serotypes are less variable than API biotypes since colony-to-colony variation of serotype does not occur.


Comparison of media and techniques for detection of group A streptococci in throat swab specimens. Detection of group A streptococci in primary throat cultures was compared by using aerobic and anerobic incubation with selective nonselective media. Sheep blood agar plates incubated anaerobically detected 98% of the group A streptococci, whereas aerobically incubated blood agar plates which had been stabbed at the time of inoculation detected only 63%. Blood agar plates containing sulfamethoxazole and trimethoprim (23.75 and 1.25 mirograms per ml, respectively) detected only 70% of group A streptocci when incubated aerobically and 84% when incubated anaerobically.


Comparison of the API 20E and Oxi/Ferm systems in identification of nonfermentative and oxidase-positive fermentative bacteria. The API 20E and Oxi/Ferm systems were tested in parallel to identify nonfermentative bacteria and oxidase-positive fermentative bacteria. Test strains consisted of consecutive clinical isolates, with stock cultures used to supplement those species infrequently recovered. The two microsystems, as well as tubes of triple sugar iron, motility, cetrimide, and oxidative glucose media, were inoculated by each worker for each organism. Identification of each isolate was by the protocol of the manufacturers, with supplemental tests and flagella stains performed when necessary. Concurrent identification was undertaken with a conventional system against which the results of the two systems were compared for accuracy. There was a 95.3% accuracy in identification by the Oxi-Ferm system and 88.9% by the API system. Almost one-fourth of all identification attempts with the API required computer assistance, and most of these were for oxidase positive bacteria. Because of this, and because the API system showed greater accuracy in identification of the oxidase-negative bacteria, it seems best suited for identification of these organisms (P. maltophilia, A. anitratus, and A. lwoffi). The Oxi/Ferm system is technically less cumbersome than the API and is well suited for both groups of organisms.


Isolation of Clostridium botulinum from Honey. Methods for the isolation of Clostridium botulinum from honey samples are described. A total of 9 of 90 honey samples were positive for C. botulinum; 6 of the positive samples had been fed to babies who developed infant botulism.


Rapid subtyping of influenza A virus isolates by membrane fluorescence. During the winter of 1977-1978 three influenza A virus serotypes (A/Vic/3/75, A/Texas/1/77 [both H3N2], and A/USSR/90/77 [H1N1]) circulated in Denver, offering us the opportunity to apply fluorescent antibody techniques to the specific identification of these viruses. Surface antigens of infected, unfixed primary monkey kidney cells were stained in suspension by an indirect immunofluorescence technique with anti-H3N2 and anti-H1N1 antisera. In tests of cells infected with known viruses, the members of the H3N2 family could not be distinguished from one another, but were easily distinguished from H1N1 strains. A total of 101 hemadsorption-positive clinical specimens were evaluated over a 6-month period. Forty-five of 48 influenza A H3N2 and 24 of 29 H1N1 specimens confirmed by hemagglutination inhibition were correctly identified by membrane fluorescence of cultured cells, with no misidentifications among influenza strains and with 1 false positive among 24 non-influenza isolates. The average time to identification by this technique was 4 days compared to 7 days by hemagglutination inhibition. Live cell membrane fluorescence is a simple, rapid, and accurate method for identifying and grouping influenza A viruses.


Invasive strain of Escherichia coli belonging to O group 29. A strain of Escherichia coli isolated from a child with diarrhea was biochemically similar and antigenically identical (O and K antigens) to the standard strain of E. coli O group 29 and was positive in the Serény test for invasiveness, which suggests that it can cause a Shigella-like illness.


Compact anaerobic glove box for hospitals and research laboratories. An anaerobic glove box designed for moderate-sized hospitals or research laboratories is described. The compact vinyl and metal enclosure becomes anaerobic rapidly after inflation, requires no vacuum pump, and provides incubator space equivalent to 15 standard anaerobic jars.


Detection of pigment precursors in white clinical strains of Serratia marcescens. Filter paper strips containing a pigment precursor extracted from Serratia marcescens strain 933 were used to determine whether white, clinical S. marcescens strains could form pigment syntrophically. In all, 114 strains (113 of clinical origin) were tested, and 99% were found to develop colors ranging from violets to pinks.


Experimental glomerulonephritis in the isolated perfused rat kidney. The development of immune deposits on the subepithelial surface of the glomerular capillary wall was studied in isolated rat kidneys perfused at controlled perfusion pressure, pH, temperature, and flow rates with recirculating oxygenated perfusate containing bovine serum albumin (BSA) in buffer and sheep antibody to rat proximal tubular epithelial cell brush border antigen (Fx1A). Control kidney were perfused with equal concentrations of non-antibody immunoglobulin (Ig)G. Renal function was monitored by measuring inulin clearance, sodium reabsorption, and urine flow as well as BSA excretion and fractional clearance. Perfused kidneys were studied by light, immunofluorescence, and electron microscopy. All kidneys perfused with anti-Fx1A developed diffuse, finely granular deposits of IgG along the glomerular capillary wall by immunofluorescence. Electron microscopy revealed these deposits to be localized exclusively in the subepithelial space and slit pores. Similar deposits were produced in a nonrecirculating perfusion system, thereby excluding the formation of immune complexes in the perfusate caused by renal release of tubular antigen. Control kidneys perfused with nonantibody IgG did not develop glomerular immune deposits. Renal function and BSA excretion were the same in experimental and control kidneys. Glomerular deposits in antibody perfused kidneys were indistinguishable from deposits in rats injected with anti-Fx1A or immunized with Fx1A to produce autologous immune complex nephropathy. These studies demonstrate that subepithelial immune deposits can be produced in the isolated rat kidney by perfusion with specific antibody to Fx1A in the absence of circulating immune complexes. In this model deposits result from in situ complex formation rather than circulating immune complex deposition.


The modification of biophysical and endotoxic properties of bacterial lipopolysaccharides by serum. Normal rabbit serum reduces the buoyant density of lipopolysaccharide (LPS) from Escherichia coli 0111:B4 (d = 1.44 g/cm3) and Salmonella minnesota R595 (d = 1.38 g/cm3) to a value less than g/cm3. This density shift is associated with the inhibition of a number of endotoxic activities of the LPS; namely, the pyrogenic activity, the ability to produce an immediate neutropenia in rabbits, lethality in adrenalectomized mice, and anticomplementary activity. A qualitatively similar change in buoyant density was observed to occur after intravenous injection of the LPS into rabbits. Preliminary evidence suggests that the density shift does not occur as a result of the degradation of the glycolipid backbone of the LPS. These data suggest that the interactions of LPS with plasma (or serum) components leading to reduction in buoyant density may account for a major pathway of LPS detoxification.


Role of secretory events in modulating human neutrophil chemotaxis. The relationship between neutrophil polymorphonuclear leukocyte (PMN) locomotion and the exocytosis of neutrophil cytoplasmic granules was studied by assessing these processes in cells migrating through micropore filters and by measuring the effects of degranulating stimuli on PMN chemotaxis, orientation, adhesiveness, and ability to bind the chemoattractant f-Met-Leu-[3H]Phe. Studies of cells migrating through cellulose nitrate filters indicated that concentrations of f-Met-Leu-Phe optimal for exocytosis were greater than those optimal for chemotaxis and actually inhibited cell migration. In other studies incubation of PMNs with concentrations of secretagogues causing exocytosis of 30% or greater PMN lysozyme increased cell adhesiveness and inhibited chemotaxis. PMNs that had secreted more than 30% lysozyme appeared round, did not orient in a gradient of chemoattractant, and were capable of significantly less f-Met-Leu-[3H]Phe binding than were control cells. The decreased binding of f-Met-Leu-Phe was not associated with hydrolysis of chemotactic peptide by washed cells, although peptide hydrolysis was caused by cell products secreted extracellularly after vigorous exocytosis. In contrast, when only 10--15% cellular lysozyme was released f-Met-Leu-Phe binding was enhanced significantly and there was no depression of chemotaxis. The data indicate limited exocytosis of intracellular granule contents is associated with increased availability of PMN cehmotactic factor receptors. Vigorous exocytosis is associated with inactivation of chemotactic responsiveness related to increase cell adhesiveness, decreased PMN binding of chemotactic factors, and to hydrolysis of chemoattractants by factors secreted extracellularly.


Adenosine deaminase deficiency: disappearance of adenine deoxynucleotides from a patient's erythrocytes after successful marrow transplantation. Accumulation of adenine deoxynucleotides (dATP and dADP) in the erythrocytes of a patient with adenosine deaminase (ADA) deficiency was confirmed. The patient, now 18 mo old, was treated with a bone marrow transplantation from his HLA identical sister at 7 mo of age. Before and after the transplant, his erythrocyte and lymphocyte ADA activities, as well as his erythrocyte nucleotide profiles, were measured. 10 wk after the marrow transplant, no ADA activity could be detected in his erythrocytes, whereas there was a mixture of donor and patient lymphocytes as measured by ADA assays and karyotyping. At the same time, both dATP and dADP had disappeared from his erythrocytes, which were entirely of patient origin. These findings indicate that partial engraftment of donor lymphocytes into an ADA-deficient patient is capable of "correcting" alterations of deoxynucleotide concentrations in the patient's ADA-deficient erythrocytes.


Selective binding site for [3H]prostacyclin on platelets. Prostacyclin (PGI(2)) is the most potent, naturally occurring inhibitor of platelet aggregation known. To determine whether PGI(2) is bound by platelets, high specific activity [9-(3)H]PGI(2) was synthesized by iodination and subsequent base treatment of the labeled precursor [9-(3)H]prostaglandin (PG)F(2alpha) methyl ester. Binding experiments were performed at room temperature with normal citrated human platelet-rich plasma that contained [(14)C]sucrose or [(14)C]PGF(1alpha) as an internal marker for the extracellular space. Binding of [(3)H]PGI(2) plateaued within 2 min and this bond radioactivity could be displaced rapidly by excess nonradioactive PGI(2). Scatchard analysis of concentration-dependent binding yielded a hyperbolic plot which appeared to be caused by the existence of two classes of binding sites. The higher affinity class has a dissociation constant of 12.1+/-2.7 nM and a capacity of 93 (+/-21)sites per platelet. The lower affinity class had a dissociation constant of 0.909+/-.236 muM and a capacity of 2,700+/-700 sites per platelet. The relative ability of PGI(2), PGE(1), PGE(2), and 6-keto-PGF(1alpha) to displace [(3)H]PGI(2) initially bound to the higher affinity class of sites were 100:5:<0.3: <0.3. These relative abilities parallel the relative potencies of these compounds as inhibitors of ADP-induced platelet aggregation in vitro. However PGD(2), which is more potent than PGE(1) as an inhibitor of aggregation, did not displace bound [(3)H]PGI(2). The higher affinity binding site for PGI(2) appears to be the specific receptor through which PGI(2) exerts its effect on platelets.


Motility and adhesiveness in human neutrophils. Effects of chemotactic factors. Human peripheral blood neutrophils (PMN) obtained from healthy adults were examined in vitro with techniques adapted to assess the effects of chemotactic factors (CF) on cellular configuration and adhesiveness. The results were compared with those that use certain conventional techniques for assessing chemotaxis and chemokinesis. Exposure of PMN to N-formyl-l-methionyl-l-phenylalanine (f-Met-Phe), zymosan-activated serum, bacterial chemotactic factor, or a low molecular weight chemotactic factor from activated serum (C5a) in the absence of a gradient resulted in a change in cellular shape from a spherical to a polarized configuration in a high percentage of cells. This occurred rapidly in suspension, under conditions designed to exclude a role for cell adhesiveness, and was reversible upon removal of the CF. Restimulation of cells with the CF resulted in reappearance of the polarized configuration to the same extent as on initial stimulation with one exception: f-Met-Phe pretreated cells failed to respond to f-Met-Phe, though they responded fully to the other CF. Each CF caused a significant increase in PMN attachment to protein-coated glass. This enhanced adhesiveness was not reversible upon removal of the CF when the cells were treated under conditions shown to produce chemotactic deactivation. Cells treated under these conditions also exhibited significantly reduced motility on glass and in micropore filters in the absence of a gradient of CF. Bacterial chemotactic factor, even at high concentrations, failed to produce deactivation and did not cause a sustained enhancement of adhesiveness.


Administration of gonadal steroids to the castrated male rat prevents a decrease in the release of gonadotropin-releasing hormone from the incubated hypothalamus. The influence of testosterone on gonadotropin-releasing hormone (GnRH) secretion was assessed indirectly by altering the serum testosterone concentration of male rats and measuring GnRH release from their incubated hypothalami 1 wk later.GnRH release from hypothalami of castrated rats was 13.4+/-1.2 (SE) pg/h, compared to 35.3+/-3.8 pg/h from hypothalami of intact rats (P < 0.001). GnRH release from the hypothalami of castrated rats treated with testosterone propionate, 100 or 500 mug daily, was 25.0+/-3.4 pg/h and 27.9+/-3.6 pg/h, which is significantly greater (P < 0.05 and P < 0.01, respectively) than that from hypothalami of castrated rats treated only with sesame oil.A similar decrease in GnRH release from hypothalami of hypophysectomized rats and prevention of this decrease by treating the hypophysectomized rats with testosterone propionate is evidence that the observed effects of testosterone are not mediated via luteinizing hormone and(or) follicle-stimulating hormone secretion. Treatment of castrated rats with either dihydrotestosterone propionate or estradiol benzoate also prevented the decrease in GnRH release from the hypothalami of castrated rats. We conclude that testosterone, dihydrotestosterone, and estradiol all prevent the decrease in GnRH release from hypothalami of castrated rats treated with these steroids. The possibility exists that these steroids may also maintain GnRH secretion in vivo.


Platelet and blood vessel arachidonate metabolism and interactions. Exogenous arachidonate addition to intact platelets, in the absence or the presence of blood vessel microsomes, results in the production of thromboxane B(2) (the stable degradation product of thromboxane A(2)) only. Prostaglandin (PG) endoperoxides are released from intact platelets only when thromboxane synthetase is inhibited. Thus, addition of exogenous arachidonate to imidazole-pretreated platelets in the presence of bovine aorta microsomes (source of prostacyclin synthetase) results predominantly in the synthesis of 6-keto-PGF(1alpha) (the stable degradation product of prostacyclin). Strips of intact aorta were removed from aspirin-treated rabbits, thus the isolated blood vessels were unable to convert endogenous or exogenous arachidonate to prostacyclin. Human platelets, with [(14)C]arachidonate-labeled phospholipids, adhered to the blood vessel segments and released some thromboxane B(2). The subsequent addition of thrombin facilitated the release of endogenous arachidonate and thromboxane, but no labeled 6-keto-PGF(1alpha) was detectable. There is therefore no direct chemical evidence of PG-endoperoxide release from human platelets during either aggregation or adhesion, which therefore precludes the possibility that blood vessels use platelet PG-endoperoxide for prostacyclin synthesis. Imidazole inhibited the thromboxane synthetase in the labeled platelets, and thereafter thrombin stimulation resulted in the release of platelet-derived, labeled PG-endoperoxides that were converted to labeled prostacyclin by the vascular prostacyclin synthetase. The latter result suggests a potential antithrombotic therapeutic benefit might be achieved using an effective thromboxane synthetase inhibitor.


Discrepancies between the doses of cholecystokinin or caerulein-stimulating exocrine and endocrine responses in perfused isolated rat pancreas. The effects of highly purified natural porcine cholecystokinin (CCK) and synthetic caerulein on the rate of flow of pancreatic juice, the rate of output of amylase, and the rate of release of immunoreactive insulin (IRI) and immunoreactive glucagon (IRG) were simultaneously investigated in the isolated perfused rat pancreas. The maximal flow rate of pancreatic juice was obtained with concentrations of CCK ranging from 0.5 to 10 mU/ml, whereas amylase output was maximal at CCK concentrations from 1 to 10 mU/ml. Caerulein at concentrations of 0.05-1 ng/ml induced a similar maximal flow rate and amylase secretion. Supramaximal stimulatory concentrations of these peptides resulted in lower rates of release of fluid and amylase than with the maximally effective concentrations. Stimulation of IRI and IRG release was elicited only with concentrations of peptides supramaximal for effects on the exocrine responses. The demonstration of very similar discrepancies between the doses of caerulein required to elicit maximal exocrine responses and those required to elicit endocrine responses provide strong evidence that the pattern of the effect of the porcine CCK is accounted for by CCK itself. Although caerulein had no influence on IRI response when superimposed on 100 or 150 mg/100 ml glucose stimulation, preperfusion of caerulein led to a significant enhancement of IRI response to a subsequent glucose stimulation in both phases. The augmentation effect was completely separate from the direct IRI-stimulating effect of caerulein, because the CCK-like peptide requires no glucose for insulinotropic action. Because the concentrations of the peptides necessary for stimulation of endocrine responses were inhibitory in their effects on exocrine responses, it may be inferred that it is unlikely that the endocrine effect is physiologically important, though the results of caerulein for augmenting glucose-stimulated IRI release suggests a possible role for CCK in carbohydrate metabolism.


Recovery of endothelial cell prostacyclin production after inhibition by low doses of aspirin. Endothelial cells synthesize prostacyclin (PGI(2)), an unstable prostaglandin that inhibits platelet aggregation and serotonin release. Because cyclooxygenase, which is necessary for synthesis of PGI(2), is inactivated by aspirin, we examined the effect of aspirin on PGI(2) production by cultured human endothelial cells. Endothelial cells synthesize PGI(2) (20.1+/-7.2 ng/10(6) cells, mean+/-SD) when stimulated with 20 muM sodium arachidonate for 2 min. PGI(2) production is inhibited by low-dose aspirin (5 muM); the t((1/2)) of inactivation is 6.0+/-1.3 min (mean+/-SEM, n = 3). Thus, endothelial cell cyclooxygenase is as sensitive to aspirin as the enzyme in platelets. After 1 h incubation with aspirin, endothelial cell PGI(2) production was inhibited 50% by 2.1+/-0.4 muM aspirin and was inhibited 90% by 6.2+/-0.9 muM aspirin (mean+/-SEM, n = 4). When endothelial cells were incubated with 100 muM aspirin, washed, and recultured, their ability to synthesize PGI(2) returned to control levels in 35.6+/-1.0 h (mean+/-SEM, n = 4). Recovery of endothelial PGI(2) production after aspirin depended on de novo protein synthesis because treatment with cycloheximide (3 mug/ml) inhibited recovery by 92%.These results indicate that although endothelial cell cyclooxygenase in vitro is inhibited by low concentrations of aspirin, endothelial cells rapidly resynthesize their cyclooxygenase after the aspirin is removed. This rapid resynthesis of cyclooxygenase lessens the likelihood that aspirin used in clinical doses promotes thrombosis.


Identification and quantitation of platelet-associated fibronectin antigen. Platelet-associated fibronectin antigen has been identified by radioimmunoassay and immunofluorescent techniques. In radioimmunoassay, platelet fibronectin was immunochemically indistinguishable from plasma fibronectin. Platelet and plasma fibronectin were bound and eluted from gelatin-sepharose under similar conditions. The level of platelet fibronectin in detergent extracts of washed platelets from 12 healthy adults was 2.85 +/- 1.24 microgram/10(9) platelets. Immunofluorescence with F(ab')2 fragments of immunochemically purified antifibronectin showed that all platelets stained with a discrete punctate pattern. The identification of platelet fibronectin antigen raises the possibility that this protein may participate in platelet-platelet or platelet-surface interactions.


Immunoglobulin and complement in normal skin. Cryostat sections of normal skin from 57 white adults were examined by direct and indirect immunofluorescence for immunoglobulins, complement factors, and transferrin. The results for basement membrane zone (BMX) were significantly different for the 11 face and 46 non-face biopsies: in the face, IgM was found in five, IgG in two, IgA in one, and C3 in none, whereas, in non-face, IgM was present in six, IgG in none, IgA in one, and C3 in five. The results for dermal vessel walls (DV) were not apparently different for face and non-face; in the 57 biopsies IgM was present in one, IgG in none, IgA in one, and C3 in one. The 11 biopsies from the face and 26 of the non-face biopsies were examined further. No IgD or C4 was identified, but one case (scalp) showed BMZ Clq, properdin, and transferrin, and in two cases (one face, one non-face) DV properdin was found. Cytoid bodies (IgM and IgA) were present in moderate numbers in one case; all other positive reactions were finely granular.


Platelet aggregation in chronic idiopathic thrombocytopenic purpura. A method for washing platelets by albumin density gradient separation has been modified to prepare platelet rich plasma of thrombocytopenic patients for platelet aggregation studies. The concentration procedure, consisting of centrifuging platelets into a specific gravity gradient between plasma and 40-45% aqueous solution of bovine albumin, does not affect platelet aggregation adversely. Platelet aggregation in eight patients with chronic idiopathic thrombocytopenic purpura was determined by this method. On the basis of the results the patients could clearly be divided into two groups: four patients with normal aggregation and four with a qualitative platelet defect. In contrast to the other patients, the group with an in vitro platelet functional defect all had more prolonged bleeding times and the presence of a serum antiplatelet antibody.


Marrow graft rejection and inhibition of growth in culture by serum in aplastic anaemia. The sera of 28 patients with aplastic anaemia were examined for their effect on granulocyte colony growth in soft agar. Normal sera did not affect colony growth, but 13 sera from patients with aplastic anaemia, three from multiparous women, and six from patients polytransfused for various disorders caused colony inhibition. This inhibition was not due to the presence of HLA antibodies in aplasia patients because some sera inhibited HLA compatible bone marrow, and polyspecific HLA antibodies were not found in all inhibitory sera. All patients who failed to show engraftment or who rejected their bone marrow graft within three weeks had serum inhibitory to normal bone marrow cell culture, but inhibition could not be demonstrated against autologous bone marrow cells in these patients with aplastic anaemia. The results show that patients with serum inhibitors have an increased risk of early graft rejection and suggest that this rejection is mediated by antibodies directed against bone marrow stem cells.


A new antigen on the epithelial membrane: its immunoperoxidase localisation in normal and neoplastic tissue. Using antisera raised against defatted human cream we have demonstrated a new antigen on the epithelial membrane. An indirect immunoperoxidase technique on routinely processed histological sections was used, and we describe results which show that this antigen has a widespread but highly selective distribution, being apparently localised to membranes that have a secretory function. The antigen is carried by a variety of adenocarcinomas, suggesting that its localisation may have a role in the diagnosis and differential diagnosis of malignant disease.


A formalin fixative for immunochemical and ultrastructural studies on gastrointestinal endocrine cells. Using indirect immunofluorescence, indirect immunoperoxidase, and unlabelled antibody enzyme techniques, gastrin, pancreatic glucagon, insulin, and somatostatin were localised in sections of both wax- and resin-embedded tissues that had been fixed in a buffered formalin solution. Ultrastructural preservation of the resin-embedded samples was also adequate for combined electron microscopy and light microscope immunochemistry. As the fixative concerned is stable it can be permanently available in surgical units. It is suggested, therefore, that this fixative should prove useful as an alternative to buffered formaldehyde, which must be freshly prepared from paraformaldehyde powder, in institutions where specimen collection is difficult or which have to refer cases with an endocrine involvement to other laboratories for immunochemical and fine structural examination.


Infection of total hip prostheses by Peptococcus magnus: an immunofluorescence and ELISA study of two cases. In two cases of infected total hip replacements, Peptococcus magnus was isolated in pure culture from the implant when it was removed. Fluorescent antibody and ELISA studies have shown that both patients developed an antibody response to this anaerobic coccus soon after the replacement operation. These results suggest that the organism is a true infective agent, which was probably responsible for the failure of the arthroplasty operation.


Identification of Gram-negative non-fermenters and oxidase-positive fermenters by the Oxi/Ferm tube. Since the recent introduction of the Roche Oxi/Ferm Tube to the UK two identification schemes have been developed by the manufacturer for use with the kit. We evaluated the success of these two schemes in identifying 222 predominantly culture collection strains belonging to 45 taxa of non-fermenters and nine taxa of oxidase-positive fermenters. The strains were chosen to represent all the taxa included in the two identification schemes developed by the manufacturer and we have therefore been able to assess the overall success of identification by the two schemes. Since, however, our choice of strains does not reflect their incidence in clinical material, our identification rates are not necessarily those that might be obtained in a routine clinical laboratory. The most advanced identification scheme so far developed for the Oxi/Ferm Tube (CCIS System 1977-1432) allowed 62% of the 222 strains to be correctly identified although a disturbing feature was that more of the strains that were not correctly identified were incorrectly identified (24%) rather than not identified (14%); these figures represent an improvement over the earlier identification scheme (CCIS System 1976-621-74346) for which the corresponding figures were 56%, 32%, and 12%. CCIS System 1977-1432 seems likely to give a better performance in a routine clinical laboratory than in this study since for those taxa which, we would judge from the material sent to us for identification, are most commonly seen in a routine laboratory (Acinetobacter calcoaceticus, A. lwoffii, Pseudomonas aeruginosa, P. fluorescens, P. maltophilia, P. pseudoalcaligenes, and P. putida) 89% were correctly identified, none remained unidentified, and 11% were incorrectly identified. Thirty strains, each of a different taxon, were tested in triplicate to assess the reproducibility of reactions in the Oxi/Ferm Tube.


Gastric function and histology in chronic renal failure. Gastric function and histology were investigated in 24 patients with untreated chronic renal failure. At endoscopy nine patients had oesophagitis, 12 patients were considered to have gastritis, and the duodenum appeared inflamed in 20 patients. Endoscopic biopsies were taken at standard sites in the stomach and duodenum; gastritis was found in all patients, and 17 patients had duodenitis. Stimulated acid secretion was impaired in seven out of 20 patients and acid hypersecretion was found in a further two patients. Pepsin output correlated well with acid output in these patients. Fasting serum gastrin levels were elevated in 12 of the 19 patients tested. Patients with atrophic gastritis had low acid outputs and hypergastrinaemia, and when extensive gastritis was present, the patients tended to have more severe renal failure and hyposecretion of acid. Three patients were studied again after regular haemodialysis or renal transplantation and were found to show marked endoscopic and histological improvement.


Toxocariasis: serological diagnosis by enzyme immunoassay. An enzyme-immunoassay was developed to measure the concentration of serum antibody specific for the secretory antigens released by migrating toxocaral larvae. This technique was evaluated by testing sera from healthy UK adults, and from patients with and without toxocariasis. In 922 healthy adults, 2.6% were found to have elevated specific antibody levels. Elevated values were observed twice as frequently in males as in females but showed no significant regression with age between 20 and 65 years. Of 62 patients with non-toxocaral helminthic infections, all had antitoxocaral antibody levels within the range of values observed in healthy controls and had a mean level which was not significantly elevated. All of 13 patients with clinical toxocariasis had enzyme-linked immunosorbent assay (ELISA) antibody levels above the 100th percentiles of both the healthy population and the helminth-infected group and had a significantly high mean value (p less than 0.001) more than 12 times that of the healthy or infected controls. The high degree of sensitivity and specificity of the toxocariasis enzyme-immunoassay indicates that this new test should be useful in reference immunodiagnostic applications and in large-scale seroepidemiological surveys.


Gingival bleeding in an experimental clinical trial design. An experimental clinical trial design is described in which the onset of gingival bleeding was used as an indicator of early gingivitis. Twenty-nine dental students participated in a double-blind crossover trial utilising chlorhexidine as a known plaque inhibitor. The onset of gingival bleeding was assessed in three ways, one of which yielded a statistically significant difference in favour of the active mouthrinse (P = 0.02). The method has a role in screening agents believed to be active against gingivitis.


Periodontal dressing materials.

Periodontal dressing materials. A detailed review of periodontal dressings is presented, covering physical, chemical and biological aspects. Areas requiring further research are outlined, particularly in the physico-chemical sphere; and some contra-indications to particular substances are described. It is concluded that there is a definite place for dressings, but that more knowledge is required before optimal properties can be developed.


Vascular morphology in noninflamed healed gingiva of dogs. The objective of this study was to determine if the regular vascular network previously reported as occurring in noninflamed marginal gingiva would become re-established following plaque control and treatment of gingiva by gingivectomy and scaling. Gingivectomies were performed on dogs with and without pre-experimental gingivitis in order to obtain gingival specimens healed for 4, 8 and 12 weeks. Plaque was controlled using toothbrushing and topical applications of 0.2% chlorhexidine gluconate. After 12 weeks, the animals were perfused via the carotid arteries with glutaraldehyde and Microfil latex compound. Specimens were processed for either routine histology or examination of vascular morphology in methyl salicylate cleared tissues. The absence of inflammation in healed gingiva was evaluated clinically, using the Gingival and Plaque Indices, and histologically. Results showed that in healed clinically and histologically noninflamed gingiva, the vascular morphology was established as a series of looped vessels which could readily be distinguished from the regular network of vessels described by Hock (1975) in marginal gingiva that had neither been inflamed nor resected.