Influence of calcium, magnesium, lanthanum and A23187 on the aggregation of polymorphonuclear neutrophils.

Chemotactic factors transiently aggregate human polymorphonuclear neutrophils. We have found that the aggregation induced by the chemotactic tripeptide, formyl-methionyl-leucyl-phenylalanine, depends on the extracellular calcium and magnesium concentrations and is inhibited by 1 and 10 M lanthanum chloride. The ionophore A23187 and 1 and 0.1 mM lanthanum chloride aggregate the cells. A23187-induced aggregation is dependent on extracellular calcium and magnesium whereas lanthanum-induced aggregation is not.


Effect of delta9-tetrahydrocannabinol on in vitro sensitization of mouse splenic lymphocytes.

The effects of delta9-tetrahydrocannabinol (delta9-THC) on the immune response of murine cells sensitized in vitro was determined using a plaque-forming cell (PFC) assay. Splenic lymphocytes from mice injected with delta9-THC showed a depressed immunologic response when compared with cells from control animals which were identically sensitized in vitro with sheep erythrocytes (SRBC). The direct addition of delta9-THC to the culture media altered the immunological response as demonstrated by a reduction in the number of PFC.


Study of human T and B lymphocytes with heterologous antisera. III. Immunofluorescence studies on tonsil sections.

Lymphoid cells from human palatine tonsils were identified on tissue sections by membrane or intracellular immunofluorescence (IF) staining. Used in an indirect technique, an anti-IgM and an anti-HTLA (human T lymphocyte antigen) antiserum gave complementary patterns of membrane IF, characteristic of the follicular organization. When serial sections were stained for each of the five classes, immunoglobulin-containing cells from all classes were found. Their relative frequencies were, in decreasing order: IgG: 61.6%; IgA: 17.3%; IgM: 12%; IgE: 7.5%; IgD: 1.6%. These differed from those of the gut-associated lymphoid tissue (IgA greater than IgG greater than IgM) and of the peripheral lymph nodes (IgG greater than IgM greater than IgA), but were close to those of mesenteric nodes. The absence of secretory component in tonsil was an additional difference from gut-associated lymphoid tissue and the relatively high proportion of IgE-containing cells, in the absence of recognized atopy, is another feature in common with mesenteric lymph nodes. Finally, slight differences between these results and those obtained on tonsil cell suspensions suggest that some degree of selection probably occurs during the isolation procedure.


Spleen cell changes during fatal and self-limiting malarial infections of mice.

Changes in the proportions and total numbers of splenic Thy-1.2+ cells, Ig+ cells and normoblasts were analysed during fatal Plasmodium berghei and non-fatal P. yoelii infections in mice. Thy-1.2+ and Ig+ cells were identified by rosetting techniques, and normoblasts by morphological criteria. The splenomegaly observed during these infections was found to be caused mainly by proliferation of normoblasts. An early increase in the numbers of Thy-1.2+ and Ig+ cells was detected in both infections, but in P. berghei infections these responses were subsequently suppressed. In P. yoelii infections Thy-1.2+ and Ig+ cell numbers were maintained at four to five-fold above normal levels until the mice had completely recovered. During the acute phase of P. yoelii infection it appeared that most splenic T-cells expressed surface immunoglobulin.


Competition of the actions of antigen and polyclonal B-cell activator in the induction and amplification of B-memory cell function.

Using the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a polyclonal B-cell activator (PBA) and sheep red blood cells (SRBC) as a T-dependent antigen, the correlation of the actions of PBA and T-dependent antigen on B cells in induction and amplification of immunological memory was studied. B-memory cell function, as judged by anti-SRBC responsiveness in vitro of spleen cells of CPS-K, was amplified by the secondary injection of SRBC into SRBC-primed mice, whereas it was decreased markedly by injection of CPS-K. When CPS-K was injected simultaneously with, or 1 or 2 days before the secondary injection of SRBC, B-memory cell function was also decreased markedly. On the other hand, CPS-K did not inhibit induction of B-memory cell function when injected simultaneouly with the primary injection of SRBC. However, CPS-K inhibited induction of B-memory cell function when injected 3 days before the primary injection of SRBC. The inhibition by CPS-K of amplification of B-memory cell function in response to SRBC when CPS-K was injected simultaneously with the secondary injection of SRBC occurred markedly in mice primed with SRBC 8 days or longer before the secondary injection, whereas it was not detectable in mice primed 3 days before. It is concluded that the CPS-K-mediated signal and the SRBC-mediated signal act competitively on the same subpopulations of B cells in induction and amplification of memory, and that the susceptibility of B cells to the CPS-K-mediated negative signal changes correspondingly with their maturation stage.


The elevation of adoptive responses to sheep erythrocytes in protein-deficient mice.

Plaque-forming cell responses and antibody titres to sheep red cells (SRC) were elevated in normally-fed irradiated adult mice reconstituted with spleen cells from young mice which had received a 4% albumin diet, compared with those given cells from normally-fed littermates. This was independent of the dose of cells transferred (5 x 10(6)--4 x 10(7)), the day of assay (3--10) and the duration of protein-deficiency (1 or 3 weeks). It suggests that the depressed responses produced by protein-deficient (donor) mice reported in earlier papers resulted from an impairment of reticuloendothelial function or a shortage of protein reserves, not directly associated with the spleen. As protein-deficiency (of the donor) accelerated the initiation of the adoptive response and increased the cell yields of the recipients it is considered likely that an enhancement of cell proliferation during the first 3--5 days after transfer was responsible for the elevated responses.


Use of leucocyte migration under agarose to study spontaneous and directed locomotion of leucocytes.

Three different cell attractants, together with the parallel use of the leucocyte migration agarose test (LMAT) and the leading front modification (LFM) of the Boyden chamber technique, were employed in studying whether the maximal migration of normal human polymorphonuclear leucocytes (PMNs) is higher toward an attractant (chemotaxis) than in the same attractant incorporated in the culture media (chemokinesis). Using LMAT, the maximal migration distance toward zymosan activated serum (ZAS) was found to be significantly longer than that under agarose mixed with ZAS, thus indicating a chemotactic effect exerted by ZAS. When bacterial culture filtrate (BCF) and casein were used as attractants, the corresponding difference was not significant, implying that the stimulatory effect of these substances on cell migration could be explained by increased random locomotion (chemokinesis) alone. In LFM, the migration rate was significantly higher along a casein gradient than without a gradient. Using ZAS, however, only chemokinesis could be demonstrated. BCF was found to attract PMNs into membrane filters only in the presence of human serum albumin. These observations give credence to the view that both LMAT and LFM are applicable to the in vitro assessment of chemotaxis and chemokinesis but the attractant of choice for this is different in each of the two methods.


A prospective evaluation of the leukocyte adherence inhibition test in colorectal cancer and its correlation with carcinoembryonic antigen levels.

Fifty-four patients from the surgical gastroenterology service and 22 healthy controls have been prospectively evaluated in a single-blind protocol by the LAI tube method. The LAI correctly identified 25 of 33 early colorectal patients staged as Dukes' B and C at surgery but none of the Dukes' D patients. An inverse relationship was seen between the results of the non-adherence index (NAI) and CEA levels which was most pronounced in those with advanced colorectal cancer. The majority of Dukes' B and C patients having a "false negative" LAI had a CEA level greater than 2.5 ng/ml, suggesting that more advanced disease than that seen at surgery may be present. Two of 22 normal controls gave a borderline positive NAI. Some technical problems, including the relatively short life of the tumor extracts, are discussed.


Antibody production by spleen cells of mice bearing MCA-induced tumours: search for suppressor cells.

The antibody response to sheep red blood cells of spleen cells from mice bearing a methylcholanthrene (MCA)-induced tumour was measured with the local hemolysis technique, after in vivo or in vitro immunization. The in vivo data varied according to the stage of tumour growth and were related to the splenomegaly developing during the tumour growth process. During the first 10--15 days after tumour grafting, the total number of plaque-forming cells in the spleen increase in proportion to the spleen growth and thereafter remained relatively constant although the splenomegaly still progressed. Experiments performed in vitro were used to test the antibody response of mixtures of cells from normal mice and syngeneic tumour-bearing animals. In most cases, no suppression of antibody production, due to cells from tumour-bearing hosts, could be detected. The results suggest that those cells, which suppress cell-mediated immune reactions, previously found in the spleen of animals bearing the MCA-induced tumour, do not suppress antibody production.


Longitudinal studies of chromosomal abnormalities and reticulum cell proliferation in New Zealand Black mice.

A longitudinal study of 40 New Zealand Black (NZB) mice and 20 BALB/c control animals was performed. A significant association was observed between the presence of acquired spleen-cell aneuploidy at some time during life and development of histological evidence of reticulum-cell neoplasia in individual animals. This finding is compatible with the hypothesis that aneuploid clones which arise in the spleens of aging NZBs are at least potentially neoplastic. However, no relationship between histologically neoplastic reticulum cell proliferation and aneuploidy was apparent in single splenic specimens obtained from NZB mice of various ages. This lack of association indicates that failure to detect chromosomal abnormalities on direct study of cells from a tumor cannot be taken as evidence that the neoplastic cells themselves lack such abnormalities.


Neoplastic behavior of chromosomally abnormal clones in New Zealand Black mice.

Old New Zealand Black (NZB) mice frequently develop reticulum-cell sarcoma and clones of aneuploid cells in their spleens. In order to define the relationship between neoplasms and chromosomal abnormalities, the pattern of distribution of aneuploid cells was studied to see if it corresponds to that expected of the malignancy, and chromosomally aberrant or normal spleen cells were transplanted into syngeneic newborn recipients. The results indicate that aneuploid cells arise focally but may disseminate widely in an affected mouse. A strong association was found between spleen-cell aneuploidy in a transplantation donor and both aneuploidy and histological evidence of reticulum-cell sarcoma in young recipients. Chromosomal aberrations in young recipients were always of donor origin. On the basis of these data, it seems likely that aneuploid clones which arise in the spleens of old NZB mice usually are at least potentially neoplastic.


An immunoregulatory factor associated with spleen cells from tumor-bearing animals. I. Effect on tumor growth and antibody production.

An immunopotentiating factor associated with spleen cells of C57BL/6J mice bearing the 3LL tumor is described. Supernatants of cultured spleen cells from tumor-bearing mice (TBM) augmented the generation of both 19S and 7S antibody-producing cells, when injected with sheep erythrocytes into syngeneic C57BL/6J mice. The enhancing supernatant acted both as a polyclonal activator, when injected in the absence of antigen, and as a potentiator of specific antigen-dependent humoral immune responses, when injected in the presence of antigen. It was found to augment induction of specific memory, but not memory expression. Concomitantly with their influence on humoral immune responses, TBM spleen cell supernatants enhanced tumor growth when injected, mixed with 3LL tumor cells, into syngeneic recipients. The secretion of a factor which augments antibody production was not confined to the 3LL tumor system. Spleen supernatants of C47BL mice carrying the B16 melanoma and those of C3H mice carrying the KHT sarcoma had a similar effect on antibody production. These findings suggest that an immunoregulatory factor(s) appears in spleen cells of TBM as a result of their interaction with the neoplastic tissue. This factor can potentiate production of antibodies, possibly also against tumor-associated antigens. The relevance of the immunopotentiating effects of such factor(s) to tumor growth is discussed.


Effects of oxyfedrine on left ventricular function in patients several months after myocardial infarction.

Left ventricular (LV) function was investigated by left heart catheterization at rest and during exercise in 15 men 3--5 months after acute myocardial infarction. The effect of 8 mg oxyfedrine i.v. in 10 patients was compared to placebo in 5. The administration of oxyfedrine led to a significant decrease of LV end-diastolic pressure; an increase of LV dp/dt max, heart rate, LV ejection fraction and LV stroke work; and a shift of LV function curve indicating its improvement. These results give evidence for a positive inotropic effect of this drug which could have therapeutic implications in patients with impaired LV function after myocardial infarction.


Pharmacokinetics of antimalarials and proposals for dosage regimens.

Blood level data for the antimalarials amodiaquine, chloroguanide, chloroquine, pyrimethamine, quinine and sulphadoxine have been retrieved from the literature and pharmacokinetically analyzed. Minimum, average and maximum blood level concentrations at steady state in suppressive treatment and peak concentrations in therapeutic treatment were predicted and blood level-time curves simulated. Based on the computer-predicted data, changes in dosage regimens are proposed to reduce the fluctuations between maximum and minimum levels in suppressive treatment, and for obtaining maximum peak levels in therapeutic treatment already with the loading dose.


Comparative bronchodilatory activity of cetiedil citrate monohydrate, theophylline, orciprenaline and placebo in adult asthmatics.

In 40 patients, a seven day observation period in which oral bronchodilators and corticosteroids were eliminated, cetiedil (100 mg, t.i.d.), theophylline (200 mg, t.i.d.) or orciprenaline (20 mg, t.i.d.) and placebo (100 mg Lactose, t.i.d.) were given for one week each, according to a double blind, crossover randomized code. Wheezing scores were improved by all three active drugs. Twenty five of these patients undertook a moderate degree of exercise. Cetiedil was the most effective drug in blocking exercise-induced bronchospasm of the drugs studied. At rest, FEV1/VC was improved by theophylline, while PEFR improved after orciprenaline. Cetiedil improved both parameters, significantly. In another 21 patients, cetiedil (100 mg, t.i.d.) was given, according to the 'patient blinded' method for two weeks. PEFR and Asthma Disability Scores steadily improved. Clinical effectiveness was observed as early as the 3rd or 4th day of cetiedil therapy, with maximum improvement detected on the 14th day. No major side effects or abnormalities in clinical laboratory tests were noted. Cetiedil also had a 'liquidifying effect' on bronchial mucous in these asthmatics.


A method for preparing a viable corneal endothelial layer, completely denuded of overlying stroma.

A method for denuding Descemet's membrane by dissection of all overlying stroma, while preserving the corneal endothelial layer, was developed and evaluated. The technique consists in treating the stroma with trypsin and surgically removing the softened stromal layers. With an automated thickness-measuring technique, the endothelium-Descemet preparation was found to range from 23 to 42 micron in thickness. Endothelial cell morphology was normal under specular and light-transmission microscopic examinations. Under electron microscopy, the endothelial cells appeared intact, except for an increase in the number of intracellular vacuoles. Occasionally, small portions of an intercellular space were found to be mildly dilated, but the over-all integrity of the junctional complex was intact. In vitro, the viability of the preparation was comparable with that of a cornea with all layers intact. Endothelial resting membrane potentials, measured with intracellular microelectrodes, were found to be within the normal (33 +/- 2 mV) range. It is thus possible to obtain a viable endothelial layer, completely stripped of stroma.


Experimental nephropathy induced in the rabbit by immunization with a lipopolysaccharide from Escherichia coli.

Twenty rabbits were each injected with 100 microgram of lipopolysaccharide from Escherichia coli 055 at weekly intervals for up to 15 months. The antisera showed an immunologic cross-reactivity with rabbit kidney glycoprotein. A macroscopic nephropathy was present in 14 of the 17 rabbits in which the kidneys were examined. All the rabbits showed extensive histologic lesions involving all the structures of the kidney: organized thrombosis of the arteries, extensive areas of infarction, glomerular atrophy, tubular necrosis and proliferation of young connective tissue. A marked infiltration with lymphoid cells and some plasmacytes was present. The immunologic character of this nephropathy and the immunopathogenic mechanism involved in its pathogenesis are discussed.


An indirect fluorescent antibody technique for Haemophilus ducreyi.

An indirect fluorescent antibody technique which allows the detection of antibody against or the recognition of Haemophilus ducreyi has been developed. Heat inactivated antiserum was reacted uith prepared smears of Haimophilus species and selected organisms commonly associated with penile ulcerations. In serum absorbed with Haemophilus sp no fluorescence was observed with 156 heterologous organisms, but all six strains of bacteriologically confirmed H. ducreyi gave significant reactions with the absorbed anti-WD-68 serum.


Endemic Serratia marcescens in the Veterans Administration Hospital in Pittsburgh, Pa., 1971--1976.

The incidence of infections due to Serratia marcescens increased steadily at the Veterans Administration Hospital in Pittsburgh, Pennsylvania from 1970 to mid 1975 followed by a decline extending into 1977. One hundred eighty-four Serratia marcescens isolates were collected from 123 patients over a 4-month period in 1975 and were characterized by stereotype and antibiotic sensitivity. Three-fourths of the isolates were clinically significant. Among 24 stereotypes O-:H2 predominated in the urinary tract and O-H12 in respiratory secretions. Clusters of serotypes were on occasion identified in the GU Surgery Ward and the Intensive Care Unit. Uninary isolates showed remarkable resistance to antibiotics; only two thirds were susceptible to amikacin, the most effective agent in vitro. The patients from whom Serratia was isolated were older than the general hospital population (60 vs. 53.6 years of age), were hospitalized longer than the age-corrected controls (62 vs. 34.5 days) and two-thirds of thirds of them acquired the infection in the hospital. Uninary catheterization and prior antibiotic therapy were significant risk factors for urinary infections, and prior surgery contributed to recovery of Serratia from the respiratory tract. Serotype and antibiotype differences between isolates from the urinary and those from the respiratory tract suggest that strains evolved a site specificity.


[The chemotherapy of urologic neoplasms].

The newest aspects for the use of cytostatic agents in the treatment of bladder, prostatic and testicular carcinomas are presented. Especially the two new drugs diaminodichlor cisplatinum and adriamycin are discussed as to their potential use in these diseases.


[Urologic findings in patients with kidney transplantations. Evaluation of a technic for uretero-cysto-neostomy].

74 consecutive human kidney transplants are reported. In 72 cases uretero-cysto-neostomies with an antireflux procedure were performed in a typical way. The technique, a modification of Leadbetter's uretero-sigmoido-neostomy, has been used by the author for years. The only urological complications were one proximal and one distal ureteral fistula. In 25% out of 20 reexamined patients a vesico-ureteral reflux was demonstrated by micturition cysto-urethrography. There was no correlation between urinary tract infections (25%) and vesico-ureteral reflux. The results are compared with the data given in literature.


[Vesicorenal reflux and kidney transplantation].

25 (30%) out of 85 adult patients with endstage renal disease were found to have vesicoureteral reflux. In group I with 15 patients renal insufficiency was caused by kidney disease alone. In a second group 8 patients had the diagnosis chronic pyelonephritis with well documented history of urinary tract infection. In a third group 2 patients have megaloureter-megalocystis syndrome. There was no case of uncomplicated, sterile reflux in the second and third groups, which seems to be a rare cause of renal insufficiency.


Pulmonary and systemic vasodilator effects of the newly discovered prostaglandin, PGI2.

The effects of the newly discovered bicyclic prostaglandin, prostacyclin (PGI2), on the pulmonary and systemic vascular beds were investigated in the anesthetized dog. PGI2 decreased systemic and pulmonary arterial pressures in a dose-related manner when injected into the vena cava in doses of 1--30 microgram. Since left ventricular end-diastolic, left atrial, and right atrial pressures were unchanged, and since cardiac output was increased or unchanged, pulmonary and systemic vascular resistances were decreased. PGI2 was 10 times more potent than prostaglandins E1 or E2 in decreasing aortic pressure when injected intravenously, and the effects of PGI2 on the systemic vascular bed were similar when injected into the vena cava or the left atrium. These data indicate that inactivation of PGI2 is minimal in the lung. The stable prostacyclin metabolite, 6-keto-PGF1alpha, had little hemodynamic effects, suggesting that responses to PGI2 were not due to formation of this metabolite. PGI2 produced dose-dependent increases in blood flow in the mesenteric and renal vascular beds. These data demonstrate that PGI2 has marked vasodilator activity in the pulmonary and systemic vascular beds and suggest that prostacyclin is the only known metabolite of arachidonic acid that dilates the pulmonary and systemic circulations.


Importance of local immunity in enteric infection.

Evidence suggests that protection against intestinal infections is mediated by an immune system that is unique in many ways. The predominant intestinal immunoglobulin differs in structure, function, and site of origin from those immunoglobulins found in the blood-stream. Cell-mediated intestinal immune responses also may arise separately from those of the rest of the body. Further studies are necessary to characterize the local immune response to many human bacterial, viral, and protozoal pathogens, and to clarify the mechanism of "homing" of immunocompetent cells to the large and small intestine, mammary glands, and other external mucosal surfaces.


Comparative pathologic findings of Escherichia coli infection in birds.

Of the 3 respiratory tract disease syndromes that occur in birds associated with Escherichia coli infection, acute colisepticemia, characterized by hyperemic and swollen viscera, tended to occur in young birds. Subacute fibrinopurulent serositis involving air sacs and pericardium was more common in older birds. Chronic granulomatous pneumonitis was not seen as flock epornitics but as chronic disease in birds dying in small numbers some time after one of the previously mentioned forms of the disease. Serotypes of 100 E coli isolates from turkey colibacillosis revealed most to be O1, O2, O36, or O78. Virulence of the isolates, as conducted by IV inoculation of 6-week-old turkeys, showed O78 strains to be highly virulent and O36 strains to vary in virulence.


Immunization of calves against salmonellosis.

Salmonella typhimurium bacterins, containing adequate antigenic mass, protected calves against clinical signs of salmonellosis and death. Protection against salmonellosis was correspondingly reduced when the bacterin was diluted 1:10 and 1:100. A mouse protection test revealed that 17 of 18 (94%) of the S typhimurium-containing bacterin serials produced in 1977 stimulated adequate immunity.


Interventions in diarrheas of infants and young children.

To prevent diarrhea, efforts must be directed toward decreasing environmental contamination of enteropathogens, altering exposure of persons to naturally occurring agents, or to increasing host tolerance to prevalent enteropathogens. A vaccine is feasible in the control of certain enteric infections among high risk groups: typhoid fever and cholera, in areas of hyperendemicity or during an epidemic, shigellosis in institutionalized populations that are at risk to develop illness due to known prevalent serotypes, enterotoxigenic Escherichia coli for US travelers to Latin America, and perhaps rotavirus for all children under 3 years of age. In diarrhea, the most useful form of treatment is an oral glucose/electrolyte solution, which in most cases will prevent dehydration. Lactose containing foods should be removed from the diet early in the course of diarrhea. Antimicrobial agents are necessary in the treatment of typhoid fever, are useful in the treatment of shigellosis, giardiasis, and amebiasis, and are contraindicated in intestinal salmonellosis.


Keratoplasty for bullous keratopathy with intraocular lens.

The presence of a pseudophakos does not appear to introduce any greater hazard when keratoplasty is required because of corneal edema. This, together with the histories obtained from patients requiring such keratoplasty, strongly suggests that the insult to the corneal endothelium takes place at the time of placement of the intraocular lens in the great majority of cases. Of possible further significance is the fact that five of the sixteen patients who underwent keratoplasty had had secondary procedures to stabilize or to reposition the lens following the original lens implantation. The increased trauma inherent in the intraocular lens operation has been pointed out by many authorities. The fact that twelve of the sixteen patients requiring keratoplasty had been operated by experienced, nationally known intraocular lens surgeons further emphasizes this. The immediate problem appears to be the development and refinement of surgical techniques to avoid some of the complications which have been described. The prognosis for keratoplasty in cases of bullous keratopathy with an intraocular lens has been found to be at least as good as in cases without an intraocular lens.


Barrier Deprivation Syndrome. A case report.

A case has been presented where uncomplicated intracapsular cataract extraction and smooth implantation of a Copeland lens was followed by CME, later by CED. The process was stopped by performing a penetrating corneal graft and replacing the offending lens with a Medallion two-loop Medical Workshop lens. The case seems to support Binkhorst's theory of Membrane Deprivation Syndrome and Worst's findings of Decompartmentalization of the Eye.


Combined keratoplasty, cataract extraction and intraocular lens implantation.

In summary, corneal surgery with or without simultaneous cataract extraction and intraocular lens implantation, or in the presence of an intraocular lens, is justified in view of possible good, clear grafts (80% of our cases) and vision better than 20/50 (60% of our cases).


Removal by bovine serum albumin of fatty acids from membrane vesicles and its effect on proline transport activity in Escherichia coli.

Bovine serum albumin appreciably stimulated the initial rate and the steady-state level of proline uptake in membrane vesicles, while it had no effect on the oxidase activity for ascorbate-phenazine methosulfate, on which the transport activity depends. Bovine serum albumin showed the strongest stimulatory effect on the transport system among the proteins tested. Three other proteins did not show any effect, while beta-lactoglobulin showed a weaker but appreciable effect on the transport activity. The incubation of membrane vesicles with bovine serum albumin resulted in extensive removal of fatty acids, while none of the other membrane components, including proteins and phospholipids, was removed by this treatment. Fatty acids inhibited the proline transport activity, while the inhibited activity was appreciably restored by incubation with the albumin. An experiment with radioactive fatty acids showed that exogenously-added fatty acids bound firmly to the membrane vesicles and were removed by subsequent incubation with the albumin. The incubation of membrane vesicles for several hours resulted in an increase of fatty acids with a concomitant loss of the transport activity. Subsequent incubation with the albumin resulted in the removal of fatty acids and the partial restoration of the transport activity. Based on these results, it is concluded that bovine serum albumin specifically removed fatty acids from membrane vesicles, resulting in activation of the proline transport system.


Crystallization of arginine-, formylmethionine-tyrosine-, and glycine-transfer RNAs from Escherichia coli.

Crystals as large as 0.5 X 0.3 X 0.2 mm of purified arginine-transfer RNA from Escherichia coli have been prepared by a vapour diffusion method. X-ray diffraction photographs showed that the crystals gave reflections up to 3.7 A spacing. They have a trigonal space group P31 2 1 (OR P32 2 1) and cell-dimensions a=97.2, b=97.2, c=94.8A. Crystals of a mercury derivative of this transfer RNA have also been obtained, and an X-ray diffusion photography of one of them is presented. Formylmethionine-transfer RNA from E. coli was crystallized in various forms, and the appearance of the polymorphs was found to depend upon the amount of spermine in the solution from which the crystallization took place. Crystals of tyrosine-transfer RNA and glycine-transfer RNA have also been obtained.


Formation of biologically active protein from the subunits of islets-activating protein (IAP), a new protein isolated from Bordetella pertussis.

Based on the finding reported in the preceding paper (Kanbayashi, et al.: J. Biochem) that subunits of islets-activating protein (IAP), a new protein purified from the culture media of Bordetella pertussis, were inactive as such, but regained the original biological activities when recombined, the conditions required for recovery of the biological activities were studied. Essentially the same biological activities as the native IAP were recovered when the smallest subunit, F-3, was incubated with one of the other subunits, F-1 and F-2, at a pH of around 7, at temperatures below 30 degrees C and for longer than 12 h. During the incubation, association products were formed which were isolated by gel filtration as homogenous proteins that consisted of two subunits probably in a molar ratio of 1 : 1. The native IAP (consisting of two IAP subunits including F-3) were equipotent in enhancing insulin secretory responses, in inhibiting epinephrine-induced hyperglycemia, in inducing leukocytosis and in increasing histamine sensitivity in experimental animals.


A new method for preparation of an antiserum to penicillin and its application for novel enzyme immunoassay of penicillin.

A new method for the preparation of ampicillin-BSA conjugate by a three step procedure was developed. The first step is the introduction of a maleimide residue to ampicillin by a cross-linking reagent, MBS. The second step is reductive cleavage of disulfide bonds in BSA. The third step is thioether formation between the introduced maleimide residues and the reduced thiol groups. The obtained ampicillin-BSA conjugated raised an anti-ampicillin serum in rabbits. A new reagent, MPGS, was used for enzyme labelling of ampicillin to avoid immunological cross reaction. Using the enzyme labelled ampicillin and anti-ampicillin serum, enzyme immunoassay of ampicillin was successful in detecting 4 ng to 1 mug. Cross reactivities of anti-ampicillin to ampicillin analogs were studied by the enzyme immunoassay to find that the antiserum is specific to penicillin especially to ampicillin but hardly reacts with cephalosporins or the penicilloic acid derivative of ampicillin.


The bacteriophage lambda int gene product. A filter assay for genetic recombination, purification of int, and specific binding to DNA.

Bacteriophage lambda int gene is required for the integration of viral DNA into the chromosome of Escherichia coli. We have extensively purified the product of the int gene (Int) from a lysogen of E. coli that constitutively expresses this gene. Int was assayed by its ability to promote integrative recombination of supertwisted substrate DNA in vitro using a new method based on filter trapping of a recombinant product DNA. In order to catalyze integrative recombination, Int must be supplemented by other factors that can be extracted from bacterial host cells. By itself, purified Int does not demonstrate detectable endonuclease, exonuclease, or nicking-closing activities. However, Int does make stable complexes with double-stranded lambda-DNA containing an attachment site, the region at which recombination takes place. No stable complexes are observed between Int and lambda-DNA without an attachment site or between Int and DNA containing the bacterial site of integration. Int, therefore, appears to be a specificity element that relies on additional factor(s) to provide or activate the catalytic functions required for recombination.


Purification, separation, and characterization of two molecular forms of D-1-amino-2-propanol:NAD+ oxidoreductase activity from extracts of Escherichia coli K-12.

D-1-Amino-2-propanol:NAD+ oxidoreductase activity, which catalyzes the second step in a pathway wherein L-threonine is converted to D-1-amino-2-propanol via the intermediate formation of aminoacetone, has been purified 500-fold from Escherichia coli K-12. Although the enzyme catalyzes the oxidation of certain diols as well as 1-amino-2-propanol, it is completely specific for the D-isomer of the amino alcohol and for NAD+. Two molecular forms (designated Form L and Form S) of the oxidoreductase, both of which are catalytically active, have been separated by gel filtration on Sephadex G-200; apparently, Form L is converted to Form S by dissociation (Form L leads to Form S). Molecular weight determinations indicate that the two forms of the enzyme are different not only in size but also in shape; Form L apparently is an asymmetric tetramer of Form S. The two molecular species have similar catalytic properties. Both exhibit the same pH optimum of 8.6, have nearly identical apparent Km values for substrate and cosubstrate, are equally sensitive to inhibition by p-mercuribenzoate and N-ethylmaleimide, and show the same specificity for cosubstrate. Neither form of the enzyme has an absolute requirement for added thiol compounds or divalent metal ions.


Kinetic analyses of the sugar phosphate:sugar transphosphorylation reaction catalyzed by the glucose enzyme II complex of the bacterial phosphotransferase system.

The sugar phosphate:sugar transphosphorylation reaction catalyzed by the glucose Enzyme II complex of the phosphotransferase system has been analyzed kinetically. Initial rates of phosphoryl transfer from glucose-6-P to methyl alpha-glucopyranoside were determined with butanol/urea-extracted membranes from Salmonella typhimurium strains. The kinetic mechanism was shown to be Bi-Bi Sequential, indicating that the Enzyme II possesses nonoverlapping binding sites for sugar and sugar phosphate. Binding of the two substrates appears to occur in a positively cooperative fashion. A mutant with a defective glucose Enzyme II was isolated which transported methyl alpha-glucoside and glucose with reduced maximal velocities and higher Km values. In vitro kinetic studies of the transphosphorylation reaction catalyzed by the mutant enzyme showed a decrease in maximal velocity and increases in the Km values for both the sugar and sugar phosphate substrates. These results are consistent with the conclusion that a single Enzyme II complex catalyzes both transport and transphosphorylation of its sugar substrates.


Evidence for protein kinase activities in the prokaryote Salmonella typhimurium.

Evidence for phosphorylation of proteins by protein kinases has been found in Salmonella typhimurium despite previous indications that protein kinase action is absent in prokaryotes. At least four proteins have been found to be phosphorylated. Serine and threonine phosphates have been isolated from acid hydrolysates of these proteins after in vivo and in vitro labeling. The kinases do not phosphorylate histones, casein, or phosvitin. It would appear that phosphorylation as a regulatory control exists in prokaryotes.


In vitro depolarization of Escherichia coli membrane vesicles by colicin Ia.

Conditions are reported under which membrane vesicles prepared from Escherichia coli K12 are depolarized by colicin Ia. Although incubation of membrane vesicles with active colicin Ia affects neither transport activity nor the ability of such vesicles to generate a deltapH or deltapsi, a single freeze-thaw cycle of such vesicles in the presence of colicin Ia leads to 1) retention of the colicin by the vesicles, 2) inactivation of transport activity, and 3) membrane depolarization, with a concomitant increase in the transmembrane deltapH. These effects are dependent upon the presence of active colicin Ia during the freeze-thaw cycle. These findings are consistent with our previous results showing that Ia-treated whole cells or membrane vesicles prepared from such cells are defective in their ability to generate a deltapsi, yet generate an increased deltapH (Tokuda, H., and Konisky, J. (1978) Proc. Natl. Acad. Sci. U. S. A., 75, 2579--2583). In addition to its effect on vesicles prepared from sensitive cells, we show that vesicles prepared from both colicin Ia-resistant and -tolerant cells are depolarized by colicin treatment with a concomitant increase in deltapH. It is concluded that the final target of colicin Ia is the cytoplasmic membrane. A model for the mechanism of colicin Ia action is presented in which colicin Ia binds to the specific colicin Ia outer membrane receptor and is subsequently translocated to the cytoplasmic membrane where its integration leads to the formation of ion channels.


Biosynthesis of bacterial glycogen. Isolation and characterization of the pyridoxal-P allosteric activator site and the ADP-glucose-protected pyridoxal-P binding site of Escherichia coli B ADP-glucose synthase.

[3H]Pyridoxal-P can be covalently incorporated into Escherichia coli B mutant strain AC70R1 ADP-glucose synthase by reduction with NaBH4. Two distinct lysine residues can be modified by the allosteric activator pyridoxal-P. Incorporation of [3H]pyridoxal-P in the presence of substrate ADP-glucose + MgCl2 prevents pyridoxylation of an ADP-glucose-protected site and allows modification of the allosteric activator site. Incorporation of [3H]pyridoxal-P in the presence of the allosteric effector, 1,6-hexanediol-P2, protects against pyridoxylation of the allosteric activator site and allows modification of the ADP-glucose-protected site. The activator site CNBr [3H]pyridoxyl-P peptide was purified to homogeneity in the presence of urea by Sephadex G-50 and CM-cellulose chromatography. The peptide consists of 59 residues, with a molecular weight of 6750. The NH2-terminal of the peptide has a 16-residue sequence overlap with the previously determined NH2-terminal sequence of the native enzyme. The activator site pyridoxyl-P lysine is identified as residue 38 of the native enzyme's NH2 terminus. The ADP-glucose-protected site CNBr [3H]pyridoxyl peptide was purified to homogeneity by Sephadex G-50 and DEAE-cellulose chromatography. The peptide consists of 21 residues, with a molecular weight of 2460. The sequence of this peptide has been elucidated.


Rapid solid phase isolation of 20 specific tRNAs from Escherichia coli.

A solid phase procedure has been developed for the rapid isolation of all 20 species of tRNA from Escherichia coli. The overall yields for a single preparation cycle ranged from 62 to 96%, the average being 80%. The values for the amino acid acceptor activities of the tRNA species equaled those reported in the literature for highly purified tRNAs. Starting from crude tRNA, a given tRNA species can easily be isolated in less than 2 h. One milliliter of the resin, which is reusable, is sufficient for the isolation of 200 mg of a specific tRNA. The procedure requires a bifunctional reagent, one moiety of which (--SO2Cl) reacts with the amino acid on the aminoacylated tRNA, the other, with the --SH group on the resin. Thus, only the desired tRNA species is bound to the resin; any of the other tRNAs in the filtrate can be isolated in another cycle. Raising the pH results in deacylation and release from the resin of the desired tRNA species. For tRNA Cys, it is necessary to block the --SH of cysteine prior to reaction with the bifunctional reagent. Side reactions involving the bifunctional reagent. Side reactions involving the bifunctional reagent and tRNA are either easily reversible or negligible (less than 0.01%).


The molecular mechanisms of dicarboxylic acid transport in Escherichia coli K12. The role and orientation of the two membrane-bound dicarboxylate binding proteins.

Previous communications from this laboratory have indicated that dicarboxylic acid transport in Escherichia coli is an active process, and that at least three genes are responsible for this transport system. In attempts to identify the transport components, one periplasmic binding protein and two membrane integral proteins (SBP 1 and SBP 2) were implicated to participate in the transport system in vivo. In the present communication, we demonstrate, through biochemical analysis of the transport mutants, that the two membrane transport genes, dctA and dctB, are responsible for the two membrane-bound dicarboxylate binding proteins, SBP 2 and SBP 1, respectively. We also find that the substrate recognition sites of SBP 1 and SBP 2 are exposed to the inner and outer surfaces of the membrane, respectively. This may have important implications for the role of SBP 1 and SBP 2 in the translocation process.


Multiple forms of carboxypeptidase Y from Saccharomyces cerevisiae. Kinetic demonstration of effects of carbohydrate residues on the catalytic mechanism of a glycoenzyme.

In the course of our further investigation of the active site titration of carboxypeptidase Y, using 4-nitrophenyl trimethylacetate, we have found that carboxypeptidase Y can be isolated in different molecular forms. Carboxypeptidase Y obtained from Fleischmann baker's yeast has a molecular weight of 53,000, as compared to 64,000 for an enzyme species isolated from Anheuser-Busch baker's yeast. The amino acid analyses of both enzymes were essentially identical and very similar to those reported by others. However, we have found that the molecular weight difference is due to a variation in carbohydrate content as determined by gas chromatography. When carboxypeptidase Y was isolated from a single source, Anheuser-Busch baker's yeast, we observed a smaller variation in carbohydrate content. In all cases, sugar analyses revealed only mannose and N-acetylglucosamine to be present. The effect of the enzyme's carbohydrate content on the "burst kinetics" of the 4-nitrophenyl trimethylacetate reaction has been examined. In general, the Anheuser-Busch enzyme, containing more carbohydrate than the Fleischmann enzyme, reacts with a larger apparent bimolecular rate constant, kcat/Km. On the other hand, the deacylation rate constant, k3, is affected only slightly.


The rate of adhesion of melanoma cells onto nonionic polymer surfaces.

The rates of adhesion of melanoma cells (carcinogenic) onto nonionic polymer surfaces were studied by using radioactively labeled cells and measuring the fraction of cells which adhered to the surface in a given time. Glow discharge (plasma) polymerization of 1,1,3,3-tetramethyldisiloxane and of nitrogen-acetylene-water (mole ratio 0.4:1.0:0.2) was used to modify the surface energy of the substrate. The cell adhesion rate was found to be given by Y = 1 - exp [-k0(gammas - gamma0)t], where Y is the fraction of cells adhered, -k0 is a characteristic rate constant, gammas is the total surface energy of the substrate, gamma0 is the threshold surface energy of cell adhesion, and t is time.


The effect of methylmethacrylate on bacterial phagocytosis and killing by human polymorphonuclear leukocytes.

The phagocytic and bactericidal activities of human polymorphonuclear leukocytes in the presence of methylmethacrylate were determined by in vitro techniques. It was found that methylmethacrylate in concentrations as low as 0.156 per cent significantly decreased the ability of leukocytes to phagocytose and kill strains of Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli. The evidence indicates that methylmethacrylate impairs the killing more than the phagocytosis by the leukocytes.


Controlled trial of low-dose heparin and sulfinpyrazone to prevent venous thromboembolism after operation on the hip.

A randomized, double-blind controlled trial of low-dose heparin combined with sulfinpyrazone to prevent deep-vein thrombosis after operation on the hip was carried out. In a group of seventy-three patients after arthroplasty, postoperative thrombosis of the veins of the lower limbs occurred in 51 per cent of the control patients and in 36 per cent of the treated patients. In a fracture group of thirty patients, thrombosis occurred in 75 per cent of the control patients and in 36 per cent of the treated patients. These differences are of borderline statistical significance in the fracture group and are of no statistical significance in the arthroplasty group. However, a 36 per cent incidence of venous thrombosis in the drug-treated patients is too high to justify recommendation of the regimen tested without careful monitoring of patients by tests designed to detect thrombosis. Then, appropriate anticoagulant therapy can be instituted if necessary.


The acute effect of position of immobilization on capital femoral epiphyseal blood flow. A quantitative study using the hydrogen washout technique.

Quantitative studies of femoral head blood flow by the hydrogen washout technique with the hips in various positions of immobilization were determined. Immobilization in flexion consistently resulted in the highest rate of femoral head blood flow. A position of relaxed abduction seemed to slightly, but significantly, decrease the rate of blood flow. The position of forced frog-leg abduction and forced internal rotation obliterated or drastically reduced circulation in the femoral head. We think that the ischemia of forced acute immobilization is secondary to compression of the immature cartilaginous femoral head. These experimental data further indicate the importance of the selected position of immobilization in the treatment of congenital dislocated hips as a cause of iatrogenic avascular necrosis.


Subchondral route for nutrition to articular cartilage in the rabbit. Measurement of diffusion with hydrogen gas in vivo.

The route of nutrients going to articular cartilage was studied by determining the diffusion of hydrogen molecules from the subchondral circulation to the articular cartilage in rabbits. In all immature animals there was diffusion of hydrogen from subchondral bone into articular cartilage, while in the older immature animals the results were variable. None of the mature animals showed any diffusion of hydrogen into articular cartilage. The rate of diffusion of hydrogen was significantly lower in the articular cartilage than in the subchondral bone in the immature animals while the concentrations of hydrogen in the articular cartilage were only fractions of those in the subchondral bone at the same instant. Histologically, the deep layers of immature cartilage are penetrated extensively by vascular buds from the ossified portion of the epiphysis, while in adults the articular cartilage is separated from subchondral vascular spaces by an end-plate of bone. Blood vessels penetrating into the basilar layer of articular cartilage in immature animals appear to play an important role in the nutrition of articular cartilage coming from the subchondral region.


Antibiotic-acrylic bone cement composites. Studies of gentamicin and Palacos.

Palacos bone cement with and without contained gentamicin was tested for antibacterial efficacy using a rabbit model of a knee hemiarthroplasty and a challenge with intra-articular Escherichia coli. The gentamicin-containing cement prevented infection from challenge with Escherichia coli and protected against inadvertent operative infection as well. Antibiotic concentrations in synovial fluid and urine remained in the therapeutic range for three days, whereas levels in serum were always quite low. Eight per cent of the gentamicin had leached out of the bone cement by eight days, most of it during the first day. Thereafter, extremely small amounts of gentamicin left the cement on a daily basis. The amount of antibiotic leaching out in vitro was proportional to the surface area of the cement. More eluted into serum than into saline.


Heavy meromyosin labeling of intermediate filaments in cultured connective tissue cells.

Mild treatment with trypsin causes a radical change in the heavy meromyosin (HMM) binding properties of intermediate filaments in glycerinated, myosin-extracted cultured chick embryo connective tissue cells. In non-trypsin-treated cells, HMM labeling of filaments was often indistinct and variable in its distribution. By contrast, in cells treated with trypsin (under conditions which allowed most intermediate filaments to survive), virtually all filaments, including those of intermediate size, decorated with HMM to give distinct arrowhead patterns. We suggest that most intermediate filaments in such cells contain a core of F-actin masked by trypsin-labile accessory proteins.


Microfilaments and microtubules in calcium ionophore-induced secretion of lysosomal enzymes from human polymorphonuclear leukocytes.

Human peripheral blood leukocytes (PMN) are induced to release lysosomal enzymes by the calcium ionophore A23187 in the presence but not the absence of extracellular Ca++. Whereas secretion induced by particulate or immune stimuli is accompanied by an increase in visible microtubules and is inhibitable by colchicine, secretion induced by A23187 and Ca++ was not accompanied by an increase in microtubule numbers and was not inhibited by colchicine. Ca++ did not appear to regulate microtubule assembly in these cells since resting PMN had a mean of 22.3 +/- 2.0 microtubules in the centriolar region as compared to 22.3 +/- 1.1 in ionophore-treated cells and 24.9 +/- 1.5 in cells exposed to ionophore and 1 mM Ca++. Bipolar filaments, 10 nm thick and 300--400 nm long, were numerous in the pericortical cytoplasm of cells exposed to both reagents. Microtubules in these cells were decorated with an electron-opaque fibrillar material. PMN exposed to A23187 and Ca++ were contracted in two directions at right angles to each other: (a) Contractions parallel to the plasma membrane resulted in extensive plication of the cell membrane. The cytoplasm subjacent to the plicae contained dense filamentous webs. Plication was prevented by cytochalasin B or reversed by subsequent exposure to an endocytic stimulus such as zymosan. (b) Contractions perpendicular to the plasma membrane, toward the cytocenter, resulted in the formation of vacuoles in normal PMN and of membrane invaginations in cytochalasin B-treated PMN. Whereas contractions parallel to the plasma membrane could occur in the absence of enzyme release (ionophore alone) and enzyme release could occur in the absence of such contractions (ionophore plus calcium plus cytochalasin B), contraction toward the cytocenter occurred in all experimental conditions in which significant enzyme release was obtained. Thus, lysosomal enzyme secretion in PMN involves contractile movements in the plasma membrane toward the lysosomes rather than the reverse. These calcium-mediated contractile events are mediated by cytochalasin B-insensitive microfilaments but not by microtubule assembly.


Histone localization in polytene chromosomes by immunofluorescence.

Polytene chromosomes of Chironomus thummi were treated with antisera elicited by purified calf thymus histone fractions, and the location of each histone type was visualized by the indirect immunofluorescence technique. Each of the antisera produced specific and distinct patterns of fluorescence, suggesting that it is possible to use the indirect immunofluorescence technique to study the in situ organization of each histone in the various regions of the chromosomes. H1 and H2A antisera produced diffuse fluorescence patterns in acetic acid-fixed chromosomes which become more defined in formaldehyde-fixed preparations. Antisera to H2B, H3 and H4, when reacted with either formaldehyde- or acetic acid-fixed chromosomes, produce distinct banding patterns closely resembling the banding of acetoorcein-stained or phase-contrast-differentiated chromosomal preparations. These antisera produce corresponding patterns of fluorescence for each chromosome, suggesting that the overall organization of the histones is similar in the various bands. Because the dense band regions stain more brightly with antihistone sera than the less compacted interband areas, we believe that the number of antigenic sites of chromosome-bound histones is related to the amount of DNA present, and that the accessibility of histone determinants does not differ between the bands and interbands.


Localization of vicilin peptidohydrolase in the cotyledons of mung bean seedlings by immunofluorescence microscopy.

Vicilin peptidohydrolase, the protease that hydrolyzes the reserve proteins in the cotyledons of mung bean (Vigna radiata) seedlings, has been localized intracellularly by immunofluorescence microscopy using monospecific antibodies against the enzyme and rhodamine-coupled goat-anti-rabbit immunoglobulin G's. The enzyme can first be visualized after 3 days of seedling growth and is associated with small foci within the cytoplasm of the storage parenchyma cells farthest from the vascular bundles. On the 4th day of growth, the protease is also present in the numerous large protein bodies within these cells. Vicilin peptidohydrolase is known to be synthesized de novo starting on the 3rd day of growth. Our observations are therefore consistent with the interpretation that the enzyme is synthesized in the cytoplasm and subsequently transported to the protein bodies.


Calcium control of ciliary arrest in mussel gill cells.

After several hours in 20 mM sodium phosphate and 40 mM KCI (pH 7.4) or similar simple solutions, ciliated cells exfoliate en masse from stripped gill epithelium of freshwater mussels, e.g., Elliptio complanatus. Three types of ciliated cells--lateral (L), laterofrontal (LF), and frontal (F)--can be distiniguished and counted separately in the suspensions. About one-half of the cells of each type remain motile. Motility is unaffected by addition of 10(-5) M A23187 or 10(-2) M Ca+2 added separately, but when ionophore and Ca+2 are added together, ciliary beat is largely arrested. Treatment of the cells with Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.) results in a total loss of motility as the ciliary membrane becomes disrupted. Such models can be reactivated by addition of ATP and Mg+2. All ciliated cell types are reactivated to about the same extent. At least 80% of the activity of the untreated preparation returns. Ca+2-EGTA buffers added to the reactivating solutions permit titration of free Ca+2 concentration vs. percent motility. Activity is unchanged for all cell types at Ca+2 less than 10(-7) M; at 10(-6) Ca+2, L cilia of all cell types are arrested differentially, whereas at Ca+2 greater than 10(-4) M most cilia of all cell types are arrested. We conclude: (a) that increasing cytoplasmic Ca+2 is directly responsible for ciliary arrest, (b) that the readily reversible physiological arrest response of the L cilia in the intact gill is caused by a rise in free Ca+2 in narrow limits from ca. 5 x 10(-7) M to ca. 8 x 10(-7) M, and (c) that the site which is sensitive to Ca+2 is part of the ciliary axoneme or the basal apparatus.


Alpha-actinin localization in the cleavage furrow during cytokinesis.

We used antibodies against alpha-actinin and myosin labeled directly with contrasting fluorochromes to localize these contractile proteins simultaneously in dividing chick embryo cells. During mitosis anti-alpha-actinin stains diffusely the entire cytoplasm including the mitotic spindle, while in the same cells intense antimyosin staining delineates the spindle. During cytokinesis both antibodies stain the cleavage furrow intensely, and until the midbody forms the two staining patterns in the same cell are identical at the resolution of the light microscope. Thereafter the anti-alpha-actinin staining of the furrow remains strong, but the antimyosin staining diminishes. These observations suggest that alpha-actinin participates along with actin and myosin in the membrane movements associated with cytokinesis.


Freeze-fracturing in normal vacuum reveals ringlike yeast plasmalemma structures.

The fine structure of the regular arrays of subunits seen on both plasmalemma fracture faces in resting and starved Saccharomyces cerevisiae (baker's yeast) has been compared using different freeze-fracture replication methods. Freeze-cleaving was carried out at 173 degrees, 133 degrees, and 108 degrees K under a vacuum of 2 X 10(-7) torr (2.6 X 10(- 7)mbar) or under liquid nitrogen at atmosphereic pressure. Independent of the preparation conditions (fracturing temperature, and whether cleaved under vacuum or liquid nitrogen), resting and starved yeast show a significant difference in the morphology of the subunits forming the regular arrays. The regularly arranged particles of the P face of the plasmalemma of starved yeast have a clear craterlike structure which has previously been reported to be demonstrated only by freeze-etching at very low temperatures in ultrahigh vacuum. A complementary structure is seen on the plasmalemma E face. Prolonged exposures of fracture faces under the protection of liquid nitrogen-cooled shrouds have shown that, because of the consequent drastic reduction of condensable gases in the specimen area, no detectable condensation contamination of exposed fracture faces occurs within 15 min at a specimen temperature of 108 degrees K. This shows that a complicated ultrahigh vacuum technology is not required for high resolution freeze- etching.


The induction of the acrosome reaction in guinea-pig sperm by the divalent metal cation ionophore A23187.

The divalent metal cation ionophore A23187 induces an acrosome reaction in guinea-pig sperm which is dependent on external calcium. Examination of this acrosome reaction by electron microscopy shows that it is morphologically normal. The known properties of A23187 and the morphological similarity between the acrosome reaction and the secretory discharges of other secretory cells suggests that the immediate cause of the acrosome reaction is an increase in the cytoplasmic free calcium concentration.


The activation of proteolysis in the acrosome reaction of guinea-pig sperm.

The divalent metal cation ionophore A23187 rapidly induces a normal acrosome reaction in a population of guinea-pig sperm suspended in calcium medium. In the course of the acrosome reaction, proacrosin, the zymogen precursor of the protease acrosin, is activated. Although the acrosome reaction causes exocytosis of the acrosomal contents, 'soluble' acrosin is not released in significant amounts until well after the sperm population as a whole has undergone an acrosome reaction. This suggests that proacrosin is stored within the acrosome in an insoluble form and that exocytosis of the acrosomal contents in the acrosome reaction is insufficient, by itself, to cause its immediate dissolution. Electron micrographs of sperm undergoing an A23187-induced acrosome reaction in the presence of the acrosin inhibitors benzamidine, p-amino-benzamidine and phenylmethylsulphonyl fluoride show that the acrosome reaction proceeds normally but that dispersal of the acrosomal contents is inhibited. These morphological changes are, for the most part, below the limit of resolution of the light microscope and using light microscopy to assess whether an acrosome reaction has taken place, it can be mistakenly inferred that the reaction itself is inhibited by the acrosin inhibitors. The inhibition of the dispersal of the acrosomal contents by acrosin inhibitors suggests that acrosin activity is important in solubilizing acrosin. These experimental observations, taken with the evidence that the acrosome reaction is a response to an increase in intracellular free calcium, have been taken as the basis of a proposal for the mechanism of proacrosin activation in the acrosome reaction.


The histochemical localization of acrosin in guinea-pig sperm after the acrosome reaction.

The protease acrosin is widely considered to be an essential component of a zona lysin which enables sperm to penetrate the zona pellucida of the egg. Sperm form a characteristic penetration slit little wider than the sperm head itself and this has long suggested that any zona lysin is attached to the sperm surface after an acrosome reaction. This paper provides the first ultrastructural evidence that this is the case. The protein acrosin inhibitor, Kunitz soybean trypsin inhibitor, has been covalently attached to the electron-dense marker, ferritin, and the conjugate incubated with guinea-pig sperm which have undergone an A23187-induced acrosome reaction. Electron microscopy shows that ferritin is distributed unevenly over the outer surface of the newly exposed inner acrosomal membrane but does not extend to the equatorial segment. This is further evidence that acrosin can be considered as a candidate for the role of zona lysin. The mechanism of sperm penetration of the zona is discussed in the light of these observations.


Respiratory tract epithelium in primary culture: concurrent growth and differentiation during establishment.

Studies of cellular function in the respiratory tract lining have traditionally been limited by the small tissue mass and functional diversity of the epithelium. Recent improvements in culture conditions have permitted long-term maintenance of epithelial cells derived from the upper respiratory tract of rats. The present study determined the extent to which proliferation and differentiation took place in such epithelial cell cultures. The labelling index obtained after 3H-dThd administration was approximately 100-fold higher than that of the quiescent epithelium in vivo; therefore, a large proportion of the cells were probably in the cycling population. Ultrastructural studies showed that features unique to the specialized mucous secretory cells and ciliated cells were lost rapidly with entry of these cells into the in vitro environment. With long-term maintenance, the cultures were reorganized into a stratified epithelium containing large, squamous, apical cells and small basal cells. The ultrastructural appearance of basal cells in vitro was nearly identical to that of basal cells in vivo. Squamous cells were frequently joined by tight junctions. Because hemicysts originated by detachment of squamous cells from the basal layers but not from adjacent squamous cells, they were considered to indicate stratification in the cultures. The proliferative and differentiative status of the mucociliary epithelium was altered by in vitro conditions, and came to resemble that of early squamous metaplasia in the respiratory tract epithelium.


The distribution of actin cables and microtubules in hybrids between malignant and non-malignant cells, and in tumours derived from them.

Cell fusion provides a genetic test for linkage between cellular phenotypic markers and malignancy. We have examined hybrid cells in which malignancy is suppressed and malignant segregants derived from them, to test whether the reappearance of malignancy is regularly associated with loss or abnormality of actin cables or microtubules. Immunofluorescence studies did not reveal any difference between the malignant and non-malignant hybrids in the number and distribution of microtubules that they contained. All the hybrids, whether tumorigenic or not, contained elaborate patterns of cytoplasmic microtubules. Furthermore, no consistent relationship was observed between the absence of actin cables and malignancy.


Determination of valproic acid (dipropylacetic acid) in plasma by gas-liquid chromatography.

A specific and sensitive procedure for the determination of valproic acid at therapeutic concentrations in human plasma has been developed. The method involves a microscale extraction of the drug from acidified plasma into chloroform. Caproic acid is added as internal standard. The extract is subsequently analysed on a gas chromatography fitted with a flame ionisation detector, and quantitation achieved by the relative-peak-height technique.


Single-antigen immunofluorescence test for chlamydial antibodies.

A simple method is presented for producing large numbers of inclusions of Chlamydia trachomatis serotype L2 in cell cultures on slides for immunofluorescence antibody staining. Preliminary results with a total of 1,241 human sera from different groups were consistent with findings by earlier methods: 82% of chlamydia-positive men with nongonococcal urethritis had antibodies at titers of greater than or equal to 8, with a geometric mean titer (GMT) of 44.9; 68.5% of chlamydia-negative men with nongonococcal urethritis had titers of greater than or equal to 8, with a GMT of 38.6; 27% of male blood donors had titers of greater than or equal to 8, with a GMT of 19.6; 95.0% of chlamydia-positive women had titers of greater than or equal to 8, with a GMT of 80.3; 67.0% of chlamydia-negative partners of men with nongonococcal urethritis had titers of greater than or equal to 8, with a GMT of 47.0; 50.2% of control women had titers of greater than or equal to 8, with a GMT of 27.7% and 8.1% of children (aged 1 to 15 years) had titers of greater than or equal to 8, with a GMT of 17.8.


Counterimmunoelectrophoresis of Reiter Treponeme Axial filaments as a diagnostic test for syphilis.

Purified axial filaments from the cultivable Reiter treponeme, previously shown to share an antigenic component with pathogenic Treponemia pallidum, were evaluated as antigen in a diagnostic test for syphilis. Antibody to the filaments was revealed by counterimmunoelectrophoresis. Conditions that produced optimal results in the test were established. A total of 343 sera from normal individuals, biological false-positive reactors, and from patients in the different stages of syphilis were subjected to the test. The results indicate the test to be sensitive and highly specific for detecting treponemal antibodies in human syphilis.


Rapid presumptive identification of Cryptococcus neoformans.

Carbohydrate-containing extracts were prepared from mature yeast colonies grown on Sabouraud dextrose agar by mixing a 0.001-ml loopful of yeast cells for 30 s in phenolized saline and removing the cells by centrifugation. Extracts were prepared from 54 Cryptococcus neoformans isolates, 29 isolates of other Cryptococcus species, 16 isolates of Candida species, 2 Rhodotorula, 2 Torulopsis, and 1 Saccharomyces species. Initially the carbohydrate content of each extract was estimated (Molisch method) and adjusted to 1, 5, and 10 microgram/ml. Twofold dilutions of each extract were tested for reactivity with the cryptococcal latex agglutination reagent of Bloomfield et al. (N. Bloomfield, M.A. Gordon, and D.F. Elmendorf, Jr., Proc. Soc. Exp. Biol. Med. 114:64-67, 1963). All 54 C. neoformans extracts gave strong agglutinations (3+ to 4+) in dilutions of 1:4 or greater. None of the other yeasts produced any agglutination, except for 1 of 15 C. laurentii isolates, which showed a 1+ reaction that disappeared at a dilution of 1:4 and above. Subsequent testing established that a single extract made from 0.001 ml of yeast cells in 6 ml of phenolized saline contained less than 5 microgram of carbohydrate per ml, was suitable for a single rapid screening dilution, and eliminated any cross-reaction from the C. laurentii isolates. In our hands this method has provided a reliable differentiation of C. neoformans from other unknown yeast colonies in less than 20 min exclusive of a Molisch determination.


Time- and media-saving testing and identification of microorganisms by multipoint inoculation on undivided agar plates.

Motility and various biochemical activities of isolates of bacteria and yeasts were tested on undivided agar plates by using a simple, manually operated multipoint inoculation apparatus that allowed the analysis of 25 isolates per 9-cm-diameter petri plate. Fermentation of all 17 carbohydrates tested as well as 13 other biochemical activities commonly used for identification of bacteria were readily demonstrated by the multipoint inoculation plate method, and the results agreed very well with those of conventional tube tests. In addition to speedy inoculation and low cost of materials, the multipoint inoculation plate method offers several other advantages when compared with conventional tube tests or with some of the manufactured test kits currently available for recognizing members of the family Enterobacteriaceae.


Immune reactions in human filariasis.

Sera from cases of elephantiasis due to Wuchereria bancrofti infection promoted an intense adhesion of peripheral blood leukocytes to W. bancrofti microfilariae in vitro. A similar adhesion was also seen using sera from some normal persons living for several years in areas where filariasis is endemic. No such adhesion was evident with sera from microfilaria carriers or from normal subjects from nonendemic areas. The adhesion was complement independent and was associated with the immunoglobulin G fraction of serum. 51Cr release studies suggested the occurrence of cell-mediated cytotoxicity to W. bancrofti microfilariae in the presence of elephantiasis serum. Microfilariae of Litomosoides carinii could be isolated free of blood cells, from the blood of infected rats. In the presence of serum, or its immunoglobulin G fraction, from patients with elephantiasis, L. carinii microfilariae adhered to human peripheral blood leukocytes or rat spleen cells.


Radial passive immune hemolysis assay for detection of heat-labile enterotoxin produced by individual colonies of Escherichia coli or Vibrio cholerae.

A rapid screening test has been developed to detect heat-labile enterotoxin produced by individual colonies of Escherichia coli or Vibrio cholerae growing on media solidified with agar. The applicability of this method for isolating tox mutants of E. coli and V. cholerae has been demonstrated.


Simple device for indicating atmospheric status of anaerobic chamber entry locks.

A simple, inexpensive device is described that indicates which door of an anerobic chamber entry lock was last opened.


New hemolytic method for determination of antistreptolysin O in whole blood.

A new method is proposed for the determination of antistreptolysin O, based on the properties of streptolysin O. The reduced form of the toxin is hemolytic, whereas the oxidized form is not; this activity can be restored, however, by a reducing agent. Both forms maintain the capacity to bind specific antibodies. Unlike the classical method, the assay is performed on whole blood, using the patient's own erythrocytes as revealing agents. A procedure is proposed which limits the operation to a single dilution of a microvolume of blood (0.05 ml brought to 2 ml) and to the distribution of constant quantities in test tubes prefilled with different amounts of oxidized streptolysin O. After a few minutes, a reducing agent is added, and the reading is performed as in the classical technique after sedimentation of the erythrocytes. Correlation with the traditional method (r = 0.987) is evaluated on the basis of the results of samples from hospitalized patients.


Rapid grouping of beta-hemolytic streptococci by latex agglutination.

Latex agglutination was compared with fluorescent-antibody staining with group A conjugate and Lancefield precipitation for grouping of beta-hemolytic streptococci. Latex agglutination correctly grouped 98.8% of 82 group A streptococci and more than 95% of 187 group B, C, or G streptococci. Occasional cross-reactions occurred between groups A and C and groups B and G.


Detection of Legionnaires disease bacteria by direct immunofluorescent staining.

Antisera and fluorescein isothiocyanate conjugates prepared for five strains of the Legionnaires bacteria were tested in both homologous and heterologous staining reactions with 10 isolates of the organism from patients in seven geographic areas. The strains were related but not identical as judged by the results of direct immunofluorescence staining. The conjugates were successfully used to detect Legionnaires disease bacteria in Formalin-fixed lung scrapings, in histological sections, and in fresh lung tissue obtained at biopsy or autopsy. In addition, the labeled antibodies are valuable for staining suspected cultures of the bacterium and for searching for the source of these organisms in soil, water, and other environmental niches.  The reagents are highly specific for detecting the Legionnaires organism in clinical specimens.


Mass spectral measurement of residual oxygen in the atmosphere of an anaerobic culture chamber.

A simple and reproducible procedure has been devised for sampling gases and for analysis of the residual oxygen in the atmosphere of the anaerobic culture chamber by mass spectrometer.


Urease production from clinical isolates of beta-hemolytic Escherichia coli.

Twenty-four lactose-fermenting, urease-producing strains of beta-hemolytic Escherichia coli were isolated from a variety of clinical material. All isolates were indole positive, citrate negative, and produced the characteristic green metallic sheen on eosin-methylene blue agar.


Characterization of proinsulin-insulin intermediates in human plasma.

This work addressed the problem of heterogeneity of immunoreactive insulin (IRI) in human plasma. Subjects with normal glucose tolerance were given 75g of an oral glucose solution, followed in 30 min by an intravenous infusion of 30g of arginine over 30 min. At the end of the infusion blood was withdrawn for analysis. IRI was extracted from plasma of individual subject by immunosorbent columns and was fractionated by gel filtration, disc gel electrophoresis and isoelectric focusing. Human IRI components were identified by molecular size, immunoreactivity with a human proinsulin antibody, sensitivity to trypsin, and by comparison of electrophoretic mobility and isoelectric point with porcine pancreatic products, after suitable correction for electric charge and molecular weight differences. The pattern of IRI heterogeneity was the same among six healthy subjects. Heterogeneity of proinsulin-size IRI in circulation was more marked than that of insulin-size material. Proinsulin and desdipeptide proinsulin were present in approximately equal amounts accompanied by minor amounts of split proinsulin and monodesamido-desdipeptide proinsulin. Insulin-size IRI contained over 80% insulin. Minor amounts of monodesamidoinsulin and diarginylinsulin were observed in some cases. The types of IRI components observed in plasma are evidence in support of a physiologic role of trypsin-and carboxypeptidase B-like enzymes in the conversion of proinsulin to insulin. Moreover, this study provides a base line for investigation of abnormalities in proinsulin-to-insulin conversion that may be associated with certain pathologic states.


Thrombogenic effect of high-dose aspirin in rabbits. Relationship to inhibition of vessel wall synthesis of prostaglandin I2-like activity.

Aspirin is a promising antithrombogenic agent. It inhibits the generation of thromboxane A(2) by acetylating platelet cyclo-oxygenase. Aspirin also inhibits vessel wall production of PGI(2) which is an inhibitor of platelet aggregation, and therefore is potentially thrombotic. To investigate these two opposing effects we studied the effects of aspirin upon fibrin accretion onto experimentally induced venous thrombi in rabbits and on the PGI(2)-like activity of vessel wall using the thrombin-induced [(14)C]serotonin release assay. A 200-mg/kg dose of aspirin significantly augmented thrombus size when compared to (a) sodium salicylate administered in equal doses, (b) aspirin in a 10-mg/kg dose or (c) controls (P < 0.001). A 200-mg/kg dose of aspirin totally inhibited vessel wall PGI(2)-like activity whereas aspirin in a 10-mg/kg dose produced less inhibition, and 200 mg/kg sodium salicylate had no effect. Local instillation of tranylcypromine, an inhibitor of PGI(2) formation, also significantly augmented thrombus size compared to saline-treated controls and totally inhibited the production of PGI(2)-like activity. The thrombogenic effect of high dose aspirin was lost if an interval of 2.5 h or longer elapsed between vessel damage and drug administration, indicating that in contrast to the platelet, the effect of aspirin on vessel wall prostaglandin synthesis is relatively short-lived. It is concluded that aspirin, in doses higher than those used clinically, can augment experimental thrombosis, presumably by inhibiting the synthesis of vessel wall PGI(2).


Application of a dehydrated test strip, HEMASTIX, for the assessment of gingivitis.

In order to develop a rapid and simple method based on chemical alterations in gingival pockets during inflammation, the HEMASTIX strips were used to record the degree of gingivitis. This dehydrated test strip records the presence of both hemoglobin derived from erythrocytes and myeloperoxidase from PMN-leukocytes. The intensity of the blue color that appeared and the amount of adsorbed fluid were recorded. The correlation of the Strip Index with Gingival Index was highly significant (P is less than 0.001) and with Gingival Bleeding Index significant (P is less than 0.01). When determined both from the left side of the upper jaw and from the Ramfjord teeth, the correlation between these two areas was highly significant (P is less than 0.001) in the case of Strip Index but only slightly significant (P is less than 0.05) with Gingival Index. According to the present results the Strip Index can be used for the assessment of biochemical changes in gingival pockets resulting from the development of gingivitis.


Immunohistochemical localization of enkephalin in rat brain and spinal cord.

The distribution of immunoreactive enkephalin in rat brain and spinal cord was studied by immunoperoxidase staining using antiserum to leucine-enkephalin ([Leu5]-enkephalin) or methionine-enkephalin ([Met5]-enkephalin). Immunoreactive staining for both enkephalins was similarly observed in nerve fibers, terminals and cell bodies in many regions of the central nervous system. Staining of perikarya was detected in hypophysectomized rats or colchicine pretreated rats. The regions of localization for enkephalin fibers and terminals include in the forebrain: lateral septum, central nucleus of the amygdala, area CA2 of the hippocampus, certain regions of the cortex, corpus striatum, bed nucleus of the stria terminalis, hypothalamus including median eminence, thalamus and subthalamus; in the midbrain: nucleus interpeduncularis, periaqueductal gray and reticular formation; in the hind brain: nucleus parabrachialis, locus ceruleus, nuclei raphes, nucleus cochlearis, nucleus tractus solitarii, nucleus spinalis nervi trigemini, motor nuclei of certain cranial nerves, nucleus commissuralis and formatio reticularis; and in the spinal cord the substantia gelatinosa. In contrast enkephalin cell bodies appear sparsely distributed in the telencephalon, diencephalon, mesencephalon and rhombencephalon. The results of the histochemical staining show that certain structures which positively stain for enkephalin closely correspond to the distribution of opiate receptors in the brain and thus support the concept that the endogenous opiate peptides are involved in the perception of pain and analgesia. The localization of enkephalin in the preoptic-hypothalamic region together with the presence of enkephalin perikarya in the paraventricular and supraoptic nuclei suggest a role of enkephalin in the regulation of neuroendocrine functions.


Case report: subependymal and leptomeningeal spread of systemic malignant lymphoma demonstrated by cranial computed tomography.

Secondary involvement of the central nervous system by systemic malignant lymphoma is uncommon, although increasingly recognized. Histologically perivascular, leptomeningeal, and subependymal infiltration by tumor cells is characteristic. Less frequently, single or multiple macroscopic masses resembling other metastatic deposits are found. A patient is presented in whom periventricular and leptomeningeal involvement by systemic histiocytic lymphoma produced prominent symmetric surface oriented enhancement on computed tomography following intravenous infusion of contrast medium. Differential diagnosis is discussed.


Mixed lichen planus-lupus erythematosus disease.

Two patients are reported with clinical symptoms of systemic lupus erythematosus associated with centrally ulcerated hypertrophic plaques and lip lesions. Histological findings from various sites were features of both lupus erythematosus and lichen planus. An increase in acid mucopolysaccharides and a thickened basement membrane which were present in some biopsies were interpreted as lupus erythematosus. The deposition of immunoglobulins and fibrinogen seemed to favor lupus erythematosus but these findings may also be seen in lichen planus. Four possible interpretations of the findings are discussed. The term "mixed lichen planus--lupus erythematosus disease" may be appropriate for these patients.


The dairy goat as a model in lactation studies.

The dairy goat has been used widely in lactation research due in large part to availability, convenience in size and handling, economic considerations, and similarity to the cow as a ruminant species in general and metabolic processes. Considerable basic knowledge of lactation relating to all species has been derived from studies with goats. Although the gross composition of goat and cow milk is similar, significant differences reflect differing synthetic functions. Caution is necessary in using the goat as a model for the dairy cow where differences occur. Some are obvious such as the gross structure of the mammary gland and differing milk constituents. Others are more subtle, such as the susceptibility to metabolic diseases associated with lactation and differing rates of metabolism affecting transfer of dietary and administered materials into milk.


Hygiene in the prevention of udder infections. V. Efficacy of teat dips under experimental exposure to mastitis pathogens.

Efficacy (reduction of new intramammary infections) was determined against Staphylococcus aureus in 14 trials and against Streptococcus agalactiae in 5 trials. Against Staphylococcus aureus seven 1% iodophor products had an average efficacy of 89.9% while three .5% iodophor products averaged 76.4%. A solution of sodium dichloro-s-triazenetrione reduced infections 79.0% and 2% dodecyl benzene sulfonic acid was 50.4% effective. Efficacies of two quaternary ammonium products at .18 and .5% were 84.8 and 60.9%. A placebo vehicle and .15 M saline as teat dips reduced infections 8.9 and 9.2%. Infection rates with Streptococcus agalactiae were low; efficacy ranged from 50.5 to 100.0%, average 65.6%. The model of experimental exposure provided a better estimate of effectiveness of a teat dip than would be possible under conditions of most commercial dairying.


Hygiene in the prevention of udder infections. VI. Comparative efficacy of a teat dip under experimental and natural exposure to mastitis pathogens.

Efficacies (reduction of new intramammary infections) of a bronopol teat dip under experimental exposure to Streptococcus agalactiae and Staphylococcus aureus were 41.7 and 54.4%. Under natural exposure, efficacy was 35.0% (P greater than .05) against Streptococcus species and 50.0% against Staphylococcus aureus during the first 9 mo of a 12-mo trial when favorable milking equipment and labor conditions prevailed. Corresponding figures were 20.0 and 13.0% during the last 3 mo following onset of malfunctioning equipment and deterioration of labor services.


Split-thickness skin grafts: an experimental study of factors related to ease or difficulty of procuring satisfactory grafts of skin from man and some common laboratory animals.

Split-thickness grafts of skin of satisfactory quality were found to be more difficult to obtain from rabbits than from animals of other genera. An explanation for this was sought in the histology of the skins of the various experimental animals. Density of hair was judged to be the factor most critical for ease or difficulty in procuring satisfactory split-thickness grafts of skin, and a cluster of histologic characteristics of skin seemed to have meaning for taxonomic classification. Silver's dermatome was found to be the test instrument for taking grafts from all subjects.


Surgical management of ulcers on the lower limbs.

Ulcers on legs may result from venous hypertension or arterial insufficiency. Ulcers of either type require skin cover. Methods of achieving such cover are explored.


Effect of cooling techniques on temperature control and cutting rate for high-speed dental drills.

This paper describes a series of tests conducted to determine the effectiveness of various cooling techniques used while preparing teeth for restoration. Comparisons are presented for four cooling techniques: air stream, air-water spray, water stream from a separate hand-held syringe, and water applied through a hollow bur. The comparisons were made in two ways, temperature control and influence on cutting rate. It was demonstrated that wet cooling techniques provided better temperature control than cooling with air and also improved the rate of material removal.


An in vitro investigation of the penetrating efficacy of BIS-GMA resin pit and fissure coatings.

The frequency of complete penetration of two commercial BIS-GMA resin pit and fissure coating materials into wide and constricted fissures was assessed from photomicrographs. It was found that 334 of the 390 wide fissures examined were completely filled by the coating materials. However, in contrast, only 28 of the 650 constricted fissures examined were completely filled by these resins.


Acetylcholine and cholinesterase in submandibular glands of rats given armin and obidoxime.

The effects of obidoxime, a pyridinium oxime, on the cholinesterase activity and acetylcholine content of the submandibular glands of rats poisoned with armin, an organophosphorus anticholinesterase agent, were studied. The results indicate that obidoxime-induced reactivation of phosphorylated cholinesterase can suppress completely the increase in the concentration of acetylcholine that develops within the submandibular gland after administration of armin alone.


High-speed drill noise and hearing: audiometric survey of 70 dentists.

Many dentists believe that noise generated from high-speed drills results in a significant loss of hearing. Audiological evaluations, consisting of hearing thresholds at eight frequencies (500 to 800 Hz) and impedance audiometry, were done of 70 dentists from eight specialties. No statistical decrease in hearing thresholds appeared in either the speech or high frequencies when the dentists were compared with a normal, age-adjusted population.


Status report on acid etching procedures. Council on Dental Materials and Devices.

The introduction of acid etching techniques into many dental disciplines has resulted in safe, simple, conservative, and sometimes more economic methods of treatment. Further, in some instances, it has extended greater opportunities for treating such groups as those physically and emotionally handicapped in whom preventive and restorative procedures are often difficult. It is a significant departure from conventional, established techniques and may supersede some and may share with other established techniques an adjunctive relationship. By many, acid etching techniques are viewed with cautious optimism. It is deceptively simple. Strict adherence to the basic principles of application is important to its success.


Terbutaline's effects on the allergy skin test.

Beta adrenergic agents are known to inhibit mediator release from skin and lung mast cells in vitro and isoproterenol and epinephrine are known to impair the immediate allergic skin test reaction. We undertook to investigate the effects of aqueous and oral terbutaline on the allergy skin tests. We particularly wished to analyze the impact of terbutaline on the late allergic reaction, but we found that terbutaline mixed with ragweed antigen does not affect either the immediate or late skin test reaction. Furthermore, 5 mg of oral terbutaline does not inhibit the immediate allergic skin test.


Use of fructose, sorbitol, or xylitol as a sweetener in diabetes mellitus.

Non-nutritive sweeteners have been utilized in the diet of diabetic patients as agents to replace glucose and sucrose. Since saccharin might be removed from the market place, the nutritive sweeteners, fructose, xylitol, and sorbitol, are being considered as possible atlernatives. This review considers the effects of these nutritive sweeteners on the main dietary concerns in the diabetic diet, i.e., control of blood glucose levels, obesity, and hyperlipidemia. The potential side effects of these agents are also reviewed.


Appraisal of codeine as an analgesic in older patients.

In an investigation of a new oral analgesic agent, codeine was chosen as the reference drug because of its established reputation as an effective agent for the relief of pain. Thirty-five patients with cancer pain were studied. Their average age was 58 years, During a 5-day hospital stay they received, on each of three days, either codeine (120 mg or 60 mg) or placebo. At hourly intervals after ingestion the nurse observer collected data on pain intensity and the degree of pain relief, and the patients independently charted the hourly intensity. Statistical analysis failed to show any significant superiority of either dose of codeine over placebo. Moreover, codeine is known to have a constipating effect. Re-appraisal of the value of codeine as an analgesic agent in elderly patients seems justified.


Prentice in Lahr and Karlsruhe.

Charles F. Prentice's father was born in England and his mother in Germany. Charles' boyhood days were spent chiefly on Staten Island, New York. At age 16 he attended "Gymnasium" in Lahr, Germany. his mother's ancestral hometonw. During the three subsequent years he studied mechanical engineering at the institute now known as the University of Karlsruhe, West Germany. Illustrations include scenes in Karlsruhe and Lahr and transcripts of his academic records.


Infections following clenched-fist injury: a new perspective.

Fifteen cases of clenched-fist injury were studied by aerobic, anaerobic, and 10% CO2 cultures. Anaerobic bacteria and Eikenella corrodens were prominent pathogens, being recovered in 60% and 20% of cases, respectively. These organisms are more susceptible to penicillin than to penicillinase-resistant penicillins or cephalosporins. All clenched-fist injuries should be cultured in aerobic, anaerobic, and 10% CO2 atmospheres. We recommend the use of both penicillin and a penicillinase-resistant penicillin as initial empiric therapy.


Correlation of phaga type, biotype and source in strains of Salmonella typhimurium.

A series of 2092 cultures of Salmonella typhimurium isolated from human, animal and other sources in 57 countries were differentiated into 204 phage types and 19 primary and 147 full biotypes. Different biotypes belonged to the same phage type and different phage types to the same biotype, so the combination of typing methods differentiated strains more finely than either method alone: 574 different ;phage type/biotypes' were distinguished in 1937 cultures belonging to the 204 recognized phage types.The combination of biotyping with phage-typing was valuable in studying the phylogeny and spread of epidemic strains by distinguishing clones of different biotype within the same phage type and by confirming the relationship between cultures isolated from widely dispersed clones and that between cultures isolated before and after a clone had undergone variation in phage type, biotype, colicin type or antibiotic-sensitivity pattern.A widespread outbreak of infection with S. typhimurium phage type 141 in Scotland comprised independent dissemination of three clones of different biotypes, 1f, 9f and 31bd. During its epidemic spread in cattle in Britain between 1962 and 1969, another strain underwent variations in phage type (type 44 to type 29), biotype (type 26a to types 26d, 26bd, 26dgi, 26dz and 26i) and antibiotic sensitivity. A group of 275 non-fimbriate, non-inositol-fermenting and non-rhamnose fermenting (FIRN) strains, particularly associated with avian infections and thought to be clonal in origin, contained 27 phage types and 22 full biotypes in the primary biotypes 29-32.


Refractoriness to migration inhibitory factor of macrophages of LPS nonresponder mouse strains.

The responsiveness to macrophage migration inhibitory factor (MIF) of peritoneal exudate cells (PEC) from the LPS unresponsive C3H/HeJ and C57BL/10ScCR mice was assessed by the indirect agarose microdroplet macrophage migration inhibition assay. No migration inhibition with PEC from C3H/HeJ nor C57BL/10ScCR mice was detected, whereas PEC from both C3H/HeN and C57BL/10Sn mice were significantly inhibited by even a 1/32 dilution of MIF-containing supernatants. Responsiveness to MIF of C3H/HeJ PEC could, however, be induced. In vivo inoculations of Mycobacterium bovis, strain BCG, 7 days before in vitro assay rendered C3H/HeJ PEC responsive to MIF. The lack of responsiveness to MIF by C3H/HeJ PEC appeared related to some form of suppression, since a mixture of PEC from C3H/HeN mice with 10 to 15% PEC from C3H/HeJ mice resulted in undetectable migration inhibition at any MIF dilution. In contrast to the usual lack of responsiveness of their macrophage to MIF, C3H/HeJ mice were able to produce MIK in response to PPD as well as their counterpart C3H/HeN mice after BCG sensitization. These results demonstrate that macrophages from C3H/HeJ and C57BL/10ScCR mice are unable to be inhibited in their in vitro migration of MIF (possibly being directly or indirectly influenced by a suppressor cell), whereas lymphoid cells from at least one of these strains, the C3H/HeJ mice, can produce MIF in response to antigenic stimulation.


Guinea pig macrophage agglutination factor is antigenically distinct from migration inhibition factor and immunoglobulin.

Lymph node cells from guinea pigs with specific delayed hypersensitivity release macrophage agglutination (MAggF) and migration inhibition factors (MIF) upon exposure to antigen or concanavalin A in serum-free medium. MAggF in culture supernatants was absorbed neither by immunoabsorbents made with a rabbit anti-guinea pig lymphokine serum that removed MIF, nor by immunoabsorbents made with rabbit anti-guinea pig Ig. These results suggest that MAggF is antigenically distinct from MIF and Ig.


Induction of B cell priming by neonatal injection of mice with thymic-independent (type 2) antigens.

Mice injected at birth with the thymus-independent type 2 antigen TNP-AECM-Ficoll have augmented anti-TNP antibody responses when their spleen cells subsequently are challenged in vitro with TNP-coupled thymic independent or thymic dependent antigens. This neonatal priming effect was shown to occur in neonatal nu/nu mice and thus does not appear to require T lymphocytes. The primary explanation for the priming effect seems to be an increase of approximately 10-fold in the numbers of TNP-specific precursors of antibody-forming cells. The neonatal injection of TNP-AECM-Ficoll induces little or no antibody formation directly. It appears, therefore, that some thymic independent antigens can deliver a signal to immature B cells, which causes clonal expansion, but is unable to induce differentiation into antibody-forming cells.


A sensitive haemolytic assay of mouse complement.

Under appropriate conditions the haemolytic activity of mouse complement can be measured in a 51Cr release assay. Modifications that increase the sensitivity are the following: (a) mouse 7 S anti-SRBC antibodies as amboceptor; (b) low amount of target cells; (c) ionic strength of 0.13 M NaCl in the test medium; (d) incubation temperature of 30 degrees C; (e) incubation time of 90 min; (f) total reaction volume of 1 ml.


Detection of antisalivary duct antibody from Sjögren's syndrome by an autoradiographic method.

A new technique to detect anti-salivary duct antibody (ASDA) has been developed by using autoradiographic, rather than immunofluorescent methods. The antibody activity detected by autoradiography is probably classic ASDA. Both techniques may be consecutively performed on the same tissue section without attenuation of either. Some of the potential advantages of the radiolabelling of ASDA are pointed out, and a few preliminary experiments using the labelled antibody as a marker are presented.