Evaluation of a metal-ceramic composite hip prosthesis.

This investigation attempts to apply the concept of direct skeletal attachment via tissue ingrowth to the attachment of femoral head prostheses to the femur. Stainless steel (316L) canine femoral head prostheses with a ceramic coated stem were implanted into the femora of dogs and their results compared to those obtained by the impaction of stainless steel prostheses and acrylic-stabilized prostheses. The implants were tested in vivo for time periods ranging from 2 to 13 months, after which time the animals were sacrificed and the femora excised. The femora were sectioned with the implants in place and prepared for examination by light microscopy. Bone was in closer approximation to the ceramic coated implants than to the other two types of implantations. The uncoated and acrylic-stabilized implants were usually characterized by the presence of a relatively thick fibrous sequestering membrane.


Fatigue fractures of the femoral component of Charnley and Charnley-Müller type total hip prostheses.

A survey is given of the mechanical failures of the femoral component of Charnley and Charnley-Müller type total hip prostheses. Fractographic analysis reveals that all the prostheses have broken by metal fatigue. A review of the clinical data has been combined with a metallurgical and a mechanical study in order to assess the reasons of the mechanical failures. The following conclusions have been reached. The prosthesis should be inserted in varus position with regard to the axis of the intramedullary canal, and the prosthesis should be made of an alloy with appreciably higher yield stress and fatigue strength than the alloys of the investigated broken Charnley and Charnley-Müller type prostheses.


Composite implants for orthopedic applications: in vivo evaluation of candidate resins.

Three resins which include poly (methyl methacrylate), surgical Simplex P, and ultrahigh molecular weight polyethylene were selected to be evaluated as candidate systems for a polymer based composite for hard tissue prosthesis. Characterization of the mechanical behavior of these polymers in different environments including in vitro and in vivo storage was accomplished. As a result, conclusions were made as to which material maintained the least amount of mechanical variance as influenced by environmental effects. In vivo studies of implanted materials provided for a study of soft tissue response to each material. Conclusions were then developed as to the varying degrees of tissue reaction initiated by each material, and which resin generated the greatest tissue response with respect to the others.


Experiences with Al2O3 implantations in humans to bridge resection defects.

Various implants have been developed for the upper and lower extremities and the spine. In order to establish whether endoprostheses made of Al2O3 will show the same good biocompatibility in humans as it has in experiments with animals, endoprostheses implantations were performed in 4 patients: 2  upper arms, 1 radius, 1 vertebra body. The implants were exposed to various loads. The biomechanical conditions were considered in the construction. The results reveal that the knowledge of material properties and prior calculations make possible the development of satisfactory endoprostheses. X-ray examinations confirm these deductions. Good functionality was achieved in vivo with all implants. They were all mechanially anchored, no bone cement was used. Thus the biocompatibility of the material was not limited. A histological examination is made of one implant. The macroscopic examination showed a stable fit and no pathological reactions, X-rays revealed that the bone remained in good contact with the implant. The longest observation period was over 1 1/4 years. Present observations show that the use of Al2O3 implants, without the use of additional materials (bone cement), results in a tight fit of the implants.


Replamineform porous biomaterials for hard tissue implant applications.

By means of the newly developed Replamineform process, the unique pore microstructures found in the skeletal calcium carbonate of certain reef corals can be replicated or reproduced with high precision in a wide variety of materials suitable for hard tissue implant and prosthetic applications. The advantages of fabricating porous biomaterials with this method include closely controlled size of both the pore diameters and the diameters of the pore interconnections, and virtually complete interconnection of the uniformly spaced pores. These properties are of great importance in implant devices, because tissue ingrowth, the stimulation of new bone formation, the suppression of undesirable scar tissue, the inhibition of adverse body responses, and firm biological fixation of the implanted material all depend upon the nature of the pore-microstructure configuration. Replamineform preparation of Al2O3, TiO2, hydroxyapatite, silver, Co-Cr-Mo alloys, and polymers is described in detail, and the characterization procedures used to determine the physical and structural properties of their materials are discussed. A few of the routinely measured characteristics include (1) quantitative computerized SEM image analysis for determining the volume, size and shape distributions of the macro and microporosity and the grain size measurement of the solid; (2) nondestructive x-radiography of specimens to reveal any internal defects; (3) mechanical strength measurements of randomly selected specimens. Experimental results up to now clearly demonstrate the superiority of microstructures imparted to metals, ceramics, and polymers with the Replamineform process.


Porous, heat cured poly (methyl methacrylate) for dental implants.

The use of polyl (methyl methacrylate) for tooth replica implants, as developed by Hodosh, is described as to indications, ingredients, and fabrication technique. Laboratory testing of this material for mechanical and thermal expansion properties, and porosity content were determined as a function of foaming agent and and anorganic bone particle content.


Alumina and zirconia coated vitallium oral endosteal implants in beagles.

The purpose of this study was to assess the addition of a ceramic coating upon a Vitallium implant to increase the implant's biologic acceptability in the oral environment. The mandibular premolar teeth in 9 adult beagle dogs were removed bilaterally and these areas allowed to heal for 6 weeks. Ceramic coating with either Al2O3 or ZrO2 was carried out by flame spray deposition upon Vitallium anchor implants (9 of each), and the implants placed into the 18 healed premolar areas. Clinical and radiographic evaluation was conducted by 2 independent investigators over a 32 week period. Implants which exhibited mobility greater than II on a scale of 0 to III, at intervals of one-half, were judged unsatisfactory. After 19 weeks, all 9 Al2O3 coated implants and 5 ZrO2 coated implants were rated unsatisfactory. After 32 weeks, 4 ZrO2 coated implants were in situ with 0 or I mobility. Radiographically the width of the peri-implant space increased in direct proportion to both time and mobility. Histologic sections demonstrated encapsulating dense fibrous connective tissue which was oriented parallel to both ZrO2 and Al2O3 implants. Results suggest the zirconia used is a superior ceramic coating to the alumina. Neither seemed to increase biologic acceptability over uncoated Vitallium implants.


Tissue ingrowth of Replamineform implants.

The Replamineform process, a new technique for the fabrication of porous hard tissue implant materials which replicates the skeletal configuration of certain marine invertebrates, was used to manufacture 1 cm long by 0.5 cm diam cylinders. The inherent advantages of porous configurations obtained through this process are controlled, uniform pore size, controlled pore-microstructure ratio, and complete interconnection of pores. The specific materials studied were chrome-cobalt-molybdenum alloy, alphaA103, hydroxyapatite prepared by hydrothermal conversion, and the basic (aragonite) CaCO3 skeleton of the coral genus Porities. The implants were placed in the canellous bone of the distal femora and proximal tibiae of adult, mongrel dogs and analyzed at 8 weeks for tissue response and ingrowth. Uniformly, new bone was found to grow into the pores of these materials and become normally mineralized. These findings were determined by microradiography, scanning electron microscopy, electron microprobe analysis, and histology. No evidences of infection, rejection, or encapsulation were seen. In the case of those CaCO3 implants left in place for 1 year, there was almost complete resorption of the cylinders, with both bony trabeculae and unmineralized collagen (presumably osteoid) found at the sites of insertion.


Mechanical properties of calcia stabilized zirconia following in vivo and in vitro aging.

Aging studies were done on calcia stabilized zirconia rods of 72% theoretical density to determine the effect of actual and simulated biological environments on their strength. They were aged without stress in vitro in Ringer's solution for 1, 2 and 4weeks or in vivo in rabbits for 12 weeks. Rods aged in vitro showed mean losses in bending strength of 16, 17 and 19% respectively after 1, 2 and 4 weeks of immersion, while those aged in vivo showed a mean loss of 25%. It was concluded that the material tested would be unsatisfactory as an orthopedic replacement because of the rapid decrease in strength which occurred when exposed to actual or simulated biological media.


Two-year biocompatibility study of ORNL graphite.

A new grade of graphite-isotropic, fine-grained, and of superior strength-has been produced at the Oak Ridge National Laboratory. The possibility of using this material in  orthopedic implants is under study at the University of Illinois. As part of this program, plugs 1/8 in. in diam were inserted in holes drilled in the proximal femur of 2 groups of 8-week old male rabbits. The groups were sacrificed at the end of 1 and 2 years. A single i.m. injection of achromycin was given 2 days prior to sacrifice. Thin sections of the plugs and surrounding tissue were examined by microradiography and optical microscopy (white light, polarizing, and fluorescent). All bone was normal. Minimal evidence of irritation or fibrous encapsulation appeared.


Factors influencing the creep behavior of poly(methyl methacrylate) cements.

Room temperature compression creep and recovery tests have been performed using samples of pure poly(methyl methacrylate) (PMMA), three commercially available poly(methyl methacrylate) cements, and several experimental cements. From these tests the influence of density, mixing procedure, particle size, methyl methacrylate-styrene copolymer, barium sulfate, aquenous storage environments, residual monomer, molecular weight of the continuous phase, benzoyl peroxide, and N,N-dimethyl-paratoluidine, upon the creep behavior of PMMA cements has been determined. The important results is that lowering the porosity or residual monomer content, increasing the powder size, or adding an MMA-styrene copolymer tends to increase the creep resistance of PMMA cement.


Fractography of poly(methyl methacrylates).

For convenience in clinical manipulation, it is the practice to fabricate PMMA protheses from mixtures of powder and monomer. When the monomer is subsequently polymerized an unusual 2-phase polymeric material results in which grains of PMMA are dispersed in a matrix of the same polymer. The mechanical properties of the 2-phase materials are inferior in certain respects relative to 1-phase polymers. The purpose of the present work is to evaluate the failure of 2-phase materials by microscopical examination of their fracture surfaces. A granular microstructure was clearly distinguishable and a distinction made between materials which fail exclusively by transgranular fracture and others which additionally exhibit intergranular fracture. In order to interpret markings observed on the fracture surfaces of the complex 2-phase systems a study was made of the influence of molecular weight on the fractography of 1-phase PMMA. Molecular weight was reduced by degradation of samples by exposure to gamma-rays. The spacing of periodic rib markings on fracture surfaces was found to decrease with molecular weight and this relationship used to provide an estimate of the molecular weight of polymer in the matrix of 2-phase materials.


Biomaterials science protocols for clinical investigations on porous alumina ceramic and vitreous carbon implants.

A written protocol for the investigation of candidate surgical implant materials is quite important. Biomaterials science sections of clinical protocols have been developed for porous alumina ceramic and nonporous vitreous carbon biomaterials. Published data on the properties of the biomaterials were evaluated as related to bone replacement and augmentation. Where necessary, limited laboratory studies were conducted. If decisions could not be reached with respect to a given application, animal studies were initiated. The surgeons worked with biomaterials in the laboratory and the biomaterials scientist attended the experimental surgery procedures. Biomaterials Science Laboratory nondestructive investigations including stereomicroscopic and x-ray inspections were conducted on the vitreous carbon dental implant systems. The investigations elucidated a number of unexpected features for both implant biomaterials and the overall interaction between the different disciplines resulted in a more complete protocol for the study of these biomaterials at our Medical and Dental Center.


Dimensional behavior of curing bone cement masses.

The curing of bone cements is accompanied by release of polymerization heat and, hence, by a temperature rise of the curing cement mass. This temperature rise causes expansion of enclosed air bubbles and evaporation of the volatile monomer. An overall expansion of 3 to 5 vol % has been mentioned in the literature. It has often been stated that this expansion favours the fixation of metal endoprostheses in the marrow cavity of bone. To check for the influence of this expansion on linear dimensions of the cured cement mass we filled stainless steel cylinders with a precision bore of 22,000 +/- 0,005 mm and a length of 120 mm with bone cement. After curing of the cement in a environment of 37 degrees C the resulting cement rod was released from the cylinder and the diameter of the rod was measured at 37 degrees C. The influence of the "foaming effect" on the transverse dimensions of the rods was studied by curing the cement at 37 degrees C and 2 atm air pressure in a high-pressure-vessel. This method of curing eliminates porosity in the cement almost completely, so that curing shrinkage is to be expected rather than expansion of the cement mass. The results indicate that a volumetric expansion of the cement during curing of cylindrical rods in laboratory experiments, can be accompanied by a linear diametrical shrinkage of the cement mass. The explanation of this phenomenon is to be sought in the fact that the volumetric expansion takes place at a time when the cement is still plastic; by the formation of gas bubbles, the cement is forced in longitudinal direction into the cylinder and when the temperature of the mass has passed through a maximum, the cooling of the cement mass results in a thermal shrinkage of approximately 0.4% linearly. Extrapolating this laboratory result to clinical situation one might doubt whether the overall expansion of bone cements during curing will result in a permanent positive pressure on the walls of marrow cavity and whether it will contribute to a better fixation of endoprostheses than in the case of a, still hypothetical, nonporous cement.


Growth kinetics of gamma 2 crystals on mercury-plated Ag3Sn.

Previous work has shown that needles of gamma 2 crystals form when mercury is plated on the surface of an Ag3Sn specimen. In the present study, the growth kinetics of gamma 2 needles were investigated from room temperature to 50 degrees C for varying amounts of plated mercury. Straight-line relationships between crystal length and the square root of the time of growth showed that the growth rate is diffusion controlled. The growth rate increased with temperature up to 40 degrees C but decreased at higher temperatures. Further, as the temperature was increased, fewer gamma 2 crystals formed and their growth length decreased. This demonstrates that a number of competing factors are involved in the growth process at high temperatures.


Development of needles and sutures for microsurgery.

At present, the smallest needle commonly available is about 130 mum in diameter in combination with a suture of about 25 mum in diameter. Marked reductions in size, particularly of the needle, are advantageous for microvascular surgery. A needle of about 70 mum in diameter, welded to a suture of about 18 mum diameter, has been developed. In initial tests, it has performed satisfactorily in the repair of the carotid artery of the white rat. It is shown that the basic properties of materials severely limit further reductions in the size of needles and sutures.


Slow crack growth in acrylic bone cement.

Slow crack growth in Perspex acrylic sheet (PMMA) and Simplex acrylic bone cement in air and water has been studied from a fracture mechanics viewpoint. It has been found that the crack velocity, V, for each material depends upon the intensification of stress at the tip of the crack. Experimental measurements have been made of V as a function of the stress intensity factor, K, at the crack tip, and a derived V, K relationship has been used to predict the times-to-failure of components made from PMMA and Simplex cement. Direct measurements of time-to-failure for PMMA have shown that the predicted values give a conservative estimate of the structural lifetime of the material.


Thermodifferential analysis of ceramic implants.

Ceramic pelletes (in Al2O3 92% and in calcium aluminate) implanted into the muscles of rabbits for varying periods of time, and samples of similar material, not implanted but mixed with organic substances, have been analyzed by means of thermal differential analysis (TDA). Which hydrated calcium aluminates are formed from implants after contract with the tissue fluid, has been established. It has also been ascertained that the ceramic implants absorb organic substances from the tissue fluid, which, from a first evaluation, seem to be composed of simple molecules. The results of this initial inquiry have provided positive indications of the utility of TDA to assist in elucidating the phenomena that occur between implant and tissue.


Deposition of blood cells on collagen and cuprophane membranes.

Dogs were dialyzed with collagen and cuprophane membranes, human renal failure patients were dialyzed with cuprophane membranes, and samples of the membranes were observed by scanning electron microscopy at different time periods. Fixation was done in glutaraldehyde and cacodylate buffer, and samples were dried at the critical point. All cellular elements were well preserved and appeared viable. Cuprophane adsorbed platelets almost selectively in dog dialyses, whereas collagen adsorbed a smaller number and more heterogeneous group of cells. In contrast to these findings, when cuprophane was used in clinical dialyses, the cell population was predominantly polymorphonuclear leukocytes.


Determination of fluorocarbon in blood.

Because fluorocarbons can dissolve relatively large quantities of oxygen and carbon dioxide, there is considerable interest in utilizing them to develop new methods of extracorporael circulation, artificial red blood cells, and liquid breathing techniques. A method for the assay of fluorocarbon in blood is presented. The fluorocarbon is extracted from the blood with toluene, and fluoride is released from the fluorocarbon in the toluene extract by reaction with sodium biphenyl. The inorganic fluoride is then extracted with aqueous sodium acetate, the pH of the extract is adjusted, and the activity of the fluoride ion is read with a fluoride-specific ion electrode. The assay was effective for fluorocarbon concentrations in the range of 1 to 30 ppm.


Platelet adhesion and contact activation time tests on HEMA coated cellulose acetate membranes.

Surface modification of cellulose acetate dialysis membranes was carried out by 60Co radiation induced graft copolymerization of the hydrogel, hydroxyethyl methacrylate (HEMA). The degree of grafting was controlled by varying the HEMA monomer concentration in the grafting solution and the radiation dose. A continuous flow platelet adhesion test was designed which allows testing under conditions more closely approximating hemodialysis than other small scale in vitro tests. Platelet adhesion on treated membranes fell substantially with increasing surface HEMA concentration. The presence of HEMA on the membrane surface did not affect the membrane activated clotting times significantly.


Early tissue infiltrate in porous polyethylene implants into bone: a scanning electron microscope study.

The results of the present study demonstrate the utility of the scanning electron microscope for characterizing the ultrastructure of the initial tissue infiltrate in porous polyethylene implants. Shortly after implantation a thin noncellular fibrous-like coating was observed to form on the pore surface. The cells observed in the polyethylene pellets 3 days after implantation were generally consistent with what one would expect to see in a hematoma. As early as 14 days after implantation much of the blood clot was replaced by newly formed bone spicules. Tissue shrinkage accompanying dehydration of the specimen for scanning electron microscope study although a disadvantage occasionally proved useful in that it provided the opportunity to study the internal surface of the fibrous coating when separated from the surface of the implant. Less shrinkage was observed in implants whose pores were filled with bone spicules.


Properties of the beta-nerve growth factor receptor in development.

The cell surface receptor for beta-nerve growth factor was used as a probe to study the development of embryonic chick sensory ganglia. The ganglia were shown to lose their responsiveness to nerve growth factor in vitro between 14 and 16 days of embryonic age. This loss occurred by a decrease in the magnitude of the maximum biological response, not by a shifting of the response to higher concentrations. Binding assays for the beta-nerve growth factor receptor, using 125I-radiolabelled beta-nerve growth factor, were performed with cells from sensory ganglia 8, 12, 14, 16, 18, and 21 days of age. The assays revealed a twofold increase in the number of receptor sites per ganglion between 8 and 14 days and a sixfold drop between 14 and 16 days of embryonic life. Neither increase nor decrease was accompanied by a large change in the affinity of the receptor for the protein. Together with the results of the bioassay, the data show that the loss of biological responsiveness is correlated with and may be due to a loss of the cells' ability to bind beta-nerve growth factor. Correlation of the results of the binding assays with the known ontogeny of the chick embryo provides a hint at the role of nerve growth factor in normal development.


Inhibitor effects during the cell cycle in Chlamydomonas reinhardtii. Determination of transition points in asynchronous cultures.

A wide variety of inhibitors (drugs, antibiotics, and antimetabolites) will block cell division within an ongoing cell cycle in autotrophic cultures of Chlamydomonas reinhardtii. To determine when during the cell cycle a given inhibitor is effective in preventing cell division, a technique is described which does not rely on the use of synchronous cultures. The technique permits the measurement of transition points, the cell cycle stage at which the subsequent cell division becomes insensitive to the effects of an inhibitor. A map of transition points in the cell cycle reveals that they are grouped into two broad periods, the second and fourth quarters. In general, inhibitors which block organellar DNA, RNA, and protein synthesis have second-quarter transition points, while those which inhibit nuclear cytoplasmic macromolecular synthesis have fourth-quarter transition points. The specific grouping of these transition points into two periods suggests that the synthesis of organellar components is completed midway through the cell cycle and that the synthesis of nonorganellar components required for cell division is not completed until late in the cell cycle.


Tubulin constancy during morphological differentiation of mouse neuroblastoma cells.

Clonal cell lines N18 and N103 of the mouse neuroblastoma C1300 possess an undifferentiated neuroblast morphology under optimal growth conditions; however, when deprived of serum, N18 can be induced to extend long neurites. Although initial neurite outgrowth is rapid, very long fibers are found only after several days. Both initial outgrowths and established neurites contain microtubules; however, the number and density of these polymerized tubules increase markedly during this time. Optimum conditions have been established for assessing the colchicine-binding activity of neuroblastoma sonicates. A time-decay colchicine-binding assay was used to make a comparative study of the tubulin content of both undifferentiated and differentiated N18 as well as the nondifferentiating N103 and the rat glioma C6. Both morphologies of clone N18 possessed similar concentrations of tubulin (130-140 pmol/10(6) cells). Although cells of clone N103 contain 20% less tubulin than N18 cells, this is considerably more tubulin than is present in the glioma C6 (30 pmol/10(6) cells) which has a similar generation time. Quantitative densitometry of neuroblastoma extracts electrophoresed on SDS-polyacrylamide gels confirmed the constancy of tubulin. Radiolabeled proteins from neuroblastoma cells subjected to both growth conditions show that neurite outgrowth does not create a disproportionate demand for tubulin synthesis. Thus, the morphological differentiation of neuroblastoma cells probably reflects the regulation of tubulin storage and microtubule polymerization.


Membrane-bound ribosomes of myeloma cells. II. Kinetic studies on the entry of newly made ribosomal subunits into the free and the membrane-bound ribosomal particles.

The kinetics of appearance of newly made 60S and 40S ribosomal subunits in the free and membrane-bound ribosomal particles of P3K cells were explored by determining the specific radioactivities of their 18S and 28S RNA after various lengths of [3H]uridine pulse. Both 40S and 60S subunits enter free and membrane-bound polyribosomes at comparable rates from the cytoplasmic pool of newly made, free native subunits, the 40S subunits entering the native subunit pool and the polyribosomes slightly earlier than the 60S subunits. At all times, the specific radioactivity of the membrane-bound native 60S subunits was slightly lower than that of the polyribosomal 60S subunits. This indicates that the membrane-bound native 60S subunits are not precursors destined to enter membrane-bound polyribosomes and suggests that they result from the dissociation of ribosomes after chain termination. The results observed also suggest that the membrane-bound native 60S subunits are not reutilized before their release from the membranes, which probably takes place shortly after dissociation from their 40S subunits. The monoribosomes, both free and membrane-bound, had the lowest specific radioactivities in their subunits. Finally, a small amount of newly made native 40S subunits, containing 18S RNA of high specific radioactivity, and apparently also newly made messenger RNA were detected on the membranes. The high turnover of these membrane-bound native 40S subunits suggests that they may represent initiation complexes formed with mRNA which has just reached the membranes and which has not yet given rise to polyribosomes.


Effect of streptolysin O on erythrocyte membranes, liposomes, and lipid dispersions. A protein-cholesterol interaction.

The effect of the bacterial cytolytic toxin, streptolysin O (SLO), on rabbit erythrocyte membranes, liposomes, and lipid dispersions was examined. SLO produced no gross alterations in the major erythrocyte membrane proteins or lipids. However, when erythrocytes were treated with SLO and examined by electron microscopy, rings and "C"-shaped structures were observed in the cell membrane. The rings had an electron-dense center, 24 nm in diameter, and the overall diameter of the structure was 38 nm. Ring formation also occurred when erythrocyte membranes were fixed with glutaraldehyde and OsO4 before the addition of toxin. In contrast, rings were not seen when erythrocytes were treated with toxin at 0 degrees C, indicating that adsorption of SLO to the membrane is not sufficient for ring formation since toxin is known to bind to erythrocytes at that temperature. The ring structures were present on lecithin-cholesterol-dicetylphosphate liposomes after SLO treatment, but there was no release of the trapped, internal markers, K2CrO4 or glucose. The crucial role of cholesterol in the formation of rings and C's was demonstrated by the fact that these structures were present in toxin-treated cholesterol dispersions, but not in lecithin-dicetylphosphate dispersions nor in the SLO preparations alone. The importance of cholesterol was also shown by the finding that no rings were present in membranes or cholesterol dispersions which had been treated with digitonin before SLO was added. Although rings do not appear to be "holes" in the membrane, a model is proposed which suggests that cholesterol molecules are sequestered during ring and C-structure formation, and that this process plays a role in SLO-induced hemolysis.


Cytoplasmic transfer of chloramphenicol resistance in human tissue culture cells.

The cytoplasmic inheritance of human chloramphenicol (cap) resistance has been demonstrated by removing the nuclei of cells of the CAP-resistant HeLa strain 296-1 (enucleation) and fusing them to a CAP-sensitive HeLa strain lacking nuclear thymidine kinase. Plating the fusion products in bromodeoxyuridine and CAP resulted in the growth of about 150 colonies/10(6) parent cells plated. Permanent cell lines (cybrids) grown from such fusions have been designated HEB. A recloned HEB cybrid (HEB7A) has also been enucleated and fused to hypoxanthine phosphoribosyl transferase (HPRT)-deficient HeLa cells (S3AG1) and HPRT-deficient lymphocytes (WAL-2A). Cybrids were selected in thioguanine and CAP. In the fusion of enucleated (en) HEB7A to S3AG1, 1,200 colonies/10(6) parents were observed. Fusion of enHEB7A to WAL-2A was done in mass culture and cybrids were obtained on three separate occasions. In every case the parental controls were negative. All isolates tested from the above fusions have the CAP-resistant characteristics, in vivo and in vitro, of the enucleated parent and the nuclear characteristics of the CAP-sensitive parent, such as chromosome number, morphology, and specific isozyme and chromosome markers. Therefore, it can be concluded that CAP resistance is coded in the cytoplasm and not in the nucleus of 296-1 cells. Furthermore, this resistance can be transferred to cells of widely different origin and differentiated state. These studies represent the first genetic evidence of cytoplasmic inheritance in human cells.


Circulation and turnover of synaptic vesicle membrane in cultured fetal mammalian spinal cord neurons.

Intact neurons in cultures of fetal rodent spinal cord explants show stimulation-dependent uptake of horseradish peroxidase (HRP) into many small vesicles and occasional tubules and multivesicular bodies (MVB) at presynaptic terminals. Presynaptic terminals were allowed to take up HRP during 1 h of strychnine-enhanced stimulation of synaptic transmitter release and then "chased" in tracer-free medium either with strychnine or with 10 mM Mg++ which depresses transmitter release. Tracer-containing vesicles are lost from terminals under both chase conditions; the loss is more rapid (4-8 h) with strychnine than with 10 mM Mg++ (8-16 h). There is a parallel decrease in the numbers of labeled MVB's at terminals. Loss of tracer with 10 mM Mg++ does not appear to be due to the membrane rearrangements (exocytosis coupled to endocytosis) that presumably lead to initial tracer uptake; terminals exposed to HRP and Mg++ for up to 16 h show little tracer uptake into vesicles. Nor is the decrease likely to the due to loss of HRP enzyme activity; HRP is very stable in solution. During the chases there is a striking accumulation of HRP in perikarya that is far more extensive in cultures initially exposed to tracer with strychnine than 10 mM Mg++ regardless of chase conditions. Much of the tracer ends up in large dense bodies. These findings suggest that synaptic vesicle membrane turnover involves retrograde axonal transport of membrane to neuronal perikarya for further processing, including lysosomal degradation. The more rapid (4-8 h) loss of tracer-containing vesicles with strychnine may reflect vesicle membrane reutilization for exocytosis.


Inhibition by colchicine of fibrinogen translocation in hepatocytes.

In the rat, 8 h after intraperitoneal administration of colchicine, fibrinogen (detected by antirat fibrinogen antibodies labeled with peroxidase) accumulated in the lumina of the rough endoplasmic reticulum of the hepatocytes; 16 and 24 h after colchicine administration, fibrinogen was detected, respectively, in the lumina of the smooth endoplasmic reticulum and in the Golgi apparatus. The effect of colchicine on the cytoplasmic translocation of fibrinogen could be due to a direct action of the drug on the membranes of the endoplasmic reticulum or could be the indirect result of the disruptive action of the drug on the microtubules.


Cytoplasmic filaments and cellular wound healing in Amoeba proteus.

The flexibility and self-healing properties of animal cell surface membranes are well known. These properties have been best exploited in various micrurgical studies on living cells (2, 3), especially in amoebae (7, 20). During nuclear transplantation in amoebae, the hole in the membrane through which a nucleus passes can have a diameter of 20-30 mum, and yet such holes are quickly sealed, although some cytoplasm usually escapes during the transfer. While enucleating amoebae in previous studies, we found that if a very small portion of a nucleus was pushed through the membrane and exposed to the external medium, the amoeba expelled such a nucleus on its own accord. When this happened, a new membrane appeared to form around the embedded portion of the nucleus and no visible loss of cytoplasm occurred during nuclear extrusion. In the present study, we examined amoebae that were at different stages of expelling partially exposed nuclei, to follow the sequence of events during the apparent new membrane formation. Unexpectedly, we found that a new membrane is not formed around the nucleus from inside but a hole is sealed primarily by a constriction of the existing membrane, and that cytoplasmic filaments are responsible for the prevention of the loss of cytoplasm.


Membrane-bound ribosomes of myeloma cells. III. The role of the messenger RNA and the nascent polypeptide chain in the binding of ribosomes to membranes.

Mild ribonuclease treatment of the membrane fraction of P3K cells released three types of membrane-bound ribosomal particles: (a) all the newly made native 40S subunits detected after 2 h of [3H]uridine pulse. Since after a 3-min pulse with [35S]methionine these membrane native subunits appear to contain at least sevenfold more Met-tRNA per particle than the free native subunits, they may all be initiation complexes with mRNA molecules which have just become associated with the membranes; (b) about 50% of the ribosomes present in polyribosomes. Evidence is presented that the released ribosomes carry nascent chains about two and a half to three times shorter than those present on the ribosomes remaining bound to the membranes. It is proposed that in the membrane-bound polyribosomes of P3K cells, only the ribosomes closer to the 3' end of the mRNA molecules are directly bound, while the latest ribosomes to enter the polyribosomal structures are indirectly bound through the mRNA molecules; (c) a small number of 40S subunits of polyribosomal origin, presumably initiation complexes attached at the 5' end of mRNA molecules of polyribosomes. When the P3K cells were incubated with inhibitors acting at different steps of protein synthesis, it was found that puromycin and pactamycin decreased by about 40% the proportion of ribosomes in the membrane fraction, while cycloheximide and anisomycin had no such effect. The ribosomes remaining on the membrane fraction of puromycin-treated cells consisted of a few polyribosomes, and of an accumulation of 80S and 60S particles, which were almost entirely released by high salt treatment of the membranes. The membrane-bound ribosomes found after pactamycin treatment consisted of a few polyribosomes, with a striking accumulation of native 60S subunits and an increased number of native 40S subunits. On the basis of the observations made in this and the preceding papers, a model for the binding of ribosomes to membranes and for the ribosomal cycle on the membranes is proposed. It is suggested that ribosomal subunits exchange between free and membrane-bound polyribosomes through the cytoplasmic pool of free native subunits, and that their entry into membrane-bound ribosomes is mediated by mRNA molecules associated with membranes.


Interaction of phospholipid vesicles with cultured mammalian cells. II. Studies of mechanism.

The mechanism of interaction of artificially generated lipid vesicles (approximately 500 A diameter) with Chinese hamster V79 cells bathed in a simple balanced salt solution was investigated. The major pathways of exogenous lipid incorporation in vesicle-treated cells are vesicle-cell fusion and vesicle-cell lipid exchange. At 37 degrees C, the fusion process is dominant, while at 2 degrees C or with energy depleted cells, exchange of lipids between vesicles and cells is important. The fusion mechanism was demonstrated using vesicles of [14C]lecithin containing trapped [13H]inulin. Consistent with a fusion hypothesis, both components became cell associated at 37 degrees C in nearly the same proportions as they were present in the applied vesicles. Additional arguments in favor of vesicle-cell fusion and against phagocytosis or adsorption of intact vesicles are presented. At 2 degrees C or with inhibitor-treated cells, the [3H]inulin uptake was largely suppressed, while the lipid uptake was reduced to a lesser extent. Evidence for vesicle-cell lipid exchange was obtained using V79 cells grown on 3H precursors for cellular lipids. [14C]lecithin vesicles, incubated with such cells, showed no change in their elution properties when subjected to molecular sieve chromatography on Sepharose 4B. However, radioactivity and thin-layer chromatographic analyses revealed that a variety of cell lipiids had been exchanged into the uniamellar vesicles. Further evidence for the fusion and exchange processes was obtained using vesicles prepared from mixtures of [3H]lecithin and [14C]cholesterol. A two-step fusion mechanism consistent with the present findings is proposed as a working model for other fusion studies.


Transient erythropoietic spleen colonies: effects of erythropoietin in normal and genetically anemic W/Wv mice.

Properties of the cells (TE-CFU) that give rise within four to six days to transient endogenous erythropoietic spleen colonies in irradiated mice have been investigated. The results obtained indicate that (1) erythropoietic maturation within such colonies is highly erythropoietin-dependent, (2) the population size of TE-CFU is not erythropoietin-dependent, (3) initial exposure to a high dose of erythropoietin followed by continuing exposure to lower doses is required for maximal efficiency of colony formation by TE-CFU, (4) successful transplantation of TE-CFU has not been achieved, but they appear among the progeny of transplanted hemopoietic cells, (5) TE-CFU are defective in mice of genotype W/Wv. These findings are consistent with the view that the TE-CFU assay detects a class of early erythropoietin-sensitive progenitor cells committed to erythropoietic diffferentiation, rather than "abortive" colony formation by pluripotent stem cells.


Transfer of adenine nucleotides between the releasable and nonreleasable compartments of rabbit blood platelets.

The metabolic pool of adenine nucleotides in platelets can be labeled by incubating platelets for 1 h in vitro with [14C]adenosine or [32P]orthophosphate. When these platelets are treated with thrombin, the adenine nucleotides released are not labeled. Because of this, Holmsen's suggestion of a metabolically inert pool of granule nucleotides has been generally accepted. We have found that upon incubation of labeled rabbit platelets for longer times (up to 6 h) in vitro, or upon reinjection and reharvesting at times up to 66 h, the releasable pool of adenine nucleotides becomes labeled. Because the rates of 32p and 14C incorporation into this releasable pool are similar, it seems likely that these labels enter the granules as ATP. Equilibrium between the metabolic and granule pools is complete by 18 h. When rabbit platelets are labeled in vivo by intravenous injection of [32P]orthophosphate, peak labeling occurs between 60 and 70 h; this corresponds to their maturation time. The platelets probably incorporate 32P during their production in the megakaryocytes. The specific radioactivity of the adenine nucleotides in the releasable (granule) pool of these platelets is the same as the specific radioactivity in the nonreleasable (metabolic) pool. Since inorganic phosphate in platelets (and undoubtedly in the megakaryocytes) exchanges with inorganic phosphate in plasma, and since the radioactivity of the latter decreases rapidly, the adenine nucleotides in the two pools must exchange to maintain the same specific radioactivity. Transfer of adenine nucleotides into storage granules may represent a general phenomenon because it has been observed in the chromaffin cells of the adrenal medulla also.


Relationship of energy production to gluconeogenesis in renal cortical tubules.

Isolated tubules prepared by collagenase treatment of rat renal cortex retained their ultrastructural integrity and responded to added lactate and succinate with an increase in gluconeogenesis and respiration. Inhibition of the mitochondrial respiratory chain with rotenone, or energy conservation sites with oligomycin caused a marked reduction in respiration and ATP content thereby completely inhibiting net gluconeogenesis. Dissociation of gluconeogenesis from respiration was accomplished with quinolinic acid and hydrazine, inhibitors of gluconeogenesis. At 5 times 10(-3) M quinolinic acid, gluconeogenesis from succinate was inhibited approximately 50% and from lactate nearly 100%. This concentration of quinolinic acid did not affect oxygen uptake or the ATP content of tubules in the presence or absence of substrate. Hydrazine at 10(-3) M resulted in approximately 75% inhibition of glucose formation from succinate and complete inhibition from lactate without interfering with respiration or ATP content. The increased mitochondrial energy generation, as manifested by accelerated respiration was independent of gluconeogenesis. The unchanging cell ATP concentration with a higher respiratory rate upon addition of exogenous substrate bespeaks increased ATP turnover. ATP utilization for the substrate-induced enhancement of gluconeogenesis could not account for the increment in ATP hydrolysis.


Interaction of the fluorescent probe, 1-anilino-8-napthalene sulfonate, with the sulfate transport system of Ehrlich ascites tumor cells.

The addition of the fluorescent dye, ANS, to intact ascites tumor cells results in an enhancement of fluorescence intensity. The increase in fluorescence intensity as a function of time is biphasic which suggests that at least two processes occur. The first associated with the rapid initial rise in fluorescence represents binding to the cell surface while the second or slower phase reflects entrance of ANS into the intracellular phase. The relationship between bound and free ANS in 0.50 mM sulfate medium was used to calculate the apparent dissociation constant of ANS-membrane complex (Kd = 6.53 times 10(-5) M) and the total number of ANS binding sites (4.49 nmoles/mg dry weight). Kinetic analysis of steady state sulfate transport in the presence and absence of ANS suggests that (1) sulfate exchange can be described by Michaelis Menten type kinetics (Km = 2.05 times 10(-3) M), (2) a small fraction of surface associated ANS competitively inhibits sulfate exchange (Ki = 4.28 times 10(-6) M) and (3) the transport system has a higher affinity for ANS than for sulfate. These data are consistent with the hypothesis that inhibition of sulfate exchange is related to the direct, reversible interaction of the negatively charged sulfonate group of ANS with superficial positively charged membrane sites.


Variations in the cell-free translating apparatus of cultured animal cells as a function of time during cell growth.

Vero M3 cells, a line derived from the kidney of an African Green Monkey, display certain alterations in their protein synthetic apparatus as a function of time during a growth cycle. (Growth cycle here refers to exponential growth of unsynchronized cells in culture and their subsequent passage into the stationary phase.) The capacity of cytoplasmic extracts of these cells to promote endogeneous mRNA-mediated polypeptide synthesis or poly U-mediated polyphenylalanine synthesis declines from the second day after the initiation of the growth cycle. The ribosome sedimentation profile indicates that after the second day of growth a decrease also occurs in the total amount of ribosomes per cell, and that a shift occurs from predominantly polyribosome structures to predominantly subunits and monoribosomes structures. The activity of the translation factor, elongation factor 1, also progressively decreases after the second day of growth. Furthermore, when crude factor preparations from cells in the second day of growth (Exponential phase) and from cells in the fifth day of growth (Stationary phase) are compared for leucyl-tRNA synthetase and prolyl-tRNA synthetase activities, it is found that the extracts from fifth-day cells have significantly less activity. The activity of another enzyme, acid phosphatase, remains relatively unaffected as a function of time during the cell growth cycle. When HeLa S3 plating cells are grown under the same conditions, they do not display the same responses.


Interaction of two mechanisms regulating alkali cations in HeLa cells.

The alkali cation content of HeLa cells is independent of culture density and of whether the cells are grown in suspension or attached to the culture vessel. With a cell doubling time of 28 hours, the cell K content turns over approximately once per hour. Following partial blockade of the alkali-cation transport system with ouabain, two distinct but interrelated mechanisms operate in the cellular response: (a) an increase in intracellular Na stimulates the pump so that the short-term alteration in electrolyte compostition is less than would be expected from the fraction of pump sites inhibited, and (b) there is a cycloheximide-sensitive recovery in transport capacity reflecting a restoration of functional transport sites to their normal density on the cell surface. Experimental manipulations that mimic the effect of ouabain lead to a stimulation of transport, but they do not result in an increase in the number of ouabain-binding sites on the surface. The data are consistent with a four-to-six hour turn-over of transport sites at the surface, but there is no evidence for a speicific induction of the transport system within this short-term recovery period.


Effect of cell trypsinization on nuclear proteins of WI-38 fibroblasts in culture.

When resting confluent monolayers of WI-38 fibrolasts are trypsinized and replated at a lower density they are stimulated to proliferate again with an interval of 18 hours between replating and the onset of DNA synthesis. Trypsinization of resting cells causes a 40% loss of nuclear proteins as well as of cytoplasmic proteins. The amount of nuclear proteins remains low for the first six hours after the cells have been replated and then it increases rapidly, reaching the same level of non-trypsinized resting cells by ten hours after plating. The proteins that are lost from the nucleus immediately after trypsinization are chromatin-associated proteins and most of them are non-histone chromosomal proteins, although a modest loss of histones cannot be ruled out. The loss of non-histone chromosomal proteins from cells that have been trypsinized causes changes in the structure of chromatin that can be detected by circular dichroism and by viscosity measurements. These results show that cell trypsinization causes an extensive loss of proteins from chromatin and that the loss is restored only several hours after the cells have been replated at a lower density.


Adhesion of Phytophthora palmivora zoospores: detection and ultrastructural visualization of concanavalin A-receptor sites appearing during encystment.

The binding of concanavalin A (Con A) to the cell surface of zoospores and cysts of Phytophthora palmivora was studied by radiometry (125I-Con A), ultraviolet microscopy (fluorescein-Con A) and electron microscopy peroxidase-diaminobenzidine technique). Zoospores were found to secrete during the early stages of encystment a Con A-binding material susceptible to trypsin digestion. This glycoprotein is contained in the so-called peripheral vesicles and is probably responsible for the adhesion of the encysting zoospores to solid surfaces.


Effect of near ultraviolet and visible light on amoeba.

The near ultraviolet and visible light (VL) impinging at an intensity of 2-5 x 10(2) J s-1 m-2 for 2-5 h kills the mitotic and the early S-phase (0- to 15-min-old) amoebae. At the mid- and late S-period only a fraction of cells are killed by VL and G2 phase cells are quite resistant. Amoebae of all cell cycle stages show a delay in the first mitotic division. DNA synthesis, as measured by [3H]thymidine incorporation, is depressed in the VL-exposed early-S amoebae. A concurrent but temporary inhibition in [3H]leucine incorporation also occurs in these cells. However, no significant change in [3H]uridine incorporation has been found. To localize the site of lethal damage, nuclear transplantation studies were undertaken between the control amoebae and the amoebae treated with VL. The nucleus of a  VL-exposed early S-phase cell recovers when transplanted immediately after VL exposure into an enucleate G2 cytoplasm but dies if grafted into an enucleat S-phase cytoplasm. The therapeutic effect of the G2 cytoplasm, although at a lower level, is also evident even when the treated early S-phase nucleus is implanted 20 h later, but not after 48 h, into the G2 cytoplasm. The amoeba cytoplasm shows resistance to VL-irradiation, can accept a control nucleus from any cell cycle stage, and function normally. The G2 nucleus also remains apparently unaffected to VL exposure and can survive when it is transfered to the control cytoplasm of any cell-cycle phase. All these findings are discussed in the light of the possible existence of a repair system against VL-induced damage in the G2-phase amoeba.


Cellular uptake of soluble and aggregated ferritin: distinction between pinocytosis and phagocytosis.

Cellular uptake of ferritin amounting to 0-5 mug/mg cell protein or more can be measured colorimetrically on the basis of ferritin-iron content. 131I-serum albumin, soluble ferritin and aggregated ferritin used in equimolar concentrations are taken up differently by Sarcoma SI80 cells in culture. The net uptakes in 2 h at 37 degrees C are 0-065, 4-3 and 24-7 mug/mg cell protein or 0-93, 8-0 and 45-7 mumol, respectively. Albumin uptake is not inhibited by a 26-fold molar ferritin excess but is significantly inhibited by a 43-fold excess. The transport mechanism of the ferritins differs from that of albumin in that it is significantly inhibitable by 2 times 10(-4) M monoiodoacetate. Soluble ferritin contains small aggregates which are removed by filtration through Millipore membranes of 0-05, 0-1 and 0-22 mum. When the 0-1-mum filtrate is re-examined, uptake is no longer inhibited by iodoacetate. Since it can be inferred from other work that albumin is taken up by pinocytosis and ferritin aggregates by phagocytosis, the difference in susceptibility to inhibition is proposed as a way to distinguish pinocytosis from phagocytosis. Ferritin may form larger visible aggregates in culture medium. The transport mechanism of this aggregated ferritin differs from that of soluble unfiltered ferritin in that it causes concomitant enhancement of albumin uptake. Albumin transported by virtue of this effect becomes partially susceptible to iodoacetate. Thus, in addition to a distinction between pinocytosis and phagocytosis, our data single out 2 forms of albumin transport and 3 forms of ferritin transport.


Localization of lectins in legume cotyledons.

High-resolution techniques for the localization of lectins are described. Concanavalin A (Con A) and phytohaemagglutinin (PHA) are localized using a fluorescent method with (FITC)-labelled immunoglobulins which bind to the lectins in sections of jack and red kidney bean cotyledons. Specificity is defined by the use of specific sugar inhibitors. Both Con A and PHA are found in cytoplasmic sites. Lectins with beta-glycoside specificity are detected with red-coloured artificial carbohydrate antigens. The beta-galactosyl and beta-glucosyl antigens bind specifically to clusters of spherical bodies in the intercellular spaces, to cell wall sites, and to the periphery of the cytoplasm associated with the cell membrane.


Differences in density of Concanavalin A-binding sites due to differences in surface morphology of suspended normal and transformed 3T3 fibroblasts.

Calculations of the density of Concanavalin A (Con A)-binding sites on normal and transformed fibroblasts have, as yet, been based on the unproven assumption that suspended cells are smooth spheres. We studied the surface morphology of suspended normal and transformed fibroblasts with scanning and transmission electron microscopes, and found a large difference in surface morphology between suspended normal and transformed 3T3 cells. When this difference in surface morphology was taken into account, the estimated cell surface area of normal 3T3 cells was approximately seven times larger than that of transformed 3T3 cells. Since equal numbers of 3H-Con A molecules are bound on normal and transformed cells, the density of Con A-binding sites is approximately seven times greater on transformed than on normal 3T3 cells. The difference in density of Con A-binding sites between normal and transformed fibroblasts might be sufficient to explain the difference in agglutination response, as originally suggested by Burger, and may also be the cause of the different degrees of clustering of Con A-binding sites on the plasma membrane of these cells.


The distribution of Concanavalin A receptor sites on the membrane of chromaffin granules.

The distribution of concanavalin A (con A) receptor sites on the membranes of chromaffin granules has been investigated by binding studies using 125I-labelled con A and by electron-microscope studies using ferritin-labelled con A. In both experiments con A was observed to bind to chromaffin granule membranes but not to intact granules. The ferritin-con A particles bind to only one of the two possible surfaces of the chromaffin granule membranes. These results are in agreement with previous observations concerning the asymmetric distribution of saccharide residues on the surfaces of a number of different plasma membranes. They suggest that for the intracellular membrane of the chromaffin granule the saccharide sites, like those in plasma membranes, are not exposed to the cell cytoplasm. Further work is necessary to establish whether these sites are on the inner surface of the membrane or whether they are unmasked during the conversion of granules to membrane ghosts.


Photosensitivity and heat resistance conferred by BrdU incorporation upon a thymidine kinase-deficient mouse cell line with persistent mitochondrial enzyme activity.

The selective incorporation of 5-bromodeoxyuridine (BrdU) into mitochondrial DNA (mit-DNA) in the LM(TK-) ClID cell line, a thymidine kinase-deficient derivative of L fibroblasts with persistent mitochondrial enzyme activity, has been utilized to specifically damage mit-DNA by 'visible' light irradiation. ('Visible light' indicates the source of light used, although the components most active photochemically on BrdU-substituted DNA are in the near-visible range, 300-340 nm.) (Szybalski & Opara-Kubinski, 1965). LM(TK-) Cl ID cells, which had been grown in the presence of 30 mug/ml BrdU, were irradiated with 'visible' light. Analysis of the pre-existing mit-DNA in these cells, which had been long-term labelled with [5-3H]deoxycytidine, showed a progressive decrease, with increasing duration of irradiation, in the proportion of the closed-circular form and an increase in that of the open-circular form of mit-DNA, with the subsequent appearance of fragments of this DNA. Furthermore, there was a decrease during irradiation in the total amount of mit-DNA, which became about 35% of the non-irradiated control after 65 h irradiation. On the other hand, irradiation with 'visible' light failed to cause any quantitative or qualitative change, with respect to the non-irradiated control, in mit-DNA from cells grown in the absence of BrdU and long-term labelled with [Me-3h]thymidine. An analysis of the incorporation of [5-3H]deoxycytidine into mit-DNA of BrdU-grown cells, during a 3-h exposure of the cells to the precursor following irradiation, showed a fairly rapid decline of mit-DNA labelling; this became about 50% of the non-irradiated control after 12 h irradiation, decreasing to about 25% in the next 48 h. By contrast, no effect of irradiation was observed on the subsequent pulse-labelling of mit-DNA with [Me-3H]thymidine in cells grown in the absence of BrdU. Furthermore, no change in the size of the extracted nuclear DNA was found in irradiated BrdU-grown cells. The progressive and selective damage and destruction of mit-DNA during irradiation with 'visible' light of Cl ID cells correlate fairly well with the kinetics of loss of cell viability occurring under the same conditions, as described in the accompanying paper, strongly suggesting a link between the two phenomena.


An electron-microscope study of the intranucleolar chromatin during nucleologenesis in root meristematic cells of Allium cepa.

The various states of condensation of the chromatin material contained inside the lacunar regions of the reforming nucleolus in Allium cepa, have been investigated by means of conventional electron-microscope techniques. The observations reveal that, in the emerging early to late telophase nucleoli, the intralacunar chromatin material in question appears both in an extended and a condensed condition; from late telophase th the mid G1 period of interphase, the intralacunar chromatin material of the rapidly growing and developing nucleoli is present in an extended state only. An attempt is made to interpret these morphological findings in the light of current knowledge concerning the structural relationship of the nucleolar organizing region of the nucleolar chromosome with the interphase nucleolus in plant cells. The relevant observational evidence would be consistent with the view that the chromatin-containing lacunar regions of the reforming nucleolus in Allium cepa correspond, in fact, to cross- or oblique sections of a meandering channel through which the nucleolar organizing segment of the nucleolar chromosome passes. Assuming the applicability to intranucleolar chromatin of the general concept of condensed-inactive versus extended-active chromatin, it is concluded that gradual uncoiling and subsequent decondensation of the chromatin of the nucleolar organizing region in the form of a convoluted loop structure are key morphological and functional events associated with the process of nucleologenesis in the species investigated.


[Surgical methods in non-traumatic perforations of the colon into the free peritoneum (36 cases)].

When operating as an emergency for a perforation of the colon should one carry out a by-pass operation, colostomy or restore intestinal continuity? 36 colonic perforations are reported here, as a complication of cancer in 50 p. 100 of cases, or sigmoiditis in 38 p. 100 of cases. The perforation was situated on the sigmoid in 77 p. 100 of cases. 13 by-pass operations were carried out with a 23 p. 100 mortality, 12 colectomies without anastomosis with a 50 p. 100 mortality, and 2 colectomies with restoration of continuity, 9 p. 100 mortality. The overall mortality was 27.7 p. 100 much less than reported by other authors but, nevertheless partly due to the severity of peritonitis due to the presence of feces and, partly due to the operation adopted. The present attitude of the authors depends on the merits of each case. A large incision is made and the peritoneum thoroughly cleaned. If the surgeon is experienced, colonic resection is advisable for perforated cnacer and certain cases of sigmoiditis. Restoration of continuity depends, above all, on the anatomical condition of the colon above the lesion. Hartmann's resections or by-pass operations thus still have indications.


[Physiopathological and anatomo-clinical study of peritoneal irrigation in generalized acute peritonitis].

The objects of peritoneal wash-out and the conditions of its efficacy in acute generalised peritonitis were first defined. The authors then report the results of their physio-pathological research. The rules of intra-peritoneal wash-out then laid down. They suggest a method of wash-out perfectly adapted to the infected peritoneum. Study of the washings provides valuable information. The appearance, bacteriology and rate of wash-out, lead one to define criteria of prognosis which are useful and reliable for later treatment. These criteria may be grouped in a triad which are of value in addition to the clinical signs. The pathological consequences are then discussed. In the immediate post-operative period, there is no noxious effect on intestinal sutures, which can be attributed to the wash-out in this series of 20 cases. In the long-term, the absence of adhesions with restitutio ad integrum of the peritoneal cavity, was noted in 4 patients, and verified later by operation. The physiopathological basis and criteria of efficacy suggest that peritoneal wash-out should be used more often in modern treatment.


[Familial cancer of the parathyroid glands. Importance of angiography in the diagnosis of regional recurrences. Considerations on 2 cases].

The authors report two new cases of functional parathyroid carcinoma, the course of which varied between 5 and 7 years. The main fact was their familial character, as the disease affected two siblings. No similar case was found in the world literature. These two cases were characterised by symptoms in the bones or pancreatic symptoms which dominated the whole clinical course. Finally, the second case was studied by angiography which permitted the authors to localise a lymphnode metastasis on the opposite side. As far as treatment was concerned, the frequency and severity of local and regional spread justifies wide removal at an early stage. This attitude is necessary in any case of parathyroid tumor with an infiltrating character.


[Early postoperative intestinal obstructions. Contribution of  radiography to diagnosis of the type and to the indication for reoperation (experience in 84 cases)].

The authors report 84 cases of post-operative intestinal obstruction and demonstrate the value of straight XRays and barium meal in the diagnosis of the variety of early post-operative obstruction and the indications for operation. In 74 cases out of 82, a precise diagnosis of the mechanical or paralytic nature of the obstruction was made and the operation always carried out under best possible conditions.


[Effects of Tham, isoprenaline and propranolol on the blood flow and vascular resistance of the liver after clamping of the afferent and efferent vessels. Relation to splanchnic shock].

To determine the roles of portal circulation and the hepatic arteries during states of shock, the authors studied on 38 dogs, the effects of 30 minutes clamping of the sub-diaphragmatic part of the inferior vena cava, the portal vein and the hepatic artery, either together or successively. They measured the pressure in these various vessels, the transhepatic flow, using Xenon133, and calculated the intra-hepatic resistance. The animals were then treated with Tham, isoprenaline and propanolol. Tolerance to clamping was improved significantly when the animals were treated with the association of these drugs. The portal pressure and the systemic arterial pressure, rapidly returned to normal. This was also the case with hepatic, venous or arterial blood flow. There was a remarkable stability between the sinusoidal and biliary resistances, compared with disturbances in control groups. The already well known beneficial effect of Tham was increased by the apparently paradoxical association of isoprenaline and propranolol. In fact, with this dosage, they counter-balanced their reciprocal disadvantages. Finally, a study of blood flow and vascular resistance in the liver, suggests the existence of two components in splanchnic shock, hepatic and visceral.


[Study of 187 portacaval anastomoses. Statistical analysis of prognostic factors. II. Comparative study of the results of anastomoses with and without interruption of the portal trunk. Discussion].

The authors report a comparative retrospective study of 124 end-to-side portal anastomoses and 63 side-to-side anastomoses. The influence of a certain number of clinical and pathological factors on mortality and immediate and long-term survival, were statistically verified. The quality of the functional result result was analysed in each group of operated patients. Comparison of the results of trunkular anastomoses with or without interruption of the portal vein, showed both as regards, post-operative and late mortality, that the functional results were definitely in favor of end-to-side anastomosis. Thus side-to-side anastomoses gave 40,4 p. 100 of good results and 59,6 average or poor results, as against 60 p. 100 gave rise to portal systemic encephalopathy in 17 p. 100 of cases as against 35 p. 100 in the case of the side-to-side anastomosis. Only the sub-group of operated patients with hepatic vein thrombosis seemed to benefit from side-to-side anastomosis.


[1st clinical trials of Rhodergon velvet vascular prostheses].

The authors report a preliminary clinical and radiological trial of the use of Rhodergon velvet prosthesis for arterial replacement. Out of 9 valid cases, there was one failure, with a femoropopliteal prosthesis, explained by turbulence induced by change of direction. The average delay between operation and arteriography was 80 days.


[Retroperitoneal enteroid cyst].

The author report a case of enteroid retroperitoneal cyst treated by enucleation. Such a lesion raises mainly the problem of histological recognition and embryological interpretation for which there are two possible theories: (1) Uptake, before sealing off of the embryo, of a cell group of endoblastic origin which develops some distance from the digestive tract. (2) Retroperitoneal course of a simple teratoma of enteroid type. Such a possibility of teratoma leaves open the possibility of malignant transformation.


[Exploration and removal in focal infections of the liver (apropos of 32 resections in a 4-year period].

The surgeon can no longer be satisfied today, with a diagnosis of focal lesion of the liver. It is also necessary to give the precise localisation of the nodular image compared with the segments and vessels of the gland. The lesional topography is usually determined by coeliac arteriography but, in certain cases, complementary cavography becomes necessary. On knowledge of this hepatic angio-cartography, depends the choice of approach and the nature of the surgical operation, for there are easy methods of approach where, for example, removal may reasonably be attempted and, other segments which are difficult or impossible to approach, e.g. segments VIII and IV posteriorly. The authors report their experience of surgery of the liver between 1971 and April 1974 for 14 abscesses, 15 hydatid cysts, 5 tumours and 9 cases of trauma. They carried out 32 resections, 22 of the right lobe of the liver, 10 of the left lobe. The immediate results are studied in relation to the indications and techniques used.


[Duodenal obstruction by biliary calculus].

The authors report a case of cholecysto-duodenal fistula complicated by gallstone obstruction of the duodenal bulb, revealed by vomiting. Treatment included cholecystectomy, removal of the duodenal stone and suture of the duodenal fistual. The post-operative period was unevenful.


[Multiple fractures of the lower extremities (a propos of 50 patients)].

According to a study of 50 multiple fractures of the lower limbs, the frequency of associated lesions justifies the creation of new multiple injury units, well equipped in which may be found specialists of all branches of surgery. Although internal fixation in one  stage as an emergency, is ideal in all fractures, one should in fact be circumspect for the danger of infection should lead one to avoid carrying out internal fixation if this is not absolutely necessary.


[Colo-anal invaginations in adults].

Colo-anal intussusception is exceptional, there are only 6 published cases including ours, intussusception of the colon into the rectum is rare. It raises numerous diagnostic and surgical problems. The cause is always a tumour, usually malignant. The diagnosis is easy in the case of colo-anal intussusception, in which one may see the tumour and determine its nature. It is sometimes difficult in the case of colo-rectal intussusception, hence the interest of sigmoidoscopy and biopsy of the tumour. Surgical treatment includes simple reduction in the case of colo-rectal intussusception. On the other hand, in the case of colo-anal intussusception, one should first reduce the condition, otherwise it is very difficult to remove the tumour, but there is very frequently necrosis of the prolapsed segment, which may require removal in two stages, limited low resection of the necrotic segment and high removab after reduction. This operation is more easily carried out by two surgical teams operating simultaneously.


[Facial injuries].

In spite of a certain reduction in their numbers, facial injuries still raise frequent problems for general surgeons. Car accidents are now less commonly responsible than motorcycle accidents. In the most complex cases, one may find lesions of the soft parts and bony lesions in association. The wounds should be examined carefully, all foreign bodies removed and should only be sutured if one is certain tht the subjacent structures are undamaged, e.g. facial bony canals are often damaged in vertical wounds of the cheek situated behind the anterior border of the masseter muscle. As far as bony lesions are concerned, they rarely give rise to typical breakdowns of the suture lines described by Lefort, but more commonly cause true dislocations which are impossible to describe. Fractures are often undiagnosed owing to oedema which masks them, e.g. those of the malar bone and of the orbit and even those of the nose. This failure to diagnose them is serious for, at a later stage, surgical correction is more difficult and the prejudice is then not only esthetic but also functional, e.g. causing diplopia. Clinical examination of a patient with trauma of the face includes a series of simple gestures which a general surgeion should carry out in order to avoid failure to diagnose such lesions. Although he may not treat them all, he sould recognise them and decide which have priority in the treatment of multiple injuries. Careful inspection and palpation usually permit one to detect bony lesions which XRays then demonstrate only by careful choice of appropriate views.


[Surgical treatment of cavernomas of the portal vein. 56 cases, including 42 operated cases].

The difficulty of surgical treatment of cavernomas of the portal vein results from the anatomical conditions created by this type of portal hypertension, in spite of favourable physiological conditions due to the integrity of the hepatic parenchyma. The latter is, in fact, relative after a certain length of history. This erratic course renders difficult assessment of the efficacy of methods of treatment. In the light of 56 cases, including 42 operated cases, the authors present the indications for surgery which depend on the severity and frequency of hemorrhage and also on the patient's age. When repeated bleeding occurs, a by-pass should be attempted whenever possible, if the bleeding impedes social life or growth, or if it threatens life. Mesenterico-caval anastomosis is preferable before the age of 7 years. Spleno-renal anastomosis requires a sufficient size of splenic vein. Ligature of aesophageal varices or disconnection of the oesophagus from the stomach, may be considered in cases of moderate hemorrhage. The varices may be treated by sclerosing injections after failure of a by-pass operation or in inoperable patients. Oesophago-gastrectomy is useful but should be a last resort.


[Surgical treatment of recurrences after operations for essential varices].

Among our cases of post-operative recurrent varicose veins, we report here only those cases which required further surgery. Thus we were able to carry out a comparative study of a series of 30 patients who were re-operated on between the 16th of March 1959 and the 16th of March 1966, and 44 patients operated on under the same conditions between the 16th of March 1966 and the 16th of March 1974. The causes of recurrence were different in the two groups. Before 1966, they were mainly due to the choice of inefficacious operations. Since 1966, they are due to poor technique in resection of the junction of the femoral vein with the saphenous vein. This was the most common cause, and these failures were favoured by saphenous abnormalities. Associated with stripping of the large vessels, resection of inguinal cavernoma which prolonged the femoro-saphenous junction, was the main stage in these re-operations. It was perfectly efficacious in the treatment of varicose veins but required also resection of inguinal lymph nodes with the cavernoma. Post-operative complications, such as lymphatic fistula and oedema, led us to prefer simple isolation of the cavernoma from the deep veins, but the first and essential stage remains resection of the junction of the femoral and saphenous veins and stripping of accessory trunks.


[Retroperitoneal mesenchymomas. Remarks apropos of 19 cases].

The authors report 19 cases and review the literature. They analyse the pathological and clinical characteristics of retro-peritoneal mesenchymoma. The diagnosis of these tumours is facilitated by intravenous urography, barium enema, arteriography and bidimensional echography. Cavography is interesting to determine the venous risk. The unforeseeable course, which is always severe in these tumours, is characterised by the frequency of local relapse. The prognosis is very poor with only one 8 year survival in this series. Surgical removal is the only possible treatment, in spite of difficulty, especially during recurrences.


[Arteritis in aged patients].

Gangrene is the usual reason for admission to a surgical unit in elderly patients with peripheral vascular disease. It is often the first sign of arterial disease occurring in a foot with satisfactory skin and may be triggered off by trauma or infection. Obstruction at various levels of the profunda femoris artery, of the main leg arteries with calcification of the media, are the usual characteristics, whether the patient is diabetic or not. This explains why reconstructive vascular surgery is rarely possible but this, nevertheless, remains the best chance for obtaining healing of trophic disorders with limited removal of the toes. No failure was observed in cases of axillo-femoral or femoro-femoral by-pass. But, as in diabetics, one may, in elderly patients with gangrene, attempt to conserve weight-bearing areas thanks to medical treatment in association in some cases with lumbar sympathectomy when the patient's general condition makes this possible. The mortality is low when patients are selected for operation and depending on the surgical technique adopted, e.g. sub-cutaneous by-pass or surgery of the origin of the profunda femoris. The mortality is less than 7 p. 100 during the first month (one death in 31 vascular operations, one death in 40 sympathectomies), but the mortality during the first 6 months exceeds 20 p. 100 due to cardiac, or cerebral complications or urinary infection. The prevention of digestive symptoms, nutritional, renal or urinary complications, and electrolyte disturbances is essential together with physiotherapy and reduction in time spent in hospital, for example, the patient may be sent home and treated by the district nurse. 135 patients with arteritis, including 48 diabetics, aged over 70 years, admitted to hospital in 1971 at the Saint-Joseph hospital, illustrate these findings. 109 patients with 114 diseased limbs, had gangrene or ischemic ulcer. Out of 80 survivors, beyond 6 months, i.e. 84 limbs, in 75 cases the weight bearing areas were preserved, in 9 cases amputation was necessary but in 8 of these it was possible to carry out amputation below the knee and walking was possible. These functional results justify attempts to maintain weight bearing areas or, at least, the knee, whatever the duration of healing or amputation of a toe or of the metatarsus; in Syme's amputation of the leg, healing requires 2 to 4 months.


[Post-traumatic arterial aneurysms and arteriovenous fistulas].

The authors report 49 cases of arterial pseudo-aneurysm or post-traumatic arterio-venous fistula of the limbs, neck and face. Operations were carried out as early as possible without observing the usual 3 months delay. Restoration of arterial continuity was always attempted in the common carotid artery and the main limb vessels. Out of 35 attempts, vascular permeability was restored definitely in 22 cases, doubtfully (comparatively poor pulse) in 4 cases. In 7 cases, the functional result was good in spite of the absence of permeability of the reconstructed vessel. The series studied included one death due to arterio-venous fistula at the base of the neck, one case of partial distal gangrene and one case of secondary hemorrhage which was cured by ligature. The 14 cases of distal or collateral aneurysm were treated by resection and ligature without any post-operative complications. The proximity of the nerves adds to the difficulty of this form of surgery in the axilla, at the root of the thigh and in the parotid area.


[Occupational osteopathy of rug weavers. Apropos of 64 cases].

Carpet weaving carried out by young girls in abnormal positions for long periods, causes in the latter important bony deformities, aggravated by poor hygiene and diet. Prevention and treatment is, however, possible.


[A case of anterior tibial arteriovenous fistula after closed fracture of the leg].

This case permits one to emphasize the great rareness of arteriovenous fistula after closed fractures of the shaft of the tibia. Fistulas in this anterior tibial position are remarkably latent, cause no symptoms below the fistula nor symptoms of heart failure. Treatment by several ligatures, permitted this patient to return to work 15 days after operation without any further treatment. The patient no longer has any symptoms.


[Post-cholecystectomy pseudo-aneurysm. Spontaneous regression demonstrated by angiography].

The authors report the case of a pseudo-aneurysm of the right hepatic artery following a difficult cholecystectomy in a patient with sub-acute pancreatitis. Angiography, two years later, showed spontaneous regression of the pseudo-aneurysm and the development of chronic pancreatitis.


[An original technic of reimplantation of the papilla after accidental disinsertion during gastroduodenectomy for duodenal ulcer].

During accidental disinsertion of the papilla during surgery of duodenal ulcer, an isolated, pediculated loop of the small intestine was interposed between the head of the pancreas and the second part of the duodenum.


Classification of secondary amenorrhea based on distinct hormonal patterns.

Patients with secondary amenorrhea have been classified into 4 clincal groups. In order to further investigate these 4 groups, LH, FSH, and estradiol (E2) were measured at 15 min intervals for 4 h in 21 patients with secondary amenorrhea. Patients within each group had similar hormonal patterns, but there was a distinct variation among the groups. Three patients in group 1 (polycystic ovaries [PCO]) had elevated basal levels of only LH with marked irregular fluctuations. Seven patients in group II (hypothalamic-pituitary dysfunction) had normal basal levels of LH, FSH, and E2. Only LH showed oscillations of varying mahnitude and frequency. Eight patients in group III (hypothalamic-pituitary failure) had low or low-normal levels of LH, FSH, and low E2 with minimal or absent fluctuations. Three patients in group IV (ovarian failure) had high basal levels of FSH and LH and irregular fluctuations. This study confirmed the rationality of separating patients with secondary amenorrhea into 4 different groups. In addition, it was found that in group III patients, the total amount of LH secreted in a 4-hour period of time appears to be insufficient to stimulate E2 production from the ovary even when a single sample was found to be in the normal range.


Iodine-induced thyrotoxicosis in apparently normal thyroid glands.

Although iodine-induced thyrotoxicosis was reported to occur in patients with obvious underlying thyroid disorders, it is not known to occur in patients with apparently normal thyroid glands. From ten such cases evidence is presented that thyrotoxicosis: a) appeared during treatments by iodide or organic-iodine-containing drugs, in the absence of any past history of thyroid disorder; b) was accompanied by almost undetectable radioidine uptake which nevertheless could be activated by TSH; c) subsided spontaneously within a few weeks or months after stopping the high intake of iodine; d) and left, after a period of hypothyroidism, an apparently normal thyroid gland which had resumed normal size, function, uptake, and suppressibility.


Stimulation of the secretion of parathyroid hormone during hypoglycemic stress.

The effect and the mechanism of insulin-induced hypoglycemic stress on parathyroid hormone (PTH) secretion in man was evaluated. Porcine crystalline insulin (0.1 U/kg,iv) caused symtomatic hypoglycemia in all subjects. The maximum hypoglycemia occurred between 25 and 35 min following the insulin administration. A significant increase in the serum PTH concentration occurred promptly at 5 min following the time of maximum hypoglycemia, achieving peak levels between 5 and 15 min. Thereafter the serum PTH concentration gradually declined, reaching baseline values of 60 min. Serum cortisol levels were increased, but not until 30 min following the maximum hypoglycemia, and remained significantly elevated at 60 min. Therefore the stimulation of PTH secretion was not caused by elevated levels of serum cortisol. The serum calcium and magnesium levels remained unchanged throughout the procedure. A significant and sustained decline in the serum inorganic phosphate levels occurred following insulin administration. The results indicate that hypoglycemic stress stimulates PTH secretion, presumably via increased epinephrine levels, and further suggest that adrenergic stimuli play an important role in the physiology of PTH secretion.


Kinetics and interconversion of prednisolone and prednisone studied with new radioimmunogassays.

To determine the kinetics of metabolism and interconversion of prednisolone and prednisone, the plasma levels of these synthetic glucocorticoids were measured by new radioimmunoassays (RIA) after intravenous (iv) or oral administration to normal humans. Following the iv injection of prednisolone phosphate in 8 normal subjects, the mean half-time of prednisolone was 240+/-20 min (mean+/-SE) and the metabolic clearance rate was 82+/-7.2 1/24 h/m2. Of 10 mg of prednisone given orally to 5 normal subjects, 69+/-5% was absorbed and converted to peripheral plasma prednisolone within 8 h. The data indicate that prednisone is extensively metabolized to prednisolone before it clears the splanchnic circulation and the formation prednisone by oxidation of the 11-hydorxyl group of prednisolone appears to occur mainly in peripheral tissues.


Clinical and endocrinologic characterization of a patients with the syndrome of incomplete testicular feminization.

A 46 XY individual with male pseudohermaphroditism was investigated. The phenotype was distinctive in that the habitus was female in character, but partial fusion of the labioscrotal folds, testes, and male wolffian duct structures that terminated in the vagina were present. Müllerian structures were absent. At the expected time of puberty both feminization (breast development) and virilization (clitoral enlargement) took place. Studies of estrogen and androgen dynamics revealed plasma testosterone levels and production rates characteristic of normal men. Plasma estrogen levels and production rates were greater than those of normal men. Plasma gonadotropin levels were also high. These findings suggest that the fundamental defect in this patient is androgen resistance rather than defective androgen synthesis. Dihydrotestosterone formation from testosterone slices of epididymis and perineal skin was normal. The family history was uninformative. On endocrinologic, genetic, and phenotypic grounds the syndrome of incomplete testicular feminization can be separated from the complete form of testicular feminization and from familial incomplete male pseudohermaphroditism, Type 2. Additional studies will be required to determine whether this disorder is also distinct from the Type 1 form of familial incomplete male pseudohermaphromditism.


Effect of spironolactone on sex hormones in man.

Administration spironolactone at a dosage of 400 mg/day to healthy male volunteers for 5 days resulted in a significant rise in plasma progesterone and 17alpha-hydroxyprogesterone which persisted throughout the study. A transient increase in plasma FSH and LH concentration was observed after the second but not the third or fifth days of drug administration. There was no change in plasma concentration of testosterone, 17beta-estradiol, or prolactin. These findings are consistent with a previously-reported spironolactone-induced destruction of the microsomal enzyme cytochrome P-450, an enzyme necessary for 17-hydroxylase and desmolase activity. The results do not explain the decrease of libido, the impotence, and the gynecomastia frequently associated with spironolactone therapy in males.


Interference of spironolactone in 11-doexycorticosterone radioassays.

Uniformly elevated plasma 11-deoxycorticosterone (DOC) values are obtained in patients receiving spironolactone. This elevation is shown to be spurious, and is a result of cross-interference of spironolactine and its metabolite, canrenone, in both the DOC radioimmunoassay and competitive protein-binding radioassay.


Altered blood androgens in elderly men with prostate hyperplasia.

Plasma testosterone (T) and dihydrotestosterone (DHT) were measured in the plasma of otherwise healthy men ages 60-90 who had prostate hyperplasia. The androgens were measured by specific radioimmunoassays using paper and celite column chromatography. In the elderly subjects, plasma T was 466+/-35 (SE) ng per 100 ml which is reduced (p less than 0.05) as compared with values from younger men. In contrast, DHT levels were elevated when compared with values from men ages 20-39, 89 (53-152) versus 49 (33-74) 95% confidence ng per 100 ml. Analysis of data by the Mann Whitney U test demonstrates that the groups are different (p less than 0.001). These studies indicate that DHT levels and T/DHT ratios are altered in unstressed elderly men with BPH. Further study is necessary to delineate the source of DHT and whether these altered androgen levels are the cause of prostate hypertrophy or an unrelated effect of aging.


Effects of morphine on serum growth hormone, cortisol, prolactin and thyroid stimulating hormone in man.

Morphine administration in man results in a significant increase in serum prolactin without altering the levels of growth hormone, thyroid stimulating hormone and cortisol. Apomorphine prevented the morphine induced prolactin rise. It is suggested that the effect on prolactin is not mediated via non specific stress or changes in the thyroid stimulating hormone releasing factor, but via suppression prolactin inhibiting factor or activation of a specific prolactin releasing factor.


Cellular elongation under the influence of antibacterial agents: way to differentiate coccobacilli from cocci.

Representatives of Moraxella, Acinetobacter, and various other groups of short, gram-negative bacilli are readily distinguished from Neisseria by microscopic observation of filaments produced by the rods during growth in the presence of low concentrations of penicillins or sulfadiazine. Wet mounts of bacteria from routine antibiotic susceptibility test cultures are satisfactory for examination of morphology.


Isolation and characterization of a lactose-positive strain of Proteus morganii.

The characterization of a lactose-positive Proteus morganii is presented. The increased incidence of such atypical microorganisms emphasizes the need for a thorough examination of Escherichia coli-like colonies by clinical microbiologists.


Comparison of three procedures for biochemical testing of anaerobic bacteria.

The Analytab Products, Inc. (API), anaerobic multitest microsystem (MICRO) was compared with the Center for Disease Control conventional (CONV) thioglycolate (supplemented with hemin and vitamin K1) system and with pre-reduced anaerobically sterilized (PRAS) media as recommended by the Virginia Polytechnic Institute. Growth from a solid medium was suspended to produce standard inocula. Substrates included 16 carbohydrates, indole, urea, gelatin, and esculin. API strips were inoculated in air and incubated in GasPak (BBL) jars. MICRO tests were read at 1 and 2 days. CONV tests at 1, 2, and 7 days, and PRAS tests at 3 weeks. One hundred thirty well-characterized strains of anaerobes (76 gram-negative rods, 16 cocci, 26 gram-positive nonsporeforming rods, and 12 clostridia), including 48 reference strains, were studied. Of 2,600 tests performed, 2,085 (80.2%) showed agreement with all three methods. There was 90.9% agreement between the MICRO and CONV, 84.9% between the MICRO and PRAS, and 84.6% between the CONV and PRAS tests. All MICRO tests were reliable except for indole, which was not sensitive enough, and gelatin, which was very insensitive. The MICRO system permits performance of biochemical tests at the workbench in the average clinical laboratory without the need for expensive equipment and time-consuming procedures.


Practical aerobic membrane filtration blood culture technique: clinical blood culture trial.

During the course of preliminary clinical trials of an improved membrane filter blood culture system, filter plugging produced by a gelling of the lysing solution was observed when the patients had high leukocyte counts. A solution of streptokinase-streptodornase (Varidase, Lederle Laboratories) dissolved or prevented the gel and permitted rapid filtration without plugging. With streptokinase-streptodornase incorporated in the filtration procedure, a comparison of several culture systems was carried out on 176 blood cultures. Brucella broth with and without sodium polyanethol sulfonate, a prereduced osmotically stabilized broth, pour plates, and an improved aerobic membrane filter system were compared. The membrane filter system yielded 29 of the total of 37 positive cultures, far surpassing all other systems. Eight of these cultures were detected first by the filter technique, and 13 were positive only in this system. Nineteen of the 37 positive cultures were from patients on antimicrobial agents. Fourteen of these were detected by the filter, twice the number detected by any of the other systems.


Presumptive speciation of Streptococcus bovis and other group D streptococci from human sources by using arginine and pyruvate tests.

A simplified method for speciation of group D streptococci is described. A total of 4,156 streptococcal isolates from human clinical material was tested for ability to hydrolyze esculin in the presence of 40% bile, ferment pyruvate, hydrolyze arginine, and grow in media containing 40% bile or 6.5% NaCl. Streptococci which hydrolyzed esculin in 40% bile, but which did not hydrolyze arginine, were also tested for their ability to ferment raffinose or sorbose. Sixty percent (2,503) of the isolates hydrolyzed esculin in the presence of 40% bile and were thus presumptively identified as group D. By application of the other criteria, 84% of these were speciated as Streptococcus faecalis, 7% were speciated as S. faecium, 6% were speciated as S. bovis, 2% were speciated as S. avium, and 1% were not identified. This scheme was shown to be both reliable and practical for use in the diagnostic laboratory. S. avium and S. bovis isolates were characterized, and 18 S. bovis isolates from patients with bacterial endocarditis were compared physiologically with 151 isolates of this species from other sources.


Recognition of Pseudomonas pickettii in the clinical laboratory: biochemical characterization of 62 strains.

Pseudomonas pickettii strains were studied to determine the characteristics essential for their identification in the clinical microbiology laboratory. Preliminary investigations indicated that these glucose-oxidizing, denitrifying, gram-negative rods were quite similar to an unclassified group of clinical isolates designated VA-2. Gas liquid chromatography of trimethylsilyl derivatives of whole cell hydrolysates of P. pickettii and VA-2 strains yielded nearly identical elution profiles. The VA-2 cultures were concluded to be probable strains of P. pickettii. A method is presented for differentiating cultures of P. pickettii from other similar bacteria encountered in clinical specimens.


Evaluation of blood culture media supplemented with sucrose or with cysteine.

A total of 5,883 blood samples from patients with suspected bacteremia were inoculated concurrently into each of three media under vacuum with CO2: tryptic soy broth (TSB) with sodium polyanetholesulfonate (SPS), TSB with SPS and cysteine, and TSB with SPS and sucrose. There were 395 positive cultures, excluding presumed contaminants. No significant differences were noted with the addition of cysteine to TSB with SPS, and no streptococcal mutants requiring thiol groups were isolated. Haemophilus, Staphylococcus aureus, and bacteriodaceae were isolated more frequently (P less than 0.05) in the absence of sucrose. The addition of sucrose to TSB containing SPS did not significantly increase the rate of positivity or the time interval to detection of positivity of any group of bacteria.


Purification of Japanese encephalitis virus vaccine by zonal centrifugation.

A large-scale procedure for purification and concentration of Japanese encephalitis virus vaccine by a continuous-flow isopycnic banding technique in a sucrose density gradient solution, using a K-III zonal centrifuge rotor, is presented. The quality of zonal-purified vaccine was compared with commercial and Japanese National Institutes of Health reference vaccines for antigenicity, immunodiffusion, and allergic encephalitogenicity tests to show its high purity.


Rapid method to detect rubella immunoglobulin M and immunoglobulin A antibodies.

Immunoglobulin (Ig) G was removed from serum specimens by precipitation with gamma chain-specific anti-human IgG of rabbit origin. The remaining rubella virus-specific IgM (and IgA) antibodies were then detected by the rubella hemagglutination-inhibition test. This procedure has proven to be as reliable as estimations carried out with IgM fractions separated on a sucrose density gradient.


Biotypes of Proteus rettgeri.

Examination of 729 isolates of Proteus rettgeri showed that 674 reacted positively in tests for phenylalanine deamination, indole production, growth on citrate, and acid production from meso-inositol, and negatively for L-ornithine decarboxylation and acid production from lactose, maltose, D-xylose, and L-arabinose. Only 51 isolates differed in one, and four differed in two of these ten reactions, which were taken as the core characteristics of the species. On the basis of additional tests (acid production from salicin, L-rhamnose, D-mannitol, adonitol, and D-arabitol), the 729 isolates could be separated into five groups. Goups 1, 2, 3, and 4 could be further separated on the basis of the reaction with meso-erythritol, and group 5 could be subdivided on the basis of reaction with D-mannitol. Two metabolically distinct kinds of P. rettgeri were recognized. Isolates of the first kind (groups 1, 2, 3, and 4) each utilized both adonitol and D-arabitol and most utilized meso-erythritol. Isolates of the other kind (group 5) were negative with the three polyhydric alcohols but resembled, in their reactions, some strains of Providencia stuartii. These may be intermediates between P. rettgeri that catabolize these substrates and the Providencia.