Demonstration by light and electron microscopy of capsules on gonococci recently grown in vivo.

A study by light microscopy, using Leishman's stain alone or Leishman's stain followed by nigrosin, showed the presence of capsules on gonococci of two strains subcultured from subcutaneous chambers in guinea pigs. With the Alcian blue method of preparation for electron microscopy, gonococci of both strains recently grown in vivo showed densely stained capsules on some cells, while others in the same preparation showed only irregular masses of dense material on their surfaces with strands connecting adjacent bacteria. Treatment with antiserum, complement and conglutinin revealed irregular masses and strands of extracellular material with fixatives that did not contain Alcian blue.     

Selection from gonococci grown in vitro of a colony type with some virulence properties of organisms adapted in vivo.

Gonococci from subcutaneously implanted chambers in guinea pigs produced, on agar, more than 95% small colonies showing a "double highlight" (DH) effect in oblique reflected light combined with transmitted light. Laboratory strains of gonococci produced some DH colonies, but other showed a single highlight (SH) or no highlight (NH). Selection of DH colonies and comparison of their organisms with gonococci grown in vivo and with those from SH colonies, showed that the DH character was associated with high infectivity for guinea-pig chambers, resistance to killing by human phagocytes and heavy pilation. Furthermore, DH colonies were found in the first culture of three fresh samples of urethral pus. Thus, the DH colony characteristic may be a more reliable criterion of pathogenicity of gonococcal isolates than systems used previously. There were, however, some differences between the gonococci grown in vivo and the DH colony types. The gonococci grown in vivo and cultured once on solid medium possessed one or two antigens which differed from those of DH (or SH) colonies. They also formed smooth suspensions (which separated slowly) in saline, compared with the rough suspensions (which separated quickly) formed by gonococci from DH (or SH) colonies. Finally, the organisms grown in vivo were resistant to killing by human serum whereas the DH (and SH) colony types were susceptible; the resistance of the organisms grown in vivo was lost during one subculture on agar suggesting that the property is a phenotypic characteristic. Hence, in addition to selecting DH colony types the conditions in vivo produce organisms which differ, probably phenotypically, from cultured organisms.     

Decrease in adherence of bacteria and yeasts to human mucosal epithelial cells by noxythiolin in vitro

Adherence of buccal and vaginal isolates of Candida albicans to buccal epithelial cells and the adherence of urine isolates of Escherichia coli and Staphylococcus saprophyticus to uroepithelial cells was quantified by light microscopy. The antimicrobial agent noxythiolin reduced the adherence of these micro-organisms in both exponential and stationary growth phases. Adherence of both the blastospore and pseudohyphal forms of C. albicans was reduced. Treatment of epithelial cells and/or micro-organisms with noxythiolin resulted in decreased adherence. No anti-adherence effect was observed with formaldehyde and N-methylthiourea, the degradative products of noxythiolin.     

Glycoproteomic survey of Mammillaria gracillis tissues grown in vitro

The structure elucidation of protein-linked N-glycans in plants has raised interest in the past years due to remarkable physiological roles attributed to these modifications. However, little information about the glycoprotein patterns related to plant cell differentiation, dedifferentiation and transformation is available. In this work, the use of two-dimensional polyacrylamide gel electrophoresis in conjunction with matrix assisted laser/desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) for the characterization of carbohydrates released from plant glycoproteins is described. Proteins from different Mammillaria tissues (shoot, callus, hyperhydric regenerant, and TW tumor) were separated by 2D SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane and incubated with Con A to detect N-glycosylated proteins. To discover if the same protein can have various N-glycan structures depending on the organization status of the tissue, the selected glycoprotein spot, which was common for all investigated tissues, was excised from the gels and digested by PNGase A. The released oligosaccharides were analyzed by MALDI-TOF-MS. The results obtained in this study indicate that the N-glycosylation pattern of the protein is clearly dependent on level of plant tissue organization and can be related to the specific morphogenic status.     

The biosynthesis of intestinal mucins. The effect of salicylate on glycoprotein biosynthesis by sheep colonic and human gastric mucosal tissues in vitro

1. Incubation of sheep colonic mucosal scrapings in Krebs-Ringer buffer for 2(1/2)hr. in the presence of salicylate (15mm) resulted in decreased incorporation of radioactivity into the epithelial glycoprotein from the following labelled precursors: 16.6mum-d-[2-(14)C]glucose (83.9% inhibition), 20mum-l-[U-(14)C]threonine (82%) and (35)SO(4) (2-)(79%). Oxygen uptake measured simultaneously was diminished to 41% of the control value. 2. At lower concentrations of salicylate (e.g. 3.75mm), incorporation of 20mum-l-[U-(14)C]threonine was little affected (3-6% inhibition), whereas utilization of 4mum-d-[U-(14)C]glucose and (35)SO(4) (2-) was inhibited (41-48% and 40-59% of the control values respectively). 3. Analysis of the papain-digested glycoprotein from tissue incubations with 16.6mum-d-[2-(14)C]glucose in the presence of salicylate (3.75mm) showed large decreases in labelling of N-acetylneuraminic acid and N-glycollylneuraminic acid residues (57% and 34% of the control values respectively) and of hexosamine constituents (glucosamine, 55% inhibition; galactosamine, 33% inhibition). Labelling of neutral sugars (galactose and fucose) was relatively little affected (9 and 11% inhibition respectively). 4. Glucose 6-phosphate transaminase and glucosamine 6-phosphate acetylase in particle-free enzyme preparations of the sheep tissue were unaffected by salicylate at the above concentrations. Acetyl-CoA synthetase was markedly inhibited. 5. Human gastric mucosa (from operation), on incubation as above, had in one experiment an oxygen consumption of 9.9mul./hr./mg. dry wt. of tissue and incorporated 5mum-d-[U-(14)C]glucose (15.8% of the total radioactivity added) into bound hexosamine (20.6% of the total radioactivity incorporated), hexoses (glucose and galactose, 5.7%) and fucose (14.2%). The presence of salicylate (15mm) decreased the incorporation of 5mum-d-[U-(14)C]glucose into the glycoprotein by 74%, all sugar constituents being affected, without influence on the rate of oxygen consumption. 6. The results suggest an inhibitory effect of salicylate on glycoprotein biosynthesis at the level of the amino sugar intermediates.     

Capsulation of in vitro and in vivo grown Bacteroides species

By centrifugation on a four step Percoll density gradient cells of Bacteroides species could be separated according to the size of extracellular structure. The difference in size was visible by both light and electron microscopy. Two structures were observed on Bacteroides fragilis by electron microscopy, namely a fibrous network and an electron dense layer. An electron dense layer was visible on Bacteroides ovatus only when stained with ruthenium red. B. fragilis cells grown in the mouse peritoneal cavity did not produce a large fibrous network. An electron dense layer was observed on some cells in the presence of ruthenium red stain and cells possessing this layer were phagocytosed in vivo.     

Factors influencing human leukocyte adherence in vivo

The effects of different factors on leukocyte adherence was evaluated by using human peripheral blood before and after treatment (application). Magnesium ions, anti-inflammatory drugs and dextran have decreased neutrophil adherence. Calcium ions, propranolol, blood transfusion caused a significant decrease in adherence. These findings support the conclusion that the adherence test, which is influenced by numerous factors and whose results had a wide scatter, is not a useful tool in clinical diagnosis.     

Behavior of in vitro grown normal human mucosal epithelial cells and tumorigenic rat cells inoculated into nude mice

The present study describes the behavior of in vitro grown normal human oral mucosal epithelial cells and that of a tumorigenic epithelial cell line following subcutaneous inoculation into nude mice. A successful recovery of viable human epithelial cell inocula was seen in 25-90% of mice and there was no improvement in recovery rates after addition of fibroblasts. These inocula resulted in cyst formation lined by a 2-6 cell layer unkeratinized squamous epithelium without rete ridges. There was no increase in recovery rate or size of cysts when coinoculated with fibroblasts. The tumorigenic cell inocula were successfully recovered in all cases. Tumors established from these inocula had a low grade of differentiation and were without signs of metastasis. Inocula of tumorigenic cells showed an increased size after addition of fibroblasts to the inocula. The model may be useful in studies of interactions between inoculations of heterologous normal and pathologic cells as well as in studies of differentiation of carcinogen-treated epithelial cells.     

Growth and chlorophyll production in plant callus tissues grown in vitro

Summary Growth, nutrition and chlorophyll development were studied in chlorophyllous callus tissues isolated from the following edible angiospermous plants: carrot root, crown gall of tomato, endive embryo, leaf petiole and stem of lettuce, leaf petiole of parsley, pea stem and rose stem. Growth patterns of these tissues in vitro were sigmoid. Synthetic media produced less growth, in terms of fresh weight increase, than media containing coconut milk, a highly complex and little understood natural substance. Murashige and Skoog's synthetic medium proved useful for satisfactory growth and chlorophyll production in a number of tissues. Its usefulness was further increased by additional amounts of copper sulphate, potassium nitrate and monobasic ammonium phosphate. Increased levels of iron and magnesium inhibited growth. Incorporation of yeast extract in the tobacco-high-salts-medium produced the highest amount of growth and chlorophyll formation in endive tissue. Presence of exogenous sucrose was essential for the continued good growth of the above callus tissues in vitro. Highest amount of growth took place either in white light or in the dark. Different tissues had different responses to high or low intensities of light. Endive and carrot tissues produced in vitro were palatable to human taste. Endive tissue was particularly good as it also differentiated many small rosettes of leaves, shoots and had a mild aromatic flavor typical of the endive plants grown in nature.     

Factors influencing human leukocyte adherence in vitro

Studies were performed on factors influencing leucocyte adherence in vitro. Blood condensation was found to increase leukocyte adherence. Addition of heparin, dextran or ethanol caused a significant reduction of white blood cell count in blood samples in comparison with blood mixed with sodium EDTA or ACD solution. This suggests the existence of two granulocyte subpopulations; viz, rapidly adhering and slowly adhering. Heparin enhanced granulocyte adherence, while dextran and ethanol decreased it. Five-day storage of ACD blood led to a decrease in granulocyte adherence, while addition of heparin or histamine to ACD blood prevented this change to occur. The glucose concentration of 1,000 mg/dl augmented granulocyte adherence, while higher glucose concentrations induced its progressive fall below the control values. There was no significant change of lymphocyte adherence during the experiments.     

ATP content of Mycobacterium tuberculosis grown in vivo and in vitro

In order to determine the reason for the slow growth of Mycobacterium leprae either in a host or in vitro, the growth characteristics of Mycobacterium tuberculosis were studied. The ATP content of in vitro-grown M. tuberculosis was about 520 pg/10(6) viable organisms. The ATP levels from in vivo-derived organisms obtained from liver and spleen of mice was about 130 pg (in cases of chronic infection) and about 270 pg (in cases of acute infection). When the in vivo-derived organisms were inoculated into culture medium, the growth rates for both types of organisms, acute as well as chronic infection, were the same and the maximum growth was reached during the fifth subculture. Although the maximum ATP content for both types of organism was the same, it was attained during the 4th subculture for organisms obtained during acute infection and during the 6th subculture for those obtained during chronic infection. The comparison between the ATP content of M. leprae and of M. tuberculosis indicates the reason for the slow growth of M. leprae.     

Nuclear DNA contents of Pisum genotypes grown in vivo and in vitro

Summary The nuclear DNA content of prophase nuclei in root tips of two cultivars and two primitive lines of Pisum sativum and of Pisum fulvum have been determined, using a scanning microdensitometer. The nuclear DNA contents differed significantly between the genotypes investigated but there was no correlation with their supposed phylogenetic positions.A loss of 73% of the DNA from cells of aseptically cultured excised pea roots has been recently reported (Abbott, 1971). In marked contrast to this claim, our measurements of the nuclear 4C DNA content of root tip meristematic cells have shown that there is no significant loss in excised roots compared with attached roots.     

The intracellular survival and growth of gonococci in human phagocytes.

In reassessment of previous tests for intracellular survival, results have been confirmed and additional evidence obtained indicating that some gonococci can survive and multiply in human phagocytes. Use was made of the ability of penicillin to penetrate phagocytes and to kill only actively growing organisms. In microscopic counts on 33 urethral exudate smears, an average of 49% of gonococci were associated with polymorphonuclear phagocytes. The organisms were unevenly distributed amongst the phagocytes, with most cells uninfected and some containing large numbers. Many phagocytes also remained uninfected in tests in vitro with low gonococcal inocula although experiments with large inocula showed that most phagocytes could ingest gonococci. It is proposed that ingestion of one gonococcus may stimulate the phagocytes to take up more. Phagocytes were killed and disintegrated after ingesting large numbers of gonococci and similar effect in vivo may be responsible for the large clumps of organisms seen in urethral exudate. These results underline the probable importance in the pathogenesis of gonorrhoea of intracellular survival in phagocytes.     

Stimulatory effect of honey on multiplication of lactic acid bacteria under in vitro and in vivo conditions

The effect of honey and sucrose on lactic acid bacteria in vitro and in rat gut was studied to determine whether these organisms were affected differently by honey compared with sucrose. Under in vitro conditions, the number of Lactobacillus acidophilus and Lactobacillus plantarum counts increased 10-100 fold in the presence of honey compared with sucrose. Feeding of honey to rats also resulted in significant increase in counts of lactic acid bacteria. Although there was no significant difference in the counts of lactic acid bacteria in the small and large intestines of different groups, the honey-fed group showed a significant increase (P<0.05) in counts over the control and sucrose-fed animals. The results support the fact that consumption of honey has a beneficial effect on the physiological constitution of animals fed with it.