Humoral immunity aspects of meningococcal infection. I. A method of performing and the characteristics of the immune bacteriolysis reaction]

Immune bacteriolysis test with meningococcus, group A, was used for the purpose of serum antibody study. Meningococcus cultures with a bright orange fluorescence of the colonies in oblique illumination (the I type) proved to possess the greatest lysability. Guinea pig serum sorbed with meningococcus suspension was found to be the best source of the complement. Sera obtained after 1 to 3 days of rabbit immunization, containing mostly IgM antibodies, had the greatest bactericidal capacity. Only those fractions which contained IgM possessed bactericidal activity in the hyperimmune rabbit sera with a high IgG antibody concentration. No lytic activity was displayed against meningococcus by unfractionated hyperimmune sera.     

Staining method of poly(3-hydroxyalkanoic acids) producing bacteria by nile blue

Summary Using an ethanol solution of nile blue, we have developed an efficient method to detect the colonies of poly(3-hydroxyalkanoic acids) (PHA) producing bacteria on the agar plate. When the bacterial colonies with PHA granules were stained with nile blue, the stained colonies fluoresced bright orange on the irradiation of UV light. In the fluoresce emission spectra, fluorescence intensity increased with an increase in the PHA content of bacterial cells.Alcaligenes eutrophus andA.latus colonies with poly(3-hydroxybutyric acid) (PHB) homopolymer exhibited an emission maximum at 580nm on the excitation at 490nm. On the other hand,Pseudomonas oleovorans andP.putida with medium-chain-length (mcl-) PHA copolymers of C6, C8 and C10 units exhibited an emission maximum at 570nm.     

Traffic lights in trichodesmium. Regulation of photosynthesis for nitrogen fixation studied by chlorophyll fluorescence kinetic microscopy

We investigated interactions between photosynthesis and nitrogen fixation in the non-heterocystous marine cyanobacterium Trichodesmium IMS101 at the single-cell level by two-dimensional (imaging) microscopic measurements of chlorophyll fluorescence kinetics. Nitrogen fixation was closely associated with the appearance of cells with high basic fluorescence yield (F(0)), termed bright cells. In cultures aerated with normal air, both nitrogen fixation and bright cells appeared in the middle of the light phase. In cultures aerated with 5% oxygen, both processes occurred at a low level throughout most of the day. Under 50% oxygen, nitrogen fixation commenced at the beginning of the light phase but declined soon afterwards. Rapid reversible switches between fluorescence levels were observed, which indicated that the elevated F(0) of the bright cells originates from reversible uncoupling of the photosystem II (PSII) antenna from the PSII reaction center. Two physiologically distinct types of bright cells were observed. Type I had about double F(0) compared to the normal F(0) in the dark phase and a PSII activity, measured as variable fluorescence (F(v) = F(m) - F(0)), similar to normal non-diazotrophic cells. Correlation of type I cells with nitrogen fixation, oxygen concentration, and light suggests that this physiological state is connected to an up-regulation of the Mehler reaction, resulting in oxygen consumption despite functional PSII. Type II cells had more than three times the normal F(0) and hardly any PSII activity measurable by variable fluorescence. They did not occur under low-oxygen concentrations, but appeared under high-oxygen levels outside the diazotrophic period, suggesting that this state represents a reaction to oxidative stress not necessarily connected to nitrogen fixation. In addition to the two high-fluorescence states, cells were observed to reversibly enter a low-fluorescence state. This occurred mainly after a cell went through its bright phase and may represent a fluorescence-quenching recovery phase.     

Immunochemistry of the Cell Walls of Listeria monocytogenes

The antigenic specificity of Listeria monocytogenes types I, II, III, IVa, and IVb was studied by immunochemical techniques. Immunologically active carbohydrates of the various types were extracted from cell walls and were chemically analyzed. Types I and II contained predominantly glucosamine and rhamnose; type III, galactose, rhamnose, and glucosamine; and types IVa and IVb, glucose and galactose. Quantitative precipitin inhibition tests with purified monosaccharides indicated that the major antigenic determinant of types I and II is rhamnose. Precipitin reactions could not be detected with type III carbohydrate and homologous or heterologous antisera. The major determinants of types IVa and IVb were found to be galactose and glucose, respectively. As much as 87% inhibition of the quantitative precipitin test for types I and II was obtained with rhamnose, 72% for type IVa with galactose, and 72% for type IVb with glucose. The immunochemical basis for the antigenic specificity of L. monocytogenes types I, II, IVa, and IVb was further confirmed by using agar gel diffusion. Cross-reactions among the various type-specific carbohydrates and heterologous antisera were also studied. Type II carbohydrate was found to contain galactose and react with type IVa antisera. This reaction could be blocked by galactose. Type I carbohydrate did not contain galactose nor did it react with antiserum prepared from type IVa cells. Therefore, the somatic antigens of type I and type II L. monocytogenes, previously thought to be identical, appeared to differ. The dominant immuno-specific group in the cross-reaction between type IVb carbohydrate and type IVa antisera was found to be galactose. Type IVa absorbed antisera did not produce a significant cross-reaction with type IVb carbohydrate. The results obtained from this investigation indicate a lesser degree of antigenic relationship between type IVa and type IVb L. monocytogenes than was previously believed to exist.     

NADH-dehydrogenase reaction in combination with immunoperoxidase (PAP) staining for light microscopic observation on the interneuronal relations of the enteric nervous system of the pig

A novel procedure for a simultaneous demonstration of particular enteric nerve cell types and peptidergic nerve fibres has been developed by combining the histochemical reaction for NADH-dependent dehydrogenase and the unlabelled antibody peroxidase-antiperoxidase (PAP) method described by Sternberger. Whole-mount spreads were successively incubated in a NADH: nitroblue tetrazolium solution, fixed with a picric acid/formaldehyde mixture, dehydrated, cleared and rehydrated before processing for immunocytochemical localization of the neuropeptide by the PAP method. The nerve cells appear heavily stained by deposits of dark blue formazan, whereas the peptide-containing nerves appear bright brown. In the myenteric and submucous plexuses of the porcine small intestine the devised method allows an appropriate identification of Dogiel's type I, type II and type III neurons surrounded by varicose enkephalin-like immunoreactive fibre baskets with button-like twigs to the very surface of the ganglionic cells, suggestive of synaptic connections.     

Distinction of the two type I coppers in bovine ceruloplasmin

Redox potentials of the two type I copper ions, ‘blue copper ions’, of bovine ceruloplasmin (ferroxidase, iron(II): oxygen oxidoreductase, EC 1.16.3.1) were determined to be 370 and 390 mV (vs. NHE). These two type I copper ions were clearly differentiated during the anaerobic reduction process of oxidized ceruloplasmin and the reoxidation process of completely reduced ceruloplasmin by using absorption, circular dichroic and electron paramagnetic resonance spectroscopies. One of the blue copper ions is reduced faster and reoxidized very slowly, and is assumed to be located away from the active site of ceruloplasmin. On the other hand, the other blue copper ion, which is reduced more slowly and reoxidized rapidly, is supposed to interact with other types of coppers, such as type II (non-blue) and type III (EPR undetectable) coppers. The active site of ceruloplasmin is considered to be comprised of one type I, one type II and a pair of type III copper ions.     

Expression of mRNAs for collagens and other matrix components in dedifferentiating and redifferentiating human chondrocytes in culture

Cell cultures were initiated from epiphyseal cartilages, diaphyseal periosteum, and muscle of 16-week human fetuses. Total RNAs isolated from these cultures were analyzed for the levels of mRNAs for major fibrillar collagens, two proteoglycan core proteins and osteonectin. In standard monolayer cultures the differentiated chondrocyte phenotype was replaced by a dedifferentiated one: the mRNA levels of cartilage-specific type II collagen decreased upon subculturing, while those of types I and III collagen, and the core proteins increased. When the cells were transferred to grow in agarose, redifferentiation (reappearance of type II collagen mRNA) occurred. Fibroblasts grown from periosteum and muscle were found to contain mRNAs for types I and III collagen and proteoglycan cores. When these cells were transferred to agarose they acquired a shape indistinguishable from chondrocytes, but no type II collagen mRNA was observed.     

Ultrastructural study on long-term cultures of bone marrow cells with histamine-producing stimulating factor (HCSF)

In a previous study (Dy et al. 1981) we have demonstrated that histamine-producing cell stimulating factor (HCSF), a lymphokine released by T-cells, is present in supernatants obtained from secondary mixed lymphocyte cultures set up with cells from the donor and the recipient of a skin allograft, and that HCSF causes an increased production of histamine from target cells present in bone marrow. The most abundant source of target cells was found in the less dense layer of a discontinuous Ficoll gradient of bone marrow cells. Ultrastructural studies of this layer showed that it is composed of four types of cell: type I, immature cells; type II, mastocyte-like cells; type III, macrophages and type IV, lymphocytes. We have examined the effect of HCSF on this cell population; in long-term cultures we observed a progressive numerical decrease in type I cells, accompanied by an increase in type II cells (clearly observed as early as 48 h), leading to a pure population of mastocytes cells after 45 days of culture.     

Compartmentation and characterization of different proteoglycans in bovine arterial wall

Proteoglycans stained specifically with cuprolinic blue have been visualized in electron micrographs of bovine arterial tissue. Three differently sized proteoglycan-cuprolinic blue precipitates, designated as types I, II, and III, could be detected in the extracellular matrix. The precipitates could be distinguished by their length, width, area, topographical distribution, and their characteristic association with other matrix components. By taking into account the available biochemical data and the individual susceptibilities of the precipitates towards specific glycosaminoglycan-degrading enzymes, each type of proteoglycan-cuprolinic blue precipitate could be attributed to a proteoglycan population containing dermatan sulfate, chondroitin sulfate, or heparan sulfate as its main glycosaminoglycan component.     

Variation of cytosine methylation in 57 sweet orange cultivars

Sweet orange is an important group of citrus cultivars, which includes a number of bud sport cultivars. Little is known about the CpG methylation status of the CCGG sequences in the orange genome. In this study, methylation-sensitive amplification polymorphism (MSAP), based on the application of isoschizomers (Hpa II and Msp I), was first used to analyze cytosine methylation patterns in 57 orange cultivars that were not fully differentiated by regular DNA molecular markers. Three types of bands were generated from ten primer pairs. Type I bands were present following restriction with Eco RI + Hpa II and Eco RI + Msp I; type II or type III were present only following restriction with either Eco RI + Hpa II or with Eco RI + Msp I. The total number of these three types of bands was 802, 72, and 157, respectively. Among these, the number of polymorphic bands were 244 (30.2%), 23 (31.9%), and 32 (20.4%), in type I, II and III, respectively. The methylation patterns of these 57 cultivars are discussed and assessed by dendrograms derived from the analysis of polymorphic MSAP bands. The distribution of polymorphic bands of the above three types demonstrate the methylation patterns and frequency at the cytosine loci. We suggest that methylation events could be more frequent than demethylation events, and that the methylation patterns maybe associated with phenotypic traits.     

Fluorescence ‘off–on’ probe for lead (II) detection based on Atractylodes III CQDs and bioimaging

In this work, a type of carbon quantum dots (CQDs) with bright blue emission was readily fabricated through one-step hydrothermal treatment from Atractylodes III. We explored the surface morphology and optical properties of the CQDs using transmission electron microscopy, X-ray diffraction patterns, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy and ultraviolet–visible light spectrophotometry. The obtained CQDs possessed good photoluminescence properties, water solubility, and biocompatibility. The fluorescence quantum yield of these was 3.72%. It was found that the fluorescence intensity of CQDs was quenched by picric acid. After adding lead (II), the fluorescence could be effectively recovered. Therefore, an ‘off–on’ fluorescence probe was designed to detect lead (II) in the range 0–580 μM and the limit of detection was 0.068 μM. In addition, the experiments showed that the CQDs could be successfully used in bioimaging and as a hidden fluorescent ink.     

Three types of mouse peptidylarginine deiminase: characterization and tissue distribution.

Three types of mouse peptidylarginine deiminase were separated by DEAE-Sephacel ion-exchange column chromatography, and we propose designating them peptidylarginine deiminase type I, II, and III according to the order of elution. The type II enzyme was widely distributed in various tissues including the skeletal muscle, whereas the type I enzyme was localized in the epidermis and uterus, and the type III enzyme was detected in the epidermis and hair follicles. These enzymes were distinguished by their molecular weights and substrate specificity. The molecular weights were estimated to be approximately 54,000 (type I) and 100,000 (type II and III) by Sephacryl S-200 gel filtration column chromatography. On SDS-PAGE the type II and III enzymes gave Mr = 81,000 and Mr = 76,000, respectively. Among the substrates tested, the type I enzyme showed highest activity toward BZ-L-Arg-NH2, type II toward BZ-L-Arg-O-Et, and type III toward protamine. Western blot analysis showed that antibodies against the type II enzyme were immuno-crossreactive to the type III enzyme.     

Short pulses of antigen induce in vitro an antibody response to haptenic determinants

The cellular composition of the mouse thymus has been analysed at different ages and in different strains by using size distribution analysis in combination with preparative cell electrophoresis and bovine serum albumin (BSA) gradient centrifugation. It was possible to distinguish three major subpopulations of small lymphocytes: cell type I is small, dense and electrophoretically slow, cell type II is intermediate by all three parameters, and cell type III has the largest volume, lowest buoyant density and highest electrophoretic mobility.Cell type III was enriched in animals treated with cyclophosphamide and was practically the only cell type found in the thymus of hydrocortisone-treated mice. The data thus show that the increase of the average size of thymus cells after hydrocortisone treatment reported previously is due to a shift in relative proportions of distinct cell types with different size rather than due to a drug-induced enlargement of individual cells. The larger cell type III resides probably in the thymic medulla and carries graft-versus-host (G.v.H.) reactivity as well as other typical T cell functions. Possible functions of the smaller, probably cortical, cell types are discussed.Newborn mice were found to contain only cell types II and III, 4-week-old CBA contained I, II, and III, and adult mice were found to contain only cell types I and III.The two-dimensional distribution patterns (“finger prints”) in respect to size and electrophoretic mobility appeared to be typical for the three cell types irrespective of the age or strain of the mice tested. These physical parameters, therefore, provided relatively constant markers for the identification and characterization of distinct cellular subpopulations in the thymus. Each of these subpopulations is probably in itself heterogenous in respect to antigen specificity. It is proposed to call lymphocytes with different antigen specificity but identical physical characteristics “isotypic lymphocytes.”     

A Study on Pollen Morphology of the Genus Viburnum from China

The present study on pollen grains of the genus Viburnum Linn. Shows that: 1. The pollen characteristics are of no significance in division of sections, but each species has its own pollen characteristics. 2. The exine can be divided into three types: I. Exine semitectate, reticulate, muri psilate; II. Exine semitectate, reticulate, muri verrucate; III. Exine intectate, retipilate or pilate, the heads of pila verrucate. The evolutionary trend of the exine is III→II→I. 3. Four types of colpus margin are recognized: I. intectate; II. The colpus margin semitectate, reticulate; III. The colpus margin tectate; IV. Syncolpate at one pole, the margin tectate (only in V. farreri). 4. The ora can be divided into five types according to its membrane shape under SEM: I. The membrane of ora indistinct; II. The membrane of ora semispherical, discontinuous with colpus margin; III. The membrane of ora semispherical, continuous with colpus margin; IV. The membrane of ora semi-ellipsoidat, lolongate; V. The membrane of ora semi-ellipsoidal, lalongate, across the colpus like a bridge. 5. According to both pollen characters and inflorescence, the authors suggest that Sect. Pseudotinus be divided into two subsections, one including V. sympodiale, V. furcatum and V. latanoides, which have large sterile marginal flowers with the exine belonging to the Type I, and the other including only V. nervosum, which has no large sterr-ile marginal flowers with the exine belonging to the Type III.     

A mutant of Escherichia coli which accumulates large amounts of coproporphyrin.

A mutant of Escherichia coli which accumulates a large amount of coproporphyrin, presumably because of a block in heme biosynthesis, has been isolated after nitrosoguanidine mutagenesis. On rich media, the mutant forms colonies which give bright orange fluorescence when illuminated with ultraviolet light. The mutant appears to be similar to a Salmonella typhimurium mutant, deficient in uroporphyrinogen III cosynthase, described by Sasarman and Desrochers ((1976) J. Bacteriol. 128, 717--721). A striking property of the mutant is that coproporphyrin is retained within the cells in rich media but is almost totally excreted out of cells in minimal glucose medium.     

Stimulation of collagenase production by collagen in mammalian cell cultures.

In this study, we investigated the possible regulatory role of collagen in collagenase production by cultured human skin fibroblasts and human and rabbit synovial cells. Addition of types I, II or III collagen in solution to the culture media markedly stimulated trypsin-activable collagenase activity in these cultures. In the human cell cultures the stimulatory effect of collagen was further enhanced by a soluble factor isolated from human monocyte culture media (Dayer, Russell and Krane, 1977). Both native and denatured forms of collagen stimulated enzyme production; their relative efficacy varied among the different types. The native form of both types I and II collagen showed a greater effect on collagenase production than the corresponding denatured form, whereas with type III collagen the denatured form was more effective.     

Contrast in the Photoelectric Effect of Organic and Biochemical Surfaces: A First Step towards Selective Labeling in Photoelectron Microscopy

The photoelectric effect can provide the physical basis for a new method of mapping organic and biological surfaces. The technique, photoelectron microscopy, is similar to fluorescence microscopy using incident ultraviolet light except that photoejected electrons form the image of the specimen surface. In this work the minimum wavelengths of incident light required to produce an image were determined for the molecules 3,6-bis(dimethylamino)acridine (acridine orange) (I), benzo[a]pyrene (II), N,N,N′,N′-tetraphenylbenzidine (III), and copper phthalocyanine (IV). The photoelectron image thresholds for these compounds are 220 (I), 215 (II), 220 (III), and 240 nm (IV), all ±5 nm. Contrast of I-IV with respect to typical protein, lipid, nucleic acid, and polysaccharide surfaces was examined over the wavelength range 240-180 nm. The low magnification micrographs exhibited bright areas corresponding to I-IV but dark regions for the biochemical surfaces. The high contrast suggests the feasibility of performing extrinsic photoelectron microscopy experiments through selective labeling of sites on biological surfaces.     

THE ROLE OF UNICELLS IN THE POLYMORPHIC SCENEDESMUS ARMATUS (CHLOROPHYCEAE)1

The UTEX 2193 strain of Scenedesmus armatus (Chod.) Chod, when cultured in any of several media (whether natural or artificial, concentrated or dilute) produced a variety of colonial morphologies as well as a unicell population. Morphological expression was related to culture ape. When the initial cell density was just a feu1 hundred cells per mL. the culture first produced a unicell population, then spiny colonies, and as stationary phase was approached, spine-less colonies. Two classes of spiny colonies were detected. Type I colonies had elongate cells with the terminal cells shorter than median cells. Spines were longer than cell length. The wider, oval, grainy cells of Type II colonies were uniform m length. Spines were shorter and thicker than those on Type I colonies. Only Type I colonies produced unicells: the latter appeared as two morphs. The smaller unicell was obovoid with four delicate spines: the larger had ovate cells bearing four thicker spines. Control of unicell development in all media was achieved by carefully monitoring colony type and cell number used for the inoculum. A unicellular population developed in batch culture in defined media, both concentrated and dilute, when the initial cell density (either Type I or Type II colonies) was low (below 1000 cells-mL^?1), as well as in synchronous cultures. With higher initial cell densities, e.g. 2 × 10⁴ cells·mL^?1, the inoculum had to contain Type I colonies to produce unicells. Unicells were also produced in water from Agronomy Pond, where the strain originated. We discuss the role of unicell populations in the distribution of Scenedesmus.     

Distinct subtypes of zona pellucida morphology reflect canine oocyte viability and cumulus-oocyte complex quality

The aim of this study was to analyze surface morphology of the zona pellucida (ZP) and assess its relationship with oocyte viability, cumulus-oocyte complex (COC) quality, and oocyte donor age in dogs. Canine ovaries were sliced to release COCs for use in three experiments. In Experiment 1, oocytes from high-quality (grade I) COCs were viewed with scanning electron microscopy to visualize the zona surface. Four zonae, classified as types I, II, III, and IV, were detectable on high-quality oocytes. Most (95.5%) dog donors had oocytes with two or three ZP types. The ZP type I had a smooth compact surface with few pores. The ZP type II was less compact with many distinct circular or elliptical pores. The ZP type III had a rough surface with folds and many irregular shaped pores and hollows. The ZP type IV also had a rough surface with folds, but in addition, stringy filaments obscured the pores and hollows. The frequency of ZP type I in the oocyte population was low (2.7%), whereas ZP types II, III, and IV each occurred in approximately one-third of the oocyte population. In Experiment 2, oocytes from high-quality COCs were stained with propidium iodide (PI) before scanning electron microscopy to investigate the relationship of oocyte viability with ZP morphology. In Experiment 3, oocytes were collected from low-quality (grade 2) and high-quality (grade 1) COCs to investigate the role of COC quality on zona structure. Zonae types I and II were characteristic of PI-positive (dead) oocytes and oocytes from low-quality COCs, whereas ZP types III and IV were prevalent on PI-negative (living) oocytes and oocytes from high-quality COCs. We concluded that the heterogeneous ZP surface underwent structural rearrangements related to oocyte viability and COC quality. This warrants further investigation into ZP structure and may be useful for canine-assisted reproduction.     

Modulation of types I and III procollagen synthesis at various stages of arterial smooth muscle cell growth in vitro

Collagen synthesis was monitored in cultures of rabbit arterial smooth muscle cells (SMC). Both the rate of collagen synthesis per cell and collagen synthesis as a percent of total protein synthesis were measured at specific intervals from 1 to 14 days after inoculation of smooth muscle cells. The proportions of types I and III collagen present in the conditioned incubation medium and in the cell layer were also examined. After inoculation the cells displayed population expansion typical of SMC in which growth slowed but did not cease after the cells attained confluence. Collagen synthesis rates, expressed as [14C]hydroxyproline per cell, were eight-fold higher in preconfluent cells. In these cultures collagen accounted for more than 20% of the newly synthesized, 14C-labeled protein present as trichloroacetic acid (TCA)-insoluble material in 24 h culture media. In post-confluent cultures, this percentage was reduced to about 7% of the total protein synthesized. Synthesis rates of both collagen and non-collagen protein decreased with increasing time after inoculation. However, the rate of decline of collagen synthesis was three times greater than that seen for non-collagen protein. Early cultures synthesized relatively more type I than type III procollagen. The type I to type III ratio was highest at day 3 and declined after that time to day 14. While the synthesis of both types decreased with increasing age, type I declined at a greater rate resulting in a predominance of type III procollagen secretion by older cultures. We conclude that protein synthesis in general and collagen synthesis in particular are quantitatively and qualitatively dependent upon the growth stage of SMC in vitro.