Production of the Shwartzman Reaction with Microbial L Forms

Study of potential pathogenicity of microbial L forms was done by the localized Shwartzman reaction. Stable L forms of Proteus mirabilis served as skin preparation in rabbits for induction of Shwartzman reaction by subsequent intravenous injection of either P. mirabilis L forms or Escherichia coli endotoxin. The intensity of the reaction was positively correlated to numbers of L forms in the skin. L forms also served as the intravenous challenge. In vivo multiplication of L forms was not a prerequisite for the reaction, as it could be produced with nonviable, osmotically lysed L forms. The reaction produced with L forms in the skin was more intense than that produced with the parent bacterial form. These latter observations, coupled with the demonstration that L forms disappeared from the skin (lysed?) after 4 hr, in contrast to bacteria which were recoverable for 72 hr (duration of study), suggest release of endotoxin by L forms as a pathogenic mechanism.     

Quaternary structure of the Mr 46,000 mannose 6-phosphate specific receptor: effect of ligand, pH, and receptor concentration on the equilibrium between dimeric and tetrameric receptor forms

The Mr 46,000 mannose 6-phosphate specific receptor exists in solution as a mixture of noncovalently associated dimeric and tetrameric forms. The two quaternary forms were separated by sucrose density centrifugation, and their composition was assessed by cross-linking with bifunctional reagents followed by SDS-polyacrylamide gel electrophoresis. The dependence of equilibrium between the dimeric and tetrameric forms on pH, receptor concentration, and presence of mannose 6-phosphate was studied. The formation of tetrameric forms is favored by pH values around 7, high receptor concentration, and presence of mannose 6-phosphate ligand. Tetrameric forms bind stronger at pH 7 to phosphomannan-Sepharose 4B than dimeric forms. Both quaternary forms dissociate at the same pH from a mannose 6-phosphate affinity matrix. When starting with dimeric or tetrameric forms, the equilibrium between dimeric and tetrameric forms is reached at pH 7.5 and 4 degrees C after 6-8 days. The presence of 5 mM mannose 6-phosphate shifts the equilibrium toward tetrameric forms. At pH 4.5 and 4 degrees C, the association of dimeric to tetrameric forms is negligible, while tetrameric forms dissociate to dimeric forms within 12 h. The results demonstrate that oligomerization is an intrinsic property of MPR-46 that is affected by ligand binding, pH, and receptor concentration.     

Induction of L Forms of Haemophilus influenzae in Culture and Their Demonstration in Human Bronchial Secretions

Transitional forms and round bodies of Haemophilus influenzae were identified in sputa from patients with chronic bronchitis who were receiving penicillin therapy for H. influenzae infections. In vitro growth of L forms of this organism was induced by penicillin and glycine and was studied for comparison with development in vivo. Variant forms demonstrated in sputum were similar to variant forms observed in penicillin-induced L colonies. Recurrence of infection after cessation of therapy was related to reversion of persisting L forms to bacillary forms. That these forms were derived from H. influenzae was established by direct staining with fluoresceinlabeled specific antibody. This demonstration that transitional forms and round bodies of H. influenzae occurred in vivo suggests that L forms of bacteria may be significant in chronic or recurrent infections.     

Theory of Phyllotaxis. 2. Crystallographic Interpretation of the Interrelation between Lower and Superior Phyllotaxis Forms

Cross-opposite phyllotaxis forms are defined as superior with respect to the alternate ones and verticillate phyllotaxis forms as superior with respect to the opposite ones. Different phyllotaxis forms can be interpreted as a result of stretching of crystal-like structures of the embryo formed by dense packing of rudiments. Based on hypothetical concepts of the properties of plant rudiments and embryos, possible mechanisms of the formation of superior phyllotaxis forms from the lower ones have been analyzed. It was shown that the superior phyllotaxis forms can be considered as the results of additive summation of the lower forms. The theoretical conclusions are confirmed by the examples of polymorphic phyllotaxis in conspecific plants and by the facts of accidental splitting of superior phyllotaxis forms into the corresponding lower forms in nature and in experiment. The hexagonal-tetragonal type of phyllotaxis was theoretically predicted and found in nature. The mechanism underlying the formation of multiple forms of the helical phyllotaxis was considered.     

The multiple forms of brain acetycholinesterase. III. Implications for the histochemical demonstration of acetylcholinesterase

The multiple forms of acetylcholinesterase (AChE, E.C. 3.1.1.7) have been investigated with regard to their histochemical demonstrability. Their pattern is influenced by buffer treatment, fixation, and by incubation conditions causing aggregation and disaggregation as well as loss or inactivation of individual forms. The standard histochemical method for AChE preferentially demonstrates the high molecular forms. Most of the oligomer forms are washed out or inactivated. A selective demonstration of the highly aggregated forms is possible either by inhibition of the oligomers with diisopropylfluoridate (DFP) or by specifically dissolving them out. No reason could be found for the selective demonstration of the low molecular weight forms.     

Metabolic studies by 1H NMR of different forms of Trypanosoma cruzi as obtained by 'in vitro' culture

Abstract By culturing Trypanosoma cruzi epimastigotes in modified Grace's medium with 10% foetal bovine serum, a significant quantity of metacyclic forms could be obtained. Transformation was observed after 8 days of culture, with metacyclic forms reaching 75%. Cultured Vero cells were infected with metacyclic forms and maintained until free-amastigote forms were obtained. Additionally, amastigote-like forms could be obtained by subjecting metacyclic cultures to heat shock. Parasites were grown with glucose as the major carbon source. The metabolites produced and excreted during culture were identified by difference proton nuclear magnetic resonance spectroscopy and quantified by enzymatic methods. The final products of glucose catabolism differed not only quantitatively but also qualitatively for the three major life-cycle stages of T. cruzi . The end products of metabolism produced by epimastigote forms were mainly acetate and pyruvate and, to a lesser extend, l-alanine and ethanol. Differences between epimastigotes and metacyclic forms were only quantitative. However, free amastigotes as well as amastigote-like forms, excreted acetate, glycerol, and pyruvate and to a lesser extent succinate, but no l-alanine or ethanol.     

Multiple forms of myeloperoxidase from human neutrophilic granulocytes: evidence for differences in compartmentalization, enzymatic activity, and subunit structure

Multiple forms of myeloperoxidase from normal human neutrophilic granulocytes obtained from a single donor can be resolved by carboxymethyl (CM)-cellulose ion-exchange column chromatography into three forms (I, II, and III) designated in order of elution of adsorbed enzyme using a linear salt gradient. Selective solubilization of individual forms of the enzyme by detergent (form I) or high-ionic-strength procedures (forms II and III) suggested that these forms of the enzyme were compartmentalized differently. All three forms were purified by a combination of preferential extraction, manipulation of ionic strength, and ion-exchange and molecular sieve chromatography. Purified forms II and III had similar specific activities for a variety of substrates. Form I was less active toward several of these same substrates, most notably iodide, with a specific activity about one-half that of forms II and III. All forms had similar spectral properties characteristic of a type alpha heme. The amino acid compositions of the three forms were similar, yet significant differences were found in selected residues such as the charged amino acids. Native polyacrylamide gel electrophoresis resolved small differences in mobility between the forms which were consistent with the charge heterogeneity observed on CM-cellulose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis data were consistent with the generally accepted subunit structure of two heavy chains and two light chains. All three forms contained a small-molecular-weight subunit of Mr 11,500. Form I contained a large subunit of Mr 63,000, while forms II and III contained a corresponding subunit of Mr approximately 57,500. We conclude that heterogeneity of human myeloperoxidase is accompanied by differences in cellular compartmentalization, enzymatic activity, and subunit structure.     

The theory of phyllotaxis. II. Crystallographic interpretation of the interrelation between lower and superior phyllotaxis forms

Cross-opposite phyllotaxis forms are defined as superior with respect to the alternate ones and verticillate phyllotaxis forms as superior with respect to the opposite ones. Different phyllotaxis forms can be interpreted as a result of stretching of crystal-like structures of the embryo formed by dense packing of rudiments. Based on hypothetical concepts of the properties of plant rudiments and embryos, possible mechanisms of the formation of superior phyllotaxis forms from the lower ones have been analyzed. It was shown that the superior phyllotaxis forms can be considered as the results of additive summation of the lower forms. The theoretical conclusions are confirmed by the examples of polymorphic phyllotaxis in conspecific plants and by the facts of accidental splitting of superior phyllotaxis forms into the corresponding lower forms in nature and in experiment. The mechanisms underlying the formation of multiple forms of helical phyllotaxis have been proposed. The concept of a new type of mixed hexagonal-tetragonal phyllotaxis has been formulated and the mechanism of its formation has been considered. The forms of corn grain packaging in the corncob and leaf arrangement on the strawberry tomato stem are given as examples of true hexagonal-tetragonal phyllotaxis in nature.     

Allozyme differentiation of various chromosomal forms of the spotted suslik (Spermophilus suslicus Guld, 1770, Rodentia)]

Eleven enzyme and four nonenzyme protein systems controlled by 25 loci were electrophoretically analyzed in the allopatric karyotypic forms (2n = 34 and 2n = 36) of the spotted souslik Spermophilus suslicus. Genetic variability and differentiation for the forms with different chromosome sets were estimated. Two discriminative loci (Alb and Tf) for the studied chromosome forms were found. The UPGMA dendrogram was constructed, which summarizes genetic (allozyme) relationships found for the forms of the spotted souslik with different chromosome sets. Subdividing the species into two karyotypic forms was shown to be followed by differentiating these forms at the allozyme level.     

IX. Meaning and the Semantic Act

Shpet concludes his definition of the structure of the word as follows:

Indeed, if one accepts that morphological forms are external and agrees to call ontic forms of named things pure, then the logical forms lying between them will be inner forms with respect to both the former and the latter since, in this latter case, the "content" of an object is inner content veiled by its pure forms. It is this content, being internally logically formed, that constitutes sense. Logical forms are inner forms as forms of ideal sense, expressed and communicated; ontic forms are pure forms of real and possible corporeal content.     

Non‐pathogenic association of L‐form bacteria (pseudomonas syringae pv. phaseolicola) with bean plants (Phaseolus vulgaris L.) and its potential for biocontrol of halo blight disease

L‐forms of the halo blight pathogen, Pseudomonas syringae phaseolicola, were maintained in a medium which suppressed cell wall synthesis. These L‐forms, unlike revertants (walled forms derived from unstable L‐forms) and cell walled (parent) organisms, did not elicit a hypersensitive response in tobacco leaves. Association of L‐forms with Phaseolus vulgaris was established by seed imbibition in L‐form suspensions compared with appropriate control treatments (5% mannitol or heat‐killed cells). Seedling emergence and plant growth was not affected by L‐form imbibition. The association was detected by agglutination assays using polyclonal antibody. The L‐form association was localized to the lower shoot tissue and was progressively lost with age of plants. Plants with associated L‐forms had vigour and shoot weights equivalent to controls and showed no disease symptoms. The cell walled form could not be isolated from plants showing positive agglutination. On challenge with the pathogen, plants associated with L‐forms showed significantly less disease symptoms than controls. Stem extracts, from associated plants, were inhibitory to in vitro cultures of both L‐forms and parent forms of Ps. syr. phaseolicola. These results indicate that L‐form associations confer induced systemic resistance to bean plants and might be developed as novel biocontrol systems.     

Cross-reactivity of vector-borne metacyclic forms of Trypanosoma cruzi with mammalian and culture stages

Metacyclic forms of Trypanosoma cruzi isolated from the hindgut of infected insect vectors (Rhodnius prolixus) were found to be immunologically cross-reactive with cultured epimastigote, amastigote, and metacyclic stages of the parasite as well as with bloodstream trypomastigote forms by direct agglutination and indirect immunofluorescence techniques. Sera specific for each of these forms of the parasite systematically yielded maximal antibody titers when measured against the homologous antigen, indicating that antigenic determinants are shared by all of the developmental forms used in this work. Supporting this conclusion were the significant reductions in anti-insect-derived metacyclic antibody titer caused by absorption with any of the other life stages of T. cruzi. These results are relevant to the potential use of laboratory-grown forms of T. cruzi in vaccination against a natural infection with this parasite.     

In vitro production of metacyclic forms of Trypanosoma musculi and their infectivity in CBA mice

Bloodstream forms of Trypanosoma musculi, cultured in Schneider's drosophila medium at room temperature, multiply and differentiate through a series of developmental stages into infective metacyclic trypomastigotes in 10 days. Oral inoculation with these culture forms into CBA mice produced a parasitemia similar to that produced by intraperitoneal infection with bloodstream forms except for a three-day longer prepatent period. Attempts to induce parasitemia with bloodstream forms given orally were unsuccessful.     

Characteristics of pathogenic non-fluorescent (smooth) and non-pathogenic fluorescent (rough) forms of Pseudomonas tolaasii and Pseudomonas gingeri

Pseudomonas tolaasii and Ps. gingeri cultures isolated from naturally diseased mushrooms and cultures obtained from other workers were all observed to contain both smooth and rough colony forms. The smooth forms produced mucoid, non-fluorescent, glistening opaque colonies with entire margins. The rough forms produced non-mucoid, fluorescent, dull, translucent greenish-yellow colonies with irregular margins. Smooth forms were observed to produce a toxin and were pathogenic to mushrooms, whereas rough forms did not produce toxin and were non-pathogenic. Isolates of Ps. tolaasii were distinguishable from Ps. gingeri by various biochemical tests. In general, however, biochemical differences between the rough and smooth forms of each species could not be detected. Rough forms of Ps. tolaasii and Ps. gingeri remained stable in culture but smooth forms were unstable, tending to convert to rough forms at a very high rate.     

T4溶菌酶晶体分子堆积的研究

以不对称单位中只有一个分子的１０种不同晶型的Ｔ４溶菌酶晶体为材料,对晶体中的分子堆积进行了研究,结果表明,在溶剂含量较高的晶型中,非极性基团在接触面积中所占的比例略高于溶剂含量较低的晶型,而其极性和带电荷基团在接触面积中所占的比例略低于溶剂含量较低的晶型。溶剂含量较高的晶型多含有晶体学二重轴,二重轴相关的分子间的接触与其他接触相比,含有较少的极性相互作用。这些结果说明溶剂含量的高低可能是由不同结晶     

Multiple forms of rat-liver dihydropteridine reductase identified by their differing isoelectric points

Purified rat-liver dihydropteridine reductase is homogeneous by gel filtration (Mr approximately 51,000), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately 25,500), and native polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two identical subunits. However, analysis by isoelectric focusing has revealed three enzyme forms with approximate isoelectric points of 6.5, 5.9, and 5.7 (designated forms, I, II, and III, respectively). The three forms, isolated in 65% yield by preparative chromatofocusing, are stable in 0.05 M phosphate buffer, pH 6.8, containing 1 mM beta-mercaptoethanol and exhibit similar kinetic constants when the catalytic activities of the isolated forms are compared with quinonoid dihydrobiopterin as substrate. All forms generate complexes with the enzymatic cofactor NADH which are also detectable by IEF. When examined further by IEF under denaturing conditions in 6 M urea the enzyme demonstrates a differing subunit composition for its three forms. Two distinct subunits, designated alpha and beta, can be identified, and additional evidence suggests that the native enzyme forms I, II, and III represent the three differing dimeric combinations alpha alpha (form I), alpha beta (form II), and beta beta (form III).     

Evidence for two structurally related progesterone receptors in chick oviduct cytosol

The existence of two progesterone receptor forms present in crude cytosol of chick oviduct has been demonstrated by photoaffinity labelling using [3H]R5020. On SDS-polyacrylamide gels these two forms exhibit app. Mr-values of 79000 and 109000 corresponding to the progesterone receptor forms A and B. Peptide maps of photoaffinity-labelled steroid receptors have been established by limited proteolysis with alpha-chymotrypsin. The peptide map obtained for chick oviduct cytosol progesterone receptor crosslinked with [3H]R5020 proved to be the sum of peptides obtained from partially purified preparations of forms A and B. The peptide maps of both progesterone receptor forms were identical for peptides below the Mr-value of form A, indicating extensive homology of the two forms. A significantly different peptide pattern was observed for the rat liver glucocorticoid receptor crosslinked with [3H]triamcinolone acetonide. Prolonged proteolysis with chymotrypsin gave rise to peptides with Mr-values of 6000 and 10000 from the hormone-binding domain of progesterone and glucocorticoid receptors, respectively.     

Phosphate content of Escherichia coli alkaline phosphatase isozymes. The effect of phosphate and zinc on the separation of isozymes.

Alkaline phosphatase from Escherichia coli was isolated as two major isoenzyme forms that were separated by DEAE-cellulose chromatography. Each form contained 2 equiv of endogenous phosphate. The endogenous phosphate, although difficult to remove, readily exchanges with phosphate. The forms also were separable by polyacrylamide gel electrophoresis. Apoenzyme prepared from native enzyme by the removal of zinc (and phosphate) also contains electrophoretically distinct enzyme forms which are indistinguishable from the native forms on gel electrophoresis. The isozymes were also found to have similar affinities for inorganic phosphate and susceptibilities to inactivation by EDTA. These results are not consistent with the notion that the formation or separation of isoenzyme forms is dependent upon different amounts of bound phosphate. They are consistent with the suggestion that a difference in amino acid composition is the basis for the occurrence and separation of these isoenzymes.     

Two types of asymmetric acetylcholinesterase in chick hindlimb muscle: Developmental profiles,in Vivo and in cell culture,and recovery after inactivation

1. We have analyzed the behavior of two types of asymmetric molecular forms (A forms) of acetylcholinesterase (AChE) during development of chick hindlimb muscle, in vivo and in cell culture, and upon irreversible inactivation of peroneal muscle AChE with diisopropylfluorophosphate (DFP) in vivo. 2. In agreement with previous developmental studies on chick muscle, globular forms of AChE (G forms) are predominant in chick hindlimb at early embryonic ages, being gradually replaced by A forms as hatching (and, therefore, onset of locomotion) approaches. Of the two A-form types, AI appears and accumulates significantly earlier than AII, so that A/G and II/I ratios higher than 1 are attained only at about hatching time. 3. Cultures prepared from 11-day chick embryo hindlimb myoblasts express both types of A forms, with a combined activity of 27% of total AChE after 12 days in culture. AI forms appear again earlier and are much more abundant than type II asymmetric species through the life span of cultures. 4. All AChE activity in the peroneal muscle is irreversibly inactivated by injection of DFP in vivo. The recovery of A forms follows the same sequence described for normal development, with a delayed and slower recovery of AII forms as compared with AI. 5. Several hypotheses involving tail polypeptides or tissue target molecules, or posttranslational interconversion, are proposed to help explain the earlier appearance and accumulation of AI forms in chick muscle.     

The effect of iodoacetate on the dehydrogenation activity of smooth and rought forms of yeasts in the presence of ethanol or acetate

The possible causes of higher sensitivity of aerobic metabolism of ethanol in the smooth forms of yeasts toward the inhibitory effect of iodoacetate as compared with the rough forms, formed by spontaneous dissociation of yeast cultures, were investigated. It was found that the dehydrogenation of ethanol by cell-free preparations of both yeast forms does not possess different sensitivity toward the effect of iodoacetate although there are certain differences between the dehydrogenases of the two yeast forms (e.g. in their activity in borate). The dehydrogenation activity of intact cells of the smooth forms in the presence of ethanol is inhibited by 10^?3M iodoacetate while in the rough forms it is not affected. Similar conditions exist during dehydrogenation of acetate. These results serve as a basis for assessing certain differences in the permeability of the cell membranes of the two forms of yeasts to iodoacetate of possibly also a different effect of iodoacetate on the uptake of ethanol or acetate by intact cells of both yeast forms. The necessity for raising the pH of intact cells of smooth forms above the optimum of the enzyme involved if detectable activity levels of this enzyme are to be achieved also points to certain differences in the functional properties of the cell membranes of the two yeast forms.