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Bacillus subtilis cheRB, which encodes the chemotactic methyltransferase, has been cloned and sequenced. CheRB is a polypeptide of 256 amino acids, with a predicted molecular mass of 28 kDa. A comparison of the predicted amino acid sequence of B. subtilis CheRB with that of Escherichia coli CheRE demonstrates that the two enzymes share 31% amino acid identity. The homology was functional in that the expression of cheBB in an E. coli cheRE null mutant made the bacteria Che+. In contrast to cheRE null mutants which show a strong smooth swimming bias, cheRB null mutants were predominantly tumbly. They respond to the addition and subsequent removal of attractant. They also respond to the addition of repellent but do not adapt; they resume prestimulus bias on removal of repellent. Tethering analysis of a culture of a cheRB null mutant revealed two distinct subpopulations, each demonstrating unique behaviors. One showed a strong clockwise flagellar rotation bias, whereas the other was more random. The latter phenotype may be due to a deficiency of CheB and may reflect an interaction of CheB and CheR. Measurements of CheB activity in the cheR null mutant showed them to be only 20% of wild type levels. We conclude from this work that CheRB functions to promote adaptation to repellent stimuli in B. subtilis, whereas CheRE functions to promote adaptation to attractant stimuli in E. coli.

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The Bacillus subtilis gene encoding CheB (cheBB), the chemotactic methylesterase, has been sequenced. The 39-kDa protein which resulted from the expression of cheBB, using a T7 expression system was consistent with the predicted open reading frame. CheBB shares 39.5% identity with Escherichia coli CheBE and can complement a cheBE null mutant. CheBB is required for removal of methyl groups from the receptors upon attractant stimulus and appears to play an important role in adaptation to the addition of attractants, whereas CheBE plays an important role in adaptation to the addition of repellents. Unlike the cheBE and cheRE mutants of E. coli, which show extreme flagellar rotational biases, the unstimulated cheBB mutant showed a normal (wild type) bias. Upon addition of attractant, the cheBB null mutant showed a counter-clockwise bias that was higher than for wild type and demonstrated only partial adaptation. In the capillary assay for the attractant azetidine-2-carboxylic acid, the mutant gave a wild type response at low concentrations but a very reduced response at high concentrations. We conclude that B. subtilis has an effective methylation-independent adaptation system but must utilize the methylation system for adaptation to high concentrations of attractant.

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X-ray microanalysis of frozen-hydrated tissue sections permits direct quantitative analysis of diffusible elements in defined cellular compartments. Because the sections are hydrated, elemental concentrations can be defined as wet-weight mass fractions. Use of these techniques should also permit determination of water fraction in cellular compartments. Reliable preparative techniques provide flat, smooth, 0.5 micrometers-thick sections with little elemental and morphological disruption. The specimen support and transfer system described permits hydrated sections to be transferred to the scanning electron microscope cold stage for examination and analysis without contamination or water loss and without introduction of extraneous x-ray radiation.

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New specimen handling and analytic techniques for the application of x-ray microanalysis to studies of cell and organ biology have been recently described (Saubermann et al., 1981, J. Cell Biol. 88:257-267). Based on these techniques, absolute quantitative standardization has been established through x-ray analysis of frozen-hydrated and then dried sections of independently measured standard solutions of elements. These experiments demonstrate that the specific techniques employed have a probable error of less than 10%. Artificial electrolyte gradients established in gelatin were subjected to analysis to determine whether there was elemental displacement under non-membrane-limited conditions at the temperatures employed for sectioning (-30 degrees to -40 degrees C). No significant difference was observed between such gradients in serial sections cut at -30 degrees and -80 degrees C. Similarly, no additional ice-crystal-damage artifact was found in sections cut at -30 degrees C when compared with sections cut at -80 degrees C. Thus, in terms of ice-crystal size, gradient maintenance, and compartmental differentiation, cryosectioning at -30 degrees to -40 degrees C was not associated with redistribution incompatible with 1- to 2-micrometers spatial resolution, and absolute measurements of elemental concentration were practical within regions of this size.

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A common tattoo occurring in the mouth is caused by the insertion of amalgam filling material into the soft tissues. Fifteen amalgam tattoos were examined. Amalgam or its derivatives were found within macrophages, fibroblasts and multinuclear giant cells. Fine particles were found associated with: the basement-membranes of mucosal epithelium, of striped muscle fibres, and of muscle cells of blood vessels; collagen; elastic tissue; and the connective tissue of nerves. Amalgam consists mainly of mercury, silver and tin. The analytical results suggest that corrosion occurs and that mercury and some tin are lost from the tattoos, leaving silver and tin in macrophages and giant cells, and silver in fibroblasts and in the above-mentioned extracellular sites.

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The epithelial cells of the midgut in 1-40 day old adult female houseflies were examined by electron microscopic X-ray microalnalytic and histochemical techniques in order to study the mode of genesis, chemical nature and age-associated distribution of dense bodies. Dense bodies contain high concentration of phosphorus sulphur, chlorine, calcium, iron and copper; they are therfore termed concretions. Concretionary material is initially deposited within Golgi vesicles, lamellar bodies and residual bodies. The average size of the concretion granules and the concentration of the sequestered material increases with age, while new concretions are continually formed throughout life. With advancing age, concretions accumulate in the epithelial cells and occupy a considerable proportion of the cytoplasm in old flies. It is postulated that the concretions sequester superfluous minerals and may play an important role in the excretory system of the adult housefly.

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Electron microscopic X-ray microanalysis and atomic absorption spectrophotometry were used to determine changes in zinc concentration after myocardial ischemic injury by coronary ligature in dogs. The zinc concentration increased in the damaged myocardium especially in specific intracellular locations identified by means of the microprobe. It was concluded that the selective zinc augmentation was related to proliferative and biosynthetic reactive processes in the myocardium.

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This study shows that after intra-articular injection, aurothiomalate and colloidal gold of small (200 A) particle size were rapidly absorbed from joints while the larger, 300 A, particle size colloidal radioactive gold could not be found outside them. Larger particle size suspensions seem therefore more likely to remain localised in the joint and its lining synovium after intra-articular injection, the systemic absorption from the joint cavity diminishing with increasing particle size. It was also found that the intra-articular injection of small amounts of aurothiomalate, of colloidal gold and of colloidal radioactive gold produces identical degenerative lesions in the lining cells of the proximal convoluted tubules of the kidneys. These lesions were always found, although gold particles were demonstrated only in sampled kidney tissues of the animals injected with the soluble gold preparation whereas no gold could be detected in the tissues of animals injected with colloidal non-radioactive or radioactive gold. Electron microscopic evidence is presented to suggest the possibility that the mitochondria are the "target" organelles of the gold-induced cellular damage. Mitochondrial damage was demonstrated in liver and spleen in addition to the already described kidney damage. The correlation between structure and function of the mitochondrial changes is not clear, and ionic shifts may be both a cause and a result of damage.

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The elemental composition of different types of keratohyalin granules from the epidermis of newborn and adult rats was studied by means of an EMMA-4 analytical electron microscope, equipped with an energy-dispersive X-ray spectrometer. An absolute quantitation of the sulphur concentration in keratohyalin granules was performed. The results demonstrate that epidermal keratohyalin granules are chemically heterogeneous. A type of keratohyalin granule present in the nuclei and cytoplasm of epidermal cells from both newborn and adult rats - termed single granules - is rich in sulphur, having a content of 2-5-3-6%. Other types of keratohyalin granules, which differ in newborn and adult rats, contain a sulphur-poor component; they often have a sulphur-rich component as well. The sulphur-poor keratohyalin contains 0-6-0-9% sulphur. It is suggested that the sulphur-rich keratohyalin granules are the source of the peripheral envelope protein of cornified cells.

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Rabbits were injected with soluble gold, and cold injury was inflicted upon their extraocular muscles. Following the typical freeze-thaw damage in which the myofiber basement membrane is preserved, a polymorphonuclear granulocyte exudation occurred. At this stage only a few interstitial cells with long processes contained gold. At one and two days abundant mononuclear cells with gold were observed at the site of injury. In subsequent stages of muscle regeneration the amount of tracer decreased but myoblasts, and even fully formed myotubes, often contained detectable gold. Some data on "in vivo" gold salt kinetics and the specificity of gold localization were utilised in the study of the relation between mononuclear interstitial cells and myoblasts.

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Sites with high calcium affinity in Paramecium aurelia were identified by high calcium (5 mM) fixation and electron microscope methods. Electron-opaque deposits were observed on the cytoplasmic side of surface membranes, particularly at the basal regions of cilia and trichocyst-pellicle fusion sites. Deposits were also observed on some smooth cytomembranes, within the axoneme of cilia, and on basal bodies. The divalent cations, Mg2+, Mn2+, Sr2+, Ni2+, Ba2+, and Zn2+, could be substituted for Ca2+ in the procedure. Deposits were larger with 5 mM Sr2+. Ba2+, and Mn2+ at ciliary transverse plates and the terminal plate of basal bodies. Microprobe analysis showed that Ca and C1 were concentrated within deposits. In some analyses, S and P were detected in deposits. Also, microprobe analysis of 5 mM Mn2+-fixed P. aurelia showed that those deposits were enriched in Mn and C1 and sometimes enriched in P. Deposits were seen only when the ciliates were actively swimming at the time of fixation. Locomotory mutants having defective membrane Ca-gating mechanisms and ciliates fixed while exhibiting ciliary reversal showed no obvious differences in deposition pattern and intensity. Possible correlations between electron-opaque deposits and the locations of intramembranous particles seen by freeze-fracture studied, as well as sites where fibrillar material associate with membranes are considered. The possibility that the action sites of calcium and other divalent cations were identified is discussed.

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The electron microprobe shows that the dense bodies of human platelets have a mean P:Ca peak ratio of 1-2. After treatment with dry chloroform/methanol this falls to 0-89. These ratios vary slightly from patient to patient. The use of calcium and phosphorus standards enables these peak ratios to be converted to atomic ratios. The size of the phosphorus peak remaining after lipid extraction was given absolute terms with reference to the known quantities of adenine nucleotides and inorganic pyrophosphate in dense bodies. From the mean P:Ca atomic ratio of 1-76 the quantity of calcium in dense bodies was 0-6 mg/10(11) platelets or 2-97 mg Ca/g dry weight of platelets. This is within the published range for total platelet calcium. If all the phosphorus extracted by lipid solvents were phospholipid there would be 5-65 mg/10(11) platelets, and it would occupy most of the space inside dense bodies. The dense bodies of pig platelets contain both magnesium and calcium in a varying ratio to each other. These results are discussed in relation to control mechanisms that may influence aggregation.

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Rabbits and rats were given single injections of aurothiomalate by different routes. The animals were killed at progressive intervals, and sections from various organs were examined by electron microscopic x-ray microanalysis. Ultrastructurally, characteristic material was regularly found in vacuoles, dense and heterogeneous bodies of macrophages, hepatocytes and renal epithelial cells. Occasionally, other mesenchymal cells also contained gold. Histochemical and analytical tests showed that the gold-containing organelles were devoid of acid phosphatase activity. The generalized rapid spread, retention and selectivity of localization after a single small dose make aurothiomalate a useful marker substance for ultrastructural studies.

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The epithelium and the lumen of the Malpighian tubules of the housefly contains mineralized dense bodies called concretions. The morphological characteristics, mode of origin, nature of the sequestered elements and the age-associated changes in the distribution of concretions are reported. There are three types of concretions in the cytoplasm, which have been designated as type A, type B, and type C. Type A concretions are membrane-bound spherical structures which may arise by the gradual intravacuolar accumulation of dense material. Type B concretions appear to be related to multivesicular bodies. Type C concretions are heteromorphic and morphologically resemble the residual bodies. They show a positive localization of acid phosphatase reaction product. X-ray microanalysis of intracytoplasmic and intraluminal concretions revealed the presence of phosphorus, sulphur, chlorine, calcium, iron, zinc and copper. There was no evidence suggesting the extrusion of the intracytoplasmic concretions into the lumen of the Malpighian tubules. There is an age-associated increase in the distribution of type C concretions. It is hypothesized that the sequestration of metal ions within the concretions may provide a means for the effective excretion of these elements.

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The presence of gold in the kidney of a 58-year-old woman was confirmed by use of ultrastructural and microprobe examinations. A series of animal experiments were performed in which small single doses of gold were administered systemically and intraarticularly in rabbits. Both the colloidal suspension and the soluable salt gold sodium thiomalate (Myochrysine) were used. Selective lesions occurred in the proximal convoluted tubules and the mitochondria appeared to be the target organelle. They contained gold after gold sodium thiomalate but not after colloidal gold.

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High speed cinephotographic techniques were used to determine the pattern of fluid flow about the hispid flagellum of Ochromonas danica and to investigate the behavior of this flagellum in media of increased viscosity. The fluid currents are consistent with the hypothesis that the mastigonemes are passive, rigid, remain normal to the flagellar surface, and lie in the plane of flagellar undulation during motility.

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