Analysis of outgrowth of Bacillus subtilis spores lacking penicillin-binding protein 2a.

The loss of Bacillus subtilis penicillin-binding protein (PBP) 2a, encoded by pbpA, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore. Further analyses to define the defect in pbpA spore outgrowth have shown that (i) outgrowing pbpA spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of pbpA spores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and pbpA spores once cell elongation had been initiated; (iii) outgrowth and elongation of pbpA spores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.

Visualizing taste papillae in vivo with scanning electron microscopy of a high resolution cast.

A method using polyvinylsiloxane (PVS), a high-resolution dental impression material, to obtain negative images of lingual surfaces is described. Epoxy-resin tongue replicas made from these impressions were examined with scanning electron microscopy (SEM). This method has been developed to visualize structural details of the tongue surface of living human beings and laboratory animals. The utility of the method is demonstrated with hamster tongues, which have well-defined fungiform papillae with single taste pores, and human tongues, which have more variable surface structures. Replicas made from PVS impressions of tongues of living hamsters were compared with the same tongues after fixation. The replicas contained much of the detail present in fixed tongues. With SEM, it was possible to identify individual fungiform papillae, which contained depressions with the size and the location of hamster taste pores. Individual papillae could also be recognized in human-tongue replicas, but taste pores could not be identified with certainty. These replicas provide permanent, three-dimensional records of tongue topography that could be used to document changes due to trauma, disease and aging.

Glycosaminoglycan synthesis is depressed during mitosis and elevated during early G1.

[35S]Sulfate incorporation was measured in populations of Chinese hamster ovary cells enriched for mitotics, early G1 cells, and interphase monolayers or suspensions. Incorporation was determined by biochemical analysis of extracts and quantitative autoradiography of thick sections. 90% of [35S]sulfate was incorporated into glycosaminoglycan (GAG). Incorporation was depressed fourfold in mitotics and stimulated by from two- to three-fold in early G1 cells relative to mixed interphase cells. GAG synthesis was maintained into late G2. Thus, the rate of GAG biosynthesis was correlated temporally with the detachment and reattachment of cells to substrate. Inhibitors of protein synthesis brought about the rapid arrest of GAG biosynthesis. However, xylosides, which bypass the requirement for core protein, did not bring oligosaccharide sulfation in mitotics to interphase levels. These observations indicate an inhibition of Golgi processing and are consistent with a generalized defect of membrane vesicle-mediated transport during mitosis.

Membrane activity and topography of F-Met-Leu-Phe-Treated polymorphonuclear leukocytes. Acute and sustained responses to chemotactic peptide.

The chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (f-Met-Leu-Phe) causes a dramatic stimulation of membrane ruffling and a fluid pinocytosis in polymorphonuclear leukocytes (PMNs). These responses are maximal by 1 minute and subside within 5-10 minutes. The same immediate responses characterize cells exposed to several peptide hormones and may thus represent an essential component of target cell activation by peptides. The stimulation of the whole membrane following f-Met-Leu-Phe binding is succeeded by the development of a polarized cell shape with a posterior uropod and a broad anterior lamellipodium, both subtended by microfilaments. Membrane components and functions segregate into distinct domains on polarized PMNs. Thus, succinyl concanavalin A-receptor complexes are capped and internalized by receptor-mediated endocytosis at the uropod; the uptake by fluid pinocytosis of fluorescein-dextran is restricted to the uropod; and coated pits and coated vesicles are concentrated at the uropod. The lamellipodium excludes coated pits and lacks pinocytic activity but shows preferential binding of immunoglobulin aggregates, presumably to Fc receptors. The origin and physiologic implications of these asymmetries of membrane molecular and functional topography on polarized cells are discussed.

Microtubule assembly and conanavalin A capping in lymphocytes: reappraisal using normal and abnormal human peripheral blood cells.

We have analyzed the assembly of microtubules and the distribution of concanavalin A(Con A)-receptor complexes in the same populations of human peripheral blood T and B lymphocytes. We hoped to resolve the prolonged controversy over the relationship of microtubules to Con A cap formation in lymphocytes and to explain the abnormally high spontaneous and colchicine-induced Con A capping that was observed recently in lymphocytes from a patient with an inherited form of severe combined immunodeficiency disease (SCID) characterized by total immunologic dysfunction despite normal numbers and distribution of T and B cells. The data establish that (i) microtubule disassembly is correlated with enhanced Con A cap formation on normal human lymphocytes; (ii) T and B cells differ significantly from each other and from circulating polymorphonuclear leukocytes with respect to their capping responses after exposure to colchicine; and (iii) there is an abnormal relationship of microtubule assembly to surface topography in the functionally defective SCID cells.

Microtubule dynamics and glutathione metabolism in phagocytizing human polymorphonuclear leukocytes.

Glutathione oxidants such as tertiary butyl hydroperoxide were shown previously to prevent microtubule assembly and cause breakdown of preassembled cytoplasmic microtubules in human polymorphonuclear leukocytes. The objectives of the present study were to determine the temporal relationship between the attachment and ingestion of phagocytic particles and the assembly of microtubules, and simultaneously to quantify the levels of reduced glutathione and products of its oxidation as potential physiological regulators of assembly. Polymorphonuclear leukocytes from human peripheral blood were induced to phagocytize opsonized zymosan at 30 degrees C. Microtubule assembly was assessed in the electron microscope by direct counts of microtubules in thin sections through centrioles. Acid extracts were assayed for reduced glutathione (GSH) and oxidized glutathione (GSSG), by the sensitive enzymatic procedure of Tietze. Washed protein pellets were assayed for free sulfhydryl groups and for mixed protein disulfides with glutathione (protein-SSG) after borohydride splitting of the disulfide bond. Resting cells have few assembled microtubules. Phagocytosis induces a cycle of rapid assembly followed by disassembly. Assembly is initiated by particle contact and is maximal by 3 min of phagocytosis. Disassembly after 5-9 min of phagocytosis is preceded by a slow rise in GSSG and coincides with a rapid rise in protein-SSG. Protein-SSG also increases under conditions in which butyl hydroperoxide inhibits the assembly of microtubules that normally follows binding of concanavalin A to leukocyte cell surface receptors. No evidence for direct involvement of GSH in the induction of assembly was obtained. The formation of protein-SSG, however, emerges as a possible regulatory mechanism for the inhibition of microtubule assembly and induction of their disassembly.

Better epoxy resin embedding for electron microscopy at low relative humidity.

In the absence of other factors known to influence sectioning properties, high environmental relative humidity is shown to yield poorly embedded tissue. Humidity-related effects are avoided if the following embedding precedure is used; impregnate tissues using the following solutions 1) 70% alcohol - 5 minutes, 2) 95% alcohol - 2 x 15 minutes, 3) absolute alcohol - 3 x 20 minutes, 4) acetone - 2 x 15 minutes, 5) 1:1 mixture of acetone-epoxy resin (DDSA, 63.4 g; Araldite 502, 5.6 g; Epon 812, 39.4 g; DMP-30, 2.6 g) - 1 hour, 6) acetone-epoxy resin 1:3 - 1 hour, 7) epoxy resin - 1 hour; complete the preparation of blocks as follows 8) when tissues have been oriented in epoxy resin in flat embedding molds, place molds in one evacuated vacuum desiccator 10 cm above a 2 cm layer of Drierite for 24 hours at room temperature, 9) raise temperature to 60 C and maintain for 3 days to cure resin.