Melanin formation in barley grain occurs within plastids of pericarp and husk cells.

Melanins are a class of darkly pigmented biopolymers which are widely distributed among living organisms. The molecular and cellular mechanisms adopted by bacteria, fungi and animals to synthesize melanin, have been well described, but less is known regarding their production in plants. Here, a pair of barley near isogenic lines, bred to differ with respect to the pigmentation of the spike, was compared in order to understand the tissue and cellular location of melanin deposition. The melanic nature of the pigments purified from black spikes was confirmed by a series of solubility tests and Fourier transform infrared spectroscopy. An analysis of grains harvested at various stages of their development revealed that intracellular pigmented structures first appeared in the pericarp and the husk of black spike plants at early dough stage. The co-localization of these structures with red autofluorescence suggested that they form in chloroplast-derived plastids, here designated "melanoplasts". Differences in dynamics of plastid internal structure during grain ripening were detected between the lines by transmission electron microscopy. Both lines accumulated plastoglobuli inside plastids, which persisted in black grain pericarp tissue up to the hard dough stage, while neither plastoglobuli nor any plastids were observed in grain of the control line at this stage. The role of plastoglobuli in melanin synthesis is discussed.

Cytological Techniques to Study Cytomixis in Plant Male Meiosis.

In this chapter we describe cytological techniques to study cytomixis, a process of nuclear migration between plant cells, in squashed plant male meiocytes of Nicotiana tabacum and Secale cereale. To perform immunostaining or fluorescence in situ hybridization (FISH) on meiotic cells involved in cytomixis common protocols are modified. During preparation of specimens for subsequent cytological analysis, it is necessary not only to make DNA and proteins accessible to DNA probes and antibodies, but also to preserve cell cytoplasm. There are also some important modifications in the protocols applied for meiocytes of different plant species. Here we describe protocols for immunostaining and FISH in rigid tobacco male meiocytes with dense cytoplasm and thick callose wall, that tolerate hard squashing, and in soft rye male meiocytes, that are easily damaged upon squashing, both to study cytomixis.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein ESAT6-CFP10-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute tuberculosis. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of animals with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized animals do not develop the symptoms of acute tuberculosis and their body weight gain was five times more as compared with the non-immunized-infected animals. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis (TB) is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein CFP10-ESAT6-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute TB. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of guinea pigs with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized guinea pigs do not develop the symptoms of acute TB and their body weight gain was five times more as compared with the non-immunized infected guinea pigs. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages.

New insights into cytomixis: specific cellular features and prevalence in higher plants.

The phenomenon of intercellular migration of nuclei in plant tissues (cytomixis) was discovered over a century ago, which has been followed by numerous attempts to clarify the essence of this process as well as to determine its causes and consequences. Most attention of researchers has been paid to cytomixis in microsporogenesis, since the transfer of part of genetic material between microsporocytes may influence the ploidy level of the produced pollen and, presumably, have an evolutionary significance. This review compiles the data on cytological pattern of cytomixis and proposes a scheme as to how cytomictic channels are formed and function in angiosperms. The prevalence of cytomixis in different plant taxa is analyzed using the published data. The causes, mechanisms, and consequences of the nuclear migration between cells in plant tissues are discussed.

An ultrastructural study of cytomixis in tobacco pollen mother cells.

Intercellular chromatin migration (cytomixis) in the pollen mother cells of two tobacco (Nicotiana tabacum L.) lines was analyzed by electron microscopy during the first meiotic prophase. The maximal manifestation of cytomixis was observed in the pachytene. As a rule, several cells connected with one another by cytomictic channels wherein the nuclei migrated were observable at this stage. In the majority of cases, nuclei passed from cell to cell concurrently through several closely located cytomictic channels. Chromatin migrated between cells within the nuclear envelope, and its disintegration was unobservable. The nucleus, after passing through cytomictic channels into another cell, can be divided into individual micronuclei or, in the case of a direct contact with another nucleus, can form a nuclear bridge. It has been demonstrated that the chromatin structure after intracellular migration visually matches the chromatin structure before it passed through the cytomictic channel. No signs of pyknosis were observable in the chromatin of the micronuclei formed after cytomixis, and the synaptonemal complex was distinctly seen. The dynamics of changes in the nucleoli during cytomixis was for the first time monitored on an ultrastructural level. Possible mechanisms determining cytomixis are discussed and the significance of this process in plant development is considered.