Germin isoforms are discrete temporal markers of wheat development. Pseudogermin is a uniquely thermostable water-soluble oligomeric protein in ungerminated embryos and like germin in germinated embryos, it is incorporated into cell walls

Nascent synthesis and accumulation of germin and its mRNA mark the onset of renewed growth when wheat embryos are germinated in water. Germin is a water-soluble, pepsin-resistant protein that is not found in immature embryos, or in mature embryos before their germination. An antiserum was raised by injecting rabbits with germin that was freed of other proteins by pepsinization and gel filtration. The antiserum has been used to detect, in extracts of mature embryos from dry, ungerminated wheat grains, a protein that is antigenically related to germin. The antigenically related protein has been named pseudogermin. Pseudogermin accumulates, maximally, between 20-25-days postanthesis, then declines appreciably in amount by 30-days postanthesis, in soluble extracts of immature embryos from several wheat varieties. The antiserum was also used to identify germin and pseudogermin among the proteins extracted from cell walls and to bind immunogold to cell walls preparatory to visualizing freeze-cleaved embryos by scanning electron microscopy. Wall-associated germin accounts for about 40% of the total germin in germinating wheat embryos. Appearance of germin in the apoplast is the most conspicuous germination-related change in the distribution of cell-wall proteins. It seems that germin may act at the level of the apoplast and that pseudogermin may subsume the role of germin at low water potentials during embryogenesis. The N-terminal eicosapeptide sequences in germin and pseudogermin are very similar but SDS/PAGE analysis detects discrete differences between the mobilities of their constituent monomers as well as gross differences between the stabilities of the parent oligomers. Like germin, pseudogermin is a water-soluble, pepsin-resistant protein, but pseudogermin has unprecedented disulphide-independent thermostability properties that have never been previously reported for a water-soluble oligomeric protein. Polysaccharides that co-purify with otherwise pure specimens of germin (and pseudogermin) have been isolated for analysis and shown to be highly substituted glucuronogalactoarabinoxylans. The possible biological significance of selective and tenacious association between germin and glucuronogalactoarabinoxylans is discussed in relation to cell expansion during embryogenic and germinative development of wheat, as are some peculiarities of amino-acid sequence that suggest a possible relation between germin and a proton-specific ion pump: gastric ATPase.     

Phosphatase-negative mutants of Legionella pneumophila and their behavior in mammalian cell infection

Microbial phosphatases are known or suspected to play a role in the pathogenesis of several intracellular pathogens, including Legionella micdadei. Legionella pneumophila also possess phosphatase activities, but their possible roles in cellular infection are unknown. We generated mutants of a serogroup 1 isolate of L. pneumophila that lack the major phosphatase. Isolation of a Pho- mutant after random mutagenesis with transposon MudII4041 allowed us to dissociate the major alkaline phosphatase (pH optimum approximately 8) from a minor acid phosphatase activity. Both activities were concentrated in the bacterial periplasm. The gene encoding the major alkaline phosphatase (pho) was cloned by expression in E. coli and used to generate a site directed mutation in two L. pneumophila strains. Each parent-mutant pair was compared in a U937 cell tissue culture assay for capacity to infect, lyse, and grow within mammalian cells. Although the parental stains differed in their U937 cell cytopathicity, neither was significantly more infective than its Pho- derivative, suggesting that the alkaline phosphatase activity is not essential for cellular infection. Because they are not attenuated, Pho- mutants can be used to generate gene fusions with E. coli alkaline phosphatase to study and secretion and cellular infectivity in L. pneumophila.     

Intracellular signaling events during positive and negative selection of CD4+CD8+ thymocytes in vitro

We have used in vitro models of thymocyte positive and negative selection in conjunction with selective inhibitors of the TCR-mediated signaling cascade to investigate the intracellular signaling events that mediate these processes. We report that Ro 31.8425, a potent and selective inhibitor of protein kinase C, which blocks the activation of mature T cells in a dose-dependent fashion, has no effect on either positive or negative selection of CD4+8+ thymocytes. In contrast, cyclosporin A fails to prevent negative selection, but inhibits positive selection through a direct effect on developing thymocytes, rather than through the perturbation of stromal cell support. Thus, our data suggest that positive and negative selection may operate via distinct intracellular signaling pathways.     

Fine needle aspiration cytology of oral and pharyngeal lesions. A study of 45 cases

Forty-five patients with oral or pharyngeal swellings were subjected to fine needle aspiration cytology (FNAC) of the mucosal surface over eight years. The age of the patients ranged from 2 to 85 years. The male:female ratio was 25:20. The common sites of involvement were palate (16 cases), cheek (9), pharynx (7) and tonsillar/peritonsillar area (6). Tongue, maxilla, alveolus and lips were less frequently involved. FNAC played an important role in differentiating inflammatory from neoplastic lesions and also benign from malignant neoplasms. Fifteen cases were cytologically diagnosed as benign neoplasms and included pleomorphic adenoma (11 cases), schwannoma (2), odontogenic tumor (1) and benign neoplasm not otherwise specified (1). Sixteen cases were diagnosed as malignancies. There were seven cases of malignant salivary gland tumors and 6 of squamous cell carcinoma. Two cases were high grade non-Hodgkin's lymphomas, and one was malignant odontogenic tumor. Of the 11 inflammatory or reactive lesions, 4 were found to be harboring Actinomyces. The rate of inadequate sampling was 6.7%. Histopathology reports on excised tissue were available for 10 cases only. Seven of nine cases with adequate cytology (77.88%) showed complete agreement with histology.     

Pharmacological characterisation of excitatory synaptic transmission in the cat red nucleus in vivo

Extracellular recordings were made from the magnocellular neurones of the red nucleus (mRN) in anaesthetised cats. A study was made of the effects of selective excitatory amino acid receptor antagonists on excitatory monosynaptic responses evoked from the sensorimotor cortex (SMC) and cerebellar interpositus nucleus (IPN). Iontophoretically applied CNQX and NBQX antagonised both SMC and IPN responses whereas, D-AP5 inhibited the SMC response but was ineffective to the IPN. At currents that selectively antagonised NMDA responses, CPPene had no effect on either SMC or IPN responses. 7-chlorokynurenate inhibited both SMC and IPN responses but required currents that antagonised both AMPA and NMDA responses and was therefore acting in a non-selective manner. Iontophoretically applied glycine was inhibitory to both agonist and synaptic responses, whilst D-serine potentiated NMDA responses but did not enhance monosynaptic responses of the SMC. However in the presence of either 7-chlorokynurenate or high currents of CNQX that reduced the SMC synaptic activation of the mRN neurones, D-serine attenuated the inhibitory action of these antagonists. It is concluded that monosynaptic responses from the SMC are mediated by both NMDA and non-NMDA receptors whereas the monosynaptic responses evoked from the IPN are mediated only by non-NMDA receptors. The lack of effect of CPPene is consistent with the postulate that two NMDA receptor subtypes are present on mRN neurones.     

Hydroxyurea affects cell morphology, cation transport, and red blood cell adhesion in cultured vascular endothelial cells

Hydroxyurea (HU) significantly increases fetal hemoglobin (Hb) production and concomitantly affects passive erythrocyte K transport and cell volume in patients homozygous for Hb S, thus decreasing disease severity. Red blood cells (RBCs) with Hb S display a greater adherence to vascular endothelial cells (VECs) than do Hb A cells, thus increasing the probability of vaso-occlusive crisis. The effect of HU on the structure and function of VECs is still unknown. In the present study, HU significantly changed, in a dose-dependent manner, the morphology and monovalent cation composition of cultured VECs after incubation in normal culture medium for up to 10 days in the absence and presence of 0.3 (therapeutic dose) and 3.0 (toxic dose) mmol/L HU. Treated cells showed significant morphologic changes such as an increase in apparent cell size and the formation of multinucleated giant cells. The protein content per dish decreased by 50% and 80% at 0.3 and 3.0 mmol/L HU, respectively, accompanied by an increase in cell Na (maximum, approximately 200%) and cell K (maximum, approximately 50%) contents at about days 4 to 6 and 8 to 10, respectively. In addition, HU decreased RBC adherence to VECs in experiments with 51Cr-loaded Hb A or Hb S RBCs. The HU-induced changes in VEC morphology, cation composition, and RBC adherence may be caused or accompanied by alterations in cell membrane permeability, transformation of endothelial cells, or decreased number/density of VEC adhesion molecules. Precise mechanisms of the HU effects warrant further investigation in light of the reported beneficial effects of HU in the treatment of sickle cell anemia.     

Monoclonal gammopathy and neuropathy

There is a known coexistence between polyneuropathy and monoclonal gammopathy (immunoglobulin M, immunoglobulin G, immunoglobulin A). Antibodies to several glycoconjugates of the peripheral nervous system have been found in 50-65% of patients with immunoglobulin M monoclonal proteins, and distinct clinical syndromes have been recognized. However, if no antibodies are found, a relationship between monoclonal protein and polyneuropathy is still debatable. Therapeutic intervention is generally directed at removing the autoantibodies by reducing the number of monoclonal B-cells.     

Polyneuropathy: diagnosis and management

A polyneuropathy is characterized by a symmetrical distribution of sensory or motor abnormalities, more pronounced distally than proximally and usually more evident in the lower than in the upper limbs. Polyneuropathies may be classified on the basis of (a) clinical picture: acute/subacute/chronic, sensory/motor/autonomous, axonal degeneration/segmental demyelinization, and (b) cause: metabolic disorder, deficiency, infection, auto(dys)immunity, hereditary and toxic/iatrogenic polyneuropathy, with idiopathic polyneuropathy as the remaining group. Damaged nerves may recover as the result of spontaneous remyelinization and axonal regeneration. Treatment is particularly successful in immunomediated neuropathies. Withdrawal is often successful in intoxications and suppletion in deficiencies. Even if no treatment is possible, the diagnosis is important: the patient can be taught to accept his disease and the prognosis can be determined, in connection with possible handicaps.