Investigations into the occurrence of plant cell surface epitopes in exudate gums

A panel of monoclonal antibodies, derived for the specific recognition of developmentally regulated epitopes of the plasma membranes and cell walls of plant cells, has been characterized in relation to binding activities with samples of exudate gums. The monoclonal antibodies are shown to recognize specifically structural features present in four exudate gums—arabic, karaya, ghatti and tragacanth. Dialysis of the gums and competitive inhibitor ELISAs indicated that the monoclonal antibodies recognize both high and low molecular weight components of each gum. The spatial relationships, and possible overlap, of the arabinogalactan epitopes on the most antigenic gum, gum arabic, have been explored in more detail by means of an epitope mapping technique.     

An electron microscopic study of the baroreceptors in the internal carotid artery of the spontaneously hypertensive rat

Summary The carotid baroreceptor field of normotensive (NTR) and spontaneously hypertensive rats (SHR) examined in this study extends for about 0.5 mm along the length and about 1/3 to 1/2 of the circumference of the wall of the internal carotid artery opposite to the carotid body. The vascular wall of the baroreceptor field exhibits neither a marked dilation to form a carotid sinus nor histological differences in the intima and media compared to other parts of the carotid artery. Histologically the adventitia of the baroreceptor field is characterized by (1) an increased thickness and by less well developed elastic lamellae in comparison with other parts of the arterial wall, (2) a profuse blood and nerve supply, and (3) a richness of cellular elements. The presumptive baroreceptor terminals are localized in the inner 1/3 of the adventitia and display local enlargements that appear to show preferential association with the cell body or processes of the Schwann cell but not with other components of the adventitia. The enlargements are characterized by an accumulation of very densely packed mitochondria, and glycogen particles. No morphological alterations were noted in the baroreceptor terminals of SHR except for proliferated basal laminae that invest the terminals. Our work does not support the concept that resetting of the baroreceptors is due to degeneration of the terminals.Supported by a grant from the Edward G. Schlieder Educational Foundation. Appreciation is extended to Mrs. Lia Pedroza for technical assistance and to Dr. Edward Frohlich of the Alton Ochsner Medical Foundation for supplying the animals used in this research     

Light and electron microscopic studies of diet-induced hepatic changes in mice

Adult mice were fed a choline-deficient ethionine enriched (CDE) diet for 24, 48 or 72 h. They were then fasted for 24 or 48 h prior to sacrifice. All tissues were studied by light and electron microscopy. Animals fed the CDE diet for 24 h exhibited cells with vacuolated cytoplasm, and the accumulation of lipid in these cells was clearly abnormal. Animals fed the CDE diet for 24 h and subsequently a regular diet for 48 h displayed normal hepatocytes, suggesting that the alterations at 24 h were reversible. Following 48 or 72 h of feeding the CDE diet, abundant lipid-laden cells were observed in the hepatic lobules, and at the electron microscope level these cells were undergoing frank degeneration. Evidence indicated that changes after 48 or 72 h were irreversible.     

Interaction of CK1δ with γTuSC ensures proper microtubule assembly and spindle positioning

Casein kinase 1δ (CK1δ) family members associate with microtubule-organizing centers (MTOCs) from yeast to humans, but their mitotic roles and targets have yet to be identified. We show here that budding yeast CK1δ, Hrr25, is a γ-tubulin small complex (γTuSC) binding factor. Moreover, Hrr25''s association with γTuSC depends on its kinase activity and its noncatalytic central domain. Loss of Hrr25 kinase activity resulted in assembly of unusually long cytoplasmic microtubules and defects in spindle positioning, consistent with roles in regulation of γTuSC-mediated microtubule nucleation and the Kar9 spindle-positioning pathway, respectively. Hrr25 directly phosphorylated γTuSC proteins in vivo and in vitro, and this phosphorylation promoted γTuSC integrity and activity. Because CK1δ and γTuSC are highly conserved and present at MTOCs in diverse eukaryotes, similar regulatory mechanisms are expected to apply generally in eukaryotes.     

scFv-based fluorogen activating proteins and variable domain inhibitors as fluorescent biosensor platforms

Single chain antibodies (scFvs) are engineered proteins composed of IgG variable heavy (V_H) and variable light (V_L) domains tethered together by a flexible peptide linker. We have characterized the individual V_H or V_L domain activities of several scFvs isolated from a yeast surface-display library for their ability to bind environmentally sensitive fluorogenic dyes causing them to fluoresce. For many of the scFvs, both V_H and V_L domains are required for dye binding and fluorescence. The analysis of other scFvs, however, revealed that either the V_H or the V_L domain alone is sufficient to cause the fluorogenic dye activation. Furthermore, the inactive complementary domains in the original scFvs either contribute nothing to, or actually inhibit the activity of these active single domains. We have explored the interactions between active variable domains and inactive complementary domains by extensive variable domain swapping through in vitro gene manipulations to create hybrid scFvs. In this study, we demonstrate that significant alteration of the fluorogenic dye activation by the active V_H or V_L domains can occur by partnering with different V_H or V_L complementary domains in the scFv format. Hybrid scFvs can be generated that have fluorogen-activating domains that are completely inhibited by interactions with other domains. Such hybrid scFvs are excellent platforms for the development of several types of genetically encoded, fluorescence-generating biosensors.     

Leukotriene and prostaglandin production in rat brain synaptosomes treated with phospholipase A2 neurotoxins and enzymes

β-Bungarotoxin (β-BuTX) and notexin cause an irreversible blockade of neurotransmitter release through specific and potent effects at the presynaptic nerve terminal, however, the mechanism of action in uncertain. We examined the effects of β-BuTX and notexin on LT and PG production in rat cerebrocortical synaptosomes in order to determine if eicosanoid production might mediate or regulate the pharmacological actions of these phospholipase A₂(PLA₂) neurotoxins. The effects of the PLA₂ enzymes isolated from Naja naja atra and Naja nigricollis snake venoms (which are not presynaptic selective) on LT and PG production were compared with the effects of β-BuTX and notexin. N. n. atra PLA₂, β-BuTX, and notexin (all 50 nM) produced a time dependent rise in free fatty acids as measured in synaptic plasma membranes isolated from treated synaptosomes. Both the PLA₂ neurotoxins and enzymes stimulated LTC₄, LTB₄, and PGE₂ production, as measured by radioimmunoassay. In all cases, the PLA₂ enzymes were more potent than the PLA₂ neurotoxins. This observations correlates with their relative enzymatic potencies, as measured by free fatty acid generation. EDTA and BSA antagonized PLA₂ induced LTB₄ production and BSA also antagonized PLA₂ induced PGE₂ production. These results suggest that stimulation of eicosanoid production does not mediate the potent and specific presynaptic actions of β-BuTX and notexin.