Cytoplasmic layer

III. The inner envelope a syncytial layer showing much variation. Some workers divide this layer into two zones - zone I, a cytoplasmic layer and zone II, a gelatinous layer (Fig. 7.14). Part of this embryonic layer gives rise to the embryophore (Fig. 7.4) and also to the oncospheral membrane (Figs. 7.4, 7.11 and 7.14) (a very thin layer surrounding the oncosphere), which is often counted as a fourth layer. Additional layers, which may be further derived from the above basic envelopes have been reported in some species (e.g. H. nana 204), but it is beyond the scope of this text to discuss all the various modifications which can occur. Only those features which have a special physiological significance are discussed below. 

The presence of a subshell membrane has been noted by a number of workers (439, 440, 541), but it is often difficult to see and its embryonic origin is unknown. In H. diminuta, it is probably represented by the cytoplasmic layer (zone I see below). This membrane appears to be relatively impermeable to many substances and is unaffected by proteases, carbohydrases or lipases. It appears to be a mucopolysaccharide-protein complex and may be important in preventing premature hatching, as well as providing back up protection for the egg shell against a hostile external environment. 

Test Indicates presence of Variety" Shell Cytoplasmic layer Inner envelope Gelatinous layer Embryophore Penetration gland Hooks... 

The majority of proteins occurring in fruits and vegetables play enzymatic roles that are very important in the physiology and postmortem behavior of the crop. The protein content in vegetables is lower than 3%, except in sweet maize (above 4%). In fruits it ranges from below 1% to above 1.5%. They are found mainly in the cytoplasmic layers. 

The lipids of fruits and vegetables are, like the proteins, largely confined to the cytoplasmic layers, in which they are especially associated with the surface membranes. Their content in fruits and vegetables is always lower than 1%. Lipid and lipid-like fractions are particularly prominent in the protective tissues at the surfaces of plant parts — epidermal and corky layers. 

The egg extract will now be separated into three layers. The bottom and darkest layer contains yolk, pigmented granules, etc. the middle layer contains cytoplasmic fraction (the desired material), and the top layer contains lipid-enriched material. In order to collect the cytoplasmic layer, the lipid layer must first be disrupted, which can be accomplished by piercing the lipid layer with a PIOOO pipet tip so as to create a hole. 

Using a new PIOOO pipet tip, collect the cytoplasmic layer (straw-colored middle layer) through the hole in the lipid layer and transfer the collected cytoplasm to a new centrifuge tube on ice ( eeNote 8). 

Again, collect the cytoplasmic layer into a new prechilled centrifuge tube and add LPA, cycloheximide, and cytochalasin D to final concentrations of 10 pg/mL each. 

This clearing step may be repeated if the cytoplasmic layer still contains a significant amount of pigment or lipid material, which may result in proteolysis of p-catenin by non-Wnt pathways. Excessive spins, however, decrease the robustness of the extract to support p-catenin degradation mediated by Wnt components. 

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