Monthly Archives: May 2016

Could these be surfactant protein D fuzzy balls?

Layered intracisternal protein granules in mammalian lung, Ultimate order, the cell

It is so fun to try to figure out whether the accumulations of layered protein in the RER in type II alveolar cells are, in fact, surfactant protein A.  These images have sat on my office shelf for 30 years.  As posted a few days ago, this particular electron micrograph has intracisternal bodies or granules or RER accumulation of a layered protein (call it what you want) that were common to guinea pig type II cells, but just one profile of RER had funny round “fuzzy balls” in it.  I only saw this once.  From the beginning it was easy to see that they were not at all like ribosomes, which one might quickly think could get stuck in the bands of such a wild production of a protein, but they are the wrong size, and have a  totally different texture appearance, so ribosomes are pretty much “out of the question” as a choice. The next easiest conclusion was that they were glycogen, also electron dense, but I am not sure why there would all of a sudden be glycogen in these protein (- whatever protein it turns out to be) accumulations, and besides, glycogen is more random in its clumped appearance and is bound within membranes (one can google pictures of glycogen in the liver and see the difference).  But I remembered reading about these wacky “fuzzy balls” probably composed of surfactant protein D (Patrick Waters et al, Chapter 6, in: Target Pattern Recognition in Innate Immunity, ed. Uday Kishore, Landes Bioscience and Springer Science+Business Media, 2009) and did some images tests to determine if these could be surfactant protein D.  Fuzzy balls are the cruciform surfactant D, becomes a multimer  – 4 crucifers in the same 100 nm (others say more like 90 nm) structures.s

I just cropped out ribosomes from the identical electron micrograph as I cropped out a sample of these fuzzy round structures and compared their size, side by side.  You see that this doesn’t come out exactly right.  The proposed size for the surfactant protein D fuzzy balls is just a little bit too large, to fit nicely into the shape of what appears in my micrographs.  In addition, I was hoping that the only protein found in these intracisternal bodies was surfactant protein A, and not containing any other surfactant protein.  But the thought was intriguing.  I do not know of any reports that indicate surfactant protein A can form similar fuzzy balls, but it seems that it might be possible…. and the dimensions for a surfactant protein A fuzzy ball would be closer to the size estimated for these, per the adjacent ribosomes.

Anyway, check out the diagrams of a surfactant D fuzzy ball and a ribosome, matched with their nm markers, and the micrograph of the intracisternal body from which the fuzzy balls were derived.  If you have any interest, or information, I would love to hear about it.  Red rectangle on bottom image is the portion of the fuzzy balls used, while purple rectangle on the bottom image shows where ribosomes were used.

fuzzy_balls

Dark band of RER profile in type II alveolar cell of a guinea pig

Electron microgaphs of lung, Layered intracisternal protein granules in mammalian lung, Ultimate order, the cell

Dark band of an intracisternal body (dark bands in a perpendicular section of these intracisternal bodies span about 100 nm, but when spread out over a tangential cut, can have many more nm from the next dark layer in these inclusions. (I am calling them inclusions for want of a better word, but are rather sites of over-production and layered organization of what I think is SP-A.  It provides an alternative view of the very orderly periodicity (3,5,7,9 – depending upon orientation and how thin the section is).  These two images are identical except that I have increased the contrast, and burned (photoshop) the order which I see in these tangential portions (which likely represent the carbohydrate recognition domains of SP-A.  I thought at first these hexagons were SP-A, then began to wonder if i could find similar structures “anywhere” in “any section” as an artifact of fixation, so went to several micrographs and scanned and examined them for randomly occurring small octagonal structures about 25 nm in diameter.  There are many, but then I used the “light” banded area just as a control to the dark banding in an RER profile though to contain large amounts of organized SP-A as an internal control, and to help rule out the possibility that the fixation turns all proteins into patterned molecules (which it probably does to some degree) but which would fool one into thinking it was SP-A.  So the bottom line is that I think the hexagonal structures (about 20-25 nm in diameter (see ribosomes nearby for comparisons in size at 20 nm).

tangential_guinea_pig_ICB_dark_band

 

Unusual RER profile with SP-A? protein plus other inclusions.

Layered intracisternal protein granules in mammalian lung, Ultimate order, the cell

This particular profile of RER in a type II alveolar cell of a guinea pig has bothered me for at least 3.5 decades. The guinea pig is aged but otherwise untreated, i.e. wasn’t part of a experimental treatment protocol but was part of a study on toluene diisocyanate exposure.  This RER profile has very large cluster inclusions within the periodicity, which is very difficult to see in this particular profile because of the tangential sectioning.  I have no clue what this is, but it shows considerable substructure on enlargement of the micrograph (taken at 23,500x, with the V pole piece in an old Siemens 1A electron microscope, and the enlargement in the darkroom was 4x.  Scan was 3200 ppi.

Several areas which showed a terrific hexagonal substructure were cut and pasted, a marker for their relative nm bar scales given in each, based upon a repeat measure of local (the same micrograph) ribosomes at a pretty much standard 20 nm size.  Please help identify these (ha ha) (I do have comments turned off for obvious spam reasons) you will be able to contact me in other ways.

tangential_guinea_pig_ICB_odd_inclusions

 

Guinea pig alveolar type II cell: SP-A bouquets?

Electron microgaphs of lung, Layered intracisternal protein granules in mammalian lung, Ultimate order, the cell

Plugging away at whether these hexagonal structures found within the RER protein accumulations in some alveolar type II cells which are found on tangential sectioning of what I have called “intracisternal bodies” are really SP-A bouquets which are splayed out with a center dot being the neck and collagen portions of that molecule.  I have taken a portion of one such intracisternal body which has a small portion which is ribosome studded highlighted (using the burn tool in photoshop) a few of the hexagonal arrangements which seem to have six or so densities on the perimeter, possibly the carbohydrate recognition domain trimers of the SP-A 18-mer.

Previously posted images of those found in ferret type II alveolar cells really looked to be larger, and that is problematic.  These hexagonal structures (pictured below) are from an aged, untreated, control, guinea pig  and the bouquets seem more in line with the size of a 20 nm ribosome, a string of which I have included for assisting with perception of relative sizes.  There is a large tangentially sectioned dark band (actually two dark bands) in the image below unaltered, enlarged with the ribosomes in which I have highlighted the hexagonal molecules (using ONLY THE BURN TOOL in photoshop) to accentuate what I see as patterning. The ultimate test of this would be to take an unrelated micrograph of an unrelated RER inclusion, at the same magnification and see if i could pick out as many hexagons per nm in it as I find in a tangential section of an intracisternal body ( ha ha–sort of pedestrian but it would be a good test).

So the upper micrograph gives you a distance marker (bar=100 nm) and the upper box gives you the area of ribosomes and a portion of the intracisternal protein structure which I presume to be SP-A. The lower box gives you the identical area of as is found in the bottom panel, which has two areas of slightly darker electron density marking the tangential portions which are the dark lines of the intracisternal body periodicity.  In both enlargements (boxes) I have highlighted just a few of the very many hexagons, which are hopefully the top down view of SP-A molecules.

For those of you who are curious, a mitochondrion is off to the right, and there are also portions of two large intracisternal bodies, and half a dozen or more single – periodicity intracisternal bodies as well.  In the central intracisternal body on the right hand side shows the 100 nm periodicity (which on a completely perpendicular section contains anywhere from 3 – 9 bands).

guinea_pig_ICB_SP-A_boxesguinea_pig_ICB_SP-A_highlights

More evidence that SP-A bouquets are found in tangential sections

Electron microgaphs of lung, Layered intracisternal protein granules in mammalian lung, Ultimate order, the cell

More evidence that SP-A bouquets are found in tangential sections of the protein filled bodies found in some alveolar type II cells.  I am pretty convinced that these bodies are for the greater part surfactant protein A that takes the form of an highly organized (layered) structure that shows linear organization and period repeats that are consistent with an end to end configuration of surfactant protein A, again mirrored.  That is, bouquet end to tail end, tail end to bouquet (times 2) per 100 nm period with the central and outside lines of the periodicity representing the carbohydrate recognition domains.

Previous posts show this intracisternal protein on cross section perpendicular to the long axis of the periodicity – or layering, which is easily recognized, and quite orderly, but since the protein inclusions also show curvature and in some cases end to end exchanges in layering direction, and others even as single tubules on cross section, and there are usually  “growing ends” with ribosomes along that growing edge of the RER profile.  The number of ribosomes counted out comes to something on the order of 2 ribosomes per period, and also there are occasions (shown on this blog site) where ribosomes and the protein (presumably surfactant protein A) appear in the same configuration that one would expect from a top down view of the SP-A bouquet right adjacent to each other, it just seems that the anatomy and the protein structures fit together.

All these new data were found 35 years after the initial discovery of that structure with TEM and that initial search for what this body was, and which started back in 1980 when I queried a respiratory scholar and pediatrician then at CCHMC — who was/is a surfactant specialist — but who also was not interested.  So this study was taken off the back burner in 2016 for a little fun and replay.  I am quite sure that this RER compilation of protein represents an overproduction of surfactant protein A which behaves in the RER profile like other collectins (langerin being a great example and even showing layering patterns onto which a langrin molecule image can be placed, though producing a different orientation of periodicity than SP-A, but conceptually similar, and obviously related in the greater scheme of the way collectins are ordered within the RER.   Here is a quick animation that shows one such tangential section of an RER  profile filled with this protein, but the periodicity  is spread by a tangential orientation to the plane of the section. The darker regions of the image relate to the darker regions of the banding and those areas represent the top clusters of trimers, assembled as the 18 mer of SP-A open and with a center dot presumably representing the neck. Thee micrograph was only contrast enhanced and the burn tool used in photoshop, two scratches were edited out with the bandaid tool, otherwise pixels were unchanged from the original micrograph scanned at 3600 pixels per inch.  Images imported into CorelDRAW, exported as transparent png images, and the animation was made with Swishmax 4. (no data were changed)

Thin sections here are probably about 100 nm thick, maybe a little more, thus there is overlap between one 18 mer and the other.  It has to be determined (but looks plausible) that the hexagonal SP-A stacks up like a honey comb.

Frigidaire standalone oven and cooktop: Planned obsolescence – where is corporate conscience?

Rants

I am not alone in thinking that the big companies, those that are “too big to fail” are predators.   Not that the makers of Frigidaire are too big to fail, because I am sure if the consumers rose up against companies that quit making parts for perfectly good units just to press consumers into whole item purchases, for the company profit (greed is a better term), they would surely fail.

Why am i forced into being a contributor to landfill, to waste, to consumerism when my own conscience tells me that repair, reuse, recycle is the more “cosmos friendly” way to co-exist with nature.  I don’t understand why we, the people, let big companies dictate our future.  Makes me really irate.  So I must add to landfil 75 pounds of metal and mixed other stuff because Frigidaire no longer makes the clock/timer part for my REGC39  oven and cooktop? Shame on you big corporations.  What a truly sad commentary on corporate conscience.

Intra-cisternal protein in type II alveolar cells in dogs

Layered intracisternal protein granules in mammalian lung, Ultimate order, the cell

Still working on whether this is SP-A (surfactant protein A) in the layered protein paracrystalline type banding seen in the RER of at least three species of mammal. The most prominent of these banded protein products in the RER of alveolar type II cells seems to have occurred in guinea pigs, though they were first observed in ferrets.  These six images are high mag images where just the RER with the layered protein are highlighted (with the black arrow). Since they all came from different images and magnification is just to messy to calculate I have substituted the 20 nm ribosome as a “scale”  so the red dots are a “relative” measurement of 20 nm.  This puts all the intracisternal bodies in perspective.

The dog is different than the other two species in which the intracisternal body in RER of type II alveolar cells is seen as I have only found them with a single banding period…. whereas in guinea pigs the number of periods in a single RER profile with this protein can be very large and difficult to count because of the perpendicular and arched and oft changing directions of the layering.  However, the mean number of bands in guinea pigs, 787 periods,  n=189 RER profiles,  produced a mean of 4.1 (SD=2.9) +- 0.21 (SEM) periods per profile while in ferret of the 625 periods counted, n=123 profiles of RER, the mean number of periods per profile of RER was 2.66  (SD=4.9) +- 0.39.  Mongrel dog had very few profiles, and number of profiles was = to the number of periods (n=7).  Six of those are shown in the figure below:  they were about 100 nm thick and maybe twice that long.

dog_ICBa

More ribosomes and SP-A? in alveolar type II cells

Layered intracisternal protein granules in mammalian lung, Ultimate order, the cell

Adding some SP-A profiles (?) and ribosomes from a second tangential section through an intracisternal body.  I am more and more convinced that the major dark band seen in these profiles within the RER of some type II alveolar cells (many of which are posted in this blog) when sectioned tangentially show the bouquet of the 18-mer of SP-A splayed out giving a hexagonal- or oval-like structure.  These are not plentiful, but also not infrequent, and are just prominent enough to be given attention.  As a comparison, I have examined immediately adjacent cytoplasm and do not see this kind of arrangement of the cytoplasm, therefore it seems NOT to be an random artifact of fixation (though we all recognize that fixation induces artifact of necessity) the hexagonal patterns are found in tangentially sectioned areas of the intracisternal bodies.

ribosomes_SP-A-perhaps_more

Ribosomes and SP-A? at RER membrane

Layered intracisternal protein granules in mammalian lung, Ultimate order, the cell

This is an interesting picture. It derives from a micrograph from an untreated ferret (my animal # 2, negative 4640, block 18578, 27,900 x magnified 4 x) and scanned and processed in photoshop  using contrast, color balance, dodge and burn only.  While continuing to look for molecules that might be surfactant protein A, the round to hexagonal objects with a central density appeared sometimes to be closely linked with ribosomes at the edge of the RER membrane when the occasional opportune tangential section of an intracisternal body in a type II cell is achieved.

These round SP-A? molecules are more prominent in guinea pig micrographs, but this array along the growing edge of a profile of RER was particularly nice in that several adjacent ribosomes were adjacent to several round-to hexagonal profiles of what might be protein product.

I have highlighted the ribosomes and round structures in blue, put a red arrow pointing the a ribosome, a light red line around one such round to hexagonal structure (left in the figure below) and also shown a blue box around the original cisternal body and ribosomes (photoshopped only with contrast and brightness) (right in the figure below.  The relative sizes: ribosomes, approx 20 nm, the hexagonal structures about twice that.

Most publications give the SP-A molecule as being around 25 microns…. i think if the “bundle of flowers” typically assigned to SP-A 18-mer were to spread out at the head… that the dimension when seen from the top could closely approximate the dimension found here.  Each ribosome can be used as a guide to magnification.

ribosomes_n_spa_at_membrane

Ribosomes and SP-A perhaps?

Layered intracisternal protein granules in mammalian lung, Ultimate order, the cell

One transmission electron micrograph gave me a particularly interesting view of the intracisternal protein contents of a type II cell which has the large bodies filled with what I think is an organized layering of surfactant protein A.  This tangential section was unique in that I could find many little objects which comprised a central density and six densities surrounding it…. making it pretty interestingly similar to the “bouquet” style protein 18-mer that is touted to be representative of surfactant protein A.

I have cut and pasted out ribosomes from the membrane of that RER profile for an approximation of the size (which is pretty clearly like other ribosomes at 20 nm in diameter) and kept the sizes relative to these circular hexagonal-with-central density areas in sync.  You can see that they are just slightly larger than the ribosomes themselves, and that the estimated size fot he SP-A molecules (also given at about 20 nm) might be within tolerable limits.

The top rows of the image below are ribosomes and these circular proteins from a single tangentially cut profile of RER (cisternal body) from a guinea pig type II cell;  the bottom set is from a not quite as nice tangential section of ribosomes and the circular proteins from a cisternal body of a ferret type II cell.

Below that image is a low res pair of images (guinea pig on the left ferret on the right) from which the ribosomes and SP-A? were selected.

I included one ribosome adjacent to a round SP-A ? molecule that has its own bar=20 nm.

ribosomes_SP-A-perhapstwo_trangential_profiles