Existence of straw cells in tissues and in cultures
A wide range of tissue types and cell lines from different mammals have been surveyed for the existence of straw cells. Freshly harvested tissues of brain, heart, liver, lung and skin of young cows, and frozen tissues of normal and tumor sections of mouse prostate have straw cells in their native environments (Figure ). Tubular structures in the hundreds were identified from 100 mg of brain and heart tissues, and in the tens of thousands from liver, lung and skin tissues. Approximately 800 straw cells/μl of tissue wash fluid (100 mg tissue: 100 μl water) were quantified from bovine lung tissues (freshly slaughtered at 1 to 2 years age). In experiments with cultured cells, Human MCF7, CACO2, THP-1 and mouse D1 cells formed similar tubular shapes when dehydrated (Figure ). Initially, very fine needle-like structures that we have termed filaments protrude from the cell surface. These filaments continue to elongate and fuse with filaments from other dehydrated cells forming an integrated network. The cell body itself becomes much smaller and assumes a tube-like structure. Cells that are detachable and mobile can cooperate in a coordinated manner by forming collaborative networks to acquire and distribute water may have increased their chances for survival.
Figure 1 Tubular structures found in tissues and cultures. A. Pre-existence of tubular structures in six tissue types from fresh prepared three-month-old female calf and in frozen prostate tissues from mice. Straw cells typically possess several filament branches (more ...)
The timing of transformation varies in vitro, with the majority of structures becoming visible at complete dehydration. Typically, more than 80% of cells transform with a population of 10,000 cells in 0.5 ml medium per well. Cell density, salts, the volume of the medium and drying rate affect the amount of transformation and filamentous growth. Cells dehydrated in a 2 × volume cell free phosphate saline medium and subjected to the same dehydration conditions produce no tubular structures. This suggests a nutritional requirement for negating transformation. Because the transformation mechanism appears to be conserved across mammalian cell types to include differentiated macrophage (THP-1) and adipose cells (D1), it is reasonable to consider that dehydration exhibited a selective pressure on primitive cells. Cells that are detachable and mobile can cooperate in a coordinated manner by forming collaborative networks to acquire and distribute water may have increased their chances for survival.
SEM images of straw cells and filamentous networks in solution are shown in Figure . The filaments are spread out and connected to form networks, they originate from the tubular center, and the joints of the connections appear to be smooth and seamless. Cross-sections of the straw cells are shown in TEM images (Figure ). The samples were prepared from dehydration-transformed cells that were precipitated at 16,000 g, depending on the length of filamentous extensions; straw cells were either in the pellet or in the supernatant. The central tubular wall has a thickness of 200 nm; thirty times thicker than the lipid bilayer of a normal cell membrane. The wall structure appears dense and compact, and stained lightly with regular 1% osmium oxide fixation. The filamentous extension's wall is approximately 120 nm thick, 80 nm thinner than the central tubular wall. Unlike the tubular center, the filamentous extensions have a nearly perfect homogeneous content and no recognizable organelles.
The volume of a tube is much smaller than that of the main cell body, indicating that most of the cellular material has been condensed. Filaments, typically between 1 and 10 μm in diameter, often extend far from the location of the main cell body. In one instance, we observed filament growth to lengths of over 4 cm, an increase in approximately two thousand fold of the original length of the cell and seemingly confined only by the culturing well. Filaments were extended in multiple directions from cells along the culture flask as well as through the air. TEM images show the straw cell wall to be markedly robust, which probably indicates a state of dormancy and prevention of water loss.
Straw cell properties
Time course studies from four young animals revealed that straw cell population increases with the age of the animal in heart and lung tissues, but stays constant for brain, liver, and skin tissues (Figure ). When tissues post mortem were dehydrated in air, the straw cell population increased in brain and heart tissues. Brain tissues, when dehydrated for three days, produced straw cells comparable in number to those of liver and lung tissues at the initial stage of transformation (Figure ). On average the transformation from normal cells to a tubular structure occurs over an 8 h period.
Figure 2 Functional assays.A. The population of transformed tubular structures correlates increasingly with the age of the animal. B. Production of straw cells in postmortem tissues.C. Effect of UV-C radiation on regular and transformed cells. A lethal UV-C dose (more ...)
A lethal dose of UV-C radiation (10 min) did not affect tubular transformation of cultured cells (Figure ) or in postmortem tissues (Figure ). UV-C radiation also seems to have no effect on straw cell reversion to a normal round state (explained in 2G). During hydration, water passes rapidly through the straw cell network. Water movement through the straw cell network was estimated on a sub-second scale (Figure , the first two). Tubular structures from different cell types were connected to each other (Figure , last).
Normal cells stained positively for actin and nucleic acids (Figure , first two), whereas early tubular cell bodies were barely stainable and negative for mature straw cells and filaments (the middle panels). The tubular wall appeared to be impermeable to small molecules such as ethanol, paraformaldehyde and TritonX-100 used in the staining procedure. When rehydrated, filaments quickly disintegrate. Figure , left bottom, displays collapsed filaments stained with rabbit straw cell polyclonal antibody. Collapsed filaments in solution produced numerous round shaped dots similar in size to large as E. coli cells. Antibodies generated against purified straw cells have produced positive signals from human urine but not feces (Figure , right bottom). Transformed cells obtained from non-transformed cells in vitro were re-plated in fresh medium in new 4-chambered-wells. More than 50% of the straw cells regained their normal spherical shape and began to divide in 5 – 15 days (Figure ). Newly recovered cells were still resistant to staining indicating that they were still in a stressed state. The morphologies of these recovering structures are presented in Figure .
Figure 3 The morphology of reviving tubular structures. A. Time course images of the tubular structure into regular shaped cells over a period of 0 to 20 days. B. The life cycle of dehydration-induced straw cells. Newly emerged cell has granular surface with smaller (more ...)
Purified filaments were isolated from media and separated from lipid and protein fractions. The filaments were laid down on a glass slide for viewing with some strands being measured up to 4 cm in length (Figure ). Morphologically, the surface of the filaments is characterized by numerous hair-like structures and small circular openings (Figure ). Interestingly, the structures are somewhat similar to that of hairy roots from a plant whose purpose is to absorb water and nutrients.
The cycle of dehydration and rehydration is characterized in Figure . When rehydrated, straw cells became visible and attached to the plate bottom in 5 days. There are dark, irregular shaped bodies (day 5 and day 10 left panels) that give rise to round-shaped cells in 5 to 10 days with different morphology. Dot-shaped particles with a dimension of 1 μm (Figure , day 5, middle panel) enlarged to 10 μm size in 5 to 10 days. Some cells appeared to recover more readily than others with a more robust surface structure than cells in the panel to the right, which took a longer period of time for recovery. Cells of different dimensions developed from the matrix over the course of 30 days (day 17, left panel). When recovering cells reached a diameter of approximately 10 μm, the overall cell morphology assumed a normal non-dehydrated appearance. Seventeen days later, they were indistinguishable from normal cells (day 17, right). The dehydration-rehydration transformation cycle is summarized in Figure . We repeated this 30-day cycle for several months and observed that cells were able to cycle back and forth between the straw cell and normal morphologies in response to dehydration and rehydration, respectively. However, the number of total viable cells diminished with each successive cycle.
Chemical compositions of the filaments
Fourier-Transform Infrared Spectroscopy (FTIR) was used to examine the chemical composition of the filaments. Proteins and carbohydrates absorb infrared radiation and the intensity and characteristic bands provide the fingerprint of these molecules [9
]. Alterations in individual vibrational modes and changes in coupled vibrations can be used to assess hydrogen bonding among proteins and carbohydrates [11
]. In the filaments, several bands have been shifted relative to the control spectra of proteins (Figure ). In the fingerprint region of 900 – 1500 cm-1
, common bands can be seen between test samples and the controls around 1650 cm-1
, 1550 cm-1
and 1050 cm-1
. Bands assigned to hydroxyl stretching modes (3350 cm-1
) in the protein control samples (spectra labeled Serum and BSA) were decreased in intensity and shifted to higher frequencies (3450 cm-1
) for peaks in the same range in the test samples, which is possibly due to disrupted hydrogen bonding in the test samples. Amide I band vibrations (carbonyl stretch at 1650 cm-1
) were shifted to lower frequencies in the test samples, while the CH2
stretching modes 2850 cm-1
) appear sharper in both the absorbance spectra of the straw cells (spectrum labeled CACO) and the blue dextran (spectrum labeled BD), but broader in the spectra of the protein controls. This disparity suggests that the straw cells could contain larger amounts of carbohydrates compared to the controls. These effects mimic those of hydrated trehalose [9
]. Taken together, FTIR recorded on CACO2 and bovine straw cells, along with protein (serum, BSA) and polysaccharide (blue dextran) standards indicate that elevated carbohydrate levels most likely contribute to the chemical composition of the straw cells.
Figure 4 Chemical compositions of the filaments by. A. FTIR absorbance spectra of the straw cells (CACO2 and bovine liver), along with proteins (serum, BSA), polysaccharide (blue dextran) controls indicate the straw cells are dominated by bands characteristic (more ...)
Protein composition of the filaments was estimated on SDS/PAGE. Collapsed tube separation on SDS/PAGE showed that approximately 1% (w/w by densitometry) consisted of protein (Figure ). Two bands with the molecular weight of approximately 50 kD, along with a BSA band at 66 KD were observed. BSA appeared to be purified by a factor of 5,000 fold when compared to the initial medium having a 10% fetal bovine serum (top band). The protein bands were processed by the in-gel tryptic digestion procedure and micro-sequenced. Among the proteins identified from these two bands using the data dependent acquisition method was a human 40S ribosomal protein (P04643). Western analysis of collapsed straw cells on SDS/PAGE revealed a single band around 212 KD (Data not shown).
Mass spectrometry analysis of partially hydrolyzed filaments revealed a series of six glucose polymers; sulphated glucose polymers and N-acetylglucosamines polymers were among the polymers identified (Figure ). The polysaccharides were highly acidic and carried multiple negative charges at pH 7.0. The conditions favoring singly charged ions were optimized for detection in both positive and negative ion modes. Three linear polymers differ from each other by a glucose unit (162.06) and a fucose (146.08). A GlcNAC (203.08) monomer was seen in 1 N HCl hydrolyzed bovine liver filaments (Figure ). Among identified monomers, fucose (146.08) may be involved in branching. N-acetylglucosamines is a common unit at the reducing end of the bi-antennary structures. Three linear glucose polymers from tubular CACO2 cells differed by a neutral loss of sulphated glucose (242.0) as displayed in Figure . Serial-A sugar ions appear to be the oxygenated form of serial-B sugar ions and serial-C sugar ions were shown to carry two negative charges. These polysaccharides can be summarized in the following structure: R1 and R2 attached to C4 (Figure ). The C6 and C2 of the glucose can be present either individually or together and was found in a range of just a few to over a million, when estimated by a gel filtration experiment (data not shown). The acidic makeup of straw cells causes them to be highly hydrophilic. This is the probable reason for their presence in the supernatant of sequential treatment of (1) centrifugation at 16,000 g; (2) chloroform: methanol extraction; (3) C18 reverse-phase matrix binding, (4) 100°C, 5 min heat treatment and (5) 5%TCA precipitation.
Figure 5 Carbohydrate compositions by Mass spectrometry. A. Structural characterization by negative electrospray ionization mass spectrometry. MS analysis has been carried out on oligosaccharides obtained from partial hydrolysis of bovine liver filaments. Spectrum (more ...)
The radioisotope-labeling technique was used to determine if the straw cells were either a product of a metabolically active process involving active cellular growth or if they were a lifeless, physical process involving spontaneous polymerization of cellular materials. Radioisotope labeling of the growth medium using (1-14C) glucose determined that the incorporation of 14C glucose was more prevalent for dehydrated cells than for normal growing cells, indicating an elevated energy need and increased carbohydrate content of the straw cells (Table ). More 14C was also found in protein fractions from cultures with tubular transformations than from regular growth cultures, indicating that an active amino acid and/or protein metabolism is involved in the process. Overall, the tubular transformation process appeared to be more active metabolically than for normal growth.
Percent radioactivity recovery in cellular fractions from (1-14C) glucose labeled MCF7 cells
Collapsed filaments found in urine
The HPLC method was used to analyze urinary samples from people at different ages for total carbohydrate quantification. Information on urination volume and time lapse from last urination was collected and used to calculate the carbohydrate production per hour per person, which was in turn converted to number of straw cell structures, based on the equivalency of the carbohydrate mass per tubular structure. Urine carbohydrate levels were found to fluctuate before and after meals, but the basal carbohydrate level remained stable, suggesting that it may be proportional to the straw cell wall material secreted in urine. We discovered that the basal carbohydrate level (lowest urinary carbohydrate content) plotted against age; a polynomial correlation (R2 = 0.94) between tubular cell production and human ages ranged from 3 to 80 years old (Figure ).
Figure 6 Analysis of urinary straw cells by HPLC. Samples were collected from eight individuals whose urine sugar level was measured using methods adapted from the literature [40, 42]. Quantitative analysis was on C18 reverse phase column chromatography on the (more ...)
Urinary glycosaminoglycans (GAGs) found widely in animal urine at levels possibly associated with diseases having repeated units of sulphated glucose [13
], may be derived from these tubular straw cell structures which contain sulphated glucose polymers. Because these filaments were visually detected in urine, the total urinary polysaccharide content may reflect the degree of straw cell formation inside the body, providing an indicator of the aging process.