The rate of bulk flow from the Golgi to the plasma membrane

A truncated analog of the backbone of sphingomyelin and glycolipids was synthesized. This truncated C8C8 ceramide was soluble in water (but was still able to cross cell membranes) and was utilized by the Golgi apparatus of living cells to produce water-soluble truncated phospholipids and glycolipids that were then secreted into the medium. Sphingomyelin is synthesized in a proximal (likely the cis) Golgi compartment. At 37 degrees C in CHO cells, the sphingomyelin analog is secreted with a half time of about 10 min. With this rate of bulk flow, no special signal is needed to pass through the Golgi to the plasma membrane. At 30 degrees C the half time of secretion of a lumenal ER marker is about 18 min, and that of the truncated sphingomyelin is about 14 min. Comparison of these rates sets an upper limit of about 4 min for half of the ER to be drained into the proximal Golgi at 30 degrees C.     

Indirect hemagglutination test of Rickettsia orientalis: its specificity for reference strains, Karp, Gilliam and Kato.

Amberlite XE-64 and bovine serum albumin-treated rickettsia suspension, which was prepared from the chicken yolk sacs infected with each of 3 reference strains of Rickettsia orientalis, Karp, Gilliam and Kato, was sonicated at 10 KC for 15 min at 4 degrees C and centrifuged at 10,000 rpm for 40 min at 0 degrees C. The supernatant was used to sensitize the formalinized and tanned sheep red blood cells (SFTSRC). This antigen (2.5%) could be preserved for at least 1 wk at 4-8 degrees C and at least a month, if merthiolate (1:10,000) was added. Each SFTSRCP of 3 reference strains was found to be specific in indirect hemagglutination (IHA) reaction with each homologous immune serum and there was little cross reaction with the heterologous one. This minor cross reaction might be due to the presence of a small amount of soluble antigen in SFTSRC. No cross IHA reaction with the typhus fever immune serum was observed. The IHA test was considered to be useful for the diagnosis of scrub typhus.     

Increased flow of testicular blood plasma during local heating of the testes of rams.

After removal of the scrotal skin, one testis of each of 12 adult anaesthetized rams was kept at 33 degrees C for 60 min, then heated either to 36 degrees C for 60 min and then to 39 degrees C for 60 min, or to 36 degrees C for 120 min and then returned to 33 degrees C for 100 min, while the other testis was maintained at 33 degrees C. Flow of testicular blood plasma was measured every 10 min using the technique of dilution of sodium p-aminohippurate. When the temperature of the testis was raised to 36 degrees C, flow of blood plasma gradually increased and reached a higher than normal rate at the end of the first hour, without any further increase during the second hour. The increase in mean flow rate was 25.8 +/- 3.4% (mean +/- SEM) during the second hour at 36 degrees C, and 77.1 +/- 12.8% during the hour at 39 degrees C, compared with the respective values at 33 degrees C. No significant changes were seen in testicular lymph flow determined by collection for 10 min in four rams at 36 degrees C (60 min) and then at 39 degrees C (60 min). These results are different from those from earlier studies in which total blood flow was unchanged when the scrotum and testes were heated. The difference could be related either to lack of heating of the scrotum or to the lower temperatures used in the present study.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat-induced disturbances of intracellular movement and the consistency of the aqueous cytoplasm in HeLa S-3 cells: a laser-Doppler and proton NMR study.

Intracellular particle movements, of both saltatory and streaming types, in HeLa S-3 cells were simultaneously interrupted after 1 h exposure of cells to 43 degrees C, within 10 min at 44 degrees C and within 5 min at 45 degrees C. Intracellular movement inhibited after 15 min at 44 degrees C and 10 min at 45 degrees C was not reversible in cells rescued at 37 degrees C. Brownian motion was not observed in heat-treated cells while they were maintained at elevated temperatures, but became pronounced in blebbing which occurred shortly after they were returned to 37 degrees C. Returning these cells to 45 degrees C intensified the Brownian activity inside blebs, and rapidly induced cell lysis. The same heat-treated cells were simultaneously studied by laser-Doppler microscopy, which confirmed: a) that flow (cytoplasmic streaming) is completely arrested at 44 degrees C within 10 min, b) flow recovered in 10-15 min in cells rescued after 10-15 min at 44 degrees C, c) submicroscopic particles down to the size of water molecules had faster self-diffusion coefficients at 44 degrees C than at 37 degrees C. Proton nmr studies on cells exposed from 4 to 45 degrees C gave corrected relaxation times T1 and T2 which rose with temperature in a predictable manner. Inhibition of cellular movement at elevated temperatures was not specifically attributable to the depletion of intracellular ATP levels.     

Ascus development in two temperature-sensitive four-spore mutants of Neurospora crassa

Two nonallelic Four-spore mutants are known in which ascospore walls enclose the four immediate products of meiosis rather than the normal eight products of a postmeiotic mitosis. Expression depends on temperature. The Four-spore phenotype is expressed when the developing asci are subjected either to high temperatures (25-30 degrees C) for Fsp-1 or to low temperatures (15-20 degrees C) for Fsp-2. Heterozygous Fsp-1 X Fsp-1+ crosses make eight-spored asci at 15-20 degrees C but produce many four-spored asci at 25 degrees C and mostly four-spored asci at 30 degrees C. Homozygous Fsp-1 X Fsp-1 crosses respond similarly to increasing temperature but make 40-50% four-spored asci even at 20 degrees C. Heterozygous Fsp-2 X Fsp-2+ crosses produce almost exclusively four-spored asci at 15 degrees C but a mixture of four- and eight-spored asci at 20, 25, and 30 degrees C. Homozygous Fsp-2 X Fsp-2 crosses make all four-spored asci at 15 and 20 degrees C and a mixture of four- and eight-spored asci at 25 and 30 degrees C. When both Fsp-1 and Fsp-2 are present in a cross, either homozygous or heterozygous, no asci contain more than four ascospores at any temperature. Limited temperature-shift experiments with Fsp-1 and Fsp-2 show that the sensitive period for Four-spore expression is sometime after meiotic prophase, possibly at interphase II.     

The effects of time and temperature on flow cytometry enumerated live Cryptosporidium parvum oocysts

AIMS: Recoveries of spiked standard suspensions are used to evaluate method performance. For many applications, gamma-irradiated Cryptosporidium oocysts are appropriate. In contrast, methods that determine viability, such as Cryptosporidium cell culture, require the use of live oocysts. Oocyst standards are usually prepared at a flow cytometry laboratory for use at another laboratory, and thus the samples are shipped. The goal of this study was to evaluate the shipping and storage stability of flow cytometry enumerated live oocysts over time at three temperatures: 4 degrees C, room temperature and 37 degrees C. METHODS AND RESULTS: Replicate samples containing 100 live C. parvum oocysts were prepared by flow cytometry and stored at 4 degrees C, room temperature and 37 degrees C. These samples were counted at various time points. Significant oocyst losses were observed after storage for 1 day at 37 degrees C, 7 days at room temperature and 21 days at 4 degrees C. CONCLUSIONS: Live C. parvum oocysts internal standards should be used within 10 days of preparation, and stored and shipped at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: When evaluating method performance with live oocysts, both the storage temperature and time are critical factors for obtaining reliable and accurate results.     

Archaebacterial elongation factor Tu insensitive to pulvomycin and kirromycin

A spermine-dependent, polyphenylalanine-synthesizing cell-free system having an optimum activity at 75-85 degrees C, has been developed from the extremely thermoacidophilic archaebacterium Caldariella acidophila. The C. acidophila system is totally insensitive to the EF-Tu targeted antibiotics pulvomycin (at 40 degrees C) and kirromycin (at 47-72 degrees C) contrary to control systems derived from both mesophilic (Escherichia coli) and thermoacidophilic (Bacillus acidocaldarius) eubacteria. The archaebacterial EF-Tu-equivalent factor is also immunologically unrelated to eubacterial EF-Tu and does not cross react with antibodies against Escherichia coli EF-Tu. The pulvomycin and kirromycin reactions thus provide new phyletic markers for archaebacterial ancestry.     

Skin blood flow during incremental exercise in a thermoneutral and a hot dry environment

Eight physically fit men performed two incremental bicycle ergometer tests, one in an ambient temperature of 25 degrees C and the other at 40 degrees C. Oesophageal temperature (Tes) increased continuously throughout the tests up to 38.0 and 38.3 degrees C, respectively. In both environments, forearm blood flow (plethysmography) was linearly related to Tes above the Tes threshold for vasodilation, but at the heaviest work loads this relationship was clearly attenuated and therefore indicated skin vasoconstriction, which tended to be more pronounced at 25 degrees C. During recovery at 25 degrees C, in some subjects the forearm blood flow increased above the levels observed at the end of the graded exercise in spite of a decreasing Tes. Skin blood flow, measured by laser Doppler flow meter at the shoulder, was quantitatively different but, on average, seemed to reveal the same response pattern as the forearm blood flow. In spite of the higher level of skin blood flow in the heat, blood lactate accumulation did not differ between the two environments. The present results suggest that there is competition between skin vasoconstriction and vasodilation at heavy work rates, the former having precedence in a thermoneutral environment to increase muscle perfusion. During short-term graded exercise in a hot environment, skin vasoconstriction with other circulatory adjustments seems to be able to maintain adequate muscle perfusion at heavy work levels, but probably not during maximum exercise.     

Partitional measurement of capillary and arteriovenous anastomotic blood flow in the human finger by laser-Doppler-flowmeter

This study was made to see whether changes in blood flow through the capillaries and arteriovenous anastomoses (AVA's) of the human finger can be measured by noninvasive flowmetry. Total finger blood flow (FBF) was measured by venous occlusion plethysmography; blood flow was measured by a laser-Doppler flowmeter (ADVANCE, ALF-2100, Tokyo, Japan) using probes with optic fiber separations of 0.3 mm (LDF-0.3) and 0.7 mm (LDF-0.7). The maximum sensitivities for LDF-0.3 and LDF-0.7 were at depths of 0.8 and 1.2 mm from the tissue surface respectively. Two series of experiments were performed on separate days. In the first series the test hand was immersed in a water bath whose temperature (Tw) was 25 degrees C at an ambient temperature (Ta) of 25 degrees C. Tw was raised to 35 degrees C (local hand warming), which was then followed by an increase in Ta to 35 degrees C (whole body warming). FBF, LDF-0.3, and LDF-0.7 increased during these thermal stimulations. However, the relationship of FBF to LDF-0.3 showed two different regression lines. In contrast, the relationship of FBF to LDF-0.7 showed a single regression line. In the second series, with Ta at 35 degrees C, the test hand was immersed in a water bath at Tw 35 degrees C. Tw was then raised every 10 min by 2 degrees C steps from 35 to 41 degrees C. At Tw 39-41 degrees C, FBF and LDF-0.7 in the test hand were significantly decreased compared with those at Tw 35 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Venous return from distal regions affects heat loss from the arms and legs during exercise-induced thermal loads

To study the role of venous return from distal parts of the extremities in influencing heat loss from the more proximal parts, changes in mean skin temperature (Tsk) of the non-exercising extremities were measured by color thermography during leg and arm exercise in eight healthy subjects. Thirty minutes of either leg or arm exercise at an ambient temperature (Ta) of 20 degrees C or 30 degrees C produced a greatly increased blood flow in the hand or foot and a great increase in venous return through the superficial skin veins of the extremities. During the first 10 min of recovery from the exercise, blood flow to and venous return from the hand or foot on the tested side was occluded with a wrist or ankle cuff at a pressure of 33.3 kPa (250 mm Hg), while blood flow to the control hand or foot remained undisturbed. During the 10-min wrist occlusion, Tsk increased significantly from 28.3 degrees +/- 0.41 degrees C to 30.1 degrees +/- 0.29 degrees C in the control forearm, but remained at nearly the same level (28.0 degrees +/- 0.34 degrees C to 28.2 degrees +/- 0.25 degrees C) in the occluded forearm. In the legs, although Tsk on both sides was virtually identical (32.0 degrees +/- 0.31 degrees C, control vs 32.0 degrees +/- 0.36 degrees C, tested) before occlusion, Tsk on the control side (32.6 degrees +/- 0.27 degrees C) was significantly higher than that on the tested side (32.2 degrees +/- 0.21 degrees C) after ankle occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood flow and catecholamine concentration in bovine and caprine skin during thermal sweating

1. Blood flow and the concentrations of noradrenaline, adrenaline and dopamine were determined in the skins of cattle and goats, before, at the onset of and 3 hr after commencement of sweating induced by heat exposure (40 degrees C). 2. The onset of sweating in both cattle and goats was associated with a rise in cutaneous blood flow, which was thus independent of sweat pattern. Cutaneous blood flow was also higher at 40 degrees C than at 15 degrees C. 3. The predominant catecholamine in the skin of both species was dopamine, which in the goat increased in concentration in the warm environment. 4. There was no clear evidence of a change in the amount of any of the cutaneous catecholamines during exposure to 40 degrees C, although there was a consistent tendency for the concentrations of adrenaline in the calf and noradrenaline in the goat, to fall during the onset of sweating.     

Slow flow in axons detached from their perikarya

Slow flow was followed in unmyelinated olfactory axons, severed from their cell bodies, at 14 degrees C, 21 degrees C, and 31 degrees C. Slow flow does not stop after axotomy but rather accelerates to a value 3.3 times faster than the rates measured in an intact nerve. These velocities are equivalent to the rates of slow flow characteristic of regenerating fibers. The injury appears to have an influence on the contralateral intact nerve, where slow flow velocity increases to severed nerve values for several days before reverting to intact nerve rates. It can be hypothesized that the increase in the rate of slow flow is triggered by a factor repressed in intact nerve but released into the blood stream following injury.     

Hydrolysis of oleuropein by recombinant beta-glycosidase from hyperthermophilic archaeon Sulfolobus solfataricus immobilised on chitosan matrix

The recombinant beta-glycosidase (EcS beta gly) from Sulfolobus solfataricus was immobilised on chitosan to perform the enzymatic hydrolysis of commercial oleuropein (heterosidic ester of elenolic acid and 3,4-dihydroxy-phenylethanol (hydroxytyrosol)) at two temperatures (60 and 70 degrees C). Interestingly, on the basis of the reasonable assumption that the enzyme hydrolyses only the sugar linkage, the biotransformation produces unstable aglycone species formed by oleuropein hydrolysis that, differently from some commercially available beta-glucosidases tested, give rise to the formation of hydroxytyrosol, at the operative temperatures of the bioreactor. The results of the biotransformation at 70 degrees C showed that the main products are hydroxytyrosol, and glucose, being the oleuropein aglycone present in low amount at the end of reaction. Both in single step approach or in recycle approach the amounts of glucose and oleuropein aglycone were lightly dependent from flow rate. The amount of hydroxytyrosol, increased on decreasing the flow rate of bioreactor in recycle approach, following a non-linear trend and obtaining the highest value at a flow rate of 15 ml h-1 while in the single step approach the 3,4-dihydroxy-phenylethanol was at its maximum at higher flow rate (16 ml h-1). For the hydrolysis of the oleuropein by bioreactor at 60 degrees C we used lower molar ratio oleuropein/enzyme only by the single step approach. In these conditions it is possible to obtain high amounts of only two products (glucose and hydroxytyrosol) in short time (2 h). The stability of the bioreactor at the operative temperatures showed a t1/2 of 30 days at 70 degrees C and a t1/2 of 56 days at 60 degrees C.     

The heat-stable root-knot nematode resistance gene <Emphasis Type="Italic">Mi-9</Emphasis> from Lycopersicon peruvianum is localized on the short arm of chromosome 6

The tomato gene Mi-1 confers resistance to three species of root-knot nematodes, Meloidogyne spp. However, the resistance mediated by Mi-1 is inactive at soil temperatures above 28 degrees C. Previously, we identified and mapped a novel heat-stable nematode resistance gene from the wild species Lycopersicon peruvianum accession LA2157 on to chromosome 6. Here we report further characterization of this heat-stable resistance against three Mi-1-avirulent biotypes of Meloidogyne javanica, Meloidogyne arenaria and Meloidogyne incognita. Screening segregating F(2) and F(3) progenies, derived from an intraspecific cross between susceptible LA392 and resistant LA2157, for nematode resistance at 25 degrees C and 32 degrees C, revealed a simple dominant monogenic inheritance with all the biotypes tested. We designate this gene as Mi-9. As a first step towards cloning of Mi-9, we constructed a linkage map around this gene. A total of 216 F(2) progeny from the cross between LA392 and LA2157 were screened with M. javanica at 32 degrees C and with CT119 and Aps-1, markers that flank the genetic interval that contains the Mi-1 gene. DNA marker analysis indicated that these markers also flank Mi-9. Further mapping of recombinants with both RFLP and PCR-based markers localized Mi-9 to the short arm of chromosome 6 and within the same genetic interval that spans the Mi-1 region.     

Thermal control and design considerations for a high-performance fluid warmer

The process of warming liquids for intravenous infusion presents several technical challenges for the engineer: Typical liquid inlet temperatures can range from 5 degrees C to 20 degrees C, flow requirements can vary from essentially zero ("Keep Vein Open," or K.V.O.) to 30 L/h, and desired outlet temperature is fixed at a maximum threshold of 41 degrees C to minimize the risk of thermally mediated hemolysis. The primary challenge is developing a control technique that can tailor the energy introduced to the liquid in response to a randomly variable flow in order to achieve, but not exceed, a fixed temperature. The most difficult aspect of this challenge is preventing the transient infusate temperature from exceeding 43 degrees C, even when the power requirement varies by orders of magnitude, such as occurs when the flow suddenly decreases from maximum to zero. Many current-generation fluid warmers are optimized for operation at either low or high flow rates; we believed that it was possible to design an easy-to-use device that could achieve good performance across the entire range of flow rates. This article describes some of the methods that were used successfully to meet these challenges in the design of the Augustine Medical Ranger blood fluid warmer.     

Effect of heat stress on muscle blood flow during dynamic handgrip exercise

During exercise in a hot environment, blood flow in the exercising muscles may be reduced in favour of the cutaneous circulation. The aim of our study was to examine whether an acute heat exposure (65-70 degrees C) in sauna conditions reduces the blood flow in forearm muscles during handgrip exercise in comparison to tests at thermoneutrality (25 degrees C). Nine healthy men performed dynamic handgrip exercise of the right hand by rhythmically squeezing a water-filled rubber tube at 13% (light), and at 34% (moderate) of maximal voluntary contraction. The left arm served as a control. The muscle blood flow was estimated as the difference in plethysmographic blood flow between the exercising and the control forearm. Skin blood flow was estimated by laser Doppler flowmetry in both forearms. Oesophageal temperature averaged 36.92 (SEM 0.08) degrees C at thermoneutrality, and 37.74 (SEM 0.07) degrees C (P less than 0.01) at the end of the heat stress. The corresponding values for heart rate were 58 (SEM 2) and 99 (SEM 5) beats.min-1 (P less than 0.01), respectively. At 25 degrees C, handgrip exercise increased blood flow in the exercising forearm above the control forearm by 6.0 (SEM 0.8) ml.100 ml-1.min-1 during light exercise, and by 17.9 (SEM 2.5) ml.100 ml-1.min-1 during moderate exercise. In the heat, the increases were significantly higher: 12.5 (SEM 2.2) ml.100 ml-1.min-1 at the light exercise level (P less than 0.01), and 32.2 (SEM 5.9) ml.100 ml-1.min-1 (P less than 0.05) at the moderate exercise level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: evidence for a maturation model for the formation of lysosomes

The temperature dependence of acidification of internalized dextran by Swiss 3T3 cells was determined using dual fluorescence flow cytometry. Essentially no acidification was observed at 11 degrees C; acidification was limited to pH 6-6.5 at temperatures between 13 degrees C and 17 degrees C. In contrast, a rapid drop to pH 6-6.5 followed by acidification to pH 5-5.5 was observed at temperatures above 19 degrees C. These results confirm the biphasic nature of the acidification process (J. Cell Biol. (1984) 98: 1757-1762). The timing of exposure of material internalized by fluid-phase endocytosis to lysosomal enzymes was determined for Swiss 3T3 cells by using a fluorogenic substrate specific for Cathepsin B. Hydrolysis of the substrate, as measured by both fluorometry and flow cytometry, began within minutes of its addition to cells at 37 degrees C, and was inhibited by coincubation with leupeptin, a competitive inhibitor of the enzyme, or by weak bases, which raise the pH of acidic compartments. At temperatures between 13 degrees (and 21 degrees C, the rate of hydrolysis was reduced to 31-44% of that at 37 degrees C. Thus, in contrast to previous reports, exposure of endocytosed material to at least one lysosomal enzyme is not inhibited below 20 degrees C; the reduction in hydrolysis rate may be explained by the temperature effects on the efficiency of the enzyme. The results for acidification and proteolysis are consistent with, but do not prove, a maturation model for the formation of lysosomes. We suggest that at lower temperatures, part of the maturation involving recycling and/or concentration of the contents of the endosome is inhibited. This causes the endosome to remain as a mildly acidic, low-density organelle containing lysosomal enzymes.     

Survival of mouse morulae vitrified in an ethylene glycol-based solution after exposure to the solution at various temperatures.

Mouse morulae were exposed in one step to a vitrification solution (EFS, a modified PBS containing 40% ethylene glycol, 18% Ficoll, and 0.3-M sucrose) at various temperatures, then cooled rapidly in liquid nitrogen, and then warmed rapidly. All of the embryos exposed to the EFS solution for 0.5 min at 25 degrees C before vitrification developed in culture. However, survival rates were lower if the duration of exposure was prolonged to 2, 5, or 10 min. At lower ambient temperatures (20, 10, and 5 degrees C), high survival rates were associated with longer exposure to the EFS solution. The toxicity of the EFS solution was also lower at lower temperatures. The toxic injury of morulae was manifested as decompaction of the blastomeres. Among the three additives in the EFS solution, ethylene glycol, which can cross cell membranes, was responsible for the toxicity. The results show that the optimum time for exposure of the embryos to the EFS solution before rapid cooling varies with the ambient temperature, i.e., 0.5 min at 25 degrees C, 0.5-5 min at 20 degrees C, 2-5 min at 10 degrees C, and 2-10 min at 5 degrees C. If they are exposed for an optimum period, almost all mouse morulae can survive vitrification (94-100%).     

Short-term cryopreservation of human breast carcinoma cells for flow cytometry

A procedure is described for short-term cryopreservation of primary human tumor cells and tissue slices for later analysis by flow cytometry. Cells were mechanically dispersed into a freezing medium, which was then frozen at either -20 degrees C or -70 degrees C for delayed cell cycle analysis. The results show that a correlation coefficient of greater than 0.95 exists between cell cycle kinetic analyses performed immediately after surgical excision of the tumor and on cells frozen from 1 to 30 days at -70 degrees C in this freezing solution. Somewhat lower levels of correlation exist for cells frozen at -20 degrees C in this freezing medium. This procedure has also been successfully used to preserve freshly isolated breast carcinoma cells shipped from distant laboratories for analysis in the flow cytometer, thus expanding the data base on certain types of breast carcinoma.     

Effect of temperature on cross-bridge properties in intact frog muscle fibers

It is well known that the force developed by skeletal muscles increases with temperature. Despite the work done on this subject, the mechanism of force potentiation is still debated. Most of the published papers suggest that force enhancement is due to the increase of the individual cross-bridge force. However, reports on skinned fibers and single-molecule experiments suggest that cross-bridge force is temperature independent. The effects of temperature on cross-bridge properties in intact frog fibers were investigated in this study by applying fast stretches at various tension levels (P) on the tetanus rise at 5 degrees C and 14 degrees C to induce cross-bridge detachment. Cross-bridge number was measured from the force (critical force, P(c)) needed to detach the cross-bridge ensemble, and the average cross-bridge strain was calculated from the sarcomere elongation needed to reach P(c) (critical length, L(c)). Our results show that P(c) increased linearly with the force developed at both temperatures, but the P(c)/P ratio was considerably smaller at 14 degrees C. This means that the average force per cross bridge is greater at high temperature. This mechanism accounts for all the tetanic force enhancement. The critical length L(c) was independent of the tension developed at both temperatures but was significantly lower at high temperature suggesting that cross bridges at 14 degrees C are more strained. The increased cross-bridge strain accounts for the greater average force developed.