Transplantation of rabbit kidneys perfused with glycerol solutions at 10 degrees C.

Experiments are described in which rabbit kidneys were perfused with solutions containing 2, 3, or 4 m glycerol and then were autografted in order to measure any functional impairment. The basic perfusate contained Haemaccel (17.5 g/litre) and mannitol (111 mm) and had a total osmolality of 400 mosmol/kg. The perfusion temperature was 10 °C. Glycerol concentration was slowly increased at a rate of 30 mm min^?1 to 2, 3, or 4 m, was held at the maximum concentration for 30 min, and was then decreased to < 0.1 m at 30 mm min^?1. An immediate contralateral nephrectomy was carried out, and the function of the perfused kidney was assessed by serial measurements of blood urea and serum creatinine, by the determination of 24-hr endogenous creatinine clearance at 1 month and by histological examination. It was found that a concentration of 3 m glycerol was readily tolerated.     

Relationship between variations in pathogenicity and lag phase at 37°C of Listeria monocytogenes previously stored at 4°C

s. BUNCIC AND S.M. AVERY. 1996. Three haemolytic, pathogenic strains of Listeria monocytogenes (a reference strain, a food-derived strain and a human strain) were held at 4°C for 4 weeks in phosphate-buffered saline pH 5.5 or 7.0, with and without 0.2% potassium sorbate or 0.3% sodium acetate. The number of viable cells did not change significantly during this storage. Pathogenicity of non-growing L. monocytogenes cells for 14-d-old chick embryos was determined before and after storage. Storage at 4°C resulted in decreased pathogenicity, but effects were strain-, pH- and substrate-dependent. After 4 weeks storage at 4°C non-growing bacterial cells were transferred to Brain Heart Infusion broth and growth characteristics were determined during incubation at 37°C. Strains that showed decreased pathogenicity had significantly longer lag phases at 37°C than strains that maintained pathogenicity. It is concluded that decreased pathogenicity of L. monocytogenes stored without growth at 4°C for 4 weeks and subsequent long lag phase at 37°C are correlated.     

The water and cation content of nonmetabolizing perfused rabbit kidneys.

Rabbit kidneys treated with cyanide and iodoacetate have been perfused with solutions containing various compounds that were intended to control the passage of fluid across the capillaries or the cell membranes. Following perfusion the albumin and EDTA space of each kidney and its water and cation content was measured. Total water content increased significantly unless 7–8% ($\text{w}\text{v}$) polyethylene glycol or dextran of mean MW 6000 was present in the perfusate. The EDTA space always increased, but this may have been due, at least in part, to penetration of the intracellular space by the marker. The albumin space was increased only in the presence of hydroxyethyl starch or sucrose. All the kidneys gained sodium and lost potassium, but this effect was least with the perfusate containing dextran of MW 6000 which also gave the lowest EDTA space, a normal total water content, and a normal vascular resistance. It is suggested that the addition of low molecular weight polymers to perfusates used for organ preservation will help to control edema, which may result in improved function after transplantation.     

Promoter-independent cold-shock induction of cspA and its derepression at 37°C by mRNA stabilization

The gene for CspA, the major cold-shock protein of Escherichia coli is known to be dramatically induced upon temperature downshift. Here, we report that three-base substitutions around the Shine–Dalgarno sequence in the 159-base 5'-untranslated region of the cspA mRNA stabilizes the mRNA 150-fold, resulting in constitutive expression of cspA at 37°C. This stabilization was found to be at least partially due to resistance against RNase E degradation. The cold-shock induction of cspA was also achieved by exchanging its promoter with the non-cold-shock lpp promoter. The results presented indicate that the cspA gene is efficiently transcribed even at 37°C. However, the translation of the cspA mRNA is blocked because of its extreme instability at 37°C. The presented results also demonstrate that the cspA gene is constitutively transcribed at all temperatures; however, its expression at 37°C is prevented by destabilizing its mRNA.     

Effect of fatty acid supplementation on the lipid composition of Mycobacterium smegmatis ATCC 607, grown at 27° and 37°C

Mycobacterium smegmatis ATCC 607 was grown at 27 and 37°C, with and without exogenous unsaturated fatty acids, viz. elaidic, oleic and palmitoleic acids, added to the growth medium. The total lipid content of M. smegmatis ATCC 607 was lower at 27°C, and with added oleic acid, when compared with the controls, but higher in presence of palmitoleic acid. At 37°C no significant differences were noted in the total lipid content. In general, the total lipid content was lower with all of the fatty acid supplementations at both 27 and 37°C. The phosphatidylethanolamine content was slightly higher at 27°C in the presence of elaidic or palmitoleic acid, but was markedly lower with oleic acid supplementation at 37°C. The cardiolipin content was lower in the presence of any of the fatty acids at 27°C, and higher in the medium supplemented with elaidic or oleic acid at 37°C. The unsaturated to saturated fatty acids ratio was higher with palmitoleic acid supplementation at 27°C, but remained unchanged in cells grown at 37°C. The modifications in mycobacterial lipids are a reflection of the organism's ability to adapt to changing growth conditions.     

Loss of Viability and Metabolic Injury of Staphylococcus aureus Resulting from Storage at 1°, 3°, 5° and 7°C

Staphylococcus aureus incubated in Tryptic Soy Broth at 1°, 3°, 5° and 7°C became increasingly sensitive to Mannitol Salt Agar. Injury, as measured by salt sensitivity, decreased with increasing temperature from 1° to 7°C.     

Introduction and removal of cryoprotective agents with rabbit kidneys: assessment by transplantation

Rabbit kidneys were perfused with up to 4 M glycerol or propane-1,2-diol (propylene glycol, PG) in three vehicle solutions: one normokalemic and made hypertonic with mannitol (HP5), one hyperkalemic but without mannitol (HP6), and one hyperkalemic and with mannitol (HP7). Subsequent function was assessed by autotransplantation. Up to 3 M glycerol in HP5 was well tolerated but not in HP6 or HP7. Conversely, up to 3 M PG in HP7 was compatible with excellent post-transplant function, but the same concentration in HP5 was severely damaging. PG (4 M) in either solution was severely injurious and no kidneys survived perfusion with this concentration. Vascular resistance was well controlled by the vehicle solutions with mannitol, but it was generally higher during perfusion with the hyperkalemic HP7 compared with the normokalemic HP5. No kidneys perfused with 3 M solutions of either of the cryoprotective agents and cooled briefly to -6 degrees C without freezing had any post-transplant function, and neither did kidneys perfused with 3 M PG or 4 M glycerol tolerate slow cooling to -80 degrees C and warming. The need to optimize perfusate composition for the CPA being used is clear, and the dramatic increase in toxicity of PG when the concentration exceeds 3 M supports the suggestion that mixtures of PG and glycerol should be considered. The observation of damage at high subzero temperatures, before freezing has occurred, requires further detailed study.     

Production of exotoxins by Aeromonas spp. at 5°C

The ability of 60 strains of Aeromonas to produce enterotoxin and haemolysin after cultivation at 5°C for 7–10 d was investigated. The strains were isolated from lamb meat, offal, carcasses and faeces, and had previously been tested for their ability to produce these exotoxins at 37°C. The results showed that some strains of Aeromonas hydrophila and A. sobria were capable of producing enterotoxin and haemolysin at 5°C, but none of the A. caviae strains tested produced these two factors. Of the 30 A. hydrophila strains investigated 25 and 27 were enterotoxigenic and haemolytic respectively. Likewise, of the 24 A. sobria strains investigated 16 and 18 were enterotoxigenic and haemolytic respectively. The results indicate that certain strains of Aeromonas species, in particular A. hydrophila and A. sobria , are of potential public health significance in meats stored at refrigeration temperature.     

Reducing the time rabbit sperm are held at 5 °C negatively affects their fertilizing ability after cryopreservation

Cooling sperm to and equilibrating the sperm at 5 °C require the most time in any sperm cryopreservation protocol. Reducing the time required for these phases would simplify sperm freezing protocols and allow greater number of ejaculates to be processed and frozen in a given time. This study determined how holding rabbit sperm at 5 °C for different lengths of time (0, 10, 15, 20, 30, or 45 minutes) affected the quality of rabbit sperm, measured by in vitro assays, and if reducing the cooling time to only 10 minutes affected the fertilizing ability of the sperm. Reducing the time sperm were held at 5 °C to 10 minutes did not affect the in vitro quality of the sperm (percent motile and with intact plasma membranes), although eliminating the cooling phase completely (directly freezing the sperm from room temperature) decreased in vitro assessed sperm quality (P < 0.01). However, reducing the time sperm were held at 5 °C, from 45 to 10 minutes, negatively affected the fertilizing ability of sperm in vivo (P < 0.05). In conclusion, completely eliminating cooling rabbit sperm to 5 °C before freezing is detrimental for rabbit sperm cryosurvival, and although shortening the time sperm are held at 5 °C to 10 minutes does not reduce in vitro sperm quality, it does reduce the fertility of rabbit sperm. Therefore, the length of time rabbit sperm equilibrate at 5 °C is crucial to the fertilizing ability of rabbit sperm and must be longer than 10 minutes. Currently, it is not known if holding rabbit sperm at 5 °C for less than 45 minutes will affect sperm fertilizing ability.     

Scaling of oxygen consumption of Lake Magadi tilapia, a fish living at 37°C

Rates of oxygen consumption were measured in the geothermal, hot spring fish, Oreochromis alcalicus grahami by stopped flow respirometry. At 37° C, routine oxygen consumption followed the allometric relationship: V o₂=0.738 M ^0.75, where V o₂ is ml O₂ h ⁻¹ and M is body mass (g). This represents a routine metabolic rate for a 10 g fish at 37° C of 0.415 ml O₂ g⁻¹ h ⁻¹ (16.4 μmol O₂ g ⁻¹ h ⁻¹). Acutely increasing the temperature from 37 to 42° C significantly elevated the rate of O₂ consumption from 0.739 to 0.970 ml O₂ g ⁻¹ h ⁻¹ ( Q ₁₀=l.72). In the field, O. a. grahami was observed to be 'gulping' air from the surface of the water especially in hot springs that exceeded 40° C. O. a. grahami may utilize aerial respiration when O₂ requirements are high.     

The biosynthesis of 2-guanidinoethanol in intact mice and isolated perfused rabbit kidneys

The metabolic pathway for the synthesis of 2-guanidinoethanol (GEt) was studied in intact mice and isolated perfused rabbit kidneys. GEt excretions in 24-hr urine increased after the intraperitoneal injection of ethanolamine (EA) into mice. Perfusion of isolated rabbit kidneys with EA and L-arginine (Arg) enhanced the GEt excretion from the ureter. This enhancement was observed in an EA concentration-dependent manner under the presence of Arg. When glycine (Gly) was added to the perfusion medium together with EA and Arg, the enhancement of GEt excretion was inhibited, whereas, guanidinoacetic acid excretion was increased to the same extent as during the perfusion with Gly and Arg. These results indicate that GEt is synthesized from Arg and EA in the kidney and that this synthesis is catalyzed by Arg:Gly amidinotransferase (EC 2.1.4.1.). We also described the guanidino compound excretion levels, including levels of GEt, in the rabbit, mouse, rat, and cat. The levels varied considerably with mammalian species.     

Production of cell wall dissolving enzymes by Erwinia carotovora subsp. atroseptica in vitro at 27°C and 30°5C

Cultures of Erwinia carotovora subsp. atroseptica , grown at 27°C and 30·°C in different liquid media were assayed for activities of pectate lyase, polygalacturonase and cellulase. Total production of both pectate lyase and of polygalacturonase was 3–6 times less at 30·5°C than at 27°C; secretion of pectate lyase was similarly affected. Cellulase was cell bound and its production was not affected by the temperatures investigated. Growth, protein synthesis and protease activity were similar at the two temperatures and production of enzyme activity at 27°C and 30·5°C was independent of the growth medium.     

Dielectric properties of developing rabbit brain at 37 degrees C

The dielectric properties of developing rabbit brain were measured at 37 degrees C between 10 MHz and 18 GHz using time domain and frequency domain systems. The results show a variation with age of the dielectric properties of brain. An analysis of the data suggests that the water dispersion in the brain of newly born animals can be represented by a Debye equation. This dispersion increases in complexity with age, and there is evidence of a smaller additional relaxation process centered around 1 GHz. It is concluded that the principal contribution to this subsidiary dispersion region arises from water of hydration.