Function of rabbit kidneys in vitro at normothermia following equilibration with 3.0 M Me2SO and removal by hypertonic washout at 10 degrees C

In the present study, rabbit kidneys were assayed for function on a 37 °C in vitro perfusion system after perfusion on a 10 °C perfusion system which permits the slow introduction and removal of cryoprotectant. The final concentration of 3.0 M Me₂SO was introduced slowly at two different rates. The washout was achieved by perfusion with Me₂SO-free solutions made hypertonic with mannitol. Two regimens of washout were used: 800, 700, 600, 500, and 400 mOsm/kg; and 600, 500, and 400 mOsm/kg.During perfusion at 37 °C, the glomerular filtration rate was similar in all groups and this increased significantly in all groups with time. Protein leakage was minimal. All three Me₂SO groups showed a depressed Na reabsorption capacity, but the 800 mOsm group was the most severely affected. This was also found with glucose reabsorption. We concluded that rabbit kidneys will function well with the cryoprotectant Me₂SO up to 3 M concentration when introduced slowly and washed out with hypertonic mannitol beginning at 600 mOsm/kg. When 800 mOsm is used at the initial step, the proximal tubular function is severely affected.     

Kinetics of permeation and intracellular events associated with Me2SO permeation of rabbit kidneys during perfusion at 10 °C

The permeation kinetics of the cryoprotectant dimethylsulfoxide (Me₂SO) were investigated by the measure of total water, Me₂SO, and inulin spaces using radioactive tracers. Complete permeation of rabbit kidneys with a perfusate concentration of 3.0 M Me₂SO at 10 °C was obtained 35 min after reaching the maximum concentration when the cryoprotectant was introduced slowly. Equilibration was complete in the medulla before the cortex, probably due to the higher flow rates.Through study of the change in Me₂SO space and the inulin space, we were able to obtain evidence of cellular permeation of Me₂SO. In control kidneys, the inulin space decreased slightly during perfusion, an indication of cell swelling. Kidneys perfused with Me₂SO demonstrated a doubling of the inulin space, which did not decrease with time. The most likely explanation of this phenomenon is marked cell shrinkage, which was confirmed on histological examination and increased intraluminal tubular fluid. The picture is more complex since after intracellar equilibration, rehydration of the cell is not observed.     

Temperature Characteristics of Excitation in Space-Clamped Squid Axons

Temperature characteristics of excitability in the squid giant axon were measured for the space-clamped axon with the double sucrose gap technique. Threshold strength-duration curves were obtained for square wave current pulses from 10 µsec to 10 msec and at temperatures from 5°C to 35°C. The threshold change of potential, at which an action potential separated from a subthreshold response, averaged 17 mv at 20°C with a Q¹⁰ of 1.15. The average threshold current density at rheobase was 12 µa/cm² at 20°C with a Q¹⁰ of 2.35 compared to 2.3 obtained previously. At short times the threshold charge was 1.5·10^-8 coul/cm². This was relatively independent of temperature and occasionally showed a minimum in the temperature range. At intermediate times and all temperatures the threshold currents were less than for both the single time constant model and the two factor excitation process as developed by Hill. FitzHugh has made computer investigations of the effect of temperature on the excitation of the squid axon membrane as represented by the Hodgkin-Huxley equations. These are in general in good agreement with our experimental results.     

Intracarotid 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Administration: Biochemical and Behavioral Observations in a Primate Model of Hemiparkinsonism

Cynomolgus monkeys received intracarotid injections of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to produce a chronic unilateral model of parkinsonism. Extensive dopamine (DA) depletion was observed in the caudate nucleus and putamen on the side ipsilateral to the injection and this was associated with contralateral tremor, rigidity, and bradykinesia. A dose of 1.25 mg of MPTP caused ipsilateral DA loss of 99.4% in the caudate nucleus, 99.8% in the putamen, and 74.2% in the nucleus accumbens. A dose of 2.5 mg caused ipsilateral DA depletion of 99.3% in the caudate nucleus, 99.5% in putamen, and 90.1% in the nucleus accumbens. The unilateral aspect of the lesion was dose sensitive, with the 2.5-mg dose causing bilateral asymmetric DA depletion. Tissue concentrations of serotonin were not affected by the toxin. These findings confirm that intracarotid injection of MPTP may produce a useful primate model of hemiparkinsonism that can be associated with selective unilateral DA depletion when the appropriate dose of toxin is used.     

Properties of ts Cl mouse L cells which exhibit temperature-sensitive DNA synthesis.

ts Cl mouse L cells are temperature-sensitive (ts) in DNA synthesis. The protein involved undergoes inactivation at 38.5 °C, with an apparent half-life of 3–4 h. A variety of experimental approaches yield data indicating that the ts Cl gene product acts directly during the DNA-synthesis period, probably late during the duplication of chromosomal DNA. The specificity of the ts lesion is reflected in the fact that replication of mitochondrial DNA is unaffected for many hours after nuclear DNA synthesis is almost totally inhibited. Temperature inactivation is not due to degradation or to loss of template capacity of preformed DNA. ts Cl cells are able to enter a DNA-synthesis phase at the higher temperature, as indicated by radioautographic experiments and by studies in which cells, blocked at the permissive temperature (34 °C) in a pre-DNA synthesis phase by isoleucine deprivation, are subsequently incubated at 38.5 °C. Cells arrested early in DNA synthesis by hydroxyurea treatment at 34 °C continue such synthesis for a short interval after up-shift to 38.5 °C. However, they are then unable to complete the S phase in progress nor can they proceed into cell division. The kinetics of DNA synthesis in cells incubated at 38.5 °C and back-shifted to 34 °C are compatible with the model that the ts Cl locus encodes an S phase function.     

Alterations in Nuclear Ribonucleic Acid Metabolism Induced by Kinetin

Removal of deoxyribonucleic acid from meristematic onion root cells grown in solutions of kinetin, followed by metachromatic staining in azure B bromide, indicated the presence of appreciable amounts of ribonucleic acid in nuclei exposed to the cell division factor.     

Failure to Use Cubicles and Concentrate Dispenser by Heifers after Transfer from Rearing Accommodation to Milking Herd

Thirty-three dairy farms in the Norwegian counties of ?stfold and Akershus in which cubicle sheds had been in use for at least one year and with a herd size of less than 60 cows, were contacted and asked to participate in a study. The study focused on heifers' use of cubicles and concentrate dispenser just after being transferred from rearing accommodation to the milking herd. For each heifer, the farmer recorded cubicle use once nightly between 9 and 11 pm. The daily amount of concentrate released in the dispenser and the allotted daily ration were also recorded. The recording period was 15 consecutive days for cubicle use and 7 days for concentrate dispenser use. Cubicle refusal behaviour, i.e. lying outside the cubicles, was analysed by logistic regression using rearing accommodation of heifers, herd size, heifer age, and housing layout as independent variables, and herd as a clustering variable. On Day 2 after transfer, 34% of the heifers were showing cubicle refusal behaviour (N = 340). By Day 15 this percentage had dropped to 23. Cubicle refusal was lower throughout the whole period among heifers which used the cubicles on the 3 first days after transfer compared to those which did not. This tendency could also be detected several months later. The analysis showed cubicle refusal to be significantly associated with rearing accommodation (OR = 6.1, c.i._95%OR = 1.5–24.3, P = 0.01) and cubicle layout in the shed (OR = 0.2, c.i._95%OR = 0.0–0.7, P = 0.01). None of the tested variables were found to be significant for failure to use the concentrate dispenser, a behaviour which was less frequent than cubicle refusal. However, 8 percent of the heifers did not visit the dispenser at all throughout the 7 days of observation.     

The HopZ family of Pseudomonas syringae type III effectors require myristoylation for virulence and avirulence functions in Arabidopsis thaliana

Pseudomonas syringae utilizes the type III secretion system to translocate effector proteins into plant cells, where they can contribute to the pathogen's ability to infect and cause disease. Recognition of these effectors by resistance proteins induces defense responses that typically include a programmed cell death reaction called the hypersensitive response. The YopJ/HopZ family of type III effector proteins is a common family of effector proteins found in animal- and plant-pathogenic bacteria. The HopZ family in P. syringae includes HopZ1a(PsyA2), HopZ1b(PgyUnB647), HopZ1c(PmaE54326), HopZ2(Ppi895A) and HopZ3(PsyB728a). HopZ1a is predicted to be most similar to the ancestral hopZ allele and causes a hypersensitive response in multiple plant species, including Arabidopsis thaliana. Therefore, it has been proposed that host defense responses have driven the diversification of this effector family. In this study, we further characterized the hypersensitive response induced by HopZ1a and demonstrated that it is not dependent on known resistance genes. Further, we identified a novel virulence function for HopZ2 that requires the catalytic cysteine demonstrated to be required for protease activity. Sequence analysis of the HopZ family revealed the presence of a predicted myristoylation sequence in all members except HopZ3. We demonstrated that the myristoylation site is required for membrane localization of this effector family and contributes to the virulence and avirulence activities of HopZ2 and HopZ1a, respectively. This paper provides insight into the selective pressures driving virulence protein evolution by describing a detailed functional characterization of the diverse HopZ family of type III effectors with the model plant Arabidopsis.