Effect of cisplatin on the plasma membrane phosphatase activities in ascites sarcoma-180 cells: a cytochemical study

To study the effects of cisplatin [cis-dichlorodiammine-platinum (II)] on tumor cells in the presence or absence of the immune system, animals with ascites sarcoma-180 tumor burden were treated with therapeutic dose levels (9 mg/kg). Similarly, ascites sarcoma-180 cells were maintained in tissue culture media containing the same levels of the drug. Cell samples were taken from the animals at 12-hr intervals for 3 days, whereas samples were drawn from the tissue cultures at 15-, 30-, 45-, and 60-min and at 2-, 3-, 4-, and 5-hr intervals. Treated and untreated cells from in vitro and in vivo experiments, when checked for alkaline phosphatase, 5'-nucleotidase, Ca2+-ATPase, and Na+-K+-ATPase, show a gradual decrease in activity on the plasma membrane. It takes about 60 min for inactivation of any enzyme in vitro, whereas it takes 2 days in in vivo experiments. Quantitative analysis show alkaline phosphatase activity drops from 9.7 to 4.9 nmol in just 15 min, and drops further to 0.79 nmol after 2 hr. Inactivation of various plasma membrane enzymes, resulting in permeability changes, is probably responsible for cell death.     

Growth hormone stimulation of amino acid transport and utilization by the perfused rat liver.

The effects of growth hormone, administered in vivo or added in vitro, on amino acid transport and utilization have been studied in perfused livers of normal and hypophysectomized rats. A perfusion system employing a nonrecirculating medium was used in all of the studies. Two nonmetalbolizable amino acid analogues, alpha-aminoisobutyric acid (AIB) and 1-aminocyclopentane carboxylic acid (cycloleucine) were used to study transport. Accumulation of AIB increased linearly over a 60-min perfusion period, reaching distribution ratios of between 1 and 2 for both groups of animals. Treatment of both normal and hypophysectomized rats with growth hormone 60 min prior to the start of perfusion increased AIB distribution ratios by up to 84 and 108%, respectively. Accumulation of cycloleucine was linear for only about 20 min of perfusion and then plateaued. Steady state distribution ratios of this analogue ranged between 1 and 2 for both groups of animals. Growth hormone treatment had no apparent effect on the time necessary to reach these steady state levels, but significantly increased them in livers of both normal and hypophysectomized rats by 16 and 42%, respectively. Studies designed to analyze the kinetic properties of these hormone effects revealed that growth hormone treatment caused 2-fold i-crease in the maximum velocities of both the AIB and cycloleucine transport systems. The substrate concentration for half-maximal transport velocity was increased slightly for both systems by growth hormone. Direct effects of growth hormone were demonstrated in studies where livers of hypophysectomized rats were perfused under conditions simulationg those of experiments in which the hormone was administered in vivo. Following an initial 45-min period of perfusion the medium during the 20 min. Growth hormone added to the medium during the entire 65-min perfusion at a concentration of 1 mug per ml caused a 30% increase in the cycloleucine distribution ratio. Under similar experimental conditions growth hormone directly stimulated three hepatic pathways of amino acid utilization: (a) incorporation of [14C]valine into protein, (b) urea formation and (c) conversion of 14-C-amino-acids to labeled glucose. Intracellular concentrations of seven amino acids, including threonine, serine, proline, glycine, alanine, lysine, and arginine, were increased significantly in livers perfused with medium containing growth hormone...     

Extrahpetic distribution of sulfobromophthalein.

Plasma clearance of sulfobromophthalein (BSP) is widely used as a measure of hepatic function. Its validity depends upon its exclusive elimination from the body via bile. For example, in the present study, when BSP was administered intravenously (i.v.) to rats at four different doses (18.75, 37.5, 75, and 150 mg/kg), less than 0.5% of each dose was excreted into the urine and between 70 and 85% was excreted into the bile within 6 h after administration. It has been assumed that the distribution of BSP is limited to the blood and liver witith very little appearing in other tissues. When we measured the amount of BSP in the plasma, liver, and the bile 10 min after the i.v. administration of either a high (150 mg/kg) or a low (18.75 mg/kg) dose of BSP, only 60% of the dose was accounted for. The concentration of BSP and 12-I-labelled albumin (RISA) was measured in various tissue samples 10 min after administration of 17.5 or 150 mg of BSP or RISA per kilogram. More BSP was found in all tissues than was contained in the plasma entrapped therein. Thus, the distribution of BSP is not limited to the liver and plasma. During excretion BSP leaves other tissue (kidney, spleen, lung, etc.) and is ultimately excreted into the bile.     

Effect of Japanese sitting style (seiza) on the center of foot pressure after standing

Seiza is one of the most commonly used sitting postures in various enrichment lessons of Japanese origin. It is reported that Seiza with large knee flexion produces harmful effects on the cartilage of knee joints and hemodynamics of the lower legs. This study aimed at examining the influence of Seiza on tissue oxygenation kinetics of the lower limbs, plantar somatic and cutaneous sensation, and the center of foot pressure (COP) sway using 10 young adults. COP sway was measured for 1 min just after sitting on a chair for 10 min (pre-test), after 30-min Seiza (post-test 1), and 5 min after Seiza (post-test 2). To evaluate the COP sway, we used 4 body sway factors; unit time sway factor (F1), front-back sway factor (F2), left-right sway factor (F3) and high frequency band power spectrum factor (F4). Physiological parameters (i.e., tissue oxygenation kinetics in the lower legs and sensation on the sole) were measured during 30-min Seiza (continuously on tissue oxygenation, and at 1 min intervals on sensation), and for 1 min just before each COP test (pre-test, post-test 1 and 2).Oxygenated hemoglobin/myoglobin (Hb/Mb) concentration decreased markedly and deoxygenated Hb/Mb concentration increased markedly, resulting in reaching a plateau state at around 7 min. Tissue Hb/Mb index changed little during Seiza. Proprioceptive perception thresholds increased rapidly about 17 min after Starting Seiza. Means of 3 COP sway factors of F1, F2 and F4 were significantly higher in post-test 1 than in pre-test and post-test 2. In conclusion, a marked decrease in tissue oxygen concentration of the lower legs within 4-5 min, and an increase of proprioceptive perception thresholds in the sole at about 17 min are induced by Seiza. Although wiggle and quick body sway in the antero-posterior axis increases markedly in an upright posture just after maintaining Seiza for 30 min, sway recovers after sitting on a chair for 5 min.     

Effect of inhibiting prostaglandin synthesis on edema formation and albumin leakage during thermal trauma in the rat

Prostaglandins (PGs) participate in the inflammatory response, but the contribution of endogenously synthesized PGs to edema formation and increased vascular permeability is not known. Using a 10% scald burn in the rat, we measured water content (as percent, wet minus dry/wet weight) and 131I-RISA leakage (counts/g dry tissue) in scalded and normal skin at 30 minutes and 3 hr post injury. Four groups (10 rats/group) in each time period studied: control; scald; scald, 5 mg/kg indomethacin; scald, 10 mg/kg indomethacin. Indomethacin was administered intravenously 30 minutes before the scald; RISA was injected intravenously 30 min before termination of the study. In all indomethacin-treated groups immunoreactive plasma PGA was significantly lower (p less than 0.05) than in scalded, untreated groups. All scalded groups showed significantly higher RISA counts and water content than did the control group (p less than 0.01). At 30 min post-injury the indomethacin -treated groups did not differ from the untreated scald group (p greater than 0.20). In the 3 hour study all scalded groups had significantly higher content and RISA counts than control (p less than 0.01). Thus PGs produced during thermal trauma do not greatly contribute to the edema formation and increase in vascular permeability.     

Sexual behavior in aging male rhesus macaques: Effects of test duration on sexual performance

The sexual behavior displayed by nine old (20-year and older) rhesus (Macaca mulatta) males in 10-min tests was compared to that displayed in 1-hr tests. The tests were part of a long-term study on the decline in male sexual activity that accompanies old age. The males were paired with 10 ovariectomized, estrogentreated females in two blocks of 10 tests; each male was tested once with each female in each test block. The percentages of males that achieved intromissions and ejaculated in the two test blocks were the same (P < 0.05). Although the percentage of tests in which males displayed these behaviors was higher in the longer-test block (P > 0.05), there was a significant positive correlation of performance in 10-min tests with performance in 1-hr tests. Assuming a random distribution of contacting, mounting, intromission, and ejaculation throughout the hour, we would have predicted a significantly lower number of these behaviors in the first 10 min of the 1-hr tests than we actually observed. The mean percentages of 1-hr tests with contacting, mounting, intromission, and ejaculation was significantly lower than that of 10-min tests conducted with the same males 11 years earlier. Thus, the decline in sexual performance was not an artifact of the limited (10-min) test duration.     

The development of lysosomal apparatus. II. Incorporation,subcellular distribution,and intraparticulate hydrolysis of 131 I-albumin by liver of mice at perinatal stages

Mouse fetuses at 15th and 18th day of gestation, and newborns aging 0, 5, 10, and 15 days were injected i.p. with 131 I-albumin (RISA). The degree of incorporation by liver and the intraparticulate hydrolysis of RISA in 27,000g × 10 min. particles increased after birth. By this time, changes in subcellular distribution of RISA and marker enzymes were also observed. Following an i.p. injection of India ink, numerous hepatic cells stained with carbon particles were observed at the light microscope from day 5 of life. The results suggest that the lysosomal apparatus acquires full capacity to incorporate colloidal particles and to degrade foreign macromolecules in the first week of life.     

Modification of rat thymocyte membrane properties by hyperthermia and ionizing radiation.

Thymocytes are one the most widely used cell models for the study of radiation-induced interphase death. This cell-type was chosen for the study of hyperthermic and radiation effects on two membrane-related processes implicated in the interphase death of cells: Na+-dependent 2-aminoisobutyric acid (AIB) transport and cyclic 3'-5' adenosine monophsophate formation. The response of AIB transport to heat is dose-dependent, but the biphasic thermal response curve (AIB uptake versus time) differs fom the sigmoidal radiation response curve. Heating thymocytes for 20-30 min at 43 degrees C stimulates AIB uptake. Additional heating at 43 degrees C, however, markedly reduces AIB uptake. Despite the immediate stimulating effect of heat (30 min at 43 degrees C), the thymocyte has already developed irrepairable impairments, as demonstrated by the fractionated heating experiments. The heat-induced impairment of AIB uptake is mainly on the Na+-dependent component of neutral amino-acid transport, affecting primarily the maximal rate of uptake, i.e. Vmax. Additional evidence for heat-induced plasma membrane damage is the alteration in cAMP levels. Heating thymocytes for 30 min or longer at 43 degrees C causes a massive rise in cAMP level within the cell. This differs from thymocytes exposed to radiation where no rise in cAMP is observed.     

Derepression of amino acid transport by amino acid starvation in rat hepatoma cells.

Amino acid starvation causes an adaptive increase in the initial rate of transport of selected neutral amino acids in an established line of rat hepatoma cells in tissue culture. After a lag of 30 min, the initial rate of transport of alpha-aminoisobutyric acid (AIB) increases to a maximum after 4 to 6 h starvation of 2 to 3 times that seen in control cells. The increased rate of transport is accompanied by an increase in the Vmax and a modest decrease in the Km for this transport system, and is reversed by readdition of amino acids. The enhancement is specific for amino acids transported by the A or alanine-preferring system (AIB, glycine, proline); uptake of amino acids transported by the L or leucine-preferring system (threonine, phenylalanine, tyrosine, leucine) or the Ly+ system for dibasci amino acids (lysine) is decreased under these conditions. Amino acids which compete with AIB for transport also prevent the starvation-induced increase in AIB transport; amino acids which do not compete fail to prevent the enhancement. Paradoxically threonine, phenylalanine, tryptophan, and tyrosine, which do not compete with AIB for transport, block the enhancement of transport upon amino acid starvation. The starvation-induced enhancement of amino acid transport does not appear to be the result of a release from transinhibition. After 30 min of amino acid starvation, AIB transport is either unchanged or slightly decreased even though amino acid pools are already depleted. Furthermore, loading cells with high concentrations of a single amino acid following a period of amino acid starvation fails to prevent the enhancement of AIB transport, whereas incubation of the cells with the single amino acid for the entire duration of amino acid starvation prevents the enhancement; intracellular amino acid pools are similar under both conditions. The enhancement of amino acid transport requires concomitant RNA and protein synthesis, consistent with the view that the adaptive increase reflects an increased amount of a rate-limiting protein involved in the transport process. Dexamethasone, which dramatically inhibits AIB transport in cells incubated in amino acid-containing medium, both blocks the starvation-induced increase in AIB transport, and causes a time-dependent decrease in transport velocity in cells whose transport has previously been enhanced by starvation.     

Changes of basal and steroidal precursor-stimulated testosterone secretion in isolated Mongolian gerbil and guinea pig testes after a single episode of heating

1. In the absence of steroidal precursors, testosterone secretion by Mongolian gerbil testes incubated at 37 degrees C was 340 ng/g tissue/4 hr. Addition of 1 microgram progesterone or DHEA drastically stimulated testosterone secretion by testes incubated at 37 degrees C (progesterone: 3281 ng/g tissue/4 hr, DHEA: 4654 ng/g tissue/4 hr). 2. While neither basal nor DHEA-stimulated production of testosterone was significantly affected by a single episode of heating (43-44 C for 30 min), progesterone-stimulated testosterone secretion markedly decreased during the 4-hr incubation period. 3. In contrast, in isolated testes of adult guinea pigs, a single episode of heating (44 degrees C for 30 min) resulted in a drastic reduction of basal and precursor-stimulated testosterone production during the 4-hr incubation period. 4. From these data it appears that enzymatic activities in the testes of the two species do not have their maxima at the same temperature, but rather in each case at, or close to, the temperature prevailing in the scrotal testis.     

Effect of inhibiting prostaglandin synthesis on edema formation and albumin leakage during thermal trauma in the rat

Prostaglandins (PGs) participate in the inflammatory response, but the contribution of endogenously synthesized PGs to edema formation and increased vascular permeability is not known. Using a 10% scald burn in the rat, we measured water content (as percent, wet minus dry/wet weight) and ¹³¹I-RISA leakage (counts/g dry tissue) in scalded and normal skin at 30 minutes and 3 hr post injury. Four groups (10 rats/group) in each time period studied: control; scald; scald, 5 mg/kg indomethacin; scald, 10 mg/kg indomethacin. Indomethacin was administered intravenously 30 minutes before the scald; RISA was injected intravenously 30 min before termination of the study. In all indomethacin-treated groups immunoreactive plasma PGA was significantly lower (p < 0.05) than in scalded, untreated groups. All scalded groups showed significantly higher RISA counts and water content than did the control group (p < 0.01). At 30 min post-injury the indomethacin-treated groups did not differ from the untreated scald group (p> 0.20). In the 3 hour study all scalded groups had significantly higher content and RISA counts than control (p < 0.01). Thus PGs produced during thermal trauma do not greatly contribute to the edema formation and increase in vascular permeability.     

Inhibitory effect of alcohol on the established suckling-induced prolactin surge in lactating rats

The effect of acute alcohol infusion on the established suckling-induced prolactin surge in lactating rats was examined. Dams were implanted with an atrial catheter on Day 6 of lactation and blood sampling was done on Day 10. Following the separation of litters from dams for a 6-hr period, a baseline blood sample was removed via a catheter extension. Pups were weighed and returned to dams. Subsequent blood samples were obtained 10, 30, and 60 min after initiation of suckling. Dams were then infused with alcohol doses of 0, 0.5, 1.0, 2.0, or 2.5 g/kg body wt. Infusion (0.1 ml/min) was completed in approximately 30 min. Additional blood samples were obtained 10 30, 60, and 120 min after the termination of infusion. In a separate group of rats, pups were removed from the dam after the first 60 min of suckling and additional blood samples were obtained 40, 70, 90, and 150 min after removal of pups (corresponding to 10-, 30-, 60-, and 120-min samples for rats infused with various alcohol doses). Alcohol, when administered after the establishement of suckling-induced prolactin surge and resulting in blood alcohol levels equal to or greater than legal human intoxication levels, inhibited prolactin release. However, continued suckling for an extended period (120 min in the present study) overcame this inhibitory effect, even when the blood alcohol level was comparable to (2.0 g/kg group) or greater than (2.5 g/kg group) the human legal intoxication level. Furthermore, in rats with established prolactin surges, the patterns of prolactin decline that followed alcohol administration or pup removal were comparable, indicating that similar mechanism(s) may be involved.     

Technique-dependent variations in cerebral microvessel blood volumes and hematocrits in the rat.

To quantitate small parenchymal microvessel blood volumes in the brain, the distribution spaces of radiolabeled red blood cells (RBC) and serum albumin (RISA) were assessed in rats by different methods of tissue sampling and radioassay. Three minutes after intravenous administration of 55Fe-RBCs and/or 125I-RISA, the rats were decapitated. The brain was either immediately frozen within the skull and later removed (head-frozen group) or rapidly removed from the skull and then frozen (brain-frozen group). Radioactivity was measured either by liquid scintillation counting of tissue pieces, which contained pial plus large and small parenchymal microvessels, or by quantitative autoradiography (QAR) of tissue sections, which indicated small parenchymal microvessel blood only. In 12 of 15 areas, the RISA, RBC, and blood volumes determined by liquid scintillation counting of head-frozen tissue pieces were equal to or greater than those of brain-frozen tissue; this indicated less than or equal to 25% greater blood retention in pial and parenchymal microvessels with head freezing. At the parenchymal microvessel level (QAR assay), the distribution volumes of RBCs, RISA, and blood were similar with the two freezing techniques; hence with QAR either freezing procedure can be used to assess small parenchymal microvessel blood volumes.     

Effect of KCN and p-chloromercuribenzenesulfonic acid on the release of sucrose and 2-amino(1-14C)isobutyric acid by the seed coat of Pisum sativum

The process of sugar and amino acid release by the seed coat of Pisum sativum L. cv. Marzia was studied. Prior to measuring the release of solutes by the seed coat of developing ovules, the embryo was removed from each ovule studied. After this surgical treatment, each "empty" seed coat was filled with the appropriate solution (pH 5.5) with or without inhibitor. Both KCN and p-chloromercuribenzenesulfonic acid (PCMBS) strongly inhibited the release of sucrose and p -aminoisobutyric acid (AIB) by the seed-coat. These data support the view that phloem unloading is an energy-dependent process sensitive to the sulfhydryl group modifier PCMBS. In pulse-labelling experiments, addition of high concentrations of unlabelled sucrose (200 m M ) and AIB (25 m M ) to the solution filling the seed coat cavity did not diminish the release of labelled solutes by the unloading sites of the seed coat. This observation presents evidence against the view that phloem unloading into a strong sink is related to low sugar concentrations in the apoplast.     

A new protocol for studying the early events during liver regeneration

Hepatic amino acid transport and ornithine decarboxylase (ODC) activity were measured as early events during liver regeneration in rats adapted to a meal-feeding protocol in which food was presented during the first 2 hr of a daily 12-hr dark period. Surgeries were performed during the early hours of the light period, and food was withheld on the day following surgery to control the effect of feeding on the parameters measured. Initial experiments indicated that rats maintained on this schedule were capable of surviving partial hepatectomy. The survival rate was greater and the fat pads larger among 56-day-old than among 42-day-old rats; this indicated that animals with greater metabolic reserves were better suited for these experiments. The hepatic α-aminoisobutyric acid (α-AIB) distribution ratio and ODC activity increased above control values by 2 hr following partial hepatectomy. The α-AIB distribution ratio peaked at 10 hr after surgery and remained above control values for an additional 16 hr. In contrast, ODC activity peaked by 4 hr after surgery, followed by two smaller peaks at 10 and 20 hr. In sham-hepatectomized rats both the α-AIB distribution ratio and the ODC activity increased above control values by 3 hr after surgery, but fell to control values by 15 hr. These findings show that increases in amino acid transport and ODC activity following partial hepatectomy occur independently of feeding. The identical time course for the initiation of both of these events suggests that they result from a common effector.     

Evidence for both prelysosomal and lysosomal intermediates in endocytic pathways

Horseradish peroxidase (HRP), an enzyme internalized by fluid phase pinocytosis, has been used to study the process by which pinosome contents are delivered to lysosomes in Chinese hamster ovary cells. Pinosome contents were labeled by allowing cells to internalize HRP for 3-5 min. Following various chase times, cells were either processed for HRP and acid phosphatase (AcPase) cytochemistry or homogenized and fractionated in Percoll gradients. In Percoll gradients, pinosomes labeled by a 3-5 min HRP pulse behaved as a vesicle population more dense than plasma membrane and less dense than lysosomes. In pulse- chase experiments, internalized HRP was chased rapidly (3-6 min chase) to a density position intermediate between the "initial" pinocytic vesicle population and lysosomes. With longer chase periods, a progressive accumulation of HRP in more dense vesicles was observed. Correspondence between the HRP distribution and lysosomal marker distribution was reached after a approximately 1-h chase. By electron microscope cytochemistry of intact cells, the predominant class of HRP- positive vesicles after pulse uptakes or a 3-min chase period was characterized by a peripheral rim of reaction product and was AcPase negative. After 10-120-min chase periods, the predominant class of HRP- positive vesicles was characterized by luminal deposits and HRP activity was frequently observed in multivesicular bodies. HRP-positive vesicles after a 10- or 30-min chase were AcPase-positive. No HRP activity was detected in Golgi apparatus. Together these observations indicate that progressive processing of vesicular components of the vacuolar apparatus occurs at both a prelysosomal and lysosomal stage.     

Immunological Studies on Viral Polypeptide Synthesis in Cells Replicating Murine Sarcoma-Leukemia Virus

Antibodies to disrupted murine sarcoma-leukemia virus (MSV[MLV]) were used to study the synthesis of viral polypeptides in the transformed, virus-producing rat cell line 78A1. When cultures were labeled for 10 min with radioactive amino acids, about 9% of the total labeled proteins were precipitated with antiserum against purified MSV(MLV), and 3 to 4% were precipitated with the same antiserum after it had been absorbed with an extract from uninfected rat cells. The difference is due to the presence in the unabsorbed antiserum of antibodies to cellular proteins that are present in purified virus preparations. Intracellular viral proteins labeled with radioactive amino acids were isolated by immunoprecipitation and analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The mobilities of intracellular viral polypeptides were identical to those of the purified virion. However, labeled polypeptides having electrophoretic mobilities lower than that of the major virion polypeptide, the group-specific antigen of molecular weight 31,000, were present in higher proportion in the total cell extract and in the membrane fraction than in the virion. These polypeptides appear to be of cellular origin for they were present only in minute amounts in the immunoprecipitates obtained with the absorbed serum. After a 10-min labeling period, radioactive proteins were assembled into extracellular virions rapidly for the first 4 hr followed by a slower rate. More than 2% of the total proteins of the cell labeled in a 10-min pulse were assembled into virions at the completion of a 24-hr chase. The high-molecular-weight polypeptides with the same mobilities as those detected in the immunoprecipitate of intracellular proteins were found in virions released from cells after a 10-min pulse. A larger proportion of these high-molecular-weight proteins was detected in virions released after short chase periods (30-120 min) than after longer chase periods (6-24 hr). Two possible interpretations of these data are that the high-molecular-weight cell-derived polypeptides (i) have a turnover rate higher than that of the major virion polypeptides or (ii) are cleaved proteolytically from the virions during long incubation in the culture media.     

Insulin and exercise stimulate muscle alpha-aminoisobutyric acid transport by a Na+-K+-ATPase independent pathway

Sodium ions are required for the active transport of amino acids such as alpha-aminoisobutyric acid (AIB) into skeletal muscle. To examine the role of Na+-K+-ATPase in this phenomenon, studies were carried out using the isolated perfused rat hindquarter preparation. Perfusion for 30 min with ouabain at a dose sufficient to inhibit the Na+-K+ pump (10(-4) M) inhibited the basal rate of AIB uptake in all muscles studied by up to 80%. However, it failed to inhibit the stimulation of AIB uptake, either by insulin (200 microU/ml) or electrically-induced muscle contractions. The increase in K+ release by the hindquarter in the presence of ouabain was the same under all conditions suggesting comparable inhibition of the Na+-K+ pump. These studies suggest that the basal, but not insulin or exercise-stimulated AIB transport into muscle is acutely dependent on a functional Na+-K+ pump. They also suggest that stimulated and basal uptake of AIB involve different mechanisms.     

Nyctohemeral rhythm in melatonin response to isoproterenol in vitro: comparison of rats and Syrian hamsters

The in vitro response of melatonin synthesis was assessed by incubation of individual whole pineal glands for 4 hr without or with different concentrations of isoproterenol in the medium. Pineals were taken either at the end of the 14-hr light phase (day), or at 6 1/2 hr into the 10-hr dark phase (night) after a 30-min exposure of the animals to light just before sacrifice at night. The response was greater in pineals taken at night than in those taken at the end of the light phase in rats, but was absent at the end of the light phase in Syrian hamsters. Hamster pineals taken at night responded, though higher isoproterenol concentrations were required than in the rats. Unresponsiveness of hamster pineals during the day may explain previous failure of isoproterenol administration to stimulate pineal MEL content in this species.     

Fixation variables in horseradish peroxidase neurohistochemistry. I. The effect of fixation time and perfusion procedures upon enzyme activity.

In a series of neurohistochemical experiments the effect of aldehyde fixation upon the detection of horseradish peroxidase (HRP) was examined. These experiments demonstrated that: a) Increments in fixation of as little as 1 hr significantly decreased the number of labeled neurons; 12-hr fixation abolished HRP activity in many neuronal populations and significantly reduced the apparent size of the injection site. b) This negative fixation effect was greatest where the HRP concentration was low (e.g. in small, lightly labeled neurons) but was still evident in areas of high concentration (e.g. large, heavily labeled neurons). c) This effect was also most prominent when a less sensitive diaminobenzidine histochemical procedure was employed but was still apparent with a more sensitive benzidine dihydrochloride procedure. d) Immersion of the brain in fixative after perfusion produced a greater attenuation of HRP activity in more superficial areas. e) Immersion of the brain in buffer to terminate fixation produced a prolonged and unpredictable gradient of fixation. f) Excess, unbound fixative inhibited the histochemical reaction per se and had to be removed from the tissue but prolonged washing did not resurrect enzyme activity which was lost by fixation. To obviate these problems and optimize HRP enzyme activity a new perfusion-fixation procedure was developed. It entails 30 min fixation by perfusion which is terminated by a subsequent 30 min perfusion with cold sucrose-fuller to wash out unbound fixative. This allows the tissue to be processed immediately, produces a uniform and morphologically adequate fixation, and minimizes the negative effects of fixation on HRP enzyme activity.