ULTRASTRUCTURAL AND ENZYMATIC EVIDENCE FOR THE PRESENCE OF MICROBODIES IN THE FUNGUS ACHLYA

Ultrastructural evidence for structures resembling microbodies is presented for the fungus Achlya ambisexualis Raper. These structures are DAB positive and thus presumably contain the enzyme catalase. Activities from mycelial homogenates for. the following enzymes are given: catalase, glycolate oxidase, uricase, isocitrate lyase, malate dehydrogenase, citrate synthetase, malate synthetase and glutamate: oxaloacetate transaminase. These results suggest that Achlya contains microbodies and that they may be of the glyoxysome type. The specific activity of catalase increases substantially following initiation of antheridial hyphae by the hormone antheridiol.     

EVIDENCE FOR THE PRESENCE OF LIGNIN IN MOSS GAMETOPHYTES

Lignin has been established as a constituent of the gametophyte axes in the giant New Zealand mosses Dawsonia sp. and Dendroligotrichum sp. Isolated products comprised ca. 6-10% of the dry weight of gametophyte axes and contained 61-62% C, 6.4-6.8% H, and 5.1-7.9% OCH₃. Characteristic color reaction and ultraviolet spectra were observed, and alkaline nitrobenzene oxidation yielded perhaps 14-18% of mixed aldehydes as their 2,4-dinitrophenylhydrazones. The presence of substantial lignin in these exceptionally tall upright moss gametophytes contrasts strikingly with north temperate species such as Polytrichum, Funaria, Bryum and others, and lends support to the hypothesis that lignification is a mechanically and/or gravitationally regulated process.     

FURTHER EVIDENCE FOR THE PRESENCE OF A PANETH CELL PROGENITOR IN MOUSE INTESTINE

Photomicrographic evidence, mitotic figures and ³H-thymidine labeling among Paneth cells, is presented in support of the hypothesis that Paneth cells are replaced by cells proliferating in the bottom of the intestinal crypt rather than by migration of cells from the mid-crypt region.     

去卵巢大鼠Ru 486与垂体及子宫内膜孕激素受体结合的比较

经雌激素诱发的去卵巢大鼠在肌肉注射不同量的 Ru486(0.1mg/kg—2mg/kg)之后,垂体及子宫内膜的胞液游离孕激素受体结合位点随着 Ru486注射量的升高而逐步下降。注射 Ru486 2mg/kg 30min 后,此两组织中孕激素受体的结合位点就明显降低,2h 降到最低点,3h 开始恢复。以上实验说明 Ru486进入机体即可分别占领垂体及子宫内膜的孕激素受体的结合位点。然而 Ru486对此二组织的孕激素受体的结合作用并不完全相同,似乎子宫孕激素受体更易为 Ru486所占位,而解离也快。     

ULTRASTRUCTURAL ZONATION OF ADRENOCORTEX IN THE RAT

The fine structure of the different zones in the adrenal cortex of the adult rat has been studied under the electron microscope. Four regions mainly differentiated by the mitochondrial morphology, the lipid droplets, and the structure of the ground cytoplasm were recognized. In the glomerular zone mitochondria are thin and elongated with an abundant matrix. The inner structure is characterized by the presence of tubules of 300 A that are straight or bend at an angle and which may be grouped in parallel array giving a pseudocrystalline pattern. The wall of each tubule is a finger-like projection of the inner membrane and its cavity corresponds to the outer chamber of the mitochondrion. In the intermediary zone mitochondria are larger and irregular. The matrix is filled with convoluted tubules and vesicular elements. The lipid droplets are larger and irregular in the glomerulosa and and small in the intermedia. The ground substance is dense and contains free ribosomes in the glomerulosa and starts to be vacuolated in the intermedia. In the fasciculata mitochondria are round or oval and are filled with vesicular elements with a mean size of 450 A. Larger vesicles and more clear elements (vacuoles) are seen near the edge as if their content was diluted. Some of these vacuoles protrude on the surface. In the reticular zone mitochondria are also vesicular but frequently show signs of alteration and disruption. Dense elements recognized as microbodies are observed in the fasciculata but they increase in number in the reticularis. These results are discussed on the light of the so called zonal theory of the adrenal cortex. Two stages in the differentiation of the mitochondria are postulated. The tubular structure of the glomerulosa undergoes a process of disorientation and dilatation of the tubules to form the tubulo-vesicular elements of the intermediary zone. In a second stage of differentiation, by fragmentation of the tubules, the vesicular structure of fasciculata is formed. These findings are discussed from the viewpoint of the relationship between mitochondria and synthesis of steroid hormones. A secretory process that starts within mitochondria by the formation of vesicles and proceeds into the ground cytoplasm, as extruded and more clear vacuoles, is postulated.     

EVIDENCE FOR EXTRANORADRENERGIC DOPAMINE-β-HYDROXYLASE ACTIVITY IN RAT SALIVARY GLAND

—Three days after superior cervical ganglionectomy of adult Sprague-Dawley rats, the levels of endogenous norepinephrine, the uptake process for [³H]norepinephrine and the activity of tyrosine hydroxylase decreased 99 per cent in the ipsilateral salivary gland. In contrast, the activity of dopamine-β-hydroxylase and DOPA decarboxylase fell to 30 per cent of the activity of the contralateral innervated gland. Examination of the cofactor requirements, the characteristics of activation by cupric ion and the immunologic identity of this residual hydroxylase activity indicated that it was authentic dopamine-β-hydroxylase. The residual dopamine-β-hydroxylase in the denervated gland had the same subcellular distribution as the enzyme in the innervated salivary gland. Procedures that caused atrophy or hypertrophy of the acinar cells did not affect the total content of dopamine-β-hydroxylase in the denervated salivary gland. Chemical sympathectomy with 6-hydroxy-dopamine caused a 40 per cent decrement in the serum levels of dopamine-β-hydroxylase but a 30 per cent increase in its activity in the denervated salivary gland. Although denervation caused a complete loss of endogenous norepinephrine in the salivary gland, it resulted in only a 15 per cent decrement in the levels of endogenous octopamine and β-phenylethanolamine, two other products of dopamine-β-hydroxylase.     

EVIDENCE FOR THE HETEROGENEITY OF L-2,4-DIAMINOBUTYRIC ACID UPTAKE IN RAT CORTEX

The uptake of L-[³H]DABA by rat cerebral cortex slices was studied. Analysis of the kinetic data obtained provides evidence that DABA entry is mediated by both high and low affinity carriers. When cortical slices were incubated in the presence of equimolar [³H]DABA and [¹⁴C]GABA the ratio of entry of the two radionuclides was found to depend upon the loading concentration. The specificity of the uptake of 1 μM and 1 mM-L-DABA was examined: GABA and DABA were relatively potent inhibitors of 1 μM-DABA uptake whereas an equal concentration of histidine did not produce significant inhibition. In contrast, DABA and histidine were markedly more potent as inhibitors of 1 mM-DABA uptake than was GABA. It is concluded from these experiments that L-DABA is transported into cortical slices by a carrier which has high affinities for both DABA and GABA and by a second lower affinity carrier which prefers DABA as a substrate to GABA. On the basis of a comparison of the effects of inhibitors on [³H]DABA and [³H]GABA uptake it is estimated that approx 26% of DABA uptake at 1 μM does not occur by the high affinity carrier whereas at 1 mM-DABA this proportion rises to 62–67%.     

EVIDENCE FOR THE CLOSE ASSOCIATION OF A GLYCOPROTEIN WITH MYELIN IN RAT BRAIN

Abstract— Myelin was purified from rats which had been injected intracerebrally with radioactive fucose in order to label specifically the glycoproteins. Myelin contained a small amount of fucose-labelled glycoproteins in comparison to that in other subcellular fractions, but polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed a unique pattern of radioactive glycoproteins dominated by a major peak. The same glycoprotein was not prominent in the other subcellular fractions which were examined. This major glycoprotein in the myelin fraction was also labelled after injection with [³H]glucosamine or N -[³H]acetylmannosamine. It was the most intensely staining myelin protein when gels were treated with periodic acid-Schiff reagents, an indication that, in terms of protein-bound carbohydrate, it is the major glycoprotein in the myelin fraction. The glycoprotein was present in myelin purified from rats ranging in age from 14 days to 14 months. Extensive recycling of the myelin through the purification procedures did not significantly reduce the amount of glycoprotein in the myelin. Double label experiments with [³H]fucose and [¹⁴C]fucose were used to compare glycoproteins in myelin purified from white and grey matter, respectively, and from mixed homogenates of myelinated and unmyelinated brain. The results obtained from these experiments suggested that the glycoprotein is closely associated with myelin and that it is not in an unrelated contaminating structure. Possible locations of the glycoprotein are discussed. They include the myelin membrane itself, the oligodendroglial plasma membrane, and the axolemma of myelinated axons.     

METHYLHISTAMINE: EVIDENCE FOR SELECTIVE DEAMINATION BY MAO B IN THE RAT BRAIN IN VIVO

Abstract— A new method has been developed for the separation of histamine and its metabolites after intracisternal injection of [³H]histamine into the rat brain, involving solvent extraction and subsequent thin-layer chromatography. The effect of graded doses of the MAO inhibitors deprenil and pargyline, which at relatively low doses inhibit preferentially the B form (phenethylamine deaminating) of the enzyme, and clorgyline, which mainly inhibits the A form (serotonin, noradrenaline and dopamine deaminating) on the brain levels of intracisternally injected [³H]histamine and its labelled metabolites was studied and compared to MAO A and B activity as determined with the substrates serotonin and phenethylamine, respectively. In addition, the time-course of the effects of a single dose of pargyline (50mg/kg subcutaneously) was investigated. No [³H]imidazoleacetic acid could be detected in any of the control or treated animals. [³H]Histamine accounted for 9–12% of the total extracted radioactivity and this was not altered significantly by pretreatment with any of the MAO inhibitors up to high doses, at which both MAO A and B activities were completely inhibited. In the controls, 40–43% of the total extracted radioactivity was [³H]methylhistamine and 28–30% was [³H]methylimidazoleacetic acid. Deprenil and pargyline caused [³H]methylhistamine levels to increase in a dose-dependent manner up to about 150% of control levels and those of [³H]methylimida-zoleacetic acid to decrease concomitantly to about 10% of control levels. Clorgyline in doses up to 10 mg/kg subcutaneously (s.c.) had no effect on the levels of these two metabolites. The dose-response curves of the effects of deprenil and pargyline on [³H]methylimidazoleacetic acid levels were congruent with those of the MAOI effects on MAO B activity and not with those on MAO A activity. Pargyline (50 mg/kg s.c.) had a long lasting effect on the accumulation of [³H]methylhistamine and [³H]methylimidazoleacetic acid. Recovery occurred within 21 days, and the half-lives observed were 5.3 and 5.6 days, respectively. This compares well to the half-life for the recovery of MAO B activity reported earlier after the same dose of pargyline (5.5 days). These results suggest that methylhistamine is metabolized selectively by MAO B in rat brain. Moreover, the fact that clorgyline, at doses where phenethylamine deamination is already considerably inhibited, did not affect the deamination of methylhistamine, suggests that the latter is an even more selective substrate for MAO B than phenethylamine itself. Therefore, small doses of deprenil (0.3–3 mg/kg s.c.) or pargyline (1–3 mg/kg) can be used to influence histamine catabolism without interfering with catecholamine or serotonin deamination.     

大鼠粒细胞内质网的电镜细胞化学反应

本实验用电镜细胞化学方法观察了大鼠骨髓粒细胞发育过程中内质网的髓过氧化物酶(MPO)反应和葡萄糖-6-磷酸酶(G-6-P)反??应。结果表明:MPO除定位于内质网、核膜,还出现在高尔基体和颗粒,它是粒细胞内质网的合成产物。G-6-P只在内质网、核膜中出现,它是内质网膜的结构成分。MPO反应的超微结构定位随粒细胞发育而变,利用这种变化作标志可以划分不同发育阶段的粒细胞;G-6-P反应定位不随发育而变,但反应强度与内质网的多寡、功能状态相对应。实验还表明核膜与内质网在结构、功能上的一致性;尤其在成熟粒细胞内质网很少的情况下,核膜可能代替了内质网的功能。     

METABOLIC AND ULTRASTRUCTURAL CHANGES IN THE FROG OVARIAN FOLLICLE IN RESPONSE TO PITUITARY STIMULATION

Frog ovarian fragments were prevented from ovulating in vitro by the addition of actinomycin D up to 3 hr following pituitary stimulation; but addition of Actinomycin D 6 hr after stimulation was far less effective. Puromycin, on the other hand, effectively inhibited ovulation when added as late as 6 hr after pituitary stimulation. Although actinomycin D reduced uptake of uridine-³H, and puromycin reduced uptake of leucine-³H and lysine-¹⁴ by pituitary-stimulated ovarian tissue minus oocytes (OTMO) in vitro, it was found that pituitary stimulation did not significantly increase uptake of these compounds by OTMO. Radioautographs of ovarian follicles fixed 6 hr after the addition of pituitary extract and uridine-³H in vitro revealed increased RNA synthesis in the peritoneal surface epithelium, compared with unstimulated controls, while the ovarian sac epithelium showed no increase. Gross ultrastructural changes occurred in the peritoneal area of ovarian follicles following pituitary stimulation in vivo, including loss of collagen fibrils, and general disorganization of the connective tissue theca. Changes in the rough endoplasmic reticulum of the peritoneal epithelial cells, while frequently encountered, were less pronounced. None of these changes was observed in the ovarian sac area, or in the interfollicular region. The above data are consistent with the hypothesis that pituitary stimulation of the frog ovary results in increased synthesis of RNA and protein by the peritoneal epithelial cells, and that the protein may be collagenase.     

大鼠骨骼肌组织雄激素受体结合容量测定方法

目的 :以3H testosterone(T)为配基 ,测定大鼠骨骼肌胞浆中的雄激素受体结合容量。方法 :测定温度为 4℃ ,同位素配基的饱和浓度为 5 0 pmol/ml;肌组织以 4倍 (重量 /体积 )缓冲液稀释 ,0～ 4℃温度下 10 80 0 0×g离心 1h ;孵育 18～ 2 4h。结果 :骨骼肌中雄激素受体的Kd =2 ,8× 10 9mol/ml。单点法与多点法之间无显著区别。     

COMPARTMENTATION OF AMINO ACID METABOLISM IN THE RAT POSTERIOR PITUITARY

—Isolated rat posterior pituitary glands were incubated with [¹⁴C]glucose or [¹⁴C]acetate and the incorporation of radioactivity into several amino acids was followed. The results indicated that radioactivity was incorporated from [¹⁴C]glucose into a large pool of glutamate which appeared to be responsible for a large proportion of GABA synthesis in the gland. The specific activity of glutamine was always less than that of glutamate when [¹⁴C]glucose was the precursor employed, whereas [¹⁴C]acetate labelled a glutamate pool which had approximately the same specific activity as that of glutamine. The results are discussed with reference to the compartmentation of amino acid metabolism in the nervous system.     

PHOTOMETRIC EVIDENCE FOR THE OSMOTIC BEHAVIOR OF RAT LIVER MICROSOMES

Electron microscope observations are consistent with the interpretation that the elements of the endoplasmic reticulum are osmotically active in situ as well as after isolation. More recently, it has been reported that microsomal suspensions equilibrate almost completely with added C¹⁴-sucrose and that no osmotic behavior is evident from photometric data. These findings were considered at variance with the electron microscope data. However, equilibration with added label simply attests to a relatively high permeability, and, in addition, the photometric data need not be critical. Osmotic volume changes, measured photometrically, may be masked by concomitant events (e.g., changes in the refractive index of the test solutions at varying osmotic pressures, breakdown of the particles, and agglutination). For these reasons the photometric experiments were repeated. In this work, the reciprocal of optical density of microsomal suspensions was found to vary linearly with the reciprocal of concentration of the medium at constant refractive index. These changes probably correspond to osmotic volume changes, since the effect was found to be (a) independent of substance used and (b) osmotically reversible. The transmission of the suspension was found to vary with the refractive index of the medium, the concentration of particles, and the wavelength of incident light, according to relationships that are similar to or identical with those obtained for mitochondrial suspensions.     

大鼠垂体远侧部内分泌细胞的四重免疫组织化学染色方法

本文介绍一种显示大鼠垂体远侧部内分泌细胞的四重免疫组织化学染色方法。此法以PAP 法为基础,结合 DAB 银加强法和间接免疫金标记技术。方法比较简单,所需试剂容易得到。