THE PHYSIOLOGICAL EFFECTS OF l-TYROSINE AND l-PHENYL-ALANINE ON THE RATE AND QUALITY OF PULSATION IN THE SEVENTY-TWO HOUR EXTIRPATED EMBRYONIC CHICK HEART

1. 72 hour isolated chick hearts show an increase in pulsation rate when placed in M/1000, M/10,000, and M/50,000 l-tyrosine solutions. The optimal effect is seen in M/10,000 and M/50,000 l-tyrosine. 2. All hearts show disturbance of rhythm either in the form of irregular rhythm or heart block. 3. 62 hour isolated chick hearts are not susceptible to l-tyrosine while 96 hour hearts are markedly sensitive. 4. 72 hour isolated chick hearts placed in 1 part in 10,000 and 1 part in 50,000 l-epinephrine show approximately the same effects as were seen with l-tyrosine. 5. 72 hour isolated chick hearts placed in M/1000 and M/10,000 l-phenylalanine show an initial depression followed by an l-tyrosine effect.     

白细胞介素-2对离体大鼠心脏的作用及其机理

本实验研究免疫调节因子IL-2对心脏的生物学作用及其机制.实验结果显示,hrIL-2增加离体心脏的室性早搏个数,增加心率,影响左室发展压、左室舒张末压和冠脉流量;热失活的hrIL-2对心脏无作用;Ryanodine预处理不改变hrIL-2的致心律失常作用和增加心率的作用,但取消了hrIL-2增加左室发展压、左室舒张末压和冠脉流量的作用;Nifedipine和低钙均取消了hrIL-2的致心律失常作用,部分取消了hrIL-2增加心率、左室发展压、左室舒张末压和冠脉流量的作用.结果提示,IL-2可导致离体心脏心律失常和正性变时变力作用,其心脏作用与其正常的空间结构有关,作用机制涉及跨膜内流钙和胞浆内钙.     

CHICK BRAIN CREATINE KINASE ONTOGENY BEFORE THE ADVENT OF CREATINE AND INSULIN

The ontogeny of brain creatine kinase (CK) was studied during chick embryo development. The cytosolic activity increased 270% in 10 h from the 2nd to the 3rd days of incubation; this was followed by a plateau phase throughout development and at the end of incubation there appeared to be another increase of cytosolic and mitochondrial CK activities. Therefore, early embryonic chick brain CK is another‘constitutive’enzyme like the early embryonic chick heart CK since creatine has not been enzymatically detected in the embryo until day 4 of incubation. Insulin does not appear to stimulate the early increase of brain CK activity since the hormone is not present in the embryo until day 5 of incubation. It is likely that CK increase is associated with neuronal multiplication at early stages and possibly to neuronal maturation before hatching.     

CIRCADIAN PROFILES IN THE EMBRYONIC CHICK HEART: L-TYPE VOLTAGE-GATED CALCIUM CHANNELS AND SIGNALING PATHWAYS

Circadian clocks exist in the heart tissue and modulate multiple physiological events, from cardiac metabolism to contractile function and expression of circadian oscillator and metabolic-related genes. Ample evidence has demonstrated that there are endogenous circadian oscillators in adult mammalian cardiomyocytes. However, mammalian embryos cannot be entrained independently to light-dark (LD) cycles in vivo without any maternal influence, but circadian genes are well expressed and able to oscillate in embryonic stages. The authors took advantage of using chick embryos that are independent of maternal influences to investigate whether embryonic hearts could be entrained under LD cycles in ovo. The authors found circadian regulation of L-type voltage-gated calcium channels (L-VGCCs), the ion channels responsible for the production of cardiac muscle contraction in embryonic chick hearts. The mRNA levels and protein expression of VGCCα1C and VGCCα1D are under circadian control, and the average L-VGCC current density is significantly larger when cardiomyocytes are recorded during the night than day. The phosphorylation states of several kinases involved in insulin signaling and cardiac metabolism, including extracellular signal-regulated kinase (Erk), stress-activated protein kinase (p38), protein kinase B (Akt), and glycogen synthase kinase-3β (GSK-3β), are also under circadian control. Both Erk and p38 have been implicated in regulating cardiac contractility and in the development of various pathological states, such as cardiac hypertrophy and heart failure. Even though both Erk and phosphoinositide 3-kinase (PI3K)-Akt signaling pathways participate in complex cellular processes regarding physiological or pathological states of cardiomyocytes, the circadian oscillators in the heart regulate these pathways independently, and both pathways contribute to the circadian regulation of L-VGCCs. (Author correspondence: gko@cvm.tamu.edu)     

鸡胚与胎鼠的成纤维细胞条件培养液促进成肌细胞增殖和融合的比较

用培养过鸡胚(来亨鸡)或胎鼠(ICR小鼠)肌组织的成纤维细胞的条件培养液,定量地研究它们对胎鼠或鸡胚的成肌细胞的增殖和融合的影响。所得结果如下:(1) 胎鼠的成纤维细胞条件培养液促进胎鼠或鸡胚成肌细胞增殖,分别为对照组的2.65倍,(P<0.001)或2.35倍,(P<0.01);(2) 鸡胚的成纤维细胞条件培养液促进鸡胚或胎鼠的成肌细胞增殖,分别为对照组的2.66倍,(P<0.01)或2.17倍,(P<0.01);(3) 胎鼠的成纤维细胞条件培养液增加胎鼠或鸡胚的成肌细胞的融合率,分别为对照组的1.9倍或2.6倍;鸡胚的成纤维细胞条件培养液只增加鸡胚成肌细胞的融合率,为对照组的2.1倍,但对胎鼠成肌细胞的融合无明显的影响。 实验结果提示:成纤维细胞条件培养液促进成肌细胞的增殖,两种动物间无明显的差异,但在融合上却有一定的种属特异性。     

THE NUTRITION OF ANIMAL TISSUES CULTIVATED IN VITRO : IV. AMINO ACID REQUIREMENTS OF CHICK EMBRYONIC HEART FIBROBLASTS

1. The amino acid requirements of freshly explanted chick embryonic heart tissues cultivated in completely synthetic media have been determined, employing a nutritional depletion technique. Arginine, histidine, lysine, tyrosine, tryptophan, phenylalanine, cystine, methionine, threonine, leucine, and valine were found to be essential. Serine, isoleucine, glycine, and glutamine were found to be non-essential. Glutamic acid, aspartic acid, α-alanine, proline, and hydroxyproline were found to be inhibitory in this test system. 2. A total amino acid level of approximately 100 mg. per cent was found to be optimal and DL-amino acids were found to be non-toxic, unless used in high concentrations. 3. A comparison has been made of the amino acid requirements of various types of tissue cultures, of the chick, and of man and certain differences in these requirements have been discussed.     

THE RELATION BETWEEN THE INTRACELLULAR RIBONUCLEIC ACID DISTRIBUTION AND AMINO ACID INCORPORATION IN THE LIVER OF THE DEVELOPING CHICK EMBRYO

The RNA-P and DNA-P content of the nucleus and the RNA-P content of the whole cell of the livers of 8- to 20-day chick embryos and of adult fowls have been determined. The DNA-P content of the liver nuclei was slightly higher in the 8- and 10-day embryo than in all the other stages examined. A significant decrease in the RNA content of the cell occurred during embryonic development. The RNA content of the adult cell was the same as that of the 14- to 16-day embryo. The proportion of the cellular RNA contributed by the nucleus also decreased during development. In respect to both nuclear RNA content and distribution of RNA between nucleus and cytoplasm, the adult resembled the 8- to 12-day embryo. Examination of the fine structure of the cell showed that, as development progressed, free ribosomes decreased in number and the rough membranes increased. Slices of 8-, 14-, and 20-day embryonic livers and of adult livers were incubated with ¹⁴C-leucine, and the amount of labeled amino acid incorporated into whole tissue protein and into the proteins of the subcellular fractions was measured. Embryonic liver incorporated ¹⁴C-leucine 15 to 30 times more rapidly than adult liver. The microsomal protein was always more highly labelled than the protein in any other subcellular fraction; however, in the 8-day embryonic and the adult liver the proportion of total counts found in the nuclear fraction was considerably higher than in the 14- or 20-day embryonic liver. The significance of an apparent correlation between the proportion of the cell's RNA contributed by the nucleus and the proportion of total counts in the nuclear fraction is discussed.     

KERATIN SYNTHESIS DURING DEVELOPMENT OF THE EMBRYONIC CHICK FEATHER

The synthesis of keratin proteins during development of the embryonic chick feather was studied by quantitative gel electrophoresis of the reduced and carboxymethylated proteins. The results demonstrated a coordinated synthesis of the major keratin proteins, during and after the onset of keratin synthesis. The results from gel electrophoresis correlated well with electron microscope visualization or keratin fibrils in the developing feathers. Autoradiography at the electron microscope level indicated that the feather cells lose the ability to synthesize DNA before keratin synthesis begins, but retain the ability to synthesize RNA after keratin synthesis begins.     

大豆磷脂脂质体对再灌注心肌线粒体的影响

利用Ｌａｎｇｅｎｄｏｒｆｆ离体心脏灌流装置,研究在缺血－再灌注时补充大豆磷脂脂质体对心肌线粒体膜脂质特性和超微结构的影响。结果：在缺血－再灌注时补充大豆磷脂脂质体可提高线粒体膜磷脂含量,抑制胆固醇－磷脂摩尔比和膜脂质微粘度的增加,改善线粒体的超微结构。结果表明,补充大豆磷脂脂质体对再灌注心肌线粒体的脂质特性和超微结构的损伤性变化具有保护作用。     

DEVELOPMENTAL PATTERNS OF GANGLIOSIDES AND OF PHOSPHOLIPIDS IN CHICK RETINA AND BRAIN

Abstract— The changes in phospholipids and gangliosides during ontogenesis of chick retina have been compared with those in brain. Three phases of accumulation of ganglioside NeuNAc in the retina were detected. In contrast, brain NeuNAc rapidly increased during embryonic life until hatching, followed by a slower increase up to the adult stage. The phospholipid changes in retina and in brain occur in a-similar manner to the variations observed for gangliosides, however in retina the changes of phospholipid content are less marked than in brain, during embryonic life. There were marked changes in the retina and brain ganglioside patterns with age. G _{d 3} and G _{d 1b} decreased rapidly in per cent; correspondingly, G _{d 1a} increased during embryonic life and became the major ganglioside in place of G _{d 3}. There was a similarity between ganglioside patterns of chick retina and brain. Except for some slight variations during embryonic life, the retinal phospholipid pattern did not change noticeably.     

BIOSYNTHESIS OF COLLAGEN AND NON-COLLAGEN PROTEIN DURING DEVELOPMENT OF THE CHICK CORNEA

The relationship between the rates of increase of corneal protein fractions and incorporation of labeled precursors has been examined during embryonic and early posthatching development of the chick corneal stroma. Non-collagen protein increased gradually from 9 through 20 days of incubation. Collagen accumulated approximately logarithmically through the 19th day, the most rapid rate occurring between 13 and 20 days of incubation. The rates at which labeled amino acids are incorporated into collagen in vivo and in vitro undergo marked changes during the last week of embryonic development, corresponding closely to the rate of collagen accumulation in vivo; whereas incorporation into non-collagen protein changes much less markedly. Changes in the rate of incorporation of precursors into collagen are not due to changes in the rate of conversion of collagen from the soluble to insoluble form, or to changes in the endogenous amino acid pool size. Chick embryo corneal stroma collagen turns over very slowly, if at all. Non-collagen protein turns over more rapidly. An increase in cell number, as indicated by DNA content, does not account for the increased rate of collagen synthesis between the 9th and 16th day of incubation. It is concluded that the observed changes in collagen synthesis reflect changing activities in the individual cornea fibroblasts. These activities are comparable in the intact tissue in vivo and in isolated corneas in vitro.     

MORPHOLOGY OF ISOLATED RABBIT HEART MUSCLE MITOCHONDRIA AND THE OXIDATION OF EXTRAMITOCHONDRIAL REDUCED DIPHOSPHOPYRIDINE NUCLEOTIDE

The morphology of rabbit heart muscle mitochondria isolated in several media has been compared by electron microscopy. The internal structure of isolated mitochondria differs from that of in situ mitochondria, with the type and degree of alteration depending on the isolation medium. Examination of the isolated mitochondria after incubation revealed that additional morphological changes occurred during incubation, but these changes were less pronounced when the incubation was conducted in a complete medium containing substrate. The isolated mitochondria have been shown to be capable of catalyzing a slow aerobic oxidation of extramitochondrial reduced diphosphopyridine nucleotide. The rate of DPNH oxidation observed is sufficient to account for the ability of the mitochondria to oxidize lactate in the presence of catalytic amounts of DPNH. The suspensions used were essentially free of mitochondrial fragments, which are known to oxidize DPNH. Possible relationships of these findings to metabolism in situ are discussed. The results indicate the desirability of correlating biochemical activities with the morphology of isolated mitochondria.     

鸡胚巨噬细胞AcP酶变化及AcP酶与凋亡细胞的关系

本研究采用电镜及酶细胞化学的方法观察了鸡胚脾脏不同胚龄组巨噬细胞溶酶体酸性磷酸酶（AcP酶）的变化、凋亡实验组巨噬细胞及其AcP酶与凋亡细胞的关系。取10天、13天和17天鸡胚脾脏,按Gomori法显示AcP酶,各胚龄脾脏巨噬细胞AcP酶细胞化学反应阳性,按AcP酶染色阳性做溶酶体计数,结果显示随着胚龄的增加溶酶体数随之增加,尤以第17天组溶酶体数增加最为明显,所得数据经统计分析表明各胚龄组间溶酶体数的差异有统计学意义。凋亡实验组采用放线菌酮诱导15天鸡胚脾脏细胞凋亡,结果显示凋亡细胞为各类幼稚血细胞,以幼稚淋巴细胞为主。巨噬细胞未见凋亡,而是吞噬了大量的凋亡细胞和凋亡小体,AcP酶反应颗粒不仅出现在巨噬细胞的溶酶体、吞噬体,还见于高尔基复合体、内质网等。细胞AcP酶反应强度数字化结果表明：凋亡组酶活性显著高于对照组,差别有统计学意义,提示胚胎巨噬细胞在凋亡细胞出现时AcP酶活性增强,说明巨噬细胞吞噬和消化凋亡细胞或凋亡小体是通过AcP酶等活性物质来实现的。     

COMPARISON AND CHARACTERIZATION OF NUCLEAR ISOLATION PROCEDURES AS APPLIED TO CHICK OVIDUCT

A number of methods for the preparation of chick oviduct nuclei have been compared. Nuclei have been isolated in hypertonic sucrose and citric acid and the product has been characterized with respect to cleanliness, ultrastructure, RNA polymerase activity, RNA integrity, and chromatin composition. The study demonstrates that the choice of oviduct nuclear isolation procedure will depend markedly on the purpose for which the nuclei are required. Thus, nuclei prepared entirely in high-molarity sucrose retain the highest levels of RNA polymerase. Those prepared rapidly in the presence of citric acid retain nuclear RNA in an essentially undegraded state. Finally, a bulk preparation is described which, because of its adaptability and high yield of morphologically intact nuclei using large amounts of tissue, is ideal for use in preparing chromatin. Conditions are described by which isolated nuclei can be stored for up to 6 months and retain their morphology, chemical characteristics, and RNA polymerase activity.     

缺氧对离体鸡胚前脑神经细胞生长的影响

在缺氧(95％N_2和5％CO_2)条件下进行8d鸡胚前脑的纯神经细胞培养。培养24,48和72h后,MTT微量比色法检测显示,甲(目替)明显减少,表明神经元已遭严重损害,同时,培养液中葡萄糖被急剧地消耗。但是,即使把糖浓度提高到800—1200mg/100ml,缺氧仍造成神经细胞死亡。结果提示,胚胎脑神经细胞对缺氧敏感,胶质细胞对缺氧神经元的保护可能不在于提供糖原。     

THE TURNOVER AND SUBCELLULAR LOCALIZATION OF THE CHICK FOREBRAIN SMALL NUCLEAR RNAs

Analysis of 2 day old chick forebrain RNA on polyacrylamide gels revealed the presence of the 29S, 18S, 5.5s and 5s rRNAs, 4S tRNA and the small mol wt nuclear RNA (snRNA) species K, L, C, DD and G. The turnover of these molecules was studied following the intracerebral injection of [³H]uridine into 2 day old animals. The specific activities of these molecules declined over a 23 day time course and all displayed first-order decay kinetics. Apparent half-lives (in days) for 29S (7). 18S (S), L (24), C (6.5), DD′ (8.5), 5.5S (7), 5S (13), G (6) and 4S (7) were obtained. The subcellular localization of the snRNAs was studied in 5 day old chick forebrain. The snRNAs accounted for almost 17% of nuclear RNA, in contrast to their low levels in whole forebrain (1.2%). This was due to an approx 20-fold nuclear enrichment of C, DD′, G′ and H. Relatively low levels of K and L were present within the nuclear fraction. However, K and L, and to a lesser extent C and DD, were readily detected within the post-microsomal supernatant. Production of a pH 5 enzyme fraction from this supernatant resulted in a 2-3-fold enrichment of K, L, C and DD′ with respect to tRNA. The polysome containing fractions also displayed significant levels of L with about 1 mol of L per 30 of 5S rRNA. A new method for the determination of the subcellular distribution of the snRNAs is described. It depends upon the purity of the nuclear, microsomal and cell sap subfractions rather than upon total recoveries from such fractions. Utilizing the relative amounts of these RNA species within whole forebrain and the major subfractions, distributions (as a percentage within each fraction) were obtained. The observation of significant levels of K, L, C and DD within the cytoplasmic fractions raises some doubts about previous suggestions of an exclusively nuclear role for these molecules. It is proposed that the snRNAs may be involved in a multistage interaction within the process of eukaryotic gene expression.     

THE DIFFERENTIAL SENSITIVITY OF CULTURED CHICK MESODERMAL CELLS TO ACTINOMYCIN D

Cells were isolated from the somite mesoderm and from the unsegmented (presomite) mesoderm of early chick embryos and exposed to actinomycin D in single cell culture. Actinomycin D inhibited proliferation in cell cultures derived from the unsegmented mesoderm, although the same concentrations of this antibiotic did not inhibit cultures derived from the somite mesoderm. This differential sensitivity parallels the regionally specific necrosis and degeneration observed in the unsegmented mesoderm of intact chick embryos exposed to actinomycin D. In culture, both cell types exhibited approximately the same permeability to labeled actinomycin D and showed comparable inhibition of RNA, DNA, and protein syntheses in the presence of the antibiotic. However, freshly isolated mesodermal cells from the somite region had a higher content of RNA than did cells from the unsegmented region, and the somite cells maintained a higher rate of macromolecular synthesis in untreated cultures.     

THE ONTOGENY OF DOPAMINE-DEPENDENT INCREASE OF ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE IN THE CHICK RETINA

The adenosine 3′,5′-cyclic monophosphate level of chick embryonic retina changes during the course of development. In retinas from 6- to 15-day-old embryos the cAMP level is approximately 7 pmol/mg protein. A sharp 3-fold increase is observed between the 16th and 18th embronic day and remains constant thereafter. A dopamine-dependent increase in cAMP of the chick retina is already present in 7-day-old embryos, and by the 8th embryonic day maximal response is attained. Glutamate promotes a 2-fold stimulation. Carbachol, γ-aminobutyric acid and glycine do not cause any significant change in the level of cAMP of the embryonic tissue. Guanosine 3′,5′-cyclic monophosphate also accumulates during development. Its concentration is approx 0.5 pmol/mg protein from the 8th to the 14th embryonic day, then increases gradually until the 19th day of development when the level observed is approx 14 pmol/mg protein.     

MORPHOGENESIS OF THE COLLAGENOUS STROMA IN THE CHICK CORNEA

The embryonic chick corneal epithelium produces a highly structured acellular matrix beneath its basal surface during early development. This matrix, which contains collagen, serves as a morphogenetic template for subsequent stromal development in that the three-dimensional architecture of the adult corneal stroma is initially established, in miniature, in this epithelially derived connective tissue. Examination of the early corneal epithelium and matrix in both the light and electron microscope suggests that self assembly of the matrix may be one of several important factors in the morphogenesis of this early connective tissue.     

低氧对斑马鱼胚胎心脏和血管发育及相关基因表达的影响

研究以斑马鱼(Danio rerio)为研究模型,选择心脏和血管荧光标记的2个品系斑马鱼为实验材料,设定低氧和常氧2种水体溶氧条件,用荧光显微镜检测低氧胁迫对胚胎形态结构、心脏和血管外部形态、心率、胚胎躯干部主要血管形成的影响.研究发现低氧导致胚胎存活率低于常氧.低氧不仅滞后胚胎发育,而且造成胚胎形态异常.低氧胁迫后斑...