CERTAIN EFFECTS OF SALTS ON THE PENETRATION OF BRILLIANT CRESYL BLUE INTO NITELLA

The effect of various substances on living cells may be advantageously studied by exposing them to such substances and observing their subsequent behavior in solutions of a basic dye, brilliant cresyl blue. The rate of penetration of the basic dye, brilliant cresyl blue, is decreased when cells are exposed to salts with monovalent cations before they are placed in the dye solution (made up with borate buffer mixture). This inhibiting effect is assumed to be due to the effect of the salts on the protoplasm. This effect is not readily reversible when cells are transferred to distilled water, but it is removed by salts with bivalent or trivalent cations. In some cases it disappears in dye made up with phosphate buffer mixture, or with borate buffer mixture at the pH value in which the borax predominates, and in the case of NaCl it disappears in dye containing NaCl. No inhibiting effect is seen when cells are exposed to NaCl solution containing MgCl₂ before they are placed in the dye solution. The rate of penetration of dye is not decreased when cells are previously exposed to salts with bivalent and trivalent cations. The rate is slightly increased when cells are placed in the dye solution containing a salt with monovalent cation and probably with bivalent or trivalent cations. In the case of the bivalent and trivalent salts the increase is so slight that it may be negligible.     

ACCUMULATION OF BRILLIANT CRESYL BLUE IN THE SAP OF LIVING CELLS OF NITELLA IN THE PRESENCE OF NH3

When the living cells of Nitella are placed in a solution of brilliant cresyl blue containing NH₄Cl, the rate of accumulation of the dye in the sap is found to be lower than when the cells are placed in a solution of dye containing no NH₄Cl and this may occur without any increase in the pH value of the cell sap. This decrease is found to be primarily due to the presence of NH₃ in the sap and seems not to exist where NH₃ is present only in the external solution at the concentration used.     

USE OF BLUE LAKE AS AN INDICATOR OF BACTERIAL PENETRATION INTO EGGS

Blue Lake, an insoluble dye, was evaluated as an indicator of potential bacterial penetration into eggs. Various groups of eggs (fresh-laid; commercial; water-washed) were dipped in 0.25% (w/v) Blue Lake in 0.1% Triton X-100 solution for 2 min and incubated at room temperature up to 1 ± 24 h. Penetration was detected by counting the blue dots on shell membranes after breaking the eggs. Commercial eggs allowed the easiest penetration. All commercial eggs showed blue dots even at 2 min incubation and the average count reached 111/egg at 1 h. Water-washed eggs allowed much less penetration than commercial eggs and the counts of blue dots on those eggs were 10/egg at 2 min and 22 at 1 h. Fresh-laid eggs did not allow any penetration up to 24 h. Above results corresponded very well with the penetration study with Salmonella enteritidis and also with the morphological study of eggshell surfaces using electron microscopy where fresh-laid eggs had intact cuticle layers, but commercial eggs did not. Thus, Blue Lake dye might be used as a rapid indicator of bacterial penetration through eggshells.     

STUDIES ON NITELLA HAVING ARTIFICIAL CELL SAP I. REPLACEMENT OF THE CELL SAP WITH ARTIFICIAL SOLUTIONS

A method for replacing the cell sap of Nitella with an artificialsolution was introduced. The technique, which is a modificationof KAMIYA and KURODA'S (1, 2), is applicable not only for isotonicbut also for hypertonic or hypotonic solutions. Photometricdeterminations of K⁺, Na⁺, Ca⁺⁺ and Cl^– proved that thereplacement of the cell sap with the present method is satisfactory.The internodal cell of Nitella, whose cell sap was replacedwith an isotonic solution with a simple composition such asa mixture of KCl, NaCl and CaCl₂, can be kept living at leastfor several days, sometimes even for more than one month. (Received September 6, 1963; )     

CALCULATIONS OF BIOELECTRIC POTENTIALS : IV. SOME EFFECTS OF CALCIUM ON POTENTIALS IN NITELLA

In Nitella the substitution of KCl for NaCl changes the P.D. in a negative direction. In some cases this change is lessened by adding solid CaCl₂ to the solution of KCl. This may be due to lessening the partition coefficient of KCl or to decreasing the solubility of an organic substance which sensitizes the cell to the action of KCl. Little or no correlation exists between this effect of calcium and its ordinary antagonistic action in producing a balanced solution which preserves the life of the cell indefinitely. CaCl₂ is negative to NaCl but positive to KCl. The effects of mixtures of KCl, NaCl, and CaCl₂ are discussed. The concentration effect of a mixture of KCl + CaCl₂ shows certain peculiarities due to action currents: these resemble those found with pure KCl. These studies and others on Nitella, Valonia, and Halicystis indicate that mobilities and partition coefficients are variable and can be brought under experimental control.     

NOTE ON THE PENETRATION OF HYDROXYL IONS INTO GELATIN JELLIES

O ne of the characteristic features of living cells is that they are permeable to ammonia, but relatively impermeable to caustic soda or caustic potash. The usual method of demonstrating this fact is by staining cells with neutral red. This dye is readily taken up by living cells and is at first diffused through the cytoplasm, which thereby acquires a distinct red colour. At a later stage the dye is usually aggregated into irregular granules. When exposed to dilute solutions of ammonia the neutral red in living cells rapidly becomes yellow, indicating the penetration of the alkali. In NaOH or K. OH the colour of the dye does not change, however, until the cell begins to show obvious signs of degeneration.     

CHEMICAL RESTORATION IN NITELLA : IV. EFFECTS OF GUANIDINE

Leaching in distilled water may remove irritability and the potassium effect in Nitella but both of these may be restored by appropriate treatment with guanidine.     

FURTHER STUDIES ON THE INHIBITION OF CYPRIDINA LUMINESCENCE BY LIGHT,WITH SOME OBSERVATIONS ON METHYLENE BLUE

1. Eosin, erythrosin, rose bengale, cyanosin, acridine, and methylene blue act photodynamically on the luminescence of a Cypridina luciferin-luciferase solution. In presence of these dyes inhibition of luminescence, which without the dye occurs only in blue-violet light, takes place in green, yellow, orange, or red light, depending on the position of the absorption bands of the dye. 2. Inhibition of Cypridina luminescence without photosensitive dye in blue-violet light, or with photosensitive dye in longer wave-lengths, does not occur in absence of oxygen. Light acts by accelerating the oxidation of luciferin without luminescence. Eosin or methylene blue act by making longer wave-lengths effective, but there is no evidence that these dyes become reduced in the process. 3. The luciferin-oxyluciferin system is similar to the methylene white-methylene blue system in many ways but not exactly similar in respect to photochemical change. Oxidation of the dye is favored in acid solution, reduction in alkaline solution. However, oxidation of luciferin is favored in all pH ranges from 4 to 10 but is much more rapid in alkaline solution, either in light or darkness. There is no evidence that reduction of oxyluciferin is favored in alkaline solution. Clark''s observation that oxidation (blueing) of methylene white occurs in complete absence of oxygen has been confirmed for acid solutions. I observed no blueing in light in alkaline solution.     

THE KINETICS OF PENETRATION : XVIII. ENTRANCE OF WATER INTO IMPALED HALICYSTIS

The rate of entrance of water into impaled cells of Halicystis Osterhoutii, Blinks and Blinks, has been determined directly by measurements of the rise of sap in a capillary for dilute sea waters (containing between 90 and 30 per cent sea water). The velocity constant remains reasonably constant down to 50 per cent sea water but it decreases markedly in lower concentrations.     

COMPARATIVE STUDIES ON RESPIRATION : IV. THE EFFECT OF ETHER ON THE RESPIRATION OF WHEAT.

These experiments show that 7.3 and 3.65 per cent ether solutions cause an increase in respiration followed by a decrease. The results agree with those of Haas on Laminaria, of Gustafson on higher fungi, and of Mrs. Brooks on bacteria. They do not agree with the theory of Verworn that anesthesia is a kind of asphyxia and that it decreases respiration.     

CHANGES OF APPARENT IONIC MOBILITIES IN PROTOPLASM : IV. INFLUENCE OF GUAIACOL ON THE EFFECTS OF SODIUM AND POTASSIUM IN NITELLA

In Nitella, as in Halicystis, guaiacol increases the mobility of Na⁺ in the outer protoplasmic surface but leaves the mobility of K⁺ unaffected. This differs from the situation in Valonia where the mobility of Na⁺ is increased and that of K⁺ is decreased. The partition coefficient of Na⁺ in the outer protoplasmic surface is increased and that of K⁺ left unchanged. Recovery after the action current is delayed in the presence of guaiacol and the action curves are "square topped."     

SPECTROPHOTOMETRIC STUDIES OF PENETRATION : V. RESEMBLANCES BETWEEN THE LIVING CELL AND AN ARTIFICIAL SYSTEM IN ABSORBING METHYLENE BLUE AND TRIMETHYL THIONINE.

The rate of diffusion through the non-aqueous layer of the protoplasm depends largely on the partition coefficients mentioned above. Since these cannot be determined we have employed an artificial system in which chloroform is used in place of the non-aqueous layer of the protoplasm. The partition coefficients may be roughly determined by shaking up the aqueous solutions with chloroform and analyzing with the spectrophotometer (which is necessary with methylene blue because we are dealing with mixtures). This will show what dyes may be expected to pass through the protoplasm into the vacuole in case it behaves like the artificial system. From these results we may conclude that the artificial system and the living cell act almost alike toward methylene blue and azure B, which supports the notion of non-aqueous layers in the protoplasm. There is a close resemblance between Valonia and the artificial system in their behavior toward these dyes at pH 9.5. In the case of Nitella, on the other hand, with methylene blue solution at pH 9.2 the sap in the artificial system takes up relatively more azure B (absorption maximum at 650 mµ) than the vacuole of the living cell (655 mµ). But both take up azure B much more rapidly than methylene blue. A comparison cannot be made between the behavior of the artificial system and that of the living cell at pH 5.5 since in the latter case there arises a question of injury to cells before enough dye is collected in the sap for analysis.     

小鼠精子注入兔卵母细胞受精研究

利用显微操作仪将小鼠精子注入家兔卵母细胞的胞质内和透明带下,对鼠兔异种精卵互作和异种受精胚胎的发育进行了研究,并对注射精子的数量及卵的体外成熟时间等影响鼠兔异种显微受精的因素进行了探讨,结果如下:(1)将小鼠精子分别注入兔卵胞质内和透明带下,均能激活兔卵母细胞,导致精核解聚和原核形成;(2)小鼠精子注入兔卵胞质内和透明带下受精,杂种胚胎体外培养能发育到8-细胞期;(3)鼠兔异种受精4-细胞胚胎染色体标本制备观察结果表明,它们为正常二倍体;(4)鼠兔异种受精4-细胞胚胎的超微结构观察结果表明,它们极近似兔正常4-细胞胚胎的超微结构;(5)将小鼠精子注入兔卵透明带下,注射5—10个精子组卵的受精率(32.4%)和卵裂率(16.2%)均高于注射单个精子组的,但二组间差异不显著(P>0.05);DM 15%NCS液中体外成熟培养11—12h兔卵透明带下注入1—2个小鼠精子后的受精率(42.3%)和卵裂率(30.8%)均高于体外成熟培养24—25h组的,但二组间差异未达到显著水平(P>0.05)。     

Studies on Foliar Penetration: IV. MECHANISMS CONTROLLING THE RATE OF PENETRATION OF 2,4-DICHLOROPHENOXYACETIC ACID (2,4-D) INTO LEAVES OF PHASEOLUS VULGARIS

The effects of a wide range of metabolic inhibitors on the penetrationof 2,4-dichlorophenoxyacetic acid (2,4-D) into the leaf disksof Phaseolus vulgaris have been studied. While recognizing thelack of specificity of most inhibitors, compounds were chosenwhich are known to affect respiration, phosphorylation, photosynthesis,membrane permeability, protein synthesis, and the binding capacityof membrane systems. They were: fluoride, azide, arsenite, iodoacetate,arsenate, 2,4-dinitrophenol (DNP), 3-(3,4-dichlorophenyl), -I,I-dimethylurea (DCMU), phenylmercuric chloride, octenylsuccinicacid, decenylsuccinic acid, dimethyl sulphoxide, actinomycin-D,chloramphenicol, streptomycin, 5-fluorouracil, cycloheximide,and cetyltrimethylammoniumbromide (CTAB).At sub-toxic levelsall compounds had little or no influence on penetration in darknesssave for iodoacetate and decenylsuccinic acid, which causedsome enhanced entry at 10^-4M and 10^-3M respectively, and CTABwhich promoted penetration at concentrations known tolower thesurface tension of water.The much greater rate of penetrationof 2,4-D into disks exposed to bright light (16 000 lx) is unaffectedby fluoride, azide, DNP, octenylsuccinic acid, decenylsuccinicacid, dimethyl sulphoxide, or actinomycin-D. It is, however,progressively inhibited by increasing concentrations of arsenite,iodoacetate, arsenate, streptomycin, and 5-fluorouracil. Chloramphenicol,cycloheximide, and CTAB lower the rate of penetration at intermediateconcentrations but at high concentrations the affect is reversed.The most active inhibitors of light-induced penetration areDCMU and phenylmercuric chloride, compounds which block theproduction of ATP.These results are discussed in relation tomechanisms of transport, in particular the structureand stabilityof barriers likely to impede penetration.     

药用异喹啉生物碱的研究 Ⅳ.峨眉紫堇生物碱

从峨眉紫堇(Corydalis omeienensis)的根茎中分得十二种生物碱、经UV,IR,[α]_D,CD,~1H-NMR,~(13)C-NMR,MS及物理性质鉴定了它的结构。其中,(-)-tetrahydrocorysamine,为(-)-13-methylprotoberbrine类生物碱,为第二次从植物中获得,至今报导过的该类生物碱从天然得到的也仅此一个。     

STUDIES OF SPERMATOGENESIS IN THE HEPATICAE. IV. ON THE BLEPHAROPLAST OF BLASIA

Spermatogenesis in Blasia pusilla L. was reexamined with electron optics to help evaluate conflicting results of the two earlier light microscope studies of this species. The present micrographic resumé of spermatid transformation confirms Sharp's observation of centrosomes in very young spermatids and agrees with Woodburn's general account of spermatozoid maturation. Further, male gamete development in Blasia is shown to be closely comparable to that of Marchantia and Pellia. It is concluded that Sharp's report of blepharoplast development in Blasia by fragmentation and coalescence was based on anomalous plant material.     

TAXONOMICAL STUDIES ON ARGIDAE (HYMENOPTERA) OF CHINA IV. REVISION OF TANYPHATNIDEINI FROM CHINA WITH DESCRIPTIONS OF TWO NEW SPECIES

本文是中国三节叶蜂科三节叶蜂属新种记述之一,共记述６新种：江氏淡毛三节叶蜂ＡｒｇｅｊｉａｎｇｉＷｅｉ,ｓｐ．ｎｏｖ．,凌氏淡毛三节叶蜂Ａ．ｌｉｎｇｉＷｅｉ,ｓｐ．ｎｏｖ．,大环腹三节叶蜂Ａ．ｍｅｇａｃｉｎｃｔａＷｅｉ,ｓｐ．ｎｏｖ．,裂板淡毛三节叶蜂Ａ．ｔｈａｕｍａｔｏｐｙｇｉａＷｅｉ,ｓｐ．ｎｏｖ．,黑肩黑头三节叶蜂Ａ．ｎｉｇｒｏｃｏｌｌｉｎｉａＷｅｉ,ｓｐ．ｎｏｖ．,关氏黑头三节叶蜂Ａ．ｇｕａｎｉＷｅｉ,ｓｐ．ｎｏｖ．。新种模式标本分别保存于中南林学院昆虫标本室和中国农业大学昆虫标本室。