Comparison of Rapid Methods for Analysis of Bacterial Fatty Acids

When rapid gas-liquid chromatography methods for determination of bacterial fatty acids were compared, results showed that saponification was required for total fatty acid analysis. Transesterification with boron-trihalide reagents (BF(3)-CH(3)OH, BCl(3)-CH(3)OH) caused extensive degradation of cyclopropane acids and was less effective than saponification in releasing cellular hydroxy fatty acids. Digestion of cells with tetramethylammonium hydroxide was unsatisfactory because of extraneous gas-liquid chromatography peaks and because of lower recovery of branched-chain and hydroxy fatty acids. A simple, rapid saponification procedure which can be used for total cellular fatty acid analysis of freshly grown cells is described.     

Non-enzymatic ATP-mediated binding of hydroxymethyl derivatives of aromatic hydrocarbons to DNA

ATP mediates covalent binding of hydroxymethyl derivatives of aromatic hydrocarbons to DNA. This non-enzymatic reaction has been studied with 6-[14C]hydroxymethylbenzo[alpha]pyrene (]14C]BP-6-CH2OH) and 7-[14C]-hydroxymethylbenz[alpha]anthracene ([14C]BA-7-CH2OH) at 37 degrees C in Tris buffer (pH 7.0). While ADP mediates the reaction 25-50% as well as ATP, six other possible phosphate donors including AMP were inactive as cofactors. A complex response to ATP occurred in which low binding of BP-6-CH2OH or BA-7-CH2OH was observed at concentrations of ATP below 2.5 mM, but a greater than linear response to higher concentrations of ATP was observed until ATP was saturating. Binding of the substrates to RNA was much lower than to DNA. Fluorescence spectra of BP-6-CH2OH bound to DNA were almost identical to the spectra of 6-bromomethylbenzo[alpha]pyrene bound to DNA and free 6-methylbenzo]alpha]pyrene, indicating that ATP-mediated binding of BP-6-CH2OH to DNA occurs at the 6-methyl group. The fate of ATP and ADP in the binding reaction of BP-6-CH2OH was examined by thin layer chromatography. Loss of one phosphate group occurs during the reaction. With ATP the rate of loss is about 100-fold greater than the rate of binding of BP-6-CH2OH to DNA. This implies that the binding reaction proceeds through formation of a presumed reactive and unstable phosphate ester intermediate which then inefficiently binds to DNA.     

Loss of preservative from a tuberculin solution in rubber stoppered vials fastened with different seals

A tuberculin purified protein derivative (PPD) solution containing 0.3% phenol as a preservative was dispensed in glass vials closed with rubber stoppers fastened in three different ways, namely with Tear-off seals, Flip-off seals and partial seals. After various times of storage at 5 degrees C and 37 degrees C, the phenol content in the tuberculin solution was determined. It was found that the Flip-off seals allowed the phenol to escape at a faster rate than the Tear-off seals and that vials closed with partial seals showed the highest loss of phenol. Although these losses were much more pronounced at 37 degrees C than at 5 degrees C, the phenol content at the latter temperature was, over a period of three years, within the limits of acceptability for tuberculin products capped with Tear-off or Flip-off seals. A loss of phenol also occurred from tuberculin solution stored at -28 degrees C in vials capped with either Tear-off or partial seals. In addition to the Tear-off and Flip-off seals other seals such as the "controlled score' Flip-off seal and the Alcoa Steri-Twist cap were evaluated for their imperviousness to air. Except for the Alcoa Steri-Twist cap none of the seals we have investigated were air tight and hence entirely satisfactory to prevent losses of phenol by evaporation from tuberculin products.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell loss in bisected mouse, sheep and cow embryos

The purpose of this experiment was to determine the loss of cells during the splitting of embryos. The experiment was carried out on early, middle and late stage mouse blastocysts as well as on late stage sheep and cow blastocysts. The embryos were bisected with a metal mioroscalpel. Manipulation of the embryos was done in phosphate buffered saline (PBS) solution with 20% fetal calf serum (FCS) at +37 degrees C or in PBS solution free of Ca(++) and Mg(++) ions. To determine the number of cells present, intact embryos and demi-embryos were placed in acridine orange solution and examined under a fluorescent microscope. The average number of cells in early, middle and late stage mouse blastocysts was 39.4, 54.1 and 71.9, respectively. The loss of cells caused by splitting was 6.4, 7.6 and 9.9, respectively. The average number of cells in late stage sheep blastocysts was 115.3 and in late stage cow blastocysts it was 138.5. In sheep and cows, the loss of cells due to splitting the embryos in a PBS solution free of Ca(++) and Mg(++) ions did not differ from that observed in a PBS solution with 20% FCS. Similarly, bisection carried out at +37 degrees C did not result in lower cell losses, than that done at room temperature.     

Stereospecificity in hydroxyl radical scavenging activities of four ginsenosides produced by heat processing

The activity-guided fractionation of sun ginseng (SG, heat processed Panax ginseng C. A. Meyer at 120 degrees C) was carried out to identify its main active hydroxyl radical (*OH) scavenging components. As a result, the n-BuOH fraction mainly consisting of ginsenosides showed the strongest activity. Of several ginsenosides of SG, the *OH scavenging activities of relatively high contents of 20(S)-Rg(3), 20(R)-Rg(3), Rk(1), and Rg(5) were compared. Rg(5) and 20(S)-Rg(3) showed strong *OH scavenging IC(50) values of 0.15 and 0.44 mM, respectively, and these activities were prominently higher than each of their respective isomers. Therefore, stereospecificity exists in the *OH scavenging activities of ginsenosides produced by heat processing. Especially, the double bond at carbon-20(22) or the OH group at carbon-20 geometrically close to OH at carbon-12 is thought to increase the *OH scavenging activity of ginsenosides.     

Optically active aromatic amino acids. Part VI. Synthesis and properties of (Leu5)-enkephalin analogues containing O-methyl-L-tyrosine1 with ring substitution at position 3'.

Twelve new [Tyr(Me)1, Leu5]-enkephalin analogues with substituents at position 3' of the Tyr ring have been synthesized using traditional solution methods. The substituents were -CO2H, -CONH2, -CO2Me, -(E)-CH=NOH, -(E)-CH=NOMe and CH2OH. The analogues were C-terminated with methyl esters, amides or as free acids. In the in vitro biological assays a remarkable agonist activity to the opiate receptor mu in guinea pig ileum (GPI) relative to Leu-ENK was shown by the following: Leu-ENK, 100; [Tyr(Me)(3'-CO2Me)1, Leu-OMe5]-ENK (I), 8.1; [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OMe5]-ENK (VI), 26.2; [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OH5]-ENK (VII), 2.9; [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-NH2(5)]-ENK (VIII), 4.7; and [Tyr(Me)(3'-CH2OH)1, Leu-OMe5]-ENK (X), 5.6. The agonist effect was naltrexone- or naloxone-reversible. The masking of the hydroxyl group in (E)-hydroxyiminomethyl group of analogue (VI) by O-methylation has totally abolished its GPI agonist activity. It seems that the (E)-CH=NOH group shows affinity and plays an analogous role to the phenol group Tyr1 in leucine-enkephalin and in the tyramine group of the opiate alkaloids. The analogues: [Tyr(Me)(3'-CO2Me)1, Leu-OMe5]-ENK (I), [Tyr(Me)(3'-CO2H)1, Leu-OMe5]-ENK (II), [Tyr(Me)(3'-CO2Me)1, Leu-NH2(5)]-ENK (III), [Tyr(Me)(3'-CO2H)1, Leu-NH2(5)]-ENK (IV), [Tyr(Me)(3'-CONH2)1, Leu-NH2(5)]-ENK (V), [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OMe5]-ENK (VI), [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-OH5]-ENK (VII), [Tyr(Me)(3'-(E)-CH=NOH)1, Leu-NH2(5)]-ENK (VIII), [Tyr(Me)(3'-(E)-CH=NOMe)1, Leu-OMe5]-ENK (IX), [Tyr(Me)(3'-CH2OH)1, Leu-OMe5]-ENK (X), [Tyr(Me)(3'-CH2OH)1, Leu-OH5]-ENK (XI) and [Tyr(Me)(3'-CH2OH)1, Leu-NH2(5)]-ENK (XII) under testing had no significant agonist activity to the enkephalinergic receptor in mouse vas deferens (MVD). All methyl esters of synthesized analogues of [Leu5]-ENK showed higher activity to mu receptors than structurally identical C-terminal amides. It is a surprising result since usually C-terminate amides are stronger agonists than C-terminate esters.     

On the stability of salivary progesterone under various conditions of storage

The concentrations of progesterone in saliva of women exhibited significant decreases when the fluid was stored in plastic vials for 3 days at room temperature or 37 C. The addition of antibiotics or a variety of metabolic poisons to the saliva prior to storage did not prevent the progesterone decrement. However, the addition of albumin (2 g/dl) was protective, suggesting that the protein impeded adsorption of salivary progesterone by the plastic container. Saliva could be maintained at 37 C for 3 days in glass vials or at -20 C in plastic containers for indefinite periods without loss of progesterone titers. These data indicate that a patient under luteal function assessment may collect saliva samples in glass vials at regular intervals during the latter half of her cycle and store them in the freezer compartment of the refrigerator until shipment by mail to the laboratory for progesterone assay. With special care, plastic vials charged with albumin may also be used.     

Fatty acid composition of the tropical lichen Teloschistes flavicans and its cultivated symbionts

Fatty acid components, in both the free and combined form of the intact tropical lichen Teloschistes flavicans, and its isolated photobiont and mycobiont, were analyzed by GC-MS of derived methyl esters. Its rDNA analysis confirmed that the isolated cultured symbionts belong to the genera Trebouxia and Teloschistes, respectively. The fatty acid composition of the lichen did not correspond to those found in the isolated symbionts, suggesting that the fatty acid metabolism is markedly influenced by the symbiosis. Differences in the fatty acid composition in the lichen were observed during the summer (27 degrees C), when the main fatty acids were saturated and in the winter (22 degrees C) when an increase of unsaturated fatty acids occurred. Similar differences of composition were also observed for the cultured mycobiont at different temperatures. The increase in the unsaturation level at low temperatures would maintain the membrane fluidity. Our results are the first on the fatty acids of a tropical lichen and suggest that it is sensitive to small temperature variations, which influences its saturated and unsaturated fatty acid composition.     

A simple quantitative assay for chloramphenicol acetyltransferase by direct extraction of the labeled product into scintillation cocktail.

A simple, rapid, sensitive, quantitative, and inexpensive assay for chloramphenicol acetyltransferase (CAT) is described. The assay is based on the direct extraction of the products of the reaction into toluene-based liquid scintillation cocktail. The assay is carried out in 7-ml scintillation vials using 1 mM chloramphenicol and either 100 microM acetyl-CoA and 0.1 microCi of [3H]acetyl-CoA or 1 mM acetyl-CoA and 0.5 microCi of [3H]acetyl-CoA. After incubation, the reaction is terminated with 0.5 ml of 0.1 M sodium borate-5 M NaC, pH 9. The acetylchloramphenicols are extracted with 5 ml of 0.4% 2,5-diphenyloxazole-0.005% 1,4-bis(5-phenyloxazol-2-yl)benzene in toluene by a 30-s shaking. After a short centrifugation to clarify the layers, the vials are counted in a liquid scintillation counter. Extracted products are stable in the organic layer. Under these conditions, nearly 100% extraction of acetylchloramphenicols is shown using nonlabeled compounds and spectrophotometric methods. Using pure enzyme in the assay, linearity of activity with enzyme concentration, time, and temperature of incubation is demonstrated. Assays may even be carried out at 60 degrees C, where the enzyme activity is 3.4-fold higher than that at 23 degrees C. The increase in enzyme activity with increasing temperature is due to the increased formation of predominantly 3-acetyl and 1-acetylchloramphenicols and not to 1,3-diacetylchloramphenicol. The present assay compared very well with the standard assay using [14C]chloramphenicol and TLC. Using this assay, we measured quantitatively the CAT activity in extracts of pSV2-CAT-transfected CV-1 cells in 10 min and NIH 3T3 cell extracts in 60 min at 60 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enantiomeric resolution of asymmetric glycol by a lipase‐catalyzed transesterification reaction

The lipase catalyzed enantiomeric resolution of syn‐glycol was carried out to confirm the sector method, which can determine the absolute configuration of anti‐ and syn‐glycol from the ¹H‐NMR spectra of bis‐2‐methoxy‐2‐trifluoromethyl‐2‐phenylacetic acid (MTPA) esters. The lipase catalyzed transesterification reaction was most reactive at the C2 position (C2–OH) of (2R;3R)‐2,3‐octanediol. Both (2S;3S)‐ and (2R;3R)‐2,3‐octanediol were prepared using lipase. The ¹H‐NMR spectra of their bis‐(R)‐MTPA esters agreed well with those prepared previously via mono‐(R)‐MTPA esters. The result suggests the retention of the Mosher plane in MTPA esters possessing a hydroxyl group at the β position. The reaction rate and the stereoselectivity decreased at C2–OH with the addition of 18‐crown‐6. Chirality 11:338–342, 1999. © 1999 Wiley‐Liss, Inc.     

Escherichia coli membrane fluidity as detected by excimerization of dipyrenylpropane: sensitivity to the bacterial fatty acid profile.

A coordinated study of membrane fluidity and fatty acid composition has been carried out in Escherichia coli W3110. The lipid acyl chain profile of the bacteria, altered by growing cells in steady state at 30, 37, 42, or 45 degrees C, was determined by gas chromatography of the fatty acid methyl esters. In parallel experiments, total membranes obtained from cells of the above-mentioned cultures were labeled with dipyrenylpropane and their relative fluidity was measured on the basis of the excimer to monomer fluorescence intensity ratio of the fluorophore. It has been found that, at constant assay temperature, fluidity determined with dipyrenylpropane decreases gradually with the growth temperature increment, from 30 to 45 degrees C. Interestingly, when fatty acid composition is taken into account, fluidity increases linearly in the range under study, with the proportion of unsaturated fatty acyl chains, both variables being highly correlated (0.924 相似文献   

Carcinogenicity and metabolic profiles of 6-substituted benzo[a]pyrene derivatives on mouse skin

The ability was tested of appropriate substituents of benzo[a]pyrene (BP) at C-6 to decrease or suppress the carcinogenic activity for these BP derivatives relative to the parent compound. 8-week-old female Swiss mice in 9 groups of 30 were treated on the back with 0.2 mumol of compound in acetone 4 times weekly for 20 weeks. The following compounds were administered: BP, 6-methylbenzo[a]pyrene (BP-6-CH3), 6-hydroxymethylbenzo[a]pyrene (BP-6-CH2OH), benzo[a]pyrene-6-carboxaldehyde (BP-6-CHO), benzo[a]pyrene-6-carboxylic acid, 6-methoxybenzo[a]pyrene, 6-acetoxybenzo[a]pyrene, 6-bromobenzo[a]pyrene, and 6-iodobenzo[a]pyrene. Two additional groups received BP or BP-6-CH3 twice weekly for 20 weeks at a total dose 25% of that above. In addition, the metabolism of selected 6-substituted BP derivatives was studied, using mouse skin homogenates in vitro and mouse skin in vivo. Only four compounds were carcinogenic; the order of potency was BP greater than BP-6-CH3 greater than BP-6-CH2OH and BP-6-CHO. The difference in carcinogenicity between BP-6-CH2OH and BP-6-CHO could not be assessed by this experiment. In a further tumorigenesis experiment the carcinogenicity of BP-6-CH2OH was compared to that of BP-6 CHO, BP-6-CH3 and 6-hydroxymethylbenzo[a]pyrere sulfate ester (BP-6-CH2OSO3Na) on mouse skin. 9-week-old female Swiss mice in groups of 28 were treated at three dose levels with 0.8, 0.2 and 0.05 mumol of compounds in dioxane--dimethyl sulfoxide (75 : 25) twice weekly for 40 weeks. After 40 experimental weeks BP-6-CH2OSO3Na proved to be a more potent carcinogen than BP-6-CH2OH, which, in turn was more active than BP-6-CHO. The greater carcinogenicity of BP-6-CH3 relative to BP-6-CH2OH and BP-6-CHO is confirmed, suggesting that BP-6-CH2OH is not a proximate carcinogenic metabolite for BP-6-CH3. Since BP-6-CHO is a weaker carcinogen than BP-6-CH2OH and is efficiently reduced metabolically to BP-6-CH2OH, the latter compound may be a common proximal carcinogenic metabolite. The stronger potency of BP-6-CH2OSO3Na, compared to its alcohol, suggests that an ester of BP-6-CH2OH might be the ultimate alkylating compound reacting with cellular nucleophiles.     

Fungitoxicity of 1,4-naphthoquinones to Candida albicans and Trichophyton mentagrophytes.

Twenty-one substituted 1,4-naphthoquinones and five 8-quinolinols and copper(II) chelates were tested for antifungal activity against Candida albicans and Trichophyton mentagrophytes. Compounds containing electron-releasing or weak electron-withdrawing groups in the 2 and 3 positions of the 1,4-naphthoquinone ring were the most active against C. albicans at pH 7.0 in the presence of beef serum in the following order: 2-CH3O = 2,3-(CH3O)2 greater than 2-CH3 greater than 2-CH3S greater than 2-NH2 greater than 2,6-(CH3)2. For T. mentagrophytes under the same conditions the inhibitory 1,4-naphthoquinones contained the substituents 2-CH3O greater than 2,3-(CH3O)2 greater than 2-CH2S greater than 2-CH3 greater than 2-CH3(NaHSO3) greater than 2-NH2 greater than 2-C2H5S, 3-CH3 greater than 2,6-(CH3)2 greater than 2,3-CL2 greater than 5,8-(OH)2.     

Synthesis of D-allose fatty acid esters via lipase-catalyzed regioselective transesterification

The acylation of the rare sugar, D-allose (the C-3 epimer of D-glucose), with fatty acid vinyl esters was successfully carried out using Candida antarctica lipase in acetonitrile at 45 degrees C to give D-allose 6-alkanoates with high regioselectivity in good yields.     

Silylation reaction of dextran: effect of experimental conditions on silylation yield,regioselectivity, and chemical stability of silylated dextrans

The controlled synthesis of biodegradable copolymers of dextran grafted with aliphatic polyesters first requires the preparation of polysaccharide derivatives soluble in organic solvents. Silylation of dextran can thus lead to such organosoluble derivatives and allows the polymerization of cyclic esters initiated from the nonsilylated OH functions. Silylation of dextran was studied in DMSO by different reactants such as 1,1,1,3,3,3-hexamethyldisilazane (HMDS) in the presence of various catalysts and N,O-bis(trimethylsilyl)acetamide (BSA). According to the silylating agent and the used experimental conditions, it was possible to obtain highly or totally silylated dextrans. In parallel, an investigation of the chemical stability of the dextran chain during silylation was performed. Thus, it was found that, when used at 50 degrees C, HMDS with or without catalysts gives a relatively high silylation yield and does not alter the dextran chain length, whereas at 80 degrees C, dextran degradation was observed. BSA is a very good silylating agent, which allows reaching 100% silylation even at 50 degrees C but provokes the degradation of the polysaccharide chains. The work was completed by a study of the reactivity order of the glucosidic OH functions toward silylation reaction. This order was found to be (OH(2) > OH(4) > OH(3)) as already reported for other reactions. 2D-NMR of highly silylated dextrans demonstrated that they are constituted of both quantitatively silylated glucose units and two types of disilylated ones.     

Cryopreservation studies of Campylobacter

Seven strains of Campylobacter fetus ss. fetus, one of Campylobacter fetus ss. venerealis, and one of Campylobacter jejuni were preserved using a variety of cryopreservation methods. Organisms were frozen to -150 degrees C in a liquid nitrogen refrigerator, in the freezer compartment of a refrigerator (-20 degrees C), and in a mechanical freezer (-65 degrees C). In the latter two cases, viabilities of the organisms were compared after being frozen in Brucella Albimi broth and 10% glycerol. Viabilities were also examined after Campylobacter species were freeze-dried using rapid or slow cooling, using sucrose or skim milk as cryoprotective agents and in bulb-type vials on a manifold or batch vials. Preservation in liquid nitrogen resulted in no loss in viability after 4 years storage. When Campylobacter species were frozen at -20 degrees C, no cells were recovered after 1 month storage in Brucella Albimi broth or seven months in glycerol. A 6.5 log decrease in viability resulted after organisms were frozen at -65 degrees and subsequently stored at the same temperature for 2 years. In this case, glycerol had no protective advantage over Brucella Albimi broth. Postpreservation viability of organisms cooled slowly was two logs higher than those cooled rapidly prior to freeze-drying. When skim milk or sucrose were employed as cryoprotective agents during freeze-drying, equal viabilities resulted. Equivalent viabilities were also demonstrated when the bulb type or "batch" vials were utilized for freeze-drying. No significant differences were observed between the viabilities of the three species when a given cryopreservation method was employed.     

The effects of ionizing radiation on the peroxide content of a pure polyunsaturated lipid dispersion and of lipids and membranes derived from Acholeplasma laidlawii

Dispersions of a pure unsaturated phospholipid, dilinoleoylphosphatidyl choline, formed conjugated diene hydroperoxides when irradiated in air with 7 MeV electrons (150 Gy and 300 Gy). Peroxide formation was optimized when the dispersions were irradiated in air at 37 degrees C at a dose rate of 5 Gy/min. No significant loss of linoleic acid from the irradiated phospholipid dispersions was observed after doses of 150 or 300 Gy. Small amounts of thiobarbituric acid-reactive material were formed in irradiated unsaturated phospholipid dispersions. However, lipids or membranes isolated from 48 hour cultures of Acholeplasma laidlawii grown in media supplemented with either linoleic or linolenic acid did not appear to be peroxidized by irradiation under the same conditions.     

Laboratory-scale inactivation of African swine fever virus and swine vesicular disease virus in pig slurry

Two methods were evaluated for the inactivation of African swine fever (ASV) and swine vesicular disease (SVD) viruses in pig slurry: chemical treatment and heat treatment. The addition of NaOH or Ca(OH)2 at different concentration/time combinations at 4 degrees C and 22 degrees C was examined, as was virus stability at different temperature/time combinations. ASF virus (ASFV) was less resistant to both methods than SVD virus (SVDV). In slurry from one source, ASFV was inactivated at 65 degrees C within 1 min, whereas SVDV required at least 2 min at 65 degrees C. However, it was found that thermal inactivation depended on the characteristics of the slurry used. Addition of 1% (w/v) of NaOH or Ca(OH)2 caused the inactivation of ASFV within 150 s at 4 degrees C; 0.5% (w/v) NaOH or Ca(OH)2 required 30 min for inactivation. NaOH or Ca(OH)2 (1% (w/v)) was not effective against SVDV at 22 degrees C after 30 min, and 1.5% (w/v) NaOH or Ca(OH)2 caused inactivation of SVDV at both 4 degrees C and 22 degrees C. At higher chemical concentrations or temperatures, ASFV and SVDV inactivation was faster in slurry than in buffered medium.     

Physico-chemical studies of aluminum hydroxide in biological preparations preserved in different conditions

An observation of physico-chemical properties of 0.5% Al(OH)3 and vaccines adsorbed to it (Di, Te, Di-Te, Di-Te-Per) stored for 3-24 months in various pH (5, 7, 8) and temperatures -18 degrees, +37 degrees, +45 degrees and +65 degrees was carried out. For this purpose an analysis of sedimentation rate, microscopic observation and ++roentgenographic analysis were performed. It was found that a decrease of storage temperature of Al(OH)3 gel and vaccines down to -18 degrees C resulted in some changes in structure and morphology of this absorbent (big precipitates) and it led to a significant increase of sedimentation rate.     

Changes in acetylcholine concentration, miniature end-plate potentials and synaptic vesicles in frog neuromuscular preparations during lanthanum treatment

ACh content and synaptic ultrastructure were compared in neuromuscular preparations (sartorius muscle of Rana esculenta) incubated in control saline and in saline containing 1 mM LaCl3. ACh concentrations remained constant for 6 hr in control preparations. La3+ caused a 38% depletion of ACh within the first 30 min with subsequent recovery to 120% of control values within 3-4 hr. Recovery was prevented by hemicholinium-3. At 23 degrees C La3+ caused complete loss of synaptic vesicles: no depletion was seen at 4 degrees C. Initially MEPP frequency increased 300- to 700-fold (23 degrees C), then declined. Mean vesicle diameter did not change, but SD increased. As the frequency of MEPPs declined, the percentage of s-MEPPs greatly increased. La3+ had a postsynaptic effect which increased the amplitudes of both s-MEPPs and bell-MEPPs within a few seconds. The s-MEPP mean did not change during the course of La3+ treatment although the bell-MEPP mean usually decreased. How the decrease in synaptic vesicles, decrease in MEPP frequencies, and changes in ACh levels relate to changes in the percentage of different classes of quanta is discussed.