Assessment of adult skeletons to detect prenatal exposure to acetazolamide in mice

A skeletal variant assay system (SVAS) consisting of a group of 88 spontaneously occurring qualitative variations of the adult mouse skeleton was applied to CD-1 animals that had been exposed in utero to 0, 200, or 1,000 mg/kg/day of the sodium salt of acetazolamide dissolved in distilled water, presented by SC injection of the dam during day 8 or days 9-11 of gestation. Two separate series of experiments were performed, and skeletons were examined at postnatal 62 +/- 2 days. Variation occurred in 62 and 67 characters in the two series. Frequencies of occurrence differed from untreated (UNTD) and vehicle-treated (VEH) values of substantial numbers of variants in a dose related manner for both series in both treatment regimes as did the number of variants which showed significantly different frequencies (P less than .01) in comparisons of experimental with either UNTD or VEH. At the high doses 12 and 16 variants occurred with significantly different frequencies from UNTD in day 8 treatments in the two series, and 15 and 19 variants differed in the days 9-11 treated group. Contrasting high-dose animals with appropriate vehicle controls revealed differences in 13 and 12 variants in day 8 treatment groups and in 18 and 15 variants in days 9-11 groups. Agreement between the two series was good, especially in the D9-11 treatments. Several variants differed significantly from both UNTD and VEH in both series of experiments. Among these were a number which appeared more or less specific to acetazolamide exposure. They include: day 8 treatments--accessory parietal, frontal extension, and 27 presacral vertebrae; day 9-11 treatments--sacral fusions in dorsal processes and vertebral bodies, and caudal fusions and malformations; both sets of treatments--lumbar fusions, and fusions of the transverse processes of the sacral vertebrae. Other importantly affected variants, also seen in exposure to other compounds include: day 8 treatments--abnormal metoptic roots; day 9-11 treatments--accessory mental foramen, foramina transversaria imperfecta of the atlas, arch foramen of the fifth cervical (C) vertebra, malformed sternebrae, fossa olecrani perforata, and fewer than 30 caudal vertebrae; both treatment regimes--parted frontals, accessory transverse foramina in C3-C6, reduced articular processes on the thoracic vertebrae, and 14 ribs. By all criteria applied, the SVAS is able to detect prenatal exposure to acetazolamide in adult skeletons even in the absence of any gross morphological abnormalities.     

Prenatal ossification as an indicator of exposure to toxic agents

Following exposure to bromodeoxyuridine (BUDR), acetazolamide (ACZM), trypan blue (TRBL), cortisone (CORT), or diphenylhydantoin (DPH), alizarin-stained, cleared fetuses were examined at 18 days postcoitus for unossified cervical vertebral centra; number of ossified caudal vertebrae; number of ribs; and ossification of sternebrae, metatarsals, metacarpals, and phalangeal rows. At all teratogenic doses, in no vehicle-treated groups, and rarely in lower-dose groups, there were significant increases in frequency of unossified cervical centra, the first vertebra (C1) being most often affected, and C7 least often affected. In the high-dose CORT group, there was a significant correlation between unossified C1 and cleft palate. No association between abnormality and reduced ossification of cervical vertebrae was seen in other series examined, nor was there any correlation between litter size and abnormality. With minor complications, the number of ossified caudal vertebrae was significantly reduced after exposure at teratogenic dose levels to all compounds except DPH. Although caudal and cervical ossification were correlated with each other in those series examined, neither was correlated with abnormality. Frequency of 14 ribs was increased in BUDR, ACZM, and TRBL but not CORT or DPH. Other parameters were essentially unaffected. Significantly increased frequency of abnormality, when contrasted with untreated or vehicle-treated groups, was seen at high-dose levels in all but DPH treatments, and mortality was increased in ACZM D9-11, TRBL, and CORT. These studies show that reduced ossification of cervical centra is an excellent indicator of prenatal exposure to noxious substances, and caudal vertebrae appear to be useful as well. Increased frequency of 14 ribs occurred for all strong teratogens utilized if they were administered on day 7 or day 8 postcoitus.     

应用反向滤过及迭代重建方法评估标准放射剂量与低剂量颈椎 CT图像质量的对比研究

目的：利用反向滤过重建（filtered back-projection,FBP）及迭代重建（iterative reconstruction,IR）方法评估标准剂量及低剂量 对颈椎CT 图像质量的影响。方法：40 例受检对象行颈椎CT 检查,将其随机分为两组：标准剂量组（SD,120 kVp, 275 mAs）及低 剂量组（LD,120 kVp,150 mAs）,随机选择管电流值,所有数据均行FBP 及IR 重建。测量C3 C4 及C6 C7 椎间盘水平椎间盘、脊 神经、脊髓、韧带以及周围软组织的图像噪声值（Image noise,IN）,信噪比（signal-to-noise,SNR）及对比信噪比（contrast-to-noise, CNR）。结果：在测量的各椎间盘水平,迭代重建的信噪比及对比噪声比要明显高于反向滤过重建方法,并有效的降低了图像噪 声。低剂量迭代重建图像与标准剂量反向滤过图像相比无明显统计学意义。排除剂量及扫描层面的影响,椎间盘、脊神经及韧带 的图像质量,迭代重建评分要明显高于反向滤过重建,结果具有统计学差异;而低剂量迭代重建图像质量评分与标准剂量反向滤 过重建相比无明显差异。软组织及椎体的图像质量,迭代重建图像质量评分要低于反向滤过重建方法,结果具有统计学差异;而 低剂量迭代重建图像质量评分与标准剂量反向滤过重建相比无明显差异。整体病例图像质量评分,迭代重建方法要高于反向滤 过重建方法,低剂量迭代重建方法要高于标准剂量反向滤过重建方法。结论：应用低剂量扫描方式以及迭代重建方法进行颈椎 CT 检查可以为临床提供较好的图像质量,对于椎间盘、脊神经、脊髓显示较好,对于周围软组织以及椎体来说,图像质量相对较 差,同时可以降低大约40%的放射剂量。     

应用反向滤过及迭代重建方法评估标准放射剂量与低剂量颈椎CT图像质量的对比研究

目的：利用反向滤过重建（filtered back-projecfion,FBP）及迭代重建（iterative reconstruction,IR）方法评估标准剂量及低剂量对颈椎CT图像质量的影响。方法：40例受检对象行颈椎CT检查,将其随机分为两组：标准剂量组（SD,120kVp,275mAs）及低剂量组（LD,120kVp,150mAs）,随机选择管电流值,所有数据均行FBP及IR重建。测量C3C4及C6C7椎间盘水平椎间盘、脊神经、脊髓、韧带以及周围软组织的图像噪声值（Imagenoise,IN）,信噪比（signal—to—noise,SNR）及对比信噪比（contrast—to—noise,CNR）。结果：在测量的各椎间盘水平,迭代重建的信噪比及对比噪声比要明显高于反向滤过重建方法,并有效的降低了图像噪声。低剂量迭代重建图像与标准剂量反向滤过图像相比无明显统计学意义。排除剂量及扫描层面的影响,椎间盘、脊神经及韧带的图像质量,迭代重建评分要明显高于反向滤过重建,结果具有统计学差异;而低剂量迭代重建图像质量评分与标准剂量反向滤过重建相比无明显差异。软组织及椎体的图像质量,迭代重建图像质量评分要低于反向滤过重建方法,结果具有统计学差异;而低剂量迭代重建图像质量评分与标准剂量反向滤过重建相比无明显差异。整体病例图像质量评分,迭代重建方法要高于反向滤过重建方法,低剂量迭代重建方法要高于标准剂量反向滤过重建方法。结论：应用低剂量扫描方式以及迭代重建方法进行颈椎CT检查可以为I临床提供较好的图像质量,对于椎间盘、脊神经、脊髓显示较好,对于周围软组织以及椎体来说,图像质量相对较差,同时可以降低大约40％的放射剂量。     

The ArsR protein is a trans-acting regulatory protein

The arsR gene encodes the regulatory protein of the plasmid-encoded arsenical resistance operon. A series of in-frame fusions was constructed between the C-terminally truncated arsR gene and the coding region for the mature form of beta-lactamase (blaM). Fusions containing most of the arsR gene were still inducible by arsenicals. Fusions containing less than 102 residues of the 117-residue ArsR protein were constitutive. When a wild-type arsR gene was placed in trans, the constitutive constructs were again inducible. The results demonstrate that the ArsR protein is a trans-acting regulatory protein which controls its own expression.     

国人颈椎横突孔的形态观察与测量

本文作者用精密度为0.02毫米的游标卡尺对东北地区出土的100副成套颈椎,1200个颈椎横突孔的诸径进行了测量,得到了一系列数据,并对颈椎横突孔的形状(将它分为五型)及一些特异颈椎进行了观察,根据这些材料探讨了横突孔与椎动脉的关系。     

Studies on the mechanism of trypan blue teratogenicity in the rat developing in vivo and in vitro

Trypan blue is a potent teratogen in vivo and in vitro in the rat. Many of the abnormalities produced by trypan blue--including swollen neural tube and pericardium, subectodermal blisters, hematomas, and generalized edema--may result from altered fluid balance in and around the embryo. The present study demonstrates relationships between changes in the fluid environment around the embryo and appearance of anomalies. Rat embryos were exposed in utero or in vitro to trypan blue during the early period of organogenesis. Both exposures resulted in defects that are typical of trypan blue treatment. Osmolality of exocoelomic fluid (ECF) was measured on gestation day 10 in vivo and day 12 in vitro, both after 48 hr of exposure to trypan blue. In both cases ECF osmolality was significantly lower than controls. This was correlated with the presence of edema-related anomalies in the embryo. On gestation day 11 in vivo, three days after maternal injection of trypan blue, ECF osmolalities were significantly higher than controls; however, there was tremendous variability in this parameter in day 11 treated embryos, and some had ECF osmolalities below the control range. Increased frequency of abnormalities was correlated with abnormal ECF osmolality, below and above the control range. Trypan blue probably exerts its teratogenic effects by disturbing the function of the visceral yolk sac. The movements of an amino acid and a monosaccharide across the visceral yolk sac were measured on gestation day 12 embryos in vitro. This aspect of yolk sac function was not altered by trypan blue exposure. Ultrastructure of the visceral yolk sac was observed after trypan blue exposure in vivo and in vitro. Endodermal cells in trypan blue-treated yolk sacs contained fewer large, electron dense lysosomes than controls. These were replaced by numerous small vacuoles, which may contain trypan blue. Trypan blue causes osmotic changes in the rat embryo in vivo and in vitro. These changes are correlated with embryonic malformations. Alterations in yolk sac ultrastructure indicate that trypan blue affects the function of this membrane.     

Cytotoxicity as measured by trypan blue as a potentially confounding variable in the in vitro alkaline elution/rat hepatocyte assay

Rat hepatocytes treated in vitro with A2RA, an angiotensin II receptor antagonist, displayed increased level of DNA-strand breaks as determined by alkaline elution, without an appreciable increase in cytotoxicity as determined by a trypan blue dye exclusion assay at harvest. The alkaline elution profile appeared to have two components: a rapidly eluting component detected in the first fraction collected (often associated with DNA from dead or dying cells), followed by a more slowly eluting component detected in the subsequent fractions. Further analysis of hepatocytes treated with A2RA by pulsed-field gel electrophoresis and neutral elution revealed significant levels of DNA double-strand breaks. Electron microscopy (EM) showed pronounced damage to mitochondria; although cell blebbing was seen using both EM and light microscopy, the plasma and nuclear membranes appeared intact when examined by EM. Cellular ATP levels decreased precipitously with increasing doses of A2RA, falling to less than 10% of control values at a dose of 0.213 mM A2RA, a concentration showing 100% relative viability by trypan blue at harvest. Thus, whereas in our experience trypan blue dye exclusion accurately reflects cytotoxicity induced by the majority of test agents, in this rather unusual case, trypan blue did not accurately reflect compound-induced cytotoxicity at harvest since there was no concurrent loss of membrane integrity. However, when hepatocytes treated with A2RA were incubated for either 3 h or 20 h in the absence of compound, a sharp, dose-dependent decline in viability was observed using trypan blue dye exclusion. Together with the initial, dose-dependent drop in the alkaline elution curve, these data suggest that the observed DNA double-strand breaks arose as a consequences of endonucleolytic DNA degradation associated with cytotoxicity, rather than by a direct compound-DNA interaction. Since DNA double-strand breaks behave under alkaline denaturing conditions as two single-strand breaks and can therefore produce increases in the alkaline-elution slope values, a necessary criteria for a valid positive result in this assay is that cytotoxicity by trypan blue dye exclusion will not be greater than 30%. Our data, however, indicate that interpretation of the elution assay as a test for genotoxicity can still be confounded by the failure of the trypan blue dye exclusion assay to reflect cytotoxicity in the unusual instance when there is no concurrent, immediate loss of membrane integrity.     

Dual effect of potassium on transmitter exocytosis

The time course of exocytosis of quanta of acetylcholine induced by 20 mM K+ was studied at the frog neuromuscular junction. Images of vesicle fusion on freeze-fracture replicas were mostly localized at the active zones in resting preparations fixed in 20 mM K+. Fusions appeared also outside the active zones in preparations fixed after 1 min exposure to 20 mM K+ and were evenly distributed over the presynaptic membrane after 5 min in 20 mM K+ (even though secretion was prevented by withdrawing Ca2+ until 30 s before fixation). The mean densities of vesicle fusions were comparable in all conditions, as were the total number of quanta released during the fixation period. This indicates that fusions outside active zones represent ectopic exocytosis, slowly activated by potassium. Partial inactivation of K(+)-induced quantal release (time and concentration-dependent) was observed electrophysiologically; this may be related to the observed decrease in density of vesicle fusions along the active zones, with time. Consistently, after 5 min in 15 mM K+ fusion density at the active zones remained high. It is concluded that active zone-associated and ectopic fusions are two exocytotic processes activated with differential time courses and concentration-dependence by K+.     

Strain differences in teratogenic susceptibility to trypan blue between WM and BDIX rat strains

Female rats of WM (Wistar-Mishima)/Nem strain were mated with WM/Nem (group W) or BDIX/Nem males (group WB), and BDIX/Nem females were mated with BDIX/Nem (group B) or WM/Nem males (group BW). On day 8 of gestation, pregnant females were treated intraperitoneally with 1% aqueous solution of trypan blue at a dose of between 20 and 120 mg/kg of body weight. On day 20 of gestation, fetuses were examined for external, visceral, and skeletal malformations. In group W, fetal mortality increased dose dependently at doses higher than 20 mg/kg, and incidences of external, visceral, and skeletal malformations were significantly higher than control at doses of 30 mg/kg and more. In group B, fetal mortality and the incidence of external malformations were significantly higher than control only in the group treated with 120 mg/kg, and no significant increase of visceral and skeletal malformations was shown. It was confirmed that BDIX strain is much more resistant to trypan blue teratogenicity than WM strain. In group BW, nearly the same teratogenic effects were shown as in group W in terms of fetal mortality and incidence of malformations. However, in group WB, teratogenic effects were not so remarkable as in group BW, suggesting patroclinous effects in teratogenic susceptibility to trypan blue. In group BW, sex differences in teratogenic susceptibility were found; male fetuses were more susceptible to trypan blue than females.     

ALKALINE RIBONUCLEASE ACTIVITY INCREASE IN RAT KIDNEY CORTEX AND LIVER AFTER TRYPAN BLUE AND OTHER AZO DYES ADMINISTRATION

Acid azo dyes, most of them naphtholdisulfonic acid derivatives, were given intraperitoneally to rats and their effect on "alkaline" ribonuclease activity was studied in total homogenates of kidney cortex and liver. Acid treatment was used to release bound enzyme activity. Several of the dyes, including trypan blue, increased RNase activity in both organs 3 days after administration of single doses, while others, like Evans blue, were inactive. Activity was apparently bound to the sulfonic substitution in the 3, 6 positions in the naphthalene rings, substitutions in the benzidine rings being not critical. All of the active and most of the inactive compounds were taken up by tubule cells of kidney cortex and by reticular and parenchymal cells of liver. While the effect on both liver and kidney was obtained 1 day after trypan blue administration, RNase remained increased for only about 3 days in the first organ, and for at least a month in the second. However, repeated trypan blue doses increased liver enzyme activity for at least 9 days. Serum RNase activity was decreased after trypan blue administration. Ethionine administration together with trypan blue markedly blocked the effect of the dye on liver RNase activity; simultaneously given methionine partially reversed the action of the antimetabolite. This suggests that de novo synthesis of RNase is induced in liver by trypan blue. The action of ethionine on the kidney RNase response to trypan blue was less marked although significant; in view of the possible kidney uptake of the plasma enzyme, interpretation of this finding must be postponed. Results are discussed with reference to the mechanism of the structural specificity of the compounds used, cytological localization of the dyes and their mechanism of action on liver and kidney RNase.     

A comparative study of the inhibitory effect of trypan blue on mouse erythrocyte and C3 binding receptors of peripheral blood mononuclear cells from healthy donors and patients suffering from chronic lymphocytic leukaemia

Trypan blue has previously been shown to interact with the C3 receptor, but not with the Fc receptor. In the present work the effects of Trypan blue on mouse erythrocyte (ME) and C3 binding receptors of the peripheral blood mononuclear cells have been studied in healthy individuals and in patients with chronic lymphocytic leukemia (CLL). The resulting dose-response curves have been compared with each another. The results of statistical analysis indicate that there is no significant difference between the effects of trypan blue on the two receptors in healthy individuals. In contrast ME and C3 binding receptors differ significantly in trypan blue sensitivity in CLL patients. These data suggest that the two receptors are similar, or are situated on the cell membrane in such a way that the trypan blue bound to one of them inhibits the functioning of the other one too, but they are not the same. In the course of leukemic transformation, the trypan blue sensitivity of the ME binding receptor does not vary, whereas that of the C3 receptor is enhanced significantly.     

伊红、台盼蓝检测河蟹精子存活率的比较

对台盼蓝和伊红染色法检测河蟹(Eriocheir sinensis)精子存活率的方法进行了评价研究。结果表明,两种染色法死、活精子分别呈现出明显不同的染色特征:活精子无色透明,顶体中央凸起呈圆锥状,光镜下辐射臂及细胞边界清晰;死亡精子顶体着色,且中央有一染色较深的圆斑,核杯染色不明显,细胞体积变大,边界模糊。通过不同染色时间和不同染料浓度的比较发现,两种染色法最适染液浓度分别是0·25%的伊红和0·5%的台盼蓝,染色时间均以15min为佳。在此基础上,将新鲜精子和60℃水浴处理致死精子以不同的体积比混合,配成含致死精子比例为10%～90%的9个梯度样品,用伊红和台盼蓝分别测定各样品精子死亡率,并进行相关性分析。结果发现,各样品实测精子死亡率均略高于样品的理论死亡率,同时两种染色法实测值与样品理论值呈显著正相关(P<0·05),两种染色法之间亦呈显著正相关(P<0·05)。上述结果表明,伊红和台盼蓝可用于河蟹精子的活体染色,且两种染色法在对河蟹精子染色中具有一定的稳定性和可比性。     

Radiation sensitivity and the status of some radiation sensitivity markers in relatively sensitive lymphoid cells

The most commonly used indicators of ionizing radiation exposure are cytogenetic measures and survival parameters. All these methods have their advantages, disadvantages and uncertainties, such that better biological estimators of the absorbed dose, especially in the low dose range, are being sought. In this study we analyzed apoptosis and several proteins involved in the regulation of apoptosis as possible indicators of irradiation after relatively small doses (0.1-2 Gy) of X-rays. The studies were carried out in seven lymphoid cell lines: two mouse lymphoma L5178Y, the human pre-B cell leukemia Reh, and four human Epstein-Barr virus-transformed lymphoid cell lines (two apparently normal and two Ataxia-telangiectasia (AT)). We detected apoptosis with the in situ terminal deoxynucleotidyl transferase assay and flow cytometry, and measured the expression of several apoptotic-regulatory proteins (Bcl-2, Bax, Bclx, NF kappa B) with Western blotting. The cytokinesis-block micronucleus assay, comet assay as a measure of DNA damage, and trypan blue survival test were also done for comparison Although for the most of examined parameters of radiation sensitivity: i.e. micronucleus assay, trypan blue test and percentage of apoptosis--there were observed clear dose-effect relationships for all cell lines examined, we did not find agreement between values for these measured parameters. There are marked differences in both timing of apoptosis and percentage of apoptotic cells. Variation in the apoptotic fraction in the controls for different sets of experiments is not very pronounced. There is however considerable variation for the same parameters in irradiated cells, possibly due to their cell cycle status during irradiation, as the cultures were not synchronized. Overall, neither the numbers of apoptotic cells nor the expression of apoptosis-related proteins, nor DNA repair can serve as dose estimators or sensors for these lines, but still these parameters can give valuable supplementary information about radiation sensitivity.     

Limited lifespan in somatic cell hybrids and cybrids

The transformed phenotype is believed to be dominant in fusions between limited lifespan cells and transformed cells, based on heterokaryon experiments and on the isolation of transformed hybrids from mass cultures of fused cells. A series of fusions has been performed between limited lifespan Lesch-Nyhan fibroblast cells and a permanent HeLa cell line with a complementary genetic marker. The growth of independently isolated hybrid clones was followed in parallel with Lesch-Nyhan cells. In fusions involving Lesch-Nyhan cells which had completed about 50% of their lifespan, all hybrids initially show fibroblastic properties. Thirty-five hybrids had a limited lifespan slightly longer than Lesch-Nyhan controls. Three other hybrid clones, and all mass cultures of hybrids, showed the appearance of transformed colonies at a rate of approx. one transformant in 2 × 10₅ hybrid cells. These transformed cells showed anchorage independence, low serum requirement, chromosome loss, and have been maintained in culture for 50–100 population doublings with no signs of senescence. Fusions involving enucleated HeLa cells did not show transformation. Fusions with senescent Lesch-Nyhan cells yielded hybrids which grew beyond the normal Lesch-Nyhan cell lifespan, but which again showed limited lifespan and low frequency transformation. It is concluded that limited lifespan is expressed in a dominant manner in these fusions, and that transformation or “escape from senescence” is a low frequency event requiring the presence of the HeLa nucleus.     

Creating precise GFP fusions in plasmids using yeast homologous recombination

Using a combination of primer amplification, homologous recombination, and yeast genetics, we established a method for creating precise promoter and protein fusions in genes originating from organisms other than yeast. One major advantage of this new method is its versatility. Fusions can be produced within a target gene without constraints regarding the site of insertion. Thus, fusions can be generated within a target sequence exactly at the site desired, and all sequences upstream and downstream of the insertion site were preserved. To illustrate the general applicability of this technique, we fused the gene encoding GFP to a Caenorhabditis elegans homologue of the dishevelled gene, dsh-2. This approach is not restricted to GFP fusions but can be utilized to create fusions between almost any two sequences regardless of the source. Therefore, this method provides a flexible alternative to other PCR-mediated techniques.     

High radiosensitivity of the MOLT-4 leukaemic cell line

Radiation survival of MOLT-4, a leukaemic T-lymphocyte cell line, was measured by counting colonies formed in 0.8 per cent methyl cellulose. The survival curve was a simple exponential and showed the cells to be radiation sensitive, with D0 = 0.49 +/- 0.02 Gy and extrapolation number n = 0.92 +/- 0.09. No increase in survival as measured by colony-forming ability or trypan blue dye exclusion was seen when the dose was split into two fractions, separated by a 5 h incubation period. Electron microscopy and trypan blue dye exclusion showed that 5 h after exposure to high doses, MOLT-4 cells began to die and displayed condensed, marginated chromatin and cellular vesiculation.     

Suppression of cell-mediated tumor cell lysis and complement-induced cytotoxicity by trypan blue.

Different forms of cell-mediated cytotoxicity were suppressed in the presence of trypan blue. The systems affected included lysis of antibody-coated tumor cells by normal and C. parvum-stimulated mouse peritoneal cells and lysis of allogeneic targets by immune effector cells. The inhibition, measured in a 4-hr 51Cr release assay, was reversible and did not occur in the presence of 30% fetal calf serum or albumin. Binding between effector and target cells through Fc receptors was not affected, and lysis of allogeneic cells was inhibited at the lytic step rather than at the binding step. In contrast, lysis of sensitized erythrocytes was not inhibited by trypan blue, suggesting that lysis of these targets may not involve the steps required in tumor cell lysis. Trypan blue blocked the function of antibody before binding to target cells and also suppressed complement-induced cytolysis. Most individual complement components were susceptible to the inhibitory action of trypan blue. These results reveal an affinity of trypan blue for proteins in general that may be responsible for many of its biologic actions.     

Aged-related physiological studies comparing Candida albicans chlamydospores to yeasts.

From electron-microscopical observations, a decreased metabolic activity in 3-day-old Candida albicans chlamydospores was suggested, and progressive deterioration in chlamydospores aged 2-8 months was shown. Oxygen utilization by chlamydospore-pseudomycelium (CSP) preparations was less than that by yeast, while 3-day-old CSP preparations used significantly less O2 than 24-h CSP preparations. Amino acid incorporation was greater in yeast than in CSP preparations. Leucine incorporation by 20-h yeasts was twice that of 5-day yeasts and 5 times that of 20-h and 5-day CSP. Amino acid decarboxylation was similar in yeasts and CSP and was determined by end-product analyses to be via amino acid oxidase. Light microscopy autoradiography of [14C]leucine incorporation demonstrated that the metabolic activity in CSP preparations was due to the young growing tips of the pseudomycelium and not to mature chlamydospores. Yeasts did not take up trypan blue could be stained if first autoclaved or treated with 10% acid or 10% base. Young chlamydospores grown in the presence of trypan blue developed unstained and became permeable to the dye at 2 1/2 days. These data suggest that chlamydospores of C. albicans do not function in the classical role attributed to spores; i.e., mature chlamydospores cannot germinate, but rather age, deteriorate, and die.