Impairment of glutathione metabolism in human gastric epithelial cells treated with vacuolating cytotoxin from Helicobacter pylori.

Helicobacter pylori vacuolating cytotoxin (VacA) is believed to be one of the factors that induces gastric disease. Our previous study indicated that VacA causes a decrease in the intracellular ATP level in human gastric epithelial cells, suggesting to impair mitochondrial membrane potential followed by a decrease in energy metabolism (Kimura et al., Microb. Pathog., 1999, 26: 45--52). In the present study, we investigated whether the decrease in ATP level affects glutathione metabolism, in which its synthesis and efflux are ATP-dependent. Treatment of AZ-521 human gastric epithelial cells with 120 nM VacA for 6 h suppressed the efflux of oxidized glutathione (GSSG) in a dose- and time-dependent manner. The efflux of GSSG from the cells and glutathione (GSH) synthesis of cells treated with VacA were approximately 50 and 70% of those of the control, respectively. The turnover rate of intracellular GSH was also suppressed by VacA. Viability of the cells pretreated with VacA, then further incubated with H(2)O(2), was decreased by 50% at 6 h and 70% at 12 h. These results suggested that VacA impairs GSH metabolism in the gastric epithelial cells, which weakens the resistance of the cells against oxidative stress or cellular redox regulation by GSH.     

Helicobacter pylori VacA induces programmed necrosis in gastric epithelial cells

Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to the development of peptic ulcer disease and gastric cancer. The secreted pore-forming toxin VacA is one of the major virulence factors of H. pylori. In the current study, we show that AZ-521 human gastric epithelial cells are highly susceptible to VacA-induced cell death. Wild-type VacA causes death of these cells, whereas mutant VacA proteins defective in membrane channel formation do not. Incubation of AZ-521 cells with wild-type VacA results in cell swelling, poly(ADP-ribose) polymerase (PARP) activation, decreased intracellular ATP concentration, and lactate dehydrogenase (LDH) release. VacA-induced death of these cells is a caspase-independent process that results in cellular release of histone-binding protein high mobility group box 1 (HMGB1), a proinflammatory protein. These features are consistent with the occurrence of cell death through a programmed necrosis pathway and suggest that VacA can be included among the growing number of bacterial pore-forming toxins that induce cell death through programmed necrosis. We propose that VacA augments H. pylori-induced mucosal inflammation in the human stomach by causing programmed necrosis of gastric epithelial cells and subsequent release of proinflammatory proteins and may thereby contribute to the pathogenesis of gastric cancer and peptic ulceration.     

Clustering of Helicobacter pylori VacA in lipid rafts, mediated by its receptor, receptor-like protein tyrosine phosphatase beta, is required for intoxication in AZ-521 Cells

Helicobacter pylori vacuolating cytotoxin, VacA, induces multiple effects on epithelial cells through different cellular events: one involves pore formation, leading to vacuolation, mitochondrial damage, and apoptosis, and the second involves cell signaling, resulting in stimulation of proinflammatory responses and cell detachment. Our recent data demonstrated that VacA uses receptor-like protein tyrosine phosphatase beta (RPTPbeta) as a receptor, of which five residues (QTTQP) at positions 747 to 751 are involved in binding. In AZ-521 cells, which mainly express RPTPbeta, VacA, after binding to RPTPbeta in non-lipid raft microdomains on the cell surface, is localized with RPTPbeta in lipid rafts in a temperature- and VacA concentration-dependent process. Methyl-beta-cyclodextrin (MCD) did not block binding to RPTPbeta but inhibited translocation of VacA with RPTPbeta to lipid rafts and all subsequent events. On the other hand, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), which disrupts anion channels, did not inhibit translocation of VacA to lipid rafts or VacA-induced activation of p38 mitogen-activated protein (MAP) kinase, but inhibited VacA internalization followed by vacuolation. Thus, p38 MAP kinase activation did not appear to be required for internalization. In contrast, phosphatidylinositol-specific phospholipase C (PI-PLC) inhibited translocation, as well as p38 MAP kinase/ATF-2 activation, internalization, and VacA-induced vacuolation. Neither NPPB nor PI-PLC affected VacA binding to cells and to its receptor, RPTPbeta. Thus, receptor-dependent translocation of VacA to lipid rafts is critical for signaling pathways leading to p38 MAP kinase/ATF-2 activation and vacuolation.     

TAC-101, a benzoic acid derivative, inhibits liver metastasis of human gastrointestinal cancer and prolongs the life-span

We examined the anti-tumor effect of a novel benzoic acid derivative, TAC-101 (4-[3,5-bis(trimethylsilyl) benzamide] benzoic acid) on models with liver metastasis. Oral administration of TAC-101 significantly inhibited spontaneous liver metastasis of AZ-521 (human gastric cancer ) by orthotopic implan-tation to athymic nude mice. It also inhibited both the liver metastasis of AZ-521 induced by intrasplenic injection and the secondary lung metastasis from the liver. In addition, TAC-101 inhibited the proliferation of Co-3 (human colon adenocarcinoma) that formed a single nodule in the liver of athymic nude mice by intrahepatic implantation. The growth inhibitory effect of TAC-101 on AZ-521 experimental liver metastasis was observed when treatment was started on day 7, 14, or 21 which may correspond to the progressive stage of liver metastasis in clinical settings. Multiple administration of TAC-101 (8 mg/kg/day) significantly prolonged survival time of the animals with liver met astasis by intrasplenic injection of AZ-521 (T/C = 230%) and A549 (human lung adenocarcinoma; T/C = 186%). These effects of TAC-101 were stronger than those of 5-FU, CDDP or ATRA. Furthermore, TAC-101 inhibited the binding of AP-1 to DNA on electrophoretic mobility shift assay using nuclear extract of AZ-521 cells, although ATRA did not inhibit. These findings suggested that TAC-101 may be a candidate for a new class of anti-cancer agents for liver metastasis. © Rapid Science Ltd.     

高表达活化白细胞黏附因子对AZ-521胃癌细胞系增殖能力的影响

目的:探讨活化白细胞黏附分子(Activated leukocyte cell adhesion molecule,ALCAM),即CD166对AZ-521胃癌细胞系增殖能力的影响。方法:以pLOX-CWBmi1-CD166慢病毒质粒感染胃癌细胞株AZ-521,采用荧光定量PCR以及Western blot分别测定CD166 mRNA和蛋白表达情况;采用CCK-8测定细胞增殖情况;采用吉姆萨染色测定细胞克隆形成数。结果:pLOX-CWBmi1-CD166慢病毒质粒感染胃癌细胞株AZ-521后CD166 mRNA和蛋白水平均明显增加(P<0.05);细胞增殖能力明显增加(P<0.05);细胞克隆形成数量明显增多(P<0.05)。体内成瘤实验显示,CD166过表达小鼠肿瘤体积明显大于对照细胞(P<0.05)。结论:CD166可促进AZ-521胃癌细胞系增殖。     

Sodium azide induces necrotic cell death in rat squamous cell carcinoma SCC131

Sodium azide (NaN₃) is widely used in industry and agriculture, and also in laboratories as a potent preservative. NaN₃ induces cell death when applied to cultured cells. However, whether the mode of cell death is apoptosis or necrosis remains a subject of debate. There have been no previous reports on NaN₃-induced cell death in squamous cell carcinoma (SCC), and so we studied the mode of cell death induced by NaN₃ using the rat SCC cell line, SCC131. In this experiment, SCC131 cells died 48–72 h after NaN₃ treatment with concentrations greater than 5 mM. The NaN₃ treatment reduced the mitochondrial membrane potential and ATP content. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and DNA ladder detection assay indicated that no DNA fragmentation occurred. In addition, phosphatidyl serine did not appear on the cell surface, according to the findings of dye-uptake bioassay and flow cytometric analysis of Annexin V labeling. Electron microscopic analysis revealed that the NaN₃-treated cells showed mitochondrial swelling and rupture of the cell membrane. In conclusion, NaN₃ induces necrotic cell death in SCC131. This experimental model may be used in the study of necrotic cell death.     

Helicobacter pylori Membrane Vesicles Stimulate Innate Pro- and Anti-Inflammatory Responses and Induce Apoptosis in Jurkat T Cells

Persistent Helicobacter pylori infection induces chronic inflammation in the human gastric mucosa, which is associated with development of peptic ulceration, gastric atrophy, and gastric adenocarcinoma. It has been postulated that secretion of immunomodulatory molecules by H. pylori facilitates bacterial persistence, and membrane vesicles (MV), which have the potential to cross the gastric epithelial barrier, may mediate delivery of these molecules to host immune cells. However, bacterial MV effects on human immune cells remain largely uncharacterized to date. In the present study, we investigated the immunomodulatory effects of H. pylori MV with and without the vacuolating cytotoxin, VacA, which inhibits human T cell activity. We show a high degree of variability in the toxin content of vesicles between two H. pylori strains (SS1 and 60190). Vesicles from the more toxigenic 60190 strain contain more VacA (s1i1 type) than vesicles from the SS1 strain (s2i2 VacA), but engineering the SS1 strain to produce s1i1 VacA did not increase the toxin content of its vesicles. Vesicles from all strains tested, including a 60190 isogenic mutant null for VacA, strongly induced interleukin-10 (IL-10) and IL-6 production by human peripheral blood mononuclear cells independently of the infection status of the donor. Finally, we show that H. pylori MV induce T cell apoptosis and that this is enhanced by, but not completely dependent on, the carriage of VacA. Together, these findings suggest a role for H. pylori MV in the stimulation of innate pro- and anti-inflammatory responses and in the suppression of T cell immunity.     

水飞蓟素对同型半胱氨酸诱导的人脐静脉内皮细胞凋亡的抑制作用

目的：探讨水飞蓟素(SIL)对同型半胱氨酸(HCY)诱导的人脐静脉内皮细胞(HUVECs)凋亡的抑制作用及其作用机制。方法:采用MTT及LDH检测细胞活性，DNA琼脂糖凝胶电泳和流式细胞仪检测细胞凋亡的发生, 流式细胞仪检测线粒体膜电位的变化,并用荧光酶标仪测定caspase-3，-6，-9的活性。Western blotting分析相关蛋白的表达。结果:1 mmol/L HCY使HUVECs的存活率比对照组低79.5%(P<0.01)，5-20 μg/L SIL明显抑制HCY所致的HUVECs死亡(P<0.05, P<0.01)，20 μg/L SIL可使HUVEC的存活率恢复到对照组的83.7%。HCY刺激后Bcl-2与XIAP的表达显著低于对照组，Bax的表达显著高于对照组，20 μg/L SIL可使Bcl-2凋亡蛋白的改变发生逆转。SIL可明显抑制 1 mmol/L HCY引起的caspase-3、caspase-6、caspase-9的升高。SIL明显抑制 1 mmol/L HCY引起的Δψm下降和Cyto C、Smac及AIF的释放。结论:SIL能抑制HCY诱导的人脐静脉内皮细胞凋亡, 其细胞保护作用可能与降低细胞内活性氧水平, 抑制caspase-3，-6，-9的活性和维持线粒体膜电位的高能状态有关。     

Release of Helicobacter pylori vacuolating cytotoxin by both a specific secretion pathway and budding of outer membrane vesicles. Uptake of released toxin and vesicles by gastric epithelium

The mechanisms by which Helicobacter pylori releases its virulence factors are poorly known. Active secretion has been proposed for some products, including a vacuolating toxin (VacA). Outer membrane vesicles represent another mechanism by which some Gram-negative bacteria may release virulence factors. This study sought to localize VacA by immunocytochemistry in H. pylori cells, to determine whether H. pylori produces outer membrane vesicles, and to investigate whether such vesicles might constitute a vehicle for the delivery of bacterial virulence factors to the gastric mucosa. Small (50–300 nm) membrane vesicles were found in H. pylori culture media from both H. pylori strain 60190 and strain CCUG 17874. These vesicles appeared to originate from blebs arising on the bacterial outer membrane. VacA was immunolocalized in the periplasm and outer membrane of intact bacteria and also in outer membrane blebs and vesicles. Both soluble secreted VacA and VacA-containing vesicles bound to, and were internalized by, MKN28 cells and were detectable in the gastric mucosa from H. pylori-infected humans. The release of outer membrane vesicles by H. pylori may represent a mechanism, additional to secretory pathways, for the delivery of bacterial toxins and antigens to the gastric mucosa. Copyright © 1999 John Wiley & Sons, Ltd.     

Correlation of the Mitochondrial Activity of Two‐Cell Embryos Produced In Vitro and the Two‐Cell Block In Kunming and B6C3F1 Mice

The correlation between the early embryonic block to development and mitochondrial activity was investigated comparing two‐cell embryos produced in vitro from Kunming (KM) and B6C3F1 mice. One‐cell embryos were obtained from two species of hybrids (female KM mice mated with KM males and female B6C3F1 mice mated with KM males) and cultured for 84 hr in M16 media. The mitochondrial membrane potential, ATP content, and reactive oxygen species levels were measured in the resulting KM and B6C3F1 two‐cell embryos. Mitochondrial membrane potential and ATP content were also determined in KM and B6C3F1 metaphase II eggs. The results showed that the two‐cell block was observed in cultured KM embryos but not in B6C3F1 embryos. Mitochondrial membrane potential and ATP content of KM two‐cell embryos were significantly lower than in B6C3F1 two‐cell embryos (P < 0.01). Interestingly, the reactive oxygen species levels of KM two‐cell embryos were significantly lower than their B6C3F1 counterparts (P < 0.01). There was no difference in mitochondrial membrane potential and ATP content between KM and B6C3F1 metaphase II eggs. It is concluded that KM mice have an early two‐cell embryo block and that a possible “blocking” mechanism is the lower mitochondrial membrane potential and ATP content in these embryos. The results suggest a new approach for overcoming early embryonic development block, that of manipulating mitochondrial activity. Anat Rec, 292:661–669, 2009. © 2009 Wiley‐Liss, Inc.     

Altered calcium homeostasis in irreversibly injured P388D1 macrophages.

Sequestration of calcium by mitochondria is an important mechanism to maintain normal intracellular calcium homeostasis. Anoxic or toxic damage to these organelles has been postulated to disrupt intracellular calcium compartmentalization, leading to cell death. The authors examined the potential relationship between mitochondrial dysfunction, altered calcium homeostasis, and irreversible injury in a model system of silica-induced toxicity to P388D1 cells. Exposure to toxic silica particles, but not to nontoxic latex heads, disrupted mitochondrial membrane potential, increased membrane-associated calcium, elevated free cytosolic calcium, and killed 50% to 60% of the cell population after 6 to 8 hours. To test whether disruption of the mitochondrial membrane potential was sufficient to cause irreversible injury, P388D1 cells were exposed to either the proton ionophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or to the mitochondrial inhibitor, antimycin A. Over 90% of the treated cells showed depolarization of the mitochondrial membrane as indicated by the fluorescent probe rhodamine 123. Carbonyl cyanide p-trifluoromethoxyphenylbydrazone also caused an elevation in free cytosolic calcium as monitored by fura-2. However, even after 6 hours of exposure to these proton ionophores or mitochondrial inhibitors, P388D1 cells did not show increased chlorotetracycline (CTC)-induced fluorescence or loss of viability. P388D1 cells exposed to silica have been shown previously to lose 80% of their adenosine triphosphate (ATP) content. The effect of reduced ATP levels on intracellular calcium homeostasis and viability was assessed by exposing P338D1 cells to FCCP in the presence of sodium azide and 2-deoxyglucose, which reduced ATP content by more than 90%. Under these conditions, none of the cells were killed, and only 5.5% showed increased CTC-induced fluorescence after 6 hours. These data indicate that disruption of the mitochondrial membrane potential, even in combination with reduced ATP content, is not sufficient to kill P388D1 cells.     

Binding and internalization of the Helicobacter pylori vacuolating cytotoxin by epithelial cells.

Many Helicobacter pylori strains produce a cytotoxin (VacA) that induces vacuolation in epithelial cells. In this study, binding and internalization of the cytotoxin by HeLa or AGS (human gastric adenocarcinoma) cells were characterized by indirect fluorescence microscopy. Cells incubated with the cytotoxin at 4 degrees C displayed a uniform fluorescent plasma membrane signal. Preincubation of the cytotoxin with either rabbit antiserum to approximately 90-kDa H. pylori VacA or sera from H. pylori-infected persons inhibited its binding to cells and blocked its capacity to induce cytoplasmic vacuolation. Recombinant VacA fragments (approximately 34 and approximately 58 kDa), corresponding to two proteolytic cleavage products of approximately 90-kDa VacA, each bound to the plasma membrane of HeLa cells. Antiserum reactive with the approximately 58-kDa VacA fragment inhibited the binding of native H. pylori cytotoxin to cells and inhibited cytotoxin activity, whereas antiserum to the approximately 34-kDa fragment had no effect. When incubated with cells at 37 degrees C for > or = 3 h, the H. pylori cytotoxin localized intracellularly in a perinuclear location but did not localize within cytotoxin-induced vacuoles. When cells with previously bound cytotoxin were incubated with anticytotoxin serum at 4 degrees C and then shifted to 37 degrees C, vacuolation was completely inhibited. Bound cytotoxin became inaccessible to the neutralizing effects of antiserum after 60 to 120 min of incubation with cells at 37 degrees C. These data suggest a model in which (i) VacA binds to cells primarily via amino acid sequences in its 58-kDa fragment, (ii) VacA internalization occurs slowly in a temperature-dependent process, and (iii) VacA interacts with an intracellular target.     

Sodium pyruvate protects against H2O2 mediated apoptosis in human neuroblastoma cell line-SK-N-MC

Free radicals are involved in neuronal damage. The present study was aimed to investigate the protective effect of sodium pyruvate—a free radical scavenger against hydrogen peroxide (H₂O₂) induced apoptosis in human neuroblastoma cell line-SK-N-MC. On exposure to H₂O₂ (0.025 mM) cells exhibited apoptosis within 24 h, demonstrating a high caspase 3 activity by 3 h followed by cleavage of PARP that was maximum at 24 h. A break down in the mitochondrial membrane potential was observed 3 h onwards. Sodium pyruvate protected cells significantly (P<0.05) against apoptosis in a dose dependent manner as assessed for cell viability by dye exclusion method and apoptosis by TUNEL. Sodium pyruvate significantly inhibited caspase 3 activity, cleavage of PARP and breakdown of mitochondrial membrane potential. These data suggest that sodium pyruvate protects neuronal damage caused by H₂O₂.     

南海真菌代谢物1386A对胃癌MCG-803细胞增殖、凋亡和膜电位的影响

目的:研究南海真菌代谢物1386A对胃癌MCG-803细胞的作用。方法:采用MTT细胞活性检测法检测1386A对MCG-803细胞活性的影响,用集落形成实验检测1386A对MCG-803细胞增殖能力的影响,用流式细胞仪检测1386A对MCG-803细胞周期分布、凋亡、线粒体膜电位的影响。结果:1386A作用于MCG-803细胞24、48和72h后,MTT检测细胞活性的IC50分别为29.70、14.07和13.47μmol/L,1386A作用48h后集落形成实验检测MCG-803细胞增殖的IC50为3.27μmol/L,1386A作用48h后MCG-803细胞的S期比例由25.6%上升为56.8%,1386A作用24h后MCG-803细胞早期凋亡率由3.34%上升为8.39%,1386A作用24h后MCG-803细胞线粒体膜电位下降率为56.26%。结论:南海真菌代谢物1386A能明显抑制胃癌MCG-803细胞的生长,可能通过线粒体凋亡途径诱导细胞的凋亡。     

Gastric Autoantigenic Proteins in Helicobacter Pylori Infection

Purpose

This study tried to identify novel gastric autoimmune antigens that might be involved in aggravating the atrophic gastritis among patients with Helicobacter pylori infection using two-dimensional immunoblotting analysis.

Materials and Methods

Proteins from gastric mucosal antrectomy specimens and AGS cells (gastric adenocarcinoma cell lines derived from a Caucasian patient who had received no prior therapy) were 2-dimensionally immunoblotted separately with a pool of 300 sera from H. pylroi-infected patients at Gyeongsang National University Hospital.

Results

Thirty-eight autoantigenic proteins including alcohol dehydrogenase [NADP+], alpha enolase, gastrokine-1, gastric triacylglycerol lipase, heat shock 70 kDa protein 1, and peroxiredoxin-2 were identified in the gastric mucosal tissue. Fourteen autoantigenic proteins including programmed cell death 6-interacting protein, serum albumin and T-complex protein 1 subunit gamma were identified in the AGS cells. Albumin, alpha-enolase, annexin A3, cytoplasmic actin 1, heat shock cognate 71 kDa protein and leukocyte elastase inhibitor were commonly observed autoantigenic proteins in both gastric mucosal tissue and AGS cells. Alpha-enolase, glutathione S-transferase P, heat shock cognate 71 kDa protein, heat shock 70 kDa protein 1, human mitochondrial adenosine triphosphate synthase (ATP) subunit beta, mitochondrial 60 kDa heat shock protein, peroxiredoxin-2, 78 kDa glucose-regulated protein precursor, tyrosine-protein phosphatase non-receptor type 11 and Tryptophan-Aspartic acid (WD) repeat-containing protein 1 showed 60% or higher amino acid positivity.

Conclusion

These newly identified gastric autoimmune antigens might be useful in the control and prevention of gastroduodenal disorders, and might be valuable in breaking the vicious circle that exists in gastroduodenal disorders if their pathophysiological roles could be understood in the progress of chronic atrophic gastritis, gastroduodenal ulcers, intestinal metaplasia, and gastric carcinogenesis.     

Modulation of G proteins and second messenger responsiveness by steroid hormones in GH3 rat pituitary tumour cells

We have investigated the modulation of different G protein α- and β-subunit levels in prolactin (PRL) and growth hormone producing rat pituitary adenoma cells (GH₃ cells) in culture after prolonged exposure (6–48 h) to the steroid hormones 17β-oestradiol and dexamethasone. G_i-3α- and Gβ-subunits were the only G protein subunits which increased in response to 10^-6 m oestradiol (to approximately 150 and 200% of controls, respectively), while the other α-subunits investigated (G_sα, G_i-2α and G_oα) remained relatively unchanged. Thyroliberin (TRH)-and guanosine 5′-[βγ-imido]trisphosphate (Gpp(NH)p)-elicited adenylyl cyclase (AC) activities were reduced during 6–12 h of oestradiol treatment (by 60 and 20%, respectively), while the inhibitory effect of somatostatin (SRIF) increased by approximately 100%. Dexamethasone (10^-6 m) increased levels of the stimulatory G protein G_sα (to approximately 340%) and decreased levels of G_i-3α (to 25%). After 48 h, the AC response to TRH was reduced by approximately 70%, whereas the effect of the other modulators remained close to controls. We conclude that G protein subunits in GH₃ cells are subject to specific regulation by steroid hormones and that this may be important in the tuning of the responsiveness of PRL secretion to hormones in the in vivo situation.     

Virulent Shigella flexneri causes damage to mitochondria and triggers necrosis in infected human monocyte-derived macrophages

Shigella flexneri is a gram-negative bacterium that causes bacillary dysentery in humans that is characterized by an acute inflammatory response of the colon. The fate of phagocytes that are infected in vitro with virulent Shigella has been the subject of some investigation and debate. In this study we found that virulent Shigella caused a rapid increase in the cell membrane permeability of infected human monocyte-derived macrophages (HMDM) but not in the cell membrane permeability of monocytes, as demonstrated by the uptake of fluorescent vital dyes. Within 2 h of infection, 59% +/- 6% of the HMDM and 相似文献   

Sorting of B lymphoblasts based upon cell diameter provides cell populations enriched in different stages of cell cycle

Here we report analysis and correlation of changes in cell size and cycle state resulting from exposure of murine B lymphocytes to the mitogens lipopolysaccharide (LPS) and dextran sulfate (DxSO₄). Cell cycle changes are assessed by flow cytofluorometric analysis of acridine orange stained cells. Cell diameters are determined by flow cytometric analysis of the pulse-width (time of flight) of the axial light extinction signal. Results indicate that within 12 h of exposure of B cell populations to these mitogens, cells displaying increased diameter and containing increased RNA can be detected. Under these conditions, increased RNA content is considered indicative of G₀ to G₁ transition or entry into cell cycle (Darzynkiewicz et al., 1976). Progressive increases in cell size and transition through G₁, S, G₂, and M occur in parallel during 48 h of culture with mitogens. Sorting of cells based upon size followed by cell cycle analysis reveals a direct correlation between cell size and cycle phase. Specifically, cells 4.5–5.5 μm in diameter are in primarily G₀. Cells 5.5–7.0 μm in diameter are in early G₁. Populations of cells 7.0–10 consist of S, G₂, and M phase cells. The importance of this correlation is discussed in view of needs to more rigorously define B cell populations for investigations of biochemical events of and accessory cell requirements for activation of B lymphocytes.