Biomedical subjects

X Yao

Publications and source records attributed to X Yao.

At least 19 recordsLinked to original sources

PubMed2000

Close association of the N terminus of Kv1.3 with the pore region.

The Shaker superfamily encodes voltage-gated potassium (Kv) channels. The N termini of Shaker proteins are located intracellularly and contain several domains shown to regulate important aspects of channel function, such as speed of inactivation, channel assembly (T1 domain), and steady state protein level (T0 domain, amino acids 3-39 in rabbit). Mutations and/or deletion of certain amino acids in the T0 domain lead to a 13-fold amplification of Kv current as compared with wild type channels, primarily by increasing the absolute number of channel proteins present in the membrane (Segal, A. S., Yao, X., and Desir, G. V. (1999) Biochem. Biophys. Res. Commun. 254, 54-64). Although T0 mutants have kinetic properties virtually indistinguishable from wild type, they were noted to have a slightly larger single channel conductance, suggesting that the T0 domain might also interact with the pore region. In the present study we show that although T0 does not affect pore selectivity, it does modulate the binding affinity of the pore blocker, charybdotoxin. These results suggest that the N terminus of Kv1.3 is closely associated with the pore region.

Amino Acid Sequence↗

PubMed2000

Role of endothelium in thapsigargin-induced arterial responses in rat aorta.

We assessed the role of endothelium in the arterial response to thapsigargin, the Ca(2+)-ATPase inhibitor of the endoplasmic reticulum, in rat isolated aortic rings. Thapsigargin induced an endothelium-dependent relaxation of phenylephrine-contracted aortic rings with an EC(50) of 2.6+/-0.4 nM and a 75% maximum relaxation, while it was less effective against 30 mM K(+)-induced contraction. Pretreatment of aortic rings with N(G)-nitro-L-arginine methyl ester (30 microM) or methylene blue (1 microM) reduced thapsigargin-induced relaxation by approximately 85%. Thapsigargin failed to relax the endothelium-denuded rings. L-Arginine (3 mM) partially, but significantly, antagonized the effect of 30 microM N(G)-nitro-L-arginine methyl ester. Pretreatment with indomethacin (3 microM), glibenclamide (1 microM) or iberiotoxin (100 nM) did not alter the thapsigargin-induced relaxation. In contrast, pretreatment with tetrapentylammonium ions (TPA(+), 1-3 microM) or with 300 microM Ba(2+) suppressed the relaxant response to thapsigargin. TPA(+) (3 microM) also attenuated acetylcholine-induced relaxation. Thapsigargin-induced endothelium-dependent relaxation was primarily dependent on the presence of extracellular Ca(2+). Interestingly, when the tissues were exposed to very low concentrations of thapsigargin (1-3 nM) the nitric oxide-dependent relaxation induced by acetylcholine or A23187 was markedly reduced. While thapsigargin (3 nM) did not influence the relaxation induced by endothelium-independent dilators, sodium nitroprusside and verapamil. These results indicate that thapsigargin produced complex vascular effects primarily by acting on the endothelial cells. Thapsigargin causes an endothelial nitric oxide-dependent relaxation; on the other hand, it inhibits nitric oxide-mediated relaxation at the similar concentrations. Activation of TPA(+)- and Ba(2+)-sensitive but not Ca(2+)-activated or ATP-sensitive K(+) channels may be also involved in thapsigargin-induced relaxation of rat isolated aortic rings.

Animals↗

PubMed2000

Store-operated calcium entry in vascular endothelial cells is inhibited by cGMP via a protein kinase G-dependent mechanism.

Store-operated Ca(2+) entry in vascular endothelial cells not only serves to refill the intracellular Ca(2+) stores, but also acts to stimulate the synthesis of nitric oxide, a key vasodilatory factor. In this study, we examined the role of cGMP in regulating the store-operated Ca(2+) entry in aortic endothelial cells. Cyclopiazonic acid (CPA) and thapsigargin, two selective inhibitors of endoplasmic reticulum Ca(2+)-ATPase, were used to induce store-operated Ca(2+) entry. 8-Bromo-cGMP, an activator of protein kinase G, inhibited the CPA- or thapsigargin-induced Ca(2+) entry in a concentration-dependent manner. An inhibitor of protein kinase G, KT5823 (1 microM) or H-8 (10 microM), abolished the inhibitory action of 8-bromo-cGMP and resumed Ca(2+) entry. Addition of S-nitroso-N-acetylpenicillamine (a nitric oxide donor) or dipyridamole (a cGMP phosphodiesterase inhibitor) during CPA treatment elevated cellular cGMP levels, stimulated protein kinase G activity, and at the same time reduced Ca(2+) influx due to CPA. Patch clamp study confirmed the existence of a CPA-activated Ca(2+)-permeable channel sensitive to cGMP inhibition. These results suggest that cGMP via a protein kinase G-dependent mechanism may play a key role in the regulation of the store-operated Ca(2+) entry in vascular endothelial cells.

Animals↗

PubMed2000

Nitric oxide mediated endothelium-dependent relaxation induced by glibenclamide in rat isolated aorta.

OBJECTIVES: Glibenclamide was found to act as both a selective ATP-sensitive K(+) channel blocker and a vasorelaxant. The exact mechanisms underlying the relaxant effect of glibenclamide are unknown. The present study was designed to examine the role of endothelium/nitric oxide in glibenclamide-induced relaxation in rat isolated aortic rings. METHODS: A combination of experimental approaches including isometric force measurement, cell culture, Ca(2+) fluorescence measurement and radioimmunoassay were used to examine the vascular effect of glibenclamide. RESULTS: Glibenclamide induced a concentration-dependent relaxation more effectively in rings with endothelium (IC(50) of 32+/-4 microM) than those without endothelium (IC(50) of 365+/-29 microM). Incubation with N(G)-nitro-L-arginine methyl ester (L-NAME) or methylene blue significantly reduced and L-arginine (3 mM) potentiated the glibenclamide-induced relaxation. L-Arginine (3 mM) partially antagonized the effect of L-NAME. Glibenclamide (100 microM) increased the cyclic GMP content of endothelium-intact tissues. Pretreatment with N(G)-nitro-L-arginine (100 microM) or removal of endothelium significantly suppressed the effect of glibenclamide on cyclic GMP production. Glibenclamide elevated the intracellular Ca(2+) levels in cultured rat aortic endothelial cells. Glibenclamide also inhibited the endothelium-independent contractile response to 60 mM K(+) (IC(50) of 137+/-21 microM) and caused a rightward shift in the concentration-contraction curve for CaCl(2). Besides, glibenclamide inhibited phorbol-12,13-diacetate (1 microM)-induced contraction in Ca(2+)-free Krebs solution. CONCLUSION: These results indicate that glibenclamide-induced endothelium-dependent relaxation involves nitric oxide release and this effect may be related to its stimulatory effect on endothelial Ca(2+) levels. However, the glibenclamide-induced endothelium-independent relaxation may be associated with its inhibitory effect on Ca(2+) influx through Ca(2+) channels and on the protein kinase C-mediated contractile mechanism.

Animals↗

PubMed2000

Endothelium-dependent relaxation by tetraoctylammonium ions in rat isolated aortic rings.

Quaternary ammonium ions are common pharmacological blockers of K+ channels. This study examined the vasorelaxant effect of tetraoctylammonium ions (TOA+) in rat isolated aortic rings. TOA+ caused a concentration-dependent transient relaxation of endothelium-intact tissues. Pretreatment with NG-nitro-L-arginine methyl ester (L-NAME, 3x10(-5) M) or methylene blue (3 x 10(-6) M) or removal of the endothelium abolished the TOA+-induced relaxation. L-arginine (10(-3) M ) partially antagonized the effect of L-NAME. Glibenclamide (3x10(-6) M), charybdotoxin (CTX, 10(-7) M), indomethacin (10(-5) M), or atropine (3x10(-6) M) had no effect. Both TOA+ (10(-5) M)- and acetylcholine (ACh, 10(-5) M)-induced increase in tissue content of cyclic GMP was significantly attenuated by NG-nitro-L-arginine (L-NNA, 10(-4) M) and abolished in endothelium-denuded arteries. These results indicate that TOA+ induced endothelium-dependent relaxation which is likely mediated through nitric oxide but not other endothelium-derived factors. This relaxant action seems unique for TOA+ since other quaternary ammonium ions did not cause nitric oxide-dependent relaxation.

Animals↗

PubMed1999

His(73), often methylated, is an important structural determinant for actin. A mutagenic analysis of HIS(73) of yeast actin.

His(73), has been proposed to regulate the release of P(i) from the interior of actin following polymerization-dependent hydrolysis of bound ATP. Although it is a 3-methylhistidine in the vast majority of actins, His(73) is unmethylated in S. cerevisiae actin. We mutated His(73) in yeast actin to Arg, Lys, Ala, Gln, and Glu and detected no altered phenotypes associated with the mutations in vivo. However, they significantly affect actin function in vitro. Substitution of the more basic residues resulted in enhanced thermal stability, decreased rate of nucleotide exchange, and decreased susceptibility to controlled proteolysis relative to wild-type actin. The opposite effects are observed with the neutral and anionic substitutions. All mutations reduced the rate of polymerization. Molecular dynamics simulations predict a new conformation for the His(73) imidazole in the absence of a methyl group. It also predicts that Arg(73) tightens and stabilizes the actin and that Glu(73) causes a rearrangement of the bottom of actin's interdomain cleft leading possibly to our observed destabilization of actin. Considering the exterior location of His(73), this work indicates a surprisingly important role for the residue as a major structural determinant of actin and provides a clue to the impact caused by methylation of His(73).

Actins↗

PubMed1999

Donor-type chimerism determination by competitive polymerase chain reaction (PCR) in a primate model for bone marrow transplantation.

BACKGROUND: Evaluation of the outcome of successful bone marrow transplantation (BMT) and in-depth studies of transplantation biology rely increasingly on accurate detection of donor origin cells in the transplanted recipients. This study describes a quantitative competitive polymerase chain reaction (PCR) assay for accurate evaluation of chimerism after allogeneic BMT in a cynomologous primate model, based on detection of monkey Y-specific DNA. METHODS: A competitor standard was generated via PCR using a mutagenic primer that makes the competitor DNA 22 bp less than the wild type monkey Y-specific DNA. The mutated form can still be amplified by the primer pair for the detection of monkey Y-specific DNA. A fixed amount of sample subjected to chimerism detection was co-amplified with a range of competitor DNA using a touch down program and hot start PCR technique. The PCR products were analyzed by computing densitometry. The ratio of competitor/target (Y-specific) DNA for each sample pair was calculated. RESULTS: Using DNAs prepared from an artificial mixture of male and female cells, a set of standard curves has been obtained and the sensitivity of the established quantitative PCR was found to be 25 pg of male DNA, which corresponds approximately to 0.005 fg competitor DNA. A DNA sample taken from a female monkey, transplanted with purified CD34+ stem cells from a male monkey donor 26 days after BMT, was subjected to the competitive PCR with 10% male DNA as a control; the level of male DNA in this sample was calculated to be around 50%. CONCLUSIONS: This quantitative PCR assay offers both a high degree of specificity as well as a very accurate and sensitive evaluation of chimerism in a sex-mismatched monkey BMT model.

Animals↗

PubMed1999

A highly conserved 3-methylhistidine modification is absent in yeast actin.

To identify a protein histidine methyltransferase from Saccharomyces cerevisiae, we examined purified actin for the presence of the highly conserved 3-methylhistidine residue at position 73 by amino acid analysis of the whole protein and by amino acid analysis and mass spectrometry of the corresponding tryptic fragment. Surprisingly, we found that His-73 is not modified. A similar lack of modification was also found in actin from the yeast Candida albicans, while rabbit muscle actin revealed the expected 3-methylhistidine residue. Phylogenetic analysis of actin sequences suggests that this modification was introduced in evolution after the divergence of yeast from higher eukaryotic organisms, including unicellular eukaryotes such as Acanthamoeba, Dictyostelium, and Physarum, whose actins contain 3-methylhistidine. Our methodology for the analytical determination of 3-methylhistidine in actin offers an improved approach for investigating histidine methylation in proteins.

Actins↗

PubMed1999

Inherited human Caspase 10 mutations underlie defective lymphocyte and dendritic cell apoptosis in autoimmune lymphoproliferative syndrome type II.

Caspases are cysteine proteases that mediate programmed cell death in phylogenetically diverse multicellular organisms. We report here two kindreds with autoimmune lymphoproliferative syndrome (ALPS) type II, characterized by abnormal lymphocyte and dendritic cell homeostasis and immune regulatory defects, that harbor independent missense mutations in Caspase 10. These encode amino acid substitutions that decrease caspase activity and interfere with death receptor-induced apoptosis, particularly that stimulated by Fas ligand and TRAIL. These results provide evidence that inherited nonlethal caspase abnormalities cause pleiotropic apoptosis defects underlying autoimmunity in ALPS type II.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Different mutator phenotypes in Mlh1- versus Pms2-deficient mice.

Deficiencies in DNA mismatch repair (MMR) result in increased mutation rates and cancer risk in both humans and mice. Mouse strains homozygous for knockouts of either the Pms2 or Mlh1 MMR gene develop cancer but exhibit very different tumor spectra; only Mlh1(-/-) animals develop intestinal tumors. We carried out a detailed study of the microsatellite mutation spectra in each knockout strain. Five mononucleotide repeat tracts at four different chromosomal locations were studied by using single-molecule PCR or an in vivo forward mutation assay. Three dinucleotide repeat loci also were examined. Surprisingly, the mononucleotide repeat mutation frequency in Mlh1(-/-) mice was 2- to 3-fold higher than in Pms2(-/-) animals. The higher mutation frequency in Mlh1(-/-) mice may be a consequence of some residual DNA repair capacity in the Pms2(-/-) animals. Relevant to this idea, we observed that Pms2(-/-) mice exhibit almost normal levels of Mlh1p, whereas Mlh1(-/-) animals lack both Mlh1p and Pms2p. Comparison between Mlh1(-/-) animals and Mlh1(-/-) and Pms2(-/-) double knockout mice revealed little difference in mutator phenotype, suggesting that Mlh1 nullizygosity is sufficient to inactivate MMR completely. The findings may provide a basis for understanding the greater predisposition to intestinal cancer of Mlh1(-/-) mice. Small differences (2- to 3-fold) in mononucleotide repeat mutation rates may have dramatic effects on tumor development, requiring multiple genetic alterations in coding regions. Alternatively, this strain difference in tumor spectra also may be related to the consequences of the absence of Pms2p compared with the absence of both Pms2p and Mlh1p on as yet little understood cellular processes.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Solution structure of the chicken cysteine-rich protein, CRP1, a double-LIM protein implicated in muscle differentiation.

The mechanism by which the contractile machinery of muscle is assembled and maintained is not well-understood. Members of the cysteine-rich protein (CRP) family have been implicated in these processes. Three vertebrate CRPs (CRP1-3) that exhibit developmentally regulated muscle-specific expression have been identified. All three proteins are associated with the actin cytoskeleton, and one has been shown to be required for striated muscle structure and function. The vertebrate CRPs identified to date display a similar molecular architecture; each protein is comprised of two tandemly arrayed LIM domains, protein-binding motifs found in a number of proteins with roles in cell differentiation. Each LIM domain coordinates two Zn(II) ions that are bound independently in CCHC (C=Cys, H=His) and CCCC modules. Here we describe the solution structure of chicken CRP1 determined by homonuclear and 1H-15N heteronuclear magnetic resonance spectroscopy. Comparison of the structures of the two LIM domains of CRP1 reveals a high degree of similarity in their tertiary folds. In addition, the two component LIM domains represent two completely independent folding units and exhibit no apparent interactions with each other. The structural independence and spatial separation of the two LIM domains of CRP1 are compatible with an adapter or linker role for the protein.

Adaptor Proteins, Signal Transducing↗

PubMed1999

The T0 domain of rabbit KV1.3 regulates steady state channel protein level.

The Shaker superfamily encodes voltage-gated potassium (Kv) channels. The amino (N) terminus is important for channel assembly and mediates fast inactivation. We recently isolated a Kv channel from rabbit kidney, denoted rabKv1.3 (Yao et al., J. Clin. Invest. 97, 2525-2533, 1996) and found that deleting a region (T0 domain, amino acids 3-39) proximal to the T1 recognition domain (a.a 42-185) leads to a 13-fold amplification of Kv current as compared to wild type channels (Yao et al., BBRC 249, 492-498). Here we show that deleting the T0 domain affects neither single channel conductance nor channel open probability. Instead, it increases the absolute number of channel proteins present in the membrane. We conclude that the T0 domain is a previously unrecognized Shaker Kv1.3, N-terminal regulatory region that modulates steady state channel protein density in the plasma membrane.

Animals↗

PubMed1999

Rod-type cyclic nucleotide-gated cation channel is expressed in vascular endothelium and vascular smooth muscle cells.

OBJECTIVES: Ca(++)-permeable nonselective cation channels mediate the entry of extracellular Ca++ in vascular endothelium. They are also partly responsible for Ca++ entry in vascular smooth muscle cells (SMCs). The molecular identities of these channels have not been identified. The aim of this study is to examine whether rod-type nucleotide-gated nonselective cation (CNG1) channel, a channel which has been molecularly cloned, is related to the nonselective channels in vascular cells. METHODS: We used RT-PCR, molecular cloning, northern Blot and in situ hybridization to examine the expression of CNG1 mRNA in a variety of guinea pig and rat blood vessels with different diameters and in cultured vascular endothelial cells and vascular smooth muscle cells. RESULTS: We have cloned a 402-bp partial cDNA of CNG1 channel from guinea pig mesenteric arteries. RT-PCR and southern blot results indicate that the CNG1 mRNA is expressed in both cultured vascular endothelial and cultured vascular SMCs. Northern blot revealed the transcripts of approximately 3.2 kb, approximately 5.0 kb, and approximately 1.8 kb in cultured endothelial cells. In situ hybridization yielded strong labeling in endothelium layer of aorta, medium-sized mesenteric arteries, and small mesenteric arteries. CONCLUSION: Our findings suggest a potential role of CNG protein for Ca++ entry in vascular endothelium and vascular smooth muscles. The high expression of CNG1 mRNA in the endothelium of medium-sized arteries and small-sized arteries implicates a possible involvement of CNG1 protein in the regulation of blood supply to different regions and in the regulation of arterial blood pressure.

Amino Acid Sequence↗

PubMed1999

Activity of voltage-gated K+ channels is associated with cell proliferation and Ca2+ influx in carcinoma cells of colon cancer.

Cell proliferation of carcinoma cells DLD-1 derived from colon cancer as measured by [3H] thymidine incorporation was drastically reduced in the presence of 4-aminopyridine, an inhibitors of voltage-gated K channel. A number of nonspecific K+ channel inhibitors including TPeA, TEA, verapamil and diltiazem also inhibited [3H] incorporation at the concentration reported to inhibit voltage-gated K+ channels. The presence of voltage-gated K+ channels was confirmed by reverse transcription-PCR and cDNA sequencing. Charybdotoxin and iberiotoxin, inhibitors for Ca2+-sensitive K+ channel, and glibenclamide, a specific inhibitor for ATP-sensitive K+ channel, did not have effect on cell proliferation. These experiments suggested a critical role of voltage-gated K+ channels in proliferation of colon cancer cells. Mechanism of action of K+ channel activity in cell proliferation was explored by studying the relationship between the K+ channel activity and Ca2+ entry. The results from experiments indicated that K+ channel inhibitors blocked [Ca2+]i influx. Therefore, it is likely that K+ channel activity may modulate Ca2+ influx into colon cancer cells, and subsequently modulate the proliferation of these cells.

4-Aminopyridine↗

PubMed1999

Antineoplastic agents. III: Steroidal glycosides from Solanum nigrum.

By bioactivity-guided fractionation, three known steroidal glycosides, beta 2-solamargine (1), solamargine (2), and degalactotigonin (3), were isolated from Solanum nigrum. Compounds 1-3 induced morphological abnormality in Pyricularia oryzae mycelia with MMDC (minimum morphological deformation concentration) values of 63.0, 38.5, and 97.2 microM, respectively. This is the first report on the isolation of 1 from this species and on the cytotoxicity of 1-3 on six cultured human solid tumor cell lines HT-29 (colon), HCT-15 (colon), LNCaP (prostate), PC-3 (prostate), T47D (breast), and MDA-MB-231 (breast) in vitro. The cytotoxic assay indicated that 2 is the main antineoplastic agent in S. nigrum. The structures of 1-3 were elucidated on the basis of chemical evidence and spectral analysis, especially by 2D-NMR analysis.

Antineoplastic Agents, Phytogenic↗

PubMed1999

French Machado-Joseph disease patients do not exhibit gametic segregation distortion: a sperm typing analysis.

Segregation distortion has been reported to occur in a number of the trinucleotide repeat disorders. On the basis of a sperm typing study performed in patients of Japanese descent with Machado-Joseph disease (MJD), it was reported that disease alleles are preferentially transmitted during meiosis. We performed a sperm typing study of five MJD patients of French descent and analysis of the pooled data shows a ratio of mutant to normal alleles of 379:436 (46.5:53.5%), which does not support meiotic segregation distortion. To confirm these results, sperm typing analysis was also performed using a polymorphic marker, D14S1050, closely linked to the MJD1 gene. Among 910 sperm analyzed, the allele linked to the disease chromosome was detected in 50.3% of the samples and the allele linked to the normal chromosome was found in 49.6% of the sperm. The difference in frequency of these two alleles is not significant ( P = 0.8423). Likelihood-based analysis of segregation distortion in the single sperm data using the SPERMSEG program also showed no support for segregation distortion at the gamete level in this patient population. The previous report on the Japanese patients also suggested that disease allele stability may be influenced by a trans effect of an intragenic polymorphism (987 G/C) in the wild-type allele. All of the French patients were heterozygous for this polymorphism. However, analysis of the variance in repeat number in sperm from the French MJD patients overlapped significantly with the variance in repeat number observed in the C/C homozygous Japanese patients.

Alleles↗

PubMed1999

Comparison of the backbone dynamics of the apo- and holo-carboxy-terminal domain of the biotin carboxyl carrier subunit of Escherichia coli acetyl-CoA carboxylase.

The biotin carboxyl carrier protein (BCCP) is a subunit of acetyl-CoA carboxylase, a biotin-dependent enzyme that catalyzes the first committed step of fatty acid biosynthesis. In its functional cycle, this protein engages in heterologous protein-protein interactions with three distinct partners, depending on its state of post-translational modification. Apo-BCCP interacts specifically with the biotin holoenzyme synthetase, BirA, which results in the post-translational attachment of biotin to a single lysine residue on BCCP. Holo-BCCP then interacts with the biotin carboxylase subunit of acetyl-CoA carboxylase, which leads to the addition of the carboxylate group of bicarbonate to biotin. Finally, the carboxy-biotinylated form of BCCP interacts with transcarboxylase in the transfer of the carboxylate to acetyl-CoA to form malonyl-CoA. The determinants of protein-protein interaction specificity in this system are unknown. The NMR solution structure of the unbiotinylated form of an 87 residue C-terminal domain fragment (residue 70-156) of BCCP (holoBCCP87) and the crystal structure of the biotinylated form of a C-terminal fragment (residue 77-156) of BCCP from Escherichia coli acetyl-CoA carboxylase have previously been determined. Comparative analysis of these structures provided evidence for small, localized conformational changes in the biotin-binding region upon biotinylation of the protein. These structural changes may be important for regulating specific protein-protein interactions. Since the dynamic properties of proteins are correlated with local structural environments, we have determined the relaxation parameters of the backbone 15N nuclear spins of holoBCCP87, and compared these with the data obtained for the apo protein. The results indicate that upon biotinylation, the inherent mobility of the biotin-binding region and the protruding thumb, with which the biotin group interacts in the holo protein, are significantly reduced.

Acetyl-CoA Carboxylase↗

PubMed1999

Thickness and elasticity of gram-negative murein sacculi measured by atomic force microscopy.

Atomic force microscopy was used to measure the thickness of air-dried, collapsed murein sacculi from Escherichia coli K-12 and Pseudomonas aeruginosa PAO1. Air-dried sacculi from E. coli had a thickness of 3.0 nm, whereas those from P. aeruginosa were 1.5 nm thick. When rehydrated, the sacculi of both bacteria swelled to double their anhydrous thickness. Computer simulation of a section of a model single-layer peptidoglycan network in an aqueous solution with a Debye shielding length of 0.3 nm gave a mass distribution full width at half height of 2.4 nm, in essential agreement with these results. When E. coli sacculi were suspended over a narrow groove that had been etched into a silicon surface and the tip of the atomic force microscope used to depress and stretch the peptidoglycan, an elastic modulus of 2.5 x 10(7) N/m(2) was determined for hydrated sacculi; they were perfectly elastic, springing back to their original position when the tip was removed. Dried sacculi were more rigid with a modulus of 3 x 10(8) to 4 x 10(8) N/m(2) and at times could be broken by the atomic force microscope tip. Sacculi aligned over the groove with their long axis at right angles to the channel axis were more deformable than those with their long axis parallel to the groove axis, as would be expected if the peptidoglycan strands in the sacculus were oriented at right angles to the long cell axis of this gram-negative rod. Polar caps were not found to be more rigid structures but collapsed to the same thickness as the cylindrical portions of the sacculi. The elasticity of intact E. coli sacculi is such that, if the peptidoglycan strands are aligned in unison, the interstrand spacing should increase by 12% with every 1 atm increase in (turgor) pressure. Assuming an unstressed hydrated interstrand spacing of 1.3 nm (R. E. Burge, A. G. Fowler, and D. A. Reaveley, J. Mol. Biol. 117:927-953, 1977) and an internal turgor pressure of 3 to 5 atm (or 304 to 507 kPa) (A. L. Koch, Adv. Microbial Physiol. 24:301-366, 1983), the natural interstrand spacing in cells would be 1.6 to 2.0 nm. Clearly, if large macromolecules of a diameter greater than these spacings are secreted through this layer, the local ordering of the peptidoglycan must somehow be disrupted.

Cell Wall↗