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Biomedical subjects

K R Miller

Publications and source records attributed to K R Miller.

54 records · Page 3Linked to original sources
PubMed1979

A chloroplast membrane lacking photosystem II. Thylakoid stacking in the absence of the photosystem II particle.

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.

Chloroplasts↗
PubMed1979

Structure of a bacterial photosynthetic membrane.

The internal photosynthetic membranes of a photosynthetic bacterium, Rhodopseudomonas viridis, have been studied with a variety of electron microscope techniques. The membranes are composed of a sheet of apparently identical subunits arranged in a hexagonal fashion. The individual subunits repeat at a distance of 110 A. Optical transforms have been used to enhance micrographs of this ordered membrane, and the images synthesized in this way show details of each subunit. The individual subunits are asymmetric, differing slightly in appearance at the outer and inner surfaces of the membrane, and these surface patterns seem to be combined in the image of the thylakoid membrane in negative stain. These studies fix a maximum size for the photosynthetic unit of R. viridis and suggest the suitability of this membrane for further diffraction analysis.

Journal Article↗
PubMed1978

Chloroplast membrane biogenesis in Chlamydomonas: correlation between the formation of membrane components and membrane structure.

The y-1 mutant of Chlamydomonas reinhardi, when allowed to green in the presence of chloramphenicol (CAP), an inhibitor of protein synthesis on 70s ribosomes, form photosynthetic membranes which contain somewhat less chlorophyll than those of cells greened in the absence of the drug. Photosystem I and II activities are drastically reduced in the CAP-greened cells, and specific alterations in the polypeptide composition of the thylakoid membranes are also observed. We have examined the internal structure of the thylakoid membranes from cells greened in the presence and absence of CAP, and have found that the large particles observed on the exoplasmic fracture face (EF) are substantially reduced in size and number in the CAP-greened cells. This structural defect seems related to the absence of significant photo-system activities in the CAP-greened cells, despite the presence of most major membrane polypeptides. We suggest that CAP treatment results in a failure of the cell to organize functional reaction complexes, and is structurally reflected in the absence of large (EF) particles in such membranes. This defect can be repaired by allowing the affected cells to re-green in the absence of the drug, and the large particles reappear, paralleling an increase in photosynthetic activity.

Chlamydomonas↗
PubMed1977

Organization of the photosynthetic membrane in maize mesophyll and bundle sheath chloroplasts.

The freeze-fracturing technique has been used to investigate membrane architecture in the mesophyll and bundle sheath chloroplasts of Zea mays. The structural organization of mesophyll chloroplasts is virtually identical to that of other species of higher plants which have been investigated with this technique. Characteristic distributions of particles of various sizes are seen on each fracture face after membrane splitting during the fracturing process, and these distributions indicate the differentiation of the membrane system into sacked (grana) and unstacked (stroma) regions, typical of grana-containing chloroplasts. Bundle sheath chloroplasts contain very few grana, and the thylakoids of these plastids are therefore largely unstacked. Analysis of artificially unstacked mesophyll chloroplasts indicates that this difference is not merely related to the presence or absence of adhesion between adjacent thylakoids, but reflects a substantial difference in membrane substructure between mesophyll and bundle sheath photosynthetic membranes. Bundle sheath thylakoids contain virtually the same number of small (P fracture face) particles as mesophyll thylakoids, but contain only 40% as many of the larger (E fracture face) tetrameric particles. These differences, together with biochemical data indicating the comparative deficiency of bundle sheath chloroplasts in Photosystem II activity, suggest that the E face particles are related to the presence or absence of Photosystem II activity.

Cell Membrane↗
PubMed1977

Neonatal hypoglycemia resulting from islet cell adenomatosis. Successful treatment with total pancreatectomy.

A female infant developed apneic spells due to hypoglycemia at 73 hours of life. It was impossible to maintain the blood glucose level despite continuous intravenously given dextrose, cortisone, diazoxide, and a low-leucine diet. A subtotal pancreatectomy was performed but there was no evidence of islet cell adenoma. On second laparotomy, the head of the pancreas was removed, and on microscopic examination, islet cell adenomatosis was found. A good clinical recovery followed. Follow-up at age 3 years and 4 months shows apparently normal mental and physical development.

Adenoma, Islet Cell↗
PubMed1976

Analysis of the thylakoid outer surface. Coupling factor is limited to unstacked membrane regions.

The structure of the spinach thylakoid outer surface has been examined by deepetching, a technique which exposes the true surfaces of biological membranes by sublimination of frozen dilute buffer. The membrane surface is covered with large (150 A average diameter) and small (90 A average diameter) particles. Approximately 30% of the large particles can be removed under conditions reported to selectively remove carboxydismutase from the membrane surface. The remaining large particles can be removed only under conditions which cause a loss of coupling factor activity. When purified coupling factor is readded to membranes from which all coupling factor activity has been removed, large particles reappear, indicating that they represent coupling factor molecules. Since the number of particles and the amount of ATPase activity in the reconstituted and control membranes were the same, coupling factor molecules may be attached to specific binding sites. Analysis of antibody labeling experiments, enzyme assays, and experiments involving the unstacking and restacking of thylakoid membranes indicate that coupling factor is excluded from regions of membrane stacking (grana) and is present only in unstacked membrane regions. The exclusion of coupling factor from grana, which are known to be centers of intense photosynthetic activity, strongly suggests that the mechanism coupling electron transport to photophosphorylation is indirect. In addition to the large and small particles, in some cases regularly spaced ridges are visible on the outer surface after unstacking. Coupling factor binding sites seem to be excluded from regions where these structures occur.

Cell Membrane↗
PubMed1976

The light-harvesting chlorpohyll-protein complex of photosystem II. Its location in the photosynthetic membrane.

We have investigated the structure of the photosynthetic membrane in a mutant of barley known to lack a chlorophyll-binding protein. This protein is thought to channel excitation energy to photosystem II, and is known as the "light-harvesting chlorophyll-protein complex." Extensive stacking of thylakoids into grana occurs in both mutant and wild-type chloroplasts. Examination of membrane internal structure by freeze-fracturing indicates that only slight differences exist between the fracture faces of mutant and wild-type membranes. These differences are slight reductions in the size of particles visible on the EFs fracture face, and in the number of particles seen on the PFs fracture face. No differences can be detected between mutant and wild-type on the etched out surface of the membrane. In contrast, tetrameric particles visible on the etched inner surface of wild-type thylakoids are extremely difficult to recognize on similar surfaces of the mutant. These particles can be recognized on inner surfaces of the mutant membranes when they are organized into regular lattices, but these lattices show a much closer particle-to-particle spacing than similar lattices in wild-type membranes. Although several interpretations of these data are possible, these observations are consistent with the proposal that the light-harvesting chlorophyll-protein complex of photosystem II is bound to the tetramer (which is visible on the EFs face as a single particle) near the inner surface of the membrane. The large tetramer, which other studies have shown to span the thylakoid membrane, may represent an assembly of protein, lipid, and pigment comprising all the elements of the photosystem II reaction. A scheme is presented which illustrates one possibility for the light reaction across the photosynthetic membrane.

Carrier Proteins↗
PubMed1976

Chloroplast membranes of the green alga Acetabularia mediterranea. II. Topography of the chloroplast membrane.

The localization of the chlorophyll-protein complexes inside the thylakoid membrane of Acetabularia mediterranea was determined by fractionating the chloroplast membrane with EDTA and Triton X-100, by using pronase treatment, and by labeling the surface-exposed proteins with 125I. The effects of the various treatments were established by electrophoresis of the solubilized membrane fractions and electron microscopy. After EDTA and pronase treatment, the membrane structure was still intact. Only the two chlorophyll-protein complexes of 67,000 and 152,000 daltons and an additional polypeptides were found in the membrane before the EDTA and pronase treatment. The 125,000 dalton complex seems to be buried inside the lipid layer. The 23,000 dalton subunit of the 67,000 dalton complex is largely exposed to the surface of the EDTA-insoluble membrane and only the chlorophyll-binding subunit of 21,500 daltons is buried inside the lipid layer.

Acetabularia↗
PubMed1974

Freeze-fracture of microtubules and bridges in motile axostyles.

A freeze-fracture study of the motile axostyles of the flagellate protozoa Saccinobaculus and Pyrsonympha has been undertaken in order to obtain a view of the relationships of microtubules and their cross bridges not dependent on conventional preparative procedures. Reactivation studies using isolated axostyles prepared for freeze-fracture and then thawed demonstrate that we are observing the structure of a potentially functional axostyle. Cross fractures through the axostyle demonstrate more extensive interrow bridging than expected on the basis of observations of thin-sectioned material. Each microtubule has approximately sixfold bridge-binding sites with connections to as many as four interrow bridges. Measurements of microtubule diameter and spacing are significantly larger than those made from sectioned material and may indicate that conventional processing for electron microscopy results in the loss of structurally important water within the microtubule in addition to loss of intertubule material. Longitudinal fractures through the axostyle at various orientations demonstrate a minimum longitudinal periodicity of 160 A for both the spacing of the globular subunits within the microtubule wall and the spacing of the intrarow bridges. While intrarow bridges are strictly periodic and always oriented in parallel, interrow bridges are not strictly periodic and can be oriented at varying angles to the microtubule axis.

Eukaryota↗
PubMed1973

Role of coxsackievirus B4 in the pathogenesis of acute glomerulonephritis.

Coxsackievirus B(4) was isolated from the throat, nose, blood, stools and urine of a 9-year-old boy with acute glomerulonephritis and a pneumonitis. Neutralization test showed a greater than fourfold rise in the antibody titre to coxsackievirus B(4). The antistreptolysin O titre was elevated, but the complement component was within the normal range. The importance of the coxsackievirus B(4) in the pathogenesis of acute glomerulonephritis is clearly indicated; however, further investigations are needed to understand the details of the virus-kidney interaction.

Acute Disease↗
PubMedDate not supplied

Mapping the lateral distribution of photosystem II and the cytochrome b6/f complex by direct immune labeling of the thylakoid membrane.

By direct immunolabeling we have mapped the distribution of photosystem II (PS II) and cytochrome b6/f on the surfaces of photosynthetic membranes isolated from spinach. Photosynthetic membranes were attached to a support and gently disrupted to expose the occluded outer stacked surface, prior to labeling. Polyclonal antibodies against PS II intensely labeled the outer stacked surfaces while the outer nonstacked surface had minimal labeling. This confirms previous fractionation and immunolocalization studies which demonstrated that PS II is largely restricted to the stacked regions of the membrane. Inside-out membranes were also heavily labeled with PS II antibodies. Antibodies against cytochrome f were evenly distributed between the stacked and nonstacked outer surfaces and were found clumped together on the membrane outer surface. Previous fractionation and immunolocalization studies have indicated that cytochrome b6/f is located in both the stacked and nonstacked regions, but this is the first report to provide direct evidence that the complex may be clustered in the membrane. The clustering of antibodies to cytochrome b6/f supports the idea that this electron transport component exists as a multimeric complex within the membranes and that such complexes are found in both stacked and nonstacked regions of the photosynthetic membrane. No evidence was seen of any special differentiation of the marginal regions of the membrane, which link stacked and nonstacked regions.

Cytochromes↗
PubMedDate not supplied

Titration calorimetry as a binding assay for lipid-binding proteins.

Titration calorimetry has been evaluated as a method for obtaining binding constants and thermodynamic parameters for the cytosolic fatty acid- and lipid-binding proteins. An important feature of this method was its ability to accurately determine binding constants in a non-perturbing manner. The equilibrium was not perturbed, since there was no requirement to separate bound and free ligand in order to obtain binding parameters. Also, the structure of the lipid-protein complex was not perturbed, since native ligands were used rather than non-native analogues. As illustrated for liver fatty acid-binding protein, the method distinguished affinity classes whose dissociation constants differed by an order of magnitude or less. It also distinguished endothermic from exothermic binding reactions, as illustrated for the binding of two closely related bile salts to ileal lipid-binding protein. The main limitations of the method were its relatively low sensitivity and the difficulty working with highly insoluble ligands, such as cholesterol or saturated long-chain fatty acids. However, the signal-to-noise ratio was improved by manipulating the buffer conditions, as illustrated for oleate binding to rat intestinal fatty acid binding protein. Binding parameters are reported for oleate interactions with several wild-type and mutant lipid-binding proteins from intestine. Where possible, the binding parameters obtained from calorimetry were compared with results obtained from fluorescence and Lipidex binding assays of comparable systems.

Animals↗