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Biomedical subjects

M Helfrich

Publications and source records attributed to M Helfrich.

14 recordsLinked to original sources
PubMed2002

[The future of telepathology. An Internet "distributed system" with "open standards"].

With the availability of Internet, the interest in the possibilities of telepathology has increased considerably. In the foreground is thereby the need of the non-expert to bring in the opinions of experts on morphological findings by means of a fast and simple procedure. The new telepathology system iPath is in compliance with these needs. The system is based on small, but when possible independently working modules. This concept allows a simple adaptation of the system to the individual environment of the user (e.g. for different cameras, frame-grabbers, microscope steering tables etc.) and for individual needs. iPath has been in use for 6 months with various working groups. In telepathology a distinction is made between "passive" and "active" consultations but for both forms a non-expert brings in the opinion of an expert. In an active consultation both are in direct connection with each other (orally or via a chat-function), this is however not the case with a passive consultation. An active consultation can include the interactive discussion of the expert with the non-expert on images in an image database or the direct interpretation of images from a microscope by the expert. Four software modules are available for a free and as fast as possible application: (1) the module "Microscope control", (2) the module "Connector" (insertion of images directly from the microscope without a motorized microscope), (3) the module "Client-application" via the web-browser and (4) the module "Server" with a database. The server is placed in the internet and not behind a firewall. The server permanently receives information from the periphery and returns the information to the periphery on request. The only thing which the expert, the non-expert and the microscope have to know is how contact can made with the server.

Humans↗
PubMed1999

Identification of

[8-vinyl]-Protochlorophyllide a1 was isolated from a Prochloron sp. associated with the host ascidian, Lissoclinum patella. To obtain sufficient amounts for identification of the purified pigment, suitable extraction procedures and HPLC systems were developed. The structure was finally elucidated by UV-VIS and fluorescence spectroscopy, mass spectrometry and NMR (rotating-frame Overhauser enhancement spectroscopy). [8-vinyl]-Protochlorophyllide a was originally detected only as an intermediate in chlorophyll biosynthesis. Although its presence as a light-harvesting pigment was previously suggested in some prochlorophytes and eukaryotic algae, this is the first unequivocal demonstration of [8-vinyl]-protochlorophyllide a in an oxygenic phototroph. We also show that [8-vinyl]-protochlorophyllide a occurs in Prochloron species of four other ascidians as well as in Micromonas pusilla and Prochlorococcus marinus. The possible role of this pigment in photosynthesis is discussed.

Journal Article↗
PubMed1999

Protochlorophyllide b does not occur in barley etioplasts.

Barley (Hordeum vulgare L.) etioplasts were isolated, and the pigments were extracted with acetone. The extract was analyzed by HPLC. Only protochlorophyllide a and no protochlorophyllide b was detected (limit of detection < 1% of protochlorophyllide a). Protochlorophyllide b was synthesized starting from chlorophyll b and incubated with etioplast membranes and NADPH. In the light, photoconversion to chlorophyllide b was observed, apparently catalyzed by NADPH :protochlorophyllide oxidoreductase. In darkness, reduction of the analogue zinc protopheophorbide b to zinc 7-hydroxy-protopheophorbide a was observed, apparently catalyzed by chlorophyll b reductase. We conclude that protochlorophyllide b does not occur in detectable amounts in etioplasts, and even traces of it as the free pigment are metabolically unstable. Thus the direct experimental evidence contradicts the idea by Reinbothe et al. (Nature 397 (1999) 80-84) of a protochlorophyllide b-containing light-harvesting complex in barley etioplasts.

Hordeum↗
PubMed1999

Pigment-free NADPH:protochlorophyllide oxidoreductase from Avena sativa L. Purification and substrate specificity.

The enzyme NADPH:protochlorophyllide oxidoreductase (POR) is the key enzyme for light-dependent chlorophyll biosynthesis. It accumulates in dark-grown plants as the ternary enzyme-substrate complex POR-protochlorophyllide a-NADPH. Here, we describe a simple procedure for purification of pigment-free POR from etioplasts of Avena sativa seedlings. The procedure implies differential solubilization with n-octyl-beta-D-glucoside and one chromatographic step with DEAE-cellulose. We show, using pigment and protein analysis, that etioplasts contain a one-to-one complex of POR and protochlorophyllide a. The preparation of 13 analogues of protochlorophyllide a is described. The analogues differ in the side chains of the macrocycle and in part contain zinc instead of the central magnesium. Six analogues with different side chains at rings A or B are active substrates, seven analogues with different side chains at rings D or E are not accepted as substrates by POR. The kinetics of the light-dependent reaction reveals three groups of substrate analogues with a fast, medium and slow reaction. To evaluate the kinetic data, the molar extinction coefficients in the reaction buffer had to be determined. At concentrations above 2 mole substrate/mole enzyme, inhibition was found for protochlorophyllide a and for the analogues.

Avena↗
PubMed1996

Stabilization of chlorophyll a-binding apoproteins P700, CP47, CP43, D2, and D1 by chlorophyll a or Zn-pheophytin a.

Stabilization of chlorophyll a-binding apoproteins P700, CP47, CP43, D2, and D1 against proteolytic degradation has been investigated through in vitro synthesis of chlorophyll a or Zn-pheophytin a in intact etioplasts from barley. Stabilization of the apoproteins was dependent on the concentration of chlorophyll a or Zn-pheophytin a. Zn-pheophytin a was superior to chlorophyll a with respect to the concentration of pigment required for an equal yield of the stabilized chlorophyll a protein CP47, CP43, and P700 and for the total yield of chlorophyll a proteins. Zn-pheophytin a was most efficient for stabilizing CP47 and, at an increased concentration, efficient for stabilizing CP43, P700, and D1. Stabilization of apoproteins was highest after de novo synthesis of 90-300 pmol of Zn-pheophytin a or of about 400-600 pmol of chlorophyll a/4.2 x 10(7) etioplasts. The yield of stabilized chlorophyll proteins decreased at higher concentrations of Zn-pheophytin a, but was unaffected by higher concentrations of chlorophyll a.

Apoproteins↗
PubMed1996

Stabilization of the chlorophyll binding apoproteins, P700, CP47, CP43, D2, and D1, by synthesis of Zn-pheophytin a in intact etioplasts from barley.

Chlorophyll a was compared with Zn-pheophytin a for stabilization of chlorophyll binding apoproteins, P700, CP47, CP43, D2, and D1, in intact etioplasts from barley (Hordeum vulgare L.). Intact etioplasts were shown to effectively translate the chlorophyll apoproteins, to take up and esterify the exogenously added substrates, chlorophyllide a and Zn-pheophorbide a, with geranylgeraniolpyrophosphate. For stabilization of P700, CP47, D2, and D1, the product, Zn-pheophytin a, was shown to substitute for chlorophyll a. Stabilization of CP43 was selectively increased in the presence of Zn-pheophytin a. The degree of stabilization was shown to depend on the amount of newly synthesized Zn-pheophytin a and on the central atom of the chlorophyll molecule.

Chlorophyll↗
PubMed1995

Photoreduction of zinc protopheophorbide b with NADPH-protochlorophyllide oxidoreductase from etiolated wheat (Triticum aestivum L.).

A preparation of prolamellar bodies from wheat etioplasts was used as a source for NADPH-protochlorophyllide oxidoreductase (pchlide reductase). The enzyme was solubilized with Triton X-100 after reduction of the endogenous photoconvertible protochlorophyllide a to chlorophyllide a by saturating illumination. Protochlorophylls a and b, protochlorophyllide a and zinc protopheophorbide b were added to the soluble enzyme preparation to determine if they were reduced in the dark or in the light. None of the compounds were reduced (with NADPH) in the dark; however, light-dependent reduction was demonstrated with protochlorophyllide a and zinc protopheophorbide b. The yield was approximately 50% for both substrates. Photoreduction did not occur with the esterified protochlorophylls a and b. Photoreduction of zinc protopheophorbide b, the zinc analogue of protochlorophyllide b, is the first demonstration of the reduction of a chlorophyll-b-related compound by pchlide reductase.

Chlorophyll↗
PubMed1994

Chlorophyll synthetase cannot synthesize chlorophyll a'.

Chlorophyll synthetase catalyzes the last step of chlorophyll biosynthesis, namely prenylation (esterification) of chlorophyllide with phytyl diphosphate or geranylgeranyl diphosphate. During investigation of various chlorophyllide derivatives as potential substrates we observed lower esterification with increasing percentages of chlorophyllide a' in epimeric mixtures of chlorophyllides a and a'. To avoid epimerization during esterification, we studied the reaction in detail with model compounds [zinc-13(2)(R)-methoxy-pheophorbide a and zinc-13(2)(S)-methoxy-pheophorbide a, zinc-13(2)(R)-methoxy-pyropheophorbide a and zinc-chlorine6-13(1), 15(2)-dimethylester]. We conclude that compounds which have the 13(2)-carbomethoxy group at the same side of the macrocycle as the propionic side chain of ring D are neither substrates nor competitive inhibitors. Only compounds having the 13(2)-carbomethoxy group at the opposite site are substrates for the enzyme. Naturally occurring chlorophyll a' must be formed by epimerization after esterification.

Carbon-Oxygen Ligases↗
PubMed1994

Human osteoblast-like cells produce nitric oxide and express inducible nitric oxide synthase.

Nitric oxide (NO) is a short-lived free radical that plays an important regulatory role in several biological processes. Cytokines such as interleukin-1, tumor necrosis factor, and interferon-gamma have been shown to stimulate NO production in many cells types. Although these cytokines are known to have potent effects on bone remodeling and osteoblast function, the role of NO as an effector molecule in bone has been little studied. Here we investigate the effects of cytokines and calciotropic hormones on NO production by human osteoblast-like cells (hOB) and the role of NO as a modulator of osteoblast growth. Unstimulated hOB produced little NO, as reflected by measurement of nitrite concentrations in hOB-conditioned medium. NO production was not significantly altered by PTH and 1,25-dihydroxyvitamin D or human recombinant interleukin-1 beta (10 U/ml), tumor necrosis factor-alpha (25 ng/ml), and interferon-gamma (100 U/ml) individually. Combinations of all three cytokines at these concentrations, however, dramatically increased both NO generation and cGMP production. The stimulatory effect of cytokines on NO production began 12 h after exposure and was inhibited by cycloheximide, actinomycin-D, dexamethasone, and the competitive inhibitor of NO synthase L-NG-monomethylarginine. Reverse transcription/polymerase chain reaction analysis of hOB RNA, followed by direct sequencing of the amplified products, showed that hOB express the inducible, rather than the endothelial or neuronal, forms of NO synthase. Cytokine-induced increases in NO production were associated with a marked inhibition of [3H]thymidine uptake to less than 10% of that observed in control cultures. Abrogation of NO synthesis with L-NG-monomethylarginine under these conditions significantly increased [3H]thymidine uptake to approximately 20% of the control value, suggesting that NO may partly be responsible for the inhibition of osteoblast proliferation induced by these cytokines. Our data indicate that proinflammatory cytokines induce NO production in osteoblast-like cells and show and that this mediator plays a role in regulating cell growth. These findings may have important implications for the pathogenesis and management of bone loss in diseases associated with cytokine activation, such as rheumatoid arthritis.

Amino Acid Oxidoreductases↗
PubMed1993

Biochemical characterization of human osteoclast integrins. Osteoclasts express alpha v beta 3, alpha 2 beta 1, and alpha v beta 1 integrins.

The study of osteoclast integrins has been previously hampered by the lack of a source of large numbers of purified osteoclasts. Osteoclastoma, a human giant cell tumor of bone, supplied a rich source of osteoclasts within a tissue containing many diverse cell types. Osteoclastoma integrin immunostaining confirmed the presence of the integrin alpha v beta 3 complex and the alpha 2 and beta 1 integrin subunits on osteoclasts. However, weak integrin expression, for example with alpha v beta 5, was difficult to interpret. Purification with magnetic beads coated with vitronectin receptor monoclonal antibody (13C2) enabled osteoclast membranes to be isolated with high purity and yield (57%) from osteoclastoma tissue. Positively (osteoclast-enriched) selected membranes were biochemically assessed for integrin expression by immunoprecipitation and visualization by non-radioactive enhanced chemiluminescence. alpha 1, alpha 4, alpha 6, alpha 8, alpha M, alpha X, gpIIb, beta 4, beta 6, and beta 8 integrin chains were undetectable at a sensitivity of 1 ng. alpha 3, alpha 5, alpha L, beta 2, and alpha v beta 5 were found in the negatively selected osteoclastoma tissue but not in the positively purified osteoclast membranes. The presence of alpha v beta 1 and alpha 2 beta 1 dimers was demonstrated biochemically on the immunoisolated osteoclast membranes. Osteoclast alpha v beta 3 isolation by Arg-Gly-Asp (RGD) affinity chromatography for NH2-terminal amino acid sequencing confirmed that the osteoclast vitronectin receptor was identical to that previously characterized on other cell types. In situ hybridization using human alpha v riboprobes in osteoclasts from human and rodent bone further demonstrated the high level and specificity of expression of alpha v vitronectin receptor in osteoclasts.

Amino Acid Sequence↗
PubMed1992

Clinical pathology testing recommendations for nonclinical toxicity and safety studies. AACC-DACC/ASVCP Joint Task Force.

Clinical pathology testing in nonclinical toxicity and safety studies is an important part of safety assessment. In recent years, clinical laboratory testing has rapidly expanded and improved. Some government regulatory agencies provide guidelines for clinical pathology testing in nonclinical toxicity and safety studies. To improve these testing guidelines and the resultant safety assessments, the American Association for Clinical Chemistry's Division of Animal Clinical Chemistry and the American Society for Veterinary Clinical Pathology formed a joint committee to provide expert recommendations for clinical pathology testing of laboratory species involved in subchronic and chronic nonclinical toxicity and safety studies. These recommendations include technical recommendations on blood collection techniques and hematology, serum chemistry, and urinalysis tests.

Animals↗
PubMed1989

The osteoclast functional antigen, implicated in the regulation of bone resorption, is biochemically related to the vitronectin receptor.

We have defined the structure of the Osteoclast Functional Antigen (OFA) by immunological and biochemical means. OFA is an abundant surface antigen in human and animal osteoclasts and has been characterized previously by monoclonal antibodies 13C2 and 23C6, one of which mimicks the inhibitory activity of calcitonin on osteoclastic bone resorption. By the following criteria we show that OFA is a member of the integrin family of extracellular matrix receptors and is identical, or at least highly related, to the vitronectin receptor (VNR) previously isolated from placenta and melanoma cells. Immunoprecipitation analysis demonstrates that OFA from osteoclasts and a monkey kidney cell line Vero is a heterodimeric molecule of 140 kD (alpha chain) and 85 kD (beta chain) under nonreducing conditions; on reduction at least one low molecular mass (alpha') species (of approximately 30-kD size) is released, resulting in a 120/100-kD dimer. Immunoblots of OFA isolated from osteoclasts and Vero cells and VNR purified from placenta and probed with heterosera to OFA and monoclonal antibodies to platelet gp111a (VNR beta chain) show immunological cross-reactivity between the alpha chains of OFA and VNR and the use of gp111a as a beta chain by both. OFA from Vero cells binds to an Arg-Gly-Asp containing peptide (GRGDSPPK) isolating a heterodimer recognized by anti-OFA monoclonal antibodies, 13C2 and 23C6. Immunohistochemical analysis showed a similar tissue distribution in humans for the antigen recognized by anti-OFA antibodies, a monoclonal antibody, LM142, raised to melanoma VNR, polyclonal antibodies to the placental VNR and a monoclonal antibody to the presumptive VNR beta chain, platelet glycoprotein 111a. Finally, NH2 terminal amino acid sequencing showed that the amino-terminus of the monkey alpha chain was identical in the 12 assigned residues to that of human VNR alpha chain. The beta chain sequence of OFA differed at least 1 (and up to 4) positions from platelet gp111a (VNR beta) in the first 18 amino acids sequenced. These, and other, data provide the first indication of a function for the VNR and suggest that cell-cell and cell-extracellular matrix interactions involving integrins may play an important role in bone physiology.

Amino Acid Sequence↗
PubMed1980

An evaluation of a whole-blood platelet counter.

The performance of the platelet count in the hematology laboratory remains a laborious, time-consuming technic requiring separation of platelet-rich plasma, correction for leukocyte counts, lysis of erythrocytes, etc. The availability of an instrument that would count platelets in whole blood would appear to be a distinct advantage. One such instrument has been recently evaluated. The Ultra-Flo 100 Whole Blood Platelet Counter was found to be a reliable instrument for performing platelet counts on microspecimens of untreated whole blood. Counts were both accurate and precise, with no carry-over from specimen to specimen. The major advantage of the system is the speed with which platelet counts may be performed (25 sec) and results produced, a distinct advantage in those laboratories serving large outpatient hematology/oncology clinics. Six months of day-to-day experience in the laboratory has shown a favorable frequency-of-repair record, with little downtime.

Evaluation Studies as Topic↗