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Best Doctors' Founder, Dr Kenneth Falchuk, and Vice-Chairman, Evan Falchuk, contend misdiagnosis has become a global health crisis, despite medicine's “latest and greatest” technological advances. The authors explore five reasons why the wrong diagnoses and wrong treatments still occur so often today, and how their clinical teams' unique, patient-centric approach is helping to overcome misdiagnoses by ensuring people get the right diagnosis and the proper treatment – regardless of their condition, age, or where they live.

To obtain a comprehensive assessment of metabolite levels from extracts of leukocytes, we have recorded ultrahigh-resolution 1H-13C HSQC NMR spectra of cell extracts, which exhibit spectral signatures of numerous small molecules. However, conventional acquisition of such spectra is time-consuming and hampers measurements on multiple samples, which would be needed for statistical analysis of metabolite concentrations. Here we show that the measurement time can be dramatically reduced without loss of spectral quality when using nonlinear sampling (NLS) and a new high-fidelity forward maximum-entropy (FM) reconstruction algorithm. This FM reconstruction conserves all measured time-domain data points and guesses the missing data points by an iterative process. This consists of discrete Fourier transformation of the sparse time-domain data set, computation of the spectral entropy, determination of a multidimensional entropy gradient, and calculation of new values for the missing time-domain data points with a conjugate gradient approach. Since this procedure does not alter measured data points, it reproduces signal intensities with high fidelity and does not suffer from a dynamic range problem. As an example we measured a natural abundance 1H-13C HSQC spectrum of metabolites from granulocyte cell extracts. We show that a high-resolution 1H-13C HSQC spectrum with 4k complex increments recorded linearly within 3.7 days can be reconstructed from one-seventh of the increments with nearly identical spectral appearance, indistinguishable signal intensities, and comparable or even lower root-mean-square (rms) and peak noise patterns measured in signal-free areas. Thus, this approach allows recording of ultrahigh resolution 1H-13C HSQC spectra in a fraction of the time needed for recording linearly sampled spectra.

The essential role of zinc in embryogenesis was identified through studies of its presence in eggs and embryos, the effects of its deficiency and its role in metallo proteins required for organ development and formation. The Xenopus laevis oocyte zinc content varies during oogenesis. It increases from 3 to 70 ng zinc/oocyte as it progresses from stage I to VI. The oocyte zinc is derived from the maternal liver as part of a metallo-complex with vitellogenin. The latter transports the metal in plasma and into the oocyte. Once internalized, most of the zinc is stored within yolk platelets bound to lipovitellin, one of the processed products of vitellogenin. About 90% of the total zinc is associated with the yolk platelet lipovitellin while the remaining 10% is in a compartment associated with hitherto unknown molecule(s) or organelle(s) of the cytoplasm. The bi-compartmental distribution remains constant throughout embryogenesis since the embryo behaves as a closed system for zinc after fertilization. The yolk platelet zinc is used after the tadpole is hatched while we proposed that the 10% of the zinc in the non-yolk platelet pool is the one used for embryogenesis. It provides zinc to newly synthesized molecules responsible for the development of zinc-dependent organ genesis. Interference with the availability of this zinc by the chelating agent 1,10-phenanthroline results in the development of embryos that lack dorsal organs, including brain, eyes and spinal cord. The extensive teratology is proposed to be due to altered or absent zinc distribution between the cytosolic pool and zinc-transcription factors. The data identify the components of a zinc transport, storage and distribution system in a vertebrate organism.

The embryonic epidermis of stage 35 Xenopus laevis embryos is a highly differentiated structure composed of four cell types arranged in a regular architecture. Each type is distinguished by its distinct morphological characteristics. Some cells are ciliated (type 1); others have their surfaces covered by abundant, secreted vesicles of 0.1 μm diameter (type 2), or multiple linear aggregates of spherical subunits on their apical surfaces (type 3) or large secreted vesicles that emanate from prominent apical holes of 1 μm diameter (type 4). In contrast, the macroscopic appearance of embryos exposed to 10 μM 1,10-phenanthroline (OP) as well as the ultramicroscopic structure and organization of their epidermal cells are markedly altered. The most predominant cells of the embryonic epidermis are undifferentiated and of heterogeneous size. They lack any characteristic morphology and are arranged irregularly. Ghost cells are also identified. The recognizable differentiated cells are decreased in number and present in a scattered arrangement. These are identified as either type 1 or 2 cells but with ciliae that are shorter and thicker than control or with only a few vesicles larger than 0.1 μm in diameter on their surface. No cells with linear aggregates or prominent apical holes are identified. Except for the altered epidermis, the embryos do not develop any other major organs and exhibit axial abnormalities with an average dorso-anterior index of three. Thus, the chelating agent OP perturbs metal dependent processes essential for terminal differentiation that may likely account for the resultant abnormalities of embryo organogenesis and morphogenesis. Mol. Reprod. Dev. 55:75–82, 2000. © 2000 Wiley-Liss, Inc.

Zinc regulates the gene expression machinery. It affects the structure of chromatin, the template function of its DNA, the activity of numerous transcription factors and of RNA polymerases. Hence, it determines both the types of mRNA transcripts synthesized and the rate of transcription itself. Alterations in one or more of these zinc dependent processes have been proposed to account for the proliferative arrest and teratology induced by zinc deficiency. To examine this proposal, studies of zinc during X. laevis development have been initiated. The kinetics of X. laevis oocyte zinc uptake and storage and of zinc utilization during embryogenesis have been examined first. Vitellogenin carries zinc into the oocyte. Ten % of the total zinc (10 ng/egg) remains within the cytosol while 90% (90 ng/egg) is stored in the yolk platelets associated with lipovitellin. The cytosolic pool is the source of the zinc for all newly formed metalloproteins involved in embryo development. The yolk platelet zinc pool is stored for later use during early metamorphosis. It is now possible to examine zinc transfer to molecules, such as e.g. transcription factors, and the role of the metal in their function in development and organogenesis.

The x-ray absorption fine structure (XAFS) zinc K-edge steps for intact stages I,II and V,VI Xenopus laevis oocytes demonstrate that the zinc concentration is about 3 and 1 mM, respectively. However, the chi(k) function for the early stage oocytes differs markedly from that for the late one. Analysis of the XAFS data for stage I,II oocytes indicates that zinc is bound to 2.0 +/- 0.5 sulfur atoms at an average coordination distance of 2.29 +/- 0.02 angstroms and 2.0 +/- 0.5 nitrogen or oxygen (N/O) atoms at 2.02 +/- 0.02 angstroms. In marked contrast, in stage V,VI oocytes, zinc is bound to 4.1 +/- 0.4 N/O atoms at an average distance of 1.98 +/- 0.01 angstroms. Our previous studies demonstrated that 90% of the zinc in stage VI oocytes is sequestered within yolk platelets, associated with a single molecule, lipovitellin, the proteolytically processed product of vitellogenin. XAFS analysis of yolk platelets, lipovitellin, and vitellogenin demonstrates that zinc is bound to 4.0 +/- 0.5 N/O ligands at an average distance of 1.98 +/- 0.01 angstroms in each case, identical to that of stage V,VI oocytes. The higher shell contributions in the Fourier transforms indicate that two of the N/O zinc ligands are His in both stage V,VI and I,II oocytes. The results show that in stage I,II oocytes, there is a high concentration of a zinc protein whose zinc coordination site likely is composed of (His)2(Cys)2, such as, e.g., TFIIIA. As the oocytes develop, the predominant zinc species becomes one that exhibits the (His)2(N/0)2 zinc site found in lipovitellin. Hence, the ligands to the zinc atoms in intact oocytes and the changes that take place as a function of oogenesis and after their fertilization, during embryogenesis, now can be examined and explored.

Local and regional lymphadenopathy that is caused by wear particles released from a joint-replacement prosthesis is becoming increasingly recognized as a possible complication of arthroplasty. Particles generated by the mechanical wear of a prosthesis can leave the site of the implant through lymphatic vessels and become engulfed by macrophages within the local and regional lymph nodes. The accumulation of cells containing particles causes the enlargement of a lymph node and the characteristic histological appearance of sinus histiocytosis8. The distention and prominence of the lymphatic sinuses are due to the presence of large numbers of either histiocytes derived from the cells that line the sinuses or macrophages derived from circulating monocytes. Multinucleated giant cells, resulting from the fusion of macrophages or histiocytes, might also be found in the dilated sinuses. The accumulation of polyethylene, polymethylmethacrylate, and metal particles in the lymph nodes draining joints that have been replaced with a prosthesis has been found in studies of animals15,26 and humans2,3,10,14. Lymphadenopathy in patients who have had a joint replacement has been reported in a few of these studies8,17,22. The purposes of the present report are to document the development of lymphadenopathy after the dissemination of polyethylene particles in the lymphatic system in a patient who had a total hip replacement as well as in a patient who had a total knee replacement and to review the pathological response to polyethylene particles that leave the site of the implant. This report also provides a scientific basis for understanding the migration of particles from joints and a discussion of the clinical implications, such as the diagnostic confusion that can result from the finding of an enlarged lymph node. CASE 1. A forty-nine-year-old woman had …

Frog oocytes and embryos have long served as traditional subjects of embryological research providing structural and functional information for the interpretation of the biological processes underlying development. A large number of various chemical agents induce typical teratological changes in frog embryos. However, the effects of metal deficiency of the first transition and IIB series or of chelating agents specific for these metals have never been examined in the frog. Multidentate chelating agents, including 1,10-phenanthroline (OP), which coordinate metals through N, O or S donor atoms are teratogenic also but in a manner characteristic for this class of reagents and completely different from those referred to above. Exposure to 10−5 M OP causes maximal malformations with minimal mortality inducing craniofacial and skeletal abnormalities with failure of eye, head and other organ formation in 74% of frog embryos. In contrast, the non chelating analogue 1,7-phenanthroline (MP) has no effect at this concentration. A concentration of 10−3 M OP is lethal. The known characteristics of either zinc and/or iron complexes with OP as well as the concentrations of these elements in frog oocytes and embryos are consistent with the hypothesis that the teratological observations are due to an effect of OP on either zinc or iron proteins.

Vitellogenin induced by estrogen administration has been purified from the serum of Xenopus laevis. Six days after hormone injection, serum was collected and treated with 35% sat. (NH4)2SO4 to remove globulins. A single vitellogenin containing fraction was isolated by chromatography on a Mono-Q column. The protein was identified on the basis of its amino acid composition. N-terminal sequence analysis and SDS-PAGE demonstrated the presence of two forms of vitellogenin (A and B). Consistent with the conclusions drawn from the teratology which chelating agents induce in frog embryos (1), metal analysis shows that vitellogenin is a zinc protein with 1 g atom zinc/220 kDa monomer but containing no other IIB and transition metals.

Zinc is essential for vertebrate development; its deficiency results in multiple congenital malformations. Knowledge of the zinc biochemistry that underlies embryologic development is very limited. This has led us to investigate the zinc, iron, and copper contents of Xenopus laevis oocytes and embryos. Stage 1-6 oocytes, isolated from ovaries, and stage 1-40 embryos, obtained by in vitro fertilization techniques, were washed in metal-free water prior to digestion by 70% ultrapure HNO3. The metal content of the digests was analyzed by atomic absorption spectrometry. Stage 6 oocytes contain 65.8 +/- 4, 31.1 +/- 3, and 0.68 +/- 0.2 ng of zinc, iron and copper, respectively. The corresponding concentrations are 1, 0.5, and 0.01 mM in 1 microliter eggs. The metal content varies as a function of egg maturation. The zinc content increases from 3-7 to > 60 ng by stages 3 and 6, respectively. A similar pattern is noted for iron, which increases from 2-5 to 30 ng at analogous stages. In contrast, the copper content remains virtually unchanged in oocytes undergoing maturation. Importantly, the total of all three metals does not vary throughout the first 50 stages of development, when all tadpole organs are forming. Hence, the full complement of zinc, iron, and copper needed for incorporation into apoproteins during development is already present at a time when oocyte maturation is completed. The specific metalloproteins that store, donate, and accept these metals during induction and organogenesis and the alterations caused by metal deficiency can now be identified.

A 26-kDa endonuclease has been purified to homogeneity from zinc-sufficient Euglena gracilis. The protein binds to single-stranded DNA with a higher affinity than to double-stranded DNA, but it exhibits nucleolytic activity toward both. Thus, it converts supercoiled plasmid pBR322 DNA into the linear form, a property characteristic of endonucleases, and it continues to act on the linearized DNA until it is completely degraded. It also hydrolyzes heat-denatured, single-stranded calf thymus DNA. Moreover, at amounts below 1 microgram, it enhances RNA synthesis by RNA polymerase II, a characteristic observed with other DNases. Its addition to an in vitro transcription assay increases RNA synthesis up to 3-fold. The nuclease requires two metal components to carry out its enzymatic activities. It hydrolyzes DNA only in the presence of millimolar amounts of magnesium or micromolar quantities of other activating metal ions, such as manganese, zinc, or cobalt. However, even when optimal concentrations of Mg2+ are present, micromolar amounts of the metal-chelating agents OP and HQSA completely inhibit pBR322 digestion. Transcription enhancement is also inhibited completely by both chelators at concentrations that do not affect the intrinsic polymerase II activity. By atomic absorption spectrometry, the enzyme contains 1 g-atom of Zn/mol, which is the likely target of chelator action. The nuclease protein can also be isolated from zinc-deficient E. gracilis, but remarkably it then contains 1 mol of Cu/g-atom and no zinc.(ABSTRACT TRUNCATED AT 250 WORDS)

Biliverdin is required for Xenopus laevis embryo dorsal axis formation. When the tetrapyrrole is inactivated by phototransforming it with ultraviolet light prior to the first division, the embryo fails to synthesize dorsal mRNAs, such as goosecoid or chordin, yet forms increased amounts of ventral transcripts, such as Vent 1, and, consequently, develops ventralized morphology. Here we describe the metabolism of biliverdin during oogenesis and early embryogenesis. Estrogen induces frog hepatocytes to synthesize biliverdin and vitelogenin. The two molecules form a complex that is secreted into and transported in the plasma to be taken up by the oocyte as it matures through its six stages of oogenesis. In the oocyte, the biliverdin-vitellogenin complex is processed and stored in the yolk platelets. In these organelles, biliverdin is associated entirely with the lipovitellin domain of the processed vitellogenin. Once the egg is fertilized, its biliverdin content decreases over a 5-6 h period to participate in the chemical machinery required for dorsal axis formation. This participation must be initiated during the period encompassing the first embryonic mitosis. The results describe the pathway that generates, transports, and stores biliverdin as part of oogenesis, define the time course for its utilization after fertilization, and link biliverdin to the metabolism of the phosphoglycolipometalloprotein, vitellogenin.

The determinants of Xenopus laevis embryos that act before their first cell division are mandatory for the formation of mRNas required to establish the dorsal axis. Although their chemical identities are unknown, a number of their properties have long been recognized. One of the determinants is present in the cytoplasm and is sensitive to UV light. Thus, exposing stage 1 embryos to either standard 254-nm or, as shown here, to 366-nm UV light during the 0.3–0.4 time fraction of their first cycle inactivates the cytoplasmic determinant. As a consequence, both types of irradiated embryos fail to express dorsal markers, e.g., goosecoid and chordin, without affecting formation of ventral markers, e.g., Vent-1. The developmental outcome is dorsal axis-deficient morphology. We report here that biliverdin IXα, a normal constituent of cytoplasmic yolk platelets, is photo-transformed by irradiation with either 254- or 366-nm UV light and that the transformation triggers the dorsal axis deficiency. When the 254- or 366-nm UV-irradiated embryos, fated to dorsal axis deficiency, are incubated solely with μM amounts of biliverdin, they recover and form the axis. In contrast, incubation with either in vitro photo-transformed biliverdin or biliverdin IXα dimethyl ester does not induce recovery. The results define an approach to produce dorsal axis-deficient embryos by photo-transforming its biliverdin by irradiation with 366-nm UV light and identify an unsuspected role for biliverdin IXα in X. laevis embryogenesis.

Chicken alpha- and beta-lipovitellin are derived from parent vitellogenin proteins and contain four subunits (125, 80, 40, and 30 kDa) and two subunits (125 and 30 kDa), respectively. Metal analyses demonstrate both are zinc proteins containing 2.1 +/- 0.2 mol of zinc/275 kDa per alpha-lipovitellin and 1.4 +/- 0.2 mol of zinc/155 kDa per beta-lipovitellin, respectively. The subunits of beta-lipovitellin, Lv 1 (MW 125 kDa) and Lv 2 (MW 30 kDa), are separated by gel exclusion chromatography in the presence of zwittergent 3-16. Zinc elutes with Lv 1, suggesting that this subunit binds zinc in the absence of Lv 2. The subunits of alpha- and beta-lipovitellin were separated by SDS-PAGE, digested with trypsin, and mapped by reverse-phase HPLC. The peptide maps of the 125-kDa subunits from alpha- and beta-lipovitellin are essentially identical. Similar results are obtained for the 30-kDa subunits of both lipovitellins. The sequences of five and four peptides of the 125-kDa subunit of alpha- and beta-Lv, respectively, and two peptides of the 30-kDa subunit of alpha- and beta-lipovitellin were determined and match those predicted from the gene for vitellogenin II, Vtg II. Comparison of the amino acid composition of the 125- and 30-kDa subunits of alpha- and beta-lipovitellin support the conclusion that they originate from the same gene. The sequences of peptides from the 80- and 40-kDa subunits of alpha-lipovitellin have not been found in the NCBI nonredundant data bank. The 27-amino acid N-terminal sequence of the 40-kDa protein is 56% similar to the last third of the Lv 1-coding region of the Vtg II gene, suggesting it may come from an analogous region of the Vtg I gene. We propose a scheme for the precursor-product relationship of Vtg I.

Xenopus laevis vitellogenin contains 2 g-atoms (g-at) of Zn and 3 g-at of Ca/dimer, transports zinc in plasma, and plays a role in its distribution within the oocyte [Montorzi et al. (1994) Biochem. Biophys. Res. Commun. 200, 1407-1413; Montorzi et al. (1995) Biochemistry 34, 10851-10858]. We here report the dynamics and time course of Zn65-labeled vitellogenin uptake by and distribution within stages II and IV oocytes, the fate of the metal in oocytes as they progress from stages II to VI, as well as in the first two cleavage blastomeres, the blastula, and subsequent stages of the developing embryo and tadpole. Zn65 bound to vitellogenin is taken up within less than 30 min by either stage II or IV oocytes incubated under in vitro culture conditions whereas free Zn65 is not. Once internalized, Zn65 remains within the cytosol of stage II, whereas in stage IV oocytes, it is transferred within 4 h of its entry from the cytosol into yolk platelets. Nearly all of the transferred Zn65 is found within yolk platelets and their precursors where it is associated with the vitellogenin cleavage product, lipovitellin. Its distribution within the oocyte organelles differs at each stage of oogenesis. In the early stages (III-IV) most of the oocyte zinc is located first in the small endocytosed vesicles and then in multivesicular bodies. When the zinc transfer process is finalized in the late stages of oogenesis (V-VI), > 90% of the total oocyte zinc is within yolk platelets while the remainder is in the cytosol. In embryos and tadpoles, the larger of these two pools remain sequestered in yolk platelets and is inaccessible to cytosolic apoproteins throughout the entire period of embryo formation. Its redistribution to the cytosol does not begin until several days after the tadpole has hatched. The smaller pool, on the other hand, is already present in the cytosol and is, therefore, postulated to constitute the sole source of zinc required for embryogenesis.

Xenopus laevis vitellogenin is a plasma protein that contains a total of 5 mol of metal/440 kDa dimer, 2 mol of zinc, and 3 mol of calcium (Montorzi et al. (1994) Biochem. Biophys. Res. Commun. 200, 1407-1413]. There are no other group IIB or transition metals in the molecule. The zinc atoms are removed instantaneously by 1,10-phenanthroline (OP) (pK 4.8). Once internalized by receptor-mediated endocytosis, vitellogenin is cleaved into multiple polypeptides, i.e., the two lipovitellin subunits (1 and 2) plus phosvitin; these are then stored as microcrystals within yolk platelets. We here show by metal analysis of the individual proteins generated by vitellogenin processing that zinc and calcium occur in different domains of the vitellogenin polypeptide chain. All of the vitellogenin zinc is present in lipovitellin, in amounts equal to 1 mol of zinc/141 kDa. Calcium, in contrast, is detected exclusively in phosvitin which, in addition, contains 3 mol of magnesium/35 kDa, apparently acquired following vitellogenin entry into the oocyte. The zinc in lipovitellin is removed by OP in a concentration-dependent manner with a pK of 4.8, identical to that obtained for vitellogenin, and by exposure to acidic conditions (below pH 5). Following removal of zinc, the two lipovitellin subunits remain associated, suggesting that zinc is not involved in their interaction. On exposure to 1% SDS, lipovitellin does dissociate into 106 and 33 kDa subunits. The presence of stoichiometric quantities of zinc in both vitellogenin and lipovitellin calls for the study of the hitherto unrecognized biochemistry and functions of these proteins in zinc metabolism and development of the frog oocyte and embryo.

Majors topics addressed in this review on zinc physiology are ; 1) chemistry and biochemistry; 2) interface of biochemistry and physiology of zinc; 3) physiology and cell and molecular biology; 4) pathology

We carried out a double-blind prospective study of the effect of a filter on the incidence of phlebitis associated with intravenous infusion in 541 patients. A total of 277 patients received infusions through intravenous sets with 0.22-micron IVEX-HP filters, and 264 received infusions without filters. Each infusion was evaluated daily for a maximum of three days. The incidence of phlebitis on Days 1, 2, and 3 of the study was 14.3, 31.1, and 27 per cent for patients receiving infusions without filters and 6.8, 9.7, and 11.3 per cent for those receiving infusions through the filters (P less than 0.001). Thus, the incidence was reduced by approximately two thirds in the patients who received infusions through the IVEX-HP filters. We conclude that infusion-related phlebitis is a pervasive problem in hospitalized patients, and that it is usually caused by microparticulate components that are present in the infusion fluids and can be removed by in-line filtration.

Summary The RNA from zinc sufficient (+Zn) and deficient (−Zn) E. gracilis have been isolated and the three major RNA classes separated by affinity chromatography on oligo-(dT) and DBAE celluloses. The total RNA content and the ribosomal and transfer RNA fractions are the same in (+Zn) and (−Zn) cells. In (−Zn) cells, the messenger RNA fraction doubles and its base composition is altered, resulting in a two-fold increase in the G+C/A+U ratio. We have examined the role of Mn in determining these changes in RNA, since the intracellular content of this metal increases in (−Zn) cells. Increasing the Mn (II) content from 2 to 10 mM in assays with RNA polymerase from (−Zn) cells increases the incorporation of GMP relative to UMP from 1.0 to 3.5. Further, the ratios of CMP/GMP incorporation are 1.0, 2.2 and 1.2 in assays with 2, 5, 10 mM Mn (II), respectively. Thus, Mn (II) concentration can significantly alter function of RNA polymerase from (−Zn) E. gracilis cells.

Testosterone and its physiologically active metabolite 5 alpha-dihydrotestosterone are selective, allosteric inhibitors of the gamma subunit-containing isozymes of class I human alcohol dehydrogenase (ADH) with apparent Ki values for testosterone at pH 7.4 between 3.5 and 16 X 10(-6) M. Testosterone inhibition is noncompetitive with respect to ethanol, NAD+, 1,10-phenanthroline, and 4-methylpyrazole, identifying a regulatory site distinct from the catalytic site. Testosterone does not inhibit the class I isozymes composed only of alpha and/or beta subunits and only weakly inhibits the class II and III isozymes. Importantly, none of these human ADH isozymes oxidize or reduce the steroids with the delta 4 double bond or 5 alpha configuration. The allosteric effect of testosterone, restricted to the gamma subunits of human ADH, suggests unique metabolic specificities and pathways for these isozymes, apart from all others. This inhibition may ultimately be critical to an identification of their function(s). Analogous considerations of other metabolic effectors might further lead to similar insights regarding the alpha and beta subunit-containing isozymes as well as the class II and III ADH.

Histones and most other basic chromosomal proteins are not extracted from zinc-deficient (-Zn) Euglena gracilis chromatin either by 0.25 M HCl or by 0.3-0.6 M NaCl/7 M urea. Instead, a class of 3-5-kilodalton (kDa) polypeptides, which is absent in zinc-sufficient (+Zn) cells, is solubilized. These heterogeneous polypeptides are comprised of Asn, Arg, Cys, and Gln. The partial sequence of one of these, which is composed only of Arg and Asn, is Arg-Asn-Asn-Arg-Arg-Asn-Asn-Asn-Asn-Asn-. This demonstrates they are not proteolytic fragments of the histones, proteins which do not contain contiguous Arg-Asn or Asn-Asn sequences. Once -Zn chromatin is depleted of this 3-5-kDa material, nearly all of the histones and most non-histone proteins are extracted. On the other hand, if chromatin first is depleted of, and subsequently is reconstituted with, the 3-5-kDa material, the chromosomal proteins are not solubilized, as observed with intact chromatin. Histone H4 is an exception. Electrophoretic analysis of the solubilized H4 reveals that the degree to which it is acetylated in -Zn is lower than in +Zn chromatin. Jointly, these data indicate that chromosomal proteins bind much more tightly to DNA of -Zn than +Zn cells. The histone/DNA weight ratio in -Zn chromatin is 0.44 compared to 1.04 inmore » +Zn chromatin. However, the 3-5-kDa polypeptide fraction maintains the amount of total basic proteins per unit mass of DNA at approximately 1. Further, four non-histone proteins extractable with 5% HClO/sub 4/ or 0.35 M NaCl and characterized by high electrophoretic mobility have been purified from +Zn nuclei. Only one of these proteins is found in -Zn chromatin. Thus, zinc deficiency induces changes in the amounts and types of histones and non-histone proteins, as well as in their interaction with DNA. These findings are discussed in relation to recent advances in understanding of the role of zinc in replication and transcription.« less

Iron-, manganese-, or magnesium-deficiency has been induced in Euglena gracilis. Each arrests cell proliferation, decreases the intracellular content of the deficient metal, and increases that of several other metals. Light and electron microscopy of stationary phase cells reveal that Fe-deficient (-Fe) cells are similar in size and shape to control organisms. Magnesium-deficient (-Mg) cells, however, are larger, and approximately 14% are multilobed, containing 2 to 12 lobes of equal size emanating from a central region. Individual (-Mg) cells and each lobe of multilobed cells contain a single nucleus. Manganese-deficient (-Mn) organisms are morphologically more heterogeneous than (-Fe) or (-Mg) cells. Most are spherical and larger than controls. Approximately 15% are multilobed but, unlike (-Mg) cells, contain lobes of unequal size with either zero, one, or several nuclei present in each. Nuclei of (-Mn) cells differ in size and shape from those of control, (-Fe), or (-Mg) cells. All three deficient cell types accumulate large quantities of paramylon. Other cytoplasmic structures, however, appear normal. Addition of Fe, Mn, or Mg to the respective deficient stationary phase cultures reverses growth arrest and restores normal morphology. The results suggest that Fe-, Mn-, and Mg-deficiencies affect different stages of the E. gracilis cell cycle.

The three major RNA classes from zinc-sufficient [(+Zn)] and zinc-deficient [(=Zn)] Euglena gracilis have been separated by affinity chromatography on oligo(dT)- and N-[N'-[m-(dihydroxyboryl)phenyl]succinamoyl]aminoethyl (DBAE)-celluloses. The total RNA content and the ribosomal and transfer RNA fractions are the same in (+Zn) and (=Zn) cells. IN (-Zn) cells, the messenger RNA fraction increases, and its altered base composition reveals additional bases and a 2-fold increase in the (G+C)/(A+U) ratio. Since the intracellular content of manganese increases in (-Zn) cells, we have examined its role in determining these changes in RNA composition. An increase in the Mn2+ content from 1 to 10 mM in assays with RNA polymerases I and II from (+Zn) cells and those with the single RNA polymerase from (-Zn) cells decreases the ratio of UMP to CMP incorporated from 1.7 to 1.0, 2.1 to 0.8 and 3.5 to 0.4, respectively. Thus, Mn2+ concentration can significantly alter the products of the enzymatic action of RNA polymerases from both (+Zn) and (-Zn) E. gracilis cells.

E. gracilis DNA dependent RNA polymerase I has been purified to homogeneity. α-amanitin, over the concentration range 0.05 to 200 μg/ml, does not affect its activity, consistent with its being classified as an RNA polymerase I. Based on a molecular weight of 624,000 daltons the enzyme contains 2.2 g atom of Zn but no Mn, Cu, Fe, as determined by microwave excitation emission spectrometry. Zinc is essential for activity since the chelating agent, 1,10-phenanthroline, inhibits enzymatic function but its non-chelating analogue, 4,7-phenanthroline is ineffective. Thus, like the RNA polymerase II, zinc is a catalytically essential component of E. gracilis RNA polymerase I (1).

A multichannel atomic absorption spectrometer has been designed and constructed which combines the advantages of simultaneous analysis offered by emission spectroscopy with the increased sensitivity and rapid analysis of atomic absorption instruments. The source is a multielement hollow cathode tube and employs a long pathlength absorption cell previously shown to increase the sensitivity of detection of several elements by atomic absorption. A concave grating isolates monochromatic radiation from the emission of the hollow cathode tube and focuses it on the photomultipliers placed at exit slits placed at the focal plane on a Rowland circle. The method presented demonstrates the utility of the instrument and permits simultaneous analysis of zinc, copper, and cadmium. Three-quarter saturated oxine serves as a chelating agent to obviate interferences due to extraneous anions and cations. The method is suitable for the analysis of these metals in biologic fluids, as exemplified by the analysis of human serum and equine renal and human hepatic metallothioneins.

The relationship between peritubular capillary protein concentration and rate of sodium reabsorption by the rat proximal tubule was examined using free-flow recollection micropuncture techniques. Tubule fluid-to-plasma inulin ratios were measured before, during, and at successive intervals after brief (15-25 sec) intra-aortic injections (at the level of the renal artery) of colloid-free, isoncotic, and hyperoncotic solutions. Arterial hematocrit and protein concentrations were measured simultaneously in these rats. In other rats, total protein concentration of peritubular capillary blood plasma was determined before, during, and after these same infusions with a newly described submicroliter fiber-optic colorimeter. In the 15-25 sec interval necessary to infuse 2 ml of these test solutions, fractional and absolute sodium reabsorption varied directly with peritubular capillary colloid osmotic pressure, declining during infusion of colloid-free solutions, increasing during hyperoncotic infusions, and remaining unchanged during isoncotic infusions. In the subsequent 20-min interval after intra-aortic injection of these test solutions, capillary protein concentration remained at (isoncotic infusions) or returned to (colloid-free and hyperoncotic fluids) control values. Whereas reabsorption after colloid-free solutions returned to base line levels in parallel with the return in capillary protein concentration, after colloid infusions (which resulted in continued expansion of extracellular fluid volume), a progressive decline in reabsorption was observed. These results afford strong evidence that peritubular capillary colloid osmotic pressure is one important determinant of proximal sodium reabsorption. Nevertheless it is apparent that mechanisms other than or in addition to this must be invoked to explain the delayed inhibition of reabsorption that accompanies expansion of extracellular fluid volume by colloid solutions.

Mercury is distributed widely in the earth’s crust, in sea, ground and rain water. Importantly, all phyla and species naturally contain traces, present either as inorganic or organometallic compounds, or both.1,2 A biological role for the element is thus far undefined; however, it is present in rat liver chromatin3 and methyl mercury induces hepatic protein synthesis in this species.4 It has been known that mercury enters into biological life cycles; however, the awareness that inorganic mercury can be converted into organometallic compounds by bacteria and higher organisms has recently stimulated further interest both in the chemistry and toxicological potential of this element.5–9 This biological conversion of inorganic mercury into organic mercury is particularly significant since extensive industrial and agricultural usage of mercurials affects and increases its distribution in specific regions. Moreover, the burning of fossil fuels generates environmental mercury in amounts comparable to those from industrial processes.10

The effect of acute disease and ACTH infusion on serum zinc proteins was studied in serums from 156 healthy and diseased subjects. The mean (+/-2 S.D.) zinc content of 20 normal serums was 96 +/- 20 microng per 100 ml. In 87 serums from acutely ill patients the zinc ranged from 92 to 40 microng per 100 ml. The mean values for nearly all categories of disease studied were lower than normal (P is less than 0.001). Chromatography of normal serum on Sephadex G-100 separates two protein fractions, I and II, containing 37.8+/-8.8 and 76+/-10 microng of zinc per 100 ml, respectively. In serum from diseased patients the zinc in fraction I is unaltered whereas that in fraction II decreases to 29.8+/-7.5 microng per 100 ml (P is less than 0.001). ACTH administration reduces secrum zinc from 10 to 60 microng per 100 ml, the decrements being due to changes in the zinc content of fraction II. Thus, ACTH may have an important role in the reduction of zinc content associated with pathologic states.

Histones, and other basic proteins, have been isolated from zinc-sufficient (+Zn) Euglena gracilis by standard chromatographic methods. These cells contain 2.46 micrograms of histones and 1.96 micrograms of DNA per 10(6) organisms. Each of the histones, H1, H3, H2A, H2B, and H4, is present in both log- and stationary-phase +Zn cells and has been characterized according to its electrophoretic mobility and molecular weight. H1 has been further identified on the basis of its amino acid composition and its cross-reactivity with calf thymus histone H1 antibodies. Similarly, H3 has been recognized as well by its specific reaction with an H3 antibody. In contrast, log-phase zinc-deficient (-Zn) cells contain H1 and H3 while H2A, H2B, and H4 are absent. All of the histones vanish in stationary-phase-Zn organisms. The DNA content increases as the -Zn cells progress from log to stationary phase, reaching a value of 4.40 micrograms/10(6) cells, double that of comparable stationary-phase +Zn organisms. A 2000-3000-dalton polypeptide whose electrophoretic behavior differs from that of the known histones constitutes over 90% of the total basic proteins of -Zn cells. On addition of zinc to stationary -Zn cells, cell division resumes, and all the histones and other basic proteins reappear. Together with previous results, the data demonstrate that zinc significantly affects the metabolism of all major chromatin components, i.e., the RNA polymerases, DNA, and histones of E. gracilis [Vallee, B.L., & Falchuk, K.H. (1981) Philos. Trans. R. Soc. London, Ser. B 294, 185-197]. The implications of these effects of zinc on chromatin structure and function are discussed.

The purine and pyrimidine bases of E. gracilis have been separated using a high-pressure liquid chromatograph (HPLC). Each base was unambiguously identified by its characteristic elution profile and UV absorption spectrum. This method allows for the study of the base composition of mRNAs altered by pathological processes, exemplified here by the analysis and comparison of mRNAs from zinc sufficient and deficient organisms.

Zinc is essential for cellular proliferation. Zinc deficiency of Euglena gracilis results in arrest of cell division and deranges nucleic acid and protein metabolism pointing to a decisive role of zinc in transcription and translation. We have, therefore, investigated the role of zinc in the function of the DNA-dependent RNA polymerases of this organism. Two RNA polymerases from zinc sufficient organisms were purified first by affinity chromatography on a DNA cellulose column and subsequently separated on diethylaminoethyl (DEAE)-Sephadex A-25. The two fractions were characterized as polymerase I and II by their elution pattern from DEAE-Sephadex and sensitivity to alpha-amanitin. RNA polymerase II has a provisional molecular weight of 700 000 and contains an average of 2.2 g=atoms of zinc per mol of enzyme, but not Mn, Cu, or Fe, as measured by microwave emission spectroscopy. Chelating agents, such as 1,10-phenanthroline, 8-hydroxyquinoline, 8-hydroxyquinoline-5-sulfonic acid, and lomofungin, inhibit activity. In contrast, the nonchelating analogues, 1,7-and 4,7-phenanthroline, do not affect activity. Inhibition by 1,10-phenanthroline is instantaneous and fully reversible by dilution. 1,10-Phenanthroline also inhibits RNA polymerase I, suggesting a role of zinc in its function. The demonstration that RNA polymerase II is a zinc enzyme indicates the involvement of zinc in eukaryotic RNA synthesis and serves as a further basis for the definition of the role of this element in eukaryotic cell growth, division, and differentiation.

Copper complexes of 1,10-phenanthroline (OP-Cu) hydrolyze DNA [D'Aurora, V., Stern, A. M., & Sigman, D. S. (1978) Biochem. Biophys. Res. Commun. 80, 1025-1032; Marshall Pope, L., Reich, K. A., Graham, D. R., & Sigman, D. S. (1982) J. Biol. Chem. 257, 12121-12128]. This reaction has been studied to determine whether the 1,10-phenanthroline (OP) inhibition of the activity of RNA and DNA polymerases is the result of template hydrolysis or the chelation of a metal associated with and essential to the function of these enzymes. Addition of 4',6-diamino-2-phenylindole dihydrochloride (DAPI) to DNA generates a fluorescence signal with a linear increase of the intensity over a broad range of DNA concentrations from 0 to 100 micrograms/mL. The progress of hydrolysis of DNA by DNase I or OP (2 mM) is monitored by the time-dependent decrease in DAPI-induced fluorescence. In the presence of OP, the rate of hydrolysis increases as the Cu2+ concentration in the reaction mixture rises from 10(-8) to 10(-5) M. The rate differs for each nucleic acid template used; hydrolysis of poly(dA-dT) greater than denatured DNA greater than double-stranded DNA. However, millimolar amounts of OP do not hydrolyze the template even in the presence of Cu2+ (10(-6) M) when DNA is complexed with either Escherichia coli DNA polymerase I or Euglena gracilis or wheat germ RNA polymerase II. Under the same conditions, OP inhibits the activity of both varieties of RNA polymerase II with pKi's of 3.4 and 3.0, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Publisher Summary At present, the most widely employed methods are based on atomic spectrometry, the interaction of analyte atoms with electromagnetic radiation. These procedures fall into three categories: atomic emission, atomic absorption, and atomic fluorescent spectrometry. The features of the most readily available of these techniques— that is, flame and electrothermic atomic absorption spectrometries, their advantages and their application to the analysis of biological samples will be described in the following sections. In addition, methods for preparing zinc-free buffers and reagents, standards, and specific biological fluids and tissues for analysis, are discussed. The analysis of zinc in most biological samples can be carded out effectively by using either flame or electro thermal atomic absorption spectrometry. The availability of the instruments, their cost, ease of use, and range of sensitivities for zinc determination make them the methods of choice for routine use in biological work on this element. Special situations or problems may require the use of other techniques. The complexities of these other techniques, however, limit their usefulness to specialists skilled in the respective fields.

The histone content of zinc-deficient (-Zn) Euglena gracilis decreases while, concomitantly, DNA content increases and the transcription rate is reduced markedly [Mazus, B., Falchuk, K. H., & Vallee, B. L. (1983) Biochemistry (in press); Falchuk, K. H., Fawcett, D. W., & Vallee, B. L. (1975) J. Cell Sci. 17, 57-78]. The effects on major constituents of the genome have been examined by studying the rate and extent of hydrolysis of +Zn and -Zn chromatin by micrococcal nuclease, DNase I, or DNase II. The size of hydrolyzed DNA fragments suggests similarity of the +Zn E. gracilis chromatin organization to that of other eukaryotes. The major protein constituent of -Zn chromatin is a polypeptide of less than 3000 daltons whose electrophoretic mobility differs from that of any known histone components of chromatin, the latter described elsewhere (K. H. Falchuk et al., unpublished results). This protein profoundly affects the structure of -Zn chromatin, which is about 10-30-fold more resistant to micrococcal nuclease hydrolysis than +Zn chromatin. Moreover, the resultant DNA fragments [2000 base pairs (bp)], are much larger than those of +Zn cells. Under conditions which hydrolyze +Zn chromatin into DNA fragments smaller than 50 bp, only 50% of -Zn chromatin is digested into fragments less than 2000 bp, i.e., in the range of those expected for oligonucleosomes. Removal of the low molecular weight protein from -Zn chromatin reverses its enhanced resistance to nucleolysis and results in extensive hydrolysis. Conversely, addition of the low molecular weight protein to +Zn chromatin increases the resistance of this complex to digestion.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects induced by Fe, Mn, or Mg deficiency or cold shock on the DNA content and histones of Euglena gracilis have been examined and compared to those produced by Zn deficiency. The DNA content of the stationary-phase organisms used as controls is 2.1 micrograms/10(6) cells. The DNA of stationary-phase iron-deficient (-Fe), magnesium-deficient (-Mg), manganese-deficient (-Mn), zinc-deficient (-Zn), and cold-shocked (CS) cells is increased to 3.0, 4.6, 6.2, 3.8, and 3.8 micrograms/10(6) cells, respectively. The electrophoretic mobilities of proteins solubilized with 0.4 N H2SO4 from CS, -Fe, -Mg, and -Mn cells are nearly identical and are characteristic of the five histone classes, H1, H2A, H2B, H3, and H4. In contrast, no histones are found in the equivalent acid extract from -Zn cells. The effect of micrococcal nuclease on chromatin from control, CS, and -Zn cells was examined. The chromatin of CS cells is 1.2-fold while that from -Zn cells is 10-30-fold more resistant to micrococcal nuclease digestion than is the chromatin of control cells. Thus, the chromatin of cells grown in Zn-deficient conditions differs markedly from that of organisms cultured in media deficient in Fe, Mn, or Mg or exposed to cold shock.

We present a rare case of aneurysm of the pancreaticoduodenal artery that bled into the region of the head of the pancreas. Computed tomography (CT) demonstrated the presence and extent of the resultant mass (hematoma) but the angiographic examination allowed a specific diagnosis via visualization of the aneurysm. Unlike previously reported cases, the patient was treated conservatively. Follow-up arteriogram and CT showed spontaneous closure of the pancreaticoduodenal artery and aneurysm and complete resolution of the mass in the pancreatic head. These findings suggest that in some cases a conservative approach to a bleeding pancreaticoduodenal artery aneurysm may be indicated.

Both the single DNA-dependent RNA polymerase found in zinc-deficient (-Zn) Euglena gracilis and the RNA polymerase III from zinc-sufficient (+Zn) cells have been isolated by methods previously used to purify polymerases I and II [Falchuk, K. H., Mazus, B., Ulpino, L., & Vallee, B. L. (1976) Biochemistry 15, 4468; Falchuk, K. H., Mazus, B., Ulpino, L., & Vallee, B. L. (1977) Biochem. Biophys. Res. Commun. 74, 1206]. Like class II polymerases, the enzyme from -Zn organisms elutes from DNA-cellulose and phosphocellulose with 0.6 M NaCl and 0.35 M NH4Cl, respectively. It is inhibited by 8-hydroxyquinoline, 8-hydroxyquinoline-5-sulfonic acid, alpha,alpha'-bipyridyl, dipicolinic acid, and 1,10-phenanthroline (OP); 4,7-phenanthroline, the nonchelating analogue, does not inhibit. The pKI(OP) of this enzyme is identical with that of polymerase II but distinct from those of polymerases I and III. Elemental analysis confirms that zinc is the functional metal while copper, manganese, iron, and magnesium are absent. However, the -Zn enzyme is at least 4 orders of magnitude more resistant to alpha-amanitin (alpha-A) than the class II polymerase. Further, its response to alpha-A is unlike that of either polymerase I or polymerase III. Thus, -Zn cells contain a single, alpha-amanitin-resistant (alpha-Ar) RNA polymerase, whose behavior otherwise resembles that of the alpha-amanitin-sensitive polymerase II.