Measuring beyond the microscope field of view using digital images in squamous cell carcinoma of the lip

OBJECTIVE: To study the impact of using digital images for measuring the size of the tumors, assisting with the prognostic evaluation of carcinomas of the oral mucosa. STUDY DESIGN: The depth of invasion of 12 cases of squamous cell carcinoma of the lower lip was assessed through a microscope and through digital images. All measurements of depth of invasion were assessed in a direction orthogonal to the lip surface. First, assessment of depth of invasion was done at the microscope, using an eyepiece reticule with an engraved scale. Second, depth of invasion was assessed by digital images, using a program module developed to assist pathologists with linear measuring. When the depth of the tumor was larger than the field of view at the proper magnification, several images were taken to include the whole area of invasion. The images were finally mounted in a single image and the depth of invasion measured. RESULTS: The results show positive and negative differences between assessments when assessing depths of < 2 mm. At greater depths (> or = 2 mm), the difference was always negative, showing that for deep invasion, measurements of longer distances were always performed on digital images. CONCLUSION: Measurements with digital images beyond the field of view at proper magnification could sig nificantly alter the diagnostic and prognostic assessment made using the microscope.     

PDB2MultiGIF: A Web Tool to Create Animated Images of Molecules

A new web tool, PDB2MultiGIF (http://www.dkfz-heidelberg.de/spec/pdb2mgif/),which converts the topological information (atom types, 3D coordinates, molecular connectivity) of molecules (given in PDB format [1]) to a series of animated images (in GIF Format) [2] is described. The molecular visualisation program RASMOL [3] is used to generate the images.Electronic Supplementary Material available.     

The use of nucleotide phosphorothioate diastereomers to define the structure of metal-nucleotide bound to GTP-AMP and ATP-AMP phosphotransferases from beef-heart mitochondria

The diastereomers of adenosine 5'-O-[1-thio]triphosphate (ATP[alpha S]) and adenosine 5'-O-[2-thio]triphosphate (ATP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to ATP when bound to ATP-AMP phosphotransferase from beef heart mitochondria (AK2). Similarly, the diastereomers of guanosine 5'-O-[thio]triphosphate (GTP[alpha S]) and guanosine 5'-O-[2-thio]triphosphate (GTP[beta S]) were utilized to seek unambiguous assignment of Mg2+ coordination to GTP when bound to GTP-AMP phosphotransferase from beef heart mitochondria (AK3). Furthermore the diastereomers of guanosine 5'-O-[1-thio]diphosphate (GDP-[alpha S]) have been used to assign Mg2+ coordination to GDP when bound to AK3. The ratios (V for isomer Sp)/(V for isomer Rp) obtained in the presence of Mg2+ and Cd2+ are compared to those already published for ATP-AMP phosphotransferases from pig muscle (AK1) [Kalbitzer et al. (1983) Eur. J. Biochem. 133, 221-227] and from baker's yeast (AKy) [Tomasselli and Noda (1983) Eur. J. Biochem. 132, 109-115]. In all cases, coordination of Mg2+ to the beta-phosphate via the pro-R oxygen is present, as shown by reversal of specificity for the diastereomers of ATP [beta S] or GTP [beta S] respectively on changing the metal ion. In contrast, there is no reversal of specificity for the diastereomers of ATP [alpha S] or GTP[alpha S], or for GDP[alpha S] in the case of AK3 for the reverse reaction, indicating that there is no interaction of the metal with the alpha-phosphate group. The observed stereospecificity for the alpha-thiophosphate is consistent with the assumption of an interaction of the pro-R oxygen of the alpha-phosphate group with the enzyme.     

Spectroscopic analysis of [Trp3]-beta-casomorphin analogs. Comparative structure conformation-activity studies

A series of [3-tryptophan]-beta-casomorphin-5([Trp3]-beta-CM-5) analogs were investigated by circular dichroism (CD) and fluorescence spectroscopy to explore their structure-conformation properties in solution. In addition, the comparative opioid activities of these compounds were evaluated using the in vitro guinea pig ileum (GPI) and mouse vas deferens (MVD) assays. Specifically, the pentapeptide sequence of [Trp3]-beta-CM-5, H-Tyr-Pro-Trp-Pro-Gly-OH (I) was modified at Pro-2 and Pro-4 by D-Pro substitutions to provide two diastereometric analogs, [Trp3-D-Pro-4]-beta-CM-5 (II) and [D-Pro2,4,Trp3]-beta-CM-5 (III). In the GPI and MVD assays, beta-CM-5 effected IC50 values of 1.3 microM and 8.9 microM, respectively, which confirmed its known mu/delta-selectivity on these two peripheral opioid receptor subtypes. The potencies of compounds I, II, and III were 0.2, 2.0, and less than 0.005 relative to beta-CM-5 on the GPI assay. Compounds I and II exhibited pronounced mu/delta-selectivities (greater than 18.9- and 12.4-fold respectively), whereas compound III was essentially inactive in both the GPI and MVD assays. CD studies of beta-CM-5 and its [Trp3]-beta-CM-5 analogs showed striking differences in their near-UV and far-UV spectra in aqueous or organic solvents. In the far UV CD spectra, weak (20%) alpha-helicity (maximum at 193 nm and minima at 208 and 222 nm) for beta-CM-5 was obtained in trifluoroethanol (TFE); however, none of the [Trp3]-beta-CM-5 analogs showed such CD bands. Of potential relevance to gamma-turn or C7 secondary structure was the observation of a strong negative band at 245 nm for compounds II and III which was not solvent-dependent in H2O or TFE, whereas compound I showed this CD band exclusively in TFE. In the near-UV CD at 275 nm (Trp electronic transition), the relative order of intensities of this band were determined for the [Trp3]-beta-CM-5 compounds to be II greater than I greater than III, which was identical to their relative biological potencies in both the GPI and MVD assays. Fluorescence energy transfer (FET) experiments of compounds I-III provided the intramolecular distances (r) between their Tyr (donor) to Trp (acceptor) side-chains, by the F?rster method, and were as follows: [Trp3]-beta-CM-5, r = 10.6 A; [Trp3, D-Pro4]-beta-CM-5, r = 9.6 A; and [D-Pro2,4,Trp3]-beta-CM-5, r = 11.0 A.(ABSTRACT TRUNCATED AT 400 WORDS)     

Software for quantification of labeled bacteria from digital microscope images by automated image analysis

Automated image analysis software, CellC, was developed and validated for quantification of bacterial cells from digital microscope images. CellC enables automated enumeration of bacterial cells, comparison of total count and specific count images [e.g., 4',6-diamino-2-phenylindole (DAPI) and fluorescence in situ hybridization (FISH) images], and provides quantitative estimates of cell morphology. The software includes an intuitive graphical user interface that enables easy usage as well as sequential analysis of multiple images without user intervention. Validation of enumeration reveals correlation to be better than 0.98 when total bacterial counts by CellC are compared with manual enumeration, with all validated image types. The software is freely available and modifiable: the executable files and MATLAB source codes can be obtained at www. cs. tut.fi/sgn/csb/cellc.     

Role of tryptophan-74 of the recombinant kringle 2 domain of tissue-type plasminogen activator in its omega-amino acid binding properties.

The role of W74 in stabilization of the binding of omega-amino acids to the recombinant (r) kringle 2 domain (residues 180-261) of tissue-type plasminogen activator ([K2tPA]) has been assessed by examination of the binding (dissociation) constants (Kd) of epsilon-aminocaproic acid (EACA) and one of its structural analogues, 7-aminoheptanoic acid (7-AHpA), to variants of r-[K2tPA] generated by site-directed mutagenesis of the wild-type kringle domain. Two nonconservative mutations at W74 of r-[K2tPA] have been constructed, expressed, and purified, resulting in one variant molecule containing a W74L mutation (r-[K2tPA/W74L]) and another containing a W74S mutation (r-[K2tPA/W74S]). In both cases, binding of EACA and 7-AHpA was virtually eliminated in the mutated kringles. Two additional conservative mutations at W74 of r-[K2tPA] have been similarly generated, resulting in r-[K2tPA/W74F] and r-[K2tPA/W74Y]. For these mutants, binding of the same ligands to the variant recombinant kringle domain is retained, although it is significantly weaker in nature. The 1H-NMR spectra of each of the variant kringles demonstrates that all retain the general gross conformations of their wild-type counterpart but that some environmental changes of proton resonances occur at particular aromatic amino acid residues that may be involved in omega-amino acid binding. Differential scanning calorimetric analyses of each of the variant kringles suggest that none of the mutations led to substantial destabilization of their structures, again suggestive of gross conformational similarities in all r-[K2tPA] molecules constructed. We conclude that the aromatic character present at position 74 of wild-type r-[K2tPA] is of great importance to its ability to interact with omega-amino acid ligands, with tryptophan being the most effective amino acid at that position.     

On the active sites of the [NiFe] hydrogenase from Desulfovibrio gigas. M?ssbauer and redox-titration studies

The [NiFe] hydrogenase isolated from Desulfovibrio gigas was poised at different redox potentials and studied by M?ssbauer spectroscopy. The data firmly establish that this hydrogenase contains four prosthetic groups: one nickel center, one [3Fe-xS], and two [4Fe-4S] clusters. In the native enzyme, both the nickel and the [3Fe-xS] cluster are EPR-active. At low temperature (4.2 K), the [3Fe-xS] cluster exhibits a paramagnetic M?ssbauer spectrum typical for oxidized [3Fe-xS] clusters. At higher temperatures (greater than 20 K), the paramagnetic spectrum collapses into a quadrupole doublet with parameters magnitude of delta EQ magnitude of = 0.7 +/- 0.06 mm/s and delta = 0.36 +/- 0.06 mm/s, typical of high-spin Fe(III). The observed isomer shift is slightly larger than those observed for the three-iron clusters in D. gigas ferredoxin II (Huynh, B. H., Moura, J. J. G., Moura, I., Kent, T. A., LeGall, J., Xavier, A. V., and Münck, E. (1980) J. Biol. Chem. 255, 3242-3244) and in Azotobacter vinelandii ferredoxin I (Emptage, M. H., Kent, T. A., Huynh, B. H., Rawlings, J., Orme-Johnson, W. H., and Münck, E. (1980) J. Biol. Chem. 255, 1793-1796) and may indicate a different iron coordination environment. When D. gigas hydrogenase is poised at potentials lower than -80 mV (versus normal hydrogen electrode), the [3Fe-xS] cluster is reduced and becomes EPR-silent. The M?ssbauer data indicate that the reduced [3Fe-xS] cluster remains intact, i.e. it does not interconvert into a [4Fe-4S] cluster. Also, the electronic properties of the reduced [3Fe-xS] cluster suggest that it is magnetically isolated from the other paramagnetic centers.     

Kinetics of NO2 air space absorption in isolated rat lungs.

We previously showed, during quasi-steady-state exposures, that the rate of inhaled NO2 uptake displays reaction-mediated characteristics (J. Appl. Physiol. 68: 594-603, 1990). In vitro kinetic studies of pulmonary epithelial lining fluid (ELF) demonstrated that NO2 interfacial transfer into ELF exhibits first-order kinetics with respect to NO2, attains [NO2]-dependent rate saturation, and is aqueous substrate dependent (J. Appl. Physiol. 71: 1502-1510, 1991). We have extended these observations by evaluating the kinetics of NO2 gas phase disappearance in isolated ventilating rat lungs. Transient exposures (2-3/lung at 25 degrees C) employed rebreathing (NO2-air) from a non-compliant continuously stirred closed chamber. We observed that 1) NO2 uptake rate is independent of exposure period, 2) NO2 gas phase disappearance exhibited first-order kinetics [initial rate (r*) saturation occurred when [NO2] > 11 ppm], 3) the mean effective rate constant (k*) for NO2 gas phase disappearance ([NO2] < or = 11 ppm, tidal volume = 2.3 ml, functional residual capacity = 4 ml, ventilation frequency = 50/min) was 83 +/- 5 ml/min, 4) with [NO2] < or = 11 ppm, k* and r* were proportional to tidal volume, and 5) NO2 fractional uptakes were constant across [NO2] (< or = 11 ppm) and tidal volumes but exceeded quasi-steady-state observations. Preliminary data indicate that this divergence may be related to the inspired PCO2. These results suggest that NO2 reactive uptake within rebreathing isolated lungs follows first-order kinetics and displays initial rate saturation, similar to isolated ELF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of iron-sulfur clusters in lysine 2,3-aminomutase by electron paramagnetic resonance spectroscopy.

Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of L-alpha-lysine with L-beta-lysine. The purified enzyme contains iron-sulfur ([Fe-S]) clusters, pyridoxal phosphate, and Co(II) [Petrovich, R. M., Ruzicka, F. J., Reed, G. H., & Frey, P. A. (1991) J. Biol. Chem. 266, 7656-7660]. Enzymatic activity depends upon the presence and integrity of these cofactors. In addition, the enzyme is activated by S-adenosylmethionine, which participates in the transfer of a substrate hydrogen atom between carbon-3 of lysine and carbon-2 of beta-lysine [Moss, M., & Frey, P. A. (1987) J. Biol. Chem. 262, 14859-14862]. This paper describes the electron paramagnetic resonance (EPR) properties of the [Fe-S] clusters. Purified samples of the enzyme also contain low and variable levels of a stable radical. The radical spectrum is centered at g = 2.006 and is subject to inhomogeneous broadening at 10 K, with a p1/2 value of 550 +/- 100 microW. The low-temperature EPR spectrum of the [Fe-S] cluster is centered at g = 2.007 and undergoes power saturation at 10 K in a homogeneous manner, with a p1/2 of 15 +/- 2 mW. The signals are consistent with the formulation [4Fe-4S] and are adequately simulated by a rhombic spectrum, in which gxx = 2.027, gyy = 2.007, and gzz = 1.99. Treatment of the enzyme with reducing agents converts the cluster into an EPR-silent form. Oxidation of the purified enzyme by air or ferricyanide converts the [Fe-S] complex into a species with an EPR spectrum that is consistent with the formulation [3Fe-4S].(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleolar morphometry in fine needle aspiration biopsies of the prostate.

OBJECTIVE: To find efficient morphometric nucleolar features for distinguishing different prostatic atypia groups in fine needle aspiration biopsies. STUDY DESIGN: A computerized interactive morphometry program was used to outline nucleoli of prostate cells. The samples were divided into three groups: group 1 (definitely benign [n = 10] and atypical but benign [n = 13]), group 2 (moderately atypical [n = 11] or highly suspicious [n = 13]) and group 3 (definitely malignant [n = 17]). RESULTS: The analysis revealed a difference in the number of nucleoli between definitely benign samples and other atypia groups but not between the latter. Nucleolar size features were best in distinguishing between atypia groups. The sample-associated mean size features were more powerful than cell group-associated size features. CONCLUSION: The sample-associated mean area, defined from cells selected by an experienced cytologist, if larger than 2 micron 2, was strongly associated with definitely malignant samples. The same was true for the largest nucleolar area if larger than 5.0 micron 2. Morphometric nucleolar size features appeared efficient in distinguishing between definitely malignant and other samples.     

Binding of the gamma-subunit of retinal rod-outer-segment phosphodiesterase with both transducin and the catalytic subunits of phosphodiesterase.

The gamma-subunit of retinal rod-outer-segment phosphodiesterase (PDE-gamma) is a multifunctional protein which interacts directly with both of the catalytic subunits of PDE (PDE alpha/beta) and the alpha-subunit of the retinal G (guanine-nucleotide-binding)-protein transducin alpha (T alpha). We have previously reported that the PDE gamma binds to T alpha at residue nos. 24-45 [Morrison. Rider & Takemoto (1987) FEBS Lett. 222, 266-270]. In vitro this results in inhibition of T alpha GTP/GDP exchange [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. We now report that the inhibitory region of PDE gamma for PDE alpha/beta occurs at PDE gamma residues 54-87. This binding results in inhibition of either trypsin-solubilized or membrane-bound PDE alpha/beta. PDE gamma which has been treated with carboxypeptidase Y, removing the C-terminus, does not inhibit PDE alpha/beta, but does inhibit T alpha GTP/GDP exchange. Inhibition by PDE gamma can be removed by T alpha-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) addition to membranes. This results in a displacement of PDE gamma, but not in removal of this subunit from the membrane [Whalen, Bitensky & Takemoto (1990) Biochem. J. 265, 655-658]. These results suggest that low levels of T alpha-GTP[S] can result in displacement of PDE gamma from the membrane in vitro as a GTP[S]-T alpha-PDE gamma complex. Further activation by high levels of T alpha-GTP[S] occurs by displacement of PDE gamma from its inhibitory site on PDE alpha/beta, but not in removal from the membrane.     

Measles virus modulates human T-cell somatostatin receptors and their coupling to adenylyl cyclase.

The possible role of immunomodulatory peptide somatostatin (SRIF) in measles virus (MV)-induced immunopathology was addressed by analysis of SRIF receptors and their coupling to adenylyl cyclase in mitogen-stimulated Jurkat T cells and human peripheral blood mononuclear cells (PBMC). SRIF-specific receptors were assayed in semipurified membrane preparations by using SRIF14 containing iodinated tyrosine at the first position in the amino acid chain ([125I]Tyr1) as a radioligand. A determination of receptor number by saturation of radioligand binding at equilibrium showed that in Jurkat cells, MV infection led to a dramatic decrease in the total receptor number. The virus-associated disappearance of one (Ki2 = 12 +/- 4 nM [mean +/- standard error of the mean [SEM]]; n = 4) of two somatostatin binding sites identified in control Jurkat cells (Ki1 = 78 +/- 3 pM and Ki2 = 12 +/- 4 nM [mean +/- SEM]; n = 4) was also observed. Almost identical results were obtained for phytohemagglutinin-activated human PBMC. In the absence of MV infection, two somatostatin binding sites were present (Ki1 = 111 +/- 31 pM and Ki2 = 17 +/- 2 nM [mean +/- SEM]; n = 2), whereas in MV-infected cells, only the high-affinity (Ki1 = 48 +/- 15 pM [mean +/- SEM]; n = 2) binding site remained. In addition, MV infection reinforced the inhibitory effects of SRIF on adenylyl cyclase activity, since maximal inhibition at 1 microM peptide was 11% +/- 4% in control cells versus 25% +/- 3% (P < 0.05) in infected Jurkat cells. Moreover, MV infection severely impaired the capacity of adenylyl cyclase to be activated directly (by forskolin) or indirectly (via Gs protein-coupled vasoactive intestinal peptide receptor). An assessment of [methyl-3H]thymidine incorporation showed that SRIF increased proliferative responses to mitogens only in control cells, not in MV-infected cells. Altogether, our data emphasize that MV-associated alteration of SRIF transduction appears to be related to the loss of SRIF-dependent increase of mitogen-induced proliferation.     

14-membered cyclic opioids related to dermorphin and their partially retro-inverso modified analogues. I. Synthesis and biological activity.

As a continuation of our program to study structure-activity relationships of opiate peptides, we report the syntheses and biological activities of a series of 14-membered cyclic dermorphin analogues closely related to enkephalin analogue Tyr-c[D-A2bu-Gly-Phe-Leu] incorporating a phenylalanine at the third position in place of glycine. In addition to two parent dermorphin analogues Tyr-c[D-A2bu-Phe-Phe-(L and D)-Leu], four stereoisomeric retro-inverso modified analogues Tyr-c[D-A2bu-Phe-gPhe-(S and R)-mLeu] with a reversed amide bond between residues four and five, and Tyr-c[D-Glu-Phe-gPhe-(L and D)-rLeu] with two reversed amide bonds between residues four and five, and between residue five and the side chain of residue two have been synthesized. The results from the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays show that all analogues are superactive at either one or both opiate receptors and in general display higher activities as compared to the corresponding enkephalin analogues with a glycine at the third position. Results from the in vitro biological assays and conformational analysis using 1H-NMR spectroscopy (adjoining paper) will provide useful information to understand the role of the Phe3 aromatic side chain in dermorphin, and that of the Phe4 aromatic side chain in enkephalin, on opiate activity since these cyclic dermorphin analogues contain two Phe residues at both the third and fourth positions.     

Evidence that guanosine 5''-[gamma-thio]triphosphate stimulates plasma membrane Ca2+ inflow when introduced into hepatocytes.

1. Slowly hydrolysable analogues of GTP were introduced into hepatocytes by incubating the cells in the absence of Mg2+ and in the presence of ATP4-. Experiments using guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S])indicated that about 50% of the GTP[S] loaded into the cells was subsequently hydrolysed. 2. In cells loaded with GTP[S] and incubated in the absence of added extracellular Ca2+ (Ca2+o), the rate of activation of glycogen phosphorylase observed after addition of 1.3 mM-Ca2+o was 250% greater than the rate observed in unloaded cells. Smaller effects (130%) were observed in cells loaded with either guanyl-5'-yl imidodiphosphate or guanosine 5-[beta-thio]diphosphate (GDP[S]). Cells loaded with adenosine 5'-[gamma-thio]triphosphate showed no increase in glycogen phosphorylase activity on addition of Ca2+o. 3. The effect of a submaximal concentration of GTP[S] on the Ca2+-induced activation of glycogen phosphorylase was additive with that of a half-maximally effective concentration of vasopressin. GTP[S] did not increase the effect of a maximally effective concentration of the hormone. 4. Cells loaded with GTP[S] exhibited an increased initial rate of 45Ca2+ exchange measured at 1.3 mM-Ca2+o. 5. GTP[S] did not affect the amount of 45Ca2+ exchanged by cells incubated at 0.1 mM-Ca2+o or the ability of vasopressin to release 45Ca2+ from these cells. 6. It is concluded that the introduction of slowly hydrolysable analogues of GTP to the liver cell cytoplasmic space stimulates the inflow of Ca2+ across the plasma membrane through a channel similar to that activated by vasopressin.     

H2 histaminic receptors in rat cerebral cortex. 2. Inhibition of [3H]histamine by H2 antagonists

Sites labeled by [3H]histamine in homogenates of rat cerebral cortex reveal a pharmacological specificity typical of H2 receptors. Fourteen H2 antagonists inhibit the specific binding of the radioligand to the same level; Hill coefficients are near or equal to one for five compounds and markedly lower for nine. The binding patterns of individual antagonists (A) are well described by the empirical expression Y = F1K1/(K1 + [A]) + F2K2/(K2 + [A]), in which F1 and F2 sum to 1; F2 is 0 for those drugs that reveal a Hill coefficient of 1. Concentrations of A that reduce specific binding by 50% (IC50) correlate well (r = 0.991; P less than 0.00001) and show good numerical agreement with potencies reported for inhibition of the response to histamine in H2-mediated systems. The correlation is poorer when IC50 is replaced by either K1 (r = 0.973) or K2 (r = 0.921) for those antagonists that reveal both; the antihistaminic activity of the drug thus appears not to be associated preferentially with one or other class of sites. Since F2 varies from 0.16 to 0.60 among those antagonists that discern heterogeneity, the antagonist appears to determine the distribution of sites between the two classes. Moreover, a correlation among antagonists between values of K1 and K2 (r = 0.975; P = 0.00001) suggests that the apparent heterogeneity reflects different conformers within an otherwise homogeneous population. H2 antagonists appear to be noncompetitive with respect to each other and to the radioligand: one antagonist has relatively little effect on the values of K1, K2, and F2 revealed by another; also, estimates of K1 and K2 are independent of the concentration of [3H]histamine between 1.3 and 10 nM, although the radioligand exhibits an apparent dissociation constant of 3.9 nM [Steinberg, G. H., Eppel, J. G., Kandel, M., Kandel, S. I., & Wells, J. W. (1985) Biochemistry (preceding paper in this issue)].     

Synthesis, biology, NMR and conformation studies of the topographically constrained delta-opioid selective peptide analogs of [beta-iPrPhe(3)]deltorphin I.

Replacement of Phe3 in the endogenous delta-opioid selective peptide deltorphin I with four optically pure stereoisomers of the topographically constrained, highly hydrophobic novel amino acid beta-isopropylphenylalanine (beta-iPrPhe) produced four pharmacologically different deltorphin I peptidomimetics. Radiolabeled ligand-binding assays and in vitro biological evaluation indicate that the stereoconfiguration of the iPrPhe residue plays a crucial role in determining the binding affinity, bioactivity and selectivity of [beta-iPrPhe3]deltorphin I analogs: a (2S,3R) configuration of the iPrPhe3 residue in [beta-iPrPhe3]deltorphin I provided the most desirable biological properties with binding affinity (IC50 = 2 nM), bioassay potency (IC50 = 1.23 nM in MVD assay) and exceptional selectivity for the delta-opioid receptor over the mu-opioid receptor (30 000). Further conformational studies based on two-dimensional NMR and computer-assisted molecular modeling suggested a model for the possible bioactive conformation in which the Tyr1 and (2S,3R)-beta-iPrPhe3 residues adopt trans side-chain conformations, and the linear peptide backbone favors a distorted beta-turn conformation.     

Susceptibility of nude mice carrying the Fv-4 gene to Friend murine leukemia virus infection.

Fv-4 is a mouse gene that dominantly confers resistance to infection with Friend murine leukemia virus (F-MuLV) (S. Suzuki, Jpn. J. Exp. Med. 45:473-478, 1975). Despite complete resistance to ecotropic MuLV infection in mice carrying the Fv-4 gene, it is known that cells carrying the resistance gene in tissue culture do not always show resistance as extensive as that in vivo (H. Yoshikura and T. Odaka, JNCI 61:461-463, 1978). To investigate the immunological effect on resistance in vivo, we introduced the Fv-4 gene into BALB/c nude mice (Fv-4-/- nude[nu/nu]) by mating them with Fv-4 congenic BALB/c mice (Fv-4r/r nude+/+) and examined the susceptibility of the F2 progeny to F-MuLV. All BALB/c nude mice without the Fv-4 gene (Fv-4-/- nude[nu/nu]) were permissive to F-MuLV and developed erythroleukemia within 2 weeks after virus inoculation. The BALB/c nude mice with the Fv-4 gene (Fv-4r/r nude[nu/nu]) did not develop leukemia, and no or little virus was detected in the spleen 7 weeks after virus inoculation. The resistance to F-MuLV was dominant in (Fv-4 congenic BALB/c x BALB/c nude) F1 mice with the Fv-4r/- nude(nu/+) genotype as strictly as in (Fv-4 congenic BALB/c x BALB/c) F1 mice with the Fv-4r/- nude+/+ genotype. However, almost all BALB/c nude mice with the Fv-4r/- nude(nu/nu) genotype developed the disease within 7 weeks, and the virus was detected in all of their spleens even in the mice without leukemia. These results show that the resistance caused by the Fv-4 gene is recessive in nude mice and dominant in BALB/c mice. Some immunological effects, perhaps cell-mediated immunity, may play important roles in the resistance to F-MuLV infection in vivo in addition to the dosage effect of the Fv-4 product.     

An improved method for quantifying hematozoa by digital microscopy

Intensity of hematozoan infection is infrequently quantified because accurate calculations require visual counts of parasites relative to a large number of erythrocytes. Manual quantification of erythrocytes can be circumvented by using ImageJ software (developed by the National Institutes of Health) to count erythrocyte nuclei from digital images. Here we use the ratio of microscope erythrocyte counts to digital image erythrocyte counts (field:image ratio) to extrapolate erythrocyte counts from smaller digital images to the microscope's larger field of view. Field:image ratios were consistently calculated from 10 slides (resampling P = 0.049) and used to rapidly estimate intensity of infection within 50,000 or more erythrocytes. Intensity of hematozoan infection calculated from manual quantification of 2,000 erythrocytes was significantly lower (0.46 times) than intensity calculated from digital quantification of 50,000 erythrocytes (bootstrap P = 0.02). We contend that digital quantification of hematozoan infection offers a rapid and precise method to quantify infections of low to moderate intensity.     

Analysis of the microvasculature and tissue type ratios in normal vs. benign and malignant breast tissue

OBJECTIVE: To analyze the microvasculature and tissue type ratios in normal vs. benign and malignant breast tissue to establish a baseline for expected values against which future imaging studies can be benchmarked. STUDY DESIGN: Using computer-assisted techniques on immunostained breast tissue (normal [n = 28], fibrocystic [n = 37], fibroadenomas [n = 19], invasive carcinomas [n = 19]), values were obtained for microvessel density (MVD), mean vessel area (MVA), vessel orientation (shape) and epithelial:stromal ratio (E:S). Measurement reproducibility and the effects of fibroadenoma stromal hyalinization and fibrocystic disease severity were also tested. RESULTS: Value ranges for the 4 diagnostic groups were significantly different (P < .001). For invasive breast carcinomas, E:S and MVD were significantly higher (P < .001) but MVA was smaller as compared to that in fibroadenomas. Peripherally vs. centrally there was no significant difference in MVD, MVA or vessel shape in the neoplasms. Decreases in E:S and MVD correlated with fibroadenoma stromal hyalinization. Increases in E:S and MVA correlated with more severe fibrocystic disease. Correlation coefficients for measurement reproducibility were high across the diagnostic categories. CONCLUSION: This study established a specific, reproducible, computer-assisted technique and baseline of expected values for morphologic criteria in normal, benign and malignant breast tissue that may be used in the future to correlate new breast imaging responses with these underlying biologic properties.     

The prion peptide forms ion channels in planar lipid bilayers

One of the hypotheses concerning the pathogenic properties of the prion protein considers its influence on cellular ion homeostasis. Using the lipid bilayer technique, the influence of prion-derived peptides on the lipid bilayer conductance was characterized. To evaluate the physiological significance and possible pathological functions of the peptides, their effect on the membrane potential and respiration rate of hippocampal mitochondria was also studied. We used a peptide bearing the human prion protein sequence YSNQNNF (PrP [169-175]), and peptide SSQNNF (PrP [170-175]) bearing a naturally-occurring mutation in position 171 [N(r)S] linked to schizoaffective diseases in humans (Samaia, H.B., Mari, J.J., Vallada, H.P., Moura R.P., Simpson A.J.G., Brentani R.R. A prion-linked psychiatric disorder. Nature 390 (1997) 241). In this report, we show that PrP [170-175] N171S increases the conductance of planar lipid bilayers. Based on the conductance of single channel currents recorded in 500/500 mM KCl (cis/trans), we found a single channel conductance of 8 to 26 pS. The native prion peptide PrP [169-175] does not form ion channels in the lipid bilayer. Neither of the peptides significantly changed the membrane potential or respiration rate of isolated rat hippocampal mitochondria. We propose a possible mechanism for channel formation by aggregation of the prion-derived peptide.