Transition metal carbonyl labeling of proteins. A novel approach to a solid-phase two-site immunoassay using Fourier transform infrared spectroscopy. Labeling of bovine serum albumin (BSA) and anti-human thyroid stimulating hormone (hTSH) monoclonal antibodies (mAbs) was performed using (N-succinimidyl 4-pentynoate)hexacarbonyldicobalt (NSCo2(CO)6). Conditions of coupling were different depending on the protein to be labeled, denaturation of the mAbs occuring with high percentages of organic solvent in the reaction mixture. The influence of reaction time and initial concentration of NSCo2(CO)6 was examined. They were both shown to affect the final coupling rate of the metal carbonyl probe. Preservation of the immunoreactivity toward 125I-hTSH was observed for five conjugates having different NSCo2(CO)6: mAb molar ratios when compared to unmodified and peroxidase-labeled mAbs. Finally, a preliminary study of the quantitative detection of the metal carbonyl mAbs on microtiter wells was achieved using Fourier transform infrared spectroscopy.


Site-specific conjugation of chain-terminal chelating polymers to Fab' fragments of anti-CEA mAb: effect of linkage type and polymer size on conjugate biodistribution in nude mice bearing human colorectal carcinoma. Polylysine-based chelating polymers were used for site-specific modification of anti-CEA mAb Fab' fragments via their SH group distal to the antigen-binding site of the antibody molecule. Conjugation was performed using chain-terminal (pyridyldithio)propionate or 4-(p-maleimidophenyl)butyrate moieties to form reducible (S-S) or stable (S-C) bonds between a polymer and Fab' molecule, respectively. One S-S conjugate (S-S9) and two different S-C conjugates (S-C3 and S-C9) were prepared using 3- and 9-kDa molecular weight polymers. No significant loss of immunoreactivity was observed in solid-phase immunoassay, 90-95% of 111In-labeled conjugates being bound to CEA-coated Sepharose beads. After labeling with 111In, the conjugates had a specific radioactivity of 90-120 microCi/micrograms. Injected in nude mice bearing LS 174T carcinoma, the conjugates produced different biodistribution patterns. S-S9 was practically unable to accumulate in tumor and produced very rapid blood clearance of radioactivity and high uptake of radioactivity in liver, spleen, and especially kidneys (225% ID/g 24 h postinjection). S-C3 and S-C9 produced practically the same blood clearances (much slower than that of S-S9) and significant tumor uptake (9-10% ID/g at 24 h). S-C3 gave significantly lower radioactivity in spleen, skin, and bones, and cleared more rapidly from liver and kidneys. Renal uptake for S-C3 and S-C9 was rather high (45% ID/g at 24 h), but much lower than for S-S9.


Identification and characterization of a nucleotide binding site on recombinant murine granulocyte/macrophage-colony stimulating factor. Granulocyte/macrophage-colony stimulating factor (GM-CSF) is a regulatory cytokine important in the proliferative and functional activation of hematopoietic cells. It belongs to a family of 20 kDa or less acidic glycoprotein molecules found in a broad range of cellular sources. On the basis of the previously reported nucleotide-binding properties of interleukin-2 (IL-2), atrial natriuretic factor (ANF), and glucagon, the interaction of GM-CSF with nucleotides was investigated. Using radiolabeled 8-azidoadenosine-containing photoprobes of ATP ([gamma-32P]-8N3ATP) and Ap4A, the putative biological alarmone ([beta'-32P]-8N3Ap4A), we have identified a nucleotide binding site on recombinant murine GM-CSF (rmGM-CSF). Specificity of binding was demonstrated by saturation and competition experiments. Saturation of photoinsertion by [gamma-32P]-8N3ATP and [beta'-32P]-8N3Ap4A occurs with apparent Kd's of 10 and 0.7 microM, respectively. Using an immobilized Fe3+ affinity chromatography technique, developed specifically for the isolation of photolabeled peptides, a single radiolabeled peptide was isolated. It was identified as amino acids 5-14 near the N-terminus of GM-CSF. This peptide region has been shown in previous studies to be critical for biological activity. Also consistent with this observation is our finding that the photolabeled GM-CSF has lost most, if not all, of its biological activity, as determined by a cellular proliferation assay.


Use of microsphere-antibody conjugates in microparticle-enhanced nephelometric immunoassay. A new microparticle-enhanced nephelometric immunoassay has been recently described as a sensitive, accurate, and easy-to-perform competitive immunoassay for various analytes. As initially described, this test is based on the nephelometric quantification of the inhibition, by the antigen to be assayed, of immunoagglutination of microparticle-antigen conjugates. Its applicability as a competitive immunoassay is thus limited by the necessary availability of pure antigens to prepare microparticle-antigen conjugates. In this paper, we report an adaptation of this initial test, where microparticles are coated by monoclonal antibodies, eliminating the need for purified antigens. The new configurations of particle agglutination-based immunoassays described include use of these microparticle-antibody conjugates with microparticle-antigen conjugates, free antigen, and anti-mouse immunoglobulins antiserum. The feasibility of such configurations is studied with human chorionic gonadotropin hormone, human thyroid stimulating hormone, and human myoglobin as antigens. Capture of the analyte by microparticle-antibody conjugates is evidenced by inhibition of their agglutination with microparticle-antigen conjugates and by agglutination in a sandwich assay with a complementary monoclonal antibody or polyclonal antiserum. The use of a second xenogenic antibody enhances the agglutination process and increases the assay sensitivity. Microparticle-antibody conjugates may extend the applications of microparticle-enhanced nephelometric immunoassays to unavailable analytes.


1-(m-[211At]astatobenzyl)guanidine: synthesis via astato demetalation and preliminary in vitro and in vivo evaluation. No-carrier-added 1-(m-[211At]astatobenzyl)guanidine ([211At]MABG) was synthesized by astato demetalation using two different routes. The overall yield for the two-step approach from 3-(tri-n-butylstannyl)benzylamine was 13%. N-Chlorosuccinimide-mediated astato desilylation of 1-[3-(trimethylsilyl)benzyl]guanidine in acetic acid gave poor yields. In trifluoroacetic acid, the reaction worked well. The radiochemical yield was independent of reaction time and the amount of precursor used; however, the temperature of the reaction had a marked effect. Yields of 85% were obtained in 5 min at 70 degrees C using 0.5 mumol of the precursor. The percentage specific binding in vitro of [211At]MABG was nearly constant over a 2-log activity range and was comparable to that of no-carrier-added [131]MIBG. The accumulation of [211At]MABG in the heart and adrenals of normal mice was similar to that observed for no-carrier-added [131]MIBG.


Evaluation of peptide libraries: an iterative strategy to analyze the reactivity of peptide mixtures with antibodies. Peptide libraries corresponding to a presumed mixture of 50,625 tetrapeptides or 16,777,216 hexapeptides were each prepared in a single assembly by standard solid-phase peptide synthesis. By enzyme-linked immunosorbent assay, the tetrapeptide library was shown to inhibit the binding of an antiserum to FMRF amide with an FLRF capture antigen; the hexapeptide library was shown to inhibit the binding of a monoclonal antibody to a 28 amino acid peptide with the corresponding peptide capture antigen. An iterative strategy of variation was used to determine for each position in the tetra- or hexapeptides which amino acid contributed the most to activity. As a result we were able to logically select out of the tetrapeptide library the sequence FLRF and to select out of the hexapeptide library a sequence that differed from the apparent probable epitope but was twice as active. A single amino acid substitution in the logically derived sequence gave a peptide that was 35 times as active as the hexapeptide sequence in the original 28 amino acid peptide.


Acidic derivatives of homocysteine thiolactone: utility as anionic linkers. Homocysteine thiolactone (2) derivatives in which the nitrogen is acylated with groups containing acidic functionalities have been synthesized. These include the succinyl (3), the carboxymethylglutaryl (4), the 3-phosphonopropionyl (7), and the 3-sulfopropionyl (8) derivatives. These thiolactones can be used to introduce a thiol functionality into proteins such as the outer membrane protein complex of Neisseria meningitidis (OMPC) allowing conjugation with electrophilic ligands. This chemistry is the same as with N-acetylhomocysteine thiolactone (1), but their pKa values are such that at pH 7 concomitant negative charge is introduced into the conjugate. Such negative charge should neutralize some excess positive charge introduced when arginine- and lysine-rich peptides are bonded as ligands. In the case of OMPC, introduction of such positive charge appears to effect irreversible precipitation. The system has been studied using the maleimidopropionyl and bromoacetyltriarginine (9 and 10) derivatives as models. In select instances anionic spacers reduce the degree of precipitation relative to N-acetyl-homocysteine thiolactone derivatives.


Development and validation of a bioassay for interleukin-2. A reliable and precise method for the determination of IL-2 activity, based on stimulation of CTLL cell proliferation, was developed. Cells were incubated with different concentrations of IL-2 for 24 h in microtiter plates. The stimulatory effect was measured on a plate-reading spectrophotometer by reading the optical density of formazan, which is produced by viable cells from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The bioassay was designed as a four-dose parallel line test, fulfilling pharmacopoeial requirements for assay validity, and the inter-assay relative standard deviation (RSD) for a group of four experiments was 2.6%. The International Standard for human IL-2 and the Reference Reagent for Recombinant DNA-derived IL-2 were employed for potency determinations. The method was found suitable for potency assessments of pharmaceutical formulations of IL-2.


Preparation and characterization of biologically active 6'-O-(6-aminocaproyl)-4'-O-monophosphoryl lipid A and its conjugated derivative. N-tert-butyloxycarbonyl (t-Boc) protected 6-aminocaproic (Cap) anhydride was reacted with unprotected hexaacyl-4'-O-monophosphoryl lipid A (MLA) obtained from the lipopolysaccharide of Escherichia coli J5 to yield t-Boc-Cap-MLA. After a column purification step, the t-Boc group was removed by incubating the sample at low temperature in the presence of acid to yield Cap-MLA. This product was analyzed by californium plasma desorption mass spectrometry (PDMS). Purified t-Boc-Cap-MLA was further fractionated by reverse-phase high-performance liquid chromatography as its methyl ester and characterized by laser desorption mass spectrometry, PDMS, and proton nuclear magnetic resonance spectroscopy. These analyses revealed that the Cap group was selectively introduced into the 6'-position of MLA. To demonstrate that Cap-MLA can be conjugated to other compounds, it was reacted with biotin-Cap N-hydroxysuccinimide ester to yield biotin-(Cap)2-MLA. Analysis of this product by PDMS confirmed its expected molecular weight of 2171 and showed the presence of fragments containing the biotin and Cap groups. Monoclonal antibodies and streptavidin were used to show the presence of both lipid A and biotin in this conjugated product. These two novel lipid A derivatives were then tested for their bioactivities. Although both Cap-MLA and biotin-(Cap)2-MLA showed mitogenic activity using murine splenocytes, they were about 4-8 times less active than MLA at 20 micrograms/mL or less and only one-half as active at 100 micrograms/mL.(ABSTRACT TRUNCATED AT 250 WORDS)


Evaluation of the anti-tumour action and acute toxicity of kosins from Hagenia abyssinica. The kosins are phloroglucinol derivatives isolated from female flowers of Hagenia abyssinica (Rosaceae) and were tested for possible cytotoxic activity in vitro and in vivo against a panel of three transplantable murine adenocarcinomas of the colon of varying growth characteristics and morphology (MAC system). Significant reductions in colony formation were observed in vitro in MAC 15A tumour following 1, 3, 6 and 24 h exposure to all kosins (alpha-kosin, kosotoxin and protokosin). The kosins (kosotoxin and protokosin) were also found to be cytotoxic against MAC tumour cells in vivo in some cases. Kosotoxin was subjected to preliminary toxicity studies in mice. It showed no observable toxicity up to 200 mg kg-1 orally and was found to be toxic at doses in excess of 50 mg kg-1 (i.p.). A single dose of 100 mg kg-1 (i.p.) was lethal for 100% of the animals.


Synthesis and properties of an oligodeoxynucleotide modified with a pyrene derivative at the 5'-phosphate. The synthesis of an oligonucleotide (ODN) modified with pyrene (pyr) on the 5'-phosphate is described. The ODN and pyrene are joined through a linker composed of four methylene groups. Modification of the oligonucleotide was effected via condensation of the 2-cyanoethyl N,N-diisopropylphosphoramidite of 4-(1-pyrenyl)butanol (pyr-m4OPAm, 2) with the 5'-OH of an ODN. This derivative is suitable for incorporation into automated solid-phase DNA synthesis and was attached to the 5' terminus of the DNA chain through a phosphodiester linkage. The properties of the 5'-(pyr-m4)d(T)15 (3) and the duplex it formed with d(A)15 were investigated by fluorescence and absorbance spectroscopy. The pyrene fluorescence in the modified duplex was quenched 96.3% relative to an identical concentration of free 4-(1-pyrenyl)butanol. The ultraviolet spectrum of the 5'-(pyr-m4)-d(T)15 and 5'-(pyr-m4)-d(T)15-d-(A)15 modified duplex, in the 320-360-nm region, was red-shifted 6 nm relative to the free 4-(1-pyrenyl)-butanol. The Tm values of the unmodified and modified duplexes at 0.1 M NaCl were 34.9 and 41.9 degrees C, respectively. The pyrene-induced stabilization corresponds to a free energy change (delta delta G degrees) of -2.6 kcal/mol.


Determination of bis(amidinohydrazones) by micellar electrokinetic capillary chromatography. A micellar electrokinetic capillary chromatography method was developed for the separation and determination of aliphatic congeners of bis(amidinohydrazones) in standard solution. Eight bis(amidinohydrazones) could be determined in less than 15 min at an applied voltage of 22 kV, using 0.05 M sodium phosphate as buffer (pH 7.0) together with 1 mM N-cetyl-N,N,N-trimethylammonium bromide. Hydrostatic sample injection was employed. The method exhibited good repeatability and a linear range of 2.5-100 micrograms ml-1. A detection limit of 1 micrograms ml-1 was achieved. The method also allows the determination of bis(amidinohydrazones) in human serum samples.


Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand. We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system.


Identification and quantitation of tetrapeptide deamidation products by mass spectrometry. A method to quantify asparagine (Asn), aspartate (Asp) and isoaspartate (isoAsp) residues in small peptides by fast atom bombardment mass spectrometry (FAB-MS) was developed. Discrimination of isoAsp from Asp residues was accomplished by selective derivatization of isoAsp residues in acetic anhydride, D2O and pyridine. Deuteration occurred at any carbon adjacent to a free alpha-carboxyl group, through a transient oxazalone intermediate, allowing the isoAsp side chain and the C-terminus to incorporate deuterium. Thus, isoAsp-containing peptides incorporate one more deuterium than peptides with Asp and two more than Asn peptides. FAB CID-MS spectra of the Asn tetrapeptide, Thr-Asn-Ser-Tyr, were used to confirm the position of deuteration to the C-terminal residue. FAB and FAB CID-MS spectra demonstrated that the 1 amu shift in mass was not caused by derivatization induced deamidation of the Asn residue. FAB-MS spectra of deuterated peptide standards and mixtures containing deamidation products were obtained over the molecular ion region and deconvoluted using non-deuterated control spectra. Deuterium incorporation values for the Asn, Asp and iosAsp containing peptide standards were 80% mono-deuterated peptide, 95% mono-deuterated peptide and 63% di-deuterated peptide, respectively. IsoAsp to Asp ratios in an unknown mixture were obtained by a least-squares minimization of the difference between the unknown deuterated mixture and the isotopic envelopes from the deuterated standards. The mixture was found to contain 85% isoAsp peptide by FAB-MS, which agreed well with 81% isoAsp peptide when assayed by reversed-phase LC.


Reagent for introducing pyrene residues in oligonucleotides. A novel pyrenyl-containing phosphoramidite reagent, N-[4-(1-pyrenyl)butyryl]-O1-(4,4'-dimethoxytrityl)-O2- [(diisopropylamino)(2-cyanoethoxy)phosphino]-3-amino-1 ,2-propanediol (5), has been synthesized from 4-(1-pyrenyl)butanoic acid in four steps with the 52% overall yield and used to incorporate pyrene residue(s) into oligonucleotides. Oligonucleotides 6 and 7, bearing one or two pyrenes at the 5'-terminus, have been prepared by means of that reagent, characterized with fluorescence spectra, and successfully used as primers in a polymerase chain reaction.


Comparison of 9-fluorenylmethoxycarbonyl and 9-fluoreneacetyl-tagged silica-based derivatization reagents in high-performance liquid chromatography. Two silica reagents based on a 4-hydroxy-3-nitrobenzoyl backbone were synthesized and characterized with 9-fluorenylmethoxycarbonyl (FMOC) and 9-fluoreneacetyl (FA) tags. These reagents were tested by derivatization of primary and secondary amines. Derivatization conditions such as temperature, time and triethylamine catalyst were tested. The FA-tagged silica reagent showed better performance than the FMOC-tagged silica reagent by a comparison of derivatization efficiencies, stabilities of reagents, and blank reagent interferences with derivatization. Finally, cadaverine and an aliphatic amine mixture were analysed using the FA-tagged reagent by pre-column, off-line derivatization and fluorescence detection.


Large-scale synthesis of the bifunctional chelating agent 2-(p-nitrobenzyl)-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetr aacetic acid, and the determination of its enantiomeric purity by chiral chromatography. The attachment of radiometals to monoclonal antibodies for medical applications requires extreme stability under physiological conditions, with no significant release of metal. Chelators that can hold radiometals like 111In, 67Ga, and 90Y with high stability under these conditions are essential for radiotherapy or immunoscintigraphy. 2-(p-Nitrobenzyl)-1,4,7,10-tetraazacyclododecane- N,N',N'',N'''-tetraacetic acid (nitrobenzyl-DOTA) is one of the most promising bifunctional chelating agents. A large-scale synthesis of nitrobenzyl-DOTA is described. The overall yield for the nine-step synthesis sequence starting from nitrophenylalanine is 5.6%. Synthesis of nitrobenzyl-DOTA according to the new procedure yields up to approximately 10 g without special apparatus. Both enantiomers of the chiral chelate nitrobenzyl-DOTA have been prepared, and their enantiomeric purity has been checked by chiral chromatography.


Liquid chromatographic analysis of monosaccharides with phenylisocyanate derivatization. Because of the lack of sensitivity in carbohydrate analysis, HPLC pre-column derivatization techniques which give strongly UV absorbing compounds have been reported. These techniques have the disadvantage of leading to several chromatographic peaks from each reducing sugar. To enhance both the sensitivity and the selectivity of such specific separation problems, a simplex procedure was applied to optimize the phenylisocyanate derivatization of monosaccharides: a major and stable derivative was formed under the optimal conditions established. The method was extended to deoxysugars and methylglycosides. The limit of detection was 0.2-1 ng for all sugars tested.


Liquid chromatographic determination of a new catechol-O-methyltransferase inhibitor, entacapone, and its Z-isomer in human plasma and urine. Assay procedures for analysis of entacapone, (E)-2-cyano-N,N-diethyl-3-(3,4-dihydroxy-5-nitrophenyl)-propenamide++ +, and its Z-isomer in human plasma and urine are described. The methods were based on reversed-phase liquid chromatography with amperometric detection. Entacapone and its Z-isomer were extracted with n-hexane-ethyl acetate mixtures after acidification with hydrochloric acid. From urine extracts the analytes were back-extracted into phosphate buffer (pH 7.2). During sample treatment 1-2% of entacapone was changed to the Z-isomer. With recoveries exceeding 75% the relative standard deviations for within-day precision were less than 11% for plasma and less than 6% for urine at the quantitation limit (10 ng ml-1) and less than 6% for both methods at higher concentrations (20-2000 ng ml-1). The assays were specific with respect to all known metabolites and selective, sensitive and precise enough for determination of entacapone and its Z-isomer in plasma and urine down to 10 ng ml-1. The methods are thus suitable for the kind of pharmacokinetic studies exemplified in this paper.


Hypoxic-ischemic brain damage in term neonates--the relation of neurodevelopmental handicap to cranial ultrasound findings. This study presents ultrasound findings and neurodevelopmental follow-up in ten infants born at term suffering most severe grade of hypoxic-ischemic encephalopathy. Early ultrasound findings showed in nine of these ten neonates signs of cerebral edema accompanied in two children by intraventricular haemorrhage. Late ultrasound findings in all infants examined demonstrated severe cerebral atrophy, predominantly affecting the cortico-subcortical area. In three children multiple subcortical cysts were also present, corresponding to ultrasound findings of subcortical leukomalacia. Cranial computerized tomography was performed in six of the ten children, showing more precisely the predominant site of cortical atrophy, whereas in children with ultrasound findings of subcortical leukomalacia extensive low density areas in the subcortical white matter were present. All children had neurodevelopmental follow-up for between two and seven years. Six of the ten children have multiple disabilities suffering from spastic quadriparesis, epilepsy, mental retardation and/or visual disability. Among these six were all three children with subcortical leukomalacia. All the children demonstrated poor head growth and became markedly microcephalic. We consider ultrasonography to be very useful in the diagnosis of hypoxic-ischemic brain damage in term neonates as well in predicting the neurodevelopmental outcome in asphyxiated term infants.


Pneumococcal meningitis in the elderly.

Pneumococcal meningitis in the elderly. The authors reviewed 70 cases of Streptococcus pneumoniae meningitis occurring over a 5-years period (1985-1989). Clinical features, outcome and laboratory findings in elderly patients (> 60 years of age, 21 patients) were compared with those in younger patients (< 59 years of age, 49 patients). Mortality rate was 57% in elderly group vs. 20% in younger. Admission to the hospital was delayed in the elderly. Only 67.6% of them were admitted on the first two days of the illness vs. 81.6% of younger patients. Nearly 62% of them were deeply soporous or comatose on admission (Mathew-Lawson grade 3 and 4), while in the control group only 31% of patients had such severely altered mental status. Although glucose cerebrospinal fluid (CSF)/blood ratio tended to be lower in the elderly (0.09 vs 0.17) the difference did not reach statistical significance. Thrombocytes in the peripheral blood were lower in the elderly (113 x 10(9)/L vs. 148 x 10(9)/L, p < 0.05). When we compared laboratory findings in survivors and nonsurvivors from both groups, nonsurvivors had significantly lower glucose CSF/blood ratio (0.054 vs. 0.174, p = 0.008), and higher bilirubin levels in serum (27.9 vs 14.7, p = 0.003), but differences were more obvious in younger group of patients. Our results suggest that there is increased risk of death among elderly patients. It can be at least partially attributed to their later admittance to the hospital and because of that delayed start of the appropriate therapy and more severe conscious disturbances. All these factors contribute to the greater case-fatality ratio observed in elderly patients with pneumococcal meningitis.


Assessment of dementia flow chart approach to clinical diagnosis. With life spans increasing the number of demented people rises steeply which requires proper diagnosis, particularly in the early stage of the disease. Although the majority of patients suffering from dementia have Alzheimer's disease characterized by slowly progressive course, searching for treatable causes will enable effective support to a certain number of patients. Presence of only one clinical symptom and/or sign, like the memory impairment is not sufficient for diagnosing dementia owing to its nonspecificity. Proper diagnosis depends upon one's familiarity with the criteria, clinical features, associated symptoms and the natural course of the disease as well as a wide variety of different etiological factors manifested by the similar clinical picture. In spite of certain shortcomings, the flow chart system approach of diagnostic procedure is intended for more accurate and less time consuming assessment of the clinical diagnosis of dementia. Understanding and familiarity with different aspects of the clinical picture lie behind the rational approach and selection of various laboratory methods and techniques to confirm or exclude presence of the disease for the benefit of patients and to avoid unnecessary wasting of considerable amounts of financial resources.


Locked-in syndrome.

Locked-in syndrome. A patient, young fisherman, with a locked-in syndrome is reported, in whom intact consciousness, quadriplegia of spastic type, voluntary eye blinking, (de)sursumvergence and anarthria were observed. Thrombosis of the basilar artery and slightly disturbed bioelectrogenesis of the cerebral cortex were proved by clinical examination. The patient died after 41 days. At the autopsy thrombosis a. basilaris and ventrobasal pontine infarction were confirmed. Differential diagnosis of this and similar syndromes has been discussed.


Evidence from epileptic patients on the role of the right hemisphere in REM sleep. Eight highly selected epileptic patients found to have lasting right temporal or temporooccipital rhythmic focal activity during night sleep were reassessed following remission which occurred between a few days and 7 years after the initial finding. In polysomnographic EEG recording, when the patients had extensive epileptic discharges in the right temporal and temporo-occipital regions, a prolonged period of REM sleep occurred (34.1 +/- 7.06% of total sleep time). Following remission, however, disproportion had returned to normal (20.4 +/- 1.75%). These results confirm that intensive focal epileptic activity in the temporal and temporooccipital regions of the right hemisphere during sleep facilitate REM sleep thus indicating a dominant role for the right hemisphere in REM sleep and dreaming.


Rating scales and computed tomography in multi-infarct dementia. Forty patients who fulfilled the DSM-III-R criteria for multi-infarct dementia and had a score of 7 points or more on Hachinski ischemia score (HIS) were analyzed with the purpose to correlate the rating scales and CT scans. Among the examined patients there were 32 women with the average age of 68.5 +/- 9.8 years and 8 men with the average age of 68.8 +/- 10.4 years. No significant difference between sex in relation to Folstein Mini-mental state examination (MMSE), Gottfries-Brane-Steen scale (GBS) and Sandoz clinical assessment-geriatric scale (SCAG) was found. There is no correlation of GBS and SCAG on MMSE. With regression analysis a good correlation was found between GBS and SCAG, and we suggest that in such studies only one of these two scales is sufficient. CT abnormalities were found in about 77% of examined patients without difference according to sex. But, GBS score demonstrated greater disability among MID patients with abnormal CT scans than in MID patients with normal CT scans. In medical history of male MID patients completed stroke was significantly more common than among women, while the female MID patients had in their history significantly more frequent transient ischemic attack (TIA). This finding should be checked in a greater patient population. It is stressed that in everyday clinical practice it is necessary to use the diagnosis of multi-infarct dementia, e.g. to differentiate cerebral diseases according to etiology and pathogenesis.


Vimentin and glial fibrillary acidic protein expression in relation to neoplastic cell differentiation in glial tumors. In the present work the expression of vimentin and glial fibrillary acidic protein (GFAP) was determined in homotypic, transitional and heterotypic astroglial neoplastic areas and gemistocytes. The expression of these intermediate filament (IF) proteins within oligodendroglial neoplastic cells was determined as well. The intensity of vimentin and GFAP immunoreactivity as well as the number of immunoreactive cells within astroglial areas of different grades of differentiation were different. While there was no immunoreactivity within heterotypic areas, transitional areas and gemistocytes mainly show the same intensity of immunoreactivity and number of immunoreactive cells for both analyzed IF proteins. Within homotypic astroglial areas the number of GFAP positive cells and intensity of GFAP immunoreactivity were higher than the same vimentin parameters. It is well known that vimentin and GFAP may form heteropolymers both in vitro and in vivo. Transitions in vimentin/GFAP expression reflect not only normal development of astroglial cells but occur also with the induction of neoplastic process. Our results suggest that immunoreaction intensity and number of vimentin or GFAP immunoreactive cells correlates with the degree of differentiation of specific neoplastic cell populations. It is suggested that transitions in vimentin and GFAP expression occur in the course of neoplastic progression presumably by the modulation of their incorporation into the same IF system according to the degree of neoplastic cell differentiation.


Transcranial Doppler sonography in the acute cerebral ischemic disease. Transcranial Doppler sonography (TCD) is a noninvasive easy-performed method which can be repeated even in critically ill patients. It provides means to diagnose, quantify and monitor individual haemodynamic situation, and, probably, to predict the outcome of stroke. The purpose of this study was to evaluate the usefulness of the TCD in the examination of vascular lesions in the early phase of acute cerebral ischemic disease. The results showed different vascular disturbanies in otherwise homogenous group of patients. The TCD examinations were carried out in 30 patients in the early phase of stroke with presumed lesions in the middle cerebral artery (MCA) irrigation area. CT of the brain showed ischemic lesions of the MCA region in all patients. Additionally, blood flow velocity (BFV) was measured in posterior cerebral artery (PCA), anterior cerebral artery (ACA) and carotid siphon (CS) on both skull sides. On the basis of BFV in MCA, the patients could be apparently divided into three groups. In group I MCA the signal was absent. The patients from group 2 showed severely decreased, moderately decreased or nearly normal BFV in MCA. Group 3 consisted of three patients with increased BFV in MCA. In one of them, in whom a high degree of MCA stenosis was confirmed by angiography, BFV in MCA was extremely increased. The results have shown that TCD examinations in patients with acute stroke could provide additional information about vascular lesions, which are relevant to the course and, probably, the prognosis of the disease.


Brain amyloid in Alzheimer's disease--a new experimental model. New findings about the amyloid protein in the senile plaque and its precursor are presented. Special emphasis is laid on transgenic mice as a model for studying Alzheimer's disease in laboratory animals.


Cavernous angioma in cavernous sinus: case report of a rare location. A case of cavernous angioma situated in the left cavernous sinus is presented. Occasional difficulties in accessing correct preoperative diagnosis with computerized tomography and angiography were encountered. Intraoperative relationships of neural and vascular structures of the cavernous sinus and tumor mass are described. An "en mass" extirpation was achieved without further neurological damage. A possible origin of cavernous angioma from the 6th cranial nerve is discussed.


A hundred years of lumbar puncture.

A hundred years of lumbar puncture. In the years 1991 and 1992 the 100th anniversary of the announcement of the lumbar puncture method (1891) and 150th anniversary of the birth of its inventor Heinrich Irenaeus Quincke (1842) are celebrated. In the article a short review is given of the development of this method.


On the generation of form by the continuous interactions between cells and their extracellular matrix. The central issue of this essay is the problem of how multicellular organisms develop and maintain the complex architecture and intricate shape of tissues and organs. The concepts pattern formation, morphogenesis and differentiation are defined and discussed suggesting a distinction between processes that underlie uniformity (e.g. basic body plans) and those underlying inter- and intra-species variation. The initial stage of limb bone development--the formation of the mesenchymal condensation--is described in detail. On the basis of these data and many additional example from other developmental systems, the central role of continuous cell-ECM interactions in the generation of form is deduced. Evidence is provided as to the leading role of the mesenchymal-fibroblast-like cells in sculpturing tissue and organ architecture. It is proposed that a group of cells within their ECM, rather than the single cell, is the functional unit relevant to the generation of form. The continuous cell-ECM interactions lead to the generation of form not by a detailed obligate pathway, but rather by a process of 'selective stabilization' (Kirschner & Mitchison, 1986), i.e. a gradual organization into more stable structures, where existing structural configuration serve to increase the likelihood of certain configurations and reduce that of others. Data are quoted to support the notion that even cell division does not erase all the structural information imprinted in the cell. The role of the metazoan genome in morphogenesis is discussed in the light of the process of selective stabilization.


Life history and bioeconomy of the house mouse. 1. More is known about the western European house mouse, Mus (musculus) domesticus than any other non-human mammal. If laboratory and field information is combined, an extremely valuable understanding of the species' bioeconomy could be obtained. 2. The seven stages of mouse life-history are surveyed (up to birth, nest life, sex life, social structure, population statics and stability, senescence, and death), and the interactions between the changing phenotype and the environment are described. 3. These interactions can be used to build up a model of the opportunities and compromises which result in the fitness of individual mice. It is not yet possible to quantify such a model, but this should in principle be achievable.


The evolution of eutherian spermatozoa and underlying selective forces: female selection and sperm competition. We have examined sperm morphology and dimensions in Eutherian mammals. In most Eutherians, sperm heads are round or oval and spermatozoa have short tails (average sperm length about 65 microns; range = 33-121 microns). Rodents, however, clearly depart from the typical Eutherian pattern in that they show a broad array of head morphs and an extreme range of sperm dimensions (35-250 microns). In order to trace the evolutionary changes that rodent sperm have undergone, we have used phylogenetic relationships based on biogeographical, morphological, chromosomal and genic data, and we have superimposed onto them the information available on sperm traits. Analyses were carried out for five rodent groups on which enough information was available. The evolutionary trends which emerged from these studies have two main points in common: throughout evolution spermatozoa have become enlarged and morphologically more complex, and this process seems to have taken place independently in different lineages. A general model was developed which outlines the different evolutionary pathways that rodent sperm have undergone. The adaptive significance of the increase in head complexity and the elongation of the sperm tail remains obscure. We have integrated information from evolutionary, physiological and behavioural studies to address this issue. We argue that two main selective forces may have favoured these changes: female selection within the reproductive tract and sperm competition. The female tract represents a formidable barrier for spermatozoa and its provides an environment where numerous interactions take place. The extent of these barriers and the complexity of these poorly understood interactions suggest that females may be exercising a strong selection, which may enable them to favour particular types of spermatozoa or ejaculates from particular males. Throughout their evolution males must have evolved adaptations to overcome these barriers, and the conflicting interests of choosy females. Sperm competition is a potent evolutionary force among mammals, which has influenced not only the evolution of sperm numbers but also changes in sperm dimensions. Thus, sperm competition has favoured the elongation of the sperm tail, which has led to the attainment of faster swimming speed, an important factor when sperm from rival males compete to reach the ova first.


Light regulation of protein synthesis factor EF-G in pea chloroplasts. The activity of pea chloroplast elongation factor G (EF-G), a nuclear-coded protein required for the elongation cycle of chloroplast protein synthesis, is regulated in response to light. In pea seedlings germinated and grown under continuous white or red light, EF-G specific activity reaches a maximum between days 10 to 15, and then decreases. EF-G activity is almost undetectable in extracts from dark-grown seedlings. When 13-day dark-grown pea seedlings are transferred to light, EF-G specific activity reaches a higher value after 2 to 3 days than observed in seedlings grown under continuous light. The small and large subunits of ribulose bisphosphate carboxylase continue to accumulate after EF-G specific activity has reached maximum levels. Cytoplasmically synthesized components of the chloroplast protein synthetic apparatus, such as EF-G, may help coordinate cytoplasmic and nuclear events with chloroplast gene expression during light-induced chloroplast differentiation.


An mRNA that specifically accumulates in maize roots delineates a novel subset of developing cortical cells. A near full-length cDNA clone (pZRP3) corresponding to an mRNA that accumulates specifically in roots of maize was isolated. The ZRP3 mRNA is ca. 600 nucleotides in length. The amino acid sequence of the predicted polypeptide is rich in leucine (16%), proline (11%), and cysteine (8.5%). The zrp3 gene appears to be expressed exclusively in roots, whereas other ZRP3-related genes are expressed in additional organs of the maize plant. In situ hybridization shows that ZRP3 mRNA accumulation is largely confined to the cells of the cortical ground meristem. Furthermore, accumulation of this mRNA occurs within a distinct subset of cortical cells, the inner three to four cell layers.


RNP-T, a ribonucleoprotein from Arabidopsis thaliana, contains two RNP-80 motifs and a novel acidic repeat arranged in an alpha-helix conformation. We have isolated a 1148 bp long cDNA clone encoding an RNA-binding protein in Arabidopsis. Several partial cDNA clones were isolated by screening an Arabidopsis lambda gt11 expression library for the binding of DNA. One of these clones was used as a probe to isolate a full-length clone. The 329 amino acid protein, termed RNP-T, contains in its carboxy terminus two adjacent RNP-80 motifs, a previously described 80 amino acid long conserved putative RNA-binding domain. Each RNP-80 motif includes both consensus short sequences, RNP1 and RNP2, which are separated by 33 amino acids. We have identified an acidic domain of 54 amino acids, which is located amino-terminal to the RNP-80 motifs. Seven tandem repeats of a hexamer are present within this domain. This acidic domain has a potential alpha-helix conformation. We propose that the acidic patch might play a role in protein-protein interaction.


Analysis of the gibberellin-responsive promoter of a cathepsin B-like gene from wheat. A wheat gene (A121) encoding a protein with sequence similarity to mammalian cathepsin B is regulated by gibberellic acid (GA) in aleurone layers of germinating grains. To analyse the mechanism of A121 regulation, its promoter was fused to the beta-glucuronidase reporter gene (GUS) and introduced by micro-projectile bombardment into aleurone layers of oat. With 2.3 kb of promoter sequence, the GUS expression was enhanced by GA treatment. This effect was reversed by abscisic acid (ABA). This result showed for A121, like the alpha-amylase genes, that the regulation by GA and ABA was at the level of transcription. The GA responsiveness of the promoter was retained with as little as 276 bp of promoter sequence. Sequence comparison with a GA responsive promoter of an alpha-amylase gene identified the conserved element GCAACGGCAACGATGG which is required intact for full expression of both promoters. However, there was no identifiable similarity in the cathepsin-like promoter with the GA-responsive element of alpha-amylase promoters with the consensus sequence TAACAAA, suggesting that GA affects more than one mechanism of transcriptional control.


Structure and expression of the lignin O-methyltransferase gene from Zea mays L. The isolation and characterization of cDNA and homologous genomic clones encoding the lignin O-methyltransferase (OMT) from maize is reported. The cDNA clone has been isolated by differential screening of maize root cDNA library. Southern analysis indicates that a single gene codes for this protein. The genomic sequence contains a single 916 bp intron. The deduced protein sequence from DNA shares significant homology with the recently reported lignin-bispecific caffeic acid/5-hydroxyferulic OMTs from alfalfa and aspen. It also shares homology with OMTs from bovine pineal glands and a purple non-sulfur photosynthetic bacterium. The mRNA of this gene is present at different levels in distinct organs of the plant with the highest accumulation detected in the elongation zone of roots. Bacterial extracts from clones containing the maize OMT cDNA show an activity in methylation of caffeic acid to ferulic acid comparable to that existing in the plant extracts. These results indicate that the described gene encodes the caffeic acid 3-O-methyltransferase (COMT) involved in the lignin bio-synthesis of maize.


The tRNA(Tyr) multigene family of Nicotiana rustica: genome organization, sequence analyses and expression in vitro. Tobacco tRNA(Tyr) genes are mainly organized as a dispersed multigene family as shown by hybridization with a tRNA(Tyr)-specific probe to Southern blots of Eco RI-digested DNA. A Nicotiana genomic library was prepared by Eco RI digestion of nuclear DNA, ligation of the fragments into the vector lambda gtWES.lambda B and in vitro packaging. The phage library was screened with a 5'-labelled synthetic oligonucleotide complementary to nucleotides 18 to 37 of cytoplasmic tobacco tRNA(Tyr). Eleven hybridizing Eco RI fragments ranging in size from 1.7 to 7.5 kb were isolated from recombinant lambda phage and subcloned into pUC19 plasmid. Four of the sequenced tRNA(Tyr) genes code for the known tobacco tRNA1(Tyr) (G psi A) and seven code for tRNA2(Tyr) (G psi A). The two tRNA species differ in one nucleotide pair at the basis of the T psi C stem. Only one tRNA(Tyr) gene (pNtY5) contains a point mutation (T54-->A54). Comparison of the intervening sequences reveals that they differ considerably in length and sequence. Maturation of intron-containing pre-tRNAs was studied in HeLa and wheat germ extracts. All pre-tRNAs(Tyr)--with one exception--are processed and spliced in both extracts. The tRNA(Tyr) gene encoded by pNtY5 is transcribed efficiently in HeLa extract but processing of the pre-tRNA is impaired.


Cloning of ndhK from soybean chloroplasts using antibodies raised to mitochondrial complex I. A soybean shoot cDNA expression library was screened with polyclonal antibodies raised against red beet complex I and several clones were identified. One clone, consisting of a 1 kb insert, was fully sequenced. The sequence of 1025 bp was found to contain two extended open reading frames and the proteins encoded were identified as the ndhK and ndhJ products of the chloroplast genome. Nuclear, mitochondrial and chloroplast DNA was isolated and probed with a ndhK-specific probe. The chloroplast DNA contained a single copy of the cloned insert. With nuclear DNA, positively hybridising bands of 1.2, 2.7 and 3.2 kb were observed indicating that at least one gene homologous to ndhK of the chloroplast genome, is also present in the nucleus. The mitochondrial DNA did not hybridise with the ndhK probe. Western analysis of thylakoid proteins with the mitochondrial complex I antibodies revealed several bands. It is suggested that soybean contains two copies of the ndhK gene, one, on the plastid genome, coding for a subunit of a chloroplast NAD(P)H dehydrogenase, and the other, in the nucleus, coding for a subunit of mitochondrial complex I.


Protein-binding to reiterated motifs within the wheat rRNA gene promoter and upstream repeats. The binding of specific proteins in wheat nuclear extracts to the -34 to +10 region of the wheat ribosomal RNA gene promoter has been detected. DNase I footprinting revealed two binding regions, one spanning the transcription initiation site from -7 to +2, and a larger one further upstream from around -29 to -15. An oligonucleotide made to the -30 to -14 promoter region binds a protein in a sequence-specific manner. This protein also binds specifically to sites with related sequences further upstream in the promoter and in the associated intergenic spacer repeats. A consensus sequence, CATGG--GC-AAAAC, was defined from the different binding sites of the protein. These results, taken together with in vivo results on the enhanced activity of nucleoli containing ribosomal RNA genes with more intergenic spacer repeats, endorse the hypothesis that ribosomal RNA gene transcription is regulated by the extent of binding of a protein to many sites upstream of the transcriptional start site.


Translation controls the expression level of a chimaeric reporter gene. Transcriptional and translational fusions between the reading frame of the beta-D-glucuronidase gene (gusA) and the 2' as well as the 1' promoter of mannopine synthase (mas), a TR locus of Agrobacterium tumefaciens, were made. The expression of these constructs was studied in the transgenic F1 offspring of independent tobacco transformants at the protein level by assaying for GUS activity and western blot analysis of the GUS protein and at the steady-state mRNA level. In leaves, stems and roots no correlation was found between steady-state levels of GUS mRNA and enzyme activity. In older tissues significantly higher GUS activities were found. This is explained by the stable character of the GUS protein together with an accumulation of protein upon ageing. Three to ten times higher GUS activities were found for in vitro grown plants than for greenhouse-grown plants of the same offspring, despite similar levels of GUS mRNA. Roots from in vitro grown plants display three to ten times higher GUS activities than stems and leaves. In transgenic plants grown in vitro, containing a translational fusion with two AUGs in phase, the initiation of translation in leaf material occurred at both AUGs. Initiation of translation at the first AUG, however, was ten times more frequent. In contrast, initiation in roots from in vitro grown plants occurred exclusively at the second AUG.


Characterization of nucleotide sequences that interact with a nuclear protein fraction in rRNA gene of Vicia faba. A certain nucleotide sequence in the promoter region of Vicia faba rRNA genes that specifically binds to a nuclear protein fraction has been identified by using a gel retardation assay and DNase I footprinting technique. The binding site of this protein fraction is located about 60 bp upstream from the initiation site of the pre-rRNA transcript. This location does not correspond with previously reported results on maize rRNA genes. However, both of the binding sites share a bi-partite consensus sequence, TAT-G(N)xCAGG. Methylation interference experiments show that two G residues in TATG and the complementary strand of CAGG are important for specific DNA-protein interaction. Furthermore, competition analyses using point-mutated synthetic DNAs show that two G residues in CAGG are essential for this interaction. Similar sequences are found in promoter regions of other plant and animal rRNA genes. We suggest that these sequences may be a cis-control element commonly involved in rRNA transcription.


The expression of a rab-related gene, rab18, is induced by abscisic acid during the cold acclimation process of Arabidopsis thaliana (L.) Heynh. We have isolated a rab-related (responsive to ABA) gene, rab18 from Arabidopsis thaliana. The gene encodes a hydrophilic, glycine-rich protein (18.5 kDa), which contains the conserved serine- and lysine-rich domains characteristic of similar RAB proteins in other plant species. The rab18 mRNA accumulates in plants exposed to low temperature, water stress or exogenous ABA but not in plants subjected to heat shock. This stress-related accumulation of the rab18 mRNA is markedly decreased in the ABA-synthesis mutant aba-1, the ABA-response mutant abi-1 or in wild-type plants treated with the carotenoid synthesis inhibitor, fluridone. Exogenous ABA treatment can induce the rab18 mRNA in the aba-1 mutant but not in the abi-1 mutant. These results provide direct genetic evidence for the ABA-dependent regulation of the rab18 gene in A. thaliana.


Rhizobial lipo-oligosaccharides: answers and questions. Rhizobium bacteria produce certain lipo-oligosaccharides (modified chitin oligomers) after induction of nodulation (nod) gene transcription by the host plant. The function of the rhizobial nod genes in the biosynthesis of these lipo-oligosaccharides, focusing on their host specific aspects, is discussed. The lipo-oligosaccharides can elicit various responses in the host plants, like the formation of pre-infection threads and nodule meristems. Speculating on their function in plant morphogenesis the question is raised: do the rhizobial lipo-oligosaccharides resemble unknown plant signal molecules?


Presence of an N-terminal presequence in the PsaI protein of the photosystem I complex in the filamentous cyanobacterium Anabaena variabilis ATCC 29413. The psaI gene encoding the 5.2 kDa protein component (PsaI) of the photosystem I complex was cloned from the cyanobacterium Anabaena 29413. The gene is present in single copy in this cyanobacterial genome. The nucleotide sequence of a 500 bp region of the cloned DNA revealed the presence of an open reading frame encoding a 46 amino acid long polypeptide. The N-terminal 11 residues are absent in the mature polypeptide and thus represents the first identified cleavable presequence on the PsaI protein. We suggest that this presequence directs the N-terminus of the protein to the thylakoid lumen.


Identification of a second psaC gene in the cyanobacterium Synechocystis sp. PCC6803. The psaC gene encodes the 9 kDa protein subunit of photosystem I that carries the iron-sulfur centers FA and FB. The paper describes the sequence and transcription analysis of a second psaC gene (psaC2) from Synechocystis sp. PCC6803. ThepsaC2 gene is transcribed into an abundant monocistronic mRNA. The deduced amino acid sequence of PSI-C2 is very similar to the protein sequences from two other cyanobacteria (Synechococcus sp. PCC7002 and Nostoc sp. PCC8009). In contrast, the recently isolated psaC1 gene is not transcribed and the PSI-C1 amino acid sequence shows the highest similarity to the proteins from higher plants.


Molecular cloning of a novel phytochrome gene of the moss Ceratodon purpureus which encodes a putative light-regulated protein kinase. The phytochrome gene (phyCer) of the moss Ceratodon purpureus was isolated and characterized. phyCer is composed of three coding exons: exon I of 2035 bp, exon II of 300 bp and exon III of 1574 bp. The deduced polypeptide encoded by exon I and II exhibits substantial sequence homology to the conserved NH2-terminal chromophore domain of known phytochromes. In contrast, the COOH-terminal polypeptide encoded by exon III shows no sequence homology to any phytochrome molecule. phyCer most likely represents a single-copy gene and is expressed in a light-independent manner. From the DNA sequence analysis it can be deduced that the PhyCer polypeptide is composed of 1303 amino acids (including the starting Met) which predicts a molecular mass for PhyCer of 145 kDa. The polypeptide encoded in exon III exhibits striking homology within the 300 carboxy-terminal amino acids to the catalytic domain of protein kinases. The carboxy terminus of PhyCer was found to be most homologous to protein-tyrosine kinases of Dictyostelium discoideum and to the products of retroviral oncogenes which belong to the Raf-Mos serine/threonine kinase family. From the hydropathy profile PhyCer appears to be a soluble protein. The predicted structure suggests that PhyCer represents a soluble light-sensor protein kinase which is linked with a cellular phosphorylating cascade.


Expression of the pea metallothionein-like gene PsMTA in Escherichia coli and Arabidopsis thaliana and analysis of trace metal ion accumulation: implications for PsMTA function. The PsMTA gene from pea (Pisum sativum) shares similarity with metallothionein (MT) genes and related sequences have also been isolated from a number of other higher-plant species. The proteins encoded by these genes have not yet been purified from plants and their functions remain unclear although, by analogy to MT, roles in the metabolism and detoxification of metal ions have been proposed. By contrast, correlation between transcript abundance and Fe availability has led to an alternative proposal that these genes are involved in mechanisms of Fe efficiency. Phenotypic effects of constitutive PsMTA expression were examined in Escherichia coli and Arabidopsis thaliana. Copper accumulation by E. coli cells expressing recombinant PsMTA protein was approximately 8-fold greater than in control cells. No significant effects on the accumulation of Zn or Cd were detected. In segregating A. thaliana progeny, derived from a transgenic F1 parent containing the PsMTA gene under the control of a CaMV 35S promoter, 75% of individuals accumulated more Cu (several-fold in some plants) than untransformed, control plants. These data suggest that PsMTA protein binds Cu in planta and that uncoupled (constitutive) expression of the PsMTA gene causes enhanced Cu accumulation. Roots of P. sativum plants grown under conditions of low Fe availability showed elevated activity of root surface Fe(III) reductase and accumulated more Cu than roots of plants grown in an Fe-supplemented solution. Changes in the expression of MT-like genes, coincident with changes in Fe availability, are consistent with a role in Cu homoeostasis.


Glyphosate tolerance of cultured Corydalis sempervirens cells is acquired by an increased rate of transcription of 5-enolpyruvylshikimate 3-phosphate synthase as well as by a reduced turnover of the enzyme. Cell cultures of Corydalis sempervirens, tolerant to the herbicide glyphosate, have a 30-40-fold increased level of the herbicide's target enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, a ten-fold enhanced level of the corresponding mRNA but no amplification of the gene (Holländer-Czytko et al., Plant Mol Biol 11 (1988) 215-220). The increase at the transcriptional level is due to a higher rate of transcription of the gene, which was observed in run-off transcription assays with isolated nuclei. The further amplification at the protein level is the result of stabilization of the enzyme by the herbicide. In the presence of glyphosate the half-life of EPSP synthase was doubled leading to higher levels of both protein and enzyme activity. Overproduction of the enzyme in adapted cultures is stable at the transcriptional level, as cells from adapted cultures grown in the absence of glyphosate for three years still display an about ten-fold higher enzyme activity and transcript level than non-adapted cultures.


Factors influencing Agrobacterium-mediated transient expression of gusA in rice. Transient expression of GUS in rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens was characterized using binary vectors containing gusA genes that express minimal (pKIWI105 and pCNL1) or no (p35S-GUS-INT and pCNL56) GUS activity in bacteria. Four-day old seedlings obtained from seeds or immature embryos of rice were cut into shoot, root, and seed remnants and inoculated with various strains of A. tumefaciens. Transient GUS expression events were quantitated histochemically by determining the frequency of explants exhibiting blue spots indicative of GUS at four to six days after cocultivation with A. tumefaciens. A. tumefaciens strains that did not contain the gusA gene (At643) or a Ti-plasmid (At563 and At657) did not elicit any blue staining characteristic of GUS activity. Several parameters were important in obtaining efficient transient expression of GUS in rice mediated by A. tumefaciens. The growth regulator 2,4-D inhibited GUS expression if present during the seed germination period, but the presence of 6 mg/l 2,4-D during cocultivation of the explants with A. tumefaciens slightly enhanced GUS expression efficiency. All 21 rice cultivars tested expressed GUS after co-cultivation with A. tumefaciens. The GUS expression frequency was highest amongst the indica cultivars. The frequencies of GUS expression in japonica cultivars and in Oryza glaberrima cultivars (grown primarily in Africa) were generally one-half to one-third the level found for indica varieties. Leaf explants were more susceptible to A. tumefaciens-facilitated GUS expression than were roots or seed remnants. The vir genes of an agropine-type Ti-plasmid of A. tumefaciens were most effective in directing transient GUS expression in rice, whereas those of a nopaline-type and an octopine-type plasmid were less effective. We have also found that the frequency of transient expression of GUS was higher with pBIN19 as the precursor cloning vector than with pEND4K as the precursor cloning vector. Reasons for differences in effectiveness of these binary vectors are discussed. Using the conditions described here, A. tumefaciens-mediated frequencies of transient GUS expression in four-day old shoots of several rice cultivars were routinely in excess of 50%.


Mutational analysis of pea lectin. Substitution of Asn125 for Asp in the monosaccharide-binding site eliminates mannose/glucose-binding activity. As part of a strategy to determine the precise role of pea (Pisum sativum) lectin, Psl, in nodulation of pea by Rhizobium leguminosarum, mutations were introduced into the genetic determinant for pea lectin by site-directed mutagenesis using PCR. Introduction of a specific mutation, N125D, into a central area of the sugar-binding site resulted in complete loss of binding of Psl to dextran as well as of mannose/glucose-sensitive haemagglutination activity. As a control, substitution of an adjacent residue, A126V, did not have any detectable influence on sugar-binding activity. Both mutants appeared to represent normal Psl dimers with a molecular mass of about 55 kDa, in which binding of Ca2+ and Mn2+ ions was not affected. These results demonstrate that the NHD2 group of Asn125 is essential in sugar binding by Psl. To our knowledge, Psl N125D is the first mutant legume lectin which is unable to bind sugar residues. This mutant could be useful in the identification of the potential role of the lectin in the recognition of homologous symbionts.


Seed-specific repression of GUS activity in tobacco plants by antisense RNA. beta-Conglycinin, the 7S storage protein of soybean, is expressed only in seeds and is regulated predominantly by gene transcription [5]. We applied an antisense strategy to modify expression of a beta-glucuronidase (uidA or gusA) gene in seeds using a promoter from a beta-conglycinin gene. Transgenic tobacco plants harboring the gusA gene under the control of the CaMV 35S promoter were retransformed with a gene construct comprising the beta-conglycinin promoter fused to the gusA gene in the antisense orientation. Double transformants were regenerated and transformation was confirmed by Southern blot hybridization. Seed-specific repression of GUS activity was observed in lines containing high copy numbers of the antisense gusA transgene. Suppression of GUS activity was correlated with the amounts of (-) sense gusA transcript detected and concomitantly with a decrease in gusA transcript levels. Furthermore, the amount of suppression of GUS activity was greatest during mid to late stages of seed development, when expression of the alpha' promoter is high. These results indicate that suppression of GUS activity is due to expression of the antisense gene.


Multiple copies of virG enhance the transient transformation of celery, carrot and rice tissues by Agrobacterium tumefaciens. In an effort to improve the T-DNA-mediated transformation frequency of economically important crops, we investigated the possible enhancement effect of multiple copies of virG genes contained in Agrobacterium tumefaciens strains upon the transient transformation of celery, carrot and rice tissues. Four days after A. tumefaciens infection, we performed histochemical beta-glucuronidase (GUS) assays to determine the frequency of transient transformation of calli from celery and carrot, and explants from rice and celery. Additional copies of octopine- and agropine-type virG genes in A. tumefaciens strains containing an agropine-type Ti-plasmid enhanced the frequency of transient transformation of celery and rice. This enhancement ranged from 25% to five-fold, depending upon the source of the virG gene and the plant tissues inoculated. For both rice and celery, we observed a greater enhancement of transformation using A. tumefaciens strains containing additional copies of an octopine-type virG gene than with strains harboring additional copies of an agropine-type virG gene. Multiple copies of virG genes contained in A. tumefaciens strains harboring a nopaline-type Ti-plasmid had a smaller enhancing effect upon the transformation of celery tissues, and no enhancing effect upon the transformation of rice. In contrast, we obtained a three-fold increase in the transient transformation frequency of carrot calli using an A. tumefaciens strain harboring a nopaline-type Ti-plasmid and additional copies of an octopine-type virG gene. Our results show that multiple copies of virG in A. tumefaciens can greatly enhance the transient transformation frequency of celery, carrot and rice tissues, and that this enhancement is influenced by both the type of Ti-plasmid harbored by A. tumefaciens and by the infected plant species.


Nucleosomal structure and histone H1 subfractional composition of pea (Pisum sativum) root nodules, radicles and callus chromatin. Higher-order packaging of DNA in chromatin structures could be an essential step in the complex chain of events leading to activation/repression of eukaryotic gene expression. With the goal to investigate this aspect of transcriptional regulation of plant genes involved in symbiotic interactions between legumes and rhizobia we analyze here the molecular parameters of chromatin structure in functioning root nodules, callus and radicles of pea. Morphological intactness and the typical nucleosomal organization are preserved in purified nuclei isolated from all three sources. The calculated values of nucleosomal repeat changed from 185 +/- 5 bp in the nuclei of radicles to 168 +/- 5 bp and 195 +/- 6 bp in nodules and callus respectively. The observed changes are due to alterations in linker DNA lengths. The core histones are identical in all cases, but the subfractional composition of H1 linker histone is subjected to quantitative alterations. The most pronounced is the several-fold increase in content of the lowest-molecular-weight subfraction H1-6 which takes place during nodule development.


Cloning and transcription analysis of the ndh(A-I-G-E) gene cluster and the ndhD gene of the cyanobacterium Synechocystis sp. PCC6803. The plastid DNA of higher plants contains eleven reading frames that are homologous to subunits of the mitochondrial NADH-ubiquinone oxidoreductase (complex I). The genes are expressed, but a plastid NAD(P)H dehydrogenase has not yet been isolated and the function of the enzyme in plastid metabolism is unknown. Cyanobacteria also contain a NADH dehydrogenase that is homologous to the mitochondrial complex I. The enzyme is sensitive to rotenone and is located on the cytoplasmic and the thylakoid membrane. We report here the sequence of five subunits (ndhA, -I, G, -E and -D) of the NADH dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC6803. As in plastid DNA, the genes ndh(A-I-G-E) are clustered and probably constitute an operon. The ndhD gene is associated with a gene encoding an iron-sulphur protein of photosystem I (psaC) as in plastid DNA. In contrast to the situation in plastids, psaC and ndhD are not cotranscribed but transcribed from opposite strands. The deduced amino acid sequence of the cyanobacterial polypeptides is more similar to the corresponding plastid (40-68% identity) than to the corresponding mitochondrial subunits (17-39% identity). Thus, the cyanobacterial NADH-dehydrogenase provides a prokaryotic model system which is more suitable to genetic analysis than the enzyme of plastids.


Adenine depurination and inactivation of plant ribosomes by an antiviral protein of Mirabilis jalapa (MAP). Mirabilis antiviral protein (MAP) is a single-chain ribosome-inactivating protein (RIP) isolated from Mirabilis jalapa L. It depurinates the 28S-like rRNAs of prokaryotes and eukaryotes. A specific modification in the 25S rRNA of M. jalapa was found to occur during isolation of ribosomes by polyacrylamide/agarose composite gel electrophoresis. Primer extension analysis revealed the modification site to be at the adenine residue corresponding to A4324 in rat 28S rRNA. The amount of endogenous MAP seemed to be sufficient to inactivate most of the homologous ribosomes. The adenine of wheat ribosomes was also found to be removed to some extent by an endogenous RIP (tritin). However, the amount of endogenous tritin seemed to be insufficient for quantitative depurination of the homologous ribosomes. Endogenous MAP could shut down the protein synthesis of its own cells when it spreads into the cytoplasm through breaks of the cells. Therefore, we speculate that MAP is a defensive agent to induce viral resistance through the suicide of its own cells.


RNase X2, a pistil-specific ribonuclease from Petunia inflata, shares sequence similarity with solanaceous S proteins. Petunia inflata, a species with gametophytic self-incompatibility, has previously been found to contain a large number of ribonucleases in the pistil. The best characterized of the pistil ribonucleases are the products of the S alleles, the S proteins, which are thought to be involved in self-incompatibility interactions. Here we report the characterization of a gene encoding another pistil ribonuclease of P. inflata, RNase X2. Degenerate oligonucleotides, synthesized based on the amino-terminal sequence of RNase X2, were used as probes to isolate cDNA clones, one of which was in turn used as a probe to isolate genomic clones containing the gene for RNase X2, rnx2. The deduced amino acid sequence of RNase X2 shows 42% to 71% identity to the 20 solanaceous S proteins reported so far, with the highest degree of similarity being to S3 and S6 proteins of Nicotiana alata. The cDNA sequence predicts a leader peptide of 22 amino acids, suggesting that RNase X2, like S proteins, is an extracellular ribonuclease. Also, similar to the S gene, rnx2 is expressed only in the pistil, and contains a single intron comparable in size and identical in location to that of the S gene. However, rnx2 is not linked to the S locus, and, in contrast to the highly polymorphic S gene, it is monomorphic. The possible biological function of RNase X2 is discussed.


Arabidopsis thaliana carbonic anhydrase: cDNA sequence and effect of CO2 on mRNA levels. A full-length cDNA clone encoding carbonic anhydrase was isolated from an Arabidopsis thaliana (Columbia) leaf library. Comparison of the derived amino acid sequence obtained from this clone with those of pea and spinach reveals a considerable degree of identity. The carbonic anhydrase cDNA was used to probe the level of RNA encoding this protein in the leaves of plants grown in elevated CO2 (660 ppm). We have found that under these conditions the steady-state level of carbonic anhydrase mRNA was increased in comparison with control plants grown in normal atmospheric concentrations of CO2 (330 ppm). This raises the intriguing possibility that there exists in higher plants a mechanism for perceiving and responding to changes in environmental CO2 concentrations at the genetic level.


Differential expression of a chimeric CaMV-tomato proteinase Inhibitor I gene in leaves of transformed nightshade, tobacco and alfalfa plants. The open reading frame and terminator region of a wound-inducible tomato Inhibitor I gene, regulated by the CaMV 35S promoter, was stably integrated into the genomes of nightshade (Solanum nigrum), tobacco (Nicotiana tabacum), and alfalfa (Medicago sativa), using an Agrobacterium-mediated transformation system. The expression of the foreign Inhibitor I gene in leaves of each species was studied at the mRNA and protein levels. The levels of Inhibitor I protein present in leaves of each species correlated with the levels of mRNA. The average levels of both mRNA and Inhibitor I protein were highest in leaves of transgenic nightshade plants (over 125 micrograms of Inhibitor I per g tissue), less in tobacco plants (about 75 micrograms/g tissue), and lowest in leaves of transgenic alfalfa plants (below 20 micrograms/g tissue). Inhibitor I protein was observed in all tissues throughout transgenic plant species, but inhibitor concentration per gram of tissue was 2-3 times higher in young developing leaf tissues and floral organs. The differences in the expression of the CaMV-tomato Inhibitor I gene among the different plant genera suggests that either the rate of transcription of the foreign gene or the rate of degradation of the nascent Inhibitor I mRNA varies among genera. Using electron microscopy techniques, the newly synthesized pre-pro-Inhibitor I protein was shown to be correctly processed and stored as a mature Inhibitor I protein within the central vacuoles of leaves of transgenic nightshade and alfalfa. The results of these experiments suggest that maximal expression of foreign proteinase inhibitor genes, and perhaps other foreign defense genes, may require gene constructs that are fashioned with promoters and terminators that allow maximum expression in the selected plant species.


A DNA polymerase from maize axes: its purification and possible role. Three different DNA polymerase activities can be resolved by passing a protein extract from 24 h imbibed maize axes through DEAE-cellulose. These activities have been numbered 1, 2 and 3, according to their elution order. One of them, DNA polymerase 2, elutes at 100-120 mM phosphates. This enzyme was further purified by passing it through Heparin-Sepharose, Sephacryl S-300 and DNA cellulose. Purification was nearly 5000-fold. The enzyme needs Mg2+, is stimulated by K+, has an optimum pH of 7.0 and its optimum temperature is 30-37 degrees C. Specific inhibitors for different types of polymerases, such as aphidicolin, dideoxythymidine triphosphate and N-ethyl maleimide, gave intermediate values of inhibition, making impossible the definition of the type of enzyme purified by its inhibitory pattern. SDS-PAGE indicated the presence of several bands of molecular masses of 28-40, 56 and 15 kDa. Most of these bands could be visualized when proteins from crude extracts were analyzed by western blot, using an antibody against calf thymus DNA polymerase alpha. A high molecular mass (around 500 kDa) was calculated by western blot of native gels using the same antibody. Finally, specific activity of this enzyme increased 100-fold during maize germination whereas polymerase 3 virtually did not increase. Furthermore, immunoprecipitation experiments with the antipolymerase alpha-antibody showed a decrease in DNA polymerase activity by 70%. The possibility that polymerase 2 is a replicative enzyme is discussed.


Mitochondrial rps14 is a transcribed and edited pseudogene in Arabidopsis thaliana. We have isolated and analysed a 2 kb region of the mitochondrial genome of Arabidopsis thaliana (Columbia) showing a high level of nucleotide identity with the mitochondrial (mt) rps14 small-subunit ribosomal protein gene from Oenothera berteriana and Vicia faba, as well as with an open reading frame (ORF) located upstream of the nad3 locus in O. berteriana. The rps14 locus is present as a single copy in the A. thaliana mt genome and has a translational stop codon located near the initiation codon, as well as a deletion of one nucleotide that disturbs the coding sequence. The cloning and sequencing of nine amplified mt rps14 cDNAs clearly demonstrated that this gene is transcribed and that the mRNA precursors are edited at three positions, all involving C-to-U conversions. No editing events changing the stop codon and restoring the correct coding sequence were witnessed within the 9 individual cDNA clones. Therefore, we conclude that the single rps14 sequence of the mitochondrial genome from A. thaliana is in fact a pseudogene that is transcribed and edited but not translated.


Analysis of differential accumulation of winged bean Kunitz chymotrypsin inhibitor mRNA species by a sequence-specific termination method. Winged bean Kunitz chymotrypsin inhibitor (WCI) is encoded by a multigene family and accumulation of its mRNA is restricted in mid-maturation stage seeds and tuberous roots. In this paper, we analyzed the accumulation of mRNA derived from each WCI gene using a novel method: sequence-specific termination analysis. The results demonstrated that the accumulation of each WCI mRNA was differentially regulated in winged bean plants.


Primary structure of the Synechococcus PCC 7942 PAPS reductase gene. The structural gene encoding a thioredoxin-dependent 5'-phosphoadenylyl sulphate (PAPS) reductase (EC 1.8.4.-) from cyanobacterium Synechococcus PCC 7942 ('Anacystis nidulans') was detected by heterologous hybridization with the cysH gene from Escherichia coli K12. The cyanobacterial gene (further called par gene) comprised 696 nt which are 57.8% homologous to the enterobacterial gene. The putative open reading frame encoded a polypeptide consisting of 232 amino acid residues (deduced molecular weight 26,635) which showed significant homologies to the polypeptide from E. coli (50.8%) and to the polypeptide from Saccharomyces cerevisiae (30.3%). A single cysteine located at the C-terminus of the polypeptide of E. coli (Cys239) was conserved in Synechococcus. Conservation of this cysteinyl residue seems indispensable for catalysis. Complementation of a cysH-deficient mutant of E. coli by the cyanobacterial gene indicated that the cloned DNA is the structural gene of the PAPS reductase.


The psbL gene from bell pepper (Capsicum annuum): plastid RNA editing also occurs in non-photosynthetic chromoplasts. We have determined the nucleotide sequence of the plastid psbL gene from bell pepper. This gene has an ACG as a first codon. Isolation of RNA from pepper leaves and ripe fruits and subsequent sequencing of the psbL cDNA revealed that this ACG codon is post-transcriptionally edited into an AUG initiation codon in both leaves and fruits. These data indicate that the RNA editing machinery which exists in chloroplasts is still functional in chromoplasts from ripe fruits.


Isolation of genes abundantly expressed in rice anthers at the microspore stage. A cDNA library of rice (Oryza sativa L.) has been constructed from anthers at an early stage of pollen development. By differential screening of the library, we have isolated cDNAs of two genes, designated as Osc4 and Osc6, that are abundantly expressed in anthers containing tetrads and uninucleate microspores, but are not expressed in leaves or roots. Expression of Osc4 is absent in mature anthers, while Osc6 is present although the expression decays during pollen maturation. A comparison of the nucleotide and deduced amino acid sequences with those in data banks has not shown significant homology to known molecules.


New plant binary vectors with selectable markers located proximal to the left T-DNA border. Five new binary vectors have been constructed which have the following features: (1) different plant selectable markers including neomycin phosphotransferase (nptII), hygromycin phosphotransferase (hpt), dihydrofolate reductase (dhfr), phosphinothricin acetyl transferase (bar), and bleomycin resistance (ble); (2) selectable markers are located near the T-DNA left border and; (3) selectable marker and beta-glucuronidase (uidA) reporter genes are divergently organized for efficient expression, and can easily be removed or replaced as needed.


Nucleotide sequence of an osmotin-like cDNA induced in tomato during viroid infection. A cDNA library from tomato planta macho viroid (TPMV)-infected tomato was constructed. The library was screened at low stringency with a tobacco PR-R cDNA probe. An 832 bp cDNA from a mRNA present only in infected tissue was isolated. Nucleotide sequence showed high homology with the osmotin from both tobacco and tomato (NP24). This cDNA probably corresponds to the AP24 and P23 proteins previously described in tomato and induced upon fungal and viroid infection.


A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. A versatile gene expression cartridge and binary vector system was constructed for use in Agrobacterium-mediated plant transformation. The expression cartridge of the primary cloning vector, pART7, comprises of cauliflower mosaic virus Cabb B-JI isolate 35S promoter, a multiple cloning site and the transcriptional termination region of the octopine synthase gene. The entire cartridge can be removed from pART7 as a Not I fragment and introduced directly into the binary vector, pART27, recombinants being selected by blue/white screening for beta-galactosidase. pART27 carries the RK2 minimal replicon for maintenance in Agrobacterium, the ColE1 origin of replication for high-copy maintenance in Escherichia coli and the Tn7 spectinomycin/streptomycin resistance gene as a bacterial selectable marker. The organisational structure of the T-DNA of pART27 has been constructed taking into account the right to left border, 5' to 3' model of T-DNA transfer. The T-DNA carries the chimaeric kanamycin resistance gene (nopaline synthase promoter-neomycin phosphotransferase-nopaline synthase terminator) distal to the right border relative to the lacZ' region. Utilisation of these vectors in Agrobacterium-mediated transformation of tobacco demonstrated efficient T-DNA transfer to the plant genome.


Detection and temporal appearance of multiple copies of c-erb-B2 genes in advanced mammary carcinoma using fine needle biopsies and the polymerase chain reaction. Aspiration of tumor cells by the fine-needle biopsy method yields only a small number of cells, which hampers conventional molecular analysis for the presence of multiple copies of oncogenes. We have therefore adopted the polymerase chain reaction (PCR) method to study semi-quantitatively the level of the c-erb-B2 gene in human breast tumor samples. Of 39 patients with mammary carcinoma, 7 (19%) contained multiple copies of c-erb-B2 genes, whereas only two samples failed to give informative data. Next the multiple copies of c-erb-B2 genes, whereas only two samples failed to give informative data. Next the temporal appearance of multiple gene copies was examined in 20 patients with clinical stage IV disease. Tumor samples were obtained every second to third month from the same tumor lesion of each patient. None of the initial samples from each patient contained multiple copies of c-erb-B2. Of 16 patients that showed progressive clinical disease, 5 developed multiple gene copies, showing that the event occurs in clinical stage IV disease.


Chromosome in situ hybridization on formalin-fixed mammary tissue using non-isotopic, non-fluorescent probes: technical considerations and biological implications. Fluorescent in situ hybridization techniques have provided an important tool for interphase cytogenetic studies of human neoplasms. However, these techniques are difficult to use on formalin-fixed archival tissue sections. We describe here a non-fluorescent, non-isotopic in situ hybridization (ISH) approach that is easily applicable to paraffin-embedded breast tissue sections. The technical steps that must be monitored and individualized to optimize signal generation and detection are discussed. This ISH technique has several advantages over fluorescent detection methods. The signal obtained can be viewed using an ordinary light microscope and does not fade with time. More importantly, the signal is observed and analyzed in the context of tissue morphology. The technique permits detection of numerical chromosomal abnormalities not only in malignant but also in apparently normal and potentially premalignant mammary tissue. This may allow identification of focal genetic abnormalities as well as field-defects and enable analysis of their evolution during the multistep transformation to mammary neoplasm. This technique is also suitable for analysis of tumor heterogeneity and the correlation of numerical chromosomal aberrations with histologic, immunocytochemical, and clinical features of breast tumors.


Influence of age and parity on the development of the human breast. Breast cancer is heavily influenced by the reproductive history of the individual. Pregnancy has a protective effect which is attributed to differences in the degree of differentiation of the breast. The purpose of this work was to determine whether the quantity and the type of parenchymal structures present in the human breast were related to the age and parity history of a woman. Fifty-one human breast samples were obtained from bilateral or unilateral reduction mammoplasties performed in 40 parous women ranging in age from 18 to 57 years, and 11 nulliparous women ranging in age from 14 to 54 years. An average of 100 grams of tissue/specimen were processed for whole mount. A total of 650 slides were examined and 31,222 structures were classified and counted under the light microscope. The following mammary structures were identified: terminal structures (TS), and lobules (LOB) type 1, 2, and 3. Results were plotted for the total patient population and separately for nulliparous and parous women against age. The total patient population contained similar proportions of LOb1, 2, and 3 between ages 14-18, with a reduction of percentage of Lob1 and increase in Lob3 between ages 23 to the middle forties, when Lob3 decreased and Lob1 increased to 70%. Lob2 and TS did not exhibit significant changes throughout the period of life analyzed. When analyzed separately it was found that the breasts of nulliparous women were predominantly composed of Lob1, fewer Lob2, with Lob3 almost completely absent, whereas parous women had a high frequency of Lob3, which were the predominant structures until the fourth decade of life.(ABSTRACT TRUNCATED AT 250 WORDS)


Prediction of hormone responsiveness by mammographic parenchymal pattern in advanced primary breast cancer. Ninety-two women with advanced, unilateral breast cancer were classified according to the Wolfe and Nottingham classifications of mammographic parenchymal pattern (MPP). Both classifications of MPP were significantly correlated to the estrogen receptor (ER) status of the breast carcinoma, but could not be used for prediction of response to hormonal therapy. Age distribution was significantly different among Wolfe types as well as between ER groups, but a multiple regression analysis showed that both age and Wolfe pattern were significant and independent predictors of the ER status.


Patients' preferences for learning the results of mammographic examinations. The communication of diagnostic test results is an important aspect of the interaction between doctors and patients. Communication of mammogram results is of particular interest because the test is used to detect a common and potentially dangerous malignancy and because patients in the United States are able in some locations to obtain mammography at their own request, rather than being referred by a physician. We conducted a survey to learn about the preferences of a group of women at a traditional mammography center for learning the results of this commonly performed test. We asked women undergoing mammography to respond to questionnaires designed to learn: 1) How they felt about different methods of telling patients the results of mammograms; 2) How they were informed of the results of previous mammograms; 3) How they were told the results of the current mammograms. Patients indicated that if no abnormality is detected, they prefer to have their doctor call with the result, but if the study is 'abnormal' they wish to be told by their own physician in the office. Failing to notify the patient if the study is normal was the least preferred outcome. This group of patients did not express an interest in the most immediate form of notification (i.e. learning the result from the radiologist performing the test). Analysis of how patients felt about ways in which they were previously informed of the results of mammograms suggests that their reactions are influenced to a large extent by their clinical status. Patients undergoing mammography for diagnostic purposes, for example, were less pleased by a 'preferred' method (i.e. being told by their physician) than were those undergoing screening mammography. While patients have opinions about how they would prefer to be told their mammogram results, they are accepting a variety of methods of telling, if they are receiving good news. If abnormalities are found, patients prefer to be told in person by their own physician. Interpretations of surveys of patient satisfaction should be tempered by the finding that the clinical status of the patient alters their perceptions of satisfaction with this aspect of their physician's behavior. Patient preferences may change if increasing numbers of women are told their results by the radiologist.


Impaired primary antibody responses after vaccination against hepatitis B in patients with breast cancer. The response to hepatitis B (HBV) vaccination was studied in 32 breast cancer patients who were HBV antigen- and antibody-negative at the time of diagnosis. Ten of the patients included into the study received radical mastectomy without adjuvant therapy, 15 patients underwent breast-conserving surgery with subsequent irradiation with or without tamoxifen treatment, and 7 patients were treated by modified radical mastectomy plus adjuvant chemotherapy. Eight patients received the first dose of vaccine 3 months after irradiation therapy. Seven patients were first vaccinated 6 months following irradiation therapy. Seven healthy age-matched persons were vaccinated as controls. Antibody response in the patients was significantly delayed. Four weeks after the second vaccination 6 out of 7 controls showed a significant (> 10 IU/ml) HBs antibody titer, while only 6 out of 32 patients responded. In addition, HBs antibody titers were also significantly lower after the second vaccination even in those patients treated with surgery alone, although they were first vaccinated 3 months after surgery and had no residual tumors. This difference disappeared after the second booster immunization, at which time the frequency of significant antibody titers and the levels of HBs antibody titers were comparable between patients and controls. As the population of patients undergoing treatment for breast cancer is increasing, additional studies are needed to determine optimal immunization regimens in this group of patients.


Effect of hyperosmolality on alkaline phosphatase and stress-response protein 27 of MCF-7 breast cancer cells. MCF-7, a continuous cell line derived from a human breast carcinoma, exhibits very low alkaline phosphatase (ALP) activity. The enzyme is heat-stable and is inhibited by L-phenylalanine and L-phenylalanylglycylglycine, but not by L-homoarginine, 1-bromotetramisole, or levamisole. These data indicate that MCF-7 produces term-placental ALP, the oncodevelopmental enzyme form inappropriately expressed by a variety of human tumors. In contrast to human cancer cells that produce this enzyme monophenotypically, ALP activity of MCF-7 cells is not significantly increased by glucocorticoids or sodium butyrate. By comparison, exposure to hyperosmolality causes a striking increase in enzyme activity. Cycloheximide blocks this effect. The results obtained with cell-free assays were confirmed by cytochemical and immunocytochemical assays on whole cells. Because some of the agents tested in the enzyme modulation experiments affect cell proliferation, their possible effect on two stress-response proteins (srp 27 and srp 72) was also examined; specific immunocytochemical assays were used. These tests revealed that neither protein is affected by glucocorticoids; that sodium butyrate has no effect on srp 27, but alters the intracellular distribution of srp 72; and that hyperosmolality, while not significantly affecting srp 72, causes an increase in srp 27.


Factors affecting protein composition of breast secretions from nonlactating women. Breast secretions can be classified into two types according to their major protein components. Type I fluids contain Zn-alpha 2-glycoprotein, apolipoprotein D, and gross cystic disease fluid protein-15, while Type II fluids are characterized by the presence of lactoferrin, lysozyme, and alpha-lactalbumin. In this study, the polypeptide composition of breast secretions from 719 nonlactating women was evaluated by using polyacrylamide gel electrophoresis. The required amount for the analysis (1 microliter) was obtained from 50% of control women and from 75% of women with mammary disease. There were more secretors in premenopausal than in postmenopausal women, as well as in parous than in nulliparous women. Evaluation of factors affecting protein composition of breast secretions revealed that Type II fluids were found in the majority of women who had given birth in the last four years and in a high proportion of oral contraceptive users. After excluding both of these groups, Type II fluids were detected in 47% of patients with breast cancer, but only in 8% of control women and in 16% of women with benign breast diseases. Taken together, these results suggest that protein analysis of breast secretions could be an useful tool for the study of breast pathologies.


Interval cancer and survival in a randomized breast cancer screening trial in Stockholm. The aim of the present study was to analyse the survival rate in a group of breast cancers detected in the intervals between screening examinations in relation to clinically detected cancers in a non-screened population. All interval and control cancers were recruited from a randomized controlled mammography screening trial in Stockholm. The overall survival up to eight years of observation was higher in 191 patients with interval cancers than in 142 control cancers (p = 0.01). There were no significant differences between the two groups with regard to tumor size, stage distribution, or mean age, though the interval group did have a larger proportion of younger women. The similarity was confirmed by multiple regression analysis. The overall survival stage by stage was consistently higher in interval cancers. The survival rate in the true interval cancers was similar to that for those found in retrospect to have been detectable or traceable at the time of screening. No correlation was found between the length of the interval and the survival of patients with interval cancers. These results contradict the hypothesis that a high growth rate is associated with a poorer prognosis and that interval cancers are a more aggressive form of breast neoplasia.


Biochemical study of cyst fluid in human breast cystic disease: a review. Gross cystic disease of the breast may sometimes indicate an increased risk of breast cancer. Biochemical analysis of the cyst fluid could suggest which cysts are associated with breast cancer risk, as well as providing insights into the pathophysiology of this condition. The Na+/K+ ratio appears to be associated with the histological classification of the cyst. Sulfoconjugated estrogens and androgens, especially DHEA-S, are often found at high levels. A number of gross cystic disease fluid proteins (GCDFPs) have been described, and several polypeptide growth factors including EGF and IGF-I are frequently found. It is hoped that biochemical analysis of these components of breast cyst fluids will shed further light on the role of gross cysts in relation to breast cancer.


Placental isoferritin associated p43 antigen correlates with features of high differentiation in breast cancer. Placental isoferritin (PLF), an acidic isoform of ferritin, and its unique superheavy chain p43 have been recently described to be synthesized by breast cancer cell lines but not by normal breast epithelial cells. Since previous reports have demonstrated a correlation between the content of 'normal' ferritin in breast cancer tissue, degree of differentiation, and prognosis, we have tried to evaluate the correlation of p43 in the cytosol of 122 breast cancer samples with commonly applied prognostic factors and features of proliferation and differentiation. In parallel, we investigated the correlation of p43 expression in MCF-7 and T47-D breast cancer cell lines during proliferation induced by estradiol plus fetal calf serum (assessed by 3H-thymidine incorporation), compared to p43 expression in stationary non-stimulated cell cultures. The levels of p43 in breast cancer cytosols correlated significantly negatively with tumor size (p = 0.0001), histologic grading (p = 0.0038), nuclear pleomorphism (p = 0.0019), rate of mitosis (p = 0.0002), and lymphocytic reaction (p = 0.0001), and significantly directly with the estrogen receptor status (p = 0.0009). Although patients with a higher p43 content showed a trend for a better outcome (median follow-up: 61.4 months), an independent influence of the cytosolic p43 content on survival could not be confirmed by a multivariate Cox model. In accordance with the observed negative correlation of features of differentiation vs. p43 expression, induction of proliferation by estradiol plus FCS added to serum-free tissue culture medium correlated with a decrease of p43 synthesis in both cell lines. Expression of p43 in estrogen and FCS-absent media revealed also a decrease in relation to a low spontaneous proliferation. However, the drop of p43 synthesis was significantly stronger in cell lines with estrogen-stimulated proliferation. Our in vitro and cytosol results confirm recent clinical observations describing an inverse correlation of p43 synthesis with the degree of proliferation and differentiation in breast cancer. However, the pathologic mechanisms leading to this phenomenon as well as the negative correlation with lymphocytic infiltration are still unclear and need to be further elucidated.