Bridging to transplant: allogeneic heart transplantation after xenografting.

Xenografting seems to be a solution to use to bridge time intervals when desperately needed allograft cannot be obtained. A following allograft was never ventured. Nine dogs (weight, 20 to 25 kg; age, 2 years) underwent right cervical heart transplantation. Donors were silver foxes (3 to 4 kg). The animals were treated by triple-drug therapy consisting of cyclosporine, methylprednisolone, and azathioprine in clinical dosages. For control, six recipients received only allogeneic hearts and the identical immunosuppression. After rejection of the xenograft, a second allogeneic heart was anastomosed subsequently to the same right cervical vessels. Routine histologic and immunohistologic examinations were performed. Thromboxane B2 and 6-keto-prostaglandin F1 alpha were determined daily in peripheral blood. After final rejection the sensitization of the recipient was controlled by hemagglutination test. Survival time of the xenografts was 9.6 +/- 1.2 days; the subsequent allogeneic hearts under the same therapy beat for 4.5 +/- 5.0 days. The average survival time of control hearts was 18 +/- 1.9 days. The five hyperacute second allografts showed signs of humoral rejection by absence of inflammation. The release of thromboxane B2 was different in hyperacute, accelerated, or cellular rejection. In contrast to the long-functioning grafts, thromboxane B2 persisted during hyperacute rejection at a high level. However, 6-keto-prostaglandin F1 alpha showed no significant differences between longtime survivors and hyperacute rejecting hearts. After xenogeneic transplantation, all recipients showed hemagglutinating titers between 1:4 and 1:16. Allogeneic grafts have a different kinetics of rejection after xenogeneic heart transplantation compared with control hearts. Thromboxane B2 seems to be an important mediator in hyperacute rejection.(ABSTRACT TRUNCATED AT 250 WORDS)


Cardiomyoplasty to improve myocardial collateral blood supply as an alternative to transplantation in intractable angina.

In diffuse small-vessel coronary heart disease, heart transplantation may be indicated for intractable angina. As an alternative, a free striated muscle flap can constitute a source of neovessels. Those extraintracardial collaterals develop only 4 weeks after transplantation of the flap as shown by the following experiments with seven dogs. In those animals a myocardial infarction of the anterior wall was selectively produced by the Judkins technique for the injection of Sephadex microspheres. Four weeks later a free pectoralis muscle flap was transplanted onto the anterior wall of the heart. Arterial blood supply was achieved by anastomosis with the internal mammary artery. The venous flow was directed into the right atrium. Six weeks after surgery the patency of the anastomosis was studied by angiography, which showed patency in all animals. Pathologic examination of the hearts revealed a prominent capillary network penetrating into the underlying myocardium. Corrosion cast preparations showed even further penetration of the capillary network into the noninjured parts of the myocardium. Thus myocardial revascularization could be achieved by transplantation of a skeletal muscle on the heart.


Size matching in lung transplantation.

Volume concordance between donor lungs and the chest cavities of transplant recipients has important perioperative and postoperative implications. Between December 1987 and August 1991, 90 patients underwent lung transplantation in the Joint Marseilles-Montreal Lung Transplantation Program: 51 patients had double lung transplants, 19 patients had single lung transplants, and 20 patients had heart-lung transplants. There were 18 children (age range, 7 to 17 years) and 72 adults (age range, 18 to 58 years). Size matching was based on measurement of the submammary thoracic perimeter. Patient age (+/- 2 years) was also taken into consideration in children. Airway anastomoses were bronchial except for all heart-lung transplant patients and two double lung transplant patients, who had tracheal anastomoses. Occasional differences between donor and receiver bronchial diameters in children (greater donor size twice and smaller donor size once) required bronchoplasty in three instances. Healing was normal in these three instances, and no bronchial stenoses were noted. Performance of separate sutures rather than continuous running sutures on the cartilaginous anterior portion facilitated correction of airway diameter inequalities in adults. Excess volume was noted in three patients during closure of the thorax. In one patient, donor and recipient thoracic perimeters were similar. In two patients, however, donor size was greater by more than 20%. This was corrected by pneumoreduction with a surgical stapler. Lung size was decreased by 10% to 40% with use of this technique. Thoracic closure was facilitated and hemodynamic instability was thus corrected. No functional abnormalities were noted after surgery once the differences in lung size were corrected.(ABSTRACT TRUNCATED AT 250 WORDS)


Influence of interrupted pulmonary lymph drainage on antibody responses in hilar-stripped lungs.

Lung transplantation interrupts hilar lymphatics. This may have an impact on immune responses to antigens entering the lung because the antigens cannot reach the lung-associated lymph nodes where the immune response is generated. We investigated the interruption and regeneration of lymphatics and the influence of this on antibody responses after hilar stripping in rats in three experiments: (1) visual detection of regenerated hilar lymphatics by chromolymphography, (2) observation of transport of carbon particles from the lung to the lung-associated lymph nodes, and (3) assessment of antibody responses after lung immunization with sheep red blood cells. The findings showed that hilar lymphatics were interrupted by hilar stripping and regenerated from day 7 after operation. Transport of particles to the lung-associated lymph nodes was blocked during the first week after operation but returned to normal values thereafter. Serum antibody titers were absent or low in the rats immunized on days 7 and 10 after hilar stripping; subsequently antibody responses gradually recovered in 1 month. We conclude that antibody responses to antigens in hilar-stripped lungs are impaired as long as the antigens cannot be transported through lymphatics from the lung to the lung-associated lymph nodes. These findings can explain in part why pulmonary infections occur so frequently in the initial weeks after lung transplantation.


Size matching in heart transplantation.

After heart transplantation, problems may arise from hearts that are either too small or too large. Although the "classic" rule requires that the donor/recipient weight ratio mismatch measure not less than 0.8, the chronic organ shortage led to a challenge of that margin. In selected cases donor/recipient weight mismatches of up to 0.5 may be accepted. In these circumstances, heterotopic heart transplantation may be an alternative. In pediatric heart transplantation, disproportionately large hearts (donor/recipient weight mismatches of 2.0 to 3.0) may create the so-called "big-heart syndrome," with coma and general convulsions. Treatment consists of peripheral pressure control with nifedipine.


Noninvasive methods of rejection diagnosis after heart transplantation.

For clinical follow-up and prognosis in heart transplant patients, it is important to understand accurately the presence and extent of cardiac allograft rejection. Since the introduction of endomyocardial biopsy, almost 40 different noninvasive diagnostic procedures for the recognition of myocardial rejection have been proposed. However, endomyocardial biopsy is invasive and not suitable for frequent monitoring. If the pattern of rejection shows a focal distribution, false-negative results can be expected. Discrepancies between biopsy findings and allograft function are obviously possible. State-of-the-art information will be given on the most reliable noninvasive methods for rejection diagnosis, which can be differentiated from electrophysiology (fast-Fourier-transformed electrocardiography and intramyocardial electrocardiography), echocardiography, immunologic methods (cytoimmunologic monitoring, transferrin receptors, and interleukin-2 receptors), various biochemical markers (neopterines, prolactin, urinary polyamines, and beta 2-microglobulins), radioisotopic techniques (antimyosin-monoclonal antibodies, thallium, technetium, and gallium scintigraphy and indium-labeled cells), as well as magnetic resonance imaging. Thus modified and patient-adapted antirejection therapy can be provided if the decision for or against antirejection therapy is not based on biopsy findings alone but rather is confirmed along with histologic, electrophysiologic, biochemical, immunologic, and functional parameters.


Causes of exercise limitation after heart-lung transplantation.

A decade after the first successful human heart-lung transplantation and with improved recipient survival, there is now a growing interest in the quality of life achieved after this procedure. Patients undergoing heart-lung transplantation suffer from diseases that are most protracted and uniformly fatal. Before surgery, they are usually debilitated from the primary disease and long-term inactivity. Therefore several factors contribute to the maximal work capacity achievable after transplantation. They are principally unavoidable complications of allograft transplantation, mainly lung rejection, but include the toxic effects of immunosuppressant therapy. Many, however, are a function of the prolonged incapacitation and deconditioning imposed by the pretransplant illness. The relative roles of these factors in limiting posttransplant exercise tolerance in the various disease groups undergoing heart-lung transplantation are discussed.


Molecular basis of apparent isolated 17,20-lyase deficiency: compound heterozygous mutations in the C-terminal region (Arg(496)----Cys, Gln(461)----Stop) actually cause combined 17 alpha-hydroxylase/17,20-lyase deficiency.

The molecular defect in a reported case of isolated 17,20-lyase deficiency in a 46XY individual has been elucidated. The patient was found to be a compound heterozygote, carrying two different mutant alleles in the CYP17 gene. One allele contains a point mutation of arginine (CGC) to cysteine (TGC) at amino acid 496 in exon 8. The second allele contains a stop codon (TAG) in place of glutamine (CAG) at position 461 in exon 8 which is located 19 amino acids to the carboxy-terminal side of the P-450(17) alpha heme binding cysteine. COS-1 cells transfected with cDNAs containing one or the other of these mutations showed dramatically reduced 17 alpha-hydroxylase and 17,20-lyase activities relative to cells transfected with the wild type P-450(17) alpha cDNA. While the in vitro data in COS 1 cells can explain the patient's physical phenotype, with female external genitalia, it was somewhat discordant with the clinical expression of isolated 17,20-lyase deficiency with relative preservation of 17 alpha-hydroxylase activity in vivo. In addition to the expression studies of these two examples of mutants in the C-terminal region of cytochrome P-450(17) alpha, a third mutant cDNA construct containing a 4-base duplication at codon 480 previously found in patients with combined 17 alpha-hydroxylase/17,20-lyase deficiency was also expressed in COS-1 cells. This expressed protein was completely inactive with respect to both activities, supporting the biochemical findings in serum and in vitro biochemical data obtained using a testis from the patient. The results from these patients clearly indicate the importance of the C-terminal region of human P-450(17) alpha in its enzymatic activities.


Identification of five rare mutations including a novel frameshift mutation causing beta zero-thalassemia in Thai patients with beta zero-thalassemia/hemoglobin E disease.

6 out of 14 uncharacterized beta-thalassemia alleles from 187 Thai beta-thalassemia/HbE patients were identified by direct sequencing of DNA amplified by polymerase chain reaction. A novel mutation occurring from an insertion of adenosine in codon 95, which results in a shift of the reading frame with terminator at the new codon 101, was detected in one patient. In addition, two frameshift mutations not previously reported among the Thai population were also detected in 3 patients: one with a deletion of thymidine in codon 15 and two with an insertion of cytidine in codons 27/28. A frameshift mutation that occurred from a cytidine deletion in codon 41 was also found in one patient in this study. The remaining case was an amber mutation, GAG-TAG, in codon 43 in exon 2 of the beta-globin gene. These mutations bring the number of mutations known to be present in the Thai population to a total of 20, 15 of which were detected in beta-thalassemia/HbE patients.


Cleavage of type VIII collagen by human neutrophil elastase.

In this report, the susceptibility of type VIII collagen to human neutrophil elastase is compared to other extracellular matrix components. Type X collagen is degraded to specific fragments at a substrate to enzyme ratio of 5:1 after 20 h at room temperature, but type VIII collagen is almost completely degraded after only 4 h incubation at a substrate to enzyme ratio of 50:1 and partly degraded after only 15 min. Laminin, merosin and types I, III, IV and V collagen exhibit no susceptibility to neutrophil elastase under the latter conditions, while fibronectin is degraded.


Purification and identification of a CD25 expression inhibitory protein from cell-free supernatants of chronically HIV-infected promonocytic cells.

The CD25 (IL-2-R alpha) cell surface glycoprotein expressed transiently during T-cell activation is implicated in the high affinity IL-2 receptor. This paper shows that cell-free supernatants from chronically HIV-infected promonocytic cells spontaneously produce a soluble factor which inhibits CD25 expression on PHA-activated human PBMC. We purified the CD25 expression inhibitory activity by a factor 12,350, using XM50 ultrafiltration, Superose 12 molecular sieving chromatography and MonoQ anion-exchange chromatography. Then we associated this activity to one single spot (M(r) 29,000, pI 6.8) on an O'Farrell two-dimensional gel. Our data demonstrate that this protein (M(r) 29,000, pI 6.8) is released from HIV-infected promonocytic cells and suggest that this factor is a new monokine regulating the T-cell activation process.


Sequencing of the variant thyroxine-binding globulin (TBG)-San Diego reveals two nucleotide substitutions.

Thyroxine-binding globulin (TBG) is a liver glycoprotein that transports thyroid hormone in serum. In 1989, a variant TBG was reported with reduced binding affinity for thyroxine (T4) and triiodothyronine (T3) which results in low serum T4 and T3 levels. This variant, TBG-San Diego (TBG-SD), also displays reduced heat stability but has a normal isoelectric focusing pattern. We now report the sequence of the entire coding region of TBG-San Diego. It reveals two nucleotide substitutions: one located in exon 1 which results in the replacement of the normal Ser-23 (TCA) with threonine (ACA) and the other, located in exon 3, changes the normal codon 283 of TTG (leucine) with that of TTT, (phenylalanine). Allele specific amplification was used to search for both nucleotide substitutions in four affected members of the family. Results confirmed the co-segregation of these nucleotide substitutions with the TBG-SD phenotype. The substitution in codon 283 has been previously described and exists as a polymorphism in some ethnic groups or in combination with other TBG variants with different physical characteristics. Thus, it appears that the replacement of Ser-23 with threonine is responsible for the observed alterations in physical properties of TBG-San Diego.


L-dopa binding sites in rodent melanoma cells.

Rapid, saturable, specific and stereoselective binding of L-dopa to crude membranes and purified nuclei from rodent amelanotic melanoma cells is reported. Cross-linking of [3H]dopa to melanoma cell surface emphasized proteins of approx. 55, 30, 25 and less than 20 kDa. It is suggested that these binding sites may regulate melanocyte activity.


Identification of O-methyldopa in the ventricular fluid of patients with Parkinson's disease.

A newly developed liquid chromatographic system with a multiple electrode detector was used for quantification of neurochemical substances in ventricular fluid of patients with Parkinson's disease. During the analysis, an unknown peak was observed at almost the same high-performance liquid chromatography retention time as 5-hydroxytryptophan. Through the use of ion suppression techniques, voltammographic analysis and fast atom bombardment mass spectrometry, the compound was identified as O-methyldopa, the major metabolite of L-dopa.


Accelerated graft coronary artery disease: diagnosis and prevention.

Accelerated graft coronary artery disease (CAD) has become a major factor limiting survival among long-term heart transplant survivors. Currently 14%, 37%, and 50% of patients treated with triple therapy have angiographically apparent accelerated graft CAD at 1, 3, and 5 years after transplantation. Because cardiac allografts are denervated, transplant recipients generally do not experience angina pectoris. Therefore accelerated graft CAD may present as silent myocardial infarction, congestive heart failure, or ventricular arrhythmia leading to syncope or sudden death. Noninvasive tests for CAD have been insensitive for the detection of accelerated graft CAD because of the diffuse nature of the disease. Coronary arteriographic characteristics of accelerated graft CAD are a mixture of typical focal atherosclerotic lesions and unusual diffuse, concentric, and longitudinal narrowing prominent in middle to distal coronary vessels, with distal vessel obliteration and lack of collateral vessel formation. The presence and severity of accelerated graft CAD may be underestimated by routine angiography because of its diffuse and concentric nature. Quantitative arteriography has become an important technique to assess the progression of accelerated graft CAD. Intravascular ultrasound imaging can detect even earlier development of intimal thickening. CAD risk factor modification has had little impact on the overall incidence. We initiated a randomized study of diltiazem versus no calcium blocker to determine if this may prevent accelerated graft CAD. Patients have undergone early postoperative and annual quantitative coronary angiography since inception of the study.(ABSTRACT TRUNCATED AT 250 WORDS)


Primary sequence confirmation of the beta-amyloid peptide from hereditary cerebral haemorrhage with amyloidosis (Dutch type) by collisional activation of the doubly charged ion.

High-energy collisional activation of doubly charged peptide ions allows direct sequential analysis of peptides over 4 kDa, including leucine/isoleucine determinations. This technique was used to verify the sequence of an intact beta-amyloid peptide associated with hereditary cerebral haemorrhage with amyloidosis Dutch type (4.2 kDa), which resists both enzymatic digestion and Edman sequencing. Additionally, this approach shows promise for the primary sequence confirmation of a wide variety of large peptides by tandem mass spectrometry.


High-performance anion-exchange chromatography coupled with mass spectrometry for the determination of carbohydrates.

Methodology has been developed to couple high-performance anion-exchange chromatography (HPAE) with mass spectrometry utilizing the ion spray liquid chromatography/mass spectrometry (LC/MS) interface. Anion micro-membrane suppression (AMMS) has been used to remove the high concentrations of NaOH and NaOAc (10-400 mM total [Na+]) necessary for the separation of mixtures of monosaccharides and oligosaccharides. Post-suppressor addition of CH3CN/H2O solutions containing NH4OAc or LiOAc provided low-nanomole detection of the monosaccharides by selected ion monitoring of the cationized adducts. Maltooligosaccharide mixtures (three to seven residues) were separated and detected by the HPAE/AMMS LC/MS system in the full-scan mode. Low declustering potentials (35 V) in the LC/MS API source afforded intact singly and doubly charge ammoniated and diammoniated adducts of the sugars. Higher declustering potentials (65 V) produced abundant fragmentation of the ammoniated adducts. The corresponding lithiated and dilithiated species produced intact molecule ion species at higher declustering potentials. The endo H-released oligomannose species from RNase B were determined by the HPAE/AMMS LC/MS system as ammoniated adducts and resulting fragment ions with a high declustering potential (95 V) in the full-scan mode.


[The connection of medical indicators with oscillations in the natural gravitational field].

The hourly amount of 3140 hospital deaths, 2220 births by terminated labours, and 617 investigations of some elementary functions of haemodynamics and external respiration are correlated with tidal gravitation cycles calculated at the same time of day. The statistical analysis revealed significant dependence of the obtained data on local geometrical properties of 12-hour lunar tidal waves. Their distinctive signs were convexity-concavity and growing-sinking which the 12-hour lunar wave into 4 intervals. To each interval corresponded specific patterns of death and birth curves during 24-hours and divergent distribution of values of haemodynamics and external respiration. The investigation results and literature data point to the fact that periodic fluctuations of natural gravitation fields carrying the information on changes in space-time may serve as an outside time-marker (Zeitgeber) of biological rhythms.


[The effect of gravity flow changes on the periodicity of functional activity of canine adrenal cortex and thyroid glands].

Among the universal environmental factors changes of the gravity flow are not enough investigated. Relations between the dog corticoadrenal and thyroid glands functional rhythms and the properties of curvature level surfaces of the gravity flow changes were established. The suggested mechanism of the effect of gravitation flow changes includes ability of cells and ultrastructures to reflect the geometric properties of the local space.


Photoactivation of toxin conjugates.

A novel photocleavable protein cross-linking reagent has been used for conjugation of the ribosome-inactivating protein pokeweed antiviral protein from seeds of Phytolacca americana (PAP-S), with either the monoclonal antibody 5E9 directed against the human transferrin-receptor or the B-chain of ricin that binds to cell-surface oligosaccharides bearing terminal D-galactose residues. When irradiated with near-UV light (350 nm), the linker of these conjugates undergoes photolytic degradation, resulting in the release of native toxin that is fully functional. The cytotoxicities of these 5E9-PAP-S and ricin B-chain-PAP-S conjugates for HeLa cells could be enhanced by irradiating the cells with light after they had internalized the conjugates.


Structure-function relationships in indium-111 radioimmunoconjugates.

Conjugates formed by reaction of monoclonal antibody B72.3 with benzyl isothiocyanate derivatives of four amino polycarboxylate chelators (NTA, EGTA, EDTA, DTPA) were labeled with indium-111 and administered iv to athymic mice bearing antigen-positive (LS174T) and antigen-negative (A375) human tumor xenografts. Conjugate immunoreactivities, antibody dose, and xenograft size were controlled, so that the effects of varying chelate structure could be evaluated under conditions where immunological and physiological factors were effectively held constant. Tissue distribution and excretion of the radiometal at 24 and 48 h postinjection were shown to correlate directly with chelate thermodynamic stability (NTA less than EGTA less than EDTA less than DPTA). Radioactivity levels in the blood and the LS174T xenograft increased, while kidney levels and excretion levels decreased, with increasing chelate stability. The kidney was the only normal organ that accumulated non-antibody-bound 111In, uptake of radioactivity into all other tissues, and in particular the liver, being unaffected by changes in chelate structure. Mean transferrin saturation in the tumor-bearing athymic mice was found to be 65%. It is proposed that uptake of free 111In by serum transferrin is precluded in this model, leading to the observed renal localization of unbound label. Kidney:blood and kidney:LS174T activity ratios at 48 h postinjection provided the most sensitive indices of conjugate instability in vivo, spanning 50- and 20-fold ranges, respectively, between the least stable and the most stable conjugate. It is concluded that this antigen/antibody system and mouse model are well-suited to structure-function studies of immunoglobulin labels.


Convenient synthesis of bifunctional tetraaza macrocycles.

A convenient synthesis of 4-nitrobenzyl-substituted macrocyclic tetraamines and their conversion to bifunctional poly(amino carboxylate) chelating agents is described. Cyclization of (4-nitrobenzyl)-ethylenediamine with appropriate BOC-protected amino disuccinimido esters in dioxane at 90 degrees C resulted in the formation of 12- and 14-membered ring diamides in 40% and 44% yield, respectively. A 12-membered macrocyclic triamide was also prepared in 44% yield by cyclization of N-(2-aminoethyl)-4-nitrophenylalaninamide with disuccinimidyl N-(tert-butoxycarbonyl)iminodiacetate. Deprotection (HCl/dioxane) and reduction with borane gave the substituted macrocyclic amines which were then alkylated with either bromoacetic acid or tert-butyl bromoacetate. Preparation of the isothiocyanate derivatives and 14C labeled chelating agents are described. Attempts to prepare a 9-membered macrocyclic diamide using this cyclization technique resulted instead in a 20% yield of a 10:1 mixture of isomeric fused 5,6 ring acylamidines. Deprotection (HCl/dioxane) and reduction with borane gave a substituted piperazine derivative in 55% yield.


Conjugation and evaluation of 7E3 x P4B6, a chemically cross-linked bispecific F(ab')2 antibody which inhibits platelet aggregation and localizes tissue plasminogen activator to the platelet surface.

A bispecific F(ab')2 monoclonal antibody which recognizes both the platelet GPIIb/IIIa receptor and human tissue plasminogen activator was produced to target tPA to platelets for enhancement of thrombolysis. A stable, thioether-cross-linked bispecific F(ab')2 (7E3 X P4B6) combining the GPIIb/IIIa-specific monoclonal antibody 7E3, which inhibits platelet aggregation, and a nonneutralizing anti-tPA monoclonal antibody (P4B6) was produced. This was performed by coupling each of the parental Fab' moieties with the homobifunctional cross-linker bis(maleimido methyl) ether (BMME). 7E3 X P4B6 was sequentially purified using gel-filtration chromatography and hydrophobic interaction (HIC) HPLC. HIC was shown to completely resolve each of the parental F(ab')2 species from the bispecific one. 7E3 X P4B6 was shown to retain completely each of the parental immunoreactivities in GPIIb/IIIa and tPA binding EIA's. The bispecific antibody inhibited platelet aggregation in vitro at levels comparable to those for 7E3 Fab. Recruitment of tPA activity to washed human platelets was demonstrated using the S-2251 chromogenic substrate assay. 7E3 X P4B6 recruited 12-fold more tPA to the washed platelets than a mixture of the parental F(ab')2 molecules used as controls.


Site-specific modification of a fragment of a chimeric monoclonal antibody using reverse proteolysis.

We propose a novel method for the site-specific labeling of antibodies under mild conditions and give as an example the modification of an F(ab')2-like fragment of the chimeric monoclonal antibody B72.3. The F(ab')2-like fragment was produced by the action of the protease lysyl endopeptidase. Reverse proteolysis, catalyzed by the same enzyme, was then used to attach carbohydrazide specifically to the carboxyl termini of the heavy chains of the fragment. Finally, a radiolabeled chelator possessing an aldehyde group was conjugated to the modified fragment through a hydrazone linkage. The resulting site-specifically labeled F(ab')2-like fragment was characterized by gel electrophoresis and by enzymic digestion. It was found to possess immunoreactivity equivalent to that of the unmodified F(ab')2-like fragment as determined by immunofluorescence and ELISA (enzyme-linked immunosorbent assay) techniques. The advantages and disadvantages of this labeling method, which appear to be of quite general applicability, are discussed.


Photochemical cross-linking of guanosine 5'-triphosphate to phosphoenolpyruvate carboxykinase (GTP).

Mammalian phosphoenolpyruvate carboxykinase (PEPCK) specifically requires a guanosine or inosine nucleotide as a substrate; however, the structural basis for this nucleotide specificity is not yet known. Because affinity labels derived from guanosine have not yielded a stable, modified peptide in quantities sufficient for sequence analysis, we have investigated the utility of direct photochemical cross-linking of GTP to PEPCK in order to identify the nucleotide binding site. UV irradiation at a distance of 2 cm by a Mineralight lamp (330 microW/cm2) results in the attachment of [alpha-32P]GTP to PEPCK via a stable, covalent linkage in a reaction that is dependent upon GTP concentration and duration of irradiation. After 10 min of irradiation, more than 0.2 mol of [alpha-32P] GTP is incorporated per mole of PEPCK; under these conditions the GTP concentration required for half-maximal labeling is 69 microM. The substrates phosphoenolpyruvate, ITP, and GDP provide protection against photolabeling, as do Mn2+ and Mg2+. One major and one minor radioactive peptide derived from proteolytic digests of photolabeled PEPCK have been isolated and identified. The major modified peptide has been provisionally assigned to an acidic region near the C-terminus, and the minor peptide has been identified as Ser462-Lys471.


Vinylstannylated alkylating agents as prosthetic groups for radioiodination of small molecules: design, synthesis, and application to iodoallyl analogues of spiperone and diprenorphine.

The preparation and synthetic utility of p-toluenesulfonate esters of (E)- and (Z)-3-(tri-n-butylstannyl)-prop-2-en-1-ol as bifunctional reagents for radioiodination are described. These vinylstannylated alkylating agents are prepared in two steps from propargyl alcohol, and readily couple with nucleophilic functionality (amide nitrogen, secondary amine, tertiary alcohol) in good yields (48-95%) to provide derivatives of the neuroreceptor ligands spiperone and diprenorphine. Regio- and stereospecific radioiododestannylation with retention of configuration occurs under mild, no-carrier-added conditions to give the corresponding radiolabeled N- or O-iodoallyl analogues in good radiochemical yields (55-95%) with high specific radioactivities. The methodology is versatile and well-suited to selective labeling of small molecules with radioisotopes of iodine such as 125I or 123I.


Monoclonal antibody-beta-lactamase conjugates for the activation of a cephalosporin mustard prodrug.

Cephalosporin mustard (CM) was designed as an anticancer prodrug that could be activated in a site-specific manner by monoclonal antibody-beta-lactamase conjugates targeted to antigens present on tumor cell surfaces. Purified beta-lactamases from Bacillus cereus (BC beta L) and Escherichia coli (EC beta L) catalyzed the release of phenylenediamine mustard (PDM) from CM through a fragmentation reaction which occurs after the beta-lactam ring of CM is hydrolyzed. The Km and Vmax values were 5.7 microM and 201 mumol/min per mg for BC beta L and 43 microM and 29 mumol/min per mg for EC beta L, respectively. Conjugates of BC beta L were prepared by combining the F(ab')2 fragments of the maleimide-substituted monoclonal antibodies L6 and 1F5 with thiolated BC beta L. The conjugates showed little loss in enzymatic activity and bound nearly as well as the unmodified F(ab')2 monoclonal antibodies to antigens expressed on the H2981 human lung adenocarcinoma cell line (L6 positive, 1F5 negative). PDM was approximately 50-fold more cytotoxic than CM to H2981 cells. Treatment of the cells with L6-BC beta L followed by CM resulted in a level of cytotoxic activity that was comparable to that of PDM. This was most likely due to activation of CM by conjugate that bound to cell-surface antigens, since pretreatment of H2981 cells with BC beta L or 1F5-BC beta L enhanced the activity of CM to a lesser extent. Thus, we have shown that CM is a prodrug, and that it can be activated with immunological specificity by a monoclonal antibody-beta-lactamase conjugate.


Introduction of aliphatic amino and hydroxy groups to keto steroids using O-substituted hydroxylamines.

(Aminooxy)butyl- and -hexylamines and alcohols were synthesized by the Ing-Manske modification of the Gabriel synthesis. The aminooxy group of these heterobifunctional spacer reagents is a far more powerful nucleophile than the amino or hydroxy group because of the oxygen atom adjacent to the amino group (alpha-effect). The aminooxy group reacts readily with keto groups while the amino or hydroxy (or other) group remains free for further reactions. These excellent heterobifunctional spacer reagents were used here to derivatize keto steroids in an alkaline alcoholic solution. Using the described, general, and easy one-step synthesis, aliphatic amino or hydroxy groups with a spacer arm have been introduced to testosterone, 6-ketoestradiol, and cortisol.


Microparticle-enhanced nephelometric immunoassay with microsphere-antigen conjugates.

gamma-Irradiation of acrolein and other acrylic monomers allowed the synthesis of spherical polyfunctional hydrophilic microparticles in the size range of 50 to 300 nm, on which antigens (immunoglobulins G, chorionic gonadotropin hormone, prealbumin) could be covalently bound. Microsphere-antigen conjugates clustered together in the presence of specific antiserum or monoclonal antibodies and their agglutination was quantified by light-scattering measurement performed with a specially designed nephelometer. Essential factors concerning the conjugate agglutination and its quantitation (size of microsphere, amount of antigen bound on microsphere, concentration of conjugate, concentration of agglutinating reagent, angle of light-scattering observation) were successively studied. A microparticle-enhanced nephelometric immunoassay for prealbumin was finally developed as an example of application. It was based on the inhibition of the immunoagglutination of microspheres-prealbumin conjugate by free prealbumin. This prealbumin immunoassay was easy to perform (one-step assay without washing or phase separation), fast (30 min), reliable (variation coefficients ranged from 3.6% to 7.5% for within- and between-assay determination), and sensitive (1 microgram/L detected). It was correlated with conventional immunonephelometry and radial immunodiffusion (correlation coefficients, 0.98). Microparticle-enhanced nephelometric immunoassay offered many advantages over the last two methods. Its better sensitivity allowed a lower reagent consumption and a larger sample dilution (contrary to the conventional immunonephelometry, sample pretreatment and sample blank measurement were unnecessary). Its inhibition mode induced a total accuracy for sample with high analyte concentration (a risk of underevaluation in antigen excess conditions existed in all method based on a noncompetitive antigen-antibody reaction) and provided the possibility to quantify haptens.(ABSTRACT TRUNCATED AT 250 WORDS)


Preliminary results from the self-regulatory treatment of high-medication-consumption headache.

Data are presented from a prospective clinical replication series of ten consecutive high-medication headache patients who presented for nondrug treatment of their headaches. For the first eight, an attempt was made to withdraw the patients from medication, with the assistance of relaxation training, prior to entering a comprehensive self-regulatory treatment program. For the last two, drug withdrawal accompanied the treatment. Six of the ten patients showed clinically significant reductions in headache activity, which held up over follow-ups of up to 12 months. Psychological tests provide some discrimination between success and failures.


Activation of mouse macrophages by muramyl dipeptide coupled with an anti-macrophage monoclonal antibody.

A rat IgG2a monoclonal antibody (mAb3A33) directed against the mouse Mac-1 antigen was conjugated with muramyl dipeptide (MDP) by using an intermediate polymer; under such conditions 75 MDP molecules were bound to one antibody molecule. A poly(L-lysine) polymer substituted with muramyl dipeptide and 3-(2-pyridyldithio)propionyl residues were prepared, the remaining lysine epsilon-amino groups were acylated with D-gluconolactone, leading to a neutral polymer; then a few polymer conjugates were coupled to mAb3A33 via a disulfide bridge. The binding capacity of the monoclonal antibody was preserved after conjugation with MDP-polymer molecules. Mouse peritoneal macrophages, incubated for 24 h with MDP-mAb3A33 conjugate became cytostatic against P815 mastocytoma cells, whereas unconjugated mAb3A33 and MDP-bound to a nonspecific rat IgG2a were ineffective. An enhancement of the cytostatic activity induced by MDP-mAb3A33 conjugate was obtained in the presence of gamma-IFN. These results show that several tens of MDP molecules can be linked to a macrophage-specific monoclonal antibody by using a neutral intermediate polymer without impairing the binding antibody capacity and that this type of MDP conjugate can efficiently activate macrophages and therefore could be the basis of the development of new antitumor therapy.


Self-regulation of slow cortical potentials in psychiatric patients: depression.

Findings on depressive patients indicate that depressives have electrophysiological characteristics similar to those of schizophrenics, in that they exhibit reduced Contingent Negative Variation (CNV) amplitudes and more distinct Postimperative Negative Variations (PINVs) than normal controls. In a biofeedback experiment, 8 medicated male inpatients with the DSM III-R diagnosis of "Bipolar Disorder, Depressive," and "Major Depression" demonstrated no impairment in the self-regulation of Slow Cortical Potentials (SCP) in comparison to schizophrenics in terms of increasing and suppressing negativity. Continuous visual SCP feedback is presented to the patient as a horizontally moving rocket in a video game format. The direction changes of the rocket represented SCP changes at each point in time, recorded by the central EEG (based on the pretrial baseline). Depressives demonstrated SCP self-regulation across 20 sessions, although with many between-and-within variations. The 8 male controls were unable to regulate their SCPs across 5 sessions. This result contradicts other findings of our laboratory on normal controls. Motivational factors and insufficient operant reinforcement (financial reward) may have facilitated this effect.


The effects of centrally acting drugs on the EEG correlates of meditation.

The present study investigated the effects of three centrally acting drugs on the significant increase in the intermediate alpha frequency of the electroencephalogram (EEG) that accompanied meditation in a male volunteer. When compared to the EEG recorded before each of the three drugs was administered, naloxone tended to enhance the increase in the power of the intermediate alpha EEG (9.4-10.4 Hz), while diazepam tended to spread the increase to the slow (7.4-9.4 Hz) alpha EEG, and flumazenil was without much effect on the overall EEG pattern. However, these EEG changes when compared to similar changes obtained with saline administration were not significantly different from the latter. Thus, it is unlikely that the EEG correlates of meditation are causally related to the rise or fall of endogenous opioid peptides or benzodiazepinelike substances in the brain.


Activating hydroxyl groups of polymeric carriers using 4-fluorobenzenesulfonyl chloride.

4-Fluorobenzenesulfonyl chloride (fosyl chloride), due to the strong electron-withdrawing property of its fluoride atom, is found to be an excellent activating agent for the covalent attachment of biologicals to a variety of solid supports (e.g. functionalized polystyrene microspheres, Sepharose beads, or cellulose rods and hollow fibers). This reagent reacts rapidly with primary or secondary hydroxyl groups, at ambient temperature and pressure, to form 4-fluorobenzenesulfonate leaving groups. The activated solid support can be used immediately or preserved for several months without loss of activity by freeze-drying or by storage at 4 degrees C in aqueous solution at pH 5. Enzymes, antibodies, avidin, and other biologicals can be covalently attached to the activated solid phase with excellent retention of biological function. Potential therapeutic applications of the fosyl chloride chemistry for bioselective separation of human lymphocyte subsets from whole blood and tumor cells from bone marrow are presented.


Microencapsulated bovine chromaffin cells in vitro: effect of density and coseeding with a NGF-releasing cell line.

Immobilization of discrete cell clusters within a partially crosslinked matrix prevents reaggregation of primary tissues and may provide a means for long-term maintenance of encapsulated cells. Dissociated bovine adrenal chromaffin (BAC) cells were suspended throughout crosslinked polyanionic microspheres previously shown to be selectively permeable. Microcapsules approximately 500 microns in diameter were seeded with: 1) three different densities of BAC cells; and 2) BAC cells suspended in Matrigel or coseeded with a genetically modified nerve growth factor (NGF)-releasing fibroblast cell line. Each group was analyzed in vitro at 1, 4 and 8 weeks for spontaneous and potassium-evoked release of catecholamines, and maintained in vitro for up to 12 weeks for morphological observations. Over time, release of norepinephrine (NE) and epinephrine (EPI) diminished, while dopamine (DA) remained constant from the monoseeded capsules. In the coseeded group, an increase in potassium-evoked release of DA was observed from 1 to 4 weeks, and remained at that level up to 8 weeks. Encapsulated chromaffin cells retained a rounded morphology typical of undifferentiated cells. Intact chromaffin cells with well preserved and abundant secretory granules were observed ultrastructurally after 4 weeks in vitro. Small neurites from the chromaffin cells in the coseeded group were observed at 4 weeks with light microscopy, and up to 12 weeks with electron microscopy. Under static incubation conditions, 1 mM D-amphetamine resulted in a significant increase in the output of NE and DA from the coseeded capsules 8 weeks post-implantation, as compared to microcapsules loaded with chromaffin cells alone. Encapsulation within an immobilization matrix allows manipulation of the internal environment, thereby providing the ability to pre-treat cells with various factors in a non-invasive manner, which may enhance long-term cellular viability.


Fetal brain transplantation in kainic acid lesioned caudate nucleus of adult rats.

This study confirmed that bilateral kainic acid (KA) injection at the caudate produces aphagia and adipsia in rats. The reduction in food and water intake was fatal after a higher dosage of the drug. To test the effect of transplantation on the mortality rate, KA was first injected in the left caudate, in one set of rats. After a gap of three days, fetal striatal tissue was unilaterally transplanted at this lesioned site, along with a second injection of KA in the right caudate. Successful transplantation, as ascertained morphologically, did not significantly alter the mortality rate. The morphometric study revealed that the neurons of the transplant were larger in size, and their numerical density lower than those of the caudate of normal rats. Only very few neurons of the transplant developed functional connectivity with the host, as demonstrated by electrophysiological studies.


A sensitive dot immunobinding assay for serodiagnosis of African swine fever virus with application in field conditions.

The present work describes a simple dot immunobinding assay (DIA) for African swine fever virus (ASFV) antibody detection that can be used under field conditions. The assay uses nitrocellulose strips dotted with a cytoplasmic soluble antigen (CS-P) of ASFV. The nitrocellulose strips are adhered to a plastic handle. The test serum samples react with the CS-P, and antibodies are detected using a protein A-peroxidase conjugate. Both incubations are carried out at 20 C. The efficacy of the DIA as a screening test for ASFV was compared to an enzyme-linked immunosorbent assay (ELISA) and an immunoblotting (IB) test using 343 sera collected from natural African swine fever epizootics and from inapparent ASFV carriers. The DIA had comparable sensitivity to both reference techniques, and all samples positive in the ELISA and IB test were also positive in the DIA. False-positive reactions were not detected when whole blood or poorly preserved serum samples were tested by DIA. Some poorly preserved sera that were positive initially by the ELISA were no longer ELISA positive in a later run, although they were positive in IB and DIA. These positive DIA and IB test results could be caused by the differences in antibody epitope binding.


Estrogen receptors are present in neocortical transplants.

Fetal neocortical tissue was grafted into neocortical lesion cavities made in newborn rats. After two weeks survival, in vitro binding of [3H]-estradiol to cytosolic preparations provided evidence of estrogen receptors within the transplants. The observed high levels correspond to previous work demonstrating elevated estrogen receptor levels during the first postnatal week in the rat cerebral cortex.


Effect of culture media and incubation temperature on growth of selected strains of Francisella tularensis.

The rate and amount of growth of 4 field isolates and reference strain ATCC 6223 of Francisella tularensis were evaluated on isolation media with 2 different agar bases and with different supplements and incubated at 25 C, 35 C, and 42 C. Biochemical reactions on conventional differential media with and without cysteine were evaluated. Two of the field isolates and the reference strain were F. tularensis subspecies tularensis (formerly biovar tularensis or Type A), and 2 isolates were subspecies holarctica (formerly subspecies palaearctica or Type B). Bacto cystine heart blood agar supplemented with 1% hemoglobin, glucose cystine heart blood agar, and brain-heart infusion blood agar supported good growth of all 4 field strains, with the most luxuriant growth occurring on Bacto cystine heart blood agar with hemoglobin. Heart infusion blood agar and trypticase soy blood agar supported growth of the field isolates, although growth was diminished and delayed. Strain 6223 was distinctly fastidious and failed to grow on heart infusion or trypticase soy blood agars. Growth of strain 6223 was best on Bacto cystine heart blood agar with hemoglobin. The agar base did not affect growth unless the supplements became limiting, in which case Bacto agar base generally supported growth better than BiTek agar base. Incubation at 35 C was optimum for all 5 strains. Growth at 42 C was slow, with the greatest decrease in the rate and amount of growth occurring with field isolates of F. tularensis subspecies tularensis. Strain 6223 did not grow at 25 C, and the 4 field isolates grew slowly at the lower temperature.(ABSTRACT TRUNCATED AT 250 WORDS)


Successful xenografts of second trimester human fetal brain and retinal tissue in the anterior chamber of the eye of adult immunosuppressed rats.

Successful xenografting of first trimester human fetal CNS tissue and retina has been reported in the literature. We wished to test the feasibility of using the anterior chamber of the rat eye to support the development of more mature human fetal xenografts. Here we report on the successful outcome of human brain and retinal transplants. Adult host rats immunosuppressed with cyclosporin A accepted these xenografts and supported their further development. Periodic examination of the host eyes using a direct ophthalmoscope or an ophthalmic slit lamp permitted direct visual monitoring of the health and growth of the transplants. Histologically it was possible to identify neuronal, macroglial, and microglial (macrophage) cell types within the grafts. Mitotic activity and histogenetic differentiation took place. Blood vessels filled with hematic cells were commonly present within the grafts. The walls of these vessels prevented the leakage of horseradish peroxidase, suggesting the presence of a functional brain-blood barrier in the graft. These results indicate that it is possible to use a small animal model to study normal and pathological phenomena on late fetal human neural tissues. Our group has already taken advantage of the model to achieve HIV infectivity of fetal human brain outside the human body.


Pleuropneumonia caused by Actinobacillus pleuropneumoniae biotype 2 in growing and finishing pigs.

Actinobacillus pleuropneumoniae biotype 2 was isolated in pure culture or as the predominant isolate from the lungs of 9 growing and finishing pigs with pleuropneumonia. Gross and microscopic lesions resembled those caused by A. pleuropneumoniae biotype 1 serotypes (Nos. 1, 5, and 7) traditionally seen in the United States. The overall mortality rate for growing and finishing pigs on this 1,200-sow farrow-to-finish farm ranged from 0.37% to 0.84% per month from July 1990 to February 1991, and mortality due to respiratory disease ranged from 0.17% to 0.52% per month for the same period. This Actinobacillus species did not require V factor (no satellitism on blood agar with a Staphylococcus streak), was strongly beta-hemolytic, and demonstrated restriction fragment length polymorphisms in hybridization studies with A. suis, A. lignieresii, and A. equuli. Biochemically, the isolate most closely resembled A. pleuropneumoniae, and a DNA fragment considered specific for A. pleuropneumoniae biotypes 1 and 2 was demonstrated using polymerase chain reaction. Necrohemorrhagic pleuropneumonia similar to that caused by A. pleuropneumoniae biotype 1 was reproduced experimentally in 2 4-week-old pigs inoculated intratracheally with broth cultures of the A. pleuropneumoniae biotype 2. This study demonstrated the presence of A. pleuropneumoniae biotype 2 in the United States.


Viable adrenal medullary transplants in non-human primates: increasing the number of grafts.

The robust survival of stereotaxic adrenal medullary autografts in monkey brain parenchyma depends heavily on technique. One aspect of technique critical for clinical applications of CNS grafting is the problem of spreading treatment effects throughout large regions in a primate brain. Stereotaxic placement of a large number of grafts, which would address this problem, would require that later grafts retain the capacity for viability over a long period of time during the surgery, while earlier grafts are being made. In the present experiments involving 12 longtailed macaques (Macaca fascicularis), some grafts remained in medium 2.5 h before being transplanted, without apparent loss of viability. A clinically sufficient distribution of graft tissue would also require a large number of separate grafts to be derived from one adrenal medulla. One method for subdividing the gland might be to cut it into numerous very small pieces, but various dicing techniques described in this report have yielded little viable graft tissue. On the other hand, the number of ribbon grafts of the original dimensions available from a single gland is rather small; adrenal ribbons of the original dimensions were therefore further dissected. Half-length and half-width ribbons were successful, but less so than full-size ribbons in terms of proportionate viability. An assay of behavioral effectiveness, applied to all subjects described here, is presented in a separate contribution.


Prevalence and serovars of leptospira involved in equine abortions in central Kentucky during the 1990 foaling season.

A study to determine the prevalence of leptospira-induced abortions in the central Kentucky equine population during the 1990 foaling season and to determine the leptospira serovars responsible was conducted. From July 1, 1989 through June 30, 1990, 32 (4.4%) of 726 submissions (fetuses, stillborn foals, and/or placentas) were diagnosed as leptospirosis by the fluorescent antibody test and/or microscopic agglutination test. Attempts were made to isolate leptospires from the fetal tissues and/or the dam's urine in 31 of these cases. Leptospira interrogans serovar kennewicki was isolated from 11 (35.5%) and serovar grippotyphosa from 2 (6.5%) of the 31 cases. Of 12 cases that were culture negative with serologically positive fetal fluids, 8 had titers against serovar pomona, 1 against bratislava, 1 against grippotyphosa, 1 against hardjo, and 1 against both bratislava and pomona.


Comparison of six commercially available transport media for maintenance of Serpulina (Treponema) hyodysenteriae.

Two anaerobic (A1 and A2), 1 selective (S1), and 3 conventional (C1, C2, and C3) transport media formulations were compared for their capacity to maintain the viability of Serpulina (Treponema) hyodysenteriae. Initial experiments compared the recovery of S. hyodysenteriae from pure cultures held in each transport medium for 0.5, 1, 2, 3, 5, and 7 days at -40 C, 4 C, 25 C, and 36 C. Subsequent experiments compared each transport medium for maintenance of S. hyodysenteriae in fecal specimens obtained from experimentally infected pigs after holding for up to 7 days at 25 C. In each experiment, the viability of S. hyodysenteriae in each transport medium incubated at each temperature and for each period was determined by inoculating the transport medium onto either trypticase soy agar with 5% sheep blood or selective BJ agar and incubating at 42 C anaerobically. Viability and fecal flora contamination were evaluated blindly after 2-, 4-, and 6-day incubation periods. At -40 C, recovery of viable S. hyodysenteriae from pure culture did not differ among the transport media from 0.5 to 7 days, and all of the transport media consistently maintained the viability of the spirochetes for 7 days. At 4 C, the anaerobic and selective transport media maintained the viability of pure cultures of S. hyodysenteriae significantly better than did the conventional transport media group at day 7 (P = 0.019). At the same temperature, the anaerobic media maintained viability better than did the conventional media at 5 days (P less than 0.042).(ABSTRACT TRUNCATED AT 250 WORDS)


Behavioral effects of adrenal medullary transplants in non-human primates.

Small multiple "ribbon" autografts of intact adrenal medulla stereotaxically implanted at several sites throughout the striatum in longtailed macaques (Macaca fascicularis) have been shown to contain large amounts of viable glandular tissue as long as eight weeks after transplantation. Variations of technique clearly influence viability. All monkeys were maintained in specially adapted rotometer cages so that 24-hour measurements of activity and directional bias could be gathered. Lesions induced by intracerebral injection of 6-hydroxydopamine in the substantia nigra produced the expected chronic decrease in percentage of contralateral turning in most of the 24 subjects. Animals that received the longest viable ribbon grafts showed a reversal of this effect back toward base line, whereas monkeys whose grafts left little or no surviving tissue showed no behavioral improvement.


Chronic gastroenterocolitis in nine cats.

Lesions of a chronic gastroenterocolitis were found in 9 cats of the Persian breed that were euthanized after a prolonged period of bloody diarrhea. Gross lesions consisted of gastrointestinal edema with prominent Payer's patches, multiple grayish nodules, and a few irregular erosions within the colonic mucosa. Microscopically, the changes were composed of degeneration, necrosis, and proliferation of gastric glandular epithelium, dilated intestinal crypts with lymphoplasmacytic cell infiltration of the lamina propria, and in 1 cat, severe transmural necrosis of the colon. With the Giemsa stain, spiral-shaped organisms in the gastrointestinal lumen and intracellularly in the gastric and the colonic epithelium were observed. These organisms could not be cultured. Although the role of these spiral-shaped organisms was not determined, other agents that could explain the disorder were not found.


Canine leishmaniasis caused by Leishmania leishmania infantum in two Labrador retrievers.

Canine leishmaniasis, a generally fatal parasitic disease, was diagnosed in 2 dogs with a medical history of foreign travel, lymphadenopathy, emaciation, anorexia, intermittent fever, and cutaneous lesions. Clinically, hyperproteinemia, proteinuria, azotemia, and glomerulopathy were evident. Isolation of Leishmania species was done using Schneider's Drosophila medium. Syrian hamsters were used for infectivity studies. Clear taxonomic identification was done biochemically by isoenzyme analysis and comparison of zymogram banding patterns with 6 World Health Organization reference strains. Based on the geographic origin of affected dogs, clinicopathologic presentation, visceralization with hepatosplenomegaly in hamsters, and isoenzyme analysis, a diagnosis of Leishmania leishmania infantum was made. This study, representing the first taxonomic identification of an isolate from canine leishmaniasis, demonstrates the zoonotic and epidemiologic implications of this disease.


Hairy vetch (Vicia villosa Roth) poisoning in cattle: update and experimental induction of disease.

Hairy vetch poisoning (vetch-associated disease) of cattle is a generalized disease characterized pathologically by infiltration of skin and many internal organs by monocytes, lymphocytes, plasma cells, and often eosinophils and multinucleated giant cells and clinically by dermatitis, pruritus, often diarrhea, wasting, and high mortality. The disease was experimentally reproduced in an adult Angus female that had recovered from the natural disease 1 year earlier. She developed dermatitis on the 11th day of vetch feeding, and despite withdrawal from the vetch diet on the 12th day, death occurred 24 days after first day of vetch feeding. The cow developed lymphocytosis and hyperproteinemia. The results of other hematologic evaluations, blood chemical profiles, urinalysis, and cutaneous hypersensitivity tests using vetch lectin were normal. Lymphocyte blastogenesis studies with vetch lectin were not interpretable. Necropsy revealed gross lesions characteristic of the disease in the skin, heart, kidney, adrenal, and lymphoid tissues. Microscopically there was typical cellular infiltration in those organs and in the thyroid, liver, pancreas, salivary and mammary glands, urinary bladder, corpus luteum, and cerebral meninges. Cutaneous apocrine gland necrosis was present. The inflammatory reaction has qualities of a type-IV hypersensitivity reaction. Hypersensitivity may occur when constituents of the ingested plant are absorbed and act as antigens that sensitize lymphocytes and evoke the multisystemic granulomatous inflammatory response that characterizes the disease. Alternatively, vetch lectin may directly activate T lymphocytes to initiate the cellular response. Vetch-like diseases have been associated with a variety of diets that did not contain hairy vetch.(ABSTRACT TRUNCATED AT 250 WORDS)


A rapid, sensitive thin layer chromatography procedure for the detection of fumonisin B1 and B2.

A thin layer chromatography (TLC) method was developed for the detection of fumonisin B1 and B2 in corn and corn-based feedstuffs. Finely ground samples were extracted with acetonitrile:water (1:1), filtered, and applied to C18 cleanup columns. The columns were washed with 1% aqueous KCl followed by acetonitrile: 1% aqueous KCl (1:9), and the fumonisins were eluted with acetonitrile:water (7:3). The eluants were concentrated and spotted on reverse-phase C18 TLC plates along with fumonisin B1 and B2 standards, and the plates were developed in methanol: 4% aqueous KCl (3:2). The fumonisins were visualized by spraying the TLC plates successively with 0.1 M sodium borate buffer, fluorescamine, and 0.01 M boric acid. The plates were then dried and examined under longwave ultraviolet light. Fumonisin B1 and B2 appeared as bright yellowish-green fluorescent bands at Rfs of 0.5 and 0.1, respectively. The detection limit for the fumonisins on the TLC plate was 0.1 ppm in corn. Recoveries from spiked samples averaged greater than 80%. The identification of the fumonisins was confirmed by hydrolyzing the parent compounds of B1 and B2 to their respective C22 amino-alcohols and reexamining by TLC with the same visualizing reagents. This procedure was used to survey 193 corn samples collected from University of Missouri test plots in 1990 for fumonisin B1. Fumonisin B1 was detected in 15% of the corn samples.


Fumonisin toxicity in broiler chicks.

The effects of dietary fumonisin B1 were evaluated in young broiler chicks. The experimental design consisted of 5 treatments each with 9 randomly allotted male broiler chicks. Day-old chicks were fed diets containing 0 (feed control), 100, 200, 300, or 400 mg fumonisin B1/kg feed for 21 days. Response variables measured were chick performance, organ weights, serum biochemistry, and histologic parameters. Body weights and average daily gain dramatically decreased with increasing dietary fumonisin B1, and liver, proventriculus, and gizzard weights increased. Diarrhea, thymic cortical atrophy, multifocal hepatic necrosis, biliary hyperplasia, and rickets were present in chicks fed diets containing fumonisin B1. Serum calcium, cholesterol, and aspartate aminotransferase levels all increased at higher fumonisin dietary levels. Results indicate that fumonisin, from Fusarium moniliforme culture material, is toxic in young chicks.


Arsenic concentrations in tissues and body fluids of dogs on chronic low-level dietary sodium arsenite.

Twenty-four female Beagle dogs, 7-8 months old, were assigned to 4 groups. Control, low-dosage, medium-dosage, and high-dosage groups were offered 0, 1, 2, and 4 mg of sodium arsenite per kilogram of body weight per day (mg/kg/day), respectively, in their feed (equivalent to 0.0, 33.4, 66.7, and 133.4 micrograms/g in feed). On day 59, the dosage was doubled for the rest of the experiment, which ended on day 183. In general, arsenic concentrations in tissues and body fluids reflected arsenic levels in feed. Arsenic caused a dose-related decrease in food intake. Statistically significant differences in blood, liver, and kidney arsenic were detected, in most cases, between the 2 higher dosage groups and controls. The greatest differences in arsenic concentrations between groups were present in urine and hair. Results indicate that urine and hair would be the most useful specimens for chemical analysis when attempting to confirm low-level dietary inorganic arsenic exposure or poisoning.


Two models for transforming auditory signals from head-centered to eye-centered coordinates.

Two models for transforming auditory signals from head-centered to eye-centered coordinates are presented. The vector subtraction model subtracts a rate-coded eye position signal from a topographically weighted auditory target position signal to produce a rate-code of target location with respect to the eye. The rate-code is converted into a place-code through a graded synaptic weighting scheme and inhibition. The dendrite model performs a mapping of head-centered auditory space onto the dendrites of eye-centered units. Individual dendrites serve as logical comparators of target location and eye position. Both models produce a topographic map of auditory space in eye-centered coordinates like that found in the primate superior colliculus. Either type can be converted into a model for transforming visual signals from retinal to head-centered coordinates.


Quantitative measurement of two-component pH-sensitive colorimetric spectra using multilayer neural networks.

The purpose of this research was to develop a noise tolerant and faster processing approach for in vivo and in vitro spectrophotometric applications where distorted spectra are difficult to interpret quantitatively. A PC based multilayer neural network with a sigmoid activation function and a generalized delta learning rule was trained with a two component (protonated and unprotonated form) pH-dependent spectrum generated from microspectrophotometry of the vital dye neutral red (NR). The network makes use of the digitized absorption spectrum between 375 and 675 nm. The number of nodes in the input layer was determined by the required resolution. The number of output nodes determined the step size of the quantization value used to distinguish the input spectra (i.e. defined the number of distinct output steps). Mathematic analysis provided the conditions for which this network is guaranteed to converge. Simulation results showed that features of the input spectrum were successfully identified and stored in the weight matrix of the input and hidden layers. After convergent training with typical spectra, a calibration curve was constructed to interpret the output layer activity and therefore, predict interpolated pH values of unknown spectra. With its built-in redundant presentation, this approach needed no preprocessing procedures (baseline correction or intensive signal averaging) normally used in multicomponent analyses. The identification of unknown spectra with the activities of the output layer is a one step process using the convergent weight matrix. After learning from examples, real time applications can be accomplished without solving multiple linear equations as in the multiple linear regression method.(ABSTRACT TRUNCATED AT 250 WORDS)


Rate coherence and event coherence in the visual cortex: a neuronal model of object recognition.

We propose a function-oriented model of the visual cortex. The model addresses an essential task of the visual system: to detect and represent objects. These are defined as sets, which reappear in the input with invariant inner relations. A network, incorporating an idealized description of anatomical and physiological data, is presented with a movie showing various moving objects. In the course of time, as a result of Hebbian plasticity, a connection scheme develops which embodies in its forward and lateral connections the information necessary to perform the operations involved in object recognition. We demonstrate that coherent neural activity can exploit this information. Two types of coherence have to be distinguished in this respect. Rate coherence performs invariance operations and association, while event coherence accomplishes segmentation tasks. The model reproduces and explains experimental findings made both in physiological recordings from the visual cortex and in psychophysical studies.


Three-dimensional head angular velocity detection from otolith afferent signals.

Afferent signals from the otolith organs can produce compensatory eye position and velocity signals which has been described as linear vestibulo-ocular reflex (LVOR). The afferent otolith signals carry information about head orientation and changes of head orientation relative to gravity. A head orientation (tilt) related position signal can be obtained from population vector coding of tonic otolith afferent signals during static or dynamic head tilts, which in turn could produce compensatory eye position signals in the LVOR. On the other hand, eye angular velocity signals may be extracted, as proposed in this study, from the population response of tilt-velocity sensitive otolith afferents. Such afferents are shown to encode instantaneous head orientation relative to gravity at onset of a head movement and, as the movement continues, the projection of head angular velocity onto the earth-horizontal plane, indicating the instantaneous direction of movement relative to gravity. Angular velocity components along the earth-vertical direction which are not directly encoded by otolith afferents can be detected by central signal processing. Central reconstruction of 3D head angular velocity allows to obtain information about absolute head orientation in space even in the absence of semi-circular canal related information. Such information is important for generating compensatory eye movements as well as for dynamic control of posture.


An analytical short- and long-term memory model of presynaptic plasticity.

A mathematical model, called the Learning Gate Model (LGM), that describes phenomena responsible for biological synaptic plasticity, is presented. The functionality of the model are mainly based on the work of Kandel and colleagues on the most elementary forms of learning observed in the Aplysia Californica marine mollusc. In particular, emphasis is placed on the double temporal dynamics of synaptic plasticity and the temporal specificity of classical conditioning. By properly modeling the effect of the binding of Ca++ ions to the serotonin-sensitive adenylate cyclase enzyme, it is shown how a positively accelerated learning curve can be obtained for sensitization and classical conditioning. Phenomena of spontaneous recovery and second-order conditioning are reproduced through simulations. Mathematical analyses of the temporal trace of conditioned stimulus and of the Short-Term Memory steady state are also given.


Motor control of voluntary arm movements. Kinematic and modelling study.

The motor control of pointing and reaching-to-grasp movements was investigated using two different approaches (kinematic and modelling) in order to establish whether the type of control varies according to modifications of arm kinematics. Kinematic analysis of arm movements was performed on subjects' hand trajectories directed to large and small stimuli located at two different distances. The subjects were required either to grasp and to point to each stimulus. The kinematics of the subsequent movement, during which subject's hand came back to the starting position, were also studied. For both movements, kinematic analysis was performed on hand linear trajectories as well as on joint angular trajectories of shoulder and elbow. The second approach consisted in the parametric identification of the black box (ARMAX) model of the controller driving the arm movement. Such controller is hypothesized to work for the correct execution of the motor act. The order of the controller ARMAX model was analyzed with respect to the different experimental conditions (distal task, stimulus size and distance). Results from kinematic analysis showed that target distance and size influenced kinematic parameters both of angular and linear displacements. Nevertheless, the structure of the motor program was found to remain constant with distance and distal task, while it varied with precision requirements due to stimulus size. The estimated model order of the controller confirmed the invariance of the control law with regard to movement amplitude, whereas it was sensitive to target size.


Noninvasive diagnosis of coronary artery disease using a neural network algorithm.

This study examines the utility of neural networks for detecting coronary artery disease noninvasively by using the clinical examination variables and extracting useful information from the diastolic heart sounds associated with coronary occlusions. It has been widely reported that coronary stenoses produce sounds due to the turbulent blood flow in these vessels. These complex and highly attenuated signals taken from recordings made in both soundproof and noisy rooms were detected and analyzed to provide feature set based on the poles and power spectral density function (PSD) of the Autoregressive (AR) method after Adaptive Line Enhancement (ALE) method. In addition, some physical examination variables such as sex, age, body weight, smoking condition, diastolic pressure, systolic pressure and derivation from them were included in the feature vector. This feature vector was used as the input pattern to the neural network. The analysis was studied on one hundred recordings (63 abnormal, 37 normals). The network correctly identified 84% of the subjects with coronary artery disease and 89% of the normal subjects.


Independent control of joint stiffness in the framework of the equilibrium-point hypothesis.

In the framework of the equilibrium-point hypothesis, virtual trajectories and joint stiffness patterns have been reconstructed during two motor tasks practiced against a constant bias torque. One task required a voluntary increase in joint stiffness while preserving the original joint position. The other task involved fast elbow flexions over 36 degrees. Joint stiffness gradually subsided after the termination of fast movements. In both tasks, the external torque could slowly and unexpectedly change. The subjects were required not to change their motor commands if the torque changed, i.e. "to do the same no matter what the motor did". In both tasks, changes in joint stiffness were accompanied by unchanged virtual trajectories that were also independent of the absolute value of the bias torque. By contrast, the intercept of the joint compliant characteristic with the angle axis, r(t)-function, has demonstrated a clear dependence upon both the level of coactivation and external load. We assume that a template virtual trajectory is generated at a certain level of the motor hierarchy and is later scaled taking into account some commonly changing dynamic factors of the movement execution, for example, external load. The scaling leads to the generation of commands to the segmental structures that can be expressed, according to the equilibrium-point hypothesis, as changes in the thresholds of the tonic stretch reflex for corresponding muscles.


[The life experiences and the implication of caregivers in persons living with HIV].

This article focuses on a qualitative study, conducted in Montréal between March and July of 1990, whose aim was to understand the life experiences and the needs of close and supportive individuals that care for seropositive persons. The objective of the study is to help provide more direction to the scope of services dispensed by the Centre Pierre-Hénault, an organization created by a group of close supporters of HIV-infected persons. Eighteen individuals volunteered for the in-depth interviews: nine of these were caring for persons who had developed AIDS, four were providing support for persons who were HIV-positive, and the remaining five had assisted persons who passed away following AIDS-related diseases. The study shows that the risk of isolation is greater among those close supporters who provide care for HIV-positive persons. Although research does point to a number of fundamental traits that are shared among all types of supportive individuals, certain traits are specifically related to people caring for HIV-positive persons, including the taboo and the stigma that are linked to AIDS, as well as the rapid and unforeseen loss of people in their prime.


[Psychosocial intervention for persons infected with HIV].

We describe a psychosocial intervention program designed to enhance coping with the stress associated with HIV infection. The NUCARE intervention program is based on six components of coping including: cognitive reframing, problem-solving, relaxation, goal setting, social support and use of resources and services. The rationale and development of the intervention is discussed and practical case examples illustrating the benefits of each component are presented.


[Particulars of crisis intervention in seropositive persons or the existential crisis of HIV].

This article presents a vision of crisis intervention for seropositive persons following an approach inspired by existential psychology. Persons who intervene must view crisis intervention for HIV-positive persons as an exploration of the close relation that exists between life and death. They must investigate the interrelations with the dimensions who intervene must view crisis intervention for HIV-positive persons as an exploration of the close relation that exists between life and death. They must investigate the interrelations with the dimensions of time, space, world and body. They must also examine the characteristics of the seropositive experience that are specific to the different infected clienteles. The welcoming process, the urgency of the situation and the therapeutic complementarity must be considered as the main guidelines for this type of crisis intervention which, in addition, must take into account the various life experiences that are particular to each of the HIV-positive persons. This approach requires that the person who intervenes and plays a supporting role must put into question their own attitudes toward the phenomenon at hand. Finally, life with the infection can also bring certain intervenors and clients to discover and profit from a number of unsuspected benefits.


[The effects of perinatal infection with the HIV virus on the development of adaptive behavior].

This study examined the development of adaptive functioning in nine HIV-positive and nine HIV-negative children ranging in age from 3 months to 6 years, 11 months. Both groups showed normal development of adaptive behaviour prior to two years of age; after this time the HIV-positive group was impaired in all areas of functioning. The results are discussed in terms of the program needs of the HIV-infected children and their families.


Diagnosis and treatment of early melanoma. NIH Consensus Development Conference. January 27-29, 1992.

The National Institutes of Health Consensus Development Conference on Diagnosis and Treatment of Early Melanoma brought together experts in dermatology, pathology, epidemiology, public education, surveillance techniques, and potential new technologies as well as other health care professionals and the public to address (1) the clinical and histological characteristics of early melanoma; (2) the appropriate diagnosis, management, and followup of patients with early melanoma; (3) the role of dysplastic nevi and their significance; and (4) the role of education and screening in preventing melanoma morbidity and mortality. Following 2 days of presentations by experts and discussion by the audience, a consensus panel weighted the scientific evidence and prepared their consensus statement. Among their findings, the panel recommended that (1) melanoma in situ is a distinct entity effectively treated surgically with 0.5 centimeter margins; (2) thin invasive melanoma, less than 1 millimeter thick has the potential for long-term survival in more than 90 percent of patients after surgical excision with a 1 centimeter margin; (3) elective lymph node dissections and extensive staging evaluations are not recommended in early melanoma; (4) patients with early melanoma are at low risk for relapse but may be at high risk for development of subsequent melanomas and should be followed closely; (5) some family members of patients with melanoma are at increased risk for melanoma and should be enrolled in surveillance programs; and (6) education and screening programs have the potential to decrease morbidity and mortality from melanoma. The full text of the consensus panel's statement follows.


[Psychosocial factors influencing the intention of women infected with HIV to have a child: study of 12 cases].

This study documents the factors that influenced the decision of 12 women of child-bearing age whether to have a child in the three years following an infection by the human immunodeficiency virus (HIV). Three of the 12 women are intending to have a child, and their intention stems from their beliefs and positive attitudes toward having a child, their partner's positive influence, the absence of their children, their denial of the disease and the absence of symptoms within their family. For the nine other women, the factors that influenced their decision not to have a child include the sociopolitical and economical context, the fact their family is complete, the fact they are seropositive or III, their fear of transmitting the infection and the idea of losing a child to AIDS.


[Psychotherapy in child incest: the dangers of a psychic break].

There seems to be a consensus today by which a child, who is a victim of incest or sexually abused, must be directed to therapy in order that he or she provides a factual account of the traumatism and, by the same token, exorcises it. The authors deplore the widespread use of this destabilizing method. They emphasize that, for many children, recovery is achieved more successfully through "repression" (in the psychoanalytical sense of the term). After having obtained disclosure from the child, the authors argue that it is preferable for the intervenor to create the appropriate therapeutic conditions allowing the child to positively bury the event and move on with his or her life.


[The sexual needs of homosexual HIV seropositive men].

A two-step study was conducted to identify the sexual needs of a group of seropositive homosexual men. At first, there was an interview with 30 men which aimed to define their objectives of sexual health. This was followed by a questionnaire distributed among 88 seropositive men and 173 seronegative men, who evaluated the objectives stated in the first step. The statistical analysis of collected data allowed for the identification and narrowing down of four major needs: being in good health, being in love, being sexual and, to a lesser degree, being secure. Results are discussed and point to the role played by the biological, psychological and sociological uneasiness in the context of the difficulties that seropositive homosexual men have in satisfying their sexual needs. This evaluation can prove useful in the planning and the implementation of sexological services for HIV-positive persons.


[Quality of relations between gay couples and high-risk sexual behavior].

This study examines the hypotheses by which quality relationships among gay couples is closely related to the adoption of low-risk sexual behaviours, in monogamous and non-monogamous situations. Data was collected from 29 homosexual couples (58 men) of the Montréal area. The survey, which graded sexual conduct as well as adjustments made within the relationship, confirmed the author's hypothesis. Indeed, the distribution of couples according to a multifactorial risk index does in fact establish a connection between the couple's happiness and the level of risk during sexual relations within and outside the couple. These conclusions are discussed in light of existing data pertaining to the link between health and the quality of a relationship among heterosexual couples.


[Psychiatric repercussions of HIV infection. Current update].

Persons infected by the HIV virus are known to be vulnerable to psychological and neuropsychiatric complications. The state of knowledge today indicated that the organic cerebral syndromes and adaptation problems are among the most frequent complications requiring a psychiatric intervention. Affective problems represent yet another possible type of complication. The author provides an update of current knowledge on the nature of these problems, as well as their treatment.


[Spiritual support in HIV seropositivity or AIDS].

Acute life experiences ("expériences limites de l'existence") have long been considered the exclusive domain of religion. Until recently in Québec, the Catholic Church was noted for overseeing these symbolic events. Although the religious reference has not completely disappeared, the author argues that it has now undergone two changes, in terms of privatization and diversification. As such, several systems of belief, whether religious or secular, are being suggested to provide meaning to our lives. Yet, can these approaches provide any relevant support to patients in general, and to HIV-infected persons in particular, including the various organizations and individuals that provide care and assistance? The author points out that genuine support for seropositive persons or AIDS patients in their search for meaning and their fight against the negative forces in life, must involve a person's spiritual dimension. The outcome of this indispensable process does not necessarily have to lead to a traditional religious creed. However, in considering the issue of spiritual support in the context of illness and suffering, the author concludes that "belief in the search for meaning" is an imperative for all those who are concerned by AIDS.


[The evolution of child custody after the separation of the parents].

Following the separation of spouses, a family undergoes a reorganization process where the choice of a formula for child custody represents a major step, which child's living environment will depend on. Despite the importance of this form of custody, it seems, in the context of the crisis following separation, that decisions surrounding that choice are often taken haphazardly, without much consideration of various alternatives. On a different note, the family's profile at the time of the separation is bound to change following the pace of the child's and the parent's development. What may seem a relevant formula for a three year-old child may not necessarily be the case when the child turns eight. This article looks at the extent to which the child custody formula evolves to adjust itself to the changing needs of children and parents. Research focused on a sampling of 112 families separated for an average of four and a quarter years, with children aged 10 (N = 49) or adolescents of 15 years of age (N = 63). Data was compiled through a telephone questionnaire filled out by the parent who was the most committed to the child's custody. The description of the two categories of change (minor and major) was made according to the original child custody formula, the time lapse since the separation, the child's age and parental re-composition. Results point to three main trends: a) child custody arrangements do not evolve much over time; b) when there are changes, these are especially motivated by the needs of the parents; and c) the changes mainly result in reducing the frequency of contacts between the child and the parent without custody.


[Aurore, child martyr. Essay on violence done to children].

This article draws on the sad story of Aurore Gagnon, a battered child raised in rural Québec and whose turmoil was dramatized on film. By elaborating on this symbol, the author is able to generate, at least in a systemic perspective, a number of issues and outlooks that go far beyond the generalities usually associated with this tale. For instance, there is ample evidence showing that the behaviour of Aurore's stepmother, aberrant as it may be, is largely caused by a set of environmental circumstances. Of course, today's social conditions hardly resemble those of the past. Yet, given the conjunctions of certain factors, children, who are nevertheless our most valuable asset, continue to be in danger in the presence of their very own parents.


[Psychological distress in persons afflicted with HIV in Montreal].

This paper describes the HIV-related mental health concerns of a sample of 128 persons with HIV infection in Montréal who participated in a larger national survey of HIV-related mental health needs and services in Canada. We examined mental health distress in persons with HIV infection in Montréal compared to other cities in Canada, and in subgroups of HIV-infected Montrealers defined on the basis of sex, age, diagnosis, and risk factor status. Results demonstrate that although HIV infection has a strong and far reaching impact on mental health, there are differences in the types of concerns and issues that are distressing to specific groups of Montréal respondents. Uncertainty about the future and not being able to realize life goals, as well as feelings of helplessness and fears about potential adverse neurological consequences of HIV disease, were major sources of psychological distress. Feelings of depression, anxiety, and anger, as well as concerns about increasing physical disability, pain, infecting others, confidentiality, and finances were predominant concerns among specific subgroups. Differences between respondents in terms of source of income, age, and sex, and to a lesser extent diagnosis and risk factor status, were associated with varying levels of mental health distress. Although respondents in Montreal (and Vancouver) were more distressed than respondents in Toronto and Halifax, these differences appear to be due primarily to differences in age and source of income. Findings from this study will be useful to policy makers and health planners in developing services to meet the mental health needs of HIV infected adults.


[Consequences of AIDS in the lives of hemophiliacs and their caregiving families in Quebec: stress, response to stress and social support].

The following article focuses on the Québec portion of a national survey on the care needed by hemophiliacs with AIDS or having contracted the HIV virus. The survey was based on an approach that considers social support as a means to face stress. It also examined the needs of dispensers of care and relatives (whether mourning or not) of these persons. Participants revealed having experienced more stress because of an absence of support or simply negative support, than because of the physical deterioration caused by the disease. In addition, the question of confidentiality was often raised. In general, participants said they were satisfied with the support they had received, especially on the part of members of their family.


Critical analysis of objections against the biological molecular energy machines.

In the past, two important objections against McClare's idea of biological molecular energy machines were raised. One of the criticisms was concerned with the origin of energy gained in ATP cleavage and with an interpretation of McClare's "excited vibrational state." The former argument reveals a failure of the critics to comprehend McClare's approach. As to the excited vibrational state, it can be identified with nonequilibrium conformational states of the unit rather than with a single vibrational mode. The other criticism based on Brillouin's energy cost of measurement argued that reversible operation of biological molecular energy machines would be virtually impossible. Using propagation velocities of deformations of the unit's structure (instead of velocity of light), the objections against reversibility are invalidated even in the framework of the critic's approach. McClare's idea and relevant definitions are thus physically correct.


Plausible view on the biological molecular energy machines.

A qualitative picture of operation modes of biological molecular energy machines is presented. It is suggested that there is mutual control between the flow of molecular energy stored in a biological molecular energy machine and the sequence of nonequilibrium conformational states through which the machine passes in doing work. If the structure of the conformational space is favorable, the set of trajectories in this space decomposes into two families, each of which accomplishes another task. This divergence of trajectories enables to distinguish molecular objects according to differences in interaction between the machine and the object, i.e., to perform a measurement on a molecular object and process the object according to the result of that measurement.


Hydrophobic interaction between globin helices.

The interhelical interfaces have been examined in seven high-resolution globin chains. The profiles of hydrophobic contact, as measured by the residue solvent-accessible area loss upon folding, have been calculated. The seven globins studied differ in their overall loss of solvent-accessible area upon packing of their helices, the order being 1MBD greater than 1LH1 greater than 1ECD greater than 2MHBB greater than 2HHBB greater than 2HHBA greater than 2MHBA, which gives a measure of the difference in stability due to the hydrophobic interaction. The five helix-pair packings (AH, BE, BG, FH and GH) examined in detail have qualitative similarities. There are, however, substantial quantitative differences both at the equivalent residue level and at the level of overall helix-helix contact, which has significance in some models of folding. The AH pair has the most uniform area loss over the seven globins and the largest variation in accessible area loss on packing among the five helix pairs is the GH pair. The set of residues required to produce the globin fold has been deduced from the residue area losses.


Helix formation in methylated copolymers of lysine and alanine.

Random copolymers of lysine and alanine, 2:1 and 1:1, were trimethylated on the lysine amino groups to quaternary ammonium groups. Methylated and unmethylated polymers were prepared with Cl- or ClO4- as the counterion. CD spectra were measured for increasing concentration of peptide without added salt, and at constant peptide concentration in increasing NaCl or NaClO4. Unmethylated peptides, as the chloride, form alpha-helix more readily than do the methylated peptides. The opposite occurs with ClO4- as counterion. The helix-promoting effect of methylated lysine residues (ClO4- counterion) is diminished by the presence of alanine, as compared with effects when lysine is the only type of residue. The effect of methylation of proteins on helix formation may depend on the types of anionic groups with which the protein may be involved.


Early assembly pathways of type I collagen.

A method was developed for computing the free energy (delta Fi) of aggregates of type I collagen. The method was based on a treatment of Matheson and Flory describing phase equilibria of rigid rod polymers. It included a polymer-solvent interaction term that depended on near neighbor transfer energies. Extrahelical portions of the molecule were assigned local interaction energies differing from that assigned to the helix. Free energies of reaction for successive steps along assembly pathways (delta Fi-i+1) were computed. When allowance was made for specific pairing between extrahelical and helical domains, the so-called D-staggered (D = 670 A) alignment of molecules was preferred, as opposed to a nonstaggered, or nematic, alignment. Based on delta Fi-i+1 alone, it appeared that 1D-staggered oligomers arise first in assembly, followed later by addition of molecules in 4D alignment. Neither 4D dimers nor 4D-8D trimers were predicted to be major intermediates in assembly. This result is contrary to previous hypotheses. When energies of activation were included in the analysis, the prediction was less certain, and specific circumstances were identified in which 4D dimers and 4D-8D trimers were the earliest aggregated species in assembly.


Langevin dynamics of peptides: the frictional dependence of isomerization rates of N-acetylalanyl-N'-methylamide.

The rate constant for the transition between the equatorial and axial conformations of N-acetylalanyl-N'-methylamide has been determined from Langevin dynamics (LD) simulations with no explicit solvent. The isomerization rate is maximum at collision frequency gamma = 2 ps-1, shows diffusive character for gamma greater than or equal to 10 ps-1, but does not approach zero even at gamma = 0.01 ps-1. This behavior differs from that found for a one-dimensional bistable potential and indicates that both collisional energy transfer with solvent and vibrational energy transfer between internal modes are important in the dynamics of barrier crossing for this system. It is suggested that conformational searches of peptides be carried out using LD with a collision frequency that maximizes the isomerization rate (i.e., gamma approximately 2 ps-1). This method is expected to be more efficient than either molecular dynamics in vacuo (which corresponds to LD with gamma = 0) or molecular dynamics in solvent (where dynamics is largely diffusive).


Partial molar volumes of the amino acid side-chains of proteins in aqueous solution: some comments on their estimation using partial molar volumes of amino acids and small peptides.

The partial molar volumes of some amino acid side-chains were determined in two recent studies [Makhatadze, G.I., et al. (1990) Biopolymers 30, 1001, and Reading, J.F. & Hedwig, G.R. (1990) J. Chem. Soc. Faraday Trans. 86, 3117] using partial molar volume data, V02, in aqueous solution at 25 degrees C for some peptides of sequence Gly-X-Gly, where X is an amino acid. These side-chain partial molar volumes are critically compared with those obtained using V02 data for amino acids. It is concluded that side-chain partial molar volumes calculated using V02 data for the tripeptides are better estimates of side-chain partial molar volumes in proteins than are those determined using V02 data for amino acids.