[The characteristics of a Russian-made kit for the serological identification of streptococci groups A, B and C].

The first Soviet kits for the serological identification of streptococci, groups A, B, and C, on the basis of the coagglutination test were developed. Each kit was intended for 35-40 determinations. The optimum concentration of streptococci during their identification by means of the reagents making up the kit was about 1.6 x 10(9) cells/ml. The specificity of the reagents in comparison with the results of the identification of streptococci by reference methods was 97.3 +/- 0.9%. The reagents making up the kits can be presumably used for solving a number of practical problems in the epidemiological surveillance of streptococcal infection.


Multiple insulin-responsive elements regulate transcription of the GAPDH gene.

Multiple elements in the upstream region of the GAPDH gene play a role in mediating the acute and chronic effect of insulin on GAPDH gene expression. The complexity of this regulation provides many layers of control. In differentiated tissues, the transcriptional response to insulin results from the additive effects of g/TRE, IRE-A and IRE-B. The gTRE may interact with newly synthesized c-fos/c-jun heterodimer to activate GAPDH gene transcription. Studies are underway to determine whether protein synthesis inhibitors affect the regulation of GAPDH. Because there are several elements that mediate the effect, it will be difficult to determine the significance of these findings until each cis-acting factor and its binding protein can be studied in isolation. IRE-A and IRE-B act together to promote a 5- to 8-fold insulin effect on HGAPDH-CAT in H35 hepatoma cells and a 3-fold effect in 3T3 adipocytes. We have succeeded in detecting an insulin-sensitive DNA-binding protein referred to as IREA-BP with an element -480 to -435. Insulin treatment of differentiated 3T3 adipocytes for 1 hr results in a 4-fold increase in the amount of this binding protein, as estimated by the amount of 32P-labelled oligonucleotide retarded on non-denaturing PAGE (11). The effect of insulin on IRP-B is comparable. Furthermore, IREA-BP is induced during the process of fasting and refeeding rats, an important in vivo correlate with our tissue culture models (11). These observations imply that the binding proteins IREA-BP and IRP-B are essential components in the signal transduction pathway of insulin action on GAPDH gene expression in metabolically active tissues such as fat and liver. Differentiation-dependence and tissue-specificity are achieved through multiple regulatory elements and involve pre- and post-translational regulation of multiple transcription factors. IREA-BP is present in preadipocytes but activity in highly induced upon differentiation of preadipocytes to adipocytes. The IRE-B (-408 to -269) DNA binding protein is not detected in 3T3 preadipocytes. A gC/EBP like-protein takes part in the formation of this complex which may explain the inductive effect of differentiation on binding. Finally, footprint and cotransfection studies indicate that the differentiation-dependent protein C/EBP also regulates GAPDH gene transcription through a motif located within one hundred nucleotides of the promoter. We have begun to clone the IRE-A and IRE-B DNA binding proteins. An IRE-A binding protein that footprints the 3' domain of the IRE-A has been cloned.(ABSTRACT TRUNCATED AT 400 WORDS)


Comparative study of microsatellite and cytogenetic markers for detecting the origin of the nondisjoined chromosome 21 in Down syndrome.

Nondisjunction in trisomy 21 has traditionally been studied by cytogenetic heteromorphisms. Those studies assumed no crossing-over on the short arm of chromosome 21. Recently, increased accuracy of detection of the origin of nondisjunction has been demonstrated by DNA polymorphism analysis. We describe a comparative study of cytogenetic heteromorphisms and seven PCR-based DNA polymorphisms for detecting the origin of the additional chromosome 21 in 68 cases of Down syndrome. The polymorphisms studied were the highly informative microsatellites at loci D21S215, D21S120, D21S192, IFNAR, D21S156, HMG14, and D21S171. The meiotic stage of nondisjunction was assigned on the basis of the pericentromeric markers D21S215, D21S120, and D21S192. Only unequivocal cytogenetic results were compared with the results of the DNA analysis. The parental and meiotic division origin could be determined in 51% of the cases by using the cytogenetic markers and in 88% of the cases by using the DNA markers. Although there were no discrepancies between the two scoring systems regarding parental origin, there were eight discrepancies regarding meiotic stage of nondisjunction. Our results raise the possibility of recombination between the two marker systems, particularly on the short arm.


Linkage of Thomsen disease to the T-cell-receptor beta (TCRB) locus on chromosome 7q35.

The chromosomal localization of the gene for Thomsen disease, an autosomal dominant form of myotonia congenita, is unknown. Electrophysiologic data in Thomsen disease point to defects in muscle-membrane ion-channel function. A mouse model of myotonia congenita appears to result from transposon inactivation of a muscle chloride-channel gene which maps to a region of mouse chromosome 6. The linkage group containing this gene includes several loci which have human homologues on human chromosome 7q31-35 (synteny), and this is a candidate region for the Thomsen disease locus. Linkage analysis of Thomsen disease to the T-cell-receptor beta (TCRB) locus at 7q35 was carried out in four pedigrees (25 affected and 23 unaffected individuals) by using a PCR-based dinucleotide repeat polymorphism in the TCRB gene. Two-point linkage analysis between Thomsen disease and TCRB showed a maximum cumulative lod score of 3.963 at a recombination fraction of .10 (1-lod support interval .048-.275). We conclude that the Thomsen disease locus is linked to the TCRB locus in these families.


The use of carbon dioxide laser laparoscopy in the treatment of tubal ectopic pregnancies.

The purpose of this study was to assess the efficacy of the treatment of unruptured tubal ectopic pregnancies by the use of carbon dioxide laser laparoscopy. A series of 125 consecutive ectopic pregnancies were treated laparoscopically; the tubal pregnancy was removed by a laparoscopic laser technique. Preoperative assessment included monitoring beta-human chorionic gonadotropin levels, use of vaginal ultrasonography, and preoperative and postoperative hematocrit levels. Laparoscopic laser surgery was successful for removal of tubal ectopic pregnancies in all but four patients, in whom a laparotomy was required. Hematocrit levels before and after surgery were similar. The time necessary for beta-human chorionic gonadotropin to fall to nondetectable levels averaged between 3 and 4 weeks. There were five patients who had complications requiring additional surgery and/or medical treatment. The techniques are easy to learn, and the use of laparoscopic laser surgery in the treatment of tubal ectopic pregnancies appears to be a safe procedure with definite advantages for both the patient and the physician. There are decreased operating times, shorter hospital stays, and lower medical costs compared with those for major surgery. Subsequent successful intrauterine pregnancy rates are comparable to those of conservative methods previously reported.


Interferon-gamma receptors on human gestational choriocarcinoma cell lines: quantitative and functional studies.

This study was performed to further define the effects of interferon-gamma on choriocarcinoma cell lines and to determine whether variations in response among cell lines are attributable to quantitative differences in interferon-gamma receptors. Interferon-gamma receptors were quantified on BeWo, JEG-3 and Jar choriocarcinoma cell lines by a radiolabeled interferon-gamma ligand binding assay. The response of these cell lines to interferon-gamma was measured in two functional assays: a cell proliferation assay and a cell lysis assay after exposure to interferon-gamma with and without actinomycin-D. The number of interferon-gamma receptors on BeWo, Jar, and JEG-3 cells did not differ significantly (650, 560, and 420 interferon-gamma receptors per cell, respectively). Proliferation of all three choriocarcinoma cell lines was significantly inhibited to a similar extent by interferon-gamma. After treatment with interferon-gamma actinomycin-D, each choriocarcinoma cell line exhibited dose-dependent cell lysis; lysis of Jar was significantly less than that of either BeWo or JEG-3. These data further document variations in the response of choriocarcinoma cell lines to interferon-gamma and indicate that these differences are not the result of interferon-gamma receptor number but of postreceptor mechanisms.


Alzheimer's disease. Beta-amyloid precursor protein expression in the nucleus basalis of Meynert.

The nucleus basalis of Meynert (nbM) was examined using immunocytochemistry for beta-amyloid precursor protein (beta APP) expression in Alzheimer's disease (AD). In mild AD cases, light labeling of the cell body and proximal processes was observed, and small intracellular structures were labeled rarely. In the more severe cases, intense cytoplasmic beta APP labeling was seen, often along with small beta APP-positive structures. Double-labeling experiments demonstrated that in the more severe cases these small structures were also decorated by a neurofibrillary tangle (NFT) antiserum. Other neurons in the severe cases showed incorporation of beta APP into large inclusions, which were also labeled with the NFT antiserum. However, some large inclusions in the severe cases were labeled by the NFT antiserum but contained no beta APP. Extraneuronal NFTs did not show beta APP labeling and did not react with an antibody to the beta-amyloid peptide. These results suggest that increased expression of beta APP coincides with intracellular NFT formation in the nbM, but that the formation of extraneuronal NFTs results in a loss of beta APP immunoreactivity.


Effects of copper deficiency on T-cell mitogenic responsiveness and phenotypic profile of blood mononuclear cells from swine.

The effect of dietary copper deficiency on T-cell mitogenic responsiveness and phenotypic profile of blood mononuclear cells (MNC) in weaned pigs was examined. Outbred, weaned pigs were fed a semipurified diet containing adequate (6.4 mg/kg of body weight) or deficient (0.8 mg/kg) amounts of Cu. Pigs fed the low Cu diet for 10 weeks had markedly decreased concentrations of Cu in liver and plasma, and hypertrophic hearts. In vitro reactivity of MNC from Cu-deficient pigs to phytohemagglutinin and concanavalin A was significantly suppressed. This functional impairment was not associated with a decrease in the percentage of T cells, CD4 or CD8 cell subsets, or B cells. Expression of SLA-DQ and SLA-DR class II major histocompatibility complex (MHC) antigens was increased by Cu deficiency, the former significantly. Unlike rodents, in which inadequate Cu nutriture induces functional T cell deficiency that is associated with a decrease in the CD4 T-cell subset, swine fed inadequate Cu diets for 10 weeks had no changes in MNC subsets yet clearly manifested functional impairment of T-cell responses.


[Allergic drug reactions in children].

We present a retrospective study of allergic drug reactions seen in our pediatric allergy consulting room during the last 6 years. During this time, 840 patients were examined for suspected adverse drug reactions. Drug allergy was confirmed in 72 cases (8.5%). Of these cases, 29 (40.2%) were considered to be IgE mediated, or immediate hypersensitivity reactions. We have not found significant differences with regards to age, sex, atopic family history or atopia between patients with IgE mediated reactions compared to patients with allergic drug reactions of different mechanisms or to patients without drug allergies. Sulfonamides, streptomycin, beta-lactam and analgesics were the drugs most frequently involved in immediate type reactions. Among non-immediate reactions, fixed eruption by sulfonamides and contact dermatitis due to Mercurochrome were the most frequent.


Free thin paraumbilical perforator-based flaps.

A free paraumbilical perforator-based flap fed by a muscle perforator from the inferior deep epigastric artery and with no muscle was used in 13 patients. Among them, a free thin paraumbilical perforator-based flap with a thin layer of fat, to protect the subdermal plexus of the vessels, was used in seven patients. The dominant pedicle perforator of this thin flap is usually located around the umbilicus and a large flap can be obtained. Its critical length-to-breath ratio is considered to be 4:3. The advantages of this flap are a long and large vascular pedicle, rare postoperative abdominal herniation, little bulkiness of the flap, and a relatively large skin territory. The disadvantages are technical difficulties in dissection of the perforator and anatomical variation in the location of the perforator. We believe this flap largely overcomes the problems of the conventional rectus abdominis musculocutaneous flap.


Expression of developmentally defined retinal phenotypes in the histogenesis of retinoblastoma.

Retinoblastoma, the most common intraocular tumor of childhood, is a malignant neoplasm that arises during retinal development. The embryonal cell target for neoplastic transformation is not yet clearly defined. To better understand the histogenetic potential of this tumor, the expression of photoreceptor and glial cell-associated proteins were examined in 22 primary retinoblastomas. Interphotoreceptor retinol-binding protein (IRBP), cone and rod opsins were selected as the photoreceptor specific proteins due to their different temporal patterns of expression during normal retinal development. Neoplastic Müller cell differentiation, and non-neoplastic reactive astrocytes were identified using cellular retinaldehyde binding-protein (CRAlBP), and glial fibrillary acidic protein (GFAP), respectively. Photoreceptor proteins were present in 16 cases and showed different cellular patterns of expression. IRBP and cone opsin were usually abundant. Although rod opsin was clearly identified in eight tumors, its expression was more restricted than either IRBP or cone opsin. This differential pattern of expression, opposite to the normal pattern of photoreceptor gene expression in the adult retina, corresponded to a marked decrease in mRNA for rod opsin. Cone opsin and IRBP colocalized in fleurettes demonstrating that neoplastic human cone cells are capable of IRBP synthesis. Müller cell differentiation was present in 12 of the 16 cases in which photoreceptor proteins were detected. In contrast, GFAP was only present in reactive, stromal astrocytes associated with blood vessels. Our data suggest that the retinoblastoma has the histogenetic potential of the immature neural retinal epithelium which can give rise to both photoreceptor and Müller cell lineages. The differential expression of cone and rod phenotypes in retinoblastoma is consistent with the "default" mechanism of cone cell differentiation.


Abdominoplasty combined with other intraabdominal procedures.

One hundred three patients underwent abdominoplasty combined with other intraabdominal procedures including 67 tubal ligations, 34 total abdominal hysterectomies and 2 cholecystectomies, from January 1983 to July 1991. The patients were divided into two groups, those undergoing the standard or total abdominoplasty and those undergoing limited abdominoplasty with or without liposuction in delimited areas. In this series of 103 patients, we found only two minor complications and only three patients were transfused with autologous units of blood. When performed by well-schooled surgical teams, abdominoplasty may be combined with intraabdominal procedures with gratifying results.


Alterations in the cell wall of Saccharomyces cerevisiae induced by the alpha sex factor or a mutation in the cell cycle.

We performed experiments in parallel to study the rate of synthesis of cell wall polysaccharides and the activity of glycosyl transferases in Saccharomyces cerevisiae after arrest of a cdc 28 mutant in G1 phase by either addition of alpha-factor or transfer to the non-permissive temperature. Both effectors brought about similar time-dependent increases in the rate of synthesis and deposition of the cell wall polysaccharides chitin, glucan and mannan. These changes in cell wall composition were accompanied by an increase in the specific activities of glucan and chitin synthetases. This increase was inhibited by cycloheximide suggesting that it represented de novo enzyme biosynthesis and not enzyme activation. Our data are consistent with the notion that both alpha-factor and the cdc 28 mutation affect the same stage-specific function that controls the temporal expression of glycosyl transferases.


Characterization and partial purification of an insulinase from Neurospora crassa.

An insulin-binding metal- and thiol-dependent proteinase has been purified 1491-fold from high speed cytosolic fractions of the fungus Neurospora crassa. This enzyme resembles insulin-degrading enzymes (insulinases) present in mammalian cells and in Drosophila melanogaster in the following ways: (i) it degrades radiolabeled insulin with a specificity similar to that of rat muscle insulinase, as demonstrated by HPLC analysis of the degradation products; (ii) it is inhibited by bacitracin, EDTA, 1,10-phenanthroline, and the sulfhydryl-reactive compounds N-ethylmaleimide and p-chloromercuribenzoate, but not by inhibitors of serine proteases or by lysosomal protease inhibitors. Cross-linking with 125I-insulin labels a band of ca. 120 kDa, and several smaller bands which may represent degradation products. The N. crassa insulinase is stimulated by Mn2+ and strongly inhibited by Zn2+; Mn2+ can also reactivate the enzyme after inhibition by EDTA, but Zn2+ is ineffective. The N. crassa protein differs in this regard from mammalian and insect insulinases which are generally activated by both Mn2+ and Zn2+. This finding extends the apparent evolutionary conservation of these metal- and thiol-dependent proteases into the microbial realm.


F1-ATPase with cysteine instead of serine at residue 373 of the alpha subunit.

Escherichia coli strain AN718 contains the alpha S373F mutation in F1F0-ATP synthase which blocks ATP synthesis (oxidative phosphorylation) and steady-state F1-ATPase activity. The revertant strain AN718SS2 containing the mutation alpha C373 was isolated and shown to confer a phenotype of higher growth yield than that of the wild type in liquid medium containing limiting glucose, succinate, or LB. Purified F1 from strain AN718SS2 was found to have 30% of wild-type steady-state ATPase activity and 60% of wild-type oxidative phosphorylation activity. Azide sensitivity of ATPase activity and ADP-induced enhancement of bound aurovertin fluorescence, both of which are lost in alpha S373F mutant F1, were regained in alpha C373 F1. N-Ethylmaleimide (NEM) inactivated alpha C373 F1 steady-state ATPase potently but had no effect on unisite ATPase. Complete inactivation of alpha C373 F1 steady-state ATPase corresponded to incorporation of one NEM per F1 (mol/mol), in just one of the three alpha subunits. NEM-inactivated enzyme showed azide-insensitive residual ATPase activity and loss of ADP-induced enhancement of bound aurovertin fluorescence. The data confirm the view that placement at residue alpha 373 of a bulky amino acid side-chain (phenylalanyl or NEM-derivatized cysteinyl) blocks positive catalytic cooperativity in F1. The fact that NEM inhibits steady-state ATPase when only one alpha subunit of three is reacted suggests a cyclical catalytic mechanism.


Catalytic properties of Escherichia coli F1-ATPase depleted of endogenous nucleotides.

Nucleotide-depleted Escherichia coli F1 was prepared by the procedure of Wise et al. (1983, Biochem. J. 215, 343-350). This enzyme had high rates of steady-state ATPase and GTPase activity. When "unisite" ATP hydrolysis was measured using an F1/ATP concentration ratio of 10, all of the substoichiometric ATP became bound to the high-affinity catalytic site and none became bound to noncatalytic sites. The association rate constant for ATP binding was 7 x 10(5) M-1 s-1 and the KdATP was 7.9 x 10(-10) M, as compared to values of 3.8 x 10(5) M-1 s-1 and 1.9 x 10(-10) M, respectively, in native (i.e., nucleotide-replete) F1. Rate constants for bound ATP hydrolysis, ATP resynthesis, and P(i) release, and the reaction equilibrium constant, were similar in nucleotide-depleted and native F1. Therefore, we conclude that occupancy of the noncatalytic sites is not required for formation of the high-affinity catalytic site of F1 and has no significant effect on unisite catalysis. In further experiments we looked for the occurrence of inhibitory, catalytic-site-bound MgADP in E. coli F1. Such an entity has been reported for chloroplast and mitochondrial F1. However, our experiments gave no indication for inhibitory MgADP in E. coli F1.


Inhibition of glucocerebrosidase and induction of neural abnormality by cyclophellitol in mice.

Cyclophellitol, a cyclitol with an epoxide, is a novel microbial secondary metabolite that inhibits beta-glucosidase and beta-glucocerebrosidase. Daily administration of cyclophellitol induces a severe abnormality of the nervous system in mice while it has no toxicity in various cultured cells. It was shown to inhibit glucocerebrosidase in vivo significantly in mice and the content of glucocerebroside in liver, spleen, and brain was increased markedly. The enzyme activity was completely suppressed in brain, liver, spleen, kidney, and muscle. On the other hand hexosaminidase activity was not affected in all tissues. After a single administration of cyclophellitol the maximal inhibition of glucocerebrosidase was observed within 30 min in brain and liver, and the inhibition lasted for 2-4 days. A single administration of cyclophellitol also induced a severe abnormality of the nervous system known as Gaucher's-like disease in mice. Conduritol B epoxide is also known to inhibit glucocerebrosidase and induce Gaucher's like-disease in mice by repetitive injection. Cyclophellitol was shown to be more potent than conduritol B epoxide in inhibition of glucocerebrosidase and in induction of the neural abnormality.


Fasting serum insulin levels in essential hypertension. A meta-analysis.

BACKGROUND--The role of insulin in the genesis of essential hypertension remains an area of intense controversy. Most clinical evidence suggests a definite association. Several pathophysiologic mechanisms have been proposed. However, current data remain disparate and contradictory. METHODS--Meta-analysis allows data pooling of primary study findings and subsequent integration into a statistically meaningful outcome. This method was used to study the relationship in euglycemic individuals between blood pressure and fasting serum insulin level, age, body mass index, and fasting plasma glucose level. -A significant correlation was demonstrated between fasting serum insulin concentration and both systolic blood pressure and diastolic blood pressure. Age and body mass index also revealed meaningful associations. The meta-analytic correlation between plasma glucose level and both systolic blood pressure and diastolic blood pressure failed to achieve significance. CONCLUSIONS--Data from a meta-analytic review examining fasting serum insulin levels in euglycemic individuals demonstrate a significant correlation with systolic and diastolic blood pressure. This study supports the role of hyperinsulinemia in the pathogenesis of essential hypertension.


Five-minute treatments with fluorouracil, floxuridine, and mitomycin have long-term effects on human Tenon's capsule fibroblasts.

Proliferating human Tenon's capsule fibroblasts were exposed for 5 minutes to a wide range of concentrations of fluorouracil, floxuridine, and mitomycin. High concentrations of all three agents had prolonged effects on cell proliferation and morphologic characteristics compared with untreated control cells up to 36 days. The highest concentrations of both floxuridine (15,000 micrograms/mL) and mitomycin (1000 micrograms/mL) had an apparent cidal effect, reducing cell numbers below initial cell density. In contrast, although the highest concentration of fluorouracil (25,000 micrograms/mL) inhibited cell proliferation by more than 50% relative to the untreated control cells at 36 days, the cell numbers still increased fourfold compared with the initial cell density. These results demonstrate that 5-minute treatments with high concentrations of these drugs have prolonged effects on the proliferation of human Tenon's capsule fibroblasts in vitro. Single-dose regimens using high concentrations of these drugs at the time of operation may achieve results similar to those of protocols that involve repeated applications.


Lipoprotein(a) levels in chronic renal disease states, dialysis and transplantation.

Lipoprotein(a) is an independent risk factor for cardiovascular disease. Lipoprotein(a) levels were measured in 196 patients (103 Male [M]: 93 Female [F]) with chronic renal diseases and in 116 controls. Median levels of Lipoprotein(a) [Lp(a)] were found to be significantly elevated in patients with untreated chronic renal disease (285,285 mg/L; M,F; range 30-1675 mg/L) and in those treated with continuous ambulatory peritoneal dialysis (320, 603; M,F; range 50-1450) compared with controls (70,51; M,F; range 1-750; p less than 0.01 Males, p less than 0.001 Females). Lp(a) levels in patients treated by haemodialysis (133,35; M,F; range 5-685) and renal transplantation (100,95; M,F; range 10-1700) were not significantly different from controls. Lipoprotein(a) levels correlated inversely with serum albumin in the combined dialysis group (r = -0.34, p less than 0.001), and with urinary protein loss in the combined transplant and chronic renal diseases groups (r = 0.29, p less than 0.01). This correlation of Lp(a) with protein metabolism suggests a similarity with changes in other apolipoprotein-B containing lipoproteins in nephrosis. These findings may be relevant to the increased risk of atherosclerosis in patients with chronic renal disease and to their optimum mode of renal replacement therapy.


Trabecular spacing in post-menopausal Australian women with and without vertebral fractures.

Histomorphometric measurements were made from iliac crest biopsies of 32 women with vertebral fractures and 37 women without fracture. All were post-menopausal Australian women who had presented with back pain to a hospital out-patient endocrinology clinic. Bone from the fracture cases was characterised by loss of individual trabecular elements, with the remaining trabeculae being spaced further apart than those in the non-fracture women (p less than 0.0001). This resulted in a significant decrease in trabecular bone volume (p less than 0.01). In addition osteoid surface was reduced (p less than 0.01). Dynamic parameters of bone turnover were not significantly different between the two groups. These data should be useful for the assessment of iliac bone histomorphometry in Australian post-menopausal women suspected of having osteoporosis.


Troponin T isoform expression in the normal and failing human left ventricle: a correlation with myofibrillar ATPase activity.

The expression of troponin T, a thin filament regulatory protein, was examined in normal and failing left ventricles. The samples were obtained from the hearts of patients with severe heart failure who were undergoing cardiac transplantation, and from normal adult hearts that could not be used for transplantation. Western blots of the myofibrillar proteins demonstrated two isoforms, troponin T 1 (TnT1) and troponin T 2 (TnT2). TnT2 is expressed at significantly higher levels in failing hearts (p less than 0.004). Western blots of two-dimension SDS-PAGE gels resolved two dominant spots of TnT1 and of TnT2 and several minor troponin T species. Alkaline phosphatase treatment markedly decreased the sizes of the two acidic spots while increasing the two more basic spots by a comparable amount. Myofibrillar ATPase activity had an inverse and negative linear relationship (r = 0.7, p less than 0.02) with the myofibrillar percentage of total troponin T comprised of TnT2. In that heart failure in these transplant patients had multiple bases, we propose that rather than a cause of heart failure, the disease-associated changes in troponin T isoform expression are an adaptation to abnormal myocardial function.


Effects of different expression and posttranslational modifications of myosin light chains on contractility of skinned human cardiac fibers.

In the human ventricle two isoforms of the phosphorylatable myosin light chain (MPLC) are expressed. These two forms are designated with increasing acidity as LC-2 and LC-2*. In the normal human heart the relation between LC-2/LC-2*-expression is 70/30, suggesting the existence of three different myosin isoenzymes (MPLC-polymorphism) in the normal human ventricle. Both ventricular MPLC-isoforms are monophosphorylated, the LC-2 being higher phosphorylated than the LC-2*. In some patients with heart failure both MPLC isoforms were found to be completely dephosphorylated. In the human atrium a MPLC isoform is expressed which is different from the ventricular MPLC isoforms. The atrial MPLC isoform is mono- and diphosphorylated. Mono-phosphorylation of both the ventricular MPLC isoforms and the atrial MPLC isoform increased responsiveness as well as sensitivity of isometric tension generation of skinned fibers to Ca2+. Part of this effect could be explained by changing the cross-bridge-cycling rate: MPLC increased fapp, the rate-constant for the transition of cross-bridges from the non-force into the force-generating state, thus increasing the amount of force-generating cross-bridge states at a given [Ca2+]. Monophosphorylation of the MPLC isoforms did not change maximal shortening velocity.


Contractile proteins and sarcoplasmic reticulum calcium-ATPase gene expression in the hypertrophied and failing heart.

The physiology of myocardial contractility has been studied for over a century, but only recently has molecular biology provided new insights into the mechanisms responsible for the alterations of contraction and relaxation observed during cardiac hypertrophy and heart failure. Pressure and volume overload produce in the myocyte both qualitative changes characterized by protein isoform switches and quantitative changes characterized by modulation of single genes through a mechanogenic transduction the pathways of which are largely unknown. The qualitative changes involve differential expression of multigene families of contractile proteins, especially myosin heavy chain (MHC) and actin. All situations of pressure overload, or of combined pressure and volume overload activate the beta-MHC gene and deactivate the alpha-MHC one, which leads to a slower, more efficient contraction. In rat, pressure overload transitorily activates the alpha-skeletal actin gene, and both the timing and the distribution of the newly formed beta-MHC and alpha-skeletal actin mRNAs differ. We recently found that the isoactin pattern is the same in patients with end-stage heart failure as that of control human hearts. Moreover, both in rat and human, expression of isomyosins and isoactins are not coordinated, neither during ontogeny nor senescence. All this suggests the existence of several regulatory mechanisms activated during normal cardiac growth or by a mechanical trigger, and preliminary results indicate that it is possible to perform nuclear run-on assays in order to analyze the transcriptional step of these isogenes.(ABSTRACT TRUNCATED AT 250 WORDS)


Structural and functional diversity of human ventricular myosin.

The role of subcellular alterations in the process of heart failure remains ill-defined. Because contractile performance of failing heart muscle is depressed, possible alterations in the myosin molecule could be of particular relevance. There is increasing evidence that myofibrillar ATPase activity is reduced in congestive heart failure, whereas the findings on myosin ATPase are still controversial. The molecular causes of the reduced activity are currently not known. Because alpha-MHC is present only in small amounts in normal ventricles, a shift in favor of beta-MHC is of minor importance. Also immunohistochemical data on subspecies of beta-MHC seem not to provide an explanation. A new type of myosin heterogeneity was found by optimizing native polyacrylamide gel electrophoresis in the presence of pyrophosphate. Two bands (VA and VB) were observed in ventricles of patients with valvular disease. Because the two bands were detected also in normal hearts of large mammals, the existence of VA/VB cannot be diagnostic of diseased heart. However, the VA/VB ratio was influenced by the hemodynamic load, whereby the fast migrating band (VA) increased with the diastolic and systolic load. Because a relationship with the hemodynamic load was observed only in surgical muscle specimens, it appears that this heterogeneity is prone to post mortem modification. Further work is required to identify the molecular nature of this heterogeneity and to examine the therapeutic potential of a pharmacological modification of the VA/VB ratio.


Inhibition of tonoplast ATPase from etiolated mung bean seedlings by fluorescein 5'-isothiocyanate.

Fluorescein 5'-isothiocyanate (FITC) was used to modify the lysine residue in the active site of tonoplast H(+)-ATPase from etiolated mung-bean (Vigna radiata L.) seedlings. FITC caused marked inactivation of the enzyme activities of both membrane-bound and soluble ATPase and its associated H+ translocation. The SDS/PAGE pattern revealed that the FITC-binding site was in the large (A) subunit of ATPase. Inhibition could be substantially prevented by its physiological substrate ATP, pyrophosphate and nucleotides in the decreasing order: ATP greater than pyrophosphate greater than ADP greater than AMP greater than GTP greater than CTP greater than UTP. The mode of inhibition by FITC was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first-order kinetics with a Ki of 0.33 mM, a minimum inactivation half-time of 110 s, and a first-order rate constant of 0.244 s-1. A double-logarithmic plot of apparent rate constant versus FITC concentration gave a slope of 0.913, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labelling studies indicated that the incorporation of approx. 1 mol of FITC/mol of ATPase is sufficient to inhibit ATPase completely. The enhancement and blue shift of emission maxima of FITC after modification of ATPase indicated that the labelled lysine residue was located in a relatively hydrophobic domain.


Genetic evidence for an androgen-regulated epididymal secretory glutathione peroxidase whose transcript does not contain a selenocysteine codon.

Epididymal glutathione peroxidase (GPX) has been suggested as a major factor in combating loss of fertility of spermatozoa due to lipid peroxidation. We report here the isolation and sequence of putative GPX cDNAs from rat (Rattus rattus) and cynomolgus-monkey (Macaca fascicularis) epididymis, which exhibit marked sequence identity with known GPXs. In both species the cDNAs encode predicted preproteins containing 221 amino acid residues. Unlike other characterized GPX sequences, epididymal GPX mRNA does not contain a selenocysteine codon (UGA). However, sequence comparison and molecular-modelling studies suggest a high degree of structural conservation between epididymal and other GPXs. Transcripts corresponding to epididymal GPX are not detected in a variety of other tissues (liver, spleen, kidney and testis) and appear to be androgen-regulated in the epididymis.


Nucleotide sequence, organization and characterization of the atp genes and the encoded subunits of Mycoplasma gallisepticum ATPase.

The nucleotide sequence of a 7.8 kbp DNA fragment from the genome of Mycoplasma gallisepticum has been determined. The fragment contains a cluster of nine tightly linked genes coding for the subunits of the M. gallisepticum ATPase. The gene order is I (I-subunit), B (a-subunit), E (c-subunit), F (b-subunit), H (delta-subunit), A (alpha-subunit), G (gamma-subunit), D (beta-subunit) and C (epsilon-subunit). Two open reading frames were identified in the flanking regions; one (ORFU), preceding the I gene, encodes at least 110 amino acids and the other (ORFS), following the C gene, encodes at least 90 amino acids. The deduced amino acid sequences of the various subunits are presented and discussed with regard to the structure, function and differing sensitivity of the M. gallisepticum enzyme to dicyclohexylcarbodiimide and aurovertin. The alpha- and beta-subunits of the F1 portion are well conserved (51% and 65% identity with those of Escherichia coli), whereas the gamma-, delta- and epsilon-subunits, as well as the F0-subunits, show a low percentage identity. Nonetheless, the secondary structure of the F0-subunits show a high degree of similarity to the corresponding subunits of E. coli. Two very strong potential amphipathic alpha-helices are predicted in the delta-subunit and the N-terminus of the b-subunit contains two hydrophobic helical stretches. The possible roles of these structural properties in the close association of the F1 and F0 multisubunit complexes among mycoplasmas are discussed.


High-level stable expression of recombinant 5-HT1A 5-hydroxytryptamine receptors in Chinese hamster ovary cells.

The human 5-hydroxytryptamine 5-HT1A receptor gene was transfected into Chinese hamster ovary cells. A series of recombinant monoclonal cell lines expressing the receptor were isolated and the properties of one cell line that expressed receptors at a high level (2.8 pmol/mg) were studied in detail. In ligand binding assays with the selective 5-HT1A receptor agonist 2-(NN-di[3H]propylamino)-8-hydroxy-1,2,3,4-tetrahydronaphthalene ([3H]8-OH-DPAT) only a single class of saturable high-affinity binding sites was detected, with a pharmacological profile in competition experiments essentially identical to that of the 5-HT1A receptor of bovine hippocampus. [3H]8-OH-DPAT binding to the recombinant cell membranes was inhibited by GTP, showing that the receptors in the transfected cells couple to G-proteins. A series of 5-hydroxytryptamine agonists inhibited forskolin-stimulated adenylate cyclase activity in the cells and, despite the high level of receptor expression, their apparent efficacies were similar to those observed for inhibition of adenylate cyclase in brain. This recombinant cell line provides a complete model system for studying the 5-HT1A receptor and its transmembrane signalling system. The recombinant cells can also be grown in suspension culture for long periods but, whereas 5-HT1A receptor numbers and receptor regulation by guanine nucleotides are maintained in suspension-grown cells, the inhibition of adenylate cyclase by the 5-HT1A receptor is gradually lost.


Association of annexin V with prolactin in the rat anterior pituitary gland.

When pituitary extracts were subjected to non denaturing polyacrylamide gel electrophoresis, an unknown protein was found to associate with a proportion of the prolactin. This protein was dissociated from prolactin by sodium dodecyl sulfate. The protein was purified and sequenced. As the amino terminus was blocked, the amino acid sequences of three peptide fragments were determined. The obtained sequences of 41 amino acids were identical to partial sequences of a known protein, rat Annexin V. The molecular mass, 36 kDa, was also the same as the molecular weight of Annexin V. The existence of Annexin V mRNA in rat pituitary glands was also confirmed by polymerase chain reaction. These results show that Annexin V, a member of the calcium-dependent phospholipid binding proteins, is synthesized in the rat pituitary gland, and suggest its association with prolatin in the gland.


Stress related workers' compensation claims: recommendations involving records release.

1. The cost of stress claims is predicted to cripple the workers' compensation system, where stress claims are burgeoning and the average payout is twice that of a typical injury. The major reason to release medical records in a stress claim is to determine the validity of the claim arising from the job. 2. Occupational health nurses are frequently asked by the courts to reveal personal client information and may not be protected by the "nurse-client relationship" or "privileged communication." Politically, very little interest has been shown in restricting disclosure of private information. 3. Both ANA and AAOHN have adopted strong positions about safeguarding privacy. Legally, the ultimate responsibility for wrongful acts committed by the nurse falls on the individual nurse. 4. The most important reason to guard confidential health information is the basic tenet of the nurse-client relationship in which personal matters are held in confidence. To break this trust is to jeopardize the ability to provide optimal client care, which is the essence of nursing.


Needs expressed by mothers and fathers of young children with disabilities.

One aspect of family adaptation to a young child with disabilities was investigated through comparison of expressed needs of mothers and fathers and examination of the relation of those needs with selected child and family characteristics. A diverse sample of more than 400 parents completed the Family Needs Survey (Bailey & Simeonsson, 1988). A factor analysis of the items for mothers yielded six distinct factors. A confirmatory factor analysis of needs expressed by fathers indicated a structure that differed significantly from that found for mothers. Mothers expressed significantly more needs than did fathers. Although SES and disability type accounted for significant variance in needs of mothers, an examination of mean values suggested the effect to be of limited clinical significance.


Relations between mothers' expectations and the performance of their infants who have developmental handicaps.

Relations between mothers' expectations and the performance of their infants with a disability on specific developmental tasks were explored. These mothers predicted their child's performance on developmental measures with accuracy levels comparable to those of parents of preschool children without disabilities. Children whose mothers were initially the most accurate in their estimations tended to show fewer gains in their statistically adjusted Bayley Mental Development Index scores over the course of the study. Mother's initial accuracy scores were found to account for substantial additional portions of the variance in the children's Bayley performances 14 and 28 weeks later.


Object play and exploration in children with and without disabilities: a longitudinal study.

The development of object play was examined in 40 children (20 with and 20 without disabilities). The children with disabilities were assessed at ages 11, 15, 19, and 27 months; the children without disabilities, at 6, 11, 15, and 27 months. At each assessment, the children were videotaped during 20 minutes of play with their mothers in a controlled environment. Child's play level was positively related to developmental age for the disabled group at each assessment and for the nondisabled group at 6, 11, and 15 months of age. The types of object play observed in children with no disabilities were observed in the children with disabilities at comparable developmental ages. In a subsample matched for developmental age, the duration and frequency of active involvement with objects were greater for the children without disabilities than for the children with disabilities.


K-ABC profiles in children with fragile X syndrome, Down syndrome, and nonspecific mental retardation.

Etiology-specific profiles of intellectual abilities were compared in three groups of males with mental retardation using the Kaufman Assessment Battery for Children (K-ABC). Subjects included 10 males with fragile X syndrome, 10 with Down syndrome, and 10 with nonspecific mental retardation who were equated on both mental and chronological age. Across all three groups, sequential processing was lower than simultaneous processing or achievement, and particular subtests (e.g., Gestalt Closure) were relative strengths. Although boys with Down syndrome showed less extreme patterns of domain strengths and weaknesses, they showed a significant strength in the Sequential Processing Hand Movements subtest. In contrast, the Hand Movements subtest was lowest of all K-ABC subtests for males with fragile X syndrome. Implications were discussed for more fine-tuned research and intervention efforts.


Hearing loss in middle-age persons with Down syndrome.

Hearing function of 35 institutionalized persons with Down syndrome, age 35 to 62 years, was assessed by means of otoscopy, impedance audiometry, brainstem evoked response audiometry, and pure tone audiometry. Using brainstem evoked response audiometry, we determined response thresholds for 59 ears, which compares favorably with pure tone audiometry (20 ears). We found hearing losses of 20 dB to over 90 dB in 56 of these ears. Hearing loss should be considered and, whenever feasible, excluded as a contributing factor in social and mental deterioration in middle-age persons with Down syndrome.


Mothers' and fathers' perceptions of stress and coping with children who have severe disabilities.

Stress in families with children who have special needs, which has been the focus of much research interest, is usually assessed solely from a maternal perspective. In this study, the short form of the Questionnaire on Resources and Stress (QRS-F, Friedrich, Greenberg, & Crnic, 1983) was completed separately by mothers and fathers of children with severe developmental disabilities. To compare responses of mothers and fathers, we employed factor analysis of parcels using the parallel analysis criterion rather than the more traditional item level analysis with minimum eigenvalue criterion. Results indicated that the QRS-F differed only slightly in both factor structure and correlates as a function of parental gender. Overall, validity of the QRS-F for use with both mothers and fathers of children with severe disabilities was supported.


Delayed hydrophobic surfactant protein (SP-B, SP-C) expression in fetuses of streptozotocin-treated rats.

Tissues from fetuses and neonates of control and streptozotocin (STZ)-treated Sprague-Dawley rats were used to study the content and distribution of the hydrophobic surfactant protein B (SP-B) and the mRNAs for SP-B and SP-C using immunohistochemistry, RNA blotting, and tissue in situ hybridization. A dose of 50 mg/kg STZ was used to treat female rats before mating. The fetuses were sacrificed at fetal days 18 through 21 and neonates were obtained on neonatal days 1 and 2 (day of birth = end of day 22). At fetal day 18, SP-B was barely detectable by immunohistochemistry in control animals but the levels were progressively increased through gestation and easily detected by fetal day 21. At all fetal ages, SP-B was decreased in the STZ group compared with control animals. Both SP-B and SP-C mRNA were detectable at fetal day 18 in the control group and increased with advancing gestational age. In fetal lungs from the STZ group, SP-B and SP-C mRNA also showed an increase with advancing gestational age, but the levels were decreased compared with controls at fetal days 18, 20, and 21 (P less than 0.05). At fetal day 19, this difference did not achieve statistical significance. Differences between the two groups were no longer detected by neonatal days 1 and 2. The difference between the STZ and control groups, in both protein (SP-B) and mRNA (SP-B and SP-C), diminished with advancing fetal age but remained significant up to fetal day 21.(ABSTRACT TRUNCATED AT 250 WORDS)


Doppler color imaging. Peripheral arteries.

The impact of DCI on the evaluation of peripheral vascular disease is considerable. In essence, it is noninvasive angiography without the limitations of that modality. However, the pressure gradient can only be inferred, and pull-through pressure gradients are still the most accurate estimation of adequacy of flow. Nonetheless, segmental blood pressures and the peak velocities measured by duplex Doppler provide a noninvasive alternative for estimating pressure gradients. DCI imaging has greatly improved the perception of focal abnormalities and allows rapid appreciation of global hemodynamics. As further technical refinements improve resolution and sensitivity, color Doppler may eventually supplant angiography as the primary imaging modality in peripheral arterial diagnosis, reserving arteriography for interventional procedures.


Service needs of children with disabilities and their families.

A survey of 1527 families receiving the Child Disability Allowance was undertaken. There were substantial unmet needs for home help services, respite care and equipment. Satisfaction with services, particularly therapy, and respite care facilities was less than optimal. Many families also lacked knowledge about resources available to them. Improvement in the quality and availability of services is essential as is dissemination of information about available facilities.


Different accessory function for TH1 cells of bone marrow derived macrophages cultured in granulocyte macrophage colony stimulating factor or macrophage colony stimulating factor.

The ability of macrophages to stimulate immune responses is heterogeneous and may have influence on the type of the developing immune response. Therefore, in an attempt to define different functional states of mouse macrophages, we made use of the two macrophage growth factors: macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Generation of macrophages from freshly isolated bone marrow cells in the presence of GM-CSF results in a population expressing profound antigen presenting function for mouse TH1 cells, resulting in strong lymphokine production and proliferation of the T cells. Furthermore, high amounts of a novel soluble cytokine active on mouse TH1 cells are generated during the interaction of TH1 cells with macrophages elicited with GM-CSF. In contrast, macrophages grown from bone marrow cells for at least 14 days in the presence of M-CSF express only minimal antigen-presenting function for TH1 cells. Treatment of such macrophages for 24 h with either IFN-gamma or GM-CSF allows the distinction between two further functional states. Those treated with IFN-gamma efficiently presented antigen towards TH1 cells. The T cells produced large amounts of lymphokines and proliferate well. However, synthesis of the novel soluble cytokine (active on TH1 cells) was not detectable. The generation of this mediator requires a short-term treatment with GM-CSF of macrophages developed in the presence of M-CSF prior to their interaction with TH1 cells.


Optimized radiotherapy for head and neck cancer.

Optimization of radiation therapy for head and neck tumors requires the combination of several facets of radiation biology and physics. The aim is to achieve optimum tumor control while reducing normal tissue damage. Techniques have been developed to determine tumor radiosensitivity and growth characteristics. Their use as predictive assays of treatment response is gaining importance. As the range of therapeutic options (particularly altered fractionation regimens) increases, it is hoped that the ability to individually tailor patients' treatment will result in improved rates of tumor control and an improved therapeutic ratio. Optimization of treatment delivery based on three-dimensional treatment planning offers the opportunity for dose escalation studies and limitation of normal tissue morbidity. The combination of chemotherapy and radiotherapy continues to be investigated, although major advances using this strategy are unlikely.


Binding and internalization of the 163-171 fragment of human IL-1 beta.

The mechanisms of cell association of the human interleukin (IL-1 beta) immunostimulatory fragment 163-171 have been studied. The fragment was able to associate abundantly to both IL-1R- and IL-1R+ cells. Binding was strictly temperature dependent, was not saturable and could be inhibited by excess amounts of unlabelled 163-171 peptide but not by IL-1 beta, suggesting that the 163-171 fragment is not an IL-1R-binding domain of IL-1 beta. The fragment is readily internalized by cells by a cytochalasin-insensitive mechanism and it localizes mainly in the cytoplasm. It is concluded that the active domain 163-171 of IL-1 beta can be taken up by cells through a receptor-independent, temperature-dependent mechanisms and that its ability to activate cellular functions is based on IL-1R-independent intracellular pathways.


A successful regimen for the prevention of seroconversion after transplantation of a heart positive for hepatitis B surface antigen.

We report a case involving orthotopic heart transplantation in which a recipient, known to be serologically negative for hepatitis B virus infection, received a heart from a donor known before transplantation to be hepatitis B surface antigen positive. The recipient was treated prospectively with a combination of hepatitis B immune globulin and hepatitis B vaccine. After 10 months of follow-up, the recipient has not experienced seroconversion or viremia from hepatitis B virus and has remained without symptoms by both clinical and laboratory measurements.


Impaired atrial natriuretic factor systemic clearance contributes to its higher levels in uremia.

To evaluate the interaction between plasma levels and the systemic uptake of atrial natriuretic factor (ANF) with thyroid hormone levels during acute renal failure (ARF), seven groups of rats were analyzed: Group 1, Controls (C); Group 2, ARF; Group 3, filtering kidney with uremia; Group 4, ARF with thyroxine (T4) supplement (ARF + T4); Group 5, thyroidectomy (Tx); Group 6, ARF on Tx rats (Tx + ARF); Group 7, Tx + ARF supplemented with T4 (Tx + ARF + T4). Plasma creatinine (Cr), urea, T4, blood volume, and ANF were measured; ANF half-life (ANF t1/2; expressed in seconds) was calculated. Rats with ARF developed uremia (Cr, 377 +/- 58 versus 41 +/- 5 mumol/L), significant reduction in T4 (40 +/- 4 versus 89.2 +/- 6 nmol/L). elevation of ANF (287.7 +/- 35 versus 60.9 +/- 8 fmol/mL), and lengthening of ANF t1/2 (69.7 +/- 8 versus 37.2 +/- 6 s) compared with C (P less than 0.01). T4 supplements to ARF rats resulted in a lesser degree of uremia (Cr, 283 +/- 27; P less than 0.05) and normalization of ANF t1/2 (31.4 +/- 5); however, ANF levels remained higher than C (100.4 +/- 11.4 versus 60.9 +/- 8; P less than 0.01). Tx by itself did not change either parameter. The filtering kidney with uremia group developed mild uremia (Cr, 199 +/- 8), T4 fell (58 +/- 8), ANF levels rose (83.4 +/- 5.4), and ANF t1/2 was prolonged (54.5 +/- 12). Tx before ARF doubled the ANF level and lengthened ANF t1/2 similarly than in ARF. T4 addition (Tx + ARF + T4) normalized ANF t1/2 (29.8 +/- 3) in spite of a persistently high ANF (145.7 +/- 21). Blood volume did not change in any group.(ABSTRACT TRUNCATED AT 250 WORDS)


Circadian rhythms of heart rate and blood pressure after heart transplantation.

Blood pressure and heart rate were recorded over 24-hour periods on 39 occasions in 20 subjects 5 to 72 weeks after heart transplantation. All patients were receiving cyclosporine, azathioprine, and prednisolone. In 38 of the 39 records the mean nighttime heart rate was lower than the mean daytime rate, with a peak difference of 20.1 +/- 1.8 beats/min. Blood pressure responses were, however, of two patterns. In 15 of the 39 recordings (approximately 50% of patients) the mean nighttime systolic pressure was higher than the mean daytime systolic pressure; in the remainder the converse was observed. The pattern was generally consistent on repeated recordings from the same patient and was not related to time since transplantation, renal function, or other therapy. Echocardiographic/Doppler studies were available at the time of 31 of these recordings. No differences in left ventricular diameters, systolic function, or transmitral filling patterns were present between patients whose blood pressure was higher or lower at night. Left ventricular posterior wall thickness and the ratio between wall thickness and ventricular diameter at end diastole were greater in the group showing nighttime pressure falls. Blood pressure responses after heart transplantation show the presence of nighttime "dippers" and "nondippers." At least early after transplantation, however, nondipper status is not preferentially associated with the development of left ventricular hypertrophy. The mechanisms accounting for the different circadian blood pressure responses in heart transplant recipients are not known.


Interleukin-1 regulates cytokine gene expression in human mesangial cells through the interleukin-1 receptor type 1.

We have recently shown that interleukin-1 is a potent stimulus of gene expression and production of leukocyte chemotactic factors, colony-stimulating factors, and interleukin-6 in human mesangial cells in culture. Here, we sought to determine whether interleukin-1 induces its own gene expression in human mesangial cells. Interleukin-1 mRNA levels were quantitated by Northern blot analysis with total cellular RNAs isolated from human mesangial cells exposed for 6 h to medium alone or in the presence of human recombinant interleukin-1 beta (1 to 100 ng/mL). Interleukin-1 induced interleukin-1 mRNA expression in a dose-dependent manner. An additional finding of this study was that human mesangial cells constitutively express the 80 kd interleukin-1 receptor type 1 gene. When human mesangial cells were exposed to interleukin-1, interleukin-1 receptor expression was not modified. Similarly, other stimuli like tumor necrosis factor, transforming growth factor beta, or interleukin-6 did not modulate interleukin-1 receptor expression. Recombinant interleukin-1 receptor antagonist blocked the interleukin-1 mRNA as well as interleukin-6 and interleukin-8 mRNA accumulation induced by interleukin-1 beta. Lipopolysaccharide, which is a known stimulus for interleukin-1 transcription in several cell types, also induced interleukin-1 mRNA accumulation, thus indicating that lipopolysaccharide mediates interleukin-1 gene activation in human mesangial cells through an interleukin-1-independent pathway. These data support the pivotal role of interleukin-1 in regulating mesangial cell cytokine genes and may be taken to indicate the existence of an interleukin-1-mediated positive feedback loop that might control the secretion of active cytokines within the glomeruli when an immunological or inflammatory injury takes place.


Laparoscopic cholecystectomy in the heart transplant candidate with acute cholecystitis.

We report the first successful laparoscopic cholecystectomy for treatment of acute cholecystitis in a heart transplant candidate with end-stage heart disease. Eight successful cases of conventional cholecystectomy in heart transplant candidates have been reported, but convalescence after the conventional procedure is prolonged, and morbidity often interferes with a timely heart transplantation. Laparoscopic cholecystectomy is a less-invasive method for treatment of symptomatic cholelithiasis and cholecystitis and may be better tolerated in this patient population. Although further study is needed, we believe laparoscopic cholecystectomy will have applications in patients with end-stage heart disease.


Patterns of membrane CD45 isoform expression by leukaemic blasts and normal mature myeloid cells.

The membrane expression of CD45R isoforms by leukaemic blasts from 92 cases of acute myeloid (AML) and 12 cases of lymphoblastic leukaemia was analysed by single and two-colour flow cytometry. Compared to normal mature granulocytes, which invariably expressed UCHL1 (CD45RO) but not 2H4 (CD45RA), leukaemic myeloid blasts showed 2H4+ UCHL1- and 2H4- ++UCHL1- composite patterns of expression with the first of these phenotypes being associated in particular with the most immature AML subtype (M0). Similar analyses of blast cell fractions from monocytic AML variants, revealed wide variation in CD45R expression in cases of M4, whereas M5 leukaemias were typically 2H4+ ++UCHL1+ with minor 2H4- UCHL1- components. As most normal mature monocytes were also 2H4+ ++UCHL1+, this suggested that monocytic myeloid differentiation was primarily associated with the "acquisition" of UCHL1 and the development of 2H4/UCHL1 co-expressing cells. The expression of membrane CD45RA by leukaemic blasts is an abnormal characteristic and may be related to the maintenance of an undifferentiated state by malignant myeloid precursors.


Effect of interleukin-11 on cycling status and clonogenic maturation of fetal and adult hematopoietic progenitors.

A recently cloned human cytokine, interleukin-11 (IL-11), has functional similarities to IL-6. We tested the hypothesis that the hematopoietic actions of IL-11 in vitro also resemble those of IL-6. The effect of IL-11 on the cell cycle status of fetal and adult hematopoietic progenitors was assessed using serum-free incubations followed by tritiated thymidine suicide studies. Its effect on clonogenic maturation was assessed by including IL-11, either as a single agent or with subplateau or plateau concentrations of other recombinant cytokines, in cultures that contained neutralizing monoclonal antibodies directed against relevant growth factors. Similar to IL-6, IL-11 resulted in accelerated cycling of fetal colony-forming units-mixed (CFU-MIX), CFU-granulocyte macrophage (CFU-GM), and erythroid burst-forming units (BFU-E). This effect was additive to that of submaximal, but not to plateau, concentrations of IL-6. However, no effect of IL-11 was observed on cycling status of adult progenitors. As a single agent, IL-11 failed to support clonal maturation of either fetal or adult progenitors. IL-11 was additive to GM-CSF in supporting clonal maturation of CFU-GM from adult marrow but not from fetal blood. We conclude that the in vitro hematopoietic actions of IL-11 on cell cycle status of hematopoietic progenitors resemble those of IL-6. However, unlike IL-6, IL-11 as a single agent failed to support clonal maturation of fetal CFU-GM, BFU-E, and CFU-MIX.


Efficient clodronate entrapment within multilamellar and unilamellar liposomes.

Clodronate (dichloromethylene bisphosphonate) encapsulated within liposomes and administered intravenously eliminates resident macrophages within the liver and spleen. Macrophage depletion in the rat requires 20 mg of the encapsulated drug, and so far this has only been achieved using large multilamellar vesicles (MLV). Recent studies have shown that small unilamellar vesicles (SUV) when injected intravenously accumulate at inflamed joint sites in both animal models of arthritis and patients with rheumatoid arthritis; multilamellar vesicles were not able to do so. If phagocytic cells, such as macrophages, are responsible for SUV sequestration, then SUV containing clodronate may be targeted to the inflamed joint and may eliminate the macrophage population leading to reduction in the state of inflammation. We have adapted an existing technique to radiolabel clodronate with 99mTechnetium to use as a tracer to determine its encapsulation within liposomes, a technique that has advantages over other current methods. We have achieved a high-encapsulation efficiency of the drug within MLV and produced SUV containing sufficient clodronate to deplete macrophages in rats in a small enough volume to administer it intravenously as a single dose.


Both naive and memory T cells can provide help for human IgE production, but with different cytokine requirements.

Most in vitro systems for the induction of IgE production by human B cells require both IL-4 and the presence of T cells. Little is known about the mechanism of T cell help or the ability of different T cell subsets to provide this helper activity. In the present study we demonstrate that, in the presence of exogenous IL-4, anti-CD3 stimulated naive T cells (CD4+CD45RA+) are potent helper cells for human IgE production. In their presence, as little as 750 autologous B cells can produce up to 100 ng/ml IgE. This response was found over a broad range of anti-CD3 concentrations. IgE helper activity by naive T cells was inhibited by IL-2. Under all conditions tested, naive T cells were unable to provide help for IgM production. This is in contrast to activated memory T cells (CD4+CD45RO+), which are very efficient helper cells for IgM or IgE production, provided that IL-2 or IL-2 plus IL-4 are present respectively.


Activation of the p34 CDC2 protein kinase at the start of S phase in the human cell cycle.

Using a protocol for selecting cells on the basis of both size and age (with respect to the preceding mitosis), we isolated highly synchronous human G1 cells. With this procedure, we demonstrated that the p34 CDC2 kinase was activated at the start of S phase. Cyclin A synthesis began at the same time, and activation of the p34 CDC2 kinase at the start of S phase was, at least in part, due to its association with cyclin A. Furthermore, cells synchronized in late G1 by exposure to the drug mimosine contain active cyclin A/p34 CDC2 kinase, indicating that p34 CDC2 activation can occur before DNA synthesis begins. Thus, the cyclin A/CDC2 complex, which previously has been shown to be sufficient to start SV40 DNA synthesis in vitro, assembles and is activated at the start of S phase in vivo.


Comparative study of ketoconazole 2% foaming gel and betamethasone dipropionate 0.05% lotion in the treatment of seborrhoeic dermatitis in adults.

Sixty-two patients with seborrhoeic dermatitis were treated topically with a 2% ketoconazole foaming gel or with a 0.05% betamethasone dipropionate lotion in a single-blind study for 4 months. Changes in the number of Pityrosporum ovale were scored by a mycologist. The investigator rated the severity of erythema, scaling and itching of the patients' scalp, eyelashes, nasolabial folds and thorax. In addition, both the investigator and the patients evaluated the treatments globally. At the end of treatment, the response rate for ketoconazole 2% foaming gel was significantly higher than that for betamethasone dipropionate 0.05% lotion according to the global evaluation by the physician (89 vs. 62%, p less than 0.05) and the patient (89 vs. 65%, p less than 0.05). Ketoconazole was also superior to betamethasone with reference to the evolution of the symptoms, irrespective of their localization. This efficacy manifested itself by a significant reduction of the number of P. ovale on the scalp in the ketoconazole group (p less than 0.001) compared to the betamethasone group, in which the count was hardly changed during therapy. The treatment was also better tolerated in the ketoconazole group (5 vs. 16 patients with side-effects, p less than 0.001). It is concluded that ketoconazole 2% foaming gel offers an excellent alternative to local corticosteroids in the treatment of seborrhoeic dermatitis.


Refractory peptic ulcers.

Ulcers that do not heal after 8 weeks of treatment with standard-dose regimens of antiulcer drugs are considered refractory. Incidences of duodenal and gastric ulcers refractory to H2-receptor antagonists are 9% and 20%, respectively. When treated with a single daily dose of omeprazole 20 mg, duodenal ulcers have a refractory incidence of 3% and gastric ulcers, 11%. Omeprazole's benefit may result from its potent gastric antisecretory action. Acid hypersecretion is an important pathophysiologic factor associated with refractoriness to H2 antagonists. Some patients with refractory ulcer, however, have a normal pharmacologic response of decreasing intragastric acidity following administration of H2 antagonists. Two possible mechanisms may explain refractoriness in such cases: the relative preservation of daytime acidity, which is the usual pharmacologic response to standard doses of H2 antagonists, and excessive impairment of mucosal defense. The first possibility is consistent with the results that increased doses of H2 antagonists or more potent antisecretory drugs such as omeprazole do heal a subgroup of ulcers refractory to H2 antagonists. The second possibility is supported by reports that drugs that mainly enhance mucosal defense, such as misoprostol, sucralfate, and bismuth compounds, also effectively heal refractory ulcers, and that gastrointestinal prostaglandin levels are extremely low in the mucosa of such patients. Other reasons for refractoriness to omeprazole treatment include gastric hyperacidity and impaired gastric emptying that may disturb drug absorption. Infection with Helicobacter pylori might, to some extent, be involved in refractoriness to potent antisecretory drugs. Single daily doses of omeprazole 40 mg seem superior to omeprazole 20 mg or increased doses of H2 antagonists for maintenance therapy of H2 antagonist-refractory ulcers.(ABSTRACT TRUNCATED AT 250 WORDS)


[Syndrome differentiation in traditional Chinese medicine in chronic aplastic anemia; T-lymphocyte subpopulation in peripheral blood and erythrocyte C3b receptors].

In this paper, the Syndrome Differentiation in TCM and the determination on T-lymphocyte subpopulation and RBC C3b receptor were carried out by using indirect SPA assay and RBC C3b receptor ring test among 51 chronic aplastic anemia patients. The OKT3, OKT4 and OKT4/OKT8 ratio in groups of Kidney-Yin Deficiency group were lower, while the OKT8 was higher than that of controls. In the group of both Kidney-Yin and Yang Deficiency, OKT4 and OKT4/OKT8 ratio were lower. and the value of OKT8 is higher than that of controls. The OKT8 in the Kidney-Yang Deficiency group was higher. and the ratio of OKT4/OKT8 was lower than that of controls. The OKT8 in the Kidney-Yin Deficiency group among three groups increased higher than that of other two groups. while the OKT4/OKT8 ratio reduced. The OKT4/OKT8 ratio in the group of both Kidney-Yin and Yang Deficiency was higher than that of Kidney-Yang Deficiency group. Among the three groups, the rosette rates of RBC C3b receptor were lower than controls. The above-mentioned data were statistically significant. These characteristic changes could be used as a reference of microcosmic Syndrome Differentiation and also a guide for how to apply the immunosuppressant and immunopotentiator.


New polyolefin foil for 5-day storage of platelet concentrates (PC) collected by apheresis.

In a paired study 12 platelet concentrates (PC) of Fresenius AS-104 cell separator were stored in new polyolefin bags of Fresenius (LE2) and Fenwal PL-732 bags. On day 0 and after 3 and 5 days of storage pH, pO2, pCO2, cell counts, platelet morphology and aggregability, plasma glucose, lactate, LDH and beta TG were determined. The overall changes fell within the expected range. No relevant differences between the two bags could be detected, although a few parameters (pH, pCO2) are slightly but statistically/significantly different. It can be concluded that the new polyolefin bag is well suited for 5-day storage of PC's from the AS-104.


Progressive slowing of reaction time and increasing cerebrospinal fluid concentrations of quinolinic acid in HIV-infected individuals.

Neuropsychological functioning and cerebrospinal fluid concentrations of an endogenous neurotoxin, quinolinic acid (QUIN) were evaluated in 52 HIV-positive individuals (71% without constitutional symptoms) and 33 HIV-seronegative controls (including 15 psychiatric patients with adjustment disorders). Although the HIV-positive subjects did not differ from controls on standard neuropsychological tests, simple and choice reactions times (RT) were slow at initial evaluation (P less than 0.01) and became progressively slower at 6-month re-evaluation (P less than 0.05). Cerebrospinal fluid (CSF) QUIN was elevated at initial evaluation and increased during the 6-month interval (P less than 0.05). Moreover, during this 6-month interval, progressive slowing of RT was highly correlated with increasing levels of CSF QUIN (r = 0.85, df = 15, P less than 0.0001) but not with changes in mood, constitutional symptoms, or CD4 cell count. These findings suggest that RT may provide a sensitive behavioral measure of relatively early central nervous system involvement in HIV-infected individuals and that QUIN may play an important role in the pathogenesis of HIV-related neurological dysfunction.


The NMDA receptor antagonist MK-801 does not protect against serotonin depletions caused by high doses of DL-fenfluramine.

The non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine (MK-801) has been shown to block methamphetamine (MA) induced damage to the dopamine (DA) and serotonin (5HT) systems of the brain. DL-Fenfluramine (FEN) is another potential neurotoxin but its long-term depletions are more selective to the 5HT system. To determine whether MK-801 protects against damage induced by FEN, we treated rats with FEN (4 injections of 12.5 mg/kg, at 1 h intervals) in conjunction with either saline or MK-801 (2 injections of 2.5 mg/kg, administered 15 min before and 90 min after the first FEN injection). Two weeks post-treatment, MK-801 alone caused a small but significant decrease in 5HT tissue concentrations in striatum and amygdala. FEN significantly reduced 5HT in all 8 brain regions studied. MK-801 + FEN did not protect against FEN-induced 5HT depletions in nucleus accumbens/olfactory tubercle, septum, frontal cortex, somatosensory cortex or hippocampus. MK-801 + FEN enhanced 5HT depletions in striatum, hypothalamus and amygdala. The differential protective effect of MK-801 between MA and FEN are discussed in terms of a possible dopaminergic mechanism.


Involvement of glycine site associated with the NMDA receptor in hippocampal long-term potentiation and acquisition of spatial memory in rats.

The effects of 7-chlorokynurenic acid (7-Cl-Kyn), a selective antagonist at the glycine site associated with the N-methyl-D-aspartate (NMDA) receptor, on hippocampal long-term potentiation (LTP) and behavioral performances in a spatial learning task were investigated. Extracellular recordings of evoked potential (population spike) were made in rat hippocampal slices. Perfusion of 7-Cl-Kyn (10(-5) M) inhibited the induction of LTP following a tetanic stimulation (51 or 101 pulses at 100 Hz) both in the Schaffer/commissural-CA1 pyramidal cell synapses and in the perforant path-dentate granule cell synapses. Acquisition of a spatial memory in the Morris water maze was examined using rats chronically cannulated for application of drugs. The intact and vehicle-injected rats learned easily to escape onto a hidden platform with short latencies, while the rats given an injection of 7-Cl-Kyn (10(-8) mol/brain, i.c.v.) prior to every session took a longer time and a longer path to escape even after all 5 sessions of trials. Injection of 7-Cl-Kyn did not affect the swimming speed, an index of swimming ability. This is the first report providing direct evidence that endogenous glycine supports the processes of learning and memory.


DNABIND: an interactive microcomputer program searching for nucleotide sequences that may code for conserved DNA-binding protein motifs.

This paper presents a simple program for interactive searching for nucleotide sequences that may code for the helix-turn-helix, zinc finger or leucine zipper motifs in proteins. The helix-turn-helix motifs are predicted using the recently published method of Dodd and Egan, while zinc fingers and leucine zippers are searched for by our original methods. DNABIND is shown to detect all four known helix-turn-helix motifs in bacteriophage lambda genes and both zinc fingers of the adr1 gene of yeast.


The superficial buffer barrier in vascular smooth muscle.

Force development and fura-2 fluorescence were simultaneously measured in the rabbit inferior vena cava. Discharging SR Ca2+ with either caffeine or norepinephrine prior to stimulation of Ca2+ influx induced a delay of 30-70 s between the intracellular Ca2+ signal and development of force. This delay was abolished by the application of caffeine. These data support the superficial buffer barrier hypothesis, which holds that Ca2+ entry from the extracellular space proceeds via a restricted cytoplasmic region between the inner plasmalemmal surface and the peripheral sarcoplasmic reticulum (SR). Ca2+ accumulation by this SR fraction appears to be able to delay Ca2+ entry into the deeper myoplasm where it activates the myofilaments. Caffeine and thapsigargin elevated the steady-state [Ca2+]i, suggesting a contribution by the SR Ca2+ pump to Ca2+ extrusion from the cells. Norepinephrine enhanced myofilament Ca2+ sensitivity, while caffeine decreased it.


Dietary sodium induced cardiac hypertrophy.

In humans, high sodium intake not only increases the blood pressure, and thus can cause left ventricular hypertrophy (LVH), but also appears to increase LVH independent of this increase in blood pressure. In both normo- and hyper-tensive rats the hypertrophic effect of increased dietary sodium intake on the heart has been clearly established. In normotensive rats, this effect is strain and age dependent, and seems independent of hemodynamic effects of high sodium intake. In both rats and humans, dietary sodium appears to increase wall thickness, resembling pressure overload rather than an increased left ventricular diameter as expected of volume overload. The mechanisms through which high dietary sodium induces hypertrophy are still unknown. It is possible that dietary sodium increases either adrenergic stimulation and (or) enhances sensitivity for adrenergic stimulation and that this hypertrophic response mainly acts via stimulation of alpha 1-adrenergic receptors. Stimulation of the alpha 1-adrenergic receptors will increase the inositol phosphate-diacyl glycerol pathway and enhance the Na+/H+ exchange. The activity of this exchanger might play an important role in the development of dietary sodium induced cardiac hypertrophy.


Enzymatic synthesis of low molecular weight amyloses with modified terminal groups.

Low molecular weight amyloses with modified terminal groups were synthesized by cyclomaltohexaose (alpha-cyclodextrin) transfer using (1----4)-alpha-D-glucan: 4-alpha-D-(1----4)- alpha-D-glucopyranosyltransferase (cyclising) (EC 2.4.1.19) from Bacillus macerans. 4-Nitrophenyl alpha-malto-oligosaccharides d.p. 2-7 served as acceptors, and cyclomaltohexaose served as the donor. The reaction was optimized to obtain a majority of species of definite chain lengths in a range of d.p. 10-20, depending upon the chain length of the acceptor. The course of the coupling reactions, as well as the action of the enzyme in disproportionation, cyclisation, and hydrolysis of the products, were observed by h.p.l.c. analysis of the oligomer distributions. Using a 15-fold molar excess of cyclomaltohexaose and 0.5 units enzyme per mumol of acceptor at pH 5.2, the chromatograms revealed that the products of the coupling reaction were predominant during the first reaction period. By incubating the acceptors with the enzyme, but without the donor, the mechanism of disproportionation was elucidated as a transfer of malto-oligosaccharyl residues dependent upon the substrate chain length. The minimum chain length required for a direct cyclisation reaction was d.p. 7. The results were confirmed by separation and investigation of the products of hydrolysis and cyclisation, which were nonmodified alpha-malto-oligosaccharides and cyclomalto-oligosaccharides.


Elongation of both branches of biantennary backbones of oligo-(N-acetyllactosamino)glycans by human serum (1----3)-N-acetyl-beta-D- glucosaminyltransferase.

Partial reactions catalyzed by a (1----3)-N-acetyl-beta-D- glucosaminyltransferase (EC2.4.1.149), known to be present in human serum, were studied by use of biantennary "backbone" saccharides of oligo-N-acetyllactosamine-type as acceptors. Incubation of the radiolabeled blood-group I-active hexasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-Galp- (1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Galp-(1----4)-D-GlcNAc (1) and UDP-GlcNAc with serum gave first a transient 1:1 mixture of two isomeric heptasaccharides, beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D- GlcpNAc-(1----3)-[beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D- Galp-(1----4)-D-GlcNAc (2) and beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-GlcpNAc-(1----3)- beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Galp-(1----4)-D-Glc NAc (3), showing that both branches of 1 react equally well. The two heptasaccharides reacted further in the incubation mixture to form the radiolabeled octasaccharide, beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[be ta-D- GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Ga lp- (1----4)-D-GlcNAc (4); during this second reaction, the composition of the heptasaccharide mixture remained unchanged, indicating that 2 and 3 reacted at approximately equal rates. The heptasaccharides 2 and 3 could not be separated from each other, but they could be detected, identified, and quantitatively determined by stepwise enzymic degradations. Partial (1----3)-N-acetyl-beta-D-glucosaminylation reactions, carried out with another acceptor, the branched pentasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-Galp-(1----4)-beta- D- GlcpNAc-(1----6)]-beta-D-Gal (11), revealed that it reacted also equally well at both branches.(ABSTRACT TRUNCATED AT 400 WORDS)


Molecular modelling of acarviosine, the pseudo-disaccharide moiety of acarbose, and other inhibitors of alpha-amylases.

Acarbose and its homologs inhibit alpha-D-glucosidases, particularly alpha-amylases. These homologs have the same core, the pseudo-disaccharide acarviosine, linked to various numbers of glucose residues. The conformations of (R)- and (S)-acarviosine have been analysed. The potential energy maps, obtained by molecular mechanics calculations, show that acarviosine is flexible and has several important minima. One low-energy form is close to the shape assumed by the acarviosine moiety when acarbose is adsorbed on the surface of glycogen phosphorylase. Another likely conformation is the same as that inferred from n.m.r. data and HSEA calculations. The results reconcile those conflicting reports. Molecular modelling of other inhibitors of alpha-amylases, such as 4-thiomaltose and moranoline, shows that these pseudo-disaccharides can fill similar volumes of conformational space.


CGRP-immunoreactive endocrine cell proliferation in normal and hypoxic rat lung studied by immunocytochemical detection of incorporation of 5'-bromodeoxyuridine.

We have tested the suggestion that the reported increase, in hypoxic rats, in the number of lung endocrine cells immunoreactive for the regulatory peptide CGRP is caused by an accumulation of peptide within the cells which renders them more detectable, rather than by a real increase in proliferation. The incorporation of continuously infused 5'-bromodeoxyuridine (BrdU) into nuclei of CGRP-containing cells was studied by immunohistochemistry in the airway and respiratory epithelium of rats kept in a hypoxic (10% O2), normobaric conditions for 7 days and in normoxic, normobaric controls. Some CGRP-immunoreactive cells could also be labelled for BrdU. However, the ratio of the number of cells labelled with both CGRP and BrdU to the number of cells labelled with CGRP alone did not differ significantly between hypoxic and normoxic rats (7.1 +/- 0.7 and 6.1 +/- 1.2, respectively; mean +/- SEM; P = 0.49). These data strongly suggest that CGRP-containing endocrine cells or their precursors do proliferate in adult rat lung, but that the proliferation is not increased significantly in hypoxia.


A technique to access severely diseased arteries.

Cardiac catheterization performed in patients with severe peripheral vascular disease may represent a difficult technical challenge and is associated with a higher incidence of vascular complications. We describe a technique that uses angioplasty equipment to access severely diseased arteries. This technique allows a percutaneous transluminal coronary angioplasty (PTCA) guide wire to be steered across vascular lesions under direct visualization of the lumen and continuous pressure monitoring, potentially reducing the risk of vascular complications.


Effect of reduced aortic compliance on cardiac efficiency and contractile function of in situ canine left ventricle.

This study tests the hypothesis that arterial vascular stiffening adversely influences in situ left ventricular contractile function and energetic efficiency. Ten reflex-blocked anesthetized dogs underwent a bypass operation in which a Dacron graft was sewn to the ascending aorta and connected to the infrarenal abdominal aorta via a plastic conduit. Flow was directed through either native aorta or plastic conduit by placement of vascular clamps. Arterial properties were measured from aortic pressure-flow data, and ventricular function was assessed by pressure-volume (PV) relations. Coronary sinus blood was drained via an extracorporeal circuit for direct measurement of myocardial O2 consumption (MVO2). Data at multiple steady-state preload volumes were combined to derive chamber function and energetics relations. Energetic efficiency was assessed by the inverse slope of the MVO2-PV area relation. Directing flow through plastic versus native aorta resulted in a 60-80% reduction in compliance but little change in mean resistance. Arterial pulse pressure rose from 34 to 99 mm Hg (p less than 0.001). Contractile function assessed by the end-systolic PV relation, stroke work-end-diastolic volume relation, and dP/dtmax at matched end-diastolic volume did not significantly change. However, MVO2 increased by 32% (p less than 0.01) and was matched by a rise in PV area, such that the MVO2-PV area relation and efficiency was unaltered. The MVO2 required to sustain a given stroke volume, however, increased from 20% to 40%, depending on the baseline level (p less than 0.001). Thus, whereas the contractile function and efficiency of normal hearts are not altered by ejection into a stiff vascular system, the energetic cost to the heart for maintaining adequate flow is increased. This suggests a mechanism whereby human vascular stiffening may yield little functional decrement at rest but limit reserve capacity under conditions of increased demand.


Bacteriophage lambda DNA fragments replicate in the Paramecium macronucleus: absence of active copy number control.

We show that bacteriophage lambda DNA fragments microinjected into the macronucleus of the ciliated protozoan Paramecium can replicate as unit-length linear molecules. These linear DNA molecules are substrates for the addition of Paramecium telomeres by an endogenous telomerase. The linear DNA pieces can exist at copy numbers much higher than that of typical endogenous macronuclear chromosomes. We show that the copy number of injected DNA many fissions after microinjection reflects that of the original input copy number, suggesting that active control of copy number does not occur. Instead, the results suggest that injected DNA is replicated once per cell division.


Transcatheter management of cyanotic congenital heart defects: a review.

In this review, the role of transcatheter methods in the management of cyanotic congenital heart defects is discussed. In patients with interventricular right-to-left shunting secondary to pulmonary outflow tract obstruction (most commonly tetralogy of Fallot), balloon dilatation may be an effective palliative procedure in a substantial proportion of patients, obviating the need for a palliative shunt. We would recommend this if the patient's size or cardiac anatomy makes that patient an unsuitable candidate for safe total surgical correction. Infundibular myectomy with atherectomy catheter in tetralogy of Fallot patients may become a useful adjunct in the management of these infants. Cyanotic children with interatrial right-to-left shunt secondary to severe valvar pulmonary stenosis respond to balloon pulmonary valvuloplasty in a manner similar to that seen with isolated pulmonary valve stenosis. In these patients, balloon valvuloplasty is the treatment of choice and may be corrective in most cases. In patients with a narrowed Blalock-Taussig shunt, balloon angioplasty may improve pulmonary oligemia and systemic arterial hypoxemia and may obviate the need for a second systemic-to-pulmonary artery shunt. Balloon angioplasty is recommended if the patient's cardiac defect is not amenable to surgical correction at a low risk either because of the size of the patient or because of the complexity of the cyanotic heart defect. In patients with pulmonary valve atresia, initial opening of the atretic pulmonary valve by either laser or surgery with subsequent balloon dilatation is potentially beneficial in reducing the total number of surgical procedures that these children are likely to require. However, further clinical trials are needed prior to their general use.


Comparison of muscle phosphofructokinase from euthermic and hibernating Jaculus orientalis. Purification and determination of the quaternary structure.

1. The structural properties of skeletal muscle phosphofructokinase from euthermic and hibernating jerboa were compared. 2. The enzyme was purified by a rapid procedure; suspended in ammonium sulfate in the presence of ATP, it was found to be stable for three weeks. 3. A specific activity of 76 U/mg and at most 65 U/mg was obtained for the enzyme from the euthermic and hibernating jerboa, respectively. 4. The molecular weight was estimated to be 320 kDa for the oligomer and 80 kDa for the subunit. 5. A unique alanine residue was found at the C-terminal end, suggesting that the enzyme is a tetramer made of four identical subunits. 6. The tetrameric structure of phosphofructokinase was confirmed by using crosslinking with disuccinimidyl esters. 7. The kinetics of formation of the different crosslinked species were found to be in agreement with a model of the tetramer corresponding to a dihedral symmetry with isologuous contacts between protomers. 8. The same molecular characteristics and immunochemical properties were found for the enzyme extracted from the euthermic and hibernating animals.


Myosin light chain patterns in histochemically typed single fibers of the rat skeletal muscle.

1. This study was conducted to investigate the relationship between histochemical fiber types and myosin light chain patterns in rat single muscle fibers. 2. The hybrid of fast and slow light chains was observed in type I and II fibers of the soleus and type II fibers of the red portion of lateral head of the gastrocnemius muscles. 3. We also observed 7 types of light chain composition. Of the 7 types, 5 types were explainable by assuming the coexistence of isomyosins with either fast or slow light chains. However, the other 2 types could not be accounted for without hypothesizing the presence of isomyosins with promiscuous light chain distribution.


Allosteric control of 6-phosphofructo-1-kinase from rat lung.

1. The regulation of 6-phosphofructo-1-kinase (PFK) in the rat lung of normally fed (control), 72 hr-starved and streptozotocin-induced diabetic rats was investigated. 2. No significant changes in the total enzyme activities and the activity ratios [activity at 0.5 mM fructose 6-phosphate at pH 7.0/activity at pH 8.0 (v0.5/V)] of rat lung were observed between the control and 72 hr-starved or streptozotocin-induced diabetic rats. 3. Rat lung PFK was highly stimulated by fructose 2,6-bisphosphate (Fru-2,6-P2) as the affinity of the enzyme for fructose 6-phosphate was highly increased by this metabolite and the enzyme inhibition by ATP was released. 4. Although rat liver and mucosal PFK were found to be highly sensitive to stimulation by Fru-2,6-P2, lung PFK was significantly more sensitive to the stimulation by this metabolite than the other tissues. 5. The enzyme was highly inhibited by citrate and was only slightly inhibited by phosphocreatine. 6. ADP, AMP and c-AMP were shown to be activators of lung PFK with c-AMP being the most effective activator. 7. As a rate limiting enzyme of glycolysis, rat lung PFK is highly controlled by its allosteric effectors, especially Fru-2,6-P2, possibly for surfactant lipid synthesis which usually requires a high rate of glycolysis.


The decline of atrial natriuretic peptide (ANP) gene expression in older rats and the effects of ginsenoside on ANP gene expression.

1. The levels of atrial natriuretic peptide(ANP) gene expression in rat atria at 2-3 and 14-18 months of age and the effects of ginsenosides on r-ANP gene expression by determining the concentration of ANP-mRNA were investigated. The male and female rats were abdominally (i.p.) injected with aqueous solution of ginsenosides prepared from ginseng stems and leaves (G-PSL) and ginseng roots (G-PR), 50 mg/kg body wt, once a day for 7 days. Atria total RNA was extracted by the cold phenol method. The ANP-mRNA contents were determined using the Northern blot and dot blot hybridization technique with alpha-32*P-labelled r-prepro-ANP-cDNA probe. 2. The ANP-mRNA contents of 14-18 month rats were remarkably less than that of 2-3 month rats. The levels of male and female rats' atria at 14-18 months were about 15 and 60%, the content of male and female rats at 2-3 months respectively. 3. G-PSL and G-PR increased the ANP-mRNA content of male rats at 14-18 months 1- and 2-fold, respectively, whereas G-PSL and G-PR decreased the ANP-mRNA content in male rats of age 2-3 months. 4. These results revealed that the ANP gene expression declined during ontogenic ageing development and ginsenosides possessed anti-ageing effects in the heart endocrineous function aspect.


Interaction between the carboxyl groups of Asp127 and Asp129 in the active site of Escherichia coli phosphofructokinase.

The pH dependence of the enzymic properties of the phosphofructokinase from Escherichia coli was compared to those of two mutants in which one carboxyl group of the active site has been removed from either Asp127 or Asp129. All measurements of activity were made in the presence of allosteric activator ADP or GDP to eliminate any cooperative process. Asp129 is a crucial residue for the activity of phosphofructokinase since its conversion to Ser decreases the catalytic activity by 2-3 orders of magnitude in both the forward and reverse reactions, but the ionization of Asp129 is not directly related the pH dependence of phosphofructokinase activity. This pH dependence is however modified by the Asp129----Ser mutation, which decreases the pK of another residue, Asp127, by as much as pH of 1.5. The side chain of Asp127 has the catalytic role proposed earlier: its deprotonated form acts as a base in the forward reaction, and its protonated form acts as an acid in the reverse reaction. The protonated form of Asp127 is also required for the binding of fructose 1,6-bisphosphate. The electrostatic interaction between the carboxyl groups of Asp127 and Asp129 seems different in free phosphofructokinase to that in enzyme/substrate complexes, suggesting that a conformational change occurs upon substrate binding. The pH dependence of phosphofructokinase activity involves one other ionizable group with a pK of approximately 6 which does not belong to the side chains of Asp127 or Asp129.


Mapping of three unique Ca(2+)-binding sites in human annexin II.

Site-directed mutagenesis was employed to map and characterize Ca(2+)-binding sites in annexin II, a member of the annexin family of Ca(2+)- and phospholipid-binding proteins which serves as a major cellular substrate for the tyrosine kinase encoded by the src oncogene. Several single amino acid substitutions were introduced in the human annexin II and the various mutant proteins were scored for their affinity towards Ca2+ in different assays. The data support our previous finding [Thiel, C., Weber, K. and Gerke V. (1991) J. Biol. Chem. 266, 14,732-14,739] that a Ca(2+)-binding site is present in the third of the four repeat segments which comprise the 33-kDa protein core of annexin II. In addition to Gly206 and Thr207, which are localized in the highly conserved endonexin fold of the third repeat, Glu246 is involved in the formation of this site. Thus the architecture of this Ca(2+)-binding site in solution is very similar, if not identical, to that of Ca2+ sites identified recently in annexin V crystals [Huber, R., Schneider, M., Mayr, I., Römisch, J. and Paques, E.-P. (1990) FEBS Lett. 275, 15-21]. In addition to the site in repeat 3, we have mapped sites of presumably similar architecture in repeats 2 and 4 of annexin II. Again, an acidic amino acid which is located 40 residues C-terminal to the conserved glycine at position 4 of the endonexin fold is indispensable for high-affinity Ca2+ binding: Asp161 in the second and Asp321 in the fourth repeat. In contrast, repeat 1 does not contain an acidic amino acid at a corresponding position and also shows deviations from the other repeats in the sequence surrounding the conserved glycine. These results on annexin II together with the crystallographic information on annexin V reveal that annexins can differ in the position of the Ca2+ sites. Ca(2+)-binding sites of similar structure are present in repeats 2, 3, and 4 of annexin II while in annexin V they occur in repeats 1, 2, and 4. We also synthesized an annexin II derivative with mutations in all three Ca2+ sites. This molecule shows a greatly reduced affinity for the divalent cation. However, it is still able to bind Ca2+, indicating the presence of (an) additional Ca2+ site(s) of presumably different architecture.