The effect of carotid body denervation upon the respiratory response to hypoxia and hypercapnia in the duck

1. Respiratory responses to altered P_{a, O2} at constant P_{a, CO2} and altered P_{a, CO2} at constant P_{a, O2} were measured in fourteen unanaesthetized ducks 5-9 weeks old.

2. All the ducks responded to the inhalation of a few breaths of 100% O₂ with a fall in _E of between 11 and 17% of control, and to the reduction in P_{a, O2} from control levels (93-101 mm Hg) to 38-47 mm Hg with an average increase in _E of 190% of control which was potentiated by raising the level of CO₂.

3. Ventilation approximately doubled when P_{a, CO2} was raised to 55 mm Hg, the greatest sensitivity to changes in P_{a, CO2} being over the range 40-45 mm Hg.

4. Carotid body denervation was carried out in nine ducks and the respiratory responses were modified. The fall in _E with inhalation of 100% O₂ was abolished in all ducks and the rise in _E with hypoxia was abolished in six. The increase in _E as P_{a, CO2} was altered in steps was unaffected but the rate at which _E increased in response to 4% CO₂ was markedly reduced.

5. It is concluded that the chemoreceptor regulation of respiration in ducks is similar to that observed in non-diving animals.

     

Mechanical work and efficiency in level walking and running

1. The mechanical power spent to accelerate the limbs relative to the trunk in level walking and running, _int, has been measured at various `constant' speeds (3-33 km/hr) with the cinematographic procedure used by Fenn (1930a) at high speeds of running.

2. _int increases approximately as the square of the speed of walking and running. For a given speed _int is greater in walking than in running.

3. In walking above 3 km/hr, _int is greater than the power spent to accelerate and lift the centre of mass of the body at each step, _ext (measured by Cavagna, Thys & Zamboni, 1976b). In running _int < _ext up to about 20 km/hr, whereas at higher speeds _int > _ext.

4. The total work done by the muscles was calculated as W_tot = |W_int| + |W_ext|. Except that at the highest speeds of walking, the total work done per unit distance W_tot/km is greater in running than in walking.

5. The efficiency of positive work was measured from the ratio W_tot/Net energy expenditure: this is greater than 0·25 indicating that both in walking and in running the muscles utilize, during shortening, some energy stored during a previous phase of negative work (stretching).

6. In walking the efficiency reaches a maximum (0·35-0·40) at intermediate speeds, as may be expected from the properties of the contractile component of muscle. In running the efficiency increases steadily with speed (from 0·45 to 0·70-0·80) suggesting that positive work derives mainly from the passive recoil of muscle elastic elements and to a lesser extent from the active shortening of the contractile machinery. These findings are consistent with the different mechanics of the two exercises.

     

The respiratory response of the new-born lamb to inhaled CO-2 with and without accompanying hypoxia

1. The respiratory response to inhaled CO₂ was measured in twenty unanaesthetized new-born lambs aged 4 hr-10 days. Measurement of resting arterial pH, P_CO2 and plasma bicarbonate showed a non-respiratory acidosis immediately after birth which was corrected in the first 24-28 hr: thereafter, the acid—base pattern was of a compensated respiratory alkalosis.

2. When CO₂ was added to the inspired gases and resting arterial oxygen tension (P_a, _O2) was controlled, the average increase in minute ventilation () was 0·075 l.min^-1.kg^-1.mm Hg, P_a, _CO2^-1 and duplicate responses in the same lamb differed by 6-22·5%.

3. The slope of the /P_a, _CO2 line (S) varied inversely with P_a, _O2. In one lamb, severe hypoxia (P_a, _O2 = 21 mm Hg) caused a marked depression of the slope.

4. Neither the slope S nor the horizontal intercept B of the lines was related to the age of the lamb. B was not related to pH_a and only slightly affected by acute hypoxia. B was related to arterial [HCO₃^-] and values for both were reduced with the acid—base disturbances seen in the first 10 days after birth. Evidence was given which suggested that the response of the new-born lamb to inhaled CO₂ was similar to that of man acclimatized to a P_a, _O2 of 70-75 mm Hg.

5. In the lightly anaesthetized lamb, bilateral section of the sinus nerves caused a small reduction in the sensitivity to inhaled 5% CO₂ in air, an increase in the respiratory lag and a reduction in the rate at which increased.

6. It was concluded that, in the new-born lamb, the carotid chemo-receptors are involved in the response to inhaled CO₂ and that hypoxia potentiates this response.

     

Effects of Porcine Circovirus Type 2 (PCV2) Maternal Antibodies on Experimental Infection of Piglets with PCV2

To determine the effects of porcine circovirus type 2 (PCV2) maternal antibodies on and response to experimental PCV2 infection, 24 piglets were divided into four groups on the basis of the enzyme-linked immunosorbent assay titers of PCV2 maternal antibodies: group A (n = 6; sample/positive [S/P] ratio, <0.2), group B (n = 5; S/P ratio, >0.2 to <0.5), and groups C (n = 8) and D (n = 5) (S/P ratio, >0.5). Piglets in groups A, B, and C were inoculated with PCV2 at day 0 and challenged with PCV2 at day 42. Group D piglets were not exposed to PCV2 at day 0 but were challenged at day 42. Before challenge, seroconversion to PCV2 antibodies occurred in fiveofsix group A piglets, and the antibody level rose above the cutoff level in oneoffive group B piglets. Viremia was detected in fiveofsix, fouroffive, and twoofeight pigs in groups A, B, and C, respectively. After challenge, PCV2 DNA was detectable from 7 to 21 days postchallenge in the sera from sixofsix, fouroffive, threeofeight, and fiveoffive pigs in groups A, B, C, and D, respectively. The results indicated that protection against PCV2 infection conferred by maternal antibodies is titer dependent: higher titers are generally protective, but low titers are not.     

Modulation of telomerase activity by telomere DNA-binding proteins in Oxytricha

Telomere proteins protect the chromosomal terminus from nucleolytic degradation and end-to-end fusion, and they may contribute to telomere length control and the regulation of telomerase. The current studies investigate the effect of Oxytricha single-stranded telomere DNA-binding protein subunits α and β on telomerase elongation of telomeric DNA. A native agarose gel system was used to evaluate telomere DNA-binding protein complex composition, and the ability of telomerase to use these complexes as substrates was characterized. Efficient elongation occurred in the presence of the α subunit. Moreover, the α–DNA cross-linked complex was a substrate for telomerase. At higher α concentrations, two α subunits bound to the 16-nucleotide single-stranded DNA substrate and rendered it inaccessible to telomerase. The formation of this α·DNA·α complex may contribute to regulation of telomere length. The α·β·DNA ternary complex was not a substrate for telomerase. Even when telomerase was prebound to telomeric DNA, the addition of α and β inhibited elongation, suggesting that these telomere protein subunits have a greater affinity for the DNA and are able to displace telomerase. In addition, the ternary complex was not a substrate for terminal deoxynucleotidyltransferase. We conclude that the telomere protein inhibits telomerase by rendering the telomeric DNA inaccessible, thereby helping to maintain telomere length.     

Fidelity and Mutational Spectrum of Pfu DNA Polymerase on a Human Mitochondrial DNA Sequence

The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10⁻⁶ must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or “PCR noise”. Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived from Pyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) of Pfu was 6.5×10⁻⁷, and five of its more frequent mutations (hot spots) consisted of three transversions (GC→TA, AT→TA, and AT→CG), one transition (AT→GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be reduced.     

Serum Cytokine Responses during Acute Human Granulocytic Ehrlichiosis

Human granulocytic ehrlichiosis (HGE) is caused by obligate intracellular bacteria in the Ehrlichia phagocytophila group. The disease ranges from subclinical to fatal. We speculated that cell-mediated immunity would be important for recovery from and potentially in the clinical manifestations of HGE; thus, serum tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), gamma interferon (IFN-γ), IL-10, and IL-4 concentrations were studied. IFN-γ (1,035 ± 235 pg/ml [mean ± standard error of the mean]) and IL-10 (118 ± 46 pg/ml) concentrations were elevated in acute-phase sera versus convalescent sera and normal subjects (P≤0.013 and P≤0.018, respectively). TNF-α, IL-1β, and IL-4 levels were not elevated. Cytokine levels in severely and mildly affected patients were not different. HGE leads to induction of IFN-γ-dominated cell-mediated immunity associated with clinical manifestations, recovery from infection, or both.     

The Change of Total Anthocyanins in Blueberries and Their Antioxidant Effect After Drying and Freezing

This study examined the effects of freezing, storage, and cabinetdrying on the anthocyanin content and antioxidant activity ofblueberries (Vaccinium corymbosum L). Fresh samples werestored for two weeks at 5^°C while frozen samples were keptfor up to three months at −20^°C. There were two dryingtreatments, one including osmotic pretreatment followed bycabinet drying and the other involving only cabinet drying.Total anthocyanins found in fresh blueberries were 7.2 ±0.5mg/g dry matter, expressed as cyanidin 3-rutinosideequivalents. In comparison with fresh samples, total anthocyaninsin untreated and pretreated dried blueberries were significantlyreduced to 4.3 ± 0.1mg/g solid content, 41% loss, and3.7 ± 0.2mg/g solid content, 49% loss, respectively.Osmotic treatment followed by a thermal treatment had a greatereffect on anthocyanin loss than the thermal treatment alone. Incontrast, the frozen samples did not show any significantdecrease in anthocyanin level during three months of storage.Measurement of the antioxidant activity of anthocyanin extractsfrom blueberries showed there was no significant differencebetween fresh, dried, and frozen blueberries.     

Effects of Dietary Copper Loading on Livers of Rats: I. Changes in Subcellular Acid Phosphatases and Detection of an Additional Acid p-Nitrophenylphosphatase in the Cellular Supernatant During Copper Loading

Significant changes occurred in lysosomal structure and function as copper was metabolized by rat livers. Hepatic total acid p-nitrophenylphosphatase (pNPase) activity was markedly increased in copper-loaded rats, and this increase was almost completely accounted for by heat- and formalin-stable (HFS) acid pNPase. Heat- and formalin-labile acid pNPase was essentially unchanged. On a subcellular level, the microsomal and supernatant fractions reflected the greatest relative increase in HFS acid pNPase. Increases in lysosomal, HFS acid pNPase in the large-granule fractions correlated with increase in solubilized large-granule enzymes, LG I, with mol wt > 200,000. LG II, representng solubilized large-granule enzymes with mol wt < 200,000, remained unchanged. Marked increases in supernatant acid pNPase were principally accounted for by a sevenfold increase in a supernatant lysosomal-like enzyme, DEAE Pk 1, separated by DEAE cellulose chromatography. An additional enzyme, DEAE Pk 2A′, that was hardly or not detectable in normal rats, was consistently demonstrated and increased in copper-loaded rats. Serum HFS acid pNPase increased in copper-loaded rats, suggesting that the increased hepatic supernatant acid pNPase in part escaped into the circulating fluid. Copper was principally associated with cytoplasmic organelles and was highest in mitochondrial and lysosomal fractions.     

Sour Cherry (Prunus cerasus L) Anthocyanins as Ingredients for Functional Foods

In the recent years many studies on anthocyaninshave revealed their strong antioxidant activity and their possibleuse as chemotherapeutics. The finding that sour cherries(Prunus cerasus L) (also called tart cherries) containhigh levels of anthocyanins that possess strong antioxidant andanti-inflammatory properties has attracted muchattention to this species. Here we report the preliminary resultsof the induction of anthocyanin biosynthesis in sour cherrycallus cell cultures. The evaluation and characterization of thein vitro produced pigments are compared to those of theanthocyanins found in vivo in fruits of several sour cherrycultivars. Interestingly, the anthocyanin profiles found in wholefruit extracts were similar in all tested genotypes but weredifferent with respect to the callus extract. The evaluation ofantioxidant activity, performed by ORAC and TEAC assays, revealeda relatively high antioxidant capacity for the fruitextracts (from 1145 to 2592μmol TE/100g FW) and alower one for the callus extract (688μmol TE/100g FW).     

Association of TPH1 with suicidal behaviour and psychiatric disorders in the Chinese population

Tryptophan hydroxylase (TPH), the rate limiting enzyme in serotonin biosynthesis, is one of the most important regulating factors in the serotonergic system. Recently, polymorphisms of the TPH gene have been identified as being associated with suicide, but the evidence is inconsistent. To investigate the role in suicide of one of the isoforms, TPH1, we examined the association of five single nucleotide polymorphisms (SNPs) in the promoter region and in intron 7 of the TPH1 gene based on a sample from the Chinese population of 810 subjects, of whom 329 had made no suicide attempts (NSA), 297 had made suicide attempts (SA), and 184 were healthy subjects (HS). In this study, we observed statistically significant differences between NSA and HS subjects in allele distributions on one marker, −6526A (p=0.0329; odds ratio (OR) 1.36; 95% confidence interval (CI) 1.01 to 1.81). No significant difference in genotype distribution or allele frequencies of other polymorphisms was found between the suicide victims and the controls. The overall haplotype frequency was significantly different between cases and healthy controls (p=0.000024 NSA v HS; p<0.000001, SA v HS; p<0.000001, cases v HS). We found the haplotype TCAAA of −7180/−7065/−6526/218/779 to be strongly associated with suicidal behaviour and psychiatric disorders (p=0.00243; OR=1.62; 95% CI 1.17 to 2.24 and p=0.018; OR=1.41; 95% CI 1.05 to 1.91), which suggests an association of TPH1 with suicidal behaviour and indicates that TPH1 may play a significant role in the aetiology of psychiatric disorders in the Han Chinese population.     

Acute Plasmodium chabaudi chabaudi Malaria Infection Induces Antibodies Which Bind to the Surfaces of Parasitized Erythrocytes and Promote Their Phagocytosis by Macrophages In Vitro

CBA/Ca mice infected with 5 × 10⁴ Plasmodium chabaudi chabaudi AS-parasitized erythrocytes experience acute but self-limiting infections of relatively short duration. Parasitemia peaks (~40% infected erythrocytes) on day 10 or 11 and is then partially resolved over the ensuing 5 to 6 days, a period referred to as crisis. How humoral and cellular immune mechanisms contribute to parasite killing and/or clearance during crisis is controversial. Humoral immunity might be parasite variant, line, or species specific, while cellular immune responses would be relatively less specific. For P.c. chabaudi AS, parasite clearance is largely species and line specific during this time, which suggests a primary role for antibody activity. Accordingly, acute-phase plasma (APP; taken from P.c. chabaudi AS-infected mice at day 11 or 12 postinfection) was examined for the presence of parasite-specific antibody activity by enzyme-linked immunosorbent assay. Antibody binding to the surface of intact, live parasitized erythrocytes, particularly those containing mature (trophozoite and schizont) parasites, was demonstrated by immunofluorescence in APP and the immunoglobulin G (IgG)-containing fraction thereof. Unfractionated APP (from P.c. chabaudi AS-infected mice), as well as its IgG fraction, specifically mediated the opsonization and internalization of P.c. chabaudi AS-parasitized erythrocytes by macrophages in vitro. APP from another parasite line (P.c. chabaudi CB) did not mediate the same effect against P.c. chabaudi AS-parasitized erythrocytes. These results, which may represent one mechanism of parasite removal during crisis, are discussed in relation to the parasite variant, line, and species specificity of parasite clearance during this time.     

Overcoming cellular senescence in human cancer pathogenesis

Elevation of p16, the CDKN2/p16 tumor suppressor gene (TSG) product, occurs at senescence in normal human uroepithelial cells (HUC). Immortal HUCs and bladder cancer cell lines show either alteration of p16 or pRb, the product of the retinoblastoma (RB) TSG. In addition, many human cancers show p16 or pRb alteration along with other genetic alterations that we associated with immortalization, including +20q and −3p. These observations led us to hypothesize that p16 elevation plays a critical role in senescence cell cycle arrest and that overcoming this block is an important step in tumorigenesis in vivo, as well as immortalization in vitro. Using a novel approach, we tested these hypotheses in the present study by examining p16 and pRb status in primary culture (P0) and after passage in vitro of transitional cell carcinoma (TCC) biopsies that represented both superficial bladder tumors and invasive bladder cancers. We demonstrated that all superficial TCCs showed elevated p16 after limited passage in vitro and then senesced, like normal HUCs. In contrast, all muscle invasive TCCs contained either a p16 or a pRb alteration at P0 and all spontaneously bypassed senescence (P=0.001). Comparative genomic hybridization (CGH) was used to identify regions of chromosome loss or gain in all TCC samples. The application of a statistical model to the CGH data showed a high probability of elevated alteration rates of +20q11−q12 (0.99) and +8p22−pter (0.94) in the immortal muscle invasive TCCs, and of −9q (0.99) in the superficial TCCs. Three myoinvasive TCCs lost 3p13−p14. In this study, four of six myoinvasive TCCs also showed TP53 mutation that associated well with genome instability (P=0.001), as previously hypothesized. Notably, TP53 mutation, which has been used as a marker of tumor progression in many human cancers, was less significant in associating with progression in this study (P=0.04) than was p16 or pRb alteration (P=0.001). Thus, these data support a new model in which overcoming senescence plays a critical role in human cancer pathogenesis and requires at least two genetic changes that occur in several combinations that can include either p16 or pRb loss and at least one additional alteration, such as +20q11−q12, −3p13−p14, or −8p21−pter.     

Evaluation of the In Vitro Pyrogen Test System Based on Proinflammatory Cytokine Release from Human Monocytes: Comparison with a Human Whole Blood Culture Test System and with the Rabbit Pyrogen Test

The reliability of an in vitro pyrogen test system based on proinflammatory cytokine release from human monocytic cells was assessed by comparison with a test system based on a human whole blood culture as well as with the conventional rabbit pyrogen test. The human cells used as the pyrogen indicator cells were newly selected by subcloning of a human monocytic cell line, Mono-Mac-6. The selected cells, named MM6-CA8, responded to various pyrogens, including endotoxin, peptidoglycan (PG), Staphylococcus aureus Cowan 1 (SAC), and poly(I·C), with a high sensitivity and produced proinflammatory cytokines, such as interleukin 1 (IL-1), IL-6, and tumor necrosis factor alpha. Among these cytokines, IL-6 was produced most sensitively in response to traces of the pyrogens and detected in the largest quantities in the culture medium. The cytokine-producing responses of MM6-CA8 cells correlated significantly with the responses of cultured human whole blood, which represents an ex vivo culture test system reproducing pyrogen-induced cytokine production in the human body. In terms of cytokine inducibility, the pyrogens were ranked in the order endotoxin > PG > poly (I·C) > SAC in both culture systems, a ranking which almost agreed with the ranking of their pyrogenicity as assessed by the rabbit pyrogen test. These results suggest that the in vitro responsiveness of MM6-CA8 cells to various pyrogens is highly relevant for human pyrogenic reactions. Therefore, the in vitro test system is useful and reliable for detecting the presence of materials that are pyrogenic for humans.     

Possible role for Toll-like receptors in interaction of Fasciola hepatica excretory/secretory products with bovine macrophages

Alternative activation of macrophages (M) during helminth infection is a characteristic feature of the host immune response. Alternatively activated macrophages (AAM) are distinguished from others by high arginase 1 (Arg-1) activity, low nitric oxide (NO), and high interleukin 10 (IL-10) production. In murine models, these cells have been shown to possess anti-inflammatory properties. They have also been implicated in exacerbating a subsequent infection with a secondary pathogen. In this study we used cattle experimentally infected with Fasciola hepatica to monitor the kinetics of IL-4 and IL-10 over the course of infection. Using naïve M in vitro, we examined the effects of exposure to F. hepatica excretory/secretory products (FhepES) alone or in combination with IL-4. Our results suggest that FhepES may work in combination with IL-4 to produce AAM. The effects of FhepES on the subsequent responses to lipopolysaccharide (LPS) and purified protein derivative from Mycobacterium bovis (PPD-B), which are bovine Toll-like receptor 4 (TLR4) and TLR2 antagonists, respectively, were also examined. We found that M stimulated with FhepES together with LPS or PPD-B have reduced NO or gamma interferon production, respectively. The ability of FhepES to produce AAM was found to be heat labile and partially dependent on glycan residues. A possible role for TLR recognition is discussed.     

Two Spectrophotometric Assays for Dopamine Derivatives in Pharmaceutical Products and in Biological Samples of Schizophrenic Patients Using Copper Tetramine Complex and Tri-iodide Reagent

Two simple, rapid, and sensitive spectrophotometric methods areproposed for the determination of levodopa (LD). The first methodis based on coupling of 4-aminoantipyrine (4-AAP) with one of thedopamine derivatives (LD, CD) to give a new ligand that reactswith copper tetramine complex to give intenselycolored chelates. The colored products are quantifiedspectrophotometrically at 525 and 520nm for LD and CD,respectively. The optimization of the experimental conditions isdescribed. The method has been used for the determination of19.7–69.0 and 18.1–54.3μg mL⁻¹ of LDand CD, respectively. The accuracy of the method is achieved bythe values of recovery (100 ± 0.2%) and the precision issupported by the low standard deviation (SD = 0.17–0.59) andrelative standard deviation (CV = 0.4%–1.54%) values. Thesecond method is based on the formation of ion-pair iodinatedinner sphere or outer sphere colored complexes between the LDand triiodide ions at pH 5 and room temperature (23 ± 3^°C). This method has been used for the determination of LDwithin the concentration range 39.44–78.88μgmL⁻¹ with SD = 0.22–0.24 and recovery percent = 100 ± 0.3%.The sensitivity of the two methods is indicated bySandell's sensitivity of 0.014–0.019g cm⁻². Theresults of the two methods are compared with those of theofficial method. The interference of common drug additives,degradation products, and excipients was also studied. Theproposed methods were applied successfully to the determinationof the LD-CD synthetic mixture and Levocare drug. Thedetermination of LD in urine of some schizophrenic patients wasapplied with good precision and accuracy. The reliability of themethods was established by parallel determinations against theofficial British pharmacopoeia method.     

CpG Islands of the Pig

We describe an analysis of the CpG islands (CGIs) of the pig. We have used both database survey and a porcine genomic library that is enriched for CGIs. Approximately half of 41 pig genomic database sequences had CGIs with an average G+C content of 65.3%, an average CpG observed/expected frequency of 0.85, and an average size of 978 bp. Of 27 CGI library clones, 16 were nonrepetitive, nonribosomal DNA and CGI-like. CGI library clones had similar average values for G+C and CpG frequency to CGIs of database genes, and an average size of 670 bp, as MseI cuts within some islands. Library clones were also shown to be low copy number and unmethylated in genomic DNA. The presence in the library of seven previously known CGI sequences was confirmed as was the absence of one nonisland sequence. The CGI library exhibits an R-band pattern for many chromosomes in FISH analysis. The pig chromosome arms that show the most dense CGI population are homologous to segments of human chromosomes that are known to be gene rich.     

Activation of human monocytes by both human β1H and C3b

We tested whether highly purified human β1H and C3b, two proteins of the alternative pathway of complement activation, could exert an influence on the activity of human monocytes (M). The activation process of M was assessed by measurements of the respiratory burst in terms of nitro blue tetrazolium (NBT) reduction and by chemiluminescence (CL) tests. In NBT reduction experiments, we found a tendency for β1H to increase NBT reduction, while C3b was found to be rather inhibitory. In CL measurements, both β1H and C3b displayed a stimulatory effect on M, showing different time- and dose-dependency. For β1H, the maximum stimulation occurred after 15 min, whereas for C3b after 45 min. Zymosan particles which served as a positive control also showed the highest stimulation after 45 min. In dose—response experiments, β1H reached a plateau ranging from 30 to 80 μg/ml. In contrast, using C3b up to 170 μg/ml, no plateau was reached. M-depletion and enrichment studies suggested at M as being responsible for the stimulatory effects found.

The differences between NBT reduction and CL could possibly be explained by the measurement of only cell-bound reductive potentials by NBT reduction, while in CL measurements, products of the extracellular space are also assessed. Our results suggest that both human β1H and C3b are appropriate stimuli for human monocytes.

     

Vibrio cholerae Hemagglutinin/Protease Inactivates CTXφ

Pathogenic strains of Vibrio cholerae are lysogens of the filamentous phage CTX, which carries the genes for cholera toxin (ctxAB). We found that the titers of infective CTX in culture supernatants of El Tor CTX lysogens increased rapidly during exponential growth but dropped to undetectable levels late in stationary-phase growth. When CTX transducing particles were mixed with stationary-phase culture supernatants of El Tor strains, CTX infectivity was destroyed. Our data indicate that this growth phase-regulated factor, designated CDF (CTX-destroying factor), is the secreted hemagglutinin/protease (HA/P) of V. cholerae. A strain containing a disrupted hap gene, which encodes HA/P of V. cholerae, did not produce CDF activity in culture supernatants. Introduction of the HA/P-expressing plasmid pCH2 restored CDF activity. Also, CDF activity in culture supernatants of a variety of pathogenic V. cholerae isolates varied widely but correlated with the levels of secreted HA/P, as measured by immunoblotting with anti-HA/P antibody. CDF was purified from V. cholerae culture supernatants and shown to contain a 45-kDa polypeptide which bound anti-HA/P antibodies and which comigrated with HA/P in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The production of high levels of secreted HA/P by certain V. cholerae strains may be a factor in preventing CTX reinfection in natural environments and in the human host.     

A Locus for Autosomal Recessive Pseudoxanthoma Elasticum, with Penetrance of Vascular Symptoms in Carriers, Maps to Chromosome 16p13.1

Pseudoxanthoma elasticum (PXE) is a heritable systemic disorder characterized by calcification of the elastic fibers of the connective tissue. Symptoms are predominantly noted in the eye, the skin, and the cardiovascular system, resulting in visual loss, skin lesions, and life-threatening vascular disease. In this study we combined homozygosity mapping and genome scanning with 374 markers in affected individuals from a PXE family from a genetically isolated population in The Netherlands. Initial homozygosity in two or three patients was found with up to 20 markers, among which D16S292 located in 16p13.1. Upon refined and more extensive family screening of the latter region, close linkage without recombination was found with the marker D16S764 (Z_max=6.27). Despite clear autosomal recessive inheritance of the ocular symptoms in PXE, vascular symptoms appear in 40%–50% of the heterozygotes.