Membrane-associated proteins of T4-infected Escherichia coli

During the prereplicative period after the infection of Escherichia coli by phage T4, more than 50 proteins are synthesized. Many of them have been identified with their corresponding genes. Among them, at least 13 are selectively enriched in the membrane preparation. They include the products of the two rII genes, genes 39, 52 (DNA-delay), and others not yet identified. The majority of these proteins (90%) are extractable by the detergent, sarkosyl, and are possibly associated with the inner or cytoplasmic membrane. Based on their electrophoretic migration in SDS-polyacrylamide gels and on their tryptic fingerprints, these proteins are found to be phage induced. Two of the newly synthesized proteins that are selectively enriched in the cell wall or outer envelope fraction are found to be identical with two envelope proteins of the host cell. They are continually synthesized after phage infection although general host protein synthesis is shut off.     

Induction of protective immunity against Streptococcus mutans colonization after mucosal immunization with attenuated Salmonella enterica serovar typhimurium expressing an S. mutans adhesin under the control of in vivo-inducible nirB promoter

The purpose of the present study was to evaluate the effectiveness of an attenuated Salmonella enterica serovar Typhimurium vaccine strain expressing the saliva-binding region (SBR) of the Streptococcus mutans antigen I/II adhesin, either alone or linked with the mucosal adjuvant cholera toxin A2 and B subunits (CTA2/B) and under the control of the anaerobically inducible nirB promoter, in inducing a protective immune response against S. mutans infection. BALB/c mice were immunized by either the intranasal or the intragastric route with a single dose of 10(9) or 10(10) Salmonella CFU, respectively. The Salmonella vaccine strain expressing an unrelated antigen (fragment C of tetanus toxin [TetC]) was also used for immunization as a control. Samples of serum and secretion (saliva and vaginal washes) were collected prior to and following immunization and assessed for antibody activity by enzyme-linked immunosorbent assay. Anti-SBR antibodies were detected in the serum and saliva of experimental animals by week 3 after immunization. A booster immunization at week 17 after the initial immunization resulted in enhanced immune responses to the SBR. The serum immunoglobulin G subclass profiles were indicative of T helper type 1 responses against both the vector and the SBR antigen. To determine the effectiveness of these responses on the protection against S. mutans infection, mice were challenged after the second immunization with a virulent strain of S. mutans which was resistant to tetracycline and erythromycin. Prior to the challenge, mice were treated for 5 days with tetracycline, erythromycin, and penicillin. S. mutans was initially recovered from all of the challenged mice. This bacterium persisted at high levels for at least 5 weeks in control TetC-immunized or nonimmunized mice despite the reappearance of indigenous oral organisms. However, mice immunized with Salmonella clones expressing SBR or SBR-CTA2/B demonstrated a significant reduction in the number of S. mutans present in plaque compared to the control groups. These results provide evidence for the effectiveness of the Salmonella vector in delivering the SBR antigen for the induction of mucosal and systemic immune responses to SBR. Furthermore, the induction of a salivary anti-SBR response corresponded with protection against S. mutans colonization of tooth surfaces.     

Food Allergy Herbal Formula-1 (FAHF-1) blocks peanut-induced anaphylaxis in a murine model

BACKGROUND: Peanut allergy is a major cause of fatal and near-fatal anaphylactic reactions to foods. There is no curative therapy for this condition. Traditional Chinese medicines have been reported to have antiallergic properties, which might be useful for treating peanut allergy. OBJECTIVE: The purpose of this study was to investigate the effects of a Chinese herbal formula, FAHF-1, on peanut anaphylactic reactions in a mouse model of peanut allergy. METHODS: Mice were sensitized with freshly ground whole peanut in the presence of cholera toxin and boosted 1 and 3 weeks later. FAHF-1 treatment was initiated 1 week later and continued for 7 weeks. After treatment, mice were challenged with peanut, and anaphylactic symptoms, body temperatures, and plasma histamine and IgE levels were measured. T-cell proliferative responses and cytokine production were also determined. RESULTS: FAHF-1 completely blocked peanut-induced anaphylactic symptoms and markedly reduced mast cell degranulation and histamine release. Peanut-specific serum IgE levels were significantly reduced by 2 weeks of treatment at the time of challenge, and they remained lower 4 weeks after discontinuation of treatment. FAHF-1 significantly reduced peanut-induced lymphocyte proliferation as well as IL-4, IL-5, and IL-13 synthesis but not IFN-gamma synthesis. No toxic effects on liver or kidney functions were observed, nor was there any overall immune suppression. CONCLUSION: FAHF-1 protected peanut-sensitized mice from anaphylactic reactions and significantly reversed established IgE-mediated peanut allergy. This suggests that FAHF-1 might prove valuable for the treatment of peanut allergy.     

Evaluation of a PCR primer based on the isocitrate dehydrogenase gene for detection of Helicobacter pylori in feces

In order to improve detection and identification of Helicobacter pylori in highly contaminated samples, we evaluated new specific primers based on the DNA base sequence within the isocitrate dehydrogenase (icd) gene to amplify a 1,200-bp DNA segment. The specificity of the icd primer was tested against DNA derived from various bacteria, including 7 Helicobacter species and a panel of 1 gram-variable, 2 gram-positive, and 16 gram-negative bacteria, as well as DNA from houseflies and feces from H. pylori-negative patients. The primers permitted the detection of all clinical H. pylori isolates tested, but no reactions were observed with negative controls. Several procedures for DNA extraction from feces were evaluated using PCR with icd primers. The lower limits of detection of H. pylori DNA from two different sources containing the same number of H. pylori organisms, a pure culture and feces spiked with H. pylori, were established for each extraction method tested. The results were 8.0 x 10(3) CFU/ml for cultures of pure H. pylori, and 8.0 x 10(6) CFU/ml for H. pylori from feces, using the phenol-chloroform method; 8.0 x 10(2) and 7.0 x 10(3) CFU/ml, respectively, for a glass matrix and chaotropic solution protocol; 8.0 x 10(2) and 7.0 x 10(3) CFU/ml, respectively, for the QIAamp tissue kit; and 5.0 x 10(2) and 5.0 x 10(3) CFU/ml, respectively, for the XTRAX DNA extraction kit. We conclude that the use of the icd gene as a primer for PCR represents a specific and sensitive assay for detection of H. pylori in highly contaminated samples.     

A time-dependent loss of retrograde transport ability in distally axotomized rubrospinal neurons

Studies on the effect of axotomy on adult intrinsic central projection neurons have generally assumed that the severed proximal axonal stumps were still capable of retrogradely transporting tracer at varying times after injury. Failure of transport was interpreted as neuronal death, which is at odds with current understanding that central projection neurons survive distal axotomy. We used lumbar spinal cord-projecting rubrospinal neurons of the rat as a model to evaluate the ability of injured neurons to transport tracer retrogradely at different times after distal axotomy. We examined only the caudal part of the red nucleus, since rubrospinal neurons are concentrated here. In control animals, tracer applied to the rubrospinal tract at the T10 vertebral level labeled ventrolateral rubral neurons, while C3 application marked all rubral neurons. From 3 days after a T10 axotomy and tracer application, most ventrolateral neurons were no longer labeled by another tracer application at the C3 vertebral level via an axonal cut. The phenomenon was not caused by tracer toxicity, since a T10 tractotomy without tracer application also prevented these axotomized neurons from being labeled when treated similarly. Thus, neuronal retrograde transport capability was seriously retarded 3 days after a distal axotomy. Loss of retrograde transport may merely suggest that a mechanism no longer in service has been switched off, or perhaps it may insulate injured neurons from the effect of lesion site-derived factors. Using this property, we were able to localize cervical spinal cord-projecting rubrospinal neurons in the caudal red nucleus. Results show that although they concentrate in the dorsomedial region, some neurons were found to extend into the ventrolateral part of the nucleus.     

37℃凝血温度下正常人和哮喘患者血清ECP水平的测定

室温（２０℃）是广泛接受的血清ＥＣＰ测定凝血温度，但存在一些问题。设想体温３７℃可能是血清ＥＣＰ测定的有效凝血温度。为此，对６８例急性发作的哮喘患者在３７℃凝血温度下血清ＥＣＰ水平进行分析，比较凝血温度分别为２０℃和３７℃时同一患者血样本的ＥＣＰ水平变化，结果发现３７℃下哮喘患者血清ＥＣＰ水平（５０．９±３．１８μｇ／Ｌ）显著高于正常对照（１７．２７±１．３６μｇ／Ｌ，Ｐ＜０．０１）。在６８例病人中，３７℃凝血下有２８人血清ＥＣＰ水平高于正常值，而２０℃凝血下只有１７人血清ＥＣＰ水平高于正常值，两者间存在显著差异（Ｐ＜０．０５）。３７℃凝血温度下血清ＥＣＰ水平与哮喘症状计分显著相关（ｒ＝０．７７，Ｐ＜０．０１）。此外，尚测定了３７℃凝血温度下１０１位１０～５０岁的健康人血清ＥＣＰ水平，结果显示３７℃下正常人血清ＥＣＰ水平的几何均数是１７．８１μｇ／Ｌ，血清ＥＣＰ水平的正常值为７０μｇ／Ｌ以下（９５％的可信限）。本研究表明，在血清ＥＣＰ的测定中设凝血温度为３７℃不仅是有效、简化的方法，而且还可减少哮喘患者血清ＥＣＰ水平的假阴性。     

Identification of Candida albicans by specific primers of polymerase chain reaction and DNA probes

Candida albicans is a pathogenic yeast. Two sets of universal primers were used for specific identification of Candida albicans with PCR-amplified ribosomal DNA internal transcribed spacers (ITS). Among the species of Candida, the amplified ITSI and ITSII of DNA fragments were similar in size. The PCR product was purified and labeled with digoxigenin and used as DNA probe in the detection with target DNA of Candida albicans by hybridization. Two sets of specific primers (CA1 and CA2 to amplify ITSI, CA3 and CA4 to amplify ITSII) were designed by alignment of ribosomal ITS sequence of pathogenic Candida albicans with other species to detect C. albicans by PCR. The sensitivity of PCR using the specific primers to detect pure culture of C. albicans was 0.1 ng (about 10(3)-10(4) cells). If the yeast cells were mixed with two other strains, there was a 10-fold decrease in sensitivity (1 ng or 10(4)-10(5) cells) under the same PCR conditions.     

SPECT findings in the Kleine-Levin syndrome

STUDY OBJECTIVES: The Kleine-Levin Syndrome, is a rare disorder with onset during teenage years, but little is known on etiopathogenesis. Seven subjects with Kleine-Levin Syndrome accumulated over time had systematic SPECT studies during (n=5) and out (n=7) of the symptomatic period. SUBJECTS: Seven boys with symptom onset between 11 and 17 years of age and at least 2 episodes per year were followed for a mean of 6 years. METHODS: Electroencephalogram awake-asleep, computed tomography scan, and magnetic resonance imaging studies were performed before Tc-99m ECD single photon emission tomography (SPECT) obtained during day 4 or 5 (n=5) and at least 1 month away from the symptomatic period (n=7). RESULTS: All imaging tests except SPECT were normal. Hypoperfusion of both thalami were seen during the symptomatic period that completely disappeared during the asymptomatic period. Hypoperfusion in other regions were also noted in some, but not all subjects. They persisted during the asymptomatic period in 2 cases over the temporal lobe (2/7 cases), frontal lobe (1/7 cases), and basal ganglia (1/7 cases). The largest amount of persistent hypoperfusion was seen in the subject with longest clinical evolution. CONCLUSION: Hypoperfusion of the thalamus is a consistent finding during the symptomatic period, but perfusion abnormalities may persist even during the asymptomatic period. The longer the duration of the syndrome, the more extended the hypoperfusion regions during the asymptomatic period.     

Cannabinoid-induced presynaptic inhibition at the primary afferent trigeminal synapse of juvenile rat brainstem slices

Systemic or intraventricular administration of cannabinoids causes analgesic effects, but relatively little is known for their cellular mechanism. Using brainstem slices with the mandibular nerve attached, we examined the effect of cannabinoids on glutamatergic transmission in superficial trigeminal caudal nucleus of juvenile rats. The exogenous cannabinoid receptor agonist WIN 55,212-2 (WIN), as well as the endogenous agonist anandamide, hyperpolarized trigeminal caudal neurones and depressed the amplitude of excitatory postsynaptic potentials (EPSPs) or currents (EPSCs) monosynaptically evoked by stimulating mandibular nerves in a concentration-dependent manner. The inhibitory action of WIN was blocked or fully reversed by the CB₁ receptor antagonist SR 141716A. WIN had no effect on the amplitude of miniature excitatory postsynaptic currents (mEPSCs) recorded in the presence of tetrodotoxin or cadmium. The inhibitory effect of WIN on EPSCs was greater for those with longer synaptic latency, suggesting that cannabinoids have a stronger effect on C-fibre EPSPs than on Aδ-fibre EPSPs. Ba²⁺ (100 μ m ) blocked the hyperpolarizing effect of cannabinoids, but did not affect their inhibitory effect on EPSPs. The N-type Ca²⁺ channel blocker ω-conotoxin GVIA (ω-CgTX) occluded the WIN-mediated presynaptic inhibition, whereas the P/Q-type Ca²⁺ channel blocker ω-agatoxin TK (ω-Aga) had no effect. These results suggest that cannabinoids preferentially activate CB₁ receptors at the nerve terminal of small-diameter primary afferent fibres. Upon activation, CB₁ receptors may selectively inhibit presynaptic N-type Ca²⁺ channels and exocytotic release machinery, thereby attenuating the transmitter release at the trigeminal nociceptive synapses.