Identification of the clonal complexes of Staphylococcus aureus strains by determination of the conservation patterns of small genomic islets.

AIMS: To investigate the clonality of Staphylococcus aureus isolates, it is important to identify their clonal complexes (CCs) with multilocus sequence typing (MLST). However, it is expensive to carry out MLST analyses for many isolates. The aim of this study, therefore, was to develop a cost-effective method to identify CCs by determining the conservation pattern of 'small genomic islets' (SGIs). SGIs are nonconserved regions between strains and have single or multiple open-reading frames (ORFs). METHODS AND RESULTS: The whole-genome sequences of nine strains were compared in order to select 16 SGIs. The conservation patterns of the 16 SGIs (islet patterns) were investigated in 136 S. aureus isolates, which were classified into 21 CCs. The islet patterns (IPs) exhibited a one-to-one correspondence with the CCs, except for isolates belonging to CC1, CC5 and CC8. The IPs typical of strains belonging to CC1, CC5 and CC8 differed between those of sequence type 1 (ST1) and ST188 (CC1), ST5 and ST6 (CC5) and ST8 and ST239 (CC8). SIGNIFICANCE AND IMPACT OF THE STUDY: The CCs of many isolates can be identified in an easy and inexpensive manner by detecting these 16 SGIs. Emergent clones, particularly methicillin-resistant ones, can be identified by examining numerous islets by IP analysis.

Magnesium(II) and zinc(II)-protoporphyrin IX's stabilize the lowest oxygen affinity state of human hemoglobin even more strongly than deoxyheme.

Studies of oxygen equilibrium properties of Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrid hemoglobins (i.e. alpha2(Fe)beta2(M) and alpha2(M)beta2(Fe); M=Mg(II), Zn(II) (neither of these closed-shell metal ions binds oxygen or carbon monoxide)) are reported along with the X-ray crystal structures of alpha2(Fe)beta2(Mg) with and without CO bound. We found that Mg(II)-Fe(II) hybrids resemble Zn(II)-Fe(II) hybrids very closely in oxygen equilibrium properties. The Fe(II)-subunits in these hybrids bind oxygen with very low affinities, and the effect of allosteric effectors, such as proton and/or inositol hexaphosphate, is relatively small. We also found a striking similarity in spectrophotometric properties between Mg(II)-Fe(II) and Zn(II)-Fe(II) hybrids, particularly, the large spectral changes that occur specifically in the metal-containing beta subunits upon the R-T transition of the hybrids. In crystals, both alpha2(Fe)beta2(Mg) and alpha2(Fe-CO)beta2(Mg) adopt the quaternary structure of deoxyhemoglobin. These results, combined with the re-evaluation of the oxygen equilibrium properties of normal hemoglobin, low-affinity mutants, and metal substituted hybrids, point to a general tendency of human hemoglobin that when the association equilibrium constant of hemoglobin for the first binding oxygen molecule (K1) approaches 0.004 mmHg(-1), the cooperativity as well as the effect of allosteric effectors is virtually abolished. This is indicative of the existence of a distinct thermodynamic state which determines the lowest oxygen affinity of human hemoglobin. Moreover, excellent agreement between the reported oxygen affinity of deoxyhemoglobin in crystals and the lowest affinity in solution leads us to propose that the classical T structure of deoxyhemoglobin in the crystals represents the lowest affinity state in solution. We also survey the oxygen equilibrium properties of various metal-substituted hybrid hemoglobins studied over the past 20 years in our laboratory. The bulk of these data are consistent with the Perutz's trigger mechanism, in that the affinity of a metal hybrid is determined by the ionic radius of the metal, and also by the steric effect of the distal ligand, if present. However, there remains a fundamental contradiction among the oxygen equilibrium properties of the beta substituted hybrid hemoglobins.

Ontogeny of the leucine-enkephalin neuron system of the rat: immunohistochemical analysis. I. Lower brainstem.

Ontogeny of the leucine-enkephalin (L-Enk) neuron system in the lower brainstem of the rat was investigated by means of indirect immunofluorescence. L-Enk-containing cells first appear in the primordium of the medullary reticular formation just medial to the n. tractus spinalis nerve trigemini at the level of the the rostral half of the inferior olivary nucleus, in the n. cuneiformis, and in the mesencephalic reticular formation of the fetus at gestational day 16 (14-15-mm embryos). From that time onward, L-Enk-containing cells appear in various areas of the lower brainstem one after another until birth. After birth, although L-Enk-containing cells decrease slightly in number as the rats grow, L-Enk-containing fibers continue to increase in number.

Ontogeny of somatostatin-containing neuron system of the rat: immunohistochemical analysis. II. Forebrain and diencephalon.

The ontogeny of the somatostatin (SRIF) neuron system in the forebrain and diencephalon the rat was investigated by means of the indirect immunofluorescence method. SRIF-positive cells first appear in the primordium of the hypothalamus surrounding the fasciculus mammillothalamicus of the fetus at gestational day 14 (10-12-mm embryo). At gestational days 16-17 (14-17-mm embryo, SRIF-positive cells appear in the developing piriform cortex and entopeduncular nucleus. The fetus at gestational days 18-19 (17-26-mm embryos) showed a remarkable increase in the number of SRIF-positive cells and numerous groups of such cells are detectable in various forebrain and diencephalic areas such as the hypothalamic periventricular zone, zona incerta, area lateral to the commissura posterior (ACP), area between the optic tract and capsula interna, pars retrolenticularis (AOR), n. caudatus putamen, hippocampus, somatic sensory cortex, and n. accumbens, etc. At gestational day 20, SRIF-positive cells newly appear in the septum, olfactory bulb, diagonal band of Broca, claustrum, lateral preoptic area, and lateral habenular nucleus. The majority of SRIF-positive structures found in the forebrain and diencephalon increase in number during the perinatal stage (between gestational day 21 and postnatal day 2) and more or less maintain their immunoreactivity even in adult rats. However, SRIF-positive cells located in the AOR, ACP, and lateral septal area, etc., begin to decrease in number during the perinatal stage and no or only a few SRIF-containing cells are identified in these areas of infant and adult rats.

Neutralizing antibodies against HTLV-I in HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients and asymptomatic carriers.

We tested serum specimens from patients with HAM/TSP and asymptomatic HTLV-I carriers from endemic areas of Japan, Jamaica, Colombia, and Chile for neutralizing antibodies against HTLV-I. The data suggest a trend for neutralizing activity to be found more frequently in the sera from HAM/TSP patients than in sera from asymptomatic carriers. The result of this study emphasizes the importance of determining biologic properties of the envelope glycoprotein of HTLV-I.

Ontogeny of neurotensin-containing neuron system of the rat: immunohistochemical analysis--II. Lower brain stem.

The ontogeny of the neurotensin neuron system in the lower brain stem of the rat was investigated by means of indirect immunofluorescent method. Neurotensin-like immunoreactivity-containing cells first appear in the primordium of the n. tractus solitarii, n. tractus spinalis nervi trigemini, reticular formation just medial to the latter nucleus, n. reticularis parvocellularis, n. laterodorsalis tegmenti, and midbrain reticular formation of the fetus at gestational day 17. At gestional day 18, neurotensin-immunoreactive cells newly appear in the n. raphe dorsalis. Between gestational day 19 and postnatal day 7, the animals show a remarkable increase in number of immunoreactive cells and fibers in various lower brain stem areas except for n. tractus spinalis nervi trigemini and n. tractus solitarii. Moreover, during this stage, neurotensin-immunoreactive cells located in the n. prepositus hypoglossi and n. vestibularis lateralis appear for the first time at birth and postnatal day 5, respectively. Since postnatal day 7, although the majority of immunoreactive cells located in the lower brain stem decrease in number as the rats grow, immunoreactive cells in the n. tractus spinalis nervi trigemini, on the contrary, increase in number from after birth until postnatal day 10, and maintain more or less their immunoreactivity even in the adult rat. In addition, neurotensin-immunoreactive cells in the nucleus of the solitary tract increase in number during the fetal period, reach the maximum content at postnatal day 7-10, and maintain their immunoreactivity even in the adult rats. Thus, the present study demonstrated that neurotensin-like immunoreactive structures appear at a very early ontogenetical stage, suggesting that neurotensin plays an important role in the development of the lower brain stem of the rat. In addition, the present study further showed that neurotensin-immuno-reactivity shows various fluctuations during the ontogeny, suggesting multiple functions of neurotensin in the central nervous system.

Ontogeny of substance P-containing neuron system of the rat: immunohistochemical analysis--I. Forebrain and upper brain stem.

The ontogeny of substance P-containing neuron system in the forebrain and upper brain stem of the rat was investigated by means of the indirect immunofluorescence technique. Substance P-positive structures first appeared in the primordium of the epithalamus and the area which surrounded the commissura posterior of the rat fetus corresponding to gestational day 14 (10-12 mm embryos). On and after gestational day 14, substance P-positive structures gradually made their appearance in various areas of the forebrain and upper brain stem. Substance P-positive structures thus continued to increase in number and in density during the fetus and perinatal stage and showed histochemically maximum content at the stage between postnatal days 5 and 15. After then, substance P-positive neurons tended to decrease in number as the rats grew, while substance P-positive fibers maintained in general their strong immunoreactivity even in the adult rats. The present study demonstrates that substance P-positive structures appear at a very early ontogenetical stage. This suggests that substance P might play an important role in the development of the forebrain and upper brain stem in addition to its neurotransmitter or neuromodulator functions.

Protective effect of recombinant murine interferon beta against mouse hepatitis virus infection.

Recombinant murine interferon beta (rMuIFN-beta) protected susceptible C57BL/6 mice against lethal mouse hepatitis virus (MHV) infection. rMuIFN-beta was life saving if it was given intraperitoneally beginning 21 h before infection and daily thereafter for 9 days, and lengthened the survival time if given from 3 h after infection. rMuIFN-beta treatment beginning 24 h after infection was ineffective. The survival rate was dose-dependent, and the 50% effective dose of rMuIFN-beta for survival was 1780 IU per day. rMuIFN-beta pretreatment inhibited virus growth completely in the brain and moderately in the liver and spleen and prevented severe hepatic lesions. rMuIFN-beta also protected beige mice and cyclophosphamide-treated mice against MHV infection, suggesting that activation of natural killer cells or T-cells by rMuIFN-beta is not critical for protection.

Suppression of infectious virus spread to the liver by foscarnet following lethal infection of acyclovir-resistant herpes simplex virus type 2 in mice.

Patients with the acquired immune deficiency syndrome (AIDS) occasionally develop hepatitis, pneumonia or esophagitis due to herpes simplex virus type 2 (HSV-2) infection. HSV hepatitis is a rare but serious complication in liver transplantation. Acyclovir-resistant HSV strains may emerge in immunocompromised patients. Following intraperitoneal inoculation, HSV-2 induces necrotizing hepatitis in mice. We studied the virus spread and mortality following intraperitoneal inoculation of HSV-2 RK (an acyclovir-resistant recombinant virus with altered thymidine kinase activity) as compared to its parent virus 8620K. Neither the 50% lethal dose (LD50) nor the average survival time was significantly different between the two strains. Parenteral acyclovir treatment was found to be effective against 8620K but not RK infection. Parenteral foscarnet treatment was effective against both RK and 8620K, and also inhibited the spread of either virus to the liver, spinal cord and brain. Peroral foscarnet administration was found to prevent the virus growth in the liver.

Suppression of infectious virus spread and corneal opacification by the combined use of recombinant interferon beta and interleukin-10 following corneal infection with herpes simplex virus-1 in mice.

The effects of Interleukin-10 (IL-10) and recombinant murine interferon-beta (rMuIFN-beta) on experimental corneal herpes simplex virus 1 (HSV-1) inoculation in BALB/c mice were examined. The mice were inoculated with the HSV-1 strain KOS at their corneas after abrasion. IL-10 was then administered topically once a day for 10 days beginning 2 days post inoculation, while rMuIFN-beta was administered once a day for 10 days beginning 1 day post inoculation. The local viral growth in the inoculated eyes and trigeminal ganglia was reduced in the rMuIFN-beta-treated mice but not in the IL-10-treated mice. In the mice treated with both rMuIFN-beta and IL-10, the degree of both the local viral growth and corneal opacification decreased. The establishment of HSV-1 latency in the trigeminal ganglia was partially prevented by rMuIFN-beta treatment but not by IL-10 treatment. The combined use of the cytokines resulted in both the suppression of viral spread and the prevention of corneal inflammation induced by HSV-1 infection.

Successful foscarnet therapy for mucocutaneous infection with herpes simplex virus in a recipient after unrelated bone marrow transplantation.

A 36-year-old Japanese man who received an unrelated bone marrow transplant (BMT) developed severe mucocutaneous infection with herpes simplex virus (HSV) type 1 during oral acyclovir prophylaxis. The lesions progressed despite treatment with intravenous acyclovir and vidarabine. The HSV isolates were sensitive acyclovir, vidarabine and foscarnet in vitro, but peripheral CD3- or CD19-positive cells were barely detectable even 4 months after transplant. A 12-day course of treatment with foscarnet led to a rapid improvement. Foscarnet therapy should be considered for all severe HSV infections following BMT, regardless of whether or not the HSV isolates are sensitive to acyclovir.

Human papillomavirus DNA in condylomata acuminata from Japanese males.

We examined condylomata acuminata from Japanese males for the presence of human papillomavirus (HPV) genomes by Southern blot hybridization. HPV 6/11-related DNA was found in 91% (32/35) of the condylomata. HPV 6a DNA was found in 40% (14/35), HPV 6c DNA in 6% (2/35), and HPV 11a DNA in 37% (13/35). HPV 6-related DNA, which had an unusual PstI-cleavage pattern, was detected in one sample. Types and subtypes of HPV DNA in the samples studied (HPV 6a, 6c, and HPV 11a DNA) were not correlated with the patients' ages nor outcomes of the disease. HPV 16 DNA was not detected in any condyloma acuminatum.

Pathways and terminal fields of the cochlearofugal somatostatin tracts of very young rats.

The cochlearofugal somatostatin (SRIF) neuron system of very young rats (between newborn and 1 week old) was investigated by means of the indirect immunofluorescence technique of Coons. Cochlear nuclei (both dorsal and ventral cochlear nuclei) of this stage contain numerous SRIF-positive cells which are scarcely found in adult rats. Based upon the experimental findings obtained by this study, the majority of SRIF-positive fibers originating from these nuclei reach the contralateral inferior colliculus via well-established cochlearo-inferior collicular tracts such as dorsal acoustic striae, intermediate acoustic striae and trapezoid body, respectively. The possible significance of SRIF in the development of the auditory system is briefly discussed.

Internal spermatic vein prostaglandins in varicocele patients.

Blood was collected from 20 patients with varicocele (15 in the infertile group and 5 in the incidental varicocele group) at the time of high ligation by insertion of a catheter into the internal spermatic vein toward the renal side. Simultaneously, blood was obtained from the antecubital vein. The levels of cortisol, prostaglandin (PG)(A + B), PGE, and PGF in these blood samples were measured. Although no differences in cortisol and PG(A + B) levels were observed between internal spermatic and peripheral vein bloods in varicocele patients examined, PGE and PGF levels were higher in internal spermatic venous bloods than in peripheral venous bloods. Differences in PGF levels between the samples of blood from the internal spermatic vein and from the peripheral vein in cases of the infertile group were larger than those of the incidental varicocele group. PGE and PGF in internal spermatic vein blood samples were correlated. The possibility that PGE and PGF, refluxed from the renal vein to the testes via the internal spermatic vein in varicocele patients, disturbs spermatogenesis is discussed.

Somatostatin-like immunoreactivity in the facial, glossopharyngeal and vagal lobes of the carp.

A large number of somatostatin (SRIF)-containing structures was demonstrated in the vagal and glossopharyngeal lobes of the carp by means of the indirect immunofluorescent method. The present study further showed that the facial lobe is devoid of these structures. These facts indicate that SRIF may have an important role in relaying viscerosensory information from the vagal and glossopharyngeal nerve but not from the facial nerve.

New somatostatin-containing sites in the diencephalon of the neonatal rat.

Using an immunohistochemical technique, the present study demonstrated several new somatostatin-containing sites in the diencephalon of neonatal rats which have not been reported in adult animals. These areas are the area just lateral to the commissura posterior, the area between the optic tract and capsula interna, pars retrolenticularis and n. peripeduncularis.

Experimental allergic encephalomyelitis in the Lewis rat. A model of predictable relapse by cyclophosphamide.

Relapse of experimental allergic encephalomyelitis (EAE) was achieved in Lewis rats by cyclophosphamide (CY). All rats, immunized with an emulsion of guinea pig spinal cord homogenate in complete Freund's adjuvant and treated with 100 mg/kg of CY 21 days postimmunization (pi), developed moderate to severe paralysis 9-14 days following CY injection. A second relapse was observed in 4 of 11 rats reinjected with CY 49 days pi. Histologically, focal mononuclear cell infiltration with or without demyelination of the white matter of the central nervous system was observed. Cyclophosphamide administration caused transient leukopenia and T-cell defect, the resolution of which coincided with relapse of clinical EAE. Lymphocyte proliferative responses to myelin basic protein (BP) and concanavalin A (Con A) and antibody titers to BP were preserved in CY-treated rats. Adoptive transfer of EAE to naive recipients with Con A-activated spleen cells from donors with CY-induced relapse was unsuccessful.

Adoptive transfer of experimental allergic encephalomyelitis with lectin-activated spleen cells. Part 2. Studies on T cell subsets and interleukin 2 production.

Experimental allergic encephalomyelitis (EAE) can be transferred by spleen cells stimulated in vitro with D-mannose-binding lectins but not with N-acetyl-D-galactosamine (GalNAc)-binding lectins. EAE could also be passively transferred by spleen cells following incubation with wheat germ agglutinin (WGA), in which case the disease transfer was abolished by the specific hapten inhibitor, N-acetyl-D-glucosamine. In the presence of rat T cell monoclonal antibody, either W3/25 or OX-8, both concanavalin A and Wisteria floribunda agglutinin stimulated helper and suppressor T subpopulations. On the other hand, GalNAc-binding lectins were less effective than D-mannose-binding lectins in generating interleukin 2 (IL2) in the culture supernatant, whereas WGA-stimulated spleen cells did not produce IL2. Furthermore, spleen cells cultured with pure IL2 could not transfer EAE to the recipients. These data suggest that some factors distinct from IL2 are required for the differentiation of EAE-effector precursors into the final effector cells in this transfer system.

Position of immobilization after dislocation of the glenohumeral joint. A study with use of magnetic resonance imaging.

BACKGROUND: Glenohumeral dislocations often recur, probably because a Bankart lesion does not heal sufficiently during the period of immobilization. Using magnetic resonance imaging, we assessed the position of the Bankart lesion, with the arm in internal and external rotation, in shoulders that had had a dislocation. METHODS: Coaptation of a Bankart lesion was examined with use of magnetic resonance imaging, with the arm held at the side of the trunk and positioned first in internal rotation (mean, 29 degrees) and then in external rotation (mean, 35 degrees), in nineteen shoulders. Six shoulders (six patients) had had an initial anterior dislocation, and thirteen shoulders (twelve patients) had had recurrent anterior dislocation. Fast-spin-echo T2-weighted axial images were made when the dislocation had occurred less than two weeks earlier, and spin-echo T1-weighted axial images after intra-articular injection of gadolinium-diethylenetriamine pentaacetic acid were made when the dislocation had occurred more than two weeks earlier. Separation and displacement of the anteroinferior portion of the labrum from the glenoid rim were measured on the axial images, and coaptation of the anterior part of the capsule to the glenoid neck was assessed by measurement of the detached area, opening angle, and detached length. RESULTS: Separation and displacement of the labrum were both significantly less (p = 0.0047 and p = 0.0017, respectively) when the arm was in external rotation than when it was in internal rotation. The detached area and the opening angle of the anteroinferior portion of the capsule were both significantly smaller (p = 0.0003 and p < 0.0001, respectively), and the detached length was significantly shorter (p < 0.0001) with the arm in external rotation. CONCLUSION: Immobilization of the arm in external rotation better approximates the Bankart lesion to the glenoid neck than does the conventional position of internal rotation.

Isokinetic strength after tears of the supraspinatus tendon.

We measured the isokinetic strength of abduction, adduction, internal rotation, and external rotation in ten patients with full-thickness tears of the supraspinatus and ten with partial-thickness tears. The measurements were repeated after intra-articular or intrabursal injection of local anaesthetic. Pain blocks produced significant increases in strength in both full and partial-thickness tears. After the block, the strength in full-thickness tears compared with the opposite side was 67% to 81% in abduction and 67% to 78% in external rotation, both significantly smaller than those on the uninvolved side (p = 0.0064, p = 0.0170). In partial-thickness tears the strength after the block ranged from 82% to 111%, with no significant differences between the involved and uninvolved sides. The decreases in strength of 19% to 33% in abduction and 22% to 33% in external rotation after full-thickness tears appear to represent the contribution of supraspinatus to the strength of the shoulder.