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Myxoma virus is a pathogenic poxvirus that induces a lethal myxomatosis disease profile in European rabbits, which is characterized by fulminating lesions at the primary site of inoculation, rapid dissemination to secondary internal organs and peripheral external sites, and supervening gram-negative bacterial infection. Here we describe the role of a novel myxoma virus protein encoded by the M-T5 open reading frame during pathogenesis. The myxoma virus M-T5 protein possesses no significant sequence homology to nonviral proteins but is a member of a larger poxviral superfamily designated host range proteins. An M-T5- mutant virus was constructed by disruption of both copies of the M-T5 gene followed by insertion of the selectable marker p7.5Ecogpt. Although the M-T5- deletion mutant replicated with wild-type kinetics in rabbit fibroblasts, infection of a rabbit CD4+ T-cell line (RL5) with the myxoma virus M-T5- mutant virus resulted in the rapid and complete cessation of both host and viral protein synthesis, accompanied by the manifestation of all the classical features of programmed cell death. Infection of primary rabbit peripheral mononuclear cells with the myxoma virus M-T5-mutant virus resulted in the apoptotic death of nonadherent lymphocytes but not adherent monocytes. Within the European rabbit, disruption of the M-T5 open reading frame caused a dramatic attenuation of the rapidly lethal myxomatosis infection, and none of the infected rabbits displayed any of the characteristic features of myxomatosis. The two most significant histological observations in rabbits infected with the M-T5-mutant virus were (i) the lack of progression of the infection past the primary site of inoculation, coupled with the establishment of a rapid and effective inflammatory reaction, and (ii) the inability of the virus to initiate a cellular reaction within secondary immune organs. We conclude that M-T5 functions as a critical virulence factor by allowing productive infection of immune cells such as peripheral lymphocytes, thus facilitating virus dissemination to secondary tissue sites via the lymphatic channels.

Myxoma virus is a leporipoxvirus that causes a highly lethal virulent disease known as myxomatosis in the European rabbit. An important aspect of myxoma virus pathogenesis is the ability of the virus to productively infect lymphocytes and spread to secondary sites via lymphatic channels. We investigated the infection of the CD4+ T lymphoma cell line RL-5 with myxoma virus and Shope fibroma virus, a related but benign leporipoxvirus, and observed that myxoma virus, but not Shope fibroma virus, was able to productively infect RL-5 cells. We also discovered that infection of RL-5 cells with Shope fibroma virus or attenuated myxoma virus mutants containing disruptions in either the T2 or the M11L gene resulted in the rapid induction of DNA fragmentation, followed by morphological changes and loss in cell integrity characteristic of cell death by apoptosis. Purified exogenous T2 protein was unable to prevent apoptosis, suggesting that T2 functions intracellularly. Thus, myxoma virus T2, originally described as a secreted homologue of the tumor necrosis factor receptor, and M11L, a novel transmembrane species with no known cellular homologue, function to extend virus host range for replication in rabbit T lymphocytes through the inhibition of apoptosis in infected T lymphocytes.

Previous studies have shown that Streptococcus mutans NG8 possesses an endogenous surface protein-releasing enzyme (SPRE) activity that liberates its own surface proteins (S. F. Lee, Infect. Immun. 60:4032-4039, 1992). The present study was initiated to investigate the possible role of the release of surface proteins by SPRE in the detachment of biofilm cells in vitro. Initially, the characteristics of surface protein release by the strain (S. mutans BM71) used in this study were shown to be the same as those previously described for S. mutans NG8. BM71 displayed characteristics identical to those of NG8 in terms of pH optima and inhibitor sensitivity for protein release. Monolayer biofilms of S. mutans BM71 were formed on hydroxylapatite rods in a modified chemostat. Detachment of the biofilm cells was measured by viable cell counts of bacteria liberated after incubation of the biofilms in buffers. Results showed that biofilm cells were detached in a pH- dependent manner with a maximum rate of pH 5 (P = 0.016) to 6 (P = 0.002), a range similar to that for optimal surface protein release. The detachment of the biofilm cells was found to be inhibited by ZnCl2 (P = 0.002 to 0.023), which also inhibited surface protein release. Detachment was not inhibited significantly by CaCl2 (P = 0.525 to 0.784), precluding an ionic effect on inhibition by ZnCl2. The extent of detachment could be increased (P = 0.046) by the addition of an SPRE preparation from S. mutans but not heat-inactivated SPRE (P = 0.665) or SPRE in the presence of ZnCl2 (P = 0.199). Detachment was also studied by using biofilms of resting (viable but not dividing) cells. Results similar to those for biofilms formed from growing cells were obtained, indicating that cells detached from biofilms were not daughter cells. The results presented above show that monolayer biofilm cells of S. mutans under conditions of minimal shear force have the ability to detach from a surface and suggest that this detachment was mediated by an endogenous SPRE activity.

The site of action of hydantocidin was probed using Arabidopsis thaliana plants growing on agar plates. Herbicidal effects were reversed when the agar medium was supplemented with AMP, but not IMP or GMP, suggesting that hydantocidin blocked the two-step conversion of IMP to AMP in the de novo purine biosynthesis pathway. Hydantocidin itself did not inhibit adenylosuccinate synthetase or adenylosuccinate lyase isolated from Zea mays. However, a phosphorylated derivative of hydantocidin, N-acetyl-5'-phosphohydantocidin, was a potent inhibitor of the synthetase but not of the lyase. These results identify the site of action of hydantocidin and establish adenylosuccinate synthetase as an herbicide target of commercial potential.

The C terminus of the major surface protein P1 from Streptococcus mutans is composed of a hydrophilic domain, an LPNTGV motif, a hydrophobic domain, and a charged tail. These features are shared by surface proteins from many gram-positive coccal bacteria. To investigate the role of the C-terminal domains in antigen P1 surface localization, full-length and truncated P1 gene constructs, which were expressed on the shuttle vector pDL276, were transformed into the P1-negative mutant S. mutans SM3352, Streptococcus gordonii DL-1, and Enterococcus faecalis UV202. Transformants were tested for expression of P1 by enzyme-linked immunosorbent assaying and Western blotting. The results showed that full-length P1 was expressed by transformants of all three bacteria and was localized on the cell surface. A fusion protein composed of the Staphylococcus aureus fibronectin binding protein C terminus and the P1 protein N terminus was found to surface localize in S. mutans. Deletion of the entire C-terminal domains resulted in P1 being expressed in the culture supernatant. A P1 truncation, which carried only the hydrophilic domain at its C terminus, was found partially associated with the cell surface. This truncated P1 was readily removed from the isolated cell wall by hot sodium dodecyl sulfate-mercaptoethanol extraction. In contrast, the full-length P1 remained associated with the isolated cell wall after similar treatment, suggesting covalent linkages between the full-length P1 and the cell wall. The results described above showed that antigen P1 was anchored to the cell wall by its C-terminal domains probably via covalent linkages with the cell wall. The results also support a universal mechanism involving the C-terminal domains for protein surface localization among this group of gram-positive bacteria.

The mechanisms of signal transduction of c-jun induction by hydrogen peroxide are elucidated in NIH3T3 cells by using trapping agents of hydroxyl free radical or inhibitors of various protein kinases. Pre-treatment of the cell with hydroxyl radical scavenger dimethyl sulfoxide (DMSO) abolishes the H2O2-induced c-jun expression. Hydroxyl radical generation can be detected and quantified in cells treated sequentially with DMSO and H2O2 for 30 min respectively by methane sulfinic acid (MSA) production, especially that from particulate fraction. Induction of c-jun by H2O2 is also dramatically reduced by pretreating the cells with biological antioxidant vit. E. Protein tyrosine kinase activity of membrane fraction is induced by H2O2 within 5 to 10 min, which can be prevented by DMSO pre-treatment. Inhibitor of non-receptor type tyrosine kinase, herbimycin A, has inhibitory effect on H2O2-induced c-jun expression while the inhibitor of receptor type tyrosine kinase, tyrphostin 23 or inhibitor of cyclic AMP dependent protein kinase, KT 5720, has not. TPA pre-treatment that depletes protein kinase C (PKC) has no influence on the c-jun induction by H2O2. Our results suggest that the highly reactive species HO is generated after H2O2 enter cells and mediate the signal responses of H2O2 including c-jun induction and the activation of p60src tyrosine kinase might be one of the molecular events associated with the c-jun induction pathway.

Myxoma virus is a pathogenic poxvirus that induces extensive dysregulation of cellular immunity in infected European rabbits. Infection of a rabbit CD4+ T-cell line (RL-5) with myxoma virus results in dramatic reductions of cell surface levels of CD4 as monitored by flow cytometry. The virus-induced downregulation of CD4 requires early but not late viral gene expression and could not be inhibited by staurosporine, an inhibitor of protein kinase C, which effectively blocks phorbol 12-myristate-13-acetate-induced downregulation of CD4. The decrease in total cellular levels of CD4 during myxoma virus infection could be inhibited by the lysosomotrophic agent NH4Cl, suggesting a lysosomal fate for CD4 during myxoma virus infection. Steady-state levels of the CD4-associated protein tyrosine kinase p56lck remained unchanged during myxoma virus infection, suggesting that p56lck dissociates from CD4 prior to CD4 degradation in virus infected cells. Total p56lck kinase activity was unaffected during myxoma virus infection, although the amount of p56lck physically associated with CD4 declined in parallel with the loss of CD4. Thus, myxoma virus infection of CD4+ T lymphocytes triggers CD4 downregulation via a protein kinase C-independent pathway, causing the dissociation of p56lck and the degradation of CD4 in lysosomal vesicles.

Previous studies have shown that Streptococcus mutants is capable of releasing many surface protein antigens, particularly antigen P1. Antigen P1 is immunodominant and has been implicated in adherence of S. mutants to the acquired pellicles. The purpose of this study is to investigate the significance of release of this antigen by the cells. S. mutants NG8 (serotype c) was incubated with an anti-P1 rabbit immunoglobulin G (IgG) or a human colostral IgA which contains natural anti-P1 activity. Results indicated that the bound antibodies were released by the cells in a pH- and time-dependent manner. The optimal pH for release was between 6 and 8, and the release rate reached a plateau in 1 h at 37 degrees C. The release of bound antibodies was considered an active process, since heat-killed cells remained capable of antibody binding but failed to release the antibodies. The release was also dependent on the age of the culture, with early-exponential-phase cells releasing the maximum amount of bound IgG. The released IgG was isolated by polyethylene glycol precipitation and protein A-Sepharose column chromatography and found to be associated with antigen P1, indicating that the antibodies were released together with the antigen in the form of immune complexes. The binding of S. mutans by secretory IgA (SIgA) inhibited the adherence of the cells to salivary agglutinin-coated hydroxylapatite. However, when the SIgA-coated S. mutans was allowed to release the bound antibodies, the inhibitory effect of SIgA on adherence was abrogated. These results suggest that S. mutans is capable of shedding surface-bound antibodies in the form of antibody-antigen immune complexes. Such an action may be a strategy employed by the cells to counter the neutralizing effect of naturally occurring antibodies in the oral cavity.

Mutants of Bacillus thuringiensis subsp. kurstaki NTU 9 and Bt 158, which were isolated previously for using the diamondback moth as a target insect in Taiwan, were screening by either protein electrophoresis of intracellular proteins or enzyme-linked immunosorbent assay (ELISA). The optimal conditions of effective protein electrophoresis were (1) 24-hour cells harvested from nutrient broth were crashed by petite glass beads followed by centrifugation. And (2) the supernatant pretreated by heating at 60 degrees C for 2 minutes was electrophoresed with 7.5% native PAGE at 110 voltages. On ELISA, the antiserum used was obtained from rabbits immunized with Bt 158 crystal protein. Optimal antigen coating concentration of ELISA, attained by chequer-board titration method, was 10 micrograms/ml. Antigens (crystal protein) in samples were detected by competitive inhibition method with antiserum diluted to 10(4) fold. By using protein electrophoresis and ELISA methods, two isolates A 71 and BN 11, were denoted respectively as qualitative and quantitative mutants of Bacillus thuringiensis.

The role of nerve growth factor (NGF) in the modulation of herpes simplex virus (HSV) latency and reactivation was investigated in a mouse model. To determine whether NGF depletion would reactivate latent virus, concentrated anti-NGF serum antibodies were administered intraperitoneally to latently infected mice for 9 consecutive days. To determine whether NGF given prophylactically could suppress UV-B-induced viral reactivation, mice were irradiated with UV-B while being treated with NGF using diverse regimes over a 4-day period. Following intraperitoneal administration of anti-NGF antibodies, viral shedding was detected in a small number (10%) of mice, but it was not possible to pharmacologically suppress UV-B-induced viral reactivation with NGF. It would appear, therefore, that HSV latency in neurons innervating the cornea can be sustained and disrupted by physiological factors independent of NGF levels.

A patient who used contact lenses and had a history of blunt trauma developed vaccinia keratouveitis after accidental ocular autoinoculation from a recent vaccination site. Corneal and conjunctival cultures were taken for bacteria, fungi, Acanthamoeba, and viruses. Viral-like cytopathic effects became evident in tissue culture within three days. Immunofluorescence studies were negative for varicella-zoster virus, herpes simplex virus, adenovirus, measles, mumps, parainfluenza, and influenza. Pox viral particles were identified in the infected tissue cultures by electron microscopy. The Hind III restriction endonuclease profile of the viral DNA isolate was similar to the Lister strain of vaccinia virus. Ocular vaccinia may manifest as a masquerade syndrome and may mimic signs of herpes simplex virus, varicella-zoster virus, and Acanthamoeba infection. Although vaccination with vaccinia is currently limited to a few populations throughout the world, vaccinia must still be considered in the differential diagnosis of infectious keratouveitis.

Benzene is strongly suspected of being an animal and human carcinogen, but the mechanisms by which it induces tumors of lymphoid and hematopoietic organs are unknown. Production of active oxygen species from benzene metabolites [hydroquinone (HQ), catechol and 1,2,4-benzenetriol (1,2,4-BT) and related polyphenols (resorcinol, pyrogallol and phloroglucinol)] are investigated. Pyrogallol and 1,2,4-BT can produce H(2)O(2), O(-)(2) and (.)OH simultaneously, and have powerful mutagenic potential. Resorcinol and phloroglucinol cannot produce all of the active oxygen species, and show no mutagenic effects. Catechol can produce H(2)O(2), but cannot produce O(-)(2) and (.)OH, and has no mutagenic activity. These data strongly support the hypothesis that benzene metabolites can cause mutagenicity via the generation of oxygen radicals. Although HQ produces H(2)O(2) only, and less than produced by pyrogallol and 1,2,4-BT, the mutagenicity of HQ is higher. The results indicate that HQ may act via another mechanism to cause mutagenicity. In the presence of trace metal ions, the reactivity of polyphenols is increased. The biological significance of these phenomena are investigated and discussed. Copyright 1994 S. Karger AG, Basel

Using ion exchange chromatography and an ATP-dependent actin precipitation step, we have isolated three myosin-I isozymes that, together, account for most of the K+EDTA-ATPase activity recovered from extracts of Dictyostelium. The two major myosin-I isozymes, present in approximately equal amounts, had apparent molecular masses of 125 kDa on SDS gels and have been identified by amino acid sequence analysis as the products of the Dictyostelium myosin-IB (DMIB) and myosin-ID (DMID) genes. DMIB, with a specific K+EDTA-ATPase activity 10-fold higher than DMID, was responsible for most of the activity in cell extracts. The third isozyme, present in low amounts, had an apparent molecular mass of 137 kDa on SDS gels and is too large to be the product of any of the known myosin-I genes. DMIB eluted from DE53 cellulose columns as two distinct peaks (II and III). Addition of the phosphatase inhibitor okadaic acid to the extraction buffer increased the fraction of DMIB recovered from growth phase cells in peak III from 35 to 70%. DMIB isolated from peak III, but not from peak II, displayed a significant level of actin-activated MgATPase activity. These results indicate that peak III represents a phosphorylated, actin-activatable form of DMIB.

Purpose: The purpose of this study was to determine whether herpes simplex virus (HSV) transcripts are present in the corneas of patients with chronic herpetic stromal keratitis., Methods: Corneal buttons from patients with a history of stromal keratitis, but no ongoing active disease, together with positive and negative control tissues, were analyzed by in situ hybridization using single-stranded RNA probes for all three classes of viral lytic cycle transcripts as well as for the latency-associated transcripts (LATs). Tissues also were screened for presence of HSV genomic DNA using the polymerase chain reaction (PCR)., Results: HSV DNA was detected in 7 of 13 quiescent corneas by PCR, but no viral transcripts were detected in any of these corneas by in situ hybridization., Conclusions: At the level of detection afforded by in situ hybridization, HSV persistent in scarred human corneas after stromal keratitis appears to be transcriptionally dormant. This contrasts with the situation in neurons of latently infected sensory ganglia, in which LATs are present at high levels.

Surface proteins of Streptococcus mutans have been reported to be released into the culture filtrate at concentrations that vary with the growth conditions. The reason for this is not clear. The present study attempts to investigate the mechanism of the protein release. The results showed that whole cells and raffinose-stabilized protoplasts of S. mutans NG8, when incubated in buffers, were capable of releasing their surface proteins in a pH-dependent manner with optimal release at pH 5 to 6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the released proteins were very complex. Two proteins, adhesin P1, which has been previously shown to interact with a human salivary agglutinin, and glucosyltransferase have been identified among the released proteins. The release of adhesin P1 and other proteins was found to be inhibited by heat, Cu2+,Zn2+, and thiol-blocking reagents. The inhibition by heat and Cu2+ was irreversible, whereas that by the thiol-blocking reagents was reversible. EDTA, phenylmethylsulfonyl fluoride, and N-p-tosyl-L-lysyl-chloromethyl ketone had no effect on the release of P1, indicating that the release was probably not due to proteolytic activity. Adhesin P1 from Cu(2+)-inactivated S. mutans NG8 protoplasts could be released by mixing with fresh whole cells and protoplasts, but not the culture filtrate, of a P1-negative mutant of NG8, suggesting that the enzyme is located on the cell surface. This P1-releasing activity was also detected in two other strains of S. mutans and one strain each of S. gordonii, S. agalactiae, S. pneumoniae, and S. pyogenes. The biological role(s) of this enzyme activity remains to be determined. However, owing to its ability to release virulent surface proteins from the cell, it may play an important role in cell surface modulation among the pathogenic streptococci.

Fibrobacter succinogenes is an important cellulolytic bacterium found in the rumen and cecum of herbivores. Numerous attempts to introduce foreign DNA into F. succinogenes S85 have failed, suggesting the presence of genetic barriers in this organism. Results from this study clearly demonstrate that F. succinogenes S85 possesses a type II restriction endonuclease, FsuI, which recognizes the sequence 5'-GG(A/T)CC-3'. Analysis of the restriction products on sequencing gels showed that FsuI cleaves between the two deoxyguanosine residues, yielding a 3-base 5' protruding end. These data demonstrate that FsuI is an isoschizomer of AvaII. A methyltransferase activity has been identified in the cell extract of F. succinogenes S85. This activity modified DNA in vitro and protected the DNA from the restriction by FsuI and AvaII. DNA modified in vivo by a cloned methylase gene, which codes for M.Eco47II, also protected the DNA from restriction by FsuI, suggesting that FsuI is inhibited by methylation at one or both deoxycytosine residues of the recognition sequence. The methyltransferase activity in F. succinogenes S85 is likely modifying the same deoxycytosine residues, but the exact site(s) is unknown. A highly active DNase (DNase A) was also isolated from the cell extract of this organism. DNase A is an endonuclease which showed high activity on all forms of DNA (single stranded, double-stranded, linear, and circular) but no activity on RNA. In vitro, the DNase A hydrolyzed F. succinogenes S85 DNA extensively, indicating the lack of protection against hydrolysis by this enzyme. In the presence of Mg2+, DNA was hydrolyzed to fragments of 8 to 10 nucleotides in length. The presence of DNase A and the type II restriction-modification system of F. succinogenes S85 may be the barriers preventing the introduction of foreign DNA into this bacterium.

Although radionuclide hysterosalpingography (RNHSG) has been suggested as an efficient procedure for assessing function of fallopian tubes, the radiation dose to the ovaries was addressed as an important issue to be taken into consideration. We describe a modified method of RNHSG, calculating the radiation dose to the ovaries. A small dose of approximately 18.5 MBq (0.5 mCi) of [99mTc]pertechnetate was administered directly into the uterine cavity without overpressure. The accuracy of the method was 84.5% as compared with the contrast hysterosalpingography. The estimated average dose to the ovaries was 0.057 mGy/MBq (0.21 rad/mCi) or 1.08 mGy (108 mrad) per study. RNHSG is an accurate method for functional study of fallopian tube patency with low radiation dose.

Age-related senescence of T-cell mediated responses is well recognized. This study was designed to determine how aging affects the T-cell mediated Interleukin 2 (IL2) response to influenza vaccination. A group of healthy elderly individuals were compared to a control group of healthy young adults for their response to the 1990 influenza vaccine. Cultures of peripheral blood mononuclear cells (PBMC) were prepared from venous blood samples taken prevaccination (pre) and 8 and 12 weeks post-vaccination (post). PBMC cultures stimulated with inactivated A/Shanghai/16/89 (contained in the 1990 vaccine) and A/Philippine/2/82 (not contained in the vaccine) were assayed for peak IL2 activity. We find that after influenza vaccination, there was an insignificant increase in IL2 activity when PBMC from the young control group were stimulated with A/Shanghai/16/89 (pre, 5.14 U/mL/10(6) PBMC; post, 6.64 U/mL/10(6) PBMC) but there was a significant increase in IL2 activity when stimulated with A/Phillippine/2/82 (pre, 1.5 U/mL/10(6) PBMC; post, 8.3 U/mL/10(6) PBMC). In similar cultures of PBMC from the elderly group, there was a significant increase in IL2 response to both A/Shanghai/16/89 (pre, 1.6 U/mL/10(6) PBMC; post, 3.5 U/mL/10(6) PBMC) and A/Philippine/2/82 (pre, 0.86 U/mL/10(6) PBMC; post, 8.3 U/mL/10(6) PBMC). Measurements of CD4+/CD8+ populations were not affected by vaccination and were not significantly different in the two groups. Subgroup analysis of the elderly group revealed that previous influenza vaccination in 1989 did not significantly affect IL2 levels measured in the present study. This study shows that in healthy elderly, influenza vaccination effectively restores IL2 activity to normal. There appears to be an age-related decrease in the duration of T-cell memory.(ABSTRACT TRUNCATED AT 250 WORDS)

Insertional inactivation of the Streptococcus mutans spaP gene was used to construct an isogenic mutant (834) of strain NG8 (serotype c) which lacked the major cell surface-associated protein referred to as P1 (15). Results of several studies suggest that P1 is involved in the adherence of S. mutans to saliva-coated apatite surfaces. With an in vitro model system of hydroxyapatite (HA) beads coated with parotid saliva (PS) and additional HA surfaces coated with PS and in situ-formed glucan, it was observed that mutant 834 adhered poorly to the PS/HA surfaces. In contrast, both parent and mutant strains bound to the PS-glucan/HA surface. Groups of intact and desalivated rats were infected with each strain to determine relative capacities to induce dental caries. Rats were fed a highly cariogenic diet containing 56% sucrose for 3 to 5 weeks. Each strain colonized the rodent model and caused similar levels of smooth-surface caries under these dietary conditions. It was concluded that P1 influences the ability of organisms to adhere to saliva-coated surfaces and possibly affects primary colonization of the oral cavity in the absence of a glucan surface but has no effect on glucan-mediated adherence in vitro or in vivo.

The authors characterized a murine model of herpes simplex virus (HSV) reactivation in which recurrent herpetic keratitis was obtained in up to 80% of animals. Five weeks after ganglionic latency was established in National Institutes of Health inbred mice after corneal inoculation, HSV type 1 (HSV-1) was reactivated by irradiating the previously inoculated eye with ultraviolet (UV) light. Comparison of different UV wavelengths showed UVB to be optimal for reactivation, with peak viral recurrence being induced by a total exposure of approximately 250 mJ/cm2. Reactivated infectious virus generally began to appear in trigeminal ganglia 2 days postirradiation and was subsequently detectable in the cornea by both corneal swabbing and immunostaining for viral antigens. Two consecutive outbreaks of viral recurrence at the ocular surface were induced in selected animals by serial exposure to UVB. Advantages of this model over other models of recurrent keratitis are discussed.

The Knops/McCoy (Kn/McC) human erythrocyte blood group system belongs to the category of blood group Ag that generate so-called "high titer low avidity" antibodies in immunized transfusion recipients. Screening of red cells lacking certain high titer low avidity Ag demonstrated markedly diminished CR1 expression on McC(d-) and Kn/McC "null" (Kn(a-)McC(a-b-c-d-e-f-] erythrocytes. Additional testing by other methods confirmed these data, and biochemical assays demonstrated no detectable immunoreactive CR1 protein in membranes from Kn/McC null red cells. Human antisera to various Kn/McC Ag were then used to demonstrate that many of these antisera could be used to isolate a protein of identical m.w. to that isolated from the same cells using murine mAb CR1 antisera. Finally, protein isolated by using murine mAb anti-CR1 reacted specifically with anti-Kn/McC antibodies, demonstrating the identity of the Kn/McC and CR1 proteins. Thus, CR1 protein bears the human erythrocyte Kn/McC blood group Ag.

A restriction fragment length polymorphism study was undertaken to determine the extent and location of heterogeneity within spaP encoding the Mr 185,000 cell surface protein P1 (antigen I/II) of Streptococcus mutans serotype c isolates. The gene was found to be highly conserved except for a central variable (V) region predicted to encode less than 150 amino acids. Sequence analysis identified two V-region variants. These differences were independent of the geographic source of the isolates. Southern analysis using synthetic oligonucleotide probes indicated that nonretention of P1 (I/II) by some isolates is not due to a deletion of the 3'-terminal DNA necessary to encode an intact carboxy terminus.

Three methods, i.e., nonspecific radioimmunoassay (RIA; Incstar), fluorescence polarization immunoassay (FPIA; TDx Abbott), and high-performance liquid chromatography (HPLC), have been used for monitoring cyclosporine blood levels in renal transplantation patients. The levels obtained from 135 samples showed a modest correlation between RIA and HPLC, FPIA and HPLC, RIA and FRIA. The mean ratios of RIA to HPLC, FPIA to HPLC, and RIA to FPIA were 2.96, 4.14, and 0.73. The significant variations in cyclosporine levels result from the cross-reaction of antibody with some cyclosporine metabolites, by which these two methods often overestimate the true blood cyclosporine level. HPLC is a more effective and reliable method for pharmacokinetic studies and blood level monitoring of cyclosporine in clinical practice.

Pyridostigmine bromide (Pyr), the current drug of choice in the management of myasthenia gravis, has been suggested for use in Alzheimer's dementia, and as a prophylactic treatment for intoxication with organophosphate cholinesterase inhibitors. The present study was undertaken to evaluate the dose-response and time-course effects of acute oral administration of Pyr over a broad dose range (3-40 mg/kg) on the lever pressing of rats maintained under a multiple fixed-ratio (FR-20) time-out schedule of reinforcement for water reward. The drug produced a dose-dependent biphasic response depression in the overall rate of FR responding. Low doses of Pyr (less than or equal to 12 mg/kg) that caused no gross signs of toxicity only moderately decreased rates of responding, primarily due to a decrease in response rates. Whereas high doses of Pyr (greater than 24 mg/kg) which produced overt signs of peripheral cholinergic intoxication markedly suppressed overall responding, primarily due to cessation of responding. The lowest effective dose of performance disruption was 6 mg/kg, and the ED50 was calculated as 23.3 (17.9-28.7) mg/kg. The time-course data of performance disruption showed that low doses of Pyr (less than or equal to 12 mg/kg) had an onset latency within 40-80 min and a duration of 20-80 min, whereas high doses (greater than or equal to 24 mg/kg) had an onset latency of 20-40 min and a duration greater than 80 min. These results suggest the recommended human therapeutic or prophylactic regimen of 30-120.mg Pyr, orally taken each 8 hours, might adversely affect behavioral performance.

Fibrobacter succinogenes S85 is unable to grow with lactose as the source of carbohydrate, although it does exhibit low beta-galactosidase (EC 3.2.1.23) activity. Spontaneous mutants of strain S85 able to grow on lactose were isolated after spreading cells on a chemically defined agar medium with lactose as the carbohydrate source. A lactose-catabolizing isolate, designated L2, exhibited a sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profile and an immunoblot profile with polyclonal antibodies to whole cells of S85 which were identical to those observed for S85. Strain L2 exhibited both cell-associated and extracellular beta-galactosidase activity with either p-nitrophenyl-beta-D-galactopyranoside or lactose as the substrate. The cell-associated enzyme exhibited the greatest activity in the periplasmic space. Enzyme production was partially inhibited by glucose. The beta-galactosidase was activated by divalent cations and exhibited a pH optimum of 6.5. Analysis of the extracellular culture fluid revealed that glucose derived from the hydrolysis of lactose was used for growth, but galactose was not metabolized further. Cells were unable to take up the lactose analog, methyl-beta-D-thiogalactopyranoside. These data suggest that beta-galactosidase of F. succinogenes L2 cleaves lactose outside the cells and that the glucose released is catabolized while the galactose accumulates in the extracellular culture fluid.

The initial step in the purification of Dictyostelium myosin II heavy chain kinase A (MHCK A) is chromatography over phosphocellulose. Fractions containing MHCK A are pooled and chromatographed over a Mono Q column (Pharmacia LKB Biotechnology) equilibrated in 0.15 M KCl. Under these conditions MHCK A and most of the contaminating proteins elute in the flowthrough. The addition of Mg2+ and ATP to the Mono Q flowthrough results in the phosphorylation, within 15 min, of MHCK A to a level of 10 mol of phosphate per mole of 130-kDa kinase subunit. The hyperphosphorylated MHCK A binds to Mono Q columns in the presence of 0.15 M KCl and can be eluted, as a single homogeneous band, by a salt gradient to 0.35 M KCl. A similar purification procedure may prove useful for other proteins which can be highly phosphorylated. Hyperphosphorylation is shown to have no effect on the position at which MHCK A elutes from gel filtration columns (apparent M(r) greater than 700,000).

Freshly frozen conjunctival tissue from premenopausal and postmenopausal women and male subjects were processed for estrogen and progesterone receptors by using monoclonal antibodies and a peroxidase-antiperoxidase technique. No immunocytochemical staining was localized in the nuclei of the cells treated with the monoclonal antibodies to human estrogen receptor or human progesterone receptor in any of the conjunctival specimens, in contrast to the strongly positive staining in breast adenocarcinoma controls. Immunocytochemical staining disclosed no evidence for estrogen or progesterone receptors on cells of the ocular surface.

We compared two commercially available tests, a direct immunofluorescence assay and an enzyme-linked immunosorbent assay (ELISA), to viral isolation in tissue culture for the laboratory diagnosis of untreated and partially treated experimental herpes simplex virus keratitis. New Zealand albino rabbits were inoculated bilaterally with herpes simplex virus-1 McKrae strain after corneal scarification. One eye of each rabbit was treated with a 1% trifluorothymidine solution daily, starting on the third day after inoculation. The direct immunofluorescence assay showed lower sensitivity for herpes simplex virus detection than viral isolation in tissue culture for both untreated and partially treated eyes. The Herpchek ELISA demonstrated similar sensitivity to tissue culture in detecting herpes simplex virus in untreated eyes. In the treated group, however, the Herpchek ELISA showed a higher percentage of eyes positive for herpes simplex virus than did viral isolation in tissue culture. After the initiation of antiviral therapy, eyes that no longer harbor infectious virus that can be isolated in tissue culture may remain herpes simplex virus antigen-positive and thus be more amenable to laboratory diagnosis using the rapid ELISA method.

Reticulocyte counting using flow cytometry and a membrane-permeable fluorescent dye, thiazole orange, was evaluated as an alternative to the conventional manual method. The flow cytometric method was more precise (mean c.v. of 4.3%) than the imprecise manual technique (mean c.v. of 22.4%). Linearity was highly acceptable (r = 0.99) over the reticulocyte count range of 1.8-30.1%. Flow cytometry allows processing of large numbers of samples with reduction in technologist time, and the improved precision and tenfold increase in the number of cells counted should considerably improve the clinical utility of the reticulocyte count.

The gene spa P (formerly designated as spa P1) encoding the Mr 185,000 surface antigen I/II of Streptococcus mutans, serotype c (strain NG5) has been sequenced. The deduced amino acid sequence of antigen I/II (1561 residues) includes a putative signal peptide (residues 1-38), as well as a transmembrane region (residues 1537-1556). The N-terminal part of the protein (residues 39-550) is particularly rich in alanine and includes three tandem repeats of a sequence of 82 residues. This region is predicted to be alpha-helical, adopting a coiled-coil formation, and may account for the cell surface hydrophobicity associated with expression of antigen I/II. In contrast the C-terminal region (residues 800-1549) is proline-rich, favouring an extended conformation. Comparison with the sequence determined from Strep. mutans strain MT8148 showed that antigen I/II is highly conserved with the exception of a short central region (residues 750-805). N-terminal sequencing of purified antigens I and II components indicated that antigen I extends from the amino-terminus of the intact Mr 185,000 surface antigen while antigen II extends from residue 996.

BALB/c mice and Lewis rats were injected intraocularly with recombinant gamma-interferon (IFN-gamma). The distribution of Ia antigens and cellular infiltrates was studied over a 21-day time course. In mice, Ia antigens were induced on cells in the corneal stroma, corneal endothelium, iris, ciliary body, retinal pigment epithelium and cells in the retina. No detectable cellular infiltrate accompanied class II major histocompatibility (MHC) antigen induction in mice. In rats, cells in all of the intraocular tissues mentioned above were induced to express Ia antigens. However, in distinct contrast to mice, the induction of Ia antigens on intraocular structures in rats was accompanied by intense cellular inflammation composed predominantly of polymorphonuclear cells. These findings demonstrate that aberrant Ia expression on intraocular tissues by IFN-gamma is not sufficient to elicit a cellular autoimmune response in mice. The observed species-related differences in cellular immune response to class II MHC antigen induction on intraocular tissues parallels the susceptibility of mice and rats to experimental autoimmune uveitis.

To understand more fully the biological function(s) and investigate the reported cross-reactivity with heart tissue of antigen P1 (I/II) of Streptococcus mutans (serotype c), this molecular biological study of the responsible gene, spaP, was undertaken. A 5.2 kb Hin dIII fragment of strain NG5 was cloned into Escherichia coli JM109 by a shotgun procedure with pUC18 as the vector. Recombinant SM2949 expressed a P1 fusion protein under the control of the streptococcal promoter. Southern analysis revealed hybridization of pSM2949 with DNA from Strep. mutans (serotypes c, e, f), Strep. cricetus (a) and Strep. sobrinus (d), but not Strep. sobrinus (g), Strep. rattus (b) or Strep. downei (h). Recombinant (r) antigen was detected in E. coli periplasm, indicating the presence of a signal sequence. This product (of Mr 155K) showed partial identity to the native streptococcal P1 antigen by Ouchterlony double-diffusion analysis. The N-terminal 28 amino acid residues of rP1 were determined by Edman degradation analysis and an end-labelled oligonucleotide probe corresponding to residues 8-13 was used to determine the 5'-3' orientation of spaP by Southern hybridization with restriction enzyme digests of pSM2949. Rabbit antisera made against native and rP1 did not cross-react with human heart tissue. Isogenic mutants of strain NG8 were isolated after transformation with insertionally inactivated spaP. Each mutant was non-reactive with anti-P1 antisera. Selected mutants were shown to have a defective spaP gene incorporated into their chromosomal DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Of 20 strains of Clostridium spp. screened, 17 hydrolyzed larch wood xylan. Two strains of Clostridium acetobutylicum, NRRL B527 and ATCC 824, hydrolyzed xylan but failed to grow on solid media with larch xylan as the sole carbon source; however, strain ATCC 824 was subsequently found to grow on xylan under specified conditions in a chemostat. These two strains possessed cellulolytic activity and were therefore selected for further studies. In cellobiose-limited continuous cultures, strain NRRL B527 produced maximum xylanase activity at pH 5.2. Strain ATCC 824 produced higher xylanase, xylopyranosidase, and arabinofuranosidase activities in chemostat culture with xylose than with any other soluble carbon source as the limiting nutrient. The activities of these enzymes were markedly reduced when the cells were grown in the presence of excess glucose. The xylanase showed maximum activity at pH 5.8 to 6.0 and 65 degrees C. The enzyme was stable on the alkaline side of pH 5.2 but was unstable below this pH value. The extracellular xylanolytic activity from strain ATCC 824 hydrolyzed 12% of the larch wood xylan during a 24-h incubation period, yielding xylose, xylobiose, and xylotriose as the major hydrolysis products. Strain ATCC 824, after being induced to grow in batch culture in xylan medium supplemented with a low concentration of xylose, failed to grow reproducibly in unsupplemented xylan medium. A mutant obtained by mutagenesis with ethyl methanesulfonate was able to grow reproducibly in batch culture on xylan. Both the parent strain and the mutant were able to grow with xylan as the sole source of carbohydrate in continuous culture with the pH maintained at either 5.2 or 6.0. Under these conditions, the cells utilized approximately 50% of the xylan.

We examined retinal tissue from eight human immunodeficiency virus type 1 (HIV-1) seropositive patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex for evidence of dual infection with HIV-1 and cytomegalovirus. Culture demonstrated simultaneous infection with HIV-1 and cytomegalovirus in two of 13 retinal specimens. This was confirmed by both immunofluorescence and immunohistochemical staining. Moreover, coinfection of individual cells with cytomegalovirus and HIV-1 was observed by immunohistochemical staining. Infection of retina with cytomegalovirus or HIV-1 alone occurred in one and six of the 13 retinal specimens, respectively. HIV-1 antigens were present on scattered cells in all layers of the retina and on retinal vascular endothelium. HIV-1 was isolated from retinal tissue derived from eyes both with and without gross ocular lesions. Cytomegalovirus antigens were found in all layers of the retina, but not on vascular endothelial cells. The atypically rapid clinical progression of retinitis in one of the patients with dual HIV-1 and cytomegalovirus infection suggests the possibility that interactions between these two viruses may influence retinal disease in patients with AIDS.

Clostridium acetobutylicum NRRL B527 and ATCC 824 exhibited extracellular and cell-bound endoglucanase and cellobiase activities during growth in a chemically defined medium with cellobiose as the sole source of carbohydrate. For both strains, the endoglucanase was found to be mainly extracellular (70 to 90%) during growth in continuous or batch cultures with the pH maintained at 5.2, whereas the cellobiase was mainly cell associated (60 to 90%). During continuous cultivation of strain B527 with cellobiose as the limiting nutrient, maximum production of the endoglucanase and cellobiase occurred at pH values of 5.2 and 4.8, respectively. In the carbon-limited continuous cultures, strain 824 produced similar levels of endoglucanase, cellobiosidase, and cellobiase activities regardless of the carbon source used. However, in ammonium- or phosphate-limited cultures, with an excess of glucose, only 1/10 of the endoglucanase was produced, and neither cellobiosidase nor cellobiase activities were detectable. A crude extracellular enzyme preparation from strain B527 hydrolyzed carboxymethylcellulose and phosphoric acid-swollen cellulose readily and microcrystalline cellulose (A vicel) to a lesser extent. Glucose accounted for more than 90% of the reducing sugar produced by the hydrolysis of acid-swollen cellulose and Avicel. Strain B527 did not grow in medium with acid-swollen cellulose as the sole source of carbohydrate, although it grew readily on the products obtained by hydrolyzing the cellulose in vitro with a preparation of extracellular cellulase derived from the same organism.

The effect of WR-2721 on performance maintained by a fixed-ratio 20 (FR-20) schedule for water reinforcement was studied in male Sprague-Dawley rats. Graded doses of WR-2721 (range 25-100 mg/kg) were administered IP immediately prior to a 60 min test session. WR-2721 had a dose dependent monotonic disruptive effect on FR responding, with significant effects at doses of 50, 75 and 100 mg/kg. WR-2721 also decreased postsession water consumption, but only one significant effect at the highest dose (100 mg/kg). Both slopes of the dose-response regression line are parallel in effect. These data indicate that WR-2721 may affect drinking motivation, which could disrupt operant performance, and WR-2721 affects motor behavior at lower doses than those that depress "motivation" to drink. The log dose-probit analysis on the all-or-none disruptive pattern of pause of responding observed from cumulative records disclosed that the slope of this regression line (s = 1.11) was also almost identical to that of reinforcer decrement analyzed from graded dose-response relationship (s = 1.14) and shared the same estimated ED50's (58.5 and 55.6 mg/kg, respectively). A preliminary study using a variety of pharmacological interventions was also carried out to ascertain if the general functional gastrointestinal disorders produced by WR-2721 may subserve the behavioral deficits. Subcutaneous pretreatments with various selective, peripherally active, gastroprotective drugs [cimetidine (30 and 60 mg/kg), pirenzepine (5 and 10 mg/kg) and domperidone (1, 5 and 10 mg/kg)] 30 min prior to challenge with WR-2721 at dose of 100 mg/kg, demonstrated that these drugs did not yield any apparent significant attenuative effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Goldmann tonometer tips were inoculated with 5 X 10(5) IU of cell-free or cell-associated human immunodeficiency virus type 1 (lymphadenopathy virus type 1 isolate) or 10(4) plaque-forming units of herpes simplex virus type 1 (McKrae strain) or type 2 (Hicks strain). In an effort to mimic a "worst case" clinical scenario, each respective virus was allowed to air dry on the tonometer tip for 10 minutes. Inoculated tonometers were then (1) not treated, (2) wiped with a disposable (Kim-wipe) tissue or sterile gauze; (3) wiped with sterile gauze soaked with 3% hydrogen peroxide; or (4) wiped with a 70% isopropyl alcohol swab. The hydrogen peroxide treatment and the alcohol wipes both completely disinfected the tonometer tips for human immunodeficiency virus type 1 and herpes simplex virus types 1 and 2, whereas wiping with a sterile gauze or tissue was not effective. Wiping the Goldmann tonometer tip with an isopropyl alcohol swab and then allowing the alcohol to evaporate provides a ready and efficient means of inactivating these three enveloped viruses.

Two unrelated boys had a history of bilateral corneal clouding at birth following uncomplicated full-term gestations and spontaneous vaginal deliveries (without forceps). Clinical examinations disclosed bilateral corneal edema, no inflammation, and normal intraocular pressures. There was no history of similarly affected family members. The patients underwent penetrating keratoplasty at ages 4 months (patient 1) and 12 years (patient 2). Light and electron microscopic studies of the corneal buttons from both patients revealed areas of degeneration of the endothelium and separation of rounded endothelial cells. The morphologic features were strikingly similar to those in two acquired forms of corneal disorders--autoimmune endotheliopathy and "acute endotheliitis." Immunocytologic and in situ hybridization studies for herpes simplex virus were not consistent with either productive or latent corneal infection. Ultrastructural changes in Descemet's membrane reflect delayed or abnormal development of the postnatal nonbanded layer in patients 1 and 2, respectively. These suggest an intrauterine insult that resulted in endothelial dysfunction. The histologic and ultrastructural features of these two congenital cases are not typical of those seen in any of the recognized causes of congenital corneal clouding. We propose that these cases represent a unique congenital corneal endotheliopathy of undetermined origin.

The changes in plasma fibronectin and IgG, and monocyte- and lymphocyte-associated fibronectin were studied in patients undergoing elective cardiopulmonary bypass surgery. A significant fall (P = less than 0.0005) in plasma fibronectin occurred during bypass, resulting largely from hemodilution as assessed by IgG concentrations, but also related to consumption of fibronectin. Plasma levels were still reduced 48 hours following the operative procedure, despite variable amounts of blood components infused in the immediate post-bypass period. Monocyte-associated fibronectin increased significantly (P = less than 0.05) during bypass, and lymphocyte-associated fibronectin levels decreased. Our studies confirm a reduction in circulating fibronectin in cardiac surgery, with accompanying fall in lymphocyte-associated levels presumed to reflect nonspecific adsorption. In contrast, the increased binding to monocytes may be an important functional aspect requiring further investigation, together with assessment of monocyte-macrophage function, before empiric use of cryoprecipitate therapy in these patients is recommended.

A direct stereospecific HPLC assay for carprofen glucuronides in biologic fluids was developed that makes use of a reverse phase (C 18) gradient system, in which the mobile phase consisted of a mixture of acetonitrile and tetrabutylammonium hydroxide buffer (pH 2.5). Reference diastereomeric glucuronides of carprofen were purified from human urine after oral administration of the single enantiomers. When 0.1 ml of sample was used, the limit of detection for carprofen glucuronides was 50 ng/ml in plasma and 200 ng/ml in urine. Coefficients of variation did not exceed 12% for both intra and interday variability. This HPLC method is applicable to pharmacokinetic analysis for carprofen glucuronides in humans. After oral administration of the single enantiomers there was little indication of metabolic inversion. For the two enantiomers the apparent total and metabolic clearances were similar. The limited data available suggest that renal clearance of the (S)-glucuronide was greater than that of the (R)-glucuronide.

Two endoxylanases produced by C. acetobutylicum ATCC 824 were purified to homogeneity by column chromatography. Xylanase A, which has a molecular weight of 65,000, hydrolyzed larchwood xylan randomly, yielding xylohexaose, xylopentaose, xylotetraose, xylotriose, and xylobiose as end products. Xylanase B, which has a molecular weight of 29,000, also hydrolyzed xylan randomly, giving xylotriose and xylobiose as end products. Xylanase A hydrolyzed carboxymethyl cellulose with a higher specific activity than xylan. It also exhibited high activity on acid-swollen cellulose. Xylanase B showed practically no activity against either cellulose or carboxymethyl cellulose but was able to hydrolyze lichenan with a specific activity similar to that for xylan. Both xylanases had no aryl-beta-xylosidase activity. The smallest oligosaccharides degraded by xylanases A and B were xylohexaose and xylotetraose, respectively. The two xylanases demonstrated similar K(m) and V(max) values but had different pH optima and isoelectric points. Ouchterlony immunodiffusion tests showed that xylanases A and B lacked antigenic similarity.

The gene spa P (formerly designated as spa P1) encoding the Mr 185,000 surface antigen (I/II) of Streptococcus mutans, serotype c (NG5), has been sequenced. The gene (4683 bp) encodes a protein of 1561 amino acid residues including putative signal peptide (residues 1-38) and transmembrane (residues 1537-1556) sequences. The N-terminal region (60-550) has alanine-rich repeats and is predicted to be alpha-helical. However, the C-terminal region (800-1540) is proline-rich and favours an extended structure. Except for a short central variable region the sequences appear to be highly conserved for S. mutans serotype c. N-Terminal sequencing of separated antigen I and antigen II polypeptides suggests that the former represents the N-terminal and the latter the C-terminal portions of the intact antigen.

A beta-d-xylosidase from C. acetobutylicum ATCC 824 was purified by column chromatography on CM-Sepharose, hydroxylapatite, Phenyl Sepharose, and Sephadex G-200. The enzyme had an apparent molecular weight of 224,000 as estimated by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme consisted of two subunits of 85,000 and one subunit of 63,000 daltons. It exhibited optimal activity at pH 6.0 to 6.5 and 45 degrees C. the enzyme had an isoelectric point of 5.85. It hydrolyzed p-nitrophenylxyloside readily with a K(m) of 3.7 mM. The enzyme hydrolyzed xylo-oligosaccharides with chain lengths of 2 to 6 units by cleaving a single xylose from the chain end. It showed little or no activity against xylan, carboxymethyl cellulose, and other p-nitrophenylglycosides.