Fluorescent imaging in a glioma model in vivo.

BACKGROUND AND OBJECTIVE: Nile blue dyes have been shown to have affinity for tumor tissue as compared to surrounding normal tissue and to be relatively non-toxic. We have employed EtNBA, a lipophilic, fluorescent benzophenoxazine dye, in a murine model to image subcutaneous and intracranial U-87 glioma implants. STUDY DESIGN/MATERIALS AND METHODS: The imaging system used to detect fluorescence consists of a SIT video camera fitted with a zoom microscope-magnifying lens. The tumor was illuminated with a 632.8-nm diffuse beam from a helium-neon laser. The video image was processed using a Sony image processor to give real-time pseudocolor and enhanced black and white images. RESULTS: Following subcutaneous injection of the dye at doses of 2.5-5.0 mg/kg bw, we observed a gradual increase of the fluorescent signal from the tumor which peaked 1-3 hours post-injection with variable selectivity (typically 4:1) for tumor to normal surrounding tissues permitting the clear demarcation of the tumor. CONCLUSIONS: The present in vivo study demonstrates that EtNBA is a safe and effective photodiagnostic agent, able to demarcate U87-MG solid tumors in mice on a real-time basis at a concentration of 2.5-5.0 mg/kg 1-3 hours after administration.

Validation of a noninvasive, real-time imaging technology using bioluminescent Escherichia coli in the neutropenic mouse thigh model of infection.

A noninvasive, real-time detection technology was validated for qualitative and quantitative antimicrobial treatment applications. The lux gene cluster of Photorhabdus luminescens was introduced into an Escherichia coli clinical isolate, EC14, on a multicopy plasmid. This bioluminescent reporter bacterium was used to study antimicrobial effects in vitro and in vivo, using the neutropenic-mouse thigh model of infection. Bioluminescence was monitored and measured in vitro and in vivo with an intensified charge-coupled device (ICCD) camera system, and these results were compared to viable-cell determinations made using conventional plate counting methods. Statistical analysis demonstrated that in the presence or absence of antimicrobial agents (ceftazidime, tetracycline, or ciprofloxacin), a strong correlation existed between bioluminescence levels and viable cell counts in vitro and in vivo. Evaluation of antimicrobial agents in vivo could be reliably performed with either method, as each was a sound indicator of therapeutic success. Dose-dependent responses could also be detected in the neutropenic-mouse thigh model by using either bioluminescence or viable-cell counts as a marker. In addition, the ICCD technology was examined for the benefits of repeatedly monitoring the same animal during treatment studies. The ability to repeatedly measure the same animals reduced variability within the treatment experiments and allowed equal or greater confidence in determining treatment efficacy. This technology could reduce the number of animals used during such studies and has applications for the evaluation of test compounds during drug discovery.

Complete primary structure of the chicken alpha1(V) collagen chain.

Chicken alpha1(V) collagen cDNAs have been cloned by a variety of methods and positively identified. We present here the entire translated sequence of the chick polypeptide and compare selected regions to other collagen chains in the type V/XI family.

Activities of the Porphyromonas gingivalis PrtP proteinase determined by construction of prtP-deficient mutants and expression of the gene in Bacteroides species.

PrtP is a major cysteine proteinase of Porphyromonas gingivalis. The gene encoding this proteinase, prtP, was cloned into the Escherichia coli-Bacteroides shuttle vectors pFD288 and pFD340 and was expressed in Bacteroides cells, apparently under the control of its own promoter, when in pFD288, or a Bacteroides promoter present on pFD340. Proteolytically active PrtP was detected by fibrinogen zymography in cells or spent growth medium of several Bacteroides species harboring the recombinant plasmids. The proteinase was recovered from Bacteroides fragilis ATCC 25285(pFD340-prtP) cells by 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS) extraction and characterized with regard to exopeptidase specificity and sensitivity to proteinase inhibitors. Lys-amidolytic activity, but not Arg-amidolytic activity, was detected. PrtP was activated by cysteine and, to a lesser extent, dithiothreitol, and it was stimulated by glycine-containing compounds. It also was inhibited by Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and, to a lesser extent, H-D-Tyr-L-Pro-L-arginyl chloromethyl ketone (YPRCK) and was relatively insensitive to EDTA and leupeptin. Neither B. fragilis ATCC 25285(pFD340-prtP) cells nor the CHAPS extract effected hemagglutination of sheep red blood cells or collagen cleavage, but the cells did cleave gelatin. Furthermore, P. gingivalis W12, ATCC 33277, KDP110, and HG66 with knockout mutations in prtP were constructed by allelic replacement. Unlike the parent strains, the mutant strains produced beige colonies on plates containing sheep blood. These strains also were affected in their ability to effect hemagglutination, cleave collagen, and cleave a Lys-specific peptide substrate. This report presents the results of the first characterization of the PrtP proteinase clearly in the absence of any influence by other P. gingivalis proteins and describes the properties of P. gingivalis cells defective in the production of PrtP.

Photodynamic therapy of naturally occurring tumors in animals using a novel benzophenothiazine photosensitizer.

5-Ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride (EtNBS) is a novel photodynamic therapy (PDT) photosensitizer with efficacy against experimental murine tumors. In this preliminary study, dogs and cats with naturally occurring tumors were treated with EtNBS-PDT to determine safety and efficacy. Fifteen treatments were performed on 13 animals (9 treatments in 8 cats and 6 treatments in 5 dogs), generally using 400 J of 652 nm light. Two feline sublingual squamous cell carcinomas (SCCs) responded briefly (minor response). Six feline facial SCCs were treated, resulting in two partial responses and four long-term complete responses (CR). Two canine intraoral SCCs were treated; one responded minimally for 2 weeks (minor response), and one achieved long-term CR. One canine cutaneous mast cell tumor achieved CR, and one canine ocular mast cell tumor responded briefly. One canine ocular melanoma did not respond to treatment. Systemic reactions included nausea associated with photosensitizer injection in two cats and two dogs, elevated body temperatures during treatment in two dogs, elevated body temperature 2 days after PDT in one cat, and inappetance for 2 weeks in one cat. A peripheral neuropathy of undetermined cause occurred in one cat 2 weeks after PDT and resolved without treatment. Local reaction was well tolerated in 13 of 15 treatments. All animals were exposed to normal daylight after less than 5 days (mean, 3.5 days) without residual photosensitization. EtNBS-PDT is safe for dogs and cats and has activity against selected naturally occurring tumors, with an overall objective response rate (partial response + CR) of 61.5%.

Factors affecting virus photoinactivation by a series of phenothiazine dyes.

A series of four phenothiazine dyes, including methylene blue (MB), were previously tested for their ability to photoinactivate viruses in red cell suspensions. One of the dyes, 1,9-dimethyl-3-dimethylamino-7-dimethylaminophenothiazine (1,9-dimethylmethylene blue), exhibited good intracellular and extracellular virucidal activity for several RNA and DNA viruses under conditions that minimally affected red cell properties. In order to understand why the virucidal specificity of 1,9-dimethylmethylene blue was greater than other phenothiazines tested, the physical and chemical properties of the dye were compared to three other closely related analogues (MB, 1,9-dimethyl-3-diethylamino-7-dibutylaminophenothiazine [compound 4-140], 1,9-dimethyl-3-dimethylamino-7-diethylaminophenothiazine [compound 6-136]). All compounds required light and oxygen for virucidal activity and had relatively high singlet oxygen yields (> 0.5), but 1,9-dimethylmethylene blue had a singlet oxygen yield approximately 50% greater than that of MB. In addition, the hydrophobicity/hydophilicity of the compounds varied, with the partition coefficients (2-octanol : water) ranging from 0.11 for MB to 3560 for compound 4-140. The dyes had the following affinities for DNA: 1,9-dimethylmethylene blue > compound 6-136 > MB approximately compound 4-140. This order was similar to the order of activities for photoinactivation of the nonenveloped bacteriophage, R17, by the four compounds. Results with the most hydrophobic compound, 4-140, contrasted with those obtained with 1,9-dimethylmethylene blue. Compound 4-140 had a high affinity for protein and a low affinity for DNA. Although compound 4-140 and light inactivated the nonenveloped bacteriophage R17 poorly, the dye readily photoinactivated enveloped viruses in buffer. However, unlike results with 1,9-dimethylmethylene blue, viral inactivation of enveloped viruses by compound 4-140 was completely inhibited by the presence of red cells and plasma. Thus, the high affinity of 1,9-dimethylmethylene blue for DNA and the dye's efficient singlet oxygen yield suggest viral nucleic acid as a potential target, which could explain the photosensitizer's ability to inactivate viruses without adversely affecting anucleate red cells.

Type XVII collagen (BP 180) in the developing avian cornea.

PURPOSE: Previous sequence analyses of hemidesmosomal BP 180/collagen XVII cDNA from human skin and of a similar chicken corneal cDNA showed some similarities, but major differences as well. The authors examined whether, in one species, the same mRNA is present in cornea and skin. They also studied the developmental expression of the molecule and compared it to the transmembrane hemidesmosome component, alpha 6 beta 4 integrin, and to the formation of hemidesmosomes themselves. METHODS: Cornea and skin BP 180/collagen XVII cDNAs were cloned by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced. Developmental expression was evaluated by quantitative RT-PCR, immunoblotting, and immunofluorescence microscopy. alpha 6 beta 4 integrin was evaluated by immunofluorescence microscopy, and hemidesmosome formation was assessed by electron microscopy. RESULTS: The same alpha 1 (XVII) collagen/BP 180 mRNA is present in cornea and skin. The appearance of alpha 1 (XVII) collagen mRNA and protein shows similar temporal patterns of expression, with changes in the mRNA preceding those of the protein by approximately 2 days. The appearance of mature hemidesmosomes lags still further. Immunofluorescence histochemistry of alpha 1 (XVII) collagen and alpha 6 beta 4 integrin shows that their developmental appearance is regulated closely. CONCLUSIONS: The differences between human BP 180/collagen XVII and the chicken corneal molecule represent species divergence. The appearance of alpha 1 (XVII) collagen mRNA and protein is regulated closely, with the protein lagging. Mature hemidesmosomes, once present, have a low turnover rate. The developmental appearance of alpha 1 (XVII) collagen and alpha 6 beta 4 integrin are regulated closely. However, the component responsible for initiating hemidesmosome formation remains unknown.

Temporal expression of types XII and XIV collagen mRNA and protein during avian corneal development.

Using immunohistochemistry and competitive PCR for collagen types XII and XIV, we have followed the expression of these fibril-associated molecules during development of the avian cornea. By immunofluorescence histochemistry, both molecules are found in the acellular primary stroma and are therefore presumably of epithelial origin. During formation and development of the secondary corneal stroma, which is populated by mesenchymal cells, the molecules generally appear to be spatially segregated from each other. Type XIV collagen is found throughout most of the stroma, and therefore is predominantly a product of stromal fibroblasts. During subsequent compaction of the cornea, an event necessary for corneal transparency, the collagen XIV mRNA level increases dramatically, suggesting that this molecule may play a role in this event. Type XII collagen is more localized, occurring mainly in regions of the secondary stroma where matrices interface, such as where Bowman's membrane and Descemet's membrane abut the orthogonally layered collagen fibrils of the stromal matrix. These interfacial regions are highly stable areas of the cornea as determined previously by protease digestion and thermal denaturation studies. Type XII collagen may be involved in this stabilization.

Type V collagen and Bowman's membrane. Quantitation of mRNA in corneal epithelium and stroma.

Bowman's membrane is an acellular matrix of the cornea which lies between the epithelial basal lamina and the corneal stroma. By immunoelectron microscopy, we have determined that types I and V collagen are components of the collagen fibrils in Bowman's membrane of the chick cornea. Although these same components are found in the fibrils of the stroma, the fibrils of Bowman's membrane are smaller in diameter and less uniform than those of the stroma. At early stages of development, the corneal epithelium synthesizes the types I and II collagen of the primary stroma. We therefore asked whether it might also be capable of synthesizing the type V collagen found in Bowman's membrane at later stages of development. Our results, using competitive polymerase chain reaction to quantitate mRNA from avian corneal cells, indicate that the amount of alpha 1(V) collagen mRNA present in epithelia, relative to alpha 2(I) collagen mRNA, is greater than that in stromal fibroblasts. We postulate that this enables the epithelium to synthesize a higher ratio of type V to type I collagen than the stroma and that this proportionally higher amount of type V might account for the ultrastructural appearance of the fibrils in Bowman's membrane.

Novel photodynamic effects of a benzophenothiazine on two different murine sarcomas.

The photochemotherapeutic properties of a novel benzophenothiazine, 5-ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride, were assessed in vitro and in vivo against two murine mammary sarcoma models (EMT-6 and RIF). Photodynamic therapy (PDT) of EMT-6 and RIF cells following a 30-min incubation with dye (0.4 microgram/ml) and a light dose of 3.3 J/cm2 killed 87.0 and 99.6% of the cells, respectively. Over this same time period, RIF cells accumulate more than twice the amount of dye than the EMT-6 cell line [7.54 +/- 0.17 (SD) versus 3.11 +/- 0.15 nmol/10(6) cells] which probably accounts for their increased sensitivity to PDT. Conversely, in vivo, the EMT-6 tumor accumulates 3 times more dye (34.66 +/- 2.16 micrograms/g dry weight) than the RIF tumor (12.28 +/- 1.27 micrograms of dye/g) 3 h post-s.c. injection of dye (15 mg/kg). A study of the concentration dependent uptake of dye (following s.c. injection) in the tumor and plasma of mice bearing the EMT-6 tumor indicated a nonlinear relationship for both compartments. Maximum tissue uptake of dye and discrimination between tumor and skin or muscle occur 3-8 h following s.c. injection of dye. The ratios of dye in the tumor to the dye in surrounding skin and gastrocnemius muscle 8 h following dye injection were 4:1 and 8:1, respectively. At 24 h after dye injection, the dye was not detectable by absorption spectroscopy in the tumor, skin, or muscle. Decreasing the fluence rate from 200 to 50 mW/cm2 at a total light dose of 100 J/cm2 optimized the PDT effect. At 3 h following s.c. administration of dye, PDT of EMT-6 (7.5 mg of dye/kg; 50 mW/cm2; 100 J/cm2) and RIF tumors (15 mg dye/kg; 50 mW/cm2; 150 J/cm2) resulted in 100 and 70% cures, respectively. Histology at 24 and 72 h post-PDT showed minimal or no damage to the surrounding tissue (skin) while 70-90% of the tumor cells were destroyed or damaged. Moreover, 50-60% of the tumor cells isolated and cultured immediately following PDT were found to be nonviable. Similarly, the administration of 60 mg 5-ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride/kg also resulted in no damage to the skin 24 h following PDT. It is suggested that the redox properties of the dye coupled with the differing metabolic states of the tumor and skin, which increase the amount of photoactive, oxidized dye present in the tumor and decrease it in the skin, are responsible for this unique differential PDT effect.(ABSTRACT TRUNCATED AT 400 WORDS)

Photodynamic destruction of lysosomes mediated by Nile blue photosensitizers.

Previous studies have established that a number of Nile blue derivatives are potent photosensitizers and that they are localized primarily in the lysosomes. The present study examines whether the lysosome is a main target of the photocytotoxic action mediated by these sensitizers. Chosen for this study were NBS-6I and sat-NBS, which represented, respectively, derivatives with high and moderate degrees of lysosomal. This is indicated by the light-and drug-dose-dependent losses of acid phosphatase staining particles, reduction of hexosaminidase in the lysosome-containing subcellular fraction, and impairment of the lysosomes to take up and sequester acridine orange. Ultrastructurally, swollen and ruptured lysosomes were seen as one of the first evidences of cell damage mediated by these photosensitizers. However, the study also showed that sat-NBS, which is less lysosomal selective, was less effective in mediating lysosomal destruction. Also, the degree of lysosomal destruction mediated by sat-NBS did not parallel the degree of cytotoxicity generated. This implies that for derivatives that are not exclusively localized in the lysosome, other subcellular sites may also be damaged by the photodynamic action and may play a role in the photocytotoxic process.

Phototoxicity, redox behavior, and pharmacokinetics of benzophenoxazine analogues in EMT-6 murine sarcoma cells.

Structural modifications to the photoinactive benzophenoxazine Nile blue A have led to three novel derivatives which include 5-ethylamino-9-diethylaminobenzo[a]phenoxazinium (EtNBA), 5-ethylamino-9-diethylaminobenzo[a]phenothiazinium (EtNBS), and 5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium (EtNBSe) chlorides. The incorporation of sulfur and selenium into the benzophenoxazine moiety results in lipophilic, red-absorbing (650-660 nm) chromophores which possess significantly increased singlet oxygen yields (0.025 and 0.65, respectively, compared to 0.005 for EtNBA). This study examines the photosensitizing efficacies and pharmacokinetics in vitro in the EMT-6 murine mammary sarcoma cell line as well as the physicochemical, photochemical, and redox properties of these new analogues. Comparisons with Photofrin II, the only photosensitizer available clinically, were made in an attempt to high-light their different pharmacological characteristics. The photodynamic activity of the benzophenoxazine dyes correlates with their ability to generate the phototoxin singlet oxygen and increases in the following order: EtNBA < EtNBS << EtNBSe. At an extracellular dye concentration of 0.5 microM, the light dose required to kill approximately 50% of the cells was 2.0 and < 0.5 J/cm2 for the sulfur and selenium dyes, respectively. The light dose required to kill approximately 50% of the cells for both EtNBA and Photofrin II could not be determined because of their weak phototoxic effect under these conditions. At a light dose of 3.3 J/cm2, EtNBSe is approximately 1000 times more phototoxic than Photofrin II. All three benzophenoxazine derivatives are characterized by a similar uptake/efflux pattern in vitro consisting of a rapid and extensive cellular accumulation followed by a slow efflux rate. Contrary to their rapid uptake, 50% of the accumulated EtNBS and EtNBSe is retained intracellularly after a 6-h period in dye-free medium. Video-enhanced fluorescence microscopy corroborates the rapid uptake measurements as well as indicating the intracellular localization of the dyes in both living and thermally inactivated cells. Low extracellular dye concentrations (0.05 microM) result in a punctate fluorescence pattern in the perinuclear region, while higher dye concentrations (> 0.1 microM) lead to additional fluorescence in the cytoplasm, cytomembranes, and other organelles but apparently not the nucleus. Absorption spectrometry revealed that living cells rapidly reduce the dyes to their colorless leuko form (photoinactive) if oxygen is not readily available in the environment. It is shown that the cellular reduction is an enzymatic process and that an oxygen-free and cell-free medium containing both the coenzyme NADH and the hydride transfer enzyme diaphorase is capable of reducing the dyes to the colorless leuko form.(ABSTRACT TRUNCATED AT 400 WORDS)

Type XIV collagen is encoded by alternative transcripts with distinct 5' regions and is a multidomain protein with homologies to von Willebrand's factor, fibronectin, and other matrix proteins.

The combined nucleotide sequences of several overlapping cDNAs provide the first complete amino acid sequence of type XIV collagen. Independent confirmation of the deduced sequence is provided by amino acid sequencing of several tryptic peptides isolated from purified chicken skin type XIV collagen. Comparative analyses show that the amino-terminal non-triple-helical region of alpha 1(XIV) chains contains sequence motifs that are similar to alpha 1(IX) collagen, fibronectin type III repeats, and von Willebrand's factor A-domains. The results also strongly suggest that the alpha 1(XIV) collagen gene is identical to the gene encoding the matrix component previously named undulin. cDNAs covering the 5' region of alpha 1(XIV) mRNA fall into two classes with distinct sequences in their 5'-untranslated regions. We believe the two alternative sequences result from differential splicing of the primary transcript. Interestingly, one of the untranslated sequences shows a high degree of identity with the cis-regulatory translational control sequence in the 5'-untranslated region of a Drosophila ribosomal protein mRNA. We hypothesize therefore that the sequence in alpha 1(XIV) collagen may play a role in the control of alpha 1(XIV) protein synthesis.

Lysosomal localization and mechanism of uptake of Nile blue photosensitizers in tumor cells.

Nile blue derivatives have been shown to be potentially effective photosensitizers for photodynamic therapy of malignant tumors. Results of a previous study suggested that the high accumulation of these dyes in cells may be the result of dye aggregation, partition in membrane lipids, and/or sequestration in subcellular organelles. In this report, results of studies are presented from an investigation of the subcellular localization and mechanism of accumulation of these dyes in cells in vitro. A video-enhanced fluorescence microscopy was used, and a punctate pattern of fluorescence was seen, most of which was localized in the perinuclear region with extracellular dye concentrations between 1 to 100 nM. These particles resembled characteristic particles identified by standard lysosomal dyes. At higher dye concentrations (1 microM or above), fluorescence in the perinuclear region was too intense to resolve into discrete cellular structures, while fluorescence in other cellular structures including mitochondria and cytomembranes was visible. At even higher dye concentrations (10-100 microM), Nile blue derivatives were seen with a light microscope as blue particles, the size and location of which resembled the punctate fluorescence described above. Results which further suggest that the lysosome is the main site of dye localization include (a) histochemical staining of dye-loaded cells with the lysosomal marker enzyme acid phosphatase, which showed similar localization of the enzyme-staining and dye-containing particles, (b) phototreatment of dye-loaded cells which obliterated the majority of the acid phosphatase-stained particles, and (c) treatments with agents affecting the membrane pH gradient reduced the uptake and enhanced the efflux of dyes, while agents that alter cellular membrane potentials had no effect on dye accumulation. The uptake of the dyes was partially inhibited by inhibitors of oxidative phosphorylation indicating that at least part of the process is energy dependent. These findings, together with previous results showing that the cellular uptake of these dyes is highly concentrative and proportional to the extracellular dye concentration over a wide range, are consistent with the hypothesis that the dyes are mainly localized in the lysosomes via an ion-trapping mechanism. Results of the present study also suggest that the lysosomes may be an intracellular target for photodynamic killing of tumor cells mediated by Nile blue photosensitizers and that lysosomotropic photosensitization may be a strategy for effective and selective destruction of tumor cells.

Photosensitization, uptake, and retention of phenoxazine Nile blue derivatives in human bladder carcinoma cells.

The overall goal of our research is to develop effective new photosensitizers for tumor-selective photodynamic therapy. Phenoxazine dyes, including several Nile blue analogues, are known to localize selectively in animal tumors. Structural modifications yielded several series of analogues with substantially higher 1O2 yields and different photochemical and physicochemical properties. This study examined the photosensitization potency, cellular uptake, and retention of these derivatives in human bladder carcinoma cells (MGH-U1) in culture. Nile blue derivatives containing halogens and/or sulfur substitutes were selected to exhibit different 1O2 yields, pKa values, and hydrophobicities. The effectiveness of these derivatives in mediating photokilling of tumor cells in vitro corresponded well with the 1O2 yields of these compounds, indicating that structural modifications which resulted in increased 1O2 yields enhanced potency in mediating photocytotoxicity in vitro. Using derivatives (sat-NBS and sat-NBS-61) with the highest 1O2 quantum yield (0.35 and 0.821), over 90% cell kill was achieved at a sensitizer concentration of 5 x 10(-8) M, about 3 orders of magnitude more effective than hematoporphyrin derivative, the only sensitizer currently available clinically. This result suggests that some of the oxazine derivatives could potentially be effective photosensitizers. The correspondence between 1O2 yield and photosensitizing potency, together with results showing enhanced photocytotoxicity in the presence of D2O and reduced photocytotoxicity under hypoxic conditions, strongly suggests that the generation of 1O2 is a major mechanism mediating the photocytotoxic effect. The uptake of Nile blue derivatives by cells in culture exhibited a pattern of rapid initial uptake followed by a gradual increase in cellular dye contents. The uptake does not correlate directly with the individual pKa values or hydrophobicities of the derivatives, indicating that the structural modifications that increased 1O2 yields did not significantly alter the uptake and retention of Nile blue derivatives. The highly concentrative uptake by and slow efflux from dye-loaded cells were consistent with an active mechanism for the cellular accumulation of these dyes. On the other hand, the retention of the compounds was directly proportional to dye concentration in the medium over a 1000-fold range of concentrations, and the uptake could proceed at temperatures below 2 degrees C; these observations excluded endocytosis or a carrier-mediated mechanism for the uptake. The uptake was also unaffected by the presence of serum in the medium. Based on these results, we hypothesize that Nile blue derivatives transport across the cell membrane possibly as deprotonated forms and, upon entering the cell, either partition into lipophilic areas of the cell membranes and/or become sequestered in certain intracellular organelles.

Effect of vaccination with a pentavalent leptospiral vaccine on Leptospira interrogans serovar hardjo type hardjo-bovis infection of pregnant cattle.

Effectiveness of a pentavalent leptospiral vaccine to protect cattle from infection and reproductive problems caused by Leptospira interrogans serovar hardjo type hardjo-bovis was evaluated. Seven cows were vaccinated once and 8 cows were vaccinated twice with a USDA-licensed pentavalent leptospiral vaccine. Five cows were maintained as nonvaccinated controls. Cows were bred 1 to 2 months after the last vaccination. During the 4th to 6th month of gestation, all cows were challenge exposed on 4 occasions by conjunctival instillation of 10(8) serovar hardjo type hardjo-bovis organisms and on 3 occasions by conjunctival instillation of urine from a cow shedding hardjo-bovis. All control cows and 13 of 15 vaccinated cows became infected and shed leptospires in the urine. Leptospires were detected in fewer urine samples collected from vaccinated cows, compared with those collected from control cows. Four stillborn calves and 3 weak calves were born to control and vaccinated cows. Leptospires were detected in the kidneys of 11 apparently healthy calves born to vaccinated and control cows. Agglutinating antibodies were not detected in the precolostral serum of these calves.

Reclassification of North American leptospiral isolates belonging to serogroups Mini and Sejroe by restriction endonuclease analysis.

The genomes of North American strains of leptospires belonging to serogroups Mini and Sejroe were analyzed and compared with those of reference strains by cleavage with restriction endonucleases. The isolates selected for this study, when typed by the serologic method, were identified as serovars szwajizak, hardjo, and balcanica. However, the results of restriction endonuclease analysis (REA) indicated that a different classification existed. The 2 isolates typed as serovar szwajizak seem to be georgia by REA. Isolates belonging to serovars balcanica and hardjo had REA patterns that differed from both reference strains. Differences were not observed in the REA patterns between balcanica and hardjo isolates. All hardjo and balcanica isolates examined are suggested to be classified into a previously described hardjo, REA subtype hardjobovis. Using the enzyme Hha1, these isolates were subdivided into 3 subgroups. When examining the REA pattern of the 17 reference strains in serogroup Sejroe, 3 identical pairs were observed: wolffi and roumanica; sejroe and polonica; and istrica and nyanza. The REA again indicated that it will be a valuable method for the classification of leptospires.

Rapid and simplified protocol for isolation and characterization of leptospiral chromosomal DNA for taxonomy and diagnosis.

We have developed a rapid method for the isolation of leptospiral chromosomal DNA which yields DNA of a purity suitable for restriction endonuclease analysis. A small volume (15 to 20 ml) of an exponentially growing culture of leptospires yielded 2 to 4 micrograms of chromosomal DNA. In a 1-day protocol, the DNA was isolated, restricted with endonucleases, and fractionated on an agarose gel. Chromosomal DNA from dinger zones (visible subsurface zones of leptospiral growth) of first semisolid subcultures of field isolates was also isolated and characterized, thus greatly speeding up the diagnostic process.

Medication and programming in controlling the behavior of mentally retarded individuals in community settings.

Behavioral outcomes of a behavioral-chemical intervention procedure on stereotypic and non-compliant behavior were evaluated. One group (n = 22) of community-based mentally retarded clients was initially on psychotropic medication (major tranquilizers). Their dosage was either increased, decreased, or kept the same following behavioral intervention. A second group (n = 19) was placed on psychotropic medication following behavioral intervention. A nonequivalent between-groups design was employed that permitted 36 outcome combinations involving Conditions X Subject X Group. The effects of behavioral intervention, the validity of drug-intervention decision rules, drug-intervention effects, and the validity of the behavioral-chemical intervention model were evaluated. Results indicated that the behavioral-chemical intervention produced expected and desirable behavioral change as well as reduced levels of psychotropic drug usage.