Trauma-informed Approaches to Substance Use Interventions with Indigenous Peoples: A Scoping Review.

Indigenous Peoples experience disproportionately higher rates of problematic substance use. These problems are situated in a context of individual and intergenerational trauma from colonization, residential schools, and racist and discriminatory practices, policies, and services. Therefore, substance use interventions need to adopt a trauma-informed approach. We aimed to synthesize and report the current literature exploring the intersection of trauma and substance use interventions for Indigenous Peoples. Fourteen databases were searched using keywords for Indigenous Peoples, trauma, and substance use. Of the 1373 sources identified, 117 met inclusion criteria. Literature on trauma and substance use with Indigenous Peoples has increased in the last 5 years (2012-2016, n = 29; 2017-2021, n = 48), with most literature coming from the United States and Canada and focusing on historical or intergenerational trauma. Few articles focused on intersectional identities such as 2SLGBTQIA+ (n = 4), and none focused on veterans. There were limited sources (n = 25) that reported specific interventions at the intersection of trauma and substance use. These sources advocate for multi-faceted, trauma-informed, and culturally safe interventions for use with Indigenous Peoples. This scoping review illuminates gaps in the literature and highlights a need for research reporting on trauma-informed interventions for substance use with Indigenous Peoples.

Correlation between temperature rise and auto-fluorescence: ex vivo study with Er:YAG laser, Quantum Molecular Resonance scalpel and Nd:YAG laser on hard and soft tissues.

Surgical techniques of soft and hard oral tissues highly benefited from new technologies such as the Quantic Molecular Resonance (QMR) lancet, the Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG) laser and the Erbium-doped Yttrium Aluminum Garnet (Er:YAG) laser. Increasingly, these technologies replace scalpel, conventional electrosurgery and traditional rotary surgery instruments due to their proven advantages. Features such as reduction of the surgical time, more efficient bleeding control resulting in higher intra-operative visibility and improvement of postoperative course with better Quality of Life score (QoL) are highlighted in numerous studies published in the literature. The thermal rise of tissues during surgical incision, performed with other instruments rather than traditional cold blade scalpels, is not to be ignored by the operator and it must take into consideration first when choosing the surgical instrument and then throughout all the surgical act. Auto-fluorescence (AF) is a property possessed by every cell that exposed to a specific wavelength can absorbance or reflect with peculiar characteristics and its direct examination has been proposed as a non-invasive visual tool for investigation of suspicious changes in oral mucosa. At the limit of our knowledge, few studies have been published in the literature regarding tissue's temperature variations and the interest in Infra-Red temperature detection has been shown in various medicine fields and none of published studies investigated the possible correlation between temperature raise and AF variations. This ex vivo study aims to analyse and compare through the use of a thermal imaging camera and simultaneous detection of AF, the possible correlation between temperature increase and auto-fluorescence.

Mycobacterium avium subsp. paratuberculosis induces differential cytosine methylation at miR-21 transcription start site region.

Mycobacterium avium subspecies paratuberculosis (MAP), as an obligate intracellular bacterium, causes paratuberculosis (Johne's disease) in ruminants. Plus, MAP has consistently been isolated from Crohn's disease (CD) lesions in humans; a notion implying possible direct causative effect for MAP in CD development. Infections caused by MAP are refractory to treatment and in many cases the treatment does not easily resolve the infection. Studying the molecular mechanisms of host-pathogen interaction is helpful in identifying possible drug targets. In this line, it has already been shown that in macrophages infected with various bacteria, including mycobacteria, micro RNA 21 (miR-21) is upregulated, a change that results in diminished macrophages clearance ability and favours pathogens survival within the cells. However, the molecular mechanism(s) by which the intracellular bacteria induce miR-21 expression is not known. In order to verify possible effects from epigenetic changes induced by intracellular bacteria, we studied the cytosine methylation changes at the transcription start regions of miR-21 in THP-1 macrophages infected with MAP. For this purpose, genomic DNA was extracted from infected cells and the methylation status at the region of interest was evaluated by bisulfite conversion method. Our work showed that MAP directs de-methylation of the cystosines at CpG di-nucleotides in this region, while non-CpG cytosines of this region did not show significant changes. Interestingly, the CpG cytosines that were differentially methylated in the infected macrophages occur at the binding sites of the transcription factors already known to regulate miR-21 expression.

The relationship between growth hormone polymorphism and growth hormone receptor genes with milk yield and reproductive performance in Holstein dairy cows.

The aim of this study was to investigate the potential association between growth hormone GH/AluI and growth hormone receptor GHR/AluI polymorphisms with milk yield and reproductive performances in Holstein dairy cows in Iran. Blood samples of 150 Holstein cows were collected and their genomic DNA was extracted using Gene-Fanavaran DNA extracting kit. Fragments of the 428 bp of exon 5 growth hormone (GH) gene and the 342 bp of exon 10 growth hormone receptor (GHR) gene were amplified using the polymerase chain reaction (PCR) method. PCR products were digested by the AluI restriction enzyme and electrophoresed on 3% agarose gel. Continuous and categorical data were analyzed using linear mixed models through Proc MIXED and logistic regression models through Proc GENMOD of SAS software, respectively. The results showed no relationship between the examined traits and GH/AluI or GHR/AluI genes. A significant relationship was found between GH/AluI polymorphism and dystocia, but the presence of the GH-L allele reduced the incidence of dystocia. The results suggest that the GH-LL genotype reduces dystocia probably by affecting the release of growth hormone; nevertheless, further studies will be needed to examine the relationship between dystocia and GH genotypes.

Brucella melitensis and Mycobacterium tuberculosis depict overlapping gene expression patterns induced in infected THP-1 macrophages.

Pathogens infecting mammalian cells have developed various strategies to suppress and evade their hosts' defensive mechanisms. In this line, the intracellular bacteria that are able to survive and propagate within their host cells must have developed strategies to avert their host's killing attitude. Studying the interface of host-pathogen confrontation can provide valuable information for defining therapeutic approaches. Brucellosis, caused by the Brucella strains, is a zoonotic bacterial disease that affects thousands of humans and animals around the world inflicting discomfort and huge economic losses. Similar to many other intracellular dwelling bacteria, infections caused by Brucella are difficult to treat, and hence any attempt at identifying new and common therapeutic targets would prove beneficial for the purpose of curing infections caused by the intracellular bacteria. In THP-1 macrophage infected with Brucella melitensis we studied the expression levels of four host's genes, i.e. EMP2, ST8SIA4, HCP5 and FRMD5 known to be involved in pathogenesis of Mycobacterium tuberculosis. Our data showed that at this molecular level, except for FRMD5 that was downregulated, the other three genes were upregulated by B. melitensis. Brucella melitensis and M. tuberculosis go through similar intracellular processes and interestingly two of the investigated genes, i.e. EMP2 and ST4SIA8 were upregulated in THP-1 cell infected with B. melitensis similar to that reported for THP-1 cells infected with M. tuberculosis. At the host-pathogen interaction interface, this study depicts overlapping changes for different bacteria with common survival strategies; a fact that implies designing therapeutic approaches based on common targets may be possible.

Cancer stem cells converted from pluripotent stem cells and the cancerous niche.

Nowadays, the cancer stem cells are considered to be significantly responsible for growth, metastasis, invasion and recurrence of all cancer. Cancer stem cells are typically characterized by continuous proliferation and self-renewal as well as by differentiation potential, while stem cells are considered to differentiate into tissue- specific phenotype of mature cells under the influence of micro-environment. Cancer stem cells should be traced to the stem cells under the influence of a micro-environment, which induces malignant tumors. In this review, we propose this micro-environment as a 'cancerous niche' and discuss its importance on the formation and maintenance of cancer stem cells with the recent experimental results to establish cancer stem cell models from induced pluripotent stem cells. These models of cancer stem cell will provide the great advantages in cancer research and its therapeutic applications in the future.

The helminth community of Geophagus proximus (Perciformes: Cichlidae) from a tributary of the Paraná River, Ilha Solteira Reservoir, São Paulo State, Brazil.

This study aimed to evaluate the helminth parasites of Geophagus proximus from the São José dos Dourados River, a tributary of Paraná River, Ilha Solteira Reservoir, São Paulo State, Brazil. From May 2006 to May 2007, 116 G. proximus specimens were examined and seven different taxa of helminth were collected and identified: proteocephalidean plerocercoids (Cestoda); metacercariae of Austrodiplostomum compactum, Clinostomum heluans and Clinostomum sp. (Trematoda); and Raphidascaris (Sprentascaris) hypostomi, and larvae of Raphidascaris sp. and Contracaecum sp. (Nematoda). All parasites presented the typical aggregated pattern of distribution, as well as the presence of a high number of larval stages, an absence of influence of the host sex and seasonality upon community parameters, as well as a correlation between species richness and host body weight. Moreover, with the exception of A. compactum metacercariae, all helminths found in this study are reported for the first time in G. proximus.

Resposta humoral de bovinos vacinados contra as toxinas botulínicas tipos c e dem diferentes faixas etárias / Humoral response of vaccinated cattle against toxins of Clostridium botulinum types c and d at different ages

Foi verificada pelo teste de ELISA indireto a resposta humoral contra os toxoides botulínicos C e D em bovinos de diferentes idades. O estudo envolveu 90 animais, que foram divididos em três grupos (n = 30), de acordo com a sua faixa etária; inferior a 2 anos de idade (G1), entre 2 e 5 anos (G2) e superior a 5 anos (G3). Os grupos experimentais foram vacinados com duas doses de vacina antibotulínica bivalente (C e D) comercial, nos dias 0 e 42 após a primo-vacinação (booster). Na avaliação, quando realizada 30 dias após o booster, os animais do G3 apresentaram maior produção de anticorpos (p < 0,05) em relação aos demais grupos. Entre o G1 e G2 não houve diferença significativa na resposta humoral contra a toxina C, no entanto, contra a toxina D, os animais do G1 apresentaram maior produção de anticorpos. Todos os grupos produziram uma resposta significativa de anticorpos contra as toxinas botulínicas após a 2ª dose da vacina bivalente comercial, principalmente contra o tipo D.

Humoral response of vaccinated cattle against toxins of clostridium botulinum types C and D at different ages. Cattle humoral response against type C and D botulinum toxoids (indirect ELISA) was verified in animals of different ages. The animals (n = 90) were divided in three groups (n = 30): group one (G1): less than two years old; group two (G2): from 2 to 5 years old; group three (G3): more than 5 years old. The groups were vaccinated with two doses [0 and 42 days after primary vaccination (booster)] of bivalent (C and D) antibotulinum vaccine. Group three had higher antibody production (p ≤ 0.05) compared to the other groups, 30 days after the booster. There was no difference (G1 and G2; p ≥ 0.05) in the humoral response against C toxin, however, against D toxin, group one had higher antibody production. It was possible to conclude that after two doses of the commercial bivalent vaccine all groups produced a significant antibody response against botulinum toxins, especially against D type.

Novel and simple loading procedure of cisplatin into liposomes and targeting tumor endothelial cells.

Although intravenous administration of high levels of cisplatin (CDDP) are limited due to its severe side effects, efficient delivery of CDDP directly to the tumor should improve the therapeutic response while potentially by-passing significant side effects. High loading of CDDP into liposomes is one technique that could be used as a potential drug delivery system. Since cis-diamminedinitratoplatinum (CDDP3) is highly soluble in water and converts to CDDP in the presence of chloride ions, we encapsulated CDDP3 into liposomes in the absence of chloride ions and supplemented chloride ions to prepare CDDP-encapsulated liposomes (CDDP-Lip) resulting in a significantly improved loading efficiency of CDDP. We further conjugated the CDDP-Lip with Sialyl Lewis(X) (CDDP-SLX-Lip) because we previously demonstrated Sialyl Lewis(X) enhanced efficient accumulation of liposomes into tumors in vivo. CDDP-SLX-Lip treated mice showed a survival rate of 75% at 14 days even if a lethal level of CDDP was injected into mice. Loss of body weight was negligible and no histological abnormality was found in a variety of normal tissues. Accumulation of CDDP-SLX-Lip was about 6 times more than that of CDDP-Lip or CDDP. As the result, there was better antitumor activity of CDDP-SLX-Lip than that of CDDP-Lip with significantly less toxic effects in normal tissues.

First Report of Cucumber mosaic virus in Sweet Cherry in the People's Republic of China.

From the spring of 2003 to the summer of 2006, sweet cherry (Prunus avium) trees in orchards near Lvshun City, in the northeast People's Republic of China, had symptoms suggestive of those caused by Cucumber mosaic virus (CMV; genus Cucumovirus, family Bromoviridae). Symptoms included chlorotic patches or mottling on leaves that were also deformed (4). In April 2006, 20 symptomatic leaves sampled from 10 trees in each of four orchards were assayed for CMV with a CMV-specific antiserum (Agdia Inc., Elkhart, IN) in a double-antibody sandwich-ELISA. Of the 80 symptomatic leaf samples, 27 tested positive for the presence of CMV. CMV was detected in all four orchards, within which incidence varied between 0.5 and 4%. Viral nucleoproteins were purified by differential centrifugation and sucrose density gradient fractionation from symptomatic leaves. Transmission electron microscopy of nucleoproteins revealed isometric particles approximately 30 nm in diameter, which is also typical of CMV. Total RNA was also extracted from 100 mg of symptomatic tissue following a Trizol-based protocol (1). A reverse transcriptase-PCR assay with nucleocapsid gene-specific primers was then used (forward primer 5'-ATGGCGACGTCCTCGTTCA-3'; reverse primer 5'-CATCGTTCCCTTCAAAATAG-3') (3). A PCR product of approximately 633 bp was obtained. The PCR product was cloned and sequenced. The sequence (GenBank Accession No. HM996559) had 95% identity with the RNA-1 sequence from CMV 'Fny' strain in GenBank (Accession No. D00356.1). The People's Republic of China is one of the major producers of sweet cherry in Asia and the spread of CMV in China may cause significant economic losses. Thus, virus-infected material should not be used for propagation and surveys should be undertaken to determine if the aphid vectors capable of transmitting CMV are present (2).To our knowledge, this is the first report of CMV occurring in sweet cherry orchards in the People's Republic of China. References: (1) P. Chomczynski and K. Mackey. Biotechniques 19:942, 1995. (2) F. E. Gildow et al. Phytopathology 98:1233, 2008. (3) T. M. Rizzo and P. Palukaitis. J. Gen. Virol. 70:1, 1989. (4) J. Shang et al. Z. Naturforsch. C 65:73, 2010.

Anthelmintic resistance in cattle nematodes in northwestern São Paulo State, Brazil.

The present study was carried out in the northwestern region of São Paulo State, Brazil, to determine the anthelmintic resistance status in cattle naturally infected with gastrointestinal nematodes. The anthelmintics tested were levamisole phosphate (Ripercol, Fort Dodge), albendazole sulphoxide (Ricobendazole, Fort Dodge), ivermectin (Ivomec, Merial) and moxidectin (Cydectin, Fort Dodge), administered at the doses recommended by the manufacturers. From April 2002 to May 2004, 25 cattle farms were evaluated. On each farm, steers were divided into treatment and control (not treated) groups based on fecal egg counts (FEC). Between 7 and 10 days after the anthelmintics administration, fecal samples were collected from each animal for post-treatment FEC. Fecal cultures from each group were also prepared for larval identification. After treatment, mean FEC reduction (FECR) in treatment groups (compared with control groups) was assessed on each farm. FECR was lower than 90% on 23 farms after ivermectin treatment. On 19 farms, FECR of 100% was recorded following moxidectin treatment; on the remaining 6, FECR ranged from 90% to 97.2%. After albendazole treatment, FECR was higher than 90% on 20 farms and ranged from 47.4% to 84.6% on other 5. After levamisole treatment, FECR was higher than 90% on 23 farms and equal to 47.4% and 73.7% on other 2 farms. Results indicated the presence of resistant Cooperia spp. and Haemonchus spp., especially to ivermectin; on some farms, resistance to albendazole and levamisole was also observed.

Gene therapy of liver tumors with human liver-specific nanoparticles.

The development of safe and efficient liver-specific gene delivery approaches offers new perspectives for the treatment of liver disease, in particular, liver cancer. We evaluated the therapeutic potential of hepatotropic nanoparticles for gene therapy of liver tumor. These nanoparticles do not contain a viral genome and display the hepatitis B virus L antigen, which is essential to confer hepatic specificity. It has not been shown whether a therapeutic effect could be obtained using L nanoparticles in a human liver tumor xenograft model. Rats bearing human hepatic (NuE) and non-hepatic tumors were injected with L nanoparticles containing a green fluorescent protein (GFP) expression plasmid. GFP expression was observed only in NuE-derived tumors but not in the non-hepatic tumor. The potential for treatment of liver tumors was analyzed using L nanoparticles containing the herpes simplex virus thymidine kinase gene, in conjunction with ganciclovir pro-drug administration. The growth of NuE-derived tumors in L particle-injected rats was significantly suppressed, but not of the non-hepatic tumor control. In summary, this is the first demonstration that nanoparticles could be used for delivery of therapeutic genes with anti-tumor activity into human liver tumors. This intravenous delivery system may be one of the major advantages as compared to many other viral vector systems.

Efeito do pastejo rotacionado e alternado com bovinos adultos no controle da verminose em ovelhas / Effect of rotational and alternate grazing with adult cattle on the control of nematode parasites in sheep

Estudaram-se os efeitos do pastejo alternado de ovinos e bovinos e do pastejo rotacionado sobre o controle da verminose em ovelhas. Utilizou-se uma área experimental composta por três módulos de 1,67ha cada. Os módulos foram subdivididos em oito piquetes. Vinte ovelhas foram colocadas no módulo 1 e quatro bovinos adultos no módulo 2. Os animais permaneceram em cada piquete do módulo por cinco dias, totalizando 40 dias de permanência em cada módulo. Ao final desse período, as ovelhas foram transferidas para o módulo onde estavam os bovinos e estes para o módulo onde estavam os ovinos, mantendo esse esquema até o final do experimento. Um grupo-controle de 20 ovelhas foi mantido, também em sistema rotacionado, em um terceiro módulo, sem compartilhar a pastagem. As ovelhas submetidas ao manejo com bovinos apresentaram o menor grau de infecção por nematódeos gastrintestinais e os maiores valores de volume globular. O pastejo rotacionado de ovinos, sem a utilização de bovinos, não foi eficiente no controle da verminose das ovelhas. A utilização do pastejo rotacionado e alternado de ovinos e bovinos adultos exerceu efeito benéfico significativo no controle da verminose ovina.

Genotypic diversity of Salmonella Enteritidis isolates from sporadic patients in limited area during one year.

OBJECTIVES: Our aims were to investigate the genetic epidemiology of Salmonella enterica serovar Enteritidis (S. Enteritidis) isolates using computerized analysis of restriction enzyme cleavage patterns. METHODS: A total of 106 S. Enteritidis isolates which were collected in Hiroshima Prefecture, Japan in 2001 were tested by pulsed-field gel electrophoresis (PFGE) using BlnI and XbaI enzymes. PFGE profiles were analysed and compared by using Fingerprinting II software. RESULTS: BlnI PFGE analysis divided the isolates into 29 genotypes. At 90% similarity, BlnI cleavage grouped the isolates into 15 genotypes, while XbaI cleavage grouped them into only four. Two major clusters, each with a predominant genotype, were identified by BlnI cleavage at 42% similarity. In spite of the mixed circulation of the two predominant genotypes, one genotype for which a number of subtypes were detected was predominant during the first half of the year. In contrast, the other genotype, for which no variant subtypes were detected, followed during the latter half. The genotypes identified by computerized analysis matched well with those judged by visual inspection. CONCLUSIONS: The results confirmed the usefulness of PFGE performed with BlnI and of the Fingerprinting II software for the genotyping of S. Enteritidis. We think that the prevalent characteristics of the predominant genotypes detected here were related to the genetic variations of S. Enteritidis.

Promotion of beta-cell regeneration by betacellulin in ninety percent-pancreatectomized rats.

Betacellulin is thought to promote growth and differentiation of pancreatic beta-cells. We investigated the effect of betacellulin on regeneration of pancreatic beta-cells in 90%-pancreatectomized rats. Ninety percent pancreatectomy was performed in male Wistar rats and betacellulin (0.5 microg/g body weight) or saline was administered daily for 10 d starting immediately after pancreatectomy. In pancreatectomized rats, the morning-fed plasma glucose was significantly lower and the plasma insulin concentration was significantly higher in betacellulin-treated rats than those in control rats for up to 4 wk. Thirty days after pancreatectomy, a glucose tolerance test was performed. Betacellulin reduced the plasma glucose response to ip glucose loading. In control rats, the plasma insulin concentration was significantly lower and did not respond to glucose. In contrast, the plasma insulin concentration increased slightly but significantly in betacellulin-treated rats. Thirty days after pancreatectomy, the beta-cell mass was greater and the insulin content was significantly higher in betacellulin-treated rats than those in control rats. The numbers of islet cell-like cluster and bromodeoxy uridine/insulin double- positive cells in both islet cell-like cluster and islets were significantly higher in betacellulin-treated rats. These results indicate that administration of betacellulin improves glucose metabolism by promoting beta-cell regeneration in 90%-pancreatectomized rats.

Preparation of potent cytotoxic ribonucleases by cationization: enhanced cellular uptake and decreased interaction with ribonuclease inhibitor by chemical modification of carboxyl groups.

Carboxyl groups of bovine RNase A were amidated with ethylenediamine (to convert negative charges of carboxylate anions to positive ones), 2-aminoethanol (to eliminate negative charges), and taurine (to keep negative charges), respectively, by a carbodiimide reaction. Human RNase 1 was also modified with ethylenediamine. Surprisingly, the modified RNases were all cytotoxic toward 3T3-SV-40 cells despite their decreased ribonucleolytic activity. However, their enzymatic activity was not completely eliminated by the presence of excess cytosolic RNase inhibitor (RI). As for native RNase A and RNase 1 which were not cytotoxic, they were completely inactivated by RI. More interestingly, within the cytotoxic RNase derivatives, cytotoxicity correlated well with the net positive charge. RNase 1 and RNase A modified with ethylenediamine were more cytotoxic than naturally occurring cytotoxic bovine seminal RNase. An experiment using the fluorescence-labeled RNase derivatives indicated that the more cationic RNases were more efficiently adsorbed to the cells. Thus, it is suggested that the modification of carboxyl groups could change complementarity of RNase to RI and as a result endow RNase cytotoxicity and that cationization enhances the efficiency of cellular uptake of RNase so as to strengthen its cytotoxicity. The finding that an extracellular human enzyme such as RNase 1 could be effectively internalized into the cell by cationization suggests that cationization is a simple strategy for efficient delivery of a protein into cells and may open the way of the development of new therapeutics.

An enzyme-linked immunosorbent assay (ELISA) for detection of antibodies against Toxocara vitulorum in water buffaloes.

Toxocara vitulorum, a parasite of the small intestine of cattle and water buffaloes, is mainly acquired by calves via the colostrum/milk from infected cows. To understand the development of immune responses in calves, antibody levels to a soluble extract antigen (Ex) from T. vitulorum infective larvae were measured by an indirect ELISA with sera of 15 buffalo calves, which were sampled every 15 days for the first 180 days after birth and 9 buffalo cows during the perinatal period. From all serum samples examined during the first 180 days, antibody level was lowest and highest in calves at 1 day of age before and after suckling colostrum, respectively, suggesting that the origin of antibodies was the colostrum. Immediately after birth, antibody levels in suckled calves remained at high levels until day 15, began to decrease to lower levels between 15 and 30 days and remained relatively stable until 120 days. By comparing the immune responses of these animals with their parasitological status it was considered possible to determine if passively acquired or actively produced antibodies provided protection against the infection. High numbers of T. vitulorum eggs in the feces between 30 and 60 days indicated that passively acquired antibodies did not provide protection against the infection, at least during these first days, and the maximum fecal egg counts during 30-45 days were coincident with decreased antibody levels. Between 60 and 120 days, when serum antibodies were detected at reduced, but stable levels, adult nematodes were expelled from the intestines and no more T. vitulorum eggs were found, suggesting development of acquired resistance. However, the potential and functional protective role of the antibodies against T. vitulorum infection and the process of self-cure requires further investigation.

Physicochemical and immunological characterization of hepatitis B virus envelope particles exclusively consisting of the entire L (pre-S1 + pre-S2 + S) protein.

The hepatitis B virus (HBV) envelope (env) protein is composed of three regions; the 108- or 119-residue pre-S1 region involved in the direct interaction with hepatocytes, the 55-residue pre-S2 region associated with the polymerized albumin-mediated interaction, and the major 226-residue S protein region. Thus, to improve the immunogenic potency of conventional HB vaccines, development of a new vaccine containing the entire pre-S1 region in addition to pre-S2 and S is desired. We previously reported the efficient production of the HBV env L (pre-S1 + pre-S2 + S) protein in the recombinant yeast cells [J Biol Chem 267 (1992) 1953]. In this study, the HBV env L protein produced as nano-particles in yeast has been purified and characterized. By equilibrium sedimentation, an average molecular weight of L particle was estimated to be approximately 6.4 x 10(6), indicating that about 110 molecules of L proteins are assembled into an L particle. By atomic force microscopy in a moist atmosphere, the L particles were observed as large spherical particles with a diameter of 50-500 nm. The L particles were stable on short-time heating at a high temperature and long-time storage at a low temperature but rather unstable on repeated freezing and thawing and treatment with dithiothreitol. When immunized in mice, L particles elicited efficiently and simultaneously the anti-S, anti-pre-S2, and anti-pre-S1 antibodies. The ED(50) values in mice for the anti-S and anti-pre-S2 antibodies were similar to those elicited by the M (pre-S2 + S) particles. Furthermore, the anti-pre-S1 rabbit antibodies were found to recognize various segments of the pre-S1 region, including the pre-S1 (21-47) segment. These results show the high ability of L particles to induce all antibodies against HBV env proteins, hence promising the future application of L particles for the next generation HB vaccine.

Three-dimensional structure of human RNase 1 delta N7 at 1.9 A resolution.

Human pancreatic ribonuclease 1 (RNase 1) is considered to be the human counterpart of bovine pancreatic RNase A. Truncation of seven amino-acid residues from the amino-terminal sequence resulted in RNase 1 Delta N7, which has a reduced ribonucleolytic activity and a lower affinity for the human placental RNase inhibitor (PRI). This RNase 1 variant has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data to 1.9 A resolution. The molecule displays the alpha + beta folding topology typical of members of the RNase A superfamily. The main distinct features found in RNase 1 Delta N7 are basically located in three loops affecting the fitting of the enzyme to the active site of subtilisin and the shape of the B2 subsite. These changes, taken with the lack of the catalytically active residue Lys7, may explain the reduced affinity of RNase 1 Delta N7 for PRI and the low ribonucleolytic activity of the protein when compared with the native enzyme.

Molecular cloning and expression of rat betacellulin cDNA.

The cDNA encoding an entire open reading frame of rat betacellulin has been cloned from rat kidney. Expression of this cDNA in COS7 cells showed a significant amount of mitogenic activity in the culture media. Western blotting of the cell lysates suggested that the membrane-anchored precursor was cleaved to release its ectodomain very efficiently.