Effects of porcine epidemic diarrhea virus infection on Toll-like receptor expression and cytokine levels in porcine intestinal epithelial cells.

To explore the role of Toll-like receptors (TLRs) and interferon (IFN) in the innate immunity against porcine epidemic diarrhea virus (PEDV), we detected the expression of TLR genes in PEDV-infected IPEC-J2 cells by real-time PCR. We also detected the level of interferon α (IFN-α) and interferon γ (IFN-γ) by enzyme-linked immunosorbent assay (ELISA). Results showed that IPEC-J2 cells exhibited a clear pathological change after PEDV infection at 24 h. In addition, TLR7, TLR9 and TLR10 expressions were significantly upregulated in PEDV-infected IPEC-J2 cells at 24 h. Interestingly, the expression patterns of TLR2 and TLR4 were consistent at different stages of PEDV infection. The expression level of TLR3 decreased significantly with the increase of infection time, but the expression levels of TLR5 and TLR8 genes at 6 h and 12 h were significantly lower than those in the control group (p⟨0.01). There were significant correlations among the expression levels of TLR genes (p⟨0.05). Cytokine detection showed that the secretion level of IFN-α in the PEDV-infected group was significantly higher than that in the control group (p⟨0.01), and IFN-γ at 6 h and 12 h after PEDV infection was significantly higher than that in control group (p⟨0.01). Therefore, our results suggest that PEDV infection can induce innate immune responses in intestinal porcine jejunum epithelial cells, leading to changes in the expression of Toll-like receptors, and can regulate the resistance to virus infection by affecting the release levels of downstream cytokines.

Effects of porcine epidemic diarrhea virus infection on tight junction protein gene expression and morphology of the intestinal mucosa in pigs.

Tight junction proteins are important for the maintenance and repair of the intestinal mucosal barrier. The present study investigated relationships among tight junction protein gene expression, porcine epidemic diarrhea virus (PEDV) infection, and intestinal mucosal morphology in piglets. We compared the expression of six tight junction proteins (ZO-1, ZO-2, Occludin, Claudin-1, Claudin-4, and Claudin-5) between seven-day-old piglets infected with PEDV and normal piglets, as well as in PEDV-infected porcine intestinal epithelial cells (IPEC-J2). We also evaluated differences in mucosal morphology between PEDV-infected and normal piglets. The expression of six tight junction protein genes was lower in PEDV-infected piglets than in the normal animals. The expression of ZO-1, ZO-2, Occludin, and Claudin-4 in the intestine tissue was significantly lower (p⟨0.05) in PEDV-infected than in normal piglets. The expression of Claudin-5 in the jejunum was significantly lower in PEDV-infected piglets than in the normal animals (p⟨0.01). The expression of Claudin-1 and Claudin-5 genes in the ileum was significantly higher in PEDV-infected piglets than in normal piglets (p⟨0.01). Morphologically, the intestinal mucosa in PEDV-infected piglets exhibited clear pathological changes, including breakage and shedding of intestinal villi. In PEDV-infected IPEC-J2 cells, the mRNA expression of the six tight junction proteins showed a downward trend; in particular, the expression of the Occludin and Claudin-4 genes was significantly lower (p⟨0.01). These data suggest that the expression of these six tight junction proteins, especially Occludin and Claudin-4, plays an important role in maintaining the integrity of the intestinal mucosal barrier and resistance to PEDV infection in piglets.

Manipulation of group-velocity-locked vector dissipative solitons and properties of the generated high-order vector soliton structure.

Details of various composites of the projections originated from a fundamental group-velocity-locked vector dissipative soliton (GVLVDS) are both experimentally and numerically explored. By combining the projections from the orthogonal polarization components of the GVLVDS, a high-order vector soliton structure with a double-humped pulse profile along one polarization and a single-humped pulse profile along the orthogonal polarization can be observed. Moreover, by de-chirping the composite double-humped pulse, the time separation between the two humps is reduced from 15.36 ps to 1.28 ps, indicating that the frequency chirp of the GVLVDS contributes significantly to the shaping of the double-humped pulse profile.

Detection of QTLs controlling fast kernel dehydration in maize (Zea mays L.).

In order to understand the effect of grain moisture of inbred lines at the silking and physiological maturity stages on kernel dehydration rate, 59 maize inbred lines from six subgroups were selected. Grain moisture was measured and QTLs associated with kernel dehydration were mapped. A rapid dehydration evaluation and association analysis revealed eight inbred lines with faster dehydration rate, including Yuanwu 02, K36, Zhonger/O2, Lo1125, Han 49, Qi 319, Hua 160, and PH4CV. A single sequence repeat analysis using 85 pairs detected five QTLs with phenotypic variation contribution ≥10% in the permanent F2 generation populations Zheng 58 x S1776 and Chang 7-2 x K1131, which had LOD threshold values ≥ 3 in both 2013 and 2014. The chromosome region of qFkdr7b had not previously been reported and is preliminarily identified as a new major QTL. A false positive field verification of grain dehydration rate of 53 inbred lines indicated that the screening result of the rapid dehydration inbred lines by specific amplification with marker Phi114 was most similar to the field assessment result, followed by markers Phi127 and Phi029. The rapid dehydration lines selected based on primer Phi114 amplification were also similar to the field dehydration rate and can thus be used for molecular marker-assisted selection. A significant effort is needed to improve stress resistance and shorten the growth period via fast kernel dehydration in intermediate materials of the inbred lines K36, Zhonger/ O2, Lo1125, Han 49, Hua 160, and PH4CV, and further using the selected lines for new combinations.

Genetic analyses of the major and minor locus groups of bacterial wilt resistance in tobacco using a diallel cross design.

Tobacco germplasm samples with various levels of resistance to bacterial wilt were selected to construct F1 combinations of parental inbred lines and orthogonal diallel crosses using samples collected in 2009 (15 germplasms), 2010 (15 germplasms), and 2011 (16 germplasms). A total of 1/2P (P + 1) experimental materials were used for analysis. Based on the analyses of major and minor locus groups, genetic effects on the incidence rate and index of bacterial wilt in tobacco were investigated on the 15th and 25th day during the early stage. Significant effects were observed in major locus groups, but not in minor locus groups. Specifically, adjacent major locus groups (J1 = 13,056 and J1 = 13,055; J1 = 14,080 and J1 = 14,079) were detected in both the first and second analyses with considerable effects. Based on the additive effects of minor locus groups on the rate and index of bacterial wilt, the effects on the incidence rates of Yunyan 85, DB101, and RG11 as well as the effects on the disease index of the latter two germplasms reached the maximum. This was consistent with the disease resistance indicators of these tobacco varieties in the field (corresponding broad heritability >20%). Genetic homozygous dominant loci (+ +) increased the rate of bacterial wilt (susceptible), whereas homozygous recessive loci (- -) reduced the index of bacterial wilt (resistant) with considerable additive effects and low dominant effects, suggesting that the inheritance of the bacterial wilt rate and index in tobacco mainly relies on additive inheritance.

Association between polymorphisms in exons 4 and 10 of the BPI gene and immune indices in Sutai pigs.

The bactericidal/permeability-increasing protein (BPI) gene has been identified as a candidate gene for disease-resistance breeding. We evaluated whether polymorphisms in exons 4 and 10 of the BPI gene are associated with immune indices [interleukin-2 (IL-2), IL-4, IL-6, interferon-b (IFN-b), IL-10, and IL-12]. In this study, we identified one mutation (C522T) in the BPI exon 4 site and two mutations (A1060G and T1151G) in the BPI exon 10 site. Correlation analysis revealed that in the Sutai pig population, the effect of genotypes at the BPI exon 4 site on the level of IL-6 was significant (P < 0.05), with an effective genotype of CD; moreover, the effect of genotypes at the BPI exon 10 site on the level of IL-12 was significant (P < 0.05), and the effective genotype was AB. The optimal combined genotype was CD-AB, which was more effective regarding the IL-6 and IL-12 levels compared to the other combined genotypes (P < 0.05). These results indicate that single nucleotide polymorphisms and the combined genotypes of BPI exons 4 and 10 affect immune indices in Sutai pigs. Therefore, these genotypes should be further examined as effective markers for disease-resistant breeding of pigs.

Identification of BPI protein produced in different expression system and its association with Escherichia coli F18 susceptibility.

The super antibiotic bactericidal/permeability-increasing (BPI) protein is a member of a new generation of proteins that have been implicated as endotoxin-neutralizing agents. In this study, recombinant porcine BPI protein was obtained by generating porcine BPI encoding prokaryotic, eukaryotic, and yeast expression vectors. Recombinant protein expression was detected in yeast GS115, Escherichia coli, and 293-6E cells by gel electrophoresis and Western blotting. Escherichia coli F18 is the primary Gram-negative bacteria in the gut and the main pathogen leading to diarrhea and edema dis-ease in weaning piglets. Therefore, E. coli F18-resistant and -sensitive Sutai piglets were used to test differential expression of BPI protein by Western blotting and to investigate the potential correlation between BPI protein expression and E. coli F18-susceptibility. Recombinant porcine BPI protein expression was not detected in the prokaryotic and yeast expression systems; however, soluble protein was detected in the eukaryotic expression system. These data indicate the strong bacterio-static action of the BPI protein and confirm the feasibility of obtaining large amounts of recombinant porcine BPI recombinant protein using this eukaryotic expression system. In addition, the BPI protein expres-sion levels in the E. coli F18-resistant group were significantly higher than those in the sensitive group, indicating that high BPI protein ex-pression is associated with resistance to E. coli F18. Our findings pro-vide a basis for further investigations into the development of a drug designed to confer resistance to E. coli F18 in weaning piglets.

Influence of sirolimus-induced TGF-ß secretion on mouse Treg cell proliferation.

We examined the effects of co-culturing CD4+ CD25+ Treg cells with sirolimus or cyclosporin A on Treg cell proliferation and differentiation and on transforming growth factor-ß (TGF-ß) and Foxp3 expression. CD4+ CD25+ Treg cells were harvested from mononuclear cells of spleens of C57BL/6 mice using immunomagnetic beads and divided into control, sirolimus, and cyclosporine groups. Following a 96-h co-culture, Treg cells were assayed by flow cytometry. FoxP3 and TGF-ß mRNA levels and secretion were assayed by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Smad protein of the TGF-ß signaling pathway was assayed by western blot and its effect on CD4+ CD25+ FoxP3+ Treg cell proliferation was determined. Sirolimus-promoted differentiation and proliferation was examined using a TGF-ß neutralizing antibody. Sirolimus-treated CD4+ T cell TGF-ß secretion increased 2.5X over control levels (P < 0.01), but that of the cyclosporine group decreased marginally (P > 0.05). The CD4+ cell proportion decreased significantly (41.25 vs 69.22%, P < 0.01) and slightly (65.21 vs 69.22, P > 0.05) in the cyclosporine and sirolimus groups, respectively. T cell Foxp3 mRNA expression was significantly higher in the sirolimus-treated than in the cyclosporine (53.7 vs 40.2%, P < 0.05) and control groups (P < 0.01), but was significantly lower in the cyclosporine group than in controls (23.6 vs 40.2%, P < 0.01). Overall, sirolimus promoted CD4+ CD25+ Treg cell proliferation and growth in vitro, whereas cyclosporin A inhibited proliferation. Sirolimus might promote CD4+ CD25+ FoxP3+ regulatory T cell proliferation by inducing TGF-ß secretion in vivo.

Diversity and activity of sulphur-oxidizing bacteria and sulphate-reducing bacteria in landfill cover soils.

UNLABELLED: Sulphur bioconversion in landfill cover soils, including the metabolism of sulphur-oxidizing bacteria (SOB) and sulphate-reducing bacteria (SRB), is one of the important processes affecting H2 S emission from landfills. In this study, two landfills with or without landfill gas collection and utilization system were investigated to characterize the role of biotic and abiotic factors affecting diversity and activity of SOB and SRB in the landfill cover soils. The results revealed that the potential sulphur oxidation rates (SORs) and sulphate reduction rates (SRRs) varied with landfill sites and depths. SOR was significantly correlated with pH and SO4 (2-) , while SRR was significantly related with pH. The populations of both SOB and SRB were low in the acidic landfill cover soils (pH = 4.7-5.37). Cloning and terminal restriction fragment length polymorphism profiles of soxB and dsrB showed that SOB including Halothiobacillus, Thiobacillus, Thiovirga and Bradyrhizobium, and SRB including Desulfobacca, Desulforhabdus and Syntrophobacter dominated in the landfill cover soils, and their distributions were affected mainly by pH value and organic matter contents of soils. SIGNIFICANCE AND IMPACT OF THE STUDY: High diversity of sulphur-oxidizing bacteria (SOB) and sulphate-reducing bacteria (SRB) presented in the landfill cover soils. Among the physicochemical properties of soils (moisture content, pH, organic materials, SO4 (2-) , acid volatile sulphide and total sulphur), pH was the most important factor affecting the diversity and activity of SOB and SRB in the landfill cover soils. Higher pH of landfill cover soils (i.e. neutral or slight alkaline) was favourable for the growth of SOB and SRB, leading to a rapid bioconversion of sulphur. These findings are helpful to optimize sulphur biotransformation in landfill cover soils and to control odour pollution at landfills.

TLR4 gene expression in pig populations and its association with resistance to Escherichia coli F18.

TLR4 is the main recognition receptor of bacterial lipopolysaccharides, which play an important role in innate and adaptive immunity. We used real-time PCR to analyze the tissue expression profile and differential expression of TLR4 in 4 pig populations (Escherichia coli F18-resistant Sutai, E. coli F18-sensitive Sutai, Large White, Meishan), in order to determine the role that the TLR4 gene plays in resistance to E. coli F18. We found that TLR4 expressed consistently in the 4 populations, with relatively high levels in immune tissues and the highest level in the lung. Generally, the expression of TLR4 in E. coli F18-sensitive individuals was the highest, followed by that in E. coli F18-resistant, Large White and Meishan. In the spleen, lung, kidney, lymph nodes, and thymus gland, TLR4 expression is significantly higher in the E. coli F18-sensitive than in the other 3 populations; there were no significant differences among E. coli F18-resistant Sutai, Large White, and Meishan. In addition, Gene Ontology and pathway analysis showed that TLR4 takes part in the inflammatory response. We found that porcine TLR4 has consistent tissue specificity in each breed, and downregulation of expression of the TLR4 gene is related to resistance to E. coli F18 in weaning piglets.

Age-dependent expression of the BPI gene in Sutai piglets.

We compared and analyzed the expression of the BPI gene of Sutai piglets ranging from newborn to post-weaning days 8, 18, 30, and 35 by the real-time PCR method, in order to determine if it is involved in protection against disease caused by ETEC F18. There was a significant difference between 18 and 35-day expression in the jejunum. There were also significant differences between 35-day expression and expression at the other development stages in the duodenum. There were no significant differences in expression at 8, 18, and 30 days in the jejunum. We conclude that the porcine BPI gene may be the direct factor that resisted the ETEC F18 in weaning piglets, and that the resistance to ETEC F18 in weaning piglets is related to up-regulation of mRNA expression of BPI gene to a certain extent.

Mode-dependent effect of high-frequency electrical stimulation of the anterior thalamic nucleus on amygdala-kindled seizures in rats.

Deep brain stimulation (DBS) is an emerging treatment of epilepsy. Anterior nucleus of the thalamus (ANT) is considered to be an attractive target due to its close connection to the limbic structures and wide regions of neocortex. The present study aimed to investigate the effects of high frequency stimulation (HFS) targeting the ANT on amygdala-kindled seizures in Wistar rats in two different stimulation modes i.e. pre-treatment and post-treatment stimulations, mimicking the scheduled and responsive stimulations in clinical use respectively. When fully-kindled seizures were achieved by daily amygdala kindling (1 s train of 1 ms pulses at 60 Hz), HFS (15 min train of 100 µs pulses at 150 Hz and 450-800 µA) was applied in two modes for 10 days. Bilateral post-treatment with HFS reduced the incidence of generalized seizures and the mean behavioral seizure stage and shortened average afterdischarge duration (ADD) and generalized seizure duration (GSD), while bilateral pre-treatment with HFS resulted in a similar but much weaker inhibition of seizures. On the other hand, we also found the two stimulation modes both increased the afterdischarge threshold (ADT) and the differences of current intensity between ADT and generalized seizure threshold (GST) i.e. Δ(GST-ADT). However, Δ(GST-ADT) increased by at least 20 µA in bilateral post-treatment group, while less in bilateral pre-treatment group. Additionally, unilateral post-treatment with HFS failed to inhibit seizures. Our data show that anti-epileptic effect of bilateral post-treatment with HFS of ANT is much stronger than that of bilateral pre-treatment HFS, indicating bilateral responsive stimulation might be more appropriate for clinical anti-epileptic treatment of ANT HFS.

Cyclosporine or tacrolimus: which is the better partner for myfortic or cellcept?

BACKGROUND: Mycophenolic acid (MPA) pharmacokinetics using the mycophenolate mofetil (CellCept) formulation are known to differ between patients receiving tacrolimus (FK) or cyclosporine (CyA), but only limited data exist concerning concomitant use of FK or CyA with enteric-coated mycophenolate sodium (EC-MPS; Myfortic). This retrospective study compared the drug interactions with the mycophenolic acid blood levels using different immunosuppressants and their relation to graft survival. PATIENTS AND METHODS: We studied MPA levels in posttransplant sera from 298 renal transplant recipients. RESULTS: Patients receiving immunosuppression with CyA + Myfortic showed 94% at 5- and 10-year graft survivals, which were better than CyA + CellCept (75%, 63%). This combination suppressed posttransplant human leukocyte antigen (HLA) antibody development significantly (P = .03) with higher MPA levels. CONCLUSION: Patients immunosuppressed with CyA + Myfortic showed higher MPA levels and lower posttransplant HLA antibody development as well as the best graft survival. CyA + Myfortic or FK + Cellcept may be better combinations.

Wireless biopotential acquisition system for portable healthcare monitoring.

A complete biopotential acquisition system with an analogue front-end (AFE) chip is proposed for portable healthcare monitoring. A graphical user interface (GUI) is also implemented to display the extracted biopotential signals in real-time on a computer for patients or in a hospital via the internet for doctors. The AFE circuit defines the quality of the acquired biosignals. Thus, an AFE chip with low power consumption and a high common-mode rejection ratio (CMRR) was implemented in the TSMC 0.18-µm CMOS process. The measurement results show that the proposed AFE, with a core area of 0.1 mm(2), has a CMRR of 90 dB, and power consumption of 21.6 µW. Biopotential signals of electroencephalogram (EEG), electrocardiogram (ECG) and electromyogram (EMG) were measured to verify the proposed system. The board size of the proposed system is 6 cm × 2.5 cm and the weight is 30 g. The total power consumption of the proposed system is 66 mW.

Internet resources related to drug action and human response: a review.

It has been demonstrated that numerous proteins interact with drugs or their metabolites. Knowledge of these proteins is necessary to understand the mechanisms of drug action and human response. Progress in modern genetics, molecular biology, biochemistry and pharmacology is generating a comprehensive mechanistic understanding of drug-target interaction on the molecular level. This is valuable for researchers and pharmaceutical companies in their efforts to improve the efficacy of existing drugs and to discover new ones. Most recently, the integration of a systems biology approach into drug discovery processes calls for more holistic knowledge and easily accessible resources of the proteins that are important in drug action and human response. We have reviewed many publicly accessible internet resources of these proteins, according to their roles in drug action and human response, such as therapeutic effect, adverse reaction, absorption, distribution, metabolism and excretion.

Membrane bioreactor process of organic wastewater from brassylic acid manufacturing plant.

The wastewater treatment from brassylic acid manufacturing plant using membrane bioreactor (MBR) was studied. The membrane bioreactor consisted of batch-operation biological aeration tank and ultrafiltration evaluation tank. The content of test included the affection of variation operation conditions on ultrafiltration separation, the general characteristics of MBR process, and the difference comparing with the conventional biological treatment. The results are as follows: (1) among the test membrane material, polyether sulphone (PES) membrane is more suitable for the wastewater treatment; (2) when the cutoff molecular weight is among 10,000-50,000, the higher the cutoff molecular weight, the bigger the water flux is in the test; (3) under the operation pressure, water flux increases accompanying with the increasing of operation pressure; (4) the paper filtered COD concentration has more affection on the water flux than the suspended solid concentration; (5) as the volume loading of MBR increases, the accumulation of high molecule organic substance and colloid increases, the membrane permeate COD concentration and paper filtered COD concentration increase too, meanwhile the water flux reduces; (6) when the sludge retention time of activated sludge of MBR increases, the accumulation of high molecule organic substance and colloid reduces, the membrane permeate COD concentration and paper filtered COD concentration reduce too, and the water flux increases; (7) comparing with the conventional biological process, the microbial activity is higher, but the microbial species is less.

Gene structure for adenosine kinase in Chinese hamster and human: high-frequency mutants of CHO cells involve deletions of several introns and exons.

The structure for the adenosine kinase (AK) gene has been determined from Chinese hamster (CH) and human cells. The AK gene in CH is comprised of 11 exons ranging in length from 36 to 765 nt, with the majority <100 nt. The exact lengths of the intervening introns have not been determined, but most of them are indicated to be very large (>15 kb). A 6.6-kb fragment from human cells was also sequenced, and it contained only a single exon corresponding to exon 10 in CH. The BLAST searches of the subsequently released draft human genome sequence have revealed that the AK gene structure in human is identical to that in CH. In the human genome, the AK exons are distributed over four genomic clones totaling 752 kb, providing direct evidence that the AK gene in mammalian species is unusually large. In contrast to CH and human, the AK genes from several other eukaryotic organisms whose complete genomes are now known are quite small (between 1.2 and 2.5 kb) and either contain no introns (Saccharomyces cerevisiae and Schizosaccharomyces pombe) or various numbers of introns (Drosophila melanogaster [2], Caenorhabditis elegans [4], Arabidopsis thaliana [10]). Some of the intron-exon junctions in these species are in the same positions as in mammals. The AK gene in CH and human, as well as mouse, is linked upstream in a head-to-head fashion with the gene for the clathrin adaptor mu3 protein (or beta 3A subunit of the AP-3 protein complex), which is affected in type 2 Hermansky-Pudlak syndrome. These two genes are separated by <200 nt, and it is possible that they have a common or overlapping promoter(s). We have also determined the nature of the genetic alterations in two of the class A AK(-) mutants of CHO cells, which are obtained at a very high spontaneous frequency (10(-3)-10(-4)) in this cell line. Both mutants contained large deletions within the AK gene and greatly shortened AK transcripts. The cloning and sequencing of the transcripts from these mutants showed that the deletion in one of them led to the loss of exons 5 through 8, whereas in the other, all exons from 2 through 8 are deleted. The endpoints of these deletions lie in the large introns within the AK gene.

Crude protein content and amino acid composition in Taiwanese human milk.

Breast milk provides the essential nutrients for infants in readily available form. The content of nitrogen in human milk is of great importance because it relates to the growth of infants in the early stage, and the composition of nitrogenated compounds varies according to the lactational stage. Three-hundred-and-three human milk specimens were obtained from 240 healthy mothers living in two different districts in Taiwan, and 264 specimens were used for the analysis. The crude protein content, total and free amino acid compositions as well as urea content were evaluated using pooled milk samples according to different lactational stages and geographical location. The crude protein content decreased sharply from colostrum (2.51 g/100 mL) to mature milk (1.25 g/100 mL). Total amino acids account for 80-85% of the crude protein throughout the whole lactation period. Crude protein also contained 30 to 35 mg/ 100 mL urea and 41 to 48 mg/ 100 mL free amino acids as non-protein nitrogen components. The ratio of essential to non-essential amino acids remained constant throughout the lactation period in spite of a decline in amino acid content. The amino acid composition per 1 g of nitrogen varied during the lactation period. The differences of these lactational changing patterns of individual amino acids were probably reflected by variation of the protein composition during lactation. The sum of free amino acid content ranged from 43 to 50 mg/100 mL in Taipei and 40 to 45 mg/100 ml, in Kaohsiung. Although the variations of free amino acids during the lactation period differed among amino acids, glutamic acid predominated in mature milk while phosphoethanolamine was predominant in colostrum.

Cloning and some novel characteristics of mitochondrial Hsp70 from Chinese hamster cells.

The cDNA for Chinese hamster mitochondrial Hsp70 (mHsp70) was cloned and sequenced using a polymerase chain reaction probe based on conserved regions in the Hsp70 family of proteins. The encoded protein consists of 679 amino acids which includes a N-terminal mitochondrial targeting sequence of 46 amino acids. The mHsp70 protein contains several sequence signatures that are characteristics of prokaryotic and eukaryotic organellar Hsp70 homologs. In a phylogenetic tree based on Hsp70 sequences, it branches with the gram-negative proteobacteria, supporting the endosymbiotic origin of mitochondria from this group of prokaryotes. The mHsp70 cDNA was transcribed and translated in vitro and its import into isolated rat heart mitochondria was examined. The precursor mHsp70 was converted into a mature form of lower molecular mass (approximately 71 kDa) which became resistant to trypsin digestion. The import of mHsp70 into mitochondria was not observed in the presence of an uncoupler of energy metabolism or when the N-terminal presequence was lacking. The cDNA for mHsp70 was expressed in Escherichia coli and a polyclonal antibody to the purified recombinant protein was raised. The antibody shows no cross-reactivity to recombinant cytosolic Hsp70 protein and in 2-D gel blots it reacted specifically with the mHsp70 protein only. In immunofluorescence experiments, the antibody predominantly labeled mitochondria, and the observed labeling pattern was identical to that seen with a monoclonal antibody to the mitochondrial Hsp60 chaperonin. The affinity-purified antibody to mHsp70 was also employed to examine the subcellular distribution of the protein by cryoelectron microscopy and the immunogold-labeling technique. In these experiments, in addition to mitochondria, labeling with mitochondrial Hsp70 antibody was also observed on the plasma membrane and in unidentified cytoplasmic vesicles and granules. These studies raise the possibility that similar to the Hsp60 chaperonin and a number of other mitochondrial proteins, mHsp70 may have an extramitochondrial role.