Evaluation of chronic toxicological profile of herbo-mineral formulations: Shwaskas Chintamani Rasa and its marketed formulation namely Kas Shwas Hari Rasa.

BACKGROUND: Shwaskas Chintamani Rasa (SKC) and Kas Shwas Hari Rasa (KSH) are the Ayurvedic herbo-mineral formulations. These Ayurvedic formulations contain heavy metals which is the reason of concern and might bring up the safety issue. OBJECTIVE: This research article is aimed to study chronic toxicity of SKC and KSH for safety aspect in Wistar rats. MATERIAL AND METHOD: A study group of 220 healthy rats were divided into six groups. These rats were administered with SKC and KSH formulations where both the formulations were administered for 180 consecutive days. SKC was administered at doses of 58 mg/kg (equivalent to therapeutic dose i.e. TD), 145 mg/kg (2.5 TD), 290 mg/kg (5 TD) and KSH was administered at dose of 58 mg/kg (TD). According to OECD guideline 452, the effect of these formulations was examined on hematology, serum biochemistry and histopathology of various organs. RESULTS: Both the formulations did not produce any signs or symptoms of treatment related toxicity in both male and female Wistar rats at therapeutic dose (TD), 2.5 times TD and 5 times TD. CONCLUSION: Based on these findings, the NOAEL (No observed adverse effect level) for test formulations SKC and KSH tablets in male and female wistar rats concluded to be preclinically safe.

Impact of dietary deviation on disease progression and gut microbiome composition in lupus-prone SNF1 mice.

Environmental factors, including microbes and diet, play a key role in initiating autoimmunity in genetically predisposed individuals. However, the influence of gut microflora in the initiation and progression of systemic lupus erythematosus (SLE) is not well understood. In this study, we have examined the impact of drinking water pH on immune response, disease incidence and gut microbiome in a spontaneous mouse model of SLE. Our results show that (SWR × NZB) F1 (SNF1 ) mice that were given acidic pH water (AW) developed nephritis at a slower pace compared to those on neutral pH water (NW). Immunological analyses revealed that the NW-recipient mice carry relatively higher levels of circulating autoantibodies against nuclear antigen (nAg) as well as plasma cells. Importantly, 16S rRNA gene-targeted sequencing revealed that the composition of gut microbiome is significantly different between NW and AW groups of mice. In addition, analysis of cytokine and transcription factor expression revealed that immune response in the gut mucosa of NW recipient mice is dominated by T helper type 17 (Th17) and Th9-associated factors. Segmented filamentous bacteria (SFB) promote a Th17 response and autoimmunity in mouse models of arthritis and multiple sclerosis. Interestingly, however, not only was SFB colonization unaffected by the pH of drinking water, but also SFB failed to cause a profound increase in Th17 response and had no significant effect on lupus incidence. Overall, these observations show that simple dietary deviations such as the pH of drinking water can influence lupus incidence and affect the composition of gut microbiome.

Gender bias in lupus: does immune response initiated in the gut mucosa have a role?

The risk of developing systemic lupus erythematosus (SLE) is approximately nine times higher among women compared to men. However, very little is understood concerning the underlying mechanisms that contribute to this gender bias. Further, whether there is a link between immune response initiated in the gut mucosa, the progression of SLE and the associated gender bias has never been investigated. In this report, we show a potential link between the immune response of the gut mucosa and SLE and the gender bias of lupus for the first time, to our knowledge. Both plasma cell- and gut-imprinted- α4ß7 T cell frequencies were significantly higher in the spleen and gut mucosa of female (SWR × NZB)F1 (SNF1 ) mice compared to that of their male counterparts. Importantly, female SNF1 mice not only showed profoundly higher CD45(+) immune cell densities, but also carried large numbers of interleukin (IL)-17-, IL-22- and IL-9-producing cells in the lamina propria (LP) compared to their male counterparts. Intestinal mucosa of female SNF1 mice expressed higher levels of a large array of proinflammatory molecules, including type 1 interferons and Toll-like receptors 7 and 8 (TLR-7 and TLR-8), even before puberty. Our work, therefore, indicates that the gut immune system may play a role in the initiation and progression of disease in SLE and the associated gender bias.

Surveillance results of depleted uranium-exposed Gulf War I veterans: sixteen years of follow-up.

As part of a longitudinal surveillance program, 35 members of a larger cohort of 77 Gulf War I veterans who were victims of depleted uranium (DU) "friendly fire" during combat underwent a 3-day clinical assessment at the Baltimore Veterans Administration Medical Center (VAMC). The assessment included a detailed medical history, exposure history, physical examination, and laboratory studies. Spot and 24-h urine collections were obtained for renal function parameters and for urine uranium (U) measures. Blood U measures were also performed. Urine U excretion was significantly associated with DU retained shrapnel burden (8.821 mug U/g creatinine [creat.] vs. 0.005 mug U/g creat., p = .04). Blood as a U sampling matrix revealed satisfactory results for measures of total U with a high correlation with urine U results (r = .84) when urine U concentrations were >/=0.1 mug/g creatinine. However, isotopic results in blood detected DU in only half of the subcohort who had isotopic signatures for DU detectable in urine. After stratifying the cohort based on urine U concentration, the high-U group showed a trend toward higher concentrations of urine beta(2) microglobulin compared to the low-U group (81.7 v. 69.0 mug/g creat.; p = .11 respectively) and retinol binding protein (48.1 vs. 31.0 mug/g creat.; p = .07 respectively). Bone metabolism parameters showed only subtle differences between groups. Sixteen years after first exposure, this cohort continues to excrete elevated concentrations of urine U as a function of DU shrapnel burden. Although subtle trends emerge in renal proximal tubular function and bone formation, the cohort exhibits few clinically significant U-related health effects.

Health surveillance of Gulf War I veterans exposed to depleted uranium: updating the cohort.

A cohort of seventy-four 1991 Gulf War soldiers with known exposure to depleted uranium (DU) resulting from their involvement in friendly-fire incidents with DU munitions is being followed by the Baltimore Veterans Affairs Medical Center. Biennial medical surveillance visits designed to identify uranium-related changes in health have been conducted since 1993. On-going systemic exposure to DU in veterans with embedded metal fragments is indicated by elevated urine uranium (U) excretion at concentrations up to 1,000-fold higher than that seen in the normal population. Health outcome results from the subcohort of this group of veterans attending the 2005 surveillance visit were examined based on two measures of U exposure. As in previous years, current U exposure is measured by determining urine U concentration at the time of their surveillance visit. A cumulative measure of U exposure was also calculated based on each veteran's past urine U concentrations since first exposure in 1991. Using either exposure metric, results continued to show no evidence of clinically significant DU-related health effects. Urine concentrations of retinol binding protein (RBP), a biomarker of renal proximal tubule function, were not significantly different between the low vs. high U groups based on either the current or cumulative exposure metric. Continued evidence of a weak genotoxic effect from the on-going DU exposure as measured at the HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus and suggested by the fluorescent in-situ hybridization (FISH) results in peripheral blood recommends the need for continued surveillance of this population.

Health effects of subchronic exposure to diesel-water-methanol emulsion emission.

The U.S. Environmental Protection Agency's National Ambient Air Quality Standards for ozone and particulate matter (PM) require urban non-attainment areas to implement pollution-reduction strategies for anthropogenic source emissions. The type of fuel shown to decrease combustion emissions components versus traditional diesel fuel, is the diesel emulsion. The Lubrizol Corporation, in conjunction with Lovelace Respiratory Research Institute and several subcontracting laboratories, recently conducted a health assessment of the combustion emissions of PuriNOx diesel fuel emulsion (diesel-water-methanol) in rodents. Combustion emissions from either of two, 2002 model Cummins 5.9L ISB engines, were diluted with charcoal-filtered air to exposure concentrations of 125, 250 and 500 microg total PM/m3. The engines were operated on a continuous, repeating, heavy-duty certification cycle (U.S. Code of Federal Regulations, Title 40, Chapter I) using Rotella-T 15W-40 engine oil. Nitrogen oxide (NO) and PM were reduced when engines were operated on PuriNOx versus California Air Resources Board diesel fuel under these conditions. Male and female F344 rats were housed in Hazleton H2000 exposure chambers and exposed to exhaust atmospheres 6 h/day, five days/week for the first 11 weeks and seven days/week thereafter. Exposures ranged from 61 to 73 days depending on the treatment group. Indicators of general toxicity (body weight, organ weight, clinical pathology and histopathology), neurotoxicity (glial fibrillary acidic protein assay), genotoxicity (Ames assay, micronucleus, sister chromatid exchange), and reproduction and development were measured. Overall, effects observed were mild. Emulsion combustion emissions were not associated with neurotoxicity, reproductive/developmental toxicity, or in vivo genotoxicity. Small decreases in serum cholesterol in the 500-microg/m3 exposure group were observed. PM accumulation within alveolar macrophages was evident in all exposure groups. The latter findings are consistent with normal physiological responses to particle inhalation. Other statistically significant effects were present in some measured parameters of other exposed groups, but were not clearly attributed to emissions exposure. Positive mutagenic responses in several strains of Salmonella typhimurium were observed subsequent to treatment with emulsion emissions subfractions. Based on the cholesterol results, it can be concluded that the 250-microg/m3 exposure level was the no observed effect level. In general, biological findings in exposed rats and bacteria were consistent with exposure to petroleum diesel exhaust in the F344 rat and Ames assays.

Siva-1 negatively regulates NF-kappaB activity: effect on T-cell receptor-mediated activation-induced cell death (AICD).

Ligation of TCRs on stimulated T cells leads to activation-induced cell death (AICD) resulting in the downregulation of immune responses, a process essential for T-cell homeostasis. In this study, using transformed T-cell lines such as Jurkat and Do11.10 as cellular models of TCR-mediated AICD, we have demonstrated that the proapoptotic protein Siva-1 is required for TCR-induced apoptosis. Knockdown of Siva-1 rendered T cells specifically resistant to anti-CD3 but not Fas-induced apoptosis. Further, we observed that in Siva-1 knockout Jurkat cells, TCR-mediated activation of the canonical and non-canonical limbs of the NF-kappaB pathway are significantly enhanced as reflected by elevated nuclear levels of p65 and RelB, respectively. In addition, loss of endogenous Siva-1 also resulted in the enhanced expression of NF-kappaB- responsive anti-apoptotic genes such as Bcl-xL and c-FLIP. Interestingly, the c-FLIP(short) was detected only in TCR-ligated Siva-1 knockdown Jurkat cells. These results demonstrate a significant role for endogenous Siva-1, through its inhibitory effect on NF-kappaB activity, in TCR-mediated AICD with implications in peripheral tolerance, T-cell homeostasis and cancer.

Health effects of subchronic exposure to diesel-water emulsion emission.

The U.S. Environmental Protection Agency (EPA) National Ambient Air Quality Standards for ozone and particulate matter are requiring urban nonattainment areas to implement pollution-reduction strategies for anthropogenic source emissions. A type of fuel shown to decrease combustion emissions components versus traditional diesel fuels is the diesel-water emulsion. The Lubrizol Corporation in conjunction with Lovelace Respiratory Research Institute and several subcontracting laboratories recently conducted a rodent health assessment of inhaled combustion emissions of PuriNO(x) diesel fuel emulsion. Combustion emissions from either of two 2001 model Cummins 5.9-L ISB engines were diluted with charcoal-filtered air to exposure concentrations of 100, 200, and 400 microg total particulate matter/m(3). The engines were operated on a continuously repeating, heavy-duty certification cycle (U.S. Code of Federal Regulations, Title 40, Chapter I) using Rotella-T 15W-40 engine oil. Nitrogen oxide and particulate matter were reduced when engines were operated on PuriNO(x) versus California Air Resources Board diesel fuel under these conditions. Male and female F344 rats were housed in Hazleton H2000 exposure chambers and exposed to exhaust atmospheres 6 h/day, 5 days/wk for the first 11 wk and 7 days/wk threafter. Exposures ranged from 58 to 70 days, depending on the treatment group. Indicators of general toxicity (body weight, organ weight, clinical pathology, and histopathology), neurotoxicity (glial fibrillary acidic protein assay), genotoxicity (Ames assay, micronucleus, sister chromatid exchange), and reproduction and development were measured. Overall, effects observed were mild. Emulsion combustion emissions were not associated with neurotoxicity, reproductive/developmental toxicity, or in vivo genotoxicity. Small decreases in serum cholesterol and small increases in platelet values in some groups of exposed animals were observed. Particulate matter accumulation within alveolar macrophages was evident in all exposure groups. These findings are consistent with normal physiological responses to particle inhalation. Other statistically significant effects were present in some measured parameters of other exposed groups but were not clearly attributed to emissions exposure. Positive mutagenic responses in several strains of Salmonella typhimurium were observed subsequent to treatment with emulsion emissions subfractions. Based on the cholesterol and platelet results, it can be concluded that the 100 microg/m(3) exposure level was the no-observed-effect level. In general, biological findings in diesel emulsion emission-exposed animals and bacteria were consistent with exposure to petroleum diesel exhaust in the F344 rat and Ames assays.

The Siva-1 putative amphipathic helical region (SAH) is sufficient to bind to BCL-XL and sensitize cells to UV radiation induced apoptosis.

The human Siva gene is localized to chromosome 14q32-33 and gives rise to the full-length predominant form, Siva-1 and a minor alternate form, Siva-2 that appears to lack the proapoptotic properties of Siva-1. Our recent work has shown that the missing region in Siva-2 encodes a unique twenty amino acid putative amphipathic helical region (SAH, residues 36-55 in Siva-1). Despite the fact that Siva-1 does not belong to the BCL-2 family, it specifically interacts with the anti-apoptotic protein BCL-XL and sensitizes MCF7 breast cancer cells expressing BCL-XL to UV radiation induced apoptosis. Deletion mutagenesis has mapped the necessary region to the SAH in Siva-1. In this paper we demonstrate that the SAH region in Siva-1 is sufficient to specifically interact with the anti-apoptotic members of the BCL2 family such as BCL-XL and BCL-2 but not its apoptotic member BAX. Using transient transfections and direct microinjection of synthetic SAH peptides, we also demonstrate that the SAH region is sufficient to inhibit the BCL-XL mediated cell survival and render MDA-MB-231 and MCF7 breast cancer cells expressing BCL-XL highly susceptible to UV radiation induced apoptosis. The underlying mechanism of action of SAH mediated inhibition of BCL-XL (and/or BCL2) cell survival appears to be due to loss of mitochondrial integrity as reflected in enhanced cytochrome c release leading to the activation of caspase 9 and finally caspase 3.

Productivity improvement in xanthan gum fermentation using multiple substrate optimization.

A novel and more comprehensive formulation of the optimal control problem that reflects the operational requirements of a typical industrial fermentation has been proposed in this work. This formulation has been applied to a fed-batch bioreactor with three control variables, i.e., feed rates of carbon source, nitrogen source, and an oxygen source, to result in a 148.7% increase in product formation. Xanthan gum production using Xanthomonas campestris has been used as the model system for this optimization study, and the liquid-phase oxygen supply strategy has been used to supply oxygen to the fermentation. The formulated optimization problem has several constraints associated with it due to the nature of the system. A robust stochastic technique, differential evolution, has been used to solve this challenging optimization problem. The infinite dimensional optimization problem has been approximated to a finite dimensional one by control vector parametrization. The state constraints that are path constraints have been addressed by using penalty functions and by integrating them over the total duration to ensure a feasible solution. End point constraints on final working volume of the reactor and on the final residual concentrations of carbon and nitrogen sources have been included in the problem formulation. Further, the toxicity of the oxygen source, H(2)O(2), has been addressed by imposing a constraint on its maximum usable concentration. In addition, the initial volume of the bioreactor contents and feed concentrations have been handled as decision variables, which has enabled a well-grounded choice for their values from the optimization procedure; adhoc values are normally used in the industry. All results obtained by simulation have been validated experimentally with good agreements between experimental and simulated values.

Re-evaluation of the mutagenic potential of quinacrine dihydrochloride dihydrate.

Quinacrine has been used for voluntary female non-surgical sterilization for its ability to produce tubal occlusion. Safety issues regarding quinacrine have been raised because it has been shown to intercalate with DNA. Therefore, safety issues need to be resolved by appropriate toxicology studies to support a review for human transcervical use. Such toxicology studies include mutagenicity assays. Here we report an evaluation of the genotoxicity of quinacrine dihydrochloride dihydrate (QH) using a battery of assays. In the bacterial mutagenicity assay, QH was strongly positive in Salmonella typhimurium tester strain TA1537 with and without S9-activation and in S. typhimurium tester strain TA98 with S9-activation; QH was also strongly positive in Escherichia coli WP2 uvrA without S9-activation. QH was not mutagenic in S. typhimurium tester strains TA100 and TA1535 with and without S9-activation. QH was mutagenic in the mouse lymphoma assay in the absence of S9-activation. QH was clastogenic in Chinese hamster ovary (CHO) cells, with and without S9-activation. QH was negative for polyploidy in the same chromosome aberration test. Using a triple intraperitoneal injection treatment protocol in both male and female mice, QH was negative in the in vivo mouse micronucleated erythrocyte (micronucleus) assay. These results confirm that QH is mutagenic and clastogenic in vitro and suggest a potential risk to human health due to QH exposure after intrauterine exposure.

Lack of mutagenic activity of 1,6-hexamethylene diisocyanate.

1,6-Hexamethylene diisocyanate (HDI) is an aliphatic diisocyanate used in the manufacture of higher molecular weight biuret and trimer polyisocyanate resins. These resins are commonly used in polyurethane paints, resulting in potential occupational, and to a lesser extent consumer exposures. Because some isocyanates have been reported to be mutagenic, HDI was tested in the bacterial reverse mutation assay (Ames test), CHO/HGPRT gene mutation assay, and in the mouse micronucleus test, using vapor-phase exposures. Although indicators of toxicity were observed in each test, HDI did not induce mutagenic or clastogenic effects in any of the three assays.

An in vivo mouse micronucleus assay on musk ketone.

Musk ketone (3,5-dinitro-2,6-dimethyl-4-tert-butyl-acetophenone) was evaluated in an in vivo mouse micronucleus assay. Male and female mice were dosed with 250, 500 or 1000 mg musk ketone/kg body weight by a single intraperitoneal injection in corn oil. Results of the assay showed that under the conditions of this test evaluated at 24, 48 and 72 h after dosing, musk ketone did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice at any dose or any time period. Musk ketone was considered to be negative in the mouse in vivo micronucleus test as well as in a battery of previously published in vitro genotoxicity tests. Based on the total weight of evidence available, it was concluded that musk ketone does not have significant potential to act as a genotoxic carcinogen.

Isoenzymes of pyruvate dehydrogenase phosphatase. DNA-derived amino acid sequences, expression, and regulation.

Pyruvate dehydrogenase phosphatase (PDP) is one of the few mammalian phosphatases residing within the mitochondrial matrix space. It is responsible for dephosphorylation and reactivation of the pyruvate dehydrogenase complex (PDC) and, by this means, is intimately involved in the regulation of utilization of carbohydrate fuels in mammals. PDP is a dimeric enzyme consisting of catalytic and regulatory subunits. The catalytic subunit of PDP is a Mg2+-dependent enzyme homologous to the cytosolic phosphatases of the 2C family. In the present study, we isolated two cDNAs encoding for mitochondrial phosphatases. The first cDNA is highly homologous to the previously identified cDNA encoding for the catalytic subunit of PDP (PDP1). The second cDNA encodes a previously unknown catalytic subunit of PDP (PDP2). The new phosphatase, expressed as the recombinant protein in Escherichia coli, shows strict substrate specificity toward PDC and does not use phosphorylated branched chain alpha-ketoacid dehydrogenase as substrate. Like PDP1, PDP2 is a Mg2+-dependent enzyme, but its sensitivity to Mg2+ ions is almost 10-fold lower than that of PDP1. In contrast to PDP1, PDP2 is not regulated by Ca2+ ions. Instead, it is sensitive to the biological polyamine spermine, which, in turn, has no effect on the enzymatic activity of PDP1. Western blot analysis of PDP extracted from mitochondria isolated from liver and skeletal muscle revealed that PDP1 is predominantly expressed in mitochondria from skeletal muscle, whereas PDP2 is much more abundant in the liver rather than muscle mitochondria. Both isoenzymes are expressed in mitochondria from 3T3-L1 adipocytes, but the level of expression of PDP2 is considerably higher. These observations are consistent with previous findings on the enzymatic parameters of PDP in adipose tissue. Thus, our results provide the first evidence that there are at least two isoenzymes of PDP in mammals that are different with respect to tissue distribution and kinetic parameters and, therefore, are likely to be different functionally.

Characterization of a complex translocation [t(4;9;22)(p16;q34;q11)] in chronic myelogenous leukemia by fluorescence in situ hybridization technique.

A patient was referred with a high leukocyte count and diagnosed with chronic myelogenous leukemia (CML). Although practically asymptomatic since the time of diagnosis, he had a variable and inconsistent response to treatment. All of his bone marrow cells had a complex, three-way translocation, involving chromosomes 4, 9 and 22. Translocation of chromosome 4 to chromosome 9 was undetectable by routine cytogenetic techniques; however, by the fluorescence in situ hybridization technique, a three-way translocation was identified, 46,XY,t(4;9;22)(p16;q34;q11). Although, other chromosomes are frequently involved in complex or variant translocations with chromosome 9 and 22, participation of chromosome 4 is a very rare event. So far, two previous cases have been described in the literature with translocations involving chromosome 4p16. We present a third case of CML having similar break points whose clinical presentation is unusual.

Diversity of the pyruvate dehydrogenase kinase gene family in humans.

Recent evidence from this laboratory indicates that at least two isoenzymic forms of pyruvate dehydrogenase kinase (PDK1 and PDK2) may be involved in the regulation of enzymatic activity of mammalian pyruvate dehydrogenase complex by phosphorylation (Popov, K.M., Kedishvili, N.Y., Zhao, Y., Gudi, R., and Harris, R.A. (1994) J. Biol. Chem. 269, 29720-29724). The present study was undertaken to further explore the diversity of the pyruvate dehydrogenase kinase gene family. Here we report the deduced amino acid sequences of three isoenzymic forms of PDK found in humans. In terms of their primary structures, two isoenzymes identified in humans correspond to rat PDK1 and PDK2, whereas a third gene (PDK3) encodes for a new isoenzyme that shares 68% and 67% of amino acid identities with PDK1 and PDK2, respectively. PDK3 cDNA expressed in Eschierichia coli directs the synthesis of a polypeptide with a molecular mass of approximately 45,000 Da that possesses catalytic activity toward kinase-depleted pyruvate dehydrogenase. PDK3 appears to have the highest specific activity among the three isoenzymes tested as recombinant proteins. Tissue distribution of all three isoenzymes of human PDK was characterized by Northern blot analysis. The highest amount of PDK2 mRNA was found in heart and skeletal muscle, the lowest amount in placenta and lung. Brain, kidney, pancreas, and liver expressed an intermediate amount of PDK2 (brain > kidney = pancreas > liver). The tissue distribution of PDK1 mRNA differs markedly from PDK2. The message for PDK1 was expressed predominantly in heart with only modest levels of expression in other tissues (skeletal muscle > liver > pancreas > brain > placenta = lung > kidney). In contrast to PDk1 and PDK2, which are expressed in all tissues tested, the message for PDK3 was found almost exclusively in heart and skeletal muscle, indicating that PDK3 may serve specialized functions characteristic of muscle tissues. In all tissues tested thus far, the level of expression of PDK2 mRNA was essentially higher than that of PDK1 and PDK3, consistent with the idea that PDK2 is a major isoenzyme responsible for regulation of pyruvate dehydrogenase in human tissues.

Multirate state and parameter estimation in an antibiotic fermentation with delayed measurements.

This article discusses issues related to estimation and monitoring of fermentation processes that exhibit endogenous metabolism and time-varying maintenance activity. Such culture-related activities hamper the use of traditional, software sensor-based algorithms, such as the extended kalman filter (EKF). In the approach presented here, the individual effects of the endogenous decay and the true maintenance processes have been lumped to represent a modified maintenance coefficient, m(c). Model equations that relate measurable process outputs, such as the carbon dioxide evolution rate (CER) and biomass, to the observable process parameters (such as net specific growth rate and the modified maintenance coefficient) are proposed. These model equations are used in an estimator that can formally accommodate delayed, infrequent measurements of the culture states (such as the biomass) as well as frequent, culture-related secondary measurements (such as the CER). The resulting multirate software sensor-based estimation strategy is used to monitor biomass profiles as well as profiles of critical fermentation parameters, such as the specific growth for a fed-batch fermentation of Streptomyces clavuligerus.

Molecular cloning of the p45 subunit of pyruvate dehydrogenase kinase.

Purified preparations of rat heart pyruvate dehydrogenase kinase have two polypeptides with molecular weights of 48,000 (p48) and 45,000 (p45). Recently, we reported the primary structure of p48 (Popov, K. M., Kedishvili, N. Y., Zhao, Y., Shimomura, Y., Crabb, D. W., and Harris, R. A. (1993) J. Biol. Chem. 268, 26602-26606) and presented evidence that (i) it exhibits kinase activity for pyruvate dehydrogenase and (ii) it belongs to a family of mitochondrial protein kinases unique from other eukaryotic protein kinases. Here, we report the molecular cloning and deduced amino acid sequence of p45. The protein sequence of p45 has 70% identity to the protein sequence of p48. Minor differences exist throughout the protein sequences with the greatest difference occurring at the amino termini. Recombinant p45 protein, expressed in Escherichia coli and purified to homogeneity, catalyzed the phosphorylation and inactivation of kinase-depleted pyruvate dehydrogenase complex, indicating that p45 and p48 correspond to different isoforms of pyruvate dehydrogenase kinase. Northern blot analysis revealed a single hybridizing species of 2.5 kilobases. The highest level of p45 message expression was found in heart and skeletal muscle and the lowest in spleen and lung. Liver, kidney, brain, and testis express intermediate amounts of p45 mRNA. In contrast, p48 mRNA is predominantly expressed in heart, with other tissues expressing only a modest amount of this message. Tissue-specific expression of isoforms of pyruvate dehydrogenase kinase may indicate the existence of tissue-specific mechanisms for the regulation of pyruvate dehydrogenase activity.