A postpartum haemorrhage package with condom uterine balloon tamponade: a prospective multi-centre case series in Kenya, Sierra Leone, Senegal, and Nepal.

OBJECTIVE: To evaluate the effectiveness and safety of an ultra-low-cost uterine balloon tamponade package (ESM-UBT™) for facility-based management of uncontrolled postpartum haemorrhage (PPH) in Kenya, Sierra Leone, Senegal, and Nepal. DESIGN: Prospective multi-centre case series. SETTING: Facilities in resource-scarce areas of Kenya, Sierra Leone, Nepal, and Senegal. POPULATION: Women with uncontrolled postpartum haemorrhage in 307 facilities across the four countries. METHODS: A standardised ESM-UBT package was implemented in 307 facilities over 29 months (1 September 2012 to 1 February 2015). Data were collected via a multi-pronged approach including data card completion, chart reviews, and provider interviews. Beginning in August 2014, women who had previously undergone UBT placement were sought and queried regarding potential complications associated with UBT use. MAIN OUTCOME MEASURES: All-cause survival, survival from PPH, and post-UBT use complications (surgery, hospitalisation, antibiotics for pelvic infection) associated with UBT use. RESULTS: 201 UBTs were placed for uncontrolled vaginal haemorrhage refractory to all other interventions. In all, 38% (71/188) of women were either unconscious or confused at the time of UBT insertion. All-cause survival was 95% (190/201). However, 98% (160/163) of women survived uncontrolled PPH if delivery occurred at an ESM-UBT online facility. One (1/151) potential UBT-associated complication (postpartum endometritis) was identified and two improvised UBTs were placed in women with a ruptured uterus. CONCLUSIONS: These pilot data suggest that the ESM-UBT package is a clinically promising and safe method to arrest uncontrolled postpartum haemorrhage and save women's lives. The UBT was successfully placed by all levels of facility-based providers. Future studies are needed to further evaluate the effectiveness of ESM-UBT in low-resource settings. TWEETABLE ABSTRACT: Evidence for ESM-UBT as a clinically promising and safe method to arrest uncontrolled PPH and save women's lives.

First Confirmed Report of Sugar Beet Cyst Nematode, Heterodera schachtii, in North Dakota.

Sugar beet (Beta vulgaris L.) and canola (Brassica napus L.) are major crops in North Dakota, with sugar beet production primarily in the eastern part of the state in the Red River Valley and canola production across the northern half of the state. Both crops are hosts of sugar beet cyst nematode (SBCN), Heterodera schachtii Schmidt. In April 2011, soil samples were collected from four sugar beet fields belonging to three growers who believed the fields were infested with SBCN. The fields were located in a 65-km2 area in the Yellowstone Valley of western North Dakota. Cysts were extracted by sieving and Heterodera-like cysts with eggs were observed in all four soil samples. Population densities in the four fields ranged from 100 to 1,750 eggs/100 cm3 soil. Sugar beet seedlings (cv. M832224) were grown in a potting mix for 6 weeks in the greenhouse and then transferred to conetainers (type D40; volume 656 ml) containing autoclaved river sand. Conetainers were placed in sand in plastic pots immersed in a water bath at 27°C. Three plants were each infested with 800 eggs from field No. 2. After 55 days of incubation, the average number of females was 115 per plant. A similar experiment was conducted with canola cvs. Hyclass 940, Caliber 30, and Westar, which were inoculated with 500 eggs each from field No. 2. After 53 days of incubation, there was an average of 39, 20, and 30 females for each respective cultivar. Flask-shaped cysts (n = 26) from canola roots were light to dark brown; the vulval cone was ambifinestrate with dark brown, molar-shaped bullae positioned underneath the vulval bridge. Body length (excluding neck) ranged from 600 to 850 µm (mean 701.2 µm); body width, 350 to 580 µm (mean 469.2 µm); and length/width ratio, 1.2 to 1.8 (mean 1.5). Second-stage juvenile (J2) (n = 21) body length ranged from 400 to 485 µm (mean 437.1 µm); stylet length was 25 µm (no variation) with forwardly directed knobs; conical tail with rounded tip ranged from 37.5 to 55.0 µm long (mean 46.6 µm) with hyaline region from 20.0 to 32.5 µm (mean 27.3 µm); and lateral field presented four incisures. These morphometrics were used to identify H. schachtii according to Subbotin et al. (4). Confirmation of identification was by amplification and sequencing of a 28S rDNA gene fragment (1) from individual females (GenBank Accession No. JQ040526), which was 100% identical to H. schachtii 28S rDNA sequence (GenBank Accession No. GU475088). To our knowledge, this is the first confirmed report of H. schachtii in North Dakota. A 1958 report of SBCN in North Dakota (2) was not subsequently confirmed (3). Because there is extensive canola production across the northern part of the state bordering western and eastern sugar beet- production areas, canola may serve as a bridge for movement of SBCN from west to east. SBCN is a potential threat to these two important crops. References: (1) A. Amiri et al. Eur. J. Plant Pathol. 108:497, 2002. (2) F. Caveness. J. Sugar Beet Res. 10:544, 1958. (3) P. Donald and R. Hosford. Plant Dis. 64:45, 1980. (4) S. A. Subbotin et al. Systematics of Cyst Nematodes (Nematoda: Heteroderinae). Nematology Monographs and Perspectives. Vol. 8B. Brill, The Netherlands. 2010.

Characterization of the hepatic and splenic immune status and immunoglobulin synthesis in aged male mice lacking the peroxisome proliferator-activated receptor-alpha (PPARα).

It is now well established that the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPARα) is expressed in different types of immune cells and plays a pivotal role in the regulation of age-related production of inflammatory cytokines. However, the role(s) of this receptor in the regulation of immune cell homoeostasis in ageing non-lymphoid and lymphoid organs has not yet been resolved. We examine this issue here by evaluating the hepatic and splenic immune status and immunoglobulin (Ig) production in male PPARα-null mice and their wild-type littermates at one and 2 years of age. In comparison with the age-matched control animals, PPARα-null mice exhibited age-related elevations in the numbers of total, as well as of phenotypically distinct subpopulations of intrahepatic immune cells (IHIC) and splenocytes. Moreover, at 2 years of age, these alterations in hepatic immune cells were accompanied by significant increases in hepatic levels of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interferon-gamma (IFN-γ), in combination with the development of hepatic inflammatory loci containing mixtures of leucocytes. Alterations in splenocytes of old PPARα-null mice were also accompanied by increases in cellularity of both white and red pulps of the spleen. Furthermore, these same animals exhibited pronounced increases in the numbers of splenic plasma cells and enhanced production of Ig of different isotypes, including IgG1, IgG2a and IgE. Thus, our findings indicate that upon ageing, PPARα plays a crucial role in regulating the total numbers, compositions and functions of immune cells in both lymphoid and non-lymphoid immune organs of mice.

Isolation of murine intrahepatic immune cells employing a modified procedure for mechanical disruption and functional characterization of the B, T and natural killer T cells obtained.

Intrahepatic immune cells (IHIC) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. In the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of IHIC. These cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and DNase. The mechanical disruption yielded viable IHIC in considerably greater numbers than those obtained following enzymatic digestion. The IHIC isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which B, T, natural killer (NK), NK T cells, granulocytes and macrophages were the major populations (constituting 37.5%, 16.5%, 12.1%, 7.9%, 7.9% and 7.5% of the total number of cells recovered respectively). The IHIC obtained following enzymatic digestion contained markedly lower numbers of NK T cells (1.8%). The B, T and NK T cells among IHIC isolated employing mechanical disruption were found to be immunocompetent, i.e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin A and alpha-galactosylceramide respectively) and produced immunoglobulin M and interferon-gamma. Thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent IHIC for various types of investigation.

First Report of Soybean Cyst Nematode (Heterodera glycines) on Soybean in North Dakota.

During August 2003, soybean (Glycine max) plants from Richland County, North Dakota with white-to-yellow, lemon-shaped structures on the roots were brought to the North Dakota State University Plant Diagnostic Laboratory. To confirm that the structures were females of a cyst nematode, they were crushed and observed microscopically to determine if nematode eggs and second-stage juveniles were present. Morphology of the second-stage juveniles was consistent with Heterodera glycines, the soybean cyst nematode (SCN). A survey was conducted in soybean fields in 34 km2 around the field in which the samples originated. Ten of twenty fields surveyed had visible females on the roots of plants. Symptoms observed in those fields included patches of stunted, chlorotic, and dead plants. Soil samples were collected from selected areas within eight fields, eggs were extracted using standard soil sieving techniques, and egg numbers were determined. Egg numbers ranged from 550 to 20,000 eggs per 100 cm3 of soil. SCN collected from two different fields, designated as Dwight and LaMars, were used to determine their HG Type. Standardized procedures (1) were used in a growth chamber set at 27°C with 16-h days. Pots in the test were organized in a completely randomized design with three replicates; the test was repeated over time. After 30 days, females were extracted from roots and counted, and a female index (FI) was calculated for each indicator line (1). The mean number of females on susceptible standard cv. Lee 74, was 110. The Dwight SCN population had an FI of 5.3 on plant introduction (PI) 88788, 1.5 on PI 209332, 5.8 on PI 548316 (Cloud), and 0 on all other indicator lines. The LaMars population had an FI of 1.0 on PI 88788, 3.1 on PI 548316 (Cloud), and 0 on all other indicator lines. These results indicate that both SCN populations tested are HG Type 0. To our knowledge, this is the first report of SCN on soybean in North Dakota. Because other hosts of SCN, as well as soybean, are economically important in North Dakota, such as dry edible bean (Phaseolus vulgaris) and dry pea (Pisum sativum), this disease could adversely impact several commodities throughout the state. Reference: (1) T. L. Niblack et al. J. Nematol. 34:279, 2002.

Activity of the human cytochrome c1 promoter is modulated by E2F.

The human cytochrome c(1) promoter is strongly activated in transfected Drosophila SL2 cells expressing exogenous human E2F1. Transfection-deletion experiments, DNase I protection by E2F1 and gel mobility-shift experiments locate E2F1 activation sites to two regions on either side of the transcription start site. Deletion of either region prevents E2F1 activation in transfected SL2 cells, suggesting a co-operative interaction between them. E2F6, a member of the E2F family that lacks transactivation domains but contains specific suppressor domains, inhibits cytochrome c(1) promoter activity when co-transfected into HeLa cells, indicating that the E2F proteins modulate the cytochrome c(1) promoter in mammalian cells. However, E2F is not a general regulator of oxidative phosphorylation genes since three additional nuclear-encoded mitochondrial genes were unaffected by E2F1 or E2F6.

On the role of the general transcription factor Sp1 in the activation and repression of diverse mammalian oxidative phosphorylation genes.

To gain insight into the role of the general transcription factor, Sp1, in the expression of nuclear genes involved in mitochondrial biogenesis, we investigated Sp1 activation of the adenine nucleotide translocator 2, cytochrome c1, F1-ATPase beta subunit, and the mitochondria transcription factor (mtTFA) promoters transfected into Drosophila cell lines. The numbers and organization of GC elements vary in the four promoters, but the magnitude of activation by coexpressed human Sp1 was similar. A feature common to the four promoters is the presence of multiple, proximal Sp1-activating elements that account for 50% or more of the transcription activation by Sp1. The distribution and function of individual distal Sp1 elements is less defined and appear to be more promoter-specific. Finally, data from transfected Drosophila cells provide the first direct proof for the involvement of Sp1 in the negative regulation of the ANT2 promoter and as a possible participant in repression of the beta-subunit promoter. The role of Sp1 in both the positive and negative regulation of OXPHOS promoters is unique.

Enhanced mitochondrial biogenesis is associated with increased expression of the mitochondrial ATP-dependent Lon protease.

Rats bearing the Zajdela hepatoma tumor and T3-treated hypothyroid rats were used to study the role of protein degradation in the process of mitochondrial biogenesis. It was shown that the activity, protein and mRNA levels of the ATP-dependent Lon protease increased in rapidly growing Zajdela hepatoma cells. The increase in the rate of mitochondrial biogenesis by thyroid hormone was similarly accompanied by enhanced expression of the Lon protease. The results imply that mitochondrial biogenesis in mammalian cells is, at least partially, regulated by the matrix Lon protease.

Characterization of a silencer element and purification of a silencer protein that negatively regulates the human adenine nucleotide translocator 2 promoter.

Expression of adenine nucleotide translocator isoform 2 (ANT2) is growth regulated. In the present study, we report the presence of a silencer region in the human ANT2 promoter and the purification of a two-component factor that recognizes a specific hexanucleotide element, GTCCTG, of the silencer. Transfection of deletion constructs shows that ANT2 silencer activity extends over a region of at least 310 nts. However, mutating the GTCCTG element completely relieves silencing activity in the context of the human ANT2 promoter. The data suggest that the GTCCTG element might be required for maintaining silencer activity of the extended silencer region. The ANT2 silencer region cloned in front of the herpes simplex virus thymidine kinase promoter confers nearly complete inhibition to the heterologous promoter. However, unlike the ANT2 promoter, mutating the GTCCTG element restores only partial activity to the herpes simplex virus thymidine kinase promoter. A protein complex consisting of two major polypeptides of 37 and 49 kDa was isolated from HeLa nuclear extracts by affinity chromatography using the GTCCTG element as the affinity resin. Cross-linking studies and Southwestern analysis indicate that p37 binds DNA. p49 appears to be loosely associated with the p37/DNA complex but is necessary for strong binding of p37. Our data implicating the GTCCTG element directly in silencing of the ANT2 promoter, together with data from the literature reporting the presence of this element within the silencer region of several additional promoters, suggest a general role of the GTCCTG element in transcriptional silencing.

Thyroid hormone activates transcription from the promoter regions of some human nuclear-encoded genes of the oxidative phosphorylation system.

Thyroid hormone (T3) modulates the mRNA levels for cytochrome c and the adenine nucleotide translocator-2 (ANT2) in adult rat liver. Here we show that T3 activates expression of a reporter gene driven from the human cytochrome c1 and ANT2 promoters transfected into human choriocarcinoma JEG3 cells. By contrast, the human F1-ATPase beta-subunit promoter responded marginally, thus providing a pattern of differential expression similar to that earlier observed in rats in vivo. T3-activation is dependent on co-expression of the thyroid hormone receptor (TR alpha1). Co-expression of both the TR and RXR receptors had no additional effect. Transient transfection of deletion constructs showed that T3 activation is retained by the proximal regions of the cytochrome c1 and ANT2 promoters, and, in the case of cytochrome c1, is lost upon removal of a fragment containing the transcription initiator ((nucleotides) (nt) + 1 to + 100). The promoter regions supporting T3-activation of the reporter genes appear to lack strong DNA binding sites for TR and retinoid X receptor (RXR).

The role of thyroid hormone and promoter diversity in the regulation of nuclear encoded mitochondrial proteins.

Thyroid hormone regulates the in vivo expression of a selected set of rat nuclear genes encoding mitochondrial inner membrane proteins. Certain mRNAs, such as that for cytochrome c1, are increased as much as 20-50-fold, while others, such as core protein 1 of Complex III and the F1-ATPase beta-subunit do not respond. The promoter region of human cytochrome c1 also supports thyroid hormone induction of a reporter gene in transient transfection experiments. Thus, thyroid hormone regulates only selected genes, even for subunits within the same complex and in widely varying species. By contrast, growth activation of quiescent NIH3T3 cells, a second paradigm used for stimulating mitochondrial biogenesis, does not increase cytochrome c1 mRNA but does increase F1-ATPase beta-subunit mRNA. These findings suggest that nuclear OXPHOS genes are not necessarily expressed in a coordinated manner, and that multiple regulatory circuits might exist which are linked to different physiological stimuli. Analysis of the promoters of several OXPHOS genes reveals a great diversity and heterogeneity of transfactor binding elements. No single regulatory feature exists which could account for a coordinated expression of all OXPHOS genes. The potential diversity for regulating expression of nuclear OXPHOS genes raises the possibility for the existence of disease states linked to regulatory defects.

Mitogen activation of human peripheral T lymphocytes induces the formation of new cyclic AMP response element-binding protein nuclear complexes.

A large body of evidence indicates that experimental agents which raise cellular cAMP levels inhibit T cell growth and division. By contrast, many studies have reported that mitogen activation of T cells increases cAMP levels, implying a positive physiological role for cAMP in the activation process. In the present study we demonstrate that mitogen activation of human peripheral T lymphocytes induces nuclear factors that form complexes with cyclic AMP response element-binding protein (CREB). Four complexes are identified by the electrophoretic mobility shift assay, two of which are induced by mitogen activation. All four complexes contain CREB and are bound to the cAMP response element (CRE) core sequence (TGACGTCA), as indicated by antibody and oligonucleotide competition experiments. Binding of the four complexes to CRE is prevented by dephosphorylation of nuclear extracts and is restored by rephosphorylation with cAMP-dependent protein kinase or endogenous kinases. Similar complexes are detected in nuclear extracts of Jurkat cells. Mitogen induction of the electrophoretic mobility shift assay complexes is not accounted for by protein phosphorylation or by induction of CREB. Rather, the data indicate that mitogen increases the levels of a nuclear factor(s) that dimerizes with CREB. Induction of new CREB complexes implies a physiological role for cAMP in mitogen activation of T lymphocytes.

The effects of cAMP on the expression of glycolytic isozymes in activated peripheral human T lymphocytes.

Forskolin, an activator of adenylate cyclase, inhibits mitogen induction of glycolytic isozymes in human peripheral T cells. Inhibition of lactate dehydrogenase isozyme activity is correlated with lowered mRNA levels, suggesting a transcriptional block in the presence of increased cAMP. Forskolin added with the mitogen, or 12-24 h after the mitogen, strongly inhibits isozyme expression and DNA synthesis, and causes cells to accumulate in the G0 or early G1 phase of the cell cycle. The data suggest that DNA synthesis and isozyme expression are both inhibited by a cAMP-sensitive step(s) in the early activation or progression phase.

Transcript levels for nuclear-encoded mammalian mitochondrial respiratory-chain components are regulated by thyroid hormone in an uncoordinated fashion.

Thyroid hormone is one of the few known physiological regulators of mammalian mitochondrial biogenesis. Although it exerts a global effect on biogenesis, it does so by regulating the expression of a limited number of unidentified mitochondrial proteins. We have investigated these hormone-regulated proteins in rat liver. Hormone injection induced a 30-fold increase in the levels of cytochrome-c1 mRNA after 3 d. In addition, the mRNA for the growth-activated adenine-nucleotide translocator, ANT2, was increased 13-fold and that for the ATPase N,N'-dicyclohexylcarbodiimide-binding protein increased 4-5-fold. Mitochondrial transcripts of cytochrome-oxidase subunit I also increased. No changes were found in the mRNA levels for the F1-ATPase beta-subunit or cytochrome oxidase IV. A single low dose of triiodothyronine induces rapid increases in cytochrome-c1 and ANT2 mRNA species which parallel changes in the activity of the hormone-responsive malic enzyme, but are earlier than other mitochondrial biogenetic events. These data strengthen the view that thyroid hormone regulates synthesis of specific components within each respiratory-chain complex and that these products apparently play key roles in inner-membrane biogenesis and assembly. The significance of ANT2 induction is also discussed with respect to the rapid respiratory response induced by thyroid hormone.

Differential regulation of the transcript levels of some nuclear-encoded and mitochondrial-encoded respiratory-chain components in response to growth activation.

Biogenesis of mammalian mitochondria requires the participation of both nuclear and mitochondrial genes. In order to study the expression and coordination of these two sets of genes, serum-deprived, quiescent NIH 3T3 cells were activated by serum addition. The steady-state levels of the transcripts for two growth-response genes (the mitochondrial adenine-nucleotide translocator and non-mitochondrial beta-actin), one nuclear-encoded respiratory-chain component (F1-ATPase beta-subunit) and the mitochondrial-encoded subunit I of cytochrome oxidase decreased significantly in quiescent cells and were rapidly restored with similar kinetics after addition of serum. The transcripts for two additional nuclear-encoded mitochondrial genes (cytochrome c1 and cytochrome oxidase subunit IV) did not respond to serum deprivation or growth activation. These results imply that mitochondrial biogenesis is at least partially regulated through growth-dependent mechanisms. Furthermore, the expression of nuclear genes encoding mitochondrial respiratory-chain components does not appear to be tightly coordinated, suggesting the existence of multiple control circuits.

Expression of glycolytic isoenzymes in activated human peripheral lymphocytes: cell cycle analysis using flow cytometry.

Human peripheral lymphocytes activated with concanavalin A and phorbol myristate ester exhibit an increase in glycolysis on a time-course similar to that for DNA synthesis. Elevated glycolysis is accompanied by increased specific activities of the glycolytic enzymes. Increased enzyme activities are accounted for by the appearance of specific isoenzyme forms (muscle forms) normally expressed in rapidly growing tumor cells or in growth-stimulated cells. In the present study we analyzed the expression of the glycolytic isoenzymes during cell cycle progression of activated human lymphocytes using two-parameter (DNA and protein) flow cytometry. Time-course studies and analysis of subpopulations prepared by elutriation centrifugation showed that the inducible isoenzymes are expressed at low levels or not at all in G0 cells. They are expressed first during the G0 to G1 transition or in early G1. However, expression increases throughout G1, reaching a maximum in S-phase. Thus, induction of glycolytic isoenzymes provides an excellent marker of T-cell activation and progression toward DNA synthesis.

Hexokinase bound to rat brain mitochondria uses externally added ATP more efficiently than internally generated ATP.

The rates of glucose phosphorylation by bound hexokinase were investigated in mitochondria isolated from rat brain. Initial rates obtained either with ATP generated from oxidative phosphorylation or with ATP added externally were compared. Our results show that the external ATP supports a 2-3-fold higher hexokinase activity than does ATP generated by oxidative phosphorylation under Stage 3 conditions. ATP formed by mitochondrial creatine kinase in the presence of creatine phosphate also supports higher initial rates of glucose phosphorylation than does oxidative phosphorylation. The data suggest that concentrations of ATP present in the cytosol of normal tissue will probably maintain higher rates of glucose phosphorylation than ATP being exported directly from the mitochondrial matrix at maximal State 3 rates.

ADP is a substrate for the AAUAAA-directed poly(A) addition reaction catalyzed by HeLa cell nuclear extracts.

The specific poly(A) addition reaction catalyzed by crude nuclear extracts from HeLa cells can use ADP as efficiently as ATP as the donor of AMP residues. Both the ADP- and ATP-supported reactions require an intact upstream polyadenylation signal sequence element (AAUAAA). The mutated signal sequence (AACAAA) supports neither reaction. The ADP-supported poly(A) addition reaction can be resolved by glycerol gradient centrifugation of the crude nuclear extract into two components which are active when recombined but are inactive individually. The ATP-supported poly(A) addition is reconstituted by recombining the same gradient fractions, but the activity is lower than that supported by ADP, suggesting that an ATP-specific factor has been removed. A 150 mM KCl fraction DEAE-Sepharose of the nuclear extract, also devoid of the ATP-supported poly(A) addition reaction, retains a normal ADP-supported reaction. Together, these data show that ADP is a substrate for polyadenylation, and suggest that different factors might be required to induce ADP- or ATP-specificity in the poly(A) addition reaction.

Expression of a new set of glycolytic isozymes in activated human peripheral lymphocytes.

The combination of phorbol 12-myristate 13-acetate (PMA) and concanavalin A induces the expression of a new set of glycolytic isozymes in human peripheral lymphocytes. The induced isozyme for each enzyme tested (hexokinase, phosphofructokinase, enolase, pyruvate kinase and lactate dehydrogenase) is usually the muscle form, which is often associated with rapidly dividing tumor cells. Increases in a specific isozyme can account for the 2-5-fold increase in specific activities of the enzyme induced by Con A plus PMA. Increased specific activities and the appearance of the new isozyme forms both occur relatively late, and are probably associated with the G1 or S phase of the cell cycle.

Synthesis and targeting of hexokinase to mitochondria in hepatoma cells.

The synthesis and turnover of hexokinase has been measured in Zajdela hepatoma ascites cells labeled for short periods with [35S]methionine. Digitonin fractionation of the labeled cells into a soluble and a membrane fraction showed that only a small part of the newly labeled hexokinase is transferred to mitochondrial binding sites. The soluble enzyme disappears, however, with a half-life of less than 2 h. Glucose had no effect on the stability of the soluble enzyme in intact cells. Our experiments suggest that Zajdela cell hexokinase is synthesized in excess of binding sites and that the excess enzyme is not stable.