To overcome the skin's barrier properties that block transdermal delivery of most drugs, arrays of microscopic needles have been microfabricated primarily out of silicon or metal. This study addresses microneedles made of biocompatible and biodegradable polymers, which are expected to improve safety and manufacturability. To make biodegradable polymer microneedles with sharp tips, micro-electromechanical masking and etching were adapted to produce beveled- and chisel-tip microneedles and a new fabrication method was developed to produce tapered-cone microneedles using an in situ lens-based lithographic approach. To replicate microfabricated master structures, PDMS micromolds were generated and a novel vacuum-based method was developed to fill the molds with polylactic acid, polyglycolic acid, and their co-polymers. Mechanical testing of the resulting needles measured the force at which needles broke during axial loading and found that this failure force increased with Young's modulus of the material and needle base diameter and decreased with needle length. Failure forces were generally much larger than the forces needed to insert microneedles into skin, indicating that biodegradable polymers can have satisfactory mechanical properties for microneedles. Finally, arrays of polymer microneedles were shown to increase permeability of human cadaver skin to a low-molecular weight tracer, calcein, and a macromolecular protein, bovine serum albumin, by up to three orders of magnitude. Altogether, these results indicate that biodegradable polymer microneedles can be fabricated with an appropriate geometry and sufficient strength to insert into skin, and thereby dramatically increase transdermal transport of molecules.

Wheat germ RNA polymerase II was used to raise monoclonal antibodies (mAbs) that cross-react with the largest subunit of calf thymus RNA polymerase II. Most of these mAbs were of the IgM isotype and were shown to react with a synthetic peptide containing the consensus sequence for the C-terminal heptapeptide repeat that has been found on the largest subunit of RNA polymerase II from a variety of eukaryotic organisms. A representative mAb (3WG2) was tested for its effect on transcription in both in vitro and in vivo systems. Antibody 3WG2 did not affect the transcription (elongation) of wheat germ RNA polymerase II on denatured calf thymus DNA. When HeLa cell nuclear extracts were preincubated with the mAb, run-off transcription from a promoter that contains a TATA box (the adenovirus-2 major late promoter) and from a promoter that does not contain a TATA box (the murine dihydrofolate reductase gene promoter = dhfr) was inhibited. Transcription from these promoters was also inhibited by the synthetic peptide containing the consensus sequence when it was conjugated to bovine serum albumin. HeLa cell nuclear extract in which the endogenous RNA polymerase II had been inhibited by the specific mAb was used to examine the ability of added mammalian RNA polymerase II that lacks the C-terminal domain to accurately transcribe specific genes. When calf thymus RNA polymerase II that lacked the C-terminal domain was added back to the inhibited extract, a discrete transcript that was initiated correctly was obtained with the adenovirus-2 major late promoter; however, no discrete transcript was observed from the mouse dhfr gene promoter. When injected into Xenopus laevis oocytes, antibody 3WG2 inhibited transcription of the human histone H2b gene (contains a TATA box) and the human U1 small nuclear RNA gene (does not contain a TATA box), but did not inhibit transcription from RNA polymerase I or RNA polymerase III promoters. These results indicate that the C-terminal heptapeptide repeat plays a critical role in promoter-directed transcription, although enzyme that lacks this domain can initiate from some promoters in vitro.

Diverse studies of three cytoplasmic proteins of Escherichia coli--SecB, trigger factor and GroEL--have suggested that they can maintain precursor proteins in a conformation which is competent for membrane translocation. These proteins have been termed 'chaperones'. Using purified chaperone proteins and precursor protein substrates, we find that each of these chaperones can stabilize proOmpA for translocation and for the translocation-ATPase. These chaperones bind to proOmpA to form isolable complexes. SecB and GroEL will also form complexes with another exported protein, prePhoE. In contrast, these chaperones do not form stable complexes with a variety of soluble proteins such as SecA protein, bovine serum albumin, ovalbumin or ribonuclease A. While chaperones may transiently interact with soluble proteins to catalyze their folding, the stable interaction between chaperones and presecretory proteins, maintaining an open conformation which is essential for translocation, may commit these proteins to the secretion pathway.

We identified and characterized two regions of the human c-myc protein that target proteins into the nucleus. Using mutant c-myc proteins and proteins that fuse portions of c-myc to chicken muscle pyruvate kinase, we found that residues 320 to 328 (PAAKRVKLD; peptide M1) induced complete nuclear localization, and their removal from c-myc resulted in mutant proteins that distributed in both the nucleus and cytoplasm but retained rat embryo cell cotransforming activity. Residues 364 to 374 (RQRRNELKRSP; peptide M2) induced only partial nuclear targeting, and their removal from c-myc resulted in mutant proteins that remained nuclear but were cotransformationally inactive. We conjugated synthetic peptides containing M1 or M2 to human serum albumin and microinjected the conjugate into the cytoplasm of Vero cells. The peptide containing M1 caused rapid and complete nuclear accumulation, whereas that containing M2 caused slower and only partial nuclear localization. Thus, M1 functions as the nuclear localization signal of c-myc, and M2 serves some other and essential function.

OBJECTIVE

Hepatorenal syndrome is common in patients with advanced cirrhosis and constitutes a major problem in liver transplantation. There is no effective medical treatment for hepatorenal syndrome.

METHODS

Forty-six patients with cirrhosis and hepatorenal syndrome, hospitalized in a tertiary care center, were randomly assigned to receive either terlipressin (1-2 mg/4 hour, intravenously), a vasopressin analogue, and albumin (1 g/kg followed by 20-40 g/day) (n = 23) or albumin alone (n = 23) for a maximum of 15 days. Primary outcomes were improvement of renal function and survival at 3 months.

RESULTS

Improvement of renal function occurred in 10 patients (43.5%) treated with terlipressin and albumin compared with 2 patients (8.7%) treated with albumin alone (P = .017). Independent predictive factors of improvement of renal function were baseline urine volume, serum creatinine and leukocyte count, and treatment with terlipressin and albumin. Survival at 3 months was not significantly different between the 2 groups (terlipressin and albumin: 27% vs albumin 19%, P = .7). Independent predictive factors of 3-month survival were baseline model for end-stage liver disease score and improvement of renal function. Cardiovascular complications occurred in 4 patients treated with albumin alone and in 10 patients treated with terlipressin and albumin, yet permanent terlipressin withdrawal was required in only 3 cases.

CONCLUSIONS

As compared with albumin, treatment with terlipressin and albumin is effective in improving renal function in patients with cirrhosis and hepatorenal syndrome. Further studies with large sample sizes should be performed to test whether the improvement of renal function translates into a survival benefit.

OBJECTIVE

Nutritional intervention with branched-chain amino acid (BCAA) is reported to increase serum albumin concentration in patients with decompensated cirrhosis. However, a definite conclusion on whether it can improve patients' survival has not yet been reached. The present study aimed to test possibilities of improving survival of patients with decompensated cirrhosis by using a BCAA preparation that is suitable for long-term oral administration.

METHODS

A multicenter, randomized, and nutrient intake-controlled trial on the comparative effects of BCAA orally administered at 12 g/day for 2 years versus diet therapy with defined daily food intake (1.0-1.4 g protein kg(-1) day(-1) including BCAA preparation and 25-35 kcal kg(-1) day(-1)) was conducted in 646 patients with decompensated cirrhosis. The primary end point was a composite of death by any cause, development of liver cancer, rupture of esophageal varices, or progress of hepatic failure (event-free survival). The secondary end points were serum albumin concentration and health-related quality of life (QOL) measured by Short Form-36 questionnaire.

RESULTS

The incidence of events comprising the primary end point significantly decreased in the BCAA group as compared with the diet group (hazard ratio, 0.67; 95% confidence interval, 0.49-0.93; P = .015; median observation period, 445 days). Serum albumin concentration increased significantly in the BCAA group as compared with the diet group (P = .018). The "general health perception" domain in Short Form-36 measures was also improved (P = .003). Patients' adherence to the prescription was favorable.

CONCLUSIONS

Oral supplementation with a BCAA preparation that can be administered for a long period improves event-free survival, serum albumin concentration, and QOL in patients with decompensated cirrhosis with an adequate daily food intake.

OBJECTIVE

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by increased total kidney volume (TKV) and renal failure. This study aimed to determine if height-adjusted TKV (htTKV) predicts the onset of renal insufficiency.

METHODS

This prospective, observational, longitudinal, multicenter study included 241 adults with ADPKD and preserved renal function. Magnetic resonance imaging and iothalamate clearance were used to measure htTKV and GFR, respectively. The association between baseline htTKV and the attainment of stage 3 CKD (GFR <60 ml/min per 1.73 m(2)) during follow-up was determined.

RESULTS

After a mean follow-up of 7.9 years, stage 3 CKD was attained in 30.7% of the enrollees. Using baseline htTKV, negative correlations with GFR increased from -0.22 at baseline to -0.65 at year 8. In multivariable analysis, a baseline htTKV increase of 100 cc/m significantly predicted the development of CKD within 8 years with an odds ratio of 1.48 (95% confidence interval: 1.29, 1.70). In receiver operator characteristic curve analysis, baseline htTKV of 600 cc/m most accurately defined the risk of developing stage 3 CKD within 8 years with an area under the curve of 0.84 (95% confidence interval: 0.79, 0.90). htTKV was a better predictor than baseline age, serum creatinine, BUN, urinary albumin, or monocyte chemotactic protein-1 excretion (P<0.05).

CONCLUSIONS

Baseline htTKV ≥600 cc/m predicted the risk of developing renal insufficiency in ADPKD patients at high risk for renal disease progression within 8 years of follow-up, qualifying htTKV as a prognostic biomarker in ADPKD.

BACKGROUND

Indoxyl sulphate (IS) and p-cresyl sulphate (PCS) are uraemic toxins that have similar protein binding, dialytic clearance and proinflammatory features. However, only a few prospective studies have evaluated possible associations between these two retained solutes and renal disease progression in chronic kidney disease (CKD) patients.

METHODS

This prospective observational study evaluated independent associations between serum total IS and PCS with renal progression in a selected cohort of patients having different stages of CKD. Baseline PCS and IS were correlated with renal progression [defined as decrements in estimated glomerular filtration rate (eGFR)>> 50% from baseline or progression to end-stage renal disease (ESRD)] and death during a follow-up period of 24 months.

RESULTS

Of 268 patients, 35 (13.1%) had renal progression and 14 (5.2%) died after a mean follow-up of 21 ± 3 months. Univariate Cox regression analysis followed by multivariate analysis showed that high-serum PCS levels were associated with renal progression and all-cause mortality independent of age, gender, diabetes status, albumin levels, serum IS, serum creatinine, Ca × P product, intact parathyroid hormone, haemoglobin or high-sensitivity C-reactive protein level. Serum IS was only associated with renal progression; however, the predictive power of serum IS was weakened when serum PCS was also present in the analytical model.

CONCLUSIONS

In addition to traditional and uraemia-related risk factors such as renal function, serum IS and PCS levels may help in predicting the risk of renal progression in patients having different stages of CKD.

A method is described by which nuclei associated with some cytoplasm can be rapidly prepared from a suspension of cells. The method involves the use of lysolecithin and bovine serum albumin. Oocytes of Xenopus laevis were injected with about 200 nuclei perpared from human HeLa cells by this method. Nuclei were deposited in oocyte cytoplasm, in the oocyte nucleus, or in the dispersed contents of a ruptured oocyte nucleus. Injected HeLa nuclei enlarge up to several hundred times in volume in the course of a few days. Their enlargement is associated with chromatin dispersion, increased binding of an acidic dye, and with the reduction in size, and eventual disappearance, of nucleoli. The amount of HeLa nucleus enlargement is much greater when the oocyte nucleus is ruptured. The fate of injected nuclei was followed by the use of HeLa nuclei whose DNA had been previously labelled with [3H]thymidine. Labelled DNA does not pass from injected HeLa nuclei into the oocyte nucleus. Injected nuclei appear not to fuse with each other or with the oocyte nucleus. Nuclei prepared by the above method look morphologically healthy in oocytes cultured in vitro for up to one month after nuclear injection. Nuclei prepared by other methods, such as those involving the use of detergents, undergo deterioration within a few days after injection into oocytes.

Limited proteolysis is a widely occurring mechanism in protein biosynthesis. Protein precursors can be classified according to their functions, localization within cell compartments, and enzymic cleavage mechanisms. The presecretory proteins represent an important class of very rapidly turning over precursors which play an early role in the sequestration of secretory products and whose cleavage appears to be intimately associated with structures formed at the ribosome-membrane junction during protein synthesis. A model is proposed which predicts that the prepeptide forms a beta-pleated sheet structure with other components of the membrane which results in the transfer of a loop of peptide across the microsomal membrane. Proinsulin is representative of the general class of proproteins that are processed post-translationally within their secretory cells either during the formation and maturation of secretory granules (peptides hormones and neurotransmitters, serum albumins) or during the assembly of macromolecular structures (virus capsules, membrane-associated enzyme complexes). The former group are cleaved by Golgi-associated proteases having tryptic and carboxypeptidase B-like specificity. Some precursors are secreted as such and processed extracellularly either in the circulation or at special sites (procollagens, zymogens, provenoms, vitellogenins).

Immunization of CBA and C57 mice with a branched, multichain synthetic polypeptide, poly (tyr,glu)-poly DL-ala--poly lys, ((T,G)-A--L), in Freund's complete adjuvant results in a tenfold or more difference in the antigen-binding capacity of sera from the two strains, although they respond equally to bovine serum albumin. Immunization of CBA x C57 F(1), F(1) x CBA, and F(1) x C57 mice reveals definite genetic control of the response to (T,G)-A--L, which appears to be due to a single major genetic factor, with perhaps one or more modifying factors. Immunization of CBA and C57 mice with (H,G)-A--L, a synthetic polypeptide in which histidine replaces tyrosine, gives the opposite result, CBA's respond and C57's do not. From this, it appears that the genetic control of the response to (T,G)-A--L is specific for the antigenic determinant. The implications of these results are discussed.

Hepatocellular carcinoma (HCC) is one of the major cancers in the world. There is a striking variation in HCC incidence rates between various countries, with a highest-to-lowest ratio of 112.5 for males and 54.7 for females. The high-risk populations are clustered in sub-Saharan Africa and eastern Asia. The male-to-female ratio for HCC ranges from < 1 to 6.4 and mostly from 2 to 4. There exist significant variations in the incidence of HCC among different ethnic groups living in the same area and among migrants of the same ethnic groups living in different areas. The age curves of HCC are significantly different in various countries, suggesting variability in exposure to risk factors. Chronic carriers of hepatitis B and C viruses (HBV and HCV, respectively) have an increased risk of HCC. The relative and attributable HCC risk of HBV and HCV carrier status varies in different countries. There exists a synergistic interaction on HCC between the two viruses. Aflatoxin exposure, cigarette smoking, heavy alcohol consumption, low vegetable intake, inorganic arsenic ingestion, radioactive thorium dioxide exposure, iron overload and the use of oral contraceptives and anabolic steroids have been documented as HCC risk factors. Recent molecular epidemiological studies have shown that low serum retinol levels as well as elevated serum levels of testosterone, neu oncoprotein and aflatoxin B1-albumin adduct are associated with an increased HCC risk. There is a synergistic interaction on HCC between chronic HBV infection and aflatoxin exposure. Familial aggregation of HCC exists and a major susceptibility gene of HCC has been hypothesized. Patients of some genetic diseases are at an increased risk of HCC. The genetic polymorphisms of cytochrome P450 2E1 and 2D6 and arylamine N-acetyltransferase 2 are associated with the development of HCC. A dose-response relationship between aflatoxin exposure and HCC has been observed among chronic HBV carriers who have null genotypes of glutathione S-transferase M1 or T1, but not among those who have non-null genotypes. Human hepatocarcinogenesis is a multistage process with the involvement of a multifactorial aetiology. Gene-environment interactions are involved in the development of HCC in humans.

1. A new simple method is described for quantitating the adhesiveness of circulating polymorphonuclear leucocytes, or granulocytes, to the walls of blood vessels. The cheek pouch of anaesthetized hamsters or a small part of the mesentery of anaesthetized mice were prepared for continuous microscopic observation of selected venules. Those granulocytes which moved sufficiently slowly to be individually visible were counted for 1 or 2 min periods as they rolled past a selected point on one side of a vessel. The velocity distribution of these cells was determined by analysing films. Films were used also to measure mean blood flow velocity in the venules by observing embolizing platelet thrombi induced by the iontophoretic application of adenosine diphosphate. Emigration of granulocytes into the tissues was quantitated by enumerating them in standard areas of stained histological sections.2. In control experiments with hamster cheek pouch venules, the rolling granulocyte count usually passed through a maximum shortly after the preparation was set up and then fell to a low constant value. In mouse mesentery venules the count remained at a low approximately constant value from the beginning for at least 3 hr.3. The mean velocity of blood flow in the venules was between 900 and 200 mu/sec. All rolling granulocytes moved much more slowly; in hamster cheek pouch venules the mean velocity was about 20 mu/sec and in mouse mesentery venules about 10 mu/sec. Around these means the velocity distribution of individual cells was narrow.4. Rolling of granulocytes was abolished by superfusing ethylenediamine tetra-acetate (EDTA, 0.1 M) suggesting that the phenomenon depends on calcium or magnesium ions.5. Agents were applied locally to the observed venules. Human serum albumin, trypsin or histamine in high concentrations did not affect the rolling granulocyte count.6. The rolling granulocyte count was increased during the application of Hammarsten casein or Escherichia coli culture filtrate which are chemotactic to granulocytes in vitro. These agents did not cause alterations in mean blood flow velocity in the observed venules which might have accounted for the effect on the rolling granulocyte counts. When E. coli culture filtrate was perfused through mouse intestine the increase in rolling granulocyte count in the draining venous blood was proportional to the logarithm of the concentration of filtrate.7. The rolling granulocyte count was also increased by the local application of plasma globulin permeability factor or lymph node permeability factor.8. Granulocyte counts in standard histological sections showed no significant increases in control preparations but considerable increases following the application of Hammarsten casein.

Gene inactivation studies have shown that members of the GATA family of transcription factors are critical for endoderm differentiation in mice, flies and worms, yet how these proteins function in such a conserved developmental context has not been understood. We use in vivo footprinting of mouse embryonic endoderm cells to show that a DNA-binding site for GATA factors is occupied on a liver-specific, transcriptional enhancer of the serum albumin gene. GATA site occupancy occurs in gut endoderm cells at their pluripotent stage: the cells have the potential to initiate tissue development but they have not yet been committed to express albumin or other tissue-specific genes. The GATA-4 isoform accounts for about half of the nuclear GATA-factor-binding activity in the endoderm. GATA site occupancy persists during hepatic development and is necessary for the activity of albumin gene enhancer. Thus, GATA factors in the endoderm are among the first to bind essential regulatory sites in chromatin. Binding occurs prior to activation of gene expression, changes in cell morphology or functional commitment that would indicate differentiation. We suggest that GATA factors at target sites in chromatin may generally help potentiate gene expression and tissue specification in metazoan endoderm development.

BACKGROUND

Vitamin D status has been hypothesized to play a role in musculoskeletal function. Using data from the InCHIANTI study, we examined the association between vitamin D status and physical performance.

METHODS

A representative sample of 976 persons aged 65 years or older at study baseline were included. Physical performance was assessed using a short physical performance battery (SPPB) and handgrip strength. Multiple linear regression was used to examine the association between vitamin D (serum 25OHD), parathyroid hormone (PTH), and physical performance adjusting for sociodemographic variables, behavioral characteristics, body mass index, season, cognition, health conditions, creatinine, hemoglobin, and albumin.

RESULTS

Approximately 28.8% of women and 13.6% of men had vitamin D levels indicative of deficiency (serum 25OHD < 25.0 nmol/L) and 74.9% of women and 51.0% of men had vitamin D levels indicative of vitamin D insufficiency (serum 25OHD < 50.0 nmol/L). Vitamin D levels were significantly associated with SPPB score in men (beta coefficient [standard error (SE)]: 0.38 [0.18], p =.04) and handgrip strength in men (2.44 [0.84], p =.004) and women (1.33 [0.53], p =.01). Men and women with serum 25OHD < 25.0 nmol/L had significantly lower SPPB scores whereas those with serum 25OHD < 50 nmol/L had significantly lower handgrip strength than those with serum 25OHD>> or =25 and>> or =50 nmol/L, respectively (p <.05). PTH was significantly associated with handgrip strength only (p =.01).

CONCLUSIONS

Vitamin D status was inversely associated with poor physical performance. Given the high prevalence of vitamin D deficiency in older populations, additional studies examining the association between vitamin D status and physical function are needed.

BACKGROUND

The National Veterans Affairs Surgical Risk Study was designed to collect reliable, valid data on patient risk and outcomes for major surgery in the Veterans Health Administration and to report comparative risk-adjusted postoperative mortality rates for surgical services in Veterans Health Administration.

METHODS

This cohort study was conducted in 44 Veterans Affairs Medical Centers. Included were 87,078 major noncardiac operations performed under general, spinal, or epidural anesthesia between October 1, 1991, and December 31, 1993. The main outcomes measure was all-cause mortality within 30 days after the index procedure. Multivariable logistic regression risk-adjustment models for all operations and for eight surgical subspecialties were developed. Risk-adjusted surgical mortality rates were expressed as observed-to-expected ratios and were compared with unadjusted 30-day postoperative mortality rates.

RESULTS

Patient risk factors predictive of postoperative mortality included serum albumin level, American Society of Anesthesia class, emergency operation, and 31 additional preoperative variables. Considerable variability in unadjusted mortality rates for all operations was observed across the 44 hospitals (1.2-5.4%). After risk adjustment, observed-to-expected ratios ranged from 0.49 to 1.53. Rank order correlation of the hospitals by unadjusted and risk-adjusted mortality rates for all operations was 0.64. Ninety-three percent of the hospitals changed rank after risk adjustment, 50% by more than 5 and 25% by more than 10.

CONCLUSIONS

The Department of Veterans Affairs has successfully implemented a system for the prospective collection and comparative reporting of risk-adjusted postoperative mortality rates after major noncardiac operations. Risk adjustment had an appreciable impact on the rank ordering of the hospitals and provided a means for monitoring and potentially improving the quality of surgical care.

BACKGROUND

Albuminuria is a risk factor for chronic kidney disease progression, end-stage renal disease, cardiovascular events, and mortality. Animal studies suggested that vitamin D insufficiency may contribute to the pathogenesis of albuminuria.

METHODS

Cross-sectional study.

METHODS

15,068 adults participating in the Third National Health and Nutrition Examination Survey.

METHODS

Serum 25-hydroxyvitamin D concentration, examined in quartiles.

METHODS

Albuminuria, defined using established sex-specific cutoff values for urine albumin-creatinine ratio (25 to 2,999 mg/g for women, 17 to 2,999 mg/g for men).

RESULTS

A stepwise increase in the prevalence of albuminuria was observed with decreasing quartiles of vitamin D concentration: 8.9%, 11.5%, 13.7%, and 15.8% (P < 0.001). Adjusting for age, sex, race/ethnicity, region and season of measurement, smoking status, body mass index, and estimated glomerular filtration rate, relative risks for albuminuria by decreasing quartile of vitamin D concentration were 1.00 (reference group), 1.14 (95% confidence interval, 0.95 to 1.37), 1.22 (95% confidence interval, 1.03 to 1.45), and 1.37 (95% confidence interval, 1.10 to 1.71; P = 0.006). Additionally adjusting for blood pressure and diabetes mellitus, these risks were somewhat attenuated and retained statistical significance.

CONCLUSIONS

The cross-sectional design of this study does not allow demonstration of temporal or causal relationships between vitamin D and albuminuria.

CONCLUSIONS

Additional studies are needed to clarify the relationship of vitamin D with albuminuria and determine whether vitamin D therapy prevents or improves markers of kidney and cardiovascular disease.

An improved method is described for extraction from rat myocardium of an enzyme that cleaves the third component of complement (C3) into leukotactic fragments. Direct cleavage of C3 has been shown by the use of a radiolabeled preparation of human C3. The enzyme elutes slightly beyond (retarded to) an albumin marker in gel filtration. After surgical ligation of a coronary artery, leukotactic activity can be extracted from infarcted rat myocardium. Described in terms of its time of appearance in infarcted tissue, the leukotactic activity has been identified as cleavage products of rat C3. The C3 fragments are of low molecular weight and heterogenous in size. Rats, if treated with the C3 inactivator (isolated from cobra venom) so as to ablate serum C3, fail to develop leukotactic activity in the infarcted myocardium. In turn, few if any of the usual intense accumulations of neutrophils in response to infected myocardium are seen during the first 48 hr after coronary artery ligation. These studies suggest a nonimmunologic role for C3 in the mediation of the acute inflammatory response in nonspecific tissue injury.

The Solt-Farber protocol, in the absence of an initiating agent, was used to examine the precursor-product relationship between oval cells and hepatocytes in rat liver. The animals were administered 2-acetylaminofluorene (AAF) by gavage for 2 wk combined with partial hepatectomy 1 wk after administering AAF Two dose levels of AAF were used: 9- and 21-mg total dose for animals in Groups I and II, respectively. [3H]Thymidine was administered i.p. to one-half of the animals at Day 6 post-partial hepatectomy. Animals were sacrificed 7, 9, 11, and 13 days after surgery. Only oval cells became labeled on Day 7 in both groups. On Day 9 both labeled oval cells and labeled basophilic hepatocytes were present in Group I, whereas in Group II only oval cells remained labeled. On Days 11 and 13 both oval cells and basophilic hepatocytes were labeled in both groups. The total amount of radioactivity in Group II livers remained the same on Day 9 when only labeled oval cells were present and on Days 11 and 13 when both labeled oval cells and labeled basophilic hepatocytes were present. The calculated half-life for basophilic hepatocytes was about 50 h. The differentiation of oval cells into basophilic hepatocytes was delayed in Group II as compared to Group I, and the higher dose of AAF also induced the formation of both intestinal metaplasia and bile duct formation. In situ hybridization with an alpha-fetoprotein probe showed a strong expression in groups of typical oval cells and in cells arranged in duct-like structures. In addition a transient expression of AFP was also observed in the areas of basophilic hepatocytes 9 to 11 days after partial hepatectomy. Administration of AAF decreased the level of albumin mRNA in preexisting hepatocytes and caused a significant decrease of serum albumin. In contrast, oval cells showed a strong albumin expression, and basophilic hepatocytes formed islands of albumin-expressing cells. Oval cells and the foci of early basophilic hepatocytes lacked glucose-6-phosphatase activity. At Day 13 significant numbers of basophilic hepatocytes were positive for glucose-6-phosphatase. Oval cells were strongly gamma-glutamyltranspeptidase positive, whereas the foci of basophilic hepatocytes were negative for gamma-glutamyltranspeptidase. Only occasionally were transiently gamma-glutamyltranspeptidase-positive hepatocytes observed in basophilic foci. In summary our data indicate that oval cells can differentiate to hepatocytes and may have an important physiological function as a source of major serum proteins when hepatocytes are unable to synthesize these proteins.

Human serum albumin (HSA) is one of the most abundant proteins in the circulatory system and plays a key role in the transport of fatty acids, metabolites, and drugs. For many drugs, binding to serum albumin is a critical determinant of their distribution and pharmacokinetics; however, there have as yet been no high resolution crystal structures published of drug-albumin complexes. Here we describe high resolution crystal structures of HSA with two of the most widely used general anesthetics, propofol and halothane. In addition, we describe a crystal structure of HSA complexed with both halothane and the fatty acid, myristate. We show that the intravenous anesthetic propofol binds at two discrete sites on HSA in preformed pockets that have been shown to accommodate fatty acids. Similarly we show that the inhalational agent halothane binds (at concentrations in the pharmacologically relevant range) at three sites that are also fatty acid binding loci. At much higher halothane concentrations, we have identified additional sites that are occupied. All of the higher affinity anesthetic binding sites are amphiphilic in nature, with both polar and apolar parts, and anesthetic binding causes only minor changes in local structure.

BACKGROUND

There are numerous studies concerning the natural history and prognostic factors in cirrhosis, the results of which are useful in selecting liver transplant candidates. However, little attention has been paid to the prognostic significance of hepatic encephalopathy despite the high frequency of this complication.

METHODS

We reviewed the charts of 111 cirrhotic patients who developed a first episode of acute hepatic encephalopathy to determine their survival probability and to identify prognostic factors.

RESULTS

During follow-up (12+/-17 months), 82 (74%) patients died. The survival probability was 42% at 1 year of follow-up and 23% at 3 years. With univariate analyses followed by a multivariate analysis, 7 out of 30 clinical and standard laboratory variables were significantly associated with poor prognosis: male sex, increased serum bilirubin, alkaline phosphatase, potassium and blood urea nitrogen, and decreased serum albumin and prothrombin activity. Patients were classified into two groups according to a prognostic index calculated from these 7 variables. Survival probability at 1 and 3 years was 73% and 38%, respectively, in patients with a low prognostic index, and 10% and 3% in patients with a high prognostic index.

CONCLUSIONS

Hepatic encephalopathy is associated with short survival in cirrhotic patients. Although these patients can be classified into several groups with a different prognosis, the survival probability in every group is lower than that currently expected after liver transplantation. Therefore, cirrhotic patients developing a first episode of acute hepatic encephalopathy should be considered as potential candidates for this therapeutic procedure.

Reperfusion of ischemic tissue induces an acute inflammatory response that can result in necrosis and irreversible cell injury to both local vascular endothelium and parenchyma. To examine the pathogenesis of ischemia/reperfusion injury, we have used mice deficient in complement components C3, C4, or serum immunoglobulin in a hindlimb model of ischemia. We found that mice homozygous deficient in C3 or C4 were equally protected against reperfusion injury based on a significant reduction in leakage of radiolabeled albumin out of the vasculature. This demonstrates that classical pathway complement is an important factor in the initiation of inflammation following reperfusion. Furthermore, mice deficient in serum immunoglobulin were equally protected and this protection could be reversed by reconstitution with serum from normal mice. Thus, this report describes a novel mechanism for reperfusion injury that involves antibody deposition and activation of complement leading to inflammation permeability.

Recent work with approaches like recombinant mutants and X-ray crystallography has given much new information about the ligand-binding properties of human serum albumin (HSA). The information increases the understanding of this unique transport and depot protein and could give a structural basis for the possible construction of therapeutic agents with altered HSA-binding properties. A tabulation of high-affinity binding sites for both endogenous and exogenous compounds has been made; it could be useful for the above-mentioned purpose, but it could also be of value when trying to predict potential drug interactions at the protein-binding level. Drug displacement is not always a complication to therapy; it can be used to increase the biological effect of a drug. However, due to rebinding at other sites, the increase in the free concentration of a displaced ligand can be less than expected. Drugs and drug metabolites can also interact covalently with HSA; thiol-containing drugs often bind to the single free cysteine residue of HSA, and glucuronidated drugs react irreversibly with other residues of the protein. Reversible binding of ligands is often stereospecific, and therefore immobilized HSA can be used to separate drug isomers. Albumin-containing dialysates are useful for extracorporeal removal of endogenous toxins and in the treatment of drug overdoses. HSA has different types of hydrolytic activities, which also can be stereospecific. The esterase-like property seems especially useful in converting prodrugs to active drugs in plasma.

BACKGROUND

A reliable and sensitive complex I assay is an essential tool for the diagnosis of mitochondrial disorders, but current spectrophotometric assays suffer from low sensitivity, low specificity, or both. This deficiency is mainly due to the poor solubility of coenzyme-Q analogs and reaction mixture turbidity caused by the relatively high concentrations of tissue extract that are often required to measure complex I.

METHODS

We developed a new spectrophotometric assay to measure complex I in mitochondrial fractions and applied it to muscle and cultured fibroblasts. The method is based on measuring 2,6-dichloroindophenol reduction by electrons accepted from decylubiquinol, reduced after oxidation of NADH by complex I. The assay thus is designed to avoid nonspecific NADH oxidation because electrons produced in these reactions are not accepted by decylubiquinone, resulting in high rotenone sensitivity.

RESULTS

The assay was linear with time and amount of mitochondria. The K(m) values for NADH and 2,6-dichloroindophenol in muscle mitochondria were 0.04 and 0.017 mmol/L, respectively. The highest complex I activities were measured with 0.07 mmol/L decylubiquinone and 3.5 g/L bovine serum albumin. The latter was an essential component of the reaction mixture, increasing the solubility of decylubiquinone and rotenone. In patients with previously diagnosed complex I deficiencies, the new assay detected the complex I deficiencies in both muscle and fibroblasts.

CONCLUSIONS

This spectrophotometric assay is reproducible, sensitive, and specific for complex I activity because of its high rotenone sensitivity, and it can be applied successfully to the diagnosis of complex I deficiencies.