The gamma-crystallin proteins consist of two topologically equivalent domains, each built up out of two similar motifs. They are encoded by a gene family, which already contained five members before the divergence of rodents and primates. A further gene duplication took place in each lineage. To analyze the pattern of evolution within this gene family, the coding sequences of six human genes, six rat genes, and four mouse genes were compared. Between species, a uniform rate of evolution of all regions of the protein is seen. The ratio of synonymous to nonsynonymous substitution in the human/rat or human/mouse comparison is much lower than the ratio when rat and mouse are compared indicating that the gamma-crystallin proteins are better conserved in the rodent lineage. Within species, the regions encoding the two external motifs I and III of the protein show a greater extent of nonsynonymous substitution than the regions encoding the two internal protein motifs II and IV. The low extent of synonymous substitution between the second exons (encoding motifs I and II) of the rat gamma-crystallin genes suggests the frequent occurrence of gene conversion. In contrast, a high extent of synonymous substitution is found in exon 3 (encoding motifs III and IV) of the rat genes. The same phenomenon is seen within the human gene family. The frequencies of occurrence of the various dinucleotides deviate less from those predicted from the frequencies of occurrence of each individual nucleotide in the second exons than in the third exons. The sequences of the third exons are significantly depleted in CpG, ApA, and GpT and enriched in CpT and GpA.

The ataxia telangiectasia cell line, AT5BIVA, exhibited low repair fidelity measured by the reconstitution of transfected linear plasmid. This assay involves transfecting a linear plasmid containing two selectable marker genes: one gene (neo) is undamaged and marks transfection and the other gene (gpt) is cleaved to test functional repair. The proportion of transfected cells which have a functionally intact gpt gene gives a measure of repair fidelity. Southern analysis of individual transfected clones showed that integrated plasmids in AT5BIVA had a high frequency of sequence rearrangement. Blunt or staggered-ended termini of a linear plasmid did not determine the type of misrepair. A variety of sizes of deletions and sequence insertions were found at and around the cleavage site. Loss of intact sequence occurred similarly following transfection by linear or circular plasmid (misrepair or rearrangement error). This suggests that the action of excess exonuclease activity upon, or lack of protection of, exposed DNA termini is not the sole mechanism of misrepair. Erroneous rearrangement of circular plasmid could involve any location along the plasmid. Rearrangement of transfected circular plasmid occurred in multiple copies of the same abnormal size, suggesting that error-prone recombination rather than degradation of presumed nicked circular plasmid was the underlying mechanism. It is hypothesized that misrepair in ataxia-telangiectasia arises by error-prone recombination.

Nickel is an established human and animal carcinogen, but efforts to demonstrate its mutagenicity in a number of cell types have not been successful. In this report we describe the mutational response to nickel compounds in the G12 cell line, an hprt deficient V79 cell line containing a single copy of the E. coli gpt gene. This cell line has a low spontaneous background, making it suitable for assessment of mutagenic responses to environmental contaminants. When G12 cells were treated with insoluble particles of crystalline nickel sulfide < 5 microns in diameter, a strong, dose-dependent mutagenic response was observed up to 80 times the spontaneous background. Of 48 mutant gpt(-) clones isolated that were induced by insoluble nickel, all were capable of DNA amplification of the gpt sequences by polymerase chain reaction (PCR). The ability to produce full-length PCR products is an indication that large deletions of gene sequences have not occurred. When G12 cells were treated with soluble nickel sulfate, the mutational response was not significantly increased over the spontaneous background. This difference in mutagenic response reflects a large difference in the mutagenic potential of soluble and insoluble nickel compounds, which reflects the carcinogenic potential of these forms of nickel.

OBJECTIVE

To describe the rationale, development, and preliminary acceptability of a Group Parent Training Program (GPT) as an alternative approach for the treatment of adolescent eating disorders.

METHODS

Sixteen families participated in a 16-session group treatment. After four months, parents were administered a treatment satisfaction questionnaire.

RESULTS

Parent response to the intervention was positive. All parents indicated GPT was essential for the management of their child, improved general parenting skills, improved their own self-care, and decreased the burden they experienced as a result of managing their child's illness.

CONCLUSIONS

Given the magnitude of task demands placed on a family for the management of adolescent eating disorders, there is the need for the development of effective intervention strategies that ease the stress of illness management for parents. Preliminary satisfaction data from GPT are promising and argue for a more systematic test of this intervention.

In the present study, we investigated the relationship between serum leukocyte cell-derived chemotaxin2 (LECT2) levels and liver function in patients with acute liver failure, and its use as a prognostic indicator. We studied six acute liver failure patients (two women, four men; 49.8 +/- 20.7 years old) admitted to our hospital in 2002. These patients had diagnoses of fulminant hepatitis due to acute liver failure (1) from congestive heart failure; (2) from portal venous gas, and (3) from postoperative disseminated intravascular coagulation (DIC). We measured serum LECT2, GOT, and GPT levels, the last two being inversely proportionate to the serum LECT2 levels. When the serum GPT levels peaked, the serum LECT2 levels were the lowest. When the liver function recovered, serum LECT2 levels increased. Three of four patients died due to liver failure, one to congestive heart failure. Maximum serum LECT2 levels among the expired group were significantly lower than those among the alive group (0.96 +/- 0.8 ng/mL vs 12.9 +/- 4.3 ng/mL). Serum LECT2 levels may be a prognostic indicator of recovery from liver failure. The present study suggests that in clinical medicine LECT2 participates in regeneration after injury of hepatocytes.

The LEC rat, which suffers from hereditary hepatitis, was examined for elucidation of its clinicopathological characteristics during development of the acute phase of hepatitis by quantitative analyses of histological observations of the liver in combination with laboratory data on various serum enzymes. The progression of acute hepatitis in the LEC rat was observed to begin insidiously early in life, i.e., a few enlarged hepatocytes and Councilman bodies appeared at around 8 weeks of age without clinical signs. Furthermore, it was revealed that the acute phase of hepatitis started with a remarkable increase of Councilman bodies, large nuclei and hepatocytes in mitosis in the liver 3 to 4 weeks before the onset of fulminant hepatitis, which is characterized by the elevation of serum enzyme activities such as GOT, GPT and gamma-GTP, and the onset of jaundice. From those observations, three stages were proposed for the progression of acute hepatitis in the LEC rat.

Maskless photolithographic peptide synthesis was performed on a glass chip using an automated peptide array synthesizer system. The peptide array synthesizer was built in a closed box, which contained optical and fluidic systems. The conditions for peptide synthesis were fully controlled by a computer program. For the peptide synthesis on a glass chip, 20 NVOC-protected amino acids were synthesized. The coupling efficiencies of two model peptide sequences were examined on ACA/APTS and PEG/CHI/GPTS chips. PEG/CHI/GPTS chip gave higher average stepwise yields of GIYWHHY (94%) and YIYGSFK (98%) than those of ACA/APTS chip. To quantify peptide-protein binding affinity, HPQ- or HPM-containing pentapeptides were synthesized on a PEG/CHI/GPTS chip and the binding event of Cy3 labeled-streptavidin was quantified. The peptide sequence of IQHPQ showed highest binding affinity with Cy3 labeled-streptavidin. The results demonstrated that the photolithographic peptide array synthesis method efficiently quantified the binding activities of protein-peptide interactions and it can be used for additional biological assay applications.

Fibroblasts from a patient with xeroderma pigmentosum complementation group D were treated with Simian virus 40 to establish a transformed cell line suitable for studies of DNA-mediated gene transfer. After progressing through 2 crises, a stable line, XP6Be(SV40), was established and cultured for more than 1 year. This line retains the characteristic xeroderma pigmentosum ultraviolet hypersensitivity and is able to complement a SV40-transformed group A line when fused and assayed for ultraviolet radiation inhibition of colony-forming ability. XP6Be(SV40) expressed high levels of transfected chloramphenicol acetyltransferase activity (0.1 nmole X mg-1 X min-1) in a transient expression assay, showed stable expression of transfected gpt or neo genes (frequency 1-20 X 10(-5)), and permitted replication of the mutagenesis shuttle vector plasmid, pZ189. Ultraviolet treatment (500 J X m-2) of pZ189 prior to replication in XP6Be(SV40) resulted in a large reduction in plasmid yield (5% survival) and a 60-fold increase in the mutation frequency, reflecting the reduced ability of these cells to repair ultraviolet-damaged transfecting DNA. This cell line provides the opportunity to utilize transfection studies in cells with the xeroderma pigmentosum group D defect in excision repair.

Soluble HLA Class I antigens in sera (serum-HLA Class I, s-HLA Class I) of patients with chronic hepatitis (CH) were measured with an enzyme-linked double determinant immunoassay (E-DDIA). The mean titers of s-HLA Class I antigens of patients with CPH (mean +/- standard deviation, 2.22 +/- 1.60), CAH2A (2.24 +/- 1.65) or CAH2B (2.73 +/- 1.46) were significantly higher than that of normal subjects (0.36 +/- 0.27) (P less than 0.01). The titer of s-HLA Class I correlated significantly with the level of serum glutamic pyruvic transaminase (s-GPT) (r = 0.73), and weakly with serum level of beta 2-microglobulin (r = 0.43). In patients with chronic hepatitis type B (CH-B) treated with human lymphoblastoid interferon alpha (IFN-alpha), the titer of s-HLA Class I antigens increased. The increased level of s-HLA Class I antigens in the clinical course of chronic hepatitis may be caused by their release from necrotizing hepatocytes which have acquired the expression of HLA Class I antigens on the cell-surface membrane during viral infection.

OBJECTIVE

To delineate the clinical manifestations in different age groups and to define the viral load in patients with Epstein-Barr virus-associated infectious mononucleosis (EBV-associated IM).

METHODS

We reviewed data on 69 children with EBV-associated IM from November 2001 to October 2005. Clinical features were evaluated among four age groups: <3 years, 3 to 5 years, 6 to 9 years and 10 to 18 years. EBV viral load was measured by quantitative real-time polymerase chain reaction (PCR) in 13 patients with 15 specimens.

RESULTS

Majority of the children were younger than 7 years of age (76.8%) and the male-to-female ratio was 1.6:1. The symptoms and signs included fever (91.3%), tonsillopharyngitis (88.4%), lymphadenopathy (78.3%) and hepatitis (75.4%). The younger age group had higher monocyte count, lower occurrence of hepatitis, and lower glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) levels than the older age group. The median (range) EBV viral load of peripheral blood mononuclear cells (PBMCs) and plasma in IM patients was 738 (0-7455) copies/mug DNA and 51 (0-957) copies/mL plasma, respectively. The PBMC detection rate was high in the early (within 10 days after onset) and late phase (>10 days after onset) [90-100%]. The plasma detection rate in the early phase (66.7%) was higher than that in the late phase (40%).

CONCLUSIONS

The younger age group of EBV-associated IM patients had higher monocyte count, lower occurrence of hepatitis, and lower GOT and GPT levels than the older age group. The PBMC detection rate was almost equally high in both the early and late phases, while the plasma detection rate was higher in the early phase. Quantitative real-time PCR of EBV DNA is useful for diagnosing and monitoring EBV-associated IM, especially in younger children.

To determine hepatic diseases in obese children, biochemically and histologically, 11 obese patients with abnormal serum transaminase activities were subjected to this study. Fat accumulation in the liver was semiquantitatively graded, and histologically the 11 patients were classified into four groups; fatty liver, fatty hepatitis, fatty fibrosis and fatty cirrhosis. All patients had fat deposition in liver specimens, the grade of which did not significantly correlate with the degree of obesity. The grade of fat deposition in the liver specimens also did not significantly correlate with either serum transaminase activities or GOT/GPT ratio. Five patients were grouped into the fatty liver group, three into the fatty hepatitis group, and the remaining three patients into the fatty fibrosis group. However, no significant differences were found among the three histologically classified groups in terms of serum transaminase activities or GOT/GPT ratio. The usefulness of serum transaminase activities and GOT/GPT ratio was limited in predicting the severity of fat deposition or histological abnormality in pediatric obese patients.

The pyrimido-pyrimidine BIBX 1382 BS inhibits the intracellular tyrosine kinase domain of the epidermal growth factor receptor (EGFR), thus specifically reverting the aberrant enzymatic activity from overexpressed and constitutively activated EGFR. A phase I and pharmacokinetic study of this new specific molecule was carried out. After initially performing an accelerated titration design from the first toxicities onwards, a modified Fibonacci scheme was used to escalate the daily oral dose. The following dosages and cycles (defined as treatment during 28 days) were applied: 25 mg: 6; 50 mg: 3; 100 mg: 6; 200 mg: 7; 150 mg: 3. Over a 10 months accrual phase, 11 patients (pts) (7 females, 4 males) with a median age of 63 years (range 50-73 years), World Health Organization Performance Status (WHO PS) 0:5 pts, 1:6 pts and miscellaneous solid tumours were entered. The median number of cycles applied per pt was 2 (range 1-7). Reversible, dose-dependent increase of liver enzymes (maximal Common Toxicity Criteria (CTC) grades: gamma-glutamyl transferase (GGT): 4, aspartate aminotransferase (GOT): 3, alanine aminotransferase (GPT): 3, alkaline phosphatase (AP): 3, bilirubin: 3) occurred. Oral medication yielded plasma levels far below those expected to be efficacious. In conclusion, target plasma levels could not be reached via the oral route at a reasonable dosage. Meanwhile, a preclinically unknown metabolite was identified from the urine of one patient. Subsequently, this metabolite was found to be abundant in patient plasma. The metabolite was demonstrated to be pharmacologically inactive. Due to a dose-limiting increase of liver enzymes, low bioavailability of BIBX 1382 BS and the detection of a pharmacologically inactive metabolite, this trial was discontinued.

The uptake of nanoparticles by aquatic organisms such as fish has raised concerns about the possible adverse effects of nanoparticles (NPs). In this study, we aimed to evaluate the toxicological effects in juvenile common carp exposed to zinc oxide nanoparticles (ZnO-NPs) for 12 weeks. The carp were exposed to 0 (control), 0.1, 0.3, 0.8, and 2.4mg/L of ZnO-NPs under a flow-through exposure system. Fish were sampled at 0, 4, 8, and 12 weeks to test for zinc in the test water and blood, and biochemistry analysis; further, they were sampled at 12 weeks to observe ultrastructural changes in the liver, kidney, and gill. In the organic serum, changes in the glutamic pyruvic transaminase/alanine aminotransferase (GPT/ALT) and glutamic oxaloacetic transaminase/aspartate aminotransferase (GOT/AST) levels were significant, but changes in the lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) levels were not significantly different across all exposure periods. In the inorganic serum, the magnesium (Mg), inorganic phosphorus (IP), sodium (Na(+)), and chloride (Cl(-)) levels were significantly different in the exposure group and across exposure periods. However, calcium (Ca) and potassium (K(+)) levels were not significantly different. In the enzyme serum, the glucose (GLU) level significantly increased for the highest exposure group, but the total cholesterol (TCHO), triglyceride (Tg), and total protein (TP) levels were not significantly different during the exposure period. Ultrastructural changes in the liver induced changes in the black granules (of various sizes) in the lysosomes, indistinct nucleus membrane, and non-spherical nucleus. In the kidney, some mild changes were observed in the size and number of the lysosomes in the renal tubule. Desquamation and hypertrophy of pavement epithelial cells and vacuolation in the cytoplasm of the chloride cells were observed in the gill. Nanoparticles were also observed in the red blood cells, cytoplasm of all tissues, and glomerulus of the kidney. The observed changes in the serum and tissues may provide useful information regarding environmental conditions and risk assessments of aquatic organisms.

The glucose-6-phosphate/phosphate translocator (<em>GPT</em>) acts as an importer of carbon into the plastid. Despite the potential importance of <em>GPT</em> for storage in crop seeds, its regulatory role in biosynthetic pathways that are active during seed development is poorly understood. We have isolated <em>GPT</em>1 from Vicia narbonensis and studied its role in seed development using a transgenic approach based on the seed-specific legumin promoter LeB4. <em>GPT</em>1 is highly expressed in vegetative sink tissues, flowers and young seeds. In the embryo, localized upregulation of <em>GPT</em>1 at the onset of storage coincides with the onset of starch accumulation. Embryos of transgenic plants expressing antisense <em>GPT</em>1 showed a significant reduction (up to 55%) in the specific transport rate of glucose-6-phosphate as determined using proteoliposomes prepared from embryos. Furthermore, amyloplasts developed later and were smaller in size, while the expression of genes encoding plastid-specific translocators and proteins involved in starch biosynthesis was decreased. Metabolite analysis and stable isotope labelling demonstrated that starch biosynthesis was also reduced, although storage protein biosynthesis increased. This metabolic shift was characterized by upregulation of genes related to nitrogen uptake and protein storage, morphological variation of the protein-storing vacuoles, and a crude protein content of mature seeds of transgenics that was up to 30% higher than in wild-type. These findings provide evidence that (1) the prevailing level of <em>GPT</em>1 abundance/activity is rate-limiting for the synthesis of starch in developing seeds, (2) <em>GPT</em>1 exerts a controlling function on assimilate partitioning into storage protein, and (3) <em>GPT</em>1 is essential for the differentiation of embryonic plastids and seed maturation.

Exposure to contaminants in Great Lakes fish has been linked to impaired neuropsychological functioning in children, but neurological function of exposed adults has not been evaluated. This report describes a cross-sectional analysis of the effects of PCB/DDE exposure from contaminated fish on fine motor function in older adults. The subjects were 50-90-year-old Michigan residents who were members of a previously established study cohort. Fisheaters ate 24 lbs or more of sport-caught Lake Michigan fish/year at the time they were originally recruited in 1980-1982. Age- and sex-matched non-fisheaters ate 6 or fewer lbs/year. Outcome measures were scores on the Static Motor Steadiness Test (SMST) and Grooved Pegboard Test (GPT). PCB/DDE exposure was determined through serum analyses performed at the time of recruitment into the present study in 1993-1995. Because of the high correlation between serum PCB and DDE levels in this sample (Spearman r=0.64, P<0.0001), the effects of the two contaminants were assessed jointly using a single derived exposure variable=Low=both PCB and DDE at or below the medians of their respective distributions, intermediate=PCB and/or DDE in the third quartile, and high=PCB and/or DDE in the upper quartile. In unadjusted analyses, high exposure to PCBs/DDE was associated with significantly poorer performance on the GPT (P=0.03). However, in the multiple regression model, age and gender emerged as the most significant factors affecting GPT scores, and exposure to PCB/DDE was not significant. Performance on the SMST was not related to PCB/DDE exposure in initial unadjusted analyses, but performance with the dominant hand was marginally (P=0.052) associated with exposure in the final model. Scores on the SMST improved slightly as PCB/DDE exposure increased. A similar trend was not observed for the nondominant hand (P=0.46). These findings suggest that PCB/DDE exposure from Great Lakes fish has not significantly impaired hand steadiness or visual-motor coordination in this sample of older adults.

The adaptive response is one of the major repair pathways in Escherichia coli that removes DNA alkylation damage. To investigate the role of the adaptive response in mutagenesis, the E. coli gpt forward mutation assay system was used to determine the mutation spectrum of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in MNNG-adapted and unadapted GP120 (wild-type) and unadapted PJ5 (ada-5) cells. We observed that 34/37 mutations in the unadapted GP120 cells, 38/40 mutations in the adapted GP120 cells, and 10/10 mutations in the PJ5 cells were GC----AT transitions. The remaining 3/37 mutations in the unadapted GP120 cells were large insertions. The remaining 2/40 mutations in the adapted GP120 cells were transversions with one a GC----CG and the other an AT----CG. A surrounding sequence specificity of mutagenesis was observed for the GC----AT transitions in both the unadapted (GP120 and PJ5) and adapted (GP120) cells, with 70% of the unadapted PJ5, 68% of the unadapted GP120, and 61% of the adapted GP120 mutations occurring at the middle G of the sequence 5'--GG(A or T)--3'. Both strains also displayed a statistically significant preference for mutagenesis at guanine bases in the non-transcribed strand. The overall distribution of mutated sites in the gpt gene in adapted and unadapted cells was similar, although the rate of mutations at certain sites appeared different. These minor differences could result from either non-uniform repair of alkylation damage at different sites on the DNA, or altered processing of the alkylated bases to mutations in the adapted state.(ABSTRACT TRUNCATED AT 250 WORDS)

The white shrimp, Litopenaeus vannamei, a globally important cultured prawn species, is an ideal animal for studying the impairment caused by the effects of heavy metals that are often detected in coastal areas. In this study, L. vannamei was exposed to different concentrations of cadmium (Cd) and zinc (Zn) for up to 28 d. Histopathological alterations in the hepatopancreas were observed in L. vannamei after long-term exposure to Cd and Zn. Hepatopancreatic injury was further confirmed by the inductions of two biochemical markers, hemolymphatic glutamate-oxalacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT). It was notable that L. vannamei was able to repair its hepatopancreas from the damage caused by Zn, which was evidenced by the results of the histopathological observations, determinations of tissue metal concentrations, and examination of GOT and GPT levels.

1. The expression of soluble GPT (E.C. 2. 6. 1. 2) was analysed in seventeen independent rat hepatoma/human somatic cell hybrids and in forty-one subclones derived from two of these hybrids. 2. As judged by electrophoretic mobility, twelve hybrid clones expressed rat GPT activity only and three expressed strong rat and weak human GPT activity but no heteromeric isozymes. In the remaining two hybrids, only human GPT was demonstrable. 3. The segregation of GPT and marker enzymes in the primary hybrid cells and the subclones suggests that the human structural GPT locus is on chromosome 8. 4. The re-expression of rat GPT in segregating subclones derived from two primary hybrids which had extinguished this function, could not be correlated with presence or absence of any particular human chromosome(s).

In a double blind study carried out under standard conditions at two treatment centers silymarin, 2 sugar-coated tablets 70 mg three times daily, showed a definite therapeutic influence on the characteristic increased serum levels of bilirubin, GOT and GPT associated with acute viral hepatitis. The above mentioned values in 28 patients treated with silymarin were compared with those in 29 patients treated with placebo. The laboratory parameters in the silymarin group regressed more than in the placebo group after the 5th day of treatment. The number of patients having attained normal values after 3 weeks' treatment was higher in the silymarin group than in the placebo group. A statistical comparison revealed a difference between bilirubin and GOT values in the placebo and silymarin groups and a definite trend in the regression of GPT values in favour of silymarin. The course of the immune reaction in HBS Ag patients was not influenced by silymarin. As already proved by other investigators, the use of silymarin in acute viral hepatitis can lead to an accelerated regression in pathological values, thus indicating its use in the treatment of this liver disease.

Fifteen heavy drinkers with the histological features of chronic hepatitis were studied. Chronic hepatitis observed in heavy drinkers can be divided into two categories. One is caused by alcohol, and the other is not etiologically related to alcohol. Chronic hepatitis caused by alcohol showed a definite improvement of clinical features following abstinence, as well as significantly high serum GOT/GPT ratios and high glutamate dehydrogenase activities on admission. These clinical features are distinctly different from chronic hepatitis without etiological relation to alcohol and resemble the other types of alcoholic liver injury. The leukocyte migration inhibition test by ethanol was more frequently positive in chronic hepatitis induced by alcohol than in the other types of alcoholic liver injury except for alcoholic hepatitis. Histological characteristics of the liver in chronic hepatitis induced by alcohol included the coexistence of features of both chronic hepatitis and alcoholic fibrosis. Three of four cases of chronic hepatitis induced by alcohol developed cirrhosis during the follow-up period. These results suggest that chronic hepatitis induced by alcohol is a type of alcoholic liver disease with an immunopathological etiology. It is a step toward the development of liver cirrhosis.

The bclA gene codes for the protein backbone of the exosporium glycoprotein BclA of B. anthracis. BclA has a central collagen-like region formed by polymorphic GXX repeats and conserved amino- and carboxy-termini. It is noted here that the bclA gene is also present in the genome of Bacillus cereus and Bacillus thuringiensis. There is considerable size heterogeneity among the BclA proteins, both for species and strains, due to different numbers of GPT repeats and [GPT]5GDTGTT repeats (BclA repeats). PCR products that included the entire variable region were analyzed by conventional agarose gel electrophoresis and by micro-channel fluidics (MCF) LabChip to assess differences in molecular weight (MW). Both methods provided discrimination at the strain level for B. cereus group organisms. Results obtained by MCF electrophoresis were superior to conventional agarose gel analysis demonstrating improved reproducibility and much faster analysis time. The expression of a carbohydrate-rich exosporium (corresponding to BclA) in other members of the B. cereus group, in addition to B. anthracis, was also demonstrated ultra-structurally. Analysis of sequence variability within the bclA gene CLR revealed even greater potential for strain and species identification.

Till date, no bioartificial liver (BAL) procedure has obtained FDA approval or widespread clinical acceptance, mainly because of multifactorial limitations such as the use of microscale or undefined biomaterials, indirect and lower oxygenation levels in liver cells, short-term undesirable functions, and a lack of 3D interaction of growth factor/cytokine signaling in liver cells. To overcome preclinical limitations, primary rat liver cells were cultured on a naturally self-assembling peptide nanoscaffold (SAPN) in a clinically relevant bioreactor for up to 35 days, under 3D interaction with suitable growth factors and cytokine signaling agents, alone or combination (e.g., Group I: EPO, Group II: Activin A, Group III: IL-6, Group IV: BMP-4, Group V: BMP4 + EPO, Group VI: EPO + IL-6, Group VII: BMP4 + IL-6, Group VIII: Activin A + EPO, Group IX: IL-6 + Activin A, Group X: Activin A + BMP4, Group XI: EPO + Activin A + BMP-4 + IL-6 + HGF, and Group XII: Control). Major liver specific functions such as albumin secretion, urea metabolism, ammonia detoxification, phase contrast microscopy, immunofluorescence of liver specific markers (Albumin and CYP3A1), mitochondrial status, glutamic oxaloacetic transaminase (GOT) activity, glutamic pyruvic transaminase (GPT) activity, and cell membrane stability by the lactate dehydrogenase (LDH) test were also examined and compared with the control over time. In addition, we examined the drug biotransformation potential of a diazepam drug in a two-compartment model (cell matrix phase and supernatant), which is clinically important. This present study demonstrates an optimized 3D signaling/scaffolding in a preclinical BAL model, as well as preclinical drug screening for better drug development.