It has been shown in animal models that hepatocytes and cholangiocytes can derive from bone marrow cells. We have investigated whether such a process occurs in humans. Archival autopsy and biopsy liver specimens were obtained from 2 female recipients of therapeutic bone marrow transplantations with male donors and from 4 male recipients of orthotopic liver transplantations from female donors. Immunohistochemical staining with monoclonal antibody CAM5.2, specific for cytokeratins 8, 18, and 19, gave typical strong staining of hepatocytes, cholangiocytes, and ductular reactions in all tissues, to the exclusion of all nonepithelial cells. Slides were systematically photographed and then restained by fluorescence in situ hybridization (FISH) for X and Y chromosomes. Using morphologic criteria, field-by-field comparison of the fluorescent images with the prior photomicrographs, and persistence of the diaminiobenzidene (DAB) stain through the FISH protease digestion, Y-positive hepatocytes and cholangiocytes could be identified in male control liver tissue and in all study specimens. Cell counts were adjusted based on the number of Y-positive cells in the male control liver to correct for partial sampling of nuclei in the 3-micron thin tissue sections. Adjusted Y-positive hepatocyte and cholangiocyte engraftment ranged from 4% to 43% and from 4% to 38%, respectively, in study specimens, with the peak values being found in a case of fibrosing cholestatic recurrent hepatitis C in one of the liver transplant recipients. We therefore show that in humans, hepatocytes and cholangiocytes can be derived from extrahepatic circulating stem cells, probably of bone marrow origin, and such "transdifferentiation can replenish large numbers of hepatic parenchymal cells.

Questionnaire and biochemical measures of smoking were studied in 211 hospital outpatients. Eleven different tests of smoke intake were compared for their ability to categorize smokers and nonsmokers correctly. The concentration of cotinine, whether measured in plasma, saliva, or urine, was the best indicator of smoking, with sensitivity of 96-97 per cent and specificity of 99-100 per cent. Thiocyanate provided the poorest discrimination. Carbon monoxide measured as blood carboxyhaemoglobin or in expired air gave sensitivity and specificity of about 90 per cent. Sensitivities of the tests were little affected by the presence among the claimed nonsmokers of a group of 21 "deceivers" who concealed their smoking. It is concluded that cotinine is the measure of choice, but for most clinical applications carbon monoxide provides an acceptable degree of discrimination and is considerably cheaper and simpler to apply.

Promoter DNA sequences from a petunia chlorophyll a/b binding protein gene were fused to octopine synthase DNA sequences and the resulting chimaeric genes were introduced into petunia and tobacco cells. Populations of transformed regenerated petunia plants containing the chimaeric genes were examined so that the expression of any particular construction could be compared between independent transformants. Substantial variation was observed between transformants in the level of chimaeric gene expression. In general, transcriptional fusions in which a linker sequence interrupted the 5'-untranslated region gave rise to less chimaeric mRNA accumulation than a translational fusion. In the most actively expressing transformants the amount of mRNA from the introduced chimaeric genes was half that of the endogenous wild-type gene. Transcription initiated at the same place in the chimaeric and endogenous genes. Construction of the translational cab/ocs fusion caused three amino acid changes in the octopine synthase protein and functional octopine synthase enzyme was absent from plants in which mRNA for the chimaeric gene was abundantly expressed.

Voltage-dependent sodium channels were studied in dissociated cerebellar Purkinje neurons from rats. In whole-cell recordings, a tetrodotoxin (TTX)-sensitive inward current was elicited when the membrane was repolarized to voltages between -60 and -20 mV after depolarizations to +30 mV long enough to produce maximal inactivation. At -40 mV, this "resurgent" current peaked in 8 msec and decayed with a time constant of 30 msec. With 50 mM sodium as a charge carrier, the resurgent current was on average approximately 120 pA. CA3 pyramidal neurons had no such current. The current may reflect recovery of inactivated channels through open states, because in Purkinje neurons (but not CA3 neurons) there was partial recovery from inactivation at -40 mV, coinciding with the rise of resurgent current. In single-channel recordings, individual channels gave openings corresponding to resurgent and conventional transient current. Action potentials were recorded from dissociated neurons under current clamp to investigate the role of the resurgent current in action potential formation. Purkinje neurons fired spontaneously at approximately 30 Hz. Hyperpolarization to -85 mV prevented spontaneous firing, and brief depolarization then induced all-or-none firing of conglomerate action potentials comprising three to four spikes. When conglomerate action potentials were used as command voltages in voltage-clamp experiments, TTX-sensitive sodium current was elicited between spikes. The falling phase of an action potential is similar to voltage patterns that activate resurgent sodium current, and thus, resurgent sodium current likely contributes to the formation of conglomerate action potentials in Purkinje neurons.

Total hip replacement using porous-coated cobalt-chrome femoral implants designed for biological fixation has been evaluated in 307 patients after two years and in 89 patients after five years. Histological study of 11 retrieved specimens showed bone ingrowth in nine and fibrous tissue fixation in two. Fixation by bone ingrowth occurred in 93% of the cases in which a press fit of the stem at the isthmus was achieved, but in only 69% of those without a press fit. The clinical results at two years were excellent. The incidence of pain and limp was much lower when there was either a press fit of the stem or radiographic evidence of bone ingrowth. Factors such as age, sex, and the disease process did not influence the clinical results. Most cases showed only slight resorptive remodelling of the upper femur, but in a few cases with a larger, more rigid stem, more extensive bone loss occurred. The results after five years showed no deterioration with time. Fixation by the ingrowth of bone or of fibrous tissue both appeared to be stable, but bone ingrowth gave better clinical results.

Neutrophils are prominent participants in the joint inflammation of human rheumatoid arthritis (RA) patients, but the extent of their role in the inductive phase of joint inflammation is unknown. In the K/BxN mouse RA model, transfer of autoreactive Ig from the K/BxN mouse into mice induces a rapid and profound joint-specific inflammatory response reminiscent of human RA. We observed that after K/BxN serum transfer, the earliest clinical signs of inflammation in the ankle joint correlated with the presence of neutrophils in the synovial regions of recipient mouse ankle joints. In this study, we investigated the role of neutrophils in the early inflammatory response to transferred arthritogenic serum from the K/BxN transgenic mouse. Mice were treated with a neutrophil-depleting mAb before and following transfer of arthritogenic serum and scored for clinical indications of inflammation and severity of swelling in ankle joints and front paws. In the absence of neutrophils, mice were completely resistant to the inflammatory effects of K/BxN serum. Importantly, depletion of neutrophils in diseased recipient mice up to 5 days after serum transfer reversed the inflammatory reaction in the joints. Transfer of serum into mice deficient in the generation of nitrogen or oxygen radicals (inducible NO synthase 2 or gp91(phox) genes, respectively) gave normal inflammatory responses, indicating that neither pathway is essential for disease induction. These studies have identified a critical role for neutrophils in initiating and maintaining inflammatory processes in the joint.

HIV-1 reservoirs preclude virus eradication in patients receiving highly active antiretroviral therapy (HAART). The best characterized reservoir is a small, difficult-to-quantify pool of resting memory CD4(+) T cells carrying latent but replication-competent viral genomes. Because strategies targeting this latent reservoir are now being tested in clinical trials, well-validated high-throughput assays that quantify this reservoir are urgently needed. Here we compare eleven different approaches for quantitating persistent HIV-1 in 30 patients on HAART, using the original viral outgrowth assay for resting CD4(+) T cells carrying inducible, replication-competent viral genomes as a standard for comparison. PCR-based assays for cells containing HIV-1 DNA gave infected cell frequencies at least 2 logs higher than the viral outgrowth assay, even in subjects who started HAART during acute/early infection. This difference may reflect defective viral genomes. The ratio of infected cell frequencies determined by viral outgrowth and PCR-based assays varied dramatically between patients. Although strong correlations with the viral outgrowth assay could not be formally excluded for most assays, correlations achieved statistical significance only for integrated HIV-1 DNA in peripheral blood mononuclear cells and HIV-1 RNA/DNA ratio in rectal CD4(+) T cells. Residual viremia was below the limit of detection in many subjects and did not correlate with the viral outgrowth assays. The dramatic differences in infected cell frequencies and the lack of a precise correlation between culture and PCR-based assays raise the possibility that the successful clearance of latently infected cells may be masked by a larger and variable pool of cells with defective proviruses. These defective proviruses are detected by PCR but may not be affected by reactivation strategies and may not require eradication to accomplish an effective cure. A molecular understanding of the discrepancy between infected cell frequencies measured by viral outgrowth versus PCR assays is an urgent priority in HIV-1 cure research.

Green fluorescence protein (GFP)-based fluorescence resonance energy transfer (FRET) is increasingly used in investigation of inter- and intramolecular interactions in living cells. In this report, we present a modified method for FRET quantification in cultured cells using conventional fluorescence microscopy. To reliably measure FRET, three positive control constructs in which a cyan fluorescence protein and a yellow fluorescence protein were linked by peptides of 15, 24, or 37 amino acid residues were prepared. FRET was detected using a spectrofluorometer, a laser scanning confocal microscope, and an inverted fluorescence microscope. Three calculation methods for FRET quantification using fluorescence microscopes were compared. By normalization against expression levels of GFP fusion proteins, the modified method gave consistent FRET values that could be compared among different cells with varying protein expression levels. Whole-cell global analysis using this method allowed FRET measurement with high spatial resolutions. Using such a procedure, the interaction of synaptic proteins syntaxin and the synaptosomal associated protein of 25 kDa (SNAP-25) was examined in PC12 cells, which showed strong FRET on plasma membranes. These results demonstrate the effectiveness of the modified method for FRET measurement in live cell systems.

OBJECTIVE

This study reports the results of the first phase of a national study to determine the prevalence of diabetes and prediabetes (impaired fasting glucose and/or impaired glucose tolerance) in India.

METHODS

A total of 363 primary sampling units (188 urban, 175 rural), in three states (Tamilnadu, Maharashtra and Jharkhand) and one union territory (Chandigarh) of India were sampled using a stratified multistage sampling design to survey individuals aged ≥ 20 years. The prevalence rates of diabetes and prediabetes were assessed by measurement of fasting and 2 h post glucose load capillary blood glucose.

RESULTS

Of the 16,607 individuals selected for the study, 14,277 (86%) participated, of whom 13,055 gave blood samples. The weighted prevalence of diabetes (both known and newly diagnosed) was 10.4% in Tamilnadu, 8.4% in Maharashtra, 5.3% in Jharkhand, and 13.6% in Chandigarh. The prevalences of prediabetes (impaired fasting glucose and/or impaired glucose tolerance) were 8.3%, 12.8%, 8.1% and 14.6% respectively. Multiple logistic regression analysis showed that age, male sex, family history of diabetes, urban residence, abdominal obesity, generalised obesity, hypertension and income status were significantly associated with diabetes. Significant risk factors for prediabetes were age, family history of diabetes, abdominal obesity, hypertension and income status.

CONCLUSIONS

We estimate that, in 2011, Maharashtra will have 6 million individuals with diabetes and 9.2 million with prediabetes, Tamilnadu will have 4.8 million with diabetes and 3.9 million with prediabetes, Jharkhand will have 0.96 million with diabetes and 1.5 million with prediabetes, and Chandigarh will have 0.12 million with diabetes and 0.13 million with prediabetes. Projections for the whole of India would be 62.4 million people with diabetes and 77.2 million people with prediabetes.

This two-part meta-analysis combined data from eight prospective randomized trials designed to compare the nutritional efficacy of early enteral (TEN) and parenteral (TPN) nutrition in high-risk surgical patients. The combined data gave sufficient patient numbers (TEN, n = 118; TPN, n = 112) to adequately address whether route of substrate delivery affected septic complication incidence. Phase I (dropouts excluded) meta-analysis confirmed data homogeneity across study sites, that TEN and TPN groups were comparable, and that significantly fewer TEN patients experienced septic complications (TEN, 18%; TPN, 35%; p = 0.01). Phase II meta-analysis, an intent-to-treat analysis (dropouts included), confirmed that fewer TEN patients developed septic complications. Further breakdown by patient type showed that all trauma and blunt trauma subgroups had the most significant reduction in septic complications when fed enterally. In conclusion, this meta-analysis attests to the feasibility of early postoperative TEN in high-risk surgical patients and that these patients have reduced septic morbidity rates compared with those administered TPN.

Since the tumor necrosis factor alpha (TNF-alpha) gene was found to be located in the central major histocompatibility complex (MHC) there has been much speculation concerning a genetic association between particular TNF alleles and disease susceptibility. A relationship between the MHC haplotype A1, B8, DR3, TNF-alpha expression levels and susceptibility to autoimmune disease has been suggested by several groups. The identification of the -308 polymorphism and its association with the HLA A1, B8, DR3 haplotype have led to speculation that the polymorphism may play a role in the altered expression of TNF-alpha. We have demonstrated that the region (-323 to -285) encompassing -308 in the TNF2 allele binds nuclear factors differently to the same region in the promoter of the more common TNF1 allele. The G/A -308 polymorphism affected the affinity of factor binding and resulted in a factor binding to TNF2 but not TNF1. The observed differential binding was shown to be functional, with the 38bp region from TNF2 causing a two-fold greater activity of a heterologous promoter over that due to the same region in TNF1. To further substantiate the functional consequences of the TNF-alpha -308 polymorphism, we analysed both allelic forms of the TNF-alpha promoter region (-993 to +110) in a transient transfection assay, using luciferase as a reporter gene. The results showed that when present with the 3'UTR the -308A allelic form gave a two-fold greater level of transcription than the 308G form in PMA-stimulated Jurkat and U937 cells. This suggests that the -308 G/A polymorphism may play a role in the altered TNF-alpha gene expression observed in individuals with the HLA A1, B8, DR3 haplotype.

1. An acid ninhydrin reagent was found to react specifically in forming a pink product (E(max.) 560mmu) with cysteine. 2. The method was highly sensitive for the determination of cysteine (in 28.0x10(3)). Homocysteine, glutathione, proline, ornithine and other naturally occurring amino acids tested did not give a similar reaction. 3. The reaction product was stable for at least 3-4hr. at room temperature and the extinction was proportional to the concentration in the range 0.05-0.5mumole of cysteine. 4. The acid ninhydrin reagent also gave yellow products (E(max.) 370-404mmu) with tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine and indol-3-ylacetic acid. 5. The method was applied for the determination of cysteine in perchloric acid extracts of rat brain, liver and blood.

The interleukin 2 receptor gamma chain (IL-2R gamma) is a component of the receptors for IL-2, IL-4, IL-7, and IL-15. Mutations in IL-2R gamma in man appear responsible for the X chromosome-linked immunodeficiency SCIDX1, characterized by a defect in T-cell and natural killer (NK)-cell differentiation with the presence of poorly functioning B cells. To explore at which level IL-2R gamma affects lymphoid development in vivo, we have analyzed mice derived from embryonic stem (ES) cells with mutant IL-2R gamma loci generated by Cre/loxP-mediated recombination. In the peripheral blood of chimeric animals, lymphoid cells derived from IL-2R gamma- ES cells were not detected, although control ES cells carrying an IL-2R gamma gene with embedded loxP sites gave rise to T-, B-, and NK-cell lineages. Germline IL-2R gamma-deficient male animals, however, developed some mature splenic B and T cells, although the absolute number of lymphocytes was almost 10-fold reduced. In contrast, there was a complete disappearance of NK cells (over 350-fold reduction). Development of gut-associated intraepithelial lymphocytes was also severely diminished, and Peyer's patches were not detected. In vitro mitogenic responses of thymocytes, IL-4-directed immunoglobulin class switch of splenocytes, and NK activity were defective. Thus, IL-2R gamma facilitates mainstream B- and T-cell generation and function and also appears to be essential for NK-cell development.

OBJECTIVE

To derive a regression equation that estimates metabolic equivalent (MET) from accelerometer counts, and to define thresholds of accelerometer counts that can be used to delineate sedentary, light, moderate, and vigorous activity in adolescent girls.

METHODS

Seventy-four healthy 8th grade girls, age 13 - 14 yr, were recruited from urban areas of Baltimore, MD, Minneapolis/St. Paul, MN, and Columbia, SC, to participate in the study. Accelerometer and oxygen consumption (.-)VO(2)) data for 10 activities that varied in intensity from sedentary (e.g., TV watching) to vigorous (e.g., running) were collected. While performing these activities, the girls wore two accelerometers, a heart rate monitor and a Cosmed K4b2 portable metabolic unit for measurement of (.-)VO(2). A random-coefficients model was used to estimate the relationship between accelerometer counts and (.-)VO(2). Activity thresholds were defined by minimizing the false positive and false negative classifications.

RESULTS

The activities provided a wide range in (.-)VO(2) (3 - 36 mL x kg x min) with a correspondingly wide range in accelerometer counts (1- 3928 counts x 30 s). The regression line for MET score versus counts was MET = 2.01 +/- 0.00171 (counts x 30 s) (mixed model R = 0.84, SEE = 1.36). A threshold of 1500 counts x 30 s defined the lower end of the moderate intensity (approximately 4.6 METs) range of physical activity. That cutpoint distinguished between slow and brisk walking, and gave the lowest number of false positive and false negative classifications. The threshold ranges for sedentary, light, moderate, and vigorous physical activity were found to be 0 - 50, 51- 1499, 1500 - 2600, and >2600 counts x 30 s, respectively.

CONCLUSIONS

The developed equation and these activity thresholds can be used for prediction of MET score from accelerometer counts and participation in various intensities of physical activity in adolescent girls.

Human induced pluripotent stem cells (iPSCs) present exciting opportunities for studying development and for in vitro disease modeling. However, reported variability in the behavior of iPSCs has called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate. Under standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to that of human embryonic stem cells (ESCs). Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all 16 iPSC lines passed a stringent test of differentiation capacity despite variations in karyotype and in the expression of early pluripotency markers and transgenes. This iPSC and ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.

Twenty clones were isolated from cultured Aedes albopictus (Singh) cells in the presence of anti-Chikungunya (CHIK) virus serum. Each clone was tested for its yields of Dengue (DEN) viruses, types 1, 2, 3 and 4, and also CHIK virus. Clone C6 showed the highest yield of each virus tested. Forty-three clones obtained by recloning C6 in the presence of anti-DEN sera showed almost the same virus yields as C6. One of the clones, C6/36, showed mild to extensive cytopathic effects several days after virus infection, in contrast to the original uncloned (SAAR) cells. Fluorescent antibody staining revealed that the amount of virus antigen accumulated in the cytoplasm was almost the same in every cell in the case of clone C6/36, while it was highly heterogeneous for uncloned SAAR cells. Growth curves of the viruses indicated that clone C6/36 gave a significantly higher yield for each virus than uncloned SAAR cells up to 7 days after infection. Virus sensitivity of the C6/36 clone did not change by growing the cells with the medium used for uncloned SAAR cells, nor did the virus sensitivity of uncloned cells increase in medium used for clone C6/36. However, the C6/36 clone became resistant to CHIK virus, but not to DEN or Sindbis viruses, after incubation with the medium used for another A. albopictus cell line (SAAK). The transfer of the specific resistance to CHIK may be mediated by some latent virus related to CHIK.

1. By subdividing ventral roots and supplying stimulating pulses to different groups of motor units in rotation, smooth contractions of soleus could be obtained with low rates of stimulation.2. Isometric tension was recorded with different rates of stimulation, and at different muscle lengths.3. Longitudinal histological sections were cut from muscles fixed at different lengths, and sarcomeres were measured. Mean sarcomere lengths in soleus could then be related to the angle at the ankle.4. At high rates of stimulation the maximum active tension was obtained at a length corresponding to an angle of about 60 degrees at the ankle, and a mean sarcomere length of about 2.8 mu. The isometric tension fell only slightly on shortening the muscle to a length equivalent to 100 degrees , and a mean sarcomere length about 2.3 mu. Further shortening caused a marked fall in tension.5. There was a reciprocal relationship between stimulus rate and muscle length; when the muscle was long low rates of stimulation gave near maximal tension, whereas at short lengths the maximum tension was reached only when the stimulus rate was very high. It is suggested that stimulating pulses activate the contractile machinery of the muscle more effectively at long than at short muscle lengths.6. When at low rates of stimulation pulses were distributed among the motor units in rotation to give a smooth contraction, the tension rose higher than during the unfused tetanus that accompanied synchronous stimulation of the same motor units at the same rate. It is suggested that in an unfused tetanus internal movement of the muscle reduces the tension below that developed in a truly isometric state.7. The rate of rise of tension in an isometric tetanus varied with both muscle length and rate of stimulation. At each stimulus rate there was a range of lengths in which the isometric tension developed slowly, this was the same length range in which, at that stimulus rate, the length tension curve was steep.

The merits of formaldehyde, formaldehyde-glutaraldehyde combinations, and glutaraldehyde in phosphate buffers have been compared as fixatives that will give easy and satisfactory preservation of tissues for routine automated histologic processing and yet keep them suitable for electron microscopical studies after prolonged storage at room temperature. We recommend a combination of 4% commercial formaldehyde and 1% glutaraldehyde in a buffer of 176 mOsm/liter. Tissue sections should not exceed 3 mm in width, and tissues to be examined by electron microscopy should be taken from the outside of the tissue sections. All special stains performed at light microscopical level gave satisfactory results. The fixative should be stored at 4 C and is stable for at least three months. The superior cross-linking features of glutaraldehyde are retained, while the concentration of glutaraldehyde is low enough not to substantially obscure the PAS reaction.

Direct microinjection of DNA by glass micropipettes was used to introduce the Herpes simplex virus thymidine kinase gene into cultured mammalian cells. When DNA was delivered directly into the nuclei of LMTK-, a mouse cell line deficient in thymidine kinase activity, 50--100% of the cells expressed TK enzymatic activity. In contrast, no TK activity could be detected when the DNA was injected into the cytoplasm. The number of injected LMTK- cells capable of indefinite growth in a TK+ selective medium (that is, transformants) depended on the nature of the plasmid DNA into which the HSV-TK gene was inserted. One cell in 500-1000 cells which received nuclear injections with pBR322/TK DNA gave rise to a viable colony when grown in HAT medium (that is, a TK+ selective medium). The transformation frequency increased to one in five injected cells when specific SV40 DNA sequences were also introduced into the HSV-TK plasmid. With the microinjection procedure transformation frequency was relatively insensitive to DNA concentration and did not depend on co-injecting with a carrier DNA. Most of the transformants were stable in nonselective medium as soon as they could be tested.

Bone morphogenetic proteins (BMPs) are secreted proteins that interact with cell-surface receptors and are believed to play a variety of important roles during vertebrate embryogenesis. Bmpr, also known as ALK-3 and Brk-1, encodes a type I transforming growth factor-beta (TGF-beta) family receptor for BMP-2 and BMP-4. Bmpr is expressed ubiquitously during early mouse embryogenesis and in most adult mouse tissues. To study the function of Bmpr during mammalian development, we generated Bmpr-mutant mice. After embryonic day 9.5 (E9.5), no homozygous mutants were recovered from heterozygote matings. Homozygous mutants with morphological defects were first detected at E7.0 and were smaller than normal. Morphological and molecular examination demonstrated that no mesoderm had formed in the mutant embryos. The growth characteristics of homozygous mutant blastocysts cultured in vitro were indistinguishable from those of controls; however, embryonic ectoderm (epiblast) cell proliferation was reduced in all homozygous mutants at E6.5 before morphological abnormalities had become prominent. Teratomas arising from E7.0 mutant embryos contained derivatives from all three germ layers but were smaller and gave rise to fewer mesodermal cell types, such as muscle and cartilage, than controls. These results suggest that signaling through this type I BMP-2/4 receptor is not necessary for preimplantation or for initial postimplantation development but may be essential for the inductive events that lead to the formation of mesoderm during gastrulation and later for the differentiation of a subset of mesodermal cell types.

The sequence of small-subunit rRNA varies in an orderly manner across phylogenetic lines and contains segments that are conserved at the species, genus, or kingdom level. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, we amplified bacterial 16S ribosomal DNA sequences with the polymerase chain reaction. Priming sites were located at the extreme 5' end, the extreme 3' end, and the center of 16S ribosomal DNA. The isolates tested with these primers included members of the genera Staphylococcus, Coxiella, Rickettsia, Clostridium, Neisseria, Mycobacterium, Bilophila, Eubacterium, Fusobacterium, and Lactobacillus and the family Enterobacteriaceae. Initially, the yields from the reactions were erratic because the primers were self-complementary at the 3' ends. Revised primers that were not self-complementary gave more reproducible results. With the latter primers, 0.4 pg of Escherichia coli DNA consistently gave a visible band after amplification. This method should be useful for increasing the amounts of bacterial 16S ribosomal DNA sequences for the purposes of sequencing and probing. It should have a broad range of applications, including the detection and identification of known pathogens that are difficult to culture. This approach may make it possible to identify new, nonculturable bacterial pathogens.

We have expressed six previously cloned isoforms of human microtubule-associated tau protein in Escherichia coli and purified them to homogeneity in a biologically active form. They range from 352 to 441 amino acids in length and differ from each other by the presence of three or four tandem repeats in the carboxy-terminal half and by the presence or absence of 29 or 58 amino acid inserts in the amino-terminus. When mixed together they gave a set of six bands on SDS-PAGE gels with apparent molecular weights of 48-67 kd and with a characteristic pattern of spacings. Four of these bands aligned with the major tau bands found in adult human cerebral cortex following perchloric acid extraction and alkaline phosphatase treatment. They consisted of isoforms with three repeats and no insertions, four repeats and no amino-terminal insertions and three- and four-repeat containing isoforms with the 29 amino acid insertion. In fetal human brain extracts treated with alkaline phosphatase one of the two major tau bands aligned with the three-repeat containing isoform with no insertions, whereas the molecular nature of the second major tau band remains to be established. The recombinant tau isoforms were biologically active at micromolar concentrations, as assessed by their ability to promote microtubule assembly. The rates of assembly were 2.5-3.0 times faster for isoforms containing four repeats when compared with three-repeat containing isoforms, with no significant contribution by the amino-terminal insertions.

Nonalcohol-induced fatty liver is widely believed to be a benign condition with little or no risk of disease progression. There have been occasional reports of progression to cirrhosis but none in the absence of preexisting fibrosis on the index biopsy specimen even when co-existing hepatitis was present (steatohepatitis). From our histological database (1978 to 1985), we identified 161 patients with fatty liver seen at our institution and traced the case notes of 156. One hundred five patients were initially excluded as having an alcohol-induced cause, and the remaining 51 either were seen in the clinic (37) or had died, in which cases copies of their death certificates were obtained (14). A further 7 patients were excluded after clinic attendance gave evidence of alcohol excess and another 4 after review of their initial biopsy showed the presence of fibrosis or steatohepatitis. The apparent cause of the steatosis in the 40 included patients with strictly nonalcohol-induced pure fatty liver was obesity in 12, diabetes in 4 (1 obese patient), and cachexia associated with extrahepatic malignancy in 6. Four of the remaining 19 had serological evidence of an autoimmune disorder, but none of these had any clinical or histological features of autoimmune liver disease. Nine patients had evidence of hyperlipidemia, 3 of whom were also obese. At a median follow-up of 11 years (7 to 16), 12 of 26 living patients had abnormal results of liver blood tests and had repeat liver biopsies performed. None had progressed to steatohepatitis or cirrhosis; 1 obese patient had developed mild fibrosis 9.8 years after her index biopsy.(ABSTRACT TRUNCATED AT 250 WORDS)

There are few reports describing experimental models of the growth and metastasis of human breast carcinomas. This article discusses the tumorigenic and metastatic properties of two estrogen receptor-negative breast carcinomas injected into nude mice. Tumor growth in the mammary fatpad (m.f.p.) and the subcutis was compared in female nude mice. The injection of 10(5) viable cells of two human breast carcinoma cell lines (MDA-MB-231 and MDA-MB-435) gave a 100% tumor take rate in the m.f.p., whereas only 40% of the s.c. injections produced tumors and these occurred several weeks after the appearance of the m.f.p. tumors. Thus, the m.f.p. of nude mice is a favorable site for the growth of human breast carcinomas. MDA-MB-435 tumors produced distant metastases in 80% to 100% of recipients. The most common sites for metastasis were the lymph nodes and lungs, with a lower incidence of metastases in muscle (chest wall and thigh), heart, and brain. New variant cell lines were isolated from metastases in the lungs, brain, and heart. All the cell lines were tumorigenic in the m.f.p., and the lung- and heart-derived metastasis lines produced slightly more lung metastases than the original cell line. However, the brain metastasis variant produced significantly fewer lung metastases. Intravenous inoculation of the spontaneous metastasis-derived cell lines produced few lung colonies. Only cell variants isolated from experimental lung metastases showed enhanced lung colonization potential when reinjected i.v. Our results suggest that the estrogen receptor-negative MDA-MB-435 cell line injected in the m.f.p. of nude mice could be a valuable tool for analysis of the cellular and molecular basis of the metastasis of advanced breast cancer.