The trans-activator protein Tax of human T-cell leukemia virus type 1 (HTLV-1) activates the viral 21-base-pair (bp) enhancer in the long terminal repeat and has been suggested to associate indirectly with the enhancer DNA. To demonstrate this, we used DNA-affinity precipitation assay and detected the Tax protein in 21-bp DNA-protein complexes isolated from HTLV-1-infected cells. To identify cellular components in the complexes, we tested various 21-bp DNA-binding proteins by gel electrophoretic mobility-shift assay. Each binding protein gave a shifted band of each 21-bp DNA-protein complex, and exogenously added Tax protein further shifted these bands of cAMP-responsive element (CRE) binding protein (CREB) and CRE modulator but did not shift other bands. Anti-Tax antibodies blocked formation of the complex, indicating complex formations of [Tax-CREB(or CRE modulator)-21-bp DNA]. The formations of these complexes paralleled the functional activities of Tax mutants. Furthermore, the Tax-CREB complex was detected in a nuclear extract of HTLV-1-infected cells, and the Tax-CREB-21-bp-DNA complex was demonstrated as a major component of Tax complexes containing the 21-bp DNA probe. These observations indicate that Tax protein binds to CREB and CRE modulator and the complexes then bind to the 21-bp enhancer, suggesting that the complex binding to the enhancer mediates trans-activation of transcription.

Full-length (42 kDa) CCAAT/enhancer binding protein alpha (C/EBP alpha) (p42) has been implicated in the transcriptional activation of adipocyte genes including the 422(aP2) and C/EBP alpha genes during differentiation of 3T3-L1 preadipocytes. We have identified a 30-kDa isoform (p30) of C/EBP alpha that is expressed by 3T3-L1 adipocytes, mouse adipose tissue, and rat liver. In vitro translation of wild-type C/EBP alpha mRNA or transient transfection with a wild-type C/EBP alpha vector gave rise to similar levels of p42 and p30. Mutational analysis revealed that p30 is an alternative translation product initiated at the third in-frame methionine codon of the C/EBP alpha message. p30C/EBP alpha binds to the C/EBP sites within and activates reporter gene expression driven by the 422(aP2) and C/EBP alpha gene promoters. Although transfection of 3T3-L1 preadipocytes with a strong p30C/EBP alpha expression vector is insufficient to induce differentiation, this vector advances the differentiation program. Unlike p42C/EBP alpha, which inhibits cell proliferation, p30C/EBP alpha is not antimitotic. Thus, the N-terminal 12-kDa segment of full-length C/EBP alpha contains an amino acid sequence necessary for antimitotic activity. During differentiation of 3T3-L1 preadipocytes and during hepatocyte development, the cellular p42C/EBP alpha/p30C/EBP alpha ratio changes, raising the possibility of a regulatory role.

Complaints regarding, and morbidity of, osteoporosis are caused by fractures which are associated with pain and decrease of physical function, social function, and well-being. These are aspects of quality of life. Health-related quality of life covers physical, mental, and social well-being. Quality of life may be measured for evaluation of treatment effects in clinical trials, for the assessment of the burden of the disease of osteoporosis, and for estimates of the cost-effectiveness of different treatment scenarios in health care policy. Quality of life has been measured in patients with osteoporosis with generic questionnaires such as SF-36 and EQ-5D, which can be used in many diseases, or with one of the six available osteoporotic-specific questionnaires, e.g., Qualeffo-41 or OPAQ. Every questionnaire has to be validated to assess psychometric properties and discrimination power between patients with osteoporosis and control subjects. The value attached to specific health states (utility) can be assessed with some generic instruments or by systematic questioning of the patient, e.g., the time-trade-off method. This results in one value for health status ranging from 0 (death) to 1 (perfect health). Utility values can be used to calculate loss of quality-adjusted life years (QALY). Most data have been obtained in patients with prevalent vertebral fractures. Scores of specific and generic questionnaires showed significant loss of quality of life with prevalent vertebral fractures. In addition, studies with Qualeffo-41 and OPAQ showed a deteriorating quality of life with increasing number of vertebral fractures. Lumbar fractures had more impact on quality of life than thoracic fractures. Incident vertebral fractures were also associated with a decrease of quality of life especially in the physical function domain. This applied to clinical incident vertebral fractures as well as to subclinical fractures to a lesser degree. Loss of quality of life following hip fracture has been documented with generic and osteoporosis-specific questionnaires. A considerable loss was observed in the 1st year with some improvement in the 2nd year, but not to baseline values. Quality of life depended on comorbidity, mobility, activities of daily life (ADL)-independence, and fracture complaints. Utility loss has been observed following hip fracture, especially disabling hip fracture, hip and vertebral fracture combined, or multiple vertebral fractures. Utility following osteoporotic fractures has been valued by patients, the healthy elderly, and panels of experts. The healthy elderly gave the worse quality-of-life scores (lower utility) to various hip fractures than patients with hip fractures themselves. In conclusion, suitable instruments exist for measuring quality of life in patients with osteoporotic fractures. These instruments are useful for clinical trials and for assessment of the burden of disease.

A rapid sequencing method for ribosomal RNA was applied to the resolution of evolutionary relationships among Metazoa. Representatives of 22 classes in 10 animal phyla were used to infer phylogenetic relationships, based on evolutionary distances determined from pairwise comparisons of the 18S ribosomal RNA sequences. The classical Eumetazoa are divided into two groups. Cnidarians arose from a protist ancestry different from the second group, the Bilateria. Within the Bilateria, an early split gave rise to Platyhelminthes (flatworms) and the coelomate lineage. Coelomates are thus monophyletic, and they radiated rapidly into four groups: chordates, echinoderms, arthropods, and eucoelomate protostomes.

Use of the specific-locus test to measure the frequency of transmitted gene mutations induced in mouse spermatogonia has shown ethylnitrosourea to be by far the most potent mutagen yet discovered in the mouse. The dose used, 250 mg/kg, gave a mutation rate 5 times as high as had been obtained with 600 R, the most effective acute dose of x-rays. Compared to procarbazine, heretofore the most mutagenic chemical known in the mouse, ethylnitrosourea proved to be 15 times more mutagenic than the peak effect obtained with the most effective dose of procarbazine. Because of its high mutagenicity, ethylnitrosourea can serve as a model compound in exploring the effect of such factors as dose response, dose fractionation, sex, and cell stage on the mutagenic action of a chemical. Ethylnitrosourea is clearly the mutagen of choice for the production of any kind of desired new gene mutations in the mouse.

Two hybridoma cell lines were obtained, A1 and B6, which produced monoclonal antibodies reacting with the 44,000-MW C-terminal domain of the adenovirus type 5 DNA-binding protein (DBP). Clone A1 antibodies reacted with the native form of the DBP, but failed to recognize this protein after denaturation (by exposure to sodium dodecylsulfate, or production of the DBP at 39.5 degrees by H5ts107, a temperature-sensitive DBP mutant). Clone B6 antibodies bound to both the native and denatured forms of the DBP. Immunofluorescent staining of wild-type-virus infected cells revealed the DBP located in discrete nuclear patches. A1 and B6 antibodies detected this patched localization of the DBP in nuclei of H5ts107-infected cells grown at 32 degrees. However, at the nonpermissive temperature of 39.5 degrees, A1 antibodies failed to detect the DBP, and B6 antibodies gave a uniform nuclear fluorescent distribution of the DBP. Thus the nuclear pattern of localization for the DBP synthesized by H5ts107 was temperature dependent in this mutant.

Mutations in the rpoB gene of Escherichia coli result in resistance to the antibiotic rifampicin (Rif(r)) by altering the beta subunit of RNA polymerase. Previous studies have identified 39 single base substitutions in the rpoB gene that lead to Rif(r) at 37 degrees C and an additional two mutations that result in temperature sensitive cells. We have extended this work and identified an additional 30 single base substitutions that result in the Rif(r) phenotype. With these mutations the rpoB/Rif(r) system now allows the monitoring of 69 base substitutions at 37 degrees at 37 sites (base pairs) distributed among 24 coding positions. Each of the six possible base substitutions is represented by 8-17 mutations. More than 90% of the mutations are within a small enough region of the rpoB gene to allow PCR amplification with a single pair of oligonucleotide primers, followed by sequencing with a single primer, leading to rapid analysis of numerous mutations. The remaining mutations can be monitored using an additional primer pair. To calibrate this system we sequenced over 500 mutations in rpoB occurring spontaneously or generated by different mutagens and mutators with known specificity. These results show that rpoB/Rif(r) is an accurate and easy to employ detection system, and offers the advantage of allowing analysis of mutations occurring on the chromosome rather than on an extrachromosomal element. The mutS, mutT, mutY, M mutators, as well as the mutagenic agents ethyl methanesulfonate (EMS), ultraviolet (UV) irradiation, 2-aminopurine (2AP), 5-azacytidine (5AZ), and cisplatin (CPT) gave results predicted by their characterized specificities. The number of different sequence contexts is sufficient to reveal significant hotspots among the spontaneous mutS, 2-aminopurine, ultraviolet light, 5-azacytidine, and cisplatin mutational spectra. The cisplatin distribution is particularly striking, with 68% of the mutations resulting from an A:T->>T:A transversion at a single site. Because of the conservation of key regions of RNA polymerase among many microorganisms, using the Rif(r)/rpoB system may be a general method for studying mutational processes in microorganisms without well developed genetic systems.

Vibrio parahaemolyticus O3:K6 strains responsible for the increase in the number of cases of diarrhea in Calcutta, India, beginning in February 1996 and those isolated from Southeast Asian travelers beginning in 1995 were shown to belong to a unique clone characterized by possession of the tdh gene but not the trh gene and by unique arbitrarily primed PCR (AP-PCR) profiles (J. Okuda, M. Ishibashi, E. Hayakawa, T. Nishino, Y. Takeda, A. K. Mukhopadhyay, S. Garg, S. K. Bhattacharya, G. B. Nair, and M. Nishibuchi, J. Clin. Microbiol. 35:3150-3155, 1997). Evidence supporting a hypothesis that this clone emerged only recently and is spreading to many countries was obtained in this study. Of 227 strains isolated in a hospital in Bangladesh between 1977 and 1998, only 22 strains isolated between 1996 and 1998 belonged to the new O3:K6 clone (defined by the serovar, the tdh and trh typing, and AP-PCR profiles). The O3:K6 strains isolated from clinical sources in Taiwan, Laos, Japan, Thailand, Korea, and the United States between 1997 and 1998 were also shown to belong to the new O3:K6 clone. The clonality of the new O3:K6 strains was also confirmed by analysis of the toxRS sequence, which has been shown to be useful for phylogenetic analysis of the members of the genus Vibrio. The toxRS sequences of the representative strains of the new O3:K6 clone differed from those of the O3:K6 strains isolated before 1995 at least at 7 base positions within a 1,346-bp region. A new PCR method targeted to 2 of the base positions unique to the new O3:K6 clone was developed. This PCR method could clearly differentiate all 172 strains belonging to the new O3:K6 clone from other O3:K6 strains isolated earlier. One hundred sixty-six strains belonging to 28 serovars other than O3:K6 were also examined by the new PCR method. The tdh-positive and trh-lacking strains that belonged to the O4:K68 and O1:K untypeable serovars and were isolated in three countries and from international travelers beginning in 1997 gave positive results. The AP-PCR profiles of these strains were nearly identical to those of the new O3:K6 clone, and their toxRS sequences were 100% identical to that of the new O3:K6 clone. The results suggest that these strains may have diverged from the new O3:K6 clone by alteration of the O:K antigens. In conclusion, this study presents strong evidence for the first pandemicity in the history of V. parahaemolyticus and reports a novel toxRS-targeted PCR method that will be useful in epidemiological investigation of the cases associated with the current pandemic spread.

The Pax5 gene coding for the transcription factor BSAP has an essential role in B lymphopoiesis and midbrain development. Here we present a detailed analysis of the B-cell phenotype of Pax5 mutant mice that revealed a differential dependency of fetal and adult B lymphopoiesis on this transcriptional regulator. B-cell development is arrested in the bone marrow at the early pro-B (pre-BI) cell stage, which is characterized by expression of the early markers c-kit, CD43, lambda5, VpreB, and HSA and the absence of the later markers CD25 and BP-1. These pre-BI cells fail to express the BSAP target gene CD19 and are capable of long-term proliferation in vitro in the presence of stromal cells and IL-7. B-lymphoid progenitors could not be detected in the fetal liver of Pax5 mutant embryos. However, Pax5-deficient fetal liver cells gave rise to the development of pre-BI cells in bone marrow on transplantation into lethally irradiated mice. These data indicate different functions of Pax5 in the distinctive microenvironments of fetal liver and adult bone marrow. As shown by PCR analyses, the pre-BI cells in Pax5-deficient bone marrow have undergone D(H)-to-J(H) rearrangement of the immunoglobulin heavy-chain locus at normal frequency. In contrast, V(H)-to-D(H)J(H) rearrangements were reduced approximately 50-fold in Pax5-deficient pre-BI cells, suggesting a role for Pax5 in the developmental pathway controlling V-to-DJ recombination.

The current human whole-cell vaccine is ineffective against pneumonic plague caused by typical F1 capsule positive (F1+) strains of Yersinia pestis. The authors found this vaccine to also be ineffective against F1-negative (F1-) Y. pestis strains, which have been isolated from a human case and from rodents. For these reasons, the authors developed a recombinant vaccine composed of a fusion protein of F1 with a second protective immunogen, V antigen. This vaccine protected experimental mice against pneumonic as well as bubonic plague produced by either an F1+ or F1- strain of Y. pestis, gave better protection than F1 or V alone against the F1+ strain, and may provide the basis for an improved human plague vaccine.

Liver stiffness, noninvasively measured by transient elastography, correlates well with liver fibrosis stage. The aim of this prospective study was to evaluate the liver stiffness measurement (LSM) as a predictor of hepatocellular carcinoma (HCC) development among patients with chronic hepatitis C. Between December 2004 and June 2005, a total of 984 HCV-RNA positive patients, without HCC or a past history of it, visited the University of Tokyo Hospital. LSM was performed successfully in 866 patients, who gave informed consent. During the follow-up period (mean, 3.0 years), HCC developed in 77 patients (2.9% per 1 person-year). The cumulative incidence rates of HCC at 1, 2, and 3 years were 2.4%, 6.0%, and 8.9%, respectively. Adjusting for other significant factors for HCC development, patients with higher LSM were revealed to be at a significantly higher risk, with a hazard ratio, as compared to LSM < or =10 kPa, of 16.7 (95% confidence interval [CI], 3.71-75.2; P < 0.001) when LSM 10.1-15 kPa, 20.9 (95% CI, 4.43-98.8; P < 0.001) when LSM 15.1-20 kPa, 25.6 (95%CI, 5.21-126.1; P < 0.001) when LSM 20.1-25 kPa, and 45.5 (95% CI, 9.75-212.3; P < 0.001) when LSM >25 kPa.

CONCLUSIONS

This prospective study has shown the association between LSM and the risk of HCC development in patients with hepatitis C. The utility of LSM is not limited to a surrogate for liver biopsy but can be applied as an indicator of the wide range of the risk of HCC development.

High-grade serous ovarian carcinoma (HGSOC) is a lethal disease for which improved screening and treatment strategies are urgently needed. Progress in these areas is impeded by our poor understanding of HGSOC pathogenesis. Most ovarian cancer research is based on the hypothesis that HGSOC arises from ovarian surface epithelial cells. However, recent studies suggest that >50% of high-grade serous carcinomas involving the ovary likely arise from fallopian tube epithelium. Therefore, limiting HGSOC research to modeling based on ovarian surface epithelium alone is inadequate. To address the need for a fallopian tube-based model of HGSOC, we have developed a system for studying human fallopian tube secretory epithelial cell (FTSEC) transformation. Our model is based on (i) immortalization of FTSECs isolated from primary samples of normal, nondiseased human fallopian tubes, (ii) transformation of FTSECs with defined genetic elements, and (iii) xenograft-based tumorigenic assays. We use our model to show that FTSECs immortalized with human telomerase reverse transcriptase (hTERT) plus SV40 large T and small T antigens are transformed by either oncogenic Ras (H-Ras(V12)) or c-Myc expression, leading to increased proliferation, clonogenicity, and anchorage-independent growth. Additionally, we demonstrate that FTSECs remain susceptible to c-Myc-mediated transformation in the absence of viral oncoproteins, by replacing SV40 large T and small T antigens with sh-p53, mutant CDK4 (CDK4(R24C)), and sh-PP2A-B56γ. Importantly, all transformed FTSECs gave rise to high-grade Müllerian carcinomas that were grossly, histologically, immunophenotypically, and genomically similar to human HGSOC. With this model, we will now be able to assess the transformative effects of specific genetic alterations on FTSECs in order to characterize their respective roles in HGSOC development.

1. We have purified the AMP-activated protein kinase 4800-fold from rat liver. The acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase kinase activities copurify through all six purification steps and are inactivated with similar kinetics by treatment with the reactive ATP analogue fluorosulphonylbenzoyladenosine. 2. The final preparation contains several polypeptides detectable by SDS/polyacrylamide gel electrophoresis, but only one of these, with an apparent molecular mass of 63 kDa, is labelled using [14C]fluorosulphonylbenzoyladenosine. This is also the only polypeptide in the preparation that becomes significantly labelled during incubation with [gamma 32P]ATP. This autophosphorylation reaction did not affect the AMP-stimulated kinase activity. 3. In the absence of AMP the purified kinase has apparent Km values for ATP and acetyl-CoA carboxylase of 86 microM and 1.9 microM respectively. AMP increases the Vmax 3-5-fold without a significant change in the Km for either protein or ATP substrates. 4. The response to AMP depends on the ATP concentration in the assay, but at a near-physiological ATP concentration the half-maximal effect of AMP occurs at 14 microM. Studies with a range of nucleoside monophosphates and diphosphates, and AMP analogues showed that the allosteric activation by AMP was very specific. ADP gave a small stimulation at low concentrations but was inhibitory at high concentrations. 5. These results show that the AMP-activated protein kinase is the major HMG-CoA reductase kinase detectable in rat liver under our assay conditions and that it is therefore likely to be identical to previously described HMG-CoA reductase kinase(s) which are activated by adenine nucleotides and phosphorylation. The AMP-binding and catalytic domains of the kinase are located on a 63-kDa polypeptide which is subject to autophosphorylation.

We determined whether the formation of the hepatic primordium in the rat is associated with the expression of liver-specific markers. Further, we examined the origin of intra- and extrahepatic bile ducts and tried to establish whether there are cell types in the developing liver that might correspond to "stem-like" cells ("oval cells") that proliferate during carcinogenesis and toxic injury in adult livers. Using in situ hybridization and immunohistochemical methods, we show that alpha-fetoprotein (AFP) mRNA is detected in cells of the ventral foregut at 10.5 days of development and that the protein is first detected 1 day later. Thus, AFP transcription occurs before liver morphogenesis, and translation of the protein is first detected when liver cords are being formed, indicating that AFP expression in endodermal cells signals their commitment toward the liver lineage. Although albumin is considered a trait of differentiated hepatocytes, its mRNA was first detected just 1 day later than the AFP message. An analysis of the expression of lineage-specific cytokeratins (cytokeratins 7, 9, 18, and 19), surface markers, and histochemical determination of gamma-glutamyl transferase activity and glycogen revealed that (a) hepatoblasts undergo gradual maturation throughout liver development, (b) AFP- and albumin-containing hepatoblasts gave rise to intra- and extrahepatic bile ducts, and (c) hepatoblasts forming primitive intrahepatic bile ducts during liver development have markers similar to those expressed by stem-like cells that proliferate during liver carcinogenesis.

1. Nitrogenase from the facultative anaerobe Klebsiella pneumoniae was resolved into two protein components resembling those obtained from other nitrogen-fixing bacteria. 2. Both proteins were purified to homogeneity as shown by the criteria of disc electrophoresis and ultracentrifugal analysis. 3. The larger component had a mol.wt. of 218000 and contained one Mo atom, 17Fe atoms and 17 acid-labile sulphide groups/mol; it contained two types of subunit, present in equal amounts, of mol.wts. 50000 and 60000. All the common amino acids were present, with a predominance of acidic residues. The apparent partial specific volume was 0.73; ultracentrifugal analysis gave s(0) (20,w)=11.0S and D(0) (20,w)=4.94x10(-7)cm(2)/s. The specific activities (nmol of product formed/min per mg of protein) when assayed with the second nitrogenase component were 1500 for H(2) evolution, 380 for N(2) reduction, 1200 for acetylene reduction and 5400 for ATP hydrolysis. The reduced protein showed electron-paramagnetic-resonance signals at g=4.3, 3.7 and 2.015; the Mössbauer spectrum of the reduced protein consisted of at least three doublets. The u.v. spectra of the oxidized and reduced proteins were identical. On oxidation the absorbance increased generally throughout the visible region and a shoulder at 430nm appeared. The circular-dichroism spectra of both the oxidized and reduced proteins were the same, consisting mainly of a negative trough at 220nm. 4. The smaller component had mol.wt. 66800 and contained four Fe atoms and four acid-labile sulphide groups in a molecule comprising two subunits each of mol.wt. 34600. All common amino acids except tryptophan were present, with a predominance of acidic residues. The apparent partial specific volume calculated from the amino acid analysis was 0.732, which was significantly higher than that obtained from density measurements (0.69); ultracentrifugal analysis gave s(0) (20,w)=4.8S and D(0) (20,w)=5.55x10(-7)cm(2)/s. The specific activities (nmol of product formed/min per mg of protein) were 1050 for H(2) evolution, 275 for N(2) reduction, 980 for acetylene reduction and 4350 for ATP hydrolysis. The protein was not cold-labile. The reduced protein showed electron-paramagnetic-resonance signals in the g=1.94 region. The Mössbauer spectrum of the reduced protein consisted of a doublet at 77 degrees K. The u.v. spectra of reduced and O(2)-inactivated proteins were identical, and inactivation by O(2) generally increased the absorbance in the visible region and resulted in a shoulder at 460nm. The circular-dichroism spectra exhibited a negative trough at 220nm and inactivation by O(2) decreased the depth of the trough. 5. The reduction of N(2) and acetylene, and H(2) evolution, were maximal at a 1:1 molar ratio of the Fe-containing protein to the Mo-Fe-containing protein; excess of the Mo-Fe-containing protein was inhibitory. All reductions were accompanied by H(2) evolution. The combined proteins had no ATP-independent hydrogenase activity.

1. Capsazepine is a synthetic analogue of the sensory neurone excitotoxin, capsaicin. The present study shows the capsazepine acts as a competitive antagonist of capsaicin. 2. Capsazepine (10 microM) reversibly reduced or abolished the current response to capsaicin (500 nM) of voltage-clamped dorsal root ganglion (DRG) neurones from rats. In contrast, the responses to 50 microM gamma-aminobutyric acid (GABA) and 5 microM adenosine 5'-triphosphate (ATP) were unaffected. 3. The effects of capsazepine were examined quantitatively with radioactive ion flux experiments. Capsazepine inhibited the capsaicin (500 nM)-induced 45Ca2+ uptake in cultures of rat DRG neurones with an IC50 of 420 +/- 46 nM (mean +/- s.e.mean, n = 6). The 45Ca2+ uptake evoked by resiniferatoxin (RTX), a potent capsaicin-like agonist was also inhibited. (Log concentration)-effect curves for RTX (0.3 nM-1 microM) were shifted in a competitive manner by capsazepine. The Schild plot of the data had a slope of 1.08 +/- 0.15 (s.e.) and gave an apparent Kd estimate for capsazepine of 220 nM (95% confidence limits, 57-400 nM). 4. Capsazepine also inhibited the capsaicin- and RTX-evoked efflux of 86Rb+ from cultured DRG neurones. The inhibition appeared to be competitive and Schild plots yielded apparent Kd estimates of 148 nM (95% confidence limits, 30-332 nM) with capsaicin as the agonist and 107 nM (95% confidence limits, 49-162 nM) with RTX as agonist. 5. A similar competitive inhibition by capsazepine was seen for capsaicin-induced [14C]-guanidinium efflux from segments of adult rat vagus nerves (apparent Kd = 690 nM; 95% confidence limits, 63 nM-1.45 microM). No significant difference was noted in the apparent Kd estimates for capsazepine in assays on cultured DRG neurones and vagus nerve as shown by the overlap in the 95% confidence limits.6. Capsazepine, at concentrations up to 1O microM, had no significant effects on the efflux of 86Rb+ from cultured DRG neurones evoked either by depolarization with high (50 mM) K' solutions or by acidification of the external medium to pH 5.0-5.6. Similarly capsazepine had no significant effect on he depolarization (50 mM KCl)-induced efflux of [14C]-guanidinium from vagus nerve preparations.7. Ruthenium Red was also tested for antagonism against capsaicin evoked ['4C]-guanidinium release from vague nerves and capsaicin induced 45Ca2" uptake in cultures of DRG neurones. In contrast to capsazepine the inhibition by Ruthenium Red (10-500nM in DRG and 0.5-10microM in vagus nerve experiments) was not consistent with a competitive antagonism, but rather suggested a more complex,non-competitive inhibition.

1. Rates of insulin release, glucose utilization (measured as [(3)H]water formation from [5-(3)H]glucose) and glucose oxidation (measured as (14)CO(2) formation from [1-(14)C]- or [6-(14)C]-glucose) were determined in mouse pancreatic islets incubated in vitro, and were used to estimate the rate of oxidation of glucose by the pentose cycle pathway under various conditions. Rates of oxidation of [U-(14)C]ribose and [U-(14)C]xylitol were also measured. 2. Insulin secretion was stimulated fivefold when the medium glucose concentration was raised from 3.3 to 16.7mm in the absence of caffeine; in the presence of caffeine (5mm) a similar increase in glucose concentration evoked a much larger (30-fold) increase in insulin release. Glucose utilization was also increased severalfold as the intracellular glucose concentration was raised over this range, particularly between 5 and 11mm, but the rate of oxidation of glucose via the pentose cycle was not increased. 3. Glucosamine (20mm) inhibited glucose-stimulated insulin release and glucose utilization but not glucose metabolism via the pentose cycle. No evidence was obtained for any selective effect on the metabolism of glucose via the pentose cycle of tolbutamide, glibenclamide, dibutyryl 3':5'-cyclic AMP, glucagon, caffeine, theophylline, ouabain, adrenaline, colchicine, mannoheptulose or iodoacetamide. Phenazine methosulphate (5mum) increased pentose-cycle flux but inhibited glucose-stimulated insulin release. 4. No formation of (14)CO(2) from [U-(14)C]ribose could be detected: [U-(14)C]xylitol gave rise to small amounts of (14)CO(2). Ribose and xylitol had no effect on the rate of oxidation of glucose; ribitol and xylitol had no effect on the rate of glucose utilization. Ribose, ribitol and xylitol did not stimulate insulin release under conditions in which glucose produced a large stimulation. 5. It is concluded that in normal mouse islets glucose metabolism via the pentose cycle does not play a primary role in insulin-secretory responses.

Peptides containing the tripeptide sequence Arg-Gly-Asp can duplicate or inhibit the cell attachment-promoting effects of fibronectin and vitronectin. Peptides analogous to a prototype peptide, Gly-Arg-Gly-Asp-Ser-Pro-Cys, the sequence of which was taken from the cell attachment site of fibronectin, were assayed for their relative abilities to inhibit the attachment of cells to a fibronectin or vitronectin substrate. A peptide having the L-Arg residue replaced with D-Arg showed no difference in this capacity, whereas substituting Gly with D-Ala or L-Asp with D-Asp resulted in completely inactive peptides. Replacement of L-Ser with D-Ser drastically reduced the influence that the resulting peptide had on the vitronectin interaction, but this peptide showed little difference in its effect on the binding of cells to fibronectin when compared with the prototype peptide. Furthermore, substitution of the Ser with L-Asn resulted in a peptide that had an apparent increased preference for the fibronectin receptor and decreased preference for the vitronectin receptor. Conversely, threonine in this position gave a peptide with increased preference for the vitronectin receptor, whereas L-Pro in this position gave a completely inactive peptide. Finally, by cyclicizing the prototype peptide to restrict its conformational flexibility, a peptide was obtained that was a much improved inhibitor of attachment of cells to vitronectin and yet nearly inactive with respect to the interactions of cells with fibronectin substrates. These studies lend support to the hypothesis that different Arg-Gly-Asp-directed adhesion receptors can recognize differences in the conformation and environment of the Arg-Gly-Asp tripeptide, and they establish the feasibility of obtaining synthetic probes that are more selective for individual receptors than are the peptides modeled after the natural sequences of adhesive extracellular matrix molecules.

Excellent normal ranges for plasma lipid and lipoprotein cholesterol levels have been developed by the Lipid Research Clinics program, standardized by the Centers for Disease Control (CDC). However these values were generated by methods not currently in use in most clinical chemistry laboratories. Automated enzymatic methods for cholesterol, triglycerides and free glycerol determinations, as well as a dextran sulfate-Mg2+ procedure for separation of high density lipoproteins (HDL) with standardization are described. Similar methods for the measurement of unesterified cholesterol and phospholipids are also given. Serum pools for total cholesterol with values ranging from 1220-3490 mg/l produced coefficients of variation (CV) less than or equal to 2.85%; reference values for low total cholesterol samples in a range of 280-727 mg/l gave CV of 4.35% or less; HDL cholesterol reference values of 265-640 mg/l yielded CV less than or equal to 3.70%; and values for triglycerides between 0.74 and 3.05 mmol/l gave CV of 4.22% or less through three to eight testing cycles (9-24 mth). These data indicate that long term CDC standardization of total cholesterol, triglycerides, and HDL cholesterol can be obtained with automated enzymatic methods utilizing commercially available reagents.

Human mast cells are known to arise from a CD34(+)/c-kit(+) progenitor cell population that also gives rise to neutrophils, eosinophils, basophils, and monocytes. To further characterize cells within the CD34(+)/c-kit(+) population that yield mast cells, this progenitor was additionally sorted for CD13, a myeloid marker known to appear early on rodent mast cells and cultured human mast cells, but not expressed or expressed at low levels on human tissue mast cells; and cultured in recombinant human (rh) stem cell factor (rhSCF), rh interleukin-3 (rhIL-3; first week only), and rhIL-6. Initial sorts revealed that although the majority of cells in culture arose from the CD34(+)/c-kit(+)/CD13(-) cell population, mast cells arose from a CD34(+)/c-kit(+)/CD13(+) progenitor cell that also gave rise to a population of monocytes. Sequential sorting confirmed that CD34(+)/c-kit(+)/CD13(+) cells in CD34(+)/c-kit(+)/CD13(-) sorts gave rise to the few mast cells observed in CD13(-) sorted cells. CD34(+)/c-kit(+)/CD13(+) cells plated as single cells in the presence of various cytokine combinations gave rise to pure mast cell, monocyte, or mixed mast cell/monocyte progeny. Addition of either rh granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or rhIL-5 to the CD34(+)/c-kit(+)/CD13(+) progenitor cell population cultured in rhSCF, rhIL-3, and rhIL-6 did increase the number of total cells cultured and in the case of rhIL-5, did increase total mast cell numbers. Neither rhGM-CSF or rhIL-5 led to additional cell populations, ie, even with the addition of rhGM-CSF or rhIL-5, only mast cells and monocytes grew from CD34(+)/c-kit(+)/CD13(+) cells. Thus, human mast cells and a population of monocytes arise from precursor cells that express CD34, c-kit, and CD13; and within which, are mast cell, monocyte, and mast/monocyte (bipotential) precursors.

Lithium dodecyl sulfate/polyacrylamide gel electrophoresis of Chlamydomonas reinhardtii thylakoid membranes at room temperature gave two chlorophyll-protein complexes, CP I and CP II, as had been reported previously. However, when the electrophoresis was performed at 4 degrees C, there was an increase in the amount of chlorophyll associated with CP I and CP II, and in addition, three other chlorophyll-protein complexes appeared. Two of these complexes, designated CP III and CP IV, were characterized and found to be similar in their compositions. Each complex contains four to five molecules of chlorophyll a, one molecule of beta-carotene, and one polypeptide chain. The apoprotein of CP III is polypeptide 5 (M(r) 50,000) and that of CP IV is polypeptide 6 (M(r) 47,000); the two polypeptides are structurally unrelated. Chlorophyll-protein complexes similar to C. reinhardtii CP III and CP IV were also detected in higher plants (e.g., Pisum sativum). The apoproteins of the higher plant complexes are immunochemically related to those of the C. reinhardtii complexes, as shown by crossed immunoelectrophoresis. Absorption spectra of CP III and CP IV at -196 degrees C revealed a component at 682 nm. This observation, together with the previous results on photosystem II mutants [Chua, N.-H. & Bennoun, P. (1975) Proc. Natl. Acad. Sci. USA 72, 2175-2179], provides indirect evidence that CP III and CP IV may be involved in the primary photochemistry of photosystem II.

The merits of microwave-based antigen retrieval methods for diagnostic immunohistochemistry have recently been emphasized. As such technology has drawbacks for busy routine laboratories, we have investigated an alternative method of heat-mediated antigen retrieval (HMAR), using a domestic pressure cooker as a source of superheating. A simple protocol, with superheating in 0.01 M citrate buffer (pH 6.0) for 2 min, gave results comparable to those obtainable in a microwave oven. The range of antigens retrieved and the antigens for which heating was ineffective matched the published microwave experience. The advantages included the speed of treatment, the reproducibility of results with large batches of slides, the ability to use metal slide racks, and economy of time and equipment costs.

The acetylcholine receptor from the electric ray Torpedo californica is composed of five subunits; two are identical and the other three are structurally related to them. Microsequence analysis of the four polypeptides demonstrates amino acid homology among the subunits. Further sequence analysis of both membrane-bound and Triton-solubilized, chromatographically purified receptor gave the stoichiometry of the four subunits (40,000:50,000:60,000:65,000 daltons) as 2:1:1:1, indicating that this protein is a pentameric complex with a molecular weight of 255,000 daltons. Genealogical analysis suggests that divergence from a common ancestral gene occurred early in the evolution of the receptor. This shared ancestry argues that each of the four subunits plays a functional role in the receptor's physiological action.

The axonal projections of the ventral subiculum to the bed nucleus of the stria terminalis (BST) were examined in the rat with the anterograde neuronal tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). Axons originating in the ventral subiculum coursed to the BST through either the fimbria-fornix, or a pathway involving the stria terminalis via the amygdala. Ventral subicular axons gave rise to dense terminal networks that were preferentially distributed in medial and ventral subregions of the BST. The distribution of subicular fibers and terminals was examined in relation to BST neurons that project to the hypothalamic paraventricular nucleus (PVN). In these cases, discrete iontophoretic injections of the retrograde tracer Fluoro-gold were made in the PVN, with PHA-L delivered to the ipsilateral ventral subiculum. An immunocytochemical double-labeling protocol was then employed for the simultaneous detection of PHA-L and Fluoro-gold, and provided light microscopic evidence for subicular input to PVN-projecting cells located within the BST. In a second series of experiments, the gamma-amino butyric acid (GABA)ergic nature of the BST was examined by in situ hybridization histochemistry for detection of transcripts encoding GAD67 mRNA. The studies revealed that a high proportion of BST neurons express GAD67 transcripts. Also, experiments combining Fluoro-gold tracing with GAD67 in situ hybridization suggested that a proportion of PVN-projecting neurons in the BST are GABAergic. Taken together, the results of these sets of studies suggest that the inhibitory influences of the hippocampus on the PVN might be relayed through specific portions of the BST. These findings may have important implications for our understanding of the neural regulation of the hypothalamic-pituitary-adrenal axis.