Molecular basis for the binding of SH3 ligands with non-peptide elements identified by combinatorial synthesis.

BACKGROUND: Protein-structure-based combinatorial chemistry has recently been used to discover several ligands containing non-peptide binding elements to the Src SH3 domain. The encoded library used has the form Cap-M1-M2-M3-PLPPLP, in which the Cap and Mi's are composed of a diverse set of organic monomers. The PLPPLP portion provided a structural bias directing the non-peptide fragment Cap-M1-M2-M3 to the SH3 specificity pocket. Fifteen ligands were selected from > 1.1 million distinct compounds. The structural basis for selection was unknown. RESULTS: The solution structures of the Src SH3 domain complexed with two ligands containing non-peptide elements selected from the library were determined by multidimensional NMR spectroscopy. The non-peptide moieties of the ligands interact with the specificity pocket of Src SH3 domain differently from peptides complexed with SH3 domains. Structural information about the ligands was used to design various homologs, whose affinities for the SH3 domain were measured. The results provide a structural basis for understanding the selection of a few optimal ligands from a large library. CONCLUSIONS: The cycle of protein-structure-based combinatorial chemistry followed by structure determination of the few highest affinity ligands provides a powerful new tool for the field of molecular recognition.

Human peripheral blood polymorphonuclear leukocytes at the ovulatory period are in an activated state.

The ovulatory process has been compared with inflammation. We investigated the state of female peripheral polymorphonuclear leukocytes (PMN) during menstrual cycles, and found that PMN contained high levels of superoxide, hydrogen peroxide and nitric oxide (NO) during at the peri-ovulatory period. Assuming the cause of this elevation to be a luteinizing hormone (LH), the surge of which preceded the ovulation, we examined the responsiveness of PMN to pituitary LH. The results revealed that this hormone elevated dose-dependently the production of reactive oxygen intermediates (ROI). Furthermore, we demonstrated the mRNA expression of LH receptors and their presence on PMN. The data indicated that the LH surge before on the ovulatory day resulted in general activation of PMN, suggesting that this state of PMN may be a necessary step for initiation of ovulation, rather than a defensive role against infection.

Histologic growth pattern of hepatocellular carcinoma: relationship to orcein (hepatitis B surface antigen)-positive cells in cancer tissue.

The histologic pattern of tumor growth at tumor-nontumor boundaries was studied in 60 livers bearing hepatocellular carcinoma. Three growth patterns, arbitrarily described as "sinusoidal," "replacing," and "encapsulated," were distinguished. Cancer cells growing in the sinusoids between liver cell cords (sinusoidal pattern) were anaplastic; those growing in an expansile fashion and acquiring a fibrous capsule (encapsulated pattern) were most differentiated; and those growing into the cord of liver cells and replacing them (replacing pattern) were differentiated to an intermediate degree. There was certain relation between the histologic growth patterns and gross morphologic features of the tumors. Test results for 20 of the 60 cases were positive for serum hepatitis B surface antigen (HBsAg), and the livers also contained orcein-positive cells. Orcein-positive cells were frequently seen at the border between tumor and parenchyma. Cells containing HBsAg as an orcein-positive inclusion were present in cancer tissue in three cases. When serial sections were made from such areas and stained alternately with hematoxylin and eosin and orcein, it was found that these cells were hepatocytes blended with cancer cells. This phenomenon was related to the growth pattern of tumor cells. Orcein-positive cells were never found in metastatic lesions.

[Case study of interleukin-1 beta, tumor necrosis factor alpha and interleukin-6 production peripheral blood monocytes in patients with diabetes mellitus complicated by pulmonary tuberculosis].

Patients with diabetes mellitus (DM) show an increased susceptibility to bacterial infections due to the presence of neutrophil dysfunction. Susceptibility to tuberculosis has also been reported in such patients, however, the reason remains unclear. This study measured the production of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha) and interleukin-6 (IL-6) by the peripheral monocytes of patients diagnosed with pulmonary tuberculosis accompanied by DM (TB+DM) and patients without DM complications (TB) using age-matched, healthy control subjects for comparison. Also examined was the relationship between cytokine production and DM control. The results were as follows: (1) The production of IL-1 beta, TNF alpha and IL-6 in TB patients was significantly higher than that observed in the healthy control subjects. (2) The production of IL-1 beta, TNF alpha and IL-6 in TB+DM patients was significantly lower than that observed in the TB patients. (3) The production of IL-1 beta and TNF alpha in TB+DM patients with poor control was significantly lower than that observed in the patients with good control. (4) The TNF alpha production had a significant inverse correlation to HbA1c in the TB+DM patients. This study demonstrated that the production of cytokines is impaired in TB+DM patients and suggests a close correlation between tuberculosis immunity and DM.

[Interaction between interleukin-1 and tumor necrosis factor productions by peripheral blood monocytes and nutritional disturbance in active pulmonary tuberculosis].

Malnutrition is frequently observed in patients with pulmonary tuberculosis. We have already reported the nutritional disturbance in those patients by comprehensive nutritional assessment. But the mechanism of this nutritional disturbance remains unclear. We anticipated that cytokines contributed to the nutritional disturbance. To elucidate this mechanism we measured the productions of interleukin-1 (IL-1) and tumor necrosis factor (TNF) by peripheral blood monocytes, and correlated them with nutritional parameters in those patients. These cytokines had been reported to mediate metabolic alterations in inflammatory process. Subjects were 45 patients with bacteriologically confirmed pulmonary tuberculosis and their controls matched by age and sex. Adherent monocyte at 0.5 x 10(6)/ml were stimulated by lipopolysaccharide (LPS), and the culture supernatant was measured by ELISA for IL-1 and TNF. In order to assess nutritional status we measured serum albumin, transferrin, prealbumin, retinol binding protein, branched chain amino acid (BCAA)/aromatic amino acid (AAA) ratio as amino acid imbalance index, % ideal body weight (%IBW), % arm muscle circumference (% AMC) as muscle mass index, % triceps skin fold thickness (% TSF), as fat store index. The results were as follows: (1) Patients with active pulmonary tuberculosis were confirmed to be malnourished in visceral proteins, plasma amino acid, and anthropometric indices. (2) In patients with moderate or mild nutritional depletion the production of IL-1 and TNF was higher than that in healthy controls, and significantly correlated inversely with the nutritional parameters. (3) In patients with severe nutritional depletion the production of IL-1 and TNF was lower than that in healthy controls.(ABSTRACT TRUNCATED AT 250 WORDS)

[Reduction in mean cerebral blood flow measurements using 99mTc-ECD-SPECT during normal aging].

Mean cerebral blood flow (mCBF) was measured by SPECT using the 99mTc-ECD-Patlak-Plot method in a selected group of 61 normal non-hospitalized subjects aged 51 to 91 years. The mCBF values showed 48.4 +/- 4.7 ml/100 g/min in aged 50-59 years group, 49.9 +/- 5.9 ml/100 g/min in aged 60-69 years group, 46.4 +/- 6.5 ml/100 g/min in aged 70-79 group, 38.0 +/- 3.7 ml/100 g/min in aged 80-89 years group, 38.9 ml/100 g/min in aged 90-99 years group. There was a statistically significant reduction of mCBF with advancing age (R = -0.41; p = 0.001). Women have significantly higher mCBF values than men up to aged 70 years group. In this study, there was no significant laterality in the mCBF between right and left hemispheres in all decade groups. The history of hypertension, alcohol consumption, and cigarette smoking failed to show significant difference in the mCBF values. The present study shows that normal aging is associated with mCBF reduction.

The relationship between natural killer cell activity and delayed-type hypersensitivity reaction to 2,4-dinitrochlorbenzene in the spectrum of chronic, intractable pulmonary tuberculosis.

We examined natural killer (NK) cell activity and delayed-type hypersensitivity reaction to 2,4-dinitrochlorbenzene (DNCB) in the spectrum of chronic, intractable pulmonary tuberculosis. A high reactive group as defined by high NK cell activity and positive reaction to DNCB were characterised by stable clinical courses assessed by radiographical and clinical changes. A low reactive group defined by non-augmented NK cell activity and negative reaction to DNCB were characterised by progressive clinical courses. Although far advanced radiographic lesions were observed more frequently in the low reactive group and moderately advanced lesions more frequently in the high reactive group, there was not a significant statistical trend.

Hepatocellular carcinoma presenting as intrabile duct tumor growth: a clinicopathologic study of 24 cases.

We describe the features of 24 cases of hepatocellular carcinoma (HCC) with prominent intrabile duct tumor growth seen among 238 autopsy and 21 surgical cases of HCC. Progressive obstructive jaundice occurred during the course of most cases and was the presenting sign in nine. A fluctuating rise and fall of the total bilirubin was seen in two cases. The average survival time of the cases was significantly shorter than that of HCC patients without intrabile duct tumor growth (Mann-Whitney's U-test, one-tailed, P less than 0.05). The average survival time after the development of severe jaundice (total bilirubin over 10 mg/dl) was only 16 days. Intrabile duct tumor casts were located in the hepatic and/or the common bile ducts in 19 cases (79%) and in five cases were seen in the peripheral (medium to small-sized) bile ducts. Hemobilia developed in five cases (21%) and was regarded as the immediate cause of death in one. Grossly all the cases presented infiltrative or mixed (infiltrative and nodular) growth pattern. Intrabile duct tumor growth and associated marked obstructive jaundice may frequently herald the terminal phase of HCC in certain patients. In our series, approximately 40% of patients with HCC and significant jaundice had gross evidence of extensive intraductal tumor growth. In the absence of intraductal tumor growth, jaundice in HCC usually was seen in a setting of progressive terminal hepatic failure.

Crackles in asbestos workers: auscultation and lung sound analysis.

We surveyed 270 asbestos factory workers and 222 control subjects matched for age to assess the usefulness of listening for fine discontinuous adventitious lung sound (fine crackles) in detection of the early stages of interstitial fibrosis of asbestosis. Fine crackles at both bases were heard more frequently in asbestos workers (32.2%) than in controls (4.5%) (P less than 0.01). The presence of bilateral basal crackles was related to the duration of asbestos exposure and occurred before abnormalities could be detected by chest radiography. Two observers examined 74 workers randomly chosen from among 270 asbestos workers and found inter-observer agreement to be high (91.9%). There was close agreement between findings on chest auscultation and sound recordings.

Orally active cephalosporins: synthesis, structure-activity relationships and oral absorption of 3-[(E) and (Z)-2-substituted vinyl]-cephalosporins.

A series of 7beta-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoacetamid o]-3-[(E)- and (Z)-2-substituted vinyl]-3-cephem-4-carboxylic acids was designed and synthesized using palladium-catalyzed coupling reactions of a 3-methanesulfonyloxy-3-cephem and an E substituted vinyl stannane or Wittig reaction of a 3-triphenylphosphoniummethyl cephem and an aldehyde as a key step. These compounds were evaluated for in vitro antibacterial activity and oral absorption in rats. A number of them exhibited excellent antibacterial activity against both gram-positive and gram-negative bacteria including Haemophilus influenzae. Among them, FR86524 (2j). having a (Z)-2-(3-pyridyl)vinyl moiety at the C-3 position, had the most well balanced activity. Although FR86254 exhibited low oral absorption, the pivaloyloxymethyl ester (23) of FR86524 showed improved oral absorption.

Synthesis and antibacterial activity of novel 4-pyrrolidinylthio carbapenems Part IV. 2-Alkyl substituents containing cationic heteroaromatics linked via a C-C bond.

The synthesis and biological activity of a novel series of 2-alkyl-4-pyrrolidinylthio-beta-methylcarbapenems containing a variety of cationic heteroaromatic substituents linked via a C-C bond is described. As a result of these studies, we selected FR21818 (In) as a candidate compound for development. FR21818 exhibited a well balanced spectrum of antibacterial activity, including Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA), excellent urinary recovery, good stability against renal dehydropeptidase-I (DHP-I). no antigenicity and mutagenicity, weak toxicities, and good efficacy and therapeutic effect on mice systemic infections. Affinities to PBP's, permeability of outer membrane, and plasma levels in mice, dog, and cynomolgous monkey of FR21818 are also reported.

Synthesis and antibacterial activity of novel 4-pyrrolidinylthio carbapenems. Part III: Novel 2-alkyl substituents containing cationic heteroaromatics linked via a C-N bond.

The synthesis and biological activity of a novel series of 2-alkyl-4-pyrrolidinylthio-beta-methylcarbapenems containing a variety of cationic heteroaromatic substituents is described. As a result of these studies, we uncovered a relationship between in vitro antibacterial activity and the length of the alkyl spacer part, and discovered FR20950 (1c), containing a two methylene spacer moiety and an imidazolio group, which possesses a balanced spectrum of antibacterial activity, including Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, FR20950 exhibited excellent urinary recovery, and comparable stability against renal dehydropeptidase-I (DHP-I) to Biapenem. DHP-I stability could be improved by introduction of a substituent on to the imidazole ring.