Interaction of dinitrophenyl-pepstatins with human cathepsin D and with anti-dinitrophenyl antibody. Development of potential reagents for the localization in vivo of active proteinases at sites of tissue injury.

Extracellular cathepsin D has been observed by various cytochemical methods at sites of tissue injury. However, the role of this enzyme in connective tissue matrix degradation is uncertain because there are no histochemical methods for determining whether or not the cathepsin D is active at such sites in living tissues. We considered that the combined use of a labelled tight-binding inhibitor with immunoprecipitation of the enzymes might overcome this problem. We have explored the application of derivatives of the inhibitor pepstatin, as only active cathepsin D binds pepstatin tightly. A series of N-pepstatinyl-N'-dinitrophenyl-alpha, omega-diaminoalkanes were synthesized with alkyl-chain lengths of two, four and six carbon atoms. These compounds were tight-binding inhibitors of human cathepsin D. In fluorescence-quenching titrations the dinitrophenyl groups were also fully available to bind high-affinity anti-dinitrophenyl antibody. It was shown by immunodiffusion in gels and by gel permeation chromatography that N-pepstatinyl-N'-dinitrophenyl-1,6-diaminohexane was a bifunction inhibitor able to bind cathepsin D and anti-dinitrophenyl antibody at the same time.     

The effect of human placental lactogen upon the metabolism of rat and human adipose tissue.

The metabolic effects of human placental lactogen (HPL) on rat and human white fat were tested in vitro. When tested against rat tissue, HPL resembled insulin in stimulating uptake of glucose and incorporation of [14C] glucose into CO2, triglyceride and glycogen, but differed from insulin in stimulating glycerol release and in failing to stimulate the incorporation of [14C] The stimulation of [14C] glucose incorporation and the inhibition of glycerol release by insulin were antagonized by HPL. The effects of HPL on human white fat resembled those on rat white fat,except that glycerol release was not stimulated in human tissue. The possible role of HPL in causing the diabetogenic stress of pregnancy is discussed in the light of these findings.     

The spatiotemporal transfer function of the Limulus lateral eye

The dynamics of the Limulus retina may be well described by the spatiotemporal transfer function, which measures the response of the eye to moving sinusoidal gratings. We consider a model for this system, which incorporates an excitatory generator potential, and self- and lateral inhibitory processes. Procedures are described which allow estimation of parameters for the model consistent with the empirical transfer function data. Transfer functions calculated from the model show good agreement with laboratory measurements, and may be used to predict accurately the response of the eye to arbitrary moving stimuli. The model allows convenient interpretation of the transfer function measurements in terms of physiological processes which underly the response of the Limulus retina.     

Removal of carbohydrate from influenza A virus and its hemagglutinin and the effect on biological activities.

Treatment of influenza virus and its purified hemagglutining with glycosidases from Diplococcus pneumoniae, which included beta-galactosidase, beta-N-acetylglucosminidase, and endoglycosidase D, released amino and neutral sugars from the virus and these as well as large oligosaccharides from the purified hemagglutinin. The released glucosamine-containing oligosaccharides were of one discrete size. Large oligosaccharides not removed by the glycosidases were found on the virus as well as the hemagglutinin. Some oligosaccharides on the virus were inaccessible to the enzymes, since they could be removed only from the purified hemagglutinin. Approximately 50% of the hemagglutinin carbohydrates could be removed without effect on hemagglutinating activity. Similarly, removal of 20 to 25% of the carbohydrates from intact virus particles did not alter infectivity.     

Bound Form Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana

The synthesis of H₂O-soluble and NaOH-hydrolyzable bound forms of indole-3-acetic acid (IAA) in petiole slices of Nicotiana glauca, Nicotiana langsdorffii, and their tumorous and nontumorous hybrids in the presence of exogenous ¹⁴C-IAA was investigated. The synthesis of conjugates progressively increased during 6 hours of incubation in ¹⁴C-IAA. The results showed that the rate of synthesis of IAA conjugates was higher in tumorous hybrids supplied exogenous IAA than in the parental species similarly supplied, and the rate of synthesis was higher in amphidiploid tumor plants than in a nontumorous mutant. It was also found that after 10 to 12 hours of incubation, 45% of the IAA taken up by F1 hybrids was in conjugated form whereas only 10 to 25% of the IAA taken up by a nontumorous mutant, N. langsdorffii, or N. glauca was conjugated. An F1 hybrid and an amphidiploid hybrid were found equally efficient in conjugating exogenously supplied IAA. It is postulated on the basis of these and other findings that IAA conjugates play an important role in tumorigenesis in Nicotiana.     

Metronidazole in prevention and treatment of bacteroides infections in elective colonic surgery.

A double-blind randomised trial was carried out among 46 patients undergoing elective colonic surgery; 27 patients received prophylactic metronidazole and 19 received placebo. Anaerobic infections did not develop in any of the metronidazole-treated patients, but did develop in 11 (58%) of 19 controls who were subsequently successfully treated with metronidazole.     

A human 15-kDa IFN-induced protein induces the secretion of IFN-gamma.

A 15,000 molecular weight protein (15-kDa), induced and secreted by human PBMC after treatment with IFN-alpha or -beta, was assessed for its ability to modulate cellular function. Although it had no effect on growth or 2'5'-A synthetase activity in Daudi, U-937, or HL-60 cells, when incubated with fresh human PBMC, LPS-induced monocyte cytotoxicity against WEHI-164 target cells was augmented. This stimulation was inhibited by both an antibody against TNF-alpha and a rabbit polyclonal antiserum to the 15-kDa protein. Furthermore, when the 15-kDa protein was added to PBMC an increase in GTP cyclohydrolase I activity, as assessed by neopterin secretion, resulted. Neopterin secretion by PBMC in response to the 15-kDa was increased in a dose-responsive manner up to more than sixfold over baseline, with a 15-kDa concentration of less than 10 ng/ml effective. The 15-kDa protein also stimulated indoleamine 2,3-dioxygenase (IDO) activity in fresh, human PBMC. Induction of neopterin secretion and IDO activity was inhibited by a polyclonal antiserum to 15-kDa. LPS-induced cytotoxic activity was not augmented by 15-kDa pretreatment of purified monocytes, indicating the need for the presence of a second cell population and the indirect action of the 15-kDa on the induction of monocyte activities. When PBMC or purified CD3+ cells, but not purified CD14+ cells, were incubated with the 15-kDa protein, secretion of a factor was induced that resulted in the induction of IDO activity in PMA-differentiated THP-1 cells. An antibody to IFN-gamma, but not IFN-alpha, inhibited the induction of IDO activity by this secreted factor. In addition, antiserum to the 15-kDa blocked the secretion of IFN-gamma from the CD3+ cells. Thus, a 15-kDa product of IFN-alpha- and IFN-beta-treated monocytes and lymphocytes can stimulate secretion of IFN-gamma from CD3+ cells.     

Evidence that most of the radioimmunoassayable inhibin secreted by the corpus luteum of the common marmoset monkey is of a non-dimeric form.

Although recent data for several species of primate, including human and marmoset, indicate that the corpus luteum secretes high levels of radioimmunoassayable inhibin, the nature of the immunoreactive (ir) inhibin detected has not been established. In this study, plasma ir-inhibin levels during the ovarian cycle of the marmoset (n = 12 animals) were measured by alpha-subunit-directed inhibin RIA, and values were compared with those estimated by a recently developed two-site immunoradiometric assay (IRMA) specific for inhibin alpha-beta dimer. Consistent with earlier data, plasma levels of ir-inhibin measured by RIA (overall mean value 133 +/- 7 ng/ml; n = 171) reached values 4-fold higher (p less than 0.001) during the luteal phase (222 +/- 20 ng/ml) than during the follicular phase (58 +/- 8 ng/ml), being directly correlated with plasma progesterone levels (r = 0.65; p less than 0.001). In contrast, plasma ir-inhibin levels estimated by IRMA were substantially lower than those measured by RIA (overall mean value 9.62 +/- 1.08 ng/ml; n = 171) and did not vary significantly during the cycle. Administration of a luteolytic dose of cloprostenol during the late luteal phase/early pregnancy led to an abrupt fall in plasma concentrations of progesterone (95%) and alpha-inhibin measured by RIA (82%), whereas dimeric inhibin levels remained unchanged. Analysis of marmoset luteal extracts (n = 5) by RIA, IRMA, and inhibin bioassay yielded inhibin estimates of 102.6 +/- 21.0, 0.632 +/- 0.103, and less than 2.0 ng/mg, respectively, thus confirming that only a very small proportion of the inhibin produced was dimeric (i.e., bioactive) in nature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of cinnamyl alcohol dehydrogenase from tobacco stems

Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. In this paper, we report the purification of CAD to homogeneity from tobacco (Nicotiana tabacum) stems. The enzyme is low in abundance, comprising approximately 0.05% of total soluble cell protein. A simple and efficient purification procedure for CAD was developed. It employs three chromatography steps, including two affinity matrices, Blue Sepharose and 2′5′ ADP-Sepharose. The purified enzyme has a specific cofactor requirement for NADP and has high affinity for coniferyl alcohol (K_m = 12 micromolar) and coniferaldehyde (K_m = 0.3 micromolar). Two different sized polypeptide subunits of 42.5 and 44 kilodaltons were identified and separated by reverse-phase HPLC. Peptide mapping and amino acid composition analysis of the polypeptides showed that they are closely related, although not identical.