Calcium release in smooth muscle

In smooth muscle, maintenance of the contractile response is due to Ca2+ influx through two types of Ca2+ channel, a voltage-dependent Ca2+ channel and a receptor-linked Ca2+ channel. However, a more transient contraction can be obtained by release of Ca2+ from a cellular store, possibly the sarcoplasmic reticulum. In spike generating smooth muscle (e.g., guinea-pig taenia caeci), spike discharges may trigger the release of cellular Ca2+ by activating a Ca2+-induced Ca2+ release mechanism. Caffeine directly activates this mechanism in the absence of a triggered Ca2+ influx. In contrast to this, maintained depolarization may not only release but also refill the Ca2+ store. Drug-receptor interactions also release Ca2+ from a cellular store. This release may be elicited with inositol trisphosphate produced by receptor-linked phosphoinositide turnover. In non-spike generating smooth muscle (e.g., rabbit thoracic aorta), maintained membrane depolarization does not release but, instead, fills the Ca2+ store. However, caffeine and receptor-agonists release the Ca2+ store - possibly by activating the Ca2+-induced Ca2+ release mechanism and phosphoinositide turnover, respectively. The Ca2+ store in smooth muscle is filled by Ca2+ entry through voltage dependent Ca2+ channels and also by resting Ca2+ influx in the absence of receptor-agonists. The Ca2+ entering the cells through these pathways may be accumulated by the Ca2+ store and may activate the contractile filaments.     

Immunogenic and antigenic epitopes of Ig. XXV. Monoclonal antibodies that differentiate the Mcg+/Mcg- and Oz+/Oz- C region isotypes of human lambda L chains

The C region of human lambda L chains is specified by multiple C lambda genes of which three--C lambda 1, C lambda 2, and C lambda 3--encode for the isotypes designated Mcg+, Kern- Oz-, and Kern- Oz+, respectively. The Mcg, Kern, and Oz factors have been characterized by sequence differences involving specific C lambda amino acid residues. They have also been recognized serologically by polyclonal antisera but, with rare exception, these reagents are no longer available. We have obtained two murine anti-human lambda-chain mAb, 14G1 and 14D1, that recognize antigenic determinants specific for the C lambda isotypes Mcg and Oz, respectively. These antisera have been used to classify as Mcg+/Mcg- or Oz+/Oz- monoclonal lambda-chains (Bence Jones proteins) and intact Ig lambda proteins. There was complete concordance between the chemical and serologic assignment of lambda-chains as Mcg+/Mcg- or as Oz+/Oz-; no single protein expressed both isotypes. There was no evident association between the C region isotype Mcg or Oz and the V region subgroup of the protein tested. However, our finding that four of seven amyloid-associated lambda VI Bence Jones proteins were Oz+ suggests a predominant expression of the C lambda 3 gene product among proteins of this uncommon V lambda subgroup.     

Regional assignment of the gene for human liver/bone/kidney alkaline phosphatase to chromosome 1p36.1-p34

We have used three different methods to map the human liver/bone/kidney alkaline phosphatase (ALPL) locus: (1) Southern blot analysis of DNA derived from a panel of human-rodent somatic cell hybrids; (2) in situ hybridization to human chromosomes; and (3) genetic linkage analysis. Our results indicate that the ALPL locus maps to human chromosome bands 1p36.1-p34 and is genetically linked to the Rh (maximum lod score of 15.66 at a recombination value of 0.10) and fucosidase A (maximum lod score of 8.24 at a recombination value of 0.02) loci. These results, combined with restriction fragment length polymorphisms identified by ALPL DNA probes, provide a useful marker for gene mapping studies involving the short arm of chromosome 1. In addition, our results help to elucidate further the structure and evolution of the human alkaline phosphatase multigene enzyme family.     

Receptor activation of G proteins

G proteins are a highly conserved family of membrane-associated proteins composed of alpha, beta, and gamma subunits. The alpha subunit, which is unique for each G protein, binds GDP or GTP. Receptors such as those for beta- and alpha-adrenergic catecholamines, muscarinic agonists, and the retinal photoreceptor rhodopsin, catalyze the exchange of GDP for GTP binding to the alpha subunit of a specific G protein. G alpha.GTP regulates appropriate effector enzymes such as adenylyl cyclase or the cyclic GMP phosphodiesterase. The beta gamma-subunit complex of G proteins is required for efficient receptor-catalyzed alpha subunit guanine nucleotide exchange and also functions as an attenuator of alpha subunit activation of effector enzymes. Recent elucidation of both receptor and G protein primary sequence has allowed structural predictions and new experimental approaches to study the mechanism of receptor-catalyzed G protein regulation of specific effector systems and the control of cell function including metabolism, secretion, and growth.     

Nerve cell and nematocyte production in Hydra is deregulated by lithium ions

Summary LiCl, a well-known vegetalising agent, interferes with the commitment of stem cells to nerve cells and nematocytes in Hydra attenuata. Treatment with 20 mM LiCl inhibits commitment to nerve cells, treatment with 1 mM LiCl inhibits commitment to nematocytes. However, LiCl does not prevent stem cells committed to the nematocyte pathway from dividing and differentiating into nests of nematocytes. Following LiCl treatment, determination to nerve cells and nematocytes is triggered again. Commitment to nerve cells is strongly stimulated within the first 3 h following pulse treatment with LiCl if the animals have been fed immediately prior to treatment. In Hydra exposed to LiCl for 10 days the stem cell density is reduced by at least 90% of the initial value, and nematocytes are almost completely missing, whereas the density of nerve cells is within the normal range in animals with normal morphology. Animals which developed a transverse constriction in the middle of the body axis contain a 1.7-fold higher nerve cell density in the lower part than is observed in control animals.     

Studies on the effect of NAD(H) on nitrogenase activity in Rhodospirillum rubrum

The effect of NAD(P) and analogs of this nucleotide on nitrogenase activity in Rhodospirillum rubrum has been studied. Addition of NAD⁺ to nitrogen fixing Rsp. rubrum leads to inhibition of nitrogenase. NADP⁺ has the same effect but NADH or analogs modified in the nicotinamide portion do not cause inhibition. In contrast to ammonium ions, addition of NAD⁺ leads to inhibition of nitrogenase in cells that have been N-starved under argon. The inhibitory effect of NAD⁺ is more pronounced at lower light intensities. Addition of NAD⁺ also leads to inhibition of glutamine synthetase, a phenomenon also occurring when “switchoff” is produced by the addition of effectors such as ammonium ions or glutamine. It is also shown that NAD⁺ is taken up by Rsp. rubrum cells.     

Correlation between manganese-deficiency,loss of respiratory chain complex I activity and citric acid production in Aspergillus niger

The correlation between manganese deficiency, loss of mitochondrial respiratory chain NADH: ubiquinone oxidoreductase (complex I) activity and citric acid overproduction in the Aspergillus niger strain B 60 was analysed. With increasing manganese-supplementation of the production medium the loss of complex I activity and the production of citric acid was reduced. Addition of manganese during growth stopped further loss of complex I activity and further increase of citric acid production. A possible causality between complex I deficiency and citric acid overproduction is discussed.     

Digital chemiluminescence imaging of DNA sequencing blots using a charge-coupled device camera.

Digital chemiluminescence imaging with a cryogenically cooled charge-coupled device (CCD) camera is used to visualize DNA sequencing fragments covalently bound to a blotting membrane. The detection is based on DNA hybridization with an alkaline phosphatase(AP) labeled oligodeoxyribonucleotide probe and AP triggered chemiluminescence of the substrate 3-(2'-spiro-adamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD). The detection using a direct AP-oligonucleotide conjugate is compared to the secondary detection of biotinylated oligonucleotides with respect to their sensitivity and nonspecific binding to the nylon membrane by quantitative imaging. Using the direct oligonucleotide-AP conjugate as a hybridization probe, sub-attomol (0.5 pg of 2.7 kb pUC plasmid DNA) quantities of membrane bound DNA are detectable with 30 min CCD exposures. Detection using the biotinylated probe in combination with streptavidin-AP was found to be background limited by nonspecific binding of streptavidin-AP and the oligo(biotin-11-dUTP) label in equal proportions. In contrast, the nonspecific background of AP-labeled oligonucleotide is indistinguishable from that seen with 5'-32P-label, in that respect making AP an ideal enzymatic label. The effect of hybridization time, probe concentration, and presence of luminescence enhancers on the detection of plasmid DNA were investigated.     

The relationship between short-term memory capacity and EEG power spectral density

Multiplying memory span by mental speed, we obtain the information entropy of short-term memory capacity, which is rate-limiting for cognitive functions and corresponds with EEG power spectral density. The number of EEG harmonics (n = 1, 2,, 9) is identical with memory span, and the eigenvalues of the EEG impulse response are represented by the zero-crossings up to the convolved fundamental, the P300. In analogy to quantum mechanics the brain seems to be an ideal detector simply measuring the energy of wave forms. No matter what the stimulus is and how the brain behaves, the metric of signal and memory can always be understood as a superposition of n states of different energy and their eigenvalues.     

Chemo-adoptive immunotherapy of nude mice implanted with human colorectal carcinoma and melanoma cell lines

Summary The antitumor effects of chemotherapy, recombinant human interleukin-2 (IL-2), recombinant human interferon A/D (IFN), allogeneic human lymphokine-activated killer (LAK) cells, and antitumor monoclonal antibody (mAb), administered alone and in various combinations, were tested in athymic nude mice carrying human tumor xenografts. Treatment began 6–18 days after i.v. or i.p. inoculation of colorectal carcinoma or melanoma cell lines, when macroscopic growths were evident. Chemotherapy consisted of two or three courses of 5-fluorouracil (5-FU) or dacarbazine. IL-2 and/or IFN were administered three to five times weekly for 1–3 weeks, usually starting 2–5 days after chemotherapy. Human LAK cells were infused once or twice weekly for 2 or 3 weeks concurrently with IL-2. In some experiments, murine anticolorectal carcinoma mAb (SF25) was administered. In both tumor systems, chemotherapy alone or immunotherapy alone (IL-2, IL-2 + LAK cells, IFN, IL-2 + IFN ± LAK cells) had little or no therapeutic effects. Additive effects were obtained by combining chemotherapy with IL-2 and LAK cells or with IL-2 and IFN. In the majority of the experiments, the most effective combination was chemotherapy + IL-2 + IFN + LAK cells. Treatment with mAb was beneficial in the colorectal carcinoma system when combined with 5-FU + IL-2 or 5-FU + IL-2 + IFN. Homing experiments with radiolabeled human and mouse LAK cells injected i.v. showed increased early accumulation in the liver and lungs, whereas freshly explanted mouse splenocytes localized mostly in the spleen and liver. The tissue distribution pattern of human LAK cells was similar in normal and tumor-bearing mice (with lung metastases). These findings suggest that combination of chemotherapy with cytokines and LAK cells can be partially effective for advanced solid human tumors even in the absence of the host's T-cell immune response. Preliminary experiments showed that tumor-specific, anti-melanoma T-cell clones were effective in local (s.c.) tumor growth inhibition (Winn assay) following coinjection with the autologous tumor cells.