Selective changes of neurotransmitter receptors in middle-aged gerbil brain

Age-related alterations in major neurotransmitter receptors and voltage dependent calcium channels were analyzed by receptor autoradiography in the gerbil brain. [³H]Quinuclidinyl benzilate (QNB). [³H]cyclohexyladenosine (CHA), [³H]muscimol, [³H]MK-801, [³H]SCH 23390, [³H]naloxone, and [³H]PN200-110 were used to label muscarinic acetylcholine receptors, adenosine A₁ receptors, γ-aminobutyric acid_A (GABA_A) receptors, (NMDA) receptors, dopamine D₁ receptors, opioid receptors, and voltage dependent calcium channels, respectively. In middle-aged gerbils (16 months old), the hippocampus exhibited a significant elevation in [³H]QNB, [³H]MK-801, [³H]SCH 23390, [³H]naloxone, and [³H]PN200-110 binding, whereas [³H]CHA and [³H]muscimol binding showed a significant reduction in this area, compared with that of young animals (1 month). On the other hand, the cerebellum showed a significant alteration in [³H]QNB, [³H]CHA, and [³H]naloxone binding and the striatum also exhibited a significant alteration in [³H]SCH 23390 and [³H]CHA binding in middle-aged gerbils. The neocortex showed a significant elevation only in [³H]CHA binding in middle-aged animals. The nucleus accumbens and thalamus also showed a significant alteration only in [³H]muscimol binding. However, the hypothalamus and substantia nigra exhibited no significant alteration in these bindings in middle-aged gerbils. These results demonstrate the age-related alterations of various neurotransmitter receptors and voltage dependent calcium channels in most brain regions. Furthermore, they suggest that the hippocampus is most susceptible to aging processes and is altered at an early stage of senescence.     

A Novel Method for Generating Nested Deletions Using the in Vitro Bacteriophage T3 DNA Packaging System

To sequence a DNA segment inserted into a cosmid vector underthe directed sequencing strategy, we established a simple andrapid method for generating nested deletions which uses thein vitro packaging system of bacteriophage T3 DNA. The principleis based on the previous finding that this system can translocateany linear double-stranded DNA up to 40 kb into the phage capsidin a time-dependent manner and the encapsulated DNA becomesDNase-resistant. For this purpose, we constructed a cosmid vectorthat carries two different antibiotic selection markers at bothsides of the multiple cloning site, and after insertion of aDNA segment, the clone was linearized by -terminase at the cossite. After the packaging reaction in vitro followed by DNasetreatment, the encapsulated DNA was introduced into Escherichiacoli cells to give clones with unidirectional deletions by differentialantibiotic selection. Restriction and sequence analyses of deletionclones demonstrated that an ordered set of clones with nesteddeletions, ranging from less than 1 kb to 25 kb, was createdfrom either the end of the DNA segment. Thus, nested deletionclones that cover the entire region of a 40-kb cosmid insertcan be obtained by a single packaging reaction, and its restrictionmap can be simultaneously obtained.     

Partitioning behavior and enrichment of proteins with reversed micellar extraction: I. forward extraction of proteins from aqueous to reversed micellar phase

Protein extractions using aerosol OT (AOT)-isooctane reverse micelle solutions have been studied to explore the potential for separating and enriching proteins with the reversed micellar extraction. The effects of pH, ionic strength, and different cations of chlorides in a bulk aqueous phase and of AOT concentration in an organic phase on the partitioning of lysozyme and myoglobin and the solubilization of water are presented in detail. The extraction of lysozyme was affected by the concentration of potassium or barium but was almost independent of that of sodium or calcium, whose ionic diameter is smaller than that of potassium and barium. For the extraction of myoglobin, however, the effect of barium concentration was not appreciable. Lysozyme could be enriched into the reversed micellar phase up to 30 times the aqueous feed concentration. (c) 1993 John Wiley & Sons, Inc.     

Condition for the appearance of the metastable P beta' phase in fully hydrated phosphatidylcholines as studied by small-angle x-ray diffraction.

In the ripple phase of fully hydrated multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC), two kinds of small-angle x-ray diffraction profiles are observed on cooling through the main transition. One is a seemingly normal profile similar to that observed on heating and the other is the superposition of the diffraction profiles for the primary (normal) and the secondary ripple structures. We found that the profile obtained depended on the cooling rate. Increasing the cooling rate from 0.1 degrees C/min to 1 degrees C/min caused the peaks originating from the secondary ripple structure to diminish. After a cooling scan at 43 degrees C/min, the profile became similar to that of the normal ripple structure, although a trace of the secondary ripple structure remains. The results are interpreted in terms of the rise and fall of three-dimensional correlated domains composed of both primary and secondary ripple structures. At slow cooling rates, correlated domains of both kinds of ripple structures develop. As the cooling rate is increased, the domain of the primary ripple structure remains correlated, while that of the secondary ripple structure becomes less correlated. In addition, the multipeak profile appears even at rapid cooling rates, if the final low temperature lies just below the Tm for the main transition. This results suggests that formation of the correlated domains of the secondary ripple structure requires a certain time interval during which the DPPC vesicles experience the temperature just below the main transition. The secondary ripple structure takes place in phosphatidylcholines having more than 15 carbons in each hydrocarbon chain upon cooling through the main transition.     

Beneficial effect of ACE inhibitor in congestive heart failure

The effects of captopril, an angiotensin-converting enzyme inhibitor, on congestive heart failure (CHF) were investigated in animal and clinical studies. Congestive heart failure was induced in rats by a combination of pressure and volume overload. Cardiac pressure overload was induced by constricting one renal artery (Goldblatt rat) and volume overload was induced by aorto-caval fistula. Captopril (100 mg/kg/day) was then administered for 14 weeks. Isometric contraction was assessed using isolated left ventricular papillary muscles. The maximum developed tension and the maximum rate of increase in tension (dT/dtmax) were decreased in untreated rats with CHF and improved in captopriltreated rats. The left ventricular myosin isoenzyme pattern was shifted towards V3 in untreated rats with CHF, and was shifted back towards V1 in the captopril-treated rats. In the clinical study, captopril (37.5–75 mg/day) was administered to patients with cardiomyopathy for 12 months. There was no effect on left ventricular mass in hypertrophic cardiomyopathy, although systolic anterior motion of the mitral valve disappeared in one patient. In dilated cardiomyopathy, however, left ventricular mass tended to decrease. These results indicate that captopril has a beneficial effect in congestive heart failure.     

Abnormalities of ADP/ATP carrier protein in J-2-N cardiomyopathic hamsters

ADP/ATP carrier protein (AAC) is located in the mitochondrial inner membrane and has an important function in mitochondrial energy supply. This protein transports ATP to the cytoplasm and counter transports ADP into the mitochondria. J-2-N cardiomyopathic hamsters were investigated to determine the AAC content in cardiac mitochondria. After recording an electrocardiogram and collecting blood, the cardiac mitochondria were isolated. The mitochondrial membranes were labelled with eosin-5-maleimide (EMA) and separated on SDS polyacrylamide gels. The position of the AAC component was identified by exposing the gel under UV light, and the AAC content was determined by densitometry after staining with Coomassie blue. The AAC content ratio was significantly decreased in both 10-week-old and 1-year survived J-2-N hamsters when compared to control Golden hamster. Among 10-week-old J-2-N hamsters, the decrease in the AAC content ratio was more marked for the animals with more severe myocardial damage. The H⁺-ATPase activities of mitochondrial membrane were higher in 10-week-old J-2-N hamsters than in control hamsters. These results suggest that the decrease of AAC in J-2-N hamster plays an important role in the pathogenesis of cardiomyopathy in J-2-N hamsters.     

Localization of spectrin isoforms in the adult mouse heart

The distribution of two isoforms of spectrin in the adult mouse heart was investigated by Western blotting and immunocytochemistry by use of monospecific antibodies to erythrocyte spectrin and nonerythroid brain spectrin (240/235). Western blotting revealed proteins analogous to both isoforms of -spectrin in adult heart. Light-microscopic immunocytochemistry indicated that erythroid spectrin was distributed throughout the myocardium, with immunofluorescence localized to plasma membranes, Z-lines, and intercalated discs. Antibodies to brain spectrin (240/235) exhibited staining throughout the heart, with a generally diffuse distribution except for the prominent immunoreactivity associated with the intercalated discs. Nonerythroid spectrin immunofluorescence was detected in the endothelial cells of the endocardium and the mesothelial cell lining of the epicardium. Erythrocyte spectrin was not detected in the endocardium or the epicardium. The identification and localization of spectrin isoforms in the mammalian heart suggest the importance of spectrin proteins in the structural integrity and proper function of cardiac cells and tissues. This is the first demonstration of two different -spectrin subunits in the mammalian heart.     

Cytokine regulation of cell-to-cell interactions in lymphokine-activated killer cell cytotoxicity in vitro

The permanent pancreas carcinoma cell line, PCI-24, was developed in order to analyse cytokine regulation on pancreas carcinoma and lymphokine-activated killer (LAK) cell interaction. PCI cells expressed ICAM-1 and HLA-ABC, but not HLA-DR antigens. PCI cells showed augmented ICAM-1 and HLA-ABC expression when incubated with interferon (IFN) and tumour necrosis factor . A similar but weak augmentary effect on the HLA-ABC and ICAM-1 surface expression was seen with interleukin-1 treatment. Natural attachment of LAK to PCI cells was augmented by recombinant IFN in close association with ICAM-1 up-regulation on PCI cells. In addition, natural attachment was significantly inhibited by anti-LFA-1 and anti-ICAM-1 antibody treatments. Cytotoxicity of the LAK cells against PCI cells was also significantly inhibited with the same treatment. Thus, the attachment of LAK cells to PCI cells through LFA-1/ICAM-1 molecules appeared to be essential for the cytotoxicity for PCI cells. Pretreatment of PCI cells, but not of LAK cells, with IFN or other cytokines resulted in a decrease of susceptibility for LAK cell cytotoxicity. The decreased susceptibility inversely correlated with HLA-ABC expression on the PCI cells. The collective evidence indicates that, although LAK cell attachment to pancreas carcinoma cells through the LFA-1/ICAM-1 molecule is augmented by IFN, IFN treatment of pancreas carcinoma cells reduces LAK cell cytotoxicity possibly through an increase in HLA-ABC or a regulation of molecules closely associated to HLA-ABC expression.     

Chemotaxis away from thiocyanic and isothiocyanic esters in Pseudomonas aeruginosa

Abstract A novel mycoplasmal species designated as Mycoplasma penetrans has recently been isolated from patients infected with human immunodeficiency virus. The 16S rRNA gene from this mycoplasma was cloned and its nucleotide sequence determined. This sequence was aligned with previously published homologous sequences from several mycoplasmas and with related Gram-positive bacteria and a phylogenetic tree was constructed. The results indicate that M. penetrans belongs to the evolutionary group Pneumoniae.     

Regulation of L-alanine-initiated germination ofBacillus subtilis spores by alanine racemase

Summary Germination ofBacillus subtilis spores was initiated by L-Ala and competitively inhibited by D-Ala, suggesting the presence of an alanine receptor. The spores showed alanine racemase activity in the spore coat. To investigate the role of alanine racemase (L D) on germination, net racemase activity was determined using diphenylamine as a germination inhibitor and germination was measured using D-penicillamine as a racemase inhibitor. Apparent affinity of L-Ala to the germinant receptor was more than 1000 times higher than that to the racemase. Germination increased in the presence of D-penicillamine, when the concentration of L-Ala was low and that of spores was high. Racemase activity was optimal at 65°C at pH 9.0 and germination at 43°C at pH 7.2. Under unfavorable growth conditions such as high population of spores in limited nutrients, high temperature and high pH, spore alanine racemase converted the germinant actively to the inhibitor and this conversion may regulate germination for survival of the population.