Nodulation studies on legumes exotic to Australia: Hedysarum coronarium

Abstract Symbiotic experiments in glasshouse, controlled environment cabinet, and field were conducted with four lines of sulla ( Hedysarum coronarium ) and 15 strains of Rhizobium spp. This plant is highly Rhizobium -specific and appropriate strains are most unlikely to occur naturally in Australia. Under several sets of experimental conditions, H. coronarium nodulated abundantly and effectively with homologous rhizobia introduced from Spain and Italy. The optimum temperature for nitrogen fixation was relatively low (approx. 21°C) but significant interactions between line of host, strain of rhizobia, and growth temperature were frequent. The rhizobia were persistent in soil.     

Presence of human papillomavirus DNA sequences in cervical intraepithelial neoplasia.

Twenty two patients referred to a district colposcopy clinic because of an abnormal cervical cytology report or a suspicious cervix and found to have a cervical epithelial abnormality were studied. The techniques of cytology, histology, immunohistochemistry, and DNA-DNA hybridisation were used to detect infection by human papillomavirus. Using an indirect immunoalkaline phosphatase technique human papillomavirus antigen was found in biopsy specimens from six of the 22 patients and DNA of papillomavirus type 6 in biopsy specimens from 13 of these women, including four out of six whose histological diagnosis was cervical intraepithelial neoplasia grade 3. In eight cases where cytological, colposcopical, and histological investigations all indicated the presence of wart virus infection, papillomavirus type 6 DNA was found in seven. Papillomavirus type 6 DNA was found in more than half of the proved cases of cervical intraepithelial neoplasia. The presence of this viral DNA in women with no cervical abnormality is to be studied.     

Adenovirus E1A makes two distinct contacts with the retinoblastoma protein.

Two regions near the amino terminus of the adenovirus E1A protein, which were first identified by sequence conservation among various adenovirus serotypes, have been shown by genetic studies to be essential for E1A-mediated transformation. These same regions are also required for interaction with a number of cellular proteins, including the retinoblastoma protein (pRB). Using synthetic peptides corresponding to portions of these conserved regions, we show that each region can bind independently to pRB. These interactions were observed in both competition and binding assays. In both types of assay, region 2 peptides (E1A amino acids 115 to 132) bound pRB with higher affinity than did region 1 peptides (E1A amino acids 37 to 54), while a peptide combining region 1 and 2 sequences consistently provided the highest-affinity interaction. Cross-blocking experiments using region 1 peptides and region 2 peptides suggested that these two regions of E1A make distinct contacts with pRB. These data support the notion that the pRB-binding domain of E1A contains at least two functional elements.     

Bovine pyruvate kinase isozymes and hybrid isozymes. Electrophoretic studies and tissue distribution.

Electrophoresis of various bovine tissue extracts revealed, in addition to the three major homotetrameric isozymes of pyruvate kinase (K₄, L₄, and M₄), numerous intermediate bands that behave electrophoretically as hybrid isozymes. Kidney, for example, contains both K-L and K-M hybrid sets. Representative hybrids from each set, tentatively identified as K₂L₂ and K₃M, were isolated from kidney by ionexchange chromatography and their subunit compositions were confirmed by dissociation and subsequent reassociation into new hybrid sets. All of the tissues examined that contain type K₄ also have substantial quantities of K-M hybrids, establishing the presence of the type M isozyme in a great many tissues other than striated muscle and brain, where it is most abundant. In addition, small quantities of K subunits apparently are produced even in striated muscle, which previously had been thought to contain only M₄. The pattern of hybrids and enzyme specific activities differ markedly within tissues from the same organ, as shown by dissection of the heart and great vessels. Aortic smooth muscle has a fairly uniform distribution of K-M hybrids, while cardiac muscle has mostly M₄ with a little KM₃. Connective tissue from heart valves, on the other hand, has a five-membered set dominated by K₃M, while Purkinje fibers have a five-membered set dominated by KM₃. The occurrence of K-M hybrids in these and many other tissues indicates that the distribution of mammalian pyruvate kinase isozymes is much more complex than previously reported.     

The reversibility of skeletal muscle pyruvate kinase and an assessment of its capacity to support glyconeogenesis.

The kinetics of pyruvate phosphorylation by rabbit skeletal muscle pyruvate kinase (EC 2.7.1.40) has been studied with a coupled assay using P-enolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (EC 1.1.1.37). The reaction sequence is (See journal for formula). Although the equilibrium of the pyruvate kinase reaction by itself strongly favors pyruvate production, the over-all equilibrium of this coupled system favors the depletion of pyruvate, thus greatly reducing the problem of back reaction during the assay. In addition, the oxidation of NADH by malate dehydrogenase makes it possible to monitor the system with a spectrophotometer. The Michaelis constant of pyruvate kinase was found to be 0.9 mM for ATP and 7 mM for pyruvate, values that agree reasonably well with earlier studies using direct assays. However, the maximum velocity is about 6 mumol of pyruvate phosphorylated/min/mg of enzyme, which is very much faster than that indicated by earlier studies. These results suggest that the metabolic significance of the reverse reaction of muscle pyruvate kinase may have been underestimated. In particular, the data given here suggest that its rate in vivo is probably comparable to the observed rate of glycogen synthesis from lactate, making possible glyconeogenesis in muscle by pyruvate kinase reversal without the need for an enzymatic bypass of the kind employed by liver and kidney.     

Intercellular communication in the rat anterior pituitary gland: an in vivo and in vitro study

The concept of "stimulus-secretion coupling" suggested by Douglas and co-workers to explain the events related to monamine discharge by the adrenal medulla (5, 7) may be applied to other endocrine tissues, such as adrenal cortex (36), pancreatic islets (4), and magnocellular hypothalamic neurons (6), which exhibit a similar ion-dependent process of hormone elaboration. In addition, they share another feature, that of joining neighbor cells via membrane junctions (12, 26, and Fletcher, unpublished observation). Given this, and the reports that hormone secretion by the pars distalis also involves a secretagogue-induced decrease in membrane bioelectric potential accompanied by a rise in cellular [Ca++] (27, 34, 41), it was appropriate to test the possibility that cells of the anterior pituitary gland are united by junctions.     

PYRUVATE KINASE ISOZYMES IN NEURONS, GLIA, NEUROBLASTOMA, AND GLIOBLASTOMA

Abstract– The distribution of pyruvate kinase isozymes (EC 2.7.1.40) was examined in cells and tissues from the central and peripheral nervous system of the rat. Most tissues contain significant quantities of both the K₄ (fetal type) and M₄ (skeletal muscle type) isozymes plus tetrameric hybrids comprised of various combination of the type M and type K subunits. Retina, for example, contains a five-mem-bered hybrid set weighted toward K₄, while sciatic nerve and spinal cord have patterns very similar to that of adult brain, consisting predominantly of M₄ with small amounts of K₄ and K-M hybrids. This adult pattern is achieved by a gradual shift from a hybrid set dominated by K₄ in fetal life, to the pattern at birth at which time the two most prominent bands were M₄ and K₂M₂, and finally to the adult pattern by about 28 days after birth. Neurons and glial cells were isolated from rat and mouse brains at the various developmental levels. The pyruvate kinase isozyme patterns in the two cell types were similar to each other and to the patterns seen in whole brain homogenates at all ages, indicating similar rates of isozymic maturation in the two cell types. The correlation of maturation with pyruvate kinase isozyme patterns was further tested in cultures of malignant cell lines. A K-M hybrid set, weighted toward K₄, was seen in two clonal lines of mouse neuroblastoma under normal culture conditions. However, lowering the serum concentration in the culture medium or adding bromodeoxyuridine caused a shift in the patterns toward type M as the cells differentiated, mimicking in part the in vivo maturation of normal cells. On the other hand, a rapidly growing and poorly differentiated line of rat glioblastoma had only K₄ under all conditions examined.