Isolation and characterization of adult rat hearts cells.

Adult rat heart muscle cells were isolated after simultaneous perfusion of multiple (two to eight) hearts with buffered salt solutions containing collagenase and hyaluronidase. Yields (35 to 50% of ventricular weight with approximately 70% viability) are quantitatively suitable for metabolic studies. Viability has been determined by the ability of intact cells to exclude trypan blue and the inability of intact cells to oxidize exogenous succinate. Micrographs show that the fine structure of the isolated cells is well ordered. Cell concentrations of glycogen, glucose 6-phosphate, citrate, and various enzymes were similar to those of intact heart. ATP and creatine phosphate concentrations were lower than in whole hearts. Adenosine 3′,5′-monophosphate concentrations were somewhat elevated. Deoxyribonucleic acid was lower than in whole tissue. The isolated cells retain certain metabolic control mechanisms. The uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, increased oxygen consumption severalfold, whereas exogenous ADP had no effect on respiration. Under anaerobic conditions the rates of glucose utilization and lactate production were faster than in the presence of oxygen, indicating retention of the Pasteur effect. The addition of glucose and insulin caused a decrease in oxygen uptake or the Crabtree effect. Exogenously added pyruvate decreased glycolytic flux and produced a pronounced increase in intracellular citrate and glucose 6-phosphate. Isoproterenol stimulated adenylate cyclase activity of the isolated cells at the same concentrations effective with intact heart preparations. Isoproterenol and glucagon caused the activation of phosphorylase. The cells deteriorated as a function of incubation time, as indicated by a decrease in ATP content and a loss of lactate dehydrogenase into the medium. Cell deterioration was greatly accelerated by Ca²⁺ at concentrations greater than 10^?5m.     

Chromosomal localization of transfected genes by a combination of hot banding and fluorescence in situ hybridization.

We describe the combination of hot banding with fluorescence in situ hybridization as a rapid and efficient method to identify integration sites of transfected DNA sequences in chromosomes. As a test system we used SW480 EJ2, a clonal cell line obtained after transfection of SW480 with pSV2neoEJ, a plasmid containing a point-mutated, c-Ha-RAS oncogene. Nick-translated probes were compared with random primed-labeled probes to evaluate their relative efficiency in fluorescence in situ hybridization. The fluorescence signals were quantified in interphase nuclei by confocal scanning laser microscopy. Nick-translated probes were found to yield better results. Hot banding followed by fluorescence in situ hybridization localized the integration site of pSV2neoEJ in SW480 EJ2 at the site of a translocation on a marker chromosome Xp+. The combination of fluorescence in situ hybridization and hot banding can be used to (a) rapidly and efficiently analyze integration sites in large numbers of transfectants, (b) assess the clonality of transfected cell lines, and (c) localize the site of integration of transfected genes in the recipient genome.     

Characteristics of hamadryas-like male baboons living in anubis baboon troops in the Awash hybrid zone, Ethiopia.

In the twenty-two years since the Awash baboon hybrid zone was first described, about 25-30 hamadryas or hamadryas-like hybrid males have taken up residence in anubis baboon groups. Most enter as mature adults, though some may immigrate as juvenile followers. Long-term tracking of known immigrants indicates that they may reside for four years or more in their adopted troop, during which time they establish a series of comparatively short-term harems, and probably sire hybrid offspring. "Abductions" of anubis females to hamadryas troops, as postulated by early observers, were not seen, though their occurrence cannot be ruled out. Younger, more recent immigrants tend to be "purer" hamadryas in phenotype, perhaps reflecting a more distant origin, or a change in the composition of neighboring hamadryas populations. Coexisting hamadryas and anubis show no signs of reproductive isolation by behavioral barriers, and evidently belong to a single "recognition species."     

Post-transfusion malaria in thalassaemia patients

A total of 125 beta-thalassaemia patients receiving repeated blood transfusions were screened by Giemsa stain, Acridine-orange stain and antigen detection for evidence of malaria infection on each visit. A total of 8 (6.4%) of the patients developed post-transfusion malaria (PMT) as confirmed by tracing the infected blood donors. A high incidence of PTM in thalassaemia patients appears to be due to the use of fresh blood and the high frequency of blood transfusions required by these patients. Antigen detection using monoclonal antibody was found to be more sensitive for diagnosis of PTM and for screening suspected donors than the conventional blood smear examination methods and is therefore recommended for routine blood donor screening to rule out malaria infection.     

The intracellular distribution of adenylate kinase in the leaves of spinach,wheat and barley

Of the total adenylate-kinase activity in 10-d-old barley and wheat leaves, 40–50% is localised in the chloroplasts, while in mature spinach leaves 50–70% of the enzyme is chloroplastic. The extra-chloroplastic adenylate-kinase activity is associated with the mitochondria, very little, if any, is freely soluble in the cytoplasm. The adenylate pool of the cytoplasm could have access to adenylate-kinase activity in the intermitochondrial space because of the free permeation of adenylates across the outer mitochondrial membrane. Thus the adenylate pool of the cytoplasm could be subject to adenylate-kinase equilibrium. The mitochondrial adenylate kinase appeared to the localised exclusively in the intermembrane space.     

Base sequence studies of 300 nucleotide renatured repeated human DNA clones

A band of 300 nucleotide long duplex DNA is released by treating renatured repeated human DNA with the single strand-specific endonuclease S₁. Since many of the interspersed repeated sequences in human DNA are 300 nucleotides long, this band should be enriched in such repeats. We have determined the nucleotide sequences of 15 clones constructed from these 300 nucleotide S₁-resistant repeats. Ten of these cloned sequences are members of the Alu family of interspersed repeats. These ten sequences share a recognizable consensus sequence from which individual clones have an average divergence of 12.8%. The 300 nucleotide Alu family consensus sequence has a dimeric structure and was evidently formed from a head to tail duplication of an ancestral monomeric sequence. Three of the remaining clones are variations on a simple pentanucleotide sequence previously reported for human satellite III DNA. Two of the 15 clones have distinct and complex sequences and may represent other families of interspersed repeated sequences.     

Molecular evidence for Gondwanan origins of multiple lineages within a diverse Australasian gecko radiation

Aim Gondwanan lineages are a prominent component of the Australian terrestrial biota. However, most squamate (lizard and snake) lineages in Australia appear to be derived from relatively recent dispersal from Asia (< 30 Ma) and in situ diversification, subsequent to the isolation of Australia from other Gondwanan landmasses. We test the hypothesis that the Australian radiation of diplodactyloid geckos (families Carphodactylidae, Diplodactylidae and Pygopodidae), in contrast to other endemic squamate groups, has a Gondwanan origin and comprises multiple lineages that originated before the separation of Australia from Antarctica. Location Australasia. Methods Bayesian (beast ) and penalized likelihood rate smoothing (PLRS) (r 8s ) molecular dating methods and two long nuclear DNA sequences (RAG‐1 and c‐mos) were used to estimate a timeframe for divergence events among 18 genera and 30 species of Australian diplodactyloids. Results At least five lineages of Australian diplodactyloid geckos are estimated to have originated > 34 Ma (pre‐Oligocene) and basal splits among the Australian diplodactyloids occurred c. 70 Ma. However, most extant generic and intergeneric diversity within diplodactyloid lineages appears to post‐date the late Oligocene (< 30 Ma). Main conclusions Basal divergences within the diplodactyloids significantly pre‐date the final break‐up of East Gondwana, indicating that the group is one of the most ancient extant endemic vertebrate radiations east of Wallace’s Line. At least five Australian lineages of diplodactyloid gecko are each as old or older than other well‐dated Australian squamate radiations (e.g. elapid snakes and agamids). The limbless Pygopodidae (morphologically the most aberrant living geckos) appears to have radiated before Australia was occupied by potential ecological analogues. However, in spite of the great age of the diplodactyloid radiation, most extant diversity appears to be of relatively recent origin, a pattern that is shared with other Australian squamate lineages.