Differential analysis of animal bone marrow by flow cytometry.

A simple procedure was developed for rapid analysis of animal bone marrow by flow cytometry using the lipophilic cationic dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)]. The batch process allows differentiation of bone marrow cells into lymphoid, erythroid, and myeloid populations and enables classification of erythroid and myeloid cells into proliferating and maturing subpopulations. From these data, myeloid:erythroid (M:E) ratios and maturation indices for erythroid and myeloid cells (EMI and MMI, respectively) can be derived. This procedure provides the opportunity to analyze bone marrow quantitatively and offers distinct advantages to current manual methods in terms of simplicity, throughput, and reproducibility. The method has been tested successfully using marrow from Wistar rats, B6C3F1 mice, beagle dogs, and cynomolgus monkeys. This technique facilitates the evaluation of bone marrow samples taken from preclinical safety studies or from animal colonies of large size.     

Relatedness of penicillin-binding protein 1a genes from different clones of penicillin-resistant Streptococcus pneumoniae isolated in South Africa and Spain.

Penicillin-resistant strains of Streptococcus pneumoniae have been common in South Africa and Spain for several years. Multilocus enzyme electrophoresis identified one clone of capsular type 6B which was prevalent in Spain and another clone of type 23F that was present in both countries. Genes for penicillin-binding proteins (PBPs) in penicillin-resistant strains are often mosaics where parts of the pneumococcal genes are replaced by homologous genes from other species. We have compared the mosaic structures of the PBP 1a genes from the two clones as well as from genetically distinct South African isolates. Four classes of mosaic PBP 1a genes were found that contained blocks of sequences divergent by 6-22% from those of sensitive genes; two classes contained sequences coming from more than one external source. Data are presented showing that the PBP 1a genes from the 23F and the 6B clone are related, and that the two PBP 1a genes from the South African isolates are also related. We suggest that the type 23F clone originated in Spain prior to distribution into other continents.     

GTPase-activating protein SH2-SH3 domains induce gene expression in a Ras-dependent fashion.

The p21ras GTPase-activating protein (GAP) is thought to function as both a negative regulator and a downstream target of p21ras. Here, we have investigated the role of GAP by using a transient expression assay with a fos luciferase reporter plasmid. We used GAP deletion mutants that lack the domain involved in interaction with p21ras and encode essentially only the SH2-SH3 domains. When these GAP deletion mutants were expressed, we observed a marked induction of fos promoter activity similar to induction by activated p21ras. Expression of a full-length GAP construct had no effect on the activity of the fos promoter. Activation of the fos promoter by these GAP SH2-SH3 regions was inhibited by cotransfection of a dominant inhibitory mutant of p21ras, Ras(Asn-17). Thus, the induction of gene expression by GAP SH2-SH3 domains is dependent on p21ras activity. Moreover, induction of fos promoter activity by GAP SH2-SH3 domains is increased severalfold after cotransfection of an activated mutant of p21ras, Ras(Leu-61), or insulin stimulation of A14 cells, both leading to an increase in the levels of GTP-bound p21ras. The combined effect of Ras(Leu-61) and the GAP deletion mutants was not inhibited by Ras(Asn-17), indicating that GAP SH2-SH3 domains do not function to activate endogenous p21ras but cooperate with another signal coming from active p21ras. These data suggest that GAP SH2-SH3 domains serve to induce gene expression by p21ras but that additional signals coming from p21ras are required for them to function.     

Hypertrophy without increased isometric strength after weight training

Eight men (20-23 years) weight trained 3 days.week-1 for 19 weeks. Training sessions consisted of six sets of a leg press exercise (simultaneous hip and knee extension and ankle plantar flexion) on a weight machine, the last three sets with the heaviest weight that could be used for 7-20 repetitions. In comparison to a control group (n = 6) only the trained group increased (P less than 0.01) weight lifting performance (heaviest weight lifted for one repetition, 29%), and left and right knee extensor cross-sectional area (CAT scanning and computerized planimetry, 11%, P less than 0.05). In contrast, training caused no increase in maximal voluntary isometric knee extension strength, electrically evoked knee extensor peak twitch torque, and knee extensor motor unit activation (interpolated twitch method). These data indicate that a moderate but significant amount of hypertrophy induced by weight training does not necessarily increase performance in an isometric strength task different from the training task but involving the same muscle group. The failure of evoked twitch torque to increase despite hypertrophy may further indicate that moderate hypertrophy in the early stage of strength training may not necessarily cause an increase in intrinsic muscle force generating capacity.     

Comparative efficiencies of bispecific F(ab′γ)2 and chimeric mouse/human IgG antibodies in recruiting cellular effectors for cytotoxicity via Fcγ receptors

Summary The three forms of Fc receptor carried by monocytes (FcRI, II) and natural killer (NK) cells (FcRIII) are all capable of mediating cell lysis. Here we compare the use of F(ab)₂ bispecific antibodies, specifically targetting individual FcR, and chimeric IgG mouse/human antibodies which are capable of targetting all FcR, for their ability to mediate target cell destruction. The derivatives are prepared by linking hinge sulphydryl residues via tandem thioether bonds, using a bismaleimide crosslinker: Fab from an anti-FcR mAb linked to Fab from a common anti-target mAb (BsAb), or Fab from the common anti-target mouse antibody linked to human Fc (FabFc or bisFabFc). All the derivatives targetting chick red blood cells gave efficient lysis, although different effector cell donors yielded differences in both the lytic levels achieved and the comparative efficiencies of derivatives. In contrast, significant lysis of the guinea pig lymphoblastic leukaemia, L₂C, regularly resulted only via the anti-FcRIII BsAb and the chimeric derivatives. These results suggest that the chimeric, Fc-containing derivatives mediate tumour cell lysis principally through FcRIII on NK cells. This is in contrast to the situation with the chick red blood cells where the chimeric derivatives appear capable of lysing erythrocytes by utilizing either monocytes or NK cells, because significant (50%) lysis occurred with effector cell populations magnetically depleted through either FcRII or FcRIII. A major difference between these two types of antibody derivative was their ability to function in the presence of high concentrations of normal human Fc. The lysis mediated by BsAb reactive with FcRI or II was unaffected by the presence of human Fc at 2.5 mg/ml (a concentration comparable with that yielded by IgG in plasma) whereas the BsAb recognizing FcRIII and all the Fc-containing derivatives were completely inhibited.This work has been supported by Tenovus, the Cancer Research Campaign, the Leukaemia Research Fund, Italfarmaco, Milano, Italy and the Imperial Cancer Research Fund     

Change of speciation of Cu(II) in growth medium due to uptake of ammonia byPseudomonas testosteroni during growth

Growth ofPseudomonas testosteroni in a medium containing 1mm Cu(II) causes a color change from blue to green. The spectrum of the supernatant solution from the blue culture shows an absorption at 660 nm, identical to that of 1mm [Cu(II)] in the medium. The green supernatant solution shows a UV absorption, which tails into the visible and so is responsible for the green color, and ad-d absorption at 720 nm. The absorption at 660 nm for the blue supernatant solution is probably due to [Cu(NH₃)₃(H₂O)₃]²⁺. Growth of the organism causes loss of ammonia and a speciation change to [Cu(NH₃)₂(H₂O)₄]²⁺, with a shift in absorption maximum from 660 to 720 nm. These conclusions are based upon the spectra of known aquaammine complexes of Cu(II) and calculations of the speciation of Cu(II) before and after growth. Change in metal speciation owing to nutrient uptake by an organism does not appear to have been recognized previously.     

Prenatal diagnosis of 21-hydroxylase deficiency congenital adrenal hyperplasia using the polymerase chain reaction

Summary We present an improved method for the prenatal diagnosis of congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. The polymerase chain reaction (PCR) was used to analyze DNA from an affected index case, the parents, and a cultured chorionic villus sample, for point mutations in the steroid 21-hydroxylase (CYP21) gene. We can predict that the fetus is an unaffected carrier.     

Molecular mimicry and the autoimmune response to the peripheral nerve myelin PO glycoprotein

In the Lewis rat immunisation with the myelin PO glycoprotein can induce an inflammatory demyelinating disease of the peripheral nervous system, experimental allergic neuritis (EAN), which has many clinical and histopathological parallels with the human disease the Guillain-Barre syndrome. In view of the reported association of GBS with a number of infectious agents we have investigated whether molecular mimicry may occur between microbial antigens and the PO protein that could possibly trigger a similar pathogenic autoimmune response in man. A computer search of the available protein sequence data bases identified several absolute sequence homologies between PO and viral proteins that involve five or more consecutive amino acid residues. Four of these sequence homologies involved viral pathogens previously associated with the Guillain-Barre syndrome, namely Epstein-Barr virus (EBV), cytomegalovirus (CMV), Varicella zoster virus (VZV) and human immunodeficiency virus I (HIV I). Although, sequence homologies were also found between viral peptides and the neuritogenic determinants of PO, residues 56–71 and 180–199, these homologies proved incapable of eliciting EAN in the Lewis rat. These observations are discussed with reference to the role that molecular mimicry between T cell epitopes on pathogen derived antigens and the PO protein may play in the pathogenesis of the Guillain-Barre syndrome.Abbreviations EAN Experimental allergic neuritis - EAE experimental allergic encephalomyelitis - PNS peripheral nervous system - CNS central nervous system - MBP myelin basic protein - GBS Guillain Barre syndrome - CFA complete Freund's Adjuvant - LPC lysophosphatidyl choline - VZV Varicella zoster virus - CMV cytomegalovirus - EBV Epstein Barr virus - HIV I human immunodeficiency virus I Special issue dedicated to Dr. Alan N. Davison     

Structure and expression of a gene from Arabidopsis thaliana encoding a protein related to SNF1 protein kinase

The AKin10 gene from Arabidopsis thaliana encoding a putative Ser/Thr protein kinase (PK) has been isolated and characterized. The AKin10-encoding gene is located on a genomic 5.4-kb BamHI fragment and contains ten introns, one being located in the 5' untranslated region. The deduced amino acid sequence of AKin10 is 65% identical over the catalytic domain to the yeast PK (SNF1). SNF1 is essential for the derepression of many glucose-repressible genes, including Suc2 which encodes invertase. Southern blot hybridization experiments suggested the presence of one copy of the gene per haploid genome of A. thaliana. Northern hybridization experiments indicated that this gene is expressed in roots, shoots and leaves. AKin10 may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism in higher plants.     

Spring migration of Atlantic mackerel,Scomber scombrus,in relation to water temperature through Cabot Strait (Gulf of St. Lawrence)

Synopsis Sea surface temperatures across Cabot Strait (Gulf of St. Lawrence) ranged from 6 to 9°C on June 3, 1989, but only from 3 to 5°C on June 2, 1990. Periods of peak commercial landings of mackerel in eastern Cape Breton Island extended from May 22 to June 3 in 1989, and from May 28 to June 2 in 1990. In late May 1990, Atlantic mackerel were captured with a purse-seiner during exploratory fishing in Cabot Strait; commercial quantities were caught in water as cold as 2.8°C. The presence of mackerel in water a full 4°C colder than its reported lower tolerance limit indicates that the development of the 7°C isotherm is not a requirement for the vernal appearing of mackerel. The overlap of the periods of peak commercial landings between 1989 and 1990, despite: marked differences in warming chronology, suggests that the movements of mackerel are not as closely linked to water temperature as previously reported. The fish's thermal preferences could be subordinate to their reproductive requirements at this stage of their spawning migration.