Rec dependence of mu transposition from P22-transduced fragments.

Derivatives of bacteriophage Mu carrying a lac operon and a selectable drug resistance element (Mu d phages) are frequently used tools of bacterial genetics. Mu d prophages used in this way can be treated as transposons, in that the inserted material can be transduced from one strain to another by general transducing phages, such as P1 and P22. When a Mu d prophage is transduced into a new recipient by P1 or P22, the Mu d element can transpose from the transduced fragment into the bacterial chromosome. Transposition of the Mu d element from a P22-transduced fragment shows several striking differences from transposition of a Mu d genome injected by a Mu virion. First, the frequency of transposition from a transduced fragment is greatly enhanced by a P22 helper genome. Second, transposition requires the host recA, B, and C functions. Transposition of Mu following injection by a Mu virion is rec independent. While the basis of these observations is not understood, we suggest that the Mu X protein, a 65-kilodalton protein injected by a Mu virion and required for Mu transposition, may not be packaged by P22. We suggest that the effects seen reflect the behavior of a Mu genome in the absence of the X protein.     

Characterization of an endogenous substrate related to insulin and insulin-like growth factor-I receptors in lizard brain

Lizard insulin receptors are evolutionarily highly conserved. Wheat germ agglutinin-purified brain membranes demonstrate the presence of an endogenous substrate (pp 105) for both the insulin and insulin-like growth factor-I receptors. Both insulin and I-insulin-like growth factor-I stimulate the phosphorylation of this endogenous substrate in a dose-dependent manner. Following insulin-stimulated autophosphorylation of the beta subunit, there is a lag period of about 5 min prior to observable phosphorylation of the endogenous substrate. Phosphoamino acid analysis of both the beta subunit as well as pp 105 reveal primarily phosphotyrosine in both the basal as well as the stimulated state.     

Detection of terminal N-linked N-acetylglucosamine residues in the Golgi apparatus using galactosyltransferase and endoglucosaminidase F/peptide N-glycosidase F: adaptation of a biochemical approach to electron microscopy

Purified human milk beta-N-acetylglucosaminide beta 1, 4 galactosyltransferase (EC 2.4.1.38) was used to galactosylate N-acetylglucosamine (GlcNAc) residues present in ultra-thin sections of Lowicryl K4M-embedded rat and pig liver. Both endogenous galactose and galactosylated transferase products could be revealed by Ricinus communis lectin I-gold complexes (RcL I-g15). Without galactosyltransferase (GT) treatment, labeling for galactose (gal) was limited to the trans region of rat and pig hepatocyte Golgi apparatus. After exposure to GT, additional labeling was found over cis Golgi apparatus cisternae. RcL I-g15 labeling was sensitive to a purified preparation of endoglucosaminidase F/peptide N-glycosidase F (at pH 9). This indicates that endogenous gal and gal transferred by GT to terminal GlcNAc residues are present N-linked oligosaccharides. The RcL I-g15 labeling produced by GT was insensitive to extensive washing with solutions containing either EDTA and urea or SDS and 2-mercaptoethanol or 0.1 M GlcNAc. Substrate inhibition studies showed that 50 mM GlcNAc specifically inhibited the additional RcL I-g15 labeling produced by GT. The use of purified glycosyltransferases therefore appears to allow specific detection of oligosaccharide substrates and their high resolution localization in thin sections by electron microscopy.     

Barbiturate effects on hippocampal excitatory synaptic responses are selective and pathway specific

Barbiturate actions on excitatory synaptic responses in CA 1 and dentate regions of hippocampal slices were studied to determine whether different effects occur on anatomically distinct synaptic pathways. Pentobarbital facilitated transmission between stratum radiatum inputs and CA 1 neurons at low concentrations (0.02-0.08 mM) and produced postsynaptic depression at higher concentrations. Only depression was observed for stratum oriens inputs to CA 1 and perforant path inputs to dentate granulae neurons. The (+) isomer of pentobarbital was approximately four times more potent than the (-) isomer of racemic mixture. Phenobarbital (0.04-0.12 mM) produced only depression of synaptic responses in CA 1 and dentate pathways. Comparison of effect on field excitatory postsynaptic potentials and population spike responses indicated that the barbiturates act at selective and pathway-specific sites. The results provide further evidence for specific cellular and membrane recognition sites for barbiturate action.     

Biochemical characterization of polypeptide components involved in neurite fasciculation and elongation

Polypeptide components and carbohydrate linkage types of F11 antigen and G4 antigen, two chick cell-surface glycoproteins implicated in neurite fasciculation and elongation [Rathjen, F.G., Wolff, J.M., Bonhoeffer, F. and Rutishauser, U. (1987) J. Cell Biol. 104, 343-353], have been studied in comparison to mouse L1 antigen. Tryptic fingerprint analysis does not reveal any relation of the 130-kDa components of G4 or F11 antigens to each other or to neural cell-adhesion molecules. The 180/190-kDa component of G4 antigen comprises parts of the 130-kDa and 80/65-kDa components and shares a sequence corresponding to the amino terminus of the G4 130-kDa component as shown serologically with anti-peptide sera. This closely parallels the relationship found for mouse L1 antigen components. In contrast, the F11 170-kDa component is different from the F11 130-kDa component, as shown serologically and by fingerprint analysis. A combination of chemical and enzymatic deglycosylation methods reveals that while O-glycosylation cannot be detected F11 130-kDa, G4 130-kDa and L1 140-kDa components contain N-linked carbohydrates. Endoglycosidase H treatment shows that the oligosaccharides present in the G4 130-kDa component and mouse L1 are mostly of the complex type, while the F11 130-kDa component consists of two populations, one containing mainly complex-type carbohydrates and a second containing high-mannose/hybrid-type carbohydrates.     

Prevention of non-specific interactions of gold-labeled reagents on tissue sections

The protein A-gold technique is amongst the most useful labeling techniques available for light and electron microscopic immunolabeling. Some electron microscopic studies, however, have suggested that protein A-gold, and other protein-gold complexes as well, may bind non-specifically to certain tissue structures, particularly in skin, creating a specious pattern of labeling. We utilized the protein A-gold technique with antiserum to both involucrin and keratin under a variety of conditions to document the specificity of labeling. When the standard conditions were followed, the protein A-gold technique produces highly specific results. These conditions include: 1. the blocking of unreacted aldehyde groups by amination; 2. the blocking of non-specific binding sites on tissue sections by preincubation with inert proteins; and 3. the use of proper concentration of the protein A-gold complex. However, non-specific labeling could be produced if the three components of the standard protocol were omitted. In particular, the use of too concentrated protein A-gold lead to non-specific labeling. We report here also updated working protocols for antigen detection with protein A-gold on semithin Lowicryl K4M and paraffin sections which provide optimal staining results.     

Modeling rendille household herd composition

Previous analysis of Rendille household herd composition revealed a transition from camel to cattle ownership for sedentary impoverished Rendille pastoralists of northern Kenya. In an attempt to delineate determinants of livestock holdings, logistic regression analysis of 112 household herds from the Rendille settlement of Korr, Marsabit District, Kenya was undertaken. Results indicated that household wealth, measured in present livestock holdings, past drought losses, and livestock sales, formed better predictors of cattle ownership than did household characteristics pertaining to labor supply, wage earners, age-set membership, and birth order of household head. These results are discussed in light of pastoral strategies designed to minimize risk.     

Drought and economic differentiation among Ariaal pastoralists of Kenya

This paper examines the effects of the 1984 drought upon household wealth differences in a community of Ariaal pastoralists of northern Kenya. The database consists of 1985 post-drought livestock counts and informants' statements of species-specific drought loss, compared to 1976 livestock counts on the same 38 households. The analysis confirms the hypothesis that the drought resulted in increased household wealth inequalities. It is suggested that the combination of differential herd growth, differential participation in the cash market, and differential loss to the drought has contributed to a polarization within Ariaal of rich and poor, resulting in rural proletarianization and urban migration.