Biomedical subjects

B V Zemelman

Publications and source records attributed to B V Zemelman.

9 recordsLinked to original sources

PubMed2001

Genetic schemes and schemata in neurophysiology.

Information in nervous systems is often carried by neural ensembles--groups of neurons in transient functional linkage--and written in a code that involves the spatial locations of active neurons or synapses and the times at which activity occurs. Even in favorable neuroanatomical circumstances, studying neural ensemble function presents a serious experimental challenge. One recent strategy to overcome this challenge relies on protein-based sensors that provide direct optical images of neural activity, and on protein-based effectors that interfere with it. Because these molecules are encodable in DNA, they can be introduced into intact animals by genetic manipulation, and their expression pattern can be tailored to include--exclusively and at the same time comprehensively--the neurons of interest. Circumscribed populations of neurons can thus be studied in virtual isolation at defined stages of intact neural pathways.

Animals↗

PubMed1998

PRIM: proximity imaging of green fluorescent protein-tagged polypeptides.

We report a serendipitous discovery that extends the impressive catalog of reporter functions performed by green fluorescent protein (GFP) or its derivatives. When two GFP molecules are brought into proximity, changes in the relative intensities of green fluorescence emitted upon excitation at 395 vs. 475 nm result. These spectral changes provide a sensitive ratiometric index of the extent of self-association that can be exploited to quantitatively image homo-oligomerization or clustering processes of GFP-tagged proteins in vivo. The method, which we term proximity imaging (PRIM), complements fluorescence resonance energy transfer between a blue fluorescent protein donor and a GFP acceptor, a powerful method for imaging proximity relationships between different proteins. However, unlike fluorescence resonance energy transfer (which is a spectral interaction), PRIM depends on direct contact between two GFP modules, which can lead to structural perturbations and concomitant spectral changes within a module. Moreover, the precise spatial arrangement of the GFP molecules within a given dimer determines the magnitude and direction of the spectral change. We have used PRIM to detect FK1012-induced dimerization of GFP fused to FK506-binding protein and clustering of glycosylphosphatidylinositol-anchored GFP at cell surfaces.

Amino Acid Sequence↗

PubMed1998

Gos1p, a Saccharomyces cerevisiae SNARE protein involved in Golgi transport.

Specific transport between secretory compartments requires that vesicular carriers contain targeting proteins known as SNAREs. Ten v-SNAREs have been identified in the genome of the yeast Saccharomyces cerevisiae by sequence analysis. We report here the characterization of Gos1p, a v-SNARE localized to the Golgi compartment and likely homolog of the mammalian protein GOS-28/GS28. Gos1p is a type II membrane protein with characteristic SNARE sequence hallmarks and is functionally a SNARE protein. Gos1p was originally identified as a 28 kDa protein in an immunoprecipitate of the cis-Golgi t-SNARE Sed5p. This interaction between Sed5p and Gos1p is direct as demonstrated by in vitro binding with recombinant proteins. Deletion of GOS1 results in viable haploids with modest growth and secretory defects. Close examination of the secretory phenotype of GOS1-disrupted cells suggests that Gos1p may play a role in multiple transport steps, specifically ER-Golgi and/or intra-Golgi transport.

Biological Transport↗

PubMed1998

SNAREpins: minimal machinery for membrane fusion.

Recombinant v- and t-SNARE proteins reconstituted into separate lipid bilayer vesicles assemble into SNAREpins-SNARE complexes linking two membranes. This leads to spontaneous fusion of the docked membranes at physiological temperature. Docked unfused intermediates can accumulate at lower temperatures and can fuse when brought to physiological temperature. A supply of unassembled v- and t-SNAREs is needed for these intermediates to form, but not for the fusion that follows. These data imply that SNAREpins are the minimal machinery for cellular membrane fusion.

Bacterial Proteins↗

PubMed1994

Herpes simplex virus type 1 recombination: the Uc-DR1 region is required for high-level a-sequence-mediated recombination.

The a sequences of herpes simplex virus type 1 are believed to be the cis sites for inversion events that generate four isomeric forms of the viral genome. Using an assay that measures deletion of a beta-galactosidase gene positioned between two directly repeated sequences in plasmids transiently maintained in Vero cells, we had found that the a sequence is more recombinogenic than another sequence of similar size. To investigate the basis for the enhanced recombination mediated by the a sequence, we examined plasmids containing direct repeats of approximately 350 bp from a variety of sources and with a wide range of G+C content. We observed that all of these plasmids show similar recombination frequencies (3 to 4%) in herpes simplex virus type 1-infected cells. However, recombination between directly repeated a sequences occurs at twice this frequency (6 to 10%). In addition, we find that insertion of a cleavage site for an a-sequence-specific endonuclease into the repeated sequences does not appreciably increase the frequency of recombination, indicating that the presence of endonuclease cleavage sites within the a sequence does not account for its recombinogenicity. Finally, by replacing segments of the a sequence with DNA fragments of similar length, we have determined that only the 95-bp Uc-DR1 segment is indispensable for high-level a-sequence-mediated recombination.

Animals↗

PubMed1992

Recombination in vitro between herpes simplex virus type 1 a sequences.

We have partially purified an activity from extracts of cells infected with herpes simplex virus type 1 that mediates recombination between repeated copies of the 317-base-pair a sequence of herpes simplex virus type 1. Recombination leads to deletion of a lacZ indicator gene situated between two directly repeated copies of the a sequence and is scored by transformation of lacZ- Escherichia coli. The two products of the reaction can be observed directly by restriction enzyme digestion and Southern blot analysis. The recombinase activity is also detectable, but at a lower level, in uninfected cell extracts. The DNA substrate must contain the two a sequences arranged in direct orientation to generate the lacZ deletion. However, when the a sequences are arranged in inverted orientation, an inversion results. A substrate with two homologous sequences of size and G + C content similar to the a sequence undergoes recombination at a much lower frequency. The reaction requires a divalent cation (Mg2+ or Mn2+) but not ATP or any other nucleoside triphosphate. The simple requirements and specificity for the a sequence suggest that the recombination may proceed by a site-specific mechanism.

Animals↗

PubMed1992

Radiation-reduced hybrids for the myotonic dystrophy locus.

The myotonic dystrophy (DM) gene maps to the long arm of human chromosome 19 and is flanked by markers ERCC1 and D19S51. Also mapping to this region is the polio virus receptor gene (PVS). To produce more markers for this interval, we have constructed radiation-reduced hybrids by selecting for the retention of ERCC1 and for the loss of PVS. One of the cell lines produced has been characterized extensively and contains about 2 Mb of human DNA derived exclusively from chromosome 19, and includes ERCC1 and D19S51. Phage libraries constructed from DNA of this cell line have been screened and several new markers identified, including two for which cDNAs have been isolated. These represent candidate genes for DM. The new markers have also been used to extend the long-range restriction map of this region.

Base Sequence↗

PubMed1992

Expression and developmental regulation of Na+,K+ adenosine triphosphatase in the rat small intestine.

The Na+,K(+)-ATPase ion pump plays a critical role in fluid and electrolyte physiology of the small intestine. Here we show that, of the three known alpha isotypes (alpha 1, alpha 2, and alpha 3) of the sodium pump found in the rat, only alpha 1 is expressed in the small intestine. The expression of this isotype, considered at the level of mRNA, is under developmental control, with the adult intestine exhibiting approximately a threefold increase in alpha 1 message over the neonate. Cortisone treatment of the neonate results in near-adult levels of alpha 1 mRNA expression. An increase in the abundance of alpha 1 isotype parallels the changes in its mRNA expression. beta subunit mRNA is expressed coordinately with the alpha 1 subunit mRNA. A four- to five-fold rise in the Na+,K(+)-ATPase activity is also developmentally induced.

Age Factors↗

PubMed1989

Host response to Escherichia coli heat-labile enterotoxin via two microvillus membrane receptors in the rat intestine.

The responsiveness of enterocytes to Escherichia coli heat-labile enterotoxin (LT) was studied in the small intestine of 6- to 7-week-old rats. Dose-effect analysis showed the dose required for a 50% maximal LT-induced secretory response to be at 8 nM. After the well-documented glycolipid GM1 receptor was blocked with the cholera toxin B subunit, LT still activated the second messenger cascade, measured in terms of heightened cellular adenylate cyclase activity, and caused fluid to be secreted into ligated intestinal loops. Furthermore, Scatchard analysis of binding kinetics suggested that LT bound to two receptor sites on the intestinal microvillus membrane. The toxin also bound to delipidated membrane but was competitively inhibited by a galactose-specific lectin, RCA60, suggesting that the additional receptor is a galactoglycoprotein. Western blot analysis of toxin binding to membrane proteins revealed a group of binding components around 85 to 150 kilodaltons. When measured at 2.2 nM LT, approximately 70% of LT-binding activity took place through a high-affinity (Kd1, 0.38 nM) GM1 receptor and 30% of LT-binding activity took place through a low-affinity (Kd2, 3.3 nM) glycoprotein receptor. These results suggest that LT functions through two microvillus membrane receptors in the mature rat small intestine.

Adenylyl Cyclases↗