Search PubMed⌕ Search

Biomedical subjects

L S Goldstein

Publications and source records attributed to L S Goldstein.

At least 73 records · Page 4Linked to original sources
PubMed1997

alpha-Spectrin is required for ovarian follicle monolayer integrity in Drosophila melanogaster.

To understand the role of the spectrin-based membrane skeleton in generating epithelial polarity, we characterized the distribution of membrane skeletal components in Drosophila ovarian follicle cells and in somatic clones of mutant cells that lack alpha-spectrin. Immunolocalization data reveal that wild-type follicle cells contain two populations of spectrin heterodimers: a network of alphabeta heterodimers concentrated on the lateral plasma membrane and an alphabetaH population targeted to the apical surface. Induction of somatic clones lacking alpha-spectrin leads to follicle cell hyperplasia. Surprisingly, elimination of alpha-spectrin from follicle cells does not appear to prevent the assembly of conventional beta-spectrin and ankyrin at the lateral domain of the follicle cell plasma membrane. However, the alpha-subunit is essential for the correct localization of betaH-spectrin to the apical surface. As a consequence of disrupting the apical membrane skeleton a distinct sub population of follicle cells undergoes unregulated proliferation which leads to the loss of monolayer organization and disruption of the anterior-posterior axis of the oocyte. These results suggest that the spectrin-based membrane skeleton is required in a developmental pathway that controls follicle cell monolayer integrity and proliferation.

Animals↗
PubMed1997

Is direct collection of pleural fluid into a heparinized syringe important for determination of pleural pH? A brief report.

INTRODUCTION: It has long been believed that pleural fluid must be directly aspirated into a heparinized syringe to obtain an accurate value. Many operators aspirate 30 to 60 mL of pleural fluid into a syringe without heparin, and then place 1 mL into a heparinized syringe from which the pH is determined. We postulated that this technique does not cause a clinically significant difference in pleural pH values. METHODS: Patients undergoing thoracentesis in the outpatient clinic, general ward, and medical ICU were eligible. After the initial entry of the needle into the pleural space, a heparinized syringe was used to obtain pleural fluid for pH determination. A 60-mL syringe was then used to aspirate additional pleural fluid for biochemical analysis and culture. At the end of the procedure, a second aliquot of pleural fluid was placed into a heparinized syringe for pH determination. A difference of 0.1 in pH was taken as clinically important. RESULTS: Twenty-one pleural fluid samples were obtained from 20 patients. Pleural fluid pH determinations were within 0.1 in all but one patient. The mean pH for the directly collected group was 7.39 (25%: 7.35; 75%: 7.45). The mean for the indirectly collected group was 7.41 (25%: 7.35; 75%: 7.45). The difference between the two means (0.02; 95% confidence interval, 0.0368 to 0.00131) was statistically significant but clinically unimportant (p=0.037). CONCLUSIONS: Pleural fluid can be collected in a large syringe and then placed into a heparinized syringe to assess pH. This is useful information because the use of just one syringe saves time and reduces the risk of iatrogenic complications.

Aged↗
PubMed1996

Going mobile: microtubule motors and chromosome segregation.

Proper chromosome segregation in eukaryotes depends upon the mitotic and meiotic spindles, which assemble at the time of cell division and then disassemble upon its completion. These spindles are composed in large part of microtubules, which either generate force by controlled polymerization and depolymerization or transduce force generated by molecular microtubule motors. In this review, we discuss recent insights into chromosome segregation mechanisms gained from the analyses of force generation during meiosis and mitosis. These analyses have demonstrated that members of the kinesin superfamily and the dynein family are essential in all organisms for proper chromosome and spindle behavior. It is also apparent that forces generated by microtubule polymerization and depolymerization are capable of generating forces sufficient for chromosome movement in vitro; whether they do so in vivo is as yet unclear. An important realization that has emerged is that some spindle activities can be accomplished by more than one motor so that functional redundancy is evident. In addition, some meiotic or mitotic movements apparently occur through the cooperative action of independent semiredundant processes. Finally, the molecular characterization of kinesin-related proteins has revealed that variations both in primary sequence and in associations with other proteins can produce motor complexes that may use a variety of mechanisms to transduce force in association with microtubules. Much remains to be learned about the regulation of these activities and the coordination of opposing and cooperative events involved in chromosome segregation; this set of problems represents one of the most important future frontiers of research.

Animals↗
PubMed1996

Receptor tyrosine phosphatases are required for motor axon guidance in the Drosophila embryo.

The receptor tyrosine phosphatases DPTP69D and DPTP99A are expressed on motor axons in Drosophila embryos. In mutant embryos lacking DPTP69D protein, motor neuron growth cones stop growing before reaching their muscle targets, or follow incorrect pathways that bypass these muscles. Mutant embryos lacking DPTP99A are indistinguishable from wild type. Motor axon defects in dptp69D dptp99A double mutant embryos, however, are much more severe than in embryos lacking only DPTP69D. Our results demonstrate that DPTP69D and DPTP99A are required for motor axon guidance and that they have partially redundant functions during development of the neuro-muscular system.

Animals↗
PubMed1996

Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein.

We have used time-lapse laser scanning confocal microscopy to directly examine microtubule reorganization during meiotic spindle assembly in living Drosophila oocytes. These studies indicate that the bipolarity of the meiosis I spindle is not the result of a duplication and separation of centrosomal microtubule organizing centers (MTOCs). Instead, microtubules first associate with a tight chromatin mass, and then bundle to form a bipolar spindle that lacks asters. Analysis of mutant oocytes indicates that the Non-Claret Disjunctional (NCD) kinesin-like protein is required for normal spindle assembly kinetics and stabilization of the spindle during metaphase arrest. Immunolocalization analyses demonstrate that NCD is associated with spindle microtubules, and that the centrosomal components gamma-tubulin, CP-190, and CP-60 are not concentrated at the meiotic spindle poles. Based on these observations, we propose that microtubule bundling by the NCD kinesin-like protein promotes assembly of a stable bipolar spindle in the absence of typical MTOCs.

Animals↗
PubMed1996

Novel susceptibility locus for mouse hepatomas: evidence for a conserved tumor suppressor gene.

We have identified previously a putative tumor suppressor gene (TSG) locus at human chromosome (hchr) 7q31 showing that it is altered in a variety of human epithelial tumors. To determine whether this TSG is conserved in mice, we studied loss of heterozygosity (LOH) in chemically induced mouse liver adenomas. The LOH analysis was performed by polymerase chain reaction amplification of 17 (CA)n microsatellite repeats on mouse chromosome (mchr) 6 A2-C3. Ninety-six of 106 cases (90.6%) had LOH at D6Mit50, and 89.5% had LOH at D6Mit179. These two loci are 0.2 cM apart on mchr 6A2. Another high-LOH site was found in the C3 band. The high incidence of LOH in the 7q-homologous segment of mchr 6 indicates that the human TSG is conserved and is involved in the development of hepatomas.

Animals↗
PubMed1995

DNA binding and meiotic chromosomal localization of the Drosophila nod kinesin-like protein.

The Drosophila no distributive disjunction (nod) gene encodes a kinesin-like protein that has been proposed to push chromosomes toward the metaphase plate during female meiosis. We report that the nonmotor domain of the nod protein can mediate direct binding to DNA. Using an antiserum prepared against bacterially expressed nod protein, we show that during prometaphase nod protein is localized on oocyte chromosomes and is not restricted to either specific chromosomal regions or to the kinetochore. Thus, motor-based chromosome-microtubule interactions are not limited to the centromere, but extend along the chromosome arms, providing a molecular explanation for the polar ejection force.

Amino Acid Sequence↗
PubMed1995

Drosophila development requires spectrin network formation.

The head-end associations of spectrin give rise to tetramers and make it possible for the molecule to form networks. We analyzed the head-end associations of Drosophila spectrin in vitro and in vivo. Immunoprecipitation assays using protein fragments synthesized in vitro from recombinant DNA showed that interchain binding at the head end was mediated by segment 0-1 of alpha-spectrin and segment 18 of beta-spectrin. Point mutations equivalent to erythroid spectrin mutations that are responsible for human hemolytic anemias diminished Drosophila spectrin head-end interchain binding in vitro. To test the in vivo consequence of deficient head-end interchain binding, we introduced constructs expressing head-end interchain binding mutant alpha-spectrin into the Drosophila genome and tested for rescue of an alpha-spectrin null mutation. An alpha-spectrin minigene lacking the codons for head-end interchain binding failed to rescue the lethality of the null mutant, whereas a minigene with a point mutation in these codons overcame the lethality of the null mutant in a temperature-dependent manner. The rescued flies were viable and fertile at 25 degrees C, but they became sterile because of defects in oogenesis when shifted to 29 degrees C. At 29 degrees C, egg chamber tissue disruption and cell shape changes were evident, even though the mutant spectrin remained stably associated with cell membranes. Our results show that spectrin's capacity to form a network is a crucial aspect of its function in nonerythroid cells.

Amino Acid Sequence↗
PubMed1995

Motor activity and mitotic spindle localization of the Drosophila kinesin-like protein KLP61F.

The KLP61F gene product is essential for Drosophila development. Mutations in KLP61F display a mitotic arrest phenotype caused by a failure in the proper separation of duplicated centrosomes (Heck et al., 1993). Sequence analysis of KLP61F identified it as a member of the bimC family of kinesin-like microtubule motor proteins. Here we report that KLP61F is distinct from KRP130, a kinesin-like protein recently purified from Drosophila embryos and suggested to be the product of the KLP61F gene (Cole et al., 1994). We also characterized recombinant KLP61F and found that it possesses microtubule-stimulated ATPase and microtubule translocation activities in vitro. In addition, we have used an affinity-purified, KLP61F-specific antiserum to localize native KLP61F and an epitope-tagged KLP61F fusion protein during various stages of mitosis in Drosophila syncytial blastoderm embryos. From early prophase through anaphase, KLP61F is coincident with the distribution of tubulin. Together these results confirm the existence of multiple bimC-like kinesins in Drosophila and suggest that KLP61F function is intrinsic to the mitotic spindle.

Adenosine Triphosphatases↗
PubMed1995

An inverse PCR screen for the detection of P element insertions in cloned genomic intervals in Drosophila melanogaster.

We developed a screening approach that utilizes an inverse polymerase chain reaction (PCR) to detect P element insertions in or near previously cloned genes in Drosophila melanogaster. We used this approach in a large scale genetic screen in which P elements were mobilized from sites on the X chromosome to new autosomal locations. Mutagenized flies were combined in pools, and our screening approach was used to generate probes corresponding to the sequences flanking each site of insertion. These probes then were used for hybridization to cloned genomic intervals, allowing individuals carrying insertions in them to be detected. We used the same approach to perform repeated rounds of sib-selection to generate stable insertion lines. We screened 16,100 insert bearing individuals and recovered 11 insertions in five intervals containing genes encoding members of the kinesin superfamily in Drosophila melanogaster. In addition, we recovered an insertion in the region including the Larval Serum Protein-2 gene. Examination by Southern hybridization confirms that the lines we recovered represent genuine insertions in the corresponding genomic intervals. Our data indicates that this approach will be very efficient both for P element mutagenesis of new genomic regions and for detection and recovery of "local" P element transposition events. In addition, our data constitutes a survey of preferred P element insertion sites in the Drosophila genome and suggests that insertion sites that are mutable at a rate of approximately 10(-4) are distributed every 40-50 kb.

Animals↗
PubMed1995

Structural features involved in force generation in the kinesin superfamily.

In this article, I discuss our current understanding of the structural features of the microtubule-dependent motor kinesin and its relatives, which are needed for force generation. It has become clear that the motor domain itself is likely to be only about 340 amino acids, and that direction of movement is also controlled within this region. There is reason, however, to be suspicious that elements in the tail that are located immediately adjacent to the motor region influence, or in some way contribute to, normal motor activity. Finally, I describe recent work designed to identify the regions involved in the kinesin-microtubule interaction.

Animals↗
PubMed1994

Characterization of the KLP68D kinesin-like protein in Drosophila: possible roles in axonal transport.

This paper describes the molecular and biochemical properties of KLP68D, a new kinesin-like motor protein in Drosophila melanogaster. Sequence analysis of a full-length cDNA encoding KLP68D demonstrates that this protein has a domain that shares significant sequence identity with the entire 340-amin acid kinesin heavy chain motor domain. Sequences extending beyond the motor domain predict a region of alpha-helical coiled-coil followed by a globular "tail" region; there is significant sequence similarity between the alpha-helical coiled-coil region of the KLP68D protein and similar regions of the KIF3 protein of mouse and the KRP85 protein of sea urchin. This finding suggests that all three proteins may be members of the same family, and that they all perform related functions. KLP68D protein produced in Escherichia coli is, like kinesin itself, a plus-end directed microtubule motor. In situ hybridization analysis of KLP68D RNA in Drosophila embryos indicates that the KLP68D gene is expressed primarily in the central nervous system and in a subset of the peripheral nervous system during embryogenesis. Thus, KLP68D may be used for anterograde axonal transport and could conceivably move cargoes in fly neurons different than those moved by kinesin heavy chain or other plus-end directed motors.

Amino Acid Sequence↗
PubMed1993

The Drosophila kinesin light chain. Primary structure and interaction with kinesin heavy chain.

Kinesin light chain (KLC) complexes with the kinesin heavy chain (KHC) to form native kinesin. Proposed functions of KLC include coupling of cargo to KHC or modulation of KHC ATPase activity. In this paper we use the KHC tail, which binds specifically to KLC in blot overlays, as a probe to clone a cDNA encoding KLC from a Drosophila expression library. The identified clone encodes a protein with 70% amino acid identity to rat KLC. Drosophila KLC is predicted to form an alpha-helical coiled-coil between residues 34 and 129, followed by five imperfect tandem repeats of unknown function and a sixth shorter motif. These repeats are highly conserved across species. The Drosophila KLC gene is located at 69D on the third chromosome and is widely expressed, with 1.8-kb transcripts in most tissues, and slightly smaller transcripts in gonads. Finally, we present evidence that the heptad repeats of KLC are required for interaction with the KHC tail. Since the KHC tail used in our assay includes about 20 heptad repeats, this result suggests that KHC and KLC interact via coiled-coils. Such an interaction could provide stability to the KHC-KLC complex in vivo.

Amino Acid Sequence↗
PubMed1993

Direction of microtubule movement is an intrinsic property of the motor domains of kinesin heavy chain and Drosophila ncd protein.

The kinesin heavy chain and the ncd (non-claret disjunctional) gene product of Drosophila are microtubule-associated motor proteins related by sequence similarity within an approximately 340-aa domain. Despite the sequence similarity, the kinesin heavy chain and ncd protein move in opposite directions on microtubules. To investigate the molecular basis for direction of movement, we created a series of truncated kinesin heavy chain and ncd proteins. We found that the conserved domain of both proteins has microtubule motor activity, although the efficiency with which ATP hydrolysis is coupled to microtubule movement declines dramatically with increasing truncation. Further, the direction of movement is intrinsic to the conserved motor domains, rather than being a consequence of domain organization or adjacent sequences.

Animals↗