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Biomedical subjects

J W Little

Publications and source records attributed to J W Little.

At least 37 records · Page 2Linked to original sources
PubMed1998

Mutations affecting cooperative DNA binding of phage HK022 CI repressor.

Cooperative protein-DNA interactions play critical roles in gene regulation in all organisms. Among the best-studied cooperative interactions is that of phage lambda repressor, which binds cooperatively to two adjacent operators. Similar cooperative interactions are also shown by several other lambdoid phage repressors, including HK022 CI repressor, which we study here. This protein has a much higher degree of cooperativity than seen with lambda repressor, and previous evidence has suggested that cooperativity may play roles in HK022 gene regulation that have no parallel in lambda. We have isolated several cooperativity or Coop- mutations in HK022 cI. These mutant proteins were partially defective in vivo for binding to two adjacent operators, but normal or nearly so for binding to a single operator. Two mutations showed mutual suppression, in that the double mutation had wild-type behavior. Analysis of several purified mutant proteins showed that they were also defective for cooperative binding in vitro. Unexpectedly, the mutant proteins showed an altered pattern of in vitro binding to DNA at non-operator sites. Several of them also increased the rate of specific repressor cleavage. We propose a conformational model in which the various functions of the wild-type protein are carried out by differing conformations; these conformations are normally in balance, and the mutations perturb this balance.

Bacteriophage lambda↗
PubMed1998

The spacing between binding sites controls the mode of cooperative DNA-protein interactions: implications for evolution of regulatory circuitry.

The CI repressors of lambdoid phages bind cooperatively to adjacent binding sites. Although these binding sites are found at complex operators containing three binding sites, cooperative binding occurs only between dimers bound at two of the sites, a mode of binding termed pairwise cooperativity. At the level of regulation, pairwise cooperativity allows the proper operation of the genetic switch. At the mechanistic level, it has been proposed to result from steric distortion of the complex, such that a protein dimer bound to a central site cannot contact both flanking dimers because it "leans" towards one of the two sites. We have tested this model using the CI repressor of phage HK022. Previous work suggested that reducing the spacing between adjacent operators might allow cooperative interaction among three dimers, a mode of cooperativity we term extended. Using a set of synthetic templates, we have shown that reducing the spacing allows extended cooperativity among three binding sites. Accordingly, the mode of cooperativity changes qualitatively in response to changes in the inter-site spacing. That is, the change in spacing has major functional consequences for the properties of the complex. We suggest that such changes can play important roles in the evolution of gene regulatory circuitry and of other processes involving nucleoprotein complexes.

Bacteriophage lambda↗
PubMed1998

Posttraumatic syringomyelia: profound neuronal loss, yet preserved function.

Posttraumatic syrinxes may extend many cord segments rostral to a spinal cord injury (SCI) and significantly dilate the spinal cord, yet few neurologic deficits may be noted. Careful physical examination may reveal ascending loss of pain and temperature without evident functional motor decline. We present a 49-year-old man with T4 paraplegia and a large posttraumatic syrinx who died 3 weeks after syringoperitoneal shunting. Neuropathologic study revealed a large bilateral syrinx cavity from T1 to C6 that tapered to a small unilateral syrinx at C2. Light microscopy of sections from T1 to C2 showed massive loss of intermediate to intermedio-lateral gray neurons and moderate reduction of motoneurons at T1 to C6 levels. Despite these findings, manual muscle testing results remained normal for wrist extensors and elbow extensors, and the patient continued to perform independent sliding board transfers. We conclude that this large progressive syrinx did not merely dissect neural elements apart but caused extensive neuronal damage. Loss of interneurons was evident in spinal segments with preserved strength and function. Possible mechanisms to explain the relatively minimal clinical deficits in view of the neuronal loss are discussed.

Activities of Daily Living↗
PubMed1997

Axonal sprouting following incomplete spinal cord injury: an experimental model.

Recovery of function following incomplete spinal cord injury may in part result from growth of new connections by spared descending pathways. It has been difficult to demonstrate such anatomical reorganization with traditional anatomic techniques. This study utilizes an immunocytochemical method to demonstrate axonal growth cones within the lumbar spinal cord in rats recovering from an incomplete midthoracic spinal cord injury. Adult rats underwent subtotal section of the midthoracic cord sparing the left lateral funiculus and a portion of the left ventral funiculus. Light microscope immunocytochemistry was performed on sections of lumbar spinal cord with antibodies to identify sprouting axons. These antibodies were used to determine the distribution of growth cones on both sides of the lumbar spinal cord in experimental and control animals. Growth cones were first observed three days after the spinal cord lesion. Specific labeling, similar in appearance to previous reports of growth cone identification, was apparent within the immediate gray and ventral horns on both sides of the cord. These data support the hypothesis of collateral sprouting distal to the lesion site following incomplete spinal cord injury. It further supports the idea that recovery of function following incomplete spinal cord injury is, in part, mediated by spared descending pathways.

Animals↗
PubMed1996

Mutant LexA proteins with specific defects in autodigestion.

In self-processing biochemical reactions, a protein or RNA molecule specifically modifies its own structure. Many such reactions are regulated in response to the needs of the cell by an interaction with another effector molecule. In the system we study here, specific cleavage of the Escherichia coli LexA repressor, LexA cleaves itself in vitro at a slow rate, but in vivo cleavage requires interaction with an activated form of RecA protein. RecA acts indirectly as a coprotease to stimulate LexA autodigestion. We describe here a new class of lexA mutants, lexA (Adg-; for autodigestion-defective) mutants, termed Adg- for brevity. Adg- mutants specifically interfered with the ability of LexA to autodigest but left intact its ability to undergo RecA-mediated cleavage. The data are consistent with a conformational model in which RecA favors a reactive conformation capable of undergoing cleavage. To our knowledge, this is the first example of a mutation in a regulated self-processing reaction that impairs the rate of self-processing without markedly affecting the stimulated reaction. Had wild-type lexA carried such a substitution, discovery of its self-processing would have been difficult; we suggest that, in other systems, a slow rate of self-processing has prevented recognition that a reaction is of this nature.

Amino Acid Sequence↗
PubMed1996

Spinal cord infarction: varying degrees of upper and lower motoneuron dysfunction.

Five patients with spinal cord infarction underwent electrophysiologic evaluation. Two subjects with complete paralysis had absent compound muscle action potentials (M-responses), suggesting complete loss of lower motoneurons (LMN). Three subjects with incomplete cord infarction had preserved M-responses, reduced voluntary recruitment and abnormally slow motor-unit firing rates during maximal effort, suggesting upper motoneuron (UMN) weakness. These five patients demonstrate a range of neuronal damage after cord ischemia. With severe cord infarction, there is LMN degeneration and paralysis. With partial cord infarction, there is selective interneuron loss, resulting in UMN weakness. Electrodiagnostic evaluation can help determine prognosis for motor recovery after spinal cord infarction.

Action Potentials↗
PubMed1994

Spared descending pathways mediate locomotor recovery after subtotal spinal cord injury.

The location of pathways responsible for locomotor recovery after incomplete spinal cord injury has been studied in adult rats. Animals were allowed to recover from subtotal mid-thoracic cord section which spared left lateral and ventral funiculus fibers. Subsequent lumbar commissurotomy or left thoracic cord hemisection abolished the locomotor recovery. Spared descending fibers passing through the initial incomplete cord lesion appear to mediate locomotor recovery.

Animals↗
PubMed1994

Cooperative DNA-protein interactions. Effects of changing the spacing between adjacent binding sites.

Cooperative binding of specific DNA-binding proteins plays crucial roles in gene regulatory circuitry, and is a model system for interactions between proteins bound to DNA. We have studied coliphage HK022 repressor, which binds to two adjacent operators with a cooperativity parameter of approximately 2000. We examined the effect of changing the spacing between these two operators on cooperativity and on the conformation of the complex. Maximum cooperativity was seen with the wild-type spacing; considerable cooperativity was retained for most spacing variants, but was abolished when the operators lay on opposite faces of the DNA helix. Most spacing variants conferred changes in the conformation of the DNA-protein complex. Our data indicate that the pairwise cooperativity observed with the wild-type spacing results from a conformation that prevents protein-protein contacts with flanking bound dimers. We conclude that protein-DNA complexes involving the same specific binding sites and the same protein molecules can adopt many different conformations, depending on the spacing between the binding sites. This conclusion may be broadly applicable to protein-DNA interactions in other systems.

Allosteric Regulation↗
PubMed1994

M/RMS: an EMG method for quantifying upper motoneuron and functional weakness.

A new method for quantifying upper motoneuron (UMN) weakness using standard EMG equipment is presented and compared to the twitch occlusion method. Control subjects and patients with stroke, spinal cord injury (SCI), and peripheral nerve injury (PNI) were evaluated. Force, EMG, and twitch force from nerve electrical stimulation were recorded during isometric contractions of the biceps and triceps. Ratios of the elicited CMAP amplitude (M)/voluntary-root-mean-square EMG activity (RMS) and of the voluntary contraction twitch force (Ti)/the resting twitch force (Tmax) were the two measures of UMN weakness studied. Both ratios are linearly correlated with force for controls, log M/RMS (r = 0.96) and Ti/Tmax (r = 0.86). Log M/RMS ratio was abnormally high (> mean + 2 SD; i.e., > 1.09) for weak muscles affected by stroke and incomplete SCI, but was normal in muscles weakened by PNI. An elevated M/RMS ratio, may aid in quantification of deficient supraspinal activation from UMN or functional weakness.

Adult↗
PubMed1994

Relation of changes in radionuclide measures of effective renal plasma flow and glomerular filtration rate in spinal cord injury patients.

The association of changes in radionuclide measures of effective renal plasma flow (ERPF) and glomerular filtration rate (GFR) in relation to the time since spinal cord injury (SCI) was investigated in 30 male SCI patients ages 28-72 (mean 49), with intervals from injury ranging from two to 48 years. ERPF and GFR were determined from plasma clearance curves following bolus injections of I-131 orthoiodohippurate (OIH) and Tc-99m diethylenetriaminepentaacetic acid (DTPA) respectively, and were compared with published age-adjusted normal values. Fifteen patients had decreased ERPF and 13 had decreased GFR, with decline in ERPF tending to precede a similar change in GFR. Among 18 patients < or = 20 years post-SCI, GFR was abnormal in six (33 percent), while ERPF was decreased in 10 (56 percent). In twenty-four patients (80 percent), GFR and ERPF were either both normal (n = 13) or both abnormal (n = 11), but filtration fraction (FF = GFR/ERPF) was increased to > 0.23 in 18 patients, including nine with normal GFR. Only 2 patients had normal ERPF/abnormal GFR, both of whom were > 35 years post-SCI and had FF < 0.20. In the SCI population, decreases in ERPF tend to precede similar changes in GFR, with resultant increase in FF in the majority of patients.

Adult↗
PubMed1994

Hydronephrosis due to improper condom catheter use.

This report concerns a patient with bilateral hydronephrosis due to a tight external condom catheter retention strap. Urodynamics with fluoroscopy and cystoscopy failed to demonstrate bladder outlet obstruction, urethral stricture, uretero-vesicle junction abnormality, or other explanations for the bilateral hydronephrosis. The hydronephrosis resolved when the external condom catheter retention strap was eliminated. The authors emphasize the importance of patient education to prevent this problem and encourage alertness by clinicians to assess appropriateness of external condom catheter use.

Aged↗
PubMed1994

Managing dental patients with joint prostheses.

A literature review explored the possible association of late infections of major joint prostheses and dental bacteremias. The review showed transient dental bacteremias had little or no role in causing late infections of prosthetic joint replacements. However, patients with certain systemic conditions or complications with their prostheses may be at greater risk for infection after transient dental bacteremias.

American Dental Association↗
PubMed1994

Normal limits of side-to-side H-reflex amplitude variability.

Our hypothesis was that side-to-side difference in the tibial H-reflex amplitude can aid in the diagnosis of unilateral S1 radiculopathy with an otherwise normal side-to-side H-reflex latency difference. We studied 47 healthy subjects, (mean +/- SD, 35 +/- 10 years) using methods similar to those described by Braddom and Johnson. Only those with side-to-side latency difference less than 1.5 msec were included (n = 45). Current was increased in 0.5 to 1.0 mA increments until the H-reflex amplitude maximized and then fell; on each side the response with the maximal peak-to-peak amplitude was measured for peak-to-peak amplitude and onset latency. The amplitude on the side of the smaller response was expressed as a ratio of the contralateral amplitude; this mean value (+/- SD) was 0.74 +/- 0.17. We conclude that a side-to-side amplitude ratio smaller than 0.4 (mean - 2 SD) in the face of a normal side-to-side latency difference is probably abnormal.

Adult↗
PubMed1994

Electrodiagnosis of upper limb weakness in acute quadriplegia.

Clinical and neuropathologic observations after cervical spinal cord injury suggest varying involvement of gray and white matter. The resulting upper limb weakness may reflect varying degrees of upper motoneuron (UMN) and/or lower motoneuron (LMN) involvement. This study uses electrophysiologic measures, including compound muscle action potential (M response) amplitude, root mean square (RMS) of the surface electromyographic activity during voluntary muscle contractions and the firing rate of motor units, to distinguish UMN and LMN weakness in upper extremities after acute quadriplegia. M response amplitude did not correlate with strength; many muscles had large M responses given their strength. These muscles manifest: (1) high M/RMS ratios (ratio of electrically elicited to voluntarily recruited electromyographic activity) and (2) slow firing rates of single motor units during maximal isometric contractions. For muscles with normal M amplitudes, M/RMS ratio correlates inversely with strength. For muscles with normal M/RMS ratios, M amplitude correlates positively with strength. Cluster analysis was used to distinguish UMN, LMN or Mixed types of weakness. Distinguishing these different types of weakness in acute quadriplegia may allow individualized rehabilitation for the type of weakness present.

Action Potentials↗
PubMed1993

LexA and lambda Cl repressors as enzymes: specific cleavage in an intermolecular reaction.

During the SOS response, LexA repressor is inactivated by specific cleavage. Although cleavage requires RecA protein in vivo, RecA acts indirectly as a coprotease by stimulating an inherent self-cleavage activity of LexA. In lambda lysogens, cleavage of lambda Cl repressor in a similar but far slower reaction results in prophage induction. We describe an intermolecular cleavage reaction in which the C-terminal fragment of LexA acted as an enzyme to cleave other molecules of LexA. The C-terminal fragment of lambda repressor cleaved the LexA substrates about as efficiently as did the LexA enzyme, suggesting that the slow rate of Cl self-cleavage results from a weak interaction between its cleavage site and the active site.

Bacterial Proteins↗
PubMed1993

Highly cooperative DNA binding by the coliphage HK022 repressor.

The CI repressor protein from the temperate lambdoid phage HK022 was purified to near homogeneity and used in DNase I footprinting analyses to identify six binding sites in this phage. All these sites contained homologous 15 bp inverted repeats. Three of these 15 bp inverted repeats were located between the cI and cro (OR1 to OR3), and the other three were 3' to the cI gene (OL1 to OL3). Two of these sites were identified as operator sites for the repressor by DNA sequence analyses of virulent phage mutants. Almost all these mutations identified lay within the 15 bp inverted repeats comprising OR1 and OR2, and almost all were in the most highly conserved positions in the operators. The majority of virulent mutants contained mutations in both OR1 and OR2. Intrinsic affinities for individual operators were measured by DNase I footprinting analyses using DNA fragments which contained a single wild-type operator adjacent to two mutant operators. Comparison of these values with the affinity observed with these sites in the wild-type operator indicated that HK022 CI repressor bound cooperativity to OR1 and OR2 with a cooperativity parameter, omega, of almost 2000. Cooperative binding occurred in an alternative pairwise fashion, as previously seen with lambda CI repressor. In addition to cooperative binding between two adjacent operators, the repressor also increased the affinity for adjacent non-specific DNA sites, resulting in a periodic pattern of binding termed "phasing". This phasing pattern extended beyond regions predicted for pair-wise interaction, but was significantly decreased on a template with two adjacent operators, suggesting that pairwise cooperativity interfered with phasing.

Bacteriophage lambda↗
PubMed1993

Silent periods after electromagnetic stimulation of the motor cortex.

Electromagnetic stimulation has been used to excite tracts in the human motor cortex, but little has been done to study inhibitory effects. In three normal subjects we have noted that an electromagnetic stimulation applied to the motor cortex during maximum voluntary contraction produces silent periods lasting up to 300 ms in the contralateral abductor pollicis brevis. Duration of the silent period is directly proportional to the intensity of the stimulus. Abductor hallucis has similar silent periods lasting 150 to 200 ms, although proximal upper and lower limb muscles have shorter silent periods. F and H waves are easily elicited during this silent period suggesting that the alpha motoneuron is still excitable. This silent period may result, at least in part, from a synchronous volley of inhibitory postsynaptic potentials induced by the electromagnetic stimulator at the cortical level. This technique may prove useful in further delineating cortical inhibitory function.

Adult↗