Packed protein bilayers in the 0.90 A resolution structure of a designed alpha helical bundle.

A 12-residue peptide designed to form an alpha-helix and self-associate into an antiparallel 4-alpha-helical bundle yields a 0.9 A crystal structure revealing unanticipated features. The structure was determined by direct phasing with the "Shake-and-Bake" program, and contains four crystallographically distinct 12-mer peptide molecules plus solvent for a total of 479 atoms. The crystal is formed from nearly ideal alpha-helices hydrogen bonded head-to-tail into columns, which in turn pack side-by-side into sheets spanning the width of the crystal. Within each sheet, the alpha-helices run antiparallel and are closely spaced (9-10 A center-to-center). The sheets are more loosely packed against each other (13-14 A between helix centers). Each sheet is amphiphilic: apolar leucine side chains project from one face, charged lysine and glutamate side chains from the other face. The sheets are stacked with two polar faces opposing and two apolar faces opposing. The result is a periodic biomaterial composed of packed protein bilayers, with alternating polar and apolar interfaces. All of the 30 water molecules in the unit cell lie in the polar interface or between the stacked termini of helices. A section through the sheet reveals that the helices packed at the apolar interface resemble the four-alpha-helical bundle of the design, but the helices overhang parts of the adjacent bundles, and the helix crossing angles are less steep than intended (7-11 degrees rather than 18 degrees).

Atomic solvation parameters applied to molecular dynamics of proteins in solution.

A solvation energy function for use in the molecular simulation of proteins is proposed. It is based on the accessible surface areas of atoms in the protein and on atomic solvation parameters derived from empirical vapor-to-water free energies of transfer of amino acid side-chain analogs. The energy function and its derivatives were added to the CHARMM molecular simulation program (Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., States, D.J., Swaminathan, S., & Karplus, M., 1983, J. Comput. Chem. 4(2), 187-217). The effect of the added energy term was evaluated by 110 ps of molecular dynamics on the 26-residue protein melittin. The melittin monomer and tetramer were studied both with and without the added term. With the added energy term the monomer partially unfolded, while the secondary structure of the tetramer was preserved, in agreement with reported experiments (Brown, L.R., Lauterwein, J., & Wuethrich, K., 1980, Biochim. Biophys. Acta 622(2), 231-244; Lauterwein, J., Brown, L.R., & Wuethrich, K., 1980, Biochim. Biophys. Acta 622(2), 219-230).

Where metal ions bind in proteins.

The environments of metal ions (Li+, Na+, K+, Ag+, Cs+, Mg2+, Ca2+, Mn2+, Cu2+, Zn2+) in proteins and other metal-host molecules have been examined. Regardless of the metal and its precise pattern of ligation to the protein, there is a common qualitative feature to the binding site: the metal is ligated by a shell of hydrophilic atomic groups (containing oxygen, nitrogen, or sulfur atoms) and this hydrophilic shell is embedded within a larger shell of hydrophobic atomic groups (containing carbon atoms). That is, metals bind at centers of high hydrophobicity contrast. This qualitative observation can be described analytically by the hydrophobicity contrast function, C, evaluated from the structure. This function is large and positive for a sphere of hydrophilic atomic groups (characterized by atomic solvation parameters, delta sigma, having values less than 0) at the center of a larger sphere of hydrophobic atomic groups (characterized by delta sigma greater than 0). In the 23 metal-binding molecules we have examined, the maximum values of the contrast function lie near to observed metal binding sites. This suggests that the hydrophobicity contrast function may be useful for locating, characterizing, and designing metal binding sites in proteins.

Crystal structure of alpha 1: implications for protein design.

X-ray diffraction shows the structure of a synthetic protein model, formed from noncovalent self-association of a 12-residue peptide and of sulfate ions at low pH. This peptide is a fragment of a 16-residue polypeptide that was designed to form an amphiphilic alpha helix with a ridge of Leu residues along one helical face. By interdigitation of the leucines of four such helices, the design called for self-association into a four-alpha-helical bundle. The crystal structure (2.7 angstrom resolution; R factor = 0.215) reveals a structure more complex than the design, with both a tetramer and a hexamer. The alpha-helical tetramer with leucine interior has more oblique crossing angles than most four-alpha-helical bundles; the hexamer has a globular hydrophobic core of 12 leucine residues and three associated sulfate ions. Computational analysis suggests that the hexameric association is tighter than the tetrameric one. The consistency of the structure with the design is discussed, as well as the divergence.

Single-stage hypospadias repair using the operating microscope.

Because of the continuing trend toward earlier repair of hypospadias, improved visualization of tissues is important. We have reviewed 33 consecutive hypospadias repairs performed using the operating microscope. Complications included three strictures (9.1%) and five fistulae (15.1%). The microscope proved most useful in resident teaching, allowing both surgeon and assistant to visualize optimally the tissues.

Effect of plasma proteins and buffer in flushing solutions on rat kidney preservation by cold storage.

The isolated rat kidney perfused at 37 C was used to evaluate the effect of adding plasma proteins to, and varying osmolality of, cold-storage flushing solutions with or without buffering. Addition of albumin improved immediate poststorage kidney function (glomerular filtration rate [GFR], fractional sodium reabsorption, and fractional protein clearance) of all flushing solutions tested after 6 hr and 24 hr of storage. At 6 hr, these improvements also correlated with less weight gain. Flushing solutions containing citrate and sulfate produced significantly better return of function after 24 hr of cold storage than Krebs' or Collins'-derived solutions. Osmolality was unimportant with solutions containing citrate. Collins' solution with reduced MgSO4 yielded better poststorage function than conventional solution. An all-citrate isotonic solution buffered with 15 mmol THAM preserved poststorage function at 48 hr better than a similarly buffered solution containing both citrate and sulfate. Loss of dry weight during storage and subsequent perfusion appeared to correlate, in these experiments, with loss of poststorage function. The isolated rat kidney provides discrimination among various flushing solutions. The technique might be useful in the assay of additional variables that might affect the quality of kidney preservation.

Effect of delayed graft function and ALG on the circaseptan (about 7-day) rhythm of human renal allograft rejection.

Postimplantation records of 157 kidney transplant recipients with first rejection episodes within 50 days of surgery were studied. Of these 36 had living-related and 121 cadaver donors. Recipients of cadaver donor kidneys were divided into four subgroups: with and without postoperative acute renal failure (ARF), and with and without approximately two weeks of immunosuppression by antilymphoblast globulin (ALG) added to conventional therapy. All recipients with immediate function without ALG showed evidence of periodicity in probability of occurrence of rejection that was highly significant for a 7-day period beginning at the time of surgery. The remaining groups showed less significant periodicity or no significant periodicity beginning at the time of surgery, but they did show a highly significant circaseptan rhythm of rejection episodes beginning with cessation of ALG treatment or with onset of diuresis following ARF in the absence of ALG. It is suggested that clinical manifestation of the immunologic attack of recipient upon graft has an intrinsic development period of about 7 days beginning with implantation. However, initiation of the first period may be blocked by ALG or by low renal blood flow during ARF.

Some effects of preservation and acute renal failure on function and proteinuria of renal transplants.

We assessed the effects of preservation on subsequent graft function by measuring and comparing creatinine and fractional protein clearances in 18 live-related (LR) and 38 cadaver-donor (CD) grafts, the latter selected on the basis of short warm ischemia times (less than 5 mins) and stable donor hemodynamic status prior to organ recovery. CD recipients with immediate graft function had lower initial creatinine clearances and greater fractional protein clearances than LR recipients. The role of preservation in producing greater fractional protein clearance was suggested by the observation of time-dependent increasing proteinuria during continuous hypothermic perfusion of four human CD kidneys and the significant correlation between the initial degree of proteinuria at onset of diuresis and the duration of cold preservation of those CD kidneys with immediate function. One-half of CD grafts, however, manifested acute renal failure (ARF) after implantation and in these grafts no correlation between cold preservation duration and fractional protein clearance at onset of diuresis was noted. Development of ARF in CD grafts was associated with still lower creatinine clearances, but higher fractional protein clearances during the first week of diuresis. These effects of preservation and those of preceding ARF on fractional protein clearances were no longer noted 2 wk after onset of diuresis in nonrejecting CD grafts. These observations suggest the presence of preservation-induced injury to grafts even when "immediate function" occurs.

Diagnostic and prognostic significance of an increase in fractional protein clearance ratio before and during rejection of renal transplant.

We measured prospectively changes in fractional protein clearance ratio (CPr/CCr) in 21 live-related (LR) and 41 cadaver donor (CD) renal transplants before and during onset of first rejections. Fifty-three recipients manifested a rejection within the first post-transplant month. Fractional protein clearance increased in all patients during rejection. An increase in CPr/CCr prior to other evidence of impending rejection, and therefore clinically useful, required at least a 10-day rejection-free interval dated from onset of diuresis (whether diuresis was immediate or delayed by acute tubular necrosis (ATN)). Twenty-three of 25 nonantilymphocyte globulin (ALG)-treated CD transplants manifested clinical and laboratory signs of the first rejection episode prior to the 10th day of diuresis compared with 5 of 21 LR and none of 16 ALG-treated CD transplants. Persistence of elevated CPr/CCr despite treatment forecast graft loss (11 of 13), whereas a decrease in this ratio was associated with ultimate reversal of the rejection process.

Diurnal circadian rhythms of electrolyte excretion and filtration rate in end-stage renal disease.

Diurnal excretory rhythms of Na, K, Cl and PO4 were measured in consecutive 3-hour urine collection periods in 2 subjects with advanced renal failure, one of whom was hypertensive, and had been edematous for 4 and 8 days, respectively. Inulin clearance was measured over a 39-hour span in the hypertensive patient. As compared with normal subjects studied under similar conditions, the amplitudes of all rhythms were depressed, the amplitude of K especially so. The rhythm of PO4 excretion was phase-shifted (late AM acrophase) in both patients. The rhythms of Na, Cl and K were phase-shifted in the hypertensive but not the normotensive patient. Ca and Mg rhythms measured in the normotensive subject were within normal limits of amplitude and phase, coinciding with those of Na and Cl. Amplitude of inulin clearance in the hypertensive patient was normal but with reversed phase.

Extracellular fluid of the kidney preserved by the Collins technique.

Hilar drainage fluid of dog kidneys was analyzed as an approximation to renal extracellular fluid after preservation by flushing with chilled high K-low Na solution (Collins C4) followed by ice-cold storage for 24 and 48 hr in a bath of flushing medium. Compared with the medium, Na and Cl were increased to 30 mM/liter and K decreased slightly to 93 mM/liter. Glucose decreased, whereas lactate, lactic dehydrogenase, and creatine phosphokinase increased by significant amounts in both the drainage fluid and bath. The inulin space of the undrained kidney average 37% of wet weight. Calculated intracellular Na and Cl concentrations averaged 50 and 37 mM/kg cell water while K remained within normal limits. A significant fraction of red blood cells retained during initial flushing entered the effluent during storage. Bath and effluent composition of a human cadaver kidney approximated those of a dog.

Diurnal circadian rhythms of renal function and electrolyte excretion in heart failure.

Diurnal cycles of glomerular filtration rate (GFR), effective renal plasma flow (ERPF), and of excretion rates of sodium, potassium, magnesium, chloride and phosphate were measured in a 22 year old man with moderately severe heart failure under standardized conditions. Cycles of GFR, ERPF and excretion of potassium, chloride, and phosphate were indistinguishable from those of normals. The phases of the sodium and probably the magnesium excretory cycles were reversed from normal. The significance of some of the observations is discussed.